44 age groups. This could be due to the fact that as the child grows older the habit of feeding beef increases.\\nMultiple infections occurred in 55 individuals making 6.4% of the total examined subjects and 16.1% of those who had intestinal parasites. The level of double infections with intestinal parasites determined in the present study (6.2%) was much lower than what was reported from southwest Ethiopia portraying a double infection of 35.8% among urban communities [19]. The possible difference in the socio-demographic condition of the study population and the environmental condition might explain the observed difference in double infection in the two study localities.\\nThis study did not assess opportunistic intestinal parasitic infections due to lack of laboratory facilities in our department. Although important risk factors such as age, sex and altitude were considered, some risk factors were not evaluated in the current study. Apart from these limitations our study has the following strengths. It is the first of its kind in the area i.e. pattern of intestinal parasitism was not studied earlier than this current study in the study area. Moreover, all the participants from each kebele were sampled at one specific time in the sampling period to avoid seasonal biases. In addition, the sample was collected from the entire population of highland and lowland dwellers by giving equal probability for each individual in selected kebeles which reflects the real prevalence of intestinal parasites in Gamo area. Furthermore, standard laboratory techniques were used; all laboratory tasks followed standard procedures; and quality control mechanisms were employed at each step of the study.\",\n \"The high prevalence of intestinal parasitic infections among the lowland and highland dwellers in Gamo area indicated that parasitic infections are considerable public health problems. The present study has also revealed that E. histolytica/dispar and Giardia as common protozoan while A. lumberciod, hookworm and T. trichiura as common helminths that cause parasitic infection with varying magnitude in the study area. Enhancing socioeconomic status, improving sanitation facilities, instilling health education and promoting ways of keeping personal hygiene can be good strategies to control these infections in the area.\",\n \"The authors declare that they have no competing interests.\",\n \"TW Conception of the research idea, designing, collection of data, data analysis, interpretation, and manuscript drafting. TT1 Designing, collection of data and manuscript reviewing. BS Collection of data and manuscript reviewing. TT Data analysis, interpretation and manuscript drafting. All authors read and approved the final manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2458/13/151/prepub\\n\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,null,"results","discussion","conclusions",null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null\n]","isConversational":false},"keywords":{"kind":"list like","value":["Control strategies","Intestinal parasites","Parasitism","Protozoan"],"string":"[\n \"Control strategies\",\n \"Intestinal parasites\",\n \"Parasitism\",\n \"Protozoan\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Background:\nIntestinal parasitic infestation represents a large and serious medical and public health problem in developing countries. It is estimated that some 3.5 billion people are affected, and that 450 million are ill as a result of these infections, the majority being children [1]. Apart from causing morbidity and mortality, infection with intestinal parasites has known to cause iron deficiency anemia, growth retardation in children and other physical and mental health problems [2]. Furthermore, chronic intestinal parasitic infections have become the subject of speculation and investigation in relation to the spreading and severity of other infectious diseases of viral origin, tuberculosis and malaria [3-6].\nSeveral factors like climatic conditions, poor sanitation, unsafe drinking water, and lack of toilet facilities are the main contributors to the high prevalence of intestinal parasites in the tropical and sub-tropical countries [7]. In addition, intestinal parasitic agents increase in polluted environments such as refuse heaps, gutters and sewage units in and around human dwelling and living conditions of the people in crowded or unhealthy situations [8]. Hence, a better understanding of the above factors, as well as how social, cultural, behavioral and community awareness affect the epidemiology and control of intestinal parasites may help to design effective control strategies for these diseases [9,10].\nIntestinal parasites are widely distributed in Ethiopia largely due to the low level of environmental and personal hygiene, contamination of food and drinking water that results from improper disposal of human excreta [11,12]. In addition, lack of awareness of simple health promotion practices is also a contributing factor [13]. According to the Ethiopian Ministry of Health [14] more than half a million annual visits of the outpatient services of the health institutions are due to intestinal parasitic infections. However, this report may be an underestimate, because most of the health institutions lack appropriate diagnostic methods to detect low levels of parasite burden. In addition, some of the diagnostic methods for specific intestinal parasites, especially for the newly emerging opportunistic intestinal parasites, are not available to peripheral health institutions.\nPrevious studies gave due attention to the distributions of intestinal parasites in different altitudes, community groups such as school children or other groups confined to camps [15-18]. Hence, the pattern of intestinal parasitism in a community with diverse groups of people as a whole was not illustrated particularly in the study area. The purpose of this study was to assess the magnitude and patterns of intestinal parasitism in highland and lowland dwellers, Gamo area, South Ethiopia.\n\nMethods:\n Study area A community-based cross-sectional study was conducted between September 2010 and July 2011 in four selected areas of Gamo Gofa Zone administrative sub-division: Lante, Kolla Shelle, Dorze and Geressie kebeles. The zone is located at 505 kms South of Addis Ababa and has a total area of 12, 581.4 square kms. The general elevation of the zone ranges from 600 to 3300 m above sea level. The highland and lowland areas of the zone are characterized by an average annual rain fall of 1166 mm and 900 mm, respectively. The topography of the land is characterized by an undulating feature that makes the existence of different climatic zones in the area possible.\nTwo of the study ‘kebeles’ (these are the smallest officially acknowledged administrative vicinities in the zone): Dorze and Geressie kebeles are positioned on the highland areas where as Lante and Kolla Shelle kebeles are situated on lowland settings of the zone. They are located a short distance from the zonal administrative center, Arba Minch. Dorze and Lante are located at 30 kms and 22 kms to the north, where as Geressie and Kolla Shelle are located at 56 kms and 28.6 kms to the south of Arba Minch, respectively. In all study kebeles healthcare is provided by health centers and private clinics which are staffed by few health officers, nurses and laboratory technicians. Though it is irregular and non-continuous, the control measures for intestinal parasites include health education, deworming of under five year children and treatment of drinking water.\nA community-based cross-sectional study was conducted between September 2010 and July 2011 in four selected areas of Gamo Gofa Zone administrative sub-division: Lante, Kolla Shelle, Dorze and Geressie kebeles. The zone is located at 505 kms South of Addis Ababa and has a total area of 12, 581.4 square kms. The general elevation of the zone ranges from 600 to 3300 m above sea level. The highland and lowland areas of the zone are characterized by an average annual rain fall of 1166 mm and 900 mm, respectively. The topography of the land is characterized by an undulating feature that makes the existence of different climatic zones in the area possible.\nTwo of the study ‘kebeles’ (these are the smallest officially acknowledged administrative vicinities in the zone): Dorze and Geressie kebeles are positioned on the highland areas where as Lante and Kolla Shelle kebeles are situated on lowland settings of the zone. They are located a short distance from the zonal administrative center, Arba Minch. Dorze and Lante are located at 30 kms and 22 kms to the north, where as Geressie and Kolla Shelle are located at 56 kms and 28.6 kms to the south of Arba Minch, respectively. In all study kebeles healthcare is provided by health centers and private clinics which are staffed by few health officers, nurses and laboratory technicians. Though it is irregular and non-continuous, the control measures for intestinal parasites include health education, deworming of under five year children and treatment of drinking water.\n Sample size and sampling techniques The sample size was determined using the single proportion population formula. It was calculated based on a prevalence of 83% [19] with a margin of error of 0.05 and a confidence level of 95%. The design effect was calculated by taking the intraclass correlation for the statistic (i.e.1%) in parasitic infection in highland and lowland areas of Ethiopia. A design effect of 4 was used to allow for multistage sampling. The calculated study sample size was 867. The study sites were divided into two regions based on altitude and from each altitude two kebeles were randomly selected. The households were selected using systematic sampling method and the study individuals were chosen using a simple random sampling method. The calculated sample size was divided to each kebele based on population size. As a result, 464 (192 from Lante and 272 from Kolla Shelle) samples were obtained from lowland and 403 (124 from Dorze and 279 from Geressie) samples were obtained from highland settings.\nThe sample size was determined using the single proportion population formula. It was calculated based on a prevalence of 83% [19] with a margin of error of 0.05 and a confidence level of 95%. The design effect was calculated by taking the intraclass correlation for the statistic (i.e.1%) in parasitic infection in highland and lowland areas of Ethiopia. A design effect of 4 was used to allow for multistage sampling. The calculated study sample size was 867. The study sites were divided into two regions based on altitude and from each altitude two kebeles were randomly selected. The households were selected using systematic sampling method and the study individuals were chosen using a simple random sampling method. The calculated sample size was divided to each kebele based on population size. As a result, 464 (192 from Lante and 272 from Kolla Shelle) samples were obtained from lowland and 403 (124 from Dorze and 279 from Geressie) samples were obtained from highland settings.\n Stool collection and processing About 2 g of fresh fecal samples were collected from each consenting study subject and placed in separate labeled clean plastic stool containers. At the time of collection, date of sampling, the name of the participant, age, sex and consistency of the stool (formed, soft, semi-soft and watery) were recorded for each subject on a recording format. A portion of stool was examined at field by direct wet mount with saline (0.85% sodium chloride solution) to observe motile intestinal parasites and trophozoites under light microscope at 100× and 400× magnifications. The remaining part was preserved with 10% formalin in the ratio of 1 g of stool to 3 ml of formalin for later examination at Arba Minch University. Lugol’s iodine staining technique was also done to observe cysts of the intestinal protozoan parasites. A portion of preserved stool sample was processed by formol-ether concentration method as described by Ritchie [20], with some modification. In brief the stool sample was sieved with cotton gauze and transferred to 15 ml centrifuge tube. Then 8 ml of 10% formalin and 3 ml of diethyl ether was added and centrifuged for 2 min at 2000 rpm. The supernatant was discarded and the residues were transferred to microscopic slides and observed under light microscope at 100× and 400× magnifications for the presence of cysts and ova of the parasites. The presence of parasites was confirmed when observed by any of the methods above.\nAbout 2 g of fresh fecal samples were collected from each consenting study subject and placed in separate labeled clean plastic stool containers. At the time of collection, date of sampling, the name of the participant, age, sex and consistency of the stool (formed, soft, semi-soft and watery) were recorded for each subject on a recording format. A portion of stool was examined at field by direct wet mount with saline (0.85% sodium chloride solution) to observe motile intestinal parasites and trophozoites under light microscope at 100× and 400× magnifications. The remaining part was preserved with 10% formalin in the ratio of 1 g of stool to 3 ml of formalin for later examination at Arba Minch University. Lugol’s iodine staining technique was also done to observe cysts of the intestinal protozoan parasites. A portion of preserved stool sample was processed by formol-ether concentration method as described by Ritchie [20], with some modification. In brief the stool sample was sieved with cotton gauze and transferred to 15 ml centrifuge tube. Then 8 ml of 10% formalin and 3 ml of diethyl ether was added and centrifuged for 2 min at 2000 rpm. The supernatant was discarded and the residues were transferred to microscopic slides and observed under light microscope at 100× and 400× magnifications for the presence of cysts and ova of the parasites. The presence of parasites was confirmed when observed by any of the methods above.\n Quality control Before starting the actual work, quality of reagents and instruments were checked by experienced laboratory technologist. The specimens were also checked for serial number, quality and procedures of collection. To eliminate observer bias, each stool sample was examined by two laboratory technicians. The technicians were not informed about the health and other status of the study participants. In cases where the results were discordant, a third senior reader was used. The result of the third expert reader was considered the final result.\nBefore starting the actual work, quality of reagents and instruments were checked by experienced laboratory technologist. The specimens were also checked for serial number, quality and procedures of collection. To eliminate observer bias, each stool sample was examined by two laboratory technicians. The technicians were not informed about the health and other status of the study participants. In cases where the results were discordant, a third senior reader was used. The result of the third expert reader was considered the final result.\n Data analysis Statistical analysis was performed with SPSS software version 16. Chi-square (χ2) was used to verify possible association between infection and exposure to different factors. Probability values were considered to be statistically significant when the calculated P-value was equal to or less than 0.05.\nStatistical analysis was performed with SPSS software version 16. Chi-square (χ2) was used to verify possible association between infection and exposure to different factors. Probability values were considered to be statistically significant when the calculated P-value was equal to or less than 0.05.\n Ethical clearance The study was reviewed and approved by ethical committee of Arba Minch University. The ethical considerations were addressed by treating positive individuals using standard drugs under the supervision of a local nurse. The objective of the study was explained to kebele leaders and dwellers; and written consent was sought from parents or guardians of the selected children during stool sample collection.\nThe study was reviewed and approved by ethical committee of Arba Minch University. The ethical considerations were addressed by treating positive individuals using standard drugs under the supervision of a local nurse. The objective of the study was explained to kebele leaders and dwellers; and written consent was sought from parents or guardians of the selected children during stool sample collection.\n\nStudy area:\nA community-based cross-sectional study was conducted between September 2010 and July 2011 in four selected areas of Gamo Gofa Zone administrative sub-division: Lante, Kolla Shelle, Dorze and Geressie kebeles. The zone is located at 505 kms South of Addis Ababa and has a total area of 12, 581.4 square kms. The general elevation of the zone ranges from 600 to 3300 m above sea level. The highland and lowland areas of the zone are characterized by an average annual rain fall of 1166 mm and 900 mm, respectively. The topography of the land is characterized by an undulating feature that makes the existence of different climatic zones in the area possible.\nTwo of the study ‘kebeles’ (these are the smallest officially acknowledged administrative vicinities in the zone): Dorze and Geressie kebeles are positioned on the highland areas where as Lante and Kolla Shelle kebeles are situated on lowland settings of the zone. They are located a short distance from the zonal administrative center, Arba Minch. Dorze and Lante are located at 30 kms and 22 kms to the north, where as Geressie and Kolla Shelle are located at 56 kms and 28.6 kms to the south of Arba Minch, respectively. In all study kebeles healthcare is provided by health centers and private clinics which are staffed by few health officers, nurses and laboratory technicians. Though it is irregular and non-continuous, the control measures for intestinal parasites include health education, deworming of under five year children and treatment of drinking water.\n\nSample size and sampling techniques:\nThe sample size was determined using the single proportion population formula. It was calculated based on a prevalence of 83% [19] with a margin of error of 0.05 and a confidence level of 95%. The design effect was calculated by taking the intraclass correlation for the statistic (i.e.1%) in parasitic infection in highland and lowland areas of Ethiopia. A design effect of 4 was used to allow for multistage sampling. The calculated study sample size was 867. The study sites were divided into two regions based on altitude and from each altitude two kebeles were randomly selected. The households were selected using systematic sampling method and the study individuals were chosen using a simple random sampling method. The calculated sample size was divided to each kebele based on population size. As a result, 464 (192 from Lante and 272 from Kolla Shelle) samples were obtained from lowland and 403 (124 from Dorze and 279 from Geressie) samples were obtained from highland settings.\n\nStool collection and processing:\nAbout 2 g of fresh fecal samples were collected from each consenting study subject and placed in separate labeled clean plastic stool containers. At the time of collection, date of sampling, the name of the participant, age, sex and consistency of the stool (formed, soft, semi-soft and watery) were recorded for each subject on a recording format. A portion of stool was examined at field by direct wet mount with saline (0.85% sodium chloride solution) to observe motile intestinal parasites and trophozoites under light microscope at 100× and 400× magnifications. The remaining part was preserved with 10% formalin in the ratio of 1 g of stool to 3 ml of formalin for later examination at Arba Minch University. Lugol’s iodine staining technique was also done to observe cysts of the intestinal protozoan parasites. A portion of preserved stool sample was processed by formol-ether concentration method as described by Ritchie [20], with some modification. In brief the stool sample was sieved with cotton gauze and transferred to 15 ml centrifuge tube. Then 8 ml of 10% formalin and 3 ml of diethyl ether was added and centrifuged for 2 min at 2000 rpm. The supernatant was discarded and the residues were transferred to microscopic slides and observed under light microscope at 100× and 400× magnifications for the presence of cysts and ova of the parasites. The presence of parasites was confirmed when observed by any of the methods above.\n\nQuality control:\nBefore starting the actual work, quality of reagents and instruments were checked by experienced laboratory technologist. The specimens were also checked for serial number, quality and procedures of collection. To eliminate observer bias, each stool sample was examined by two laboratory technicians. The technicians were not informed about the health and other status of the study participants. In cases where the results were discordant, a third senior reader was used. The result of the third expert reader was considered the final result.\n\nData analysis:\nStatistical analysis was performed with SPSS software version 16. Chi-square (χ2) was used to verify possible association between infection and exposure to different factors. Probability values were considered to be statistically significant when the calculated P-value was equal to or less than 0.05.\n\nEthical clearance:\nThe study was reviewed and approved by ethical committee of Arba Minch University. The ethical considerations were addressed by treating positive individuals using standard drugs under the supervision of a local nurse. The objective of the study was explained to kebele leaders and dwellers; and written consent was sought from parents or guardians of the selected children during stool sample collection.\n\nResults:\nA total of 867 study participants were selected for investigation. However, 10(1.2%) were excluded because of inability to provide specimen. For this reason a total of 858 individuals were included in the study (Table 1). Three hundred forty two of the study individuals were found to have single or multiple intestinal parasitic infections, which make the overall prevalence 39.9%. Of the entire positive samples for the parasite, 188 were female participants and 154 were male participants with female to male ratio of 1:0.8 (Table 1). The mean age of the participants was 25 ± 19.\n\nFrequency distribution of sex, age group and altitude of the study subjects in Gamo area\nKey: * Lante and Kolla Shelle.\n** Dorze and Gereessie.\nDifferent types of parasites including protozoans, trematode, cestodes and nematodes were detected from the stool samples of study participants. Prevalence of Entamoeba histolytica/dispar (E. histolytica/dispar) was the highest 98(11.4%), followed by Giardia lamblia (G. lamblia) 91(10.6%), Ascaris lumbricoides (A. lumbricoides) 67(7.8%), Strongyloides stercoralis (S. stercoralis) 51(5.9%), hookworm 42(4.9%), Trichuris trichiura (T. trichiura) 24(2.8%), Taenia species (Taenia spp.) 18(2.1%), Hymenolepis nana (H. nana) 7(0.6%) and Schistosoma mansoni (S. mansoni) 1(0.12%), in that order. The majorities of the positive cases were single infections (83.9%) and double infections (15.5%). Of the triple infected persons, one was coinfected with E. histolytica/dispar, G. lamblia and Taenia spp. and the other with E. histolytica/dispar, S. stercoralis and hookworm (Figure 1).\n\nSingle and mixed infections among residents of Gamo area, Gamo Gofa Zone, South Ethiopia.\nThe prevalence of infection with different intestinal helminths and protozoan parasites for lowland (Lante and Kolla Shelle) and highland (Dorze and Gressie) is shown in Table 2. Out of 459 stool samples collected from lowland area, 174(37.9%) were positive for at least one parasite. Similarly, of the 399 stool samples collected from highland area, 169(42.3%) were positive for at least one parasite. No statistically significant difference was observed (P = 0.185) between presence of intestinal parasites and altitude (Tables 1 and 2). However, G. lamblia (P < 0.001) and hookworm (P = 0.002) were significantly more prevalent in lowland areas whereas A. lumbricoides (P < 0.001) and T. trichiura (P < 0.001) were significantly more prevalent in highland areas, but S. stercoralis was exclusive to lowland areas (Table 2).\n\nPrevalence of intestinal parasites among study subjects in lowland and highland dwellers in Gamo area\nKey: χ2 and P-value values represent lowland and highland comparison.\n* Represent statistically significant difference (P < 0.05).\nAs observed in Table 2, of 190 and 269 stool samples collected from Lante and Kolla Shelle, 66(34.7%) and 108(40.1%) were found positive for at least one parasite, respectively. Similarly, out of 123 and 276 fecal samples collected from Dorze and Geressie sites, 50(40.7%) and 118(42.8%) were infected with one or more intestinal parasites. The overall difference in parasite prevalence was no statistically significant among the study sites, (P = 0.38). However, E. histolytica/dispar prevalence was significantly higher in Geressie (P < 0.001) than other study areas. A. lumbricoides and T. trichiura were significantly higher in Dorze (P < 0.005) and S. mansoni was detected in Lante alone.\nThe distribution of infection among female and male participants is shown in Table 3. Female participants showed the highest infection rate (41.0%), followed closely by male participants (38.6%) (Table 1). The calculated P-value (0.48) indicates that the difference in the prevalence of intestinal parasites between female and male participants was statistically not significant. However, A. lumbricoides infection was significantly higher (P = 0.004) among female participants than male participants (Table 3).\n\nSex related prevalence of intestinal parasites among study subjects in Gamo area\nKey: * Represent statistically significant difference (P < 0.05).\nTo see variations in age group, the study population was divided into 4 age groups: birth to 4 years, 5 to 14 years, 15–44 years, and over 44 years. The distribution of infection among study subjects in the different age groups is shown in Table 4. The overall infection rate was highest among the 15–44 years age group (42.3%) followed by above 44 years age group (39.7%). Only 29.4% of children from birth to 4 years age group were infected. The difference in the prevalence of intestinal parasites was insignificant (P = 0.228) among the age groups (Table 1). Each parasite was also not associated with the age of the participant (P > 0.005) except T. trichiura which shown significantly high (P = 0.007) prevalence among the age group of 5–14 years (Table 4).\n\nAge related prevalence of intestinal parasites among study subjects in Gamo area\nKey: < 4 years = Preschool children, 5–14 years = School children, 15–44 years = Reproductive age, > 44 years = elder.\n* Represent statistically significant difference (P < 0.05).\n\nDiscussion:\nPrimary objectives of epidemiological studies on the prevalence of infection of intestinal parasites in different regions/localities are to identify high-risk communities and formulate appropriate intervention. In line with this view, the present study attempted to assess the prevalence of different intestinal parasitic infections in highland and lowland dwellers in Gamo area, Gamo Gofa Zone and then recommend appropriate intervention.\nThe results of the study showed the occurrence of several intestinal parasites of public health importance among inhabitants in four kebeles found in Gamo area of Gamo Gofa Zone, South Nations. The overall prevalence of 39.9% with one or more intestinal parasites found in this study was much lower than what was reported (82.8%) from residents of four villages in southwestern Ethiopia by Yeneneh [21]; (83.8%) from school-children around Lake Langano by Legesse and Erko [22] and from that of Mengistu and collegues [19] (83%) from urban dwellers in southwest Ethiopia. However, the prevalence in our study was slightly higher compared to other community-based studies conducted in Saudi Arabia by Al-Shammari et al.[23] showing an overall prevalence of 32.2%. The possible explanations for the discrepancy between the present and previous study finding might be the result of variation in sampling techniques used, the difference in the quality of drinking water source, and variation in the environmental condition of the different study localities.\nThe prevalence of E. histolytica/dispar and G. lamblia infection in this study was 11.4% and 10.6%, respectively. These are within the range of the nation-wide prevalence rate for amoebiasis and giardiasis [24]. However, the present finding was relatively higher than that reported from urban dwellers in southwest Ethiopia, where Mengistu et al.[19] recorded 3.1% and 3.6% for amoebiasis and giardiasis, respectively [19]. The prevalence of giardiasis was also higher than that of Birrie and Erko report of 3.1% among non-school children [25]. Giardia cysts have been isolated from water supplies in different parts of the world [26,27]. Epidemic giardiasis may be related to drinking water [28]. The present study was also conducted in a rural area that may share the mentioned risk factors.\nThe level of ascariasis observed in this study (7.8%) of fecal samples was far lower than that reported from urban dwellers in Jimma, where Mengistu et al.[19] recorded 41.0% prevalence. The prevalence of hookworm infection was 42(4.9%). The rate is lower than the previous community based study in Jimma, where Mengistu and colleagues recorded 17.5% prevalence [19]. However, the present findings on prevalence of S. stercoralis, H. nana, and Taenia spp. was not much different from the findings of previous studies reported by Woldemichael et al., McConnel and Armstrong [18,24].\nAlthough significant difference was not observed, the prevalence of intestinal parasites infections was slightly lower among lowland dwellers (37.9%) than highland dwellers (42.3%). The existence of relatively low prevalence of human intestinal parasites in lowlands is due to low prevalence of A. lumbricoides and T. trichiura. A nation-wide study conducted on ascariasis in Ethiopia has indicated a low prevalence of ascariasis in the low and dry areas of the country [29]. In agreement to this report, the present study showed relatively low prevalence of ascariasis (0.7%) and trichuriasis (0.2%) among lowland dwellers. This might be because of proper toilet facilities, apposite hand washing habit and better awareness about health in lowland dwellers than highland dwellers in our study area.\nThough the reason for high prevalence of G. lamblia in lowland area is unclear, the high prevalence of hookworm infection might be contributed to low shoe wearing habit during irrigation. According to the geo climatic type of area, significant highest prevalence of S. stercoralis infection was found in lowland. The explanation of this is that prevalence of S. stercoralis infection is parallel with hookworm infection [30]. This may lead us to a conclusion that geo climatic factors, such as soil textures, farming ecosystem, temperature, humidity and rainfall essentially influence the infection of S. stercoralis as the lowlands are located near Lake Abaya.\nFrom the four study sites, S. mansoni was reported only from Lante kebele. However, the prevalence of S. mansoni (0.12) was much lower as compared to 14.8% reported by Mengistu et al.[19]. Although this study did not evaluate the migratory history of the study participants, the existence of such low level infection rate may imply the relocation of infected individual from endemic foci. Nevertheless, the presence of schistosome-infected dweller in Lante kebele represents a risk for the introduction of a new transmission focus where the snail hosts might be available. Hence, extensive study on the transmission foci in the area is recommended to take timely intervention.\nVariations that might have occurred due to gender and age group differences were also examined in this current study. Variation of results due to gender differences was not observed in the study. The result was similar to a study conducted in the central part of Turkey [31]. But A. lumbricoides infection was significantly higher among female participants than male participants in this study. Though significant difference among age groups was not observed in the current study, the prevalence of infection was higher in reproductive age group. This might be due to the fact that this is the age group of working people who are engaged in agriculture and most likely exposed to infection through contaminated soil, water and food. The rate of infection by Taenia spp. was also higher in 15–44 and > 44 age groups. This could be due to the fact that as the child grows older the habit of feeding beef increases.\nMultiple infections occurred in 55 individuals making 6.4% of the total examined subjects and 16.1% of those who had intestinal parasites. The level of double infections with intestinal parasites determined in the present study (6.2%) was much lower than what was reported from southwest Ethiopia portraying a double infection of 35.8% among urban communities [19]. The possible difference in the socio-demographic condition of the study population and the environmental condition might explain the observed difference in double infection in the two study localities.\nThis study did not assess opportunistic intestinal parasitic infections due to lack of laboratory facilities in our department. Although important risk factors such as age, sex and altitude were considered, some risk factors were not evaluated in the current study. Apart from these limitations our study has the following strengths. It is the first of its kind in the area i.e. pattern of intestinal parasitism was not studied earlier than this current study in the study area. Moreover, all the participants from each kebele were sampled at one specific time in the sampling period to avoid seasonal biases. In addition, the sample was collected from the entire population of highland and lowland dwellers by giving equal probability for each individual in selected kebeles which reflects the real prevalence of intestinal parasites in Gamo area. Furthermore, standard laboratory techniques were used; all laboratory tasks followed standard procedures; and quality control mechanisms were employed at each step of the study.\n\nConclusion:\nThe high prevalence of intestinal parasitic infections among the lowland and highland dwellers in Gamo area indicated that parasitic infections are considerable public health problems. The present study has also revealed that E. histolytica/dispar and Giardia as common protozoan while A. lumberciod, hookworm and T. trichiura as common helminths that cause parasitic infection with varying magnitude in the study area. Enhancing socioeconomic status, improving sanitation facilities, instilling health education and promoting ways of keeping personal hygiene can be good strategies to control these infections in the area.\n\nCompeting interests:\nThe authors declare that they have no competing interests.\n\nAuthors’ contributions:\nTW Conception of the research idea, designing, collection of data, data analysis, interpretation, and manuscript drafting. TT1 Designing, collection of data and manuscript reviewing. BS Collection of data and manuscript reviewing. TT Data analysis, interpretation and manuscript drafting. All authors read and approved the final manuscript.\n\nPre-publication history:\nThe pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2458/13/151/prepub\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nEpidemiological information on the prevalence of intestinal parasitic infections in different regions is a prerequisite to develop appropriate control strategies. Therefore, this present study was conducted to assess the magnitude and pattern of intestinal parasitism in highland and lowland dwellers in Gamo area, South Ethiopia.\n\nMethods:\nCommunity-based cross-sectional study was conducted between September 2010 and July 2011 at Lante, Kolla Shelle, Dorze and Geressie kebeles of Gamo Gofa Zone, South Ethiopia. The study sites and study participants were selected using multistage sampling method. Data were gathered through house-to-house survey. A total of 858 stool specimens were collected and processed using direct wet mount and formol-ether concentration techniques for the presence of parasite.\n\nResults:\nOut of the total examined subjects, 342(39.9%) were found positive for at least one intestinal parasite. The prevalence of Entamoeba histolytica/dispar was the highest 98(11.4%), followed by Giardia lamblia 91(10.6%), Ascaris lumbricoides 67(7.8%), Strongyloides stercoralis 51(5.9%), hookworm 42(4.9%), Trichuris trichiura 24(2.8%), Taenia species 18(2.1%), Hymenolepis nana 7(0.6%) and Schistosoma mansoni 1(0.12%). No statistically significant difference was observed in the prevalence of intestinal parasitic infections among lowland (37.9%) and highland dwellers (42.3%) (P = 0.185). The prevalence of intestinal parasitic infection was not significantly different among the study sites but it was relatively higher in Geressie (42.8%) than other kebeles. Sex was not associated with parasitic infections (P = 0.481). No statistically significant difference of infection was observed among the age groups (P = 0.228) but it was higher in reproductive age group.\n\nConclusions:\nThe high prevalence of intestinal parasitic infections among the lowland and highland dwellers in Gamo area indicated that parasitic infections are important public health problems. Thus, infection control measures and the development of awareness strategies to improve sanitation and health education should be considered."},"background_conclusion_text":{"kind":"string","value":"Background:\nIntestinal parasitic infestation represents a large and serious medical and public health problem in developing countries. It is estimated that some 3.5 billion people are affected, and that 450 million are ill as a result of these infections, the majority being children [1]. Apart from causing morbidity and mortality, infection with intestinal parasites has known to cause iron deficiency anemia, growth retardation in children and other physical and mental health problems [2]. Furthermore, chronic intestinal parasitic infections have become the subject of speculation and investigation in relation to the spreading and severity of other infectious diseases of viral origin, tuberculosis and malaria [3-6].\nSeveral factors like climatic conditions, poor sanitation, unsafe drinking water, and lack of toilet facilities are the main contributors to the high prevalence of intestinal parasites in the tropical and sub-tropical countries [7]. In addition, intestinal parasitic agents increase in polluted environments such as refuse heaps, gutters and sewage units in and around human dwelling and living conditions of the people in crowded or unhealthy situations [8]. Hence, a better understanding of the above factors, as well as how social, cultural, behavioral and community awareness affect the epidemiology and control of intestinal parasites may help to design effective control strategies for these diseases [9,10].\nIntestinal parasites are widely distributed in Ethiopia largely due to the low level of environmental and personal hygiene, contamination of food and drinking water that results from improper disposal of human excreta [11,12]. In addition, lack of awareness of simple health promotion practices is also a contributing factor [13]. According to the Ethiopian Ministry of Health [14] more than half a million annual visits of the outpatient services of the health institutions are due to intestinal parasitic infections. However, this report may be an underestimate, because most of the health institutions lack appropriate diagnostic methods to detect low levels of parasite burden. In addition, some of the diagnostic methods for specific intestinal parasites, especially for the newly emerging opportunistic intestinal parasites, are not available to peripheral health institutions.\nPrevious studies gave due attention to the distributions of intestinal parasites in different altitudes, community groups such as school children or other groups confined to camps [15-18]. Hence, the pattern of intestinal parasitism in a community with diverse groups of people as a whole was not illustrated particularly in the study area. The purpose of this study was to assess the magnitude and patterns of intestinal parasitism in highland and lowland dwellers, Gamo area, South Ethiopia.\n\nConclusion:\nThe high prevalence of intestinal parasitic infections among the lowland and highland dwellers in Gamo area indicated that parasitic infections are considerable public health problems. The present study has also revealed that E. histolytica/dispar and Giardia as common protozoan while A. lumberciod, hookworm and T. trichiura as common helminths that cause parasitic infection with varying magnitude in the study area. Enhancing socioeconomic status, improving sanitation facilities, instilling health education and promoting ways of keeping personal hygiene can be good strategies to control these infections in the area."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nEpidemiological information on the prevalence of intestinal parasitic infections in different regions is a prerequisite to develop appropriate control strategies. Therefore, this present study was conducted to assess the magnitude and pattern of intestinal parasitism in highland and lowland dwellers in Gamo area, South Ethiopia.\n\nMethods:\nCommunity-based cross-sectional study was conducted between September 2010 and July 2011 at Lante, Kolla Shelle, Dorze and Geressie kebeles of Gamo Gofa Zone, South Ethiopia. The study sites and study participants were selected using multistage sampling method. Data were gathered through house-to-house survey. A total of 858 stool specimens were collected and processed using direct wet mount and formol-ether concentration techniques for the presence of parasite.\n\nResults:\nOut of the total examined subjects, 342(39.9%) were found positive for at least one intestinal parasite. The prevalence of Entamoeba histolytica/dispar was the highest 98(11.4%), followed by Giardia lamblia 91(10.6%), Ascaris lumbricoides 67(7.8%), Strongyloides stercoralis 51(5.9%), hookworm 42(4.9%), Trichuris trichiura 24(2.8%), Taenia species 18(2.1%), Hymenolepis nana 7(0.6%) and Schistosoma mansoni 1(0.12%). No statistically significant difference was observed in the prevalence of intestinal parasitic infections among lowland (37.9%) and highland dwellers (42.3%) (P = 0.185). The prevalence of intestinal parasitic infection was not significantly different among the study sites but it was relatively higher in Geressie (42.8%) than other kebeles. Sex was not associated with parasitic infections (P = 0.481). No statistically significant difference of infection was observed among the age groups (P = 0.228) but it was higher in reproductive age group.\n\nConclusions:\nThe high prevalence of intestinal parasitic infections among the lowland and highland dwellers in Gamo area indicated that parasitic infections are important public health problems. Thus, infection control measures and the development of awareness strategies to improve sanitation and health education should be considered."},"whole_article_text_length":{"kind":"number","value":6022,"string":"6,022"},"whole_article_abstract_length":{"kind":"number","value":383,"string":"383"},"other_sections_lengths":{"kind":"list like","value":[481,284,183,269,92,52,65,10,58,16],"string":"[\n 481,\n 284,\n 183,\n 269,\n 92,\n 52,\n 65,\n 10,\n 58,\n 16\n]","isConversational":false},"num_sections":{"kind":"number","value":14,"string":"14"},"most_frequent_words":{"kind":"list like","value":["study","intestinal","prevalence","parasites","stool","area","infection","intestinal parasites","lowland","sample"],"string":"[\n \"study\",\n \"intestinal\",\n \"prevalence\",\n \"parasites\",\n \"stool\",\n \"area\",\n \"infection\",\n \"intestinal parasites\",\n \"lowland\",\n \"sample\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["intestinal parasitic infections","intestinal parasites help","intestinal parasites especially","intestinal parasites level","prevalence intestinal parasitic"],"string":"[\n \"intestinal parasitic infections\",\n \"intestinal parasites help\",\n \"intestinal parasites especially\",\n \"intestinal parasites level\",\n \"prevalence intestinal parasitic\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Control strategies | Intestinal parasites | Parasitism | Protozoan [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Control strategies | Intestinal parasites | Parasitism | Protozoan [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Control strategies | Intestinal parasites | Parasitism | Protozoan [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Control strategies | Intestinal parasites | Parasitism | Protozoan [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Control strategies | Intestinal parasites | Parasitism | Protozoan [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Control strategies | Intestinal parasites | Parasitism | Protozoan [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Age Distribution | Child | Child, Preschool | Cross-Sectional Studies | Ethiopia | Feces | Female | Humans | Infant | Infant, Newborn | Intestinal Diseases, Parasitic | Male | Middle Aged | Prevalence | Sex Distribution | Young Adult [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Age Distribution | Child | Child, Preschool | Cross-Sectional Studies | Ethiopia | Feces | Female | Humans | Infant | Infant, Newborn | Intestinal Diseases, Parasitic | Male | Middle Aged | Prevalence | Sex Distribution | Young Adult [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Age Distribution | Child | Child, Preschool | Cross-Sectional Studies | Ethiopia | Feces | Female | Humans | Infant | Infant, Newborn | Intestinal Diseases, Parasitic | Male | Middle Aged | Prevalence | Sex Distribution | Young Adult [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Age Distribution | Child | Child, Preschool | Cross-Sectional Studies | Ethiopia | Feces | Female | Humans | Infant | Infant, Newborn | Intestinal Diseases, Parasitic | Male | Middle Aged | Prevalence | Sex Distribution | Young Adult [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Age Distribution | Child | Child, Preschool | Cross-Sectional Studies | Ethiopia | Feces | Female | Humans | Infant | Infant, Newborn | Intestinal Diseases, Parasitic | Male | Middle Aged | Prevalence | Sex Distribution | Young Adult [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Age Distribution | Child | Child, Preschool | Cross-Sectional Studies | Ethiopia | Feces | Female | Humans | Infant | Infant, Newborn | Intestinal Diseases, Parasitic | Male | Middle Aged | Prevalence | Sex Distribution | Young Adult [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] intestinal parasitic infections | intestinal parasites help | intestinal parasites especially | intestinal parasites level | prevalence intestinal parasitic [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] intestinal parasitic infections | intestinal parasites help | intestinal parasites especially | intestinal parasites level | prevalence intestinal parasitic [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] intestinal parasitic infections | intestinal parasites help | intestinal parasites especially | intestinal parasites level | prevalence intestinal parasitic [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] intestinal parasitic infections | intestinal parasites help | intestinal parasites especially | intestinal parasites level | prevalence intestinal parasitic [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] intestinal parasitic infections | intestinal parasites help | intestinal parasites especially | intestinal parasites level | prevalence intestinal parasitic [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] intestinal parasitic infections | intestinal parasites help | intestinal parasites especially | intestinal parasites level | prevalence intestinal parasitic [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] study | intestinal | prevalence | parasites | stool | area | infection | intestinal parasites | lowland | sample [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] study | intestinal | prevalence | parasites | stool | area | infection | intestinal parasites | lowland | sample [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] study | intestinal | prevalence | parasites | stool | area | infection | intestinal parasites | lowland | sample [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] study | intestinal | prevalence | parasites | stool | area | infection | intestinal parasites | lowland | sample [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] study | intestinal | prevalence | parasites | stool | area | infection | intestinal parasites | lowland | sample [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] study | intestinal | prevalence | parasites | stool | area | infection | intestinal parasites | lowland | sample [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] intestinal | intestinal parasites | parasites | health | institutions | health institutions | intestinal parasitic | addition | people | lack [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] stool | kms | study | sample | zone | kebeles | size | calculated | ml | sampling [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] years | table | age | participants | prevalence | difference | 44 years | parasites | study | group [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] infections | common | parasitic | area | parasitic infections | health | good strategies | ways keeping | socioeconomic status improving sanitation | socioeconomic status improving [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] study | intestinal | parasites | stool | prevalence | area | health | sample | infection | intestinal parasites [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] study | intestinal | parasites | stool | prevalence | area | health | sample | infection | intestinal parasites [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Gamo | South Ethiopia [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] between September 2010 and July 2011 | Dorze | Geressie | Gofa Zone | South Ethiopia ||| ||| ||| 858 | wet mount [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 342(39.9% | at least one ||| Entamoeba | 98(11.4% | Giardia | 91(10.6% | Ascaris | 67(7.8% | Strongyloides | 51(5.9% | 42(4.9% | Trichuris | 24(2.8% | Taenia | 18(2.1% | Hymenolepis | 7(0.6% | Schistosoma | 1(0.12% ||| 37.9% | 42.3% | 0.185 ||| Geressie | 42.8% ||| 0.481 ||| 0.228 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Gamo ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Gamo | South Ethiopia ||| between September 2010 and July 2011 | Dorze | Geressie | Gofa Zone | South Ethiopia ||| ||| ||| 858 | wet mount ||| 342(39.9% | at least one ||| Entamoeba | 98(11.4% | Giardia | 91(10.6% | Ascaris | 67(7.8% | Strongyloides | 51(5.9% | 42(4.9% | Trichuris | 24(2.8% | Taenia | 18(2.1% | Hymenolepis | 7(0.6% | Schistosoma | 1(0.12% ||| 37.9% | 42.3% | 0.185 ||| Geressie | 42.8% ||| 0.481 ||| 0.228 ||| Gamo ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Gamo | South Ethiopia ||| between September 2010 and July 2011 | Dorze | Geressie | Gofa Zone | South Ethiopia ||| ||| ||| 858 | wet mount ||| 342(39.9% | at least one ||| Entamoeba | 98(11.4% | Giardia | 91(10.6% | Ascaris | 67(7.8% | Strongyloides | 51(5.9% | 42(4.9% | Trichuris | 24(2.8% | Taenia | 18(2.1% | Hymenolepis | 7(0.6% | Schistosoma | 1(0.12% ||| 37.9% | 42.3% | 0.185 ||| Geressie | 42.8% ||| 0.481 ||| 0.228 ||| Gamo ||| [SUMMARY]"}}},{"rowIdx":50,"cells":{"title":{"kind":"string","value":"The etiological roles of miRNAs, lncRNAs, and circRNAs in neuropathic pain: A narrative review."},"pmid":{"kind":"string","value":"35808924"},"background_abstract":{"kind":"string","value":"Non-coding RNAs (ncRNAs) are involved in neuropathic pain development. Herein, we systematically searched for neuropathic pain-related ncRNAs expression changes, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular non-coding RNAs (circRNAs)."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We searched two databases, PubMed and GeenMedical, for relevant studies."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Peripheral nerve injury or noxious stimuli can induce extensive changes in the expression of ncRNAs. For example, higher serum miR-132-3p, -146b-5p, and -384 was observed in neuropathic pain patients. Either sciatic nerve ligation, dorsal root ganglion (DRG) transaction, or ventral root transection (VRT) could upregulate miR-21 and miR-31 while downregulating miR-668 and miR-672 in the injured DRG. lncRNAs, such as early growth response 2-antisense-RNA (Egr2-AS-RNA) and Kcna2-AS-RNA, were upregulated in Schwann cells and inflicted DRG after nerve injury, respectively. Dysregulated circRNA homeodomain-interacting protein kinase 3 (circHIPK3) in serum and the DRG, abnormally expressed lncRNAs X-inactive specific transcript (XIST), nuclear enriched abundant transcript 1 (NEAT1), small nucleolar RNA host gene 1 (SNHG1), as well as ciRS-7, zinc finger protein 609 (cirZNF609), circ_0005075, and circAnks1a in the spinal cord were suggested to participate in neuropathic pain development. Dysregulated miRNAs contribute to neuropathic pain via neuroinflammation, autophagy, abnormal ion channel expression, regulating pain-related mediators, protein kinases, structural proteins, neurotransmission excitatory-inhibitory imbalances, or exosome miRNA-mediated neuron-glia communication. In addition, lncRNAs and circRNAs are essential in neuropathic pain by acting as antisense RNA and miRNA sponges, epigenetically regulating pain-related molecules expression, or modulating miRNA processing."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Numerous dysregulated ncRNAs have been suggested to participate in neuropathic pain development. However, there is much work to be done before ncRNA-based analgesics can be clinically used for various reasons such as conservation among species, proper delivery, stability, and off-target effects."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Ganglia, Spinal","Humans","MicroRNAs","Neuralgia","RNA, Circular","RNA, Long Noncoding"],"string":"[\n \"Ganglia, Spinal\",\n \"Humans\",\n \"MicroRNAs\",\n \"Neuralgia\",\n \"RNA, Circular\",\n \"RNA, Long Noncoding\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"9396192"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"According to the International Association for the Study of Pain, neuropathic pain is the most severe chronic pain condition triggered by a lesion or disease of the somatosensory system. It is characteristic of hyperalgesia, allodynia, or spontaneous pain.\n1\n Neuropathic pain can have peripheral and central origins, with the former including neuropathic pain after peripheral nerve injury, trigeminal neuralgia, postherpetic neuralgia, painful radiculopathy, and painful polyneuropathy. In contrast, central neuropathic pain includes neuropathic pain after spinal cord or brain injury, multiple sclerosis, and chronic central post‐stroke pain. Approximately 7%–10% of the general population will experience neuropathic pain, with the majority not having satisfactory pain relief with current therapies, leading to great suffering for individuals and enormous economic and social burdens.\n2\n\n\nThe exact molecular mechanisms underlying neuropathic pain remain unclear, and elucidating them is crucial for developing mechanism‐based treatment strategies. One proposed mechanism involves altered gene or protein expression along the pain processing pathways. Therefore, understanding how genes or proteins are dysregulated may help us to find a way to normalize these abnormalities and treat neuropathic pain.\nIn recent years, accumulating evidence has suggested the essential role of non‐coding RNAs (ncRNAs) in various physiological and pathological procedures, such as embryonic development, inflammation, tumors, and respiratory and cardiovascular diseases.\n3\n ncRNAs have no protein‐coding potential, but they can govern gene or protein expression with diverse mechanisms. ncRNAs are extensively distributed in the peripheral and central nervous systems, including pain‐related structures.\n4\n Broad abnormal ncRNAs expression is observed following peripheral stimulation. These abnormalities are related to hyperalgesia during chronic pain development. The available data indicate that ncRNAs may be essential for hyperalgesia. In this review, we focus on microRNA (miRNA), long non‐coding RNA (lncRNA), and circular non‐coding RNA (circRNA) expression changes in neuropathic pain. Other types of ncRNAs are seldom reported in neuropathic pain and, thus, are not discussed herein. Notably, we will pay attention to their etiological role in the development of neuropathic pain and the current challenges and considerations for miRNA‐, lncRNA‐, and circRNA‐based therapeutics for neuropathic pain."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"null"},"results_text":{"kind":"null"},"conclusions_title":{"kind":"string","value":"CONCLUSION"},"conclusions_text":{"kind":"string","value":"Recent human and animal studies have identified accumulating dysregulated miRNAs and lncRNAs in the serum or along pain processing pathways following peripheral nerve injury or noxious stimulation. Experimental studies have validated their essential role in neuropathic pain. miRNAs contribute to neuropathic pain development via neuroinflammation, autophagy, abnormal ion channel expression, regulating pain‐related mediators, protein kinases, structural proteins, neurotransmission excitatory–‐inhibitory imbalances, and exosome miRNA‐mediated neuron–glia communication. Meanwhile, lncRNAs and circRNAs are crucial in neuropathic pain development by acting as AS RNA and miRNA sponges, epigenetically regulating pain‐related molecules expression, or modulating miRNA processing. However, more work is required before miRNA‐ and lncRNA‐based analgesics can be clinically used for various reasons, including their conservation among species, proper delivery, stability, off‐target effects, and potential activation of the immune system."},"other_sections_titles":{"kind":"list like","value":["INTRODUCTION","\nBIOGENESIS AND FUNCTION OF miRNAs, lncRNAs, AND circRNAs\n","\nPERIPHERAL NERVE INJURY OR NOXIOUS STIMULI INDUCE EXTENSIVE miRNAs, lncRNAs, AND circRNAs EXPRESSION CHANGES\n","\nmiRNAs expression changes","\nlncRNAs and circRNAs expression changes","\nSTUDYING miRNA NEUROPATHIC PAIN MECHANISMS\n","\nmiRNAs regulate neuroinflammation in neuropathic pain development","Autophagy","The contribution of miRNAs to ion channel expression in neuropathic pain","\nmiRNAs regulate pain‐related mediators, protein kinases, and structural proteins","Neurotransmission excitatory–inhibitory imbalances","\nEV miRNAs‐mediated neuron–glia communication","\nSTUDYING lncRNAs AND circRNAs NEUROPATHIC PAIN MECHANISMS\n","\nlncRNAs act as AS RNA\n","\nlncRNAs act as miRNA sponges","\nlncRNAs epigenetically regulate pain‐related molecules expression","\nlncRNAs modulate miRNA processing","\nCHALLENGES AND CONSIDERATIONS IN DELIVERING miRNA‐AND lncRNAs‐BASED THERAPETUTICS\n","AUTHOR CONTRIBUTIONS"],"string":"[\n \"INTRODUCTION\",\n \"\\nBIOGENESIS AND FUNCTION OF miRNAs, lncRNAs, AND circRNAs\\n\",\n \"\\nPERIPHERAL NERVE INJURY OR NOXIOUS STIMULI INDUCE EXTENSIVE miRNAs, lncRNAs, AND circRNAs EXPRESSION CHANGES\\n\",\n \"\\nmiRNAs expression changes\",\n \"\\nlncRNAs and circRNAs expression changes\",\n \"\\nSTUDYING miRNA NEUROPATHIC PAIN MECHANISMS\\n\",\n \"\\nmiRNAs regulate neuroinflammation in neuropathic pain development\",\n \"Autophagy\",\n \"The contribution of miRNAs to ion channel expression in neuropathic pain\",\n \"\\nmiRNAs regulate pain‐related mediators, protein kinases, and structural proteins\",\n \"Neurotransmission excitatory–inhibitory imbalances\",\n \"\\nEV miRNAs‐mediated neuron–glia communication\",\n \"\\nSTUDYING lncRNAs AND circRNAs NEUROPATHIC PAIN MECHANISMS\\n\",\n \"\\nlncRNAs act as AS RNA\\n\",\n \"\\nlncRNAs act as miRNA sponges\",\n \"\\nlncRNAs epigenetically regulate pain‐related molecules expression\",\n \"\\nlncRNAs modulate miRNA processing\",\n \"\\nCHALLENGES AND CONSIDERATIONS IN DELIVERING miRNA‐AND lncRNAs‐BASED THERAPETUTICS\\n\",\n \"AUTHOR CONTRIBUTIONS\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["According to the International Association for the Study of Pain, neuropathic pain is the most severe chronic pain condition triggered by a lesion or disease of the somatosensory system. It is characteristic of hyperalgesia, allodynia, or spontaneous pain.\n1\n Neuropathic pain can have peripheral and central origins, with the former including neuropathic pain after peripheral nerve injury, trigeminal neuralgia, postherpetic neuralgia, painful radiculopathy, and painful polyneuropathy. In contrast, central neuropathic pain includes neuropathic pain after spinal cord or brain injury, multiple sclerosis, and chronic central post‐stroke pain. Approximately 7%–10% of the general population will experience neuropathic pain, with the majority not having satisfactory pain relief with current therapies, leading to great suffering for individuals and enormous economic and social burdens.\n2\n\n\nThe exact molecular mechanisms underlying neuropathic pain remain unclear, and elucidating them is crucial for developing mechanism‐based treatment strategies. One proposed mechanism involves altered gene or protein expression along the pain processing pathways. Therefore, understanding how genes or proteins are dysregulated may help us to find a way to normalize these abnormalities and treat neuropathic pain.\nIn recent years, accumulating evidence has suggested the essential role of non‐coding RNAs (ncRNAs) in various physiological and pathological procedures, such as embryonic development, inflammation, tumors, and respiratory and cardiovascular diseases.\n3\n ncRNAs have no protein‐coding potential, but they can govern gene or protein expression with diverse mechanisms. ncRNAs are extensively distributed in the peripheral and central nervous systems, including pain‐related structures.\n4\n Broad abnormal ncRNAs expression is observed following peripheral stimulation. These abnormalities are related to hyperalgesia during chronic pain development. The available data indicate that ncRNAs may be essential for hyperalgesia. In this review, we focus on microRNA (miRNA), long non‐coding RNA (lncRNA), and circular non‐coding RNA (circRNA) expression changes in neuropathic pain. Other types of ncRNAs are seldom reported in neuropathic pain and, thus, are not discussed herein. Notably, we will pay attention to their etiological role in the development of neuropathic pain and the current challenges and considerations for miRNA‐, lncRNA‐, and circRNA‐based therapeutics for neuropathic pain.","Typically, miRNA production involves three steps: cropping, exporting, and dicing. First, the miRNA gene is transcribed in the nucleus, mainly by RNA polymerase II.\n5\n The resulting primary miRNA transcript (pri‐miRNA) is several kilobases (kb) in length, with a specific stem–loop structure that harbors mature miRNAs in the stem. The mature miRNA is ‘cropped’ by Drosha and its interactor DGCR8 (DiGeorge syndrome critical region 8), which cleaves pri‐miRNA at the stem to produce pre‐miRNA,\n6\n 60–70 nucleotides (nt) in length with a hairpin structure. Then, exportin‐5 recognizes and exports pre‐miRNA from the nucleus to the cytoplasm.\n7\n In the cytoplasm, ribonuclease III (RNAse III), termed Dicer, further cleaves the pre‐miRNA to release double‐stranded miRNA with a length of ~22 nt.\n8\n This miRNA is unwound by an unknown helicase or cleaved by Argonaute (Ago) to form the RNA‐induced silencing complex.\n9\n One strand in the RNA duplex remains with Ago as a mature miRNA, and the other is degraded. The seed sequence of the miRNA incompletely or entirely combines with the target mRNA sequence, resulting in target mRNAs degradation or transcriptional regulation.\n10\n\n\nUnlike miRNAs, lncRNAs are mRNAs‐like transcripts ranging in length from 200 nt to 100 kb that lack prominent open reading frames.\n11\n The lncRNA cellular mechanism is highly related to their intracellular localization. lncRNAs control chromatin functions, transcription, and RNA processing in the nucleus and affect mRNA stability, translation, and cellular signaling in the cytoplasm.\n12\n Compared to lncRNAs, circRNAs are more stable because a single circRNA molecular ends can be covalently linked compared to linear RNA. circRNAs are evolutionarily conserved molecules that are essential in the post‐transcriptional modification of gene expression by acting as miRNAs sponges or interacting with transcription or translational machinery. Numerous lncRNAs and circRNAs are distributed within pain‐related regions and dysregulated after peripheral noxious stimulation. Moreover, functional studies have indicated that miRNAs, lncRNAs, and circRNAs participate in neuropathic pain development by regulating diverse pain‐related genes along the pain processing pathways.","\nmiRNAs expression changes Microarray and deep‐sequencing analyses revealed that nerve injury or noxious stimuli could induce broad changes in miRNA expression in serum or along the pain processing pathways, including the perineal nerve, dorsal root ganglion (DRG), spinal cord, and supraspinal regions. For normal pain signal transmission, nerve injury or noxious stimuli are detected by nociceptors in the DRG or trigeminal ganglion (TG) and then transmitted to upstream neurons in the spinal dorsal horn. Subsequently, nociceptive stimuli are integrated, processed, and further transmitted ascending to specific supraspinal brain regions.\nHuman studies have identified as many as 1134 differentially expressed (DE) genes in the serum of individuals with or without neuropathic pain after spinal cord injury (SCI).\n13\n miR‐204‐5p, ‐519d‐3p, ‐20b‐5p, and ‐6838‐5p might act as promising biomarkers and intervention targets for preventing and therapizing neuropathic pain after SCI. In trigeminal neuralgia individuals, serum miR‐132‐3p, ‐146b‐5p, ‐155‐5p, and ‐384 levels were prominently increased compared with healthy controls.\n14\n Patients with painful peripheral neuropathy had higher miR‐21 level in the serum and sural nerve compared with healthy controls. Meanwhile, miR‐155 was reduced in the serum and inflicted lower leg skin.\n15\n Animal and human studies showed that miR‐30c‐5p was upregulated in the serum and cerebro spinal fluid (CSF) of sciatic nerve injury rats and neuropathic pain patients with chronic peripheral ischemia. The high expression of miR‐30c‐5p, together with other clinical parameters, might be used to predict neuropathic pain development in patients with chronic peripheral ischemia.\n16\n Additionally, an animal study showed that plasma‐derived DE extracellular vesicle (EV) miRNAs regulated processes that are essential for neuropathic pain development. Most DE EV miRNAs for inflammation suppression were downregulated, potentially acting as biomarkers and targets in neuropathic pain treatment.\n17\n\n\nIn addition, numerous DE miRNAs have been identified in the DRG. One week after spared nerve injury (SNI), 33 and 39 miRNAs in the DRG were upregulated and downregulated, respectively, with most DE miRNAs related to axon guidance, focal adhesion, Ras and Wnt signaling pathways.\n18\n Furthermore, nerve injury‐induced miRNAs expression was dynamic and time‐dependent,\n19\n, \n20\n implicating multiple regulatory mechanisms in neuropathic pain initiation and development. Nerve injury redistributes miRNAs from a uniform style within the DRG soma of non‐allodynic animals to preferential localization to peripheral neurons in allodynic animals.\n21\n Furthermore, either sciatic nerve ligation (SNL), DRG transaction (DRT), or ventral root transection (VRT) could upregulate miR‐21 and miR‐31 while downregulating miR‐668 and miR‐672 in the injured DRG,\n22\n implying that these miRNAs could be therapeutic targets for treating diverse types of neuropathic pain.\nThe spinal cord dorsal horn relays and modulates pain signals from the peripheral nociceptors to the supraspinal regions. In the spinal cord, numerous miRNA expression changes have been observed in chronic constriction injury (CCI) and diabetic neuropathic pain (DNP) rodents.\n23\n, \n24\n, \n25\n, \n26\n Previous research has suggested some miRNAs may be related to neuropathic pain development, for example, miR‐500, ‐221, and ‐21,\n25\n thus, acting as potential targets in its treatment.\n27\n\n\nCytoscape software constructed the miRNA–target gene regulatory network in the supraspinal region, including the nucleus accumbens (NAc), medial prefrontal cortex, and periaqueductal gray, between SNI and sham rats. Finally, four essential DE genes, includingCXCR2, IL12B, TNFSF8, and GRK1, and five miRNAs, including miR‐208a‐5p, −7688‐3p, −344f‐3p, −135b‐3p, and ‐135a‐2‐3p, were identified, indicating their essential roles in neuropathic pain pathogenesis.\n28\n Furthermore, in the prelimbic cortex of SNI rats, the DE miRNA–mRNA network pointed to molecules associated with inflammation.\n29\n DE miRNAs were also observed in the bilateral hippocampus of CCI rats. However, no significant difference was observed bilaterally in the hippocampus.\n30\n (Figure 1).\nmicroRNAs (miRNAs) expression change following peripheral nerve injury or noxious stimuli SCI, spinal cord injury; CSF, cerebrol spinal fluid; DE, differential expressed; EV, extravascular vesicle; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury; DRT, DRG transection; VRT, ventral root transection\nMicroarray and deep‐sequencing analyses revealed that nerve injury or noxious stimuli could induce broad changes in miRNA expression in serum or along the pain processing pathways, including the perineal nerve, dorsal root ganglion (DRG), spinal cord, and supraspinal regions. For normal pain signal transmission, nerve injury or noxious stimuli are detected by nociceptors in the DRG or trigeminal ganglion (TG) and then transmitted to upstream neurons in the spinal dorsal horn. Subsequently, nociceptive stimuli are integrated, processed, and further transmitted ascending to specific supraspinal brain regions.\nHuman studies have identified as many as 1134 differentially expressed (DE) genes in the serum of individuals with or without neuropathic pain after spinal cord injury (SCI).\n13\n miR‐204‐5p, ‐519d‐3p, ‐20b‐5p, and ‐6838‐5p might act as promising biomarkers and intervention targets for preventing and therapizing neuropathic pain after SCI. In trigeminal neuralgia individuals, serum miR‐132‐3p, ‐146b‐5p, ‐155‐5p, and ‐384 levels were prominently increased compared with healthy controls.\n14\n Patients with painful peripheral neuropathy had higher miR‐21 level in the serum and sural nerve compared with healthy controls. Meanwhile, miR‐155 was reduced in the serum and inflicted lower leg skin.\n15\n Animal and human studies showed that miR‐30c‐5p was upregulated in the serum and cerebro spinal fluid (CSF) of sciatic nerve injury rats and neuropathic pain patients with chronic peripheral ischemia. The high expression of miR‐30c‐5p, together with other clinical parameters, might be used to predict neuropathic pain development in patients with chronic peripheral ischemia.\n16\n Additionally, an animal study showed that plasma‐derived DE extracellular vesicle (EV) miRNAs regulated processes that are essential for neuropathic pain development. Most DE EV miRNAs for inflammation suppression were downregulated, potentially acting as biomarkers and targets in neuropathic pain treatment.\n17\n\n\nIn addition, numerous DE miRNAs have been identified in the DRG. One week after spared nerve injury (SNI), 33 and 39 miRNAs in the DRG were upregulated and downregulated, respectively, with most DE miRNAs related to axon guidance, focal adhesion, Ras and Wnt signaling pathways.\n18\n Furthermore, nerve injury‐induced miRNAs expression was dynamic and time‐dependent,\n19\n, \n20\n implicating multiple regulatory mechanisms in neuropathic pain initiation and development. Nerve injury redistributes miRNAs from a uniform style within the DRG soma of non‐allodynic animals to preferential localization to peripheral neurons in allodynic animals.\n21\n Furthermore, either sciatic nerve ligation (SNL), DRG transaction (DRT), or ventral root transection (VRT) could upregulate miR‐21 and miR‐31 while downregulating miR‐668 and miR‐672 in the injured DRG,\n22\n implying that these miRNAs could be therapeutic targets for treating diverse types of neuropathic pain.\nThe spinal cord dorsal horn relays and modulates pain signals from the peripheral nociceptors to the supraspinal regions. In the spinal cord, numerous miRNA expression changes have been observed in chronic constriction injury (CCI) and diabetic neuropathic pain (DNP) rodents.\n23\n, \n24\n, \n25\n, \n26\n Previous research has suggested some miRNAs may be related to neuropathic pain development, for example, miR‐500, ‐221, and ‐21,\n25\n thus, acting as potential targets in its treatment.\n27\n\n\nCytoscape software constructed the miRNA–target gene regulatory network in the supraspinal region, including the nucleus accumbens (NAc), medial prefrontal cortex, and periaqueductal gray, between SNI and sham rats. Finally, four essential DE genes, includingCXCR2, IL12B, TNFSF8, and GRK1, and five miRNAs, including miR‐208a‐5p, −7688‐3p, −344f‐3p, −135b‐3p, and ‐135a‐2‐3p, were identified, indicating their essential roles in neuropathic pain pathogenesis.\n28\n Furthermore, in the prelimbic cortex of SNI rats, the DE miRNA–mRNA network pointed to molecules associated with inflammation.\n29\n DE miRNAs were also observed in the bilateral hippocampus of CCI rats. However, no significant difference was observed bilaterally in the hippocampus.\n30\n (Figure 1).\nmicroRNAs (miRNAs) expression change following peripheral nerve injury or noxious stimuli SCI, spinal cord injury; CSF, cerebrol spinal fluid; DE, differential expressed; EV, extravascular vesicle; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury; DRT, DRG transection; VRT, ventral root transection\n\nlncRNAs and circRNAs expression changes Patients with type 2 DNP had a prominent higher expression of serum lncRNA NONRATT021972 and more severe neuropathic pain symptoms.\n31\n LINC01119 and LINC02447 in the peripheral blood of SCI patients were identified in pain pathways that were important for neuropathic pain development.\n32\n\n\nMicroarray analysis revealed that a nerve injury could induce time‐dependent lncRNA expression changes in the sciatic nerve.\n33\n lncRNA H19 was persistently upregulated in Schwann cells along the peripheral nerve, proximal and distal to the injured site.\n34\n In the DRG, the transcriptomic analysis identified 86 known and 26 novel lncRNAs genes to be DE after spared sciatic nerve injury.\n35\n Of these, rno‐Cntnap2 and AC111653.1 were essential in peripheral nerve regeneration and were involved in neuropathic pain.\nNext‐generation RNA sequencing showed 134 lncRNAs and 188 circRNAs were prominently changed 14 days after SNI in the spinal cord.\n36\n In addition, microarray analysis identified 1481 and 1096 DE lncRNAs and mRNAs, respectively, in the spinal cord dorsal horn of DNP rats. Of these, 289 neighboring and 57 overlapping lncRNA‐mRNA pairs, including ENSMUST00000150952‐Mbp and AK081017‐Usp15, have been suggested to participate in neuropathic pain development.\n37\n\n\ncircRNAs have characteristic circularized structures following the backsplicing of exons from antisense RNAs (AS RNAs) or mRNAs and, thus, are highly stable. Most circRNAs are highly conserved among species and lack translation potential, despite cap‐independent translation. A recent study showed that 363 and 106 circRNAs were significantly dysregulated in the ipsilateral dorsal horn after nerve injury.\n38\n (Figure 2).\nLong non‐coding RNA (lncRNAs) and circular non‐coding RNAs (circRNAs) expression change following peripheral nerve injury or noxious stimuli DNP, diabetic neuropathic pain; SCI, spinal cord injury; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury\nPatients with type 2 DNP had a prominent higher expression of serum lncRNA NONRATT021972 and more severe neuropathic pain symptoms.\n31\n LINC01119 and LINC02447 in the peripheral blood of SCI patients were identified in pain pathways that were important for neuropathic pain development.\n32\n\n\nMicroarray analysis revealed that a nerve injury could induce time‐dependent lncRNA expression changes in the sciatic nerve.\n33\n lncRNA H19 was persistently upregulated in Schwann cells along the peripheral nerve, proximal and distal to the injured site.\n34\n In the DRG, the transcriptomic analysis identified 86 known and 26 novel lncRNAs genes to be DE after spared sciatic nerve injury.\n35\n Of these, rno‐Cntnap2 and AC111653.1 were essential in peripheral nerve regeneration and were involved in neuropathic pain.\nNext‐generation RNA sequencing showed 134 lncRNAs and 188 circRNAs were prominently changed 14 days after SNI in the spinal cord.\n36\n In addition, microarray analysis identified 1481 and 1096 DE lncRNAs and mRNAs, respectively, in the spinal cord dorsal horn of DNP rats. Of these, 289 neighboring and 57 overlapping lncRNA‐mRNA pairs, including ENSMUST00000150952‐Mbp and AK081017‐Usp15, have been suggested to participate in neuropathic pain development.\n37\n\n\ncircRNAs have characteristic circularized structures following the backsplicing of exons from antisense RNAs (AS RNAs) or mRNAs and, thus, are highly stable. Most circRNAs are highly conserved among species and lack translation potential, despite cap‐independent translation. A recent study showed that 363 and 106 circRNAs were significantly dysregulated in the ipsilateral dorsal horn after nerve injury.\n38\n (Figure 2).\nLong non‐coding RNA (lncRNAs) and circular non‐coding RNAs (circRNAs) expression change following peripheral nerve injury or noxious stimuli DNP, diabetic neuropathic pain; SCI, spinal cord injury; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury","Microarray and deep‐sequencing analyses revealed that nerve injury or noxious stimuli could induce broad changes in miRNA expression in serum or along the pain processing pathways, including the perineal nerve, dorsal root ganglion (DRG), spinal cord, and supraspinal regions. For normal pain signal transmission, nerve injury or noxious stimuli are detected by nociceptors in the DRG or trigeminal ganglion (TG) and then transmitted to upstream neurons in the spinal dorsal horn. Subsequently, nociceptive stimuli are integrated, processed, and further transmitted ascending to specific supraspinal brain regions.\nHuman studies have identified as many as 1134 differentially expressed (DE) genes in the serum of individuals with or without neuropathic pain after spinal cord injury (SCI).\n13\n miR‐204‐5p, ‐519d‐3p, ‐20b‐5p, and ‐6838‐5p might act as promising biomarkers and intervention targets for preventing and therapizing neuropathic pain after SCI. In trigeminal neuralgia individuals, serum miR‐132‐3p, ‐146b‐5p, ‐155‐5p, and ‐384 levels were prominently increased compared with healthy controls.\n14\n Patients with painful peripheral neuropathy had higher miR‐21 level in the serum and sural nerve compared with healthy controls. Meanwhile, miR‐155 was reduced in the serum and inflicted lower leg skin.\n15\n Animal and human studies showed that miR‐30c‐5p was upregulated in the serum and cerebro spinal fluid (CSF) of sciatic nerve injury rats and neuropathic pain patients with chronic peripheral ischemia. The high expression of miR‐30c‐5p, together with other clinical parameters, might be used to predict neuropathic pain development in patients with chronic peripheral ischemia.\n16\n Additionally, an animal study showed that plasma‐derived DE extracellular vesicle (EV) miRNAs regulated processes that are essential for neuropathic pain development. Most DE EV miRNAs for inflammation suppression were downregulated, potentially acting as biomarkers and targets in neuropathic pain treatment.\n17\n\n\nIn addition, numerous DE miRNAs have been identified in the DRG. One week after spared nerve injury (SNI), 33 and 39 miRNAs in the DRG were upregulated and downregulated, respectively, with most DE miRNAs related to axon guidance, focal adhesion, Ras and Wnt signaling pathways.\n18\n Furthermore, nerve injury‐induced miRNAs expression was dynamic and time‐dependent,\n19\n, \n20\n implicating multiple regulatory mechanisms in neuropathic pain initiation and development. Nerve injury redistributes miRNAs from a uniform style within the DRG soma of non‐allodynic animals to preferential localization to peripheral neurons in allodynic animals.\n21\n Furthermore, either sciatic nerve ligation (SNL), DRG transaction (DRT), or ventral root transection (VRT) could upregulate miR‐21 and miR‐31 while downregulating miR‐668 and miR‐672 in the injured DRG,\n22\n implying that these miRNAs could be therapeutic targets for treating diverse types of neuropathic pain.\nThe spinal cord dorsal horn relays and modulates pain signals from the peripheral nociceptors to the supraspinal regions. In the spinal cord, numerous miRNA expression changes have been observed in chronic constriction injury (CCI) and diabetic neuropathic pain (DNP) rodents.\n23\n, \n24\n, \n25\n, \n26\n Previous research has suggested some miRNAs may be related to neuropathic pain development, for example, miR‐500, ‐221, and ‐21,\n25\n thus, acting as potential targets in its treatment.\n27\n\n\nCytoscape software constructed the miRNA–target gene regulatory network in the supraspinal region, including the nucleus accumbens (NAc), medial prefrontal cortex, and periaqueductal gray, between SNI and sham rats. Finally, four essential DE genes, includingCXCR2, IL12B, TNFSF8, and GRK1, and five miRNAs, including miR‐208a‐5p, −7688‐3p, −344f‐3p, −135b‐3p, and ‐135a‐2‐3p, were identified, indicating their essential roles in neuropathic pain pathogenesis.\n28\n Furthermore, in the prelimbic cortex of SNI rats, the DE miRNA–mRNA network pointed to molecules associated with inflammation.\n29\n DE miRNAs were also observed in the bilateral hippocampus of CCI rats. However, no significant difference was observed bilaterally in the hippocampus.\n30\n (Figure 1).\nmicroRNAs (miRNAs) expression change following peripheral nerve injury or noxious stimuli SCI, spinal cord injury; CSF, cerebrol spinal fluid; DE, differential expressed; EV, extravascular vesicle; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury; DRT, DRG transection; VRT, ventral root transection","Patients with type 2 DNP had a prominent higher expression of serum lncRNA NONRATT021972 and more severe neuropathic pain symptoms.\n31\n LINC01119 and LINC02447 in the peripheral blood of SCI patients were identified in pain pathways that were important for neuropathic pain development.\n32\n\n\nMicroarray analysis revealed that a nerve injury could induce time‐dependent lncRNA expression changes in the sciatic nerve.\n33\n lncRNA H19 was persistently upregulated in Schwann cells along the peripheral nerve, proximal and distal to the injured site.\n34\n In the DRG, the transcriptomic analysis identified 86 known and 26 novel lncRNAs genes to be DE after spared sciatic nerve injury.\n35\n Of these, rno‐Cntnap2 and AC111653.1 were essential in peripheral nerve regeneration and were involved in neuropathic pain.\nNext‐generation RNA sequencing showed 134 lncRNAs and 188 circRNAs were prominently changed 14 days after SNI in the spinal cord.\n36\n In addition, microarray analysis identified 1481 and 1096 DE lncRNAs and mRNAs, respectively, in the spinal cord dorsal horn of DNP rats. Of these, 289 neighboring and 57 overlapping lncRNA‐mRNA pairs, including ENSMUST00000150952‐Mbp and AK081017‐Usp15, have been suggested to participate in neuropathic pain development.\n37\n\n\ncircRNAs have characteristic circularized structures following the backsplicing of exons from antisense RNAs (AS RNAs) or mRNAs and, thus, are highly stable. Most circRNAs are highly conserved among species and lack translation potential, despite cap‐independent translation. A recent study showed that 363 and 106 circRNAs were significantly dysregulated in the ipsilateral dorsal horn after nerve injury.\n38\n (Figure 2).\nLong non‐coding RNA (lncRNAs) and circular non‐coding RNAs (circRNAs) expression change following peripheral nerve injury or noxious stimuli DNP, diabetic neuropathic pain; SCI, spinal cord injury; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury","miRNAs contribute to the development of neuropathic pain via diverse mechanisms, such as neuroinflammation, autophagy, abnormal ion channel expression, regulating pain‐related mediators, protein kinases, structural proteins, neurotransmission excitatory–inhibitory imbalances, and exosome miRNA‐mediated neuron–glia communication (Table 1).\nmiRNAs expression change for the development of neuropathic pain\nKv1.1, Kv1.4, Kv3.4, Kv4.3, Kv7.5,\nDPP10, Navβ1 ↓\np‐p38 MAPK ↑\nAbbreviations: ACC, anterior cingulate cortex; BACE1, beta‐site amyloid precursor protein‐cleaving enzyme 1; BDNF, brain‐derived neurotrophic factor; CCI, chronic constriction injury; CCR1, C–C chemokine receptor 1; CSF, cerebro spinal fluid; CSF1, colony‐stimulating factor‐1; CXCR4, chemokine CXC receptor 4; DNMT3a, DNA methyltransferase 3a; DNP, diabetic neuropathic pain; DRG, dorsal root ganglion; EV, extracellular vesicle; EZH2, enhancer of zeste homolog 2; GRK2, G protein‐coupled receptor kinases 2; IGF‐1/IGF‐1R, insulin‐like growth factor‐1/insulin‐like growth factor‐1 receptor; KCNMA1, calcium‐activated potassium channel subunit α‐1; MeCP2, methyl cytosine–guanine dinucleotide (CpG)‐binding protein 2; MIP‐1a, macrophage inflammatory protein‐1 alpha; NEFL, neurofilament light polypeptide; NF‐κB, nuclear factor – kappa B; NGF, nerve growth factor; NLRP3, NOD‐like receptor protein 3; NOX2, NADPH oxidase 2; NP, neuropathic pain; pSNL, partial sciatic nerve ligation; RAP1A, Ras‐related protein 1A; RASA1, RAS P21 protein activator 1; ROS, reactive oxygen species; S1PR1, sphingosine‐1‐phosphate receptor 1; SCI, spinal cord injury; Scn2b, β2 subunit of the voltage‐gated sodium channel; SIRT1, histone deacetylase sirtuin 1; SNL, spinal nerve ligation; SOCS1, suppressor of cytokine signaling 1; STAT3, signal transducer and activator of transcription 3; TLR4, toll‐like receptor 4; TNFR1, TNF receptor 1; TRPA1, transient receptor potential ankyrin 1; TXNIP, thioredoxin‐interacting protein; VTR, ventral root transection; ZEB1, zinc finger E‐box binding homeobox 1.\n\nmiRNAs regulate neuroinflammation in neuropathic pain development miRNA‐based epigenetic regulation is essential in neuroinflammation. miRNAs are predicted to regulate diverse neuroinflammation‐related targets along the pain processing pathways.\nAmong patients with neuropathic pain, miR‐101 decreased in the serum and sural nerve, which is related to nuclear factor‐kappa B (NF‐κB) signaling activation.\n39\n Meanwhile, serum miR‐124a and miR‐155 expression was upregulated. They were identified to inhibit histone deacetylase sirtuin 1 (SIRT1) in primary human cluster of differentiation 4 (CD4) (+) cells and induce their differentiation toward regulatory T cells (Tregs), thus reducing pain‐related inflammation.\n40\n Such miRNA–target interactions may act as an endogenous protective mechanism for neuropathic pain.\nmiR‐7a, expressed in small‐sized nociceptive DRG neurons, is downregulated after nerve injury, as reported, targeting neurofilament light polypeptides (NEFLs).\n41\n NEFL encodes a neuronal protein vital for neurofilament formation and increases signal transducer and activator of transcription 3 (STAT3) phosphorylation, which is highly related to cell differentiation and neuroinflammation. In diabetic peripheral neuropathic mice, miR‐590‐3p was downregulated to disinhibit Ras‐related protein 1A (RAP1A) in the DRG tissue and inhibit neural T cells infiltration.\n42\n Thus, exogenous miR‐590‐3p may be a potential alternative for neuropathic pain treatment. CCI downregulated miR‐140 and miR‐144 expression in the DRG. Intrathecally injected miR‐140 and miR‐144 agomir decreased inflammatory factor secretion and ameliorated hyperalgesia by targeting sphingosine‐1‐phosphate receptor 1 (S1PR1) and RAS P21 protein activator 1 (RASA1), respectively.\n43\n, \n44\n\n\nmiRNAs are essential in neuropathic pain development via neuroinflammation‐related mechanisms in the spinal cord. miR‐130a‐3p targets and downregulates insulin‐like growth factor‐1/insulin‐like growth factor‐1 receptor (IGF‐1/IGF‐1R) expression to alleviate SCI‐induced neuropathic pain by mitigating microglial activation and NF‐κB phosphorylation.\n45\n miR‐378 was decreased in CCI rats, inhibiting neuropathic pain development by targeting the enhancer of zeste homolog 2 (EZH2).\n46\n EZH2 promotes neuropathic pain by increasing tumor necrosis factor‐alpha (TNF‐α), interleukin (IL)‐1β, and monocyte chemoattractant protein‐1 (MCP‐1) production. Intrathecally injecting miRNA‐138 lentivirus can remarkably alleviate neuropathic pain in partial sciatic nerve ligation (pSNL) rats by suppressing toll‐like receptor 4 (TLR4) and macrophage inflammatory protein‐1 alpha (MIP‐1α)/C‐C chemokine receptor 1 (CCR1) signaling pathways.\n47\n\n\nmiR‐15a/16 targets and downregulates G protein‐coupled receptor kinases 2 (GRK2) to disinhibit p38‐MAPK (mitogen‐activated protein kinase) and NF‐κB, contributing to neuroinflammation after CCI.\n48\n GRK2‐deficient mice present a pro‐inflammatory phenotype in spinal cord microglia/macrophages, restored by miR‐124.\n49\n SNL increased DNA methyltransferase 3a (DNMT3a) expression related to hypermethylation of the miR‐214‐3p promoter, resulting in miR‐214‐3p expression reduction, which enhanced astrocyte reactivity, colony‐stimulating factor‐1 (CSF1), and interleukin 6 (IL‐6) production, and hyperalgesia in rats.\n50\n Electro‐acupuncture attenuated SCI by inhibiting Nav1.3 and Bax in the injured spinal cord through miR‐214 upregulation.\n51\n Downregulated miR‐128 was reported to contribute to neuropathic pain via p38 or zinc finger E‐box binding homeobox 1 (ZEB1) activation in the spinal cord.\n52\n, \n53\n Meanwhile, ZEB1 was also targeted by miR‐200b/miR‐429, orchestrating neuropathic pain development.\n54\n\n\nIn a pSNL mouse model, nerve injury significantly reduced miR‐23a expression in spinal glial cells, concomitant with the upregulation of its target chemokine, CXC receptor 4 (CXCR4). In naïve mice, either miR‐23a downregulation or CXCR4 upregulation could active the thioredoxin‐interacting protein (TXNIP)/NOD‐like receptor protein 3 (NLRP3) inflammasome axis. Both intrathecal miR‐23a mimics and spinal CXCR4 downregulation by a lentivirus inhibited TXNIP or NLRP3 upregulation to alleviate hyperalgesia.\n55\n\n\nNotably, one miRNA was shown to have distinctive targets in different animal models of the spinal cord. For example, miR‐155 downregulation targeted and upregulated NADPH oxidase 2 (NOX2) expression to induce reactive oxygen species (ROS) production after SCI, presenting a pro‐inflammatory phenotype in microglia/macrophages.\n56\n The ability to induce glial polarization was also observed in cultured BV‐2 microglia.\n57\n In bortezomib‐induced neuropathic pain rats, downregulated miR‐155 upregulated TNF receptor 1 (TNFR1) expression, which activated its downstream signaling pathways, including p38‐MAPK, c‐Jun N‐terminal kinase (JNK), and transient receptor potential ankyrin 1 (TRPA1).\n58\n Therefore, it is suggested that miR‐155 might act as an intervention target for neuropathic pain. As expected, treatment with ibuprofen and L‐arginine delayed the behavioral pain changes while inhibiting spinal miR‐155 and NO.\n59\n miR‐155‐5p is also known to destabilize the blood–nerve barrier and expression of tight junction proteins, such as claudin‐1 and zonula occludens‐1 (ZO‐1). Tissue plasminogen activator (tPA) could transiently open such barriers to facilitate topically applying analgesics, via miR‐155‐5p upregulation.\n60\n However, we also noted that CCI upregulated but did not downregulate spinal cord miR‐155, and miR‐155 inhibition enhanced suppressor of cytokine signaling 1 (SOCS1) expression to inactivate inflammation via NF‐κB and p38‐MAPK inhibition.\n61\n In oxaliplatin‐induced peripheral neuropathic pain, spinal cord miR‐155 expression was also upregulated, and intrathecally injecting the miR‐155 inhibitor attenuated hyperalgesia in rats, possibly by inhibiting oxidative stress–TRPA1 pathways.\n62\n The underlying mechanisms of such distinctive miR‐155 expression change are still unknown and require further research into different pain models.\nmiRNA‐based epigenetic regulation is essential in neuroinflammation. miRNAs are predicted to regulate diverse neuroinflammation‐related targets along the pain processing pathways.\nAmong patients with neuropathic pain, miR‐101 decreased in the serum and sural nerve, which is related to nuclear factor‐kappa B (NF‐κB) signaling activation.\n39\n Meanwhile, serum miR‐124a and miR‐155 expression was upregulated. They were identified to inhibit histone deacetylase sirtuin 1 (SIRT1) in primary human cluster of differentiation 4 (CD4) (+) cells and induce their differentiation toward regulatory T cells (Tregs), thus reducing pain‐related inflammation.\n40\n Such miRNA–target interactions may act as an endogenous protective mechanism for neuropathic pain.\nmiR‐7a, expressed in small‐sized nociceptive DRG neurons, is downregulated after nerve injury, as reported, targeting neurofilament light polypeptides (NEFLs).\n41\n NEFL encodes a neuronal protein vital for neurofilament formation and increases signal transducer and activator of transcription 3 (STAT3) phosphorylation, which is highly related to cell differentiation and neuroinflammation. In diabetic peripheral neuropathic mice, miR‐590‐3p was downregulated to disinhibit Ras‐related protein 1A (RAP1A) in the DRG tissue and inhibit neural T cells infiltration.\n42\n Thus, exogenous miR‐590‐3p may be a potential alternative for neuropathic pain treatment. CCI downregulated miR‐140 and miR‐144 expression in the DRG. Intrathecally injected miR‐140 and miR‐144 agomir decreased inflammatory factor secretion and ameliorated hyperalgesia by targeting sphingosine‐1‐phosphate receptor 1 (S1PR1) and RAS P21 protein activator 1 (RASA1), respectively.\n43\n, \n44\n\n\nmiRNAs are essential in neuropathic pain development via neuroinflammation‐related mechanisms in the spinal cord. miR‐130a‐3p targets and downregulates insulin‐like growth factor‐1/insulin‐like growth factor‐1 receptor (IGF‐1/IGF‐1R) expression to alleviate SCI‐induced neuropathic pain by mitigating microglial activation and NF‐κB phosphorylation.\n45\n miR‐378 was decreased in CCI rats, inhibiting neuropathic pain development by targeting the enhancer of zeste homolog 2 (EZH2).\n46\n EZH2 promotes neuropathic pain by increasing tumor necrosis factor‐alpha (TNF‐α), interleukin (IL)‐1β, and monocyte chemoattractant protein‐1 (MCP‐1) production. Intrathecally injecting miRNA‐138 lentivirus can remarkably alleviate neuropathic pain in partial sciatic nerve ligation (pSNL) rats by suppressing toll‐like receptor 4 (TLR4) and macrophage inflammatory protein‐1 alpha (MIP‐1α)/C‐C chemokine receptor 1 (CCR1) signaling pathways.\n47\n\n\nmiR‐15a/16 targets and downregulates G protein‐coupled receptor kinases 2 (GRK2) to disinhibit p38‐MAPK (mitogen‐activated protein kinase) and NF‐κB, contributing to neuroinflammation after CCI.\n48\n GRK2‐deficient mice present a pro‐inflammatory phenotype in spinal cord microglia/macrophages, restored by miR‐124.\n49\n SNL increased DNA methyltransferase 3a (DNMT3a) expression related to hypermethylation of the miR‐214‐3p promoter, resulting in miR‐214‐3p expression reduction, which enhanced astrocyte reactivity, colony‐stimulating factor‐1 (CSF1), and interleukin 6 (IL‐6) production, and hyperalgesia in rats.\n50\n Electro‐acupuncture attenuated SCI by inhibiting Nav1.3 and Bax in the injured spinal cord through miR‐214 upregulation.\n51\n Downregulated miR‐128 was reported to contribute to neuropathic pain via p38 or zinc finger E‐box binding homeobox 1 (ZEB1) activation in the spinal cord.\n52\n, \n53\n Meanwhile, ZEB1 was also targeted by miR‐200b/miR‐429, orchestrating neuropathic pain development.\n54\n\n\nIn a pSNL mouse model, nerve injury significantly reduced miR‐23a expression in spinal glial cells, concomitant with the upregulation of its target chemokine, CXC receptor 4 (CXCR4). In naïve mice, either miR‐23a downregulation or CXCR4 upregulation could active the thioredoxin‐interacting protein (TXNIP)/NOD‐like receptor protein 3 (NLRP3) inflammasome axis. Both intrathecal miR‐23a mimics and spinal CXCR4 downregulation by a lentivirus inhibited TXNIP or NLRP3 upregulation to alleviate hyperalgesia.\n55\n\n\nNotably, one miRNA was shown to have distinctive targets in different animal models of the spinal cord. For example, miR‐155 downregulation targeted and upregulated NADPH oxidase 2 (NOX2) expression to induce reactive oxygen species (ROS) production after SCI, presenting a pro‐inflammatory phenotype in microglia/macrophages.\n56\n The ability to induce glial polarization was also observed in cultured BV‐2 microglia.\n57\n In bortezomib‐induced neuropathic pain rats, downregulated miR‐155 upregulated TNF receptor 1 (TNFR1) expression, which activated its downstream signaling pathways, including p38‐MAPK, c‐Jun N‐terminal kinase (JNK), and transient receptor potential ankyrin 1 (TRPA1).\n58\n Therefore, it is suggested that miR‐155 might act as an intervention target for neuropathic pain. As expected, treatment with ibuprofen and L‐arginine delayed the behavioral pain changes while inhibiting spinal miR‐155 and NO.\n59\n miR‐155‐5p is also known to destabilize the blood–nerve barrier and expression of tight junction proteins, such as claudin‐1 and zonula occludens‐1 (ZO‐1). Tissue plasminogen activator (tPA) could transiently open such barriers to facilitate topically applying analgesics, via miR‐155‐5p upregulation.\n60\n However, we also noted that CCI upregulated but did not downregulate spinal cord miR‐155, and miR‐155 inhibition enhanced suppressor of cytokine signaling 1 (SOCS1) expression to inactivate inflammation via NF‐κB and p38‐MAPK inhibition.\n61\n In oxaliplatin‐induced peripheral neuropathic pain, spinal cord miR‐155 expression was also upregulated, and intrathecally injecting the miR‐155 inhibitor attenuated hyperalgesia in rats, possibly by inhibiting oxidative stress–TRPA1 pathways.\n62\n The underlying mechanisms of such distinctive miR‐155 expression change are still unknown and require further research into different pain models.\nAutophagy miRNA‐related autophagy is involved in neuropathic pain regulation. As reported, miR‐15a downregulated and stimulated AKT serine/threonine kinase 3 (AKT3), and inhibited autophagy post‐CCI.\n63\n Impaired autophagy participates in neuropathic pain development. Intrathecal miR‐15a agomir prominently suppressed AKT3 expression, induced autophagy, and attenuated CCI‐induced neuropathic pain. Similar to miR‐15a, miR‐145 and miR‐20b‐5p contributed to neuropathic pain regulation via protein kinase B (AKT)‐related autophagy pathways.\n64\n, \n65\n Meanwhile, miR‐195 in the spinal cord was observed to upregulate post‐SNL, targeting and inhibiting Autophagy‐Related 14 (ATG14) and its autophagy activation.\n66\n The miR‐195 inhibitor activated autophagy and suppressed neuroinflammation in vivo and in vitro.\nmiRNA‐related autophagy is involved in neuropathic pain regulation. As reported, miR‐15a downregulated and stimulated AKT serine/threonine kinase 3 (AKT3), and inhibited autophagy post‐CCI.\n63\n Impaired autophagy participates in neuropathic pain development. Intrathecal miR‐15a agomir prominently suppressed AKT3 expression, induced autophagy, and attenuated CCI‐induced neuropathic pain. Similar to miR‐15a, miR‐145 and miR‐20b‐5p contributed to neuropathic pain regulation via protein kinase B (AKT)‐related autophagy pathways.\n64\n, \n65\n Meanwhile, miR‐195 in the spinal cord was observed to upregulate post‐SNL, targeting and inhibiting Autophagy‐Related 14 (ATG14) and its autophagy activation.\n66\n The miR‐195 inhibitor activated autophagy and suppressed neuroinflammation in vivo and in vitro.\nThe contribution of miRNAs to ion channel expression in neuropathic pain Another class of miRNA‐based regulation focuses on ion channels, including sodium, potassium, and calcium channels, and transient receptor potential (TRP) channels, to modulate action potential production, firing rate, and neurotransmitter release.\nTheoretically, miRNAs can simultaneously have multiple target mRNAs because precise matching is not a prerequisite for inhibiting the target sequence. For example, other than NEFL,\n41\n miR‐7a targets the β2 subunit of the voltage‐gated sodium channel (Scn2b) with post‐transcriptional regulation to induce nociceptive DRG neurons hyperexcitability.\n67\n Other than miR‐7a, the other known miRNA‐ targets involved in sodium channels expression include miR‐30b, ‐96, ‐384‐5p and their target Scn3a (encoding Nav1.3),\n68\n, \n69\n, \n70\n miR‐30b‐5p and its target Scn8a (encoding Nav1.6),\n71\n and miR‐182 and ‐30b and their target Scn9a (encoding Nav1.7).\n72\n, \n73\n\n\nA miRNA cluster is a polycistronic gene containing several miRNAs derived from a single primary or nascent transcript. Approximately 40% of miRNAs are predicted to form clusters, but their significance is still unknown. miR‐17‐92 is a miRNA cluster that includes six different members, of which miR‐18a, ‐19a, ‐19b, and ‐92a upregulation induces allodynia. The predicted targets of the miR‐17‐92 cluster encompass genes encoding diverse voltage‐gated potassium channels and their regulatory subunits, including Kv1.1, Kv1.4, Kv3.4, Kv4.3, Kv7.5, dipeptidyl peptidase 10 (DPP10), and Navβ1.\n74\n CCI upregulated miR‐137 to target and downregulate Kcna2, which encodes Kv1.2 in the DRG and spinal dorsal horn.\n75\n By contrast, CCI decreased miR‐183‐5p expression in the DRG, and the predicted target gene TREK‐1, a subunit of the 2P‐domain K+ channel, was increased.\n76\n\n\nFurthermore, the miRNA‐183 cluster (miR‐183, part of miR‐96/182/183) regulates either basal mechanical or neuropathic pain.\n77\n The miR‐183 cluster targets Cacna2d1 and Cacna2d2, which encode auxiliary voltage‐gated calcium channel subunits α2δ‐1 and α2δ‐2, to affect nociceptor excitability. Nerve injury downregulated miR‐103 in the spinal cord, which simultaneously targeted and inhibited Cacna1c, Cacna2d1, and Cacnb1 and encoded the Cav1.2‐α1, α2δ1, and β1 subunits of the voltage‐gated calcium channels macromolecular complex Cav1.2 L‐type calcium channel, respectively.\n78\n Intrathecal miR‐103 successfully relieved neuropathic pain.\nIn addition to ion channels, TRP channels are ligand‐gated ion channels that promote painful sensations.\n79\n In the DRG, TRPA1 participates in miR‐141‐5p alleviated oxaliplatin‐induced neuropathic pain.\n80\n In SNI mice, miR‐449a ameliorated neuropathic pain by decreasing the activity of TRPA1 and calcium‐activated potassium channel subunit α‐1 (KCNMA1), thus acting as a potential therapeutic alternative for treating neuropathic pain.\n81\n\n\nAnother class of miRNA‐based regulation focuses on ion channels, including sodium, potassium, and calcium channels, and transient receptor potential (TRP) channels, to modulate action potential production, firing rate, and neurotransmitter release.\nTheoretically, miRNAs can simultaneously have multiple target mRNAs because precise matching is not a prerequisite for inhibiting the target sequence. For example, other than NEFL,\n41\n miR‐7a targets the β2 subunit of the voltage‐gated sodium channel (Scn2b) with post‐transcriptional regulation to induce nociceptive DRG neurons hyperexcitability.\n67\n Other than miR‐7a, the other known miRNA‐ targets involved in sodium channels expression include miR‐30b, ‐96, ‐384‐5p and their target Scn3a (encoding Nav1.3),\n68\n, \n69\n, \n70\n miR‐30b‐5p and its target Scn8a (encoding Nav1.6),\n71\n and miR‐182 and ‐30b and their target Scn9a (encoding Nav1.7).\n72\n, \n73\n\n\nA miRNA cluster is a polycistronic gene containing several miRNAs derived from a single primary or nascent transcript. Approximately 40% of miRNAs are predicted to form clusters, but their significance is still unknown. miR‐17‐92 is a miRNA cluster that includes six different members, of which miR‐18a, ‐19a, ‐19b, and ‐92a upregulation induces allodynia. The predicted targets of the miR‐17‐92 cluster encompass genes encoding diverse voltage‐gated potassium channels and their regulatory subunits, including Kv1.1, Kv1.4, Kv3.4, Kv4.3, Kv7.5, dipeptidyl peptidase 10 (DPP10), and Navβ1.\n74\n CCI upregulated miR‐137 to target and downregulate Kcna2, which encodes Kv1.2 in the DRG and spinal dorsal horn.\n75\n By contrast, CCI decreased miR‐183‐5p expression in the DRG, and the predicted target gene TREK‐1, a subunit of the 2P‐domain K+ channel, was increased.\n76\n\n\nFurthermore, the miRNA‐183 cluster (miR‐183, part of miR‐96/182/183) regulates either basal mechanical or neuropathic pain.\n77\n The miR‐183 cluster targets Cacna2d1 and Cacna2d2, which encode auxiliary voltage‐gated calcium channel subunits α2δ‐1 and α2δ‐2, to affect nociceptor excitability. Nerve injury downregulated miR‐103 in the spinal cord, which simultaneously targeted and inhibited Cacna1c, Cacna2d1, and Cacnb1 and encoded the Cav1.2‐α1, α2δ1, and β1 subunits of the voltage‐gated calcium channels macromolecular complex Cav1.2 L‐type calcium channel, respectively.\n78\n Intrathecal miR‐103 successfully relieved neuropathic pain.\nIn addition to ion channels, TRP channels are ligand‐gated ion channels that promote painful sensations.\n79\n In the DRG, TRPA1 participates in miR‐141‐5p alleviated oxaliplatin‐induced neuropathic pain.\n80\n In SNI mice, miR‐449a ameliorated neuropathic pain by decreasing the activity of TRPA1 and calcium‐activated potassium channel subunit α‐1 (KCNMA1), thus acting as a potential therapeutic alternative for treating neuropathic pain.\n81\n\n\n\nmiRNAs regulate pain‐related mediators, protein kinases, and structural proteins miRNAs regulate diverse pain‐related mediators, protein kinases, and structural proteins along the pain processing pathways. For HIV‐associated symptomatic distal sensory polyneuropathy and neuropathic pain, serum miR‐455‐3p acted as a potential biomarker, possibly targeting multiple genes involved in peripheral neuropathic pain, such as nerve growth factor (NGF) and related genes.\n82\n Brain‐derived neurotrophic factor (BDNF), another well‐recognized pain mediator, is a common miR‐1, ‐183, and ‐206 target.\n83\n, \n84\n, \n85\n After nerve injury, several miRNAs, including miR‐19a, ‐301, and ‐132, were downregulated, and their target methyl cytosine–guanine dinucleotide (CpG)‐binding protein 2 (MeCP2) expression was increased, leading to concomitant BDNF upregulation.\n86\n miR‐30c‐5p was upregulated in the serum and CSF of patients with chronic peripheral ischemia.\n16\n A miR‐30c‐5p inhibitor intraventricular injection postponed neuropathic pain development and fully reversed hyperalgesia in rodents. Transforming growth factor beta (TGF‐β) participates in the effects of miR‐30c‐5p, which refers to the endogenous opioid analgesic system.\n16\n\n\nmiRNAs dysregulate specific pain‐related protein kinases. For example, in DNP rats, miR‐133a‐3p was dysregulated in the sciatic nerve, interacting with p‐p38 MAPK to participate in the development of neuropathic pain.\n87\n Via a methyl CpG‐binding domain and transcriptional repressor domain, MeCP2 acts as a transcriptional repressor, and its overexpression improves neuropathic pain, potentiating an anti‐nociceptive effect of MeCP2. The authors also noted that MeCP2 expression changed post‐transcriptionally, and its mRNA level did not significantly change after SNI. However, the protein level was upregulated. During the development period of neuropathic pain, phospho‐cAMP response element‐binding protein (p‐CREB) elevated rapidly but returned 3–7 days after SNI, concomitant with miR‐132 downregulation, which targets MeCP2 and inhibits its expression post‐transcriptionally.\n88\n SNL decreased miR‐200b and miR‐429 in NAc neurons, along with an upregulation of their target (DNMT3a).\n89\n Further mechanism studies found that DNMT3a in the NAc was expressed in NR1 immunoreactive neurons, suggesting the dysregulation of ‘mesolimbic motivation circuitry’ for neuropathic pain development.\nThe CCI induced time‐dependent miR‐1 downregulation in injured sciatic nerves. This change in expression was related to the upregulation and translocation of the miR‐1‐targeted connexin 43 (Cx43), the major connexin of astrocytes.\n83\n miR‐1 mimics could reduce Cx43 expression in cultured human glioblastoma cells. However, miR‐1 mimics intraneural transfection failed to alter Cx43 protein expression and did not improve pain behavior. The authors attributed such treatment failure to insufficient inhibition of miR‐1 for Cx43 via intraneural injection. Alternatively, there were regulatory mechanisms for Cx43 in vivo other than miR‐1.\n90\n However, miR‐1 in the DRG was DE according to peripheral nerve injury type. CCI, pSNL, and sural nerve injury downregulated miR‐1. However, axotomy of the sciatic nerve and tibial nerve injury increased its expression in the DRG.\n83\n, \n91\n, \n92\n As previously described, miR‐1 was also upregulated following capsaicin treatment and bone cancer pain.\n91\n, \n93\n Additionally, miR‐1 downregulation inhibited bone cancer pain. Collectively, the action of miR‐1 on pain seems to be complex and stimulus‐dependent.\nNotably, specific miRNAs can promote neuropathic pain development via diverse mechanisms. For example, beta‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1), a membrane protease essential for myelination, was downregulated after miR‐15b overexpression in vitro or in the DRG of chemotherapy‐related neuropathic pain rats.\n94\n BACE1‐mediated neuregulin 1 reduction decreases nerve conduction velocity. In addition, BACE1 modulated Navβ2 subunit expression and neuronal activity and regulated inflammation‐related TNFR expression.\nmiRNAs regulate diverse pain‐related mediators, protein kinases, and structural proteins along the pain processing pathways. For HIV‐associated symptomatic distal sensory polyneuropathy and neuropathic pain, serum miR‐455‐3p acted as a potential biomarker, possibly targeting multiple genes involved in peripheral neuropathic pain, such as nerve growth factor (NGF) and related genes.\n82\n Brain‐derived neurotrophic factor (BDNF), another well‐recognized pain mediator, is a common miR‐1, ‐183, and ‐206 target.\n83\n, \n84\n, \n85\n After nerve injury, several miRNAs, including miR‐19a, ‐301, and ‐132, were downregulated, and their target methyl cytosine–guanine dinucleotide (CpG)‐binding protein 2 (MeCP2) expression was increased, leading to concomitant BDNF upregulation.\n86\n miR‐30c‐5p was upregulated in the serum and CSF of patients with chronic peripheral ischemia.\n16\n A miR‐30c‐5p inhibitor intraventricular injection postponed neuropathic pain development and fully reversed hyperalgesia in rodents. Transforming growth factor beta (TGF‐β) participates in the effects of miR‐30c‐5p, which refers to the endogenous opioid analgesic system.\n16\n\n\nmiRNAs dysregulate specific pain‐related protein kinases. For example, in DNP rats, miR‐133a‐3p was dysregulated in the sciatic nerve, interacting with p‐p38 MAPK to participate in the development of neuropathic pain.\n87\n Via a methyl CpG‐binding domain and transcriptional repressor domain, MeCP2 acts as a transcriptional repressor, and its overexpression improves neuropathic pain, potentiating an anti‐nociceptive effect of MeCP2. The authors also noted that MeCP2 expression changed post‐transcriptionally, and its mRNA level did not significantly change after SNI. However, the protein level was upregulated. During the development period of neuropathic pain, phospho‐cAMP response element‐binding protein (p‐CREB) elevated rapidly but returned 3–7 days after SNI, concomitant with miR‐132 downregulation, which targets MeCP2 and inhibits its expression post‐transcriptionally.\n88\n SNL decreased miR‐200b and miR‐429 in NAc neurons, along with an upregulation of their target (DNMT3a).\n89\n Further mechanism studies found that DNMT3a in the NAc was expressed in NR1 immunoreactive neurons, suggesting the dysregulation of ‘mesolimbic motivation circuitry’ for neuropathic pain development.\nThe CCI induced time‐dependent miR‐1 downregulation in injured sciatic nerves. This change in expression was related to the upregulation and translocation of the miR‐1‐targeted connexin 43 (Cx43), the major connexin of astrocytes.\n83\n miR‐1 mimics could reduce Cx43 expression in cultured human glioblastoma cells. However, miR‐1 mimics intraneural transfection failed to alter Cx43 protein expression and did not improve pain behavior. The authors attributed such treatment failure to insufficient inhibition of miR‐1 for Cx43 via intraneural injection. Alternatively, there were regulatory mechanisms for Cx43 in vivo other than miR‐1.\n90\n However, miR‐1 in the DRG was DE according to peripheral nerve injury type. CCI, pSNL, and sural nerve injury downregulated miR‐1. However, axotomy of the sciatic nerve and tibial nerve injury increased its expression in the DRG.\n83\n, \n91\n, \n92\n As previously described, miR‐1 was also upregulated following capsaicin treatment and bone cancer pain.\n91\n, \n93\n Additionally, miR‐1 downregulation inhibited bone cancer pain. Collectively, the action of miR‐1 on pain seems to be complex and stimulus‐dependent.\nNotably, specific miRNAs can promote neuropathic pain development via diverse mechanisms. For example, beta‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1), a membrane protease essential for myelination, was downregulated after miR‐15b overexpression in vitro or in the DRG of chemotherapy‐related neuropathic pain rats.\n94\n BACE1‐mediated neuregulin 1 reduction decreases nerve conduction velocity. In addition, BACE1 modulated Navβ2 subunit expression and neuronal activity and regulated inflammation‐related TNFR expression.\nNeurotransmission excitatory–inhibitory imbalances Neurotransmission excitatory and inhibitory imbalances in the spinal cord also contribute to neuropathic pain development. For example, miR‐500 increased to regulate glutamic acid decarboxylase 67 (GAD67) expression and target the specific site of Gad1 in the dorsal horn.\n95\n GAD67 expression reduction inhibited the function of GABAergic neurons and the resultant inhibitory synaptic transmission dysregulation contributed to neuropathic pain development. In addition, miR‐23b is crucial for improving neuropathic pain in the injured spinal cord by downregulating its target gene, Nox4, which further normalizes glutamic acid decarboxylase 65/67 (GAD65/67) expression and protects GABAergic neurons from apoptosis.\n96\n Microarray analysis showed that miR‐539 was prominently reduced in the contralateral anterior cingulate cortex (ACC) after CCI, which is related to enhanced NR2B protein expression. Injecting miR‐539 mimics into the contralateral ACC attenuated CCI‐evoked mechanical hyperalgesia, suggesting that the N‐methyl‐D‐aspartate (NMDA) receptor NR2B subunit regulates neuropathic pain.\n97\n\n\nNeurotransmission excitatory and inhibitory imbalances in the spinal cord also contribute to neuropathic pain development. For example, miR‐500 increased to regulate glutamic acid decarboxylase 67 (GAD67) expression and target the specific site of Gad1 in the dorsal horn.\n95\n GAD67 expression reduction inhibited the function of GABAergic neurons and the resultant inhibitory synaptic transmission dysregulation contributed to neuropathic pain development. In addition, miR‐23b is crucial for improving neuropathic pain in the injured spinal cord by downregulating its target gene, Nox4, which further normalizes glutamic acid decarboxylase 65/67 (GAD65/67) expression and protects GABAergic neurons from apoptosis.\n96\n Microarray analysis showed that miR‐539 was prominently reduced in the contralateral anterior cingulate cortex (ACC) after CCI, which is related to enhanced NR2B protein expression. Injecting miR‐539 mimics into the contralateral ACC attenuated CCI‐evoked mechanical hyperalgesia, suggesting that the N‐methyl‐D‐aspartate (NMDA) receptor NR2B subunit regulates neuropathic pain.\n97\n\n\n\nEV miRNAs‐mediated neuron–glia communication We mainly focused on exosomal miR‐21 with regard to EV miRNAs in neuropathic pain. As aforementioned, SNL, DRT, or VRT could upregulate miR‐21 in the injured DRG.\n22\n Notably, miR‐21 was also increased in serum exosomes from nerve‐ligated mice.\n98\n In another in‐depth study, macrophages readily took up pure sensory neuron‐derived exosomes encompassing miR‐21‐5p to promote a pro‐inflammatory phenotype.\n99\n Either intrathecal miR‐21‐5p antagomir or miR‐21 conditional deletion in sensory neurons ameliorated hyperalgesia and macrophage recruitment in the DRG.\nUnlike the miRNA target gene inhibitory mechanism, miR‐21 acts as an endogenous toll‐like receptor 8 (TLR8) ligand, leading to neuropathic pain development.\n100\n TLR8, a nucleic acid‐sensing receptor, is located in the endosomes and lysosomes, leading to ERK‐mediated inflammatory mediator production and neuronal activation after SNL. Although miR‐21 and TLR8 are only distributed in small‐ and medium‐sized neurons, miR‐21 can also be derived from large‐sized neurons and reach TLR8 in the endosomes of other types of neurons.\n101\n Similar to miR‐21, DRG sensory neurons secreted miR‐23a‐enriched EVs following nerve injury and were taken up by macrophages to enhance M1 polarization in vitro. A20, an NF‐κB signaling pathway inhibitor, is a verified miR‐23a target gene.\n102\n Moreover, intrathecally delivering EVs‐miR‐23a antagomir attenuated neuropathic hyperalgesia and reduced M1 macrophages by inhibiting A20 to activate NF‐κB signaling.\nWe mainly focused on exosomal miR‐21 with regard to EV miRNAs in neuropathic pain. As aforementioned, SNL, DRT, or VRT could upregulate miR‐21 in the injured DRG.\n22\n Notably, miR‐21 was also increased in serum exosomes from nerve‐ligated mice.\n98\n In another in‐depth study, macrophages readily took up pure sensory neuron‐derived exosomes encompassing miR‐21‐5p to promote a pro‐inflammatory phenotype.\n99\n Either intrathecal miR‐21‐5p antagomir or miR‐21 conditional deletion in sensory neurons ameliorated hyperalgesia and macrophage recruitment in the DRG.\nUnlike the miRNA target gene inhibitory mechanism, miR‐21 acts as an endogenous toll‐like receptor 8 (TLR8) ligand, leading to neuropathic pain development.\n100\n TLR8, a nucleic acid‐sensing receptor, is located in the endosomes and lysosomes, leading to ERK‐mediated inflammatory mediator production and neuronal activation after SNL. Although miR‐21 and TLR8 are only distributed in small‐ and medium‐sized neurons, miR‐21 can also be derived from large‐sized neurons and reach TLR8 in the endosomes of other types of neurons.\n101\n Similar to miR‐21, DRG sensory neurons secreted miR‐23a‐enriched EVs following nerve injury and were taken up by macrophages to enhance M1 polarization in vitro. A20, an NF‐κB signaling pathway inhibitor, is a verified miR‐23a target gene.\n102\n Moreover, intrathecally delivering EVs‐miR‐23a antagomir attenuated neuropathic hyperalgesia and reduced M1 macrophages by inhibiting A20 to activate NF‐κB signaling.","miRNA‐based epigenetic regulation is essential in neuroinflammation. miRNAs are predicted to regulate diverse neuroinflammation‐related targets along the pain processing pathways.\nAmong patients with neuropathic pain, miR‐101 decreased in the serum and sural nerve, which is related to nuclear factor‐kappa B (NF‐κB) signaling activation.\n39\n Meanwhile, serum miR‐124a and miR‐155 expression was upregulated. They were identified to inhibit histone deacetylase sirtuin 1 (SIRT1) in primary human cluster of differentiation 4 (CD4) (+) cells and induce their differentiation toward regulatory T cells (Tregs), thus reducing pain‐related inflammation.\n40\n Such miRNA–target interactions may act as an endogenous protective mechanism for neuropathic pain.\nmiR‐7a, expressed in small‐sized nociceptive DRG neurons, is downregulated after nerve injury, as reported, targeting neurofilament light polypeptides (NEFLs).\n41\n NEFL encodes a neuronal protein vital for neurofilament formation and increases signal transducer and activator of transcription 3 (STAT3) phosphorylation, which is highly related to cell differentiation and neuroinflammation. In diabetic peripheral neuropathic mice, miR‐590‐3p was downregulated to disinhibit Ras‐related protein 1A (RAP1A) in the DRG tissue and inhibit neural T cells infiltration.\n42\n Thus, exogenous miR‐590‐3p may be a potential alternative for neuropathic pain treatment. CCI downregulated miR‐140 and miR‐144 expression in the DRG. Intrathecally injected miR‐140 and miR‐144 agomir decreased inflammatory factor secretion and ameliorated hyperalgesia by targeting sphingosine‐1‐phosphate receptor 1 (S1PR1) and RAS P21 protein activator 1 (RASA1), respectively.\n43\n, \n44\n\n\nmiRNAs are essential in neuropathic pain development via neuroinflammation‐related mechanisms in the spinal cord. miR‐130a‐3p targets and downregulates insulin‐like growth factor‐1/insulin‐like growth factor‐1 receptor (IGF‐1/IGF‐1R) expression to alleviate SCI‐induced neuropathic pain by mitigating microglial activation and NF‐κB phosphorylation.\n45\n miR‐378 was decreased in CCI rats, inhibiting neuropathic pain development by targeting the enhancer of zeste homolog 2 (EZH2).\n46\n EZH2 promotes neuropathic pain by increasing tumor necrosis factor‐alpha (TNF‐α), interleukin (IL)‐1β, and monocyte chemoattractant protein‐1 (MCP‐1) production. Intrathecally injecting miRNA‐138 lentivirus can remarkably alleviate neuropathic pain in partial sciatic nerve ligation (pSNL) rats by suppressing toll‐like receptor 4 (TLR4) and macrophage inflammatory protein‐1 alpha (MIP‐1α)/C‐C chemokine receptor 1 (CCR1) signaling pathways.\n47\n\n\nmiR‐15a/16 targets and downregulates G protein‐coupled receptor kinases 2 (GRK2) to disinhibit p38‐MAPK (mitogen‐activated protein kinase) and NF‐κB, contributing to neuroinflammation after CCI.\n48\n GRK2‐deficient mice present a pro‐inflammatory phenotype in spinal cord microglia/macrophages, restored by miR‐124.\n49\n SNL increased DNA methyltransferase 3a (DNMT3a) expression related to hypermethylation of the miR‐214‐3p promoter, resulting in miR‐214‐3p expression reduction, which enhanced astrocyte reactivity, colony‐stimulating factor‐1 (CSF1), and interleukin 6 (IL‐6) production, and hyperalgesia in rats.\n50\n Electro‐acupuncture attenuated SCI by inhibiting Nav1.3 and Bax in the injured spinal cord through miR‐214 upregulation.\n51\n Downregulated miR‐128 was reported to contribute to neuropathic pain via p38 or zinc finger E‐box binding homeobox 1 (ZEB1) activation in the spinal cord.\n52\n, \n53\n Meanwhile, ZEB1 was also targeted by miR‐200b/miR‐429, orchestrating neuropathic pain development.\n54\n\n\nIn a pSNL mouse model, nerve injury significantly reduced miR‐23a expression in spinal glial cells, concomitant with the upregulation of its target chemokine, CXC receptor 4 (CXCR4). In naïve mice, either miR‐23a downregulation or CXCR4 upregulation could active the thioredoxin‐interacting protein (TXNIP)/NOD‐like receptor protein 3 (NLRP3) inflammasome axis. Both intrathecal miR‐23a mimics and spinal CXCR4 downregulation by a lentivirus inhibited TXNIP or NLRP3 upregulation to alleviate hyperalgesia.\n55\n\n\nNotably, one miRNA was shown to have distinctive targets in different animal models of the spinal cord. For example, miR‐155 downregulation targeted and upregulated NADPH oxidase 2 (NOX2) expression to induce reactive oxygen species (ROS) production after SCI, presenting a pro‐inflammatory phenotype in microglia/macrophages.\n56\n The ability to induce glial polarization was also observed in cultured BV‐2 microglia.\n57\n In bortezomib‐induced neuropathic pain rats, downregulated miR‐155 upregulated TNF receptor 1 (TNFR1) expression, which activated its downstream signaling pathways, including p38‐MAPK, c‐Jun N‐terminal kinase (JNK), and transient receptor potential ankyrin 1 (TRPA1).\n58\n Therefore, it is suggested that miR‐155 might act as an intervention target for neuropathic pain. As expected, treatment with ibuprofen and L‐arginine delayed the behavioral pain changes while inhibiting spinal miR‐155 and NO.\n59\n miR‐155‐5p is also known to destabilize the blood–nerve barrier and expression of tight junction proteins, such as claudin‐1 and zonula occludens‐1 (ZO‐1). Tissue plasminogen activator (tPA) could transiently open such barriers to facilitate topically applying analgesics, via miR‐155‐5p upregulation.\n60\n However, we also noted that CCI upregulated but did not downregulate spinal cord miR‐155, and miR‐155 inhibition enhanced suppressor of cytokine signaling 1 (SOCS1) expression to inactivate inflammation via NF‐κB and p38‐MAPK inhibition.\n61\n In oxaliplatin‐induced peripheral neuropathic pain, spinal cord miR‐155 expression was also upregulated, and intrathecally injecting the miR‐155 inhibitor attenuated hyperalgesia in rats, possibly by inhibiting oxidative stress–TRPA1 pathways.\n62\n The underlying mechanisms of such distinctive miR‐155 expression change are still unknown and require further research into different pain models.","miRNA‐related autophagy is involved in neuropathic pain regulation. As reported, miR‐15a downregulated and stimulated AKT serine/threonine kinase 3 (AKT3), and inhibited autophagy post‐CCI.\n63\n Impaired autophagy participates in neuropathic pain development. Intrathecal miR‐15a agomir prominently suppressed AKT3 expression, induced autophagy, and attenuated CCI‐induced neuropathic pain. Similar to miR‐15a, miR‐145 and miR‐20b‐5p contributed to neuropathic pain regulation via protein kinase B (AKT)‐related autophagy pathways.\n64\n, \n65\n Meanwhile, miR‐195 in the spinal cord was observed to upregulate post‐SNL, targeting and inhibiting Autophagy‐Related 14 (ATG14) and its autophagy activation.\n66\n The miR‐195 inhibitor activated autophagy and suppressed neuroinflammation in vivo and in vitro.","Another class of miRNA‐based regulation focuses on ion channels, including sodium, potassium, and calcium channels, and transient receptor potential (TRP) channels, to modulate action potential production, firing rate, and neurotransmitter release.\nTheoretically, miRNAs can simultaneously have multiple target mRNAs because precise matching is not a prerequisite for inhibiting the target sequence. For example, other than NEFL,\n41\n miR‐7a targets the β2 subunit of the voltage‐gated sodium channel (Scn2b) with post‐transcriptional regulation to induce nociceptive DRG neurons hyperexcitability.\n67\n Other than miR‐7a, the other known miRNA‐ targets involved in sodium channels expression include miR‐30b, ‐96, ‐384‐5p and their target Scn3a (encoding Nav1.3),\n68\n, \n69\n, \n70\n miR‐30b‐5p and its target Scn8a (encoding Nav1.6),\n71\n and miR‐182 and ‐30b and their target Scn9a (encoding Nav1.7).\n72\n, \n73\n\n\nA miRNA cluster is a polycistronic gene containing several miRNAs derived from a single primary or nascent transcript. Approximately 40% of miRNAs are predicted to form clusters, but their significance is still unknown. miR‐17‐92 is a miRNA cluster that includes six different members, of which miR‐18a, ‐19a, ‐19b, and ‐92a upregulation induces allodynia. The predicted targets of the miR‐17‐92 cluster encompass genes encoding diverse voltage‐gated potassium channels and their regulatory subunits, including Kv1.1, Kv1.4, Kv3.4, Kv4.3, Kv7.5, dipeptidyl peptidase 10 (DPP10), and Navβ1.\n74\n CCI upregulated miR‐137 to target and downregulate Kcna2, which encodes Kv1.2 in the DRG and spinal dorsal horn.\n75\n By contrast, CCI decreased miR‐183‐5p expression in the DRG, and the predicted target gene TREK‐1, a subunit of the 2P‐domain K+ channel, was increased.\n76\n\n\nFurthermore, the miRNA‐183 cluster (miR‐183, part of miR‐96/182/183) regulates either basal mechanical or neuropathic pain.\n77\n The miR‐183 cluster targets Cacna2d1 and Cacna2d2, which encode auxiliary voltage‐gated calcium channel subunits α2δ‐1 and α2δ‐2, to affect nociceptor excitability. Nerve injury downregulated miR‐103 in the spinal cord, which simultaneously targeted and inhibited Cacna1c, Cacna2d1, and Cacnb1 and encoded the Cav1.2‐α1, α2δ1, and β1 subunits of the voltage‐gated calcium channels macromolecular complex Cav1.2 L‐type calcium channel, respectively.\n78\n Intrathecal miR‐103 successfully relieved neuropathic pain.\nIn addition to ion channels, TRP channels are ligand‐gated ion channels that promote painful sensations.\n79\n In the DRG, TRPA1 participates in miR‐141‐5p alleviated oxaliplatin‐induced neuropathic pain.\n80\n In SNI mice, miR‐449a ameliorated neuropathic pain by decreasing the activity of TRPA1 and calcium‐activated potassium channel subunit α‐1 (KCNMA1), thus acting as a potential therapeutic alternative for treating neuropathic pain.\n81\n\n","miRNAs regulate diverse pain‐related mediators, protein kinases, and structural proteins along the pain processing pathways. For HIV‐associated symptomatic distal sensory polyneuropathy and neuropathic pain, serum miR‐455‐3p acted as a potential biomarker, possibly targeting multiple genes involved in peripheral neuropathic pain, such as nerve growth factor (NGF) and related genes.\n82\n Brain‐derived neurotrophic factor (BDNF), another well‐recognized pain mediator, is a common miR‐1, ‐183, and ‐206 target.\n83\n, \n84\n, \n85\n After nerve injury, several miRNAs, including miR‐19a, ‐301, and ‐132, were downregulated, and their target methyl cytosine–guanine dinucleotide (CpG)‐binding protein 2 (MeCP2) expression was increased, leading to concomitant BDNF upregulation.\n86\n miR‐30c‐5p was upregulated in the serum and CSF of patients with chronic peripheral ischemia.\n16\n A miR‐30c‐5p inhibitor intraventricular injection postponed neuropathic pain development and fully reversed hyperalgesia in rodents. Transforming growth factor beta (TGF‐β) participates in the effects of miR‐30c‐5p, which refers to the endogenous opioid analgesic system.\n16\n\n\nmiRNAs dysregulate specific pain‐related protein kinases. For example, in DNP rats, miR‐133a‐3p was dysregulated in the sciatic nerve, interacting with p‐p38 MAPK to participate in the development of neuropathic pain.\n87\n Via a methyl CpG‐binding domain and transcriptional repressor domain, MeCP2 acts as a transcriptional repressor, and its overexpression improves neuropathic pain, potentiating an anti‐nociceptive effect of MeCP2. The authors also noted that MeCP2 expression changed post‐transcriptionally, and its mRNA level did not significantly change after SNI. However, the protein level was upregulated. During the development period of neuropathic pain, phospho‐cAMP response element‐binding protein (p‐CREB) elevated rapidly but returned 3–7 days after SNI, concomitant with miR‐132 downregulation, which targets MeCP2 and inhibits its expression post‐transcriptionally.\n88\n SNL decreased miR‐200b and miR‐429 in NAc neurons, along with an upregulation of their target (DNMT3a).\n89\n Further mechanism studies found that DNMT3a in the NAc was expressed in NR1 immunoreactive neurons, suggesting the dysregulation of ‘mesolimbic motivation circuitry’ for neuropathic pain development.\nThe CCI induced time‐dependent miR‐1 downregulation in injured sciatic nerves. This change in expression was related to the upregulation and translocation of the miR‐1‐targeted connexin 43 (Cx43), the major connexin of astrocytes.\n83\n miR‐1 mimics could reduce Cx43 expression in cultured human glioblastoma cells. However, miR‐1 mimics intraneural transfection failed to alter Cx43 protein expression and did not improve pain behavior. The authors attributed such treatment failure to insufficient inhibition of miR‐1 for Cx43 via intraneural injection. Alternatively, there were regulatory mechanisms for Cx43 in vivo other than miR‐1.\n90\n However, miR‐1 in the DRG was DE according to peripheral nerve injury type. CCI, pSNL, and sural nerve injury downregulated miR‐1. However, axotomy of the sciatic nerve and tibial nerve injury increased its expression in the DRG.\n83\n, \n91\n, \n92\n As previously described, miR‐1 was also upregulated following capsaicin treatment and bone cancer pain.\n91\n, \n93\n Additionally, miR‐1 downregulation inhibited bone cancer pain. Collectively, the action of miR‐1 on pain seems to be complex and stimulus‐dependent.\nNotably, specific miRNAs can promote neuropathic pain development via diverse mechanisms. For example, beta‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1), a membrane protease essential for myelination, was downregulated after miR‐15b overexpression in vitro or in the DRG of chemotherapy‐related neuropathic pain rats.\n94\n BACE1‐mediated neuregulin 1 reduction decreases nerve conduction velocity. In addition, BACE1 modulated Navβ2 subunit expression and neuronal activity and regulated inflammation‐related TNFR expression.","Neurotransmission excitatory and inhibitory imbalances in the spinal cord also contribute to neuropathic pain development. For example, miR‐500 increased to regulate glutamic acid decarboxylase 67 (GAD67) expression and target the specific site of Gad1 in the dorsal horn.\n95\n GAD67 expression reduction inhibited the function of GABAergic neurons and the resultant inhibitory synaptic transmission dysregulation contributed to neuropathic pain development. In addition, miR‐23b is crucial for improving neuropathic pain in the injured spinal cord by downregulating its target gene, Nox4, which further normalizes glutamic acid decarboxylase 65/67 (GAD65/67) expression and protects GABAergic neurons from apoptosis.\n96\n Microarray analysis showed that miR‐539 was prominently reduced in the contralateral anterior cingulate cortex (ACC) after CCI, which is related to enhanced NR2B protein expression. Injecting miR‐539 mimics into the contralateral ACC attenuated CCI‐evoked mechanical hyperalgesia, suggesting that the N‐methyl‐D‐aspartate (NMDA) receptor NR2B subunit regulates neuropathic pain.\n97\n\n","We mainly focused on exosomal miR‐21 with regard to EV miRNAs in neuropathic pain. As aforementioned, SNL, DRT, or VRT could upregulate miR‐21 in the injured DRG.\n22\n Notably, miR‐21 was also increased in serum exosomes from nerve‐ligated mice.\n98\n In another in‐depth study, macrophages readily took up pure sensory neuron‐derived exosomes encompassing miR‐21‐5p to promote a pro‐inflammatory phenotype.\n99\n Either intrathecal miR‐21‐5p antagomir or miR‐21 conditional deletion in sensory neurons ameliorated hyperalgesia and macrophage recruitment in the DRG.\nUnlike the miRNA target gene inhibitory mechanism, miR‐21 acts as an endogenous toll‐like receptor 8 (TLR8) ligand, leading to neuropathic pain development.\n100\n TLR8, a nucleic acid‐sensing receptor, is located in the endosomes and lysosomes, leading to ERK‐mediated inflammatory mediator production and neuronal activation after SNL. Although miR‐21 and TLR8 are only distributed in small‐ and medium‐sized neurons, miR‐21 can also be derived from large‐sized neurons and reach TLR8 in the endosomes of other types of neurons.\n101\n Similar to miR‐21, DRG sensory neurons secreted miR‐23a‐enriched EVs following nerve injury and were taken up by macrophages to enhance M1 polarization in vitro. A20, an NF‐κB signaling pathway inhibitor, is a verified miR‐23a target gene.\n102\n Moreover, intrathecally delivering EVs‐miR‐23a antagomir attenuated neuropathic hyperalgesia and reduced M1 macrophages by inhibiting A20 to activate NF‐κB signaling.","lncRNAs and circRNAs are essential in neuropathic pain development by acting as AS RNA or miRNA sponges, epigenetically regulating pain‐related molecules expression, or modulating miRNA processing (Table 2).\nLong non‐coding RNAs (lncRNAs) and circular non‐coding RNAs (circRNAs) expression change for the development of neuropathic pain\nAbbreviations: AQP4, aquaporin 4; AS RNA, antisense RNA; CCI, chronic constriction injury; CDK4, cyclin‐dependent kinase 4; CDK6, cyclin‐dependent kinase 6; DNP, diabetic neuropathic pain; DRG, dorsal root ganglion; ENO1, enolase 1; EphB1, ephrin type‐b 1; EZH2, enhancer of zeste homolog 2; HMGB1, high mobility group box 1; NAT, natural antisense transcript; NOTCH2, NOTCH receptor 2; NP, neuropathic pain; pSNL, partial sciatic nerve ligation; pSTAT3, phosphorylated signal transducer and activator of transcription 3; SCI, spinal cord injury; SGC, satellite glia cell; SNL, spinal nerve ligation; STAT3, signal transducer and activator of transcription 3; TLR5, toll‐like receptor 5; TNFAIP1, tumor necrosis factor alpha‐induced protein 1; TRPV1, transient receptor potential vanilloid 1; XIST, X‐inactive specific transcript; ZEB1, zinc finger E‐box binding homeobox 1.\n\nlncRNAs act as AS RNA\n After peripheral nerve injury, Egr2‐AS‐RNA is upregulated in Schwann cells.\n103\n On the early growth response 2 (Egr2) promoter, Egr2‐AS‐RNA recruits an epigenetic silencing complex to downregulate Egr2, essential for peripheral myelination. Ectopic Egr2‐AS‐RNA expression in DRG cultures downregulates Egr2 mRNA and induces demyelination. In vivo, Egr2‐AS‐RNA inhibition reverts Egr2‐related gene expression and delays demyelination.\nVoltage‐gated potassium channel (Kv) Kcna2‐AS‐RNA is an endogenous, highly conserved, and widely explored lncRNA in neuropathic pain.\n104\n It is a natural antisense transcript (NAT), distributed in the cytoplasm and targets Kcna2 mRNA, which encodes the pain regulation‐related membrane Kv1.2 subunit. Kcna2‐AS‐RNA was time‐dependently upregulated in the inflicted rat DRG after nerve injury. It decreased the total Kv current, upregulated DRG neurons excitability, and produced neuropathic pain symptoms.\nThe complementary strand of DNA opposite the Scn9a gene encodes Scn9a NAT, another antisense lncRNA expressed in the DRG.\n105\n Scn9a NAT is suggested to be a negative regulator of Scn9a mRNA. Scn9a NAT overexpression inhibited Scn9a mRNA, its encoded protein Nav1.7, and Nav1.7 currents in DRG neurons. However, Scn9a NAT and Scn9a mRNA levels did not significantly change in the injured DRG until 2 weeks after nerve injury. More work is required to determine whether NAT can confer analgesia and reduce pain in the neuropathic pain state.\nAfter peripheral nerve injury, Egr2‐AS‐RNA is upregulated in Schwann cells.\n103\n On the early growth response 2 (Egr2) promoter, Egr2‐AS‐RNA recruits an epigenetic silencing complex to downregulate Egr2, essential for peripheral myelination. Ectopic Egr2‐AS‐RNA expression in DRG cultures downregulates Egr2 mRNA and induces demyelination. In vivo, Egr2‐AS‐RNA inhibition reverts Egr2‐related gene expression and delays demyelination.\nVoltage‐gated potassium channel (Kv) Kcna2‐AS‐RNA is an endogenous, highly conserved, and widely explored lncRNA in neuropathic pain.\n104\n It is a natural antisense transcript (NAT), distributed in the cytoplasm and targets Kcna2 mRNA, which encodes the pain regulation‐related membrane Kv1.2 subunit. Kcna2‐AS‐RNA was time‐dependently upregulated in the inflicted rat DRG after nerve injury. It decreased the total Kv current, upregulated DRG neurons excitability, and produced neuropathic pain symptoms.\nThe complementary strand of DNA opposite the Scn9a gene encodes Scn9a NAT, another antisense lncRNA expressed in the DRG.\n105\n Scn9a NAT is suggested to be a negative regulator of Scn9a mRNA. Scn9a NAT overexpression inhibited Scn9a mRNA, its encoded protein Nav1.7, and Nav1.7 currents in DRG neurons. However, Scn9a NAT and Scn9a mRNA levels did not significantly change in the injured DRG until 2 weeks after nerve injury. More work is required to determine whether NAT can confer analgesia and reduce pain in the neuropathic pain state.\n\nlncRNAs act as miRNA sponges lncRNAs may act as miRNA sponges to form the lncRNA‐miRNA‐mRNA axis and regulate target gene expression. For example, the lncRNA X‐inactive specific transcript (XIST) is upregulated in the dorsal horn of the spinal cord after CCI. By sponging miR‐137, ‐150, ‐154‐5p, and ‐544, XIST can inhibit the expression of corresponding targets, including TNF‐α‐induced protein 1 (TNFAIP1), ZEB1, TLR5, and STAT3.\n106\n, \n107\n, \n108\n, \n109\n TNFAIP1 activates the NF‐κB signaling pathway, while ZEB1, TLR5, and STAT3 are crucial in the neuroinflammatory response. XIST inhibition markedly ameliorates neuropathic pain development. In addition, lncRNA nuclear enriched abundant transcript 1 (NEAT1) was upregulated in the spinal cord dorsal horn to form NEAT1‐miR‐381‐high mobility group box 1 (HMGB1) and NEAT1–miR‐128‐3p–aquaporin 4 (AQP4) axes following CCI and SCI, respectively.\n110\n, \n111\n NEAT1 downregulation inhibited IL‐6, IL‐1β, and TNF‐α to improve neuropathic pain.\nAlthough the function of circRNAs as miRNA sponges is still largely unknown, they have been termed as competing endogenous RNAs that bind to target miRNAs and regulate their function.\n112\n For example, circHIPK3 is a circRNA highly enriched in serum from DNP patients and in the DRG from DNP rats.\n113\n circHIPK3 sponges miR‐124 to promote neuroinflammation, pointing to the involvement of the circHIPK3–miR‐124 axis during DNP. circRNA ciRS‐7 participates in neuropathic pain progression by sponging miR‐135a‐5p to regulate autophagy and inflammation in the spinal cord of CCI rats.\n114\n Besides, cirZNF609–miR‐22‐3p‐enolase 1 (ENO1) and circ_0005075–miR‐151a‐3p‐NOTCH receptor 2 (NOTCH2) regulatory axes upregulate inflammatory factor expression and promote neuropathic pain development in the spinal cord after CCI.\n115\n, \n116\n\n\nlncRNAs may act as miRNA sponges to form the lncRNA‐miRNA‐mRNA axis and regulate target gene expression. For example, the lncRNA X‐inactive specific transcript (XIST) is upregulated in the dorsal horn of the spinal cord after CCI. By sponging miR‐137, ‐150, ‐154‐5p, and ‐544, XIST can inhibit the expression of corresponding targets, including TNF‐α‐induced protein 1 (TNFAIP1), ZEB1, TLR5, and STAT3.\n106\n, \n107\n, \n108\n, \n109\n TNFAIP1 activates the NF‐κB signaling pathway, while ZEB1, TLR5, and STAT3 are crucial in the neuroinflammatory response. XIST inhibition markedly ameliorates neuropathic pain development. In addition, lncRNA nuclear enriched abundant transcript 1 (NEAT1) was upregulated in the spinal cord dorsal horn to form NEAT1‐miR‐381‐high mobility group box 1 (HMGB1) and NEAT1–miR‐128‐3p–aquaporin 4 (AQP4) axes following CCI and SCI, respectively.\n110\n, \n111\n NEAT1 downregulation inhibited IL‐6, IL‐1β, and TNF‐α to improve neuropathic pain.\nAlthough the function of circRNAs as miRNA sponges is still largely unknown, they have been termed as competing endogenous RNAs that bind to target miRNAs and regulate their function.\n112\n For example, circHIPK3 is a circRNA highly enriched in serum from DNP patients and in the DRG from DNP rats.\n113\n circHIPK3 sponges miR‐124 to promote neuroinflammation, pointing to the involvement of the circHIPK3–miR‐124 axis during DNP. circRNA ciRS‐7 participates in neuropathic pain progression by sponging miR‐135a‐5p to regulate autophagy and inflammation in the spinal cord of CCI rats.\n114\n Besides, cirZNF609–miR‐22‐3p‐enolase 1 (ENO1) and circ_0005075–miR‐151a‐3p‐NOTCH receptor 2 (NOTCH2) regulatory axes upregulate inflammatory factor expression and promote neuropathic pain development in the spinal cord after CCI.\n115\n, \n116\n\n\n\nlncRNAs epigenetically regulate pain‐related molecules expression In addition to acting as a miRNA sponge, lncRNAs can epigenetically regulate the expression of pain‐related molecules. For example, peripheral nerve injury decreases the expression of DRG‐specifically enriched lncRNAs (DS‐lncRNAs) in the injured DRG. Restoring DS‐lncRNAs blocks nerve injury‐induced increases in euchromatic histone lysine N‐methyltransferase 2 (Ehmt2) mRNA and its encoding protein G9a, reverses G9a‐related decreases in opioid receptors and Kcna2 in the injured DRG, and ameliorates nerve injury‐induced pain hypersensitivity.\n117\n In addition, transcriptome screening in the DRG of DNP rats\n118\n has identified diverse dysregulated lncRNAs, including uc.48+,\n119\n BC168687,\n120\n, \n121\n NONRATT021972,\n31\n, \n122\n, \n123\n MRAK009713,\n124\n and Lncenc1.\n125\n The expression of uc.48+, BC168687, NONRATT021972, MRAK009713, and Lncenc1 was prominently higher in the DRG of neuropathic pain rats.\n119\n, \n120\n, \n121\n, \n122\n, \n123\n, \n124\n, \n125\n Blocking the upregulation via intrathecal or intravenous administration of the corresponding small‐interfering RNA (siRNA) may alleviate neuropathic pain by inhibiting the excitatory transmission mediated by purinergic receptors,\n119\n, \n120\n, \n122\n, \n123\n, \n124\n TNF‐α‐related pathways,\n31\n transient receptor potential vanilloid 1 (TRPV1),\n121\n or EZH2.\n125\n\n\nAs demonstrated by a luciferase assay and RNA‐binding protein immunoprecipitation, lncRNA small nucleolar RNA host gene 1 (SNHG1) can induce neuropathic pain in the spinal cord by binding to the promoter region of cyclin‐dependent kinase 4 (CDK4), stimulating its expression.\n126\n SNHG1 knockdown alleviated neuropathic pain development, and SNHG1 overexpression was able to induce neuropathic pain. Similarly, lncRNA PKIA‐AS1 participates in SNL‐induced neuropathic pain by downregulating DNA methyltransferase 1‐catalyzed cyclin‐dependent kinase 6 (DNMT1‐catalyzed) CDK6 promoter methylation and regulating CDK6.\n127\n Cyclin‐dependent kinases (CDKs) transcriptionally enhanced pro‐inflammatory gene expression during the G1 cell phase. Furthermore, CDK6 recruitment to the nuclear chromatin fraction by cytokines is related to NF‐κB, STAT, and activator protein 1 (AP‐1) activation to induce neuroinflammation.\n128\n Other than the DRG, lncRNA Kcna2‐AS‐RNA was also highly expressed in the spinal cord of postherpetic neuralgia rats, and its downregulation alleviated neuropathic pain by reducing phospho‐STAT3 (pSTAT3) translocation from the cytoplasm to the nucleus and then inhibiting spinal astrocytes activation.\n129\n\n\nIn addition to acting as a miRNA sponge, lncRNAs can epigenetically regulate the expression of pain‐related molecules. For example, peripheral nerve injury decreases the expression of DRG‐specifically enriched lncRNAs (DS‐lncRNAs) in the injured DRG. Restoring DS‐lncRNAs blocks nerve injury‐induced increases in euchromatic histone lysine N‐methyltransferase 2 (Ehmt2) mRNA and its encoding protein G9a, reverses G9a‐related decreases in opioid receptors and Kcna2 in the injured DRG, and ameliorates nerve injury‐induced pain hypersensitivity.\n117\n In addition, transcriptome screening in the DRG of DNP rats\n118\n has identified diverse dysregulated lncRNAs, including uc.48+,\n119\n BC168687,\n120\n, \n121\n NONRATT021972,\n31\n, \n122\n, \n123\n MRAK009713,\n124\n and Lncenc1.\n125\n The expression of uc.48+, BC168687, NONRATT021972, MRAK009713, and Lncenc1 was prominently higher in the DRG of neuropathic pain rats.\n119\n, \n120\n, \n121\n, \n122\n, \n123\n, \n124\n, \n125\n Blocking the upregulation via intrathecal or intravenous administration of the corresponding small‐interfering RNA (siRNA) may alleviate neuropathic pain by inhibiting the excitatory transmission mediated by purinergic receptors,\n119\n, \n120\n, \n122\n, \n123\n, \n124\n TNF‐α‐related pathways,\n31\n transient receptor potential vanilloid 1 (TRPV1),\n121\n or EZH2.\n125\n\n\nAs demonstrated by a luciferase assay and RNA‐binding protein immunoprecipitation, lncRNA small nucleolar RNA host gene 1 (SNHG1) can induce neuropathic pain in the spinal cord by binding to the promoter region of cyclin‐dependent kinase 4 (CDK4), stimulating its expression.\n126\n SNHG1 knockdown alleviated neuropathic pain development, and SNHG1 overexpression was able to induce neuropathic pain. Similarly, lncRNA PKIA‐AS1 participates in SNL‐induced neuropathic pain by downregulating DNA methyltransferase 1‐catalyzed cyclin‐dependent kinase 6 (DNMT1‐catalyzed) CDK6 promoter methylation and regulating CDK6.\n127\n Cyclin‐dependent kinases (CDKs) transcriptionally enhanced pro‐inflammatory gene expression during the G1 cell phase. Furthermore, CDK6 recruitment to the nuclear chromatin fraction by cytokines is related to NF‐κB, STAT, and activator protein 1 (AP‐1) activation to induce neuroinflammation.\n128\n Other than the DRG, lncRNA Kcna2‐AS‐RNA was also highly expressed in the spinal cord of postherpetic neuralgia rats, and its downregulation alleviated neuropathic pain by reducing phospho‐STAT3 (pSTAT3) translocation from the cytoplasm to the nucleus and then inhibiting spinal astrocytes activation.\n129\n\n\n\nlncRNAs modulate miRNA processing Specific lncRNAs are essential in neuropathic pain by modulating miRNA processing. For example, the transcribed ultraconserved lncRNA uc.153 was prominently increased in the spinal cord of rats with CCI neuropathic pain. uc.153 knockdown reversed CCI‐induced pain behaviors and spinal neuronal hypersensitivity. Mechanistically, uc.153 negatively modulated Dicer‐mediated pre‐miR‐182‐5p processing and inhibited maturation. Meanwhile, spinal miR‐182‐5p downregulation increased the expression of its target, ephrin type‐b (EphB1), and p‐NR2B (phosphorylated N‐methyl‐D‐aspartate receptor (NMDAR) 2B subunit) expression and facilitated hyperalgesia.\n130\n\n\nCollectively, lncRNAs are crucial in neuropathic pain via diverse mechanisms. Notably, one lncRNA may act with more than one mechanism to regulate target gene expression. For example, SNL upregulated circAnks1a in the cytoplasm and nucleus. circAnks1a enhances the interplay between Y‐box‐binding protein 1 (YBX1) and transportin‐1 to facilitate YBX1 nuclear translocation in the cytoplasm. Meanwhile, in the nucleus, circAnks1a combines with the Vegfb promoter and recruits YBX1 to the Vegfb promoter to enhance its transcription. Additionally, cytoplasmic circAnks1a sponges miR‐324‐3p to regulate vascular endothelial growth factor B (VEGFB) expression. VEGFB binding to its receptor results in the activation of various downstream targets, including p38‐MAPK, PKB/AKT (protein kinase B), extracellular signal‐regulated kinase (ERK)/MAPK, and phosphoinositide 3‐kinase (PI3K).\n131\n Therefore, VEGFB upregulation increases dorsal horn neuron excitability and contributes to pain hypersensitivity after nerve injury.\n132\n\n\nWe also noted that specific lncRNAs were DE following various nerve injuries. For example, lncRNA Malat1 was upregulated after CCI to sponge miR‐206, and Malat1 suppression delayed neuropathic pain progression via miR‐206‐ZEB2 axis‐mediated neuroinflammation inhibition.\n133\n Conversely, in rats with complete brachial plexus avulsion‐induced neuropathic pain, Malat1 decreased in spinal cord neurons, and such downregulation increased neuronal spontaneous electrical activity via calcium flux regulation.\n134\n The reason for such distinctive Malat1 expression following different pain models is still unclear, and further research is required.\nSpecific lncRNAs are essential in neuropathic pain by modulating miRNA processing. For example, the transcribed ultraconserved lncRNA uc.153 was prominently increased in the spinal cord of rats with CCI neuropathic pain. uc.153 knockdown reversed CCI‐induced pain behaviors and spinal neuronal hypersensitivity. Mechanistically, uc.153 negatively modulated Dicer‐mediated pre‐miR‐182‐5p processing and inhibited maturation. Meanwhile, spinal miR‐182‐5p downregulation increased the expression of its target, ephrin type‐b (EphB1), and p‐NR2B (phosphorylated N‐methyl‐D‐aspartate receptor (NMDAR) 2B subunit) expression and facilitated hyperalgesia.\n130\n\n\nCollectively, lncRNAs are crucial in neuropathic pain via diverse mechanisms. Notably, one lncRNA may act with more than one mechanism to regulate target gene expression. For example, SNL upregulated circAnks1a in the cytoplasm and nucleus. circAnks1a enhances the interplay between Y‐box‐binding protein 1 (YBX1) and transportin‐1 to facilitate YBX1 nuclear translocation in the cytoplasm. Meanwhile, in the nucleus, circAnks1a combines with the Vegfb promoter and recruits YBX1 to the Vegfb promoter to enhance its transcription. Additionally, cytoplasmic circAnks1a sponges miR‐324‐3p to regulate vascular endothelial growth factor B (VEGFB) expression. VEGFB binding to its receptor results in the activation of various downstream targets, including p38‐MAPK, PKB/AKT (protein kinase B), extracellular signal‐regulated kinase (ERK)/MAPK, and phosphoinositide 3‐kinase (PI3K).\n131\n Therefore, VEGFB upregulation increases dorsal horn neuron excitability and contributes to pain hypersensitivity after nerve injury.\n132\n\n\nWe also noted that specific lncRNAs were DE following various nerve injuries. For example, lncRNA Malat1 was upregulated after CCI to sponge miR‐206, and Malat1 suppression delayed neuropathic pain progression via miR‐206‐ZEB2 axis‐mediated neuroinflammation inhibition.\n133\n Conversely, in rats with complete brachial plexus avulsion‐induced neuropathic pain, Malat1 decreased in spinal cord neurons, and such downregulation increased neuronal spontaneous electrical activity via calcium flux regulation.\n134\n The reason for such distinctive Malat1 expression following different pain models is still unclear, and further research is required.","After peripheral nerve injury, Egr2‐AS‐RNA is upregulated in Schwann cells.\n103\n On the early growth response 2 (Egr2) promoter, Egr2‐AS‐RNA recruits an epigenetic silencing complex to downregulate Egr2, essential for peripheral myelination. Ectopic Egr2‐AS‐RNA expression in DRG cultures downregulates Egr2 mRNA and induces demyelination. In vivo, Egr2‐AS‐RNA inhibition reverts Egr2‐related gene expression and delays demyelination.\nVoltage‐gated potassium channel (Kv) Kcna2‐AS‐RNA is an endogenous, highly conserved, and widely explored lncRNA in neuropathic pain.\n104\n It is a natural antisense transcript (NAT), distributed in the cytoplasm and targets Kcna2 mRNA, which encodes the pain regulation‐related membrane Kv1.2 subunit. Kcna2‐AS‐RNA was time‐dependently upregulated in the inflicted rat DRG after nerve injury. It decreased the total Kv current, upregulated DRG neurons excitability, and produced neuropathic pain symptoms.\nThe complementary strand of DNA opposite the Scn9a gene encodes Scn9a NAT, another antisense lncRNA expressed in the DRG.\n105\n Scn9a NAT is suggested to be a negative regulator of Scn9a mRNA. Scn9a NAT overexpression inhibited Scn9a mRNA, its encoded protein Nav1.7, and Nav1.7 currents in DRG neurons. However, Scn9a NAT and Scn9a mRNA levels did not significantly change in the injured DRG until 2 weeks after nerve injury. More work is required to determine whether NAT can confer analgesia and reduce pain in the neuropathic pain state.","lncRNAs may act as miRNA sponges to form the lncRNA‐miRNA‐mRNA axis and regulate target gene expression. For example, the lncRNA X‐inactive specific transcript (XIST) is upregulated in the dorsal horn of the spinal cord after CCI. By sponging miR‐137, ‐150, ‐154‐5p, and ‐544, XIST can inhibit the expression of corresponding targets, including TNF‐α‐induced protein 1 (TNFAIP1), ZEB1, TLR5, and STAT3.\n106\n, \n107\n, \n108\n, \n109\n TNFAIP1 activates the NF‐κB signaling pathway, while ZEB1, TLR5, and STAT3 are crucial in the neuroinflammatory response. XIST inhibition markedly ameliorates neuropathic pain development. In addition, lncRNA nuclear enriched abundant transcript 1 (NEAT1) was upregulated in the spinal cord dorsal horn to form NEAT1‐miR‐381‐high mobility group box 1 (HMGB1) and NEAT1–miR‐128‐3p–aquaporin 4 (AQP4) axes following CCI and SCI, respectively.\n110\n, \n111\n NEAT1 downregulation inhibited IL‐6, IL‐1β, and TNF‐α to improve neuropathic pain.\nAlthough the function of circRNAs as miRNA sponges is still largely unknown, they have been termed as competing endogenous RNAs that bind to target miRNAs and regulate their function.\n112\n For example, circHIPK3 is a circRNA highly enriched in serum from DNP patients and in the DRG from DNP rats.\n113\n circHIPK3 sponges miR‐124 to promote neuroinflammation, pointing to the involvement of the circHIPK3–miR‐124 axis during DNP. circRNA ciRS‐7 participates in neuropathic pain progression by sponging miR‐135a‐5p to regulate autophagy and inflammation in the spinal cord of CCI rats.\n114\n Besides, cirZNF609–miR‐22‐3p‐enolase 1 (ENO1) and circ_0005075–miR‐151a‐3p‐NOTCH receptor 2 (NOTCH2) regulatory axes upregulate inflammatory factor expression and promote neuropathic pain development in the spinal cord after CCI.\n115\n, \n116\n\n","In addition to acting as a miRNA sponge, lncRNAs can epigenetically regulate the expression of pain‐related molecules. For example, peripheral nerve injury decreases the expression of DRG‐specifically enriched lncRNAs (DS‐lncRNAs) in the injured DRG. Restoring DS‐lncRNAs blocks nerve injury‐induced increases in euchromatic histone lysine N‐methyltransferase 2 (Ehmt2) mRNA and its encoding protein G9a, reverses G9a‐related decreases in opioid receptors and Kcna2 in the injured DRG, and ameliorates nerve injury‐induced pain hypersensitivity.\n117\n In addition, transcriptome screening in the DRG of DNP rats\n118\n has identified diverse dysregulated lncRNAs, including uc.48+,\n119\n BC168687,\n120\n, \n121\n NONRATT021972,\n31\n, \n122\n, \n123\n MRAK009713,\n124\n and Lncenc1.\n125\n The expression of uc.48+, BC168687, NONRATT021972, MRAK009713, and Lncenc1 was prominently higher in the DRG of neuropathic pain rats.\n119\n, \n120\n, \n121\n, \n122\n, \n123\n, \n124\n, \n125\n Blocking the upregulation via intrathecal or intravenous administration of the corresponding small‐interfering RNA (siRNA) may alleviate neuropathic pain by inhibiting the excitatory transmission mediated by purinergic receptors,\n119\n, \n120\n, \n122\n, \n123\n, \n124\n TNF‐α‐related pathways,\n31\n transient receptor potential vanilloid 1 (TRPV1),\n121\n or EZH2.\n125\n\n\nAs demonstrated by a luciferase assay and RNA‐binding protein immunoprecipitation, lncRNA small nucleolar RNA host gene 1 (SNHG1) can induce neuropathic pain in the spinal cord by binding to the promoter region of cyclin‐dependent kinase 4 (CDK4), stimulating its expression.\n126\n SNHG1 knockdown alleviated neuropathic pain development, and SNHG1 overexpression was able to induce neuropathic pain. Similarly, lncRNA PKIA‐AS1 participates in SNL‐induced neuropathic pain by downregulating DNA methyltransferase 1‐catalyzed cyclin‐dependent kinase 6 (DNMT1‐catalyzed) CDK6 promoter methylation and regulating CDK6.\n127\n Cyclin‐dependent kinases (CDKs) transcriptionally enhanced pro‐inflammatory gene expression during the G1 cell phase. Furthermore, CDK6 recruitment to the nuclear chromatin fraction by cytokines is related to NF‐κB, STAT, and activator protein 1 (AP‐1) activation to induce neuroinflammation.\n128\n Other than the DRG, lncRNA Kcna2‐AS‐RNA was also highly expressed in the spinal cord of postherpetic neuralgia rats, and its downregulation alleviated neuropathic pain by reducing phospho‐STAT3 (pSTAT3) translocation from the cytoplasm to the nucleus and then inhibiting spinal astrocytes activation.\n129\n\n","Specific lncRNAs are essential in neuropathic pain by modulating miRNA processing. For example, the transcribed ultraconserved lncRNA uc.153 was prominently increased in the spinal cord of rats with CCI neuropathic pain. uc.153 knockdown reversed CCI‐induced pain behaviors and spinal neuronal hypersensitivity. Mechanistically, uc.153 negatively modulated Dicer‐mediated pre‐miR‐182‐5p processing and inhibited maturation. Meanwhile, spinal miR‐182‐5p downregulation increased the expression of its target, ephrin type‐b (EphB1), and p‐NR2B (phosphorylated N‐methyl‐D‐aspartate receptor (NMDAR) 2B subunit) expression and facilitated hyperalgesia.\n130\n\n\nCollectively, lncRNAs are crucial in neuropathic pain via diverse mechanisms. Notably, one lncRNA may act with more than one mechanism to regulate target gene expression. For example, SNL upregulated circAnks1a in the cytoplasm and nucleus. circAnks1a enhances the interplay between Y‐box‐binding protein 1 (YBX1) and transportin‐1 to facilitate YBX1 nuclear translocation in the cytoplasm. Meanwhile, in the nucleus, circAnks1a combines with the Vegfb promoter and recruits YBX1 to the Vegfb promoter to enhance its transcription. Additionally, cytoplasmic circAnks1a sponges miR‐324‐3p to regulate vascular endothelial growth factor B (VEGFB) expression. VEGFB binding to its receptor results in the activation of various downstream targets, including p38‐MAPK, PKB/AKT (protein kinase B), extracellular signal‐regulated kinase (ERK)/MAPK, and phosphoinositide 3‐kinase (PI3K).\n131\n Therefore, VEGFB upregulation increases dorsal horn neuron excitability and contributes to pain hypersensitivity after nerve injury.\n132\n\n\nWe also noted that specific lncRNAs were DE following various nerve injuries. For example, lncRNA Malat1 was upregulated after CCI to sponge miR‐206, and Malat1 suppression delayed neuropathic pain progression via miR‐206‐ZEB2 axis‐mediated neuroinflammation inhibition.\n133\n Conversely, in rats with complete brachial plexus avulsion‐induced neuropathic pain, Malat1 decreased in spinal cord neurons, and such downregulation increased neuronal spontaneous electrical activity via calcium flux regulation.\n134\n The reason for such distinctive Malat1 expression following different pain models is still unclear, and further research is required.","There are two main strategies for modulating miRNA function during pain treatment: upregulation or downregulation of specific miRNAs. miRNA mimics or virus‐based constructs were used to upregulate miRNA expression. miRNA inhibitors, miRNA sponges, or inhibition of a particular miRNA‐mRNA interaction has been applied to downregulate specific miRNAs. The design of lncRNA‐based therapeutics includes diverse approaches, such as post‐transcriptional inhibition of lncRNAs by antisense oligonucleotides or siRNA and steric blockades of lncRNA–protein interactions by small molecules and morpholino.\n135\n\n\nHowever, there is much work to be done before miRNA‐ and lncRNA‐based analgesics can be clinically used for various reasons, including their conservation among species, proper delivery, stability, off‐target effects, and potential activation of the immune system.\nFirst, most available findings regarding the efficacy of miRNAs or lncRNAs during neuropathic pain are based on animal but not human studies. Whether such results can be extrapolated to humans and their translational potential is still unknown because of conservation among species.\nSecond, the blood–brain barrier (BBB) is a practical challenge for delivering RNA‐based therapeutics into the central nervous system (CNS) via intravenous injection. Viral vectors, polypeptides, aptamers, and particular chemical modifications have been developed. Supplementing cholesterol molecules to the sense strand of a miRNA mimic or inhibitor has proven to be an efficient strategy. Cholesterol‐conjugated siRNAs showed better silencing potency than unconjugated siRNAs and presented a high efficacy for delivery to oligodendrocytes in the CNS.\n136\n Another proven method is the immunoliposome, a combination of liposomes, receptor‐targeted monoclonal antibodies, and the target molecules.\n137\n An immunoliposome nanocomplex has been reported to deliver therapeutic nucleic acids across the BBB into the deep brain by transferrin receptors.\n138\n In addition, intrathecal injections are a feasible approach in animal studies for neuropathic pain treatment. As reported, miR‐146a attenuated neuropathic pain partially by inhibiting TNF receptor‐associated factor 6 (TRAF6) and its downstream phospho‐JNK/C–C motif chemokine ligand 2 (pJNK/CCL2) signaling in the spinal cord.\n139\n Intrathecal injection of miR‐146a‐5p encapsulated nanoparticles provided an analgesic effect via NF‐κB and p38‐MAPK inhibition in spinal microglia.\n140\n In recent years, poly (D, L‐lactic‐co‐glycolic acid) (PLGA)‐nanoparticles have been applied to deliver siRNAs and plasmids into the spinal cord to treat neuropathic pain in rats.\n141\n PLGA copolymer is a promising US Food and Drug Administration (FDA)‐approved gene transmission material because of its biodegradability and biocompatibility in humans.\n142\n Intrathecal treatments with C‐X3‐C motif chemokine receptor 1 (CX3CR1), p38, p66shc siRNA encapsulated PLGA nanoparticles, or forkhead box P3 (Foxp3) plasmid‐encapsulated PLGA nanoparticles inhibit microglial activation and hyperalgesia in SNL rats.\n141\n, \n143\n, \n144\n, \n145\n\n\nExosomes are another promising delivery carrier for treating neuropathic pain. Exosomes are natural membranous microvesicles that carry RNAs, with the advantage of being efficient, cell‐free, and nonimmunogenicity. Intravenous injecting neuron‐targeted exosomes delivered the carried siRNAs to neurons, microglia, and oligodendrocytes to knock down specific gene expression in mice brains.\n146\n This approach enabled cell‐specific delivery of the siRNA‐cargo across the BBB. Mesenchymal stem cells (MSCs) are pluripotent stem cells with immunomodulatory, anti‐inflammatory, and nutritional properties. The treatment efficacy of exosomes derived from MSCs has been proven in neuropathic pain.\n147\n Intrathecal, local, or subcutaneous application of exosomes obtained from human umbilical cord MSCs could mitigate nerve injury‐induced hyperalgesia.\n148\n, \n149\n, \n150\n Furthermore, immunofluorescence results showed that most intrathecally injected exosomes could be found in injured peripheral axons, the DRG, and the spinal dorsal horn, potentiating a homing ability of exosomes.\n148\n\n\nThird, although miRNAs are relatively stable in vivo, they have long‐lasting efficacy and a high resistance to nucleolytic degradation compared to mRNAs. Particular chemical modifications are still required to prolong its half‐life or increase its stability, for example, by generating locked‐nucleic acids (LNAs). LNA modifications increase the RNA affinity of antisense oligonucleotides, presenting an excellent miRNA inhibitory activity at a low dosage.\n151\n Additionally, the LNA technique was used to synthesize highly stable aptamers.\n152\n\n\nFourth, the off‐target effect is another consideration. One miRNA may regulate multiple genes, and the off‐target potential of one miRNA may produce undesirable side effects.\n153\n, \n154\n\n\nFinally, activation of the immune system is a potential adverse event. Specific CpG motifs in oligonucleotides could trigger nonspecific immunological activity.\n155\n Therefore, RNA‐based therapeutics might also produce specific immune reactions after producing antibodies against the oligonucleotides.","Ming Jiang: Investigation, Methodology. Yelong Wang: Manuscript preparation. Jing Wang: Figures and tables preparation. Shanwu Feng: Supervision, Funding acquisition. Xian Wang: Conceptualization, Funding acquisition."],"string":"[\n \"According to the International Association for the Study of Pain, neuropathic pain is the most severe chronic pain condition triggered by a lesion or disease of the somatosensory system. It is characteristic of hyperalgesia, allodynia, or spontaneous pain.\\n1\\n Neuropathic pain can have peripheral and central origins, with the former including neuropathic pain after peripheral nerve injury, trigeminal neuralgia, postherpetic neuralgia, painful radiculopathy, and painful polyneuropathy. In contrast, central neuropathic pain includes neuropathic pain after spinal cord or brain injury, multiple sclerosis, and chronic central post‐stroke pain. Approximately 7%–10% of the general population will experience neuropathic pain, with the majority not having satisfactory pain relief with current therapies, leading to great suffering for individuals and enormous economic and social burdens.\\n2\\n\\n\\nThe exact molecular mechanisms underlying neuropathic pain remain unclear, and elucidating them is crucial for developing mechanism‐based treatment strategies. One proposed mechanism involves altered gene or protein expression along the pain processing pathways. Therefore, understanding how genes or proteins are dysregulated may help us to find a way to normalize these abnormalities and treat neuropathic pain.\\nIn recent years, accumulating evidence has suggested the essential role of non‐coding RNAs (ncRNAs) in various physiological and pathological procedures, such as embryonic development, inflammation, tumors, and respiratory and cardiovascular diseases.\\n3\\n ncRNAs have no protein‐coding potential, but they can govern gene or protein expression with diverse mechanisms. ncRNAs are extensively distributed in the peripheral and central nervous systems, including pain‐related structures.\\n4\\n Broad abnormal ncRNAs expression is observed following peripheral stimulation. These abnormalities are related to hyperalgesia during chronic pain development. The available data indicate that ncRNAs may be essential for hyperalgesia. In this review, we focus on microRNA (miRNA), long non‐coding RNA (lncRNA), and circular non‐coding RNA (circRNA) expression changes in neuropathic pain. Other types of ncRNAs are seldom reported in neuropathic pain and, thus, are not discussed herein. Notably, we will pay attention to their etiological role in the development of neuropathic pain and the current challenges and considerations for miRNA‐, lncRNA‐, and circRNA‐based therapeutics for neuropathic pain.\",\n \"Typically, miRNA production involves three steps: cropping, exporting, and dicing. First, the miRNA gene is transcribed in the nucleus, mainly by RNA polymerase II.\\n5\\n The resulting primary miRNA transcript (pri‐miRNA) is several kilobases (kb) in length, with a specific stem–loop structure that harbors mature miRNAs in the stem. The mature miRNA is ‘cropped’ by Drosha and its interactor DGCR8 (DiGeorge syndrome critical region 8), which cleaves pri‐miRNA at the stem to produce pre‐miRNA,\\n6\\n 60–70 nucleotides (nt) in length with a hairpin structure. Then, exportin‐5 recognizes and exports pre‐miRNA from the nucleus to the cytoplasm.\\n7\\n In the cytoplasm, ribonuclease III (RNAse III), termed Dicer, further cleaves the pre‐miRNA to release double‐stranded miRNA with a length of ~22 nt.\\n8\\n This miRNA is unwound by an unknown helicase or cleaved by Argonaute (Ago) to form the RNA‐induced silencing complex.\\n9\\n One strand in the RNA duplex remains with Ago as a mature miRNA, and the other is degraded. The seed sequence of the miRNA incompletely or entirely combines with the target mRNA sequence, resulting in target mRNAs degradation or transcriptional regulation.\\n10\\n\\n\\nUnlike miRNAs, lncRNAs are mRNAs‐like transcripts ranging in length from 200 nt to 100 kb that lack prominent open reading frames.\\n11\\n The lncRNA cellular mechanism is highly related to their intracellular localization. lncRNAs control chromatin functions, transcription, and RNA processing in the nucleus and affect mRNA stability, translation, and cellular signaling in the cytoplasm.\\n12\\n Compared to lncRNAs, circRNAs are more stable because a single circRNA molecular ends can be covalently linked compared to linear RNA. circRNAs are evolutionarily conserved molecules that are essential in the post‐transcriptional modification of gene expression by acting as miRNAs sponges or interacting with transcription or translational machinery. Numerous lncRNAs and circRNAs are distributed within pain‐related regions and dysregulated after peripheral noxious stimulation. Moreover, functional studies have indicated that miRNAs, lncRNAs, and circRNAs participate in neuropathic pain development by regulating diverse pain‐related genes along the pain processing pathways.\",\n \"\\nmiRNAs expression changes Microarray and deep‐sequencing analyses revealed that nerve injury or noxious stimuli could induce broad changes in miRNA expression in serum or along the pain processing pathways, including the perineal nerve, dorsal root ganglion (DRG), spinal cord, and supraspinal regions. For normal pain signal transmission, nerve injury or noxious stimuli are detected by nociceptors in the DRG or trigeminal ganglion (TG) and then transmitted to upstream neurons in the spinal dorsal horn. Subsequently, nociceptive stimuli are integrated, processed, and further transmitted ascending to specific supraspinal brain regions.\\nHuman studies have identified as many as 1134 differentially expressed (DE) genes in the serum of individuals with or without neuropathic pain after spinal cord injury (SCI).\\n13\\n miR‐204‐5p, ‐519d‐3p, ‐20b‐5p, and ‐6838‐5p might act as promising biomarkers and intervention targets for preventing and therapizing neuropathic pain after SCI. In trigeminal neuralgia individuals, serum miR‐132‐3p, ‐146b‐5p, ‐155‐5p, and ‐384 levels were prominently increased compared with healthy controls.\\n14\\n Patients with painful peripheral neuropathy had higher miR‐21 level in the serum and sural nerve compared with healthy controls. Meanwhile, miR‐155 was reduced in the serum and inflicted lower leg skin.\\n15\\n Animal and human studies showed that miR‐30c‐5p was upregulated in the serum and cerebro spinal fluid (CSF) of sciatic nerve injury rats and neuropathic pain patients with chronic peripheral ischemia. The high expression of miR‐30c‐5p, together with other clinical parameters, might be used to predict neuropathic pain development in patients with chronic peripheral ischemia.\\n16\\n Additionally, an animal study showed that plasma‐derived DE extracellular vesicle (EV) miRNAs regulated processes that are essential for neuropathic pain development. Most DE EV miRNAs for inflammation suppression were downregulated, potentially acting as biomarkers and targets in neuropathic pain treatment.\\n17\\n\\n\\nIn addition, numerous DE miRNAs have been identified in the DRG. One week after spared nerve injury (SNI), 33 and 39 miRNAs in the DRG were upregulated and downregulated, respectively, with most DE miRNAs related to axon guidance, focal adhesion, Ras and Wnt signaling pathways.\\n18\\n Furthermore, nerve injury‐induced miRNAs expression was dynamic and time‐dependent,\\n19\\n, \\n20\\n implicating multiple regulatory mechanisms in neuropathic pain initiation and development. Nerve injury redistributes miRNAs from a uniform style within the DRG soma of non‐allodynic animals to preferential localization to peripheral neurons in allodynic animals.\\n21\\n Furthermore, either sciatic nerve ligation (SNL), DRG transaction (DRT), or ventral root transection (VRT) could upregulate miR‐21 and miR‐31 while downregulating miR‐668 and miR‐672 in the injured DRG,\\n22\\n implying that these miRNAs could be therapeutic targets for treating diverse types of neuropathic pain.\\nThe spinal cord dorsal horn relays and modulates pain signals from the peripheral nociceptors to the supraspinal regions. In the spinal cord, numerous miRNA expression changes have been observed in chronic constriction injury (CCI) and diabetic neuropathic pain (DNP) rodents.\\n23\\n, \\n24\\n, \\n25\\n, \\n26\\n Previous research has suggested some miRNAs may be related to neuropathic pain development, for example, miR‐500, ‐221, and ‐21,\\n25\\n thus, acting as potential targets in its treatment.\\n27\\n\\n\\nCytoscape software constructed the miRNA–target gene regulatory network in the supraspinal region, including the nucleus accumbens (NAc), medial prefrontal cortex, and periaqueductal gray, between SNI and sham rats. Finally, four essential DE genes, includingCXCR2, IL12B, TNFSF8, and GRK1, and five miRNAs, including miR‐208a‐5p, −7688‐3p, −344f‐3p, −135b‐3p, and ‐135a‐2‐3p, were identified, indicating their essential roles in neuropathic pain pathogenesis.\\n28\\n Furthermore, in the prelimbic cortex of SNI rats, the DE miRNA–mRNA network pointed to molecules associated with inflammation.\\n29\\n DE miRNAs were also observed in the bilateral hippocampus of CCI rats. However, no significant difference was observed bilaterally in the hippocampus.\\n30\\n (Figure 1).\\nmicroRNAs (miRNAs) expression change following peripheral nerve injury or noxious stimuli SCI, spinal cord injury; CSF, cerebrol spinal fluid; DE, differential expressed; EV, extravascular vesicle; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury; DRT, DRG transection; VRT, ventral root transection\\nMicroarray and deep‐sequencing analyses revealed that nerve injury or noxious stimuli could induce broad changes in miRNA expression in serum or along the pain processing pathways, including the perineal nerve, dorsal root ganglion (DRG), spinal cord, and supraspinal regions. For normal pain signal transmission, nerve injury or noxious stimuli are detected by nociceptors in the DRG or trigeminal ganglion (TG) and then transmitted to upstream neurons in the spinal dorsal horn. Subsequently, nociceptive stimuli are integrated, processed, and further transmitted ascending to specific supraspinal brain regions.\\nHuman studies have identified as many as 1134 differentially expressed (DE) genes in the serum of individuals with or without neuropathic pain after spinal cord injury (SCI).\\n13\\n miR‐204‐5p, ‐519d‐3p, ‐20b‐5p, and ‐6838‐5p might act as promising biomarkers and intervention targets for preventing and therapizing neuropathic pain after SCI. In trigeminal neuralgia individuals, serum miR‐132‐3p, ‐146b‐5p, ‐155‐5p, and ‐384 levels were prominently increased compared with healthy controls.\\n14\\n Patients with painful peripheral neuropathy had higher miR‐21 level in the serum and sural nerve compared with healthy controls. Meanwhile, miR‐155 was reduced in the serum and inflicted lower leg skin.\\n15\\n Animal and human studies showed that miR‐30c‐5p was upregulated in the serum and cerebro spinal fluid (CSF) of sciatic nerve injury rats and neuropathic pain patients with chronic peripheral ischemia. The high expression of miR‐30c‐5p, together with other clinical parameters, might be used to predict neuropathic pain development in patients with chronic peripheral ischemia.\\n16\\n Additionally, an animal study showed that plasma‐derived DE extracellular vesicle (EV) miRNAs regulated processes that are essential for neuropathic pain development. Most DE EV miRNAs for inflammation suppression were downregulated, potentially acting as biomarkers and targets in neuropathic pain treatment.\\n17\\n\\n\\nIn addition, numerous DE miRNAs have been identified in the DRG. One week after spared nerve injury (SNI), 33 and 39 miRNAs in the DRG were upregulated and downregulated, respectively, with most DE miRNAs related to axon guidance, focal adhesion, Ras and Wnt signaling pathways.\\n18\\n Furthermore, nerve injury‐induced miRNAs expression was dynamic and time‐dependent,\\n19\\n, \\n20\\n implicating multiple regulatory mechanisms in neuropathic pain initiation and development. Nerve injury redistributes miRNAs from a uniform style within the DRG soma of non‐allodynic animals to preferential localization to peripheral neurons in allodynic animals.\\n21\\n Furthermore, either sciatic nerve ligation (SNL), DRG transaction (DRT), or ventral root transection (VRT) could upregulate miR‐21 and miR‐31 while downregulating miR‐668 and miR‐672 in the injured DRG,\\n22\\n implying that these miRNAs could be therapeutic targets for treating diverse types of neuropathic pain.\\nThe spinal cord dorsal horn relays and modulates pain signals from the peripheral nociceptors to the supraspinal regions. In the spinal cord, numerous miRNA expression changes have been observed in chronic constriction injury (CCI) and diabetic neuropathic pain (DNP) rodents.\\n23\\n, \\n24\\n, \\n25\\n, \\n26\\n Previous research has suggested some miRNAs may be related to neuropathic pain development, for example, miR‐500, ‐221, and ‐21,\\n25\\n thus, acting as potential targets in its treatment.\\n27\\n\\n\\nCytoscape software constructed the miRNA–target gene regulatory network in the supraspinal region, including the nucleus accumbens (NAc), medial prefrontal cortex, and periaqueductal gray, between SNI and sham rats. Finally, four essential DE genes, includingCXCR2, IL12B, TNFSF8, and GRK1, and five miRNAs, including miR‐208a‐5p, −7688‐3p, −344f‐3p, −135b‐3p, and ‐135a‐2‐3p, were identified, indicating their essential roles in neuropathic pain pathogenesis.\\n28\\n Furthermore, in the prelimbic cortex of SNI rats, the DE miRNA–mRNA network pointed to molecules associated with inflammation.\\n29\\n DE miRNAs were also observed in the bilateral hippocampus of CCI rats. However, no significant difference was observed bilaterally in the hippocampus.\\n30\\n (Figure 1).\\nmicroRNAs (miRNAs) expression change following peripheral nerve injury or noxious stimuli SCI, spinal cord injury; CSF, cerebrol spinal fluid; DE, differential expressed; EV, extravascular vesicle; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury; DRT, DRG transection; VRT, ventral root transection\\n\\nlncRNAs and circRNAs expression changes Patients with type 2 DNP had a prominent higher expression of serum lncRNA NONRATT021972 and more severe neuropathic pain symptoms.\\n31\\n LINC01119 and LINC02447 in the peripheral blood of SCI patients were identified in pain pathways that were important for neuropathic pain development.\\n32\\n\\n\\nMicroarray analysis revealed that a nerve injury could induce time‐dependent lncRNA expression changes in the sciatic nerve.\\n33\\n lncRNA H19 was persistently upregulated in Schwann cells along the peripheral nerve, proximal and distal to the injured site.\\n34\\n In the DRG, the transcriptomic analysis identified 86 known and 26 novel lncRNAs genes to be DE after spared sciatic nerve injury.\\n35\\n Of these, rno‐Cntnap2 and AC111653.1 were essential in peripheral nerve regeneration and were involved in neuropathic pain.\\nNext‐generation RNA sequencing showed 134 lncRNAs and 188 circRNAs were prominently changed 14 days after SNI in the spinal cord.\\n36\\n In addition, microarray analysis identified 1481 and 1096 DE lncRNAs and mRNAs, respectively, in the spinal cord dorsal horn of DNP rats. Of these, 289 neighboring and 57 overlapping lncRNA‐mRNA pairs, including ENSMUST00000150952‐Mbp and AK081017‐Usp15, have been suggested to participate in neuropathic pain development.\\n37\\n\\n\\ncircRNAs have characteristic circularized structures following the backsplicing of exons from antisense RNAs (AS RNAs) or mRNAs and, thus, are highly stable. Most circRNAs are highly conserved among species and lack translation potential, despite cap‐independent translation. A recent study showed that 363 and 106 circRNAs were significantly dysregulated in the ipsilateral dorsal horn after nerve injury.\\n38\\n (Figure 2).\\nLong non‐coding RNA (lncRNAs) and circular non‐coding RNAs (circRNAs) expression change following peripheral nerve injury or noxious stimuli DNP, diabetic neuropathic pain; SCI, spinal cord injury; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury\\nPatients with type 2 DNP had a prominent higher expression of serum lncRNA NONRATT021972 and more severe neuropathic pain symptoms.\\n31\\n LINC01119 and LINC02447 in the peripheral blood of SCI patients were identified in pain pathways that were important for neuropathic pain development.\\n32\\n\\n\\nMicroarray analysis revealed that a nerve injury could induce time‐dependent lncRNA expression changes in the sciatic nerve.\\n33\\n lncRNA H19 was persistently upregulated in Schwann cells along the peripheral nerve, proximal and distal to the injured site.\\n34\\n In the DRG, the transcriptomic analysis identified 86 known and 26 novel lncRNAs genes to be DE after spared sciatic nerve injury.\\n35\\n Of these, rno‐Cntnap2 and AC111653.1 were essential in peripheral nerve regeneration and were involved in neuropathic pain.\\nNext‐generation RNA sequencing showed 134 lncRNAs and 188 circRNAs were prominently changed 14 days after SNI in the spinal cord.\\n36\\n In addition, microarray analysis identified 1481 and 1096 DE lncRNAs and mRNAs, respectively, in the spinal cord dorsal horn of DNP rats. Of these, 289 neighboring and 57 overlapping lncRNA‐mRNA pairs, including ENSMUST00000150952‐Mbp and AK081017‐Usp15, have been suggested to participate in neuropathic pain development.\\n37\\n\\n\\ncircRNAs have characteristic circularized structures following the backsplicing of exons from antisense RNAs (AS RNAs) or mRNAs and, thus, are highly stable. Most circRNAs are highly conserved among species and lack translation potential, despite cap‐independent translation. A recent study showed that 363 and 106 circRNAs were significantly dysregulated in the ipsilateral dorsal horn after nerve injury.\\n38\\n (Figure 2).\\nLong non‐coding RNA (lncRNAs) and circular non‐coding RNAs (circRNAs) expression change following peripheral nerve injury or noxious stimuli DNP, diabetic neuropathic pain; SCI, spinal cord injury; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury\",\n \"Microarray and deep‐sequencing analyses revealed that nerve injury or noxious stimuli could induce broad changes in miRNA expression in serum or along the pain processing pathways, including the perineal nerve, dorsal root ganglion (DRG), spinal cord, and supraspinal regions. For normal pain signal transmission, nerve injury or noxious stimuli are detected by nociceptors in the DRG or trigeminal ganglion (TG) and then transmitted to upstream neurons in the spinal dorsal horn. Subsequently, nociceptive stimuli are integrated, processed, and further transmitted ascending to specific supraspinal brain regions.\\nHuman studies have identified as many as 1134 differentially expressed (DE) genes in the serum of individuals with or without neuropathic pain after spinal cord injury (SCI).\\n13\\n miR‐204‐5p, ‐519d‐3p, ‐20b‐5p, and ‐6838‐5p might act as promising biomarkers and intervention targets for preventing and therapizing neuropathic pain after SCI. In trigeminal neuralgia individuals, serum miR‐132‐3p, ‐146b‐5p, ‐155‐5p, and ‐384 levels were prominently increased compared with healthy controls.\\n14\\n Patients with painful peripheral neuropathy had higher miR‐21 level in the serum and sural nerve compared with healthy controls. Meanwhile, miR‐155 was reduced in the serum and inflicted lower leg skin.\\n15\\n Animal and human studies showed that miR‐30c‐5p was upregulated in the serum and cerebro spinal fluid (CSF) of sciatic nerve injury rats and neuropathic pain patients with chronic peripheral ischemia. The high expression of miR‐30c‐5p, together with other clinical parameters, might be used to predict neuropathic pain development in patients with chronic peripheral ischemia.\\n16\\n Additionally, an animal study showed that plasma‐derived DE extracellular vesicle (EV) miRNAs regulated processes that are essential for neuropathic pain development. Most DE EV miRNAs for inflammation suppression were downregulated, potentially acting as biomarkers and targets in neuropathic pain treatment.\\n17\\n\\n\\nIn addition, numerous DE miRNAs have been identified in the DRG. One week after spared nerve injury (SNI), 33 and 39 miRNAs in the DRG were upregulated and downregulated, respectively, with most DE miRNAs related to axon guidance, focal adhesion, Ras and Wnt signaling pathways.\\n18\\n Furthermore, nerve injury‐induced miRNAs expression was dynamic and time‐dependent,\\n19\\n, \\n20\\n implicating multiple regulatory mechanisms in neuropathic pain initiation and development. Nerve injury redistributes miRNAs from a uniform style within the DRG soma of non‐allodynic animals to preferential localization to peripheral neurons in allodynic animals.\\n21\\n Furthermore, either sciatic nerve ligation (SNL), DRG transaction (DRT), or ventral root transection (VRT) could upregulate miR‐21 and miR‐31 while downregulating miR‐668 and miR‐672 in the injured DRG,\\n22\\n implying that these miRNAs could be therapeutic targets for treating diverse types of neuropathic pain.\\nThe spinal cord dorsal horn relays and modulates pain signals from the peripheral nociceptors to the supraspinal regions. In the spinal cord, numerous miRNA expression changes have been observed in chronic constriction injury (CCI) and diabetic neuropathic pain (DNP) rodents.\\n23\\n, \\n24\\n, \\n25\\n, \\n26\\n Previous research has suggested some miRNAs may be related to neuropathic pain development, for example, miR‐500, ‐221, and ‐21,\\n25\\n thus, acting as potential targets in its treatment.\\n27\\n\\n\\nCytoscape software constructed the miRNA–target gene regulatory network in the supraspinal region, including the nucleus accumbens (NAc), medial prefrontal cortex, and periaqueductal gray, between SNI and sham rats. Finally, four essential DE genes, includingCXCR2, IL12B, TNFSF8, and GRK1, and five miRNAs, including miR‐208a‐5p, −7688‐3p, −344f‐3p, −135b‐3p, and ‐135a‐2‐3p, were identified, indicating their essential roles in neuropathic pain pathogenesis.\\n28\\n Furthermore, in the prelimbic cortex of SNI rats, the DE miRNA–mRNA network pointed to molecules associated with inflammation.\\n29\\n DE miRNAs were also observed in the bilateral hippocampus of CCI rats. However, no significant difference was observed bilaterally in the hippocampus.\\n30\\n (Figure 1).\\nmicroRNAs (miRNAs) expression change following peripheral nerve injury or noxious stimuli SCI, spinal cord injury; CSF, cerebrol spinal fluid; DE, differential expressed; EV, extravascular vesicle; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury; DRT, DRG transection; VRT, ventral root transection\",\n \"Patients with type 2 DNP had a prominent higher expression of serum lncRNA NONRATT021972 and more severe neuropathic pain symptoms.\\n31\\n LINC01119 and LINC02447 in the peripheral blood of SCI patients were identified in pain pathways that were important for neuropathic pain development.\\n32\\n\\n\\nMicroarray analysis revealed that a nerve injury could induce time‐dependent lncRNA expression changes in the sciatic nerve.\\n33\\n lncRNA H19 was persistently upregulated in Schwann cells along the peripheral nerve, proximal and distal to the injured site.\\n34\\n In the DRG, the transcriptomic analysis identified 86 known and 26 novel lncRNAs genes to be DE after spared sciatic nerve injury.\\n35\\n Of these, rno‐Cntnap2 and AC111653.1 were essential in peripheral nerve regeneration and were involved in neuropathic pain.\\nNext‐generation RNA sequencing showed 134 lncRNAs and 188 circRNAs were prominently changed 14 days after SNI in the spinal cord.\\n36\\n In addition, microarray analysis identified 1481 and 1096 DE lncRNAs and mRNAs, respectively, in the spinal cord dorsal horn of DNP rats. Of these, 289 neighboring and 57 overlapping lncRNA‐mRNA pairs, including ENSMUST00000150952‐Mbp and AK081017‐Usp15, have been suggested to participate in neuropathic pain development.\\n37\\n\\n\\ncircRNAs have characteristic circularized structures following the backsplicing of exons from antisense RNAs (AS RNAs) or mRNAs and, thus, are highly stable. Most circRNAs are highly conserved among species and lack translation potential, despite cap‐independent translation. A recent study showed that 363 and 106 circRNAs were significantly dysregulated in the ipsilateral dorsal horn after nerve injury.\\n38\\n (Figure 2).\\nLong non‐coding RNA (lncRNAs) and circular non‐coding RNAs (circRNAs) expression change following peripheral nerve injury or noxious stimuli DNP, diabetic neuropathic pain; SCI, spinal cord injury; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury\",\n \"miRNAs contribute to the development of neuropathic pain via diverse mechanisms, such as neuroinflammation, autophagy, abnormal ion channel expression, regulating pain‐related mediators, protein kinases, structural proteins, neurotransmission excitatory–inhibitory imbalances, and exosome miRNA‐mediated neuron–glia communication (Table 1).\\nmiRNAs expression change for the development of neuropathic pain\\nKv1.1, Kv1.4, Kv3.4, Kv4.3, Kv7.5,\\nDPP10, Navβ1 ↓\\np‐p38 MAPK ↑\\nAbbreviations: ACC, anterior cingulate cortex; BACE1, beta‐site amyloid precursor protein‐cleaving enzyme 1; BDNF, brain‐derived neurotrophic factor; CCI, chronic constriction injury; CCR1, C–C chemokine receptor 1; CSF, cerebro spinal fluid; CSF1, colony‐stimulating factor‐1; CXCR4, chemokine CXC receptor 4; DNMT3a, DNA methyltransferase 3a; DNP, diabetic neuropathic pain; DRG, dorsal root ganglion; EV, extracellular vesicle; EZH2, enhancer of zeste homolog 2; GRK2, G protein‐coupled receptor kinases 2; IGF‐1/IGF‐1R, insulin‐like growth factor‐1/insulin‐like growth factor‐1 receptor; KCNMA1, calcium‐activated potassium channel subunit α‐1; MeCP2, methyl cytosine–guanine dinucleotide (CpG)‐binding protein 2; MIP‐1a, macrophage inflammatory protein‐1 alpha; NEFL, neurofilament light polypeptide; NF‐κB, nuclear factor – kappa B; NGF, nerve growth factor; NLRP3, NOD‐like receptor protein 3; NOX2, NADPH oxidase 2; NP, neuropathic pain; pSNL, partial sciatic nerve ligation; RAP1A, Ras‐related protein 1A; RASA1, RAS P21 protein activator 1; ROS, reactive oxygen species; S1PR1, sphingosine‐1‐phosphate receptor 1; SCI, spinal cord injury; Scn2b, β2 subunit of the voltage‐gated sodium channel; SIRT1, histone deacetylase sirtuin 1; SNL, spinal nerve ligation; SOCS1, suppressor of cytokine signaling 1; STAT3, signal transducer and activator of transcription 3; TLR4, toll‐like receptor 4; TNFR1, TNF receptor 1; TRPA1, transient receptor potential ankyrin 1; TXNIP, thioredoxin‐interacting protein; VTR, ventral root transection; ZEB1, zinc finger E‐box binding homeobox 1.\\n\\nmiRNAs regulate neuroinflammation in neuropathic pain development miRNA‐based epigenetic regulation is essential in neuroinflammation. miRNAs are predicted to regulate diverse neuroinflammation‐related targets along the pain processing pathways.\\nAmong patients with neuropathic pain, miR‐101 decreased in the serum and sural nerve, which is related to nuclear factor‐kappa B (NF‐κB) signaling activation.\\n39\\n Meanwhile, serum miR‐124a and miR‐155 expression was upregulated. They were identified to inhibit histone deacetylase sirtuin 1 (SIRT1) in primary human cluster of differentiation 4 (CD4) (+) cells and induce their differentiation toward regulatory T cells (Tregs), thus reducing pain‐related inflammation.\\n40\\n Such miRNA–target interactions may act as an endogenous protective mechanism for neuropathic pain.\\nmiR‐7a, expressed in small‐sized nociceptive DRG neurons, is downregulated after nerve injury, as reported, targeting neurofilament light polypeptides (NEFLs).\\n41\\n NEFL encodes a neuronal protein vital for neurofilament formation and increases signal transducer and activator of transcription 3 (STAT3) phosphorylation, which is highly related to cell differentiation and neuroinflammation. In diabetic peripheral neuropathic mice, miR‐590‐3p was downregulated to disinhibit Ras‐related protein 1A (RAP1A) in the DRG tissue and inhibit neural T cells infiltration.\\n42\\n Thus, exogenous miR‐590‐3p may be a potential alternative for neuropathic pain treatment. CCI downregulated miR‐140 and miR‐144 expression in the DRG. Intrathecally injected miR‐140 and miR‐144 agomir decreased inflammatory factor secretion and ameliorated hyperalgesia by targeting sphingosine‐1‐phosphate receptor 1 (S1PR1) and RAS P21 protein activator 1 (RASA1), respectively.\\n43\\n, \\n44\\n\\n\\nmiRNAs are essential in neuropathic pain development via neuroinflammation‐related mechanisms in the spinal cord. miR‐130a‐3p targets and downregulates insulin‐like growth factor‐1/insulin‐like growth factor‐1 receptor (IGF‐1/IGF‐1R) expression to alleviate SCI‐induced neuropathic pain by mitigating microglial activation and NF‐κB phosphorylation.\\n45\\n miR‐378 was decreased in CCI rats, inhibiting neuropathic pain development by targeting the enhancer of zeste homolog 2 (EZH2).\\n46\\n EZH2 promotes neuropathic pain by increasing tumor necrosis factor‐alpha (TNF‐α), interleukin (IL)‐1β, and monocyte chemoattractant protein‐1 (MCP‐1) production. Intrathecally injecting miRNA‐138 lentivirus can remarkably alleviate neuropathic pain in partial sciatic nerve ligation (pSNL) rats by suppressing toll‐like receptor 4 (TLR4) and macrophage inflammatory protein‐1 alpha (MIP‐1α)/C‐C chemokine receptor 1 (CCR1) signaling pathways.\\n47\\n\\n\\nmiR‐15a/16 targets and downregulates G protein‐coupled receptor kinases 2 (GRK2) to disinhibit p38‐MAPK (mitogen‐activated protein kinase) and NF‐κB, contributing to neuroinflammation after CCI.\\n48\\n GRK2‐deficient mice present a pro‐inflammatory phenotype in spinal cord microglia/macrophages, restored by miR‐124.\\n49\\n SNL increased DNA methyltransferase 3a (DNMT3a) expression related to hypermethylation of the miR‐214‐3p promoter, resulting in miR‐214‐3p expression reduction, which enhanced astrocyte reactivity, colony‐stimulating factor‐1 (CSF1), and interleukin 6 (IL‐6) production, and hyperalgesia in rats.\\n50\\n Electro‐acupuncture attenuated SCI by inhibiting Nav1.3 and Bax in the injured spinal cord through miR‐214 upregulation.\\n51\\n Downregulated miR‐128 was reported to contribute to neuropathic pain via p38 or zinc finger E‐box binding homeobox 1 (ZEB1) activation in the spinal cord.\\n52\\n, \\n53\\n Meanwhile, ZEB1 was also targeted by miR‐200b/miR‐429, orchestrating neuropathic pain development.\\n54\\n\\n\\nIn a pSNL mouse model, nerve injury significantly reduced miR‐23a expression in spinal glial cells, concomitant with the upregulation of its target chemokine, CXC receptor 4 (CXCR4). In naïve mice, either miR‐23a downregulation or CXCR4 upregulation could active the thioredoxin‐interacting protein (TXNIP)/NOD‐like receptor protein 3 (NLRP3) inflammasome axis. Both intrathecal miR‐23a mimics and spinal CXCR4 downregulation by a lentivirus inhibited TXNIP or NLRP3 upregulation to alleviate hyperalgesia.\\n55\\n\\n\\nNotably, one miRNA was shown to have distinctive targets in different animal models of the spinal cord. For example, miR‐155 downregulation targeted and upregulated NADPH oxidase 2 (NOX2) expression to induce reactive oxygen species (ROS) production after SCI, presenting a pro‐inflammatory phenotype in microglia/macrophages.\\n56\\n The ability to induce glial polarization was also observed in cultured BV‐2 microglia.\\n57\\n In bortezomib‐induced neuropathic pain rats, downregulated miR‐155 upregulated TNF receptor 1 (TNFR1) expression, which activated its downstream signaling pathways, including p38‐MAPK, c‐Jun N‐terminal kinase (JNK), and transient receptor potential ankyrin 1 (TRPA1).\\n58\\n Therefore, it is suggested that miR‐155 might act as an intervention target for neuropathic pain. As expected, treatment with ibuprofen and L‐arginine delayed the behavioral pain changes while inhibiting spinal miR‐155 and NO.\\n59\\n miR‐155‐5p is also known to destabilize the blood–nerve barrier and expression of tight junction proteins, such as claudin‐1 and zonula occludens‐1 (ZO‐1). Tissue plasminogen activator (tPA) could transiently open such barriers to facilitate topically applying analgesics, via miR‐155‐5p upregulation.\\n60\\n However, we also noted that CCI upregulated but did not downregulate spinal cord miR‐155, and miR‐155 inhibition enhanced suppressor of cytokine signaling 1 (SOCS1) expression to inactivate inflammation via NF‐κB and p38‐MAPK inhibition.\\n61\\n In oxaliplatin‐induced peripheral neuropathic pain, spinal cord miR‐155 expression was also upregulated, and intrathecally injecting the miR‐155 inhibitor attenuated hyperalgesia in rats, possibly by inhibiting oxidative stress–TRPA1 pathways.\\n62\\n The underlying mechanisms of such distinctive miR‐155 expression change are still unknown and require further research into different pain models.\\nmiRNA‐based epigenetic regulation is essential in neuroinflammation. miRNAs are predicted to regulate diverse neuroinflammation‐related targets along the pain processing pathways.\\nAmong patients with neuropathic pain, miR‐101 decreased in the serum and sural nerve, which is related to nuclear factor‐kappa B (NF‐κB) signaling activation.\\n39\\n Meanwhile, serum miR‐124a and miR‐155 expression was upregulated. They were identified to inhibit histone deacetylase sirtuin 1 (SIRT1) in primary human cluster of differentiation 4 (CD4) (+) cells and induce their differentiation toward regulatory T cells (Tregs), thus reducing pain‐related inflammation.\\n40\\n Such miRNA–target interactions may act as an endogenous protective mechanism for neuropathic pain.\\nmiR‐7a, expressed in small‐sized nociceptive DRG neurons, is downregulated after nerve injury, as reported, targeting neurofilament light polypeptides (NEFLs).\\n41\\n NEFL encodes a neuronal protein vital for neurofilament formation and increases signal transducer and activator of transcription 3 (STAT3) phosphorylation, which is highly related to cell differentiation and neuroinflammation. In diabetic peripheral neuropathic mice, miR‐590‐3p was downregulated to disinhibit Ras‐related protein 1A (RAP1A) in the DRG tissue and inhibit neural T cells infiltration.\\n42\\n Thus, exogenous miR‐590‐3p may be a potential alternative for neuropathic pain treatment. CCI downregulated miR‐140 and miR‐144 expression in the DRG. Intrathecally injected miR‐140 and miR‐144 agomir decreased inflammatory factor secretion and ameliorated hyperalgesia by targeting sphingosine‐1‐phosphate receptor 1 (S1PR1) and RAS P21 protein activator 1 (RASA1), respectively.\\n43\\n, \\n44\\n\\n\\nmiRNAs are essential in neuropathic pain development via neuroinflammation‐related mechanisms in the spinal cord. miR‐130a‐3p targets and downregulates insulin‐like growth factor‐1/insulin‐like growth factor‐1 receptor (IGF‐1/IGF‐1R) expression to alleviate SCI‐induced neuropathic pain by mitigating microglial activation and NF‐κB phosphorylation.\\n45\\n miR‐378 was decreased in CCI rats, inhibiting neuropathic pain development by targeting the enhancer of zeste homolog 2 (EZH2).\\n46\\n EZH2 promotes neuropathic pain by increasing tumor necrosis factor‐alpha (TNF‐α), interleukin (IL)‐1β, and monocyte chemoattractant protein‐1 (MCP‐1) production. Intrathecally injecting miRNA‐138 lentivirus can remarkably alleviate neuropathic pain in partial sciatic nerve ligation (pSNL) rats by suppressing toll‐like receptor 4 (TLR4) and macrophage inflammatory protein‐1 alpha (MIP‐1α)/C‐C chemokine receptor 1 (CCR1) signaling pathways.\\n47\\n\\n\\nmiR‐15a/16 targets and downregulates G protein‐coupled receptor kinases 2 (GRK2) to disinhibit p38‐MAPK (mitogen‐activated protein kinase) and NF‐κB, contributing to neuroinflammation after CCI.\\n48\\n GRK2‐deficient mice present a pro‐inflammatory phenotype in spinal cord microglia/macrophages, restored by miR‐124.\\n49\\n SNL increased DNA methyltransferase 3a (DNMT3a) expression related to hypermethylation of the miR‐214‐3p promoter, resulting in miR‐214‐3p expression reduction, which enhanced astrocyte reactivity, colony‐stimulating factor‐1 (CSF1), and interleukin 6 (IL‐6) production, and hyperalgesia in rats.\\n50\\n Electro‐acupuncture attenuated SCI by inhibiting Nav1.3 and Bax in the injured spinal cord through miR‐214 upregulation.\\n51\\n Downregulated miR‐128 was reported to contribute to neuropathic pain via p38 or zinc finger E‐box binding homeobox 1 (ZEB1) activation in the spinal cord.\\n52\\n, \\n53\\n Meanwhile, ZEB1 was also targeted by miR‐200b/miR‐429, orchestrating neuropathic pain development.\\n54\\n\\n\\nIn a pSNL mouse model, nerve injury significantly reduced miR‐23a expression in spinal glial cells, concomitant with the upregulation of its target chemokine, CXC receptor 4 (CXCR4). In naïve mice, either miR‐23a downregulation or CXCR4 upregulation could active the thioredoxin‐interacting protein (TXNIP)/NOD‐like receptor protein 3 (NLRP3) inflammasome axis. Both intrathecal miR‐23a mimics and spinal CXCR4 downregulation by a lentivirus inhibited TXNIP or NLRP3 upregulation to alleviate hyperalgesia.\\n55\\n\\n\\nNotably, one miRNA was shown to have distinctive targets in different animal models of the spinal cord. For example, miR‐155 downregulation targeted and upregulated NADPH oxidase 2 (NOX2) expression to induce reactive oxygen species (ROS) production after SCI, presenting a pro‐inflammatory phenotype in microglia/macrophages.\\n56\\n The ability to induce glial polarization was also observed in cultured BV‐2 microglia.\\n57\\n In bortezomib‐induced neuropathic pain rats, downregulated miR‐155 upregulated TNF receptor 1 (TNFR1) expression, which activated its downstream signaling pathways, including p38‐MAPK, c‐Jun N‐terminal kinase (JNK), and transient receptor potential ankyrin 1 (TRPA1).\\n58\\n Therefore, it is suggested that miR‐155 might act as an intervention target for neuropathic pain. As expected, treatment with ibuprofen and L‐arginine delayed the behavioral pain changes while inhibiting spinal miR‐155 and NO.\\n59\\n miR‐155‐5p is also known to destabilize the blood–nerve barrier and expression of tight junction proteins, such as claudin‐1 and zonula occludens‐1 (ZO‐1). Tissue plasminogen activator (tPA) could transiently open such barriers to facilitate topically applying analgesics, via miR‐155‐5p upregulation.\\n60\\n However, we also noted that CCI upregulated but did not downregulate spinal cord miR‐155, and miR‐155 inhibition enhanced suppressor of cytokine signaling 1 (SOCS1) expression to inactivate inflammation via NF‐κB and p38‐MAPK inhibition.\\n61\\n In oxaliplatin‐induced peripheral neuropathic pain, spinal cord miR‐155 expression was also upregulated, and intrathecally injecting the miR‐155 inhibitor attenuated hyperalgesia in rats, possibly by inhibiting oxidative stress–TRPA1 pathways.\\n62\\n The underlying mechanisms of such distinctive miR‐155 expression change are still unknown and require further research into different pain models.\\nAutophagy miRNA‐related autophagy is involved in neuropathic pain regulation. As reported, miR‐15a downregulated and stimulated AKT serine/threonine kinase 3 (AKT3), and inhibited autophagy post‐CCI.\\n63\\n Impaired autophagy participates in neuropathic pain development. Intrathecal miR‐15a agomir prominently suppressed AKT3 expression, induced autophagy, and attenuated CCI‐induced neuropathic pain. Similar to miR‐15a, miR‐145 and miR‐20b‐5p contributed to neuropathic pain regulation via protein kinase B (AKT)‐related autophagy pathways.\\n64\\n, \\n65\\n Meanwhile, miR‐195 in the spinal cord was observed to upregulate post‐SNL, targeting and inhibiting Autophagy‐Related 14 (ATG14) and its autophagy activation.\\n66\\n The miR‐195 inhibitor activated autophagy and suppressed neuroinflammation in vivo and in vitro.\\nmiRNA‐related autophagy is involved in neuropathic pain regulation. As reported, miR‐15a downregulated and stimulated AKT serine/threonine kinase 3 (AKT3), and inhibited autophagy post‐CCI.\\n63\\n Impaired autophagy participates in neuropathic pain development. Intrathecal miR‐15a agomir prominently suppressed AKT3 expression, induced autophagy, and attenuated CCI‐induced neuropathic pain. Similar to miR‐15a, miR‐145 and miR‐20b‐5p contributed to neuropathic pain regulation via protein kinase B (AKT)‐related autophagy pathways.\\n64\\n, \\n65\\n Meanwhile, miR‐195 in the spinal cord was observed to upregulate post‐SNL, targeting and inhibiting Autophagy‐Related 14 (ATG14) and its autophagy activation.\\n66\\n The miR‐195 inhibitor activated autophagy and suppressed neuroinflammation in vivo and in vitro.\\nThe contribution of miRNAs to ion channel expression in neuropathic pain Another class of miRNA‐based regulation focuses on ion channels, including sodium, potassium, and calcium channels, and transient receptor potential (TRP) channels, to modulate action potential production, firing rate, and neurotransmitter release.\\nTheoretically, miRNAs can simultaneously have multiple target mRNAs because precise matching is not a prerequisite for inhibiting the target sequence. For example, other than NEFL,\\n41\\n miR‐7a targets the β2 subunit of the voltage‐gated sodium channel (Scn2b) with post‐transcriptional regulation to induce nociceptive DRG neurons hyperexcitability.\\n67\\n Other than miR‐7a, the other known miRNA‐ targets involved in sodium channels expression include miR‐30b, ‐96, ‐384‐5p and their target Scn3a (encoding Nav1.3),\\n68\\n, \\n69\\n, \\n70\\n miR‐30b‐5p and its target Scn8a (encoding Nav1.6),\\n71\\n and miR‐182 and ‐30b and their target Scn9a (encoding Nav1.7).\\n72\\n, \\n73\\n\\n\\nA miRNA cluster is a polycistronic gene containing several miRNAs derived from a single primary or nascent transcript. Approximately 40% of miRNAs are predicted to form clusters, but their significance is still unknown. miR‐17‐92 is a miRNA cluster that includes six different members, of which miR‐18a, ‐19a, ‐19b, and ‐92a upregulation induces allodynia. The predicted targets of the miR‐17‐92 cluster encompass genes encoding diverse voltage‐gated potassium channels and their regulatory subunits, including Kv1.1, Kv1.4, Kv3.4, Kv4.3, Kv7.5, dipeptidyl peptidase 10 (DPP10), and Navβ1.\\n74\\n CCI upregulated miR‐137 to target and downregulate Kcna2, which encodes Kv1.2 in the DRG and spinal dorsal horn.\\n75\\n By contrast, CCI decreased miR‐183‐5p expression in the DRG, and the predicted target gene TREK‐1, a subunit of the 2P‐domain K+ channel, was increased.\\n76\\n\\n\\nFurthermore, the miRNA‐183 cluster (miR‐183, part of miR‐96/182/183) regulates either basal mechanical or neuropathic pain.\\n77\\n The miR‐183 cluster targets Cacna2d1 and Cacna2d2, which encode auxiliary voltage‐gated calcium channel subunits α2δ‐1 and α2δ‐2, to affect nociceptor excitability. Nerve injury downregulated miR‐103 in the spinal cord, which simultaneously targeted and inhibited Cacna1c, Cacna2d1, and Cacnb1 and encoded the Cav1.2‐α1, α2δ1, and β1 subunits of the voltage‐gated calcium channels macromolecular complex Cav1.2 L‐type calcium channel, respectively.\\n78\\n Intrathecal miR‐103 successfully relieved neuropathic pain.\\nIn addition to ion channels, TRP channels are ligand‐gated ion channels that promote painful sensations.\\n79\\n In the DRG, TRPA1 participates in miR‐141‐5p alleviated oxaliplatin‐induced neuropathic pain.\\n80\\n In SNI mice, miR‐449a ameliorated neuropathic pain by decreasing the activity of TRPA1 and calcium‐activated potassium channel subunit α‐1 (KCNMA1), thus acting as a potential therapeutic alternative for treating neuropathic pain.\\n81\\n\\n\\nAnother class of miRNA‐based regulation focuses on ion channels, including sodium, potassium, and calcium channels, and transient receptor potential (TRP) channels, to modulate action potential production, firing rate, and neurotransmitter release.\\nTheoretically, miRNAs can simultaneously have multiple target mRNAs because precise matching is not a prerequisite for inhibiting the target sequence. For example, other than NEFL,\\n41\\n miR‐7a targets the β2 subunit of the voltage‐gated sodium channel (Scn2b) with post‐transcriptional regulation to induce nociceptive DRG neurons hyperexcitability.\\n67\\n Other than miR‐7a, the other known miRNA‐ targets involved in sodium channels expression include miR‐30b, ‐96, ‐384‐5p and their target Scn3a (encoding Nav1.3),\\n68\\n, \\n69\\n, \\n70\\n miR‐30b‐5p and its target Scn8a (encoding Nav1.6),\\n71\\n and miR‐182 and ‐30b and their target Scn9a (encoding Nav1.7).\\n72\\n, \\n73\\n\\n\\nA miRNA cluster is a polycistronic gene containing several miRNAs derived from a single primary or nascent transcript. Approximately 40% of miRNAs are predicted to form clusters, but their significance is still unknown. miR‐17‐92 is a miRNA cluster that includes six different members, of which miR‐18a, ‐19a, ‐19b, and ‐92a upregulation induces allodynia. The predicted targets of the miR‐17‐92 cluster encompass genes encoding diverse voltage‐gated potassium channels and their regulatory subunits, including Kv1.1, Kv1.4, Kv3.4, Kv4.3, Kv7.5, dipeptidyl peptidase 10 (DPP10), and Navβ1.\\n74\\n CCI upregulated miR‐137 to target and downregulate Kcna2, which encodes Kv1.2 in the DRG and spinal dorsal horn.\\n75\\n By contrast, CCI decreased miR‐183‐5p expression in the DRG, and the predicted target gene TREK‐1, a subunit of the 2P‐domain K+ channel, was increased.\\n76\\n\\n\\nFurthermore, the miRNA‐183 cluster (miR‐183, part of miR‐96/182/183) regulates either basal mechanical or neuropathic pain.\\n77\\n The miR‐183 cluster targets Cacna2d1 and Cacna2d2, which encode auxiliary voltage‐gated calcium channel subunits α2δ‐1 and α2δ‐2, to affect nociceptor excitability. Nerve injury downregulated miR‐103 in the spinal cord, which simultaneously targeted and inhibited Cacna1c, Cacna2d1, and Cacnb1 and encoded the Cav1.2‐α1, α2δ1, and β1 subunits of the voltage‐gated calcium channels macromolecular complex Cav1.2 L‐type calcium channel, respectively.\\n78\\n Intrathecal miR‐103 successfully relieved neuropathic pain.\\nIn addition to ion channels, TRP channels are ligand‐gated ion channels that promote painful sensations.\\n79\\n In the DRG, TRPA1 participates in miR‐141‐5p alleviated oxaliplatin‐induced neuropathic pain.\\n80\\n In SNI mice, miR‐449a ameliorated neuropathic pain by decreasing the activity of TRPA1 and calcium‐activated potassium channel subunit α‐1 (KCNMA1), thus acting as a potential therapeutic alternative for treating neuropathic pain.\\n81\\n\\n\\n\\nmiRNAs regulate pain‐related mediators, protein kinases, and structural proteins miRNAs regulate diverse pain‐related mediators, protein kinases, and structural proteins along the pain processing pathways. For HIV‐associated symptomatic distal sensory polyneuropathy and neuropathic pain, serum miR‐455‐3p acted as a potential biomarker, possibly targeting multiple genes involved in peripheral neuropathic pain, such as nerve growth factor (NGF) and related genes.\\n82\\n Brain‐derived neurotrophic factor (BDNF), another well‐recognized pain mediator, is a common miR‐1, ‐183, and ‐206 target.\\n83\\n, \\n84\\n, \\n85\\n After nerve injury, several miRNAs, including miR‐19a, ‐301, and ‐132, were downregulated, and their target methyl cytosine–guanine dinucleotide (CpG)‐binding protein 2 (MeCP2) expression was increased, leading to concomitant BDNF upregulation.\\n86\\n miR‐30c‐5p was upregulated in the serum and CSF of patients with chronic peripheral ischemia.\\n16\\n A miR‐30c‐5p inhibitor intraventricular injection postponed neuropathic pain development and fully reversed hyperalgesia in rodents. Transforming growth factor beta (TGF‐β) participates in the effects of miR‐30c‐5p, which refers to the endogenous opioid analgesic system.\\n16\\n\\n\\nmiRNAs dysregulate specific pain‐related protein kinases. For example, in DNP rats, miR‐133a‐3p was dysregulated in the sciatic nerve, interacting with p‐p38 MAPK to participate in the development of neuropathic pain.\\n87\\n Via a methyl CpG‐binding domain and transcriptional repressor domain, MeCP2 acts as a transcriptional repressor, and its overexpression improves neuropathic pain, potentiating an anti‐nociceptive effect of MeCP2. The authors also noted that MeCP2 expression changed post‐transcriptionally, and its mRNA level did not significantly change after SNI. However, the protein level was upregulated. During the development period of neuropathic pain, phospho‐cAMP response element‐binding protein (p‐CREB) elevated rapidly but returned 3–7 days after SNI, concomitant with miR‐132 downregulation, which targets MeCP2 and inhibits its expression post‐transcriptionally.\\n88\\n SNL decreased miR‐200b and miR‐429 in NAc neurons, along with an upregulation of their target (DNMT3a).\\n89\\n Further mechanism studies found that DNMT3a in the NAc was expressed in NR1 immunoreactive neurons, suggesting the dysregulation of ‘mesolimbic motivation circuitry’ for neuropathic pain development.\\nThe CCI induced time‐dependent miR‐1 downregulation in injured sciatic nerves. This change in expression was related to the upregulation and translocation of the miR‐1‐targeted connexin 43 (Cx43), the major connexin of astrocytes.\\n83\\n miR‐1 mimics could reduce Cx43 expression in cultured human glioblastoma cells. However, miR‐1 mimics intraneural transfection failed to alter Cx43 protein expression and did not improve pain behavior. The authors attributed such treatment failure to insufficient inhibition of miR‐1 for Cx43 via intraneural injection. Alternatively, there were regulatory mechanisms for Cx43 in vivo other than miR‐1.\\n90\\n However, miR‐1 in the DRG was DE according to peripheral nerve injury type. CCI, pSNL, and sural nerve injury downregulated miR‐1. However, axotomy of the sciatic nerve and tibial nerve injury increased its expression in the DRG.\\n83\\n, \\n91\\n, \\n92\\n As previously described, miR‐1 was also upregulated following capsaicin treatment and bone cancer pain.\\n91\\n, \\n93\\n Additionally, miR‐1 downregulation inhibited bone cancer pain. Collectively, the action of miR‐1 on pain seems to be complex and stimulus‐dependent.\\nNotably, specific miRNAs can promote neuropathic pain development via diverse mechanisms. For example, beta‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1), a membrane protease essential for myelination, was downregulated after miR‐15b overexpression in vitro or in the DRG of chemotherapy‐related neuropathic pain rats.\\n94\\n BACE1‐mediated neuregulin 1 reduction decreases nerve conduction velocity. In addition, BACE1 modulated Navβ2 subunit expression and neuronal activity and regulated inflammation‐related TNFR expression.\\nmiRNAs regulate diverse pain‐related mediators, protein kinases, and structural proteins along the pain processing pathways. For HIV‐associated symptomatic distal sensory polyneuropathy and neuropathic pain, serum miR‐455‐3p acted as a potential biomarker, possibly targeting multiple genes involved in peripheral neuropathic pain, such as nerve growth factor (NGF) and related genes.\\n82\\n Brain‐derived neurotrophic factor (BDNF), another well‐recognized pain mediator, is a common miR‐1, ‐183, and ‐206 target.\\n83\\n, \\n84\\n, \\n85\\n After nerve injury, several miRNAs, including miR‐19a, ‐301, and ‐132, were downregulated, and their target methyl cytosine–guanine dinucleotide (CpG)‐binding protein 2 (MeCP2) expression was increased, leading to concomitant BDNF upregulation.\\n86\\n miR‐30c‐5p was upregulated in the serum and CSF of patients with chronic peripheral ischemia.\\n16\\n A miR‐30c‐5p inhibitor intraventricular injection postponed neuropathic pain development and fully reversed hyperalgesia in rodents. Transforming growth factor beta (TGF‐β) participates in the effects of miR‐30c‐5p, which refers to the endogenous opioid analgesic system.\\n16\\n\\n\\nmiRNAs dysregulate specific pain‐related protein kinases. For example, in DNP rats, miR‐133a‐3p was dysregulated in the sciatic nerve, interacting with p‐p38 MAPK to participate in the development of neuropathic pain.\\n87\\n Via a methyl CpG‐binding domain and transcriptional repressor domain, MeCP2 acts as a transcriptional repressor, and its overexpression improves neuropathic pain, potentiating an anti‐nociceptive effect of MeCP2. The authors also noted that MeCP2 expression changed post‐transcriptionally, and its mRNA level did not significantly change after SNI. However, the protein level was upregulated. During the development period of neuropathic pain, phospho‐cAMP response element‐binding protein (p‐CREB) elevated rapidly but returned 3–7 days after SNI, concomitant with miR‐132 downregulation, which targets MeCP2 and inhibits its expression post‐transcriptionally.\\n88\\n SNL decreased miR‐200b and miR‐429 in NAc neurons, along with an upregulation of their target (DNMT3a).\\n89\\n Further mechanism studies found that DNMT3a in the NAc was expressed in NR1 immunoreactive neurons, suggesting the dysregulation of ‘mesolimbic motivation circuitry’ for neuropathic pain development.\\nThe CCI induced time‐dependent miR‐1 downregulation in injured sciatic nerves. This change in expression was related to the upregulation and translocation of the miR‐1‐targeted connexin 43 (Cx43), the major connexin of astrocytes.\\n83\\n miR‐1 mimics could reduce Cx43 expression in cultured human glioblastoma cells. However, miR‐1 mimics intraneural transfection failed to alter Cx43 protein expression and did not improve pain behavior. The authors attributed such treatment failure to insufficient inhibition of miR‐1 for Cx43 via intraneural injection. Alternatively, there were regulatory mechanisms for Cx43 in vivo other than miR‐1.\\n90\\n However, miR‐1 in the DRG was DE according to peripheral nerve injury type. CCI, pSNL, and sural nerve injury downregulated miR‐1. However, axotomy of the sciatic nerve and tibial nerve injury increased its expression in the DRG.\\n83\\n, \\n91\\n, \\n92\\n As previously described, miR‐1 was also upregulated following capsaicin treatment and bone cancer pain.\\n91\\n, \\n93\\n Additionally, miR‐1 downregulation inhibited bone cancer pain. Collectively, the action of miR‐1 on pain seems to be complex and stimulus‐dependent.\\nNotably, specific miRNAs can promote neuropathic pain development via diverse mechanisms. For example, beta‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1), a membrane protease essential for myelination, was downregulated after miR‐15b overexpression in vitro or in the DRG of chemotherapy‐related neuropathic pain rats.\\n94\\n BACE1‐mediated neuregulin 1 reduction decreases nerve conduction velocity. In addition, BACE1 modulated Navβ2 subunit expression and neuronal activity and regulated inflammation‐related TNFR expression.\\nNeurotransmission excitatory–inhibitory imbalances Neurotransmission excitatory and inhibitory imbalances in the spinal cord also contribute to neuropathic pain development. For example, miR‐500 increased to regulate glutamic acid decarboxylase 67 (GAD67) expression and target the specific site of Gad1 in the dorsal horn.\\n95\\n GAD67 expression reduction inhibited the function of GABAergic neurons and the resultant inhibitory synaptic transmission dysregulation contributed to neuropathic pain development. In addition, miR‐23b is crucial for improving neuropathic pain in the injured spinal cord by downregulating its target gene, Nox4, which further normalizes glutamic acid decarboxylase 65/67 (GAD65/67) expression and protects GABAergic neurons from apoptosis.\\n96\\n Microarray analysis showed that miR‐539 was prominently reduced in the contralateral anterior cingulate cortex (ACC) after CCI, which is related to enhanced NR2B protein expression. Injecting miR‐539 mimics into the contralateral ACC attenuated CCI‐evoked mechanical hyperalgesia, suggesting that the N‐methyl‐D‐aspartate (NMDA) receptor NR2B subunit regulates neuropathic pain.\\n97\\n\\n\\nNeurotransmission excitatory and inhibitory imbalances in the spinal cord also contribute to neuropathic pain development. For example, miR‐500 increased to regulate glutamic acid decarboxylase 67 (GAD67) expression and target the specific site of Gad1 in the dorsal horn.\\n95\\n GAD67 expression reduction inhibited the function of GABAergic neurons and the resultant inhibitory synaptic transmission dysregulation contributed to neuropathic pain development. In addition, miR‐23b is crucial for improving neuropathic pain in the injured spinal cord by downregulating its target gene, Nox4, which further normalizes glutamic acid decarboxylase 65/67 (GAD65/67) expression and protects GABAergic neurons from apoptosis.\\n96\\n Microarray analysis showed that miR‐539 was prominently reduced in the contralateral anterior cingulate cortex (ACC) after CCI, which is related to enhanced NR2B protein expression. Injecting miR‐539 mimics into the contralateral ACC attenuated CCI‐evoked mechanical hyperalgesia, suggesting that the N‐methyl‐D‐aspartate (NMDA) receptor NR2B subunit regulates neuropathic pain.\\n97\\n\\n\\n\\nEV miRNAs‐mediated neuron–glia communication We mainly focused on exosomal miR‐21 with regard to EV miRNAs in neuropathic pain. As aforementioned, SNL, DRT, or VRT could upregulate miR‐21 in the injured DRG.\\n22\\n Notably, miR‐21 was also increased in serum exosomes from nerve‐ligated mice.\\n98\\n In another in‐depth study, macrophages readily took up pure sensory neuron‐derived exosomes encompassing miR‐21‐5p to promote a pro‐inflammatory phenotype.\\n99\\n Either intrathecal miR‐21‐5p antagomir or miR‐21 conditional deletion in sensory neurons ameliorated hyperalgesia and macrophage recruitment in the DRG.\\nUnlike the miRNA target gene inhibitory mechanism, miR‐21 acts as an endogenous toll‐like receptor 8 (TLR8) ligand, leading to neuropathic pain development.\\n100\\n TLR8, a nucleic acid‐sensing receptor, is located in the endosomes and lysosomes, leading to ERK‐mediated inflammatory mediator production and neuronal activation after SNL. Although miR‐21 and TLR8 are only distributed in small‐ and medium‐sized neurons, miR‐21 can also be derived from large‐sized neurons and reach TLR8 in the endosomes of other types of neurons.\\n101\\n Similar to miR‐21, DRG sensory neurons secreted miR‐23a‐enriched EVs following nerve injury and were taken up by macrophages to enhance M1 polarization in vitro. A20, an NF‐κB signaling pathway inhibitor, is a verified miR‐23a target gene.\\n102\\n Moreover, intrathecally delivering EVs‐miR‐23a antagomir attenuated neuropathic hyperalgesia and reduced M1 macrophages by inhibiting A20 to activate NF‐κB signaling.\\nWe mainly focused on exosomal miR‐21 with regard to EV miRNAs in neuropathic pain. As aforementioned, SNL, DRT, or VRT could upregulate miR‐21 in the injured DRG.\\n22\\n Notably, miR‐21 was also increased in serum exosomes from nerve‐ligated mice.\\n98\\n In another in‐depth study, macrophages readily took up pure sensory neuron‐derived exosomes encompassing miR‐21‐5p to promote a pro‐inflammatory phenotype.\\n99\\n Either intrathecal miR‐21‐5p antagomir or miR‐21 conditional deletion in sensory neurons ameliorated hyperalgesia and macrophage recruitment in the DRG.\\nUnlike the miRNA target gene inhibitory mechanism, miR‐21 acts as an endogenous toll‐like receptor 8 (TLR8) ligand, leading to neuropathic pain development.\\n100\\n TLR8, a nucleic acid‐sensing receptor, is located in the endosomes and lysosomes, leading to ERK‐mediated inflammatory mediator production and neuronal activation after SNL. Although miR‐21 and TLR8 are only distributed in small‐ and medium‐sized neurons, miR‐21 can also be derived from large‐sized neurons and reach TLR8 in the endosomes of other types of neurons.\\n101\\n Similar to miR‐21, DRG sensory neurons secreted miR‐23a‐enriched EVs following nerve injury and were taken up by macrophages to enhance M1 polarization in vitro. A20, an NF‐κB signaling pathway inhibitor, is a verified miR‐23a target gene.\\n102\\n Moreover, intrathecally delivering EVs‐miR‐23a antagomir attenuated neuropathic hyperalgesia and reduced M1 macrophages by inhibiting A20 to activate NF‐κB signaling.\",\n \"miRNA‐based epigenetic regulation is essential in neuroinflammation. miRNAs are predicted to regulate diverse neuroinflammation‐related targets along the pain processing pathways.\\nAmong patients with neuropathic pain, miR‐101 decreased in the serum and sural nerve, which is related to nuclear factor‐kappa B (NF‐κB) signaling activation.\\n39\\n Meanwhile, serum miR‐124a and miR‐155 expression was upregulated. They were identified to inhibit histone deacetylase sirtuin 1 (SIRT1) in primary human cluster of differentiation 4 (CD4) (+) cells and induce their differentiation toward regulatory T cells (Tregs), thus reducing pain‐related inflammation.\\n40\\n Such miRNA–target interactions may act as an endogenous protective mechanism for neuropathic pain.\\nmiR‐7a, expressed in small‐sized nociceptive DRG neurons, is downregulated after nerve injury, as reported, targeting neurofilament light polypeptides (NEFLs).\\n41\\n NEFL encodes a neuronal protein vital for neurofilament formation and increases signal transducer and activator of transcription 3 (STAT3) phosphorylation, which is highly related to cell differentiation and neuroinflammation. In diabetic peripheral neuropathic mice, miR‐590‐3p was downregulated to disinhibit Ras‐related protein 1A (RAP1A) in the DRG tissue and inhibit neural T cells infiltration.\\n42\\n Thus, exogenous miR‐590‐3p may be a potential alternative for neuropathic pain treatment. CCI downregulated miR‐140 and miR‐144 expression in the DRG. Intrathecally injected miR‐140 and miR‐144 agomir decreased inflammatory factor secretion and ameliorated hyperalgesia by targeting sphingosine‐1‐phosphate receptor 1 (S1PR1) and RAS P21 protein activator 1 (RASA1), respectively.\\n43\\n, \\n44\\n\\n\\nmiRNAs are essential in neuropathic pain development via neuroinflammation‐related mechanisms in the spinal cord. miR‐130a‐3p targets and downregulates insulin‐like growth factor‐1/insulin‐like growth factor‐1 receptor (IGF‐1/IGF‐1R) expression to alleviate SCI‐induced neuropathic pain by mitigating microglial activation and NF‐κB phosphorylation.\\n45\\n miR‐378 was decreased in CCI rats, inhibiting neuropathic pain development by targeting the enhancer of zeste homolog 2 (EZH2).\\n46\\n EZH2 promotes neuropathic pain by increasing tumor necrosis factor‐alpha (TNF‐α), interleukin (IL)‐1β, and monocyte chemoattractant protein‐1 (MCP‐1) production. Intrathecally injecting miRNA‐138 lentivirus can remarkably alleviate neuropathic pain in partial sciatic nerve ligation (pSNL) rats by suppressing toll‐like receptor 4 (TLR4) and macrophage inflammatory protein‐1 alpha (MIP‐1α)/C‐C chemokine receptor 1 (CCR1) signaling pathways.\\n47\\n\\n\\nmiR‐15a/16 targets and downregulates G protein‐coupled receptor kinases 2 (GRK2) to disinhibit p38‐MAPK (mitogen‐activated protein kinase) and NF‐κB, contributing to neuroinflammation after CCI.\\n48\\n GRK2‐deficient mice present a pro‐inflammatory phenotype in spinal cord microglia/macrophages, restored by miR‐124.\\n49\\n SNL increased DNA methyltransferase 3a (DNMT3a) expression related to hypermethylation of the miR‐214‐3p promoter, resulting in miR‐214‐3p expression reduction, which enhanced astrocyte reactivity, colony‐stimulating factor‐1 (CSF1), and interleukin 6 (IL‐6) production, and hyperalgesia in rats.\\n50\\n Electro‐acupuncture attenuated SCI by inhibiting Nav1.3 and Bax in the injured spinal cord through miR‐214 upregulation.\\n51\\n Downregulated miR‐128 was reported to contribute to neuropathic pain via p38 or zinc finger E‐box binding homeobox 1 (ZEB1) activation in the spinal cord.\\n52\\n, \\n53\\n Meanwhile, ZEB1 was also targeted by miR‐200b/miR‐429, orchestrating neuropathic pain development.\\n54\\n\\n\\nIn a pSNL mouse model, nerve injury significantly reduced miR‐23a expression in spinal glial cells, concomitant with the upregulation of its target chemokine, CXC receptor 4 (CXCR4). In naïve mice, either miR‐23a downregulation or CXCR4 upregulation could active the thioredoxin‐interacting protein (TXNIP)/NOD‐like receptor protein 3 (NLRP3) inflammasome axis. Both intrathecal miR‐23a mimics and spinal CXCR4 downregulation by a lentivirus inhibited TXNIP or NLRP3 upregulation to alleviate hyperalgesia.\\n55\\n\\n\\nNotably, one miRNA was shown to have distinctive targets in different animal models of the spinal cord. For example, miR‐155 downregulation targeted and upregulated NADPH oxidase 2 (NOX2) expression to induce reactive oxygen species (ROS) production after SCI, presenting a pro‐inflammatory phenotype in microglia/macrophages.\\n56\\n The ability to induce glial polarization was also observed in cultured BV‐2 microglia.\\n57\\n In bortezomib‐induced neuropathic pain rats, downregulated miR‐155 upregulated TNF receptor 1 (TNFR1) expression, which activated its downstream signaling pathways, including p38‐MAPK, c‐Jun N‐terminal kinase (JNK), and transient receptor potential ankyrin 1 (TRPA1).\\n58\\n Therefore, it is suggested that miR‐155 might act as an intervention target for neuropathic pain. As expected, treatment with ibuprofen and L‐arginine delayed the behavioral pain changes while inhibiting spinal miR‐155 and NO.\\n59\\n miR‐155‐5p is also known to destabilize the blood–nerve barrier and expression of tight junction proteins, such as claudin‐1 and zonula occludens‐1 (ZO‐1). Tissue plasminogen activator (tPA) could transiently open such barriers to facilitate topically applying analgesics, via miR‐155‐5p upregulation.\\n60\\n However, we also noted that CCI upregulated but did not downregulate spinal cord miR‐155, and miR‐155 inhibition enhanced suppressor of cytokine signaling 1 (SOCS1) expression to inactivate inflammation via NF‐κB and p38‐MAPK inhibition.\\n61\\n In oxaliplatin‐induced peripheral neuropathic pain, spinal cord miR‐155 expression was also upregulated, and intrathecally injecting the miR‐155 inhibitor attenuated hyperalgesia in rats, possibly by inhibiting oxidative stress–TRPA1 pathways.\\n62\\n The underlying mechanisms of such distinctive miR‐155 expression change are still unknown and require further research into different pain models.\",\n \"miRNA‐related autophagy is involved in neuropathic pain regulation. As reported, miR‐15a downregulated and stimulated AKT serine/threonine kinase 3 (AKT3), and inhibited autophagy post‐CCI.\\n63\\n Impaired autophagy participates in neuropathic pain development. Intrathecal miR‐15a agomir prominently suppressed AKT3 expression, induced autophagy, and attenuated CCI‐induced neuropathic pain. Similar to miR‐15a, miR‐145 and miR‐20b‐5p contributed to neuropathic pain regulation via protein kinase B (AKT)‐related autophagy pathways.\\n64\\n, \\n65\\n Meanwhile, miR‐195 in the spinal cord was observed to upregulate post‐SNL, targeting and inhibiting Autophagy‐Related 14 (ATG14) and its autophagy activation.\\n66\\n The miR‐195 inhibitor activated autophagy and suppressed neuroinflammation in vivo and in vitro.\",\n \"Another class of miRNA‐based regulation focuses on ion channels, including sodium, potassium, and calcium channels, and transient receptor potential (TRP) channels, to modulate action potential production, firing rate, and neurotransmitter release.\\nTheoretically, miRNAs can simultaneously have multiple target mRNAs because precise matching is not a prerequisite for inhibiting the target sequence. For example, other than NEFL,\\n41\\n miR‐7a targets the β2 subunit of the voltage‐gated sodium channel (Scn2b) with post‐transcriptional regulation to induce nociceptive DRG neurons hyperexcitability.\\n67\\n Other than miR‐7a, the other known miRNA‐ targets involved in sodium channels expression include miR‐30b, ‐96, ‐384‐5p and their target Scn3a (encoding Nav1.3),\\n68\\n, \\n69\\n, \\n70\\n miR‐30b‐5p and its target Scn8a (encoding Nav1.6),\\n71\\n and miR‐182 and ‐30b and their target Scn9a (encoding Nav1.7).\\n72\\n, \\n73\\n\\n\\nA miRNA cluster is a polycistronic gene containing several miRNAs derived from a single primary or nascent transcript. Approximately 40% of miRNAs are predicted to form clusters, but their significance is still unknown. miR‐17‐92 is a miRNA cluster that includes six different members, of which miR‐18a, ‐19a, ‐19b, and ‐92a upregulation induces allodynia. The predicted targets of the miR‐17‐92 cluster encompass genes encoding diverse voltage‐gated potassium channels and their regulatory subunits, including Kv1.1, Kv1.4, Kv3.4, Kv4.3, Kv7.5, dipeptidyl peptidase 10 (DPP10), and Navβ1.\\n74\\n CCI upregulated miR‐137 to target and downregulate Kcna2, which encodes Kv1.2 in the DRG and spinal dorsal horn.\\n75\\n By contrast, CCI decreased miR‐183‐5p expression in the DRG, and the predicted target gene TREK‐1, a subunit of the 2P‐domain K+ channel, was increased.\\n76\\n\\n\\nFurthermore, the miRNA‐183 cluster (miR‐183, part of miR‐96/182/183) regulates either basal mechanical or neuropathic pain.\\n77\\n The miR‐183 cluster targets Cacna2d1 and Cacna2d2, which encode auxiliary voltage‐gated calcium channel subunits α2δ‐1 and α2δ‐2, to affect nociceptor excitability. Nerve injury downregulated miR‐103 in the spinal cord, which simultaneously targeted and inhibited Cacna1c, Cacna2d1, and Cacnb1 and encoded the Cav1.2‐α1, α2δ1, and β1 subunits of the voltage‐gated calcium channels macromolecular complex Cav1.2 L‐type calcium channel, respectively.\\n78\\n Intrathecal miR‐103 successfully relieved neuropathic pain.\\nIn addition to ion channels, TRP channels are ligand‐gated ion channels that promote painful sensations.\\n79\\n In the DRG, TRPA1 participates in miR‐141‐5p alleviated oxaliplatin‐induced neuropathic pain.\\n80\\n In SNI mice, miR‐449a ameliorated neuropathic pain by decreasing the activity of TRPA1 and calcium‐activated potassium channel subunit α‐1 (KCNMA1), thus acting as a potential therapeutic alternative for treating neuropathic pain.\\n81\\n\\n\",\n \"miRNAs regulate diverse pain‐related mediators, protein kinases, and structural proteins along the pain processing pathways. For HIV‐associated symptomatic distal sensory polyneuropathy and neuropathic pain, serum miR‐455‐3p acted as a potential biomarker, possibly targeting multiple genes involved in peripheral neuropathic pain, such as nerve growth factor (NGF) and related genes.\\n82\\n Brain‐derived neurotrophic factor (BDNF), another well‐recognized pain mediator, is a common miR‐1, ‐183, and ‐206 target.\\n83\\n, \\n84\\n, \\n85\\n After nerve injury, several miRNAs, including miR‐19a, ‐301, and ‐132, were downregulated, and their target methyl cytosine–guanine dinucleotide (CpG)‐binding protein 2 (MeCP2) expression was increased, leading to concomitant BDNF upregulation.\\n86\\n miR‐30c‐5p was upregulated in the serum and CSF of patients with chronic peripheral ischemia.\\n16\\n A miR‐30c‐5p inhibitor intraventricular injection postponed neuropathic pain development and fully reversed hyperalgesia in rodents. Transforming growth factor beta (TGF‐β) participates in the effects of miR‐30c‐5p, which refers to the endogenous opioid analgesic system.\\n16\\n\\n\\nmiRNAs dysregulate specific pain‐related protein kinases. For example, in DNP rats, miR‐133a‐3p was dysregulated in the sciatic nerve, interacting with p‐p38 MAPK to participate in the development of neuropathic pain.\\n87\\n Via a methyl CpG‐binding domain and transcriptional repressor domain, MeCP2 acts as a transcriptional repressor, and its overexpression improves neuropathic pain, potentiating an anti‐nociceptive effect of MeCP2. The authors also noted that MeCP2 expression changed post‐transcriptionally, and its mRNA level did not significantly change after SNI. However, the protein level was upregulated. During the development period of neuropathic pain, phospho‐cAMP response element‐binding protein (p‐CREB) elevated rapidly but returned 3–7 days after SNI, concomitant with miR‐132 downregulation, which targets MeCP2 and inhibits its expression post‐transcriptionally.\\n88\\n SNL decreased miR‐200b and miR‐429 in NAc neurons, along with an upregulation of their target (DNMT3a).\\n89\\n Further mechanism studies found that DNMT3a in the NAc was expressed in NR1 immunoreactive neurons, suggesting the dysregulation of ‘mesolimbic motivation circuitry’ for neuropathic pain development.\\nThe CCI induced time‐dependent miR‐1 downregulation in injured sciatic nerves. This change in expression was related to the upregulation and translocation of the miR‐1‐targeted connexin 43 (Cx43), the major connexin of astrocytes.\\n83\\n miR‐1 mimics could reduce Cx43 expression in cultured human glioblastoma cells. However, miR‐1 mimics intraneural transfection failed to alter Cx43 protein expression and did not improve pain behavior. The authors attributed such treatment failure to insufficient inhibition of miR‐1 for Cx43 via intraneural injection. Alternatively, there were regulatory mechanisms for Cx43 in vivo other than miR‐1.\\n90\\n However, miR‐1 in the DRG was DE according to peripheral nerve injury type. CCI, pSNL, and sural nerve injury downregulated miR‐1. However, axotomy of the sciatic nerve and tibial nerve injury increased its expression in the DRG.\\n83\\n, \\n91\\n, \\n92\\n As previously described, miR‐1 was also upregulated following capsaicin treatment and bone cancer pain.\\n91\\n, \\n93\\n Additionally, miR‐1 downregulation inhibited bone cancer pain. Collectively, the action of miR‐1 on pain seems to be complex and stimulus‐dependent.\\nNotably, specific miRNAs can promote neuropathic pain development via diverse mechanisms. For example, beta‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1), a membrane protease essential for myelination, was downregulated after miR‐15b overexpression in vitro or in the DRG of chemotherapy‐related neuropathic pain rats.\\n94\\n BACE1‐mediated neuregulin 1 reduction decreases nerve conduction velocity. In addition, BACE1 modulated Navβ2 subunit expression and neuronal activity and regulated inflammation‐related TNFR expression.\",\n \"Neurotransmission excitatory and inhibitory imbalances in the spinal cord also contribute to neuropathic pain development. For example, miR‐500 increased to regulate glutamic acid decarboxylase 67 (GAD67) expression and target the specific site of Gad1 in the dorsal horn.\\n95\\n GAD67 expression reduction inhibited the function of GABAergic neurons and the resultant inhibitory synaptic transmission dysregulation contributed to neuropathic pain development. In addition, miR‐23b is crucial for improving neuropathic pain in the injured spinal cord by downregulating its target gene, Nox4, which further normalizes glutamic acid decarboxylase 65/67 (GAD65/67) expression and protects GABAergic neurons from apoptosis.\\n96\\n Microarray analysis showed that miR‐539 was prominently reduced in the contralateral anterior cingulate cortex (ACC) after CCI, which is related to enhanced NR2B protein expression. Injecting miR‐539 mimics into the contralateral ACC attenuated CCI‐evoked mechanical hyperalgesia, suggesting that the N‐methyl‐D‐aspartate (NMDA) receptor NR2B subunit regulates neuropathic pain.\\n97\\n\\n\",\n \"We mainly focused on exosomal miR‐21 with regard to EV miRNAs in neuropathic pain. As aforementioned, SNL, DRT, or VRT could upregulate miR‐21 in the injured DRG.\\n22\\n Notably, miR‐21 was also increased in serum exosomes from nerve‐ligated mice.\\n98\\n In another in‐depth study, macrophages readily took up pure sensory neuron‐derived exosomes encompassing miR‐21‐5p to promote a pro‐inflammatory phenotype.\\n99\\n Either intrathecal miR‐21‐5p antagomir or miR‐21 conditional deletion in sensory neurons ameliorated hyperalgesia and macrophage recruitment in the DRG.\\nUnlike the miRNA target gene inhibitory mechanism, miR‐21 acts as an endogenous toll‐like receptor 8 (TLR8) ligand, leading to neuropathic pain development.\\n100\\n TLR8, a nucleic acid‐sensing receptor, is located in the endosomes and lysosomes, leading to ERK‐mediated inflammatory mediator production and neuronal activation after SNL. Although miR‐21 and TLR8 are only distributed in small‐ and medium‐sized neurons, miR‐21 can also be derived from large‐sized neurons and reach TLR8 in the endosomes of other types of neurons.\\n101\\n Similar to miR‐21, DRG sensory neurons secreted miR‐23a‐enriched EVs following nerve injury and were taken up by macrophages to enhance M1 polarization in vitro. A20, an NF‐κB signaling pathway inhibitor, is a verified miR‐23a target gene.\\n102\\n Moreover, intrathecally delivering EVs‐miR‐23a antagomir attenuated neuropathic hyperalgesia and reduced M1 macrophages by inhibiting A20 to activate NF‐κB signaling.\",\n \"lncRNAs and circRNAs are essential in neuropathic pain development by acting as AS RNA or miRNA sponges, epigenetically regulating pain‐related molecules expression, or modulating miRNA processing (Table 2).\\nLong non‐coding RNAs (lncRNAs) and circular non‐coding RNAs (circRNAs) expression change for the development of neuropathic pain\\nAbbreviations: AQP4, aquaporin 4; AS RNA, antisense RNA; CCI, chronic constriction injury; CDK4, cyclin‐dependent kinase 4; CDK6, cyclin‐dependent kinase 6; DNP, diabetic neuropathic pain; DRG, dorsal root ganglion; ENO1, enolase 1; EphB1, ephrin type‐b 1; EZH2, enhancer of zeste homolog 2; HMGB1, high mobility group box 1; NAT, natural antisense transcript; NOTCH2, NOTCH receptor 2; NP, neuropathic pain; pSNL, partial sciatic nerve ligation; pSTAT3, phosphorylated signal transducer and activator of transcription 3; SCI, spinal cord injury; SGC, satellite glia cell; SNL, spinal nerve ligation; STAT3, signal transducer and activator of transcription 3; TLR5, toll‐like receptor 5; TNFAIP1, tumor necrosis factor alpha‐induced protein 1; TRPV1, transient receptor potential vanilloid 1; XIST, X‐inactive specific transcript; ZEB1, zinc finger E‐box binding homeobox 1.\\n\\nlncRNAs act as AS RNA\\n After peripheral nerve injury, Egr2‐AS‐RNA is upregulated in Schwann cells.\\n103\\n On the early growth response 2 (Egr2) promoter, Egr2‐AS‐RNA recruits an epigenetic silencing complex to downregulate Egr2, essential for peripheral myelination. Ectopic Egr2‐AS‐RNA expression in DRG cultures downregulates Egr2 mRNA and induces demyelination. In vivo, Egr2‐AS‐RNA inhibition reverts Egr2‐related gene expression and delays demyelination.\\nVoltage‐gated potassium channel (Kv) Kcna2‐AS‐RNA is an endogenous, highly conserved, and widely explored lncRNA in neuropathic pain.\\n104\\n It is a natural antisense transcript (NAT), distributed in the cytoplasm and targets Kcna2 mRNA, which encodes the pain regulation‐related membrane Kv1.2 subunit. Kcna2‐AS‐RNA was time‐dependently upregulated in the inflicted rat DRG after nerve injury. It decreased the total Kv current, upregulated DRG neurons excitability, and produced neuropathic pain symptoms.\\nThe complementary strand of DNA opposite the Scn9a gene encodes Scn9a NAT, another antisense lncRNA expressed in the DRG.\\n105\\n Scn9a NAT is suggested to be a negative regulator of Scn9a mRNA. Scn9a NAT overexpression inhibited Scn9a mRNA, its encoded protein Nav1.7, and Nav1.7 currents in DRG neurons. However, Scn9a NAT and Scn9a mRNA levels did not significantly change in the injured DRG until 2 weeks after nerve injury. More work is required to determine whether NAT can confer analgesia and reduce pain in the neuropathic pain state.\\nAfter peripheral nerve injury, Egr2‐AS‐RNA is upregulated in Schwann cells.\\n103\\n On the early growth response 2 (Egr2) promoter, Egr2‐AS‐RNA recruits an epigenetic silencing complex to downregulate Egr2, essential for peripheral myelination. Ectopic Egr2‐AS‐RNA expression in DRG cultures downregulates Egr2 mRNA and induces demyelination. In vivo, Egr2‐AS‐RNA inhibition reverts Egr2‐related gene expression and delays demyelination.\\nVoltage‐gated potassium channel (Kv) Kcna2‐AS‐RNA is an endogenous, highly conserved, and widely explored lncRNA in neuropathic pain.\\n104\\n It is a natural antisense transcript (NAT), distributed in the cytoplasm and targets Kcna2 mRNA, which encodes the pain regulation‐related membrane Kv1.2 subunit. Kcna2‐AS‐RNA was time‐dependently upregulated in the inflicted rat DRG after nerve injury. It decreased the total Kv current, upregulated DRG neurons excitability, and produced neuropathic pain symptoms.\\nThe complementary strand of DNA opposite the Scn9a gene encodes Scn9a NAT, another antisense lncRNA expressed in the DRG.\\n105\\n Scn9a NAT is suggested to be a negative regulator of Scn9a mRNA. Scn9a NAT overexpression inhibited Scn9a mRNA, its encoded protein Nav1.7, and Nav1.7 currents in DRG neurons. However, Scn9a NAT and Scn9a mRNA levels did not significantly change in the injured DRG until 2 weeks after nerve injury. More work is required to determine whether NAT can confer analgesia and reduce pain in the neuropathic pain state.\\n\\nlncRNAs act as miRNA sponges lncRNAs may act as miRNA sponges to form the lncRNA‐miRNA‐mRNA axis and regulate target gene expression. For example, the lncRNA X‐inactive specific transcript (XIST) is upregulated in the dorsal horn of the spinal cord after CCI. By sponging miR‐137, ‐150, ‐154‐5p, and ‐544, XIST can inhibit the expression of corresponding targets, including TNF‐α‐induced protein 1 (TNFAIP1), ZEB1, TLR5, and STAT3.\\n106\\n, \\n107\\n, \\n108\\n, \\n109\\n TNFAIP1 activates the NF‐κB signaling pathway, while ZEB1, TLR5, and STAT3 are crucial in the neuroinflammatory response. XIST inhibition markedly ameliorates neuropathic pain development. In addition, lncRNA nuclear enriched abundant transcript 1 (NEAT1) was upregulated in the spinal cord dorsal horn to form NEAT1‐miR‐381‐high mobility group box 1 (HMGB1) and NEAT1–miR‐128‐3p–aquaporin 4 (AQP4) axes following CCI and SCI, respectively.\\n110\\n, \\n111\\n NEAT1 downregulation inhibited IL‐6, IL‐1β, and TNF‐α to improve neuropathic pain.\\nAlthough the function of circRNAs as miRNA sponges is still largely unknown, they have been termed as competing endogenous RNAs that bind to target miRNAs and regulate their function.\\n112\\n For example, circHIPK3 is a circRNA highly enriched in serum from DNP patients and in the DRG from DNP rats.\\n113\\n circHIPK3 sponges miR‐124 to promote neuroinflammation, pointing to the involvement of the circHIPK3–miR‐124 axis during DNP. circRNA ciRS‐7 participates in neuropathic pain progression by sponging miR‐135a‐5p to regulate autophagy and inflammation in the spinal cord of CCI rats.\\n114\\n Besides, cirZNF609–miR‐22‐3p‐enolase 1 (ENO1) and circ_0005075–miR‐151a‐3p‐NOTCH receptor 2 (NOTCH2) regulatory axes upregulate inflammatory factor expression and promote neuropathic pain development in the spinal cord after CCI.\\n115\\n, \\n116\\n\\n\\nlncRNAs may act as miRNA sponges to form the lncRNA‐miRNA‐mRNA axis and regulate target gene expression. For example, the lncRNA X‐inactive specific transcript (XIST) is upregulated in the dorsal horn of the spinal cord after CCI. By sponging miR‐137, ‐150, ‐154‐5p, and ‐544, XIST can inhibit the expression of corresponding targets, including TNF‐α‐induced protein 1 (TNFAIP1), ZEB1, TLR5, and STAT3.\\n106\\n, \\n107\\n, \\n108\\n, \\n109\\n TNFAIP1 activates the NF‐κB signaling pathway, while ZEB1, TLR5, and STAT3 are crucial in the neuroinflammatory response. XIST inhibition markedly ameliorates neuropathic pain development. In addition, lncRNA nuclear enriched abundant transcript 1 (NEAT1) was upregulated in the spinal cord dorsal horn to form NEAT1‐miR‐381‐high mobility group box 1 (HMGB1) and NEAT1–miR‐128‐3p–aquaporin 4 (AQP4) axes following CCI and SCI, respectively.\\n110\\n, \\n111\\n NEAT1 downregulation inhibited IL‐6, IL‐1β, and TNF‐α to improve neuropathic pain.\\nAlthough the function of circRNAs as miRNA sponges is still largely unknown, they have been termed as competing endogenous RNAs that bind to target miRNAs and regulate their function.\\n112\\n For example, circHIPK3 is a circRNA highly enriched in serum from DNP patients and in the DRG from DNP rats.\\n113\\n circHIPK3 sponges miR‐124 to promote neuroinflammation, pointing to the involvement of the circHIPK3–miR‐124 axis during DNP. circRNA ciRS‐7 participates in neuropathic pain progression by sponging miR‐135a‐5p to regulate autophagy and inflammation in the spinal cord of CCI rats.\\n114\\n Besides, cirZNF609–miR‐22‐3p‐enolase 1 (ENO1) and circ_0005075–miR‐151a‐3p‐NOTCH receptor 2 (NOTCH2) regulatory axes upregulate inflammatory factor expression and promote neuropathic pain development in the spinal cord after CCI.\\n115\\n, \\n116\\n\\n\\n\\nlncRNAs epigenetically regulate pain‐related molecules expression In addition to acting as a miRNA sponge, lncRNAs can epigenetically regulate the expression of pain‐related molecules. For example, peripheral nerve injury decreases the expression of DRG‐specifically enriched lncRNAs (DS‐lncRNAs) in the injured DRG. Restoring DS‐lncRNAs blocks nerve injury‐induced increases in euchromatic histone lysine N‐methyltransferase 2 (Ehmt2) mRNA and its encoding protein G9a, reverses G9a‐related decreases in opioid receptors and Kcna2 in the injured DRG, and ameliorates nerve injury‐induced pain hypersensitivity.\\n117\\n In addition, transcriptome screening in the DRG of DNP rats\\n118\\n has identified diverse dysregulated lncRNAs, including uc.48+,\\n119\\n BC168687,\\n120\\n, \\n121\\n NONRATT021972,\\n31\\n, \\n122\\n, \\n123\\n MRAK009713,\\n124\\n and Lncenc1.\\n125\\n The expression of uc.48+, BC168687, NONRATT021972, MRAK009713, and Lncenc1 was prominently higher in the DRG of neuropathic pain rats.\\n119\\n, \\n120\\n, \\n121\\n, \\n122\\n, \\n123\\n, \\n124\\n, \\n125\\n Blocking the upregulation via intrathecal or intravenous administration of the corresponding small‐interfering RNA (siRNA) may alleviate neuropathic pain by inhibiting the excitatory transmission mediated by purinergic receptors,\\n119\\n, \\n120\\n, \\n122\\n, \\n123\\n, \\n124\\n TNF‐α‐related pathways,\\n31\\n transient receptor potential vanilloid 1 (TRPV1),\\n121\\n or EZH2.\\n125\\n\\n\\nAs demonstrated by a luciferase assay and RNA‐binding protein immunoprecipitation, lncRNA small nucleolar RNA host gene 1 (SNHG1) can induce neuropathic pain in the spinal cord by binding to the promoter region of cyclin‐dependent kinase 4 (CDK4), stimulating its expression.\\n126\\n SNHG1 knockdown alleviated neuropathic pain development, and SNHG1 overexpression was able to induce neuropathic pain. Similarly, lncRNA PKIA‐AS1 participates in SNL‐induced neuropathic pain by downregulating DNA methyltransferase 1‐catalyzed cyclin‐dependent kinase 6 (DNMT1‐catalyzed) CDK6 promoter methylation and regulating CDK6.\\n127\\n Cyclin‐dependent kinases (CDKs) transcriptionally enhanced pro‐inflammatory gene expression during the G1 cell phase. Furthermore, CDK6 recruitment to the nuclear chromatin fraction by cytokines is related to NF‐κB, STAT, and activator protein 1 (AP‐1) activation to induce neuroinflammation.\\n128\\n Other than the DRG, lncRNA Kcna2‐AS‐RNA was also highly expressed in the spinal cord of postherpetic neuralgia rats, and its downregulation alleviated neuropathic pain by reducing phospho‐STAT3 (pSTAT3) translocation from the cytoplasm to the nucleus and then inhibiting spinal astrocytes activation.\\n129\\n\\n\\nIn addition to acting as a miRNA sponge, lncRNAs can epigenetically regulate the expression of pain‐related molecules. For example, peripheral nerve injury decreases the expression of DRG‐specifically enriched lncRNAs (DS‐lncRNAs) in the injured DRG. Restoring DS‐lncRNAs blocks nerve injury‐induced increases in euchromatic histone lysine N‐methyltransferase 2 (Ehmt2) mRNA and its encoding protein G9a, reverses G9a‐related decreases in opioid receptors and Kcna2 in the injured DRG, and ameliorates nerve injury‐induced pain hypersensitivity.\\n117\\n In addition, transcriptome screening in the DRG of DNP rats\\n118\\n has identified diverse dysregulated lncRNAs, including uc.48+,\\n119\\n BC168687,\\n120\\n, \\n121\\n NONRATT021972,\\n31\\n, \\n122\\n, \\n123\\n MRAK009713,\\n124\\n and Lncenc1.\\n125\\n The expression of uc.48+, BC168687, NONRATT021972, MRAK009713, and Lncenc1 was prominently higher in the DRG of neuropathic pain rats.\\n119\\n, \\n120\\n, \\n121\\n, \\n122\\n, \\n123\\n, \\n124\\n, \\n125\\n Blocking the upregulation via intrathecal or intravenous administration of the corresponding small‐interfering RNA (siRNA) may alleviate neuropathic pain by inhibiting the excitatory transmission mediated by purinergic receptors,\\n119\\n, \\n120\\n, \\n122\\n, \\n123\\n, \\n124\\n TNF‐α‐related pathways,\\n31\\n transient receptor potential vanilloid 1 (TRPV1),\\n121\\n or EZH2.\\n125\\n\\n\\nAs demonstrated by a luciferase assay and RNA‐binding protein immunoprecipitation, lncRNA small nucleolar RNA host gene 1 (SNHG1) can induce neuropathic pain in the spinal cord by binding to the promoter region of cyclin‐dependent kinase 4 (CDK4), stimulating its expression.\\n126\\n SNHG1 knockdown alleviated neuropathic pain development, and SNHG1 overexpression was able to induce neuropathic pain. Similarly, lncRNA PKIA‐AS1 participates in SNL‐induced neuropathic pain by downregulating DNA methyltransferase 1‐catalyzed cyclin‐dependent kinase 6 (DNMT1‐catalyzed) CDK6 promoter methylation and regulating CDK6.\\n127\\n Cyclin‐dependent kinases (CDKs) transcriptionally enhanced pro‐inflammatory gene expression during the G1 cell phase. Furthermore, CDK6 recruitment to the nuclear chromatin fraction by cytokines is related to NF‐κB, STAT, and activator protein 1 (AP‐1) activation to induce neuroinflammation.\\n128\\n Other than the DRG, lncRNA Kcna2‐AS‐RNA was also highly expressed in the spinal cord of postherpetic neuralgia rats, and its downregulation alleviated neuropathic pain by reducing phospho‐STAT3 (pSTAT3) translocation from the cytoplasm to the nucleus and then inhibiting spinal astrocytes activation.\\n129\\n\\n\\n\\nlncRNAs modulate miRNA processing Specific lncRNAs are essential in neuropathic pain by modulating miRNA processing. For example, the transcribed ultraconserved lncRNA uc.153 was prominently increased in the spinal cord of rats with CCI neuropathic pain. uc.153 knockdown reversed CCI‐induced pain behaviors and spinal neuronal hypersensitivity. Mechanistically, uc.153 negatively modulated Dicer‐mediated pre‐miR‐182‐5p processing and inhibited maturation. Meanwhile, spinal miR‐182‐5p downregulation increased the expression of its target, ephrin type‐b (EphB1), and p‐NR2B (phosphorylated N‐methyl‐D‐aspartate receptor (NMDAR) 2B subunit) expression and facilitated hyperalgesia.\\n130\\n\\n\\nCollectively, lncRNAs are crucial in neuropathic pain via diverse mechanisms. Notably, one lncRNA may act with more than one mechanism to regulate target gene expression. For example, SNL upregulated circAnks1a in the cytoplasm and nucleus. circAnks1a enhances the interplay between Y‐box‐binding protein 1 (YBX1) and transportin‐1 to facilitate YBX1 nuclear translocation in the cytoplasm. Meanwhile, in the nucleus, circAnks1a combines with the Vegfb promoter and recruits YBX1 to the Vegfb promoter to enhance its transcription. Additionally, cytoplasmic circAnks1a sponges miR‐324‐3p to regulate vascular endothelial growth factor B (VEGFB) expression. VEGFB binding to its receptor results in the activation of various downstream targets, including p38‐MAPK, PKB/AKT (protein kinase B), extracellular signal‐regulated kinase (ERK)/MAPK, and phosphoinositide 3‐kinase (PI3K).\\n131\\n Therefore, VEGFB upregulation increases dorsal horn neuron excitability and contributes to pain hypersensitivity after nerve injury.\\n132\\n\\n\\nWe also noted that specific lncRNAs were DE following various nerve injuries. For example, lncRNA Malat1 was upregulated after CCI to sponge miR‐206, and Malat1 suppression delayed neuropathic pain progression via miR‐206‐ZEB2 axis‐mediated neuroinflammation inhibition.\\n133\\n Conversely, in rats with complete brachial plexus avulsion‐induced neuropathic pain, Malat1 decreased in spinal cord neurons, and such downregulation increased neuronal spontaneous electrical activity via calcium flux regulation.\\n134\\n The reason for such distinctive Malat1 expression following different pain models is still unclear, and further research is required.\\nSpecific lncRNAs are essential in neuropathic pain by modulating miRNA processing. For example, the transcribed ultraconserved lncRNA uc.153 was prominently increased in the spinal cord of rats with CCI neuropathic pain. uc.153 knockdown reversed CCI‐induced pain behaviors and spinal neuronal hypersensitivity. Mechanistically, uc.153 negatively modulated Dicer‐mediated pre‐miR‐182‐5p processing and inhibited maturation. Meanwhile, spinal miR‐182‐5p downregulation increased the expression of its target, ephrin type‐b (EphB1), and p‐NR2B (phosphorylated N‐methyl‐D‐aspartate receptor (NMDAR) 2B subunit) expression and facilitated hyperalgesia.\\n130\\n\\n\\nCollectively, lncRNAs are crucial in neuropathic pain via diverse mechanisms. Notably, one lncRNA may act with more than one mechanism to regulate target gene expression. For example, SNL upregulated circAnks1a in the cytoplasm and nucleus. circAnks1a enhances the interplay between Y‐box‐binding protein 1 (YBX1) and transportin‐1 to facilitate YBX1 nuclear translocation in the cytoplasm. Meanwhile, in the nucleus, circAnks1a combines with the Vegfb promoter and recruits YBX1 to the Vegfb promoter to enhance its transcription. Additionally, cytoplasmic circAnks1a sponges miR‐324‐3p to regulate vascular endothelial growth factor B (VEGFB) expression. VEGFB binding to its receptor results in the activation of various downstream targets, including p38‐MAPK, PKB/AKT (protein kinase B), extracellular signal‐regulated kinase (ERK)/MAPK, and phosphoinositide 3‐kinase (PI3K).\\n131\\n Therefore, VEGFB upregulation increases dorsal horn neuron excitability and contributes to pain hypersensitivity after nerve injury.\\n132\\n\\n\\nWe also noted that specific lncRNAs were DE following various nerve injuries. For example, lncRNA Malat1 was upregulated after CCI to sponge miR‐206, and Malat1 suppression delayed neuropathic pain progression via miR‐206‐ZEB2 axis‐mediated neuroinflammation inhibition.\\n133\\n Conversely, in rats with complete brachial plexus avulsion‐induced neuropathic pain, Malat1 decreased in spinal cord neurons, and such downregulation increased neuronal spontaneous electrical activity via calcium flux regulation.\\n134\\n The reason for such distinctive Malat1 expression following different pain models is still unclear, and further research is required.\",\n \"After peripheral nerve injury, Egr2‐AS‐RNA is upregulated in Schwann cells.\\n103\\n On the early growth response 2 (Egr2) promoter, Egr2‐AS‐RNA recruits an epigenetic silencing complex to downregulate Egr2, essential for peripheral myelination. Ectopic Egr2‐AS‐RNA expression in DRG cultures downregulates Egr2 mRNA and induces demyelination. In vivo, Egr2‐AS‐RNA inhibition reverts Egr2‐related gene expression and delays demyelination.\\nVoltage‐gated potassium channel (Kv) Kcna2‐AS‐RNA is an endogenous, highly conserved, and widely explored lncRNA in neuropathic pain.\\n104\\n It is a natural antisense transcript (NAT), distributed in the cytoplasm and targets Kcna2 mRNA, which encodes the pain regulation‐related membrane Kv1.2 subunit. Kcna2‐AS‐RNA was time‐dependently upregulated in the inflicted rat DRG after nerve injury. It decreased the total Kv current, upregulated DRG neurons excitability, and produced neuropathic pain symptoms.\\nThe complementary strand of DNA opposite the Scn9a gene encodes Scn9a NAT, another antisense lncRNA expressed in the DRG.\\n105\\n Scn9a NAT is suggested to be a negative regulator of Scn9a mRNA. Scn9a NAT overexpression inhibited Scn9a mRNA, its encoded protein Nav1.7, and Nav1.7 currents in DRG neurons. However, Scn9a NAT and Scn9a mRNA levels did not significantly change in the injured DRG until 2 weeks after nerve injury. More work is required to determine whether NAT can confer analgesia and reduce pain in the neuropathic pain state.\",\n \"lncRNAs may act as miRNA sponges to form the lncRNA‐miRNA‐mRNA axis and regulate target gene expression. For example, the lncRNA X‐inactive specific transcript (XIST) is upregulated in the dorsal horn of the spinal cord after CCI. By sponging miR‐137, ‐150, ‐154‐5p, and ‐544, XIST can inhibit the expression of corresponding targets, including TNF‐α‐induced protein 1 (TNFAIP1), ZEB1, TLR5, and STAT3.\\n106\\n, \\n107\\n, \\n108\\n, \\n109\\n TNFAIP1 activates the NF‐κB signaling pathway, while ZEB1, TLR5, and STAT3 are crucial in the neuroinflammatory response. XIST inhibition markedly ameliorates neuropathic pain development. In addition, lncRNA nuclear enriched abundant transcript 1 (NEAT1) was upregulated in the spinal cord dorsal horn to form NEAT1‐miR‐381‐high mobility group box 1 (HMGB1) and NEAT1–miR‐128‐3p–aquaporin 4 (AQP4) axes following CCI and SCI, respectively.\\n110\\n, \\n111\\n NEAT1 downregulation inhibited IL‐6, IL‐1β, and TNF‐α to improve neuropathic pain.\\nAlthough the function of circRNAs as miRNA sponges is still largely unknown, they have been termed as competing endogenous RNAs that bind to target miRNAs and regulate their function.\\n112\\n For example, circHIPK3 is a circRNA highly enriched in serum from DNP patients and in the DRG from DNP rats.\\n113\\n circHIPK3 sponges miR‐124 to promote neuroinflammation, pointing to the involvement of the circHIPK3–miR‐124 axis during DNP. circRNA ciRS‐7 participates in neuropathic pain progression by sponging miR‐135a‐5p to regulate autophagy and inflammation in the spinal cord of CCI rats.\\n114\\n Besides, cirZNF609–miR‐22‐3p‐enolase 1 (ENO1) and circ_0005075–miR‐151a‐3p‐NOTCH receptor 2 (NOTCH2) regulatory axes upregulate inflammatory factor expression and promote neuropathic pain development in the spinal cord after CCI.\\n115\\n, \\n116\\n\\n\",\n \"In addition to acting as a miRNA sponge, lncRNAs can epigenetically regulate the expression of pain‐related molecules. For example, peripheral nerve injury decreases the expression of DRG‐specifically enriched lncRNAs (DS‐lncRNAs) in the injured DRG. Restoring DS‐lncRNAs blocks nerve injury‐induced increases in euchromatic histone lysine N‐methyltransferase 2 (Ehmt2) mRNA and its encoding protein G9a, reverses G9a‐related decreases in opioid receptors and Kcna2 in the injured DRG, and ameliorates nerve injury‐induced pain hypersensitivity.\\n117\\n In addition, transcriptome screening in the DRG of DNP rats\\n118\\n has identified diverse dysregulated lncRNAs, including uc.48+,\\n119\\n BC168687,\\n120\\n, \\n121\\n NONRATT021972,\\n31\\n, \\n122\\n, \\n123\\n MRAK009713,\\n124\\n and Lncenc1.\\n125\\n The expression of uc.48+, BC168687, NONRATT021972, MRAK009713, and Lncenc1 was prominently higher in the DRG of neuropathic pain rats.\\n119\\n, \\n120\\n, \\n121\\n, \\n122\\n, \\n123\\n, \\n124\\n, \\n125\\n Blocking the upregulation via intrathecal or intravenous administration of the corresponding small‐interfering RNA (siRNA) may alleviate neuropathic pain by inhibiting the excitatory transmission mediated by purinergic receptors,\\n119\\n, \\n120\\n, \\n122\\n, \\n123\\n, \\n124\\n TNF‐α‐related pathways,\\n31\\n transient receptor potential vanilloid 1 (TRPV1),\\n121\\n or EZH2.\\n125\\n\\n\\nAs demonstrated by a luciferase assay and RNA‐binding protein immunoprecipitation, lncRNA small nucleolar RNA host gene 1 (SNHG1) can induce neuropathic pain in the spinal cord by binding to the promoter region of cyclin‐dependent kinase 4 (CDK4), stimulating its expression.\\n126\\n SNHG1 knockdown alleviated neuropathic pain development, and SNHG1 overexpression was able to induce neuropathic pain. Similarly, lncRNA PKIA‐AS1 participates in SNL‐induced neuropathic pain by downregulating DNA methyltransferase 1‐catalyzed cyclin‐dependent kinase 6 (DNMT1‐catalyzed) CDK6 promoter methylation and regulating CDK6.\\n127\\n Cyclin‐dependent kinases (CDKs) transcriptionally enhanced pro‐inflammatory gene expression during the G1 cell phase. Furthermore, CDK6 recruitment to the nuclear chromatin fraction by cytokines is related to NF‐κB, STAT, and activator protein 1 (AP‐1) activation to induce neuroinflammation.\\n128\\n Other than the DRG, lncRNA Kcna2‐AS‐RNA was also highly expressed in the spinal cord of postherpetic neuralgia rats, and its downregulation alleviated neuropathic pain by reducing phospho‐STAT3 (pSTAT3) translocation from the cytoplasm to the nucleus and then inhibiting spinal astrocytes activation.\\n129\\n\\n\",\n \"Specific lncRNAs are essential in neuropathic pain by modulating miRNA processing. For example, the transcribed ultraconserved lncRNA uc.153 was prominently increased in the spinal cord of rats with CCI neuropathic pain. uc.153 knockdown reversed CCI‐induced pain behaviors and spinal neuronal hypersensitivity. Mechanistically, uc.153 negatively modulated Dicer‐mediated pre‐miR‐182‐5p processing and inhibited maturation. Meanwhile, spinal miR‐182‐5p downregulation increased the expression of its target, ephrin type‐b (EphB1), and p‐NR2B (phosphorylated N‐methyl‐D‐aspartate receptor (NMDAR) 2B subunit) expression and facilitated hyperalgesia.\\n130\\n\\n\\nCollectively, lncRNAs are crucial in neuropathic pain via diverse mechanisms. Notably, one lncRNA may act with more than one mechanism to regulate target gene expression. For example, SNL upregulated circAnks1a in the cytoplasm and nucleus. circAnks1a enhances the interplay between Y‐box‐binding protein 1 (YBX1) and transportin‐1 to facilitate YBX1 nuclear translocation in the cytoplasm. Meanwhile, in the nucleus, circAnks1a combines with the Vegfb promoter and recruits YBX1 to the Vegfb promoter to enhance its transcription. Additionally, cytoplasmic circAnks1a sponges miR‐324‐3p to regulate vascular endothelial growth factor B (VEGFB) expression. VEGFB binding to its receptor results in the activation of various downstream targets, including p38‐MAPK, PKB/AKT (protein kinase B), extracellular signal‐regulated kinase (ERK)/MAPK, and phosphoinositide 3‐kinase (PI3K).\\n131\\n Therefore, VEGFB upregulation increases dorsal horn neuron excitability and contributes to pain hypersensitivity after nerve injury.\\n132\\n\\n\\nWe also noted that specific lncRNAs were DE following various nerve injuries. For example, lncRNA Malat1 was upregulated after CCI to sponge miR‐206, and Malat1 suppression delayed neuropathic pain progression via miR‐206‐ZEB2 axis‐mediated neuroinflammation inhibition.\\n133\\n Conversely, in rats with complete brachial plexus avulsion‐induced neuropathic pain, Malat1 decreased in spinal cord neurons, and such downregulation increased neuronal spontaneous electrical activity via calcium flux regulation.\\n134\\n The reason for such distinctive Malat1 expression following different pain models is still unclear, and further research is required.\",\n \"There are two main strategies for modulating miRNA function during pain treatment: upregulation or downregulation of specific miRNAs. miRNA mimics or virus‐based constructs were used to upregulate miRNA expression. miRNA inhibitors, miRNA sponges, or inhibition of a particular miRNA‐mRNA interaction has been applied to downregulate specific miRNAs. The design of lncRNA‐based therapeutics includes diverse approaches, such as post‐transcriptional inhibition of lncRNAs by antisense oligonucleotides or siRNA and steric blockades of lncRNA–protein interactions by small molecules and morpholino.\\n135\\n\\n\\nHowever, there is much work to be done before miRNA‐ and lncRNA‐based analgesics can be clinically used for various reasons, including their conservation among species, proper delivery, stability, off‐target effects, and potential activation of the immune system.\\nFirst, most available findings regarding the efficacy of miRNAs or lncRNAs during neuropathic pain are based on animal but not human studies. Whether such results can be extrapolated to humans and their translational potential is still unknown because of conservation among species.\\nSecond, the blood–brain barrier (BBB) is a practical challenge for delivering RNA‐based therapeutics into the central nervous system (CNS) via intravenous injection. Viral vectors, polypeptides, aptamers, and particular chemical modifications have been developed. Supplementing cholesterol molecules to the sense strand of a miRNA mimic or inhibitor has proven to be an efficient strategy. Cholesterol‐conjugated siRNAs showed better silencing potency than unconjugated siRNAs and presented a high efficacy for delivery to oligodendrocytes in the CNS.\\n136\\n Another proven method is the immunoliposome, a combination of liposomes, receptor‐targeted monoclonal antibodies, and the target molecules.\\n137\\n An immunoliposome nanocomplex has been reported to deliver therapeutic nucleic acids across the BBB into the deep brain by transferrin receptors.\\n138\\n In addition, intrathecal injections are a feasible approach in animal studies for neuropathic pain treatment. As reported, miR‐146a attenuated neuropathic pain partially by inhibiting TNF receptor‐associated factor 6 (TRAF6) and its downstream phospho‐JNK/C–C motif chemokine ligand 2 (pJNK/CCL2) signaling in the spinal cord.\\n139\\n Intrathecal injection of miR‐146a‐5p encapsulated nanoparticles provided an analgesic effect via NF‐κB and p38‐MAPK inhibition in spinal microglia.\\n140\\n In recent years, poly (D, L‐lactic‐co‐glycolic acid) (PLGA)‐nanoparticles have been applied to deliver siRNAs and plasmids into the spinal cord to treat neuropathic pain in rats.\\n141\\n PLGA copolymer is a promising US Food and Drug Administration (FDA)‐approved gene transmission material because of its biodegradability and biocompatibility in humans.\\n142\\n Intrathecal treatments with C‐X3‐C motif chemokine receptor 1 (CX3CR1), p38, p66shc siRNA encapsulated PLGA nanoparticles, or forkhead box P3 (Foxp3) plasmid‐encapsulated PLGA nanoparticles inhibit microglial activation and hyperalgesia in SNL rats.\\n141\\n, \\n143\\n, \\n144\\n, \\n145\\n\\n\\nExosomes are another promising delivery carrier for treating neuropathic pain. Exosomes are natural membranous microvesicles that carry RNAs, with the advantage of being efficient, cell‐free, and nonimmunogenicity. Intravenous injecting neuron‐targeted exosomes delivered the carried siRNAs to neurons, microglia, and oligodendrocytes to knock down specific gene expression in mice brains.\\n146\\n This approach enabled cell‐specific delivery of the siRNA‐cargo across the BBB. Mesenchymal stem cells (MSCs) are pluripotent stem cells with immunomodulatory, anti‐inflammatory, and nutritional properties. The treatment efficacy of exosomes derived from MSCs has been proven in neuropathic pain.\\n147\\n Intrathecal, local, or subcutaneous application of exosomes obtained from human umbilical cord MSCs could mitigate nerve injury‐induced hyperalgesia.\\n148\\n, \\n149\\n, \\n150\\n Furthermore, immunofluorescence results showed that most intrathecally injected exosomes could be found in injured peripheral axons, the DRG, and the spinal dorsal horn, potentiating a homing ability of exosomes.\\n148\\n\\n\\nThird, although miRNAs are relatively stable in vivo, they have long‐lasting efficacy and a high resistance to nucleolytic degradation compared to mRNAs. Particular chemical modifications are still required to prolong its half‐life or increase its stability, for example, by generating locked‐nucleic acids (LNAs). LNA modifications increase the RNA affinity of antisense oligonucleotides, presenting an excellent miRNA inhibitory activity at a low dosage.\\n151\\n Additionally, the LNA technique was used to synthesize highly stable aptamers.\\n152\\n\\n\\nFourth, the off‐target effect is another consideration. One miRNA may regulate multiple genes, and the off‐target potential of one miRNA may produce undesirable side effects.\\n153\\n, \\n154\\n\\n\\nFinally, activation of the immune system is a potential adverse event. Specific CpG motifs in oligonucleotides could trigger nonspecific immunological activity.\\n155\\n Therefore, RNA‐based therapeutics might also produce specific immune reactions after producing antibodies against the oligonucleotides.\",\n \"Ming Jiang: Investigation, Methodology. Yelong Wang: Manuscript preparation. Jing Wang: Figures and tables preparation. Shanwu Feng: Supervision, Funding acquisition. Xian Wang: Conceptualization, Funding acquisition.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","\nBIOGENESIS AND FUNCTION OF miRNAs, lncRNAs, AND circRNAs\n","\nPERIPHERAL NERVE INJURY OR NOXIOUS STIMULI INDUCE EXTENSIVE miRNAs, lncRNAs, AND circRNAs EXPRESSION CHANGES\n","\nmiRNAs expression changes","\nlncRNAs and circRNAs expression changes","\nSTUDYING miRNA NEUROPATHIC PAIN MECHANISMS\n","\nmiRNAs regulate neuroinflammation in neuropathic pain development","Autophagy","The contribution of miRNAs to ion channel expression in neuropathic pain","\nmiRNAs regulate pain‐related mediators, protein kinases, and structural proteins","Neurotransmission excitatory–inhibitory imbalances","\nEV miRNAs‐mediated neuron–glia communication","\nSTUDYING lncRNAs AND circRNAs NEUROPATHIC PAIN MECHANISMS\n","\nlncRNAs act as AS RNA\n","\nlncRNAs act as miRNA sponges","\nlncRNAs epigenetically regulate pain‐related molecules expression","\nlncRNAs modulate miRNA processing","\nCHALLENGES AND CONSIDERATIONS IN DELIVERING miRNA‐AND lncRNAs‐BASED THERAPETUTICS\n","CONCLUSION","AUTHOR CONTRIBUTIONS","CONFLICT OF INTERESTS"],"string":"[\n \"INTRODUCTION\",\n \"\\nBIOGENESIS AND FUNCTION OF miRNAs, lncRNAs, AND circRNAs\\n\",\n \"\\nPERIPHERAL NERVE INJURY OR NOXIOUS STIMULI INDUCE EXTENSIVE miRNAs, lncRNAs, AND circRNAs EXPRESSION CHANGES\\n\",\n \"\\nmiRNAs expression changes\",\n \"\\nlncRNAs and circRNAs expression changes\",\n \"\\nSTUDYING miRNA NEUROPATHIC PAIN MECHANISMS\\n\",\n \"\\nmiRNAs regulate neuroinflammation in neuropathic pain development\",\n \"Autophagy\",\n \"The contribution of miRNAs to ion channel expression in neuropathic pain\",\n \"\\nmiRNAs regulate pain‐related mediators, protein kinases, and structural proteins\",\n \"Neurotransmission excitatory–inhibitory imbalances\",\n \"\\nEV miRNAs‐mediated neuron–glia communication\",\n \"\\nSTUDYING lncRNAs AND circRNAs NEUROPATHIC PAIN MECHANISMS\\n\",\n \"\\nlncRNAs act as AS RNA\\n\",\n \"\\nlncRNAs act as miRNA sponges\",\n \"\\nlncRNAs epigenetically regulate pain‐related molecules expression\",\n \"\\nlncRNAs modulate miRNA processing\",\n \"\\nCHALLENGES AND CONSIDERATIONS IN DELIVERING miRNA‐AND lncRNAs‐BASED THERAPETUTICS\\n\",\n \"CONCLUSION\",\n \"AUTHOR CONTRIBUTIONS\",\n \"CONFLICT OF INTERESTS\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["According to the International Association for the Study of Pain, neuropathic pain is the most severe chronic pain condition triggered by a lesion or disease of the somatosensory system. It is characteristic of hyperalgesia, allodynia, or spontaneous pain.\n1\n Neuropathic pain can have peripheral and central origins, with the former including neuropathic pain after peripheral nerve injury, trigeminal neuralgia, postherpetic neuralgia, painful radiculopathy, and painful polyneuropathy. In contrast, central neuropathic pain includes neuropathic pain after spinal cord or brain injury, multiple sclerosis, and chronic central post‐stroke pain. Approximately 7%–10% of the general population will experience neuropathic pain, with the majority not having satisfactory pain relief with current therapies, leading to great suffering for individuals and enormous economic and social burdens.\n2\n\n\nThe exact molecular mechanisms underlying neuropathic pain remain unclear, and elucidating them is crucial for developing mechanism‐based treatment strategies. One proposed mechanism involves altered gene or protein expression along the pain processing pathways. Therefore, understanding how genes or proteins are dysregulated may help us to find a way to normalize these abnormalities and treat neuropathic pain.\nIn recent years, accumulating evidence has suggested the essential role of non‐coding RNAs (ncRNAs) in various physiological and pathological procedures, such as embryonic development, inflammation, tumors, and respiratory and cardiovascular diseases.\n3\n ncRNAs have no protein‐coding potential, but they can govern gene or protein expression with diverse mechanisms. ncRNAs are extensively distributed in the peripheral and central nervous systems, including pain‐related structures.\n4\n Broad abnormal ncRNAs expression is observed following peripheral stimulation. These abnormalities are related to hyperalgesia during chronic pain development. The available data indicate that ncRNAs may be essential for hyperalgesia. In this review, we focus on microRNA (miRNA), long non‐coding RNA (lncRNA), and circular non‐coding RNA (circRNA) expression changes in neuropathic pain. Other types of ncRNAs are seldom reported in neuropathic pain and, thus, are not discussed herein. Notably, we will pay attention to their etiological role in the development of neuropathic pain and the current challenges and considerations for miRNA‐, lncRNA‐, and circRNA‐based therapeutics for neuropathic pain.","Typically, miRNA production involves three steps: cropping, exporting, and dicing. First, the miRNA gene is transcribed in the nucleus, mainly by RNA polymerase II.\n5\n The resulting primary miRNA transcript (pri‐miRNA) is several kilobases (kb) in length, with a specific stem–loop structure that harbors mature miRNAs in the stem. The mature miRNA is ‘cropped’ by Drosha and its interactor DGCR8 (DiGeorge syndrome critical region 8), which cleaves pri‐miRNA at the stem to produce pre‐miRNA,\n6\n 60–70 nucleotides (nt) in length with a hairpin structure. Then, exportin‐5 recognizes and exports pre‐miRNA from the nucleus to the cytoplasm.\n7\n In the cytoplasm, ribonuclease III (RNAse III), termed Dicer, further cleaves the pre‐miRNA to release double‐stranded miRNA with a length of ~22 nt.\n8\n This miRNA is unwound by an unknown helicase or cleaved by Argonaute (Ago) to form the RNA‐induced silencing complex.\n9\n One strand in the RNA duplex remains with Ago as a mature miRNA, and the other is degraded. The seed sequence of the miRNA incompletely or entirely combines with the target mRNA sequence, resulting in target mRNAs degradation or transcriptional regulation.\n10\n\n\nUnlike miRNAs, lncRNAs are mRNAs‐like transcripts ranging in length from 200 nt to 100 kb that lack prominent open reading frames.\n11\n The lncRNA cellular mechanism is highly related to their intracellular localization. lncRNAs control chromatin functions, transcription, and RNA processing in the nucleus and affect mRNA stability, translation, and cellular signaling in the cytoplasm.\n12\n Compared to lncRNAs, circRNAs are more stable because a single circRNA molecular ends can be covalently linked compared to linear RNA. circRNAs are evolutionarily conserved molecules that are essential in the post‐transcriptional modification of gene expression by acting as miRNAs sponges or interacting with transcription or translational machinery. Numerous lncRNAs and circRNAs are distributed within pain‐related regions and dysregulated after peripheral noxious stimulation. Moreover, functional studies have indicated that miRNAs, lncRNAs, and circRNAs participate in neuropathic pain development by regulating diverse pain‐related genes along the pain processing pathways.","\nmiRNAs expression changes Microarray and deep‐sequencing analyses revealed that nerve injury or noxious stimuli could induce broad changes in miRNA expression in serum or along the pain processing pathways, including the perineal nerve, dorsal root ganglion (DRG), spinal cord, and supraspinal regions. For normal pain signal transmission, nerve injury or noxious stimuli are detected by nociceptors in the DRG or trigeminal ganglion (TG) and then transmitted to upstream neurons in the spinal dorsal horn. Subsequently, nociceptive stimuli are integrated, processed, and further transmitted ascending to specific supraspinal brain regions.\nHuman studies have identified as many as 1134 differentially expressed (DE) genes in the serum of individuals with or without neuropathic pain after spinal cord injury (SCI).\n13\n miR‐204‐5p, ‐519d‐3p, ‐20b‐5p, and ‐6838‐5p might act as promising biomarkers and intervention targets for preventing and therapizing neuropathic pain after SCI. In trigeminal neuralgia individuals, serum miR‐132‐3p, ‐146b‐5p, ‐155‐5p, and ‐384 levels were prominently increased compared with healthy controls.\n14\n Patients with painful peripheral neuropathy had higher miR‐21 level in the serum and sural nerve compared with healthy controls. Meanwhile, miR‐155 was reduced in the serum and inflicted lower leg skin.\n15\n Animal and human studies showed that miR‐30c‐5p was upregulated in the serum and cerebro spinal fluid (CSF) of sciatic nerve injury rats and neuropathic pain patients with chronic peripheral ischemia. The high expression of miR‐30c‐5p, together with other clinical parameters, might be used to predict neuropathic pain development in patients with chronic peripheral ischemia.\n16\n Additionally, an animal study showed that plasma‐derived DE extracellular vesicle (EV) miRNAs regulated processes that are essential for neuropathic pain development. Most DE EV miRNAs for inflammation suppression were downregulated, potentially acting as biomarkers and targets in neuropathic pain treatment.\n17\n\n\nIn addition, numerous DE miRNAs have been identified in the DRG. One week after spared nerve injury (SNI), 33 and 39 miRNAs in the DRG were upregulated and downregulated, respectively, with most DE miRNAs related to axon guidance, focal adhesion, Ras and Wnt signaling pathways.\n18\n Furthermore, nerve injury‐induced miRNAs expression was dynamic and time‐dependent,\n19\n, \n20\n implicating multiple regulatory mechanisms in neuropathic pain initiation and development. Nerve injury redistributes miRNAs from a uniform style within the DRG soma of non‐allodynic animals to preferential localization to peripheral neurons in allodynic animals.\n21\n Furthermore, either sciatic nerve ligation (SNL), DRG transaction (DRT), or ventral root transection (VRT) could upregulate miR‐21 and miR‐31 while downregulating miR‐668 and miR‐672 in the injured DRG,\n22\n implying that these miRNAs could be therapeutic targets for treating diverse types of neuropathic pain.\nThe spinal cord dorsal horn relays and modulates pain signals from the peripheral nociceptors to the supraspinal regions. In the spinal cord, numerous miRNA expression changes have been observed in chronic constriction injury (CCI) and diabetic neuropathic pain (DNP) rodents.\n23\n, \n24\n, \n25\n, \n26\n Previous research has suggested some miRNAs may be related to neuropathic pain development, for example, miR‐500, ‐221, and ‐21,\n25\n thus, acting as potential targets in its treatment.\n27\n\n\nCytoscape software constructed the miRNA–target gene regulatory network in the supraspinal region, including the nucleus accumbens (NAc), medial prefrontal cortex, and periaqueductal gray, between SNI and sham rats. Finally, four essential DE genes, includingCXCR2, IL12B, TNFSF8, and GRK1, and five miRNAs, including miR‐208a‐5p, −7688‐3p, −344f‐3p, −135b‐3p, and ‐135a‐2‐3p, were identified, indicating their essential roles in neuropathic pain pathogenesis.\n28\n Furthermore, in the prelimbic cortex of SNI rats, the DE miRNA–mRNA network pointed to molecules associated with inflammation.\n29\n DE miRNAs were also observed in the bilateral hippocampus of CCI rats. However, no significant difference was observed bilaterally in the hippocampus.\n30\n (Figure 1).\nmicroRNAs (miRNAs) expression change following peripheral nerve injury or noxious stimuli SCI, spinal cord injury; CSF, cerebrol spinal fluid; DE, differential expressed; EV, extravascular vesicle; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury; DRT, DRG transection; VRT, ventral root transection\nMicroarray and deep‐sequencing analyses revealed that nerve injury or noxious stimuli could induce broad changes in miRNA expression in serum or along the pain processing pathways, including the perineal nerve, dorsal root ganglion (DRG), spinal cord, and supraspinal regions. For normal pain signal transmission, nerve injury or noxious stimuli are detected by nociceptors in the DRG or trigeminal ganglion (TG) and then transmitted to upstream neurons in the spinal dorsal horn. Subsequently, nociceptive stimuli are integrated, processed, and further transmitted ascending to specific supraspinal brain regions.\nHuman studies have identified as many as 1134 differentially expressed (DE) genes in the serum of individuals with or without neuropathic pain after spinal cord injury (SCI).\n13\n miR‐204‐5p, ‐519d‐3p, ‐20b‐5p, and ‐6838‐5p might act as promising biomarkers and intervention targets for preventing and therapizing neuropathic pain after SCI. In trigeminal neuralgia individuals, serum miR‐132‐3p, ‐146b‐5p, ‐155‐5p, and ‐384 levels were prominently increased compared with healthy controls.\n14\n Patients with painful peripheral neuropathy had higher miR‐21 level in the serum and sural nerve compared with healthy controls. Meanwhile, miR‐155 was reduced in the serum and inflicted lower leg skin.\n15\n Animal and human studies showed that miR‐30c‐5p was upregulated in the serum and cerebro spinal fluid (CSF) of sciatic nerve injury rats and neuropathic pain patients with chronic peripheral ischemia. The high expression of miR‐30c‐5p, together with other clinical parameters, might be used to predict neuropathic pain development in patients with chronic peripheral ischemia.\n16\n Additionally, an animal study showed that plasma‐derived DE extracellular vesicle (EV) miRNAs regulated processes that are essential for neuropathic pain development. Most DE EV miRNAs for inflammation suppression were downregulated, potentially acting as biomarkers and targets in neuropathic pain treatment.\n17\n\n\nIn addition, numerous DE miRNAs have been identified in the DRG. One week after spared nerve injury (SNI), 33 and 39 miRNAs in the DRG were upregulated and downregulated, respectively, with most DE miRNAs related to axon guidance, focal adhesion, Ras and Wnt signaling pathways.\n18\n Furthermore, nerve injury‐induced miRNAs expression was dynamic and time‐dependent,\n19\n, \n20\n implicating multiple regulatory mechanisms in neuropathic pain initiation and development. Nerve injury redistributes miRNAs from a uniform style within the DRG soma of non‐allodynic animals to preferential localization to peripheral neurons in allodynic animals.\n21\n Furthermore, either sciatic nerve ligation (SNL), DRG transaction (DRT), or ventral root transection (VRT) could upregulate miR‐21 and miR‐31 while downregulating miR‐668 and miR‐672 in the injured DRG,\n22\n implying that these miRNAs could be therapeutic targets for treating diverse types of neuropathic pain.\nThe spinal cord dorsal horn relays and modulates pain signals from the peripheral nociceptors to the supraspinal regions. In the spinal cord, numerous miRNA expression changes have been observed in chronic constriction injury (CCI) and diabetic neuropathic pain (DNP) rodents.\n23\n, \n24\n, \n25\n, \n26\n Previous research has suggested some miRNAs may be related to neuropathic pain development, for example, miR‐500, ‐221, and ‐21,\n25\n thus, acting as potential targets in its treatment.\n27\n\n\nCytoscape software constructed the miRNA–target gene regulatory network in the supraspinal region, including the nucleus accumbens (NAc), medial prefrontal cortex, and periaqueductal gray, between SNI and sham rats. Finally, four essential DE genes, includingCXCR2, IL12B, TNFSF8, and GRK1, and five miRNAs, including miR‐208a‐5p, −7688‐3p, −344f‐3p, −135b‐3p, and ‐135a‐2‐3p, were identified, indicating their essential roles in neuropathic pain pathogenesis.\n28\n Furthermore, in the prelimbic cortex of SNI rats, the DE miRNA–mRNA network pointed to molecules associated with inflammation.\n29\n DE miRNAs were also observed in the bilateral hippocampus of CCI rats. However, no significant difference was observed bilaterally in the hippocampus.\n30\n (Figure 1).\nmicroRNAs (miRNAs) expression change following peripheral nerve injury or noxious stimuli SCI, spinal cord injury; CSF, cerebrol spinal fluid; DE, differential expressed; EV, extravascular vesicle; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury; DRT, DRG transection; VRT, ventral root transection\n\nlncRNAs and circRNAs expression changes Patients with type 2 DNP had a prominent higher expression of serum lncRNA NONRATT021972 and more severe neuropathic pain symptoms.\n31\n LINC01119 and LINC02447 in the peripheral blood of SCI patients were identified in pain pathways that were important for neuropathic pain development.\n32\n\n\nMicroarray analysis revealed that a nerve injury could induce time‐dependent lncRNA expression changes in the sciatic nerve.\n33\n lncRNA H19 was persistently upregulated in Schwann cells along the peripheral nerve, proximal and distal to the injured site.\n34\n In the DRG, the transcriptomic analysis identified 86 known and 26 novel lncRNAs genes to be DE after spared sciatic nerve injury.\n35\n Of these, rno‐Cntnap2 and AC111653.1 were essential in peripheral nerve regeneration and were involved in neuropathic pain.\nNext‐generation RNA sequencing showed 134 lncRNAs and 188 circRNAs were prominently changed 14 days after SNI in the spinal cord.\n36\n In addition, microarray analysis identified 1481 and 1096 DE lncRNAs and mRNAs, respectively, in the spinal cord dorsal horn of DNP rats. Of these, 289 neighboring and 57 overlapping lncRNA‐mRNA pairs, including ENSMUST00000150952‐Mbp and AK081017‐Usp15, have been suggested to participate in neuropathic pain development.\n37\n\n\ncircRNAs have characteristic circularized structures following the backsplicing of exons from antisense RNAs (AS RNAs) or mRNAs and, thus, are highly stable. Most circRNAs are highly conserved among species and lack translation potential, despite cap‐independent translation. A recent study showed that 363 and 106 circRNAs were significantly dysregulated in the ipsilateral dorsal horn after nerve injury.\n38\n (Figure 2).\nLong non‐coding RNA (lncRNAs) and circular non‐coding RNAs (circRNAs) expression change following peripheral nerve injury or noxious stimuli DNP, diabetic neuropathic pain; SCI, spinal cord injury; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury\nPatients with type 2 DNP had a prominent higher expression of serum lncRNA NONRATT021972 and more severe neuropathic pain symptoms.\n31\n LINC01119 and LINC02447 in the peripheral blood of SCI patients were identified in pain pathways that were important for neuropathic pain development.\n32\n\n\nMicroarray analysis revealed that a nerve injury could induce time‐dependent lncRNA expression changes in the sciatic nerve.\n33\n lncRNA H19 was persistently upregulated in Schwann cells along the peripheral nerve, proximal and distal to the injured site.\n34\n In the DRG, the transcriptomic analysis identified 86 known and 26 novel lncRNAs genes to be DE after spared sciatic nerve injury.\n35\n Of these, rno‐Cntnap2 and AC111653.1 were essential in peripheral nerve regeneration and were involved in neuropathic pain.\nNext‐generation RNA sequencing showed 134 lncRNAs and 188 circRNAs were prominently changed 14 days after SNI in the spinal cord.\n36\n In addition, microarray analysis identified 1481 and 1096 DE lncRNAs and mRNAs, respectively, in the spinal cord dorsal horn of DNP rats. Of these, 289 neighboring and 57 overlapping lncRNA‐mRNA pairs, including ENSMUST00000150952‐Mbp and AK081017‐Usp15, have been suggested to participate in neuropathic pain development.\n37\n\n\ncircRNAs have characteristic circularized structures following the backsplicing of exons from antisense RNAs (AS RNAs) or mRNAs and, thus, are highly stable. Most circRNAs are highly conserved among species and lack translation potential, despite cap‐independent translation. A recent study showed that 363 and 106 circRNAs were significantly dysregulated in the ipsilateral dorsal horn after nerve injury.\n38\n (Figure 2).\nLong non‐coding RNA (lncRNAs) and circular non‐coding RNAs (circRNAs) expression change following peripheral nerve injury or noxious stimuli DNP, diabetic neuropathic pain; SCI, spinal cord injury; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury","Microarray and deep‐sequencing analyses revealed that nerve injury or noxious stimuli could induce broad changes in miRNA expression in serum or along the pain processing pathways, including the perineal nerve, dorsal root ganglion (DRG), spinal cord, and supraspinal regions. For normal pain signal transmission, nerve injury or noxious stimuli are detected by nociceptors in the DRG or trigeminal ganglion (TG) and then transmitted to upstream neurons in the spinal dorsal horn. Subsequently, nociceptive stimuli are integrated, processed, and further transmitted ascending to specific supraspinal brain regions.\nHuman studies have identified as many as 1134 differentially expressed (DE) genes in the serum of individuals with or without neuropathic pain after spinal cord injury (SCI).\n13\n miR‐204‐5p, ‐519d‐3p, ‐20b‐5p, and ‐6838‐5p might act as promising biomarkers and intervention targets for preventing and therapizing neuropathic pain after SCI. In trigeminal neuralgia individuals, serum miR‐132‐3p, ‐146b‐5p, ‐155‐5p, and ‐384 levels were prominently increased compared with healthy controls.\n14\n Patients with painful peripheral neuropathy had higher miR‐21 level in the serum and sural nerve compared with healthy controls. Meanwhile, miR‐155 was reduced in the serum and inflicted lower leg skin.\n15\n Animal and human studies showed that miR‐30c‐5p was upregulated in the serum and cerebro spinal fluid (CSF) of sciatic nerve injury rats and neuropathic pain patients with chronic peripheral ischemia. The high expression of miR‐30c‐5p, together with other clinical parameters, might be used to predict neuropathic pain development in patients with chronic peripheral ischemia.\n16\n Additionally, an animal study showed that plasma‐derived DE extracellular vesicle (EV) miRNAs regulated processes that are essential for neuropathic pain development. Most DE EV miRNAs for inflammation suppression were downregulated, potentially acting as biomarkers and targets in neuropathic pain treatment.\n17\n\n\nIn addition, numerous DE miRNAs have been identified in the DRG. One week after spared nerve injury (SNI), 33 and 39 miRNAs in the DRG were upregulated and downregulated, respectively, with most DE miRNAs related to axon guidance, focal adhesion, Ras and Wnt signaling pathways.\n18\n Furthermore, nerve injury‐induced miRNAs expression was dynamic and time‐dependent,\n19\n, \n20\n implicating multiple regulatory mechanisms in neuropathic pain initiation and development. Nerve injury redistributes miRNAs from a uniform style within the DRG soma of non‐allodynic animals to preferential localization to peripheral neurons in allodynic animals.\n21\n Furthermore, either sciatic nerve ligation (SNL), DRG transaction (DRT), or ventral root transection (VRT) could upregulate miR‐21 and miR‐31 while downregulating miR‐668 and miR‐672 in the injured DRG,\n22\n implying that these miRNAs could be therapeutic targets for treating diverse types of neuropathic pain.\nThe spinal cord dorsal horn relays and modulates pain signals from the peripheral nociceptors to the supraspinal regions. In the spinal cord, numerous miRNA expression changes have been observed in chronic constriction injury (CCI) and diabetic neuropathic pain (DNP) rodents.\n23\n, \n24\n, \n25\n, \n26\n Previous research has suggested some miRNAs may be related to neuropathic pain development, for example, miR‐500, ‐221, and ‐21,\n25\n thus, acting as potential targets in its treatment.\n27\n\n\nCytoscape software constructed the miRNA–target gene regulatory network in the supraspinal region, including the nucleus accumbens (NAc), medial prefrontal cortex, and periaqueductal gray, between SNI and sham rats. Finally, four essential DE genes, includingCXCR2, IL12B, TNFSF8, and GRK1, and five miRNAs, including miR‐208a‐5p, −7688‐3p, −344f‐3p, −135b‐3p, and ‐135a‐2‐3p, were identified, indicating their essential roles in neuropathic pain pathogenesis.\n28\n Furthermore, in the prelimbic cortex of SNI rats, the DE miRNA–mRNA network pointed to molecules associated with inflammation.\n29\n DE miRNAs were also observed in the bilateral hippocampus of CCI rats. However, no significant difference was observed bilaterally in the hippocampus.\n30\n (Figure 1).\nmicroRNAs (miRNAs) expression change following peripheral nerve injury or noxious stimuli SCI, spinal cord injury; CSF, cerebrol spinal fluid; DE, differential expressed; EV, extravascular vesicle; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury; DRT, DRG transection; VRT, ventral root transection","Patients with type 2 DNP had a prominent higher expression of serum lncRNA NONRATT021972 and more severe neuropathic pain symptoms.\n31\n LINC01119 and LINC02447 in the peripheral blood of SCI patients were identified in pain pathways that were important for neuropathic pain development.\n32\n\n\nMicroarray analysis revealed that a nerve injury could induce time‐dependent lncRNA expression changes in the sciatic nerve.\n33\n lncRNA H19 was persistently upregulated in Schwann cells along the peripheral nerve, proximal and distal to the injured site.\n34\n In the DRG, the transcriptomic analysis identified 86 known and 26 novel lncRNAs genes to be DE after spared sciatic nerve injury.\n35\n Of these, rno‐Cntnap2 and AC111653.1 were essential in peripheral nerve regeneration and were involved in neuropathic pain.\nNext‐generation RNA sequencing showed 134 lncRNAs and 188 circRNAs were prominently changed 14 days after SNI in the spinal cord.\n36\n In addition, microarray analysis identified 1481 and 1096 DE lncRNAs and mRNAs, respectively, in the spinal cord dorsal horn of DNP rats. Of these, 289 neighboring and 57 overlapping lncRNA‐mRNA pairs, including ENSMUST00000150952‐Mbp and AK081017‐Usp15, have been suggested to participate in neuropathic pain development.\n37\n\n\ncircRNAs have characteristic circularized structures following the backsplicing of exons from antisense RNAs (AS RNAs) or mRNAs and, thus, are highly stable. Most circRNAs are highly conserved among species and lack translation potential, despite cap‐independent translation. A recent study showed that 363 and 106 circRNAs were significantly dysregulated in the ipsilateral dorsal horn after nerve injury.\n38\n (Figure 2).\nLong non‐coding RNA (lncRNAs) and circular non‐coding RNAs (circRNAs) expression change following peripheral nerve injury or noxious stimuli DNP, diabetic neuropathic pain; SCI, spinal cord injury; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury","miRNAs contribute to the development of neuropathic pain via diverse mechanisms, such as neuroinflammation, autophagy, abnormal ion channel expression, regulating pain‐related mediators, protein kinases, structural proteins, neurotransmission excitatory–inhibitory imbalances, and exosome miRNA‐mediated neuron–glia communication (Table 1).\nmiRNAs expression change for the development of neuropathic pain\nKv1.1, Kv1.4, Kv3.4, Kv4.3, Kv7.5,\nDPP10, Navβ1 ↓\np‐p38 MAPK ↑\nAbbreviations: ACC, anterior cingulate cortex; BACE1, beta‐site amyloid precursor protein‐cleaving enzyme 1; BDNF, brain‐derived neurotrophic factor; CCI, chronic constriction injury; CCR1, C–C chemokine receptor 1; CSF, cerebro spinal fluid; CSF1, colony‐stimulating factor‐1; CXCR4, chemokine CXC receptor 4; DNMT3a, DNA methyltransferase 3a; DNP, diabetic neuropathic pain; DRG, dorsal root ganglion; EV, extracellular vesicle; EZH2, enhancer of zeste homolog 2; GRK2, G protein‐coupled receptor kinases 2; IGF‐1/IGF‐1R, insulin‐like growth factor‐1/insulin‐like growth factor‐1 receptor; KCNMA1, calcium‐activated potassium channel subunit α‐1; MeCP2, methyl cytosine–guanine dinucleotide (CpG)‐binding protein 2; MIP‐1a, macrophage inflammatory protein‐1 alpha; NEFL, neurofilament light polypeptide; NF‐κB, nuclear factor – kappa B; NGF, nerve growth factor; NLRP3, NOD‐like receptor protein 3; NOX2, NADPH oxidase 2; NP, neuropathic pain; pSNL, partial sciatic nerve ligation; RAP1A, Ras‐related protein 1A; RASA1, RAS P21 protein activator 1; ROS, reactive oxygen species; S1PR1, sphingosine‐1‐phosphate receptor 1; SCI, spinal cord injury; Scn2b, β2 subunit of the voltage‐gated sodium channel; SIRT1, histone deacetylase sirtuin 1; SNL, spinal nerve ligation; SOCS1, suppressor of cytokine signaling 1; STAT3, signal transducer and activator of transcription 3; TLR4, toll‐like receptor 4; TNFR1, TNF receptor 1; TRPA1, transient receptor potential ankyrin 1; TXNIP, thioredoxin‐interacting protein; VTR, ventral root transection; ZEB1, zinc finger E‐box binding homeobox 1.\n\nmiRNAs regulate neuroinflammation in neuropathic pain development miRNA‐based epigenetic regulation is essential in neuroinflammation. miRNAs are predicted to regulate diverse neuroinflammation‐related targets along the pain processing pathways.\nAmong patients with neuropathic pain, miR‐101 decreased in the serum and sural nerve, which is related to nuclear factor‐kappa B (NF‐κB) signaling activation.\n39\n Meanwhile, serum miR‐124a and miR‐155 expression was upregulated. They were identified to inhibit histone deacetylase sirtuin 1 (SIRT1) in primary human cluster of differentiation 4 (CD4) (+) cells and induce their differentiation toward regulatory T cells (Tregs), thus reducing pain‐related inflammation.\n40\n Such miRNA–target interactions may act as an endogenous protective mechanism for neuropathic pain.\nmiR‐7a, expressed in small‐sized nociceptive DRG neurons, is downregulated after nerve injury, as reported, targeting neurofilament light polypeptides (NEFLs).\n41\n NEFL encodes a neuronal protein vital for neurofilament formation and increases signal transducer and activator of transcription 3 (STAT3) phosphorylation, which is highly related to cell differentiation and neuroinflammation. In diabetic peripheral neuropathic mice, miR‐590‐3p was downregulated to disinhibit Ras‐related protein 1A (RAP1A) in the DRG tissue and inhibit neural T cells infiltration.\n42\n Thus, exogenous miR‐590‐3p may be a potential alternative for neuropathic pain treatment. CCI downregulated miR‐140 and miR‐144 expression in the DRG. Intrathecally injected miR‐140 and miR‐144 agomir decreased inflammatory factor secretion and ameliorated hyperalgesia by targeting sphingosine‐1‐phosphate receptor 1 (S1PR1) and RAS P21 protein activator 1 (RASA1), respectively.\n43\n, \n44\n\n\nmiRNAs are essential in neuropathic pain development via neuroinflammation‐related mechanisms in the spinal cord. miR‐130a‐3p targets and downregulates insulin‐like growth factor‐1/insulin‐like growth factor‐1 receptor (IGF‐1/IGF‐1R) expression to alleviate SCI‐induced neuropathic pain by mitigating microglial activation and NF‐κB phosphorylation.\n45\n miR‐378 was decreased in CCI rats, inhibiting neuropathic pain development by targeting the enhancer of zeste homolog 2 (EZH2).\n46\n EZH2 promotes neuropathic pain by increasing tumor necrosis factor‐alpha (TNF‐α), interleukin (IL)‐1β, and monocyte chemoattractant protein‐1 (MCP‐1) production. Intrathecally injecting miRNA‐138 lentivirus can remarkably alleviate neuropathic pain in partial sciatic nerve ligation (pSNL) rats by suppressing toll‐like receptor 4 (TLR4) and macrophage inflammatory protein‐1 alpha (MIP‐1α)/C‐C chemokine receptor 1 (CCR1) signaling pathways.\n47\n\n\nmiR‐15a/16 targets and downregulates G protein‐coupled receptor kinases 2 (GRK2) to disinhibit p38‐MAPK (mitogen‐activated protein kinase) and NF‐κB, contributing to neuroinflammation after CCI.\n48\n GRK2‐deficient mice present a pro‐inflammatory phenotype in spinal cord microglia/macrophages, restored by miR‐124.\n49\n SNL increased DNA methyltransferase 3a (DNMT3a) expression related to hypermethylation of the miR‐214‐3p promoter, resulting in miR‐214‐3p expression reduction, which enhanced astrocyte reactivity, colony‐stimulating factor‐1 (CSF1), and interleukin 6 (IL‐6) production, and hyperalgesia in rats.\n50\n Electro‐acupuncture attenuated SCI by inhibiting Nav1.3 and Bax in the injured spinal cord through miR‐214 upregulation.\n51\n Downregulated miR‐128 was reported to contribute to neuropathic pain via p38 or zinc finger E‐box binding homeobox 1 (ZEB1) activation in the spinal cord.\n52\n, \n53\n Meanwhile, ZEB1 was also targeted by miR‐200b/miR‐429, orchestrating neuropathic pain development.\n54\n\n\nIn a pSNL mouse model, nerve injury significantly reduced miR‐23a expression in spinal glial cells, concomitant with the upregulation of its target chemokine, CXC receptor 4 (CXCR4). In naïve mice, either miR‐23a downregulation or CXCR4 upregulation could active the thioredoxin‐interacting protein (TXNIP)/NOD‐like receptor protein 3 (NLRP3) inflammasome axis. Both intrathecal miR‐23a mimics and spinal CXCR4 downregulation by a lentivirus inhibited TXNIP or NLRP3 upregulation to alleviate hyperalgesia.\n55\n\n\nNotably, one miRNA was shown to have distinctive targets in different animal models of the spinal cord. For example, miR‐155 downregulation targeted and upregulated NADPH oxidase 2 (NOX2) expression to induce reactive oxygen species (ROS) production after SCI, presenting a pro‐inflammatory phenotype in microglia/macrophages.\n56\n The ability to induce glial polarization was also observed in cultured BV‐2 microglia.\n57\n In bortezomib‐induced neuropathic pain rats, downregulated miR‐155 upregulated TNF receptor 1 (TNFR1) expression, which activated its downstream signaling pathways, including p38‐MAPK, c‐Jun N‐terminal kinase (JNK), and transient receptor potential ankyrin 1 (TRPA1).\n58\n Therefore, it is suggested that miR‐155 might act as an intervention target for neuropathic pain. As expected, treatment with ibuprofen and L‐arginine delayed the behavioral pain changes while inhibiting spinal miR‐155 and NO.\n59\n miR‐155‐5p is also known to destabilize the blood–nerve barrier and expression of tight junction proteins, such as claudin‐1 and zonula occludens‐1 (ZO‐1). Tissue plasminogen activator (tPA) could transiently open such barriers to facilitate topically applying analgesics, via miR‐155‐5p upregulation.\n60\n However, we also noted that CCI upregulated but did not downregulate spinal cord miR‐155, and miR‐155 inhibition enhanced suppressor of cytokine signaling 1 (SOCS1) expression to inactivate inflammation via NF‐κB and p38‐MAPK inhibition.\n61\n In oxaliplatin‐induced peripheral neuropathic pain, spinal cord miR‐155 expression was also upregulated, and intrathecally injecting the miR‐155 inhibitor attenuated hyperalgesia in rats, possibly by inhibiting oxidative stress–TRPA1 pathways.\n62\n The underlying mechanisms of such distinctive miR‐155 expression change are still unknown and require further research into different pain models.\nmiRNA‐based epigenetic regulation is essential in neuroinflammation. miRNAs are predicted to regulate diverse neuroinflammation‐related targets along the pain processing pathways.\nAmong patients with neuropathic pain, miR‐101 decreased in the serum and sural nerve, which is related to nuclear factor‐kappa B (NF‐κB) signaling activation.\n39\n Meanwhile, serum miR‐124a and miR‐155 expression was upregulated. They were identified to inhibit histone deacetylase sirtuin 1 (SIRT1) in primary human cluster of differentiation 4 (CD4) (+) cells and induce their differentiation toward regulatory T cells (Tregs), thus reducing pain‐related inflammation.\n40\n Such miRNA–target interactions may act as an endogenous protective mechanism for neuropathic pain.\nmiR‐7a, expressed in small‐sized nociceptive DRG neurons, is downregulated after nerve injury, as reported, targeting neurofilament light polypeptides (NEFLs).\n41\n NEFL encodes a neuronal protein vital for neurofilament formation and increases signal transducer and activator of transcription 3 (STAT3) phosphorylation, which is highly related to cell differentiation and neuroinflammation. In diabetic peripheral neuropathic mice, miR‐590‐3p was downregulated to disinhibit Ras‐related protein 1A (RAP1A) in the DRG tissue and inhibit neural T cells infiltration.\n42\n Thus, exogenous miR‐590‐3p may be a potential alternative for neuropathic pain treatment. CCI downregulated miR‐140 and miR‐144 expression in the DRG. Intrathecally injected miR‐140 and miR‐144 agomir decreased inflammatory factor secretion and ameliorated hyperalgesia by targeting sphingosine‐1‐phosphate receptor 1 (S1PR1) and RAS P21 protein activator 1 (RASA1), respectively.\n43\n, \n44\n\n\nmiRNAs are essential in neuropathic pain development via neuroinflammation‐related mechanisms in the spinal cord. miR‐130a‐3p targets and downregulates insulin‐like growth factor‐1/insulin‐like growth factor‐1 receptor (IGF‐1/IGF‐1R) expression to alleviate SCI‐induced neuropathic pain by mitigating microglial activation and NF‐κB phosphorylation.\n45\n miR‐378 was decreased in CCI rats, inhibiting neuropathic pain development by targeting the enhancer of zeste homolog 2 (EZH2).\n46\n EZH2 promotes neuropathic pain by increasing tumor necrosis factor‐alpha (TNF‐α), interleukin (IL)‐1β, and monocyte chemoattractant protein‐1 (MCP‐1) production. Intrathecally injecting miRNA‐138 lentivirus can remarkably alleviate neuropathic pain in partial sciatic nerve ligation (pSNL) rats by suppressing toll‐like receptor 4 (TLR4) and macrophage inflammatory protein‐1 alpha (MIP‐1α)/C‐C chemokine receptor 1 (CCR1) signaling pathways.\n47\n\n\nmiR‐15a/16 targets and downregulates G protein‐coupled receptor kinases 2 (GRK2) to disinhibit p38‐MAPK (mitogen‐activated protein kinase) and NF‐κB, contributing to neuroinflammation after CCI.\n48\n GRK2‐deficient mice present a pro‐inflammatory phenotype in spinal cord microglia/macrophages, restored by miR‐124.\n49\n SNL increased DNA methyltransferase 3a (DNMT3a) expression related to hypermethylation of the miR‐214‐3p promoter, resulting in miR‐214‐3p expression reduction, which enhanced astrocyte reactivity, colony‐stimulating factor‐1 (CSF1), and interleukin 6 (IL‐6) production, and hyperalgesia in rats.\n50\n Electro‐acupuncture attenuated SCI by inhibiting Nav1.3 and Bax in the injured spinal cord through miR‐214 upregulation.\n51\n Downregulated miR‐128 was reported to contribute to neuropathic pain via p38 or zinc finger E‐box binding homeobox 1 (ZEB1) activation in the spinal cord.\n52\n, \n53\n Meanwhile, ZEB1 was also targeted by miR‐200b/miR‐429, orchestrating neuropathic pain development.\n54\n\n\nIn a pSNL mouse model, nerve injury significantly reduced miR‐23a expression in spinal glial cells, concomitant with the upregulation of its target chemokine, CXC receptor 4 (CXCR4). In naïve mice, either miR‐23a downregulation or CXCR4 upregulation could active the thioredoxin‐interacting protein (TXNIP)/NOD‐like receptor protein 3 (NLRP3) inflammasome axis. Both intrathecal miR‐23a mimics and spinal CXCR4 downregulation by a lentivirus inhibited TXNIP or NLRP3 upregulation to alleviate hyperalgesia.\n55\n\n\nNotably, one miRNA was shown to have distinctive targets in different animal models of the spinal cord. For example, miR‐155 downregulation targeted and upregulated NADPH oxidase 2 (NOX2) expression to induce reactive oxygen species (ROS) production after SCI, presenting a pro‐inflammatory phenotype in microglia/macrophages.\n56\n The ability to induce glial polarization was also observed in cultured BV‐2 microglia.\n57\n In bortezomib‐induced neuropathic pain rats, downregulated miR‐155 upregulated TNF receptor 1 (TNFR1) expression, which activated its downstream signaling pathways, including p38‐MAPK, c‐Jun N‐terminal kinase (JNK), and transient receptor potential ankyrin 1 (TRPA1).\n58\n Therefore, it is suggested that miR‐155 might act as an intervention target for neuropathic pain. As expected, treatment with ibuprofen and L‐arginine delayed the behavioral pain changes while inhibiting spinal miR‐155 and NO.\n59\n miR‐155‐5p is also known to destabilize the blood–nerve barrier and expression of tight junction proteins, such as claudin‐1 and zonula occludens‐1 (ZO‐1). Tissue plasminogen activator (tPA) could transiently open such barriers to facilitate topically applying analgesics, via miR‐155‐5p upregulation.\n60\n However, we also noted that CCI upregulated but did not downregulate spinal cord miR‐155, and miR‐155 inhibition enhanced suppressor of cytokine signaling 1 (SOCS1) expression to inactivate inflammation via NF‐κB and p38‐MAPK inhibition.\n61\n In oxaliplatin‐induced peripheral neuropathic pain, spinal cord miR‐155 expression was also upregulated, and intrathecally injecting the miR‐155 inhibitor attenuated hyperalgesia in rats, possibly by inhibiting oxidative stress–TRPA1 pathways.\n62\n The underlying mechanisms of such distinctive miR‐155 expression change are still unknown and require further research into different pain models.\nAutophagy miRNA‐related autophagy is involved in neuropathic pain regulation. As reported, miR‐15a downregulated and stimulated AKT serine/threonine kinase 3 (AKT3), and inhibited autophagy post‐CCI.\n63\n Impaired autophagy participates in neuropathic pain development. Intrathecal miR‐15a agomir prominently suppressed AKT3 expression, induced autophagy, and attenuated CCI‐induced neuropathic pain. Similar to miR‐15a, miR‐145 and miR‐20b‐5p contributed to neuropathic pain regulation via protein kinase B (AKT)‐related autophagy pathways.\n64\n, \n65\n Meanwhile, miR‐195 in the spinal cord was observed to upregulate post‐SNL, targeting and inhibiting Autophagy‐Related 14 (ATG14) and its autophagy activation.\n66\n The miR‐195 inhibitor activated autophagy and suppressed neuroinflammation in vivo and in vitro.\nmiRNA‐related autophagy is involved in neuropathic pain regulation. As reported, miR‐15a downregulated and stimulated AKT serine/threonine kinase 3 (AKT3), and inhibited autophagy post‐CCI.\n63\n Impaired autophagy participates in neuropathic pain development. Intrathecal miR‐15a agomir prominently suppressed AKT3 expression, induced autophagy, and attenuated CCI‐induced neuropathic pain. Similar to miR‐15a, miR‐145 and miR‐20b‐5p contributed to neuropathic pain regulation via protein kinase B (AKT)‐related autophagy pathways.\n64\n, \n65\n Meanwhile, miR‐195 in the spinal cord was observed to upregulate post‐SNL, targeting and inhibiting Autophagy‐Related 14 (ATG14) and its autophagy activation.\n66\n The miR‐195 inhibitor activated autophagy and suppressed neuroinflammation in vivo and in vitro.\nThe contribution of miRNAs to ion channel expression in neuropathic pain Another class of miRNA‐based regulation focuses on ion channels, including sodium, potassium, and calcium channels, and transient receptor potential (TRP) channels, to modulate action potential production, firing rate, and neurotransmitter release.\nTheoretically, miRNAs can simultaneously have multiple target mRNAs because precise matching is not a prerequisite for inhibiting the target sequence. For example, other than NEFL,\n41\n miR‐7a targets the β2 subunit of the voltage‐gated sodium channel (Scn2b) with post‐transcriptional regulation to induce nociceptive DRG neurons hyperexcitability.\n67\n Other than miR‐7a, the other known miRNA‐ targets involved in sodium channels expression include miR‐30b, ‐96, ‐384‐5p and their target Scn3a (encoding Nav1.3),\n68\n, \n69\n, \n70\n miR‐30b‐5p and its target Scn8a (encoding Nav1.6),\n71\n and miR‐182 and ‐30b and their target Scn9a (encoding Nav1.7).\n72\n, \n73\n\n\nA miRNA cluster is a polycistronic gene containing several miRNAs derived from a single primary or nascent transcript. Approximately 40% of miRNAs are predicted to form clusters, but their significance is still unknown. miR‐17‐92 is a miRNA cluster that includes six different members, of which miR‐18a, ‐19a, ‐19b, and ‐92a upregulation induces allodynia. The predicted targets of the miR‐17‐92 cluster encompass genes encoding diverse voltage‐gated potassium channels and their regulatory subunits, including Kv1.1, Kv1.4, Kv3.4, Kv4.3, Kv7.5, dipeptidyl peptidase 10 (DPP10), and Navβ1.\n74\n CCI upregulated miR‐137 to target and downregulate Kcna2, which encodes Kv1.2 in the DRG and spinal dorsal horn.\n75\n By contrast, CCI decreased miR‐183‐5p expression in the DRG, and the predicted target gene TREK‐1, a subunit of the 2P‐domain K+ channel, was increased.\n76\n\n\nFurthermore, the miRNA‐183 cluster (miR‐183, part of miR‐96/182/183) regulates either basal mechanical or neuropathic pain.\n77\n The miR‐183 cluster targets Cacna2d1 and Cacna2d2, which encode auxiliary voltage‐gated calcium channel subunits α2δ‐1 and α2δ‐2, to affect nociceptor excitability. Nerve injury downregulated miR‐103 in the spinal cord, which simultaneously targeted and inhibited Cacna1c, Cacna2d1, and Cacnb1 and encoded the Cav1.2‐α1, α2δ1, and β1 subunits of the voltage‐gated calcium channels macromolecular complex Cav1.2 L‐type calcium channel, respectively.\n78\n Intrathecal miR‐103 successfully relieved neuropathic pain.\nIn addition to ion channels, TRP channels are ligand‐gated ion channels that promote painful sensations.\n79\n In the DRG, TRPA1 participates in miR‐141‐5p alleviated oxaliplatin‐induced neuropathic pain.\n80\n In SNI mice, miR‐449a ameliorated neuropathic pain by decreasing the activity of TRPA1 and calcium‐activated potassium channel subunit α‐1 (KCNMA1), thus acting as a potential therapeutic alternative for treating neuropathic pain.\n81\n\n\nAnother class of miRNA‐based regulation focuses on ion channels, including sodium, potassium, and calcium channels, and transient receptor potential (TRP) channels, to modulate action potential production, firing rate, and neurotransmitter release.\nTheoretically, miRNAs can simultaneously have multiple target mRNAs because precise matching is not a prerequisite for inhibiting the target sequence. For example, other than NEFL,\n41\n miR‐7a targets the β2 subunit of the voltage‐gated sodium channel (Scn2b) with post‐transcriptional regulation to induce nociceptive DRG neurons hyperexcitability.\n67\n Other than miR‐7a, the other known miRNA‐ targets involved in sodium channels expression include miR‐30b, ‐96, ‐384‐5p and their target Scn3a (encoding Nav1.3),\n68\n, \n69\n, \n70\n miR‐30b‐5p and its target Scn8a (encoding Nav1.6),\n71\n and miR‐182 and ‐30b and their target Scn9a (encoding Nav1.7).\n72\n, \n73\n\n\nA miRNA cluster is a polycistronic gene containing several miRNAs derived from a single primary or nascent transcript. Approximately 40% of miRNAs are predicted to form clusters, but their significance is still unknown. miR‐17‐92 is a miRNA cluster that includes six different members, of which miR‐18a, ‐19a, ‐19b, and ‐92a upregulation induces allodynia. The predicted targets of the miR‐17‐92 cluster encompass genes encoding diverse voltage‐gated potassium channels and their regulatory subunits, including Kv1.1, Kv1.4, Kv3.4, Kv4.3, Kv7.5, dipeptidyl peptidase 10 (DPP10), and Navβ1.\n74\n CCI upregulated miR‐137 to target and downregulate Kcna2, which encodes Kv1.2 in the DRG and spinal dorsal horn.\n75\n By contrast, CCI decreased miR‐183‐5p expression in the DRG, and the predicted target gene TREK‐1, a subunit of the 2P‐domain K+ channel, was increased.\n76\n\n\nFurthermore, the miRNA‐183 cluster (miR‐183, part of miR‐96/182/183) regulates either basal mechanical or neuropathic pain.\n77\n The miR‐183 cluster targets Cacna2d1 and Cacna2d2, which encode auxiliary voltage‐gated calcium channel subunits α2δ‐1 and α2δ‐2, to affect nociceptor excitability. Nerve injury downregulated miR‐103 in the spinal cord, which simultaneously targeted and inhibited Cacna1c, Cacna2d1, and Cacnb1 and encoded the Cav1.2‐α1, α2δ1, and β1 subunits of the voltage‐gated calcium channels macromolecular complex Cav1.2 L‐type calcium channel, respectively.\n78\n Intrathecal miR‐103 successfully relieved neuropathic pain.\nIn addition to ion channels, TRP channels are ligand‐gated ion channels that promote painful sensations.\n79\n In the DRG, TRPA1 participates in miR‐141‐5p alleviated oxaliplatin‐induced neuropathic pain.\n80\n In SNI mice, miR‐449a ameliorated neuropathic pain by decreasing the activity of TRPA1 and calcium‐activated potassium channel subunit α‐1 (KCNMA1), thus acting as a potential therapeutic alternative for treating neuropathic pain.\n81\n\n\n\nmiRNAs regulate pain‐related mediators, protein kinases, and structural proteins miRNAs regulate diverse pain‐related mediators, protein kinases, and structural proteins along the pain processing pathways. For HIV‐associated symptomatic distal sensory polyneuropathy and neuropathic pain, serum miR‐455‐3p acted as a potential biomarker, possibly targeting multiple genes involved in peripheral neuropathic pain, such as nerve growth factor (NGF) and related genes.\n82\n Brain‐derived neurotrophic factor (BDNF), another well‐recognized pain mediator, is a common miR‐1, ‐183, and ‐206 target.\n83\n, \n84\n, \n85\n After nerve injury, several miRNAs, including miR‐19a, ‐301, and ‐132, were downregulated, and their target methyl cytosine–guanine dinucleotide (CpG)‐binding protein 2 (MeCP2) expression was increased, leading to concomitant BDNF upregulation.\n86\n miR‐30c‐5p was upregulated in the serum and CSF of patients with chronic peripheral ischemia.\n16\n A miR‐30c‐5p inhibitor intraventricular injection postponed neuropathic pain development and fully reversed hyperalgesia in rodents. Transforming growth factor beta (TGF‐β) participates in the effects of miR‐30c‐5p, which refers to the endogenous opioid analgesic system.\n16\n\n\nmiRNAs dysregulate specific pain‐related protein kinases. For example, in DNP rats, miR‐133a‐3p was dysregulated in the sciatic nerve, interacting with p‐p38 MAPK to participate in the development of neuropathic pain.\n87\n Via a methyl CpG‐binding domain and transcriptional repressor domain, MeCP2 acts as a transcriptional repressor, and its overexpression improves neuropathic pain, potentiating an anti‐nociceptive effect of MeCP2. The authors also noted that MeCP2 expression changed post‐transcriptionally, and its mRNA level did not significantly change after SNI. However, the protein level was upregulated. During the development period of neuropathic pain, phospho‐cAMP response element‐binding protein (p‐CREB) elevated rapidly but returned 3–7 days after SNI, concomitant with miR‐132 downregulation, which targets MeCP2 and inhibits its expression post‐transcriptionally.\n88\n SNL decreased miR‐200b and miR‐429 in NAc neurons, along with an upregulation of their target (DNMT3a).\n89\n Further mechanism studies found that DNMT3a in the NAc was expressed in NR1 immunoreactive neurons, suggesting the dysregulation of ‘mesolimbic motivation circuitry’ for neuropathic pain development.\nThe CCI induced time‐dependent miR‐1 downregulation in injured sciatic nerves. This change in expression was related to the upregulation and translocation of the miR‐1‐targeted connexin 43 (Cx43), the major connexin of astrocytes.\n83\n miR‐1 mimics could reduce Cx43 expression in cultured human glioblastoma cells. However, miR‐1 mimics intraneural transfection failed to alter Cx43 protein expression and did not improve pain behavior. The authors attributed such treatment failure to insufficient inhibition of miR‐1 for Cx43 via intraneural injection. Alternatively, there were regulatory mechanisms for Cx43 in vivo other than miR‐1.\n90\n However, miR‐1 in the DRG was DE according to peripheral nerve injury type. CCI, pSNL, and sural nerve injury downregulated miR‐1. However, axotomy of the sciatic nerve and tibial nerve injury increased its expression in the DRG.\n83\n, \n91\n, \n92\n As previously described, miR‐1 was also upregulated following capsaicin treatment and bone cancer pain.\n91\n, \n93\n Additionally, miR‐1 downregulation inhibited bone cancer pain. Collectively, the action of miR‐1 on pain seems to be complex and stimulus‐dependent.\nNotably, specific miRNAs can promote neuropathic pain development via diverse mechanisms. For example, beta‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1), a membrane protease essential for myelination, was downregulated after miR‐15b overexpression in vitro or in the DRG of chemotherapy‐related neuropathic pain rats.\n94\n BACE1‐mediated neuregulin 1 reduction decreases nerve conduction velocity. In addition, BACE1 modulated Navβ2 subunit expression and neuronal activity and regulated inflammation‐related TNFR expression.\nmiRNAs regulate diverse pain‐related mediators, protein kinases, and structural proteins along the pain processing pathways. For HIV‐associated symptomatic distal sensory polyneuropathy and neuropathic pain, serum miR‐455‐3p acted as a potential biomarker, possibly targeting multiple genes involved in peripheral neuropathic pain, such as nerve growth factor (NGF) and related genes.\n82\n Brain‐derived neurotrophic factor (BDNF), another well‐recognized pain mediator, is a common miR‐1, ‐183, and ‐206 target.\n83\n, \n84\n, \n85\n After nerve injury, several miRNAs, including miR‐19a, ‐301, and ‐132, were downregulated, and their target methyl cytosine–guanine dinucleotide (CpG)‐binding protein 2 (MeCP2) expression was increased, leading to concomitant BDNF upregulation.\n86\n miR‐30c‐5p was upregulated in the serum and CSF of patients with chronic peripheral ischemia.\n16\n A miR‐30c‐5p inhibitor intraventricular injection postponed neuropathic pain development and fully reversed hyperalgesia in rodents. Transforming growth factor beta (TGF‐β) participates in the effects of miR‐30c‐5p, which refers to the endogenous opioid analgesic system.\n16\n\n\nmiRNAs dysregulate specific pain‐related protein kinases. For example, in DNP rats, miR‐133a‐3p was dysregulated in the sciatic nerve, interacting with p‐p38 MAPK to participate in the development of neuropathic pain.\n87\n Via a methyl CpG‐binding domain and transcriptional repressor domain, MeCP2 acts as a transcriptional repressor, and its overexpression improves neuropathic pain, potentiating an anti‐nociceptive effect of MeCP2. The authors also noted that MeCP2 expression changed post‐transcriptionally, and its mRNA level did not significantly change after SNI. However, the protein level was upregulated. During the development period of neuropathic pain, phospho‐cAMP response element‐binding protein (p‐CREB) elevated rapidly but returned 3–7 days after SNI, concomitant with miR‐132 downregulation, which targets MeCP2 and inhibits its expression post‐transcriptionally.\n88\n SNL decreased miR‐200b and miR‐429 in NAc neurons, along with an upregulation of their target (DNMT3a).\n89\n Further mechanism studies found that DNMT3a in the NAc was expressed in NR1 immunoreactive neurons, suggesting the dysregulation of ‘mesolimbic motivation circuitry’ for neuropathic pain development.\nThe CCI induced time‐dependent miR‐1 downregulation in injured sciatic nerves. This change in expression was related to the upregulation and translocation of the miR‐1‐targeted connexin 43 (Cx43), the major connexin of astrocytes.\n83\n miR‐1 mimics could reduce Cx43 expression in cultured human glioblastoma cells. However, miR‐1 mimics intraneural transfection failed to alter Cx43 protein expression and did not improve pain behavior. The authors attributed such treatment failure to insufficient inhibition of miR‐1 for Cx43 via intraneural injection. Alternatively, there were regulatory mechanisms for Cx43 in vivo other than miR‐1.\n90\n However, miR‐1 in the DRG was DE according to peripheral nerve injury type. CCI, pSNL, and sural nerve injury downregulated miR‐1. However, axotomy of the sciatic nerve and tibial nerve injury increased its expression in the DRG.\n83\n, \n91\n, \n92\n As previously described, miR‐1 was also upregulated following capsaicin treatment and bone cancer pain.\n91\n, \n93\n Additionally, miR‐1 downregulation inhibited bone cancer pain. Collectively, the action of miR‐1 on pain seems to be complex and stimulus‐dependent.\nNotably, specific miRNAs can promote neuropathic pain development via diverse mechanisms. For example, beta‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1), a membrane protease essential for myelination, was downregulated after miR‐15b overexpression in vitro or in the DRG of chemotherapy‐related neuropathic pain rats.\n94\n BACE1‐mediated neuregulin 1 reduction decreases nerve conduction velocity. In addition, BACE1 modulated Navβ2 subunit expression and neuronal activity and regulated inflammation‐related TNFR expression.\nNeurotransmission excitatory–inhibitory imbalances Neurotransmission excitatory and inhibitory imbalances in the spinal cord also contribute to neuropathic pain development. For example, miR‐500 increased to regulate glutamic acid decarboxylase 67 (GAD67) expression and target the specific site of Gad1 in the dorsal horn.\n95\n GAD67 expression reduction inhibited the function of GABAergic neurons and the resultant inhibitory synaptic transmission dysregulation contributed to neuropathic pain development. In addition, miR‐23b is crucial for improving neuropathic pain in the injured spinal cord by downregulating its target gene, Nox4, which further normalizes glutamic acid decarboxylase 65/67 (GAD65/67) expression and protects GABAergic neurons from apoptosis.\n96\n Microarray analysis showed that miR‐539 was prominently reduced in the contralateral anterior cingulate cortex (ACC) after CCI, which is related to enhanced NR2B protein expression. Injecting miR‐539 mimics into the contralateral ACC attenuated CCI‐evoked mechanical hyperalgesia, suggesting that the N‐methyl‐D‐aspartate (NMDA) receptor NR2B subunit regulates neuropathic pain.\n97\n\n\nNeurotransmission excitatory and inhibitory imbalances in the spinal cord also contribute to neuropathic pain development. For example, miR‐500 increased to regulate glutamic acid decarboxylase 67 (GAD67) expression and target the specific site of Gad1 in the dorsal horn.\n95\n GAD67 expression reduction inhibited the function of GABAergic neurons and the resultant inhibitory synaptic transmission dysregulation contributed to neuropathic pain development. In addition, miR‐23b is crucial for improving neuropathic pain in the injured spinal cord by downregulating its target gene, Nox4, which further normalizes glutamic acid decarboxylase 65/67 (GAD65/67) expression and protects GABAergic neurons from apoptosis.\n96\n Microarray analysis showed that miR‐539 was prominently reduced in the contralateral anterior cingulate cortex (ACC) after CCI, which is related to enhanced NR2B protein expression. Injecting miR‐539 mimics into the contralateral ACC attenuated CCI‐evoked mechanical hyperalgesia, suggesting that the N‐methyl‐D‐aspartate (NMDA) receptor NR2B subunit regulates neuropathic pain.\n97\n\n\n\nEV miRNAs‐mediated neuron–glia communication We mainly focused on exosomal miR‐21 with regard to EV miRNAs in neuropathic pain. As aforementioned, SNL, DRT, or VRT could upregulate miR‐21 in the injured DRG.\n22\n Notably, miR‐21 was also increased in serum exosomes from nerve‐ligated mice.\n98\n In another in‐depth study, macrophages readily took up pure sensory neuron‐derived exosomes encompassing miR‐21‐5p to promote a pro‐inflammatory phenotype.\n99\n Either intrathecal miR‐21‐5p antagomir or miR‐21 conditional deletion in sensory neurons ameliorated hyperalgesia and macrophage recruitment in the DRG.\nUnlike the miRNA target gene inhibitory mechanism, miR‐21 acts as an endogenous toll‐like receptor 8 (TLR8) ligand, leading to neuropathic pain development.\n100\n TLR8, a nucleic acid‐sensing receptor, is located in the endosomes and lysosomes, leading to ERK‐mediated inflammatory mediator production and neuronal activation after SNL. Although miR‐21 and TLR8 are only distributed in small‐ and medium‐sized neurons, miR‐21 can also be derived from large‐sized neurons and reach TLR8 in the endosomes of other types of neurons.\n101\n Similar to miR‐21, DRG sensory neurons secreted miR‐23a‐enriched EVs following nerve injury and were taken up by macrophages to enhance M1 polarization in vitro. A20, an NF‐κB signaling pathway inhibitor, is a verified miR‐23a target gene.\n102\n Moreover, intrathecally delivering EVs‐miR‐23a antagomir attenuated neuropathic hyperalgesia and reduced M1 macrophages by inhibiting A20 to activate NF‐κB signaling.\nWe mainly focused on exosomal miR‐21 with regard to EV miRNAs in neuropathic pain. As aforementioned, SNL, DRT, or VRT could upregulate miR‐21 in the injured DRG.\n22\n Notably, miR‐21 was also increased in serum exosomes from nerve‐ligated mice.\n98\n In another in‐depth study, macrophages readily took up pure sensory neuron‐derived exosomes encompassing miR‐21‐5p to promote a pro‐inflammatory phenotype.\n99\n Either intrathecal miR‐21‐5p antagomir or miR‐21 conditional deletion in sensory neurons ameliorated hyperalgesia and macrophage recruitment in the DRG.\nUnlike the miRNA target gene inhibitory mechanism, miR‐21 acts as an endogenous toll‐like receptor 8 (TLR8) ligand, leading to neuropathic pain development.\n100\n TLR8, a nucleic acid‐sensing receptor, is located in the endosomes and lysosomes, leading to ERK‐mediated inflammatory mediator production and neuronal activation after SNL. Although miR‐21 and TLR8 are only distributed in small‐ and medium‐sized neurons, miR‐21 can also be derived from large‐sized neurons and reach TLR8 in the endosomes of other types of neurons.\n101\n Similar to miR‐21, DRG sensory neurons secreted miR‐23a‐enriched EVs following nerve injury and were taken up by macrophages to enhance M1 polarization in vitro. A20, an NF‐κB signaling pathway inhibitor, is a verified miR‐23a target gene.\n102\n Moreover, intrathecally delivering EVs‐miR‐23a antagomir attenuated neuropathic hyperalgesia and reduced M1 macrophages by inhibiting A20 to activate NF‐κB signaling.","miRNA‐based epigenetic regulation is essential in neuroinflammation. miRNAs are predicted to regulate diverse neuroinflammation‐related targets along the pain processing pathways.\nAmong patients with neuropathic pain, miR‐101 decreased in the serum and sural nerve, which is related to nuclear factor‐kappa B (NF‐κB) signaling activation.\n39\n Meanwhile, serum miR‐124a and miR‐155 expression was upregulated. They were identified to inhibit histone deacetylase sirtuin 1 (SIRT1) in primary human cluster of differentiation 4 (CD4) (+) cells and induce their differentiation toward regulatory T cells (Tregs), thus reducing pain‐related inflammation.\n40\n Such miRNA–target interactions may act as an endogenous protective mechanism for neuropathic pain.\nmiR‐7a, expressed in small‐sized nociceptive DRG neurons, is downregulated after nerve injury, as reported, targeting neurofilament light polypeptides (NEFLs).\n41\n NEFL encodes a neuronal protein vital for neurofilament formation and increases signal transducer and activator of transcription 3 (STAT3) phosphorylation, which is highly related to cell differentiation and neuroinflammation. In diabetic peripheral neuropathic mice, miR‐590‐3p was downregulated to disinhibit Ras‐related protein 1A (RAP1A) in the DRG tissue and inhibit neural T cells infiltration.\n42\n Thus, exogenous miR‐590‐3p may be a potential alternative for neuropathic pain treatment. CCI downregulated miR‐140 and miR‐144 expression in the DRG. Intrathecally injected miR‐140 and miR‐144 agomir decreased inflammatory factor secretion and ameliorated hyperalgesia by targeting sphingosine‐1‐phosphate receptor 1 (S1PR1) and RAS P21 protein activator 1 (RASA1), respectively.\n43\n, \n44\n\n\nmiRNAs are essential in neuropathic pain development via neuroinflammation‐related mechanisms in the spinal cord. miR‐130a‐3p targets and downregulates insulin‐like growth factor‐1/insulin‐like growth factor‐1 receptor (IGF‐1/IGF‐1R) expression to alleviate SCI‐induced neuropathic pain by mitigating microglial activation and NF‐κB phosphorylation.\n45\n miR‐378 was decreased in CCI rats, inhibiting neuropathic pain development by targeting the enhancer of zeste homolog 2 (EZH2).\n46\n EZH2 promotes neuropathic pain by increasing tumor necrosis factor‐alpha (TNF‐α), interleukin (IL)‐1β, and monocyte chemoattractant protein‐1 (MCP‐1) production. Intrathecally injecting miRNA‐138 lentivirus can remarkably alleviate neuropathic pain in partial sciatic nerve ligation (pSNL) rats by suppressing toll‐like receptor 4 (TLR4) and macrophage inflammatory protein‐1 alpha (MIP‐1α)/C‐C chemokine receptor 1 (CCR1) signaling pathways.\n47\n\n\nmiR‐15a/16 targets and downregulates G protein‐coupled receptor kinases 2 (GRK2) to disinhibit p38‐MAPK (mitogen‐activated protein kinase) and NF‐κB, contributing to neuroinflammation after CCI.\n48\n GRK2‐deficient mice present a pro‐inflammatory phenotype in spinal cord microglia/macrophages, restored by miR‐124.\n49\n SNL increased DNA methyltransferase 3a (DNMT3a) expression related to hypermethylation of the miR‐214‐3p promoter, resulting in miR‐214‐3p expression reduction, which enhanced astrocyte reactivity, colony‐stimulating factor‐1 (CSF1), and interleukin 6 (IL‐6) production, and hyperalgesia in rats.\n50\n Electro‐acupuncture attenuated SCI by inhibiting Nav1.3 and Bax in the injured spinal cord through miR‐214 upregulation.\n51\n Downregulated miR‐128 was reported to contribute to neuropathic pain via p38 or zinc finger E‐box binding homeobox 1 (ZEB1) activation in the spinal cord.\n52\n, \n53\n Meanwhile, ZEB1 was also targeted by miR‐200b/miR‐429, orchestrating neuropathic pain development.\n54\n\n\nIn a pSNL mouse model, nerve injury significantly reduced miR‐23a expression in spinal glial cells, concomitant with the upregulation of its target chemokine, CXC receptor 4 (CXCR4). In naïve mice, either miR‐23a downregulation or CXCR4 upregulation could active the thioredoxin‐interacting protein (TXNIP)/NOD‐like receptor protein 3 (NLRP3) inflammasome axis. Both intrathecal miR‐23a mimics and spinal CXCR4 downregulation by a lentivirus inhibited TXNIP or NLRP3 upregulation to alleviate hyperalgesia.\n55\n\n\nNotably, one miRNA was shown to have distinctive targets in different animal models of the spinal cord. For example, miR‐155 downregulation targeted and upregulated NADPH oxidase 2 (NOX2) expression to induce reactive oxygen species (ROS) production after SCI, presenting a pro‐inflammatory phenotype in microglia/macrophages.\n56\n The ability to induce glial polarization was also observed in cultured BV‐2 microglia.\n57\n In bortezomib‐induced neuropathic pain rats, downregulated miR‐155 upregulated TNF receptor 1 (TNFR1) expression, which activated its downstream signaling pathways, including p38‐MAPK, c‐Jun N‐terminal kinase (JNK), and transient receptor potential ankyrin 1 (TRPA1).\n58\n Therefore, it is suggested that miR‐155 might act as an intervention target for neuropathic pain. As expected, treatment with ibuprofen and L‐arginine delayed the behavioral pain changes while inhibiting spinal miR‐155 and NO.\n59\n miR‐155‐5p is also known to destabilize the blood–nerve barrier and expression of tight junction proteins, such as claudin‐1 and zonula occludens‐1 (ZO‐1). Tissue plasminogen activator (tPA) could transiently open such barriers to facilitate topically applying analgesics, via miR‐155‐5p upregulation.\n60\n However, we also noted that CCI upregulated but did not downregulate spinal cord miR‐155, and miR‐155 inhibition enhanced suppressor of cytokine signaling 1 (SOCS1) expression to inactivate inflammation via NF‐κB and p38‐MAPK inhibition.\n61\n In oxaliplatin‐induced peripheral neuropathic pain, spinal cord miR‐155 expression was also upregulated, and intrathecally injecting the miR‐155 inhibitor attenuated hyperalgesia in rats, possibly by inhibiting oxidative stress–TRPA1 pathways.\n62\n The underlying mechanisms of such distinctive miR‐155 expression change are still unknown and require further research into different pain models.","miRNA‐related autophagy is involved in neuropathic pain regulation. As reported, miR‐15a downregulated and stimulated AKT serine/threonine kinase 3 (AKT3), and inhibited autophagy post‐CCI.\n63\n Impaired autophagy participates in neuropathic pain development. Intrathecal miR‐15a agomir prominently suppressed AKT3 expression, induced autophagy, and attenuated CCI‐induced neuropathic pain. Similar to miR‐15a, miR‐145 and miR‐20b‐5p contributed to neuropathic pain regulation via protein kinase B (AKT)‐related autophagy pathways.\n64\n, \n65\n Meanwhile, miR‐195 in the spinal cord was observed to upregulate post‐SNL, targeting and inhibiting Autophagy‐Related 14 (ATG14) and its autophagy activation.\n66\n The miR‐195 inhibitor activated autophagy and suppressed neuroinflammation in vivo and in vitro.","Another class of miRNA‐based regulation focuses on ion channels, including sodium, potassium, and calcium channels, and transient receptor potential (TRP) channels, to modulate action potential production, firing rate, and neurotransmitter release.\nTheoretically, miRNAs can simultaneously have multiple target mRNAs because precise matching is not a prerequisite for inhibiting the target sequence. For example, other than NEFL,\n41\n miR‐7a targets the β2 subunit of the voltage‐gated sodium channel (Scn2b) with post‐transcriptional regulation to induce nociceptive DRG neurons hyperexcitability.\n67\n Other than miR‐7a, the other known miRNA‐ targets involved in sodium channels expression include miR‐30b, ‐96, ‐384‐5p and their target Scn3a (encoding Nav1.3),\n68\n, \n69\n, \n70\n miR‐30b‐5p and its target Scn8a (encoding Nav1.6),\n71\n and miR‐182 and ‐30b and their target Scn9a (encoding Nav1.7).\n72\n, \n73\n\n\nA miRNA cluster is a polycistronic gene containing several miRNAs derived from a single primary or nascent transcript. Approximately 40% of miRNAs are predicted to form clusters, but their significance is still unknown. miR‐17‐92 is a miRNA cluster that includes six different members, of which miR‐18a, ‐19a, ‐19b, and ‐92a upregulation induces allodynia. The predicted targets of the miR‐17‐92 cluster encompass genes encoding diverse voltage‐gated potassium channels and their regulatory subunits, including Kv1.1, Kv1.4, Kv3.4, Kv4.3, Kv7.5, dipeptidyl peptidase 10 (DPP10), and Navβ1.\n74\n CCI upregulated miR‐137 to target and downregulate Kcna2, which encodes Kv1.2 in the DRG and spinal dorsal horn.\n75\n By contrast, CCI decreased miR‐183‐5p expression in the DRG, and the predicted target gene TREK‐1, a subunit of the 2P‐domain K+ channel, was increased.\n76\n\n\nFurthermore, the miRNA‐183 cluster (miR‐183, part of miR‐96/182/183) regulates either basal mechanical or neuropathic pain.\n77\n The miR‐183 cluster targets Cacna2d1 and Cacna2d2, which encode auxiliary voltage‐gated calcium channel subunits α2δ‐1 and α2δ‐2, to affect nociceptor excitability. Nerve injury downregulated miR‐103 in the spinal cord, which simultaneously targeted and inhibited Cacna1c, Cacna2d1, and Cacnb1 and encoded the Cav1.2‐α1, α2δ1, and β1 subunits of the voltage‐gated calcium channels macromolecular complex Cav1.2 L‐type calcium channel, respectively.\n78\n Intrathecal miR‐103 successfully relieved neuropathic pain.\nIn addition to ion channels, TRP channels are ligand‐gated ion channels that promote painful sensations.\n79\n In the DRG, TRPA1 participates in miR‐141‐5p alleviated oxaliplatin‐induced neuropathic pain.\n80\n In SNI mice, miR‐449a ameliorated neuropathic pain by decreasing the activity of TRPA1 and calcium‐activated potassium channel subunit α‐1 (KCNMA1), thus acting as a potential therapeutic alternative for treating neuropathic pain.\n81\n\n","miRNAs regulate diverse pain‐related mediators, protein kinases, and structural proteins along the pain processing pathways. For HIV‐associated symptomatic distal sensory polyneuropathy and neuropathic pain, serum miR‐455‐3p acted as a potential biomarker, possibly targeting multiple genes involved in peripheral neuropathic pain, such as nerve growth factor (NGF) and related genes.\n82\n Brain‐derived neurotrophic factor (BDNF), another well‐recognized pain mediator, is a common miR‐1, ‐183, and ‐206 target.\n83\n, \n84\n, \n85\n After nerve injury, several miRNAs, including miR‐19a, ‐301, and ‐132, were downregulated, and their target methyl cytosine–guanine dinucleotide (CpG)‐binding protein 2 (MeCP2) expression was increased, leading to concomitant BDNF upregulation.\n86\n miR‐30c‐5p was upregulated in the serum and CSF of patients with chronic peripheral ischemia.\n16\n A miR‐30c‐5p inhibitor intraventricular injection postponed neuropathic pain development and fully reversed hyperalgesia in rodents. Transforming growth factor beta (TGF‐β) participates in the effects of miR‐30c‐5p, which refers to the endogenous opioid analgesic system.\n16\n\n\nmiRNAs dysregulate specific pain‐related protein kinases. For example, in DNP rats, miR‐133a‐3p was dysregulated in the sciatic nerve, interacting with p‐p38 MAPK to participate in the development of neuropathic pain.\n87\n Via a methyl CpG‐binding domain and transcriptional repressor domain, MeCP2 acts as a transcriptional repressor, and its overexpression improves neuropathic pain, potentiating an anti‐nociceptive effect of MeCP2. The authors also noted that MeCP2 expression changed post‐transcriptionally, and its mRNA level did not significantly change after SNI. However, the protein level was upregulated. During the development period of neuropathic pain, phospho‐cAMP response element‐binding protein (p‐CREB) elevated rapidly but returned 3–7 days after SNI, concomitant with miR‐132 downregulation, which targets MeCP2 and inhibits its expression post‐transcriptionally.\n88\n SNL decreased miR‐200b and miR‐429 in NAc neurons, along with an upregulation of their target (DNMT3a).\n89\n Further mechanism studies found that DNMT3a in the NAc was expressed in NR1 immunoreactive neurons, suggesting the dysregulation of ‘mesolimbic motivation circuitry’ for neuropathic pain development.\nThe CCI induced time‐dependent miR‐1 downregulation in injured sciatic nerves. This change in expression was related to the upregulation and translocation of the miR‐1‐targeted connexin 43 (Cx43), the major connexin of astrocytes.\n83\n miR‐1 mimics could reduce Cx43 expression in cultured human glioblastoma cells. However, miR‐1 mimics intraneural transfection failed to alter Cx43 protein expression and did not improve pain behavior. The authors attributed such treatment failure to insufficient inhibition of miR‐1 for Cx43 via intraneural injection. Alternatively, there were regulatory mechanisms for Cx43 in vivo other than miR‐1.\n90\n However, miR‐1 in the DRG was DE according to peripheral nerve injury type. CCI, pSNL, and sural nerve injury downregulated miR‐1. However, axotomy of the sciatic nerve and tibial nerve injury increased its expression in the DRG.\n83\n, \n91\n, \n92\n As previously described, miR‐1 was also upregulated following capsaicin treatment and bone cancer pain.\n91\n, \n93\n Additionally, miR‐1 downregulation inhibited bone cancer pain. Collectively, the action of miR‐1 on pain seems to be complex and stimulus‐dependent.\nNotably, specific miRNAs can promote neuropathic pain development via diverse mechanisms. For example, beta‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1), a membrane protease essential for myelination, was downregulated after miR‐15b overexpression in vitro or in the DRG of chemotherapy‐related neuropathic pain rats.\n94\n BACE1‐mediated neuregulin 1 reduction decreases nerve conduction velocity. In addition, BACE1 modulated Navβ2 subunit expression and neuronal activity and regulated inflammation‐related TNFR expression.","Neurotransmission excitatory and inhibitory imbalances in the spinal cord also contribute to neuropathic pain development. For example, miR‐500 increased to regulate glutamic acid decarboxylase 67 (GAD67) expression and target the specific site of Gad1 in the dorsal horn.\n95\n GAD67 expression reduction inhibited the function of GABAergic neurons and the resultant inhibitory synaptic transmission dysregulation contributed to neuropathic pain development. In addition, miR‐23b is crucial for improving neuropathic pain in the injured spinal cord by downregulating its target gene, Nox4, which further normalizes glutamic acid decarboxylase 65/67 (GAD65/67) expression and protects GABAergic neurons from apoptosis.\n96\n Microarray analysis showed that miR‐539 was prominently reduced in the contralateral anterior cingulate cortex (ACC) after CCI, which is related to enhanced NR2B protein expression. Injecting miR‐539 mimics into the contralateral ACC attenuated CCI‐evoked mechanical hyperalgesia, suggesting that the N‐methyl‐D‐aspartate (NMDA) receptor NR2B subunit regulates neuropathic pain.\n97\n\n","We mainly focused on exosomal miR‐21 with regard to EV miRNAs in neuropathic pain. As aforementioned, SNL, DRT, or VRT could upregulate miR‐21 in the injured DRG.\n22\n Notably, miR‐21 was also increased in serum exosomes from nerve‐ligated mice.\n98\n In another in‐depth study, macrophages readily took up pure sensory neuron‐derived exosomes encompassing miR‐21‐5p to promote a pro‐inflammatory phenotype.\n99\n Either intrathecal miR‐21‐5p antagomir or miR‐21 conditional deletion in sensory neurons ameliorated hyperalgesia and macrophage recruitment in the DRG.\nUnlike the miRNA target gene inhibitory mechanism, miR‐21 acts as an endogenous toll‐like receptor 8 (TLR8) ligand, leading to neuropathic pain development.\n100\n TLR8, a nucleic acid‐sensing receptor, is located in the endosomes and lysosomes, leading to ERK‐mediated inflammatory mediator production and neuronal activation after SNL. Although miR‐21 and TLR8 are only distributed in small‐ and medium‐sized neurons, miR‐21 can also be derived from large‐sized neurons and reach TLR8 in the endosomes of other types of neurons.\n101\n Similar to miR‐21, DRG sensory neurons secreted miR‐23a‐enriched EVs following nerve injury and were taken up by macrophages to enhance M1 polarization in vitro. A20, an NF‐κB signaling pathway inhibitor, is a verified miR‐23a target gene.\n102\n Moreover, intrathecally delivering EVs‐miR‐23a antagomir attenuated neuropathic hyperalgesia and reduced M1 macrophages by inhibiting A20 to activate NF‐κB signaling.","lncRNAs and circRNAs are essential in neuropathic pain development by acting as AS RNA or miRNA sponges, epigenetically regulating pain‐related molecules expression, or modulating miRNA processing (Table 2).\nLong non‐coding RNAs (lncRNAs) and circular non‐coding RNAs (circRNAs) expression change for the development of neuropathic pain\nAbbreviations: AQP4, aquaporin 4; AS RNA, antisense RNA; CCI, chronic constriction injury; CDK4, cyclin‐dependent kinase 4; CDK6, cyclin‐dependent kinase 6; DNP, diabetic neuropathic pain; DRG, dorsal root ganglion; ENO1, enolase 1; EphB1, ephrin type‐b 1; EZH2, enhancer of zeste homolog 2; HMGB1, high mobility group box 1; NAT, natural antisense transcript; NOTCH2, NOTCH receptor 2; NP, neuropathic pain; pSNL, partial sciatic nerve ligation; pSTAT3, phosphorylated signal transducer and activator of transcription 3; SCI, spinal cord injury; SGC, satellite glia cell; SNL, spinal nerve ligation; STAT3, signal transducer and activator of transcription 3; TLR5, toll‐like receptor 5; TNFAIP1, tumor necrosis factor alpha‐induced protein 1; TRPV1, transient receptor potential vanilloid 1; XIST, X‐inactive specific transcript; ZEB1, zinc finger E‐box binding homeobox 1.\n\nlncRNAs act as AS RNA\n After peripheral nerve injury, Egr2‐AS‐RNA is upregulated in Schwann cells.\n103\n On the early growth response 2 (Egr2) promoter, Egr2‐AS‐RNA recruits an epigenetic silencing complex to downregulate Egr2, essential for peripheral myelination. Ectopic Egr2‐AS‐RNA expression in DRG cultures downregulates Egr2 mRNA and induces demyelination. In vivo, Egr2‐AS‐RNA inhibition reverts Egr2‐related gene expression and delays demyelination.\nVoltage‐gated potassium channel (Kv) Kcna2‐AS‐RNA is an endogenous, highly conserved, and widely explored lncRNA in neuropathic pain.\n104\n It is a natural antisense transcript (NAT), distributed in the cytoplasm and targets Kcna2 mRNA, which encodes the pain regulation‐related membrane Kv1.2 subunit. Kcna2‐AS‐RNA was time‐dependently upregulated in the inflicted rat DRG after nerve injury. It decreased the total Kv current, upregulated DRG neurons excitability, and produced neuropathic pain symptoms.\nThe complementary strand of DNA opposite the Scn9a gene encodes Scn9a NAT, another antisense lncRNA expressed in the DRG.\n105\n Scn9a NAT is suggested to be a negative regulator of Scn9a mRNA. Scn9a NAT overexpression inhibited Scn9a mRNA, its encoded protein Nav1.7, and Nav1.7 currents in DRG neurons. However, Scn9a NAT and Scn9a mRNA levels did not significantly change in the injured DRG until 2 weeks after nerve injury. More work is required to determine whether NAT can confer analgesia and reduce pain in the neuropathic pain state.\nAfter peripheral nerve injury, Egr2‐AS‐RNA is upregulated in Schwann cells.\n103\n On the early growth response 2 (Egr2) promoter, Egr2‐AS‐RNA recruits an epigenetic silencing complex to downregulate Egr2, essential for peripheral myelination. Ectopic Egr2‐AS‐RNA expression in DRG cultures downregulates Egr2 mRNA and induces demyelination. In vivo, Egr2‐AS‐RNA inhibition reverts Egr2‐related gene expression and delays demyelination.\nVoltage‐gated potassium channel (Kv) Kcna2‐AS‐RNA is an endogenous, highly conserved, and widely explored lncRNA in neuropathic pain.\n104\n It is a natural antisense transcript (NAT), distributed in the cytoplasm and targets Kcna2 mRNA, which encodes the pain regulation‐related membrane Kv1.2 subunit. Kcna2‐AS‐RNA was time‐dependently upregulated in the inflicted rat DRG after nerve injury. It decreased the total Kv current, upregulated DRG neurons excitability, and produced neuropathic pain symptoms.\nThe complementary strand of DNA opposite the Scn9a gene encodes Scn9a NAT, another antisense lncRNA expressed in the DRG.\n105\n Scn9a NAT is suggested to be a negative regulator of Scn9a mRNA. Scn9a NAT overexpression inhibited Scn9a mRNA, its encoded protein Nav1.7, and Nav1.7 currents in DRG neurons. However, Scn9a NAT and Scn9a mRNA levels did not significantly change in the injured DRG until 2 weeks after nerve injury. More work is required to determine whether NAT can confer analgesia and reduce pain in the neuropathic pain state.\n\nlncRNAs act as miRNA sponges lncRNAs may act as miRNA sponges to form the lncRNA‐miRNA‐mRNA axis and regulate target gene expression. For example, the lncRNA X‐inactive specific transcript (XIST) is upregulated in the dorsal horn of the spinal cord after CCI. By sponging miR‐137, ‐150, ‐154‐5p, and ‐544, XIST can inhibit the expression of corresponding targets, including TNF‐α‐induced protein 1 (TNFAIP1), ZEB1, TLR5, and STAT3.\n106\n, \n107\n, \n108\n, \n109\n TNFAIP1 activates the NF‐κB signaling pathway, while ZEB1, TLR5, and STAT3 are crucial in the neuroinflammatory response. XIST inhibition markedly ameliorates neuropathic pain development. In addition, lncRNA nuclear enriched abundant transcript 1 (NEAT1) was upregulated in the spinal cord dorsal horn to form NEAT1‐miR‐381‐high mobility group box 1 (HMGB1) and NEAT1–miR‐128‐3p–aquaporin 4 (AQP4) axes following CCI and SCI, respectively.\n110\n, \n111\n NEAT1 downregulation inhibited IL‐6, IL‐1β, and TNF‐α to improve neuropathic pain.\nAlthough the function of circRNAs as miRNA sponges is still largely unknown, they have been termed as competing endogenous RNAs that bind to target miRNAs and regulate their function.\n112\n For example, circHIPK3 is a circRNA highly enriched in serum from DNP patients and in the DRG from DNP rats.\n113\n circHIPK3 sponges miR‐124 to promote neuroinflammation, pointing to the involvement of the circHIPK3–miR‐124 axis during DNP. circRNA ciRS‐7 participates in neuropathic pain progression by sponging miR‐135a‐5p to regulate autophagy and inflammation in the spinal cord of CCI rats.\n114\n Besides, cirZNF609–miR‐22‐3p‐enolase 1 (ENO1) and circ_0005075–miR‐151a‐3p‐NOTCH receptor 2 (NOTCH2) regulatory axes upregulate inflammatory factor expression and promote neuropathic pain development in the spinal cord after CCI.\n115\n, \n116\n\n\nlncRNAs may act as miRNA sponges to form the lncRNA‐miRNA‐mRNA axis and regulate target gene expression. For example, the lncRNA X‐inactive specific transcript (XIST) is upregulated in the dorsal horn of the spinal cord after CCI. By sponging miR‐137, ‐150, ‐154‐5p, and ‐544, XIST can inhibit the expression of corresponding targets, including TNF‐α‐induced protein 1 (TNFAIP1), ZEB1, TLR5, and STAT3.\n106\n, \n107\n, \n108\n, \n109\n TNFAIP1 activates the NF‐κB signaling pathway, while ZEB1, TLR5, and STAT3 are crucial in the neuroinflammatory response. XIST inhibition markedly ameliorates neuropathic pain development. In addition, lncRNA nuclear enriched abundant transcript 1 (NEAT1) was upregulated in the spinal cord dorsal horn to form NEAT1‐miR‐381‐high mobility group box 1 (HMGB1) and NEAT1–miR‐128‐3p–aquaporin 4 (AQP4) axes following CCI and SCI, respectively.\n110\n, \n111\n NEAT1 downregulation inhibited IL‐6, IL‐1β, and TNF‐α to improve neuropathic pain.\nAlthough the function of circRNAs as miRNA sponges is still largely unknown, they have been termed as competing endogenous RNAs that bind to target miRNAs and regulate their function.\n112\n For example, circHIPK3 is a circRNA highly enriched in serum from DNP patients and in the DRG from DNP rats.\n113\n circHIPK3 sponges miR‐124 to promote neuroinflammation, pointing to the involvement of the circHIPK3–miR‐124 axis during DNP. circRNA ciRS‐7 participates in neuropathic pain progression by sponging miR‐135a‐5p to regulate autophagy and inflammation in the spinal cord of CCI rats.\n114\n Besides, cirZNF609–miR‐22‐3p‐enolase 1 (ENO1) and circ_0005075–miR‐151a‐3p‐NOTCH receptor 2 (NOTCH2) regulatory axes upregulate inflammatory factor expression and promote neuropathic pain development in the spinal cord after CCI.\n115\n, \n116\n\n\n\nlncRNAs epigenetically regulate pain‐related molecules expression In addition to acting as a miRNA sponge, lncRNAs can epigenetically regulate the expression of pain‐related molecules. For example, peripheral nerve injury decreases the expression of DRG‐specifically enriched lncRNAs (DS‐lncRNAs) in the injured DRG. Restoring DS‐lncRNAs blocks nerve injury‐induced increases in euchromatic histone lysine N‐methyltransferase 2 (Ehmt2) mRNA and its encoding protein G9a, reverses G9a‐related decreases in opioid receptors and Kcna2 in the injured DRG, and ameliorates nerve injury‐induced pain hypersensitivity.\n117\n In addition, transcriptome screening in the DRG of DNP rats\n118\n has identified diverse dysregulated lncRNAs, including uc.48+,\n119\n BC168687,\n120\n, \n121\n NONRATT021972,\n31\n, \n122\n, \n123\n MRAK009713,\n124\n and Lncenc1.\n125\n The expression of uc.48+, BC168687, NONRATT021972, MRAK009713, and Lncenc1 was prominently higher in the DRG of neuropathic pain rats.\n119\n, \n120\n, \n121\n, \n122\n, \n123\n, \n124\n, \n125\n Blocking the upregulation via intrathecal or intravenous administration of the corresponding small‐interfering RNA (siRNA) may alleviate neuropathic pain by inhibiting the excitatory transmission mediated by purinergic receptors,\n119\n, \n120\n, \n122\n, \n123\n, \n124\n TNF‐α‐related pathways,\n31\n transient receptor potential vanilloid 1 (TRPV1),\n121\n or EZH2.\n125\n\n\nAs demonstrated by a luciferase assay and RNA‐binding protein immunoprecipitation, lncRNA small nucleolar RNA host gene 1 (SNHG1) can induce neuropathic pain in the spinal cord by binding to the promoter region of cyclin‐dependent kinase 4 (CDK4), stimulating its expression.\n126\n SNHG1 knockdown alleviated neuropathic pain development, and SNHG1 overexpression was able to induce neuropathic pain. Similarly, lncRNA PKIA‐AS1 participates in SNL‐induced neuropathic pain by downregulating DNA methyltransferase 1‐catalyzed cyclin‐dependent kinase 6 (DNMT1‐catalyzed) CDK6 promoter methylation and regulating CDK6.\n127\n Cyclin‐dependent kinases (CDKs) transcriptionally enhanced pro‐inflammatory gene expression during the G1 cell phase. Furthermore, CDK6 recruitment to the nuclear chromatin fraction by cytokines is related to NF‐κB, STAT, and activator protein 1 (AP‐1) activation to induce neuroinflammation.\n128\n Other than the DRG, lncRNA Kcna2‐AS‐RNA was also highly expressed in the spinal cord of postherpetic neuralgia rats, and its downregulation alleviated neuropathic pain by reducing phospho‐STAT3 (pSTAT3) translocation from the cytoplasm to the nucleus and then inhibiting spinal astrocytes activation.\n129\n\n\nIn addition to acting as a miRNA sponge, lncRNAs can epigenetically regulate the expression of pain‐related molecules. For example, peripheral nerve injury decreases the expression of DRG‐specifically enriched lncRNAs (DS‐lncRNAs) in the injured DRG. Restoring DS‐lncRNAs blocks nerve injury‐induced increases in euchromatic histone lysine N‐methyltransferase 2 (Ehmt2) mRNA and its encoding protein G9a, reverses G9a‐related decreases in opioid receptors and Kcna2 in the injured DRG, and ameliorates nerve injury‐induced pain hypersensitivity.\n117\n In addition, transcriptome screening in the DRG of DNP rats\n118\n has identified diverse dysregulated lncRNAs, including uc.48+,\n119\n BC168687,\n120\n, \n121\n NONRATT021972,\n31\n, \n122\n, \n123\n MRAK009713,\n124\n and Lncenc1.\n125\n The expression of uc.48+, BC168687, NONRATT021972, MRAK009713, and Lncenc1 was prominently higher in the DRG of neuropathic pain rats.\n119\n, \n120\n, \n121\n, \n122\n, \n123\n, \n124\n, \n125\n Blocking the upregulation via intrathecal or intravenous administration of the corresponding small‐interfering RNA (siRNA) may alleviate neuropathic pain by inhibiting the excitatory transmission mediated by purinergic receptors,\n119\n, \n120\n, \n122\n, \n123\n, \n124\n TNF‐α‐related pathways,\n31\n transient receptor potential vanilloid 1 (TRPV1),\n121\n or EZH2.\n125\n\n\nAs demonstrated by a luciferase assay and RNA‐binding protein immunoprecipitation, lncRNA small nucleolar RNA host gene 1 (SNHG1) can induce neuropathic pain in the spinal cord by binding to the promoter region of cyclin‐dependent kinase 4 (CDK4), stimulating its expression.\n126\n SNHG1 knockdown alleviated neuropathic pain development, and SNHG1 overexpression was able to induce neuropathic pain. Similarly, lncRNA PKIA‐AS1 participates in SNL‐induced neuropathic pain by downregulating DNA methyltransferase 1‐catalyzed cyclin‐dependent kinase 6 (DNMT1‐catalyzed) CDK6 promoter methylation and regulating CDK6.\n127\n Cyclin‐dependent kinases (CDKs) transcriptionally enhanced pro‐inflammatory gene expression during the G1 cell phase. Furthermore, CDK6 recruitment to the nuclear chromatin fraction by cytokines is related to NF‐κB, STAT, and activator protein 1 (AP‐1) activation to induce neuroinflammation.\n128\n Other than the DRG, lncRNA Kcna2‐AS‐RNA was also highly expressed in the spinal cord of postherpetic neuralgia rats, and its downregulation alleviated neuropathic pain by reducing phospho‐STAT3 (pSTAT3) translocation from the cytoplasm to the nucleus and then inhibiting spinal astrocytes activation.\n129\n\n\n\nlncRNAs modulate miRNA processing Specific lncRNAs are essential in neuropathic pain by modulating miRNA processing. For example, the transcribed ultraconserved lncRNA uc.153 was prominently increased in the spinal cord of rats with CCI neuropathic pain. uc.153 knockdown reversed CCI‐induced pain behaviors and spinal neuronal hypersensitivity. Mechanistically, uc.153 negatively modulated Dicer‐mediated pre‐miR‐182‐5p processing and inhibited maturation. Meanwhile, spinal miR‐182‐5p downregulation increased the expression of its target, ephrin type‐b (EphB1), and p‐NR2B (phosphorylated N‐methyl‐D‐aspartate receptor (NMDAR) 2B subunit) expression and facilitated hyperalgesia.\n130\n\n\nCollectively, lncRNAs are crucial in neuropathic pain via diverse mechanisms. Notably, one lncRNA may act with more than one mechanism to regulate target gene expression. For example, SNL upregulated circAnks1a in the cytoplasm and nucleus. circAnks1a enhances the interplay between Y‐box‐binding protein 1 (YBX1) and transportin‐1 to facilitate YBX1 nuclear translocation in the cytoplasm. Meanwhile, in the nucleus, circAnks1a combines with the Vegfb promoter and recruits YBX1 to the Vegfb promoter to enhance its transcription. Additionally, cytoplasmic circAnks1a sponges miR‐324‐3p to regulate vascular endothelial growth factor B (VEGFB) expression. VEGFB binding to its receptor results in the activation of various downstream targets, including p38‐MAPK, PKB/AKT (protein kinase B), extracellular signal‐regulated kinase (ERK)/MAPK, and phosphoinositide 3‐kinase (PI3K).\n131\n Therefore, VEGFB upregulation increases dorsal horn neuron excitability and contributes to pain hypersensitivity after nerve injury.\n132\n\n\nWe also noted that specific lncRNAs were DE following various nerve injuries. For example, lncRNA Malat1 was upregulated after CCI to sponge miR‐206, and Malat1 suppression delayed neuropathic pain progression via miR‐206‐ZEB2 axis‐mediated neuroinflammation inhibition.\n133\n Conversely, in rats with complete brachial plexus avulsion‐induced neuropathic pain, Malat1 decreased in spinal cord neurons, and such downregulation increased neuronal spontaneous electrical activity via calcium flux regulation.\n134\n The reason for such distinctive Malat1 expression following different pain models is still unclear, and further research is required.\nSpecific lncRNAs are essential in neuropathic pain by modulating miRNA processing. For example, the transcribed ultraconserved lncRNA uc.153 was prominently increased in the spinal cord of rats with CCI neuropathic pain. uc.153 knockdown reversed CCI‐induced pain behaviors and spinal neuronal hypersensitivity. Mechanistically, uc.153 negatively modulated Dicer‐mediated pre‐miR‐182‐5p processing and inhibited maturation. Meanwhile, spinal miR‐182‐5p downregulation increased the expression of its target, ephrin type‐b (EphB1), and p‐NR2B (phosphorylated N‐methyl‐D‐aspartate receptor (NMDAR) 2B subunit) expression and facilitated hyperalgesia.\n130\n\n\nCollectively, lncRNAs are crucial in neuropathic pain via diverse mechanisms. Notably, one lncRNA may act with more than one mechanism to regulate target gene expression. For example, SNL upregulated circAnks1a in the cytoplasm and nucleus. circAnks1a enhances the interplay between Y‐box‐binding protein 1 (YBX1) and transportin‐1 to facilitate YBX1 nuclear translocation in the cytoplasm. Meanwhile, in the nucleus, circAnks1a combines with the Vegfb promoter and recruits YBX1 to the Vegfb promoter to enhance its transcription. Additionally, cytoplasmic circAnks1a sponges miR‐324‐3p to regulate vascular endothelial growth factor B (VEGFB) expression. VEGFB binding to its receptor results in the activation of various downstream targets, including p38‐MAPK, PKB/AKT (protein kinase B), extracellular signal‐regulated kinase (ERK)/MAPK, and phosphoinositide 3‐kinase (PI3K).\n131\n Therefore, VEGFB upregulation increases dorsal horn neuron excitability and contributes to pain hypersensitivity after nerve injury.\n132\n\n\nWe also noted that specific lncRNAs were DE following various nerve injuries. For example, lncRNA Malat1 was upregulated after CCI to sponge miR‐206, and Malat1 suppression delayed neuropathic pain progression via miR‐206‐ZEB2 axis‐mediated neuroinflammation inhibition.\n133\n Conversely, in rats with complete brachial plexus avulsion‐induced neuropathic pain, Malat1 decreased in spinal cord neurons, and such downregulation increased neuronal spontaneous electrical activity via calcium flux regulation.\n134\n The reason for such distinctive Malat1 expression following different pain models is still unclear, and further research is required.","After peripheral nerve injury, Egr2‐AS‐RNA is upregulated in Schwann cells.\n103\n On the early growth response 2 (Egr2) promoter, Egr2‐AS‐RNA recruits an epigenetic silencing complex to downregulate Egr2, essential for peripheral myelination. Ectopic Egr2‐AS‐RNA expression in DRG cultures downregulates Egr2 mRNA and induces demyelination. In vivo, Egr2‐AS‐RNA inhibition reverts Egr2‐related gene expression and delays demyelination.\nVoltage‐gated potassium channel (Kv) Kcna2‐AS‐RNA is an endogenous, highly conserved, and widely explored lncRNA in neuropathic pain.\n104\n It is a natural antisense transcript (NAT), distributed in the cytoplasm and targets Kcna2 mRNA, which encodes the pain regulation‐related membrane Kv1.2 subunit. Kcna2‐AS‐RNA was time‐dependently upregulated in the inflicted rat DRG after nerve injury. It decreased the total Kv current, upregulated DRG neurons excitability, and produced neuropathic pain symptoms.\nThe complementary strand of DNA opposite the Scn9a gene encodes Scn9a NAT, another antisense lncRNA expressed in the DRG.\n105\n Scn9a NAT is suggested to be a negative regulator of Scn9a mRNA. Scn9a NAT overexpression inhibited Scn9a mRNA, its encoded protein Nav1.7, and Nav1.7 currents in DRG neurons. However, Scn9a NAT and Scn9a mRNA levels did not significantly change in the injured DRG until 2 weeks after nerve injury. More work is required to determine whether NAT can confer analgesia and reduce pain in the neuropathic pain state.","lncRNAs may act as miRNA sponges to form the lncRNA‐miRNA‐mRNA axis and regulate target gene expression. For example, the lncRNA X‐inactive specific transcript (XIST) is upregulated in the dorsal horn of the spinal cord after CCI. By sponging miR‐137, ‐150, ‐154‐5p, and ‐544, XIST can inhibit the expression of corresponding targets, including TNF‐α‐induced protein 1 (TNFAIP1), ZEB1, TLR5, and STAT3.\n106\n, \n107\n, \n108\n, \n109\n TNFAIP1 activates the NF‐κB signaling pathway, while ZEB1, TLR5, and STAT3 are crucial in the neuroinflammatory response. XIST inhibition markedly ameliorates neuropathic pain development. In addition, lncRNA nuclear enriched abundant transcript 1 (NEAT1) was upregulated in the spinal cord dorsal horn to form NEAT1‐miR‐381‐high mobility group box 1 (HMGB1) and NEAT1–miR‐128‐3p–aquaporin 4 (AQP4) axes following CCI and SCI, respectively.\n110\n, \n111\n NEAT1 downregulation inhibited IL‐6, IL‐1β, and TNF‐α to improve neuropathic pain.\nAlthough the function of circRNAs as miRNA sponges is still largely unknown, they have been termed as competing endogenous RNAs that bind to target miRNAs and regulate their function.\n112\n For example, circHIPK3 is a circRNA highly enriched in serum from DNP patients and in the DRG from DNP rats.\n113\n circHIPK3 sponges miR‐124 to promote neuroinflammation, pointing to the involvement of the circHIPK3–miR‐124 axis during DNP. circRNA ciRS‐7 participates in neuropathic pain progression by sponging miR‐135a‐5p to regulate autophagy and inflammation in the spinal cord of CCI rats.\n114\n Besides, cirZNF609–miR‐22‐3p‐enolase 1 (ENO1) and circ_0005075–miR‐151a‐3p‐NOTCH receptor 2 (NOTCH2) regulatory axes upregulate inflammatory factor expression and promote neuropathic pain development in the spinal cord after CCI.\n115\n, \n116\n\n","In addition to acting as a miRNA sponge, lncRNAs can epigenetically regulate the expression of pain‐related molecules. For example, peripheral nerve injury decreases the expression of DRG‐specifically enriched lncRNAs (DS‐lncRNAs) in the injured DRG. Restoring DS‐lncRNAs blocks nerve injury‐induced increases in euchromatic histone lysine N‐methyltransferase 2 (Ehmt2) mRNA and its encoding protein G9a, reverses G9a‐related decreases in opioid receptors and Kcna2 in the injured DRG, and ameliorates nerve injury‐induced pain hypersensitivity.\n117\n In addition, transcriptome screening in the DRG of DNP rats\n118\n has identified diverse dysregulated lncRNAs, including uc.48+,\n119\n BC168687,\n120\n, \n121\n NONRATT021972,\n31\n, \n122\n, \n123\n MRAK009713,\n124\n and Lncenc1.\n125\n The expression of uc.48+, BC168687, NONRATT021972, MRAK009713, and Lncenc1 was prominently higher in the DRG of neuropathic pain rats.\n119\n, \n120\n, \n121\n, \n122\n, \n123\n, \n124\n, \n125\n Blocking the upregulation via intrathecal or intravenous administration of the corresponding small‐interfering RNA (siRNA) may alleviate neuropathic pain by inhibiting the excitatory transmission mediated by purinergic receptors,\n119\n, \n120\n, \n122\n, \n123\n, \n124\n TNF‐α‐related pathways,\n31\n transient receptor potential vanilloid 1 (TRPV1),\n121\n or EZH2.\n125\n\n\nAs demonstrated by a luciferase assay and RNA‐binding protein immunoprecipitation, lncRNA small nucleolar RNA host gene 1 (SNHG1) can induce neuropathic pain in the spinal cord by binding to the promoter region of cyclin‐dependent kinase 4 (CDK4), stimulating its expression.\n126\n SNHG1 knockdown alleviated neuropathic pain development, and SNHG1 overexpression was able to induce neuropathic pain. Similarly, lncRNA PKIA‐AS1 participates in SNL‐induced neuropathic pain by downregulating DNA methyltransferase 1‐catalyzed cyclin‐dependent kinase 6 (DNMT1‐catalyzed) CDK6 promoter methylation and regulating CDK6.\n127\n Cyclin‐dependent kinases (CDKs) transcriptionally enhanced pro‐inflammatory gene expression during the G1 cell phase. Furthermore, CDK6 recruitment to the nuclear chromatin fraction by cytokines is related to NF‐κB, STAT, and activator protein 1 (AP‐1) activation to induce neuroinflammation.\n128\n Other than the DRG, lncRNA Kcna2‐AS‐RNA was also highly expressed in the spinal cord of postherpetic neuralgia rats, and its downregulation alleviated neuropathic pain by reducing phospho‐STAT3 (pSTAT3) translocation from the cytoplasm to the nucleus and then inhibiting spinal astrocytes activation.\n129\n\n","Specific lncRNAs are essential in neuropathic pain by modulating miRNA processing. For example, the transcribed ultraconserved lncRNA uc.153 was prominently increased in the spinal cord of rats with CCI neuropathic pain. uc.153 knockdown reversed CCI‐induced pain behaviors and spinal neuronal hypersensitivity. Mechanistically, uc.153 negatively modulated Dicer‐mediated pre‐miR‐182‐5p processing and inhibited maturation. Meanwhile, spinal miR‐182‐5p downregulation increased the expression of its target, ephrin type‐b (EphB1), and p‐NR2B (phosphorylated N‐methyl‐D‐aspartate receptor (NMDAR) 2B subunit) expression and facilitated hyperalgesia.\n130\n\n\nCollectively, lncRNAs are crucial in neuropathic pain via diverse mechanisms. Notably, one lncRNA may act with more than one mechanism to regulate target gene expression. For example, SNL upregulated circAnks1a in the cytoplasm and nucleus. circAnks1a enhances the interplay between Y‐box‐binding protein 1 (YBX1) and transportin‐1 to facilitate YBX1 nuclear translocation in the cytoplasm. Meanwhile, in the nucleus, circAnks1a combines with the Vegfb promoter and recruits YBX1 to the Vegfb promoter to enhance its transcription. Additionally, cytoplasmic circAnks1a sponges miR‐324‐3p to regulate vascular endothelial growth factor B (VEGFB) expression. VEGFB binding to its receptor results in the activation of various downstream targets, including p38‐MAPK, PKB/AKT (protein kinase B), extracellular signal‐regulated kinase (ERK)/MAPK, and phosphoinositide 3‐kinase (PI3K).\n131\n Therefore, VEGFB upregulation increases dorsal horn neuron excitability and contributes to pain hypersensitivity after nerve injury.\n132\n\n\nWe also noted that specific lncRNAs were DE following various nerve injuries. For example, lncRNA Malat1 was upregulated after CCI to sponge miR‐206, and Malat1 suppression delayed neuropathic pain progression via miR‐206‐ZEB2 axis‐mediated neuroinflammation inhibition.\n133\n Conversely, in rats with complete brachial plexus avulsion‐induced neuropathic pain, Malat1 decreased in spinal cord neurons, and such downregulation increased neuronal spontaneous electrical activity via calcium flux regulation.\n134\n The reason for such distinctive Malat1 expression following different pain models is still unclear, and further research is required.","There are two main strategies for modulating miRNA function during pain treatment: upregulation or downregulation of specific miRNAs. miRNA mimics or virus‐based constructs were used to upregulate miRNA expression. miRNA inhibitors, miRNA sponges, or inhibition of a particular miRNA‐mRNA interaction has been applied to downregulate specific miRNAs. The design of lncRNA‐based therapeutics includes diverse approaches, such as post‐transcriptional inhibition of lncRNAs by antisense oligonucleotides or siRNA and steric blockades of lncRNA–protein interactions by small molecules and morpholino.\n135\n\n\nHowever, there is much work to be done before miRNA‐ and lncRNA‐based analgesics can be clinically used for various reasons, including their conservation among species, proper delivery, stability, off‐target effects, and potential activation of the immune system.\nFirst, most available findings regarding the efficacy of miRNAs or lncRNAs during neuropathic pain are based on animal but not human studies. Whether such results can be extrapolated to humans and their translational potential is still unknown because of conservation among species.\nSecond, the blood–brain barrier (BBB) is a practical challenge for delivering RNA‐based therapeutics into the central nervous system (CNS) via intravenous injection. Viral vectors, polypeptides, aptamers, and particular chemical modifications have been developed. Supplementing cholesterol molecules to the sense strand of a miRNA mimic or inhibitor has proven to be an efficient strategy. Cholesterol‐conjugated siRNAs showed better silencing potency than unconjugated siRNAs and presented a high efficacy for delivery to oligodendrocytes in the CNS.\n136\n Another proven method is the immunoliposome, a combination of liposomes, receptor‐targeted monoclonal antibodies, and the target molecules.\n137\n An immunoliposome nanocomplex has been reported to deliver therapeutic nucleic acids across the BBB into the deep brain by transferrin receptors.\n138\n In addition, intrathecal injections are a feasible approach in animal studies for neuropathic pain treatment. As reported, miR‐146a attenuated neuropathic pain partially by inhibiting TNF receptor‐associated factor 6 (TRAF6) and its downstream phospho‐JNK/C–C motif chemokine ligand 2 (pJNK/CCL2) signaling in the spinal cord.\n139\n Intrathecal injection of miR‐146a‐5p encapsulated nanoparticles provided an analgesic effect via NF‐κB and p38‐MAPK inhibition in spinal microglia.\n140\n In recent years, poly (D, L‐lactic‐co‐glycolic acid) (PLGA)‐nanoparticles have been applied to deliver siRNAs and plasmids into the spinal cord to treat neuropathic pain in rats.\n141\n PLGA copolymer is a promising US Food and Drug Administration (FDA)‐approved gene transmission material because of its biodegradability and biocompatibility in humans.\n142\n Intrathecal treatments with C‐X3‐C motif chemokine receptor 1 (CX3CR1), p38, p66shc siRNA encapsulated PLGA nanoparticles, or forkhead box P3 (Foxp3) plasmid‐encapsulated PLGA nanoparticles inhibit microglial activation and hyperalgesia in SNL rats.\n141\n, \n143\n, \n144\n, \n145\n\n\nExosomes are another promising delivery carrier for treating neuropathic pain. Exosomes are natural membranous microvesicles that carry RNAs, with the advantage of being efficient, cell‐free, and nonimmunogenicity. Intravenous injecting neuron‐targeted exosomes delivered the carried siRNAs to neurons, microglia, and oligodendrocytes to knock down specific gene expression in mice brains.\n146\n This approach enabled cell‐specific delivery of the siRNA‐cargo across the BBB. Mesenchymal stem cells (MSCs) are pluripotent stem cells with immunomodulatory, anti‐inflammatory, and nutritional properties. The treatment efficacy of exosomes derived from MSCs has been proven in neuropathic pain.\n147\n Intrathecal, local, or subcutaneous application of exosomes obtained from human umbilical cord MSCs could mitigate nerve injury‐induced hyperalgesia.\n148\n, \n149\n, \n150\n Furthermore, immunofluorescence results showed that most intrathecally injected exosomes could be found in injured peripheral axons, the DRG, and the spinal dorsal horn, potentiating a homing ability of exosomes.\n148\n\n\nThird, although miRNAs are relatively stable in vivo, they have long‐lasting efficacy and a high resistance to nucleolytic degradation compared to mRNAs. Particular chemical modifications are still required to prolong its half‐life or increase its stability, for example, by generating locked‐nucleic acids (LNAs). LNA modifications increase the RNA affinity of antisense oligonucleotides, presenting an excellent miRNA inhibitory activity at a low dosage.\n151\n Additionally, the LNA technique was used to synthesize highly stable aptamers.\n152\n\n\nFourth, the off‐target effect is another consideration. One miRNA may regulate multiple genes, and the off‐target potential of one miRNA may produce undesirable side effects.\n153\n, \n154\n\n\nFinally, activation of the immune system is a potential adverse event. Specific CpG motifs in oligonucleotides could trigger nonspecific immunological activity.\n155\n Therefore, RNA‐based therapeutics might also produce specific immune reactions after producing antibodies against the oligonucleotides.","Recent human and animal studies have identified accumulating dysregulated miRNAs and lncRNAs in the serum or along pain processing pathways following peripheral nerve injury or noxious stimulation. Experimental studies have validated their essential role in neuropathic pain. miRNAs contribute to neuropathic pain development via neuroinflammation, autophagy, abnormal ion channel expression, regulating pain‐related mediators, protein kinases, structural proteins, neurotransmission excitatory–‐inhibitory imbalances, and exosome miRNA‐mediated neuron–glia communication. Meanwhile, lncRNAs and circRNAs are crucial in neuropathic pain development by acting as AS RNA and miRNA sponges, epigenetically regulating pain‐related molecules expression, or modulating miRNA processing. However, more work is required before miRNA‐ and lncRNA‐based analgesics can be clinically used for various reasons, including their conservation among species, proper delivery, stability, off‐target effects, and potential activation of the immune system.","Ming Jiang: Investigation, Methodology. Yelong Wang: Manuscript preparation. Jing Wang: Figures and tables preparation. Shanwu Feng: Supervision, Funding acquisition. Xian Wang: Conceptualization, Funding acquisition.","The authors declare no competing interest in this review."],"string":"[\n \"According to the International Association for the Study of Pain, neuropathic pain is the most severe chronic pain condition triggered by a lesion or disease of the somatosensory system. It is characteristic of hyperalgesia, allodynia, or spontaneous pain.\\n1\\n Neuropathic pain can have peripheral and central origins, with the former including neuropathic pain after peripheral nerve injury, trigeminal neuralgia, postherpetic neuralgia, painful radiculopathy, and painful polyneuropathy. In contrast, central neuropathic pain includes neuropathic pain after spinal cord or brain injury, multiple sclerosis, and chronic central post‐stroke pain. Approximately 7%–10% of the general population will experience neuropathic pain, with the majority not having satisfactory pain relief with current therapies, leading to great suffering for individuals and enormous economic and social burdens.\\n2\\n\\n\\nThe exact molecular mechanisms underlying neuropathic pain remain unclear, and elucidating them is crucial for developing mechanism‐based treatment strategies. One proposed mechanism involves altered gene or protein expression along the pain processing pathways. Therefore, understanding how genes or proteins are dysregulated may help us to find a way to normalize these abnormalities and treat neuropathic pain.\\nIn recent years, accumulating evidence has suggested the essential role of non‐coding RNAs (ncRNAs) in various physiological and pathological procedures, such as embryonic development, inflammation, tumors, and respiratory and cardiovascular diseases.\\n3\\n ncRNAs have no protein‐coding potential, but they can govern gene or protein expression with diverse mechanisms. ncRNAs are extensively distributed in the peripheral and central nervous systems, including pain‐related structures.\\n4\\n Broad abnormal ncRNAs expression is observed following peripheral stimulation. These abnormalities are related to hyperalgesia during chronic pain development. The available data indicate that ncRNAs may be essential for hyperalgesia. In this review, we focus on microRNA (miRNA), long non‐coding RNA (lncRNA), and circular non‐coding RNA (circRNA) expression changes in neuropathic pain. Other types of ncRNAs are seldom reported in neuropathic pain and, thus, are not discussed herein. Notably, we will pay attention to their etiological role in the development of neuropathic pain and the current challenges and considerations for miRNA‐, lncRNA‐, and circRNA‐based therapeutics for neuropathic pain.\",\n \"Typically, miRNA production involves three steps: cropping, exporting, and dicing. First, the miRNA gene is transcribed in the nucleus, mainly by RNA polymerase II.\\n5\\n The resulting primary miRNA transcript (pri‐miRNA) is several kilobases (kb) in length, with a specific stem–loop structure that harbors mature miRNAs in the stem. The mature miRNA is ‘cropped’ by Drosha and its interactor DGCR8 (DiGeorge syndrome critical region 8), which cleaves pri‐miRNA at the stem to produce pre‐miRNA,\\n6\\n 60–70 nucleotides (nt) in length with a hairpin structure. Then, exportin‐5 recognizes and exports pre‐miRNA from the nucleus to the cytoplasm.\\n7\\n In the cytoplasm, ribonuclease III (RNAse III), termed Dicer, further cleaves the pre‐miRNA to release double‐stranded miRNA with a length of ~22 nt.\\n8\\n This miRNA is unwound by an unknown helicase or cleaved by Argonaute (Ago) to form the RNA‐induced silencing complex.\\n9\\n One strand in the RNA duplex remains with Ago as a mature miRNA, and the other is degraded. The seed sequence of the miRNA incompletely or entirely combines with the target mRNA sequence, resulting in target mRNAs degradation or transcriptional regulation.\\n10\\n\\n\\nUnlike miRNAs, lncRNAs are mRNAs‐like transcripts ranging in length from 200 nt to 100 kb that lack prominent open reading frames.\\n11\\n The lncRNA cellular mechanism is highly related to their intracellular localization. lncRNAs control chromatin functions, transcription, and RNA processing in the nucleus and affect mRNA stability, translation, and cellular signaling in the cytoplasm.\\n12\\n Compared to lncRNAs, circRNAs are more stable because a single circRNA molecular ends can be covalently linked compared to linear RNA. circRNAs are evolutionarily conserved molecules that are essential in the post‐transcriptional modification of gene expression by acting as miRNAs sponges or interacting with transcription or translational machinery. Numerous lncRNAs and circRNAs are distributed within pain‐related regions and dysregulated after peripheral noxious stimulation. Moreover, functional studies have indicated that miRNAs, lncRNAs, and circRNAs participate in neuropathic pain development by regulating diverse pain‐related genes along the pain processing pathways.\",\n \"\\nmiRNAs expression changes Microarray and deep‐sequencing analyses revealed that nerve injury or noxious stimuli could induce broad changes in miRNA expression in serum or along the pain processing pathways, including the perineal nerve, dorsal root ganglion (DRG), spinal cord, and supraspinal regions. For normal pain signal transmission, nerve injury or noxious stimuli are detected by nociceptors in the DRG or trigeminal ganglion (TG) and then transmitted to upstream neurons in the spinal dorsal horn. Subsequently, nociceptive stimuli are integrated, processed, and further transmitted ascending to specific supraspinal brain regions.\\nHuman studies have identified as many as 1134 differentially expressed (DE) genes in the serum of individuals with or without neuropathic pain after spinal cord injury (SCI).\\n13\\n miR‐204‐5p, ‐519d‐3p, ‐20b‐5p, and ‐6838‐5p might act as promising biomarkers and intervention targets for preventing and therapizing neuropathic pain after SCI. In trigeminal neuralgia individuals, serum miR‐132‐3p, ‐146b‐5p, ‐155‐5p, and ‐384 levels were prominently increased compared with healthy controls.\\n14\\n Patients with painful peripheral neuropathy had higher miR‐21 level in the serum and sural nerve compared with healthy controls. Meanwhile, miR‐155 was reduced in the serum and inflicted lower leg skin.\\n15\\n Animal and human studies showed that miR‐30c‐5p was upregulated in the serum and cerebro spinal fluid (CSF) of sciatic nerve injury rats and neuropathic pain patients with chronic peripheral ischemia. The high expression of miR‐30c‐5p, together with other clinical parameters, might be used to predict neuropathic pain development in patients with chronic peripheral ischemia.\\n16\\n Additionally, an animal study showed that plasma‐derived DE extracellular vesicle (EV) miRNAs regulated processes that are essential for neuropathic pain development. Most DE EV miRNAs for inflammation suppression were downregulated, potentially acting as biomarkers and targets in neuropathic pain treatment.\\n17\\n\\n\\nIn addition, numerous DE miRNAs have been identified in the DRG. One week after spared nerve injury (SNI), 33 and 39 miRNAs in the DRG were upregulated and downregulated, respectively, with most DE miRNAs related to axon guidance, focal adhesion, Ras and Wnt signaling pathways.\\n18\\n Furthermore, nerve injury‐induced miRNAs expression was dynamic and time‐dependent,\\n19\\n, \\n20\\n implicating multiple regulatory mechanisms in neuropathic pain initiation and development. Nerve injury redistributes miRNAs from a uniform style within the DRG soma of non‐allodynic animals to preferential localization to peripheral neurons in allodynic animals.\\n21\\n Furthermore, either sciatic nerve ligation (SNL), DRG transaction (DRT), or ventral root transection (VRT) could upregulate miR‐21 and miR‐31 while downregulating miR‐668 and miR‐672 in the injured DRG,\\n22\\n implying that these miRNAs could be therapeutic targets for treating diverse types of neuropathic pain.\\nThe spinal cord dorsal horn relays and modulates pain signals from the peripheral nociceptors to the supraspinal regions. In the spinal cord, numerous miRNA expression changes have been observed in chronic constriction injury (CCI) and diabetic neuropathic pain (DNP) rodents.\\n23\\n, \\n24\\n, \\n25\\n, \\n26\\n Previous research has suggested some miRNAs may be related to neuropathic pain development, for example, miR‐500, ‐221, and ‐21,\\n25\\n thus, acting as potential targets in its treatment.\\n27\\n\\n\\nCytoscape software constructed the miRNA–target gene regulatory network in the supraspinal region, including the nucleus accumbens (NAc), medial prefrontal cortex, and periaqueductal gray, between SNI and sham rats. Finally, four essential DE genes, includingCXCR2, IL12B, TNFSF8, and GRK1, and five miRNAs, including miR‐208a‐5p, −7688‐3p, −344f‐3p, −135b‐3p, and ‐135a‐2‐3p, were identified, indicating their essential roles in neuropathic pain pathogenesis.\\n28\\n Furthermore, in the prelimbic cortex of SNI rats, the DE miRNA–mRNA network pointed to molecules associated with inflammation.\\n29\\n DE miRNAs were also observed in the bilateral hippocampus of CCI rats. However, no significant difference was observed bilaterally in the hippocampus.\\n30\\n (Figure 1).\\nmicroRNAs (miRNAs) expression change following peripheral nerve injury or noxious stimuli SCI, spinal cord injury; CSF, cerebrol spinal fluid; DE, differential expressed; EV, extravascular vesicle; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury; DRT, DRG transection; VRT, ventral root transection\\nMicroarray and deep‐sequencing analyses revealed that nerve injury or noxious stimuli could induce broad changes in miRNA expression in serum or along the pain processing pathways, including the perineal nerve, dorsal root ganglion (DRG), spinal cord, and supraspinal regions. For normal pain signal transmission, nerve injury or noxious stimuli are detected by nociceptors in the DRG or trigeminal ganglion (TG) and then transmitted to upstream neurons in the spinal dorsal horn. Subsequently, nociceptive stimuli are integrated, processed, and further transmitted ascending to specific supraspinal brain regions.\\nHuman studies have identified as many as 1134 differentially expressed (DE) genes in the serum of individuals with or without neuropathic pain after spinal cord injury (SCI).\\n13\\n miR‐204‐5p, ‐519d‐3p, ‐20b‐5p, and ‐6838‐5p might act as promising biomarkers and intervention targets for preventing and therapizing neuropathic pain after SCI. In trigeminal neuralgia individuals, serum miR‐132‐3p, ‐146b‐5p, ‐155‐5p, and ‐384 levels were prominently increased compared with healthy controls.\\n14\\n Patients with painful peripheral neuropathy had higher miR‐21 level in the serum and sural nerve compared with healthy controls. Meanwhile, miR‐155 was reduced in the serum and inflicted lower leg skin.\\n15\\n Animal and human studies showed that miR‐30c‐5p was upregulated in the serum and cerebro spinal fluid (CSF) of sciatic nerve injury rats and neuropathic pain patients with chronic peripheral ischemia. The high expression of miR‐30c‐5p, together with other clinical parameters, might be used to predict neuropathic pain development in patients with chronic peripheral ischemia.\\n16\\n Additionally, an animal study showed that plasma‐derived DE extracellular vesicle (EV) miRNAs regulated processes that are essential for neuropathic pain development. Most DE EV miRNAs for inflammation suppression were downregulated, potentially acting as biomarkers and targets in neuropathic pain treatment.\\n17\\n\\n\\nIn addition, numerous DE miRNAs have been identified in the DRG. One week after spared nerve injury (SNI), 33 and 39 miRNAs in the DRG were upregulated and downregulated, respectively, with most DE miRNAs related to axon guidance, focal adhesion, Ras and Wnt signaling pathways.\\n18\\n Furthermore, nerve injury‐induced miRNAs expression was dynamic and time‐dependent,\\n19\\n, \\n20\\n implicating multiple regulatory mechanisms in neuropathic pain initiation and development. Nerve injury redistributes miRNAs from a uniform style within the DRG soma of non‐allodynic animals to preferential localization to peripheral neurons in allodynic animals.\\n21\\n Furthermore, either sciatic nerve ligation (SNL), DRG transaction (DRT), or ventral root transection (VRT) could upregulate miR‐21 and miR‐31 while downregulating miR‐668 and miR‐672 in the injured DRG,\\n22\\n implying that these miRNAs could be therapeutic targets for treating diverse types of neuropathic pain.\\nThe spinal cord dorsal horn relays and modulates pain signals from the peripheral nociceptors to the supraspinal regions. In the spinal cord, numerous miRNA expression changes have been observed in chronic constriction injury (CCI) and diabetic neuropathic pain (DNP) rodents.\\n23\\n, \\n24\\n, \\n25\\n, \\n26\\n Previous research has suggested some miRNAs may be related to neuropathic pain development, for example, miR‐500, ‐221, and ‐21,\\n25\\n thus, acting as potential targets in its treatment.\\n27\\n\\n\\nCytoscape software constructed the miRNA–target gene regulatory network in the supraspinal region, including the nucleus accumbens (NAc), medial prefrontal cortex, and periaqueductal gray, between SNI and sham rats. Finally, four essential DE genes, includingCXCR2, IL12B, TNFSF8, and GRK1, and five miRNAs, including miR‐208a‐5p, −7688‐3p, −344f‐3p, −135b‐3p, and ‐135a‐2‐3p, were identified, indicating their essential roles in neuropathic pain pathogenesis.\\n28\\n Furthermore, in the prelimbic cortex of SNI rats, the DE miRNA–mRNA network pointed to molecules associated with inflammation.\\n29\\n DE miRNAs were also observed in the bilateral hippocampus of CCI rats. However, no significant difference was observed bilaterally in the hippocampus.\\n30\\n (Figure 1).\\nmicroRNAs (miRNAs) expression change following peripheral nerve injury or noxious stimuli SCI, spinal cord injury; CSF, cerebrol spinal fluid; DE, differential expressed; EV, extravascular vesicle; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury; DRT, DRG transection; VRT, ventral root transection\\n\\nlncRNAs and circRNAs expression changes Patients with type 2 DNP had a prominent higher expression of serum lncRNA NONRATT021972 and more severe neuropathic pain symptoms.\\n31\\n LINC01119 and LINC02447 in the peripheral blood of SCI patients were identified in pain pathways that were important for neuropathic pain development.\\n32\\n\\n\\nMicroarray analysis revealed that a nerve injury could induce time‐dependent lncRNA expression changes in the sciatic nerve.\\n33\\n lncRNA H19 was persistently upregulated in Schwann cells along the peripheral nerve, proximal and distal to the injured site.\\n34\\n In the DRG, the transcriptomic analysis identified 86 known and 26 novel lncRNAs genes to be DE after spared sciatic nerve injury.\\n35\\n Of these, rno‐Cntnap2 and AC111653.1 were essential in peripheral nerve regeneration and were involved in neuropathic pain.\\nNext‐generation RNA sequencing showed 134 lncRNAs and 188 circRNAs were prominently changed 14 days after SNI in the spinal cord.\\n36\\n In addition, microarray analysis identified 1481 and 1096 DE lncRNAs and mRNAs, respectively, in the spinal cord dorsal horn of DNP rats. Of these, 289 neighboring and 57 overlapping lncRNA‐mRNA pairs, including ENSMUST00000150952‐Mbp and AK081017‐Usp15, have been suggested to participate in neuropathic pain development.\\n37\\n\\n\\ncircRNAs have characteristic circularized structures following the backsplicing of exons from antisense RNAs (AS RNAs) or mRNAs and, thus, are highly stable. Most circRNAs are highly conserved among species and lack translation potential, despite cap‐independent translation. A recent study showed that 363 and 106 circRNAs were significantly dysregulated in the ipsilateral dorsal horn after nerve injury.\\n38\\n (Figure 2).\\nLong non‐coding RNA (lncRNAs) and circular non‐coding RNAs (circRNAs) expression change following peripheral nerve injury or noxious stimuli DNP, diabetic neuropathic pain; SCI, spinal cord injury; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury\\nPatients with type 2 DNP had a prominent higher expression of serum lncRNA NONRATT021972 and more severe neuropathic pain symptoms.\\n31\\n LINC01119 and LINC02447 in the peripheral blood of SCI patients were identified in pain pathways that were important for neuropathic pain development.\\n32\\n\\n\\nMicroarray analysis revealed that a nerve injury could induce time‐dependent lncRNA expression changes in the sciatic nerve.\\n33\\n lncRNA H19 was persistently upregulated in Schwann cells along the peripheral nerve, proximal and distal to the injured site.\\n34\\n In the DRG, the transcriptomic analysis identified 86 known and 26 novel lncRNAs genes to be DE after spared sciatic nerve injury.\\n35\\n Of these, rno‐Cntnap2 and AC111653.1 were essential in peripheral nerve regeneration and were involved in neuropathic pain.\\nNext‐generation RNA sequencing showed 134 lncRNAs and 188 circRNAs were prominently changed 14 days after SNI in the spinal cord.\\n36\\n In addition, microarray analysis identified 1481 and 1096 DE lncRNAs and mRNAs, respectively, in the spinal cord dorsal horn of DNP rats. Of these, 289 neighboring and 57 overlapping lncRNA‐mRNA pairs, including ENSMUST00000150952‐Mbp and AK081017‐Usp15, have been suggested to participate in neuropathic pain development.\\n37\\n\\n\\ncircRNAs have characteristic circularized structures following the backsplicing of exons from antisense RNAs (AS RNAs) or mRNAs and, thus, are highly stable. Most circRNAs are highly conserved among species and lack translation potential, despite cap‐independent translation. A recent study showed that 363 and 106 circRNAs were significantly dysregulated in the ipsilateral dorsal horn after nerve injury.\\n38\\n (Figure 2).\\nLong non‐coding RNA (lncRNAs) and circular non‐coding RNAs (circRNAs) expression change following peripheral nerve injury or noxious stimuli DNP, diabetic neuropathic pain; SCI, spinal cord injury; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury\",\n \"Microarray and deep‐sequencing analyses revealed that nerve injury or noxious stimuli could induce broad changes in miRNA expression in serum or along the pain processing pathways, including the perineal nerve, dorsal root ganglion (DRG), spinal cord, and supraspinal regions. For normal pain signal transmission, nerve injury or noxious stimuli are detected by nociceptors in the DRG or trigeminal ganglion (TG) and then transmitted to upstream neurons in the spinal dorsal horn. Subsequently, nociceptive stimuli are integrated, processed, and further transmitted ascending to specific supraspinal brain regions.\\nHuman studies have identified as many as 1134 differentially expressed (DE) genes in the serum of individuals with or without neuropathic pain after spinal cord injury (SCI).\\n13\\n miR‐204‐5p, ‐519d‐3p, ‐20b‐5p, and ‐6838‐5p might act as promising biomarkers and intervention targets for preventing and therapizing neuropathic pain after SCI. In trigeminal neuralgia individuals, serum miR‐132‐3p, ‐146b‐5p, ‐155‐5p, and ‐384 levels were prominently increased compared with healthy controls.\\n14\\n Patients with painful peripheral neuropathy had higher miR‐21 level in the serum and sural nerve compared with healthy controls. Meanwhile, miR‐155 was reduced in the serum and inflicted lower leg skin.\\n15\\n Animal and human studies showed that miR‐30c‐5p was upregulated in the serum and cerebro spinal fluid (CSF) of sciatic nerve injury rats and neuropathic pain patients with chronic peripheral ischemia. The high expression of miR‐30c‐5p, together with other clinical parameters, might be used to predict neuropathic pain development in patients with chronic peripheral ischemia.\\n16\\n Additionally, an animal study showed that plasma‐derived DE extracellular vesicle (EV) miRNAs regulated processes that are essential for neuropathic pain development. Most DE EV miRNAs for inflammation suppression were downregulated, potentially acting as biomarkers and targets in neuropathic pain treatment.\\n17\\n\\n\\nIn addition, numerous DE miRNAs have been identified in the DRG. One week after spared nerve injury (SNI), 33 and 39 miRNAs in the DRG were upregulated and downregulated, respectively, with most DE miRNAs related to axon guidance, focal adhesion, Ras and Wnt signaling pathways.\\n18\\n Furthermore, nerve injury‐induced miRNAs expression was dynamic and time‐dependent,\\n19\\n, \\n20\\n implicating multiple regulatory mechanisms in neuropathic pain initiation and development. Nerve injury redistributes miRNAs from a uniform style within the DRG soma of non‐allodynic animals to preferential localization to peripheral neurons in allodynic animals.\\n21\\n Furthermore, either sciatic nerve ligation (SNL), DRG transaction (DRT), or ventral root transection (VRT) could upregulate miR‐21 and miR‐31 while downregulating miR‐668 and miR‐672 in the injured DRG,\\n22\\n implying that these miRNAs could be therapeutic targets for treating diverse types of neuropathic pain.\\nThe spinal cord dorsal horn relays and modulates pain signals from the peripheral nociceptors to the supraspinal regions. In the spinal cord, numerous miRNA expression changes have been observed in chronic constriction injury (CCI) and diabetic neuropathic pain (DNP) rodents.\\n23\\n, \\n24\\n, \\n25\\n, \\n26\\n Previous research has suggested some miRNAs may be related to neuropathic pain development, for example, miR‐500, ‐221, and ‐21,\\n25\\n thus, acting as potential targets in its treatment.\\n27\\n\\n\\nCytoscape software constructed the miRNA–target gene regulatory network in the supraspinal region, including the nucleus accumbens (NAc), medial prefrontal cortex, and periaqueductal gray, between SNI and sham rats. Finally, four essential DE genes, includingCXCR2, IL12B, TNFSF8, and GRK1, and five miRNAs, including miR‐208a‐5p, −7688‐3p, −344f‐3p, −135b‐3p, and ‐135a‐2‐3p, were identified, indicating their essential roles in neuropathic pain pathogenesis.\\n28\\n Furthermore, in the prelimbic cortex of SNI rats, the DE miRNA–mRNA network pointed to molecules associated with inflammation.\\n29\\n DE miRNAs were also observed in the bilateral hippocampus of CCI rats. However, no significant difference was observed bilaterally in the hippocampus.\\n30\\n (Figure 1).\\nmicroRNAs (miRNAs) expression change following peripheral nerve injury or noxious stimuli SCI, spinal cord injury; CSF, cerebrol spinal fluid; DE, differential expressed; EV, extravascular vesicle; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury; DRT, DRG transection; VRT, ventral root transection\",\n \"Patients with type 2 DNP had a prominent higher expression of serum lncRNA NONRATT021972 and more severe neuropathic pain symptoms.\\n31\\n LINC01119 and LINC02447 in the peripheral blood of SCI patients were identified in pain pathways that were important for neuropathic pain development.\\n32\\n\\n\\nMicroarray analysis revealed that a nerve injury could induce time‐dependent lncRNA expression changes in the sciatic nerve.\\n33\\n lncRNA H19 was persistently upregulated in Schwann cells along the peripheral nerve, proximal and distal to the injured site.\\n34\\n In the DRG, the transcriptomic analysis identified 86 known and 26 novel lncRNAs genes to be DE after spared sciatic nerve injury.\\n35\\n Of these, rno‐Cntnap2 and AC111653.1 were essential in peripheral nerve regeneration and were involved in neuropathic pain.\\nNext‐generation RNA sequencing showed 134 lncRNAs and 188 circRNAs were prominently changed 14 days after SNI in the spinal cord.\\n36\\n In addition, microarray analysis identified 1481 and 1096 DE lncRNAs and mRNAs, respectively, in the spinal cord dorsal horn of DNP rats. Of these, 289 neighboring and 57 overlapping lncRNA‐mRNA pairs, including ENSMUST00000150952‐Mbp and AK081017‐Usp15, have been suggested to participate in neuropathic pain development.\\n37\\n\\n\\ncircRNAs have characteristic circularized structures following the backsplicing of exons from antisense RNAs (AS RNAs) or mRNAs and, thus, are highly stable. Most circRNAs are highly conserved among species and lack translation potential, despite cap‐independent translation. A recent study showed that 363 and 106 circRNAs were significantly dysregulated in the ipsilateral dorsal horn after nerve injury.\\n38\\n (Figure 2).\\nLong non‐coding RNA (lncRNAs) and circular non‐coding RNAs (circRNAs) expression change following peripheral nerve injury or noxious stimuli DNP, diabetic neuropathic pain; SCI, spinal cord injury; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury\",\n \"miRNAs contribute to the development of neuropathic pain via diverse mechanisms, such as neuroinflammation, autophagy, abnormal ion channel expression, regulating pain‐related mediators, protein kinases, structural proteins, neurotransmission excitatory–inhibitory imbalances, and exosome miRNA‐mediated neuron–glia communication (Table 1).\\nmiRNAs expression change for the development of neuropathic pain\\nKv1.1, Kv1.4, Kv3.4, Kv4.3, Kv7.5,\\nDPP10, Navβ1 ↓\\np‐p38 MAPK ↑\\nAbbreviations: ACC, anterior cingulate cortex; BACE1, beta‐site amyloid precursor protein‐cleaving enzyme 1; BDNF, brain‐derived neurotrophic factor; CCI, chronic constriction injury; CCR1, C–C chemokine receptor 1; CSF, cerebro spinal fluid; CSF1, colony‐stimulating factor‐1; CXCR4, chemokine CXC receptor 4; DNMT3a, DNA methyltransferase 3a; DNP, diabetic neuropathic pain; DRG, dorsal root ganglion; EV, extracellular vesicle; EZH2, enhancer of zeste homolog 2; GRK2, G protein‐coupled receptor kinases 2; IGF‐1/IGF‐1R, insulin‐like growth factor‐1/insulin‐like growth factor‐1 receptor; KCNMA1, calcium‐activated potassium channel subunit α‐1; MeCP2, methyl cytosine–guanine dinucleotide (CpG)‐binding protein 2; MIP‐1a, macrophage inflammatory protein‐1 alpha; NEFL, neurofilament light polypeptide; NF‐κB, nuclear factor – kappa B; NGF, nerve growth factor; NLRP3, NOD‐like receptor protein 3; NOX2, NADPH oxidase 2; NP, neuropathic pain; pSNL, partial sciatic nerve ligation; RAP1A, Ras‐related protein 1A; RASA1, RAS P21 protein activator 1; ROS, reactive oxygen species; S1PR1, sphingosine‐1‐phosphate receptor 1; SCI, spinal cord injury; Scn2b, β2 subunit of the voltage‐gated sodium channel; SIRT1, histone deacetylase sirtuin 1; SNL, spinal nerve ligation; SOCS1, suppressor of cytokine signaling 1; STAT3, signal transducer and activator of transcription 3; TLR4, toll‐like receptor 4; TNFR1, TNF receptor 1; TRPA1, transient receptor potential ankyrin 1; TXNIP, thioredoxin‐interacting protein; VTR, ventral root transection; ZEB1, zinc finger E‐box binding homeobox 1.\\n\\nmiRNAs regulate neuroinflammation in neuropathic pain development miRNA‐based epigenetic regulation is essential in neuroinflammation. miRNAs are predicted to regulate diverse neuroinflammation‐related targets along the pain processing pathways.\\nAmong patients with neuropathic pain, miR‐101 decreased in the serum and sural nerve, which is related to nuclear factor‐kappa B (NF‐κB) signaling activation.\\n39\\n Meanwhile, serum miR‐124a and miR‐155 expression was upregulated. They were identified to inhibit histone deacetylase sirtuin 1 (SIRT1) in primary human cluster of differentiation 4 (CD4) (+) cells and induce their differentiation toward regulatory T cells (Tregs), thus reducing pain‐related inflammation.\\n40\\n Such miRNA–target interactions may act as an endogenous protective mechanism for neuropathic pain.\\nmiR‐7a, expressed in small‐sized nociceptive DRG neurons, is downregulated after nerve injury, as reported, targeting neurofilament light polypeptides (NEFLs).\\n41\\n NEFL encodes a neuronal protein vital for neurofilament formation and increases signal transducer and activator of transcription 3 (STAT3) phosphorylation, which is highly related to cell differentiation and neuroinflammation. In diabetic peripheral neuropathic mice, miR‐590‐3p was downregulated to disinhibit Ras‐related protein 1A (RAP1A) in the DRG tissue and inhibit neural T cells infiltration.\\n42\\n Thus, exogenous miR‐590‐3p may be a potential alternative for neuropathic pain treatment. CCI downregulated miR‐140 and miR‐144 expression in the DRG. Intrathecally injected miR‐140 and miR‐144 agomir decreased inflammatory factor secretion and ameliorated hyperalgesia by targeting sphingosine‐1‐phosphate receptor 1 (S1PR1) and RAS P21 protein activator 1 (RASA1), respectively.\\n43\\n, \\n44\\n\\n\\nmiRNAs are essential in neuropathic pain development via neuroinflammation‐related mechanisms in the spinal cord. miR‐130a‐3p targets and downregulates insulin‐like growth factor‐1/insulin‐like growth factor‐1 receptor (IGF‐1/IGF‐1R) expression to alleviate SCI‐induced neuropathic pain by mitigating microglial activation and NF‐κB phosphorylation.\\n45\\n miR‐378 was decreased in CCI rats, inhibiting neuropathic pain development by targeting the enhancer of zeste homolog 2 (EZH2).\\n46\\n EZH2 promotes neuropathic pain by increasing tumor necrosis factor‐alpha (TNF‐α), interleukin (IL)‐1β, and monocyte chemoattractant protein‐1 (MCP‐1) production. Intrathecally injecting miRNA‐138 lentivirus can remarkably alleviate neuropathic pain in partial sciatic nerve ligation (pSNL) rats by suppressing toll‐like receptor 4 (TLR4) and macrophage inflammatory protein‐1 alpha (MIP‐1α)/C‐C chemokine receptor 1 (CCR1) signaling pathways.\\n47\\n\\n\\nmiR‐15a/16 targets and downregulates G protein‐coupled receptor kinases 2 (GRK2) to disinhibit p38‐MAPK (mitogen‐activated protein kinase) and NF‐κB, contributing to neuroinflammation after CCI.\\n48\\n GRK2‐deficient mice present a pro‐inflammatory phenotype in spinal cord microglia/macrophages, restored by miR‐124.\\n49\\n SNL increased DNA methyltransferase 3a (DNMT3a) expression related to hypermethylation of the miR‐214‐3p promoter, resulting in miR‐214‐3p expression reduction, which enhanced astrocyte reactivity, colony‐stimulating factor‐1 (CSF1), and interleukin 6 (IL‐6) production, and hyperalgesia in rats.\\n50\\n Electro‐acupuncture attenuated SCI by inhibiting Nav1.3 and Bax in the injured spinal cord through miR‐214 upregulation.\\n51\\n Downregulated miR‐128 was reported to contribute to neuropathic pain via p38 or zinc finger E‐box binding homeobox 1 (ZEB1) activation in the spinal cord.\\n52\\n, \\n53\\n Meanwhile, ZEB1 was also targeted by miR‐200b/miR‐429, orchestrating neuropathic pain development.\\n54\\n\\n\\nIn a pSNL mouse model, nerve injury significantly reduced miR‐23a expression in spinal glial cells, concomitant with the upregulation of its target chemokine, CXC receptor 4 (CXCR4). In naïve mice, either miR‐23a downregulation or CXCR4 upregulation could active the thioredoxin‐interacting protein (TXNIP)/NOD‐like receptor protein 3 (NLRP3) inflammasome axis. Both intrathecal miR‐23a mimics and spinal CXCR4 downregulation by a lentivirus inhibited TXNIP or NLRP3 upregulation to alleviate hyperalgesia.\\n55\\n\\n\\nNotably, one miRNA was shown to have distinctive targets in different animal models of the spinal cord. For example, miR‐155 downregulation targeted and upregulated NADPH oxidase 2 (NOX2) expression to induce reactive oxygen species (ROS) production after SCI, presenting a pro‐inflammatory phenotype in microglia/macrophages.\\n56\\n The ability to induce glial polarization was also observed in cultured BV‐2 microglia.\\n57\\n In bortezomib‐induced neuropathic pain rats, downregulated miR‐155 upregulated TNF receptor 1 (TNFR1) expression, which activated its downstream signaling pathways, including p38‐MAPK, c‐Jun N‐terminal kinase (JNK), and transient receptor potential ankyrin 1 (TRPA1).\\n58\\n Therefore, it is suggested that miR‐155 might act as an intervention target for neuropathic pain. As expected, treatment with ibuprofen and L‐arginine delayed the behavioral pain changes while inhibiting spinal miR‐155 and NO.\\n59\\n miR‐155‐5p is also known to destabilize the blood–nerve barrier and expression of tight junction proteins, such as claudin‐1 and zonula occludens‐1 (ZO‐1). Tissue plasminogen activator (tPA) could transiently open such barriers to facilitate topically applying analgesics, via miR‐155‐5p upregulation.\\n60\\n However, we also noted that CCI upregulated but did not downregulate spinal cord miR‐155, and miR‐155 inhibition enhanced suppressor of cytokine signaling 1 (SOCS1) expression to inactivate inflammation via NF‐κB and p38‐MAPK inhibition.\\n61\\n In oxaliplatin‐induced peripheral neuropathic pain, spinal cord miR‐155 expression was also upregulated, and intrathecally injecting the miR‐155 inhibitor attenuated hyperalgesia in rats, possibly by inhibiting oxidative stress–TRPA1 pathways.\\n62\\n The underlying mechanisms of such distinctive miR‐155 expression change are still unknown and require further research into different pain models.\\nmiRNA‐based epigenetic regulation is essential in neuroinflammation. miRNAs are predicted to regulate diverse neuroinflammation‐related targets along the pain processing pathways.\\nAmong patients with neuropathic pain, miR‐101 decreased in the serum and sural nerve, which is related to nuclear factor‐kappa B (NF‐κB) signaling activation.\\n39\\n Meanwhile, serum miR‐124a and miR‐155 expression was upregulated. They were identified to inhibit histone deacetylase sirtuin 1 (SIRT1) in primary human cluster of differentiation 4 (CD4) (+) cells and induce their differentiation toward regulatory T cells (Tregs), thus reducing pain‐related inflammation.\\n40\\n Such miRNA–target interactions may act as an endogenous protective mechanism for neuropathic pain.\\nmiR‐7a, expressed in small‐sized nociceptive DRG neurons, is downregulated after nerve injury, as reported, targeting neurofilament light polypeptides (NEFLs).\\n41\\n NEFL encodes a neuronal protein vital for neurofilament formation and increases signal transducer and activator of transcription 3 (STAT3) phosphorylation, which is highly related to cell differentiation and neuroinflammation. In diabetic peripheral neuropathic mice, miR‐590‐3p was downregulated to disinhibit Ras‐related protein 1A (RAP1A) in the DRG tissue and inhibit neural T cells infiltration.\\n42\\n Thus, exogenous miR‐590‐3p may be a potential alternative for neuropathic pain treatment. CCI downregulated miR‐140 and miR‐144 expression in the DRG. Intrathecally injected miR‐140 and miR‐144 agomir decreased inflammatory factor secretion and ameliorated hyperalgesia by targeting sphingosine‐1‐phosphate receptor 1 (S1PR1) and RAS P21 protein activator 1 (RASA1), respectively.\\n43\\n, \\n44\\n\\n\\nmiRNAs are essential in neuropathic pain development via neuroinflammation‐related mechanisms in the spinal cord. miR‐130a‐3p targets and downregulates insulin‐like growth factor‐1/insulin‐like growth factor‐1 receptor (IGF‐1/IGF‐1R) expression to alleviate SCI‐induced neuropathic pain by mitigating microglial activation and NF‐κB phosphorylation.\\n45\\n miR‐378 was decreased in CCI rats, inhibiting neuropathic pain development by targeting the enhancer of zeste homolog 2 (EZH2).\\n46\\n EZH2 promotes neuropathic pain by increasing tumor necrosis factor‐alpha (TNF‐α), interleukin (IL)‐1β, and monocyte chemoattractant protein‐1 (MCP‐1) production. Intrathecally injecting miRNA‐138 lentivirus can remarkably alleviate neuropathic pain in partial sciatic nerve ligation (pSNL) rats by suppressing toll‐like receptor 4 (TLR4) and macrophage inflammatory protein‐1 alpha (MIP‐1α)/C‐C chemokine receptor 1 (CCR1) signaling pathways.\\n47\\n\\n\\nmiR‐15a/16 targets and downregulates G protein‐coupled receptor kinases 2 (GRK2) to disinhibit p38‐MAPK (mitogen‐activated protein kinase) and NF‐κB, contributing to neuroinflammation after CCI.\\n48\\n GRK2‐deficient mice present a pro‐inflammatory phenotype in spinal cord microglia/macrophages, restored by miR‐124.\\n49\\n SNL increased DNA methyltransferase 3a (DNMT3a) expression related to hypermethylation of the miR‐214‐3p promoter, resulting in miR‐214‐3p expression reduction, which enhanced astrocyte reactivity, colony‐stimulating factor‐1 (CSF1), and interleukin 6 (IL‐6) production, and hyperalgesia in rats.\\n50\\n Electro‐acupuncture attenuated SCI by inhibiting Nav1.3 and Bax in the injured spinal cord through miR‐214 upregulation.\\n51\\n Downregulated miR‐128 was reported to contribute to neuropathic pain via p38 or zinc finger E‐box binding homeobox 1 (ZEB1) activation in the spinal cord.\\n52\\n, \\n53\\n Meanwhile, ZEB1 was also targeted by miR‐200b/miR‐429, orchestrating neuropathic pain development.\\n54\\n\\n\\nIn a pSNL mouse model, nerve injury significantly reduced miR‐23a expression in spinal glial cells, concomitant with the upregulation of its target chemokine, CXC receptor 4 (CXCR4). In naïve mice, either miR‐23a downregulation or CXCR4 upregulation could active the thioredoxin‐interacting protein (TXNIP)/NOD‐like receptor protein 3 (NLRP3) inflammasome axis. Both intrathecal miR‐23a mimics and spinal CXCR4 downregulation by a lentivirus inhibited TXNIP or NLRP3 upregulation to alleviate hyperalgesia.\\n55\\n\\n\\nNotably, one miRNA was shown to have distinctive targets in different animal models of the spinal cord. For example, miR‐155 downregulation targeted and upregulated NADPH oxidase 2 (NOX2) expression to induce reactive oxygen species (ROS) production after SCI, presenting a pro‐inflammatory phenotype in microglia/macrophages.\\n56\\n The ability to induce glial polarization was also observed in cultured BV‐2 microglia.\\n57\\n In bortezomib‐induced neuropathic pain rats, downregulated miR‐155 upregulated TNF receptor 1 (TNFR1) expression, which activated its downstream signaling pathways, including p38‐MAPK, c‐Jun N‐terminal kinase (JNK), and transient receptor potential ankyrin 1 (TRPA1).\\n58\\n Therefore, it is suggested that miR‐155 might act as an intervention target for neuropathic pain. As expected, treatment with ibuprofen and L‐arginine delayed the behavioral pain changes while inhibiting spinal miR‐155 and NO.\\n59\\n miR‐155‐5p is also known to destabilize the blood–nerve barrier and expression of tight junction proteins, such as claudin‐1 and zonula occludens‐1 (ZO‐1). Tissue plasminogen activator (tPA) could transiently open such barriers to facilitate topically applying analgesics, via miR‐155‐5p upregulation.\\n60\\n However, we also noted that CCI upregulated but did not downregulate spinal cord miR‐155, and miR‐155 inhibition enhanced suppressor of cytokine signaling 1 (SOCS1) expression to inactivate inflammation via NF‐κB and p38‐MAPK inhibition.\\n61\\n In oxaliplatin‐induced peripheral neuropathic pain, spinal cord miR‐155 expression was also upregulated, and intrathecally injecting the miR‐155 inhibitor attenuated hyperalgesia in rats, possibly by inhibiting oxidative stress–TRPA1 pathways.\\n62\\n The underlying mechanisms of such distinctive miR‐155 expression change are still unknown and require further research into different pain models.\\nAutophagy miRNA‐related autophagy is involved in neuropathic pain regulation. As reported, miR‐15a downregulated and stimulated AKT serine/threonine kinase 3 (AKT3), and inhibited autophagy post‐CCI.\\n63\\n Impaired autophagy participates in neuropathic pain development. Intrathecal miR‐15a agomir prominently suppressed AKT3 expression, induced autophagy, and attenuated CCI‐induced neuropathic pain. Similar to miR‐15a, miR‐145 and miR‐20b‐5p contributed to neuropathic pain regulation via protein kinase B (AKT)‐related autophagy pathways.\\n64\\n, \\n65\\n Meanwhile, miR‐195 in the spinal cord was observed to upregulate post‐SNL, targeting and inhibiting Autophagy‐Related 14 (ATG14) and its autophagy activation.\\n66\\n The miR‐195 inhibitor activated autophagy and suppressed neuroinflammation in vivo and in vitro.\\nmiRNA‐related autophagy is involved in neuropathic pain regulation. As reported, miR‐15a downregulated and stimulated AKT serine/threonine kinase 3 (AKT3), and inhibited autophagy post‐CCI.\\n63\\n Impaired autophagy participates in neuropathic pain development. Intrathecal miR‐15a agomir prominently suppressed AKT3 expression, induced autophagy, and attenuated CCI‐induced neuropathic pain. Similar to miR‐15a, miR‐145 and miR‐20b‐5p contributed to neuropathic pain regulation via protein kinase B (AKT)‐related autophagy pathways.\\n64\\n, \\n65\\n Meanwhile, miR‐195 in the spinal cord was observed to upregulate post‐SNL, targeting and inhibiting Autophagy‐Related 14 (ATG14) and its autophagy activation.\\n66\\n The miR‐195 inhibitor activated autophagy and suppressed neuroinflammation in vivo and in vitro.\\nThe contribution of miRNAs to ion channel expression in neuropathic pain Another class of miRNA‐based regulation focuses on ion channels, including sodium, potassium, and calcium channels, and transient receptor potential (TRP) channels, to modulate action potential production, firing rate, and neurotransmitter release.\\nTheoretically, miRNAs can simultaneously have multiple target mRNAs because precise matching is not a prerequisite for inhibiting the target sequence. For example, other than NEFL,\\n41\\n miR‐7a targets the β2 subunit of the voltage‐gated sodium channel (Scn2b) with post‐transcriptional regulation to induce nociceptive DRG neurons hyperexcitability.\\n67\\n Other than miR‐7a, the other known miRNA‐ targets involved in sodium channels expression include miR‐30b, ‐96, ‐384‐5p and their target Scn3a (encoding Nav1.3),\\n68\\n, \\n69\\n, \\n70\\n miR‐30b‐5p and its target Scn8a (encoding Nav1.6),\\n71\\n and miR‐182 and ‐30b and their target Scn9a (encoding Nav1.7).\\n72\\n, \\n73\\n\\n\\nA miRNA cluster is a polycistronic gene containing several miRNAs derived from a single primary or nascent transcript. Approximately 40% of miRNAs are predicted to form clusters, but their significance is still unknown. miR‐17‐92 is a miRNA cluster that includes six different members, of which miR‐18a, ‐19a, ‐19b, and ‐92a upregulation induces allodynia. The predicted targets of the miR‐17‐92 cluster encompass genes encoding diverse voltage‐gated potassium channels and their regulatory subunits, including Kv1.1, Kv1.4, Kv3.4, Kv4.3, Kv7.5, dipeptidyl peptidase 10 (DPP10), and Navβ1.\\n74\\n CCI upregulated miR‐137 to target and downregulate Kcna2, which encodes Kv1.2 in the DRG and spinal dorsal horn.\\n75\\n By contrast, CCI decreased miR‐183‐5p expression in the DRG, and the predicted target gene TREK‐1, a subunit of the 2P‐domain K+ channel, was increased.\\n76\\n\\n\\nFurthermore, the miRNA‐183 cluster (miR‐183, part of miR‐96/182/183) regulates either basal mechanical or neuropathic pain.\\n77\\n The miR‐183 cluster targets Cacna2d1 and Cacna2d2, which encode auxiliary voltage‐gated calcium channel subunits α2δ‐1 and α2δ‐2, to affect nociceptor excitability. Nerve injury downregulated miR‐103 in the spinal cord, which simultaneously targeted and inhibited Cacna1c, Cacna2d1, and Cacnb1 and encoded the Cav1.2‐α1, α2δ1, and β1 subunits of the voltage‐gated calcium channels macromolecular complex Cav1.2 L‐type calcium channel, respectively.\\n78\\n Intrathecal miR‐103 successfully relieved neuropathic pain.\\nIn addition to ion channels, TRP channels are ligand‐gated ion channels that promote painful sensations.\\n79\\n In the DRG, TRPA1 participates in miR‐141‐5p alleviated oxaliplatin‐induced neuropathic pain.\\n80\\n In SNI mice, miR‐449a ameliorated neuropathic pain by decreasing the activity of TRPA1 and calcium‐activated potassium channel subunit α‐1 (KCNMA1), thus acting as a potential therapeutic alternative for treating neuropathic pain.\\n81\\n\\n\\nAnother class of miRNA‐based regulation focuses on ion channels, including sodium, potassium, and calcium channels, and transient receptor potential (TRP) channels, to modulate action potential production, firing rate, and neurotransmitter release.\\nTheoretically, miRNAs can simultaneously have multiple target mRNAs because precise matching is not a prerequisite for inhibiting the target sequence. For example, other than NEFL,\\n41\\n miR‐7a targets the β2 subunit of the voltage‐gated sodium channel (Scn2b) with post‐transcriptional regulation to induce nociceptive DRG neurons hyperexcitability.\\n67\\n Other than miR‐7a, the other known miRNA‐ targets involved in sodium channels expression include miR‐30b, ‐96, ‐384‐5p and their target Scn3a (encoding Nav1.3),\\n68\\n, \\n69\\n, \\n70\\n miR‐30b‐5p and its target Scn8a (encoding Nav1.6),\\n71\\n and miR‐182 and ‐30b and their target Scn9a (encoding Nav1.7).\\n72\\n, \\n73\\n\\n\\nA miRNA cluster is a polycistronic gene containing several miRNAs derived from a single primary or nascent transcript. Approximately 40% of miRNAs are predicted to form clusters, but their significance is still unknown. miR‐17‐92 is a miRNA cluster that includes six different members, of which miR‐18a, ‐19a, ‐19b, and ‐92a upregulation induces allodynia. The predicted targets of the miR‐17‐92 cluster encompass genes encoding diverse voltage‐gated potassium channels and their regulatory subunits, including Kv1.1, Kv1.4, Kv3.4, Kv4.3, Kv7.5, dipeptidyl peptidase 10 (DPP10), and Navβ1.\\n74\\n CCI upregulated miR‐137 to target and downregulate Kcna2, which encodes Kv1.2 in the DRG and spinal dorsal horn.\\n75\\n By contrast, CCI decreased miR‐183‐5p expression in the DRG, and the predicted target gene TREK‐1, a subunit of the 2P‐domain K+ channel, was increased.\\n76\\n\\n\\nFurthermore, the miRNA‐183 cluster (miR‐183, part of miR‐96/182/183) regulates either basal mechanical or neuropathic pain.\\n77\\n The miR‐183 cluster targets Cacna2d1 and Cacna2d2, which encode auxiliary voltage‐gated calcium channel subunits α2δ‐1 and α2δ‐2, to affect nociceptor excitability. Nerve injury downregulated miR‐103 in the spinal cord, which simultaneously targeted and inhibited Cacna1c, Cacna2d1, and Cacnb1 and encoded the Cav1.2‐α1, α2δ1, and β1 subunits of the voltage‐gated calcium channels macromolecular complex Cav1.2 L‐type calcium channel, respectively.\\n78\\n Intrathecal miR‐103 successfully relieved neuropathic pain.\\nIn addition to ion channels, TRP channels are ligand‐gated ion channels that promote painful sensations.\\n79\\n In the DRG, TRPA1 participates in miR‐141‐5p alleviated oxaliplatin‐induced neuropathic pain.\\n80\\n In SNI mice, miR‐449a ameliorated neuropathic pain by decreasing the activity of TRPA1 and calcium‐activated potassium channel subunit α‐1 (KCNMA1), thus acting as a potential therapeutic alternative for treating neuropathic pain.\\n81\\n\\n\\n\\nmiRNAs regulate pain‐related mediators, protein kinases, and structural proteins miRNAs regulate diverse pain‐related mediators, protein kinases, and structural proteins along the pain processing pathways. For HIV‐associated symptomatic distal sensory polyneuropathy and neuropathic pain, serum miR‐455‐3p acted as a potential biomarker, possibly targeting multiple genes involved in peripheral neuropathic pain, such as nerve growth factor (NGF) and related genes.\\n82\\n Brain‐derived neurotrophic factor (BDNF), another well‐recognized pain mediator, is a common miR‐1, ‐183, and ‐206 target.\\n83\\n, \\n84\\n, \\n85\\n After nerve injury, several miRNAs, including miR‐19a, ‐301, and ‐132, were downregulated, and their target methyl cytosine–guanine dinucleotide (CpG)‐binding protein 2 (MeCP2) expression was increased, leading to concomitant BDNF upregulation.\\n86\\n miR‐30c‐5p was upregulated in the serum and CSF of patients with chronic peripheral ischemia.\\n16\\n A miR‐30c‐5p inhibitor intraventricular injection postponed neuropathic pain development and fully reversed hyperalgesia in rodents. Transforming growth factor beta (TGF‐β) participates in the effects of miR‐30c‐5p, which refers to the endogenous opioid analgesic system.\\n16\\n\\n\\nmiRNAs dysregulate specific pain‐related protein kinases. For example, in DNP rats, miR‐133a‐3p was dysregulated in the sciatic nerve, interacting with p‐p38 MAPK to participate in the development of neuropathic pain.\\n87\\n Via a methyl CpG‐binding domain and transcriptional repressor domain, MeCP2 acts as a transcriptional repressor, and its overexpression improves neuropathic pain, potentiating an anti‐nociceptive effect of MeCP2. The authors also noted that MeCP2 expression changed post‐transcriptionally, and its mRNA level did not significantly change after SNI. However, the protein level was upregulated. During the development period of neuropathic pain, phospho‐cAMP response element‐binding protein (p‐CREB) elevated rapidly but returned 3–7 days after SNI, concomitant with miR‐132 downregulation, which targets MeCP2 and inhibits its expression post‐transcriptionally.\\n88\\n SNL decreased miR‐200b and miR‐429 in NAc neurons, along with an upregulation of their target (DNMT3a).\\n89\\n Further mechanism studies found that DNMT3a in the NAc was expressed in NR1 immunoreactive neurons, suggesting the dysregulation of ‘mesolimbic motivation circuitry’ for neuropathic pain development.\\nThe CCI induced time‐dependent miR‐1 downregulation in injured sciatic nerves. This change in expression was related to the upregulation and translocation of the miR‐1‐targeted connexin 43 (Cx43), the major connexin of astrocytes.\\n83\\n miR‐1 mimics could reduce Cx43 expression in cultured human glioblastoma cells. However, miR‐1 mimics intraneural transfection failed to alter Cx43 protein expression and did not improve pain behavior. The authors attributed such treatment failure to insufficient inhibition of miR‐1 for Cx43 via intraneural injection. Alternatively, there were regulatory mechanisms for Cx43 in vivo other than miR‐1.\\n90\\n However, miR‐1 in the DRG was DE according to peripheral nerve injury type. CCI, pSNL, and sural nerve injury downregulated miR‐1. However, axotomy of the sciatic nerve and tibial nerve injury increased its expression in the DRG.\\n83\\n, \\n91\\n, \\n92\\n As previously described, miR‐1 was also upregulated following capsaicin treatment and bone cancer pain.\\n91\\n, \\n93\\n Additionally, miR‐1 downregulation inhibited bone cancer pain. Collectively, the action of miR‐1 on pain seems to be complex and stimulus‐dependent.\\nNotably, specific miRNAs can promote neuropathic pain development via diverse mechanisms. For example, beta‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1), a membrane protease essential for myelination, was downregulated after miR‐15b overexpression in vitro or in the DRG of chemotherapy‐related neuropathic pain rats.\\n94\\n BACE1‐mediated neuregulin 1 reduction decreases nerve conduction velocity. In addition, BACE1 modulated Navβ2 subunit expression and neuronal activity and regulated inflammation‐related TNFR expression.\\nmiRNAs regulate diverse pain‐related mediators, protein kinases, and structural proteins along the pain processing pathways. For HIV‐associated symptomatic distal sensory polyneuropathy and neuropathic pain, serum miR‐455‐3p acted as a potential biomarker, possibly targeting multiple genes involved in peripheral neuropathic pain, such as nerve growth factor (NGF) and related genes.\\n82\\n Brain‐derived neurotrophic factor (BDNF), another well‐recognized pain mediator, is a common miR‐1, ‐183, and ‐206 target.\\n83\\n, \\n84\\n, \\n85\\n After nerve injury, several miRNAs, including miR‐19a, ‐301, and ‐132, were downregulated, and their target methyl cytosine–guanine dinucleotide (CpG)‐binding protein 2 (MeCP2) expression was increased, leading to concomitant BDNF upregulation.\\n86\\n miR‐30c‐5p was upregulated in the serum and CSF of patients with chronic peripheral ischemia.\\n16\\n A miR‐30c‐5p inhibitor intraventricular injection postponed neuropathic pain development and fully reversed hyperalgesia in rodents. Transforming growth factor beta (TGF‐β) participates in the effects of miR‐30c‐5p, which refers to the endogenous opioid analgesic system.\\n16\\n\\n\\nmiRNAs dysregulate specific pain‐related protein kinases. For example, in DNP rats, miR‐133a‐3p was dysregulated in the sciatic nerve, interacting with p‐p38 MAPK to participate in the development of neuropathic pain.\\n87\\n Via a methyl CpG‐binding domain and transcriptional repressor domain, MeCP2 acts as a transcriptional repressor, and its overexpression improves neuropathic pain, potentiating an anti‐nociceptive effect of MeCP2. The authors also noted that MeCP2 expression changed post‐transcriptionally, and its mRNA level did not significantly change after SNI. However, the protein level was upregulated. During the development period of neuropathic pain, phospho‐cAMP response element‐binding protein (p‐CREB) elevated rapidly but returned 3–7 days after SNI, concomitant with miR‐132 downregulation, which targets MeCP2 and inhibits its expression post‐transcriptionally.\\n88\\n SNL decreased miR‐200b and miR‐429 in NAc neurons, along with an upregulation of their target (DNMT3a).\\n89\\n Further mechanism studies found that DNMT3a in the NAc was expressed in NR1 immunoreactive neurons, suggesting the dysregulation of ‘mesolimbic motivation circuitry’ for neuropathic pain development.\\nThe CCI induced time‐dependent miR‐1 downregulation in injured sciatic nerves. This change in expression was related to the upregulation and translocation of the miR‐1‐targeted connexin 43 (Cx43), the major connexin of astrocytes.\\n83\\n miR‐1 mimics could reduce Cx43 expression in cultured human glioblastoma cells. However, miR‐1 mimics intraneural transfection failed to alter Cx43 protein expression and did not improve pain behavior. The authors attributed such treatment failure to insufficient inhibition of miR‐1 for Cx43 via intraneural injection. Alternatively, there were regulatory mechanisms for Cx43 in vivo other than miR‐1.\\n90\\n However, miR‐1 in the DRG was DE according to peripheral nerve injury type. CCI, pSNL, and sural nerve injury downregulated miR‐1. However, axotomy of the sciatic nerve and tibial nerve injury increased its expression in the DRG.\\n83\\n, \\n91\\n, \\n92\\n As previously described, miR‐1 was also upregulated following capsaicin treatment and bone cancer pain.\\n91\\n, \\n93\\n Additionally, miR‐1 downregulation inhibited bone cancer pain. Collectively, the action of miR‐1 on pain seems to be complex and stimulus‐dependent.\\nNotably, specific miRNAs can promote neuropathic pain development via diverse mechanisms. For example, beta‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1), a membrane protease essential for myelination, was downregulated after miR‐15b overexpression in vitro or in the DRG of chemotherapy‐related neuropathic pain rats.\\n94\\n BACE1‐mediated neuregulin 1 reduction decreases nerve conduction velocity. In addition, BACE1 modulated Navβ2 subunit expression and neuronal activity and regulated inflammation‐related TNFR expression.\\nNeurotransmission excitatory–inhibitory imbalances Neurotransmission excitatory and inhibitory imbalances in the spinal cord also contribute to neuropathic pain development. For example, miR‐500 increased to regulate glutamic acid decarboxylase 67 (GAD67) expression and target the specific site of Gad1 in the dorsal horn.\\n95\\n GAD67 expression reduction inhibited the function of GABAergic neurons and the resultant inhibitory synaptic transmission dysregulation contributed to neuropathic pain development. In addition, miR‐23b is crucial for improving neuropathic pain in the injured spinal cord by downregulating its target gene, Nox4, which further normalizes glutamic acid decarboxylase 65/67 (GAD65/67) expression and protects GABAergic neurons from apoptosis.\\n96\\n Microarray analysis showed that miR‐539 was prominently reduced in the contralateral anterior cingulate cortex (ACC) after CCI, which is related to enhanced NR2B protein expression. Injecting miR‐539 mimics into the contralateral ACC attenuated CCI‐evoked mechanical hyperalgesia, suggesting that the N‐methyl‐D‐aspartate (NMDA) receptor NR2B subunit regulates neuropathic pain.\\n97\\n\\n\\nNeurotransmission excitatory and inhibitory imbalances in the spinal cord also contribute to neuropathic pain development. For example, miR‐500 increased to regulate glutamic acid decarboxylase 67 (GAD67) expression and target the specific site of Gad1 in the dorsal horn.\\n95\\n GAD67 expression reduction inhibited the function of GABAergic neurons and the resultant inhibitory synaptic transmission dysregulation contributed to neuropathic pain development. In addition, miR‐23b is crucial for improving neuropathic pain in the injured spinal cord by downregulating its target gene, Nox4, which further normalizes glutamic acid decarboxylase 65/67 (GAD65/67) expression and protects GABAergic neurons from apoptosis.\\n96\\n Microarray analysis showed that miR‐539 was prominently reduced in the contralateral anterior cingulate cortex (ACC) after CCI, which is related to enhanced NR2B protein expression. Injecting miR‐539 mimics into the contralateral ACC attenuated CCI‐evoked mechanical hyperalgesia, suggesting that the N‐methyl‐D‐aspartate (NMDA) receptor NR2B subunit regulates neuropathic pain.\\n97\\n\\n\\n\\nEV miRNAs‐mediated neuron–glia communication We mainly focused on exosomal miR‐21 with regard to EV miRNAs in neuropathic pain. As aforementioned, SNL, DRT, or VRT could upregulate miR‐21 in the injured DRG.\\n22\\n Notably, miR‐21 was also increased in serum exosomes from nerve‐ligated mice.\\n98\\n In another in‐depth study, macrophages readily took up pure sensory neuron‐derived exosomes encompassing miR‐21‐5p to promote a pro‐inflammatory phenotype.\\n99\\n Either intrathecal miR‐21‐5p antagomir or miR‐21 conditional deletion in sensory neurons ameliorated hyperalgesia and macrophage recruitment in the DRG.\\nUnlike the miRNA target gene inhibitory mechanism, miR‐21 acts as an endogenous toll‐like receptor 8 (TLR8) ligand, leading to neuropathic pain development.\\n100\\n TLR8, a nucleic acid‐sensing receptor, is located in the endosomes and lysosomes, leading to ERK‐mediated inflammatory mediator production and neuronal activation after SNL. Although miR‐21 and TLR8 are only distributed in small‐ and medium‐sized neurons, miR‐21 can also be derived from large‐sized neurons and reach TLR8 in the endosomes of other types of neurons.\\n101\\n Similar to miR‐21, DRG sensory neurons secreted miR‐23a‐enriched EVs following nerve injury and were taken up by macrophages to enhance M1 polarization in vitro. A20, an NF‐κB signaling pathway inhibitor, is a verified miR‐23a target gene.\\n102\\n Moreover, intrathecally delivering EVs‐miR‐23a antagomir attenuated neuropathic hyperalgesia and reduced M1 macrophages by inhibiting A20 to activate NF‐κB signaling.\\nWe mainly focused on exosomal miR‐21 with regard to EV miRNAs in neuropathic pain. As aforementioned, SNL, DRT, or VRT could upregulate miR‐21 in the injured DRG.\\n22\\n Notably, miR‐21 was also increased in serum exosomes from nerve‐ligated mice.\\n98\\n In another in‐depth study, macrophages readily took up pure sensory neuron‐derived exosomes encompassing miR‐21‐5p to promote a pro‐inflammatory phenotype.\\n99\\n Either intrathecal miR‐21‐5p antagomir or miR‐21 conditional deletion in sensory neurons ameliorated hyperalgesia and macrophage recruitment in the DRG.\\nUnlike the miRNA target gene inhibitory mechanism, miR‐21 acts as an endogenous toll‐like receptor 8 (TLR8) ligand, leading to neuropathic pain development.\\n100\\n TLR8, a nucleic acid‐sensing receptor, is located in the endosomes and lysosomes, leading to ERK‐mediated inflammatory mediator production and neuronal activation after SNL. Although miR‐21 and TLR8 are only distributed in small‐ and medium‐sized neurons, miR‐21 can also be derived from large‐sized neurons and reach TLR8 in the endosomes of other types of neurons.\\n101\\n Similar to miR‐21, DRG sensory neurons secreted miR‐23a‐enriched EVs following nerve injury and were taken up by macrophages to enhance M1 polarization in vitro. A20, an NF‐κB signaling pathway inhibitor, is a verified miR‐23a target gene.\\n102\\n Moreover, intrathecally delivering EVs‐miR‐23a antagomir attenuated neuropathic hyperalgesia and reduced M1 macrophages by inhibiting A20 to activate NF‐κB signaling.\",\n \"miRNA‐based epigenetic regulation is essential in neuroinflammation. miRNAs are predicted to regulate diverse neuroinflammation‐related targets along the pain processing pathways.\\nAmong patients with neuropathic pain, miR‐101 decreased in the serum and sural nerve, which is related to nuclear factor‐kappa B (NF‐κB) signaling activation.\\n39\\n Meanwhile, serum miR‐124a and miR‐155 expression was upregulated. They were identified to inhibit histone deacetylase sirtuin 1 (SIRT1) in primary human cluster of differentiation 4 (CD4) (+) cells and induce their differentiation toward regulatory T cells (Tregs), thus reducing pain‐related inflammation.\\n40\\n Such miRNA–target interactions may act as an endogenous protective mechanism for neuropathic pain.\\nmiR‐7a, expressed in small‐sized nociceptive DRG neurons, is downregulated after nerve injury, as reported, targeting neurofilament light polypeptides (NEFLs).\\n41\\n NEFL encodes a neuronal protein vital for neurofilament formation and increases signal transducer and activator of transcription 3 (STAT3) phosphorylation, which is highly related to cell differentiation and neuroinflammation. In diabetic peripheral neuropathic mice, miR‐590‐3p was downregulated to disinhibit Ras‐related protein 1A (RAP1A) in the DRG tissue and inhibit neural T cells infiltration.\\n42\\n Thus, exogenous miR‐590‐3p may be a potential alternative for neuropathic pain treatment. CCI downregulated miR‐140 and miR‐144 expression in the DRG. Intrathecally injected miR‐140 and miR‐144 agomir decreased inflammatory factor secretion and ameliorated hyperalgesia by targeting sphingosine‐1‐phosphate receptor 1 (S1PR1) and RAS P21 protein activator 1 (RASA1), respectively.\\n43\\n, \\n44\\n\\n\\nmiRNAs are essential in neuropathic pain development via neuroinflammation‐related mechanisms in the spinal cord. miR‐130a‐3p targets and downregulates insulin‐like growth factor‐1/insulin‐like growth factor‐1 receptor (IGF‐1/IGF‐1R) expression to alleviate SCI‐induced neuropathic pain by mitigating microglial activation and NF‐κB phosphorylation.\\n45\\n miR‐378 was decreased in CCI rats, inhibiting neuropathic pain development by targeting the enhancer of zeste homolog 2 (EZH2).\\n46\\n EZH2 promotes neuropathic pain by increasing tumor necrosis factor‐alpha (TNF‐α), interleukin (IL)‐1β, and monocyte chemoattractant protein‐1 (MCP‐1) production. Intrathecally injecting miRNA‐138 lentivirus can remarkably alleviate neuropathic pain in partial sciatic nerve ligation (pSNL) rats by suppressing toll‐like receptor 4 (TLR4) and macrophage inflammatory protein‐1 alpha (MIP‐1α)/C‐C chemokine receptor 1 (CCR1) signaling pathways.\\n47\\n\\n\\nmiR‐15a/16 targets and downregulates G protein‐coupled receptor kinases 2 (GRK2) to disinhibit p38‐MAPK (mitogen‐activated protein kinase) and NF‐κB, contributing to neuroinflammation after CCI.\\n48\\n GRK2‐deficient mice present a pro‐inflammatory phenotype in spinal cord microglia/macrophages, restored by miR‐124.\\n49\\n SNL increased DNA methyltransferase 3a (DNMT3a) expression related to hypermethylation of the miR‐214‐3p promoter, resulting in miR‐214‐3p expression reduction, which enhanced astrocyte reactivity, colony‐stimulating factor‐1 (CSF1), and interleukin 6 (IL‐6) production, and hyperalgesia in rats.\\n50\\n Electro‐acupuncture attenuated SCI by inhibiting Nav1.3 and Bax in the injured spinal cord through miR‐214 upregulation.\\n51\\n Downregulated miR‐128 was reported to contribute to neuropathic pain via p38 or zinc finger E‐box binding homeobox 1 (ZEB1) activation in the spinal cord.\\n52\\n, \\n53\\n Meanwhile, ZEB1 was also targeted by miR‐200b/miR‐429, orchestrating neuropathic pain development.\\n54\\n\\n\\nIn a pSNL mouse model, nerve injury significantly reduced miR‐23a expression in spinal glial cells, concomitant with the upregulation of its target chemokine, CXC receptor 4 (CXCR4). In naïve mice, either miR‐23a downregulation or CXCR4 upregulation could active the thioredoxin‐interacting protein (TXNIP)/NOD‐like receptor protein 3 (NLRP3) inflammasome axis. Both intrathecal miR‐23a mimics and spinal CXCR4 downregulation by a lentivirus inhibited TXNIP or NLRP3 upregulation to alleviate hyperalgesia.\\n55\\n\\n\\nNotably, one miRNA was shown to have distinctive targets in different animal models of the spinal cord. For example, miR‐155 downregulation targeted and upregulated NADPH oxidase 2 (NOX2) expression to induce reactive oxygen species (ROS) production after SCI, presenting a pro‐inflammatory phenotype in microglia/macrophages.\\n56\\n The ability to induce glial polarization was also observed in cultured BV‐2 microglia.\\n57\\n In bortezomib‐induced neuropathic pain rats, downregulated miR‐155 upregulated TNF receptor 1 (TNFR1) expression, which activated its downstream signaling pathways, including p38‐MAPK, c‐Jun N‐terminal kinase (JNK), and transient receptor potential ankyrin 1 (TRPA1).\\n58\\n Therefore, it is suggested that miR‐155 might act as an intervention target for neuropathic pain. As expected, treatment with ibuprofen and L‐arginine delayed the behavioral pain changes while inhibiting spinal miR‐155 and NO.\\n59\\n miR‐155‐5p is also known to destabilize the blood–nerve barrier and expression of tight junction proteins, such as claudin‐1 and zonula occludens‐1 (ZO‐1). Tissue plasminogen activator (tPA) could transiently open such barriers to facilitate topically applying analgesics, via miR‐155‐5p upregulation.\\n60\\n However, we also noted that CCI upregulated but did not downregulate spinal cord miR‐155, and miR‐155 inhibition enhanced suppressor of cytokine signaling 1 (SOCS1) expression to inactivate inflammation via NF‐κB and p38‐MAPK inhibition.\\n61\\n In oxaliplatin‐induced peripheral neuropathic pain, spinal cord miR‐155 expression was also upregulated, and intrathecally injecting the miR‐155 inhibitor attenuated hyperalgesia in rats, possibly by inhibiting oxidative stress–TRPA1 pathways.\\n62\\n The underlying mechanisms of such distinctive miR‐155 expression change are still unknown and require further research into different pain models.\",\n \"miRNA‐related autophagy is involved in neuropathic pain regulation. As reported, miR‐15a downregulated and stimulated AKT serine/threonine kinase 3 (AKT3), and inhibited autophagy post‐CCI.\\n63\\n Impaired autophagy participates in neuropathic pain development. Intrathecal miR‐15a agomir prominently suppressed AKT3 expression, induced autophagy, and attenuated CCI‐induced neuropathic pain. Similar to miR‐15a, miR‐145 and miR‐20b‐5p contributed to neuropathic pain regulation via protein kinase B (AKT)‐related autophagy pathways.\\n64\\n, \\n65\\n Meanwhile, miR‐195 in the spinal cord was observed to upregulate post‐SNL, targeting and inhibiting Autophagy‐Related 14 (ATG14) and its autophagy activation.\\n66\\n The miR‐195 inhibitor activated autophagy and suppressed neuroinflammation in vivo and in vitro.\",\n \"Another class of miRNA‐based regulation focuses on ion channels, including sodium, potassium, and calcium channels, and transient receptor potential (TRP) channels, to modulate action potential production, firing rate, and neurotransmitter release.\\nTheoretically, miRNAs can simultaneously have multiple target mRNAs because precise matching is not a prerequisite for inhibiting the target sequence. For example, other than NEFL,\\n41\\n miR‐7a targets the β2 subunit of the voltage‐gated sodium channel (Scn2b) with post‐transcriptional regulation to induce nociceptive DRG neurons hyperexcitability.\\n67\\n Other than miR‐7a, the other known miRNA‐ targets involved in sodium channels expression include miR‐30b, ‐96, ‐384‐5p and their target Scn3a (encoding Nav1.3),\\n68\\n, \\n69\\n, \\n70\\n miR‐30b‐5p and its target Scn8a (encoding Nav1.6),\\n71\\n and miR‐182 and ‐30b and their target Scn9a (encoding Nav1.7).\\n72\\n, \\n73\\n\\n\\nA miRNA cluster is a polycistronic gene containing several miRNAs derived from a single primary or nascent transcript. Approximately 40% of miRNAs are predicted to form clusters, but their significance is still unknown. miR‐17‐92 is a miRNA cluster that includes six different members, of which miR‐18a, ‐19a, ‐19b, and ‐92a upregulation induces allodynia. The predicted targets of the miR‐17‐92 cluster encompass genes encoding diverse voltage‐gated potassium channels and their regulatory subunits, including Kv1.1, Kv1.4, Kv3.4, Kv4.3, Kv7.5, dipeptidyl peptidase 10 (DPP10), and Navβ1.\\n74\\n CCI upregulated miR‐137 to target and downregulate Kcna2, which encodes Kv1.2 in the DRG and spinal dorsal horn.\\n75\\n By contrast, CCI decreased miR‐183‐5p expression in the DRG, and the predicted target gene TREK‐1, a subunit of the 2P‐domain K+ channel, was increased.\\n76\\n\\n\\nFurthermore, the miRNA‐183 cluster (miR‐183, part of miR‐96/182/183) regulates either basal mechanical or neuropathic pain.\\n77\\n The miR‐183 cluster targets Cacna2d1 and Cacna2d2, which encode auxiliary voltage‐gated calcium channel subunits α2δ‐1 and α2δ‐2, to affect nociceptor excitability. Nerve injury downregulated miR‐103 in the spinal cord, which simultaneously targeted and inhibited Cacna1c, Cacna2d1, and Cacnb1 and encoded the Cav1.2‐α1, α2δ1, and β1 subunits of the voltage‐gated calcium channels macromolecular complex Cav1.2 L‐type calcium channel, respectively.\\n78\\n Intrathecal miR‐103 successfully relieved neuropathic pain.\\nIn addition to ion channels, TRP channels are ligand‐gated ion channels that promote painful sensations.\\n79\\n In the DRG, TRPA1 participates in miR‐141‐5p alleviated oxaliplatin‐induced neuropathic pain.\\n80\\n In SNI mice, miR‐449a ameliorated neuropathic pain by decreasing the activity of TRPA1 and calcium‐activated potassium channel subunit α‐1 (KCNMA1), thus acting as a potential therapeutic alternative for treating neuropathic pain.\\n81\\n\\n\",\n \"miRNAs regulate diverse pain‐related mediators, protein kinases, and structural proteins along the pain processing pathways. For HIV‐associated symptomatic distal sensory polyneuropathy and neuropathic pain, serum miR‐455‐3p acted as a potential biomarker, possibly targeting multiple genes involved in peripheral neuropathic pain, such as nerve growth factor (NGF) and related genes.\\n82\\n Brain‐derived neurotrophic factor (BDNF), another well‐recognized pain mediator, is a common miR‐1, ‐183, and ‐206 target.\\n83\\n, \\n84\\n, \\n85\\n After nerve injury, several miRNAs, including miR‐19a, ‐301, and ‐132, were downregulated, and their target methyl cytosine–guanine dinucleotide (CpG)‐binding protein 2 (MeCP2) expression was increased, leading to concomitant BDNF upregulation.\\n86\\n miR‐30c‐5p was upregulated in the serum and CSF of patients with chronic peripheral ischemia.\\n16\\n A miR‐30c‐5p inhibitor intraventricular injection postponed neuropathic pain development and fully reversed hyperalgesia in rodents. Transforming growth factor beta (TGF‐β) participates in the effects of miR‐30c‐5p, which refers to the endogenous opioid analgesic system.\\n16\\n\\n\\nmiRNAs dysregulate specific pain‐related protein kinases. For example, in DNP rats, miR‐133a‐3p was dysregulated in the sciatic nerve, interacting with p‐p38 MAPK to participate in the development of neuropathic pain.\\n87\\n Via a methyl CpG‐binding domain and transcriptional repressor domain, MeCP2 acts as a transcriptional repressor, and its overexpression improves neuropathic pain, potentiating an anti‐nociceptive effect of MeCP2. The authors also noted that MeCP2 expression changed post‐transcriptionally, and its mRNA level did not significantly change after SNI. However, the protein level was upregulated. During the development period of neuropathic pain, phospho‐cAMP response element‐binding protein (p‐CREB) elevated rapidly but returned 3–7 days after SNI, concomitant with miR‐132 downregulation, which targets MeCP2 and inhibits its expression post‐transcriptionally.\\n88\\n SNL decreased miR‐200b and miR‐429 in NAc neurons, along with an upregulation of their target (DNMT3a).\\n89\\n Further mechanism studies found that DNMT3a in the NAc was expressed in NR1 immunoreactive neurons, suggesting the dysregulation of ‘mesolimbic motivation circuitry’ for neuropathic pain development.\\nThe CCI induced time‐dependent miR‐1 downregulation in injured sciatic nerves. This change in expression was related to the upregulation and translocation of the miR‐1‐targeted connexin 43 (Cx43), the major connexin of astrocytes.\\n83\\n miR‐1 mimics could reduce Cx43 expression in cultured human glioblastoma cells. However, miR‐1 mimics intraneural transfection failed to alter Cx43 protein expression and did not improve pain behavior. The authors attributed such treatment failure to insufficient inhibition of miR‐1 for Cx43 via intraneural injection. Alternatively, there were regulatory mechanisms for Cx43 in vivo other than miR‐1.\\n90\\n However, miR‐1 in the DRG was DE according to peripheral nerve injury type. CCI, pSNL, and sural nerve injury downregulated miR‐1. However, axotomy of the sciatic nerve and tibial nerve injury increased its expression in the DRG.\\n83\\n, \\n91\\n, \\n92\\n As previously described, miR‐1 was also upregulated following capsaicin treatment and bone cancer pain.\\n91\\n, \\n93\\n Additionally, miR‐1 downregulation inhibited bone cancer pain. Collectively, the action of miR‐1 on pain seems to be complex and stimulus‐dependent.\\nNotably, specific miRNAs can promote neuropathic pain development via diverse mechanisms. For example, beta‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1), a membrane protease essential for myelination, was downregulated after miR‐15b overexpression in vitro or in the DRG of chemotherapy‐related neuropathic pain rats.\\n94\\n BACE1‐mediated neuregulin 1 reduction decreases nerve conduction velocity. In addition, BACE1 modulated Navβ2 subunit expression and neuronal activity and regulated inflammation‐related TNFR expression.\",\n \"Neurotransmission excitatory and inhibitory imbalances in the spinal cord also contribute to neuropathic pain development. For example, miR‐500 increased to regulate glutamic acid decarboxylase 67 (GAD67) expression and target the specific site of Gad1 in the dorsal horn.\\n95\\n GAD67 expression reduction inhibited the function of GABAergic neurons and the resultant inhibitory synaptic transmission dysregulation contributed to neuropathic pain development. In addition, miR‐23b is crucial for improving neuropathic pain in the injured spinal cord by downregulating its target gene, Nox4, which further normalizes glutamic acid decarboxylase 65/67 (GAD65/67) expression and protects GABAergic neurons from apoptosis.\\n96\\n Microarray analysis showed that miR‐539 was prominently reduced in the contralateral anterior cingulate cortex (ACC) after CCI, which is related to enhanced NR2B protein expression. Injecting miR‐539 mimics into the contralateral ACC attenuated CCI‐evoked mechanical hyperalgesia, suggesting that the N‐methyl‐D‐aspartate (NMDA) receptor NR2B subunit regulates neuropathic pain.\\n97\\n\\n\",\n \"We mainly focused on exosomal miR‐21 with regard to EV miRNAs in neuropathic pain. As aforementioned, SNL, DRT, or VRT could upregulate miR‐21 in the injured DRG.\\n22\\n Notably, miR‐21 was also increased in serum exosomes from nerve‐ligated mice.\\n98\\n In another in‐depth study, macrophages readily took up pure sensory neuron‐derived exosomes encompassing miR‐21‐5p to promote a pro‐inflammatory phenotype.\\n99\\n Either intrathecal miR‐21‐5p antagomir or miR‐21 conditional deletion in sensory neurons ameliorated hyperalgesia and macrophage recruitment in the DRG.\\nUnlike the miRNA target gene inhibitory mechanism, miR‐21 acts as an endogenous toll‐like receptor 8 (TLR8) ligand, leading to neuropathic pain development.\\n100\\n TLR8, a nucleic acid‐sensing receptor, is located in the endosomes and lysosomes, leading to ERK‐mediated inflammatory mediator production and neuronal activation after SNL. Although miR‐21 and TLR8 are only distributed in small‐ and medium‐sized neurons, miR‐21 can also be derived from large‐sized neurons and reach TLR8 in the endosomes of other types of neurons.\\n101\\n Similar to miR‐21, DRG sensory neurons secreted miR‐23a‐enriched EVs following nerve injury and were taken up by macrophages to enhance M1 polarization in vitro. A20, an NF‐κB signaling pathway inhibitor, is a verified miR‐23a target gene.\\n102\\n Moreover, intrathecally delivering EVs‐miR‐23a antagomir attenuated neuropathic hyperalgesia and reduced M1 macrophages by inhibiting A20 to activate NF‐κB signaling.\",\n \"lncRNAs and circRNAs are essential in neuropathic pain development by acting as AS RNA or miRNA sponges, epigenetically regulating pain‐related molecules expression, or modulating miRNA processing (Table 2).\\nLong non‐coding RNAs (lncRNAs) and circular non‐coding RNAs (circRNAs) expression change for the development of neuropathic pain\\nAbbreviations: AQP4, aquaporin 4; AS RNA, antisense RNA; CCI, chronic constriction injury; CDK4, cyclin‐dependent kinase 4; CDK6, cyclin‐dependent kinase 6; DNP, diabetic neuropathic pain; DRG, dorsal root ganglion; ENO1, enolase 1; EphB1, ephrin type‐b 1; EZH2, enhancer of zeste homolog 2; HMGB1, high mobility group box 1; NAT, natural antisense transcript; NOTCH2, NOTCH receptor 2; NP, neuropathic pain; pSNL, partial sciatic nerve ligation; pSTAT3, phosphorylated signal transducer and activator of transcription 3; SCI, spinal cord injury; SGC, satellite glia cell; SNL, spinal nerve ligation; STAT3, signal transducer and activator of transcription 3; TLR5, toll‐like receptor 5; TNFAIP1, tumor necrosis factor alpha‐induced protein 1; TRPV1, transient receptor potential vanilloid 1; XIST, X‐inactive specific transcript; ZEB1, zinc finger E‐box binding homeobox 1.\\n\\nlncRNAs act as AS RNA\\n After peripheral nerve injury, Egr2‐AS‐RNA is upregulated in Schwann cells.\\n103\\n On the early growth response 2 (Egr2) promoter, Egr2‐AS‐RNA recruits an epigenetic silencing complex to downregulate Egr2, essential for peripheral myelination. Ectopic Egr2‐AS‐RNA expression in DRG cultures downregulates Egr2 mRNA and induces demyelination. In vivo, Egr2‐AS‐RNA inhibition reverts Egr2‐related gene expression and delays demyelination.\\nVoltage‐gated potassium channel (Kv) Kcna2‐AS‐RNA is an endogenous, highly conserved, and widely explored lncRNA in neuropathic pain.\\n104\\n It is a natural antisense transcript (NAT), distributed in the cytoplasm and targets Kcna2 mRNA, which encodes the pain regulation‐related membrane Kv1.2 subunit. Kcna2‐AS‐RNA was time‐dependently upregulated in the inflicted rat DRG after nerve injury. It decreased the total Kv current, upregulated DRG neurons excitability, and produced neuropathic pain symptoms.\\nThe complementary strand of DNA opposite the Scn9a gene encodes Scn9a NAT, another antisense lncRNA expressed in the DRG.\\n105\\n Scn9a NAT is suggested to be a negative regulator of Scn9a mRNA. Scn9a NAT overexpression inhibited Scn9a mRNA, its encoded protein Nav1.7, and Nav1.7 currents in DRG neurons. However, Scn9a NAT and Scn9a mRNA levels did not significantly change in the injured DRG until 2 weeks after nerve injury. More work is required to determine whether NAT can confer analgesia and reduce pain in the neuropathic pain state.\\nAfter peripheral nerve injury, Egr2‐AS‐RNA is upregulated in Schwann cells.\\n103\\n On the early growth response 2 (Egr2) promoter, Egr2‐AS‐RNA recruits an epigenetic silencing complex to downregulate Egr2, essential for peripheral myelination. Ectopic Egr2‐AS‐RNA expression in DRG cultures downregulates Egr2 mRNA and induces demyelination. In vivo, Egr2‐AS‐RNA inhibition reverts Egr2‐related gene expression and delays demyelination.\\nVoltage‐gated potassium channel (Kv) Kcna2‐AS‐RNA is an endogenous, highly conserved, and widely explored lncRNA in neuropathic pain.\\n104\\n It is a natural antisense transcript (NAT), distributed in the cytoplasm and targets Kcna2 mRNA, which encodes the pain regulation‐related membrane Kv1.2 subunit. Kcna2‐AS‐RNA was time‐dependently upregulated in the inflicted rat DRG after nerve injury. It decreased the total Kv current, upregulated DRG neurons excitability, and produced neuropathic pain symptoms.\\nThe complementary strand of DNA opposite the Scn9a gene encodes Scn9a NAT, another antisense lncRNA expressed in the DRG.\\n105\\n Scn9a NAT is suggested to be a negative regulator of Scn9a mRNA. Scn9a NAT overexpression inhibited Scn9a mRNA, its encoded protein Nav1.7, and Nav1.7 currents in DRG neurons. However, Scn9a NAT and Scn9a mRNA levels did not significantly change in the injured DRG until 2 weeks after nerve injury. More work is required to determine whether NAT can confer analgesia and reduce pain in the neuropathic pain state.\\n\\nlncRNAs act as miRNA sponges lncRNAs may act as miRNA sponges to form the lncRNA‐miRNA‐mRNA axis and regulate target gene expression. For example, the lncRNA X‐inactive specific transcript (XIST) is upregulated in the dorsal horn of the spinal cord after CCI. By sponging miR‐137, ‐150, ‐154‐5p, and ‐544, XIST can inhibit the expression of corresponding targets, including TNF‐α‐induced protein 1 (TNFAIP1), ZEB1, TLR5, and STAT3.\\n106\\n, \\n107\\n, \\n108\\n, \\n109\\n TNFAIP1 activates the NF‐κB signaling pathway, while ZEB1, TLR5, and STAT3 are crucial in the neuroinflammatory response. XIST inhibition markedly ameliorates neuropathic pain development. In addition, lncRNA nuclear enriched abundant transcript 1 (NEAT1) was upregulated in the spinal cord dorsal horn to form NEAT1‐miR‐381‐high mobility group box 1 (HMGB1) and NEAT1–miR‐128‐3p–aquaporin 4 (AQP4) axes following CCI and SCI, respectively.\\n110\\n, \\n111\\n NEAT1 downregulation inhibited IL‐6, IL‐1β, and TNF‐α to improve neuropathic pain.\\nAlthough the function of circRNAs as miRNA sponges is still largely unknown, they have been termed as competing endogenous RNAs that bind to target miRNAs and regulate their function.\\n112\\n For example, circHIPK3 is a circRNA highly enriched in serum from DNP patients and in the DRG from DNP rats.\\n113\\n circHIPK3 sponges miR‐124 to promote neuroinflammation, pointing to the involvement of the circHIPK3–miR‐124 axis during DNP. circRNA ciRS‐7 participates in neuropathic pain progression by sponging miR‐135a‐5p to regulate autophagy and inflammation in the spinal cord of CCI rats.\\n114\\n Besides, cirZNF609–miR‐22‐3p‐enolase 1 (ENO1) and circ_0005075–miR‐151a‐3p‐NOTCH receptor 2 (NOTCH2) regulatory axes upregulate inflammatory factor expression and promote neuropathic pain development in the spinal cord after CCI.\\n115\\n, \\n116\\n\\n\\nlncRNAs may act as miRNA sponges to form the lncRNA‐miRNA‐mRNA axis and regulate target gene expression. For example, the lncRNA X‐inactive specific transcript (XIST) is upregulated in the dorsal horn of the spinal cord after CCI. By sponging miR‐137, ‐150, ‐154‐5p, and ‐544, XIST can inhibit the expression of corresponding targets, including TNF‐α‐induced protein 1 (TNFAIP1), ZEB1, TLR5, and STAT3.\\n106\\n, \\n107\\n, \\n108\\n, \\n109\\n TNFAIP1 activates the NF‐κB signaling pathway, while ZEB1, TLR5, and STAT3 are crucial in the neuroinflammatory response. XIST inhibition markedly ameliorates neuropathic pain development. In addition, lncRNA nuclear enriched abundant transcript 1 (NEAT1) was upregulated in the spinal cord dorsal horn to form NEAT1‐miR‐381‐high mobility group box 1 (HMGB1) and NEAT1–miR‐128‐3p–aquaporin 4 (AQP4) axes following CCI and SCI, respectively.\\n110\\n, \\n111\\n NEAT1 downregulation inhibited IL‐6, IL‐1β, and TNF‐α to improve neuropathic pain.\\nAlthough the function of circRNAs as miRNA sponges is still largely unknown, they have been termed as competing endogenous RNAs that bind to target miRNAs and regulate their function.\\n112\\n For example, circHIPK3 is a circRNA highly enriched in serum from DNP patients and in the DRG from DNP rats.\\n113\\n circHIPK3 sponges miR‐124 to promote neuroinflammation, pointing to the involvement of the circHIPK3–miR‐124 axis during DNP. circRNA ciRS‐7 participates in neuropathic pain progression by sponging miR‐135a‐5p to regulate autophagy and inflammation in the spinal cord of CCI rats.\\n114\\n Besides, cirZNF609–miR‐22‐3p‐enolase 1 (ENO1) and circ_0005075–miR‐151a‐3p‐NOTCH receptor 2 (NOTCH2) regulatory axes upregulate inflammatory factor expression and promote neuropathic pain development in the spinal cord after CCI.\\n115\\n, \\n116\\n\\n\\n\\nlncRNAs epigenetically regulate pain‐related molecules expression In addition to acting as a miRNA sponge, lncRNAs can epigenetically regulate the expression of pain‐related molecules. For example, peripheral nerve injury decreases the expression of DRG‐specifically enriched lncRNAs (DS‐lncRNAs) in the injured DRG. Restoring DS‐lncRNAs blocks nerve injury‐induced increases in euchromatic histone lysine N‐methyltransferase 2 (Ehmt2) mRNA and its encoding protein G9a, reverses G9a‐related decreases in opioid receptors and Kcna2 in the injured DRG, and ameliorates nerve injury‐induced pain hypersensitivity.\\n117\\n In addition, transcriptome screening in the DRG of DNP rats\\n118\\n has identified diverse dysregulated lncRNAs, including uc.48+,\\n119\\n BC168687,\\n120\\n, \\n121\\n NONRATT021972,\\n31\\n, \\n122\\n, \\n123\\n MRAK009713,\\n124\\n and Lncenc1.\\n125\\n The expression of uc.48+, BC168687, NONRATT021972, MRAK009713, and Lncenc1 was prominently higher in the DRG of neuropathic pain rats.\\n119\\n, \\n120\\n, \\n121\\n, \\n122\\n, \\n123\\n, \\n124\\n, \\n125\\n Blocking the upregulation via intrathecal or intravenous administration of the corresponding small‐interfering RNA (siRNA) may alleviate neuropathic pain by inhibiting the excitatory transmission mediated by purinergic receptors,\\n119\\n, \\n120\\n, \\n122\\n, \\n123\\n, \\n124\\n TNF‐α‐related pathways,\\n31\\n transient receptor potential vanilloid 1 (TRPV1),\\n121\\n or EZH2.\\n125\\n\\n\\nAs demonstrated by a luciferase assay and RNA‐binding protein immunoprecipitation, lncRNA small nucleolar RNA host gene 1 (SNHG1) can induce neuropathic pain in the spinal cord by binding to the promoter region of cyclin‐dependent kinase 4 (CDK4), stimulating its expression.\\n126\\n SNHG1 knockdown alleviated neuropathic pain development, and SNHG1 overexpression was able to induce neuropathic pain. Similarly, lncRNA PKIA‐AS1 participates in SNL‐induced neuropathic pain by downregulating DNA methyltransferase 1‐catalyzed cyclin‐dependent kinase 6 (DNMT1‐catalyzed) CDK6 promoter methylation and regulating CDK6.\\n127\\n Cyclin‐dependent kinases (CDKs) transcriptionally enhanced pro‐inflammatory gene expression during the G1 cell phase. Furthermore, CDK6 recruitment to the nuclear chromatin fraction by cytokines is related to NF‐κB, STAT, and activator protein 1 (AP‐1) activation to induce neuroinflammation.\\n128\\n Other than the DRG, lncRNA Kcna2‐AS‐RNA was also highly expressed in the spinal cord of postherpetic neuralgia rats, and its downregulation alleviated neuropathic pain by reducing phospho‐STAT3 (pSTAT3) translocation from the cytoplasm to the nucleus and then inhibiting spinal astrocytes activation.\\n129\\n\\n\\nIn addition to acting as a miRNA sponge, lncRNAs can epigenetically regulate the expression of pain‐related molecules. For example, peripheral nerve injury decreases the expression of DRG‐specifically enriched lncRNAs (DS‐lncRNAs) in the injured DRG. Restoring DS‐lncRNAs blocks nerve injury‐induced increases in euchromatic histone lysine N‐methyltransferase 2 (Ehmt2) mRNA and its encoding protein G9a, reverses G9a‐related decreases in opioid receptors and Kcna2 in the injured DRG, and ameliorates nerve injury‐induced pain hypersensitivity.\\n117\\n In addition, transcriptome screening in the DRG of DNP rats\\n118\\n has identified diverse dysregulated lncRNAs, including uc.48+,\\n119\\n BC168687,\\n120\\n, \\n121\\n NONRATT021972,\\n31\\n, \\n122\\n, \\n123\\n MRAK009713,\\n124\\n and Lncenc1.\\n125\\n The expression of uc.48+, BC168687, NONRATT021972, MRAK009713, and Lncenc1 was prominently higher in the DRG of neuropathic pain rats.\\n119\\n, \\n120\\n, \\n121\\n, \\n122\\n, \\n123\\n, \\n124\\n, \\n125\\n Blocking the upregulation via intrathecal or intravenous administration of the corresponding small‐interfering RNA (siRNA) may alleviate neuropathic pain by inhibiting the excitatory transmission mediated by purinergic receptors,\\n119\\n, \\n120\\n, \\n122\\n, \\n123\\n, \\n124\\n TNF‐α‐related pathways,\\n31\\n transient receptor potential vanilloid 1 (TRPV1),\\n121\\n or EZH2.\\n125\\n\\n\\nAs demonstrated by a luciferase assay and RNA‐binding protein immunoprecipitation, lncRNA small nucleolar RNA host gene 1 (SNHG1) can induce neuropathic pain in the spinal cord by binding to the promoter region of cyclin‐dependent kinase 4 (CDK4), stimulating its expression.\\n126\\n SNHG1 knockdown alleviated neuropathic pain development, and SNHG1 overexpression was able to induce neuropathic pain. Similarly, lncRNA PKIA‐AS1 participates in SNL‐induced neuropathic pain by downregulating DNA methyltransferase 1‐catalyzed cyclin‐dependent kinase 6 (DNMT1‐catalyzed) CDK6 promoter methylation and regulating CDK6.\\n127\\n Cyclin‐dependent kinases (CDKs) transcriptionally enhanced pro‐inflammatory gene expression during the G1 cell phase. Furthermore, CDK6 recruitment to the nuclear chromatin fraction by cytokines is related to NF‐κB, STAT, and activator protein 1 (AP‐1) activation to induce neuroinflammation.\\n128\\n Other than the DRG, lncRNA Kcna2‐AS‐RNA was also highly expressed in the spinal cord of postherpetic neuralgia rats, and its downregulation alleviated neuropathic pain by reducing phospho‐STAT3 (pSTAT3) translocation from the cytoplasm to the nucleus and then inhibiting spinal astrocytes activation.\\n129\\n\\n\\n\\nlncRNAs modulate miRNA processing Specific lncRNAs are essential in neuropathic pain by modulating miRNA processing. For example, the transcribed ultraconserved lncRNA uc.153 was prominently increased in the spinal cord of rats with CCI neuropathic pain. uc.153 knockdown reversed CCI‐induced pain behaviors and spinal neuronal hypersensitivity. Mechanistically, uc.153 negatively modulated Dicer‐mediated pre‐miR‐182‐5p processing and inhibited maturation. Meanwhile, spinal miR‐182‐5p downregulation increased the expression of its target, ephrin type‐b (EphB1), and p‐NR2B (phosphorylated N‐methyl‐D‐aspartate receptor (NMDAR) 2B subunit) expression and facilitated hyperalgesia.\\n130\\n\\n\\nCollectively, lncRNAs are crucial in neuropathic pain via diverse mechanisms. Notably, one lncRNA may act with more than one mechanism to regulate target gene expression. For example, SNL upregulated circAnks1a in the cytoplasm and nucleus. circAnks1a enhances the interplay between Y‐box‐binding protein 1 (YBX1) and transportin‐1 to facilitate YBX1 nuclear translocation in the cytoplasm. Meanwhile, in the nucleus, circAnks1a combines with the Vegfb promoter and recruits YBX1 to the Vegfb promoter to enhance its transcription. Additionally, cytoplasmic circAnks1a sponges miR‐324‐3p to regulate vascular endothelial growth factor B (VEGFB) expression. VEGFB binding to its receptor results in the activation of various downstream targets, including p38‐MAPK, PKB/AKT (protein kinase B), extracellular signal‐regulated kinase (ERK)/MAPK, and phosphoinositide 3‐kinase (PI3K).\\n131\\n Therefore, VEGFB upregulation increases dorsal horn neuron excitability and contributes to pain hypersensitivity after nerve injury.\\n132\\n\\n\\nWe also noted that specific lncRNAs were DE following various nerve injuries. For example, lncRNA Malat1 was upregulated after CCI to sponge miR‐206, and Malat1 suppression delayed neuropathic pain progression via miR‐206‐ZEB2 axis‐mediated neuroinflammation inhibition.\\n133\\n Conversely, in rats with complete brachial plexus avulsion‐induced neuropathic pain, Malat1 decreased in spinal cord neurons, and such downregulation increased neuronal spontaneous electrical activity via calcium flux regulation.\\n134\\n The reason for such distinctive Malat1 expression following different pain models is still unclear, and further research is required.\\nSpecific lncRNAs are essential in neuropathic pain by modulating miRNA processing. For example, the transcribed ultraconserved lncRNA uc.153 was prominently increased in the spinal cord of rats with CCI neuropathic pain. uc.153 knockdown reversed CCI‐induced pain behaviors and spinal neuronal hypersensitivity. Mechanistically, uc.153 negatively modulated Dicer‐mediated pre‐miR‐182‐5p processing and inhibited maturation. Meanwhile, spinal miR‐182‐5p downregulation increased the expression of its target, ephrin type‐b (EphB1), and p‐NR2B (phosphorylated N‐methyl‐D‐aspartate receptor (NMDAR) 2B subunit) expression and facilitated hyperalgesia.\\n130\\n\\n\\nCollectively, lncRNAs are crucial in neuropathic pain via diverse mechanisms. Notably, one lncRNA may act with more than one mechanism to regulate target gene expression. For example, SNL upregulated circAnks1a in the cytoplasm and nucleus. circAnks1a enhances the interplay between Y‐box‐binding protein 1 (YBX1) and transportin‐1 to facilitate YBX1 nuclear translocation in the cytoplasm. Meanwhile, in the nucleus, circAnks1a combines with the Vegfb promoter and recruits YBX1 to the Vegfb promoter to enhance its transcription. Additionally, cytoplasmic circAnks1a sponges miR‐324‐3p to regulate vascular endothelial growth factor B (VEGFB) expression. VEGFB binding to its receptor results in the activation of various downstream targets, including p38‐MAPK, PKB/AKT (protein kinase B), extracellular signal‐regulated kinase (ERK)/MAPK, and phosphoinositide 3‐kinase (PI3K).\\n131\\n Therefore, VEGFB upregulation increases dorsal horn neuron excitability and contributes to pain hypersensitivity after nerve injury.\\n132\\n\\n\\nWe also noted that specific lncRNAs were DE following various nerve injuries. For example, lncRNA Malat1 was upregulated after CCI to sponge miR‐206, and Malat1 suppression delayed neuropathic pain progression via miR‐206‐ZEB2 axis‐mediated neuroinflammation inhibition.\\n133\\n Conversely, in rats with complete brachial plexus avulsion‐induced neuropathic pain, Malat1 decreased in spinal cord neurons, and such downregulation increased neuronal spontaneous electrical activity via calcium flux regulation.\\n134\\n The reason for such distinctive Malat1 expression following different pain models is still unclear, and further research is required.\",\n \"After peripheral nerve injury, Egr2‐AS‐RNA is upregulated in Schwann cells.\\n103\\n On the early growth response 2 (Egr2) promoter, Egr2‐AS‐RNA recruits an epigenetic silencing complex to downregulate Egr2, essential for peripheral myelination. Ectopic Egr2‐AS‐RNA expression in DRG cultures downregulates Egr2 mRNA and induces demyelination. In vivo, Egr2‐AS‐RNA inhibition reverts Egr2‐related gene expression and delays demyelination.\\nVoltage‐gated potassium channel (Kv) Kcna2‐AS‐RNA is an endogenous, highly conserved, and widely explored lncRNA in neuropathic pain.\\n104\\n It is a natural antisense transcript (NAT), distributed in the cytoplasm and targets Kcna2 mRNA, which encodes the pain regulation‐related membrane Kv1.2 subunit. Kcna2‐AS‐RNA was time‐dependently upregulated in the inflicted rat DRG after nerve injury. It decreased the total Kv current, upregulated DRG neurons excitability, and produced neuropathic pain symptoms.\\nThe complementary strand of DNA opposite the Scn9a gene encodes Scn9a NAT, another antisense lncRNA expressed in the DRG.\\n105\\n Scn9a NAT is suggested to be a negative regulator of Scn9a mRNA. Scn9a NAT overexpression inhibited Scn9a mRNA, its encoded protein Nav1.7, and Nav1.7 currents in DRG neurons. However, Scn9a NAT and Scn9a mRNA levels did not significantly change in the injured DRG until 2 weeks after nerve injury. More work is required to determine whether NAT can confer analgesia and reduce pain in the neuropathic pain state.\",\n \"lncRNAs may act as miRNA sponges to form the lncRNA‐miRNA‐mRNA axis and regulate target gene expression. For example, the lncRNA X‐inactive specific transcript (XIST) is upregulated in the dorsal horn of the spinal cord after CCI. By sponging miR‐137, ‐150, ‐154‐5p, and ‐544, XIST can inhibit the expression of corresponding targets, including TNF‐α‐induced protein 1 (TNFAIP1), ZEB1, TLR5, and STAT3.\\n106\\n, \\n107\\n, \\n108\\n, \\n109\\n TNFAIP1 activates the NF‐κB signaling pathway, while ZEB1, TLR5, and STAT3 are crucial in the neuroinflammatory response. XIST inhibition markedly ameliorates neuropathic pain development. In addition, lncRNA nuclear enriched abundant transcript 1 (NEAT1) was upregulated in the spinal cord dorsal horn to form NEAT1‐miR‐381‐high mobility group box 1 (HMGB1) and NEAT1–miR‐128‐3p–aquaporin 4 (AQP4) axes following CCI and SCI, respectively.\\n110\\n, \\n111\\n NEAT1 downregulation inhibited IL‐6, IL‐1β, and TNF‐α to improve neuropathic pain.\\nAlthough the function of circRNAs as miRNA sponges is still largely unknown, they have been termed as competing endogenous RNAs that bind to target miRNAs and regulate their function.\\n112\\n For example, circHIPK3 is a circRNA highly enriched in serum from DNP patients and in the DRG from DNP rats.\\n113\\n circHIPK3 sponges miR‐124 to promote neuroinflammation, pointing to the involvement of the circHIPK3–miR‐124 axis during DNP. circRNA ciRS‐7 participates in neuropathic pain progression by sponging miR‐135a‐5p to regulate autophagy and inflammation in the spinal cord of CCI rats.\\n114\\n Besides, cirZNF609–miR‐22‐3p‐enolase 1 (ENO1) and circ_0005075–miR‐151a‐3p‐NOTCH receptor 2 (NOTCH2) regulatory axes upregulate inflammatory factor expression and promote neuropathic pain development in the spinal cord after CCI.\\n115\\n, \\n116\\n\\n\",\n \"In addition to acting as a miRNA sponge, lncRNAs can epigenetically regulate the expression of pain‐related molecules. For example, peripheral nerve injury decreases the expression of DRG‐specifically enriched lncRNAs (DS‐lncRNAs) in the injured DRG. Restoring DS‐lncRNAs blocks nerve injury‐induced increases in euchromatic histone lysine N‐methyltransferase 2 (Ehmt2) mRNA and its encoding protein G9a, reverses G9a‐related decreases in opioid receptors and Kcna2 in the injured DRG, and ameliorates nerve injury‐induced pain hypersensitivity.\\n117\\n In addition, transcriptome screening in the DRG of DNP rats\\n118\\n has identified diverse dysregulated lncRNAs, including uc.48+,\\n119\\n BC168687,\\n120\\n, \\n121\\n NONRATT021972,\\n31\\n, \\n122\\n, \\n123\\n MRAK009713,\\n124\\n and Lncenc1.\\n125\\n The expression of uc.48+, BC168687, NONRATT021972, MRAK009713, and Lncenc1 was prominently higher in the DRG of neuropathic pain rats.\\n119\\n, \\n120\\n, \\n121\\n, \\n122\\n, \\n123\\n, \\n124\\n, \\n125\\n Blocking the upregulation via intrathecal or intravenous administration of the corresponding small‐interfering RNA (siRNA) may alleviate neuropathic pain by inhibiting the excitatory transmission mediated by purinergic receptors,\\n119\\n, \\n120\\n, \\n122\\n, \\n123\\n, \\n124\\n TNF‐α‐related pathways,\\n31\\n transient receptor potential vanilloid 1 (TRPV1),\\n121\\n or EZH2.\\n125\\n\\n\\nAs demonstrated by a luciferase assay and RNA‐binding protein immunoprecipitation, lncRNA small nucleolar RNA host gene 1 (SNHG1) can induce neuropathic pain in the spinal cord by binding to the promoter region of cyclin‐dependent kinase 4 (CDK4), stimulating its expression.\\n126\\n SNHG1 knockdown alleviated neuropathic pain development, and SNHG1 overexpression was able to induce neuropathic pain. Similarly, lncRNA PKIA‐AS1 participates in SNL‐induced neuropathic pain by downregulating DNA methyltransferase 1‐catalyzed cyclin‐dependent kinase 6 (DNMT1‐catalyzed) CDK6 promoter methylation and regulating CDK6.\\n127\\n Cyclin‐dependent kinases (CDKs) transcriptionally enhanced pro‐inflammatory gene expression during the G1 cell phase. Furthermore, CDK6 recruitment to the nuclear chromatin fraction by cytokines is related to NF‐κB, STAT, and activator protein 1 (AP‐1) activation to induce neuroinflammation.\\n128\\n Other than the DRG, lncRNA Kcna2‐AS‐RNA was also highly expressed in the spinal cord of postherpetic neuralgia rats, and its downregulation alleviated neuropathic pain by reducing phospho‐STAT3 (pSTAT3) translocation from the cytoplasm to the nucleus and then inhibiting spinal astrocytes activation.\\n129\\n\\n\",\n \"Specific lncRNAs are essential in neuropathic pain by modulating miRNA processing. For example, the transcribed ultraconserved lncRNA uc.153 was prominently increased in the spinal cord of rats with CCI neuropathic pain. uc.153 knockdown reversed CCI‐induced pain behaviors and spinal neuronal hypersensitivity. Mechanistically, uc.153 negatively modulated Dicer‐mediated pre‐miR‐182‐5p processing and inhibited maturation. Meanwhile, spinal miR‐182‐5p downregulation increased the expression of its target, ephrin type‐b (EphB1), and p‐NR2B (phosphorylated N‐methyl‐D‐aspartate receptor (NMDAR) 2B subunit) expression and facilitated hyperalgesia.\\n130\\n\\n\\nCollectively, lncRNAs are crucial in neuropathic pain via diverse mechanisms. Notably, one lncRNA may act with more than one mechanism to regulate target gene expression. For example, SNL upregulated circAnks1a in the cytoplasm and nucleus. circAnks1a enhances the interplay between Y‐box‐binding protein 1 (YBX1) and transportin‐1 to facilitate YBX1 nuclear translocation in the cytoplasm. Meanwhile, in the nucleus, circAnks1a combines with the Vegfb promoter and recruits YBX1 to the Vegfb promoter to enhance its transcription. Additionally, cytoplasmic circAnks1a sponges miR‐324‐3p to regulate vascular endothelial growth factor B (VEGFB) expression. VEGFB binding to its receptor results in the activation of various downstream targets, including p38‐MAPK, PKB/AKT (protein kinase B), extracellular signal‐regulated kinase (ERK)/MAPK, and phosphoinositide 3‐kinase (PI3K).\\n131\\n Therefore, VEGFB upregulation increases dorsal horn neuron excitability and contributes to pain hypersensitivity after nerve injury.\\n132\\n\\n\\nWe also noted that specific lncRNAs were DE following various nerve injuries. For example, lncRNA Malat1 was upregulated after CCI to sponge miR‐206, and Malat1 suppression delayed neuropathic pain progression via miR‐206‐ZEB2 axis‐mediated neuroinflammation inhibition.\\n133\\n Conversely, in rats with complete brachial plexus avulsion‐induced neuropathic pain, Malat1 decreased in spinal cord neurons, and such downregulation increased neuronal spontaneous electrical activity via calcium flux regulation.\\n134\\n The reason for such distinctive Malat1 expression following different pain models is still unclear, and further research is required.\",\n \"There are two main strategies for modulating miRNA function during pain treatment: upregulation or downregulation of specific miRNAs. miRNA mimics or virus‐based constructs were used to upregulate miRNA expression. miRNA inhibitors, miRNA sponges, or inhibition of a particular miRNA‐mRNA interaction has been applied to downregulate specific miRNAs. The design of lncRNA‐based therapeutics includes diverse approaches, such as post‐transcriptional inhibition of lncRNAs by antisense oligonucleotides or siRNA and steric blockades of lncRNA–protein interactions by small molecules and morpholino.\\n135\\n\\n\\nHowever, there is much work to be done before miRNA‐ and lncRNA‐based analgesics can be clinically used for various reasons, including their conservation among species, proper delivery, stability, off‐target effects, and potential activation of the immune system.\\nFirst, most available findings regarding the efficacy of miRNAs or lncRNAs during neuropathic pain are based on animal but not human studies. Whether such results can be extrapolated to humans and their translational potential is still unknown because of conservation among species.\\nSecond, the blood–brain barrier (BBB) is a practical challenge for delivering RNA‐based therapeutics into the central nervous system (CNS) via intravenous injection. Viral vectors, polypeptides, aptamers, and particular chemical modifications have been developed. Supplementing cholesterol molecules to the sense strand of a miRNA mimic or inhibitor has proven to be an efficient strategy. Cholesterol‐conjugated siRNAs showed better silencing potency than unconjugated siRNAs and presented a high efficacy for delivery to oligodendrocytes in the CNS.\\n136\\n Another proven method is the immunoliposome, a combination of liposomes, receptor‐targeted monoclonal antibodies, and the target molecules.\\n137\\n An immunoliposome nanocomplex has been reported to deliver therapeutic nucleic acids across the BBB into the deep brain by transferrin receptors.\\n138\\n In addition, intrathecal injections are a feasible approach in animal studies for neuropathic pain treatment. As reported, miR‐146a attenuated neuropathic pain partially by inhibiting TNF receptor‐associated factor 6 (TRAF6) and its downstream phospho‐JNK/C–C motif chemokine ligand 2 (pJNK/CCL2) signaling in the spinal cord.\\n139\\n Intrathecal injection of miR‐146a‐5p encapsulated nanoparticles provided an analgesic effect via NF‐κB and p38‐MAPK inhibition in spinal microglia.\\n140\\n In recent years, poly (D, L‐lactic‐co‐glycolic acid) (PLGA)‐nanoparticles have been applied to deliver siRNAs and plasmids into the spinal cord to treat neuropathic pain in rats.\\n141\\n PLGA copolymer is a promising US Food and Drug Administration (FDA)‐approved gene transmission material because of its biodegradability and biocompatibility in humans.\\n142\\n Intrathecal treatments with C‐X3‐C motif chemokine receptor 1 (CX3CR1), p38, p66shc siRNA encapsulated PLGA nanoparticles, or forkhead box P3 (Foxp3) plasmid‐encapsulated PLGA nanoparticles inhibit microglial activation and hyperalgesia in SNL rats.\\n141\\n, \\n143\\n, \\n144\\n, \\n145\\n\\n\\nExosomes are another promising delivery carrier for treating neuropathic pain. Exosomes are natural membranous microvesicles that carry RNAs, with the advantage of being efficient, cell‐free, and nonimmunogenicity. Intravenous injecting neuron‐targeted exosomes delivered the carried siRNAs to neurons, microglia, and oligodendrocytes to knock down specific gene expression in mice brains.\\n146\\n This approach enabled cell‐specific delivery of the siRNA‐cargo across the BBB. Mesenchymal stem cells (MSCs) are pluripotent stem cells with immunomodulatory, anti‐inflammatory, and nutritional properties. The treatment efficacy of exosomes derived from MSCs has been proven in neuropathic pain.\\n147\\n Intrathecal, local, or subcutaneous application of exosomes obtained from human umbilical cord MSCs could mitigate nerve injury‐induced hyperalgesia.\\n148\\n, \\n149\\n, \\n150\\n Furthermore, immunofluorescence results showed that most intrathecally injected exosomes could be found in injured peripheral axons, the DRG, and the spinal dorsal horn, potentiating a homing ability of exosomes.\\n148\\n\\n\\nThird, although miRNAs are relatively stable in vivo, they have long‐lasting efficacy and a high resistance to nucleolytic degradation compared to mRNAs. Particular chemical modifications are still required to prolong its half‐life or increase its stability, for example, by generating locked‐nucleic acids (LNAs). LNA modifications increase the RNA affinity of antisense oligonucleotides, presenting an excellent miRNA inhibitory activity at a low dosage.\\n151\\n Additionally, the LNA technique was used to synthesize highly stable aptamers.\\n152\\n\\n\\nFourth, the off‐target effect is another consideration. One miRNA may regulate multiple genes, and the off‐target potential of one miRNA may produce undesirable side effects.\\n153\\n, \\n154\\n\\n\\nFinally, activation of the immune system is a potential adverse event. Specific CpG motifs in oligonucleotides could trigger nonspecific immunological activity.\\n155\\n Therefore, RNA‐based therapeutics might also produce specific immune reactions after producing antibodies against the oligonucleotides.\",\n \"Recent human and animal studies have identified accumulating dysregulated miRNAs and lncRNAs in the serum or along pain processing pathways following peripheral nerve injury or noxious stimulation. Experimental studies have validated their essential role in neuropathic pain. miRNAs contribute to neuropathic pain development via neuroinflammation, autophagy, abnormal ion channel expression, regulating pain‐related mediators, protein kinases, structural proteins, neurotransmission excitatory–‐inhibitory imbalances, and exosome miRNA‐mediated neuron–glia communication. Meanwhile, lncRNAs and circRNAs are crucial in neuropathic pain development by acting as AS RNA and miRNA sponges, epigenetically regulating pain‐related molecules expression, or modulating miRNA processing. However, more work is required before miRNA‐ and lncRNA‐based analgesics can be clinically used for various reasons, including their conservation among species, proper delivery, stability, off‐target effects, and potential activation of the immune system.\",\n \"Ming Jiang: Investigation, Methodology. Yelong Wang: Manuscript preparation. Jing Wang: Figures and tables preparation. Shanwu Feng: Supervision, Funding acquisition. Xian Wang: Conceptualization, Funding acquisition.\",\n \"The authors declare no competing interest in this review.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,"conclusions",null,"COI-statement"],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"conclusions\",\n null,\n \"COI-statement\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["circRNAs","lncRNAs","mechanism study","miRNAs","neuropathic pain"],"string":"[\n \"circRNAs\",\n \"lncRNAs\",\n \"mechanism study\",\n \"miRNAs\",\n \"neuropathic pain\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nAccording to the International Association for the Study of Pain, neuropathic pain is the most severe chronic pain condition triggered by a lesion or disease of the somatosensory system. It is characteristic of hyperalgesia, allodynia, or spontaneous pain.\n1\n Neuropathic pain can have peripheral and central origins, with the former including neuropathic pain after peripheral nerve injury, trigeminal neuralgia, postherpetic neuralgia, painful radiculopathy, and painful polyneuropathy. In contrast, central neuropathic pain includes neuropathic pain after spinal cord or brain injury, multiple sclerosis, and chronic central post‐stroke pain. Approximately 7%–10% of the general population will experience neuropathic pain, with the majority not having satisfactory pain relief with current therapies, leading to great suffering for individuals and enormous economic and social burdens.\n2\n\n\nThe exact molecular mechanisms underlying neuropathic pain remain unclear, and elucidating them is crucial for developing mechanism‐based treatment strategies. One proposed mechanism involves altered gene or protein expression along the pain processing pathways. Therefore, understanding how genes or proteins are dysregulated may help us to find a way to normalize these abnormalities and treat neuropathic pain.\nIn recent years, accumulating evidence has suggested the essential role of non‐coding RNAs (ncRNAs) in various physiological and pathological procedures, such as embryonic development, inflammation, tumors, and respiratory and cardiovascular diseases.\n3\n ncRNAs have no protein‐coding potential, but they can govern gene or protein expression with diverse mechanisms. ncRNAs are extensively distributed in the peripheral and central nervous systems, including pain‐related structures.\n4\n Broad abnormal ncRNAs expression is observed following peripheral stimulation. These abnormalities are related to hyperalgesia during chronic pain development. The available data indicate that ncRNAs may be essential for hyperalgesia. In this review, we focus on microRNA (miRNA), long non‐coding RNA (lncRNA), and circular non‐coding RNA (circRNA) expression changes in neuropathic pain. Other types of ncRNAs are seldom reported in neuropathic pain and, thus, are not discussed herein. Notably, we will pay attention to their etiological role in the development of neuropathic pain and the current challenges and considerations for miRNA‐, lncRNA‐, and circRNA‐based therapeutics for neuropathic pain.\n\n\nBIOGENESIS AND FUNCTION OF miRNAs, lncRNAs, AND circRNAs\n:\nTypically, miRNA production involves three steps: cropping, exporting, and dicing. First, the miRNA gene is transcribed in the nucleus, mainly by RNA polymerase II.\n5\n The resulting primary miRNA transcript (pri‐miRNA) is several kilobases (kb) in length, with a specific stem–loop structure that harbors mature miRNAs in the stem. The mature miRNA is ‘cropped’ by Drosha and its interactor DGCR8 (DiGeorge syndrome critical region 8), which cleaves pri‐miRNA at the stem to produce pre‐miRNA,\n6\n 60–70 nucleotides (nt) in length with a hairpin structure. Then, exportin‐5 recognizes and exports pre‐miRNA from the nucleus to the cytoplasm.\n7\n In the cytoplasm, ribonuclease III (RNAse III), termed Dicer, further cleaves the pre‐miRNA to release double‐stranded miRNA with a length of ~22 nt.\n8\n This miRNA is unwound by an unknown helicase or cleaved by Argonaute (Ago) to form the RNA‐induced silencing complex.\n9\n One strand in the RNA duplex remains with Ago as a mature miRNA, and the other is degraded. The seed sequence of the miRNA incompletely or entirely combines with the target mRNA sequence, resulting in target mRNAs degradation or transcriptional regulation.\n10\n\n\nUnlike miRNAs, lncRNAs are mRNAs‐like transcripts ranging in length from 200 nt to 100 kb that lack prominent open reading frames.\n11\n The lncRNA cellular mechanism is highly related to their intracellular localization. lncRNAs control chromatin functions, transcription, and RNA processing in the nucleus and affect mRNA stability, translation, and cellular signaling in the cytoplasm.\n12\n Compared to lncRNAs, circRNAs are more stable because a single circRNA molecular ends can be covalently linked compared to linear RNA. circRNAs are evolutionarily conserved molecules that are essential in the post‐transcriptional modification of gene expression by acting as miRNAs sponges or interacting with transcription or translational machinery. Numerous lncRNAs and circRNAs are distributed within pain‐related regions and dysregulated after peripheral noxious stimulation. Moreover, functional studies have indicated that miRNAs, lncRNAs, and circRNAs participate in neuropathic pain development by regulating diverse pain‐related genes along the pain processing pathways.\n\n\nPERIPHERAL NERVE INJURY OR NOXIOUS STIMULI INDUCE EXTENSIVE miRNAs, lncRNAs, AND circRNAs EXPRESSION CHANGES\n:\n\nmiRNAs expression changes Microarray and deep‐sequencing analyses revealed that nerve injury or noxious stimuli could induce broad changes in miRNA expression in serum or along the pain processing pathways, including the perineal nerve, dorsal root ganglion (DRG), spinal cord, and supraspinal regions. For normal pain signal transmission, nerve injury or noxious stimuli are detected by nociceptors in the DRG or trigeminal ganglion (TG) and then transmitted to upstream neurons in the spinal dorsal horn. Subsequently, nociceptive stimuli are integrated, processed, and further transmitted ascending to specific supraspinal brain regions.\nHuman studies have identified as many as 1134 differentially expressed (DE) genes in the serum of individuals with or without neuropathic pain after spinal cord injury (SCI).\n13\n miR‐204‐5p, ‐519d‐3p, ‐20b‐5p, and ‐6838‐5p might act as promising biomarkers and intervention targets for preventing and therapizing neuropathic pain after SCI. In trigeminal neuralgia individuals, serum miR‐132‐3p, ‐146b‐5p, ‐155‐5p, and ‐384 levels were prominently increased compared with healthy controls.\n14\n Patients with painful peripheral neuropathy had higher miR‐21 level in the serum and sural nerve compared with healthy controls. Meanwhile, miR‐155 was reduced in the serum and inflicted lower leg skin.\n15\n Animal and human studies showed that miR‐30c‐5p was upregulated in the serum and cerebro spinal fluid (CSF) of sciatic nerve injury rats and neuropathic pain patients with chronic peripheral ischemia. The high expression of miR‐30c‐5p, together with other clinical parameters, might be used to predict neuropathic pain development in patients with chronic peripheral ischemia.\n16\n Additionally, an animal study showed that plasma‐derived DE extracellular vesicle (EV) miRNAs regulated processes that are essential for neuropathic pain development. Most DE EV miRNAs for inflammation suppression were downregulated, potentially acting as biomarkers and targets in neuropathic pain treatment.\n17\n\n\nIn addition, numerous DE miRNAs have been identified in the DRG. One week after spared nerve injury (SNI), 33 and 39 miRNAs in the DRG were upregulated and downregulated, respectively, with most DE miRNAs related to axon guidance, focal adhesion, Ras and Wnt signaling pathways.\n18\n Furthermore, nerve injury‐induced miRNAs expression was dynamic and time‐dependent,\n19\n, \n20\n implicating multiple regulatory mechanisms in neuropathic pain initiation and development. Nerve injury redistributes miRNAs from a uniform style within the DRG soma of non‐allodynic animals to preferential localization to peripheral neurons in allodynic animals.\n21\n Furthermore, either sciatic nerve ligation (SNL), DRG transaction (DRT), or ventral root transection (VRT) could upregulate miR‐21 and miR‐31 while downregulating miR‐668 and miR‐672 in the injured DRG,\n22\n implying that these miRNAs could be therapeutic targets for treating diverse types of neuropathic pain.\nThe spinal cord dorsal horn relays and modulates pain signals from the peripheral nociceptors to the supraspinal regions. In the spinal cord, numerous miRNA expression changes have been observed in chronic constriction injury (CCI) and diabetic neuropathic pain (DNP) rodents.\n23\n, \n24\n, \n25\n, \n26\n Previous research has suggested some miRNAs may be related to neuropathic pain development, for example, miR‐500, ‐221, and ‐21,\n25\n thus, acting as potential targets in its treatment.\n27\n\n\nCytoscape software constructed the miRNA–target gene regulatory network in the supraspinal region, including the nucleus accumbens (NAc), medial prefrontal cortex, and periaqueductal gray, between SNI and sham rats. Finally, four essential DE genes, includingCXCR2, IL12B, TNFSF8, and GRK1, and five miRNAs, including miR‐208a‐5p, −7688‐3p, −344f‐3p, −135b‐3p, and ‐135a‐2‐3p, were identified, indicating their essential roles in neuropathic pain pathogenesis.\n28\n Furthermore, in the prelimbic cortex of SNI rats, the DE miRNA–mRNA network pointed to molecules associated with inflammation.\n29\n DE miRNAs were also observed in the bilateral hippocampus of CCI rats. However, no significant difference was observed bilaterally in the hippocampus.\n30\n (Figure 1).\nmicroRNAs (miRNAs) expression change following peripheral nerve injury or noxious stimuli SCI, spinal cord injury; CSF, cerebrol spinal fluid; DE, differential expressed; EV, extravascular vesicle; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury; DRT, DRG transection; VRT, ventral root transection\nMicroarray and deep‐sequencing analyses revealed that nerve injury or noxious stimuli could induce broad changes in miRNA expression in serum or along the pain processing pathways, including the perineal nerve, dorsal root ganglion (DRG), spinal cord, and supraspinal regions. For normal pain signal transmission, nerve injury or noxious stimuli are detected by nociceptors in the DRG or trigeminal ganglion (TG) and then transmitted to upstream neurons in the spinal dorsal horn. Subsequently, nociceptive stimuli are integrated, processed, and further transmitted ascending to specific supraspinal brain regions.\nHuman studies have identified as many as 1134 differentially expressed (DE) genes in the serum of individuals with or without neuropathic pain after spinal cord injury (SCI).\n13\n miR‐204‐5p, ‐519d‐3p, ‐20b‐5p, and ‐6838‐5p might act as promising biomarkers and intervention targets for preventing and therapizing neuropathic pain after SCI. In trigeminal neuralgia individuals, serum miR‐132‐3p, ‐146b‐5p, ‐155‐5p, and ‐384 levels were prominently increased compared with healthy controls.\n14\n Patients with painful peripheral neuropathy had higher miR‐21 level in the serum and sural nerve compared with healthy controls. Meanwhile, miR‐155 was reduced in the serum and inflicted lower leg skin.\n15\n Animal and human studies showed that miR‐30c‐5p was upregulated in the serum and cerebro spinal fluid (CSF) of sciatic nerve injury rats and neuropathic pain patients with chronic peripheral ischemia. The high expression of miR‐30c‐5p, together with other clinical parameters, might be used to predict neuropathic pain development in patients with chronic peripheral ischemia.\n16\n Additionally, an animal study showed that plasma‐derived DE extracellular vesicle (EV) miRNAs regulated processes that are essential for neuropathic pain development. Most DE EV miRNAs for inflammation suppression were downregulated, potentially acting as biomarkers and targets in neuropathic pain treatment.\n17\n\n\nIn addition, numerous DE miRNAs have been identified in the DRG. One week after spared nerve injury (SNI), 33 and 39 miRNAs in the DRG were upregulated and downregulated, respectively, with most DE miRNAs related to axon guidance, focal adhesion, Ras and Wnt signaling pathways.\n18\n Furthermore, nerve injury‐induced miRNAs expression was dynamic and time‐dependent,\n19\n, \n20\n implicating multiple regulatory mechanisms in neuropathic pain initiation and development. Nerve injury redistributes miRNAs from a uniform style within the DRG soma of non‐allodynic animals to preferential localization to peripheral neurons in allodynic animals.\n21\n Furthermore, either sciatic nerve ligation (SNL), DRG transaction (DRT), or ventral root transection (VRT) could upregulate miR‐21 and miR‐31 while downregulating miR‐668 and miR‐672 in the injured DRG,\n22\n implying that these miRNAs could be therapeutic targets for treating diverse types of neuropathic pain.\nThe spinal cord dorsal horn relays and modulates pain signals from the peripheral nociceptors to the supraspinal regions. In the spinal cord, numerous miRNA expression changes have been observed in chronic constriction injury (CCI) and diabetic neuropathic pain (DNP) rodents.\n23\n, \n24\n, \n25\n, \n26\n Previous research has suggested some miRNAs may be related to neuropathic pain development, for example, miR‐500, ‐221, and ‐21,\n25\n thus, acting as potential targets in its treatment.\n27\n\n\nCytoscape software constructed the miRNA–target gene regulatory network in the supraspinal region, including the nucleus accumbens (NAc), medial prefrontal cortex, and periaqueductal gray, between SNI and sham rats. Finally, four essential DE genes, includingCXCR2, IL12B, TNFSF8, and GRK1, and five miRNAs, including miR‐208a‐5p, −7688‐3p, −344f‐3p, −135b‐3p, and ‐135a‐2‐3p, were identified, indicating their essential roles in neuropathic pain pathogenesis.\n28\n Furthermore, in the prelimbic cortex of SNI rats, the DE miRNA–mRNA network pointed to molecules associated with inflammation.\n29\n DE miRNAs were also observed in the bilateral hippocampus of CCI rats. However, no significant difference was observed bilaterally in the hippocampus.\n30\n (Figure 1).\nmicroRNAs (miRNAs) expression change following peripheral nerve injury or noxious stimuli SCI, spinal cord injury; CSF, cerebrol spinal fluid; DE, differential expressed; EV, extravascular vesicle; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury; DRT, DRG transection; VRT, ventral root transection\n\nlncRNAs and circRNAs expression changes Patients with type 2 DNP had a prominent higher expression of serum lncRNA NONRATT021972 and more severe neuropathic pain symptoms.\n31\n LINC01119 and LINC02447 in the peripheral blood of SCI patients were identified in pain pathways that were important for neuropathic pain development.\n32\n\n\nMicroarray analysis revealed that a nerve injury could induce time‐dependent lncRNA expression changes in the sciatic nerve.\n33\n lncRNA H19 was persistently upregulated in Schwann cells along the peripheral nerve, proximal and distal to the injured site.\n34\n In the DRG, the transcriptomic analysis identified 86 known and 26 novel lncRNAs genes to be DE after spared sciatic nerve injury.\n35\n Of these, rno‐Cntnap2 and AC111653.1 were essential in peripheral nerve regeneration and were involved in neuropathic pain.\nNext‐generation RNA sequencing showed 134 lncRNAs and 188 circRNAs were prominently changed 14 days after SNI in the spinal cord.\n36\n In addition, microarray analysis identified 1481 and 1096 DE lncRNAs and mRNAs, respectively, in the spinal cord dorsal horn of DNP rats. Of these, 289 neighboring and 57 overlapping lncRNA‐mRNA pairs, including ENSMUST00000150952‐Mbp and AK081017‐Usp15, have been suggested to participate in neuropathic pain development.\n37\n\n\ncircRNAs have characteristic circularized structures following the backsplicing of exons from antisense RNAs (AS RNAs) or mRNAs and, thus, are highly stable. Most circRNAs are highly conserved among species and lack translation potential, despite cap‐independent translation. A recent study showed that 363 and 106 circRNAs were significantly dysregulated in the ipsilateral dorsal horn after nerve injury.\n38\n (Figure 2).\nLong non‐coding RNA (lncRNAs) and circular non‐coding RNAs (circRNAs) expression change following peripheral nerve injury or noxious stimuli DNP, diabetic neuropathic pain; SCI, spinal cord injury; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury\nPatients with type 2 DNP had a prominent higher expression of serum lncRNA NONRATT021972 and more severe neuropathic pain symptoms.\n31\n LINC01119 and LINC02447 in the peripheral blood of SCI patients were identified in pain pathways that were important for neuropathic pain development.\n32\n\n\nMicroarray analysis revealed that a nerve injury could induce time‐dependent lncRNA expression changes in the sciatic nerve.\n33\n lncRNA H19 was persistently upregulated in Schwann cells along the peripheral nerve, proximal and distal to the injured site.\n34\n In the DRG, the transcriptomic analysis identified 86 known and 26 novel lncRNAs genes to be DE after spared sciatic nerve injury.\n35\n Of these, rno‐Cntnap2 and AC111653.1 were essential in peripheral nerve regeneration and were involved in neuropathic pain.\nNext‐generation RNA sequencing showed 134 lncRNAs and 188 circRNAs were prominently changed 14 days after SNI in the spinal cord.\n36\n In addition, microarray analysis identified 1481 and 1096 DE lncRNAs and mRNAs, respectively, in the spinal cord dorsal horn of DNP rats. Of these, 289 neighboring and 57 overlapping lncRNA‐mRNA pairs, including ENSMUST00000150952‐Mbp and AK081017‐Usp15, have been suggested to participate in neuropathic pain development.\n37\n\n\ncircRNAs have characteristic circularized structures following the backsplicing of exons from antisense RNAs (AS RNAs) or mRNAs and, thus, are highly stable. Most circRNAs are highly conserved among species and lack translation potential, despite cap‐independent translation. A recent study showed that 363 and 106 circRNAs were significantly dysregulated in the ipsilateral dorsal horn after nerve injury.\n38\n (Figure 2).\nLong non‐coding RNA (lncRNAs) and circular non‐coding RNAs (circRNAs) expression change following peripheral nerve injury or noxious stimuli DNP, diabetic neuropathic pain; SCI, spinal cord injury; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury\n\n\nmiRNAs expression changes:\nMicroarray and deep‐sequencing analyses revealed that nerve injury or noxious stimuli could induce broad changes in miRNA expression in serum or along the pain processing pathways, including the perineal nerve, dorsal root ganglion (DRG), spinal cord, and supraspinal regions. For normal pain signal transmission, nerve injury or noxious stimuli are detected by nociceptors in the DRG or trigeminal ganglion (TG) and then transmitted to upstream neurons in the spinal dorsal horn. Subsequently, nociceptive stimuli are integrated, processed, and further transmitted ascending to specific supraspinal brain regions.\nHuman studies have identified as many as 1134 differentially expressed (DE) genes in the serum of individuals with or without neuropathic pain after spinal cord injury (SCI).\n13\n miR‐204‐5p, ‐519d‐3p, ‐20b‐5p, and ‐6838‐5p might act as promising biomarkers and intervention targets for preventing and therapizing neuropathic pain after SCI. In trigeminal neuralgia individuals, serum miR‐132‐3p, ‐146b‐5p, ‐155‐5p, and ‐384 levels were prominently increased compared with healthy controls.\n14\n Patients with painful peripheral neuropathy had higher miR‐21 level in the serum and sural nerve compared with healthy controls. Meanwhile, miR‐155 was reduced in the serum and inflicted lower leg skin.\n15\n Animal and human studies showed that miR‐30c‐5p was upregulated in the serum and cerebro spinal fluid (CSF) of sciatic nerve injury rats and neuropathic pain patients with chronic peripheral ischemia. The high expression of miR‐30c‐5p, together with other clinical parameters, might be used to predict neuropathic pain development in patients with chronic peripheral ischemia.\n16\n Additionally, an animal study showed that plasma‐derived DE extracellular vesicle (EV) miRNAs regulated processes that are essential for neuropathic pain development. Most DE EV miRNAs for inflammation suppression were downregulated, potentially acting as biomarkers and targets in neuropathic pain treatment.\n17\n\n\nIn addition, numerous DE miRNAs have been identified in the DRG. One week after spared nerve injury (SNI), 33 and 39 miRNAs in the DRG were upregulated and downregulated, respectively, with most DE miRNAs related to axon guidance, focal adhesion, Ras and Wnt signaling pathways.\n18\n Furthermore, nerve injury‐induced miRNAs expression was dynamic and time‐dependent,\n19\n, \n20\n implicating multiple regulatory mechanisms in neuropathic pain initiation and development. Nerve injury redistributes miRNAs from a uniform style within the DRG soma of non‐allodynic animals to preferential localization to peripheral neurons in allodynic animals.\n21\n Furthermore, either sciatic nerve ligation (SNL), DRG transaction (DRT), or ventral root transection (VRT) could upregulate miR‐21 and miR‐31 while downregulating miR‐668 and miR‐672 in the injured DRG,\n22\n implying that these miRNAs could be therapeutic targets for treating diverse types of neuropathic pain.\nThe spinal cord dorsal horn relays and modulates pain signals from the peripheral nociceptors to the supraspinal regions. In the spinal cord, numerous miRNA expression changes have been observed in chronic constriction injury (CCI) and diabetic neuropathic pain (DNP) rodents.\n23\n, \n24\n, \n25\n, \n26\n Previous research has suggested some miRNAs may be related to neuropathic pain development, for example, miR‐500, ‐221, and ‐21,\n25\n thus, acting as potential targets in its treatment.\n27\n\n\nCytoscape software constructed the miRNA–target gene regulatory network in the supraspinal region, including the nucleus accumbens (NAc), medial prefrontal cortex, and periaqueductal gray, between SNI and sham rats. Finally, four essential DE genes, includingCXCR2, IL12B, TNFSF8, and GRK1, and five miRNAs, including miR‐208a‐5p, −7688‐3p, −344f‐3p, −135b‐3p, and ‐135a‐2‐3p, were identified, indicating their essential roles in neuropathic pain pathogenesis.\n28\n Furthermore, in the prelimbic cortex of SNI rats, the DE miRNA–mRNA network pointed to molecules associated with inflammation.\n29\n DE miRNAs were also observed in the bilateral hippocampus of CCI rats. However, no significant difference was observed bilaterally in the hippocampus.\n30\n (Figure 1).\nmicroRNAs (miRNAs) expression change following peripheral nerve injury or noxious stimuli SCI, spinal cord injury; CSF, cerebrol spinal fluid; DE, differential expressed; EV, extravascular vesicle; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury; DRT, DRG transection; VRT, ventral root transection\n\n\nlncRNAs and circRNAs expression changes:\nPatients with type 2 DNP had a prominent higher expression of serum lncRNA NONRATT021972 and more severe neuropathic pain symptoms.\n31\n LINC01119 and LINC02447 in the peripheral blood of SCI patients were identified in pain pathways that were important for neuropathic pain development.\n32\n\n\nMicroarray analysis revealed that a nerve injury could induce time‐dependent lncRNA expression changes in the sciatic nerve.\n33\n lncRNA H19 was persistently upregulated in Schwann cells along the peripheral nerve, proximal and distal to the injured site.\n34\n In the DRG, the transcriptomic analysis identified 86 known and 26 novel lncRNAs genes to be DE after spared sciatic nerve injury.\n35\n Of these, rno‐Cntnap2 and AC111653.1 were essential in peripheral nerve regeneration and were involved in neuropathic pain.\nNext‐generation RNA sequencing showed 134 lncRNAs and 188 circRNAs were prominently changed 14 days after SNI in the spinal cord.\n36\n In addition, microarray analysis identified 1481 and 1096 DE lncRNAs and mRNAs, respectively, in the spinal cord dorsal horn of DNP rats. Of these, 289 neighboring and 57 overlapping lncRNA‐mRNA pairs, including ENSMUST00000150952‐Mbp and AK081017‐Usp15, have been suggested to participate in neuropathic pain development.\n37\n\n\ncircRNAs have characteristic circularized structures following the backsplicing of exons from antisense RNAs (AS RNAs) or mRNAs and, thus, are highly stable. Most circRNAs are highly conserved among species and lack translation potential, despite cap‐independent translation. A recent study showed that 363 and 106 circRNAs were significantly dysregulated in the ipsilateral dorsal horn after nerve injury.\n38\n (Figure 2).\nLong non‐coding RNA (lncRNAs) and circular non‐coding RNAs (circRNAs) expression change following peripheral nerve injury or noxious stimuli DNP, diabetic neuropathic pain; SCI, spinal cord injury; SNL, spinal nerve ligation; DRG, dorsal root ganglion; SNI, spared nerve injury; CCI, chronic constriction injury\n\n\nSTUDYING miRNA NEUROPATHIC PAIN MECHANISMS\n:\nmiRNAs contribute to the development of neuropathic pain via diverse mechanisms, such as neuroinflammation, autophagy, abnormal ion channel expression, regulating pain‐related mediators, protein kinases, structural proteins, neurotransmission excitatory–inhibitory imbalances, and exosome miRNA‐mediated neuron–glia communication (Table 1).\nmiRNAs expression change for the development of neuropathic pain\nKv1.1, Kv1.4, Kv3.4, Kv4.3, Kv7.5,\nDPP10, Navβ1 ↓\np‐p38 MAPK ↑\nAbbreviations: ACC, anterior cingulate cortex; BACE1, beta‐site amyloid precursor protein‐cleaving enzyme 1; BDNF, brain‐derived neurotrophic factor; CCI, chronic constriction injury; CCR1, C–C chemokine receptor 1; CSF, cerebro spinal fluid; CSF1, colony‐stimulating factor‐1; CXCR4, chemokine CXC receptor 4; DNMT3a, DNA methyltransferase 3a; DNP, diabetic neuropathic pain; DRG, dorsal root ganglion; EV, extracellular vesicle; EZH2, enhancer of zeste homolog 2; GRK2, G protein‐coupled receptor kinases 2; IGF‐1/IGF‐1R, insulin‐like growth factor‐1/insulin‐like growth factor‐1 receptor; KCNMA1, calcium‐activated potassium channel subunit α‐1; MeCP2, methyl cytosine–guanine dinucleotide (CpG)‐binding protein 2; MIP‐1a, macrophage inflammatory protein‐1 alpha; NEFL, neurofilament light polypeptide; NF‐κB, nuclear factor – kappa B; NGF, nerve growth factor; NLRP3, NOD‐like receptor protein 3; NOX2, NADPH oxidase 2; NP, neuropathic pain; pSNL, partial sciatic nerve ligation; RAP1A, Ras‐related protein 1A; RASA1, RAS P21 protein activator 1; ROS, reactive oxygen species; S1PR1, sphingosine‐1‐phosphate receptor 1; SCI, spinal cord injury; Scn2b, β2 subunit of the voltage‐gated sodium channel; SIRT1, histone deacetylase sirtuin 1; SNL, spinal nerve ligation; SOCS1, suppressor of cytokine signaling 1; STAT3, signal transducer and activator of transcription 3; TLR4, toll‐like receptor 4; TNFR1, TNF receptor 1; TRPA1, transient receptor potential ankyrin 1; TXNIP, thioredoxin‐interacting protein; VTR, ventral root transection; ZEB1, zinc finger E‐box binding homeobox 1.\n\nmiRNAs regulate neuroinflammation in neuropathic pain development miRNA‐based epigenetic regulation is essential in neuroinflammation. miRNAs are predicted to regulate diverse neuroinflammation‐related targets along the pain processing pathways.\nAmong patients with neuropathic pain, miR‐101 decreased in the serum and sural nerve, which is related to nuclear factor‐kappa B (NF‐κB) signaling activation.\n39\n Meanwhile, serum miR‐124a and miR‐155 expression was upregulated. They were identified to inhibit histone deacetylase sirtuin 1 (SIRT1) in primary human cluster of differentiation 4 (CD4) (+) cells and induce their differentiation toward regulatory T cells (Tregs), thus reducing pain‐related inflammation.\n40\n Such miRNA–target interactions may act as an endogenous protective mechanism for neuropathic pain.\nmiR‐7a, expressed in small‐sized nociceptive DRG neurons, is downregulated after nerve injury, as reported, targeting neurofilament light polypeptides (NEFLs).\n41\n NEFL encodes a neuronal protein vital for neurofilament formation and increases signal transducer and activator of transcription 3 (STAT3) phosphorylation, which is highly related to cell differentiation and neuroinflammation. In diabetic peripheral neuropathic mice, miR‐590‐3p was downregulated to disinhibit Ras‐related protein 1A (RAP1A) in the DRG tissue and inhibit neural T cells infiltration.\n42\n Thus, exogenous miR‐590‐3p may be a potential alternative for neuropathic pain treatment. CCI downregulated miR‐140 and miR‐144 expression in the DRG. Intrathecally injected miR‐140 and miR‐144 agomir decreased inflammatory factor secretion and ameliorated hyperalgesia by targeting sphingosine‐1‐phosphate receptor 1 (S1PR1) and RAS P21 protein activator 1 (RASA1), respectively.\n43\n, \n44\n\n\nmiRNAs are essential in neuropathic pain development via neuroinflammation‐related mechanisms in the spinal cord. miR‐130a‐3p targets and downregulates insulin‐like growth factor‐1/insulin‐like growth factor‐1 receptor (IGF‐1/IGF‐1R) expression to alleviate SCI‐induced neuropathic pain by mitigating microglial activation and NF‐κB phosphorylation.\n45\n miR‐378 was decreased in CCI rats, inhibiting neuropathic pain development by targeting the enhancer of zeste homolog 2 (EZH2).\n46\n EZH2 promotes neuropathic pain by increasing tumor necrosis factor‐alpha (TNF‐α), interleukin (IL)‐1β, and monocyte chemoattractant protein‐1 (MCP‐1) production. Intrathecally injecting miRNA‐138 lentivirus can remarkably alleviate neuropathic pain in partial sciatic nerve ligation (pSNL) rats by suppressing toll‐like receptor 4 (TLR4) and macrophage inflammatory protein‐1 alpha (MIP‐1α)/C‐C chemokine receptor 1 (CCR1) signaling pathways.\n47\n\n\nmiR‐15a/16 targets and downregulates G protein‐coupled receptor kinases 2 (GRK2) to disinhibit p38‐MAPK (mitogen‐activated protein kinase) and NF‐κB, contributing to neuroinflammation after CCI.\n48\n GRK2‐deficient mice present a pro‐inflammatory phenotype in spinal cord microglia/macrophages, restored by miR‐124.\n49\n SNL increased DNA methyltransferase 3a (DNMT3a) expression related to hypermethylation of the miR‐214‐3p promoter, resulting in miR‐214‐3p expression reduction, which enhanced astrocyte reactivity, colony‐stimulating factor‐1 (CSF1), and interleukin 6 (IL‐6) production, and hyperalgesia in rats.\n50\n Electro‐acupuncture attenuated SCI by inhibiting Nav1.3 and Bax in the injured spinal cord through miR‐214 upregulation.\n51\n Downregulated miR‐128 was reported to contribute to neuropathic pain via p38 or zinc finger E‐box binding homeobox 1 (ZEB1) activation in the spinal cord.\n52\n, \n53\n Meanwhile, ZEB1 was also targeted by miR‐200b/miR‐429, orchestrating neuropathic pain development.\n54\n\n\nIn a pSNL mouse model, nerve injury significantly reduced miR‐23a expression in spinal glial cells, concomitant with the upregulation of its target chemokine, CXC receptor 4 (CXCR4). In naïve mice, either miR‐23a downregulation or CXCR4 upregulation could active the thioredoxin‐interacting protein (TXNIP)/NOD‐like receptor protein 3 (NLRP3) inflammasome axis. Both intrathecal miR‐23a mimics and spinal CXCR4 downregulation by a lentivirus inhibited TXNIP or NLRP3 upregulation to alleviate hyperalgesia.\n55\n\n\nNotably, one miRNA was shown to have distinctive targets in different animal models of the spinal cord. For example, miR‐155 downregulation targeted and upregulated NADPH oxidase 2 (NOX2) expression to induce reactive oxygen species (ROS) production after SCI, presenting a pro‐inflammatory phenotype in microglia/macrophages.\n56\n The ability to induce glial polarization was also observed in cultured BV‐2 microglia.\n57\n In bortezomib‐induced neuropathic pain rats, downregulated miR‐155 upregulated TNF receptor 1 (TNFR1) expression, which activated its downstream signaling pathways, including p38‐MAPK, c‐Jun N‐terminal kinase (JNK), and transient receptor potential ankyrin 1 (TRPA1).\n58\n Therefore, it is suggested that miR‐155 might act as an intervention target for neuropathic pain. As expected, treatment with ibuprofen and L‐arginine delayed the behavioral pain changes while inhibiting spinal miR‐155 and NO.\n59\n miR‐155‐5p is also known to destabilize the blood–nerve barrier and expression of tight junction proteins, such as claudin‐1 and zonula occludens‐1 (ZO‐1). Tissue plasminogen activator (tPA) could transiently open such barriers to facilitate topically applying analgesics, via miR‐155‐5p upregulation.\n60\n However, we also noted that CCI upregulated but did not downregulate spinal cord miR‐155, and miR‐155 inhibition enhanced suppressor of cytokine signaling 1 (SOCS1) expression to inactivate inflammation via NF‐κB and p38‐MAPK inhibition.\n61\n In oxaliplatin‐induced peripheral neuropathic pain, spinal cord miR‐155 expression was also upregulated, and intrathecally injecting the miR‐155 inhibitor attenuated hyperalgesia in rats, possibly by inhibiting oxidative stress–TRPA1 pathways.\n62\n The underlying mechanisms of such distinctive miR‐155 expression change are still unknown and require further research into different pain models.\nmiRNA‐based epigenetic regulation is essential in neuroinflammation. miRNAs are predicted to regulate diverse neuroinflammation‐related targets along the pain processing pathways.\nAmong patients with neuropathic pain, miR‐101 decreased in the serum and sural nerve, which is related to nuclear factor‐kappa B (NF‐κB) signaling activation.\n39\n Meanwhile, serum miR‐124a and miR‐155 expression was upregulated. They were identified to inhibit histone deacetylase sirtuin 1 (SIRT1) in primary human cluster of differentiation 4 (CD4) (+) cells and induce their differentiation toward regulatory T cells (Tregs), thus reducing pain‐related inflammation.\n40\n Such miRNA–target interactions may act as an endogenous protective mechanism for neuropathic pain.\nmiR‐7a, expressed in small‐sized nociceptive DRG neurons, is downregulated after nerve injury, as reported, targeting neurofilament light polypeptides (NEFLs).\n41\n NEFL encodes a neuronal protein vital for neurofilament formation and increases signal transducer and activator of transcription 3 (STAT3) phosphorylation, which is highly related to cell differentiation and neuroinflammation. In diabetic peripheral neuropathic mice, miR‐590‐3p was downregulated to disinhibit Ras‐related protein 1A (RAP1A) in the DRG tissue and inhibit neural T cells infiltration.\n42\n Thus, exogenous miR‐590‐3p may be a potential alternative for neuropathic pain treatment. CCI downregulated miR‐140 and miR‐144 expression in the DRG. Intrathecally injected miR‐140 and miR‐144 agomir decreased inflammatory factor secretion and ameliorated hyperalgesia by targeting sphingosine‐1‐phosphate receptor 1 (S1PR1) and RAS P21 protein activator 1 (RASA1), respectively.\n43\n, \n44\n\n\nmiRNAs are essential in neuropathic pain development via neuroinflammation‐related mechanisms in the spinal cord. miR‐130a‐3p targets and downregulates insulin‐like growth factor‐1/insulin‐like growth factor‐1 receptor (IGF‐1/IGF‐1R) expression to alleviate SCI‐induced neuropathic pain by mitigating microglial activation and NF‐κB phosphorylation.\n45\n miR‐378 was decreased in CCI rats, inhibiting neuropathic pain development by targeting the enhancer of zeste homolog 2 (EZH2).\n46\n EZH2 promotes neuropathic pain by increasing tumor necrosis factor‐alpha (TNF‐α), interleukin (IL)‐1β, and monocyte chemoattractant protein‐1 (MCP‐1) production. Intrathecally injecting miRNA‐138 lentivirus can remarkably alleviate neuropathic pain in partial sciatic nerve ligation (pSNL) rats by suppressing toll‐like receptor 4 (TLR4) and macrophage inflammatory protein‐1 alpha (MIP‐1α)/C‐C chemokine receptor 1 (CCR1) signaling pathways.\n47\n\n\nmiR‐15a/16 targets and downregulates G protein‐coupled receptor kinases 2 (GRK2) to disinhibit p38‐MAPK (mitogen‐activated protein kinase) and NF‐κB, contributing to neuroinflammation after CCI.\n48\n GRK2‐deficient mice present a pro‐inflammatory phenotype in spinal cord microglia/macrophages, restored by miR‐124.\n49\n SNL increased DNA methyltransferase 3a (DNMT3a) expression related to hypermethylation of the miR‐214‐3p promoter, resulting in miR‐214‐3p expression reduction, which enhanced astrocyte reactivity, colony‐stimulating factor‐1 (CSF1), and interleukin 6 (IL‐6) production, and hyperalgesia in rats.\n50\n Electro‐acupuncture attenuated SCI by inhibiting Nav1.3 and Bax in the injured spinal cord through miR‐214 upregulation.\n51\n Downregulated miR‐128 was reported to contribute to neuropathic pain via p38 or zinc finger E‐box binding homeobox 1 (ZEB1) activation in the spinal cord.\n52\n, \n53\n Meanwhile, ZEB1 was also targeted by miR‐200b/miR‐429, orchestrating neuropathic pain development.\n54\n\n\nIn a pSNL mouse model, nerve injury significantly reduced miR‐23a expression in spinal glial cells, concomitant with the upregulation of its target chemokine, CXC receptor 4 (CXCR4). In naïve mice, either miR‐23a downregulation or CXCR4 upregulation could active the thioredoxin‐interacting protein (TXNIP)/NOD‐like receptor protein 3 (NLRP3) inflammasome axis. Both intrathecal miR‐23a mimics and spinal CXCR4 downregulation by a lentivirus inhibited TXNIP or NLRP3 upregulation to alleviate hyperalgesia.\n55\n\n\nNotably, one miRNA was shown to have distinctive targets in different animal models of the spinal cord. For example, miR‐155 downregulation targeted and upregulated NADPH oxidase 2 (NOX2) expression to induce reactive oxygen species (ROS) production after SCI, presenting a pro‐inflammatory phenotype in microglia/macrophages.\n56\n The ability to induce glial polarization was also observed in cultured BV‐2 microglia.\n57\n In bortezomib‐induced neuropathic pain rats, downregulated miR‐155 upregulated TNF receptor 1 (TNFR1) expression, which activated its downstream signaling pathways, including p38‐MAPK, c‐Jun N‐terminal kinase (JNK), and transient receptor potential ankyrin 1 (TRPA1).\n58\n Therefore, it is suggested that miR‐155 might act as an intervention target for neuropathic pain. As expected, treatment with ibuprofen and L‐arginine delayed the behavioral pain changes while inhibiting spinal miR‐155 and NO.\n59\n miR‐155‐5p is also known to destabilize the blood–nerve barrier and expression of tight junction proteins, such as claudin‐1 and zonula occludens‐1 (ZO‐1). Tissue plasminogen activator (tPA) could transiently open such barriers to facilitate topically applying analgesics, via miR‐155‐5p upregulation.\n60\n However, we also noted that CCI upregulated but did not downregulate spinal cord miR‐155, and miR‐155 inhibition enhanced suppressor of cytokine signaling 1 (SOCS1) expression to inactivate inflammation via NF‐κB and p38‐MAPK inhibition.\n61\n In oxaliplatin‐induced peripheral neuropathic pain, spinal cord miR‐155 expression was also upregulated, and intrathecally injecting the miR‐155 inhibitor attenuated hyperalgesia in rats, possibly by inhibiting oxidative stress–TRPA1 pathways.\n62\n The underlying mechanisms of such distinctive miR‐155 expression change are still unknown and require further research into different pain models.\nAutophagy miRNA‐related autophagy is involved in neuropathic pain regulation. As reported, miR‐15a downregulated and stimulated AKT serine/threonine kinase 3 (AKT3), and inhibited autophagy post‐CCI.\n63\n Impaired autophagy participates in neuropathic pain development. Intrathecal miR‐15a agomir prominently suppressed AKT3 expression, induced autophagy, and attenuated CCI‐induced neuropathic pain. Similar to miR‐15a, miR‐145 and miR‐20b‐5p contributed to neuropathic pain regulation via protein kinase B (AKT)‐related autophagy pathways.\n64\n, \n65\n Meanwhile, miR‐195 in the spinal cord was observed to upregulate post‐SNL, targeting and inhibiting Autophagy‐Related 14 (ATG14) and its autophagy activation.\n66\n The miR‐195 inhibitor activated autophagy and suppressed neuroinflammation in vivo and in vitro.\nmiRNA‐related autophagy is involved in neuropathic pain regulation. As reported, miR‐15a downregulated and stimulated AKT serine/threonine kinase 3 (AKT3), and inhibited autophagy post‐CCI.\n63\n Impaired autophagy participates in neuropathic pain development. Intrathecal miR‐15a agomir prominently suppressed AKT3 expression, induced autophagy, and attenuated CCI‐induced neuropathic pain. Similar to miR‐15a, miR‐145 and miR‐20b‐5p contributed to neuropathic pain regulation via protein kinase B (AKT)‐related autophagy pathways.\n64\n, \n65\n Meanwhile, miR‐195 in the spinal cord was observed to upregulate post‐SNL, targeting and inhibiting Autophagy‐Related 14 (ATG14) and its autophagy activation.\n66\n The miR‐195 inhibitor activated autophagy and suppressed neuroinflammation in vivo and in vitro.\nThe contribution of miRNAs to ion channel expression in neuropathic pain Another class of miRNA‐based regulation focuses on ion channels, including sodium, potassium, and calcium channels, and transient receptor potential (TRP) channels, to modulate action potential production, firing rate, and neurotransmitter release.\nTheoretically, miRNAs can simultaneously have multiple target mRNAs because precise matching is not a prerequisite for inhibiting the target sequence. For example, other than NEFL,\n41\n miR‐7a targets the β2 subunit of the voltage‐gated sodium channel (Scn2b) with post‐transcriptional regulation to induce nociceptive DRG neurons hyperexcitability.\n67\n Other than miR‐7a, the other known miRNA‐ targets involved in sodium channels expression include miR‐30b, ‐96, ‐384‐5p and their target Scn3a (encoding Nav1.3),\n68\n, \n69\n, \n70\n miR‐30b‐5p and its target Scn8a (encoding Nav1.6),\n71\n and miR‐182 and ‐30b and their target Scn9a (encoding Nav1.7).\n72\n, \n73\n\n\nA miRNA cluster is a polycistronic gene containing several miRNAs derived from a single primary or nascent transcript. Approximately 40% of miRNAs are predicted to form clusters, but their significance is still unknown. miR‐17‐92 is a miRNA cluster that includes six different members, of which miR‐18a, ‐19a, ‐19b, and ‐92a upregulation induces allodynia. The predicted targets of the miR‐17‐92 cluster encompass genes encoding diverse voltage‐gated potassium channels and their regulatory subunits, including Kv1.1, Kv1.4, Kv3.4, Kv4.3, Kv7.5, dipeptidyl peptidase 10 (DPP10), and Navβ1.\n74\n CCI upregulated miR‐137 to target and downregulate Kcna2, which encodes Kv1.2 in the DRG and spinal dorsal horn.\n75\n By contrast, CCI decreased miR‐183‐5p expression in the DRG, and the predicted target gene TREK‐1, a subunit of the 2P‐domain K+ channel, was increased.\n76\n\n\nFurthermore, the miRNA‐183 cluster (miR‐183, part of miR‐96/182/183) regulates either basal mechanical or neuropathic pain.\n77\n The miR‐183 cluster targets Cacna2d1 and Cacna2d2, which encode auxiliary voltage‐gated calcium channel subunits α2δ‐1 and α2δ‐2, to affect nociceptor excitability. Nerve injury downregulated miR‐103 in the spinal cord, which simultaneously targeted and inhibited Cacna1c, Cacna2d1, and Cacnb1 and encoded the Cav1.2‐α1, α2δ1, and β1 subunits of the voltage‐gated calcium channels macromolecular complex Cav1.2 L‐type calcium channel, respectively.\n78\n Intrathecal miR‐103 successfully relieved neuropathic pain.\nIn addition to ion channels, TRP channels are ligand‐gated ion channels that promote painful sensations.\n79\n In the DRG, TRPA1 participates in miR‐141‐5p alleviated oxaliplatin‐induced neuropathic pain.\n80\n In SNI mice, miR‐449a ameliorated neuropathic pain by decreasing the activity of TRPA1 and calcium‐activated potassium channel subunit α‐1 (KCNMA1), thus acting as a potential therapeutic alternative for treating neuropathic pain.\n81\n\n\nAnother class of miRNA‐based regulation focuses on ion channels, including sodium, potassium, and calcium channels, and transient receptor potential (TRP) channels, to modulate action potential production, firing rate, and neurotransmitter release.\nTheoretically, miRNAs can simultaneously have multiple target mRNAs because precise matching is not a prerequisite for inhibiting the target sequence. For example, other than NEFL,\n41\n miR‐7a targets the β2 subunit of the voltage‐gated sodium channel (Scn2b) with post‐transcriptional regulation to induce nociceptive DRG neurons hyperexcitability.\n67\n Other than miR‐7a, the other known miRNA‐ targets involved in sodium channels expression include miR‐30b, ‐96, ‐384‐5p and their target Scn3a (encoding Nav1.3),\n68\n, \n69\n, \n70\n miR‐30b‐5p and its target Scn8a (encoding Nav1.6),\n71\n and miR‐182 and ‐30b and their target Scn9a (encoding Nav1.7).\n72\n, \n73\n\n\nA miRNA cluster is a polycistronic gene containing several miRNAs derived from a single primary or nascent transcript. Approximately 40% of miRNAs are predicted to form clusters, but their significance is still unknown. miR‐17‐92 is a miRNA cluster that includes six different members, of which miR‐18a, ‐19a, ‐19b, and ‐92a upregulation induces allodynia. The predicted targets of the miR‐17‐92 cluster encompass genes encoding diverse voltage‐gated potassium channels and their regulatory subunits, including Kv1.1, Kv1.4, Kv3.4, Kv4.3, Kv7.5, dipeptidyl peptidase 10 (DPP10), and Navβ1.\n74\n CCI upregulated miR‐137 to target and downregulate Kcna2, which encodes Kv1.2 in the DRG and spinal dorsal horn.\n75\n By contrast, CCI decreased miR‐183‐5p expression in the DRG, and the predicted target gene TREK‐1, a subunit of the 2P‐domain K+ channel, was increased.\n76\n\n\nFurthermore, the miRNA‐183 cluster (miR‐183, part of miR‐96/182/183) regulates either basal mechanical or neuropathic pain.\n77\n The miR‐183 cluster targets Cacna2d1 and Cacna2d2, which encode auxiliary voltage‐gated calcium channel subunits α2δ‐1 and α2δ‐2, to affect nociceptor excitability. Nerve injury downregulated miR‐103 in the spinal cord, which simultaneously targeted and inhibited Cacna1c, Cacna2d1, and Cacnb1 and encoded the Cav1.2‐α1, α2δ1, and β1 subunits of the voltage‐gated calcium channels macromolecular complex Cav1.2 L‐type calcium channel, respectively.\n78\n Intrathecal miR‐103 successfully relieved neuropathic pain.\nIn addition to ion channels, TRP channels are ligand‐gated ion channels that promote painful sensations.\n79\n In the DRG, TRPA1 participates in miR‐141‐5p alleviated oxaliplatin‐induced neuropathic pain.\n80\n In SNI mice, miR‐449a ameliorated neuropathic pain by decreasing the activity of TRPA1 and calcium‐activated potassium channel subunit α‐1 (KCNMA1), thus acting as a potential therapeutic alternative for treating neuropathic pain.\n81\n\n\n\nmiRNAs regulate pain‐related mediators, protein kinases, and structural proteins miRNAs regulate diverse pain‐related mediators, protein kinases, and structural proteins along the pain processing pathways. For HIV‐associated symptomatic distal sensory polyneuropathy and neuropathic pain, serum miR‐455‐3p acted as a potential biomarker, possibly targeting multiple genes involved in peripheral neuropathic pain, such as nerve growth factor (NGF) and related genes.\n82\n Brain‐derived neurotrophic factor (BDNF), another well‐recognized pain mediator, is a common miR‐1, ‐183, and ‐206 target.\n83\n, \n84\n, \n85\n After nerve injury, several miRNAs, including miR‐19a, ‐301, and ‐132, were downregulated, and their target methyl cytosine–guanine dinucleotide (CpG)‐binding protein 2 (MeCP2) expression was increased, leading to concomitant BDNF upregulation.\n86\n miR‐30c‐5p was upregulated in the serum and CSF of patients with chronic peripheral ischemia.\n16\n A miR‐30c‐5p inhibitor intraventricular injection postponed neuropathic pain development and fully reversed hyperalgesia in rodents. Transforming growth factor beta (TGF‐β) participates in the effects of miR‐30c‐5p, which refers to the endogenous opioid analgesic system.\n16\n\n\nmiRNAs dysregulate specific pain‐related protein kinases. For example, in DNP rats, miR‐133a‐3p was dysregulated in the sciatic nerve, interacting with p‐p38 MAPK to participate in the development of neuropathic pain.\n87\n Via a methyl CpG‐binding domain and transcriptional repressor domain, MeCP2 acts as a transcriptional repressor, and its overexpression improves neuropathic pain, potentiating an anti‐nociceptive effect of MeCP2. The authors also noted that MeCP2 expression changed post‐transcriptionally, and its mRNA level did not significantly change after SNI. However, the protein level was upregulated. During the development period of neuropathic pain, phospho‐cAMP response element‐binding protein (p‐CREB) elevated rapidly but returned 3–7 days after SNI, concomitant with miR‐132 downregulation, which targets MeCP2 and inhibits its expression post‐transcriptionally.\n88\n SNL decreased miR‐200b and miR‐429 in NAc neurons, along with an upregulation of their target (DNMT3a).\n89\n Further mechanism studies found that DNMT3a in the NAc was expressed in NR1 immunoreactive neurons, suggesting the dysregulation of ‘mesolimbic motivation circuitry’ for neuropathic pain development.\nThe CCI induced time‐dependent miR‐1 downregulation in injured sciatic nerves. This change in expression was related to the upregulation and translocation of the miR‐1‐targeted connexin 43 (Cx43), the major connexin of astrocytes.\n83\n miR‐1 mimics could reduce Cx43 expression in cultured human glioblastoma cells. However, miR‐1 mimics intraneural transfection failed to alter Cx43 protein expression and did not improve pain behavior. The authors attributed such treatment failure to insufficient inhibition of miR‐1 for Cx43 via intraneural injection. Alternatively, there were regulatory mechanisms for Cx43 in vivo other than miR‐1.\n90\n However, miR‐1 in the DRG was DE according to peripheral nerve injury type. CCI, pSNL, and sural nerve injury downregulated miR‐1. However, axotomy of the sciatic nerve and tibial nerve injury increased its expression in the DRG.\n83\n, \n91\n, \n92\n As previously described, miR‐1 was also upregulated following capsaicin treatment and bone cancer pain.\n91\n, \n93\n Additionally, miR‐1 downregulation inhibited bone cancer pain. Collectively, the action of miR‐1 on pain seems to be complex and stimulus‐dependent.\nNotably, specific miRNAs can promote neuropathic pain development via diverse mechanisms. For example, beta‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1), a membrane protease essential for myelination, was downregulated after miR‐15b overexpression in vitro or in the DRG of chemotherapy‐related neuropathic pain rats.\n94\n BACE1‐mediated neuregulin 1 reduction decreases nerve conduction velocity. In addition, BACE1 modulated Navβ2 subunit expression and neuronal activity and regulated inflammation‐related TNFR expression.\nmiRNAs regulate diverse pain‐related mediators, protein kinases, and structural proteins along the pain processing pathways. For HIV‐associated symptomatic distal sensory polyneuropathy and neuropathic pain, serum miR‐455‐3p acted as a potential biomarker, possibly targeting multiple genes involved in peripheral neuropathic pain, such as nerve growth factor (NGF) and related genes.\n82\n Brain‐derived neurotrophic factor (BDNF), another well‐recognized pain mediator, is a common miR‐1, ‐183, and ‐206 target.\n83\n, \n84\n, \n85\n After nerve injury, several miRNAs, including miR‐19a, ‐301, and ‐132, were downregulated, and their target methyl cytosine–guanine dinucleotide (CpG)‐binding protein 2 (MeCP2) expression was increased, leading to concomitant BDNF upregulation.\n86\n miR‐30c‐5p was upregulated in the serum and CSF of patients with chronic peripheral ischemia.\n16\n A miR‐30c‐5p inhibitor intraventricular injection postponed neuropathic pain development and fully reversed hyperalgesia in rodents. Transforming growth factor beta (TGF‐β) participates in the effects of miR‐30c‐5p, which refers to the endogenous opioid analgesic system.\n16\n\n\nmiRNAs dysregulate specific pain‐related protein kinases. For example, in DNP rats, miR‐133a‐3p was dysregulated in the sciatic nerve, interacting with p‐p38 MAPK to participate in the development of neuropathic pain.\n87\n Via a methyl CpG‐binding domain and transcriptional repressor domain, MeCP2 acts as a transcriptional repressor, and its overexpression improves neuropathic pain, potentiating an anti‐nociceptive effect of MeCP2. The authors also noted that MeCP2 expression changed post‐transcriptionally, and its mRNA level did not significantly change after SNI. However, the protein level was upregulated. During the development period of neuropathic pain, phospho‐cAMP response element‐binding protein (p‐CREB) elevated rapidly but returned 3–7 days after SNI, concomitant with miR‐132 downregulation, which targets MeCP2 and inhibits its expression post‐transcriptionally.\n88\n SNL decreased miR‐200b and miR‐429 in NAc neurons, along with an upregulation of their target (DNMT3a).\n89\n Further mechanism studies found that DNMT3a in the NAc was expressed in NR1 immunoreactive neurons, suggesting the dysregulation of ‘mesolimbic motivation circuitry’ for neuropathic pain development.\nThe CCI induced time‐dependent miR‐1 downregulation in injured sciatic nerves. This change in expression was related to the upregulation and translocation of the miR‐1‐targeted connexin 43 (Cx43), the major connexin of astrocytes.\n83\n miR‐1 mimics could reduce Cx43 expression in cultured human glioblastoma cells. However, miR‐1 mimics intraneural transfection failed to alter Cx43 protein expression and did not improve pain behavior. The authors attributed such treatment failure to insufficient inhibition of miR‐1 for Cx43 via intraneural injection. Alternatively, there were regulatory mechanisms for Cx43 in vivo other than miR‐1.\n90\n However, miR‐1 in the DRG was DE according to peripheral nerve injury type. CCI, pSNL, and sural nerve injury downregulated miR‐1. However, axotomy of the sciatic nerve and tibial nerve injury increased its expression in the DRG.\n83\n, \n91\n, \n92\n As previously described, miR‐1 was also upregulated following capsaicin treatment and bone cancer pain.\n91\n, \n93\n Additionally, miR‐1 downregulation inhibited bone cancer pain. Collectively, the action of miR‐1 on pain seems to be complex and stimulus‐dependent.\nNotably, specific miRNAs can promote neuropathic pain development via diverse mechanisms. For example, beta‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1), a membrane protease essential for myelination, was downregulated after miR‐15b overexpression in vitro or in the DRG of chemotherapy‐related neuropathic pain rats.\n94\n BACE1‐mediated neuregulin 1 reduction decreases nerve conduction velocity. In addition, BACE1 modulated Navβ2 subunit expression and neuronal activity and regulated inflammation‐related TNFR expression.\nNeurotransmission excitatory–inhibitory imbalances Neurotransmission excitatory and inhibitory imbalances in the spinal cord also contribute to neuropathic pain development. For example, miR‐500 increased to regulate glutamic acid decarboxylase 67 (GAD67) expression and target the specific site of Gad1 in the dorsal horn.\n95\n GAD67 expression reduction inhibited the function of GABAergic neurons and the resultant inhibitory synaptic transmission dysregulation contributed to neuropathic pain development. In addition, miR‐23b is crucial for improving neuropathic pain in the injured spinal cord by downregulating its target gene, Nox4, which further normalizes glutamic acid decarboxylase 65/67 (GAD65/67) expression and protects GABAergic neurons from apoptosis.\n96\n Microarray analysis showed that miR‐539 was prominently reduced in the contralateral anterior cingulate cortex (ACC) after CCI, which is related to enhanced NR2B protein expression. Injecting miR‐539 mimics into the contralateral ACC attenuated CCI‐evoked mechanical hyperalgesia, suggesting that the N‐methyl‐D‐aspartate (NMDA) receptor NR2B subunit regulates neuropathic pain.\n97\n\n\nNeurotransmission excitatory and inhibitory imbalances in the spinal cord also contribute to neuropathic pain development. For example, miR‐500 increased to regulate glutamic acid decarboxylase 67 (GAD67) expression and target the specific site of Gad1 in the dorsal horn.\n95\n GAD67 expression reduction inhibited the function of GABAergic neurons and the resultant inhibitory synaptic transmission dysregulation contributed to neuropathic pain development. In addition, miR‐23b is crucial for improving neuropathic pain in the injured spinal cord by downregulating its target gene, Nox4, which further normalizes glutamic acid decarboxylase 65/67 (GAD65/67) expression and protects GABAergic neurons from apoptosis.\n96\n Microarray analysis showed that miR‐539 was prominently reduced in the contralateral anterior cingulate cortex (ACC) after CCI, which is related to enhanced NR2B protein expression. Injecting miR‐539 mimics into the contralateral ACC attenuated CCI‐evoked mechanical hyperalgesia, suggesting that the N‐methyl‐D‐aspartate (NMDA) receptor NR2B subunit regulates neuropathic pain.\n97\n\n\n\nEV miRNAs‐mediated neuron–glia communication We mainly focused on exosomal miR‐21 with regard to EV miRNAs in neuropathic pain. As aforementioned, SNL, DRT, or VRT could upregulate miR‐21 in the injured DRG.\n22\n Notably, miR‐21 was also increased in serum exosomes from nerve‐ligated mice.\n98\n In another in‐depth study, macrophages readily took up pure sensory neuron‐derived exosomes encompassing miR‐21‐5p to promote a pro‐inflammatory phenotype.\n99\n Either intrathecal miR‐21‐5p antagomir or miR‐21 conditional deletion in sensory neurons ameliorated hyperalgesia and macrophage recruitment in the DRG.\nUnlike the miRNA target gene inhibitory mechanism, miR‐21 acts as an endogenous toll‐like receptor 8 (TLR8) ligand, leading to neuropathic pain development.\n100\n TLR8, a nucleic acid‐sensing receptor, is located in the endosomes and lysosomes, leading to ERK‐mediated inflammatory mediator production and neuronal activation after SNL. Although miR‐21 and TLR8 are only distributed in small‐ and medium‐sized neurons, miR‐21 can also be derived from large‐sized neurons and reach TLR8 in the endosomes of other types of neurons.\n101\n Similar to miR‐21, DRG sensory neurons secreted miR‐23a‐enriched EVs following nerve injury and were taken up by macrophages to enhance M1 polarization in vitro. A20, an NF‐κB signaling pathway inhibitor, is a verified miR‐23a target gene.\n102\n Moreover, intrathecally delivering EVs‐miR‐23a antagomir attenuated neuropathic hyperalgesia and reduced M1 macrophages by inhibiting A20 to activate NF‐κB signaling.\nWe mainly focused on exosomal miR‐21 with regard to EV miRNAs in neuropathic pain. As aforementioned, SNL, DRT, or VRT could upregulate miR‐21 in the injured DRG.\n22\n Notably, miR‐21 was also increased in serum exosomes from nerve‐ligated mice.\n98\n In another in‐depth study, macrophages readily took up pure sensory neuron‐derived exosomes encompassing miR‐21‐5p to promote a pro‐inflammatory phenotype.\n99\n Either intrathecal miR‐21‐5p antagomir or miR‐21 conditional deletion in sensory neurons ameliorated hyperalgesia and macrophage recruitment in the DRG.\nUnlike the miRNA target gene inhibitory mechanism, miR‐21 acts as an endogenous toll‐like receptor 8 (TLR8) ligand, leading to neuropathic pain development.\n100\n TLR8, a nucleic acid‐sensing receptor, is located in the endosomes and lysosomes, leading to ERK‐mediated inflammatory mediator production and neuronal activation after SNL. Although miR‐21 and TLR8 are only distributed in small‐ and medium‐sized neurons, miR‐21 can also be derived from large‐sized neurons and reach TLR8 in the endosomes of other types of neurons.\n101\n Similar to miR‐21, DRG sensory neurons secreted miR‐23a‐enriched EVs following nerve injury and were taken up by macrophages to enhance M1 polarization in vitro. A20, an NF‐κB signaling pathway inhibitor, is a verified miR‐23a target gene.\n102\n Moreover, intrathecally delivering EVs‐miR‐23a antagomir attenuated neuropathic hyperalgesia and reduced M1 macrophages by inhibiting A20 to activate NF‐κB signaling.\n\n\nmiRNAs regulate neuroinflammation in neuropathic pain development:\nmiRNA‐based epigenetic regulation is essential in neuroinflammation. miRNAs are predicted to regulate diverse neuroinflammation‐related targets along the pain processing pathways.\nAmong patients with neuropathic pain, miR‐101 decreased in the serum and sural nerve, which is related to nuclear factor‐kappa B (NF‐κB) signaling activation.\n39\n Meanwhile, serum miR‐124a and miR‐155 expression was upregulated. They were identified to inhibit histone deacetylase sirtuin 1 (SIRT1) in primary human cluster of differentiation 4 (CD4) (+) cells and induce their differentiation toward regulatory T cells (Tregs), thus reducing pain‐related inflammation.\n40\n Such miRNA–target interactions may act as an endogenous protective mechanism for neuropathic pain.\nmiR‐7a, expressed in small‐sized nociceptive DRG neurons, is downregulated after nerve injury, as reported, targeting neurofilament light polypeptides (NEFLs).\n41\n NEFL encodes a neuronal protein vital for neurofilament formation and increases signal transducer and activator of transcription 3 (STAT3) phosphorylation, which is highly related to cell differentiation and neuroinflammation. In diabetic peripheral neuropathic mice, miR‐590‐3p was downregulated to disinhibit Ras‐related protein 1A (RAP1A) in the DRG tissue and inhibit neural T cells infiltration.\n42\n Thus, exogenous miR‐590‐3p may be a potential alternative for neuropathic pain treatment. CCI downregulated miR‐140 and miR‐144 expression in the DRG. Intrathecally injected miR‐140 and miR‐144 agomir decreased inflammatory factor secretion and ameliorated hyperalgesia by targeting sphingosine‐1‐phosphate receptor 1 (S1PR1) and RAS P21 protein activator 1 (RASA1), respectively.\n43\n, \n44\n\n\nmiRNAs are essential in neuropathic pain development via neuroinflammation‐related mechanisms in the spinal cord. miR‐130a‐3p targets and downregulates insulin‐like growth factor‐1/insulin‐like growth factor‐1 receptor (IGF‐1/IGF‐1R) expression to alleviate SCI‐induced neuropathic pain by mitigating microglial activation and NF‐κB phosphorylation.\n45\n miR‐378 was decreased in CCI rats, inhibiting neuropathic pain development by targeting the enhancer of zeste homolog 2 (EZH2).\n46\n EZH2 promotes neuropathic pain by increasing tumor necrosis factor‐alpha (TNF‐α), interleukin (IL)‐1β, and monocyte chemoattractant protein‐1 (MCP‐1) production. Intrathecally injecting miRNA‐138 lentivirus can remarkably alleviate neuropathic pain in partial sciatic nerve ligation (pSNL) rats by suppressing toll‐like receptor 4 (TLR4) and macrophage inflammatory protein‐1 alpha (MIP‐1α)/C‐C chemokine receptor 1 (CCR1) signaling pathways.\n47\n\n\nmiR‐15a/16 targets and downregulates G protein‐coupled receptor kinases 2 (GRK2) to disinhibit p38‐MAPK (mitogen‐activated protein kinase) and NF‐κB, contributing to neuroinflammation after CCI.\n48\n GRK2‐deficient mice present a pro‐inflammatory phenotype in spinal cord microglia/macrophages, restored by miR‐124.\n49\n SNL increased DNA methyltransferase 3a (DNMT3a) expression related to hypermethylation of the miR‐214‐3p promoter, resulting in miR‐214‐3p expression reduction, which enhanced astrocyte reactivity, colony‐stimulating factor‐1 (CSF1), and interleukin 6 (IL‐6) production, and hyperalgesia in rats.\n50\n Electro‐acupuncture attenuated SCI by inhibiting Nav1.3 and Bax in the injured spinal cord through miR‐214 upregulation.\n51\n Downregulated miR‐128 was reported to contribute to neuropathic pain via p38 or zinc finger E‐box binding homeobox 1 (ZEB1) activation in the spinal cord.\n52\n, \n53\n Meanwhile, ZEB1 was also targeted by miR‐200b/miR‐429, orchestrating neuropathic pain development.\n54\n\n\nIn a pSNL mouse model, nerve injury significantly reduced miR‐23a expression in spinal glial cells, concomitant with the upregulation of its target chemokine, CXC receptor 4 (CXCR4). In naïve mice, either miR‐23a downregulation or CXCR4 upregulation could active the thioredoxin‐interacting protein (TXNIP)/NOD‐like receptor protein 3 (NLRP3) inflammasome axis. Both intrathecal miR‐23a mimics and spinal CXCR4 downregulation by a lentivirus inhibited TXNIP or NLRP3 upregulation to alleviate hyperalgesia.\n55\n\n\nNotably, one miRNA was shown to have distinctive targets in different animal models of the spinal cord. For example, miR‐155 downregulation targeted and upregulated NADPH oxidase 2 (NOX2) expression to induce reactive oxygen species (ROS) production after SCI, presenting a pro‐inflammatory phenotype in microglia/macrophages.\n56\n The ability to induce glial polarization was also observed in cultured BV‐2 microglia.\n57\n In bortezomib‐induced neuropathic pain rats, downregulated miR‐155 upregulated TNF receptor 1 (TNFR1) expression, which activated its downstream signaling pathways, including p38‐MAPK, c‐Jun N‐terminal kinase (JNK), and transient receptor potential ankyrin 1 (TRPA1).\n58\n Therefore, it is suggested that miR‐155 might act as an intervention target for neuropathic pain. As expected, treatment with ibuprofen and L‐arginine delayed the behavioral pain changes while inhibiting spinal miR‐155 and NO.\n59\n miR‐155‐5p is also known to destabilize the blood–nerve barrier and expression of tight junction proteins, such as claudin‐1 and zonula occludens‐1 (ZO‐1). Tissue plasminogen activator (tPA) could transiently open such barriers to facilitate topically applying analgesics, via miR‐155‐5p upregulation.\n60\n However, we also noted that CCI upregulated but did not downregulate spinal cord miR‐155, and miR‐155 inhibition enhanced suppressor of cytokine signaling 1 (SOCS1) expression to inactivate inflammation via NF‐κB and p38‐MAPK inhibition.\n61\n In oxaliplatin‐induced peripheral neuropathic pain, spinal cord miR‐155 expression was also upregulated, and intrathecally injecting the miR‐155 inhibitor attenuated hyperalgesia in rats, possibly by inhibiting oxidative stress–TRPA1 pathways.\n62\n The underlying mechanisms of such distinctive miR‐155 expression change are still unknown and require further research into different pain models.\n\nAutophagy:\nmiRNA‐related autophagy is involved in neuropathic pain regulation. As reported, miR‐15a downregulated and stimulated AKT serine/threonine kinase 3 (AKT3), and inhibited autophagy post‐CCI.\n63\n Impaired autophagy participates in neuropathic pain development. Intrathecal miR‐15a agomir prominently suppressed AKT3 expression, induced autophagy, and attenuated CCI‐induced neuropathic pain. Similar to miR‐15a, miR‐145 and miR‐20b‐5p contributed to neuropathic pain regulation via protein kinase B (AKT)‐related autophagy pathways.\n64\n, \n65\n Meanwhile, miR‐195 in the spinal cord was observed to upregulate post‐SNL, targeting and inhibiting Autophagy‐Related 14 (ATG14) and its autophagy activation.\n66\n The miR‐195 inhibitor activated autophagy and suppressed neuroinflammation in vivo and in vitro.\n\nThe contribution of miRNAs to ion channel expression in neuropathic pain:\nAnother class of miRNA‐based regulation focuses on ion channels, including sodium, potassium, and calcium channels, and transient receptor potential (TRP) channels, to modulate action potential production, firing rate, and neurotransmitter release.\nTheoretically, miRNAs can simultaneously have multiple target mRNAs because precise matching is not a prerequisite for inhibiting the target sequence. For example, other than NEFL,\n41\n miR‐7a targets the β2 subunit of the voltage‐gated sodium channel (Scn2b) with post‐transcriptional regulation to induce nociceptive DRG neurons hyperexcitability.\n67\n Other than miR‐7a, the other known miRNA‐ targets involved in sodium channels expression include miR‐30b, ‐96, ‐384‐5p and their target Scn3a (encoding Nav1.3),\n68\n, \n69\n, \n70\n miR‐30b‐5p and its target Scn8a (encoding Nav1.6),\n71\n and miR‐182 and ‐30b and their target Scn9a (encoding Nav1.7).\n72\n, \n73\n\n\nA miRNA cluster is a polycistronic gene containing several miRNAs derived from a single primary or nascent transcript. Approximately 40% of miRNAs are predicted to form clusters, but their significance is still unknown. miR‐17‐92 is a miRNA cluster that includes six different members, of which miR‐18a, ‐19a, ‐19b, and ‐92a upregulation induces allodynia. The predicted targets of the miR‐17‐92 cluster encompass genes encoding diverse voltage‐gated potassium channels and their regulatory subunits, including Kv1.1, Kv1.4, Kv3.4, Kv4.3, Kv7.5, dipeptidyl peptidase 10 (DPP10), and Navβ1.\n74\n CCI upregulated miR‐137 to target and downregulate Kcna2, which encodes Kv1.2 in the DRG and spinal dorsal horn.\n75\n By contrast, CCI decreased miR‐183‐5p expression in the DRG, and the predicted target gene TREK‐1, a subunit of the 2P‐domain K+ channel, was increased.\n76\n\n\nFurthermore, the miRNA‐183 cluster (miR‐183, part of miR‐96/182/183) regulates either basal mechanical or neuropathic pain.\n77\n The miR‐183 cluster targets Cacna2d1 and Cacna2d2, which encode auxiliary voltage‐gated calcium channel subunits α2δ‐1 and α2δ‐2, to affect nociceptor excitability. Nerve injury downregulated miR‐103 in the spinal cord, which simultaneously targeted and inhibited Cacna1c, Cacna2d1, and Cacnb1 and encoded the Cav1.2‐α1, α2δ1, and β1 subunits of the voltage‐gated calcium channels macromolecular complex Cav1.2 L‐type calcium channel, respectively.\n78\n Intrathecal miR‐103 successfully relieved neuropathic pain.\nIn addition to ion channels, TRP channels are ligand‐gated ion channels that promote painful sensations.\n79\n In the DRG, TRPA1 participates in miR‐141‐5p alleviated oxaliplatin‐induced neuropathic pain.\n80\n In SNI mice, miR‐449a ameliorated neuropathic pain by decreasing the activity of TRPA1 and calcium‐activated potassium channel subunit α‐1 (KCNMA1), thus acting as a potential therapeutic alternative for treating neuropathic pain.\n81\n\n\n\n\nmiRNAs regulate pain‐related mediators, protein kinases, and structural proteins:\nmiRNAs regulate diverse pain‐related mediators, protein kinases, and structural proteins along the pain processing pathways. For HIV‐associated symptomatic distal sensory polyneuropathy and neuropathic pain, serum miR‐455‐3p acted as a potential biomarker, possibly targeting multiple genes involved in peripheral neuropathic pain, such as nerve growth factor (NGF) and related genes.\n82\n Brain‐derived neurotrophic factor (BDNF), another well‐recognized pain mediator, is a common miR‐1, ‐183, and ‐206 target.\n83\n, \n84\n, \n85\n After nerve injury, several miRNAs, including miR‐19a, ‐301, and ‐132, were downregulated, and their target methyl cytosine–guanine dinucleotide (CpG)‐binding protein 2 (MeCP2) expression was increased, leading to concomitant BDNF upregulation.\n86\n miR‐30c‐5p was upregulated in the serum and CSF of patients with chronic peripheral ischemia.\n16\n A miR‐30c‐5p inhibitor intraventricular injection postponed neuropathic pain development and fully reversed hyperalgesia in rodents. Transforming growth factor beta (TGF‐β) participates in the effects of miR‐30c‐5p, which refers to the endogenous opioid analgesic system.\n16\n\n\nmiRNAs dysregulate specific pain‐related protein kinases. For example, in DNP rats, miR‐133a‐3p was dysregulated in the sciatic nerve, interacting with p‐p38 MAPK to participate in the development of neuropathic pain.\n87\n Via a methyl CpG‐binding domain and transcriptional repressor domain, MeCP2 acts as a transcriptional repressor, and its overexpression improves neuropathic pain, potentiating an anti‐nociceptive effect of MeCP2. The authors also noted that MeCP2 expression changed post‐transcriptionally, and its mRNA level did not significantly change after SNI. However, the protein level was upregulated. During the development period of neuropathic pain, phospho‐cAMP response element‐binding protein (p‐CREB) elevated rapidly but returned 3–7 days after SNI, concomitant with miR‐132 downregulation, which targets MeCP2 and inhibits its expression post‐transcriptionally.\n88\n SNL decreased miR‐200b and miR‐429 in NAc neurons, along with an upregulation of their target (DNMT3a).\n89\n Further mechanism studies found that DNMT3a in the NAc was expressed in NR1 immunoreactive neurons, suggesting the dysregulation of ‘mesolimbic motivation circuitry’ for neuropathic pain development.\nThe CCI induced time‐dependent miR‐1 downregulation in injured sciatic nerves. This change in expression was related to the upregulation and translocation of the miR‐1‐targeted connexin 43 (Cx43), the major connexin of astrocytes.\n83\n miR‐1 mimics could reduce Cx43 expression in cultured human glioblastoma cells. However, miR‐1 mimics intraneural transfection failed to alter Cx43 protein expression and did not improve pain behavior. The authors attributed such treatment failure to insufficient inhibition of miR‐1 for Cx43 via intraneural injection. Alternatively, there were regulatory mechanisms for Cx43 in vivo other than miR‐1.\n90\n However, miR‐1 in the DRG was DE according to peripheral nerve injury type. CCI, pSNL, and sural nerve injury downregulated miR‐1. However, axotomy of the sciatic nerve and tibial nerve injury increased its expression in the DRG.\n83\n, \n91\n, \n92\n As previously described, miR‐1 was also upregulated following capsaicin treatment and bone cancer pain.\n91\n, \n93\n Additionally, miR‐1 downregulation inhibited bone cancer pain. Collectively, the action of miR‐1 on pain seems to be complex and stimulus‐dependent.\nNotably, specific miRNAs can promote neuropathic pain development via diverse mechanisms. For example, beta‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1), a membrane protease essential for myelination, was downregulated after miR‐15b overexpression in vitro or in the DRG of chemotherapy‐related neuropathic pain rats.\n94\n BACE1‐mediated neuregulin 1 reduction decreases nerve conduction velocity. In addition, BACE1 modulated Navβ2 subunit expression and neuronal activity and regulated inflammation‐related TNFR expression.\n\nNeurotransmission excitatory–inhibitory imbalances:\nNeurotransmission excitatory and inhibitory imbalances in the spinal cord also contribute to neuropathic pain development. For example, miR‐500 increased to regulate glutamic acid decarboxylase 67 (GAD67) expression and target the specific site of Gad1 in the dorsal horn.\n95\n GAD67 expression reduction inhibited the function of GABAergic neurons and the resultant inhibitory synaptic transmission dysregulation contributed to neuropathic pain development. In addition, miR‐23b is crucial for improving neuropathic pain in the injured spinal cord by downregulating its target gene, Nox4, which further normalizes glutamic acid decarboxylase 65/67 (GAD65/67) expression and protects GABAergic neurons from apoptosis.\n96\n Microarray analysis showed that miR‐539 was prominently reduced in the contralateral anterior cingulate cortex (ACC) after CCI, which is related to enhanced NR2B protein expression. Injecting miR‐539 mimics into the contralateral ACC attenuated CCI‐evoked mechanical hyperalgesia, suggesting that the N‐methyl‐D‐aspartate (NMDA) receptor NR2B subunit regulates neuropathic pain.\n97\n\n\n\n\nEV miRNAs‐mediated neuron–glia communication:\nWe mainly focused on exosomal miR‐21 with regard to EV miRNAs in neuropathic pain. As aforementioned, SNL, DRT, or VRT could upregulate miR‐21 in the injured DRG.\n22\n Notably, miR‐21 was also increased in serum exosomes from nerve‐ligated mice.\n98\n In another in‐depth study, macrophages readily took up pure sensory neuron‐derived exosomes encompassing miR‐21‐5p to promote a pro‐inflammatory phenotype.\n99\n Either intrathecal miR‐21‐5p antagomir or miR‐21 conditional deletion in sensory neurons ameliorated hyperalgesia and macrophage recruitment in the DRG.\nUnlike the miRNA target gene inhibitory mechanism, miR‐21 acts as an endogenous toll‐like receptor 8 (TLR8) ligand, leading to neuropathic pain development.\n100\n TLR8, a nucleic acid‐sensing receptor, is located in the endosomes and lysosomes, leading to ERK‐mediated inflammatory mediator production and neuronal activation after SNL. Although miR‐21 and TLR8 are only distributed in small‐ and medium‐sized neurons, miR‐21 can also be derived from large‐sized neurons and reach TLR8 in the endosomes of other types of neurons.\n101\n Similar to miR‐21, DRG sensory neurons secreted miR‐23a‐enriched EVs following nerve injury and were taken up by macrophages to enhance M1 polarization in vitro. A20, an NF‐κB signaling pathway inhibitor, is a verified miR‐23a target gene.\n102\n Moreover, intrathecally delivering EVs‐miR‐23a antagomir attenuated neuropathic hyperalgesia and reduced M1 macrophages by inhibiting A20 to activate NF‐κB signaling.\n\n\nSTUDYING lncRNAs AND circRNAs NEUROPATHIC PAIN MECHANISMS\n:\nlncRNAs and circRNAs are essential in neuropathic pain development by acting as AS RNA or miRNA sponges, epigenetically regulating pain‐related molecules expression, or modulating miRNA processing (Table 2).\nLong non‐coding RNAs (lncRNAs) and circular non‐coding RNAs (circRNAs) expression change for the development of neuropathic pain\nAbbreviations: AQP4, aquaporin 4; AS RNA, antisense RNA; CCI, chronic constriction injury; CDK4, cyclin‐dependent kinase 4; CDK6, cyclin‐dependent kinase 6; DNP, diabetic neuropathic pain; DRG, dorsal root ganglion; ENO1, enolase 1; EphB1, ephrin type‐b 1; EZH2, enhancer of zeste homolog 2; HMGB1, high mobility group box 1; NAT, natural antisense transcript; NOTCH2, NOTCH receptor 2; NP, neuropathic pain; pSNL, partial sciatic nerve ligation; pSTAT3, phosphorylated signal transducer and activator of transcription 3; SCI, spinal cord injury; SGC, satellite glia cell; SNL, spinal nerve ligation; STAT3, signal transducer and activator of transcription 3; TLR5, toll‐like receptor 5; TNFAIP1, tumor necrosis factor alpha‐induced protein 1; TRPV1, transient receptor potential vanilloid 1; XIST, X‐inactive specific transcript; ZEB1, zinc finger E‐box binding homeobox 1.\n\nlncRNAs act as AS RNA\n After peripheral nerve injury, Egr2‐AS‐RNA is upregulated in Schwann cells.\n103\n On the early growth response 2 (Egr2) promoter, Egr2‐AS‐RNA recruits an epigenetic silencing complex to downregulate Egr2, essential for peripheral myelination. Ectopic Egr2‐AS‐RNA expression in DRG cultures downregulates Egr2 mRNA and induces demyelination. In vivo, Egr2‐AS‐RNA inhibition reverts Egr2‐related gene expression and delays demyelination.\nVoltage‐gated potassium channel (Kv) Kcna2‐AS‐RNA is an endogenous, highly conserved, and widely explored lncRNA in neuropathic pain.\n104\n It is a natural antisense transcript (NAT), distributed in the cytoplasm and targets Kcna2 mRNA, which encodes the pain regulation‐related membrane Kv1.2 subunit. Kcna2‐AS‐RNA was time‐dependently upregulated in the inflicted rat DRG after nerve injury. It decreased the total Kv current, upregulated DRG neurons excitability, and produced neuropathic pain symptoms.\nThe complementary strand of DNA opposite the Scn9a gene encodes Scn9a NAT, another antisense lncRNA expressed in the DRG.\n105\n Scn9a NAT is suggested to be a negative regulator of Scn9a mRNA. Scn9a NAT overexpression inhibited Scn9a mRNA, its encoded protein Nav1.7, and Nav1.7 currents in DRG neurons. However, Scn9a NAT and Scn9a mRNA levels did not significantly change in the injured DRG until 2 weeks after nerve injury. More work is required to determine whether NAT can confer analgesia and reduce pain in the neuropathic pain state.\nAfter peripheral nerve injury, Egr2‐AS‐RNA is upregulated in Schwann cells.\n103\n On the early growth response 2 (Egr2) promoter, Egr2‐AS‐RNA recruits an epigenetic silencing complex to downregulate Egr2, essential for peripheral myelination. Ectopic Egr2‐AS‐RNA expression in DRG cultures downregulates Egr2 mRNA and induces demyelination. In vivo, Egr2‐AS‐RNA inhibition reverts Egr2‐related gene expression and delays demyelination.\nVoltage‐gated potassium channel (Kv) Kcna2‐AS‐RNA is an endogenous, highly conserved, and widely explored lncRNA in neuropathic pain.\n104\n It is a natural antisense transcript (NAT), distributed in the cytoplasm and targets Kcna2 mRNA, which encodes the pain regulation‐related membrane Kv1.2 subunit. Kcna2‐AS‐RNA was time‐dependently upregulated in the inflicted rat DRG after nerve injury. It decreased the total Kv current, upregulated DRG neurons excitability, and produced neuropathic pain symptoms.\nThe complementary strand of DNA opposite the Scn9a gene encodes Scn9a NAT, another antisense lncRNA expressed in the DRG.\n105\n Scn9a NAT is suggested to be a negative regulator of Scn9a mRNA. Scn9a NAT overexpression inhibited Scn9a mRNA, its encoded protein Nav1.7, and Nav1.7 currents in DRG neurons. However, Scn9a NAT and Scn9a mRNA levels did not significantly change in the injured DRG until 2 weeks after nerve injury. More work is required to determine whether NAT can confer analgesia and reduce pain in the neuropathic pain state.\n\nlncRNAs act as miRNA sponges lncRNAs may act as miRNA sponges to form the lncRNA‐miRNA‐mRNA axis and regulate target gene expression. For example, the lncRNA X‐inactive specific transcript (XIST) is upregulated in the dorsal horn of the spinal cord after CCI. By sponging miR‐137, ‐150, ‐154‐5p, and ‐544, XIST can inhibit the expression of corresponding targets, including TNF‐α‐induced protein 1 (TNFAIP1), ZEB1, TLR5, and STAT3.\n106\n, \n107\n, \n108\n, \n109\n TNFAIP1 activates the NF‐κB signaling pathway, while ZEB1, TLR5, and STAT3 are crucial in the neuroinflammatory response. XIST inhibition markedly ameliorates neuropathic pain development. In addition, lncRNA nuclear enriched abundant transcript 1 (NEAT1) was upregulated in the spinal cord dorsal horn to form NEAT1‐miR‐381‐high mobility group box 1 (HMGB1) and NEAT1–miR‐128‐3p–aquaporin 4 (AQP4) axes following CCI and SCI, respectively.\n110\n, \n111\n NEAT1 downregulation inhibited IL‐6, IL‐1β, and TNF‐α to improve neuropathic pain.\nAlthough the function of circRNAs as miRNA sponges is still largely unknown, they have been termed as competing endogenous RNAs that bind to target miRNAs and regulate their function.\n112\n For example, circHIPK3 is a circRNA highly enriched in serum from DNP patients and in the DRG from DNP rats.\n113\n circHIPK3 sponges miR‐124 to promote neuroinflammation, pointing to the involvement of the circHIPK3–miR‐124 axis during DNP. circRNA ciRS‐7 participates in neuropathic pain progression by sponging miR‐135a‐5p to regulate autophagy and inflammation in the spinal cord of CCI rats.\n114\n Besides, cirZNF609–miR‐22‐3p‐enolase 1 (ENO1) and circ_0005075–miR‐151a‐3p‐NOTCH receptor 2 (NOTCH2) regulatory axes upregulate inflammatory factor expression and promote neuropathic pain development in the spinal cord after CCI.\n115\n, \n116\n\n\nlncRNAs may act as miRNA sponges to form the lncRNA‐miRNA‐mRNA axis and regulate target gene expression. For example, the lncRNA X‐inactive specific transcript (XIST) is upregulated in the dorsal horn of the spinal cord after CCI. By sponging miR‐137, ‐150, ‐154‐5p, and ‐544, XIST can inhibit the expression of corresponding targets, including TNF‐α‐induced protein 1 (TNFAIP1), ZEB1, TLR5, and STAT3.\n106\n, \n107\n, \n108\n, \n109\n TNFAIP1 activates the NF‐κB signaling pathway, while ZEB1, TLR5, and STAT3 are crucial in the neuroinflammatory response. XIST inhibition markedly ameliorates neuropathic pain development. In addition, lncRNA nuclear enriched abundant transcript 1 (NEAT1) was upregulated in the spinal cord dorsal horn to form NEAT1‐miR‐381‐high mobility group box 1 (HMGB1) and NEAT1–miR‐128‐3p–aquaporin 4 (AQP4) axes following CCI and SCI, respectively.\n110\n, \n111\n NEAT1 downregulation inhibited IL‐6, IL‐1β, and TNF‐α to improve neuropathic pain.\nAlthough the function of circRNAs as miRNA sponges is still largely unknown, they have been termed as competing endogenous RNAs that bind to target miRNAs and regulate their function.\n112\n For example, circHIPK3 is a circRNA highly enriched in serum from DNP patients and in the DRG from DNP rats.\n113\n circHIPK3 sponges miR‐124 to promote neuroinflammation, pointing to the involvement of the circHIPK3–miR‐124 axis during DNP. circRNA ciRS‐7 participates in neuropathic pain progression by sponging miR‐135a‐5p to regulate autophagy and inflammation in the spinal cord of CCI rats.\n114\n Besides, cirZNF609–miR‐22‐3p‐enolase 1 (ENO1) and circ_0005075–miR‐151a‐3p‐NOTCH receptor 2 (NOTCH2) regulatory axes upregulate inflammatory factor expression and promote neuropathic pain development in the spinal cord after CCI.\n115\n, \n116\n\n\n\nlncRNAs epigenetically regulate pain‐related molecules expression In addition to acting as a miRNA sponge, lncRNAs can epigenetically regulate the expression of pain‐related molecules. For example, peripheral nerve injury decreases the expression of DRG‐specifically enriched lncRNAs (DS‐lncRNAs) in the injured DRG. Restoring DS‐lncRNAs blocks nerve injury‐induced increases in euchromatic histone lysine N‐methyltransferase 2 (Ehmt2) mRNA and its encoding protein G9a, reverses G9a‐related decreases in opioid receptors and Kcna2 in the injured DRG, and ameliorates nerve injury‐induced pain hypersensitivity.\n117\n In addition, transcriptome screening in the DRG of DNP rats\n118\n has identified diverse dysregulated lncRNAs, including uc.48+,\n119\n BC168687,\n120\n, \n121\n NONRATT021972,\n31\n, \n122\n, \n123\n MRAK009713,\n124\n and Lncenc1.\n125\n The expression of uc.48+, BC168687, NONRATT021972, MRAK009713, and Lncenc1 was prominently higher in the DRG of neuropathic pain rats.\n119\n, \n120\n, \n121\n, \n122\n, \n123\n, \n124\n, \n125\n Blocking the upregulation via intrathecal or intravenous administration of the corresponding small‐interfering RNA (siRNA) may alleviate neuropathic pain by inhibiting the excitatory transmission mediated by purinergic receptors,\n119\n, \n120\n, \n122\n, \n123\n, \n124\n TNF‐α‐related pathways,\n31\n transient receptor potential vanilloid 1 (TRPV1),\n121\n or EZH2.\n125\n\n\nAs demonstrated by a luciferase assay and RNA‐binding protein immunoprecipitation, lncRNA small nucleolar RNA host gene 1 (SNHG1) can induce neuropathic pain in the spinal cord by binding to the promoter region of cyclin‐dependent kinase 4 (CDK4), stimulating its expression.\n126\n SNHG1 knockdown alleviated neuropathic pain development, and SNHG1 overexpression was able to induce neuropathic pain. Similarly, lncRNA PKIA‐AS1 participates in SNL‐induced neuropathic pain by downregulating DNA methyltransferase 1‐catalyzed cyclin‐dependent kinase 6 (DNMT1‐catalyzed) CDK6 promoter methylation and regulating CDK6.\n127\n Cyclin‐dependent kinases (CDKs) transcriptionally enhanced pro‐inflammatory gene expression during the G1 cell phase. Furthermore, CDK6 recruitment to the nuclear chromatin fraction by cytokines is related to NF‐κB, STAT, and activator protein 1 (AP‐1) activation to induce neuroinflammation.\n128\n Other than the DRG, lncRNA Kcna2‐AS‐RNA was also highly expressed in the spinal cord of postherpetic neuralgia rats, and its downregulation alleviated neuropathic pain by reducing phospho‐STAT3 (pSTAT3) translocation from the cytoplasm to the nucleus and then inhibiting spinal astrocytes activation.\n129\n\n\nIn addition to acting as a miRNA sponge, lncRNAs can epigenetically regulate the expression of pain‐related molecules. For example, peripheral nerve injury decreases the expression of DRG‐specifically enriched lncRNAs (DS‐lncRNAs) in the injured DRG. Restoring DS‐lncRNAs blocks nerve injury‐induced increases in euchromatic histone lysine N‐methyltransferase 2 (Ehmt2) mRNA and its encoding protein G9a, reverses G9a‐related decreases in opioid receptors and Kcna2 in the injured DRG, and ameliorates nerve injury‐induced pain hypersensitivity.\n117\n In addition, transcriptome screening in the DRG of DNP rats\n118\n has identified diverse dysregulated lncRNAs, including uc.48+,\n119\n BC168687,\n120\n, \n121\n NONRATT021972,\n31\n, \n122\n, \n123\n MRAK009713,\n124\n and Lncenc1.\n125\n The expression of uc.48+, BC168687, NONRATT021972, MRAK009713, and Lncenc1 was prominently higher in the DRG of neuropathic pain rats.\n119\n, \n120\n, \n121\n, \n122\n, \n123\n, \n124\n, \n125\n Blocking the upregulation via intrathecal or intravenous administration of the corresponding small‐interfering RNA (siRNA) may alleviate neuropathic pain by inhibiting the excitatory transmission mediated by purinergic receptors,\n119\n, \n120\n, \n122\n, \n123\n, \n124\n TNF‐α‐related pathways,\n31\n transient receptor potential vanilloid 1 (TRPV1),\n121\n or EZH2.\n125\n\n\nAs demonstrated by a luciferase assay and RNA‐binding protein immunoprecipitation, lncRNA small nucleolar RNA host gene 1 (SNHG1) can induce neuropathic pain in the spinal cord by binding to the promoter region of cyclin‐dependent kinase 4 (CDK4), stimulating its expression.\n126\n SNHG1 knockdown alleviated neuropathic pain development, and SNHG1 overexpression was able to induce neuropathic pain. Similarly, lncRNA PKIA‐AS1 participates in SNL‐induced neuropathic pain by downregulating DNA methyltransferase 1‐catalyzed cyclin‐dependent kinase 6 (DNMT1‐catalyzed) CDK6 promoter methylation and regulating CDK6.\n127\n Cyclin‐dependent kinases (CDKs) transcriptionally enhanced pro‐inflammatory gene expression during the G1 cell phase. Furthermore, CDK6 recruitment to the nuclear chromatin fraction by cytokines is related to NF‐κB, STAT, and activator protein 1 (AP‐1) activation to induce neuroinflammation.\n128\n Other than the DRG, lncRNA Kcna2‐AS‐RNA was also highly expressed in the spinal cord of postherpetic neuralgia rats, and its downregulation alleviated neuropathic pain by reducing phospho‐STAT3 (pSTAT3) translocation from the cytoplasm to the nucleus and then inhibiting spinal astrocytes activation.\n129\n\n\n\nlncRNAs modulate miRNA processing Specific lncRNAs are essential in neuropathic pain by modulating miRNA processing. For example, the transcribed ultraconserved lncRNA uc.153 was prominently increased in the spinal cord of rats with CCI neuropathic pain. uc.153 knockdown reversed CCI‐induced pain behaviors and spinal neuronal hypersensitivity. Mechanistically, uc.153 negatively modulated Dicer‐mediated pre‐miR‐182‐5p processing and inhibited maturation. Meanwhile, spinal miR‐182‐5p downregulation increased the expression of its target, ephrin type‐b (EphB1), and p‐NR2B (phosphorylated N‐methyl‐D‐aspartate receptor (NMDAR) 2B subunit) expression and facilitated hyperalgesia.\n130\n\n\nCollectively, lncRNAs are crucial in neuropathic pain via diverse mechanisms. Notably, one lncRNA may act with more than one mechanism to regulate target gene expression. For example, SNL upregulated circAnks1a in the cytoplasm and nucleus. circAnks1a enhances the interplay between Y‐box‐binding protein 1 (YBX1) and transportin‐1 to facilitate YBX1 nuclear translocation in the cytoplasm. Meanwhile, in the nucleus, circAnks1a combines with the Vegfb promoter and recruits YBX1 to the Vegfb promoter to enhance its transcription. Additionally, cytoplasmic circAnks1a sponges miR‐324‐3p to regulate vascular endothelial growth factor B (VEGFB) expression. VEGFB binding to its receptor results in the activation of various downstream targets, including p38‐MAPK, PKB/AKT (protein kinase B), extracellular signal‐regulated kinase (ERK)/MAPK, and phosphoinositide 3‐kinase (PI3K).\n131\n Therefore, VEGFB upregulation increases dorsal horn neuron excitability and contributes to pain hypersensitivity after nerve injury.\n132\n\n\nWe also noted that specific lncRNAs were DE following various nerve injuries. For example, lncRNA Malat1 was upregulated after CCI to sponge miR‐206, and Malat1 suppression delayed neuropathic pain progression via miR‐206‐ZEB2 axis‐mediated neuroinflammation inhibition.\n133\n Conversely, in rats with complete brachial plexus avulsion‐induced neuropathic pain, Malat1 decreased in spinal cord neurons, and such downregulation increased neuronal spontaneous electrical activity via calcium flux regulation.\n134\n The reason for such distinctive Malat1 expression following different pain models is still unclear, and further research is required.\nSpecific lncRNAs are essential in neuropathic pain by modulating miRNA processing. For example, the transcribed ultraconserved lncRNA uc.153 was prominently increased in the spinal cord of rats with CCI neuropathic pain. uc.153 knockdown reversed CCI‐induced pain behaviors and spinal neuronal hypersensitivity. Mechanistically, uc.153 negatively modulated Dicer‐mediated pre‐miR‐182‐5p processing and inhibited maturation. Meanwhile, spinal miR‐182‐5p downregulation increased the expression of its target, ephrin type‐b (EphB1), and p‐NR2B (phosphorylated N‐methyl‐D‐aspartate receptor (NMDAR) 2B subunit) expression and facilitated hyperalgesia.\n130\n\n\nCollectively, lncRNAs are crucial in neuropathic pain via diverse mechanisms. Notably, one lncRNA may act with more than one mechanism to regulate target gene expression. For example, SNL upregulated circAnks1a in the cytoplasm and nucleus. circAnks1a enhances the interplay between Y‐box‐binding protein 1 (YBX1) and transportin‐1 to facilitate YBX1 nuclear translocation in the cytoplasm. Meanwhile, in the nucleus, circAnks1a combines with the Vegfb promoter and recruits YBX1 to the Vegfb promoter to enhance its transcription. Additionally, cytoplasmic circAnks1a sponges miR‐324‐3p to regulate vascular endothelial growth factor B (VEGFB) expression. VEGFB binding to its receptor results in the activation of various downstream targets, including p38‐MAPK, PKB/AKT (protein kinase B), extracellular signal‐regulated kinase (ERK)/MAPK, and phosphoinositide 3‐kinase (PI3K).\n131\n Therefore, VEGFB upregulation increases dorsal horn neuron excitability and contributes to pain hypersensitivity after nerve injury.\n132\n\n\nWe also noted that specific lncRNAs were DE following various nerve injuries. For example, lncRNA Malat1 was upregulated after CCI to sponge miR‐206, and Malat1 suppression delayed neuropathic pain progression via miR‐206‐ZEB2 axis‐mediated neuroinflammation inhibition.\n133\n Conversely, in rats with complete brachial plexus avulsion‐induced neuropathic pain, Malat1 decreased in spinal cord neurons, and such downregulation increased neuronal spontaneous electrical activity via calcium flux regulation.\n134\n The reason for such distinctive Malat1 expression following different pain models is still unclear, and further research is required.\n\n\nlncRNAs act as AS RNA\n:\nAfter peripheral nerve injury, Egr2‐AS‐RNA is upregulated in Schwann cells.\n103\n On the early growth response 2 (Egr2) promoter, Egr2‐AS‐RNA recruits an epigenetic silencing complex to downregulate Egr2, essential for peripheral myelination. Ectopic Egr2‐AS‐RNA expression in DRG cultures downregulates Egr2 mRNA and induces demyelination. In vivo, Egr2‐AS‐RNA inhibition reverts Egr2‐related gene expression and delays demyelination.\nVoltage‐gated potassium channel (Kv) Kcna2‐AS‐RNA is an endogenous, highly conserved, and widely explored lncRNA in neuropathic pain.\n104\n It is a natural antisense transcript (NAT), distributed in the cytoplasm and targets Kcna2 mRNA, which encodes the pain regulation‐related membrane Kv1.2 subunit. Kcna2‐AS‐RNA was time‐dependently upregulated in the inflicted rat DRG after nerve injury. It decreased the total Kv current, upregulated DRG neurons excitability, and produced neuropathic pain symptoms.\nThe complementary strand of DNA opposite the Scn9a gene encodes Scn9a NAT, another antisense lncRNA expressed in the DRG.\n105\n Scn9a NAT is suggested to be a negative regulator of Scn9a mRNA. Scn9a NAT overexpression inhibited Scn9a mRNA, its encoded protein Nav1.7, and Nav1.7 currents in DRG neurons. However, Scn9a NAT and Scn9a mRNA levels did not significantly change in the injured DRG until 2 weeks after nerve injury. More work is required to determine whether NAT can confer analgesia and reduce pain in the neuropathic pain state.\n\n\nlncRNAs act as miRNA sponges:\nlncRNAs may act as miRNA sponges to form the lncRNA‐miRNA‐mRNA axis and regulate target gene expression. For example, the lncRNA X‐inactive specific transcript (XIST) is upregulated in the dorsal horn of the spinal cord after CCI. By sponging miR‐137, ‐150, ‐154‐5p, and ‐544, XIST can inhibit the expression of corresponding targets, including TNF‐α‐induced protein 1 (TNFAIP1), ZEB1, TLR5, and STAT3.\n106\n, \n107\n, \n108\n, \n109\n TNFAIP1 activates the NF‐κB signaling pathway, while ZEB1, TLR5, and STAT3 are crucial in the neuroinflammatory response. XIST inhibition markedly ameliorates neuropathic pain development. In addition, lncRNA nuclear enriched abundant transcript 1 (NEAT1) was upregulated in the spinal cord dorsal horn to form NEAT1‐miR‐381‐high mobility group box 1 (HMGB1) and NEAT1–miR‐128‐3p–aquaporin 4 (AQP4) axes following CCI and SCI, respectively.\n110\n, \n111\n NEAT1 downregulation inhibited IL‐6, IL‐1β, and TNF‐α to improve neuropathic pain.\nAlthough the function of circRNAs as miRNA sponges is still largely unknown, they have been termed as competing endogenous RNAs that bind to target miRNAs and regulate their function.\n112\n For example, circHIPK3 is a circRNA highly enriched in serum from DNP patients and in the DRG from DNP rats.\n113\n circHIPK3 sponges miR‐124 to promote neuroinflammation, pointing to the involvement of the circHIPK3–miR‐124 axis during DNP. circRNA ciRS‐7 participates in neuropathic pain progression by sponging miR‐135a‐5p to regulate autophagy and inflammation in the spinal cord of CCI rats.\n114\n Besides, cirZNF609–miR‐22‐3p‐enolase 1 (ENO1) and circ_0005075–miR‐151a‐3p‐NOTCH receptor 2 (NOTCH2) regulatory axes upregulate inflammatory factor expression and promote neuropathic pain development in the spinal cord after CCI.\n115\n, \n116\n\n\n\n\nlncRNAs epigenetically regulate pain‐related molecules expression:\nIn addition to acting as a miRNA sponge, lncRNAs can epigenetically regulate the expression of pain‐related molecules. For example, peripheral nerve injury decreases the expression of DRG‐specifically enriched lncRNAs (DS‐lncRNAs) in the injured DRG. Restoring DS‐lncRNAs blocks nerve injury‐induced increases in euchromatic histone lysine N‐methyltransferase 2 (Ehmt2) mRNA and its encoding protein G9a, reverses G9a‐related decreases in opioid receptors and Kcna2 in the injured DRG, and ameliorates nerve injury‐induced pain hypersensitivity.\n117\n In addition, transcriptome screening in the DRG of DNP rats\n118\n has identified diverse dysregulated lncRNAs, including uc.48+,\n119\n BC168687,\n120\n, \n121\n NONRATT021972,\n31\n, \n122\n, \n123\n MRAK009713,\n124\n and Lncenc1.\n125\n The expression of uc.48+, BC168687, NONRATT021972, MRAK009713, and Lncenc1 was prominently higher in the DRG of neuropathic pain rats.\n119\n, \n120\n, \n121\n, \n122\n, \n123\n, \n124\n, \n125\n Blocking the upregulation via intrathecal or intravenous administration of the corresponding small‐interfering RNA (siRNA) may alleviate neuropathic pain by inhibiting the excitatory transmission mediated by purinergic receptors,\n119\n, \n120\n, \n122\n, \n123\n, \n124\n TNF‐α‐related pathways,\n31\n transient receptor potential vanilloid 1 (TRPV1),\n121\n or EZH2.\n125\n\n\nAs demonstrated by a luciferase assay and RNA‐binding protein immunoprecipitation, lncRNA small nucleolar RNA host gene 1 (SNHG1) can induce neuropathic pain in the spinal cord by binding to the promoter region of cyclin‐dependent kinase 4 (CDK4), stimulating its expression.\n126\n SNHG1 knockdown alleviated neuropathic pain development, and SNHG1 overexpression was able to induce neuropathic pain. Similarly, lncRNA PKIA‐AS1 participates in SNL‐induced neuropathic pain by downregulating DNA methyltransferase 1‐catalyzed cyclin‐dependent kinase 6 (DNMT1‐catalyzed) CDK6 promoter methylation and regulating CDK6.\n127\n Cyclin‐dependent kinases (CDKs) transcriptionally enhanced pro‐inflammatory gene expression during the G1 cell phase. Furthermore, CDK6 recruitment to the nuclear chromatin fraction by cytokines is related to NF‐κB, STAT, and activator protein 1 (AP‐1) activation to induce neuroinflammation.\n128\n Other than the DRG, lncRNA Kcna2‐AS‐RNA was also highly expressed in the spinal cord of postherpetic neuralgia rats, and its downregulation alleviated neuropathic pain by reducing phospho‐STAT3 (pSTAT3) translocation from the cytoplasm to the nucleus and then inhibiting spinal astrocytes activation.\n129\n\n\n\n\nlncRNAs modulate miRNA processing:\nSpecific lncRNAs are essential in neuropathic pain by modulating miRNA processing. For example, the transcribed ultraconserved lncRNA uc.153 was prominently increased in the spinal cord of rats with CCI neuropathic pain. uc.153 knockdown reversed CCI‐induced pain behaviors and spinal neuronal hypersensitivity. Mechanistically, uc.153 negatively modulated Dicer‐mediated pre‐miR‐182‐5p processing and inhibited maturation. Meanwhile, spinal miR‐182‐5p downregulation increased the expression of its target, ephrin type‐b (EphB1), and p‐NR2B (phosphorylated N‐methyl‐D‐aspartate receptor (NMDAR) 2B subunit) expression and facilitated hyperalgesia.\n130\n\n\nCollectively, lncRNAs are crucial in neuropathic pain via diverse mechanisms. Notably, one lncRNA may act with more than one mechanism to regulate target gene expression. For example, SNL upregulated circAnks1a in the cytoplasm and nucleus. circAnks1a enhances the interplay between Y‐box‐binding protein 1 (YBX1) and transportin‐1 to facilitate YBX1 nuclear translocation in the cytoplasm. Meanwhile, in the nucleus, circAnks1a combines with the Vegfb promoter and recruits YBX1 to the Vegfb promoter to enhance its transcription. Additionally, cytoplasmic circAnks1a sponges miR‐324‐3p to regulate vascular endothelial growth factor B (VEGFB) expression. VEGFB binding to its receptor results in the activation of various downstream targets, including p38‐MAPK, PKB/AKT (protein kinase B), extracellular signal‐regulated kinase (ERK)/MAPK, and phosphoinositide 3‐kinase (PI3K).\n131\n Therefore, VEGFB upregulation increases dorsal horn neuron excitability and contributes to pain hypersensitivity after nerve injury.\n132\n\n\nWe also noted that specific lncRNAs were DE following various nerve injuries. For example, lncRNA Malat1 was upregulated after CCI to sponge miR‐206, and Malat1 suppression delayed neuropathic pain progression via miR‐206‐ZEB2 axis‐mediated neuroinflammation inhibition.\n133\n Conversely, in rats with complete brachial plexus avulsion‐induced neuropathic pain, Malat1 decreased in spinal cord neurons, and such downregulation increased neuronal spontaneous electrical activity via calcium flux regulation.\n134\n The reason for such distinctive Malat1 expression following different pain models is still unclear, and further research is required.\n\n\nCHALLENGES AND CONSIDERATIONS IN DELIVERING miRNA‐AND lncRNAs‐BASED THERAPETUTICS\n:\nThere are two main strategies for modulating miRNA function during pain treatment: upregulation or downregulation of specific miRNAs. miRNA mimics or virus‐based constructs were used to upregulate miRNA expression. miRNA inhibitors, miRNA sponges, or inhibition of a particular miRNA‐mRNA interaction has been applied to downregulate specific miRNAs. The design of lncRNA‐based therapeutics includes diverse approaches, such as post‐transcriptional inhibition of lncRNAs by antisense oligonucleotides or siRNA and steric blockades of lncRNA–protein interactions by small molecules and morpholino.\n135\n\n\nHowever, there is much work to be done before miRNA‐ and lncRNA‐based analgesics can be clinically used for various reasons, including their conservation among species, proper delivery, stability, off‐target effects, and potential activation of the immune system.\nFirst, most available findings regarding the efficacy of miRNAs or lncRNAs during neuropathic pain are based on animal but not human studies. Whether such results can be extrapolated to humans and their translational potential is still unknown because of conservation among species.\nSecond, the blood–brain barrier (BBB) is a practical challenge for delivering RNA‐based therapeutics into the central nervous system (CNS) via intravenous injection. Viral vectors, polypeptides, aptamers, and particular chemical modifications have been developed. Supplementing cholesterol molecules to the sense strand of a miRNA mimic or inhibitor has proven to be an efficient strategy. Cholesterol‐conjugated siRNAs showed better silencing potency than unconjugated siRNAs and presented a high efficacy for delivery to oligodendrocytes in the CNS.\n136\n Another proven method is the immunoliposome, a combination of liposomes, receptor‐targeted monoclonal antibodies, and the target molecules.\n137\n An immunoliposome nanocomplex has been reported to deliver therapeutic nucleic acids across the BBB into the deep brain by transferrin receptors.\n138\n In addition, intrathecal injections are a feasible approach in animal studies for neuropathic pain treatment. As reported, miR‐146a attenuated neuropathic pain partially by inhibiting TNF receptor‐associated factor 6 (TRAF6) and its downstream phospho‐JNK/C–C motif chemokine ligand 2 (pJNK/CCL2) signaling in the spinal cord.\n139\n Intrathecal injection of miR‐146a‐5p encapsulated nanoparticles provided an analgesic effect via NF‐κB and p38‐MAPK inhibition in spinal microglia.\n140\n In recent years, poly (D, L‐lactic‐co‐glycolic acid) (PLGA)‐nanoparticles have been applied to deliver siRNAs and plasmids into the spinal cord to treat neuropathic pain in rats.\n141\n PLGA copolymer is a promising US Food and Drug Administration (FDA)‐approved gene transmission material because of its biodegradability and biocompatibility in humans.\n142\n Intrathecal treatments with C‐X3‐C motif chemokine receptor 1 (CX3CR1), p38, p66shc siRNA encapsulated PLGA nanoparticles, or forkhead box P3 (Foxp3) plasmid‐encapsulated PLGA nanoparticles inhibit microglial activation and hyperalgesia in SNL rats.\n141\n, \n143\n, \n144\n, \n145\n\n\nExosomes are another promising delivery carrier for treating neuropathic pain. Exosomes are natural membranous microvesicles that carry RNAs, with the advantage of being efficient, cell‐free, and nonimmunogenicity. Intravenous injecting neuron‐targeted exosomes delivered the carried siRNAs to neurons, microglia, and oligodendrocytes to knock down specific gene expression in mice brains.\n146\n This approach enabled cell‐specific delivery of the siRNA‐cargo across the BBB. Mesenchymal stem cells (MSCs) are pluripotent stem cells with immunomodulatory, anti‐inflammatory, and nutritional properties. The treatment efficacy of exosomes derived from MSCs has been proven in neuropathic pain.\n147\n Intrathecal, local, or subcutaneous application of exosomes obtained from human umbilical cord MSCs could mitigate nerve injury‐induced hyperalgesia.\n148\n, \n149\n, \n150\n Furthermore, immunofluorescence results showed that most intrathecally injected exosomes could be found in injured peripheral axons, the DRG, and the spinal dorsal horn, potentiating a homing ability of exosomes.\n148\n\n\nThird, although miRNAs are relatively stable in vivo, they have long‐lasting efficacy and a high resistance to nucleolytic degradation compared to mRNAs. Particular chemical modifications are still required to prolong its half‐life or increase its stability, for example, by generating locked‐nucleic acids (LNAs). LNA modifications increase the RNA affinity of antisense oligonucleotides, presenting an excellent miRNA inhibitory activity at a low dosage.\n151\n Additionally, the LNA technique was used to synthesize highly stable aptamers.\n152\n\n\nFourth, the off‐target effect is another consideration. One miRNA may regulate multiple genes, and the off‐target potential of one miRNA may produce undesirable side effects.\n153\n, \n154\n\n\nFinally, activation of the immune system is a potential adverse event. Specific CpG motifs in oligonucleotides could trigger nonspecific immunological activity.\n155\n Therefore, RNA‐based therapeutics might also produce specific immune reactions after producing antibodies against the oligonucleotides.\n\nCONCLUSION:\nRecent human and animal studies have identified accumulating dysregulated miRNAs and lncRNAs in the serum or along pain processing pathways following peripheral nerve injury or noxious stimulation. Experimental studies have validated their essential role in neuropathic pain. miRNAs contribute to neuropathic pain development via neuroinflammation, autophagy, abnormal ion channel expression, regulating pain‐related mediators, protein kinases, structural proteins, neurotransmission excitatory–‐inhibitory imbalances, and exosome miRNA‐mediated neuron–glia communication. Meanwhile, lncRNAs and circRNAs are crucial in neuropathic pain development by acting as AS RNA and miRNA sponges, epigenetically regulating pain‐related molecules expression, or modulating miRNA processing. However, more work is required before miRNA‐ and lncRNA‐based analgesics can be clinically used for various reasons, including their conservation among species, proper delivery, stability, off‐target effects, and potential activation of the immune system.\n\nAUTHOR CONTRIBUTIONS:\nMing Jiang: Investigation, Methodology. Yelong Wang: Manuscript preparation. Jing Wang: Figures and tables preparation. Shanwu Feng: Supervision, Funding acquisition. Xian Wang: Conceptualization, Funding acquisition.\n\nCONFLICT OF INTERESTS:\nThe authors declare no competing interest in this review.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nNon-coding RNAs (ncRNAs) are involved in neuropathic pain development. Herein, we systematically searched for neuropathic pain-related ncRNAs expression changes, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular non-coding RNAs (circRNAs).\n\nMethods:\nWe searched two databases, PubMed and GeenMedical, for relevant studies.\n\nResults:\nPeripheral nerve injury or noxious stimuli can induce extensive changes in the expression of ncRNAs. For example, higher serum miR-132-3p, -146b-5p, and -384 was observed in neuropathic pain patients. Either sciatic nerve ligation, dorsal root ganglion (DRG) transaction, or ventral root transection (VRT) could upregulate miR-21 and miR-31 while downregulating miR-668 and miR-672 in the injured DRG. lncRNAs, such as early growth response 2-antisense-RNA (Egr2-AS-RNA) and Kcna2-AS-RNA, were upregulated in Schwann cells and inflicted DRG after nerve injury, respectively. Dysregulated circRNA homeodomain-interacting protein kinase 3 (circHIPK3) in serum and the DRG, abnormally expressed lncRNAs X-inactive specific transcript (XIST), nuclear enriched abundant transcript 1 (NEAT1), small nucleolar RNA host gene 1 (SNHG1), as well as ciRS-7, zinc finger protein 609 (cirZNF609), circ_0005075, and circAnks1a in the spinal cord were suggested to participate in neuropathic pain development. Dysregulated miRNAs contribute to neuropathic pain via neuroinflammation, autophagy, abnormal ion channel expression, regulating pain-related mediators, protein kinases, structural proteins, neurotransmission excitatory-inhibitory imbalances, or exosome miRNA-mediated neuron-glia communication. In addition, lncRNAs and circRNAs are essential in neuropathic pain by acting as antisense RNA and miRNA sponges, epigenetically regulating pain-related molecules expression, or modulating miRNA processing.\n\nConclusions:\nNumerous dysregulated ncRNAs have been suggested to participate in neuropathic pain development. However, there is much work to be done before ncRNA-based analgesics can be clinically used for various reasons such as conservation among species, proper delivery, stability, and off-target effects."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nAccording to the International Association for the Study of Pain, neuropathic pain is the most severe chronic pain condition triggered by a lesion or disease of the somatosensory system. It is characteristic of hyperalgesia, allodynia, or spontaneous pain.\n1\n Neuropathic pain can have peripheral and central origins, with the former including neuropathic pain after peripheral nerve injury, trigeminal neuralgia, postherpetic neuralgia, painful radiculopathy, and painful polyneuropathy. In contrast, central neuropathic pain includes neuropathic pain after spinal cord or brain injury, multiple sclerosis, and chronic central post‐stroke pain. Approximately 7%–10% of the general population will experience neuropathic pain, with the majority not having satisfactory pain relief with current therapies, leading to great suffering for individuals and enormous economic and social burdens.\n2\n\n\nThe exact molecular mechanisms underlying neuropathic pain remain unclear, and elucidating them is crucial for developing mechanism‐based treatment strategies. One proposed mechanism involves altered gene or protein expression along the pain processing pathways. Therefore, understanding how genes or proteins are dysregulated may help us to find a way to normalize these abnormalities and treat neuropathic pain.\nIn recent years, accumulating evidence has suggested the essential role of non‐coding RNAs (ncRNAs) in various physiological and pathological procedures, such as embryonic development, inflammation, tumors, and respiratory and cardiovascular diseases.\n3\n ncRNAs have no protein‐coding potential, but they can govern gene or protein expression with diverse mechanisms. ncRNAs are extensively distributed in the peripheral and central nervous systems, including pain‐related structures.\n4\n Broad abnormal ncRNAs expression is observed following peripheral stimulation. These abnormalities are related to hyperalgesia during chronic pain development. The available data indicate that ncRNAs may be essential for hyperalgesia. In this review, we focus on microRNA (miRNA), long non‐coding RNA (lncRNA), and circular non‐coding RNA (circRNA) expression changes in neuropathic pain. Other types of ncRNAs are seldom reported in neuropathic pain and, thus, are not discussed herein. Notably, we will pay attention to their etiological role in the development of neuropathic pain and the current challenges and considerations for miRNA‐, lncRNA‐, and circRNA‐based therapeutics for neuropathic pain.\n\nCONCLUSION:\nRecent human and animal studies have identified accumulating dysregulated miRNAs and lncRNAs in the serum or along pain processing pathways following peripheral nerve injury or noxious stimulation. Experimental studies have validated their essential role in neuropathic pain. miRNAs contribute to neuropathic pain development via neuroinflammation, autophagy, abnormal ion channel expression, regulating pain‐related mediators, protein kinases, structural proteins, neurotransmission excitatory–‐inhibitory imbalances, and exosome miRNA‐mediated neuron–glia communication. Meanwhile, lncRNAs and circRNAs are crucial in neuropathic pain development by acting as AS RNA and miRNA sponges, epigenetically regulating pain‐related molecules expression, or modulating miRNA processing. However, more work is required before miRNA‐ and lncRNA‐based analgesics can be clinically used for various reasons, including their conservation among species, proper delivery, stability, off‐target effects, and potential activation of the immune system."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nNon-coding RNAs (ncRNAs) are involved in neuropathic pain development. Herein, we systematically searched for neuropathic pain-related ncRNAs expression changes, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular non-coding RNAs (circRNAs).\n\nMethods:\nWe searched two databases, PubMed and GeenMedical, for relevant studies.\n\nResults:\nPeripheral nerve injury or noxious stimuli can induce extensive changes in the expression of ncRNAs. For example, higher serum miR-132-3p, -146b-5p, and -384 was observed in neuropathic pain patients. Either sciatic nerve ligation, dorsal root ganglion (DRG) transaction, or ventral root transection (VRT) could upregulate miR-21 and miR-31 while downregulating miR-668 and miR-672 in the injured DRG. lncRNAs, such as early growth response 2-antisense-RNA (Egr2-AS-RNA) and Kcna2-AS-RNA, were upregulated in Schwann cells and inflicted DRG after nerve injury, respectively. Dysregulated circRNA homeodomain-interacting protein kinase 3 (circHIPK3) in serum and the DRG, abnormally expressed lncRNAs X-inactive specific transcript (XIST), nuclear enriched abundant transcript 1 (NEAT1), small nucleolar RNA host gene 1 (SNHG1), as well as ciRS-7, zinc finger protein 609 (cirZNF609), circ_0005075, and circAnks1a in the spinal cord were suggested to participate in neuropathic pain development. Dysregulated miRNAs contribute to neuropathic pain via neuroinflammation, autophagy, abnormal ion channel expression, regulating pain-related mediators, protein kinases, structural proteins, neurotransmission excitatory-inhibitory imbalances, or exosome miRNA-mediated neuron-glia communication. In addition, lncRNAs and circRNAs are essential in neuropathic pain by acting as antisense RNA and miRNA sponges, epigenetically regulating pain-related molecules expression, or modulating miRNA processing.\n\nConclusions:\nNumerous dysregulated ncRNAs have been suggested to participate in neuropathic pain development. However, there is much work to be done before ncRNA-based analgesics can be clinically used for various reasons such as conservation among species, proper delivery, stability, and off-target effects."},"whole_article_text_length":{"kind":"number","value":18773,"string":"18,773"},"whole_article_abstract_length":{"kind":"number","value":398,"string":"398"},"other_sections_lengths":{"kind":"list like","value":[403,401,2370,826,353,5917,1000,130,510,682,170,254,3089,253,338,465,362,868,37],"string":"[\n 403,\n 401,\n 2370,\n 826,\n 353,\n 5917,\n 1000,\n 130,\n 510,\n 682,\n 170,\n 254,\n 3089,\n 253,\n 338,\n 465,\n 362,\n 868,\n 37\n]","isConversational":false},"num_sections":{"kind":"number","value":21,"string":"21"},"most_frequent_words":{"kind":"list like","value":["mir","pain","neuropathic","neuropathic pain","expression","nerve","spinal","drg","injury","mirna"],"string":"[\n \"mir\",\n \"pain\",\n \"neuropathic\",\n \"neuropathic pain\",\n \"expression\",\n \"nerve\",\n \"spinal\",\n \"drg\",\n \"injury\",\n \"mirna\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["pain regulation protein","related genes pain","genes pain processing","neuropathic pain pathogenesis","neuropathic pain mechanisms"],"string":"[\n \"pain regulation protein\",\n \"related genes pain\",\n \"genes pain processing\",\n \"neuropathic pain pathogenesis\",\n \"neuropathic pain mechanisms\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"null"},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] circRNAs | lncRNAs | mechanism study | miRNAs | neuropathic pain [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"null"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] circRNAs | lncRNAs | mechanism study | miRNAs | neuropathic pain [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] circRNAs | lncRNAs | mechanism study | miRNAs | neuropathic pain [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] circRNAs | lncRNAs | mechanism study | miRNAs | neuropathic pain [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Ganglia, Spinal | Humans | MicroRNAs | Neuralgia | RNA, Circular | RNA, Long Noncoding [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"null"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Ganglia, Spinal | Humans | MicroRNAs | Neuralgia | RNA, Circular | RNA, Long Noncoding [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Ganglia, Spinal | Humans | MicroRNAs | Neuralgia | RNA, Circular | RNA, Long Noncoding [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Ganglia, Spinal | Humans | MicroRNAs | Neuralgia | RNA, Circular | RNA, Long Noncoding [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] pain regulation protein | related genes pain | genes pain processing | neuropathic pain pathogenesis | neuropathic pain mechanisms 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injury | mirna [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"null"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] mir | pain | neuropathic | neuropathic pain | expression | nerve | spinal | drg | injury | mirna [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] mir | pain | neuropathic | neuropathic pain | expression | nerve | spinal | drg | injury | mirna [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] mir | pain | neuropathic | neuropathic pain | expression | nerve | spinal | drg | injury | mirna [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] pain | ncrnas | neuropathic | neuropathic pain | central | coding | non coding | non | peripheral central | pain peripheral [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"null"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] pain | regulating pain related | regulating pain | mirna | regulating | studies | pain related | lncrnas | role neuropathic pain mirnas | role neuropathic pain [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] mir | pain | neuropathic | neuropathic pain | expression | nerve | spinal | drg | mirna | injury [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] mir | pain | neuropathic | neuropathic pain | expression | nerve | spinal | drg | mirna | injury [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"null"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| two | PubMed | GeenMedical ||| ||| ncRNAs ||| ||| VRT | miR-668 ||| 2 | Kcna2-AS-RNA | Schwann ||| 3 | circHIPK3 | DRG | XIST | 1 | NEAT1 | RNA | 1 | SNHG1 | 609 ||| ||| RNA ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| two | PubMed | GeenMedical ||| ||| ncRNAs ||| ||| VRT | miR-668 ||| 2 | Kcna2-AS-RNA | Schwann ||| 3 | circHIPK3 | DRG | XIST | 1 | NEAT1 | RNA | 1 | SNHG1 | 609 ||| ||| RNA ||| ||| [SUMMARY]"}}},{"rowIdx":51,"cells":{"title":{"kind":"string","value":"Lectin histochemistry of the olfactory mucosa of Korean native cattle, "},"pmid":{"kind":"string","value":"36448434"},"background_abstract":{"kind":"string","value":"The olfactory mucosa (OM) is crucial for odorant perception in the main olfactory system. The terminal carbohydrates of glycoconjugates influence chemoreception in the olfactory epithelium (OE)."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"The OM of neonate and adult Korean native cattle was evaluated using histological, immunohistochemical, and lectin histochemical methods."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Histologically, the OM in both neonates and adults consists of the olfactory epithelium and the lamina propria. Additionally, using periodic acid Schiff and Alcian blue (pH 2.5), the mucus specificity of the Bowman's gland duct and acini in the lamina propria was determined. Immunohistochemistry demonstrated that mature and immature olfactory sensory neurons of OEs express the olfactory marker protein and growth associated protein-43, respectively. Lectin histochemistry indicated that numerous glycoconjugates, including as N-acetylglucosamine, mannose, galactose, N-acetylgalactosamine, complex type N-glycan, and fucose groups, were expressed at varied levels in the different cell types in the OMs of neonates and adults at varying levels. According to our observations, the cattle possessed a well-developed olfactory system, and the expression patterns of glycoconjugates in neonatal and adult OMs varied considerably."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"This is the first study to describe the morphological assessment of the OM of Korean native cattle with a focus on lectin histochemistry. The findings suggest that glycoconjugates may play a role in olfactory chemoreception, and that their labeling properties may be closely related to OM development and maturity."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Cattle","Animals","Lectins","Galactose","Olfactory Mucosa","Republic of Korea"],"string":"[\n \"Cattle\",\n \"Animals\",\n \"Lectins\",\n \"Galactose\",\n \"Olfactory Mucosa\",\n \"Republic of Korea\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"9715387"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Olfaction is important for animals because it allows them to explore their surroundings for odorous signals from food sources and environments, as well as detect chemical compounds that influence social interaction and reproductive behavior [1]. This perception is mediated by two chemosensory systems: the main olfactory system and the vomeronasal system. The olfactory mucosa (OM), positioned at the caudal/posterior roof of the nasal cavity, and the vomeronasal organ (VNO), located at the base of the nasal septum or on the roof of the mouth, are the organs comprising these two systems [23]. Traditionally, the OM and VNO have been considered functionally and anatomically distinct, with the OM detecting conventional volatile odorants and the VNO receiving pheromones [45]. These two organs share some common features but differ in neuron types, primary structures of receptor proteins, physiological pathways, and central neuroanatomical projections into the brain [6]. However, despite the anatomical and functional differences, previous studies indicate that the OM and VNO play a synergistic role in the regulation of various olfactory-induced behaviors and reproductive and social interactions in mammals [15789].\nThe OM is critical for chemical signal acquisition in the main olfactory system, conveying signals to the main olfactory bulb [4]. In mammals, the OM is composed of the olfactory epithelium (OE) and the lamina propria. The OE is predominantly composed of chemosensory neurons, supporting cells, and basal cells [10]. The lamina propria is constituted by loose connective tissue in which olfactory nerve axon bundles, Bowman's glands, and blood and lymph vessels [1011]. In mammals, odorant reception occurs via the chemosensory neurons of the OE, which contain dendrites that extend beyond the apical surface, where the cilia protrude into the mucus layer, and a basally projecting axon process [12]. Glycoconjugates (terminal carbohydrates) have been shown to play a crucial role in the chemoreception of the OE [13].\nCell surfaces are densely packed with a diverse array of glycoconjugates, each of which provides considerable biological information [14]. Lectins are naturally occurring glycoconjugate-binding molecules, and a large number of purified lectins are regarded as the primary analytical tool for studying glycoconjugates in the olfactory system [131516]. Specific lectin binding is closely related to the olfactory neuron function, as evidenced by the abundance of glycoconjugates in the mucosensory compartment of the chemosensory epithelia, where receptor-specific events associated with transduction occur [13]. Numerous animals, including rats [1718], mice [1920], marmosets [21], sheep [1522], camels [22], horses [23], and humans [24], express the carbohydrate (lectin-binding) moiety in their OM.\nDespite the fact that cattle are one of the best species for biochemical investigations on olfaction [25], there is little published information on the immunohistochemical and lectin histochemical properties of the OM in bovines. We first evaluated the histological characteristics and lectin histochemistry of the OM in Korean native cattle, and then we compared neonate to adult OMs to determine the features associated with maturity. To our knowledge, this is the first study that provides a comprehensive outlook on the expression levels of lectin-binding glycoconjugates in bovine OM."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"Histological findings of the OM In neonatal (Fig. 1A) and adult cattle (Fig. 1B), the OM was composed of the OE and the lamina propria. The OE is predominantly constituted of basal cells, receptor cells, and supporting cells. The nuclei of the basal, receptor, and supporting cells were located in the basal, middle, and apical regions, respectively, of the OE (Fig. 1A and B). The basal cells were round, pallid, and had little nuclei. The nuclei of the supporting cells were elongated and spindle-shaped, but the nuclei of receptor cells were more rounded. While neonatal and adult OMs displayed comparable histological features, adult OMs appeared thicker than neonatal OMs.\nThe lamina propria included Bowman’s glands and olfactory axon bundles. Bowman’s glands were composed of clustered acinar cells that were connected through ducts to the apical surface (Fig. 1A and B). The Bowman’s glandular ducts and/or acini stained positively for PAS (Fig. 1C and E) and Alcian blue stains (pH 2.5; Fig. 1D and F) in neonates (Fig. 1C and D) and adults (Fig. 1E and F); however, the reactivity of duct cells was relatively lower than that of gland cells. Additionally, the histological features of the OM did not differ between males and females at both ages.\nIn neonatal (Fig. 1A) and adult cattle (Fig. 1B), the OM was composed of the OE and the lamina propria. The OE is predominantly constituted of basal cells, receptor cells, and supporting cells. The nuclei of the basal, receptor, and supporting cells were located in the basal, middle, and apical regions, respectively, of the OE (Fig. 1A and B). The basal cells were round, pallid, and had little nuclei. The nuclei of the supporting cells were elongated and spindle-shaped, but the nuclei of receptor cells were more rounded. While neonatal and adult OMs displayed comparable histological features, adult OMs appeared thicker than neonatal OMs.\nThe lamina propria included Bowman’s glands and olfactory axon bundles. Bowman’s glands were composed of clustered acinar cells that were connected through ducts to the apical surface (Fig. 1A and B). The Bowman’s glandular ducts and/or acini stained positively for PAS (Fig. 1C and E) and Alcian blue stains (pH 2.5; Fig. 1D and F) in neonates (Fig. 1C and D) and adults (Fig. 1E and F); however, the reactivity of duct cells was relatively lower than that of gland cells. Additionally, the histological features of the OM did not differ between males and females at both ages.\nImmunohistochemical analysis of OMP in the OM In neonatal (Fig. 2A) and adult (Fig. 2B) cattle, OMP, a marker for mature olfactory sensory neurons, immunoreactivity was identified in the majority of the mature receptor cells in the middle region of the OE, with dendrites reaching to the OE free border, but not in basal and supporting cells. Their reactivity was more prominent in adults. GAP-43, a marker of immature olfactory sensory neurons, was found in certain receptor cells in the basal region of the OE, and some dendrites were immunopositive for GAP-43 (Fig. 2C and D). In addition, OMP and GAP-43 immunoreactivities were observed in the nerve bundles of the lamina propria (Fig. 2A and C).\nOMP, olfactory marker protein; GAP-43, growth associated protein-43; OM, olfactory mucosa; OE, olfactory epithelium; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.\nIn neonatal (Fig. 2A) and adult (Fig. 2B) cattle, OMP, a marker for mature olfactory sensory neurons, immunoreactivity was identified in the majority of the mature receptor cells in the middle region of the OE, with dendrites reaching to the OE free border, but not in basal and supporting cells. Their reactivity was more prominent in adults. GAP-43, a marker of immature olfactory sensory neurons, was found in certain receptor cells in the basal region of the OE, and some dendrites were immunopositive for GAP-43 (Fig. 2C and D). In addition, OMP and GAP-43 immunoreactivities were observed in the nerve bundles of the lamina propria (Fig. 2A and C).\nOMP, olfactory marker protein; GAP-43, growth associated protein-43; OM, olfactory mucosa; OE, olfactory epithelium; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.\nLectin histochemistry in the OM The intensity values of 21 lectin-binding sites in the OE and lamina propria of the OM of Korean native cattle are summarized in Tables 2 and 3, respectively.\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\naApical perinuclear labeling.\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\nN-acetylglucosamine-binding lectins All lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\nOM, olfactory mucosa.\nAll lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\nOM, olfactory mucosa.\nMannose-binding lectins ConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\nConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\nGalactose-binding lectins In the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\nIn the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\nN-acetylgalactosamine-binding lectins All the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\nOM, olfactory mucosa.\nAll the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\nOM, olfactory mucosa.\nComplex type N-glycan-binding lectins In the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\nIn the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\nFucose-binding lectin In the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\nIn the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\nThe intensity values of 21 lectin-binding sites in the OE and lamina propria of the OM of Korean native cattle are summarized in Tables 2 and 3, respectively.\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\naApical perinuclear labeling.\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\nN-acetylglucosamine-binding lectins All lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\nOM, olfactory mucosa.\nAll lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\nOM, olfactory mucosa.\nMannose-binding lectins ConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\nConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\nGalactose-binding lectins In the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\nIn the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\nN-acetylgalactosamine-binding lectins All the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\nOM, olfactory mucosa.\nAll the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\nOM, olfactory mucosa.\nComplex type N-glycan-binding lectins In the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\nIn the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\nFucose-binding lectin In the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\nIn the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3)."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Tissue preparation","Histological examination","Mucus histochemistry","Immunohistochemistry","Lectin histochemistry","Histological findings of the OM","Immunohistochemical analysis of OMP in the OM","Lectin histochemistry in the OM","N-acetylglucosamine-binding lectins","Mannose-binding lectins","Galactose-binding lectins","N-acetylgalactosamine-binding lectins","Complex type N-glycan-binding lectins","Fucose-binding lectin"],"string":"[\n \"Tissue preparation\",\n \"Histological examination\",\n \"Mucus histochemistry\",\n \"Immunohistochemistry\",\n \"Lectin histochemistry\",\n \"Histological findings of the OM\",\n \"Immunohistochemical analysis of OMP in the OM\",\n \"Lectin histochemistry in the OM\",\n \"N-acetylglucosamine-binding lectins\",\n \"Mannose-binding lectins\",\n \"Galactose-binding lectins\",\n \"N-acetylgalactosamine-binding lectins\",\n \"Complex type N-glycan-binding lectins\",\n \"Fucose-binding lectin\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Three neonatal OM samples (1 to 3 day old) and three adult OM samples (2.5-year-old) of Korean native cattle (Hanwoo, Bos taurus coreanae) were obtained from local farms and a local slaughterhouse in Chungcheongbuk-do, South Korea, respectively. Both sexes of all animals were utilized in this study (male: four neonates and five adults; female: three neonates and three adults). The OM used in this study belongs to a small area on the caudal roof of the nasal cavity, where it completely covered the ethmoturbinates and caudal portions of the dorsal and middle nasal conchae (Fig. 1A). Morphological exams of the nasal cavity indicated that none of them had underlying respiratory diseases. All experimental and animal handling procedures were conducted in accordance with the guidelines of Institutional Animal Care and Use Committee of Chonnam National University (26 October 2021; CNU IACUC-YB-2021-131).\nOM, olfactory mucosa; BD, Bowman’s glandular duct; BG, Bowman’s gland; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.","For light microscopic examination, the ethmoturbinates were removed immediately after death, cut into 5-cm-thick cross sections, and fixed for 3–5 days in 10% buffered formalin. Following fixation, the ethmoturbinates were trimmed and decalcified through multiple solution changes of sodium citrate-formic acid solution. When a needle could readily penetrate the bone with very little force, the decalcification process was stopped. The samples were then washed for 24 h with running tap water, dehydrated in a succession of ethanol concentrations (70%, 80%, 90%, 95%, and 100%), cleaned in xylene, embedded in paraffin, and sectioned into 4 μm slices. Following deparaffinization, the sections were utilized for hematoxylin and eosin staining, mucus and lectin histochemistry, and immunohistochemistry.","The acidic mucin was distinguished from the neutral epithelial mucin using periodic acid Schiff (PAS) and Alcian blue (pH 2.5) staining. For PAS staining, the sections were subjected to 0.5% periodic acid for 15 min, then washed and incubated in Schiff reagent for 30 min. After 10 min of washing with running tap water, the sections were counterstained with hematoxylin. For Alcian blue staining, the sections were first incubated in 3% acetic acid for 3 min, followed by 30 min in 1% Alcian blue solution in 3% acetic acid (pH 2.5). After 1 min of washing with running tap water, the sections were counterstained with neutral red.","To retrieve the antigens, the sections were heated for 1 h at 90°C in citrate buffer (0.01 M, pH 6.0). After cooling, the sections were treated for 20 min with 0.3% hydrogen peroxide in distilled water to suppress the endogenous peroxidase activity. To avoid non-specific binding, the sections were treated with normal goat serum (Vectastain Elite ABC kit; Vector Laboratories, USA) for 1 h before being incubated overnight at 4°C with rabbit monoclonal anti-olfactory marker protein (OMP) (1:1,000 dilution, Cat. No. ab183947; Abcam, UK) and rabbit polyclonal anti-growth associated protein-43 (GAP-43) (1:1,000 dilution, Cat. No. PA5-34943; ThermoFisher Scientific, USA) antibodies. The primary antibodies were excluded from the procedure as negative controls. The sections were rinsed with phosphate-buffered saline (PBS), treated with biotinylated goat anti-rabbit IgG (Vectastain Elite ABC kit) for 1 h, and then reacted with the avidin-biotin-peroxidase complex (Vectastain Elite ABC kit) for 1 h at room temperature (RT). Immunoreactivity was detected using a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories), followed by counterstaining with hematoxylin.","Biotinylated lectin kits I, II, and III (Cat. No. BK-1000, BK-2000, and BK-3000) were acquired from Vector Laboratories. The acronyms, sources, and specificities of the used lectins are shown in Table 1. On the basis of their binding specificity and inhibitory sugars, lectins were categorized as N-acetylglucosamine, mannose, galactose, N-acetylgalactosamine, complex type N-glycan, and fucose-binding lectins. For competitive inhibition, the following sugars were purchased from Sigma-Aldrich (USA) and Vector Laboratories (Table 1): N-acetyl-D-glucosamine (β-D–GlcNAc; Sigma-Aldrich), Chitin Hydrolysate (Vector Laboratories), α-methyl mannoside/α-methyl glucoside (Sigma-Aldrich), lactose (Galβ1, 4Glc; Sigma-Aldrich), N-acetyl-D-galactosamine (α-D-GalNAc; Sigma-Aldrich), melibiose (Galα1, 6Glc; Sigma-Aldrich), and β-D-galactose (Sigma-Aldrich).\nFuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose.\naThe acronyms and specificities of the 21 lectins were obtained from the data sheep (Vector laboratory) and grouped, as shown in a previous paper [23].\nTo eliminate the endogenous peroxidase activity, the sections were treated with 0.3% hydrogen peroxide in methanol. To prevent non-specific reactions, the sections were rinsed with PBS and then incubated with 1% bovine serum albumin in PBS. The sections were then treated overnight at 4°C with biotinylated lectins and reacted for 45 min at RT with an avidin-biotin-peroxidase complex (Vectastain Elite ABC kit). The sections were rinsed with PBS, treated with a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories), and counterstained with hematoxylin. Negative controls for lectin histochemistry were generated by removing the biotinylated lectins and preincubating the lectins with appropriate inhibitors for 1 h at RT.","In neonatal (Fig. 1A) and adult cattle (Fig. 1B), the OM was composed of the OE and the lamina propria. The OE is predominantly constituted of basal cells, receptor cells, and supporting cells. The nuclei of the basal, receptor, and supporting cells were located in the basal, middle, and apical regions, respectively, of the OE (Fig. 1A and B). The basal cells were round, pallid, and had little nuclei. The nuclei of the supporting cells were elongated and spindle-shaped, but the nuclei of receptor cells were more rounded. While neonatal and adult OMs displayed comparable histological features, adult OMs appeared thicker than neonatal OMs.\nThe lamina propria included Bowman’s glands and olfactory axon bundles. Bowman’s glands were composed of clustered acinar cells that were connected through ducts to the apical surface (Fig. 1A and B). The Bowman’s glandular ducts and/or acini stained positively for PAS (Fig. 1C and E) and Alcian blue stains (pH 2.5; Fig. 1D and F) in neonates (Fig. 1C and D) and adults (Fig. 1E and F); however, the reactivity of duct cells was relatively lower than that of gland cells. Additionally, the histological features of the OM did not differ between males and females at both ages.","In neonatal (Fig. 2A) and adult (Fig. 2B) cattle, OMP, a marker for mature olfactory sensory neurons, immunoreactivity was identified in the majority of the mature receptor cells in the middle region of the OE, with dendrites reaching to the OE free border, but not in basal and supporting cells. Their reactivity was more prominent in adults. GAP-43, a marker of immature olfactory sensory neurons, was found in certain receptor cells in the basal region of the OE, and some dendrites were immunopositive for GAP-43 (Fig. 2C and D). In addition, OMP and GAP-43 immunoreactivities were observed in the nerve bundles of the lamina propria (Fig. 2A and C).\nOMP, olfactory marker protein; GAP-43, growth associated protein-43; OM, olfactory mucosa; OE, olfactory epithelium; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.","The intensity values of 21 lectin-binding sites in the OE and lamina propria of the OM of Korean native cattle are summarized in Tables 2 and 3, respectively.\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\naApical perinuclear labeling.\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\nN-acetylglucosamine-binding lectins All lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\nOM, olfactory mucosa.\nAll lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\nOM, olfactory mucosa.\nMannose-binding lectins ConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\nConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\nGalactose-binding lectins In the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\nIn the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\nN-acetylgalactosamine-binding lectins All the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\nOM, olfactory mucosa.\nAll the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\nOM, olfactory mucosa.\nComplex type N-glycan-binding lectins In the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\nIn the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\nFucose-binding lectin In the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\nIn the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).","All lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\nOM, olfactory mucosa.","ConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).","In the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).","All the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\nOM, olfactory mucosa.","In the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).","In the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3)."],"string":"[\n \"Three neonatal OM samples (1 to 3 day old) and three adult OM samples (2.5-year-old) of Korean native cattle (Hanwoo, Bos taurus coreanae) were obtained from local farms and a local slaughterhouse in Chungcheongbuk-do, South Korea, respectively. Both sexes of all animals were utilized in this study (male: four neonates and five adults; female: three neonates and three adults). The OM used in this study belongs to a small area on the caudal roof of the nasal cavity, where it completely covered the ethmoturbinates and caudal portions of the dorsal and middle nasal conchae (Fig. 1A). Morphological exams of the nasal cavity indicated that none of them had underlying respiratory diseases. All experimental and animal handling procedures were conducted in accordance with the guidelines of Institutional Animal Care and Use Committee of Chonnam National University (26 October 2021; CNU IACUC-YB-2021-131).\\nOM, olfactory mucosa; BD, Bowman’s glandular duct; BG, Bowman’s gland; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.\",\n \"For light microscopic examination, the ethmoturbinates were removed immediately after death, cut into 5-cm-thick cross sections, and fixed for 3–5 days in 10% buffered formalin. Following fixation, the ethmoturbinates were trimmed and decalcified through multiple solution changes of sodium citrate-formic acid solution. When a needle could readily penetrate the bone with very little force, the decalcification process was stopped. The samples were then washed for 24 h with running tap water, dehydrated in a succession of ethanol concentrations (70%, 80%, 90%, 95%, and 100%), cleaned in xylene, embedded in paraffin, and sectioned into 4 μm slices. Following deparaffinization, the sections were utilized for hematoxylin and eosin staining, mucus and lectin histochemistry, and immunohistochemistry.\",\n \"The acidic mucin was distinguished from the neutral epithelial mucin using periodic acid Schiff (PAS) and Alcian blue (pH 2.5) staining. For PAS staining, the sections were subjected to 0.5% periodic acid for 15 min, then washed and incubated in Schiff reagent for 30 min. After 10 min of washing with running tap water, the sections were counterstained with hematoxylin. For Alcian blue staining, the sections were first incubated in 3% acetic acid for 3 min, followed by 30 min in 1% Alcian blue solution in 3% acetic acid (pH 2.5). After 1 min of washing with running tap water, the sections were counterstained with neutral red.\",\n \"To retrieve the antigens, the sections were heated for 1 h at 90°C in citrate buffer (0.01 M, pH 6.0). After cooling, the sections were treated for 20 min with 0.3% hydrogen peroxide in distilled water to suppress the endogenous peroxidase activity. To avoid non-specific binding, the sections were treated with normal goat serum (Vectastain Elite ABC kit; Vector Laboratories, USA) for 1 h before being incubated overnight at 4°C with rabbit monoclonal anti-olfactory marker protein (OMP) (1:1,000 dilution, Cat. No. ab183947; Abcam, UK) and rabbit polyclonal anti-growth associated protein-43 (GAP-43) (1:1,000 dilution, Cat. No. PA5-34943; ThermoFisher Scientific, USA) antibodies. The primary antibodies were excluded from the procedure as negative controls. The sections were rinsed with phosphate-buffered saline (PBS), treated with biotinylated goat anti-rabbit IgG (Vectastain Elite ABC kit) for 1 h, and then reacted with the avidin-biotin-peroxidase complex (Vectastain Elite ABC kit) for 1 h at room temperature (RT). Immunoreactivity was detected using a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories), followed by counterstaining with hematoxylin.\",\n \"Biotinylated lectin kits I, II, and III (Cat. No. BK-1000, BK-2000, and BK-3000) were acquired from Vector Laboratories. The acronyms, sources, and specificities of the used lectins are shown in Table 1. On the basis of their binding specificity and inhibitory sugars, lectins were categorized as N-acetylglucosamine, mannose, galactose, N-acetylgalactosamine, complex type N-glycan, and fucose-binding lectins. For competitive inhibition, the following sugars were purchased from Sigma-Aldrich (USA) and Vector Laboratories (Table 1): N-acetyl-D-glucosamine (β-D–GlcNAc; Sigma-Aldrich), Chitin Hydrolysate (Vector Laboratories), α-methyl mannoside/α-methyl glucoside (Sigma-Aldrich), lactose (Galβ1, 4Glc; Sigma-Aldrich), N-acetyl-D-galactosamine (α-D-GalNAc; Sigma-Aldrich), melibiose (Galα1, 6Glc; Sigma-Aldrich), and β-D-galactose (Sigma-Aldrich).\\nFuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose.\\naThe acronyms and specificities of the 21 lectins were obtained from the data sheep (Vector laboratory) and grouped, as shown in a previous paper [23].\\nTo eliminate the endogenous peroxidase activity, the sections were treated with 0.3% hydrogen peroxide in methanol. To prevent non-specific reactions, the sections were rinsed with PBS and then incubated with 1% bovine serum albumin in PBS. The sections were then treated overnight at 4°C with biotinylated lectins and reacted for 45 min at RT with an avidin-biotin-peroxidase complex (Vectastain Elite ABC kit). The sections were rinsed with PBS, treated with a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories), and counterstained with hematoxylin. Negative controls for lectin histochemistry were generated by removing the biotinylated lectins and preincubating the lectins with appropriate inhibitors for 1 h at RT.\",\n \"In neonatal (Fig. 1A) and adult cattle (Fig. 1B), the OM was composed of the OE and the lamina propria. The OE is predominantly constituted of basal cells, receptor cells, and supporting cells. The nuclei of the basal, receptor, and supporting cells were located in the basal, middle, and apical regions, respectively, of the OE (Fig. 1A and B). The basal cells were round, pallid, and had little nuclei. The nuclei of the supporting cells were elongated and spindle-shaped, but the nuclei of receptor cells were more rounded. While neonatal and adult OMs displayed comparable histological features, adult OMs appeared thicker than neonatal OMs.\\nThe lamina propria included Bowman’s glands and olfactory axon bundles. Bowman’s glands were composed of clustered acinar cells that were connected through ducts to the apical surface (Fig. 1A and B). The Bowman’s glandular ducts and/or acini stained positively for PAS (Fig. 1C and E) and Alcian blue stains (pH 2.5; Fig. 1D and F) in neonates (Fig. 1C and D) and adults (Fig. 1E and F); however, the reactivity of duct cells was relatively lower than that of gland cells. Additionally, the histological features of the OM did not differ between males and females at both ages.\",\n \"In neonatal (Fig. 2A) and adult (Fig. 2B) cattle, OMP, a marker for mature olfactory sensory neurons, immunoreactivity was identified in the majority of the mature receptor cells in the middle region of the OE, with dendrites reaching to the OE free border, but not in basal and supporting cells. Their reactivity was more prominent in adults. GAP-43, a marker of immature olfactory sensory neurons, was found in certain receptor cells in the basal region of the OE, and some dendrites were immunopositive for GAP-43 (Fig. 2C and D). In addition, OMP and GAP-43 immunoreactivities were observed in the nerve bundles of the lamina propria (Fig. 2A and C).\\nOMP, olfactory marker protein; GAP-43, growth associated protein-43; OM, olfactory mucosa; OE, olfactory epithelium; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.\",\n \"The intensity values of 21 lectin-binding sites in the OE and lamina propria of the OM of Korean native cattle are summarized in Tables 2 and 3, respectively.\\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\\naApical perinuclear labeling.\\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\\nN-acetylglucosamine-binding lectins All lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\\nOM, olfactory mucosa.\\nAll lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\\nOM, olfactory mucosa.\\nMannose-binding lectins ConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\\nConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\\nGalactose-binding lectins In the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\\nIn the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\\nN-acetylgalactosamine-binding lectins All the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\\nOM, olfactory mucosa.\\nAll the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\\nOM, olfactory mucosa.\\nComplex type N-glycan-binding lectins In the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\\nIn the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\\nFucose-binding lectin In the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\\nIn the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\",\n \"All lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\\nOM, olfactory mucosa.\",\n \"ConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\",\n \"In the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\",\n \"All the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\\nOM, olfactory mucosa.\",\n \"In the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\",\n \"In the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","MATERIALS AND METHODS","Tissue preparation","Histological examination","Mucus histochemistry","Immunohistochemistry","Lectin histochemistry","RESULTS","Histological findings of the OM","Immunohistochemical analysis of OMP in the OM","Lectin histochemistry in the OM","N-acetylglucosamine-binding lectins","Mannose-binding lectins","Galactose-binding lectins","N-acetylgalactosamine-binding lectins","Complex type N-glycan-binding lectins","Fucose-binding lectin","DISCUSSION"],"string":"[\n \"INTRODUCTION\",\n \"MATERIALS AND METHODS\",\n \"Tissue preparation\",\n \"Histological examination\",\n \"Mucus histochemistry\",\n \"Immunohistochemistry\",\n \"Lectin histochemistry\",\n \"RESULTS\",\n \"Histological findings of the OM\",\n \"Immunohistochemical analysis of OMP in the OM\",\n \"Lectin histochemistry in the OM\",\n \"N-acetylglucosamine-binding lectins\",\n \"Mannose-binding lectins\",\n \"Galactose-binding lectins\",\n \"N-acetylgalactosamine-binding lectins\",\n \"Complex type N-glycan-binding lectins\",\n \"Fucose-binding lectin\",\n \"DISCUSSION\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Olfaction is important for animals because it allows them to explore their surroundings for odorous signals from food sources and environments, as well as detect chemical compounds that influence social interaction and reproductive behavior [1]. This perception is mediated by two chemosensory systems: the main olfactory system and the vomeronasal system. The olfactory mucosa (OM), positioned at the caudal/posterior roof of the nasal cavity, and the vomeronasal organ (VNO), located at the base of the nasal septum or on the roof of the mouth, are the organs comprising these two systems [23]. Traditionally, the OM and VNO have been considered functionally and anatomically distinct, with the OM detecting conventional volatile odorants and the VNO receiving pheromones [45]. These two organs share some common features but differ in neuron types, primary structures of receptor proteins, physiological pathways, and central neuroanatomical projections into the brain [6]. However, despite the anatomical and functional differences, previous studies indicate that the OM and VNO play a synergistic role in the regulation of various olfactory-induced behaviors and reproductive and social interactions in mammals [15789].\nThe OM is critical for chemical signal acquisition in the main olfactory system, conveying signals to the main olfactory bulb [4]. In mammals, the OM is composed of the olfactory epithelium (OE) and the lamina propria. The OE is predominantly composed of chemosensory neurons, supporting cells, and basal cells [10]. The lamina propria is constituted by loose connective tissue in which olfactory nerve axon bundles, Bowman's glands, and blood and lymph vessels [1011]. In mammals, odorant reception occurs via the chemosensory neurons of the OE, which contain dendrites that extend beyond the apical surface, where the cilia protrude into the mucus layer, and a basally projecting axon process [12]. Glycoconjugates (terminal carbohydrates) have been shown to play a crucial role in the chemoreception of the OE [13].\nCell surfaces are densely packed with a diverse array of glycoconjugates, each of which provides considerable biological information [14]. Lectins are naturally occurring glycoconjugate-binding molecules, and a large number of purified lectins are regarded as the primary analytical tool for studying glycoconjugates in the olfactory system [131516]. Specific lectin binding is closely related to the olfactory neuron function, as evidenced by the abundance of glycoconjugates in the mucosensory compartment of the chemosensory epithelia, where receptor-specific events associated with transduction occur [13]. Numerous animals, including rats [1718], mice [1920], marmosets [21], sheep [1522], camels [22], horses [23], and humans [24], express the carbohydrate (lectin-binding) moiety in their OM.\nDespite the fact that cattle are one of the best species for biochemical investigations on olfaction [25], there is little published information on the immunohistochemical and lectin histochemical properties of the OM in bovines. We first evaluated the histological characteristics and lectin histochemistry of the OM in Korean native cattle, and then we compared neonate to adult OMs to determine the features associated with maturity. To our knowledge, this is the first study that provides a comprehensive outlook on the expression levels of lectin-binding glycoconjugates in bovine OM.","Tissue preparation Three neonatal OM samples (1 to 3 day old) and three adult OM samples (2.5-year-old) of Korean native cattle (Hanwoo, Bos taurus coreanae) were obtained from local farms and a local slaughterhouse in Chungcheongbuk-do, South Korea, respectively. Both sexes of all animals were utilized in this study (male: four neonates and five adults; female: three neonates and three adults). The OM used in this study belongs to a small area on the caudal roof of the nasal cavity, where it completely covered the ethmoturbinates and caudal portions of the dorsal and middle nasal conchae (Fig. 1A). Morphological exams of the nasal cavity indicated that none of them had underlying respiratory diseases. All experimental and animal handling procedures were conducted in accordance with the guidelines of Institutional Animal Care and Use Committee of Chonnam National University (26 October 2021; CNU IACUC-YB-2021-131).\nOM, olfactory mucosa; BD, Bowman’s glandular duct; BG, Bowman’s gland; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.\nThree neonatal OM samples (1 to 3 day old) and three adult OM samples (2.5-year-old) of Korean native cattle (Hanwoo, Bos taurus coreanae) were obtained from local farms and a local slaughterhouse in Chungcheongbuk-do, South Korea, respectively. Both sexes of all animals were utilized in this study (male: four neonates and five adults; female: three neonates and three adults). The OM used in this study belongs to a small area on the caudal roof of the nasal cavity, where it completely covered the ethmoturbinates and caudal portions of the dorsal and middle nasal conchae (Fig. 1A). Morphological exams of the nasal cavity indicated that none of them had underlying respiratory diseases. All experimental and animal handling procedures were conducted in accordance with the guidelines of Institutional Animal Care and Use Committee of Chonnam National University (26 October 2021; CNU IACUC-YB-2021-131).\nOM, olfactory mucosa; BD, Bowman’s glandular duct; BG, Bowman’s gland; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.\nHistological examination For light microscopic examination, the ethmoturbinates were removed immediately after death, cut into 5-cm-thick cross sections, and fixed for 3–5 days in 10% buffered formalin. Following fixation, the ethmoturbinates were trimmed and decalcified through multiple solution changes of sodium citrate-formic acid solution. When a needle could readily penetrate the bone with very little force, the decalcification process was stopped. The samples were then washed for 24 h with running tap water, dehydrated in a succession of ethanol concentrations (70%, 80%, 90%, 95%, and 100%), cleaned in xylene, embedded in paraffin, and sectioned into 4 μm slices. Following deparaffinization, the sections were utilized for hematoxylin and eosin staining, mucus and lectin histochemistry, and immunohistochemistry.\nFor light microscopic examination, the ethmoturbinates were removed immediately after death, cut into 5-cm-thick cross sections, and fixed for 3–5 days in 10% buffered formalin. Following fixation, the ethmoturbinates were trimmed and decalcified through multiple solution changes of sodium citrate-formic acid solution. When a needle could readily penetrate the bone with very little force, the decalcification process was stopped. The samples were then washed for 24 h with running tap water, dehydrated in a succession of ethanol concentrations (70%, 80%, 90%, 95%, and 100%), cleaned in xylene, embedded in paraffin, and sectioned into 4 μm slices. Following deparaffinization, the sections were utilized for hematoxylin and eosin staining, mucus and lectin histochemistry, and immunohistochemistry.\nMucus histochemistry The acidic mucin was distinguished from the neutral epithelial mucin using periodic acid Schiff (PAS) and Alcian blue (pH 2.5) staining. For PAS staining, the sections were subjected to 0.5% periodic acid for 15 min, then washed and incubated in Schiff reagent for 30 min. After 10 min of washing with running tap water, the sections were counterstained with hematoxylin. For Alcian blue staining, the sections were first incubated in 3% acetic acid for 3 min, followed by 30 min in 1% Alcian blue solution in 3% acetic acid (pH 2.5). After 1 min of washing with running tap water, the sections were counterstained with neutral red.\nThe acidic mucin was distinguished from the neutral epithelial mucin using periodic acid Schiff (PAS) and Alcian blue (pH 2.5) staining. For PAS staining, the sections were subjected to 0.5% periodic acid for 15 min, then washed and incubated in Schiff reagent for 30 min. After 10 min of washing with running tap water, the sections were counterstained with hematoxylin. For Alcian blue staining, the sections were first incubated in 3% acetic acid for 3 min, followed by 30 min in 1% Alcian blue solution in 3% acetic acid (pH 2.5). After 1 min of washing with running tap water, the sections were counterstained with neutral red.\nImmunohistochemistry To retrieve the antigens, the sections were heated for 1 h at 90°C in citrate buffer (0.01 M, pH 6.0). After cooling, the sections were treated for 20 min with 0.3% hydrogen peroxide in distilled water to suppress the endogenous peroxidase activity. To avoid non-specific binding, the sections were treated with normal goat serum (Vectastain Elite ABC kit; Vector Laboratories, USA) for 1 h before being incubated overnight at 4°C with rabbit monoclonal anti-olfactory marker protein (OMP) (1:1,000 dilution, Cat. No. ab183947; Abcam, UK) and rabbit polyclonal anti-growth associated protein-43 (GAP-43) (1:1,000 dilution, Cat. No. PA5-34943; ThermoFisher Scientific, USA) antibodies. The primary antibodies were excluded from the procedure as negative controls. The sections were rinsed with phosphate-buffered saline (PBS), treated with biotinylated goat anti-rabbit IgG (Vectastain Elite ABC kit) for 1 h, and then reacted with the avidin-biotin-peroxidase complex (Vectastain Elite ABC kit) for 1 h at room temperature (RT). Immunoreactivity was detected using a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories), followed by counterstaining with hematoxylin.\nTo retrieve the antigens, the sections were heated for 1 h at 90°C in citrate buffer (0.01 M, pH 6.0). After cooling, the sections were treated for 20 min with 0.3% hydrogen peroxide in distilled water to suppress the endogenous peroxidase activity. To avoid non-specific binding, the sections were treated with normal goat serum (Vectastain Elite ABC kit; Vector Laboratories, USA) for 1 h before being incubated overnight at 4°C with rabbit monoclonal anti-olfactory marker protein (OMP) (1:1,000 dilution, Cat. No. ab183947; Abcam, UK) and rabbit polyclonal anti-growth associated protein-43 (GAP-43) (1:1,000 dilution, Cat. No. PA5-34943; ThermoFisher Scientific, USA) antibodies. The primary antibodies were excluded from the procedure as negative controls. The sections were rinsed with phosphate-buffered saline (PBS), treated with biotinylated goat anti-rabbit IgG (Vectastain Elite ABC kit) for 1 h, and then reacted with the avidin-biotin-peroxidase complex (Vectastain Elite ABC kit) for 1 h at room temperature (RT). Immunoreactivity was detected using a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories), followed by counterstaining with hematoxylin.\nLectin histochemistry Biotinylated lectin kits I, II, and III (Cat. No. BK-1000, BK-2000, and BK-3000) were acquired from Vector Laboratories. The acronyms, sources, and specificities of the used lectins are shown in Table 1. On the basis of their binding specificity and inhibitory sugars, lectins were categorized as N-acetylglucosamine, mannose, galactose, N-acetylgalactosamine, complex type N-glycan, and fucose-binding lectins. For competitive inhibition, the following sugars were purchased from Sigma-Aldrich (USA) and Vector Laboratories (Table 1): N-acetyl-D-glucosamine (β-D–GlcNAc; Sigma-Aldrich), Chitin Hydrolysate (Vector Laboratories), α-methyl mannoside/α-methyl glucoside (Sigma-Aldrich), lactose (Galβ1, 4Glc; Sigma-Aldrich), N-acetyl-D-galactosamine (α-D-GalNAc; Sigma-Aldrich), melibiose (Galα1, 6Glc; Sigma-Aldrich), and β-D-galactose (Sigma-Aldrich).\nFuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose.\naThe acronyms and specificities of the 21 lectins were obtained from the data sheep (Vector laboratory) and grouped, as shown in a previous paper [23].\nTo eliminate the endogenous peroxidase activity, the sections were treated with 0.3% hydrogen peroxide in methanol. To prevent non-specific reactions, the sections were rinsed with PBS and then incubated with 1% bovine serum albumin in PBS. The sections were then treated overnight at 4°C with biotinylated lectins and reacted for 45 min at RT with an avidin-biotin-peroxidase complex (Vectastain Elite ABC kit). The sections were rinsed with PBS, treated with a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories), and counterstained with hematoxylin. Negative controls for lectin histochemistry were generated by removing the biotinylated lectins and preincubating the lectins with appropriate inhibitors for 1 h at RT.\nBiotinylated lectin kits I, II, and III (Cat. No. BK-1000, BK-2000, and BK-3000) were acquired from Vector Laboratories. The acronyms, sources, and specificities of the used lectins are shown in Table 1. On the basis of their binding specificity and inhibitory sugars, lectins were categorized as N-acetylglucosamine, mannose, galactose, N-acetylgalactosamine, complex type N-glycan, and fucose-binding lectins. For competitive inhibition, the following sugars were purchased from Sigma-Aldrich (USA) and Vector Laboratories (Table 1): N-acetyl-D-glucosamine (β-D–GlcNAc; Sigma-Aldrich), Chitin Hydrolysate (Vector Laboratories), α-methyl mannoside/α-methyl glucoside (Sigma-Aldrich), lactose (Galβ1, 4Glc; Sigma-Aldrich), N-acetyl-D-galactosamine (α-D-GalNAc; Sigma-Aldrich), melibiose (Galα1, 6Glc; Sigma-Aldrich), and β-D-galactose (Sigma-Aldrich).\nFuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose.\naThe acronyms and specificities of the 21 lectins were obtained from the data sheep (Vector laboratory) and grouped, as shown in a previous paper [23].\nTo eliminate the endogenous peroxidase activity, the sections were treated with 0.3% hydrogen peroxide in methanol. To prevent non-specific reactions, the sections were rinsed with PBS and then incubated with 1% bovine serum albumin in PBS. The sections were then treated overnight at 4°C with biotinylated lectins and reacted for 45 min at RT with an avidin-biotin-peroxidase complex (Vectastain Elite ABC kit). The sections were rinsed with PBS, treated with a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories), and counterstained with hematoxylin. Negative controls for lectin histochemistry were generated by removing the biotinylated lectins and preincubating the lectins with appropriate inhibitors for 1 h at RT.","Three neonatal OM samples (1 to 3 day old) and three adult OM samples (2.5-year-old) of Korean native cattle (Hanwoo, Bos taurus coreanae) were obtained from local farms and a local slaughterhouse in Chungcheongbuk-do, South Korea, respectively. Both sexes of all animals were utilized in this study (male: four neonates and five adults; female: three neonates and three adults). The OM used in this study belongs to a small area on the caudal roof of the nasal cavity, where it completely covered the ethmoturbinates and caudal portions of the dorsal and middle nasal conchae (Fig. 1A). Morphological exams of the nasal cavity indicated that none of them had underlying respiratory diseases. All experimental and animal handling procedures were conducted in accordance with the guidelines of Institutional Animal Care and Use Committee of Chonnam National University (26 October 2021; CNU IACUC-YB-2021-131).\nOM, olfactory mucosa; BD, Bowman’s glandular duct; BG, Bowman’s gland; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.","For light microscopic examination, the ethmoturbinates were removed immediately after death, cut into 5-cm-thick cross sections, and fixed for 3–5 days in 10% buffered formalin. Following fixation, the ethmoturbinates were trimmed and decalcified through multiple solution changes of sodium citrate-formic acid solution. When a needle could readily penetrate the bone with very little force, the decalcification process was stopped. The samples were then washed for 24 h with running tap water, dehydrated in a succession of ethanol concentrations (70%, 80%, 90%, 95%, and 100%), cleaned in xylene, embedded in paraffin, and sectioned into 4 μm slices. Following deparaffinization, the sections were utilized for hematoxylin and eosin staining, mucus and lectin histochemistry, and immunohistochemistry.","The acidic mucin was distinguished from the neutral epithelial mucin using periodic acid Schiff (PAS) and Alcian blue (pH 2.5) staining. For PAS staining, the sections were subjected to 0.5% periodic acid for 15 min, then washed and incubated in Schiff reagent for 30 min. After 10 min of washing with running tap water, the sections were counterstained with hematoxylin. For Alcian blue staining, the sections were first incubated in 3% acetic acid for 3 min, followed by 30 min in 1% Alcian blue solution in 3% acetic acid (pH 2.5). After 1 min of washing with running tap water, the sections were counterstained with neutral red.","To retrieve the antigens, the sections were heated for 1 h at 90°C in citrate buffer (0.01 M, pH 6.0). After cooling, the sections were treated for 20 min with 0.3% hydrogen peroxide in distilled water to suppress the endogenous peroxidase activity. To avoid non-specific binding, the sections were treated with normal goat serum (Vectastain Elite ABC kit; Vector Laboratories, USA) for 1 h before being incubated overnight at 4°C with rabbit monoclonal anti-olfactory marker protein (OMP) (1:1,000 dilution, Cat. No. ab183947; Abcam, UK) and rabbit polyclonal anti-growth associated protein-43 (GAP-43) (1:1,000 dilution, Cat. No. PA5-34943; ThermoFisher Scientific, USA) antibodies. The primary antibodies were excluded from the procedure as negative controls. The sections were rinsed with phosphate-buffered saline (PBS), treated with biotinylated goat anti-rabbit IgG (Vectastain Elite ABC kit) for 1 h, and then reacted with the avidin-biotin-peroxidase complex (Vectastain Elite ABC kit) for 1 h at room temperature (RT). Immunoreactivity was detected using a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories), followed by counterstaining with hematoxylin.","Biotinylated lectin kits I, II, and III (Cat. No. BK-1000, BK-2000, and BK-3000) were acquired from Vector Laboratories. The acronyms, sources, and specificities of the used lectins are shown in Table 1. On the basis of their binding specificity and inhibitory sugars, lectins were categorized as N-acetylglucosamine, mannose, galactose, N-acetylgalactosamine, complex type N-glycan, and fucose-binding lectins. For competitive inhibition, the following sugars were purchased from Sigma-Aldrich (USA) and Vector Laboratories (Table 1): N-acetyl-D-glucosamine (β-D–GlcNAc; Sigma-Aldrich), Chitin Hydrolysate (Vector Laboratories), α-methyl mannoside/α-methyl glucoside (Sigma-Aldrich), lactose (Galβ1, 4Glc; Sigma-Aldrich), N-acetyl-D-galactosamine (α-D-GalNAc; Sigma-Aldrich), melibiose (Galα1, 6Glc; Sigma-Aldrich), and β-D-galactose (Sigma-Aldrich).\nFuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose.\naThe acronyms and specificities of the 21 lectins were obtained from the data sheep (Vector laboratory) and grouped, as shown in a previous paper [23].\nTo eliminate the endogenous peroxidase activity, the sections were treated with 0.3% hydrogen peroxide in methanol. To prevent non-specific reactions, the sections were rinsed with PBS and then incubated with 1% bovine serum albumin in PBS. The sections were then treated overnight at 4°C with biotinylated lectins and reacted for 45 min at RT with an avidin-biotin-peroxidase complex (Vectastain Elite ABC kit). The sections were rinsed with PBS, treated with a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories), and counterstained with hematoxylin. Negative controls for lectin histochemistry were generated by removing the biotinylated lectins and preincubating the lectins with appropriate inhibitors for 1 h at RT.","Histological findings of the OM In neonatal (Fig. 1A) and adult cattle (Fig. 1B), the OM was composed of the OE and the lamina propria. The OE is predominantly constituted of basal cells, receptor cells, and supporting cells. The nuclei of the basal, receptor, and supporting cells were located in the basal, middle, and apical regions, respectively, of the OE (Fig. 1A and B). The basal cells were round, pallid, and had little nuclei. The nuclei of the supporting cells were elongated and spindle-shaped, but the nuclei of receptor cells were more rounded. While neonatal and adult OMs displayed comparable histological features, adult OMs appeared thicker than neonatal OMs.\nThe lamina propria included Bowman’s glands and olfactory axon bundles. Bowman’s glands were composed of clustered acinar cells that were connected through ducts to the apical surface (Fig. 1A and B). The Bowman’s glandular ducts and/or acini stained positively for PAS (Fig. 1C and E) and Alcian blue stains (pH 2.5; Fig. 1D and F) in neonates (Fig. 1C and D) and adults (Fig. 1E and F); however, the reactivity of duct cells was relatively lower than that of gland cells. Additionally, the histological features of the OM did not differ between males and females at both ages.\nIn neonatal (Fig. 1A) and adult cattle (Fig. 1B), the OM was composed of the OE and the lamina propria. The OE is predominantly constituted of basal cells, receptor cells, and supporting cells. The nuclei of the basal, receptor, and supporting cells were located in the basal, middle, and apical regions, respectively, of the OE (Fig. 1A and B). The basal cells were round, pallid, and had little nuclei. The nuclei of the supporting cells were elongated and spindle-shaped, but the nuclei of receptor cells were more rounded. While neonatal and adult OMs displayed comparable histological features, adult OMs appeared thicker than neonatal OMs.\nThe lamina propria included Bowman’s glands and olfactory axon bundles. Bowman’s glands were composed of clustered acinar cells that were connected through ducts to the apical surface (Fig. 1A and B). The Bowman’s glandular ducts and/or acini stained positively for PAS (Fig. 1C and E) and Alcian blue stains (pH 2.5; Fig. 1D and F) in neonates (Fig. 1C and D) and adults (Fig. 1E and F); however, the reactivity of duct cells was relatively lower than that of gland cells. Additionally, the histological features of the OM did not differ between males and females at both ages.\nImmunohistochemical analysis of OMP in the OM In neonatal (Fig. 2A) and adult (Fig. 2B) cattle, OMP, a marker for mature olfactory sensory neurons, immunoreactivity was identified in the majority of the mature receptor cells in the middle region of the OE, with dendrites reaching to the OE free border, but not in basal and supporting cells. Their reactivity was more prominent in adults. GAP-43, a marker of immature olfactory sensory neurons, was found in certain receptor cells in the basal region of the OE, and some dendrites were immunopositive for GAP-43 (Fig. 2C and D). In addition, OMP and GAP-43 immunoreactivities were observed in the nerve bundles of the lamina propria (Fig. 2A and C).\nOMP, olfactory marker protein; GAP-43, growth associated protein-43; OM, olfactory mucosa; OE, olfactory epithelium; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.\nIn neonatal (Fig. 2A) and adult (Fig. 2B) cattle, OMP, a marker for mature olfactory sensory neurons, immunoreactivity was identified in the majority of the mature receptor cells in the middle region of the OE, with dendrites reaching to the OE free border, but not in basal and supporting cells. Their reactivity was more prominent in adults. GAP-43, a marker of immature olfactory sensory neurons, was found in certain receptor cells in the basal region of the OE, and some dendrites were immunopositive for GAP-43 (Fig. 2C and D). In addition, OMP and GAP-43 immunoreactivities were observed in the nerve bundles of the lamina propria (Fig. 2A and C).\nOMP, olfactory marker protein; GAP-43, growth associated protein-43; OM, olfactory mucosa; OE, olfactory epithelium; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.\nLectin histochemistry in the OM The intensity values of 21 lectin-binding sites in the OE and lamina propria of the OM of Korean native cattle are summarized in Tables 2 and 3, respectively.\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\naApical perinuclear labeling.\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\nN-acetylglucosamine-binding lectins All lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\nOM, olfactory mucosa.\nAll lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\nOM, olfactory mucosa.\nMannose-binding lectins ConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\nConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\nGalactose-binding lectins In the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\nIn the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\nN-acetylgalactosamine-binding lectins All the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\nOM, olfactory mucosa.\nAll the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\nOM, olfactory mucosa.\nComplex type N-glycan-binding lectins In the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\nIn the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\nFucose-binding lectin In the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\nIn the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\nThe intensity values of 21 lectin-binding sites in the OE and lamina propria of the OM of Korean native cattle are summarized in Tables 2 and 3, respectively.\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\naApical perinuclear labeling.\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\nN-acetylglucosamine-binding lectins All lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\nOM, olfactory mucosa.\nAll lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\nOM, olfactory mucosa.\nMannose-binding lectins ConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\nConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\nGalactose-binding lectins In the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\nIn the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\nN-acetylgalactosamine-binding lectins All the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\nOM, olfactory mucosa.\nAll the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\nOM, olfactory mucosa.\nComplex type N-glycan-binding lectins In the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\nIn the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\nFucose-binding lectin In the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\nIn the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).","In neonatal (Fig. 1A) and adult cattle (Fig. 1B), the OM was composed of the OE and the lamina propria. The OE is predominantly constituted of basal cells, receptor cells, and supporting cells. The nuclei of the basal, receptor, and supporting cells were located in the basal, middle, and apical regions, respectively, of the OE (Fig. 1A and B). The basal cells were round, pallid, and had little nuclei. The nuclei of the supporting cells were elongated and spindle-shaped, but the nuclei of receptor cells were more rounded. While neonatal and adult OMs displayed comparable histological features, adult OMs appeared thicker than neonatal OMs.\nThe lamina propria included Bowman’s glands and olfactory axon bundles. Bowman’s glands were composed of clustered acinar cells that were connected through ducts to the apical surface (Fig. 1A and B). The Bowman’s glandular ducts and/or acini stained positively for PAS (Fig. 1C and E) and Alcian blue stains (pH 2.5; Fig. 1D and F) in neonates (Fig. 1C and D) and adults (Fig. 1E and F); however, the reactivity of duct cells was relatively lower than that of gland cells. Additionally, the histological features of the OM did not differ between males and females at both ages.","In neonatal (Fig. 2A) and adult (Fig. 2B) cattle, OMP, a marker for mature olfactory sensory neurons, immunoreactivity was identified in the majority of the mature receptor cells in the middle region of the OE, with dendrites reaching to the OE free border, but not in basal and supporting cells. Their reactivity was more prominent in adults. GAP-43, a marker of immature olfactory sensory neurons, was found in certain receptor cells in the basal region of the OE, and some dendrites were immunopositive for GAP-43 (Fig. 2C and D). In addition, OMP and GAP-43 immunoreactivities were observed in the nerve bundles of the lamina propria (Fig. 2A and C).\nOMP, olfactory marker protein; GAP-43, growth associated protein-43; OM, olfactory mucosa; OE, olfactory epithelium; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.","The intensity values of 21 lectin-binding sites in the OE and lamina propria of the OM of Korean native cattle are summarized in Tables 2 and 3, respectively.\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\naApical perinuclear labeling.\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\nN-acetylglucosamine-binding lectins All lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\nOM, olfactory mucosa.\nAll lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\nOM, olfactory mucosa.\nMannose-binding lectins ConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\nConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\nGalactose-binding lectins In the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\nIn the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\nN-acetylgalactosamine-binding lectins All the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\nOM, olfactory mucosa.\nAll the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\nOM, olfactory mucosa.\nComplex type N-glycan-binding lectins In the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\nIn the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\nFucose-binding lectin In the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\nIn the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).","All lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\nOM, olfactory mucosa.","ConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).","In the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).","All the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\nOM, olfactory mucosa.","In the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).","In the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).","This is the first study that visualized individual glycoconjugate (carbohydrate residues) during the postnatal development of bovine OM. We employed specific antibodies for olfactory sensory neurons against OMP and 21 lectins specific for the glycoconjugate sugar residues. Finally, we found that the distribution and expression levels of glycoconjugates varied remarkably between the OMs of neonatal and adult cattle.\nIn the present study, histological examination revealed that the cattle OM is composed of the OE and the underlying lamina propria, which contains Bowman’s glands, bundles of olfactory axons, and ensheathing glia, as observed in other mammals, such as rats [26], sheep [15], and dogs [22]. A previous study established that age-related thickening results from a rapid increase in olfactory density and contributes to an improved odor sensitivity in domestic dogs and sheep [27]. This study evaluated differences in OM morphology between neonate and adult cattle to test the notion that mucosal refinement changes with age. Our findings indicated that adult cattle OM had a more complete structural shape than that of the neonate. Additionally, PAS and Alcian blue (pH 2.5) staining were utilized to confirm the mucus specificity of the Bowman’s gland duct/acini in the lamina propria. In neonates and adults, Bowman’s gland ducts/acini were stained positively with PAS and Alcian blue to varied degrees of intensity. These findings imply that the Bowman’s glands release neutral and acidic components in the lamina propria in both the neonatal and adult OM, which may be implicated in the perception of scents or in protection against infectious agents and particles [28].\nOMP has been implicated in olfactory transduction [29], and its expression is restricted to mature chemosensory neurons in the OE [30]. We assessed the OMP expression in the OM to verify whether it is associated with mature sensory neurons that extend their dendritic knobs to the OE mucociliary layer or free border [31]. In contrast to OMP, GAP-43 expression is detected in the basal region of the OE, indicating that GAP-43 is expressed in immature neurons [32]. In neonatal and adult cattle, mature receptor cells in the middle region and immature receptor cells in the basal region of the OE were markedly positive for OMP and GAP-43, respectively. Therefore, OMP-positive mature and GAP-43-positive immature sensory neurons may be essential for the development of the main olfactory system [3233]. However, the precise roles of OMP and GAP-43 in cattle OM should be further verified.\n\nTables 2 and 3 demonstrate the results of lectin-binding patterns in the OM of neonatal and adult cattle. In all regions of the OM, including the OE free border, receptor cells, supporting cells, basal cells, nerve bundles, and Bowman’s glands, lectin-reactive glycoconjugates were found at varying intensities. Except for BSL-II and jacalin, all lectins utilized in this study were expressed in the free border of both neonatal and adult cattle, implying that the majority of the carbohydrates detected by these lectins are present in the mucociliary layer of the OE. This finding somewhat contrasts with previously published research on sheep [15] and horses [23], wherein the free borders were labeled with 13 and 19 of the 21 lectins, respectively. Except for BSL-II and jacalin, 19 of the 21 lectins were labeled in receptor cells with varying intensities in the cattle OE, indicating that sugar residues play a role in the cell biology of receptor cells. The results of this study were consistent with those of a previous study in horses [23]. However, DBA, SJA, PHA-L, and UEA-I were found in cattle but not in horses [23]. Furthermore, similar lectins have been identified in the olfactory receptor neurons and axons of other animals [34353637], which is consistent with our findings. Among the 21 lectins, 10 and 9 lectins were labeled as supporting cells in neonates and adults, respectively, and the labeling patterns were relatively comparable to those reported previously [2238], but differed from the results obtained by a previous study in the horse [23]. In the lamina propria, 14 of the 21 lectins were found in the nerve bundles with varying intensities, whereas 3 lectins that were not found in the nerve bundles were found in the OE receptor cells. We hypothesize that sugar residue modifications cause this disparity as a result of positional changes. This event also occurred in the Bowman’s gland in the lamina propria, where the expression patterns of lectin were altered as the Bowman’s gland ducts exited through the OE. In the Bowman’s glands, the majority of lectins were expressed more strongly in acinar cells than in ductal cells, implying that the acini contain a greater variety of sugar residues than the secretory ductal epithelium.\nA previous study suggested a link between the expression pattern of DBA-binding glycoconjugates and the development of the olfactory system [36]. Similarly, previous studies hypothesized that the expression patterns of lectins, such as VVA and PNA, may be associated with the development of the OM [3940]. The majority of the lectin-binding sites in the cattle OM were identical or stronger in neonates than in adults, implying that the labeling of lectins may be closely related to the development and maturation of the OM. Thus, developmental changes in the expression patterns of lectin-binding glycoconjugates were evident in the OM of Korean native cattle. However, further functional investigations are required to determine how each glycoconjugate contributes to olfactory sensory processing.\nGlobally, cattle is important for animal protein production and may be a desirable animal model for studying olfaction and its effect on animal behavior. The Korean native cattle used in this study is part of a national breeding and selection program for beef production and thus subjected to artificial selection for intensive production in South Korea [41]. For example, in the United States, artificial insemination (AI) accounts for 50% of beef cattle breeding; in South Korea, it accounts for more than 90% [42]. AI timing is an essential component of conception, and the role of pheromones and olfaction in this process is being investigated [43]. Additional factors, such as irregular or prolonged estrous cycles and anestrus, which contribute to lower conception rates during AI, have been linked to pheromones and their receptors [44]. Consequently, olfaction may differ between Korean native cattle and other cattle according to these various factors [45]; however, information about the association between fertility-related behaviors and olfaction in Korean native cattle is limited. Still, we suggest that our data on the histology and lectin histochemistry in the OM of Korean native cattle may serve as a basis for further investigating their olfactory behavior and physiology.\nIn conclusion, we have performed a comprehensive characterization of the Korean native cattle OM at the histological, immunohistochemical, and lectin histochemical levels, thereby revealing new information about the main olfactory system. Additionally, the expression levels of glycoconjugates were considerably different between the OMs of neonatal and adult cattle, with adult OMs exhibiting higher intensities than neonates. Thus, this information can help clarify the role of the OM and its chemo-communication pathways in cattle. Further functional studies, however, are required to demonstrate the role of lectin-binding glycoconjugates in olfactory sensory processing and its associated behaviors."],"string":"[\n \"Olfaction is important for animals because it allows them to explore their surroundings for odorous signals from food sources and environments, as well as detect chemical compounds that influence social interaction and reproductive behavior [1]. This perception is mediated by two chemosensory systems: the main olfactory system and the vomeronasal system. The olfactory mucosa (OM), positioned at the caudal/posterior roof of the nasal cavity, and the vomeronasal organ (VNO), located at the base of the nasal septum or on the roof of the mouth, are the organs comprising these two systems [23]. Traditionally, the OM and VNO have been considered functionally and anatomically distinct, with the OM detecting conventional volatile odorants and the VNO receiving pheromones [45]. These two organs share some common features but differ in neuron types, primary structures of receptor proteins, physiological pathways, and central neuroanatomical projections into the brain [6]. However, despite the anatomical and functional differences, previous studies indicate that the OM and VNO play a synergistic role in the regulation of various olfactory-induced behaviors and reproductive and social interactions in mammals [15789].\\nThe OM is critical for chemical signal acquisition in the main olfactory system, conveying signals to the main olfactory bulb [4]. In mammals, the OM is composed of the olfactory epithelium (OE) and the lamina propria. The OE is predominantly composed of chemosensory neurons, supporting cells, and basal cells [10]. The lamina propria is constituted by loose connective tissue in which olfactory nerve axon bundles, Bowman's glands, and blood and lymph vessels [1011]. In mammals, odorant reception occurs via the chemosensory neurons of the OE, which contain dendrites that extend beyond the apical surface, where the cilia protrude into the mucus layer, and a basally projecting axon process [12]. Glycoconjugates (terminal carbohydrates) have been shown to play a crucial role in the chemoreception of the OE [13].\\nCell surfaces are densely packed with a diverse array of glycoconjugates, each of which provides considerable biological information [14]. Lectins are naturally occurring glycoconjugate-binding molecules, and a large number of purified lectins are regarded as the primary analytical tool for studying glycoconjugates in the olfactory system [131516]. Specific lectin binding is closely related to the olfactory neuron function, as evidenced by the abundance of glycoconjugates in the mucosensory compartment of the chemosensory epithelia, where receptor-specific events associated with transduction occur [13]. Numerous animals, including rats [1718], mice [1920], marmosets [21], sheep [1522], camels [22], horses [23], and humans [24], express the carbohydrate (lectin-binding) moiety in their OM.\\nDespite the fact that cattle are one of the best species for biochemical investigations on olfaction [25], there is little published information on the immunohistochemical and lectin histochemical properties of the OM in bovines. We first evaluated the histological characteristics and lectin histochemistry of the OM in Korean native cattle, and then we compared neonate to adult OMs to determine the features associated with maturity. To our knowledge, this is the first study that provides a comprehensive outlook on the expression levels of lectin-binding glycoconjugates in bovine OM.\",\n \"Tissue preparation Three neonatal OM samples (1 to 3 day old) and three adult OM samples (2.5-year-old) of Korean native cattle (Hanwoo, Bos taurus coreanae) were obtained from local farms and a local slaughterhouse in Chungcheongbuk-do, South Korea, respectively. Both sexes of all animals were utilized in this study (male: four neonates and five adults; female: three neonates and three adults). The OM used in this study belongs to a small area on the caudal roof of the nasal cavity, where it completely covered the ethmoturbinates and caudal portions of the dorsal and middle nasal conchae (Fig. 1A). Morphological exams of the nasal cavity indicated that none of them had underlying respiratory diseases. All experimental and animal handling procedures were conducted in accordance with the guidelines of Institutional Animal Care and Use Committee of Chonnam National University (26 October 2021; CNU IACUC-YB-2021-131).\\nOM, olfactory mucosa; BD, Bowman’s glandular duct; BG, Bowman’s gland; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.\\nThree neonatal OM samples (1 to 3 day old) and three adult OM samples (2.5-year-old) of Korean native cattle (Hanwoo, Bos taurus coreanae) were obtained from local farms and a local slaughterhouse in Chungcheongbuk-do, South Korea, respectively. Both sexes of all animals were utilized in this study (male: four neonates and five adults; female: three neonates and three adults). The OM used in this study belongs to a small area on the caudal roof of the nasal cavity, where it completely covered the ethmoturbinates and caudal portions of the dorsal and middle nasal conchae (Fig. 1A). Morphological exams of the nasal cavity indicated that none of them had underlying respiratory diseases. All experimental and animal handling procedures were conducted in accordance with the guidelines of Institutional Animal Care and Use Committee of Chonnam National University (26 October 2021; CNU IACUC-YB-2021-131).\\nOM, olfactory mucosa; BD, Bowman’s glandular duct; BG, Bowman’s gland; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.\\nHistological examination For light microscopic examination, the ethmoturbinates were removed immediately after death, cut into 5-cm-thick cross sections, and fixed for 3–5 days in 10% buffered formalin. Following fixation, the ethmoturbinates were trimmed and decalcified through multiple solution changes of sodium citrate-formic acid solution. When a needle could readily penetrate the bone with very little force, the decalcification process was stopped. The samples were then washed for 24 h with running tap water, dehydrated in a succession of ethanol concentrations (70%, 80%, 90%, 95%, and 100%), cleaned in xylene, embedded in paraffin, and sectioned into 4 μm slices. Following deparaffinization, the sections were utilized for hematoxylin and eosin staining, mucus and lectin histochemistry, and immunohistochemistry.\\nFor light microscopic examination, the ethmoturbinates were removed immediately after death, cut into 5-cm-thick cross sections, and fixed for 3–5 days in 10% buffered formalin. Following fixation, the ethmoturbinates were trimmed and decalcified through multiple solution changes of sodium citrate-formic acid solution. When a needle could readily penetrate the bone with very little force, the decalcification process was stopped. The samples were then washed for 24 h with running tap water, dehydrated in a succession of ethanol concentrations (70%, 80%, 90%, 95%, and 100%), cleaned in xylene, embedded in paraffin, and sectioned into 4 μm slices. Following deparaffinization, the sections were utilized for hematoxylin and eosin staining, mucus and lectin histochemistry, and immunohistochemistry.\\nMucus histochemistry The acidic mucin was distinguished from the neutral epithelial mucin using periodic acid Schiff (PAS) and Alcian blue (pH 2.5) staining. For PAS staining, the sections were subjected to 0.5% periodic acid for 15 min, then washed and incubated in Schiff reagent for 30 min. After 10 min of washing with running tap water, the sections were counterstained with hematoxylin. For Alcian blue staining, the sections were first incubated in 3% acetic acid for 3 min, followed by 30 min in 1% Alcian blue solution in 3% acetic acid (pH 2.5). After 1 min of washing with running tap water, the sections were counterstained with neutral red.\\nThe acidic mucin was distinguished from the neutral epithelial mucin using periodic acid Schiff (PAS) and Alcian blue (pH 2.5) staining. For PAS staining, the sections were subjected to 0.5% periodic acid for 15 min, then washed and incubated in Schiff reagent for 30 min. After 10 min of washing with running tap water, the sections were counterstained with hematoxylin. For Alcian blue staining, the sections were first incubated in 3% acetic acid for 3 min, followed by 30 min in 1% Alcian blue solution in 3% acetic acid (pH 2.5). After 1 min of washing with running tap water, the sections were counterstained with neutral red.\\nImmunohistochemistry To retrieve the antigens, the sections were heated for 1 h at 90°C in citrate buffer (0.01 M, pH 6.0). After cooling, the sections were treated for 20 min with 0.3% hydrogen peroxide in distilled water to suppress the endogenous peroxidase activity. To avoid non-specific binding, the sections were treated with normal goat serum (Vectastain Elite ABC kit; Vector Laboratories, USA) for 1 h before being incubated overnight at 4°C with rabbit monoclonal anti-olfactory marker protein (OMP) (1:1,000 dilution, Cat. No. ab183947; Abcam, UK) and rabbit polyclonal anti-growth associated protein-43 (GAP-43) (1:1,000 dilution, Cat. No. PA5-34943; ThermoFisher Scientific, USA) antibodies. The primary antibodies were excluded from the procedure as negative controls. The sections were rinsed with phosphate-buffered saline (PBS), treated with biotinylated goat anti-rabbit IgG (Vectastain Elite ABC kit) for 1 h, and then reacted with the avidin-biotin-peroxidase complex (Vectastain Elite ABC kit) for 1 h at room temperature (RT). Immunoreactivity was detected using a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories), followed by counterstaining with hematoxylin.\\nTo retrieve the antigens, the sections were heated for 1 h at 90°C in citrate buffer (0.01 M, pH 6.0). After cooling, the sections were treated for 20 min with 0.3% hydrogen peroxide in distilled water to suppress the endogenous peroxidase activity. To avoid non-specific binding, the sections were treated with normal goat serum (Vectastain Elite ABC kit; Vector Laboratories, USA) for 1 h before being incubated overnight at 4°C with rabbit monoclonal anti-olfactory marker protein (OMP) (1:1,000 dilution, Cat. No. ab183947; Abcam, UK) and rabbit polyclonal anti-growth associated protein-43 (GAP-43) (1:1,000 dilution, Cat. No. PA5-34943; ThermoFisher Scientific, USA) antibodies. The primary antibodies were excluded from the procedure as negative controls. The sections were rinsed with phosphate-buffered saline (PBS), treated with biotinylated goat anti-rabbit IgG (Vectastain Elite ABC kit) for 1 h, and then reacted with the avidin-biotin-peroxidase complex (Vectastain Elite ABC kit) for 1 h at room temperature (RT). Immunoreactivity was detected using a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories), followed by counterstaining with hematoxylin.\\nLectin histochemistry Biotinylated lectin kits I, II, and III (Cat. No. BK-1000, BK-2000, and BK-3000) were acquired from Vector Laboratories. The acronyms, sources, and specificities of the used lectins are shown in Table 1. On the basis of their binding specificity and inhibitory sugars, lectins were categorized as N-acetylglucosamine, mannose, galactose, N-acetylgalactosamine, complex type N-glycan, and fucose-binding lectins. For competitive inhibition, the following sugars were purchased from Sigma-Aldrich (USA) and Vector Laboratories (Table 1): N-acetyl-D-glucosamine (β-D–GlcNAc; Sigma-Aldrich), Chitin Hydrolysate (Vector Laboratories), α-methyl mannoside/α-methyl glucoside (Sigma-Aldrich), lactose (Galβ1, 4Glc; Sigma-Aldrich), N-acetyl-D-galactosamine (α-D-GalNAc; Sigma-Aldrich), melibiose (Galα1, 6Glc; Sigma-Aldrich), and β-D-galactose (Sigma-Aldrich).\\nFuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose.\\naThe acronyms and specificities of the 21 lectins were obtained from the data sheep (Vector laboratory) and grouped, as shown in a previous paper [23].\\nTo eliminate the endogenous peroxidase activity, the sections were treated with 0.3% hydrogen peroxide in methanol. To prevent non-specific reactions, the sections were rinsed with PBS and then incubated with 1% bovine serum albumin in PBS. The sections were then treated overnight at 4°C with biotinylated lectins and reacted for 45 min at RT with an avidin-biotin-peroxidase complex (Vectastain Elite ABC kit). The sections were rinsed with PBS, treated with a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories), and counterstained with hematoxylin. Negative controls for lectin histochemistry were generated by removing the biotinylated lectins and preincubating the lectins with appropriate inhibitors for 1 h at RT.\\nBiotinylated lectin kits I, II, and III (Cat. No. BK-1000, BK-2000, and BK-3000) were acquired from Vector Laboratories. The acronyms, sources, and specificities of the used lectins are shown in Table 1. On the basis of their binding specificity and inhibitory sugars, lectins were categorized as N-acetylglucosamine, mannose, galactose, N-acetylgalactosamine, complex type N-glycan, and fucose-binding lectins. For competitive inhibition, the following sugars were purchased from Sigma-Aldrich (USA) and Vector Laboratories (Table 1): N-acetyl-D-glucosamine (β-D–GlcNAc; Sigma-Aldrich), Chitin Hydrolysate (Vector Laboratories), α-methyl mannoside/α-methyl glucoside (Sigma-Aldrich), lactose (Galβ1, 4Glc; Sigma-Aldrich), N-acetyl-D-galactosamine (α-D-GalNAc; Sigma-Aldrich), melibiose (Galα1, 6Glc; Sigma-Aldrich), and β-D-galactose (Sigma-Aldrich).\\nFuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose.\\naThe acronyms and specificities of the 21 lectins were obtained from the data sheep (Vector laboratory) and grouped, as shown in a previous paper [23].\\nTo eliminate the endogenous peroxidase activity, the sections were treated with 0.3% hydrogen peroxide in methanol. To prevent non-specific reactions, the sections were rinsed with PBS and then incubated with 1% bovine serum albumin in PBS. The sections were then treated overnight at 4°C with biotinylated lectins and reacted for 45 min at RT with an avidin-biotin-peroxidase complex (Vectastain Elite ABC kit). The sections were rinsed with PBS, treated with a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories), and counterstained with hematoxylin. Negative controls for lectin histochemistry were generated by removing the biotinylated lectins and preincubating the lectins with appropriate inhibitors for 1 h at RT.\",\n \"Three neonatal OM samples (1 to 3 day old) and three adult OM samples (2.5-year-old) of Korean native cattle (Hanwoo, Bos taurus coreanae) were obtained from local farms and a local slaughterhouse in Chungcheongbuk-do, South Korea, respectively. Both sexes of all animals were utilized in this study (male: four neonates and five adults; female: three neonates and three adults). The OM used in this study belongs to a small area on the caudal roof of the nasal cavity, where it completely covered the ethmoturbinates and caudal portions of the dorsal and middle nasal conchae (Fig. 1A). Morphological exams of the nasal cavity indicated that none of them had underlying respiratory diseases. All experimental and animal handling procedures were conducted in accordance with the guidelines of Institutional Animal Care and Use Committee of Chonnam National University (26 October 2021; CNU IACUC-YB-2021-131).\\nOM, olfactory mucosa; BD, Bowman’s glandular duct; BG, Bowman’s gland; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.\",\n \"For light microscopic examination, the ethmoturbinates were removed immediately after death, cut into 5-cm-thick cross sections, and fixed for 3–5 days in 10% buffered formalin. Following fixation, the ethmoturbinates were trimmed and decalcified through multiple solution changes of sodium citrate-formic acid solution. When a needle could readily penetrate the bone with very little force, the decalcification process was stopped. The samples were then washed for 24 h with running tap water, dehydrated in a succession of ethanol concentrations (70%, 80%, 90%, 95%, and 100%), cleaned in xylene, embedded in paraffin, and sectioned into 4 μm slices. Following deparaffinization, the sections were utilized for hematoxylin and eosin staining, mucus and lectin histochemistry, and immunohistochemistry.\",\n \"The acidic mucin was distinguished from the neutral epithelial mucin using periodic acid Schiff (PAS) and Alcian blue (pH 2.5) staining. For PAS staining, the sections were subjected to 0.5% periodic acid for 15 min, then washed and incubated in Schiff reagent for 30 min. After 10 min of washing with running tap water, the sections were counterstained with hematoxylin. For Alcian blue staining, the sections were first incubated in 3% acetic acid for 3 min, followed by 30 min in 1% Alcian blue solution in 3% acetic acid (pH 2.5). After 1 min of washing with running tap water, the sections were counterstained with neutral red.\",\n \"To retrieve the antigens, the sections were heated for 1 h at 90°C in citrate buffer (0.01 M, pH 6.0). After cooling, the sections were treated for 20 min with 0.3% hydrogen peroxide in distilled water to suppress the endogenous peroxidase activity. To avoid non-specific binding, the sections were treated with normal goat serum (Vectastain Elite ABC kit; Vector Laboratories, USA) for 1 h before being incubated overnight at 4°C with rabbit monoclonal anti-olfactory marker protein (OMP) (1:1,000 dilution, Cat. No. ab183947; Abcam, UK) and rabbit polyclonal anti-growth associated protein-43 (GAP-43) (1:1,000 dilution, Cat. No. PA5-34943; ThermoFisher Scientific, USA) antibodies. The primary antibodies were excluded from the procedure as negative controls. The sections were rinsed with phosphate-buffered saline (PBS), treated with biotinylated goat anti-rabbit IgG (Vectastain Elite ABC kit) for 1 h, and then reacted with the avidin-biotin-peroxidase complex (Vectastain Elite ABC kit) for 1 h at room temperature (RT). Immunoreactivity was detected using a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories), followed by counterstaining with hematoxylin.\",\n \"Biotinylated lectin kits I, II, and III (Cat. No. BK-1000, BK-2000, and BK-3000) were acquired from Vector Laboratories. The acronyms, sources, and specificities of the used lectins are shown in Table 1. On the basis of their binding specificity and inhibitory sugars, lectins were categorized as N-acetylglucosamine, mannose, galactose, N-acetylgalactosamine, complex type N-glycan, and fucose-binding lectins. For competitive inhibition, the following sugars were purchased from Sigma-Aldrich (USA) and Vector Laboratories (Table 1): N-acetyl-D-glucosamine (β-D–GlcNAc; Sigma-Aldrich), Chitin Hydrolysate (Vector Laboratories), α-methyl mannoside/α-methyl glucoside (Sigma-Aldrich), lactose (Galβ1, 4Glc; Sigma-Aldrich), N-acetyl-D-galactosamine (α-D-GalNAc; Sigma-Aldrich), melibiose (Galα1, 6Glc; Sigma-Aldrich), and β-D-galactose (Sigma-Aldrich).\\nFuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose.\\naThe acronyms and specificities of the 21 lectins were obtained from the data sheep (Vector laboratory) and grouped, as shown in a previous paper [23].\\nTo eliminate the endogenous peroxidase activity, the sections were treated with 0.3% hydrogen peroxide in methanol. To prevent non-specific reactions, the sections were rinsed with PBS and then incubated with 1% bovine serum albumin in PBS. The sections were then treated overnight at 4°C with biotinylated lectins and reacted for 45 min at RT with an avidin-biotin-peroxidase complex (Vectastain Elite ABC kit). The sections were rinsed with PBS, treated with a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories), and counterstained with hematoxylin. Negative controls for lectin histochemistry were generated by removing the biotinylated lectins and preincubating the lectins with appropriate inhibitors for 1 h at RT.\",\n \"Histological findings of the OM In neonatal (Fig. 1A) and adult cattle (Fig. 1B), the OM was composed of the OE and the lamina propria. The OE is predominantly constituted of basal cells, receptor cells, and supporting cells. The nuclei of the basal, receptor, and supporting cells were located in the basal, middle, and apical regions, respectively, of the OE (Fig. 1A and B). The basal cells were round, pallid, and had little nuclei. The nuclei of the supporting cells were elongated and spindle-shaped, but the nuclei of receptor cells were more rounded. While neonatal and adult OMs displayed comparable histological features, adult OMs appeared thicker than neonatal OMs.\\nThe lamina propria included Bowman’s glands and olfactory axon bundles. Bowman’s glands were composed of clustered acinar cells that were connected through ducts to the apical surface (Fig. 1A and B). The Bowman’s glandular ducts and/or acini stained positively for PAS (Fig. 1C and E) and Alcian blue stains (pH 2.5; Fig. 1D and F) in neonates (Fig. 1C and D) and adults (Fig. 1E and F); however, the reactivity of duct cells was relatively lower than that of gland cells. Additionally, the histological features of the OM did not differ between males and females at both ages.\\nIn neonatal (Fig. 1A) and adult cattle (Fig. 1B), the OM was composed of the OE and the lamina propria. The OE is predominantly constituted of basal cells, receptor cells, and supporting cells. The nuclei of the basal, receptor, and supporting cells were located in the basal, middle, and apical regions, respectively, of the OE (Fig. 1A and B). The basal cells were round, pallid, and had little nuclei. The nuclei of the supporting cells were elongated and spindle-shaped, but the nuclei of receptor cells were more rounded. While neonatal and adult OMs displayed comparable histological features, adult OMs appeared thicker than neonatal OMs.\\nThe lamina propria included Bowman’s glands and olfactory axon bundles. Bowman’s glands were composed of clustered acinar cells that were connected through ducts to the apical surface (Fig. 1A and B). The Bowman’s glandular ducts and/or acini stained positively for PAS (Fig. 1C and E) and Alcian blue stains (pH 2.5; Fig. 1D and F) in neonates (Fig. 1C and D) and adults (Fig. 1E and F); however, the reactivity of duct cells was relatively lower than that of gland cells. Additionally, the histological features of the OM did not differ between males and females at both ages.\\nImmunohistochemical analysis of OMP in the OM In neonatal (Fig. 2A) and adult (Fig. 2B) cattle, OMP, a marker for mature olfactory sensory neurons, immunoreactivity was identified in the majority of the mature receptor cells in the middle region of the OE, with dendrites reaching to the OE free border, but not in basal and supporting cells. Their reactivity was more prominent in adults. GAP-43, a marker of immature olfactory sensory neurons, was found in certain receptor cells in the basal region of the OE, and some dendrites were immunopositive for GAP-43 (Fig. 2C and D). In addition, OMP and GAP-43 immunoreactivities were observed in the nerve bundles of the lamina propria (Fig. 2A and C).\\nOMP, olfactory marker protein; GAP-43, growth associated protein-43; OM, olfactory mucosa; OE, olfactory epithelium; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.\\nIn neonatal (Fig. 2A) and adult (Fig. 2B) cattle, OMP, a marker for mature olfactory sensory neurons, immunoreactivity was identified in the majority of the mature receptor cells in the middle region of the OE, with dendrites reaching to the OE free border, but not in basal and supporting cells. Their reactivity was more prominent in adults. GAP-43, a marker of immature olfactory sensory neurons, was found in certain receptor cells in the basal region of the OE, and some dendrites were immunopositive for GAP-43 (Fig. 2C and D). In addition, OMP and GAP-43 immunoreactivities were observed in the nerve bundles of the lamina propria (Fig. 2A and C).\\nOMP, olfactory marker protein; GAP-43, growth associated protein-43; OM, olfactory mucosa; OE, olfactory epithelium; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.\\nLectin histochemistry in the OM The intensity values of 21 lectin-binding sites in the OE and lamina propria of the OM of Korean native cattle are summarized in Tables 2 and 3, respectively.\\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\\naApical perinuclear labeling.\\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\\nN-acetylglucosamine-binding lectins All lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\\nOM, olfactory mucosa.\\nAll lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\\nOM, olfactory mucosa.\\nMannose-binding lectins ConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\\nConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\\nGalactose-binding lectins In the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\\nIn the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\\nN-acetylgalactosamine-binding lectins All the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\\nOM, olfactory mucosa.\\nAll the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\\nOM, olfactory mucosa.\\nComplex type N-glycan-binding lectins In the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\\nIn the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\\nFucose-binding lectin In the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\\nIn the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\\nThe intensity values of 21 lectin-binding sites in the OE and lamina propria of the OM of Korean native cattle are summarized in Tables 2 and 3, respectively.\\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\\naApical perinuclear labeling.\\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\\nN-acetylglucosamine-binding lectins All lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\\nOM, olfactory mucosa.\\nAll lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\\nOM, olfactory mucosa.\\nMannose-binding lectins ConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\\nConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\\nGalactose-binding lectins In the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\\nIn the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\\nN-acetylgalactosamine-binding lectins All the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\\nOM, olfactory mucosa.\\nAll the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\\nOM, olfactory mucosa.\\nComplex type N-glycan-binding lectins In the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\\nIn the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\\nFucose-binding lectin In the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\\nIn the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\",\n \"In neonatal (Fig. 1A) and adult cattle (Fig. 1B), the OM was composed of the OE and the lamina propria. The OE is predominantly constituted of basal cells, receptor cells, and supporting cells. The nuclei of the basal, receptor, and supporting cells were located in the basal, middle, and apical regions, respectively, of the OE (Fig. 1A and B). The basal cells were round, pallid, and had little nuclei. The nuclei of the supporting cells were elongated and spindle-shaped, but the nuclei of receptor cells were more rounded. While neonatal and adult OMs displayed comparable histological features, adult OMs appeared thicker than neonatal OMs.\\nThe lamina propria included Bowman’s glands and olfactory axon bundles. Bowman’s glands were composed of clustered acinar cells that were connected through ducts to the apical surface (Fig. 1A and B). The Bowman’s glandular ducts and/or acini stained positively for PAS (Fig. 1C and E) and Alcian blue stains (pH 2.5; Fig. 1D and F) in neonates (Fig. 1C and D) and adults (Fig. 1E and F); however, the reactivity of duct cells was relatively lower than that of gland cells. Additionally, the histological features of the OM did not differ between males and females at both ages.\",\n \"In neonatal (Fig. 2A) and adult (Fig. 2B) cattle, OMP, a marker for mature olfactory sensory neurons, immunoreactivity was identified in the majority of the mature receptor cells in the middle region of the OE, with dendrites reaching to the OE free border, but not in basal and supporting cells. Their reactivity was more prominent in adults. GAP-43, a marker of immature olfactory sensory neurons, was found in certain receptor cells in the basal region of the OE, and some dendrites were immunopositive for GAP-43 (Fig. 2C and D). In addition, OMP and GAP-43 immunoreactivities were observed in the nerve bundles of the lamina propria (Fig. 2A and C).\\nOMP, olfactory marker protein; GAP-43, growth associated protein-43; OM, olfactory mucosa; OE, olfactory epithelium; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.\",\n \"The intensity values of 21 lectin-binding sites in the OE and lamina propria of the OM of Korean native cattle are summarized in Tables 2 and 3, respectively.\\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\\naApical perinuclear labeling.\\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\\nN-acetylglucosamine-binding lectins All lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\\nOM, olfactory mucosa.\\nAll lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\\nOM, olfactory mucosa.\\nMannose-binding lectins ConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\\nConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\\nGalactose-binding lectins In the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\\nIn the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\\nN-acetylgalactosamine-binding lectins All the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\\nOM, olfactory mucosa.\\nAll the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\\nOM, olfactory mucosa.\\nComplex type N-glycan-binding lectins In the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\\nIn the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\\nFucose-binding lectin In the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\\nIn the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\",\n \"All lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\\nOM, olfactory mucosa.\",\n \"ConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\",\n \"In the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\",\n \"All the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\\nOM, olfactory mucosa.\",\n \"In the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\",\n \"In the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\",\n \"This is the first study that visualized individual glycoconjugate (carbohydrate residues) during the postnatal development of bovine OM. We employed specific antibodies for olfactory sensory neurons against OMP and 21 lectins specific for the glycoconjugate sugar residues. Finally, we found that the distribution and expression levels of glycoconjugates varied remarkably between the OMs of neonatal and adult cattle.\\nIn the present study, histological examination revealed that the cattle OM is composed of the OE and the underlying lamina propria, which contains Bowman’s glands, bundles of olfactory axons, and ensheathing glia, as observed in other mammals, such as rats [26], sheep [15], and dogs [22]. A previous study established that age-related thickening results from a rapid increase in olfactory density and contributes to an improved odor sensitivity in domestic dogs and sheep [27]. This study evaluated differences in OM morphology between neonate and adult cattle to test the notion that mucosal refinement changes with age. Our findings indicated that adult cattle OM had a more complete structural shape than that of the neonate. Additionally, PAS and Alcian blue (pH 2.5) staining were utilized to confirm the mucus specificity of the Bowman’s gland duct/acini in the lamina propria. In neonates and adults, Bowman’s gland ducts/acini were stained positively with PAS and Alcian blue to varied degrees of intensity. These findings imply that the Bowman’s glands release neutral and acidic components in the lamina propria in both the neonatal and adult OM, which may be implicated in the perception of scents or in protection against infectious agents and particles [28].\\nOMP has been implicated in olfactory transduction [29], and its expression is restricted to mature chemosensory neurons in the OE [30]. We assessed the OMP expression in the OM to verify whether it is associated with mature sensory neurons that extend their dendritic knobs to the OE mucociliary layer or free border [31]. In contrast to OMP, GAP-43 expression is detected in the basal region of the OE, indicating that GAP-43 is expressed in immature neurons [32]. In neonatal and adult cattle, mature receptor cells in the middle region and immature receptor cells in the basal region of the OE were markedly positive for OMP and GAP-43, respectively. Therefore, OMP-positive mature and GAP-43-positive immature sensory neurons may be essential for the development of the main olfactory system [3233]. However, the precise roles of OMP and GAP-43 in cattle OM should be further verified.\\n\\nTables 2 and 3 demonstrate the results of lectin-binding patterns in the OM of neonatal and adult cattle. In all regions of the OM, including the OE free border, receptor cells, supporting cells, basal cells, nerve bundles, and Bowman’s glands, lectin-reactive glycoconjugates were found at varying intensities. Except for BSL-II and jacalin, all lectins utilized in this study were expressed in the free border of both neonatal and adult cattle, implying that the majority of the carbohydrates detected by these lectins are present in the mucociliary layer of the OE. This finding somewhat contrasts with previously published research on sheep [15] and horses [23], wherein the free borders were labeled with 13 and 19 of the 21 lectins, respectively. Except for BSL-II and jacalin, 19 of the 21 lectins were labeled in receptor cells with varying intensities in the cattle OE, indicating that sugar residues play a role in the cell biology of receptor cells. The results of this study were consistent with those of a previous study in horses [23]. However, DBA, SJA, PHA-L, and UEA-I were found in cattle but not in horses [23]. Furthermore, similar lectins have been identified in the olfactory receptor neurons and axons of other animals [34353637], which is consistent with our findings. Among the 21 lectins, 10 and 9 lectins were labeled as supporting cells in neonates and adults, respectively, and the labeling patterns were relatively comparable to those reported previously [2238], but differed from the results obtained by a previous study in the horse [23]. In the lamina propria, 14 of the 21 lectins were found in the nerve bundles with varying intensities, whereas 3 lectins that were not found in the nerve bundles were found in the OE receptor cells. We hypothesize that sugar residue modifications cause this disparity as a result of positional changes. This event also occurred in the Bowman’s gland in the lamina propria, where the expression patterns of lectin were altered as the Bowman’s gland ducts exited through the OE. In the Bowman’s glands, the majority of lectins were expressed more strongly in acinar cells than in ductal cells, implying that the acini contain a greater variety of sugar residues than the secretory ductal epithelium.\\nA previous study suggested a link between the expression pattern of DBA-binding glycoconjugates and the development of the olfactory system [36]. Similarly, previous studies hypothesized that the expression patterns of lectins, such as VVA and PNA, may be associated with the development of the OM [3940]. The majority of the lectin-binding sites in the cattle OM were identical or stronger in neonates than in adults, implying that the labeling of lectins may be closely related to the development and maturation of the OM. Thus, developmental changes in the expression patterns of lectin-binding glycoconjugates were evident in the OM of Korean native cattle. However, further functional investigations are required to determine how each glycoconjugate contributes to olfactory sensory processing.\\nGlobally, cattle is important for animal protein production and may be a desirable animal model for studying olfaction and its effect on animal behavior. The Korean native cattle used in this study is part of a national breeding and selection program for beef production and thus subjected to artificial selection for intensive production in South Korea [41]. For example, in the United States, artificial insemination (AI) accounts for 50% of beef cattle breeding; in South Korea, it accounts for more than 90% [42]. AI timing is an essential component of conception, and the role of pheromones and olfaction in this process is being investigated [43]. Additional factors, such as irregular or prolonged estrous cycles and anestrus, which contribute to lower conception rates during AI, have been linked to pheromones and their receptors [44]. Consequently, olfaction may differ between Korean native cattle and other cattle according to these various factors [45]; however, information about the association between fertility-related behaviors and olfaction in Korean native cattle is limited. Still, we suggest that our data on the histology and lectin histochemistry in the OM of Korean native cattle may serve as a basis for further investigating their olfactory behavior and physiology.\\nIn conclusion, we have performed a comprehensive characterization of the Korean native cattle OM at the histological, immunohistochemical, and lectin histochemical levels, thereby revealing new information about the main olfactory system. Additionally, the expression levels of glycoconjugates were considerably different between the OMs of neonatal and adult cattle, with adult OMs exhibiting higher intensities than neonates. Thus, this information can help clarify the role of the OM and its chemo-communication pathways in cattle. Further functional studies, however, are required to demonstrate the role of lectin-binding glycoconjugates in olfactory sensory processing and its associated behaviors.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["intro","materials|methods",null,null,null,null,null,"results",null,null,null,null,null,null,null,null,null,"discussion"],"string":"[\n \"intro\",\n \"materials|methods\",\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"discussion\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["Glycoconjugate","immunohistochemistry","Korean native cattle (Hanwoo)","lectin histochemistry","olfactory mucosa"],"string":"[\n \"Glycoconjugate\",\n \"immunohistochemistry\",\n \"Korean native cattle (Hanwoo)\",\n \"lectin histochemistry\",\n \"olfactory mucosa\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nOlfaction is important for animals because it allows them to explore their surroundings for odorous signals from food sources and environments, as well as detect chemical compounds that influence social interaction and reproductive behavior [1]. This perception is mediated by two chemosensory systems: the main olfactory system and the vomeronasal system. The olfactory mucosa (OM), positioned at the caudal/posterior roof of the nasal cavity, and the vomeronasal organ (VNO), located at the base of the nasal septum or on the roof of the mouth, are the organs comprising these two systems [23]. Traditionally, the OM and VNO have been considered functionally and anatomically distinct, with the OM detecting conventional volatile odorants and the VNO receiving pheromones [45]. These two organs share some common features but differ in neuron types, primary structures of receptor proteins, physiological pathways, and central neuroanatomical projections into the brain [6]. However, despite the anatomical and functional differences, previous studies indicate that the OM and VNO play a synergistic role in the regulation of various olfactory-induced behaviors and reproductive and social interactions in mammals [15789].\nThe OM is critical for chemical signal acquisition in the main olfactory system, conveying signals to the main olfactory bulb [4]. In mammals, the OM is composed of the olfactory epithelium (OE) and the lamina propria. The OE is predominantly composed of chemosensory neurons, supporting cells, and basal cells [10]. The lamina propria is constituted by loose connective tissue in which olfactory nerve axon bundles, Bowman's glands, and blood and lymph vessels [1011]. In mammals, odorant reception occurs via the chemosensory neurons of the OE, which contain dendrites that extend beyond the apical surface, where the cilia protrude into the mucus layer, and a basally projecting axon process [12]. Glycoconjugates (terminal carbohydrates) have been shown to play a crucial role in the chemoreception of the OE [13].\nCell surfaces are densely packed with a diverse array of glycoconjugates, each of which provides considerable biological information [14]. Lectins are naturally occurring glycoconjugate-binding molecules, and a large number of purified lectins are regarded as the primary analytical tool for studying glycoconjugates in the olfactory system [131516]. Specific lectin binding is closely related to the olfactory neuron function, as evidenced by the abundance of glycoconjugates in the mucosensory compartment of the chemosensory epithelia, where receptor-specific events associated with transduction occur [13]. Numerous animals, including rats [1718], mice [1920], marmosets [21], sheep [1522], camels [22], horses [23], and humans [24], express the carbohydrate (lectin-binding) moiety in their OM.\nDespite the fact that cattle are one of the best species for biochemical investigations on olfaction [25], there is little published information on the immunohistochemical and lectin histochemical properties of the OM in bovines. We first evaluated the histological characteristics and lectin histochemistry of the OM in Korean native cattle, and then we compared neonate to adult OMs to determine the features associated with maturity. To our knowledge, this is the first study that provides a comprehensive outlook on the expression levels of lectin-binding glycoconjugates in bovine OM.\n\nMATERIALS AND METHODS:\nTissue preparation Three neonatal OM samples (1 to 3 day old) and three adult OM samples (2.5-year-old) of Korean native cattle (Hanwoo, Bos taurus coreanae) were obtained from local farms and a local slaughterhouse in Chungcheongbuk-do, South Korea, respectively. Both sexes of all animals were utilized in this study (male: four neonates and five adults; female: three neonates and three adults). The OM used in this study belongs to a small area on the caudal roof of the nasal cavity, where it completely covered the ethmoturbinates and caudal portions of the dorsal and middle nasal conchae (Fig. 1A). Morphological exams of the nasal cavity indicated that none of them had underlying respiratory diseases. All experimental and animal handling procedures were conducted in accordance with the guidelines of Institutional Animal Care and Use Committee of Chonnam National University (26 October 2021; CNU IACUC-YB-2021-131).\nOM, olfactory mucosa; BD, Bowman’s glandular duct; BG, Bowman’s gland; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.\nThree neonatal OM samples (1 to 3 day old) and three adult OM samples (2.5-year-old) of Korean native cattle (Hanwoo, Bos taurus coreanae) were obtained from local farms and a local slaughterhouse in Chungcheongbuk-do, South Korea, respectively. Both sexes of all animals were utilized in this study (male: four neonates and five adults; female: three neonates and three adults). The OM used in this study belongs to a small area on the caudal roof of the nasal cavity, where it completely covered the ethmoturbinates and caudal portions of the dorsal and middle nasal conchae (Fig. 1A). Morphological exams of the nasal cavity indicated that none of them had underlying respiratory diseases. All experimental and animal handling procedures were conducted in accordance with the guidelines of Institutional Animal Care and Use Committee of Chonnam National University (26 October 2021; CNU IACUC-YB-2021-131).\nOM, olfactory mucosa; BD, Bowman’s glandular duct; BG, Bowman’s gland; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.\nHistological examination For light microscopic examination, the ethmoturbinates were removed immediately after death, cut into 5-cm-thick cross sections, and fixed for 3–5 days in 10% buffered formalin. Following fixation, the ethmoturbinates were trimmed and decalcified through multiple solution changes of sodium citrate-formic acid solution. When a needle could readily penetrate the bone with very little force, the decalcification process was stopped. The samples were then washed for 24 h with running tap water, dehydrated in a succession of ethanol concentrations (70%, 80%, 90%, 95%, and 100%), cleaned in xylene, embedded in paraffin, and sectioned into 4 μm slices. Following deparaffinization, the sections were utilized for hematoxylin and eosin staining, mucus and lectin histochemistry, and immunohistochemistry.\nFor light microscopic examination, the ethmoturbinates were removed immediately after death, cut into 5-cm-thick cross sections, and fixed for 3–5 days in 10% buffered formalin. Following fixation, the ethmoturbinates were trimmed and decalcified through multiple solution changes of sodium citrate-formic acid solution. When a needle could readily penetrate the bone with very little force, the decalcification process was stopped. The samples were then washed for 24 h with running tap water, dehydrated in a succession of ethanol concentrations (70%, 80%, 90%, 95%, and 100%), cleaned in xylene, embedded in paraffin, and sectioned into 4 μm slices. Following deparaffinization, the sections were utilized for hematoxylin and eosin staining, mucus and lectin histochemistry, and immunohistochemistry.\nMucus histochemistry The acidic mucin was distinguished from the neutral epithelial mucin using periodic acid Schiff (PAS) and Alcian blue (pH 2.5) staining. For PAS staining, the sections were subjected to 0.5% periodic acid for 15 min, then washed and incubated in Schiff reagent for 30 min. After 10 min of washing with running tap water, the sections were counterstained with hematoxylin. For Alcian blue staining, the sections were first incubated in 3% acetic acid for 3 min, followed by 30 min in 1% Alcian blue solution in 3% acetic acid (pH 2.5). After 1 min of washing with running tap water, the sections were counterstained with neutral red.\nThe acidic mucin was distinguished from the neutral epithelial mucin using periodic acid Schiff (PAS) and Alcian blue (pH 2.5) staining. For PAS staining, the sections were subjected to 0.5% periodic acid for 15 min, then washed and incubated in Schiff reagent for 30 min. After 10 min of washing with running tap water, the sections were counterstained with hematoxylin. For Alcian blue staining, the sections were first incubated in 3% acetic acid for 3 min, followed by 30 min in 1% Alcian blue solution in 3% acetic acid (pH 2.5). After 1 min of washing with running tap water, the sections were counterstained with neutral red.\nImmunohistochemistry To retrieve the antigens, the sections were heated for 1 h at 90°C in citrate buffer (0.01 M, pH 6.0). After cooling, the sections were treated for 20 min with 0.3% hydrogen peroxide in distilled water to suppress the endogenous peroxidase activity. To avoid non-specific binding, the sections were treated with normal goat serum (Vectastain Elite ABC kit; Vector Laboratories, USA) for 1 h before being incubated overnight at 4°C with rabbit monoclonal anti-olfactory marker protein (OMP) (1:1,000 dilution, Cat. No. ab183947; Abcam, UK) and rabbit polyclonal anti-growth associated protein-43 (GAP-43) (1:1,000 dilution, Cat. No. PA5-34943; ThermoFisher Scientific, USA) antibodies. The primary antibodies were excluded from the procedure as negative controls. The sections were rinsed with phosphate-buffered saline (PBS), treated with biotinylated goat anti-rabbit IgG (Vectastain Elite ABC kit) for 1 h, and then reacted with the avidin-biotin-peroxidase complex (Vectastain Elite ABC kit) for 1 h at room temperature (RT). Immunoreactivity was detected using a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories), followed by counterstaining with hematoxylin.\nTo retrieve the antigens, the sections were heated for 1 h at 90°C in citrate buffer (0.01 M, pH 6.0). After cooling, the sections were treated for 20 min with 0.3% hydrogen peroxide in distilled water to suppress the endogenous peroxidase activity. To avoid non-specific binding, the sections were treated with normal goat serum (Vectastain Elite ABC kit; Vector Laboratories, USA) for 1 h before being incubated overnight at 4°C with rabbit monoclonal anti-olfactory marker protein (OMP) (1:1,000 dilution, Cat. No. ab183947; Abcam, UK) and rabbit polyclonal anti-growth associated protein-43 (GAP-43) (1:1,000 dilution, Cat. No. PA5-34943; ThermoFisher Scientific, USA) antibodies. The primary antibodies were excluded from the procedure as negative controls. The sections were rinsed with phosphate-buffered saline (PBS), treated with biotinylated goat anti-rabbit IgG (Vectastain Elite ABC kit) for 1 h, and then reacted with the avidin-biotin-peroxidase complex (Vectastain Elite ABC kit) for 1 h at room temperature (RT). Immunoreactivity was detected using a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories), followed by counterstaining with hematoxylin.\nLectin histochemistry Biotinylated lectin kits I, II, and III (Cat. No. BK-1000, BK-2000, and BK-3000) were acquired from Vector Laboratories. The acronyms, sources, and specificities of the used lectins are shown in Table 1. On the basis of their binding specificity and inhibitory sugars, lectins were categorized as N-acetylglucosamine, mannose, galactose, N-acetylgalactosamine, complex type N-glycan, and fucose-binding lectins. For competitive inhibition, the following sugars were purchased from Sigma-Aldrich (USA) and Vector Laboratories (Table 1): N-acetyl-D-glucosamine (β-D–GlcNAc; Sigma-Aldrich), Chitin Hydrolysate (Vector Laboratories), α-methyl mannoside/α-methyl glucoside (Sigma-Aldrich), lactose (Galβ1, 4Glc; Sigma-Aldrich), N-acetyl-D-galactosamine (α-D-GalNAc; Sigma-Aldrich), melibiose (Galα1, 6Glc; Sigma-Aldrich), and β-D-galactose (Sigma-Aldrich).\nFuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose.\naThe acronyms and specificities of the 21 lectins were obtained from the data sheep (Vector laboratory) and grouped, as shown in a previous paper [23].\nTo eliminate the endogenous peroxidase activity, the sections were treated with 0.3% hydrogen peroxide in methanol. To prevent non-specific reactions, the sections were rinsed with PBS and then incubated with 1% bovine serum albumin in PBS. The sections were then treated overnight at 4°C with biotinylated lectins and reacted for 45 min at RT with an avidin-biotin-peroxidase complex (Vectastain Elite ABC kit). The sections were rinsed with PBS, treated with a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories), and counterstained with hematoxylin. Negative controls for lectin histochemistry were generated by removing the biotinylated lectins and preincubating the lectins with appropriate inhibitors for 1 h at RT.\nBiotinylated lectin kits I, II, and III (Cat. No. BK-1000, BK-2000, and BK-3000) were acquired from Vector Laboratories. The acronyms, sources, and specificities of the used lectins are shown in Table 1. On the basis of their binding specificity and inhibitory sugars, lectins were categorized as N-acetylglucosamine, mannose, galactose, N-acetylgalactosamine, complex type N-glycan, and fucose-binding lectins. For competitive inhibition, the following sugars were purchased from Sigma-Aldrich (USA) and Vector Laboratories (Table 1): N-acetyl-D-glucosamine (β-D–GlcNAc; Sigma-Aldrich), Chitin Hydrolysate (Vector Laboratories), α-methyl mannoside/α-methyl glucoside (Sigma-Aldrich), lactose (Galβ1, 4Glc; Sigma-Aldrich), N-acetyl-D-galactosamine (α-D-GalNAc; Sigma-Aldrich), melibiose (Galα1, 6Glc; Sigma-Aldrich), and β-D-galactose (Sigma-Aldrich).\nFuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose.\naThe acronyms and specificities of the 21 lectins were obtained from the data sheep (Vector laboratory) and grouped, as shown in a previous paper [23].\nTo eliminate the endogenous peroxidase activity, the sections were treated with 0.3% hydrogen peroxide in methanol. To prevent non-specific reactions, the sections were rinsed with PBS and then incubated with 1% bovine serum albumin in PBS. The sections were then treated overnight at 4°C with biotinylated lectins and reacted for 45 min at RT with an avidin-biotin-peroxidase complex (Vectastain Elite ABC kit). The sections were rinsed with PBS, treated with a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories), and counterstained with hematoxylin. Negative controls for lectin histochemistry were generated by removing the biotinylated lectins and preincubating the lectins with appropriate inhibitors for 1 h at RT.\n\nTissue preparation:\nThree neonatal OM samples (1 to 3 day old) and three adult OM samples (2.5-year-old) of Korean native cattle (Hanwoo, Bos taurus coreanae) were obtained from local farms and a local slaughterhouse in Chungcheongbuk-do, South Korea, respectively. Both sexes of all animals were utilized in this study (male: four neonates and five adults; female: three neonates and three adults). The OM used in this study belongs to a small area on the caudal roof of the nasal cavity, where it completely covered the ethmoturbinates and caudal portions of the dorsal and middle nasal conchae (Fig. 1A). Morphological exams of the nasal cavity indicated that none of them had underlying respiratory diseases. All experimental and animal handling procedures were conducted in accordance with the guidelines of Institutional Animal Care and Use Committee of Chonnam National University (26 October 2021; CNU IACUC-YB-2021-131).\nOM, olfactory mucosa; BD, Bowman’s glandular duct; BG, Bowman’s gland; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.\n\nHistological examination:\nFor light microscopic examination, the ethmoturbinates were removed immediately after death, cut into 5-cm-thick cross sections, and fixed for 3–5 days in 10% buffered formalin. Following fixation, the ethmoturbinates were trimmed and decalcified through multiple solution changes of sodium citrate-formic acid solution. When a needle could readily penetrate the bone with very little force, the decalcification process was stopped. The samples were then washed for 24 h with running tap water, dehydrated in a succession of ethanol concentrations (70%, 80%, 90%, 95%, and 100%), cleaned in xylene, embedded in paraffin, and sectioned into 4 μm slices. Following deparaffinization, the sections were utilized for hematoxylin and eosin staining, mucus and lectin histochemistry, and immunohistochemistry.\n\nMucus histochemistry:\nThe acidic mucin was distinguished from the neutral epithelial mucin using periodic acid Schiff (PAS) and Alcian blue (pH 2.5) staining. For PAS staining, the sections were subjected to 0.5% periodic acid for 15 min, then washed and incubated in Schiff reagent for 30 min. After 10 min of washing with running tap water, the sections were counterstained with hematoxylin. For Alcian blue staining, the sections were first incubated in 3% acetic acid for 3 min, followed by 30 min in 1% Alcian blue solution in 3% acetic acid (pH 2.5). After 1 min of washing with running tap water, the sections were counterstained with neutral red.\n\nImmunohistochemistry:\nTo retrieve the antigens, the sections were heated for 1 h at 90°C in citrate buffer (0.01 M, pH 6.0). After cooling, the sections were treated for 20 min with 0.3% hydrogen peroxide in distilled water to suppress the endogenous peroxidase activity. To avoid non-specific binding, the sections were treated with normal goat serum (Vectastain Elite ABC kit; Vector Laboratories, USA) for 1 h before being incubated overnight at 4°C with rabbit monoclonal anti-olfactory marker protein (OMP) (1:1,000 dilution, Cat. No. ab183947; Abcam, UK) and rabbit polyclonal anti-growth associated protein-43 (GAP-43) (1:1,000 dilution, Cat. No. PA5-34943; ThermoFisher Scientific, USA) antibodies. The primary antibodies were excluded from the procedure as negative controls. The sections were rinsed with phosphate-buffered saline (PBS), treated with biotinylated goat anti-rabbit IgG (Vectastain Elite ABC kit) for 1 h, and then reacted with the avidin-biotin-peroxidase complex (Vectastain Elite ABC kit) for 1 h at room temperature (RT). Immunoreactivity was detected using a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories), followed by counterstaining with hematoxylin.\n\nLectin histochemistry:\nBiotinylated lectin kits I, II, and III (Cat. No. BK-1000, BK-2000, and BK-3000) were acquired from Vector Laboratories. The acronyms, sources, and specificities of the used lectins are shown in Table 1. On the basis of their binding specificity and inhibitory sugars, lectins were categorized as N-acetylglucosamine, mannose, galactose, N-acetylgalactosamine, complex type N-glycan, and fucose-binding lectins. For competitive inhibition, the following sugars were purchased from Sigma-Aldrich (USA) and Vector Laboratories (Table 1): N-acetyl-D-glucosamine (β-D–GlcNAc; Sigma-Aldrich), Chitin Hydrolysate (Vector Laboratories), α-methyl mannoside/α-methyl glucoside (Sigma-Aldrich), lactose (Galβ1, 4Glc; Sigma-Aldrich), N-acetyl-D-galactosamine (α-D-GalNAc; Sigma-Aldrich), melibiose (Galα1, 6Glc; Sigma-Aldrich), and β-D-galactose (Sigma-Aldrich).\nFuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose.\naThe acronyms and specificities of the 21 lectins were obtained from the data sheep (Vector laboratory) and grouped, as shown in a previous paper [23].\nTo eliminate the endogenous peroxidase activity, the sections were treated with 0.3% hydrogen peroxide in methanol. To prevent non-specific reactions, the sections were rinsed with PBS and then incubated with 1% bovine serum albumin in PBS. The sections were then treated overnight at 4°C with biotinylated lectins and reacted for 45 min at RT with an avidin-biotin-peroxidase complex (Vectastain Elite ABC kit). The sections were rinsed with PBS, treated with a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories), and counterstained with hematoxylin. Negative controls for lectin histochemistry were generated by removing the biotinylated lectins and preincubating the lectins with appropriate inhibitors for 1 h at RT.\n\nRESULTS:\nHistological findings of the OM In neonatal (Fig. 1A) and adult cattle (Fig. 1B), the OM was composed of the OE and the lamina propria. The OE is predominantly constituted of basal cells, receptor cells, and supporting cells. The nuclei of the basal, receptor, and supporting cells were located in the basal, middle, and apical regions, respectively, of the OE (Fig. 1A and B). The basal cells were round, pallid, and had little nuclei. The nuclei of the supporting cells were elongated and spindle-shaped, but the nuclei of receptor cells were more rounded. While neonatal and adult OMs displayed comparable histological features, adult OMs appeared thicker than neonatal OMs.\nThe lamina propria included Bowman’s glands and olfactory axon bundles. Bowman’s glands were composed of clustered acinar cells that were connected through ducts to the apical surface (Fig. 1A and B). The Bowman’s glandular ducts and/or acini stained positively for PAS (Fig. 1C and E) and Alcian blue stains (pH 2.5; Fig. 1D and F) in neonates (Fig. 1C and D) and adults (Fig. 1E and F); however, the reactivity of duct cells was relatively lower than that of gland cells. Additionally, the histological features of the OM did not differ between males and females at both ages.\nIn neonatal (Fig. 1A) and adult cattle (Fig. 1B), the OM was composed of the OE and the lamina propria. The OE is predominantly constituted of basal cells, receptor cells, and supporting cells. The nuclei of the basal, receptor, and supporting cells were located in the basal, middle, and apical regions, respectively, of the OE (Fig. 1A and B). The basal cells were round, pallid, and had little nuclei. The nuclei of the supporting cells were elongated and spindle-shaped, but the nuclei of receptor cells were more rounded. While neonatal and adult OMs displayed comparable histological features, adult OMs appeared thicker than neonatal OMs.\nThe lamina propria included Bowman’s glands and olfactory axon bundles. Bowman’s glands were composed of clustered acinar cells that were connected through ducts to the apical surface (Fig. 1A and B). The Bowman’s glandular ducts and/or acini stained positively for PAS (Fig. 1C and E) and Alcian blue stains (pH 2.5; Fig. 1D and F) in neonates (Fig. 1C and D) and adults (Fig. 1E and F); however, the reactivity of duct cells was relatively lower than that of gland cells. Additionally, the histological features of the OM did not differ between males and females at both ages.\nImmunohistochemical analysis of OMP in the OM In neonatal (Fig. 2A) and adult (Fig. 2B) cattle, OMP, a marker for mature olfactory sensory neurons, immunoreactivity was identified in the majority of the mature receptor cells in the middle region of the OE, with dendrites reaching to the OE free border, but not in basal and supporting cells. Their reactivity was more prominent in adults. GAP-43, a marker of immature olfactory sensory neurons, was found in certain receptor cells in the basal region of the OE, and some dendrites were immunopositive for GAP-43 (Fig. 2C and D). In addition, OMP and GAP-43 immunoreactivities were observed in the nerve bundles of the lamina propria (Fig. 2A and C).\nOMP, olfactory marker protein; GAP-43, growth associated protein-43; OM, olfactory mucosa; OE, olfactory epithelium; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.\nIn neonatal (Fig. 2A) and adult (Fig. 2B) cattle, OMP, a marker for mature olfactory sensory neurons, immunoreactivity was identified in the majority of the mature receptor cells in the middle region of the OE, with dendrites reaching to the OE free border, but not in basal and supporting cells. Their reactivity was more prominent in adults. GAP-43, a marker of immature olfactory sensory neurons, was found in certain receptor cells in the basal region of the OE, and some dendrites were immunopositive for GAP-43 (Fig. 2C and D). In addition, OMP and GAP-43 immunoreactivities were observed in the nerve bundles of the lamina propria (Fig. 2A and C).\nOMP, olfactory marker protein; GAP-43, growth associated protein-43; OM, olfactory mucosa; OE, olfactory epithelium; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.\nLectin histochemistry in the OM The intensity values of 21 lectin-binding sites in the OE and lamina propria of the OM of Korean native cattle are summarized in Tables 2 and 3, respectively.\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\naApical perinuclear labeling.\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\nN-acetylglucosamine-binding lectins All lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\nOM, olfactory mucosa.\nAll lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\nOM, olfactory mucosa.\nMannose-binding lectins ConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\nConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\nGalactose-binding lectins In the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\nIn the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\nN-acetylgalactosamine-binding lectins All the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\nOM, olfactory mucosa.\nAll the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\nOM, olfactory mucosa.\nComplex type N-glycan-binding lectins In the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\nIn the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\nFucose-binding lectin In the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\nIn the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\nThe intensity values of 21 lectin-binding sites in the OE and lamina propria of the OM of Korean native cattle are summarized in Tables 2 and 3, respectively.\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\naApical perinuclear labeling.\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\nN-acetylglucosamine-binding lectins All lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\nOM, olfactory mucosa.\nAll lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\nOM, olfactory mucosa.\nMannose-binding lectins ConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\nConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\nGalactose-binding lectins In the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\nIn the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\nN-acetylgalactosamine-binding lectins All the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\nOM, olfactory mucosa.\nAll the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\nOM, olfactory mucosa.\nComplex type N-glycan-binding lectins In the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\nIn the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\nFucose-binding lectin In the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\nIn the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\n\nHistological findings of the OM:\nIn neonatal (Fig. 1A) and adult cattle (Fig. 1B), the OM was composed of the OE and the lamina propria. The OE is predominantly constituted of basal cells, receptor cells, and supporting cells. The nuclei of the basal, receptor, and supporting cells were located in the basal, middle, and apical regions, respectively, of the OE (Fig. 1A and B). The basal cells were round, pallid, and had little nuclei. The nuclei of the supporting cells were elongated and spindle-shaped, but the nuclei of receptor cells were more rounded. While neonatal and adult OMs displayed comparable histological features, adult OMs appeared thicker than neonatal OMs.\nThe lamina propria included Bowman’s glands and olfactory axon bundles. Bowman’s glands were composed of clustered acinar cells that were connected through ducts to the apical surface (Fig. 1A and B). The Bowman’s glandular ducts and/or acini stained positively for PAS (Fig. 1C and E) and Alcian blue stains (pH 2.5; Fig. 1D and F) in neonates (Fig. 1C and D) and adults (Fig. 1E and F); however, the reactivity of duct cells was relatively lower than that of gland cells. Additionally, the histological features of the OM did not differ between males and females at both ages.\n\nImmunohistochemical analysis of OMP in the OM:\nIn neonatal (Fig. 2A) and adult (Fig. 2B) cattle, OMP, a marker for mature olfactory sensory neurons, immunoreactivity was identified in the majority of the mature receptor cells in the middle region of the OE, with dendrites reaching to the OE free border, but not in basal and supporting cells. Their reactivity was more prominent in adults. GAP-43, a marker of immature olfactory sensory neurons, was found in certain receptor cells in the basal region of the OE, and some dendrites were immunopositive for GAP-43 (Fig. 2C and D). In addition, OMP and GAP-43 immunoreactivities were observed in the nerve bundles of the lamina propria (Fig. 2A and C).\nOMP, olfactory marker protein; GAP-43, growth associated protein-43; OM, olfactory mucosa; OE, olfactory epithelium; NB, nerve bundle; OBC, olfactory basal cells; OSC, olfactory supporting cells; ORC, olfactory receptor cells.\n\nLectin histochemistry in the OM:\nThe intensity values of 21 lectin-binding sites in the OE and lamina propria of the OM of Korean native cattle are summarized in Tables 2 and 3, respectively.\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\naApical perinuclear labeling.\n−, negative staining; +, faint staining; ++, moderate staining; +++, intense staining.\nN-acetylglucosamine-binding lectins All lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\nOM, olfactory mucosa.\nAll lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\nOM, olfactory mucosa.\nMannose-binding lectins ConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\nConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\nGalactose-binding lectins In the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\nIn the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\nN-acetylgalactosamine-binding lectins All the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\nOM, olfactory mucosa.\nAll the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\nOM, olfactory mucosa.\nComplex type N-glycan-binding lectins In the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\nIn the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\nFucose-binding lectin In the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\nIn the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\n\nN-acetylglucosamine-binding lectins:\nAll lectins except BSL-II were highly present in the free border of neonatal and adult OEs (Fig. 3A-F, Table 2). In neonates and adults, most OE cells lacked BSL-II reactivity (Table 2). Neonatal OE cells (especially receptor cells) responded more strongly to DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) than adult ones, but they had no remarkable differences in s-WGA (Fig. 3A and B) and WGA labeling (Table 2). Except for BSL-II, all these lectins were present in different amounts in the nerve bundles, but they did not vary with age (Fig. 3A-F, Table 3). Neonatal Bowman’s gland ducts displayed various reactivities to these lectins although reactivity in adults was faint (Fig. 3A-F, Table 3). The intensities of s-WGA (Fig. 3A and B), BSL-II, DSL (Fig. 3C and D), LEL, and STL (Fig. 3E and F) labeling were greater in neonates than in adults in Bowman’s gland acini (Table 3).\nOM, olfactory mucosa.\n\nMannose-binding lectins:\nConA, LCA (Fig. 3G and H), and PSA were labeled with varying intensities across the OE layers, except the LCA in the supporting cells of neonates; their intensities were greater in adults than in neonates (Table 2). ConA labeling was more intense in nerve bundles and Bowman’s gland duct/acini of the lamina propria in neonates than in adults (Table 3). LCA (Fig. 3G and H) labeling was more intense in Bowman’s gland duct/acini of the lamina propria in neonates than in adults, but PSA labeling was more intense in the nerve bundles of the lamina propria in adults than in neonates (Table 3).\n\nGalactose-binding lectins:\nIn the epithelial free border, most lectins were tagged similarly in neonates and adults (Table 2), but the intensity of PNA labeling was greater in neonates than in adults (Fig. 3I and J). RCA120, PNA (Fig. 3I and J), and ECL reactivities were observed in all OE layers except for supporting cells in neonates and/or adults (Table 2). The results of BSL-I labeling of the components of the lamina propria did not differ between neonatal and adult OM (Table 3). Jacalin had no reactivity in any of the OE layers in neonates and adults (Table 2) although it was tagged in Bowman’s gland duct and acini in the lamina propria (Table 3).\n\nN-acetylgalactosamine-binding lectins:\nAll the lectins produced a reaction of different intensities in neonates and adults at the epithelial free border (Fig. 4A-F, Table 2). In receptor cells, although all these lectins were detected faintly in neonates, the intensities of VVA (Fig. 4A and B) and SBA (Fig. 4E and F) were enhanced in adults (Table 2). Additionally, except for DBA, none of these lectins reacted with neonatal basal cells, whereas all lectins were faintly labeled in adults (Table 2). In nerve bundles, DBA (Fig. 4C and D) staining was more intense in neonates than in adults, whereas other lectins were absent (Table 3). In adults, the reactivities for VVA (Fig. 4A and B), SBA (Fig. 4E and F), and SJA were moderate to intense in Bowman’s gland duct and acini but were relatively lower in adults (Table 3).\nOM, olfactory mucosa.\n\nComplex type N-glycan-binding lectins:\nIn the OE (Table 2) and lamina propria (Table 3), the reactivities of PHA-E and PHA-L (Fig. 4G and H) were extensively higher in neonates than in adults. Bowman’s gland duct and acini were positive for both lectins (Fig. 4G and H).\n\nFucose-binding lectin:\nIn the OE of neonates (Fig. 4I) and adults (Fig. 4J), UEA-I was identified in the free border and receptor cells but not in the supporting cells (Table 2). UEA-I was labeled extensively in Bowman’s gland duct/acini in the lamina propria but was relatively lower in adults than in neonates (Table 3).\n\nDISCUSSION:\nThis is the first study that visualized individual glycoconjugate (carbohydrate residues) during the postnatal development of bovine OM. We employed specific antibodies for olfactory sensory neurons against OMP and 21 lectins specific for the glycoconjugate sugar residues. Finally, we found that the distribution and expression levels of glycoconjugates varied remarkably between the OMs of neonatal and adult cattle.\nIn the present study, histological examination revealed that the cattle OM is composed of the OE and the underlying lamina propria, which contains Bowman’s glands, bundles of olfactory axons, and ensheathing glia, as observed in other mammals, such as rats [26], sheep [15], and dogs [22]. A previous study established that age-related thickening results from a rapid increase in olfactory density and contributes to an improved odor sensitivity in domestic dogs and sheep [27]. This study evaluated differences in OM morphology between neonate and adult cattle to test the notion that mucosal refinement changes with age. Our findings indicated that adult cattle OM had a more complete structural shape than that of the neonate. Additionally, PAS and Alcian blue (pH 2.5) staining were utilized to confirm the mucus specificity of the Bowman’s gland duct/acini in the lamina propria. In neonates and adults, Bowman’s gland ducts/acini were stained positively with PAS and Alcian blue to varied degrees of intensity. These findings imply that the Bowman’s glands release neutral and acidic components in the lamina propria in both the neonatal and adult OM, which may be implicated in the perception of scents or in protection against infectious agents and particles [28].\nOMP has been implicated in olfactory transduction [29], and its expression is restricted to mature chemosensory neurons in the OE [30]. We assessed the OMP expression in the OM to verify whether it is associated with mature sensory neurons that extend their dendritic knobs to the OE mucociliary layer or free border [31]. In contrast to OMP, GAP-43 expression is detected in the basal region of the OE, indicating that GAP-43 is expressed in immature neurons [32]. In neonatal and adult cattle, mature receptor cells in the middle region and immature receptor cells in the basal region of the OE were markedly positive for OMP and GAP-43, respectively. Therefore, OMP-positive mature and GAP-43-positive immature sensory neurons may be essential for the development of the main olfactory system [3233]. However, the precise roles of OMP and GAP-43 in cattle OM should be further verified.\n\nTables 2 and 3 demonstrate the results of lectin-binding patterns in the OM of neonatal and adult cattle. In all regions of the OM, including the OE free border, receptor cells, supporting cells, basal cells, nerve bundles, and Bowman’s glands, lectin-reactive glycoconjugates were found at varying intensities. Except for BSL-II and jacalin, all lectins utilized in this study were expressed in the free border of both neonatal and adult cattle, implying that the majority of the carbohydrates detected by these lectins are present in the mucociliary layer of the OE. This finding somewhat contrasts with previously published research on sheep [15] and horses [23], wherein the free borders were labeled with 13 and 19 of the 21 lectins, respectively. Except for BSL-II and jacalin, 19 of the 21 lectins were labeled in receptor cells with varying intensities in the cattle OE, indicating that sugar residues play a role in the cell biology of receptor cells. The results of this study were consistent with those of a previous study in horses [23]. However, DBA, SJA, PHA-L, and UEA-I were found in cattle but not in horses [23]. Furthermore, similar lectins have been identified in the olfactory receptor neurons and axons of other animals [34353637], which is consistent with our findings. Among the 21 lectins, 10 and 9 lectins were labeled as supporting cells in neonates and adults, respectively, and the labeling patterns were relatively comparable to those reported previously [2238], but differed from the results obtained by a previous study in the horse [23]. In the lamina propria, 14 of the 21 lectins were found in the nerve bundles with varying intensities, whereas 3 lectins that were not found in the nerve bundles were found in the OE receptor cells. We hypothesize that sugar residue modifications cause this disparity as a result of positional changes. This event also occurred in the Bowman’s gland in the lamina propria, where the expression patterns of lectin were altered as the Bowman’s gland ducts exited through the OE. In the Bowman’s glands, the majority of lectins were expressed more strongly in acinar cells than in ductal cells, implying that the acini contain a greater variety of sugar residues than the secretory ductal epithelium.\nA previous study suggested a link between the expression pattern of DBA-binding glycoconjugates and the development of the olfactory system [36]. Similarly, previous studies hypothesized that the expression patterns of lectins, such as VVA and PNA, may be associated with the development of the OM [3940]. The majority of the lectin-binding sites in the cattle OM were identical or stronger in neonates than in adults, implying that the labeling of lectins may be closely related to the development and maturation of the OM. Thus, developmental changes in the expression patterns of lectin-binding glycoconjugates were evident in the OM of Korean native cattle. However, further functional investigations are required to determine how each glycoconjugate contributes to olfactory sensory processing.\nGlobally, cattle is important for animal protein production and may be a desirable animal model for studying olfaction and its effect on animal behavior. The Korean native cattle used in this study is part of a national breeding and selection program for beef production and thus subjected to artificial selection for intensive production in South Korea [41]. For example, in the United States, artificial insemination (AI) accounts for 50% of beef cattle breeding; in South Korea, it accounts for more than 90% [42]. AI timing is an essential component of conception, and the role of pheromones and olfaction in this process is being investigated [43]. Additional factors, such as irregular or prolonged estrous cycles and anestrus, which contribute to lower conception rates during AI, have been linked to pheromones and their receptors [44]. Consequently, olfaction may differ between Korean native cattle and other cattle according to these various factors [45]; however, information about the association between fertility-related behaviors and olfaction in Korean native cattle is limited. Still, we suggest that our data on the histology and lectin histochemistry in the OM of Korean native cattle may serve as a basis for further investigating their olfactory behavior and physiology.\nIn conclusion, we have performed a comprehensive characterization of the Korean native cattle OM at the histological, immunohistochemical, and lectin histochemical levels, thereby revealing new information about the main olfactory system. Additionally, the expression levels of glycoconjugates were considerably different between the OMs of neonatal and adult cattle, with adult OMs exhibiting higher intensities than neonates. Thus, this information can help clarify the role of the OM and its chemo-communication pathways in cattle. Further functional studies, however, are required to demonstrate the role of lectin-binding glycoconjugates in olfactory sensory processing and its associated behaviors.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe olfactory mucosa (OM) is crucial for odorant perception in the main olfactory system. The terminal carbohydrates of glycoconjugates influence chemoreception in the olfactory epithelium (OE).\n\nMethods:\nThe OM of neonate and adult Korean native cattle was evaluated using histological, immunohistochemical, and lectin histochemical methods.\n\nResults:\nHistologically, the OM in both neonates and adults consists of the olfactory epithelium and the lamina propria. Additionally, using periodic acid Schiff and Alcian blue (pH 2.5), the mucus specificity of the Bowman's gland duct and acini in the lamina propria was determined. Immunohistochemistry demonstrated that mature and immature olfactory sensory neurons of OEs express the olfactory marker protein and growth associated protein-43, respectively. Lectin histochemistry indicated that numerous glycoconjugates, including as N-acetylglucosamine, mannose, galactose, N-acetylgalactosamine, complex type N-glycan, and fucose groups, were expressed at varied levels in the different cell types in the OMs of neonates and adults at varying levels. According to our observations, the cattle possessed a well-developed olfactory system, and the expression patterns of glycoconjugates in neonatal and adult OMs varied considerably.\n\nConclusions:\nThis is the first study to describe the morphological assessment of the OM of Korean native cattle with a focus on lectin histochemistry. The findings suggest that glycoconjugates may play a role in olfactory chemoreception, and that their labeling properties may be closely related to OM development and maturity."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":13049,"string":"13,049"},"whole_article_abstract_length":{"kind":"number","value":275,"string":"275"},"other_sections_lengths":{"kind":"list like","value":[218,148,128,238,394,259,180,1781,235,131,139,187,62,72],"string":"[\n 218,\n 148,\n 128,\n 238,\n 394,\n 259,\n 180,\n 1781,\n 235,\n 131,\n 139,\n 187,\n 62,\n 72\n]","isConversational":false},"num_sections":{"kind":"number","value":18,"string":"18"},"most_frequent_words":{"kind":"list like","value":["fig","table","adults","cells","neonates","lectins","oe","neonates adults","bowman","om"],"string":"[\n \"fig\",\n \"table\",\n \"adults\",\n \"cells\",\n \"neonates\",\n \"lectins\",\n \"oe\",\n \"neonates adults\",\n \"bowman\",\n \"om\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["olfactory receptor neurons","identified olfactory receptor","olfactory sensory processing","vomeronasal system olfactory","olfactory system vomeronasal"],"string":"[\n \"olfactory receptor neurons\",\n \"identified olfactory receptor\",\n \"olfactory sensory processing\",\n \"vomeronasal system olfactory\",\n \"olfactory system vomeronasal\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Glycoconjugate | immunohistochemistry | Korean native cattle (Hanwoo) | lectin histochemistry | olfactory mucosa [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Glycoconjugate | immunohistochemistry | Korean native cattle (Hanwoo) | lectin histochemistry | olfactory mucosa [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Glycoconjugate | immunohistochemistry | Korean native cattle (Hanwoo) | lectin histochemistry | olfactory mucosa [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Cattle | Animals | Lectins | Galactose | Olfactory Mucosa | Republic of Korea [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Cattle | Animals | Lectins | Galactose | Olfactory Mucosa | Republic of Korea [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Cattle | Animals | Lectins | Galactose | Olfactory Mucosa | Republic of Korea [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] olfactory receptor neurons | identified olfactory receptor | olfactory sensory processing | vomeronasal system olfactory | olfactory system vomeronasal [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] olfactory receptor neurons | identified olfactory receptor | olfactory sensory processing | vomeronasal system olfactory | olfactory system vomeronasal [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] olfactory receptor neurons | identified olfactory receptor | olfactory sensory processing | vomeronasal system olfactory | olfactory system vomeronasal [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] fig | table | adults | cells | neonates | lectins | oe | neonates adults | bowman | om [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] fig | table | adults | cells | neonates | lectins | oe | neonates adults | bowman | om [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] fig | table | adults | cells | neonates | lectins | oe | neonates adults | bowman | om [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] om | glycoconjugates | olfactory | vno | system | chemosensory | lectin | olfactory system | mammals | main [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] fig | table | adults | neonates | cells | lectins | neonates adults | oe | adults table | labeling [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] fig | table | adults | neonates | cells | lectins | oe | neonates adults | olfactory | sections [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] OM ||| Alcian | Bowman ||| protein-43 ||| Lectin ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| Korean | lectin ||| ||| OM ||| Alcian | Bowman ||| protein-43 ||| Lectin ||| ||| first | Korean | lectin ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":52,"cells":{"title":{"kind":"string","value":"Polymicrobial bloodstream infections in the neonatal intensive care unit are associated with increased mortality: a case-control study."},"pmid":{"kind":"string","value":"25022748"},"background_abstract":{"kind":"string","value":"Polymicrobial infections in adults and children are associated with increase in mortality, duration of intensive care and healthcare costs. Very few studies have characterized polymicrobial bloodstream infections in the neonatal unit. Considerable variation has been reported in incidence of polymicrobial infections and associated clinical outcomes. We characterized the risk factors and outcomes of polymicrobial bloodstream infections in our neonatal units in a tertiary hospital in North America."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"In a retrospective case control study design, we identified infants in the neonatal intensive care unit with positive blood cultures at Texas Children's Hospital, over a 16-year period from January 1, 1997 to December 31, 2012. Clinical data from online databases were available from January 2009 to December 2012. For each polymicrobial bloodstream infection (case), we matched three infants with monomicrobial bloodstream infection (control) by gestational age and birth weight."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"We identified 2007 episodes of bloodstream infections during the 16 year study period and 280 (14%) of these were polymicrobial. Coagulase-negative Staphylococcus, Enterococcus, Klebsiella and Candida were the most common microbial genera isolated from polymicrobial infections. Polymicrobial bloodstream infections were associated with more than 3-fold increase in mortality and an increase in duration of infection. Surgical intervention was a significant risk factor for polymicrobial infection."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The frequency and increased mortality emphasizes the clinical significance of polymicrobial bloodstream infections in the neonatal intensive care unit. Clinical awareness and focused research on neonatal polymicrobial infections is urgently needed."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Bacteremia","Birth Weight","Candida","Case-Control Studies","Coinfection","Female","Humans","Incidence","Infant","Infant, Newborn","Intensive Care Units, Neonatal","Klebsiella","Male","Retrospective Studies","Risk Factors","Staphylococcus","Texas","Treatment Outcome"],"string":"[\n \"Bacteremia\",\n \"Birth Weight\",\n \"Candida\",\n \"Case-Control Studies\",\n \"Coinfection\",\n \"Female\",\n \"Humans\",\n \"Incidence\",\n \"Infant\",\n \"Infant, Newborn\",\n \"Intensive Care Units, Neonatal\",\n \"Klebsiella\",\n \"Male\",\n \"Retrospective Studies\",\n \"Risk Factors\",\n \"Staphylococcus\",\n \"Texas\",\n \"Treatment Outcome\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"4226990"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Polymicrobial infections increase mortality more than 2-fold in adults and children, increase length of hospital stay and healthcare costs [1,2]. Risk factors for polymicrobial infections in children and adults include the presence of a central venous catheter, administration of parenteral nutrition, gastrointestinal pathology, especially short gut syndrome, use of broad-spectrum antibiotics and immunosuppression [1,3,4].\nNeonatal polymicrobial infections are less well characterized compared to those in children or adults. Increased survival of extremely premature infants at the edge of viability, dependence on catheters and parenteral nutrition (PN), and antibiotic therapy may predispose to polymicrobial infections in neonates. The frequency of neonatal polymicrobial bloodstream infections reported in clinical studies varies from 4 to 24% of all bloodstream infections [5-10]. A standard definition for neonatal polymicrobial infections is lacking and the incidence varies from study to study partly due to variability in definition, neonatal population and practices [11]. Few studies have focused on polymicrobial infections in neonates, especially on risk factors and clinical outcomes in the western world [10-12]. In a neonatal review, the mortality due to polymicrobial infections was 3-fold greater than that of monomicrobial infections (70% vs. 23%) [10]. Organisms that are commonly implicated in neonatal polymicrobial bloodstream infections are coagulase-negative staphylococcus (CONS), Candida spp., Staphylococcus aureus and Enterococcus spp. [1,9,13-16]. A common risk factor appears to be multi-species biofilm infections originating from indwelling medical devices, notably indwelling vascular catheters or endotracheal tubes [1,17].\nPolymicrobial bloodstream infections may be defined as multiple organisms isolated during an infectious episode including those from a single blood specimen [1] or more restrictively as isolation of more than one organism from a single blood specimen only [11,12,17]. Different definitions may partly explain the varying incidence of polymicrobial bloodstream infections reported. Bizzarro et al. (Yale, 1989-2006) and Faix et al. (Ann Arbor, 1971-1986) have reported the only two studies on neonatal polymicrobial infections from North America, [10,11]. Changing epidemiology of neonatal infections dictates a need to update and understand the epidemiology of polymicrobial infections in the hospitalized infant [11]. Lack of data on polymicrobial infections from our neonatal unit and concern of significant increase in mortality, morbidity and healthcare costs due to polymicrobial infections prompted us to investigate its frequency, risk factors and clinical outcomes at Texas Children’s Hospital (TCH)."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"We hypothesized that polymicrobial infections comprise > 5% of bloodstream infections in infants residing in the neonatal intensive care units, have identifiable risk factors and are associated with higher mortality and morbidity than monomicrobial infections. We tested our hypothesis by performing a single center, retrospective, matched case-control study during a 16 year study period. Our study protocol was approved by the Institutional Review Board at Baylor College of Medicine, Houston. We followed the STROBE guidelines in reporting this study [18].\n Identification of cases and controls We identified blood culture positive infants from the clinical microbiology database, who were admitted to the neonatal intensive care units (NICUs) III and II at TCH from January 1, 1997 to December 31, 2012. Infants more than 28 days of age in the NICU at the time of the infection and those admitted from home to the NICU with positive blood cultures were also included. The data in the clinical microbiology database is entered by the microbiologist and all positive and negative cultures including those regarded as contaminants are recorded. The tertiary NICU at TCH has approximately 1500 admissions per year including inborn and outborn transferred neonates (ranged from 1338 to 1547 during the years 2009 to 2012). Very low birth weight (VLBW, birth weight < 1500 g) infant admissions ranged from 222 to 291 infants during the years 2009 to 2012.\nWe identified blood culture positive infants from the clinical microbiology database, who were admitted to the neonatal intensive care units (NICUs) III and II at TCH from January 1, 1997 to December 31, 2012. Infants more than 28 days of age in the NICU at the time of the infection and those admitted from home to the NICU with positive blood cultures were also included. The data in the clinical microbiology database is entered by the microbiologist and all positive and negative cultures including those regarded as contaminants are recorded. The tertiary NICU at TCH has approximately 1500 admissions per year including inborn and outborn transferred neonates (ranged from 1338 to 1547 during the years 2009 to 2012). Very low birth weight (VLBW, birth weight < 1500 g) infant admissions ranged from 222 to 291 infants during the years 2009 to 2012.\n Definitions Polymicrobial bloodstream infection was defined as: i) isolation of more than one organism from a single blood culture specimen and ii) isolation of more than one organism in different blood culture specimens during the same bloodstream infectious episode. We defined ‘bloodstream infection episodes’ as time-periods associated with positive blood cultures [1] and the infection episode was considered resolved when at least two subsequent blood cultures performed every 24 hrs were negative. Usually when an organism is isolated from a blood culture specimen, blood cultures are repeated every 24 hr till two blood culture specimens are negative for organisms. Our neonatal units used only aerobic blood cultures and it is not routine to perform anaerobic blood cultures for the evaluation of neonatal sepsis. Single specimen polymicrobial infections were defined as more than one organism isolated from the same blood culture specimen. We also collected data regarding duration of the infection episode from culture positivity to culture negativity. For each polymicrobial bloodstream infection (case), we selected three gestational age matched neonates with comparable birth weights with monomicrobial bloodstream infection (control) during the time period 2009 to 2012. The matching was performed by an investigator who was blinded to neonatal risk factors and outcomes. An infant who had both polymicrobial and monomicrobial infections was included in the polymicrobial infection category for evaluation of neonatal outcomes because of the possibility that even one exposure to polymicrobial infection may increase mortality or morbidity. If an infant had multiple episodes of polymicrobial infections, the first polymicrobial infection episode was analysed. Coagulase negative staphylococcal (CONS) infections, skin flora (e.g. Micrococcus spp., Gamma Streptococci) and organisms infrequent in neonates (e.g. Bacillus spp.) were considered real infections when grown from two clinical specimens and a single culture of the above organisms were deemed contaminant and not included in the analyses.\nPolymicrobial bloodstream infection was defined as: i) isolation of more than one organism from a single blood culture specimen and ii) isolation of more than one organism in different blood culture specimens during the same bloodstream infectious episode. We defined ‘bloodstream infection episodes’ as time-periods associated with positive blood cultures [1] and the infection episode was considered resolved when at least two subsequent blood cultures performed every 24 hrs were negative. Usually when an organism is isolated from a blood culture specimen, blood cultures are repeated every 24 hr till two blood culture specimens are negative for organisms. Our neonatal units used only aerobic blood cultures and it is not routine to perform anaerobic blood cultures for the evaluation of neonatal sepsis. Single specimen polymicrobial infections were defined as more than one organism isolated from the same blood culture specimen. We also collected data regarding duration of the infection episode from culture positivity to culture negativity. For each polymicrobial bloodstream infection (case), we selected three gestational age matched neonates with comparable birth weights with monomicrobial bloodstream infection (control) during the time period 2009 to 2012. The matching was performed by an investigator who was blinded to neonatal risk factors and outcomes. An infant who had both polymicrobial and monomicrobial infections was included in the polymicrobial infection category for evaluation of neonatal outcomes because of the possibility that even one exposure to polymicrobial infection may increase mortality or morbidity. If an infant had multiple episodes of polymicrobial infections, the first polymicrobial infection episode was analysed. Coagulase negative staphylococcal (CONS) infections, skin flora (e.g. Micrococcus spp., Gamma Streptococci) and organisms infrequent in neonates (e.g. Bacillus spp.) were considered real infections when grown from two clinical specimens and a single culture of the above organisms were deemed contaminant and not included in the analyses.\n Clinical data collection Neonatal clinical data including demographics, risk factors and outcomes were identified by cross referencing our institution’s neonatal clinical database (from Vermont Oxford Network (VON) database), for a 4 year period from January 1, 2009 to December 31, 2012. We collected the following clinical data for the identified cases and controls: demographic data (gestational age, birth weight, sex, age at the start of the infection episode and whether inborn or outborn), parenteral nutrition (PN) administration and its duration, presence of intravenous catheters at the time of the infectious episode (percutaneously inserted central catheters and broviac catheters but excluded umbilical catheters). We excluded umbilical venous catheters because our neonatal policy is to replace the umbilical venous catheters in the first few days of life with percutaneously inserted central catheters (PICC) and hence most umbilical lines lasted only a few days. Also, most of the bloodstream infections occurred at a time when umbilical catheters were no longer in place. We also collected data on important clinical outcomes (mortality, length of hospital stay, bronchopulmonary dysplasia, patent ductus arteriosus (PDA), necrotizing enterocolitis (NEC, ≥ stage 2 by Bell’s classification), intraventricular hemorrhage (IVH), periventricular leucomalacia (PVL) and retinopathy of prematurity (ROP), intermittent positive pressure ventilation (IPPV) at 36 wks corrected gestational age (GA), continuous positive airway pressure (CPAP) at 36 wks corrected GA, high frequency oscillatory ventilation (HFOV), inhaled nitric oxide (INO) therapy, extracorporeal membrane oxygenation (ECMO), surgeries, direct hyperbilirubinemia (direct bilirubin > 2 mg/dl or > 15% of total bilirubin) congenital heart disease and congenital malformations. All outcomes were defined as per VON database definitions.\nNeonatal clinical data including demographics, risk factors and outcomes were identified by cross referencing our institution’s neonatal clinical database (from Vermont Oxford Network (VON) database), for a 4 year period from January 1, 2009 to December 31, 2012. We collected the following clinical data for the identified cases and controls: demographic data (gestational age, birth weight, sex, age at the start of the infection episode and whether inborn or outborn), parenteral nutrition (PN) administration and its duration, presence of intravenous catheters at the time of the infectious episode (percutaneously inserted central catheters and broviac catheters but excluded umbilical catheters). We excluded umbilical venous catheters because our neonatal policy is to replace the umbilical venous catheters in the first few days of life with percutaneously inserted central catheters (PICC) and hence most umbilical lines lasted only a few days. Also, most of the bloodstream infections occurred at a time when umbilical catheters were no longer in place. We also collected data on important clinical outcomes (mortality, length of hospital stay, bronchopulmonary dysplasia, patent ductus arteriosus (PDA), necrotizing enterocolitis (NEC, ≥ stage 2 by Bell’s classification), intraventricular hemorrhage (IVH), periventricular leucomalacia (PVL) and retinopathy of prematurity (ROP), intermittent positive pressure ventilation (IPPV) at 36 wks corrected gestational age (GA), continuous positive airway pressure (CPAP) at 36 wks corrected GA, high frequency oscillatory ventilation (HFOV), inhaled nitric oxide (INO) therapy, extracorporeal membrane oxygenation (ECMO), surgeries, direct hyperbilirubinemia (direct bilirubin > 2 mg/dl or > 15% of total bilirubin) congenital heart disease and congenital malformations. All outcomes were defined as per VON database definitions.\n Statistical analyses Data were analyzed using STATA 11, Stata Corporation, Dallas, USA. Infants with polymicrobial bloodstream infections were compared with three gestational age and birth weight matched infants with monomicrobial bloodstream infections. Continuous data were analyzed for statistical significance by the Student’s t test and categorical data were analyzed by chi squared analyses. A p value of < 0.05 was considered significant. A logistic regression analysis was performed for binary outcomes and odds ratios with 95% confidence intervals were estimated. The outcome of mortality was analyzed adjusting for ‘any surgery’ in the logistic regression model. Polymicrobial infections were used as the outcome of the logistic regression analysis for risk factors and as a covariate when assessing for neonatal outcomes. A subgroup analysis of VLBW infants was performed for mortality and risk factors in the logistic regression model.\nData were analyzed using STATA 11, Stata Corporation, Dallas, USA. Infants with polymicrobial bloodstream infections were compared with three gestational age and birth weight matched infants with monomicrobial bloodstream infections. Continuous data were analyzed for statistical significance by the Student’s t test and categorical data were analyzed by chi squared analyses. A p value of < 0.05 was considered significant. A logistic regression analysis was performed for binary outcomes and odds ratios with 95% confidence intervals were estimated. The outcome of mortality was analyzed adjusting for ‘any surgery’ in the logistic regression model. Polymicrobial infections were used as the outcome of the logistic regression analysis for risk factors and as a covariate when assessing for neonatal outcomes. A subgroup analysis of VLBW infants was performed for mortality and risk factors in the logistic regression model."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"We identified 2007 bacteremia episodes during a period of 16 years in patients admitted to the NICUs at TCH; 280 episodes (14%) were polymicrobial (Figure 1). The percentage of annual polymicrobial bacteremia varied during the study period ranging from 6.3 to 18.6%. Polymicrobial bacteremia identified in single blood specimens constituted on an average of 56% (range 40 to 75%) during the 16 year study period.\nFrequency of polymicrobial bacteremia episodes. The frequencies of neonatal bacteremia episodes are plotted against the years from 1997 to 2012. The dark shaded portion of the bars indicate polymicrobial component of the infectious episodes, which ranged from 6.3 to 18.6% and on an average of about 14% during the 16-year study period. There were significant decrease in the number of infections and polymicrobial infections between the time epochs, 1998-2009 and 2010-2012 (p < 0.01).\nMicrobes isolated from monomicrobial (controls) and polymicrobial infections (cases) were similar but varied in frequency (Table 1). Microbes isolated at greater frequencies in polymicrobial infections were Candida spp. (>3%), Enterococcus faecalis (>2-fold) and Klebsiella pneumoniae (>2-fold). The most common combinations of polymicrobial organisms were CONS and Candida spp. (n = 6) and CONS and Enterococcus faecalis (n = 3). Gram positive organisms were isolated more frequently than gram negative organisms and the proportion of gram positive to gram negative organisms were similar in both monomicrobial (gram positive 51% and gram negative 42%) and polymicrobial infections (gram positive 48% and gram negative 40%).\nMicrobiology of monomicrobial and polymicrobial bloodstream infections\nIn monomicrobial bloodstream infections, 102 organisms were isolated from 102 infectious episodes. In polymicrobial bloodstream infections, 74 organisms were isolated from 34 infectious episodes. ‘Others’ include Streptococcus salivarius, Citrobacter freundii, Clostridium tertium for monomicrobial infections and for polymicrobial infections; they were Lactobacillus, Proteus mirabilis, Streptococcus sanguinus, Micrococcus spp., Acinetobacter baumanii complex and Gamma Streptococcus. All the organisms in the ‘Others’ group was cultured at least twice from 2 different samples. Candida species, Enterococcus species and Klebsiella species were isolated at higher frequencies in polymicrobial infections than monomicrobial infections. The most common combination of polymicrobial organisms were CONS and Candida (n = 6) and CONS and Enterococcus fecalis (n = 3).\nWe compared clinical data pertaining to demographics, risk factors and clinical outcomes between infants with polymicrobial bacteremia and gestational age and birthweight matched infants with monomicrobial bacteremia (data from January 2009 to December 2012) (Table 2). We obtained neonatal data for 34 infants with polymicrobial bacteremia and 102 matched infants with monomicrobial bacteremia during that period. The demographics were similar between infants with polymicrobial compared to infants with monomicrobial bacteremia. As expected, the gestational age (30.5 vs. 30.4 wk, p = 0.90) and birth weights (1592 vs.1535 g, p = 0.77) were adequately matched between controls and cases respectively. Male sex and age at infection did not differ significantly between cases and controls.\nInfant demographics, risk factors and outcomes in cases and controls\n95% CI- 95% confidence intervals, Bwt-Birth weight, GA-gestational age, PN- parenteral nutrition, NEC- necrotizing enterocolitis, BPD-bronchopulmonary dysplasia, IPPV- Intermittent positive pressure ventilation, CPAP- continuous positive airway pressure, HFOV- High frequency oscillatory ventilation, INO- inhaled nitric oxide, ECMO- extracorporeal membrane oxygenator, PDA-patent ductus arteriosus, ROP-retinopathy of prematurity, IVH-intraventricular hemorrhage, PVL- periventricular leucomalacia, severe IVH- IVH > grade 2, severe ROP- ROP > stage 2. *p values < 0.05.\nWe observed a significant difference between cases and controls in terms of surgery other than for NEC (p = 0.04) and any surgery (including NEC surgery) (p = 0.05, borderline significance). We observed an increase in the presence of a central venous catheter (97 vs. 91%, p =0.20) and incidence of direct hyperbilirubinemia (50 vs. 32%, p = 0.06) both of which did not reach statistical significance. We did not observe any significant differences in the proportion of infants receiving PN, days on PN, the incidence of NEC, surgery for NEC, congenital heart disease or congenital malformations between cases and controls.\nWe noted a significant difference in associated mortality between controls and cases (47 vs. 20%), odds ratio adjusted for ‘any surgery’, 4.3 [95% CI, 1.8 to 10.2] (p = 0.001). A significant increase in the duration of infection (culture positivity) was noted between cases and controls (2.91 vs. 2.07 days, p = 0.02). No significant differences were observed in length of hospital stay, BPD, IPPV at 36 corrected wks, CPAP at 36 corrected wks, HFOV, ECMO, PDA, severe IVH (IVH > grade 2), PVL, severe IVH or PVL, ROP or severe ROP (ROP > stage 2) (p > 0.05). An increase in INO therapy in cases were noted (41 vs. 25%, p = 0.08) but was not statistically significant.\nIn a subgroup analysis of VLBW infants, polymicrobial infections compared to monomicrobial infections, had a significantly higher mortality (OR 6.4 [95% CI 1.3 to 30.3], longer duration of infection (OR 1.4 [95% CI 1.04 to 1.8] and higher incidence of congenital malformations (OR 17.7 [95% CI, 2.1 to 148.7)."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"The main aim of the study was to investigate the frequency of polymicrobial infections in our tertiary neonatal intensive care unit, understand its impact on neonatal outcomes and promote research in the management of these infections. Polymicrobial bacteremia is common in neonates and comprises nearly 14% of all infectious episodes. The most common organisms in neonatal polymicrobial infections are CONS, S. aureus, Enterococcus species, E. coli, and Candida species. Surgical intervention is a significant risk factor for neonatal polymicrobial infections. We observed a more than 3-fold increase in mortality in infants with polymicrobial bacteremia and a significant increase in duration of the bacteremia. Research focused to prevent and to improve clinical outcomes in neonatal polymicrobial infections is urgently needed."},"other_sections_titles":{"kind":"list like","value":["Background","Identification of cases and controls","Definitions","Clinical data collection","Statistical analyses","Abbreviations","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Identification of cases and controls\",\n \"Definitions\",\n \"Clinical data collection\",\n \"Statistical analyses\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Polymicrobial infections increase mortality more than 2-fold in adults and children, increase length of hospital stay and healthcare costs [1,2]. Risk factors for polymicrobial infections in children and adults include the presence of a central venous catheter, administration of parenteral nutrition, gastrointestinal pathology, especially short gut syndrome, use of broad-spectrum antibiotics and immunosuppression [1,3,4].\nNeonatal polymicrobial infections are less well characterized compared to those in children or adults. Increased survival of extremely premature infants at the edge of viability, dependence on catheters and parenteral nutrition (PN), and antibiotic therapy may predispose to polymicrobial infections in neonates. The frequency of neonatal polymicrobial bloodstream infections reported in clinical studies varies from 4 to 24% of all bloodstream infections [5-10]. A standard definition for neonatal polymicrobial infections is lacking and the incidence varies from study to study partly due to variability in definition, neonatal population and practices [11]. Few studies have focused on polymicrobial infections in neonates, especially on risk factors and clinical outcomes in the western world [10-12]. In a neonatal review, the mortality due to polymicrobial infections was 3-fold greater than that of monomicrobial infections (70% vs. 23%) [10]. Organisms that are commonly implicated in neonatal polymicrobial bloodstream infections are coagulase-negative staphylococcus (CONS), Candida spp., Staphylococcus aureus and Enterococcus spp. [1,9,13-16]. A common risk factor appears to be multi-species biofilm infections originating from indwelling medical devices, notably indwelling vascular catheters or endotracheal tubes [1,17].\nPolymicrobial bloodstream infections may be defined as multiple organisms isolated during an infectious episode including those from a single blood specimen [1] or more restrictively as isolation of more than one organism from a single blood specimen only [11,12,17]. Different definitions may partly explain the varying incidence of polymicrobial bloodstream infections reported. Bizzarro et al. (Yale, 1989-2006) and Faix et al. (Ann Arbor, 1971-1986) have reported the only two studies on neonatal polymicrobial infections from North America, [10,11]. Changing epidemiology of neonatal infections dictates a need to update and understand the epidemiology of polymicrobial infections in the hospitalized infant [11]. Lack of data on polymicrobial infections from our neonatal unit and concern of significant increase in mortality, morbidity and healthcare costs due to polymicrobial infections prompted us to investigate its frequency, risk factors and clinical outcomes at Texas Children’s Hospital (TCH).","We identified blood culture positive infants from the clinical microbiology database, who were admitted to the neonatal intensive care units (NICUs) III and II at TCH from January 1, 1997 to December 31, 2012. Infants more than 28 days of age in the NICU at the time of the infection and those admitted from home to the NICU with positive blood cultures were also included. The data in the clinical microbiology database is entered by the microbiologist and all positive and negative cultures including those regarded as contaminants are recorded. The tertiary NICU at TCH has approximately 1500 admissions per year including inborn and outborn transferred neonates (ranged from 1338 to 1547 during the years 2009 to 2012). Very low birth weight (VLBW, birth weight < 1500 g) infant admissions ranged from 222 to 291 infants during the years 2009 to 2012.","Polymicrobial bloodstream infection was defined as: i) isolation of more than one organism from a single blood culture specimen and ii) isolation of more than one organism in different blood culture specimens during the same bloodstream infectious episode. We defined ‘bloodstream infection episodes’ as time-periods associated with positive blood cultures [1] and the infection episode was considered resolved when at least two subsequent blood cultures performed every 24 hrs were negative. Usually when an organism is isolated from a blood culture specimen, blood cultures are repeated every 24 hr till two blood culture specimens are negative for organisms. Our neonatal units used only aerobic blood cultures and it is not routine to perform anaerobic blood cultures for the evaluation of neonatal sepsis. Single specimen polymicrobial infections were defined as more than one organism isolated from the same blood culture specimen. We also collected data regarding duration of the infection episode from culture positivity to culture negativity. For each polymicrobial bloodstream infection (case), we selected three gestational age matched neonates with comparable birth weights with monomicrobial bloodstream infection (control) during the time period 2009 to 2012. The matching was performed by an investigator who was blinded to neonatal risk factors and outcomes. An infant who had both polymicrobial and monomicrobial infections was included in the polymicrobial infection category for evaluation of neonatal outcomes because of the possibility that even one exposure to polymicrobial infection may increase mortality or morbidity. If an infant had multiple episodes of polymicrobial infections, the first polymicrobial infection episode was analysed. Coagulase negative staphylococcal (CONS) infections, skin flora (e.g. Micrococcus spp., Gamma Streptococci) and organisms infrequent in neonates (e.g. Bacillus spp.) were considered real infections when grown from two clinical specimens and a single culture of the above organisms were deemed contaminant and not included in the analyses.","Neonatal clinical data including demographics, risk factors and outcomes were identified by cross referencing our institution’s neonatal clinical database (from Vermont Oxford Network (VON) database), for a 4 year period from January 1, 2009 to December 31, 2012. We collected the following clinical data for the identified cases and controls: demographic data (gestational age, birth weight, sex, age at the start of the infection episode and whether inborn or outborn), parenteral nutrition (PN) administration and its duration, presence of intravenous catheters at the time of the infectious episode (percutaneously inserted central catheters and broviac catheters but excluded umbilical catheters). We excluded umbilical venous catheters because our neonatal policy is to replace the umbilical venous catheters in the first few days of life with percutaneously inserted central catheters (PICC) and hence most umbilical lines lasted only a few days. Also, most of the bloodstream infections occurred at a time when umbilical catheters were no longer in place. We also collected data on important clinical outcomes (mortality, length of hospital stay, bronchopulmonary dysplasia, patent ductus arteriosus (PDA), necrotizing enterocolitis (NEC, ≥ stage 2 by Bell’s classification), intraventricular hemorrhage (IVH), periventricular leucomalacia (PVL) and retinopathy of prematurity (ROP), intermittent positive pressure ventilation (IPPV) at 36 wks corrected gestational age (GA), continuous positive airway pressure (CPAP) at 36 wks corrected GA, high frequency oscillatory ventilation (HFOV), inhaled nitric oxide (INO) therapy, extracorporeal membrane oxygenation (ECMO), surgeries, direct hyperbilirubinemia (direct bilirubin > 2 mg/dl or > 15% of total bilirubin) congenital heart disease and congenital malformations. All outcomes were defined as per VON database definitions.","Data were analyzed using STATA 11, Stata Corporation, Dallas, USA. Infants with polymicrobial bloodstream infections were compared with three gestational age and birth weight matched infants with monomicrobial bloodstream infections. Continuous data were analyzed for statistical significance by the Student’s t test and categorical data were analyzed by chi squared analyses. A p value of < 0.05 was considered significant. A logistic regression analysis was performed for binary outcomes and odds ratios with 95% confidence intervals were estimated. The outcome of mortality was analyzed adjusting for ‘any surgery’ in the logistic regression model. Polymicrobial infections were used as the outcome of the logistic regression analysis for risk factors and as a covariate when assessing for neonatal outcomes. A subgroup analysis of VLBW infants was performed for mortality and risk factors in the logistic regression model.","TCH: Texas Children’s Hospital; VON: Vermont Oxford Network; CONS: Coagulase negative Staphylococci; ELBW: Extremely low birth weight; VLBW: Very low birth weight; NICU: Neonatal Intensive Care Unit; PDA: Patent ductus arteriosus; BPD: Bronchopulmonary dysplasia; PVL: Periventricular leucomalacia; NEC: Necrotizing enterocolitis; TPN: Total parenteral nutrition; ROP: Retinopathy of prematurity; IVH: Intraventricular hemorrhage; IPPV: Intermittent positive pressure ventilation; CPAP: Continuous positive airway pressure; HFOV: High frequency oscillatory ventilation; INO: Inhaled nitric oxide.","None of the authors have any financial interests to disclose or competing interest.","We state that all the authors satisfy the requirements of author as laid down in ‘Instructions to the Authors’. MP conceived the project, participated in the design, data acquisition, analysis and interpretation, drafted the initial manuscript and approved the final manuscript as submitted. DZ participated in the acquisition of microbiology data and tabulating the data. YJ for clinical data acquisition and revision of the manuscript. PR participated in microbiology data acquisition, analysis and revision of the manuscript. JV for critical intellectual input and revision of the manuscript. All authors read and approved the final manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2334/14/390/prepub\n"],"string":"[\n \"Polymicrobial infections increase mortality more than 2-fold in adults and children, increase length of hospital stay and healthcare costs [1,2]. Risk factors for polymicrobial infections in children and adults include the presence of a central venous catheter, administration of parenteral nutrition, gastrointestinal pathology, especially short gut syndrome, use of broad-spectrum antibiotics and immunosuppression [1,3,4].\\nNeonatal polymicrobial infections are less well characterized compared to those in children or adults. Increased survival of extremely premature infants at the edge of viability, dependence on catheters and parenteral nutrition (PN), and antibiotic therapy may predispose to polymicrobial infections in neonates. The frequency of neonatal polymicrobial bloodstream infections reported in clinical studies varies from 4 to 24% of all bloodstream infections [5-10]. A standard definition for neonatal polymicrobial infections is lacking and the incidence varies from study to study partly due to variability in definition, neonatal population and practices [11]. Few studies have focused on polymicrobial infections in neonates, especially on risk factors and clinical outcomes in the western world [10-12]. In a neonatal review, the mortality due to polymicrobial infections was 3-fold greater than that of monomicrobial infections (70% vs. 23%) [10]. Organisms that are commonly implicated in neonatal polymicrobial bloodstream infections are coagulase-negative staphylococcus (CONS), Candida spp., Staphylococcus aureus and Enterococcus spp. [1,9,13-16]. A common risk factor appears to be multi-species biofilm infections originating from indwelling medical devices, notably indwelling vascular catheters or endotracheal tubes [1,17].\\nPolymicrobial bloodstream infections may be defined as multiple organisms isolated during an infectious episode including those from a single blood specimen [1] or more restrictively as isolation of more than one organism from a single blood specimen only [11,12,17]. Different definitions may partly explain the varying incidence of polymicrobial bloodstream infections reported. Bizzarro et al. (Yale, 1989-2006) and Faix et al. (Ann Arbor, 1971-1986) have reported the only two studies on neonatal polymicrobial infections from North America, [10,11]. Changing epidemiology of neonatal infections dictates a need to update and understand the epidemiology of polymicrobial infections in the hospitalized infant [11]. Lack of data on polymicrobial infections from our neonatal unit and concern of significant increase in mortality, morbidity and healthcare costs due to polymicrobial infections prompted us to investigate its frequency, risk factors and clinical outcomes at Texas Children’s Hospital (TCH).\",\n \"We identified blood culture positive infants from the clinical microbiology database, who were admitted to the neonatal intensive care units (NICUs) III and II at TCH from January 1, 1997 to December 31, 2012. Infants more than 28 days of age in the NICU at the time of the infection and those admitted from home to the NICU with positive blood cultures were also included. The data in the clinical microbiology database is entered by the microbiologist and all positive and negative cultures including those regarded as contaminants are recorded. The tertiary NICU at TCH has approximately 1500 admissions per year including inborn and outborn transferred neonates (ranged from 1338 to 1547 during the years 2009 to 2012). Very low birth weight (VLBW, birth weight < 1500 g) infant admissions ranged from 222 to 291 infants during the years 2009 to 2012.\",\n \"Polymicrobial bloodstream infection was defined as: i) isolation of more than one organism from a single blood culture specimen and ii) isolation of more than one organism in different blood culture specimens during the same bloodstream infectious episode. We defined ‘bloodstream infection episodes’ as time-periods associated with positive blood cultures [1] and the infection episode was considered resolved when at least two subsequent blood cultures performed every 24 hrs were negative. Usually when an organism is isolated from a blood culture specimen, blood cultures are repeated every 24 hr till two blood culture specimens are negative for organisms. Our neonatal units used only aerobic blood cultures and it is not routine to perform anaerobic blood cultures for the evaluation of neonatal sepsis. Single specimen polymicrobial infections were defined as more than one organism isolated from the same blood culture specimen. We also collected data regarding duration of the infection episode from culture positivity to culture negativity. For each polymicrobial bloodstream infection (case), we selected three gestational age matched neonates with comparable birth weights with monomicrobial bloodstream infection (control) during the time period 2009 to 2012. The matching was performed by an investigator who was blinded to neonatal risk factors and outcomes. An infant who had both polymicrobial and monomicrobial infections was included in the polymicrobial infection category for evaluation of neonatal outcomes because of the possibility that even one exposure to polymicrobial infection may increase mortality or morbidity. If an infant had multiple episodes of polymicrobial infections, the first polymicrobial infection episode was analysed. Coagulase negative staphylococcal (CONS) infections, skin flora (e.g. Micrococcus spp., Gamma Streptococci) and organisms infrequent in neonates (e.g. Bacillus spp.) were considered real infections when grown from two clinical specimens and a single culture of the above organisms were deemed contaminant and not included in the analyses.\",\n \"Neonatal clinical data including demographics, risk factors and outcomes were identified by cross referencing our institution’s neonatal clinical database (from Vermont Oxford Network (VON) database), for a 4 year period from January 1, 2009 to December 31, 2012. We collected the following clinical data for the identified cases and controls: demographic data (gestational age, birth weight, sex, age at the start of the infection episode and whether inborn or outborn), parenteral nutrition (PN) administration and its duration, presence of intravenous catheters at the time of the infectious episode (percutaneously inserted central catheters and broviac catheters but excluded umbilical catheters). We excluded umbilical venous catheters because our neonatal policy is to replace the umbilical venous catheters in the first few days of life with percutaneously inserted central catheters (PICC) and hence most umbilical lines lasted only a few days. Also, most of the bloodstream infections occurred at a time when umbilical catheters were no longer in place. We also collected data on important clinical outcomes (mortality, length of hospital stay, bronchopulmonary dysplasia, patent ductus arteriosus (PDA), necrotizing enterocolitis (NEC, ≥ stage 2 by Bell’s classification), intraventricular hemorrhage (IVH), periventricular leucomalacia (PVL) and retinopathy of prematurity (ROP), intermittent positive pressure ventilation (IPPV) at 36 wks corrected gestational age (GA), continuous positive airway pressure (CPAP) at 36 wks corrected GA, high frequency oscillatory ventilation (HFOV), inhaled nitric oxide (INO) therapy, extracorporeal membrane oxygenation (ECMO), surgeries, direct hyperbilirubinemia (direct bilirubin > 2 mg/dl or > 15% of total bilirubin) congenital heart disease and congenital malformations. All outcomes were defined as per VON database definitions.\",\n \"Data were analyzed using STATA 11, Stata Corporation, Dallas, USA. Infants with polymicrobial bloodstream infections were compared with three gestational age and birth weight matched infants with monomicrobial bloodstream infections. Continuous data were analyzed for statistical significance by the Student’s t test and categorical data were analyzed by chi squared analyses. A p value of < 0.05 was considered significant. A logistic regression analysis was performed for binary outcomes and odds ratios with 95% confidence intervals were estimated. The outcome of mortality was analyzed adjusting for ‘any surgery’ in the logistic regression model. Polymicrobial infections were used as the outcome of the logistic regression analysis for risk factors and as a covariate when assessing for neonatal outcomes. A subgroup analysis of VLBW infants was performed for mortality and risk factors in the logistic regression model.\",\n \"TCH: Texas Children’s Hospital; VON: Vermont Oxford Network; CONS: Coagulase negative Staphylococci; ELBW: Extremely low birth weight; VLBW: Very low birth weight; NICU: Neonatal Intensive Care Unit; PDA: Patent ductus arteriosus; BPD: Bronchopulmonary dysplasia; PVL: Periventricular leucomalacia; NEC: Necrotizing enterocolitis; TPN: Total parenteral nutrition; ROP: Retinopathy of prematurity; IVH: Intraventricular hemorrhage; IPPV: Intermittent positive pressure ventilation; CPAP: Continuous positive airway pressure; HFOV: High frequency oscillatory ventilation; INO: Inhaled nitric oxide.\",\n \"None of the authors have any financial interests to disclose or competing interest.\",\n \"We state that all the authors satisfy the requirements of author as laid down in ‘Instructions to the Authors’. MP conceived the project, participated in the design, data acquisition, analysis and interpretation, drafted the initial manuscript and approved the final manuscript as submitted. DZ participated in the acquisition of microbiology data and tabulating the data. YJ for clinical data acquisition and revision of the manuscript. PR participated in microbiology data acquisition, analysis and revision of the manuscript. JV for critical intellectual input and revision of the manuscript. All authors read and approved the final manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2334/14/390/prepub\\n\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Identification of cases and controls","Definitions","Clinical data collection","Statistical analyses","Results","Discussion","Conclusions","Abbreviations","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Methods\",\n \"Identification of cases and controls\",\n \"Definitions\",\n \"Clinical data collection\",\n \"Statistical analyses\",\n \"Results\",\n \"Discussion\",\n \"Conclusions\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Polymicrobial infections increase mortality more than 2-fold in adults and children, increase length of hospital stay and healthcare costs [1,2]. Risk factors for polymicrobial infections in children and adults include the presence of a central venous catheter, administration of parenteral nutrition, gastrointestinal pathology, especially short gut syndrome, use of broad-spectrum antibiotics and immunosuppression [1,3,4].\nNeonatal polymicrobial infections are less well characterized compared to those in children or adults. Increased survival of extremely premature infants at the edge of viability, dependence on catheters and parenteral nutrition (PN), and antibiotic therapy may predispose to polymicrobial infections in neonates. The frequency of neonatal polymicrobial bloodstream infections reported in clinical studies varies from 4 to 24% of all bloodstream infections [5-10]. A standard definition for neonatal polymicrobial infections is lacking and the incidence varies from study to study partly due to variability in definition, neonatal population and practices [11]. Few studies have focused on polymicrobial infections in neonates, especially on risk factors and clinical outcomes in the western world [10-12]. In a neonatal review, the mortality due to polymicrobial infections was 3-fold greater than that of monomicrobial infections (70% vs. 23%) [10]. Organisms that are commonly implicated in neonatal polymicrobial bloodstream infections are coagulase-negative staphylococcus (CONS), Candida spp., Staphylococcus aureus and Enterococcus spp. [1,9,13-16]. A common risk factor appears to be multi-species biofilm infections originating from indwelling medical devices, notably indwelling vascular catheters or endotracheal tubes [1,17].\nPolymicrobial bloodstream infections may be defined as multiple organisms isolated during an infectious episode including those from a single blood specimen [1] or more restrictively as isolation of more than one organism from a single blood specimen only [11,12,17]. Different definitions may partly explain the varying incidence of polymicrobial bloodstream infections reported. Bizzarro et al. (Yale, 1989-2006) and Faix et al. (Ann Arbor, 1971-1986) have reported the only two studies on neonatal polymicrobial infections from North America, [10,11]. Changing epidemiology of neonatal infections dictates a need to update and understand the epidemiology of polymicrobial infections in the hospitalized infant [11]. Lack of data on polymicrobial infections from our neonatal unit and concern of significant increase in mortality, morbidity and healthcare costs due to polymicrobial infections prompted us to investigate its frequency, risk factors and clinical outcomes at Texas Children’s Hospital (TCH).","We hypothesized that polymicrobial infections comprise > 5% of bloodstream infections in infants residing in the neonatal intensive care units, have identifiable risk factors and are associated with higher mortality and morbidity than monomicrobial infections. We tested our hypothesis by performing a single center, retrospective, matched case-control study during a 16 year study period. Our study protocol was approved by the Institutional Review Board at Baylor College of Medicine, Houston. We followed the STROBE guidelines in reporting this study [18].\n Identification of cases and controls We identified blood culture positive infants from the clinical microbiology database, who were admitted to the neonatal intensive care units (NICUs) III and II at TCH from January 1, 1997 to December 31, 2012. Infants more than 28 days of age in the NICU at the time of the infection and those admitted from home to the NICU with positive blood cultures were also included. The data in the clinical microbiology database is entered by the microbiologist and all positive and negative cultures including those regarded as contaminants are recorded. The tertiary NICU at TCH has approximately 1500 admissions per year including inborn and outborn transferred neonates (ranged from 1338 to 1547 during the years 2009 to 2012). Very low birth weight (VLBW, birth weight < 1500 g) infant admissions ranged from 222 to 291 infants during the years 2009 to 2012.\nWe identified blood culture positive infants from the clinical microbiology database, who were admitted to the neonatal intensive care units (NICUs) III and II at TCH from January 1, 1997 to December 31, 2012. Infants more than 28 days of age in the NICU at the time of the infection and those admitted from home to the NICU with positive blood cultures were also included. The data in the clinical microbiology database is entered by the microbiologist and all positive and negative cultures including those regarded as contaminants are recorded. The tertiary NICU at TCH has approximately 1500 admissions per year including inborn and outborn transferred neonates (ranged from 1338 to 1547 during the years 2009 to 2012). Very low birth weight (VLBW, birth weight < 1500 g) infant admissions ranged from 222 to 291 infants during the years 2009 to 2012.\n Definitions Polymicrobial bloodstream infection was defined as: i) isolation of more than one organism from a single blood culture specimen and ii) isolation of more than one organism in different blood culture specimens during the same bloodstream infectious episode. We defined ‘bloodstream infection episodes’ as time-periods associated with positive blood cultures [1] and the infection episode was considered resolved when at least two subsequent blood cultures performed every 24 hrs were negative. Usually when an organism is isolated from a blood culture specimen, blood cultures are repeated every 24 hr till two blood culture specimens are negative for organisms. Our neonatal units used only aerobic blood cultures and it is not routine to perform anaerobic blood cultures for the evaluation of neonatal sepsis. Single specimen polymicrobial infections were defined as more than one organism isolated from the same blood culture specimen. We also collected data regarding duration of the infection episode from culture positivity to culture negativity. For each polymicrobial bloodstream infection (case), we selected three gestational age matched neonates with comparable birth weights with monomicrobial bloodstream infection (control) during the time period 2009 to 2012. The matching was performed by an investigator who was blinded to neonatal risk factors and outcomes. An infant who had both polymicrobial and monomicrobial infections was included in the polymicrobial infection category for evaluation of neonatal outcomes because of the possibility that even one exposure to polymicrobial infection may increase mortality or morbidity. If an infant had multiple episodes of polymicrobial infections, the first polymicrobial infection episode was analysed. Coagulase negative staphylococcal (CONS) infections, skin flora (e.g. Micrococcus spp., Gamma Streptococci) and organisms infrequent in neonates (e.g. Bacillus spp.) were considered real infections when grown from two clinical specimens and a single culture of the above organisms were deemed contaminant and not included in the analyses.\nPolymicrobial bloodstream infection was defined as: i) isolation of more than one organism from a single blood culture specimen and ii) isolation of more than one organism in different blood culture specimens during the same bloodstream infectious episode. We defined ‘bloodstream infection episodes’ as time-periods associated with positive blood cultures [1] and the infection episode was considered resolved when at least two subsequent blood cultures performed every 24 hrs were negative. Usually when an organism is isolated from a blood culture specimen, blood cultures are repeated every 24 hr till two blood culture specimens are negative for organisms. Our neonatal units used only aerobic blood cultures and it is not routine to perform anaerobic blood cultures for the evaluation of neonatal sepsis. Single specimen polymicrobial infections were defined as more than one organism isolated from the same blood culture specimen. We also collected data regarding duration of the infection episode from culture positivity to culture negativity. For each polymicrobial bloodstream infection (case), we selected three gestational age matched neonates with comparable birth weights with monomicrobial bloodstream infection (control) during the time period 2009 to 2012. The matching was performed by an investigator who was blinded to neonatal risk factors and outcomes. An infant who had both polymicrobial and monomicrobial infections was included in the polymicrobial infection category for evaluation of neonatal outcomes because of the possibility that even one exposure to polymicrobial infection may increase mortality or morbidity. If an infant had multiple episodes of polymicrobial infections, the first polymicrobial infection episode was analysed. Coagulase negative staphylococcal (CONS) infections, skin flora (e.g. Micrococcus spp., Gamma Streptococci) and organisms infrequent in neonates (e.g. Bacillus spp.) were considered real infections when grown from two clinical specimens and a single culture of the above organisms were deemed contaminant and not included in the analyses.\n Clinical data collection Neonatal clinical data including demographics, risk factors and outcomes were identified by cross referencing our institution’s neonatal clinical database (from Vermont Oxford Network (VON) database), for a 4 year period from January 1, 2009 to December 31, 2012. We collected the following clinical data for the identified cases and controls: demographic data (gestational age, birth weight, sex, age at the start of the infection episode and whether inborn or outborn), parenteral nutrition (PN) administration and its duration, presence of intravenous catheters at the time of the infectious episode (percutaneously inserted central catheters and broviac catheters but excluded umbilical catheters). We excluded umbilical venous catheters because our neonatal policy is to replace the umbilical venous catheters in the first few days of life with percutaneously inserted central catheters (PICC) and hence most umbilical lines lasted only a few days. Also, most of the bloodstream infections occurred at a time when umbilical catheters were no longer in place. We also collected data on important clinical outcomes (mortality, length of hospital stay, bronchopulmonary dysplasia, patent ductus arteriosus (PDA), necrotizing enterocolitis (NEC, ≥ stage 2 by Bell’s classification), intraventricular hemorrhage (IVH), periventricular leucomalacia (PVL) and retinopathy of prematurity (ROP), intermittent positive pressure ventilation (IPPV) at 36 wks corrected gestational age (GA), continuous positive airway pressure (CPAP) at 36 wks corrected GA, high frequency oscillatory ventilation (HFOV), inhaled nitric oxide (INO) therapy, extracorporeal membrane oxygenation (ECMO), surgeries, direct hyperbilirubinemia (direct bilirubin > 2 mg/dl or > 15% of total bilirubin) congenital heart disease and congenital malformations. All outcomes were defined as per VON database definitions.\nNeonatal clinical data including demographics, risk factors and outcomes were identified by cross referencing our institution’s neonatal clinical database (from Vermont Oxford Network (VON) database), for a 4 year period from January 1, 2009 to December 31, 2012. We collected the following clinical data for the identified cases and controls: demographic data (gestational age, birth weight, sex, age at the start of the infection episode and whether inborn or outborn), parenteral nutrition (PN) administration and its duration, presence of intravenous catheters at the time of the infectious episode (percutaneously inserted central catheters and broviac catheters but excluded umbilical catheters). We excluded umbilical venous catheters because our neonatal policy is to replace the umbilical venous catheters in the first few days of life with percutaneously inserted central catheters (PICC) and hence most umbilical lines lasted only a few days. Also, most of the bloodstream infections occurred at a time when umbilical catheters were no longer in place. We also collected data on important clinical outcomes (mortality, length of hospital stay, bronchopulmonary dysplasia, patent ductus arteriosus (PDA), necrotizing enterocolitis (NEC, ≥ stage 2 by Bell’s classification), intraventricular hemorrhage (IVH), periventricular leucomalacia (PVL) and retinopathy of prematurity (ROP), intermittent positive pressure ventilation (IPPV) at 36 wks corrected gestational age (GA), continuous positive airway pressure (CPAP) at 36 wks corrected GA, high frequency oscillatory ventilation (HFOV), inhaled nitric oxide (INO) therapy, extracorporeal membrane oxygenation (ECMO), surgeries, direct hyperbilirubinemia (direct bilirubin > 2 mg/dl or > 15% of total bilirubin) congenital heart disease and congenital malformations. All outcomes were defined as per VON database definitions.\n Statistical analyses Data were analyzed using STATA 11, Stata Corporation, Dallas, USA. Infants with polymicrobial bloodstream infections were compared with three gestational age and birth weight matched infants with monomicrobial bloodstream infections. Continuous data were analyzed for statistical significance by the Student’s t test and categorical data were analyzed by chi squared analyses. A p value of < 0.05 was considered significant. A logistic regression analysis was performed for binary outcomes and odds ratios with 95% confidence intervals were estimated. The outcome of mortality was analyzed adjusting for ‘any surgery’ in the logistic regression model. Polymicrobial infections were used as the outcome of the logistic regression analysis for risk factors and as a covariate when assessing for neonatal outcomes. A subgroup analysis of VLBW infants was performed for mortality and risk factors in the logistic regression model.\nData were analyzed using STATA 11, Stata Corporation, Dallas, USA. Infants with polymicrobial bloodstream infections were compared with three gestational age and birth weight matched infants with monomicrobial bloodstream infections. Continuous data were analyzed for statistical significance by the Student’s t test and categorical data were analyzed by chi squared analyses. A p value of < 0.05 was considered significant. A logistic regression analysis was performed for binary outcomes and odds ratios with 95% confidence intervals were estimated. The outcome of mortality was analyzed adjusting for ‘any surgery’ in the logistic regression model. Polymicrobial infections were used as the outcome of the logistic regression analysis for risk factors and as a covariate when assessing for neonatal outcomes. A subgroup analysis of VLBW infants was performed for mortality and risk factors in the logistic regression model.","We identified blood culture positive infants from the clinical microbiology database, who were admitted to the neonatal intensive care units (NICUs) III and II at TCH from January 1, 1997 to December 31, 2012. Infants more than 28 days of age in the NICU at the time of the infection and those admitted from home to the NICU with positive blood cultures were also included. The data in the clinical microbiology database is entered by the microbiologist and all positive and negative cultures including those regarded as contaminants are recorded. The tertiary NICU at TCH has approximately 1500 admissions per year including inborn and outborn transferred neonates (ranged from 1338 to 1547 during the years 2009 to 2012). Very low birth weight (VLBW, birth weight < 1500 g) infant admissions ranged from 222 to 291 infants during the years 2009 to 2012.","Polymicrobial bloodstream infection was defined as: i) isolation of more than one organism from a single blood culture specimen and ii) isolation of more than one organism in different blood culture specimens during the same bloodstream infectious episode. We defined ‘bloodstream infection episodes’ as time-periods associated with positive blood cultures [1] and the infection episode was considered resolved when at least two subsequent blood cultures performed every 24 hrs were negative. Usually when an organism is isolated from a blood culture specimen, blood cultures are repeated every 24 hr till two blood culture specimens are negative for organisms. Our neonatal units used only aerobic blood cultures and it is not routine to perform anaerobic blood cultures for the evaluation of neonatal sepsis. Single specimen polymicrobial infections were defined as more than one organism isolated from the same blood culture specimen. We also collected data regarding duration of the infection episode from culture positivity to culture negativity. For each polymicrobial bloodstream infection (case), we selected three gestational age matched neonates with comparable birth weights with monomicrobial bloodstream infection (control) during the time period 2009 to 2012. The matching was performed by an investigator who was blinded to neonatal risk factors and outcomes. An infant who had both polymicrobial and monomicrobial infections was included in the polymicrobial infection category for evaluation of neonatal outcomes because of the possibility that even one exposure to polymicrobial infection may increase mortality or morbidity. If an infant had multiple episodes of polymicrobial infections, the first polymicrobial infection episode was analysed. Coagulase negative staphylococcal (CONS) infections, skin flora (e.g. Micrococcus spp., Gamma Streptococci) and organisms infrequent in neonates (e.g. Bacillus spp.) were considered real infections when grown from two clinical specimens and a single culture of the above organisms were deemed contaminant and not included in the analyses.","Neonatal clinical data including demographics, risk factors and outcomes were identified by cross referencing our institution’s neonatal clinical database (from Vermont Oxford Network (VON) database), for a 4 year period from January 1, 2009 to December 31, 2012. We collected the following clinical data for the identified cases and controls: demographic data (gestational age, birth weight, sex, age at the start of the infection episode and whether inborn or outborn), parenteral nutrition (PN) administration and its duration, presence of intravenous catheters at the time of the infectious episode (percutaneously inserted central catheters and broviac catheters but excluded umbilical catheters). We excluded umbilical venous catheters because our neonatal policy is to replace the umbilical venous catheters in the first few days of life with percutaneously inserted central catheters (PICC) and hence most umbilical lines lasted only a few days. Also, most of the bloodstream infections occurred at a time when umbilical catheters were no longer in place. We also collected data on important clinical outcomes (mortality, length of hospital stay, bronchopulmonary dysplasia, patent ductus arteriosus (PDA), necrotizing enterocolitis (NEC, ≥ stage 2 by Bell’s classification), intraventricular hemorrhage (IVH), periventricular leucomalacia (PVL) and retinopathy of prematurity (ROP), intermittent positive pressure ventilation (IPPV) at 36 wks corrected gestational age (GA), continuous positive airway pressure (CPAP) at 36 wks corrected GA, high frequency oscillatory ventilation (HFOV), inhaled nitric oxide (INO) therapy, extracorporeal membrane oxygenation (ECMO), surgeries, direct hyperbilirubinemia (direct bilirubin > 2 mg/dl or > 15% of total bilirubin) congenital heart disease and congenital malformations. All outcomes were defined as per VON database definitions.","Data were analyzed using STATA 11, Stata Corporation, Dallas, USA. Infants with polymicrobial bloodstream infections were compared with three gestational age and birth weight matched infants with monomicrobial bloodstream infections. Continuous data were analyzed for statistical significance by the Student’s t test and categorical data were analyzed by chi squared analyses. A p value of < 0.05 was considered significant. A logistic regression analysis was performed for binary outcomes and odds ratios with 95% confidence intervals were estimated. The outcome of mortality was analyzed adjusting for ‘any surgery’ in the logistic regression model. Polymicrobial infections were used as the outcome of the logistic regression analysis for risk factors and as a covariate when assessing for neonatal outcomes. A subgroup analysis of VLBW infants was performed for mortality and risk factors in the logistic regression model.","We identified 2007 bacteremia episodes during a period of 16 years in patients admitted to the NICUs at TCH; 280 episodes (14%) were polymicrobial (Figure 1). The percentage of annual polymicrobial bacteremia varied during the study period ranging from 6.3 to 18.6%. Polymicrobial bacteremia identified in single blood specimens constituted on an average of 56% (range 40 to 75%) during the 16 year study period.\nFrequency of polymicrobial bacteremia episodes. The frequencies of neonatal bacteremia episodes are plotted against the years from 1997 to 2012. The dark shaded portion of the bars indicate polymicrobial component of the infectious episodes, which ranged from 6.3 to 18.6% and on an average of about 14% during the 16-year study period. There were significant decrease in the number of infections and polymicrobial infections between the time epochs, 1998-2009 and 2010-2012 (p < 0.01).\nMicrobes isolated from monomicrobial (controls) and polymicrobial infections (cases) were similar but varied in frequency (Table 1). Microbes isolated at greater frequencies in polymicrobial infections were Candida spp. (>3%), Enterococcus faecalis (>2-fold) and Klebsiella pneumoniae (>2-fold). The most common combinations of polymicrobial organisms were CONS and Candida spp. (n = 6) and CONS and Enterococcus faecalis (n = 3). Gram positive organisms were isolated more frequently than gram negative organisms and the proportion of gram positive to gram negative organisms were similar in both monomicrobial (gram positive 51% and gram negative 42%) and polymicrobial infections (gram positive 48% and gram negative 40%).\nMicrobiology of monomicrobial and polymicrobial bloodstream infections\nIn monomicrobial bloodstream infections, 102 organisms were isolated from 102 infectious episodes. In polymicrobial bloodstream infections, 74 organisms were isolated from 34 infectious episodes. ‘Others’ include Streptococcus salivarius, Citrobacter freundii, Clostridium tertium for monomicrobial infections and for polymicrobial infections; they were Lactobacillus, Proteus mirabilis, Streptococcus sanguinus, Micrococcus spp., Acinetobacter baumanii complex and Gamma Streptococcus. All the organisms in the ‘Others’ group was cultured at least twice from 2 different samples. Candida species, Enterococcus species and Klebsiella species were isolated at higher frequencies in polymicrobial infections than monomicrobial infections. The most common combination of polymicrobial organisms were CONS and Candida (n = 6) and CONS and Enterococcus fecalis (n = 3).\nWe compared clinical data pertaining to demographics, risk factors and clinical outcomes between infants with polymicrobial bacteremia and gestational age and birthweight matched infants with monomicrobial bacteremia (data from January 2009 to December 2012) (Table 2). We obtained neonatal data for 34 infants with polymicrobial bacteremia and 102 matched infants with monomicrobial bacteremia during that period. The demographics were similar between infants with polymicrobial compared to infants with monomicrobial bacteremia. As expected, the gestational age (30.5 vs. 30.4 wk, p = 0.90) and birth weights (1592 vs.1535 g, p = 0.77) were adequately matched between controls and cases respectively. Male sex and age at infection did not differ significantly between cases and controls.\nInfant demographics, risk factors and outcomes in cases and controls\n95% CI- 95% confidence intervals, Bwt-Birth weight, GA-gestational age, PN- parenteral nutrition, NEC- necrotizing enterocolitis, BPD-bronchopulmonary dysplasia, IPPV- Intermittent positive pressure ventilation, CPAP- continuous positive airway pressure, HFOV- High frequency oscillatory ventilation, INO- inhaled nitric oxide, ECMO- extracorporeal membrane oxygenator, PDA-patent ductus arteriosus, ROP-retinopathy of prematurity, IVH-intraventricular hemorrhage, PVL- periventricular leucomalacia, severe IVH- IVH > grade 2, severe ROP- ROP > stage 2. *p values < 0.05.\nWe observed a significant difference between cases and controls in terms of surgery other than for NEC (p = 0.04) and any surgery (including NEC surgery) (p = 0.05, borderline significance). We observed an increase in the presence of a central venous catheter (97 vs. 91%, p =0.20) and incidence of direct hyperbilirubinemia (50 vs. 32%, p = 0.06) both of which did not reach statistical significance. We did not observe any significant differences in the proportion of infants receiving PN, days on PN, the incidence of NEC, surgery for NEC, congenital heart disease or congenital malformations between cases and controls.\nWe noted a significant difference in associated mortality between controls and cases (47 vs. 20%), odds ratio adjusted for ‘any surgery’, 4.3 [95% CI, 1.8 to 10.2] (p = 0.001). A significant increase in the duration of infection (culture positivity) was noted between cases and controls (2.91 vs. 2.07 days, p = 0.02). No significant differences were observed in length of hospital stay, BPD, IPPV at 36 corrected wks, CPAP at 36 corrected wks, HFOV, ECMO, PDA, severe IVH (IVH > grade 2), PVL, severe IVH or PVL, ROP or severe ROP (ROP > stage 2) (p > 0.05). An increase in INO therapy in cases were noted (41 vs. 25%, p = 0.08) but was not statistically significant.\nIn a subgroup analysis of VLBW infants, polymicrobial infections compared to monomicrobial infections, had a significantly higher mortality (OR 6.4 [95% CI 1.3 to 30.3], longer duration of infection (OR 1.4 [95% CI 1.04 to 1.8] and higher incidence of congenital malformations (OR 17.7 [95% CI, 2.1 to 148.7).","We performed a case-control study of neonatal polymicrobial infections in a tertiary hospital in North America, and identified polymicrobial bloodstream infections in nearly 14% of bloodstream infections. We also probed the electronic neonatal clinical database over a four year period from January 2009 to December 2012 for relevant clinical data including risk factors and outcomes. We observed that polymicrobial bloodstream infections were associated with an increased mortality (>3-fold) and increased duration of infection compared to monomicrobial bloodstream infections. Surgical intervention excluding NEC surgery was a significant risk factor for neonatal polymicrobial infections.\nThe annual frequency of polymicrobial bloodstream infections in our study was approximately 14% over a 16 year study period. The frequency of neonatal polymicrobial infections reported in literature is variable, ranging from 3.9 to 25% [10-12,17,19-21]. Contaminated multi-dose lipid emulsions were responsible for the high frequency (25%) of polymicrobial infections in one study [20]. In most studies that report neonatal infections, the isolation of multiple organisms is often not discussed. The higher frequency of polymicrobial infections in our study compared to other reports in literature may be due to two reasons. First is the varying definition of polymicrobial infections in the different studies. We defined polymicrobial bloodstream infections as multiple organisms isolated during an infectious episode including those from a single blood specimen similar to Sutter et al., as we believe that this is a true reflection of a polymicrobial infection [1]. Other studies defined polymicrobial infections as multiple pathogens isolated from a single blood specimen, which may account for the lower incidence of polymicrobial infections [11,12,17]. Using the latter definition the annual frequency of polymicrobial bloodstream infections in our study would be approximately 7.8%, comparable to the 10% frequency reported in North American by Bizzarro et al. [11]. Secondly, increased incidence of polymicrobial bloodstream infections in our study may also relate to the large percentage of medically complex infants referred to our NICU including infants requiring surgical interventions, complex congenital heart disease, ECMO, abdominal wall defects, short gut syndrome and other congenital anomalies. This complex group of infants often requires indwelling vascular catheters for parenteral nutrition or medications for extended periods of time. The annual frequency of polymicrobial bacteremia in the neonatal intensive care unit in our study varied from 6.3 to 18.6% of all bacteremia during the 16-year study period. This annual variation may be in part due to expansion of the NICU, changes in the case-mix and referral patterns, nursing policy changes or implementation of catheter care bundles. There were significant decrease in the number of infections and polymicrobial infections between the time epochs, 1998-2009 and 2010-2012 (p < 0.01). We did not note any significant differences in the number of admissions of VLBW and ELBW infants. A dedicated vascular access team was formed in June 2009 followed by better implementation of catheter care bundles in June 2009, which along with increased compliance with hand hygiene and other infection control measures may have contributed to the decrease in infections.\nIn our study, cases and controls had similar demographics, a mean gestational age of approximately 30 weeks, birth weight of approximately 1500 g, similar male to female ratio and similar percentage being inborn. We focused on risk factors reported for polymicrobial infections in existing literature. The average age at the onset of the infections was 41 days in controls and 38 days in cases and hence all infections were in the late onset sepsis category and mostly occurred beyond the first 28 days of life. Almost all of the infections were late-onset infections and hence we did not collect data on risk factors for early onset sepsis such as maternal prolonged rupture of membranes, group B streptococcus colonization, urinary tract infection or chorioamnionitis. Subgroups of infections in neonates less than 28 days of age or those that were admitted from home were too small for meaningful comparisons. We noted a significant association of polymicrobial infections with surgery other than NEC and a trend towards association with direct jaundice. Cases had a higher incidence of central venous catheter compared to controls (97 vs. 91%, p = 0.20) but this association was not statistically significant.\nIn our case-control study, we observed more than 3-fold increase in associated mortality in neonates with polymicrobial bacteremia, but the retrospective and database oriented nature of our study precludes any conclusions of causality. Mortality due to polymicrobial infections has been reported to be at least 2-fold more than that of monomicrobial infections both in adults and children [1,2]. Faix et al. reported that mortality due to polymicrobial infections in neonates is increased almost 3-fold in his study of 15 cases of polymicrobial infections in 1971-86 [10]. More recent studies by Bizzarro et al. (study period 1989-2006), Tsai et al (study period 2004 to 2011) and Gupta et al. (1 year period in early 2000) did not report an increase in attributable mortality to polymicrobial infections in their studies [11,12,17]. The variability in mortality rates reported by different studies on polymicrobial infections may be multifactorial including differences in study design, variable definitions of polymicrobial infections, patient population, study periods, virulence of the organisms isolated or other unidentified factors [11]. The mechanisms for increased mortality in polymicrobial infections are not clear. Similar increases in mortality are observed in animal models of systemic and local polymicrobial infections [22-25]. We have observed increased catheter infection and systemic dissemination in a polymicrobial biofilm catheter infection model compared to monomicrobial infection [11]. The increased mortality may also arise from inherent host vulnerability that causes the infection in the first place (e.g. prematurity, short gut) or facilitation of one infection by the other (synergism) [26]. Polymicrobial interactions in polymicrobial environments such as a mixed-species biofilms on catheters may induce a host of synergistic mechanisms including quorum sensing, induction of virulence among others that may contribute to enhanced mortality or morbidity of the host [12].\nWe also noted an increase in the duration of infection in cases compared to controls, which may be due to increased host susceptibility or a synergistic effect of the polymicrobial infection. We did not observe any differences in length of hospital stay or other clinically relevant neonatal outcomes such as BPD, IPPV or CPAP at 36 corrected weeks, HFOV, ECMO, ROP, NEC or IVH or PVL. We observed an increase in the use of INO therapy in cases compared to controls, which was not statistically significant.\nThe organisms isolated in our study were mostly similar in both monomicrobial and polymicrobial bloodstream infections, similar in antibiotic susceptibility patterns and comparable to those organisms from polymicrobial infections reported in literature. The most common organisms isolated in both monomicrobial and polymicrobial infections were CONS, Staphylococcus aureus, Escherichia coli, Enterococcus species, Klebsiella spp. and Candida spp. Infections due to Candida spp., Enterococcus species and Klebsiella spp. increased in frequency in cases compared to controls. The most frequent polymicrobial combinations were CONS with Candida spp. and other organisms with Enterococcus faecalis, similar to reported literature [1,14,27]. Studies from Asia, by Tsai et al. and Gupta et al. report a high incidence of Gram negative infections (approximately 60%) in the polymicrobial group [12,17]. In the North American study by Bizarro et al., polymicrobial infections were preponderantly caused by Gram positive organisms (77%) similar to our study [11]. Geographic variations in organisms isolated from neonatal polymicrobial infections and their antibiotic susceptibility patterns may partly explain the variation in mortality and morbidity across the world due to polymicrobial infections. In our study we did not discern any differences in the treatment regimens used to treat polymicrobial infections and monomicrobial infections that could explain the differences in mortality. Polymicrobial infections were treated adequately for all the organisms isolated and duration of therapy was consistent with our written neonatal guidelines.\nLimitations of our study include being a retrospective, observational, case-control study. Bloodstream episodes were identified from the clinical microbiology database and hence the clinical data associated with the infection or their severity were not available. The clinical risk factors and outcomes were identified from the clinical database, which was available only for a 4 year period. Being a retrospective study, it is difficult to assign a causal relationship to the increased mortality associated with polymicrobial infections in our study. We did not assess long term developmental or growth outcomes as longitudinal assessment data were not available.\nThe human microbiome project and other microbiome studies emphasize the polymicrobial nature of organism communities in the human body [28,29]. The availability of molecular culture-independent methods for detection of sepsis may increase the identification of polymicrobial infections [30]. Guidelines or recommendations for therapeutic and preventive strategies against polymicrobial infections do not exist. Prolonged therapy with antibiotics targeting all organisms isolated in a polymicrobial infection and removing infected catheters remain the mainstay in therapy. Defining the epidemiology and clinical impact of polymicrobial infections may be the first step towards delineating optimal therapy and clinical outcomes. Preventive strategies should emphasize catheter care bundles that decrease central line associated bloodstream infections. Focused research is necessary to prevent and treat polymicrobial infections to improve clinical outcomes in the vulnerable neonate.","The main aim of the study was to investigate the frequency of polymicrobial infections in our tertiary neonatal intensive care unit, understand its impact on neonatal outcomes and promote research in the management of these infections. Polymicrobial bacteremia is common in neonates and comprises nearly 14% of all infectious episodes. The most common organisms in neonatal polymicrobial infections are CONS, S. aureus, Enterococcus species, E. coli, and Candida species. Surgical intervention is a significant risk factor for neonatal polymicrobial infections. We observed a more than 3-fold increase in mortality in infants with polymicrobial bacteremia and a significant increase in duration of the bacteremia. Research focused to prevent and to improve clinical outcomes in neonatal polymicrobial infections is urgently needed.","TCH: Texas Children’s Hospital; VON: Vermont Oxford Network; CONS: Coagulase negative Staphylococci; ELBW: Extremely low birth weight; VLBW: Very low birth weight; NICU: Neonatal Intensive Care Unit; PDA: Patent ductus arteriosus; BPD: Bronchopulmonary dysplasia; PVL: Periventricular leucomalacia; NEC: Necrotizing enterocolitis; TPN: Total parenteral nutrition; ROP: Retinopathy of prematurity; IVH: Intraventricular hemorrhage; IPPV: Intermittent positive pressure ventilation; CPAP: Continuous positive airway pressure; HFOV: High frequency oscillatory ventilation; INO: Inhaled nitric oxide.","None of the authors have any financial interests to disclose or competing interest.","We state that all the authors satisfy the requirements of author as laid down in ‘Instructions to the Authors’. MP conceived the project, participated in the design, data acquisition, analysis and interpretation, drafted the initial manuscript and approved the final manuscript as submitted. DZ participated in the acquisition of microbiology data and tabulating the data. YJ for clinical data acquisition and revision of the manuscript. PR participated in microbiology data acquisition, analysis and revision of the manuscript. JV for critical intellectual input and revision of the manuscript. All authors read and approved the final manuscript.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2334/14/390/prepub\n"],"string":"[\n \"Polymicrobial infections increase mortality more than 2-fold in adults and children, increase length of hospital stay and healthcare costs [1,2]. Risk factors for polymicrobial infections in children and adults include the presence of a central venous catheter, administration of parenteral nutrition, gastrointestinal pathology, especially short gut syndrome, use of broad-spectrum antibiotics and immunosuppression [1,3,4].\\nNeonatal polymicrobial infections are less well characterized compared to those in children or adults. Increased survival of extremely premature infants at the edge of viability, dependence on catheters and parenteral nutrition (PN), and antibiotic therapy may predispose to polymicrobial infections in neonates. The frequency of neonatal polymicrobial bloodstream infections reported in clinical studies varies from 4 to 24% of all bloodstream infections [5-10]. A standard definition for neonatal polymicrobial infections is lacking and the incidence varies from study to study partly due to variability in definition, neonatal population and practices [11]. Few studies have focused on polymicrobial infections in neonates, especially on risk factors and clinical outcomes in the western world [10-12]. In a neonatal review, the mortality due to polymicrobial infections was 3-fold greater than that of monomicrobial infections (70% vs. 23%) [10]. Organisms that are commonly implicated in neonatal polymicrobial bloodstream infections are coagulase-negative staphylococcus (CONS), Candida spp., Staphylococcus aureus and Enterococcus spp. [1,9,13-16]. A common risk factor appears to be multi-species biofilm infections originating from indwelling medical devices, notably indwelling vascular catheters or endotracheal tubes [1,17].\\nPolymicrobial bloodstream infections may be defined as multiple organisms isolated during an infectious episode including those from a single blood specimen [1] or more restrictively as isolation of more than one organism from a single blood specimen only [11,12,17]. Different definitions may partly explain the varying incidence of polymicrobial bloodstream infections reported. Bizzarro et al. (Yale, 1989-2006) and Faix et al. (Ann Arbor, 1971-1986) have reported the only two studies on neonatal polymicrobial infections from North America, [10,11]. Changing epidemiology of neonatal infections dictates a need to update and understand the epidemiology of polymicrobial infections in the hospitalized infant [11]. Lack of data on polymicrobial infections from our neonatal unit and concern of significant increase in mortality, morbidity and healthcare costs due to polymicrobial infections prompted us to investigate its frequency, risk factors and clinical outcomes at Texas Children’s Hospital (TCH).\",\n \"We hypothesized that polymicrobial infections comprise > 5% of bloodstream infections in infants residing in the neonatal intensive care units, have identifiable risk factors and are associated with higher mortality and morbidity than monomicrobial infections. We tested our hypothesis by performing a single center, retrospective, matched case-control study during a 16 year study period. Our study protocol was approved by the Institutional Review Board at Baylor College of Medicine, Houston. We followed the STROBE guidelines in reporting this study [18].\\n Identification of cases and controls We identified blood culture positive infants from the clinical microbiology database, who were admitted to the neonatal intensive care units (NICUs) III and II at TCH from January 1, 1997 to December 31, 2012. Infants more than 28 days of age in the NICU at the time of the infection and those admitted from home to the NICU with positive blood cultures were also included. The data in the clinical microbiology database is entered by the microbiologist and all positive and negative cultures including those regarded as contaminants are recorded. The tertiary NICU at TCH has approximately 1500 admissions per year including inborn and outborn transferred neonates (ranged from 1338 to 1547 during the years 2009 to 2012). Very low birth weight (VLBW, birth weight < 1500 g) infant admissions ranged from 222 to 291 infants during the years 2009 to 2012.\\nWe identified blood culture positive infants from the clinical microbiology database, who were admitted to the neonatal intensive care units (NICUs) III and II at TCH from January 1, 1997 to December 31, 2012. Infants more than 28 days of age in the NICU at the time of the infection and those admitted from home to the NICU with positive blood cultures were also included. The data in the clinical microbiology database is entered by the microbiologist and all positive and negative cultures including those regarded as contaminants are recorded. The tertiary NICU at TCH has approximately 1500 admissions per year including inborn and outborn transferred neonates (ranged from 1338 to 1547 during the years 2009 to 2012). Very low birth weight (VLBW, birth weight < 1500 g) infant admissions ranged from 222 to 291 infants during the years 2009 to 2012.\\n Definitions Polymicrobial bloodstream infection was defined as: i) isolation of more than one organism from a single blood culture specimen and ii) isolation of more than one organism in different blood culture specimens during the same bloodstream infectious episode. We defined ‘bloodstream infection episodes’ as time-periods associated with positive blood cultures [1] and the infection episode was considered resolved when at least two subsequent blood cultures performed every 24 hrs were negative. Usually when an organism is isolated from a blood culture specimen, blood cultures are repeated every 24 hr till two blood culture specimens are negative for organisms. Our neonatal units used only aerobic blood cultures and it is not routine to perform anaerobic blood cultures for the evaluation of neonatal sepsis. Single specimen polymicrobial infections were defined as more than one organism isolated from the same blood culture specimen. We also collected data regarding duration of the infection episode from culture positivity to culture negativity. For each polymicrobial bloodstream infection (case), we selected three gestational age matched neonates with comparable birth weights with monomicrobial bloodstream infection (control) during the time period 2009 to 2012. The matching was performed by an investigator who was blinded to neonatal risk factors and outcomes. An infant who had both polymicrobial and monomicrobial infections was included in the polymicrobial infection category for evaluation of neonatal outcomes because of the possibility that even one exposure to polymicrobial infection may increase mortality or morbidity. If an infant had multiple episodes of polymicrobial infections, the first polymicrobial infection episode was analysed. Coagulase negative staphylococcal (CONS) infections, skin flora (e.g. Micrococcus spp., Gamma Streptococci) and organisms infrequent in neonates (e.g. Bacillus spp.) were considered real infections when grown from two clinical specimens and a single culture of the above organisms were deemed contaminant and not included in the analyses.\\nPolymicrobial bloodstream infection was defined as: i) isolation of more than one organism from a single blood culture specimen and ii) isolation of more than one organism in different blood culture specimens during the same bloodstream infectious episode. We defined ‘bloodstream infection episodes’ as time-periods associated with positive blood cultures [1] and the infection episode was considered resolved when at least two subsequent blood cultures performed every 24 hrs were negative. Usually when an organism is isolated from a blood culture specimen, blood cultures are repeated every 24 hr till two blood culture specimens are negative for organisms. Our neonatal units used only aerobic blood cultures and it is not routine to perform anaerobic blood cultures for the evaluation of neonatal sepsis. Single specimen polymicrobial infections were defined as more than one organism isolated from the same blood culture specimen. We also collected data regarding duration of the infection episode from culture positivity to culture negativity. For each polymicrobial bloodstream infection (case), we selected three gestational age matched neonates with comparable birth weights with monomicrobial bloodstream infection (control) during the time period 2009 to 2012. The matching was performed by an investigator who was blinded to neonatal risk factors and outcomes. An infant who had both polymicrobial and monomicrobial infections was included in the polymicrobial infection category for evaluation of neonatal outcomes because of the possibility that even one exposure to polymicrobial infection may increase mortality or morbidity. If an infant had multiple episodes of polymicrobial infections, the first polymicrobial infection episode was analysed. Coagulase negative staphylococcal (CONS) infections, skin flora (e.g. Micrococcus spp., Gamma Streptococci) and organisms infrequent in neonates (e.g. Bacillus spp.) were considered real infections when grown from two clinical specimens and a single culture of the above organisms were deemed contaminant and not included in the analyses.\\n Clinical data collection Neonatal clinical data including demographics, risk factors and outcomes were identified by cross referencing our institution’s neonatal clinical database (from Vermont Oxford Network (VON) database), for a 4 year period from January 1, 2009 to December 31, 2012. We collected the following clinical data for the identified cases and controls: demographic data (gestational age, birth weight, sex, age at the start of the infection episode and whether inborn or outborn), parenteral nutrition (PN) administration and its duration, presence of intravenous catheters at the time of the infectious episode (percutaneously inserted central catheters and broviac catheters but excluded umbilical catheters). We excluded umbilical venous catheters because our neonatal policy is to replace the umbilical venous catheters in the first few days of life with percutaneously inserted central catheters (PICC) and hence most umbilical lines lasted only a few days. Also, most of the bloodstream infections occurred at a time when umbilical catheters were no longer in place. We also collected data on important clinical outcomes (mortality, length of hospital stay, bronchopulmonary dysplasia, patent ductus arteriosus (PDA), necrotizing enterocolitis (NEC, ≥ stage 2 by Bell’s classification), intraventricular hemorrhage (IVH), periventricular leucomalacia (PVL) and retinopathy of prematurity (ROP), intermittent positive pressure ventilation (IPPV) at 36 wks corrected gestational age (GA), continuous positive airway pressure (CPAP) at 36 wks corrected GA, high frequency oscillatory ventilation (HFOV), inhaled nitric oxide (INO) therapy, extracorporeal membrane oxygenation (ECMO), surgeries, direct hyperbilirubinemia (direct bilirubin > 2 mg/dl or > 15% of total bilirubin) congenital heart disease and congenital malformations. All outcomes were defined as per VON database definitions.\\nNeonatal clinical data including demographics, risk factors and outcomes were identified by cross referencing our institution’s neonatal clinical database (from Vermont Oxford Network (VON) database), for a 4 year period from January 1, 2009 to December 31, 2012. We collected the following clinical data for the identified cases and controls: demographic data (gestational age, birth weight, sex, age at the start of the infection episode and whether inborn or outborn), parenteral nutrition (PN) administration and its duration, presence of intravenous catheters at the time of the infectious episode (percutaneously inserted central catheters and broviac catheters but excluded umbilical catheters). We excluded umbilical venous catheters because our neonatal policy is to replace the umbilical venous catheters in the first few days of life with percutaneously inserted central catheters (PICC) and hence most umbilical lines lasted only a few days. Also, most of the bloodstream infections occurred at a time when umbilical catheters were no longer in place. We also collected data on important clinical outcomes (mortality, length of hospital stay, bronchopulmonary dysplasia, patent ductus arteriosus (PDA), necrotizing enterocolitis (NEC, ≥ stage 2 by Bell’s classification), intraventricular hemorrhage (IVH), periventricular leucomalacia (PVL) and retinopathy of prematurity (ROP), intermittent positive pressure ventilation (IPPV) at 36 wks corrected gestational age (GA), continuous positive airway pressure (CPAP) at 36 wks corrected GA, high frequency oscillatory ventilation (HFOV), inhaled nitric oxide (INO) therapy, extracorporeal membrane oxygenation (ECMO), surgeries, direct hyperbilirubinemia (direct bilirubin > 2 mg/dl or > 15% of total bilirubin) congenital heart disease and congenital malformations. All outcomes were defined as per VON database definitions.\\n Statistical analyses Data were analyzed using STATA 11, Stata Corporation, Dallas, USA. Infants with polymicrobial bloodstream infections were compared with three gestational age and birth weight matched infants with monomicrobial bloodstream infections. Continuous data were analyzed for statistical significance by the Student’s t test and categorical data were analyzed by chi squared analyses. A p value of < 0.05 was considered significant. A logistic regression analysis was performed for binary outcomes and odds ratios with 95% confidence intervals were estimated. The outcome of mortality was analyzed adjusting for ‘any surgery’ in the logistic regression model. Polymicrobial infections were used as the outcome of the logistic regression analysis for risk factors and as a covariate when assessing for neonatal outcomes. A subgroup analysis of VLBW infants was performed for mortality and risk factors in the logistic regression model.\\nData were analyzed using STATA 11, Stata Corporation, Dallas, USA. Infants with polymicrobial bloodstream infections were compared with three gestational age and birth weight matched infants with monomicrobial bloodstream infections. Continuous data were analyzed for statistical significance by the Student’s t test and categorical data were analyzed by chi squared analyses. A p value of < 0.05 was considered significant. A logistic regression analysis was performed for binary outcomes and odds ratios with 95% confidence intervals were estimated. The outcome of mortality was analyzed adjusting for ‘any surgery’ in the logistic regression model. Polymicrobial infections were used as the outcome of the logistic regression analysis for risk factors and as a covariate when assessing for neonatal outcomes. A subgroup analysis of VLBW infants was performed for mortality and risk factors in the logistic regression model.\",\n \"We identified blood culture positive infants from the clinical microbiology database, who were admitted to the neonatal intensive care units (NICUs) III and II at TCH from January 1, 1997 to December 31, 2012. Infants more than 28 days of age in the NICU at the time of the infection and those admitted from home to the NICU with positive blood cultures were also included. The data in the clinical microbiology database is entered by the microbiologist and all positive and negative cultures including those regarded as contaminants are recorded. The tertiary NICU at TCH has approximately 1500 admissions per year including inborn and outborn transferred neonates (ranged from 1338 to 1547 during the years 2009 to 2012). Very low birth weight (VLBW, birth weight < 1500 g) infant admissions ranged from 222 to 291 infants during the years 2009 to 2012.\",\n \"Polymicrobial bloodstream infection was defined as: i) isolation of more than one organism from a single blood culture specimen and ii) isolation of more than one organism in different blood culture specimens during the same bloodstream infectious episode. We defined ‘bloodstream infection episodes’ as time-periods associated with positive blood cultures [1] and the infection episode was considered resolved when at least two subsequent blood cultures performed every 24 hrs were negative. Usually when an organism is isolated from a blood culture specimen, blood cultures are repeated every 24 hr till two blood culture specimens are negative for organisms. Our neonatal units used only aerobic blood cultures and it is not routine to perform anaerobic blood cultures for the evaluation of neonatal sepsis. Single specimen polymicrobial infections were defined as more than one organism isolated from the same blood culture specimen. We also collected data regarding duration of the infection episode from culture positivity to culture negativity. For each polymicrobial bloodstream infection (case), we selected three gestational age matched neonates with comparable birth weights with monomicrobial bloodstream infection (control) during the time period 2009 to 2012. The matching was performed by an investigator who was blinded to neonatal risk factors and outcomes. An infant who had both polymicrobial and monomicrobial infections was included in the polymicrobial infection category for evaluation of neonatal outcomes because of the possibility that even one exposure to polymicrobial infection may increase mortality or morbidity. If an infant had multiple episodes of polymicrobial infections, the first polymicrobial infection episode was analysed. Coagulase negative staphylococcal (CONS) infections, skin flora (e.g. Micrococcus spp., Gamma Streptococci) and organisms infrequent in neonates (e.g. Bacillus spp.) were considered real infections when grown from two clinical specimens and a single culture of the above organisms were deemed contaminant and not included in the analyses.\",\n \"Neonatal clinical data including demographics, risk factors and outcomes were identified by cross referencing our institution’s neonatal clinical database (from Vermont Oxford Network (VON) database), for a 4 year period from January 1, 2009 to December 31, 2012. We collected the following clinical data for the identified cases and controls: demographic data (gestational age, birth weight, sex, age at the start of the infection episode and whether inborn or outborn), parenteral nutrition (PN) administration and its duration, presence of intravenous catheters at the time of the infectious episode (percutaneously inserted central catheters and broviac catheters but excluded umbilical catheters). We excluded umbilical venous catheters because our neonatal policy is to replace the umbilical venous catheters in the first few days of life with percutaneously inserted central catheters (PICC) and hence most umbilical lines lasted only a few days. Also, most of the bloodstream infections occurred at a time when umbilical catheters were no longer in place. We also collected data on important clinical outcomes (mortality, length of hospital stay, bronchopulmonary dysplasia, patent ductus arteriosus (PDA), necrotizing enterocolitis (NEC, ≥ stage 2 by Bell’s classification), intraventricular hemorrhage (IVH), periventricular leucomalacia (PVL) and retinopathy of prematurity (ROP), intermittent positive pressure ventilation (IPPV) at 36 wks corrected gestational age (GA), continuous positive airway pressure (CPAP) at 36 wks corrected GA, high frequency oscillatory ventilation (HFOV), inhaled nitric oxide (INO) therapy, extracorporeal membrane oxygenation (ECMO), surgeries, direct hyperbilirubinemia (direct bilirubin > 2 mg/dl or > 15% of total bilirubin) congenital heart disease and congenital malformations. All outcomes were defined as per VON database definitions.\",\n \"Data were analyzed using STATA 11, Stata Corporation, Dallas, USA. Infants with polymicrobial bloodstream infections were compared with three gestational age and birth weight matched infants with monomicrobial bloodstream infections. Continuous data were analyzed for statistical significance by the Student’s t test and categorical data were analyzed by chi squared analyses. A p value of < 0.05 was considered significant. A logistic regression analysis was performed for binary outcomes and odds ratios with 95% confidence intervals were estimated. The outcome of mortality was analyzed adjusting for ‘any surgery’ in the logistic regression model. Polymicrobial infections were used as the outcome of the logistic regression analysis for risk factors and as a covariate when assessing for neonatal outcomes. A subgroup analysis of VLBW infants was performed for mortality and risk factors in the logistic regression model.\",\n \"We identified 2007 bacteremia episodes during a period of 16 years in patients admitted to the NICUs at TCH; 280 episodes (14%) were polymicrobial (Figure 1). The percentage of annual polymicrobial bacteremia varied during the study period ranging from 6.3 to 18.6%. Polymicrobial bacteremia identified in single blood specimens constituted on an average of 56% (range 40 to 75%) during the 16 year study period.\\nFrequency of polymicrobial bacteremia episodes. The frequencies of neonatal bacteremia episodes are plotted against the years from 1997 to 2012. The dark shaded portion of the bars indicate polymicrobial component of the infectious episodes, which ranged from 6.3 to 18.6% and on an average of about 14% during the 16-year study period. There were significant decrease in the number of infections and polymicrobial infections between the time epochs, 1998-2009 and 2010-2012 (p < 0.01).\\nMicrobes isolated from monomicrobial (controls) and polymicrobial infections (cases) were similar but varied in frequency (Table 1). Microbes isolated at greater frequencies in polymicrobial infections were Candida spp. (>3%), Enterococcus faecalis (>2-fold) and Klebsiella pneumoniae (>2-fold). The most common combinations of polymicrobial organisms were CONS and Candida spp. (n = 6) and CONS and Enterococcus faecalis (n = 3). Gram positive organisms were isolated more frequently than gram negative organisms and the proportion of gram positive to gram negative organisms were similar in both monomicrobial (gram positive 51% and gram negative 42%) and polymicrobial infections (gram positive 48% and gram negative 40%).\\nMicrobiology of monomicrobial and polymicrobial bloodstream infections\\nIn monomicrobial bloodstream infections, 102 organisms were isolated from 102 infectious episodes. In polymicrobial bloodstream infections, 74 organisms were isolated from 34 infectious episodes. ‘Others’ include Streptococcus salivarius, Citrobacter freundii, Clostridium tertium for monomicrobial infections and for polymicrobial infections; they were Lactobacillus, Proteus mirabilis, Streptococcus sanguinus, Micrococcus spp., Acinetobacter baumanii complex and Gamma Streptococcus. All the organisms in the ‘Others’ group was cultured at least twice from 2 different samples. Candida species, Enterococcus species and Klebsiella species were isolated at higher frequencies in polymicrobial infections than monomicrobial infections. The most common combination of polymicrobial organisms were CONS and Candida (n = 6) and CONS and Enterococcus fecalis (n = 3).\\nWe compared clinical data pertaining to demographics, risk factors and clinical outcomes between infants with polymicrobial bacteremia and gestational age and birthweight matched infants with monomicrobial bacteremia (data from January 2009 to December 2012) (Table 2). We obtained neonatal data for 34 infants with polymicrobial bacteremia and 102 matched infants with monomicrobial bacteremia during that period. The demographics were similar between infants with polymicrobial compared to infants with monomicrobial bacteremia. As expected, the gestational age (30.5 vs. 30.4 wk, p = 0.90) and birth weights (1592 vs.1535 g, p = 0.77) were adequately matched between controls and cases respectively. Male sex and age at infection did not differ significantly between cases and controls.\\nInfant demographics, risk factors and outcomes in cases and controls\\n95% CI- 95% confidence intervals, Bwt-Birth weight, GA-gestational age, PN- parenteral nutrition, NEC- necrotizing enterocolitis, BPD-bronchopulmonary dysplasia, IPPV- Intermittent positive pressure ventilation, CPAP- continuous positive airway pressure, HFOV- High frequency oscillatory ventilation, INO- inhaled nitric oxide, ECMO- extracorporeal membrane oxygenator, PDA-patent ductus arteriosus, ROP-retinopathy of prematurity, IVH-intraventricular hemorrhage, PVL- periventricular leucomalacia, severe IVH- IVH > grade 2, severe ROP- ROP > stage 2. *p values < 0.05.\\nWe observed a significant difference between cases and controls in terms of surgery other than for NEC (p = 0.04) and any surgery (including NEC surgery) (p = 0.05, borderline significance). We observed an increase in the presence of a central venous catheter (97 vs. 91%, p =0.20) and incidence of direct hyperbilirubinemia (50 vs. 32%, p = 0.06) both of which did not reach statistical significance. We did not observe any significant differences in the proportion of infants receiving PN, days on PN, the incidence of NEC, surgery for NEC, congenital heart disease or congenital malformations between cases and controls.\\nWe noted a significant difference in associated mortality between controls and cases (47 vs. 20%), odds ratio adjusted for ‘any surgery’, 4.3 [95% CI, 1.8 to 10.2] (p = 0.001). A significant increase in the duration of infection (culture positivity) was noted between cases and controls (2.91 vs. 2.07 days, p = 0.02). No significant differences were observed in length of hospital stay, BPD, IPPV at 36 corrected wks, CPAP at 36 corrected wks, HFOV, ECMO, PDA, severe IVH (IVH > grade 2), PVL, severe IVH or PVL, ROP or severe ROP (ROP > stage 2) (p > 0.05). An increase in INO therapy in cases were noted (41 vs. 25%, p = 0.08) but was not statistically significant.\\nIn a subgroup analysis of VLBW infants, polymicrobial infections compared to monomicrobial infections, had a significantly higher mortality (OR 6.4 [95% CI 1.3 to 30.3], longer duration of infection (OR 1.4 [95% CI 1.04 to 1.8] and higher incidence of congenital malformations (OR 17.7 [95% CI, 2.1 to 148.7).\",\n \"We performed a case-control study of neonatal polymicrobial infections in a tertiary hospital in North America, and identified polymicrobial bloodstream infections in nearly 14% of bloodstream infections. We also probed the electronic neonatal clinical database over a four year period from January 2009 to December 2012 for relevant clinical data including risk factors and outcomes. We observed that polymicrobial bloodstream infections were associated with an increased mortality (>3-fold) and increased duration of infection compared to monomicrobial bloodstream infections. Surgical intervention excluding NEC surgery was a significant risk factor for neonatal polymicrobial infections.\\nThe annual frequency of polymicrobial bloodstream infections in our study was approximately 14% over a 16 year study period. The frequency of neonatal polymicrobial infections reported in literature is variable, ranging from 3.9 to 25% [10-12,17,19-21]. Contaminated multi-dose lipid emulsions were responsible for the high frequency (25%) of polymicrobial infections in one study [20]. In most studies that report neonatal infections, the isolation of multiple organisms is often not discussed. The higher frequency of polymicrobial infections in our study compared to other reports in literature may be due to two reasons. First is the varying definition of polymicrobial infections in the different studies. We defined polymicrobial bloodstream infections as multiple organisms isolated during an infectious episode including those from a single blood specimen similar to Sutter et al., as we believe that this is a true reflection of a polymicrobial infection [1]. Other studies defined polymicrobial infections as multiple pathogens isolated from a single blood specimen, which may account for the lower incidence of polymicrobial infections [11,12,17]. Using the latter definition the annual frequency of polymicrobial bloodstream infections in our study would be approximately 7.8%, comparable to the 10% frequency reported in North American by Bizzarro et al. [11]. Secondly, increased incidence of polymicrobial bloodstream infections in our study may also relate to the large percentage of medically complex infants referred to our NICU including infants requiring surgical interventions, complex congenital heart disease, ECMO, abdominal wall defects, short gut syndrome and other congenital anomalies. This complex group of infants often requires indwelling vascular catheters for parenteral nutrition or medications for extended periods of time. The annual frequency of polymicrobial bacteremia in the neonatal intensive care unit in our study varied from 6.3 to 18.6% of all bacteremia during the 16-year study period. This annual variation may be in part due to expansion of the NICU, changes in the case-mix and referral patterns, nursing policy changes or implementation of catheter care bundles. There were significant decrease in the number of infections and polymicrobial infections between the time epochs, 1998-2009 and 2010-2012 (p < 0.01). We did not note any significant differences in the number of admissions of VLBW and ELBW infants. A dedicated vascular access team was formed in June 2009 followed by better implementation of catheter care bundles in June 2009, which along with increased compliance with hand hygiene and other infection control measures may have contributed to the decrease in infections.\\nIn our study, cases and controls had similar demographics, a mean gestational age of approximately 30 weeks, birth weight of approximately 1500 g, similar male to female ratio and similar percentage being inborn. We focused on risk factors reported for polymicrobial infections in existing literature. The average age at the onset of the infections was 41 days in controls and 38 days in cases and hence all infections were in the late onset sepsis category and mostly occurred beyond the first 28 days of life. Almost all of the infections were late-onset infections and hence we did not collect data on risk factors for early onset sepsis such as maternal prolonged rupture of membranes, group B streptococcus colonization, urinary tract infection or chorioamnionitis. Subgroups of infections in neonates less than 28 days of age or those that were admitted from home were too small for meaningful comparisons. We noted a significant association of polymicrobial infections with surgery other than NEC and a trend towards association with direct jaundice. Cases had a higher incidence of central venous catheter compared to controls (97 vs. 91%, p = 0.20) but this association was not statistically significant.\\nIn our case-control study, we observed more than 3-fold increase in associated mortality in neonates with polymicrobial bacteremia, but the retrospective and database oriented nature of our study precludes any conclusions of causality. Mortality due to polymicrobial infections has been reported to be at least 2-fold more than that of monomicrobial infections both in adults and children [1,2]. Faix et al. reported that mortality due to polymicrobial infections in neonates is increased almost 3-fold in his study of 15 cases of polymicrobial infections in 1971-86 [10]. More recent studies by Bizzarro et al. (study period 1989-2006), Tsai et al (study period 2004 to 2011) and Gupta et al. (1 year period in early 2000) did not report an increase in attributable mortality to polymicrobial infections in their studies [11,12,17]. The variability in mortality rates reported by different studies on polymicrobial infections may be multifactorial including differences in study design, variable definitions of polymicrobial infections, patient population, study periods, virulence of the organisms isolated or other unidentified factors [11]. The mechanisms for increased mortality in polymicrobial infections are not clear. Similar increases in mortality are observed in animal models of systemic and local polymicrobial infections [22-25]. We have observed increased catheter infection and systemic dissemination in a polymicrobial biofilm catheter infection model compared to monomicrobial infection [11]. The increased mortality may also arise from inherent host vulnerability that causes the infection in the first place (e.g. prematurity, short gut) or facilitation of one infection by the other (synergism) [26]. Polymicrobial interactions in polymicrobial environments such as a mixed-species biofilms on catheters may induce a host of synergistic mechanisms including quorum sensing, induction of virulence among others that may contribute to enhanced mortality or morbidity of the host [12].\\nWe also noted an increase in the duration of infection in cases compared to controls, which may be due to increased host susceptibility or a synergistic effect of the polymicrobial infection. We did not observe any differences in length of hospital stay or other clinically relevant neonatal outcomes such as BPD, IPPV or CPAP at 36 corrected weeks, HFOV, ECMO, ROP, NEC or IVH or PVL. We observed an increase in the use of INO therapy in cases compared to controls, which was not statistically significant.\\nThe organisms isolated in our study were mostly similar in both monomicrobial and polymicrobial bloodstream infections, similar in antibiotic susceptibility patterns and comparable to those organisms from polymicrobial infections reported in literature. The most common organisms isolated in both monomicrobial and polymicrobial infections were CONS, Staphylococcus aureus, Escherichia coli, Enterococcus species, Klebsiella spp. and Candida spp. Infections due to Candida spp., Enterococcus species and Klebsiella spp. increased in frequency in cases compared to controls. The most frequent polymicrobial combinations were CONS with Candida spp. and other organisms with Enterococcus faecalis, similar to reported literature [1,14,27]. Studies from Asia, by Tsai et al. and Gupta et al. report a high incidence of Gram negative infections (approximately 60%) in the polymicrobial group [12,17]. In the North American study by Bizarro et al., polymicrobial infections were preponderantly caused by Gram positive organisms (77%) similar to our study [11]. Geographic variations in organisms isolated from neonatal polymicrobial infections and their antibiotic susceptibility patterns may partly explain the variation in mortality and morbidity across the world due to polymicrobial infections. In our study we did not discern any differences in the treatment regimens used to treat polymicrobial infections and monomicrobial infections that could explain the differences in mortality. Polymicrobial infections were treated adequately for all the organisms isolated and duration of therapy was consistent with our written neonatal guidelines.\\nLimitations of our study include being a retrospective, observational, case-control study. Bloodstream episodes were identified from the clinical microbiology database and hence the clinical data associated with the infection or their severity were not available. The clinical risk factors and outcomes were identified from the clinical database, which was available only for a 4 year period. Being a retrospective study, it is difficult to assign a causal relationship to the increased mortality associated with polymicrobial infections in our study. We did not assess long term developmental or growth outcomes as longitudinal assessment data were not available.\\nThe human microbiome project and other microbiome studies emphasize the polymicrobial nature of organism communities in the human body [28,29]. The availability of molecular culture-independent methods for detection of sepsis may increase the identification of polymicrobial infections [30]. Guidelines or recommendations for therapeutic and preventive strategies against polymicrobial infections do not exist. Prolonged therapy with antibiotics targeting all organisms isolated in a polymicrobial infection and removing infected catheters remain the mainstay in therapy. Defining the epidemiology and clinical impact of polymicrobial infections may be the first step towards delineating optimal therapy and clinical outcomes. Preventive strategies should emphasize catheter care bundles that decrease central line associated bloodstream infections. Focused research is necessary to prevent and treat polymicrobial infections to improve clinical outcomes in the vulnerable neonate.\",\n \"The main aim of the study was to investigate the frequency of polymicrobial infections in our tertiary neonatal intensive care unit, understand its impact on neonatal outcomes and promote research in the management of these infections. Polymicrobial bacteremia is common in neonates and comprises nearly 14% of all infectious episodes. The most common organisms in neonatal polymicrobial infections are CONS, S. aureus, Enterococcus species, E. coli, and Candida species. Surgical intervention is a significant risk factor for neonatal polymicrobial infections. We observed a more than 3-fold increase in mortality in infants with polymicrobial bacteremia and a significant increase in duration of the bacteremia. Research focused to prevent and to improve clinical outcomes in neonatal polymicrobial infections is urgently needed.\",\n \"TCH: Texas Children’s Hospital; VON: Vermont Oxford Network; CONS: Coagulase negative Staphylococci; ELBW: Extremely low birth weight; VLBW: Very low birth weight; NICU: Neonatal Intensive Care Unit; PDA: Patent ductus arteriosus; BPD: Bronchopulmonary dysplasia; PVL: Periventricular leucomalacia; NEC: Necrotizing enterocolitis; TPN: Total parenteral nutrition; ROP: Retinopathy of prematurity; IVH: Intraventricular hemorrhage; IPPV: Intermittent positive pressure ventilation; CPAP: Continuous positive airway pressure; HFOV: High frequency oscillatory ventilation; INO: Inhaled nitric oxide.\",\n \"None of the authors have any financial interests to disclose or competing interest.\",\n \"We state that all the authors satisfy the requirements of author as laid down in ‘Instructions to the Authors’. MP conceived the project, participated in the design, data acquisition, analysis and interpretation, drafted the initial manuscript and approved the final manuscript as submitted. DZ participated in the acquisition of microbiology data and tabulating the data. YJ for clinical data acquisition and revision of the manuscript. PR participated in microbiology data acquisition, analysis and revision of the manuscript. JV for critical intellectual input and revision of the manuscript. All authors read and approved the final manuscript.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2334/14/390/prepub\\n\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,"results","discussion","conclusions",null,null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null,\n null\n]","isConversational":false},"keywords":{"kind":"list like","value":["Neonate","Infant","NICU","Sepsis","Polymicrobial","Mortality"],"string":"[\n \"Neonate\",\n \"Infant\",\n \"NICU\",\n \"Sepsis\",\n \"Polymicrobial\",\n \"Mortality\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Background:\nPolymicrobial infections increase mortality more than 2-fold in adults and children, increase length of hospital stay and healthcare costs [1,2]. Risk factors for polymicrobial infections in children and adults include the presence of a central venous catheter, administration of parenteral nutrition, gastrointestinal pathology, especially short gut syndrome, use of broad-spectrum antibiotics and immunosuppression [1,3,4].\nNeonatal polymicrobial infections are less well characterized compared to those in children or adults. Increased survival of extremely premature infants at the edge of viability, dependence on catheters and parenteral nutrition (PN), and antibiotic therapy may predispose to polymicrobial infections in neonates. The frequency of neonatal polymicrobial bloodstream infections reported in clinical studies varies from 4 to 24% of all bloodstream infections [5-10]. A standard definition for neonatal polymicrobial infections is lacking and the incidence varies from study to study partly due to variability in definition, neonatal population and practices [11]. Few studies have focused on polymicrobial infections in neonates, especially on risk factors and clinical outcomes in the western world [10-12]. In a neonatal review, the mortality due to polymicrobial infections was 3-fold greater than that of monomicrobial infections (70% vs. 23%) [10]. Organisms that are commonly implicated in neonatal polymicrobial bloodstream infections are coagulase-negative staphylococcus (CONS), Candida spp., Staphylococcus aureus and Enterococcus spp. [1,9,13-16]. A common risk factor appears to be multi-species biofilm infections originating from indwelling medical devices, notably indwelling vascular catheters or endotracheal tubes [1,17].\nPolymicrobial bloodstream infections may be defined as multiple organisms isolated during an infectious episode including those from a single blood specimen [1] or more restrictively as isolation of more than one organism from a single blood specimen only [11,12,17]. Different definitions may partly explain the varying incidence of polymicrobial bloodstream infections reported. Bizzarro et al. (Yale, 1989-2006) and Faix et al. (Ann Arbor, 1971-1986) have reported the only two studies on neonatal polymicrobial infections from North America, [10,11]. Changing epidemiology of neonatal infections dictates a need to update and understand the epidemiology of polymicrobial infections in the hospitalized infant [11]. Lack of data on polymicrobial infections from our neonatal unit and concern of significant increase in mortality, morbidity and healthcare costs due to polymicrobial infections prompted us to investigate its frequency, risk factors and clinical outcomes at Texas Children’s Hospital (TCH).\n\nMethods:\nWe hypothesized that polymicrobial infections comprise > 5% of bloodstream infections in infants residing in the neonatal intensive care units, have identifiable risk factors and are associated with higher mortality and morbidity than monomicrobial infections. We tested our hypothesis by performing a single center, retrospective, matched case-control study during a 16 year study period. Our study protocol was approved by the Institutional Review Board at Baylor College of Medicine, Houston. We followed the STROBE guidelines in reporting this study [18].\n Identification of cases and controls We identified blood culture positive infants from the clinical microbiology database, who were admitted to the neonatal intensive care units (NICUs) III and II at TCH from January 1, 1997 to December 31, 2012. Infants more than 28 days of age in the NICU at the time of the infection and those admitted from home to the NICU with positive blood cultures were also included. The data in the clinical microbiology database is entered by the microbiologist and all positive and negative cultures including those regarded as contaminants are recorded. The tertiary NICU at TCH has approximately 1500 admissions per year including inborn and outborn transferred neonates (ranged from 1338 to 1547 during the years 2009 to 2012). Very low birth weight (VLBW, birth weight < 1500 g) infant admissions ranged from 222 to 291 infants during the years 2009 to 2012.\nWe identified blood culture positive infants from the clinical microbiology database, who were admitted to the neonatal intensive care units (NICUs) III and II at TCH from January 1, 1997 to December 31, 2012. Infants more than 28 days of age in the NICU at the time of the infection and those admitted from home to the NICU with positive blood cultures were also included. The data in the clinical microbiology database is entered by the microbiologist and all positive and negative cultures including those regarded as contaminants are recorded. The tertiary NICU at TCH has approximately 1500 admissions per year including inborn and outborn transferred neonates (ranged from 1338 to 1547 during the years 2009 to 2012). Very low birth weight (VLBW, birth weight < 1500 g) infant admissions ranged from 222 to 291 infants during the years 2009 to 2012.\n Definitions Polymicrobial bloodstream infection was defined as: i) isolation of more than one organism from a single blood culture specimen and ii) isolation of more than one organism in different blood culture specimens during the same bloodstream infectious episode. We defined ‘bloodstream infection episodes’ as time-periods associated with positive blood cultures [1] and the infection episode was considered resolved when at least two subsequent blood cultures performed every 24 hrs were negative. Usually when an organism is isolated from a blood culture specimen, blood cultures are repeated every 24 hr till two blood culture specimens are negative for organisms. Our neonatal units used only aerobic blood cultures and it is not routine to perform anaerobic blood cultures for the evaluation of neonatal sepsis. Single specimen polymicrobial infections were defined as more than one organism isolated from the same blood culture specimen. We also collected data regarding duration of the infection episode from culture positivity to culture negativity. For each polymicrobial bloodstream infection (case), we selected three gestational age matched neonates with comparable birth weights with monomicrobial bloodstream infection (control) during the time period 2009 to 2012. The matching was performed by an investigator who was blinded to neonatal risk factors and outcomes. An infant who had both polymicrobial and monomicrobial infections was included in the polymicrobial infection category for evaluation of neonatal outcomes because of the possibility that even one exposure to polymicrobial infection may increase mortality or morbidity. If an infant had multiple episodes of polymicrobial infections, the first polymicrobial infection episode was analysed. Coagulase negative staphylococcal (CONS) infections, skin flora (e.g. Micrococcus spp., Gamma Streptococci) and organisms infrequent in neonates (e.g. Bacillus spp.) were considered real infections when grown from two clinical specimens and a single culture of the above organisms were deemed contaminant and not included in the analyses.\nPolymicrobial bloodstream infection was defined as: i) isolation of more than one organism from a single blood culture specimen and ii) isolation of more than one organism in different blood culture specimens during the same bloodstream infectious episode. We defined ‘bloodstream infection episodes’ as time-periods associated with positive blood cultures [1] and the infection episode was considered resolved when at least two subsequent blood cultures performed every 24 hrs were negative. Usually when an organism is isolated from a blood culture specimen, blood cultures are repeated every 24 hr till two blood culture specimens are negative for organisms. Our neonatal units used only aerobic blood cultures and it is not routine to perform anaerobic blood cultures for the evaluation of neonatal sepsis. Single specimen polymicrobial infections were defined as more than one organism isolated from the same blood culture specimen. We also collected data regarding duration of the infection episode from culture positivity to culture negativity. For each polymicrobial bloodstream infection (case), we selected three gestational age matched neonates with comparable birth weights with monomicrobial bloodstream infection (control) during the time period 2009 to 2012. The matching was performed by an investigator who was blinded to neonatal risk factors and outcomes. An infant who had both polymicrobial and monomicrobial infections was included in the polymicrobial infection category for evaluation of neonatal outcomes because of the possibility that even one exposure to polymicrobial infection may increase mortality or morbidity. If an infant had multiple episodes of polymicrobial infections, the first polymicrobial infection episode was analysed. Coagulase negative staphylococcal (CONS) infections, skin flora (e.g. Micrococcus spp., Gamma Streptococci) and organisms infrequent in neonates (e.g. Bacillus spp.) were considered real infections when grown from two clinical specimens and a single culture of the above organisms were deemed contaminant and not included in the analyses.\n Clinical data collection Neonatal clinical data including demographics, risk factors and outcomes were identified by cross referencing our institution’s neonatal clinical database (from Vermont Oxford Network (VON) database), for a 4 year period from January 1, 2009 to December 31, 2012. We collected the following clinical data for the identified cases and controls: demographic data (gestational age, birth weight, sex, age at the start of the infection episode and whether inborn or outborn), parenteral nutrition (PN) administration and its duration, presence of intravenous catheters at the time of the infectious episode (percutaneously inserted central catheters and broviac catheters but excluded umbilical catheters). We excluded umbilical venous catheters because our neonatal policy is to replace the umbilical venous catheters in the first few days of life with percutaneously inserted central catheters (PICC) and hence most umbilical lines lasted only a few days. Also, most of the bloodstream infections occurred at a time when umbilical catheters were no longer in place. We also collected data on important clinical outcomes (mortality, length of hospital stay, bronchopulmonary dysplasia, patent ductus arteriosus (PDA), necrotizing enterocolitis (NEC, ≥ stage 2 by Bell’s classification), intraventricular hemorrhage (IVH), periventricular leucomalacia (PVL) and retinopathy of prematurity (ROP), intermittent positive pressure ventilation (IPPV) at 36 wks corrected gestational age (GA), continuous positive airway pressure (CPAP) at 36 wks corrected GA, high frequency oscillatory ventilation (HFOV), inhaled nitric oxide (INO) therapy, extracorporeal membrane oxygenation (ECMO), surgeries, direct hyperbilirubinemia (direct bilirubin > 2 mg/dl or > 15% of total bilirubin) congenital heart disease and congenital malformations. All outcomes were defined as per VON database definitions.\nNeonatal clinical data including demographics, risk factors and outcomes were identified by cross referencing our institution’s neonatal clinical database (from Vermont Oxford Network (VON) database), for a 4 year period from January 1, 2009 to December 31, 2012. We collected the following clinical data for the identified cases and controls: demographic data (gestational age, birth weight, sex, age at the start of the infection episode and whether inborn or outborn), parenteral nutrition (PN) administration and its duration, presence of intravenous catheters at the time of the infectious episode (percutaneously inserted central catheters and broviac catheters but excluded umbilical catheters). We excluded umbilical venous catheters because our neonatal policy is to replace the umbilical venous catheters in the first few days of life with percutaneously inserted central catheters (PICC) and hence most umbilical lines lasted only a few days. Also, most of the bloodstream infections occurred at a time when umbilical catheters were no longer in place. We also collected data on important clinical outcomes (mortality, length of hospital stay, bronchopulmonary dysplasia, patent ductus arteriosus (PDA), necrotizing enterocolitis (NEC, ≥ stage 2 by Bell’s classification), intraventricular hemorrhage (IVH), periventricular leucomalacia (PVL) and retinopathy of prematurity (ROP), intermittent positive pressure ventilation (IPPV) at 36 wks corrected gestational age (GA), continuous positive airway pressure (CPAP) at 36 wks corrected GA, high frequency oscillatory ventilation (HFOV), inhaled nitric oxide (INO) therapy, extracorporeal membrane oxygenation (ECMO), surgeries, direct hyperbilirubinemia (direct bilirubin > 2 mg/dl or > 15% of total bilirubin) congenital heart disease and congenital malformations. All outcomes were defined as per VON database definitions.\n Statistical analyses Data were analyzed using STATA 11, Stata Corporation, Dallas, USA. Infants with polymicrobial bloodstream infections were compared with three gestational age and birth weight matched infants with monomicrobial bloodstream infections. Continuous data were analyzed for statistical significance by the Student’s t test and categorical data were analyzed by chi squared analyses. A p value of < 0.05 was considered significant. A logistic regression analysis was performed for binary outcomes and odds ratios with 95% confidence intervals were estimated. The outcome of mortality was analyzed adjusting for ‘any surgery’ in the logistic regression model. Polymicrobial infections were used as the outcome of the logistic regression analysis for risk factors and as a covariate when assessing for neonatal outcomes. A subgroup analysis of VLBW infants was performed for mortality and risk factors in the logistic regression model.\nData were analyzed using STATA 11, Stata Corporation, Dallas, USA. Infants with polymicrobial bloodstream infections were compared with three gestational age and birth weight matched infants with monomicrobial bloodstream infections. Continuous data were analyzed for statistical significance by the Student’s t test and categorical data were analyzed by chi squared analyses. A p value of < 0.05 was considered significant. A logistic regression analysis was performed for binary outcomes and odds ratios with 95% confidence intervals were estimated. The outcome of mortality was analyzed adjusting for ‘any surgery’ in the logistic regression model. Polymicrobial infections were used as the outcome of the logistic regression analysis for risk factors and as a covariate when assessing for neonatal outcomes. A subgroup analysis of VLBW infants was performed for mortality and risk factors in the logistic regression model.\n\nIdentification of cases and controls:\nWe identified blood culture positive infants from the clinical microbiology database, who were admitted to the neonatal intensive care units (NICUs) III and II at TCH from January 1, 1997 to December 31, 2012. Infants more than 28 days of age in the NICU at the time of the infection and those admitted from home to the NICU with positive blood cultures were also included. The data in the clinical microbiology database is entered by the microbiologist and all positive and negative cultures including those regarded as contaminants are recorded. The tertiary NICU at TCH has approximately 1500 admissions per year including inborn and outborn transferred neonates (ranged from 1338 to 1547 during the years 2009 to 2012). Very low birth weight (VLBW, birth weight < 1500 g) infant admissions ranged from 222 to 291 infants during the years 2009 to 2012.\n\nDefinitions:\nPolymicrobial bloodstream infection was defined as: i) isolation of more than one organism from a single blood culture specimen and ii) isolation of more than one organism in different blood culture specimens during the same bloodstream infectious episode. We defined ‘bloodstream infection episodes’ as time-periods associated with positive blood cultures [1] and the infection episode was considered resolved when at least two subsequent blood cultures performed every 24 hrs were negative. Usually when an organism is isolated from a blood culture specimen, blood cultures are repeated every 24 hr till two blood culture specimens are negative for organisms. Our neonatal units used only aerobic blood cultures and it is not routine to perform anaerobic blood cultures for the evaluation of neonatal sepsis. Single specimen polymicrobial infections were defined as more than one organism isolated from the same blood culture specimen. We also collected data regarding duration of the infection episode from culture positivity to culture negativity. For each polymicrobial bloodstream infection (case), we selected three gestational age matched neonates with comparable birth weights with monomicrobial bloodstream infection (control) during the time period 2009 to 2012. The matching was performed by an investigator who was blinded to neonatal risk factors and outcomes. An infant who had both polymicrobial and monomicrobial infections was included in the polymicrobial infection category for evaluation of neonatal outcomes because of the possibility that even one exposure to polymicrobial infection may increase mortality or morbidity. If an infant had multiple episodes of polymicrobial infections, the first polymicrobial infection episode was analysed. Coagulase negative staphylococcal (CONS) infections, skin flora (e.g. Micrococcus spp., Gamma Streptococci) and organisms infrequent in neonates (e.g. Bacillus spp.) were considered real infections when grown from two clinical specimens and a single culture of the above organisms were deemed contaminant and not included in the analyses.\n\nClinical data collection:\nNeonatal clinical data including demographics, risk factors and outcomes were identified by cross referencing our institution’s neonatal clinical database (from Vermont Oxford Network (VON) database), for a 4 year period from January 1, 2009 to December 31, 2012. We collected the following clinical data for the identified cases and controls: demographic data (gestational age, birth weight, sex, age at the start of the infection episode and whether inborn or outborn), parenteral nutrition (PN) administration and its duration, presence of intravenous catheters at the time of the infectious episode (percutaneously inserted central catheters and broviac catheters but excluded umbilical catheters). We excluded umbilical venous catheters because our neonatal policy is to replace the umbilical venous catheters in the first few days of life with percutaneously inserted central catheters (PICC) and hence most umbilical lines lasted only a few days. Also, most of the bloodstream infections occurred at a time when umbilical catheters were no longer in place. We also collected data on important clinical outcomes (mortality, length of hospital stay, bronchopulmonary dysplasia, patent ductus arteriosus (PDA), necrotizing enterocolitis (NEC, ≥ stage 2 by Bell’s classification), intraventricular hemorrhage (IVH), periventricular leucomalacia (PVL) and retinopathy of prematurity (ROP), intermittent positive pressure ventilation (IPPV) at 36 wks corrected gestational age (GA), continuous positive airway pressure (CPAP) at 36 wks corrected GA, high frequency oscillatory ventilation (HFOV), inhaled nitric oxide (INO) therapy, extracorporeal membrane oxygenation (ECMO), surgeries, direct hyperbilirubinemia (direct bilirubin > 2 mg/dl or > 15% of total bilirubin) congenital heart disease and congenital malformations. All outcomes were defined as per VON database definitions.\n\nStatistical analyses:\nData were analyzed using STATA 11, Stata Corporation, Dallas, USA. Infants with polymicrobial bloodstream infections were compared with three gestational age and birth weight matched infants with monomicrobial bloodstream infections. Continuous data were analyzed for statistical significance by the Student’s t test and categorical data were analyzed by chi squared analyses. A p value of < 0.05 was considered significant. A logistic regression analysis was performed for binary outcomes and odds ratios with 95% confidence intervals were estimated. The outcome of mortality was analyzed adjusting for ‘any surgery’ in the logistic regression model. Polymicrobial infections were used as the outcome of the logistic regression analysis for risk factors and as a covariate when assessing for neonatal outcomes. A subgroup analysis of VLBW infants was performed for mortality and risk factors in the logistic regression model.\n\nResults:\nWe identified 2007 bacteremia episodes during a period of 16 years in patients admitted to the NICUs at TCH; 280 episodes (14%) were polymicrobial (Figure 1). The percentage of annual polymicrobial bacteremia varied during the study period ranging from 6.3 to 18.6%. Polymicrobial bacteremia identified in single blood specimens constituted on an average of 56% (range 40 to 75%) during the 16 year study period.\nFrequency of polymicrobial bacteremia episodes. The frequencies of neonatal bacteremia episodes are plotted against the years from 1997 to 2012. The dark shaded portion of the bars indicate polymicrobial component of the infectious episodes, which ranged from 6.3 to 18.6% and on an average of about 14% during the 16-year study period. There were significant decrease in the number of infections and polymicrobial infections between the time epochs, 1998-2009 and 2010-2012 (p < 0.01).\nMicrobes isolated from monomicrobial (controls) and polymicrobial infections (cases) were similar but varied in frequency (Table 1). Microbes isolated at greater frequencies in polymicrobial infections were Candida spp. (>3%), Enterococcus faecalis (>2-fold) and Klebsiella pneumoniae (>2-fold). The most common combinations of polymicrobial organisms were CONS and Candida spp. (n = 6) and CONS and Enterococcus faecalis (n = 3). Gram positive organisms were isolated more frequently than gram negative organisms and the proportion of gram positive to gram negative organisms were similar in both monomicrobial (gram positive 51% and gram negative 42%) and polymicrobial infections (gram positive 48% and gram negative 40%).\nMicrobiology of monomicrobial and polymicrobial bloodstream infections\nIn monomicrobial bloodstream infections, 102 organisms were isolated from 102 infectious episodes. In polymicrobial bloodstream infections, 74 organisms were isolated from 34 infectious episodes. ‘Others’ include Streptococcus salivarius, Citrobacter freundii, Clostridium tertium for monomicrobial infections and for polymicrobial infections; they were Lactobacillus, Proteus mirabilis, Streptococcus sanguinus, Micrococcus spp., Acinetobacter baumanii complex and Gamma Streptococcus. All the organisms in the ‘Others’ group was cultured at least twice from 2 different samples. Candida species, Enterococcus species and Klebsiella species were isolated at higher frequencies in polymicrobial infections than monomicrobial infections. The most common combination of polymicrobial organisms were CONS and Candida (n = 6) and CONS and Enterococcus fecalis (n = 3).\nWe compared clinical data pertaining to demographics, risk factors and clinical outcomes between infants with polymicrobial bacteremia and gestational age and birthweight matched infants with monomicrobial bacteremia (data from January 2009 to December 2012) (Table 2). We obtained neonatal data for 34 infants with polymicrobial bacteremia and 102 matched infants with monomicrobial bacteremia during that period. The demographics were similar between infants with polymicrobial compared to infants with monomicrobial bacteremia. As expected, the gestational age (30.5 vs. 30.4 wk, p = 0.90) and birth weights (1592 vs.1535 g, p = 0.77) were adequately matched between controls and cases respectively. Male sex and age at infection did not differ significantly between cases and controls.\nInfant demographics, risk factors and outcomes in cases and controls\n95% CI- 95% confidence intervals, Bwt-Birth weight, GA-gestational age, PN- parenteral nutrition, NEC- necrotizing enterocolitis, BPD-bronchopulmonary dysplasia, IPPV- Intermittent positive pressure ventilation, CPAP- continuous positive airway pressure, HFOV- High frequency oscillatory ventilation, INO- inhaled nitric oxide, ECMO- extracorporeal membrane oxygenator, PDA-patent ductus arteriosus, ROP-retinopathy of prematurity, IVH-intraventricular hemorrhage, PVL- periventricular leucomalacia, severe IVH- IVH > grade 2, severe ROP- ROP > stage 2. *p values < 0.05.\nWe observed a significant difference between cases and controls in terms of surgery other than for NEC (p = 0.04) and any surgery (including NEC surgery) (p = 0.05, borderline significance). We observed an increase in the presence of a central venous catheter (97 vs. 91%, p =0.20) and incidence of direct hyperbilirubinemia (50 vs. 32%, p = 0.06) both of which did not reach statistical significance. We did not observe any significant differences in the proportion of infants receiving PN, days on PN, the incidence of NEC, surgery for NEC, congenital heart disease or congenital malformations between cases and controls.\nWe noted a significant difference in associated mortality between controls and cases (47 vs. 20%), odds ratio adjusted for ‘any surgery’, 4.3 [95% CI, 1.8 to 10.2] (p = 0.001). A significant increase in the duration of infection (culture positivity) was noted between cases and controls (2.91 vs. 2.07 days, p = 0.02). No significant differences were observed in length of hospital stay, BPD, IPPV at 36 corrected wks, CPAP at 36 corrected wks, HFOV, ECMO, PDA, severe IVH (IVH > grade 2), PVL, severe IVH or PVL, ROP or severe ROP (ROP > stage 2) (p > 0.05). An increase in INO therapy in cases were noted (41 vs. 25%, p = 0.08) but was not statistically significant.\nIn a subgroup analysis of VLBW infants, polymicrobial infections compared to monomicrobial infections, had a significantly higher mortality (OR 6.4 [95% CI 1.3 to 30.3], longer duration of infection (OR 1.4 [95% CI 1.04 to 1.8] and higher incidence of congenital malformations (OR 17.7 [95% CI, 2.1 to 148.7).\n\nDiscussion:\nWe performed a case-control study of neonatal polymicrobial infections in a tertiary hospital in North America, and identified polymicrobial bloodstream infections in nearly 14% of bloodstream infections. We also probed the electronic neonatal clinical database over a four year period from January 2009 to December 2012 for relevant clinical data including risk factors and outcomes. We observed that polymicrobial bloodstream infections were associated with an increased mortality (>3-fold) and increased duration of infection compared to monomicrobial bloodstream infections. Surgical intervention excluding NEC surgery was a significant risk factor for neonatal polymicrobial infections.\nThe annual frequency of polymicrobial bloodstream infections in our study was approximately 14% over a 16 year study period. The frequency of neonatal polymicrobial infections reported in literature is variable, ranging from 3.9 to 25% [10-12,17,19-21]. Contaminated multi-dose lipid emulsions were responsible for the high frequency (25%) of polymicrobial infections in one study [20]. In most studies that report neonatal infections, the isolation of multiple organisms is often not discussed. The higher frequency of polymicrobial infections in our study compared to other reports in literature may be due to two reasons. First is the varying definition of polymicrobial infections in the different studies. We defined polymicrobial bloodstream infections as multiple organisms isolated during an infectious episode including those from a single blood specimen similar to Sutter et al., as we believe that this is a true reflection of a polymicrobial infection [1]. Other studies defined polymicrobial infections as multiple pathogens isolated from a single blood specimen, which may account for the lower incidence of polymicrobial infections [11,12,17]. Using the latter definition the annual frequency of polymicrobial bloodstream infections in our study would be approximately 7.8%, comparable to the 10% frequency reported in North American by Bizzarro et al. [11]. Secondly, increased incidence of polymicrobial bloodstream infections in our study may also relate to the large percentage of medically complex infants referred to our NICU including infants requiring surgical interventions, complex congenital heart disease, ECMO, abdominal wall defects, short gut syndrome and other congenital anomalies. This complex group of infants often requires indwelling vascular catheters for parenteral nutrition or medications for extended periods of time. The annual frequency of polymicrobial bacteremia in the neonatal intensive care unit in our study varied from 6.3 to 18.6% of all bacteremia during the 16-year study period. This annual variation may be in part due to expansion of the NICU, changes in the case-mix and referral patterns, nursing policy changes or implementation of catheter care bundles. There were significant decrease in the number of infections and polymicrobial infections between the time epochs, 1998-2009 and 2010-2012 (p < 0.01). We did not note any significant differences in the number of admissions of VLBW and ELBW infants. A dedicated vascular access team was formed in June 2009 followed by better implementation of catheter care bundles in June 2009, which along with increased compliance with hand hygiene and other infection control measures may have contributed to the decrease in infections.\nIn our study, cases and controls had similar demographics, a mean gestational age of approximately 30 weeks, birth weight of approximately 1500 g, similar male to female ratio and similar percentage being inborn. We focused on risk factors reported for polymicrobial infections in existing literature. The average age at the onset of the infections was 41 days in controls and 38 days in cases and hence all infections were in the late onset sepsis category and mostly occurred beyond the first 28 days of life. Almost all of the infections were late-onset infections and hence we did not collect data on risk factors for early onset sepsis such as maternal prolonged rupture of membranes, group B streptococcus colonization, urinary tract infection or chorioamnionitis. Subgroups of infections in neonates less than 28 days of age or those that were admitted from home were too small for meaningful comparisons. We noted a significant association of polymicrobial infections with surgery other than NEC and a trend towards association with direct jaundice. Cases had a higher incidence of central venous catheter compared to controls (97 vs. 91%, p = 0.20) but this association was not statistically significant.\nIn our case-control study, we observed more than 3-fold increase in associated mortality in neonates with polymicrobial bacteremia, but the retrospective and database oriented nature of our study precludes any conclusions of causality. Mortality due to polymicrobial infections has been reported to be at least 2-fold more than that of monomicrobial infections both in adults and children [1,2]. Faix et al. reported that mortality due to polymicrobial infections in neonates is increased almost 3-fold in his study of 15 cases of polymicrobial infections in 1971-86 [10]. More recent studies by Bizzarro et al. (study period 1989-2006), Tsai et al (study period 2004 to 2011) and Gupta et al. (1 year period in early 2000) did not report an increase in attributable mortality to polymicrobial infections in their studies [11,12,17]. The variability in mortality rates reported by different studies on polymicrobial infections may be multifactorial including differences in study design, variable definitions of polymicrobial infections, patient population, study periods, virulence of the organisms isolated or other unidentified factors [11]. The mechanisms for increased mortality in polymicrobial infections are not clear. Similar increases in mortality are observed in animal models of systemic and local polymicrobial infections [22-25]. We have observed increased catheter infection and systemic dissemination in a polymicrobial biofilm catheter infection model compared to monomicrobial infection [11]. The increased mortality may also arise from inherent host vulnerability that causes the infection in the first place (e.g. prematurity, short gut) or facilitation of one infection by the other (synergism) [26]. Polymicrobial interactions in polymicrobial environments such as a mixed-species biofilms on catheters may induce a host of synergistic mechanisms including quorum sensing, induction of virulence among others that may contribute to enhanced mortality or morbidity of the host [12].\nWe also noted an increase in the duration of infection in cases compared to controls, which may be due to increased host susceptibility or a synergistic effect of the polymicrobial infection. We did not observe any differences in length of hospital stay or other clinically relevant neonatal outcomes such as BPD, IPPV or CPAP at 36 corrected weeks, HFOV, ECMO, ROP, NEC or IVH or PVL. We observed an increase in the use of INO therapy in cases compared to controls, which was not statistically significant.\nThe organisms isolated in our study were mostly similar in both monomicrobial and polymicrobial bloodstream infections, similar in antibiotic susceptibility patterns and comparable to those organisms from polymicrobial infections reported in literature. The most common organisms isolated in both monomicrobial and polymicrobial infections were CONS, Staphylococcus aureus, Escherichia coli, Enterococcus species, Klebsiella spp. and Candida spp. Infections due to Candida spp., Enterococcus species and Klebsiella spp. increased in frequency in cases compared to controls. The most frequent polymicrobial combinations were CONS with Candida spp. and other organisms with Enterococcus faecalis, similar to reported literature [1,14,27]. Studies from Asia, by Tsai et al. and Gupta et al. report a high incidence of Gram negative infections (approximately 60%) in the polymicrobial group [12,17]. In the North American study by Bizarro et al., polymicrobial infections were preponderantly caused by Gram positive organisms (77%) similar to our study [11]. Geographic variations in organisms isolated from neonatal polymicrobial infections and their antibiotic susceptibility patterns may partly explain the variation in mortality and morbidity across the world due to polymicrobial infections. In our study we did not discern any differences in the treatment regimens used to treat polymicrobial infections and monomicrobial infections that could explain the differences in mortality. Polymicrobial infections were treated adequately for all the organisms isolated and duration of therapy was consistent with our written neonatal guidelines.\nLimitations of our study include being a retrospective, observational, case-control study. Bloodstream episodes were identified from the clinical microbiology database and hence the clinical data associated with the infection or their severity were not available. The clinical risk factors and outcomes were identified from the clinical database, which was available only for a 4 year period. Being a retrospective study, it is difficult to assign a causal relationship to the increased mortality associated with polymicrobial infections in our study. We did not assess long term developmental or growth outcomes as longitudinal assessment data were not available.\nThe human microbiome project and other microbiome studies emphasize the polymicrobial nature of organism communities in the human body [28,29]. The availability of molecular culture-independent methods for detection of sepsis may increase the identification of polymicrobial infections [30]. Guidelines or recommendations for therapeutic and preventive strategies against polymicrobial infections do not exist. Prolonged therapy with antibiotics targeting all organisms isolated in a polymicrobial infection and removing infected catheters remain the mainstay in therapy. Defining the epidemiology and clinical impact of polymicrobial infections may be the first step towards delineating optimal therapy and clinical outcomes. Preventive strategies should emphasize catheter care bundles that decrease central line associated bloodstream infections. Focused research is necessary to prevent and treat polymicrobial infections to improve clinical outcomes in the vulnerable neonate.\n\nConclusions:\nThe main aim of the study was to investigate the frequency of polymicrobial infections in our tertiary neonatal intensive care unit, understand its impact on neonatal outcomes and promote research in the management of these infections. Polymicrobial bacteremia is common in neonates and comprises nearly 14% of all infectious episodes. The most common organisms in neonatal polymicrobial infections are CONS, S. aureus, Enterococcus species, E. coli, and Candida species. Surgical intervention is a significant risk factor for neonatal polymicrobial infections. We observed a more than 3-fold increase in mortality in infants with polymicrobial bacteremia and a significant increase in duration of the bacteremia. Research focused to prevent and to improve clinical outcomes in neonatal polymicrobial infections is urgently needed.\n\nAbbreviations:\nTCH: Texas Children’s Hospital; VON: Vermont Oxford Network; CONS: Coagulase negative Staphylococci; ELBW: Extremely low birth weight; VLBW: Very low birth weight; NICU: Neonatal Intensive Care Unit; PDA: Patent ductus arteriosus; BPD: Bronchopulmonary dysplasia; PVL: Periventricular leucomalacia; NEC: Necrotizing enterocolitis; TPN: Total parenteral nutrition; ROP: Retinopathy of prematurity; IVH: Intraventricular hemorrhage; IPPV: Intermittent positive pressure ventilation; CPAP: Continuous positive airway pressure; HFOV: High frequency oscillatory ventilation; INO: Inhaled nitric oxide.\n\nCompeting interests:\nNone of the authors have any financial interests to disclose or competing interest.\n\nAuthors’ contributions:\nWe state that all the authors satisfy the requirements of author as laid down in ‘Instructions to the Authors’. MP conceived the project, participated in the design, data acquisition, analysis and interpretation, drafted the initial manuscript and approved the final manuscript as submitted. DZ participated in the acquisition of microbiology data and tabulating the data. YJ for clinical data acquisition and revision of the manuscript. PR participated in microbiology data acquisition, analysis and revision of the manuscript. JV for critical intellectual input and revision of the manuscript. All authors read and approved the final manuscript.\n\nPre-publication history:\nThe pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2334/14/390/prepub\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nPolymicrobial infections in adults and children are associated with increase in mortality, duration of intensive care and healthcare costs. Very few studies have characterized polymicrobial bloodstream infections in the neonatal unit. Considerable variation has been reported in incidence of polymicrobial infections and associated clinical outcomes. We characterized the risk factors and outcomes of polymicrobial bloodstream infections in our neonatal units in a tertiary hospital in North America.\n\nMethods:\nIn a retrospective case control study design, we identified infants in the neonatal intensive care unit with positive blood cultures at Texas Children's Hospital, over a 16-year period from January 1, 1997 to December 31, 2012. Clinical data from online databases were available from January 2009 to December 2012. For each polymicrobial bloodstream infection (case), we matched three infants with monomicrobial bloodstream infection (control) by gestational age and birth weight.\n\nResults:\nWe identified 2007 episodes of bloodstream infections during the 16 year study period and 280 (14%) of these were polymicrobial. Coagulase-negative Staphylococcus, Enterococcus, Klebsiella and Candida were the most common microbial genera isolated from polymicrobial infections. Polymicrobial bloodstream infections were associated with more than 3-fold increase in mortality and an increase in duration of infection. Surgical intervention was a significant risk factor for polymicrobial infection.\n\nConclusions:\nThe frequency and increased mortality emphasizes the clinical significance of polymicrobial bloodstream infections in the neonatal intensive care unit. Clinical awareness and focused research on neonatal polymicrobial infections is urgently needed."},"background_conclusion_text":{"kind":"string","value":"Background:\nPolymicrobial infections increase mortality more than 2-fold in adults and children, increase length of hospital stay and healthcare costs [1,2]. Risk factors for polymicrobial infections in children and adults include the presence of a central venous catheter, administration of parenteral nutrition, gastrointestinal pathology, especially short gut syndrome, use of broad-spectrum antibiotics and immunosuppression [1,3,4].\nNeonatal polymicrobial infections are less well characterized compared to those in children or adults. Increased survival of extremely premature infants at the edge of viability, dependence on catheters and parenteral nutrition (PN), and antibiotic therapy may predispose to polymicrobial infections in neonates. The frequency of neonatal polymicrobial bloodstream infections reported in clinical studies varies from 4 to 24% of all bloodstream infections [5-10]. A standard definition for neonatal polymicrobial infections is lacking and the incidence varies from study to study partly due to variability in definition, neonatal population and practices [11]. Few studies have focused on polymicrobial infections in neonates, especially on risk factors and clinical outcomes in the western world [10-12]. In a neonatal review, the mortality due to polymicrobial infections was 3-fold greater than that of monomicrobial infections (70% vs. 23%) [10]. Organisms that are commonly implicated in neonatal polymicrobial bloodstream infections are coagulase-negative staphylococcus (CONS), Candida spp., Staphylococcus aureus and Enterococcus spp. [1,9,13-16]. A common risk factor appears to be multi-species biofilm infections originating from indwelling medical devices, notably indwelling vascular catheters or endotracheal tubes [1,17].\nPolymicrobial bloodstream infections may be defined as multiple organisms isolated during an infectious episode including those from a single blood specimen [1] or more restrictively as isolation of more than one organism from a single blood specimen only [11,12,17]. Different definitions may partly explain the varying incidence of polymicrobial bloodstream infections reported. Bizzarro et al. (Yale, 1989-2006) and Faix et al. (Ann Arbor, 1971-1986) have reported the only two studies on neonatal polymicrobial infections from North America, [10,11]. Changing epidemiology of neonatal infections dictates a need to update and understand the epidemiology of polymicrobial infections in the hospitalized infant [11]. Lack of data on polymicrobial infections from our neonatal unit and concern of significant increase in mortality, morbidity and healthcare costs due to polymicrobial infections prompted us to investigate its frequency, risk factors and clinical outcomes at Texas Children’s Hospital (TCH).\n\nConclusions:\nThe main aim of the study was to investigate the frequency of polymicrobial infections in our tertiary neonatal intensive care unit, understand its impact on neonatal outcomes and promote research in the management of these infections. Polymicrobial bacteremia is common in neonates and comprises nearly 14% of all infectious episodes. The most common organisms in neonatal polymicrobial infections are CONS, S. aureus, Enterococcus species, E. coli, and Candida species. Surgical intervention is a significant risk factor for neonatal polymicrobial infections. We observed a more than 3-fold increase in mortality in infants with polymicrobial bacteremia and a significant increase in duration of the bacteremia. Research focused to prevent and to improve clinical outcomes in neonatal polymicrobial infections is urgently needed."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nPolymicrobial infections in adults and children are associated with increase in mortality, duration of intensive care and healthcare costs. Very few studies have characterized polymicrobial bloodstream infections in the neonatal unit. Considerable variation has been reported in incidence of polymicrobial infections and associated clinical outcomes. We characterized the risk factors and outcomes of polymicrobial bloodstream infections in our neonatal units in a tertiary hospital in North America.\n\nMethods:\nIn a retrospective case control study design, we identified infants in the neonatal intensive care unit with positive blood cultures at Texas Children's Hospital, over a 16-year period from January 1, 1997 to December 31, 2012. Clinical data from online databases were available from January 2009 to December 2012. For each polymicrobial bloodstream infection (case), we matched three infants with monomicrobial bloodstream infection (control) by gestational age and birth weight.\n\nResults:\nWe identified 2007 episodes of bloodstream infections during the 16 year study period and 280 (14%) of these were polymicrobial. Coagulase-negative Staphylococcus, Enterococcus, Klebsiella and Candida were the most common microbial genera isolated from polymicrobial infections. Polymicrobial bloodstream infections were associated with more than 3-fold increase in mortality and an increase in duration of infection. Surgical intervention was a significant risk factor for polymicrobial infection.\n\nConclusions:\nThe frequency and increased mortality emphasizes the clinical significance of polymicrobial bloodstream infections in the neonatal intensive care unit. Clinical awareness and focused research on neonatal polymicrobial infections is urgently needed."},"whole_article_text_length":{"kind":"number","value":6867,"string":"6,867"},"whole_article_abstract_length":{"kind":"number","value":283,"string":"283"},"other_sections_lengths":{"kind":"list like","value":[473,161,340,339,152,105,14,109,16],"string":"[\n 473,\n 161,\n 340,\n 339,\n 152,\n 105,\n 14,\n 109,\n 16\n]","isConversational":false},"num_sections":{"kind":"number","value":13,"string":"13"},"most_frequent_words":{"kind":"list like","value":["infections","polymicrobial","polymicrobial infections","neonatal","infection","bloodstream","data","blood","clinical","study"],"string":"[\n \"infections\",\n \"polymicrobial\",\n \"polymicrobial infections\",\n \"neonatal\",\n \"infection\",\n \"bloodstream\",\n \"data\",\n \"blood\",\n \"clinical\",\n \"study\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["outcomes neonatal polymicrobial","neonates polymicrobial bacteremia","neonatal polymicrobial bloodstream","infants polymicrobial bloodstream","infants polymicrobial infections"],"string":"[\n \"outcomes neonatal polymicrobial\",\n \"neonates polymicrobial bacteremia\",\n \"neonatal polymicrobial bloodstream\",\n \"infants polymicrobial bloodstream\",\n \"infants polymicrobial infections\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Neonate | Infant | NICU | Sepsis | Polymicrobial | Mortality [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Neonate | Infant | NICU | Sepsis | Polymicrobial | Mortality [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Neonate | Infant | NICU | Sepsis | Polymicrobial | Mortality [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Neonate | Infant | NICU | Sepsis | Polymicrobial | Mortality [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Neonate | Infant | NICU | Sepsis | Polymicrobial | Mortality [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Neonate | Infant | NICU | Sepsis | Polymicrobial | Mortality [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Bacteremia | Birth Weight | Candida | Case-Control Studies | Coinfection | Female | Humans | Incidence | Infant | Infant, Newborn | Intensive Care Units, Neonatal | Klebsiella | Male | Retrospective Studies | Risk Factors | Staphylococcus | Texas | Treatment Outcome [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Bacteremia | Birth Weight | Candida | Case-Control Studies | Coinfection | Female | Humans | Incidence | Infant | Infant, Newborn | Intensive Care Units, Neonatal | Klebsiella | Male | Retrospective Studies | Risk Factors | Staphylococcus | Texas | Treatment Outcome [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Bacteremia | Birth Weight | Candida | Case-Control Studies | Coinfection | Female | Humans | Incidence | Infant | Infant, Newborn | Intensive Care Units, Neonatal | Klebsiella | Male | Retrospective Studies | Risk Factors | Staphylococcus | Texas | Treatment Outcome [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Bacteremia | Birth Weight | Candida | Case-Control Studies | Coinfection | Female | Humans | Incidence | Infant | Infant, Newborn | Intensive Care Units, Neonatal | Klebsiella | Male | Retrospective Studies | Risk Factors | Staphylococcus | Texas | Treatment Outcome [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Bacteremia | Birth Weight | Candida | Case-Control Studies | Coinfection | Female | Humans | Incidence | Infant | Infant, Newborn | Intensive Care Units, Neonatal | Klebsiella | Male | Retrospective Studies | Risk Factors | Staphylococcus | Texas | Treatment Outcome [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Bacteremia | Birth Weight | Candida | Case-Control Studies | Coinfection | Female | Humans | Incidence | Infant | Infant, Newborn | Intensive Care Units, Neonatal | Klebsiella | Male | Retrospective Studies | Risk Factors | Staphylococcus | Texas | Treatment Outcome [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] outcomes neonatal polymicrobial | neonates polymicrobial bacteremia | neonatal polymicrobial bloodstream | infants polymicrobial bloodstream | infants polymicrobial infections [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] outcomes neonatal polymicrobial | neonates polymicrobial bacteremia | neonatal polymicrobial bloodstream | infants polymicrobial bloodstream | infants polymicrobial infections [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] outcomes neonatal polymicrobial | neonates polymicrobial bacteremia | neonatal polymicrobial bloodstream | infants polymicrobial bloodstream | infants polymicrobial infections [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] outcomes neonatal polymicrobial | neonates polymicrobial bacteremia | neonatal polymicrobial bloodstream | infants polymicrobial bloodstream | infants polymicrobial infections [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] outcomes neonatal polymicrobial | neonates polymicrobial bacteremia | neonatal polymicrobial bloodstream | infants polymicrobial bloodstream | infants polymicrobial infections [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] outcomes neonatal polymicrobial | neonates polymicrobial bacteremia | neonatal polymicrobial bloodstream | infants polymicrobial bloodstream | infants polymicrobial infections [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] infections | polymicrobial | polymicrobial infections | neonatal | infection | bloodstream | data | blood | clinical | study [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] infections | polymicrobial | polymicrobial infections | neonatal | infection | bloodstream | data | blood | clinical | study [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] infections | polymicrobial | polymicrobial infections | neonatal | infection | bloodstream | data | blood | clinical | study [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] infections | polymicrobial | polymicrobial infections | neonatal | infection | bloodstream | data | blood | clinical | study [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] infections | polymicrobial | polymicrobial infections | neonatal | infection | bloodstream | data | blood | clinical | study [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] infections | polymicrobial | polymicrobial infections | neonatal | infection | bloodstream | data | blood | clinical | study [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] infections | polymicrobial | polymicrobial infections | neonatal polymicrobial | neonatal | 10 | children | bloodstream infections | 11 | bloodstream [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] blood | infection | culture | polymicrobial | catheters | cultures | infections | blood cultures | blood culture | bloodstream [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] polymicrobial | bacteremia | gram | infections | cases | monomicrobial | controls | vs | ci | 95 ci [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] polymicrobial | infections | neonatal polymicrobial infections | bacteremia | neonatal polymicrobial | polymicrobial infections | neonatal | research | polymicrobial bacteremia | species [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] polymicrobial | infections | polymicrobial infections | neonatal | infection | blood | data | bloodstream | infants | catheters [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] polymicrobial | infections | polymicrobial infections | neonatal | infection | blood | data | bloodstream | infants | catheters [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| tertiary | North America [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Texas Children's Hospital | 16-year | January 1, 1997 to December 31, 2012 ||| January 2009 to December 2012 ||| three [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 2007 | 16 year | 280 | 14% ||| Staphylococcus | Enterococcus | Klebsiella | Candida ||| more than 3-fold ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| tertiary | North America ||| Texas Children's Hospital | 16-year | January 1, 1997 to December 31, 2012 ||| January 2009 to December 2012 ||| three ||| ||| 2007 | 16 year | 280 | 14% ||| Staphylococcus | Enterococcus | Klebsiella | Candida ||| more than 3-fold ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| tertiary | North America ||| Texas Children's Hospital | 16-year | January 1, 1997 to December 31, 2012 ||| January 2009 to December 2012 ||| three ||| ||| 2007 | 16 year | 280 | 14% ||| Staphylococcus | Enterococcus | Klebsiella | Candida ||| more than 3-fold ||| ||| ||| [SUMMARY]"}}},{"rowIdx":53,"cells":{"title":{"kind":"string","value":"Outreach onsite treatment with a simplified pangenotypic direct-acting anti-viral regimen for hepatitis C virus micro-elimination in a prison."},"pmid":{"kind":"string","value":"35110949"},"background_abstract":{"kind":"string","value":"Prisoners are at risk of hepatitis C virus (HCV) infection, especially among the people who inject drugs (PWID). We implemented an outreach strategy in combination with universal mass screening and immediate onsite treatment with a simplified pan-genotypic direct-acting antivirals (DAA) regimen, 12 wk of sofosbuvir/velpatasvir, in a PWID-dominant prison in Taiwan."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"HCV-viremic patients were recruited for onsite treatment program for HCV micro-elimination with a pangenotypic DAA regimen, 12 wk of sofosbuvir/ velpatasvir, from two cohorts in Penghu Prison, either identified by mass screen or in outpatient clinics, in September 2019. Another group of HCV-viremic patients identified sporadically in outpatient clinics before mass screening were enrolled as a control group. The primary endpoint was sustained virological response (SVR12, defined as undetectable HCV ribonucleic acid (RNA) 12 wk after end-of-treatment)."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"A total of 212 HCV-viremic subjects were recruited for HCV micro-elimination campaign; 91 patients treated with sofosbuvir/Ledipasvir or glecaprevir/ pibrentasvir before mass screening were enrolled as a control. The HCV micro-elimination group had significantly lower proportion of diabetes, hypertension, hyperlipidemia, advanced fibrosis and chronic kidney diseases, but higher levels of HCV RNA. The SVR12 rate was comparable between the HCV micro-elimination and control groups, 95.8% (203/212) vs 94.5% (86/91), respectively, in intent-to-treat analysis, and 100% (203/203) vs 98.9% (86/87), respectively, in per-protocol analysis. There was no virological failure, treatment discontinuation, and serious adverse event among sofosbuvir/velpatasvir-treated patients in the HCV micro-elimination group."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Outreach mass screening followed by immediate onsite treatment with a simplified pangenotypic DAA regimen, sofosbuvir/velpatasvir, provides successful strategies toward HCV micro-elimination among prisoners."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Antiviral Agents","Hepacivirus","Hepatitis C","Hepatitis C, Chronic","Humans","Prisons"],"string":"[\n \"Antiviral Agents\",\n \"Hepacivirus\",\n \"Hepatitis C\",\n \"Hepatitis C, Chronic\",\n \"Humans\",\n \"Prisons\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"8776526"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Hepatitis C virus (HCV) infection is a progressive and blood-borne infectious disease that can lead to end stage liver diseases, such as hepatic decompensation, liver cirrhosis, and hepatocellular carcinoma[1,2]. Iatrogenic transmission of HCV, such as blood transfusion and surgery, has decreased in developed countries. Whereas people who inject drugs (PWID) has become the major population of HCV transmission, which could consist of approximately 80% of HCV-infected patients[3]. Given that lack of vaccine available, “treatment as prevention” for HCV transmission in PWID is very important for HCV elimination.\nPrisoners are at high risk of HCV infection, with prevalence rates ranging from 3.1% to 38%[4,5]. The high prevalence of HCV infection in prisoners is resulted from unsafe lifestyles, psychiatric disorders, and social problems before they are incarcerated. Recently, PWID has been the most important risk factor of HCV infection in prisoners[6]. The anti-HCV prevalence rate could be as high as 91% among PWID prisoners[7]. Screening and eliminating HCV infection in prisoners is therefore an important social health issue.\nAccording to the American Association for the Study of Liver Diseases and European Association for the Study of the Liver (EASL) guidelines, all HCV viremic patients should be treated if life span is expected more than one year[8,9]. HCV therapeutic strategies have been revolutionized significantly because of the availability of direct-acting antivirals (DAA)[10]. Interferon (IFN)-based regimens for HCV infection have serious side effects, long therapeutic duration, and contraindications, leading to the huge gaps in HCV care cascade[11]. The current IFN-free DAA regimens provide shorter treatment duration, very high treatment efficacy and safety profiles, not only for general population[12], but also for special populations[13], such as HCV/human immunodeficiency virus (HIV) coinfected patients, hepatitis B virus (HBV)/HCV coinfected patients and patients with chronic kidney diseases in real-world clinical settings[14,15].\nWorld Health Organization (WHO) set a global goal of HCV elimination by 2030[16], and Taiwan authority is even ambitious by 2025[17]. To achieve the goal, implementation of the concept of HCV micro-elimination is regarding as an efficient and practical strategy[18]. We have proved that “universal mass screening plus outreach onsite treatment” is the key to achieve HCV micro-elimination among patients under maintenance hemodialysis[19].\nRecently, the latest EASL HCV guideline recommended simplified, genotyping/ subtyping-free, pangenotypic anti-HCV treatment, either sofosbuvir/ velpatasvir or glecaprevir/pibrentasvir, to increase the accessibility and global cure rates among patients with > 12 years, chronic hepatitis C without cirrhosis or with compensated cirrhosis, with or without HIV co-infection, whatever treatment-naïve or IFN-experienced[8].\nSince HCV treatment is not frequently administered to prisoners due to unawareness of HCV infection, difficultly management, easily loss to follow-up, and lack of hepatologist in prison[20], collaboration between hepatologists and prison authorities to carry out strategies for HCV diagnosis and treatment in prisoners in highly demanded. Herein, we implemented an outreach strategy in combination with universal mass screen and onsite treatment with a simplified pan-genotypic DAA regimen, 12 wk of sofosbuvir/velpatasvir, toward HCV micro-elimination in a PWID-dominant prison in Taiwan."},"methods_title":{"kind":"string","value":"MATERIALS AND METHODS"},"methods_text":{"kind":"string","value":"Patients linked to onsite treatment program for HCV micro-elimination HCV-viremic patients were recruited from two cohorts in Penghu Prison (Agency of Corrections, Ministry of Justice, Taiwan), a PWID-dominant prison (Figure 1).\n\nPatient flowchart of hepatitis C virus treatment with a simplified pan-genotypic directly-acting antivirals regimen in Penghu Prison. HCV: Hepatitis C virus; DAA: Directly-acting antivirals; SOL/VEL: Sofosbuvir/velpatasvir; GEL/PIB: Glecaprevir/pibrentasvir; EOTVR: Virological response at end-of-treatment; SVR12: Sustained viral response at post-treatment wk 12.\nHCV-viremic patients were recruited from two cohorts in Penghu Prison (Agency of Corrections, Ministry of Justice, Taiwan), a PWID-dominant prison (Figure 1).\n\nPatient flowchart of hepatitis C virus treatment with a simplified pan-genotypic directly-acting antivirals regimen in Penghu Prison. HCV: Hepatitis C virus; DAA: Directly-acting antivirals; SOL/VEL: Sofosbuvir/velpatasvir; GEL/PIB: Glecaprevir/pibrentasvir; EOTVR: Virological response at end-of-treatment; SVR12: Sustained viral response at post-treatment wk 12.\nHCV-viremic patients identified by a universal mass screening In September 2019, we conducted a 5 d universal mass screening of viral hepatitis in Penghu Prison. These inclusion criteria were prisoners, who were at least 20 years old, being willing to enter the study for screening of viral hepatitis. The study of mass screening was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (IRB: KMUHIRB-SV(I)-20190033). All participants provided written informed consents. A total of 1137 subjects from 1697 inmates participated the mass screening[21]. Among them, 396 (34.8%) subjects had anti-HCV seropositivity; 208 (52.5%) of the 396 subjects were seropositive for HCV ribonucleic acid (RNA) and linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\nIn September 2019, we conducted a 5 d universal mass screening of viral hepatitis in Penghu Prison. These inclusion criteria were prisoners, who were at least 20 years old, being willing to enter the study for screening of viral hepatitis. The study of mass screening was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (IRB: KMUHIRB-SV(I)-20190033). All participants provided written informed consents. A total of 1137 subjects from 1697 inmates participated the mass screening[21]. Among them, 396 (34.8%) subjects had anti-HCV seropositivity; 208 (52.5%) of the 396 subjects were seropositive for HCV ribonucleic acid (RNA) and linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\nHCV-viremic patients identified in outpatient clinics during the period of HCV mass screening Another 26 HCV-viremic subjects identified in outpatient clinics of Penghu Prison between August to December 2019 were also linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\nAll patients received pretreatment evaluation in December 2019, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions. A 12 wk, oral pan-genotypic regimen of sofosbuvir/velpatasvir 400/100 mg fixed-dose combination once daily was initiated in January-February 2020. \nAnother 26 HCV-viremic subjects identified in outpatient clinics of Penghu Prison between August to December 2019 were also linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\nAll patients received pretreatment evaluation in December 2019, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions. A 12 wk, oral pan-genotypic regimen of sofosbuvir/velpatasvir 400/100 mg fixed-dose combination once daily was initiated in January-February 2020. \nPatients identified and treated by DAAs in outpatient clinics before mass screening A total of 91 HCV-viremic patients identified in outpatient clinics of Penghu Prison and treated with DAA before mass screening from 2017 to 2019 were enrolled as a control. The selection of DAA regimens were based on physician’s discretion according to the viral genotype and criteria of reimbursement of National Health Insurance Administration, Taiwan. All patients received pretreatment evaluation, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions.\nAll participants signed informed consent forms. These enrolled inmates of our study were protected according to the guidelines of the Declaration of Helsinki. The current study of DAA therapy was approved by the Institutional Review Board of Tri-Service General Hospital (IRB: TSGHIRB 2-107-05-080).\nA total of 91 HCV-viremic patients identified in outpatient clinics of Penghu Prison and treated with DAA before mass screening from 2017 to 2019 were enrolled as a control. The selection of DAA regimens were based on physician’s discretion according to the viral genotype and criteria of reimbursement of National Health Insurance Administration, Taiwan. All patients received pretreatment evaluation, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions.\nAll participants signed informed consent forms. These enrolled inmates of our study were protected according to the guidelines of the Declaration of Helsinki. The current study of DAA therapy was approved by the Institutional Review Board of Tri-Service General Hospital (IRB: TSGHIRB 2-107-05-080).\nAssessment, monitoring and endpoints Anti-HCV antibody was determined by the third generation, commercially available immunoassay (Ax SYM HCV III; Abbott Laboratories, North Chicago, IL). HCV RNA viral loads and genotype were determined by real-time PCR assays [RealTime HCV; Abbott Molecular, Des Plaines IL, United States; detection limit: 12 IU/mL])[22]. Liver cirrhosis was defined by the presence of clinical, radiological, endoscopic or laboratory evidence of cirrhosis and/or portal hypertension or fibrosis-4 index (FIB-4) (> 6.5). Laboratory data monitoring and assessment of side effects were performed at treatment wk 2, 4, 8 and end-of-treatment (EOT), and 12 wk after EOT.\nThe primary endpoint was sustained virological response (SVR12, defined as undetectable HCV RNA throughout 12 wk of the post-treatment follow-up period).\nAnti-HCV antibody was determined by the third generation, commercially available immunoassay (Ax SYM HCV III; Abbott Laboratories, North Chicago, IL). HCV RNA viral loads and genotype were determined by real-time PCR assays [RealTime HCV; Abbott Molecular, Des Plaines IL, United States; detection limit: 12 IU/mL])[22]. Liver cirrhosis was defined by the presence of clinical, radiological, endoscopic or laboratory evidence of cirrhosis and/or portal hypertension or fibrosis-4 index (FIB-4) (> 6.5). Laboratory data monitoring and assessment of side effects were performed at treatment wk 2, 4, 8 and end-of-treatment (EOT), and 12 wk after EOT.\nThe primary endpoint was sustained virological response (SVR12, defined as undetectable HCV RNA throughout 12 wk of the post-treatment follow-up period).\nStatistical analyses. The efficacy of all DAA regimens was determined in a intent-to-treat (ITT) population (all enrolled patients with at least one dose of DAA) and a per-protocol (PP) population (subjects receiving at least one dose of DAA and retained in Penghu Prison throughout the DAA treatment and follow-up period). Safety assessments reported adverse event (AE), serious adverse event (SAE) and laboratory abnormalities in the ITT population. Continuous variables are expressed as means ± standard deviation (SD), and categorical variables are expressed as percentages. The differences of continuous variables are estimated by the Student’s t test. The differences in categorical variables are analyzed using the Chi-square test. The on-treatment and off-treatment virological response rates were analyzed in number and percentages with 95% confidence interval (CI). All data analyses were performed using the SPSS software version 18.0 (SPSS Inc., Chicago, Illinois, United States).\nThe efficacy of all DAA regimens was determined in a intent-to-treat (ITT) population (all enrolled patients with at least one dose of DAA) and a per-protocol (PP) population (subjects receiving at least one dose of DAA and retained in Penghu Prison throughout the DAA treatment and follow-up period). Safety assessments reported adverse event (AE), serious adverse event (SAE) and laboratory abnormalities in the ITT population. Continuous variables are expressed as means ± standard deviation (SD), and categorical variables are expressed as percentages. The differences of continuous variables are estimated by the Student’s t test. The differences in categorical variables are analyzed using the Chi-square test. The on-treatment and off-treatment virological response rates were analyzed in number and percentages with 95% confidence interval (CI). All data analyses were performed using the SPSS software version 18.0 (SPSS Inc., Chicago, Illinois, United States)."},"results_title":{"kind":"null"},"results_text":{"kind":"null"},"conclusions_title":{"kind":"string","value":"CONCLUSION"},"conclusions_text":{"kind":"string","value":"Our study provided evidence for the concept that simplified, genotyping/subtyping-free regimens can achieve high SVR12 rate in HCV-infected prisoners. In the future, it is possible to implement the strategy to all prisoners in our country."},"other_sections_titles":{"kind":"list like","value":["INTRODUCTION","Patients linked to onsite treatment program for HCV micro-elimination","HCV-viremic patients identified by a universal mass screening","HCV-viremic patients identified in outpatient clinics during the period of HCV mass screening","Patients identified and treated by DAAs in outpatient clinics before mass screening","Assessment, monitoring and endpoints","Statistical analyses.","RESULTS","Patient flowchart of HCV micro-elimination campaign","Patient characteristics ","Treatment efficacy","Safety profiles","DISCUSSION","CONCLUSION"],"string":"[\n \"INTRODUCTION\",\n \"Patients linked to onsite treatment program for HCV micro-elimination\",\n \"HCV-viremic patients identified by a universal mass screening\",\n \"HCV-viremic patients identified in outpatient clinics during the period of HCV mass screening\",\n \"Patients identified and treated by DAAs in outpatient clinics before mass screening\",\n \"Assessment, monitoring and endpoints\",\n \"Statistical analyses.\",\n \"RESULTS\",\n \"Patient flowchart of HCV micro-elimination campaign\",\n \"Patient characteristics \",\n \"Treatment efficacy\",\n \"Safety profiles\",\n \"DISCUSSION\",\n \"CONCLUSION\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Hepatitis C virus (HCV) infection is a progressive and blood-borne infectious disease that can lead to end stage liver diseases, such as hepatic decompensation, liver cirrhosis, and hepatocellular carcinoma[1,2]. Iatrogenic transmission of HCV, such as blood transfusion and surgery, has decreased in developed countries. Whereas people who inject drugs (PWID) has become the major population of HCV transmission, which could consist of approximately 80% of HCV-infected patients[3]. Given that lack of vaccine available, “treatment as prevention” for HCV transmission in PWID is very important for HCV elimination.\nPrisoners are at high risk of HCV infection, with prevalence rates ranging from 3.1% to 38%[4,5]. The high prevalence of HCV infection in prisoners is resulted from unsafe lifestyles, psychiatric disorders, and social problems before they are incarcerated. Recently, PWID has been the most important risk factor of HCV infection in prisoners[6]. The anti-HCV prevalence rate could be as high as 91% among PWID prisoners[7]. Screening and eliminating HCV infection in prisoners is therefore an important social health issue.\nAccording to the American Association for the Study of Liver Diseases and European Association for the Study of the Liver (EASL) guidelines, all HCV viremic patients should be treated if life span is expected more than one year[8,9]. HCV therapeutic strategies have been revolutionized significantly because of the availability of direct-acting antivirals (DAA)[10]. Interferon (IFN)-based regimens for HCV infection have serious side effects, long therapeutic duration, and contraindications, leading to the huge gaps in HCV care cascade[11]. The current IFN-free DAA regimens provide shorter treatment duration, very high treatment efficacy and safety profiles, not only for general population[12], but also for special populations[13], such as HCV/human immunodeficiency virus (HIV) coinfected patients, hepatitis B virus (HBV)/HCV coinfected patients and patients with chronic kidney diseases in real-world clinical settings[14,15].\nWorld Health Organization (WHO) set a global goal of HCV elimination by 2030[16], and Taiwan authority is even ambitious by 2025[17]. To achieve the goal, implementation of the concept of HCV micro-elimination is regarding as an efficient and practical strategy[18]. We have proved that “universal mass screening plus outreach onsite treatment” is the key to achieve HCV micro-elimination among patients under maintenance hemodialysis[19].\nRecently, the latest EASL HCV guideline recommended simplified, genotyping/ subtyping-free, pangenotypic anti-HCV treatment, either sofosbuvir/ velpatasvir or glecaprevir/pibrentasvir, to increase the accessibility and global cure rates among patients with > 12 years, chronic hepatitis C without cirrhosis or with compensated cirrhosis, with or without HIV co-infection, whatever treatment-naïve or IFN-experienced[8].\nSince HCV treatment is not frequently administered to prisoners due to unawareness of HCV infection, difficultly management, easily loss to follow-up, and lack of hepatologist in prison[20], collaboration between hepatologists and prison authorities to carry out strategies for HCV diagnosis and treatment in prisoners in highly demanded. Herein, we implemented an outreach strategy in combination with universal mass screen and onsite treatment with a simplified pan-genotypic DAA regimen, 12 wk of sofosbuvir/velpatasvir, toward HCV micro-elimination in a PWID-dominant prison in Taiwan.","HCV-viremic patients were recruited from two cohorts in Penghu Prison (Agency of Corrections, Ministry of Justice, Taiwan), a PWID-dominant prison (Figure 1).\n\nPatient flowchart of hepatitis C virus treatment with a simplified pan-genotypic directly-acting antivirals regimen in Penghu Prison. HCV: Hepatitis C virus; DAA: Directly-acting antivirals; SOL/VEL: Sofosbuvir/velpatasvir; GEL/PIB: Glecaprevir/pibrentasvir; EOTVR: Virological response at end-of-treatment; SVR12: Sustained viral response at post-treatment wk 12.","In September 2019, we conducted a 5 d universal mass screening of viral hepatitis in Penghu Prison. These inclusion criteria were prisoners, who were at least 20 years old, being willing to enter the study for screening of viral hepatitis. The study of mass screening was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (IRB: KMUHIRB-SV(I)-20190033). All participants provided written informed consents. A total of 1137 subjects from 1697 inmates participated the mass screening[21]. Among them, 396 (34.8%) subjects had anti-HCV seropositivity; 208 (52.5%) of the 396 subjects were seropositive for HCV ribonucleic acid (RNA) and linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.","Another 26 HCV-viremic subjects identified in outpatient clinics of Penghu Prison between August to December 2019 were also linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\nAll patients received pretreatment evaluation in December 2019, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions. A 12 wk, oral pan-genotypic regimen of sofosbuvir/velpatasvir 400/100 mg fixed-dose combination once daily was initiated in January-February 2020. ","A total of 91 HCV-viremic patients identified in outpatient clinics of Penghu Prison and treated with DAA before mass screening from 2017 to 2019 were enrolled as a control. The selection of DAA regimens were based on physician’s discretion according to the viral genotype and criteria of reimbursement of National Health Insurance Administration, Taiwan. All patients received pretreatment evaluation, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions.\nAll participants signed informed consent forms. These enrolled inmates of our study were protected according to the guidelines of the Declaration of Helsinki. The current study of DAA therapy was approved by the Institutional Review Board of Tri-Service General Hospital (IRB: TSGHIRB 2-107-05-080).","Anti-HCV antibody was determined by the third generation, commercially available immunoassay (Ax SYM HCV III; Abbott Laboratories, North Chicago, IL). HCV RNA viral loads and genotype were determined by real-time PCR assays [RealTime HCV; Abbott Molecular, Des Plaines IL, United States; detection limit: 12 IU/mL])[22]. Liver cirrhosis was defined by the presence of clinical, radiological, endoscopic or laboratory evidence of cirrhosis and/or portal hypertension or fibrosis-4 index (FIB-4) (> 6.5). Laboratory data monitoring and assessment of side effects were performed at treatment wk 2, 4, 8 and end-of-treatment (EOT), and 12 wk after EOT.\nThe primary endpoint was sustained virological response (SVR12, defined as undetectable HCV RNA throughout 12 wk of the post-treatment follow-up period).","The efficacy of all DAA regimens was determined in a intent-to-treat (ITT) population (all enrolled patients with at least one dose of DAA) and a per-protocol (PP) population (subjects receiving at least one dose of DAA and retained in Penghu Prison throughout the DAA treatment and follow-up period). Safety assessments reported adverse event (AE), serious adverse event (SAE) and laboratory abnormalities in the ITT population. Continuous variables are expressed as means ± standard deviation (SD), and categorical variables are expressed as percentages. The differences of continuous variables are estimated by the Student’s t test. The differences in categorical variables are analyzed using the Chi-square test. The on-treatment and off-treatment virological response rates were analyzed in number and percentages with 95% confidence interval (CI). All data analyses were performed using the SPSS software version 18.0 (SPSS Inc., Chicago, Illinois, United States).","Patient flowchart of HCV micro-elimination campaign The patient flowchart of HCV mass screen, assessment and treatment was shown in Figure 1. A total of 234 HCV-viremic patients, 208 from mass screening and 26 from outpatient clinics in Penghu Prison were assessed for eligibility of group therapy with sofosbuvir/velpatasvir in December 2019. Twenty-two patients were excluded from anti-HCV therapy due to scheduled to be released from jail (n = 16) or transferred to other jails (n = 3) within 6 mo, unwilling to receive therapy (n = 2) and prior glecaprevir/pibrentasvir treatment failure (n = 1). Finally, 212 patients were recruited for sofosbuvir/velpatasvir therapy initiated in January-February 2020.\nThe patient flowchart of HCV mass screen, assessment and treatment was shown in Figure 1. A total of 234 HCV-viremic patients, 208 from mass screening and 26 from outpatient clinics in Penghu Prison were assessed for eligibility of group therapy with sofosbuvir/velpatasvir in December 2019. Twenty-two patients were excluded from anti-HCV therapy due to scheduled to be released from jail (n = 16) or transferred to other jails (n = 3) within 6 mo, unwilling to receive therapy (n = 2) and prior glecaprevir/pibrentasvir treatment failure (n = 1). Finally, 212 patients were recruited for sofosbuvir/velpatasvir therapy initiated in January-February 2020.\nPatient characteristics The baseline characteristics of 303 HCV-viremic patients, including 212 in HCV micro-elimination campaign and 91 sporadic controls from outpatient clinics before micro-elimination campaign were listed in Table 1. They mean age was 48.4 years with male dominant (99.7%). Thirty (9.9%) had HBV coinfection. The mean FIB-4 was 1.3, with 20 (6.6%) had advanced fibrosis (FIB-4 > 3.25). Only one patient (0.3%) had liver cirrhosis. The mean HCV RNA levels was 6.5 Logs IU/mL, dominant with HCV genotype 1 (HCV-GT1, 42.2%), followed by HCV-GT6 (35.3%), HCV-GT3 (11.6%) and HCV-GT2 (10.6%). Three (1%) patients were prior IFN-experienced. The two groups had comparable characteristics in terms of age, gender, HBV co-infection, liver and renal function tests, FIB-4 score, HCV genotype distribution, and prior history of IFN-based therapy. However, the sporadic patients identified in outpatient clinics had significantly higher proportion of comorbidities, including diabetes, hypertension, hyperlipidemia and an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2, but significantly lower HCV viral loads. None of patient had decompensated cirrhosis nor liver cancer.\nBaseline characteristics of hepatitis C virus-infected patients receiving directly-acting antivirals therapy between sporadic hepatitis C virus therapy in outpatient clinics and campaign of hepatitis C virus micro-elimination in Penghu prison\n56 patients did not have body mass index information (12 patients before campaign of hepatitis C virus (HCV) micro-elimination; 44 patients in campaign of HCV micro-elimination).\n\nP < 0.05. DAA: Directly-acting antivirals; HCV: Hepatitis C virus; BMI: Body mass index; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; LC: Liver cirrhosis; FIB-4: Fibrosis-4 index; HBsAg: Hepatitis B surface antigen; eGFR: Estimated glomerular filtration rate (mL/min/1.73 m2); SOF: Sofosbuvir; VEL: Velpatasvir; LDV: Ledipasvir; GLE: Glecaprevir; PIB: Pibrentasvir; IFN: Interferon.\nThe baseline characteristics of 303 HCV-viremic patients, including 212 in HCV micro-elimination campaign and 91 sporadic controls from outpatient clinics before micro-elimination campaign were listed in Table 1. They mean age was 48.4 years with male dominant (99.7%). Thirty (9.9%) had HBV coinfection. The mean FIB-4 was 1.3, with 20 (6.6%) had advanced fibrosis (FIB-4 > 3.25). Only one patient (0.3%) had liver cirrhosis. The mean HCV RNA levels was 6.5 Logs IU/mL, dominant with HCV genotype 1 (HCV-GT1, 42.2%), followed by HCV-GT6 (35.3%), HCV-GT3 (11.6%) and HCV-GT2 (10.6%). Three (1%) patients were prior IFN-experienced. The two groups had comparable characteristics in terms of age, gender, HBV co-infection, liver and renal function tests, FIB-4 score, HCV genotype distribution, and prior history of IFN-based therapy. However, the sporadic patients identified in outpatient clinics had significantly higher proportion of comorbidities, including diabetes, hypertension, hyperlipidemia and an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2, but significantly lower HCV viral loads. None of patient had decompensated cirrhosis nor liver cancer.\nBaseline characteristics of hepatitis C virus-infected patients receiving directly-acting antivirals therapy between sporadic hepatitis C virus therapy in outpatient clinics and campaign of hepatitis C virus micro-elimination in Penghu prison\n56 patients did not have body mass index information (12 patients before campaign of hepatitis C virus (HCV) micro-elimination; 44 patients in campaign of HCV micro-elimination).\n\nP < 0.05. DAA: Directly-acting antivirals; HCV: Hepatitis C virus; BMI: Body mass index; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; LC: Liver cirrhosis; FIB-4: Fibrosis-4 index; HBsAg: Hepatitis B surface antigen; eGFR: Estimated glomerular filtration rate (mL/min/1.73 m2); SOF: Sofosbuvir; VEL: Velpatasvir; LDV: Ledipasvir; GLE: Glecaprevir; PIB: Pibrentasvir; IFN: Interferon.\nTreatment efficacy All of 212 patients in HCV micro-elimination campaign received sofosbuvir/ velpatasvir treatment; while among 91 sporadic patients with DAA therapy before HCV micro-elimination campaign, 78 (85.7%) received 12 wk of sofosbuvir/Ledipasvir and 13 (14.3%) received 8-12 wk of glecaprevir/pibrentasvir according to the Taiwan HCV guideline[12,13].\nIn ITT analysis, the overall SVR12 rate was 95.4% (289/303) with comparable SVR12 rates between sporadic HCV control group (94.5%, 86/91) and HCV micro-elimination group (95.8%, 203/212, P = 0.126, Table 2).\nVirological responses of hepatitis C virus-infected patients receiving directly-acting antivirals therapy before and during campaign of hepatitis C virus micro-elimination in Penghu prison in Penghu prison\nOne missing data.\nOne transferred; One missing data.\nTwo transferred; One released.\nFour released.\nOne relapser.\nFour transferred; Five released. HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \nDuring DAA treatment period, all of patients in sporadic HCV control group completed DAA therapy, while 3 patients in HCV micro-elimination group lost-to-follow (2 transferred; 1 released). During the post-treatment follow-up period, 4 patients in sporadic HCV control group lost-to-follow (4 released), while 6 patients in HCV micro-elimination group lost-to-follow (2 transferred; 4 released). In PP analysis, the overall SVR12 rate was 99.7% (289/290) with comparable SVR12 rates between sporadic HCV control group (98.9%, 86/87) and HCV micro-elimination group (100%, 203/203, P = 0.126, Table 2). Only one patient experienced virological failure (54 years old male, treatment-naïve, HCV-GT3 infection with baseline viral loads of 62,883 IU/mL and FIB-4 of 2.37; relapsed from a 12 wk regimen of glecaprevir/pibrentasvir).\nAll of 212 patients in HCV micro-elimination campaign received sofosbuvir/ velpatasvir treatment; while among 91 sporadic patients with DAA therapy before HCV micro-elimination campaign, 78 (85.7%) received 12 wk of sofosbuvir/Ledipasvir and 13 (14.3%) received 8-12 wk of glecaprevir/pibrentasvir according to the Taiwan HCV guideline[12,13].\nIn ITT analysis, the overall SVR12 rate was 95.4% (289/303) with comparable SVR12 rates between sporadic HCV control group (94.5%, 86/91) and HCV micro-elimination group (95.8%, 203/212, P = 0.126, Table 2).\nVirological responses of hepatitis C virus-infected patients receiving directly-acting antivirals therapy before and during campaign of hepatitis C virus micro-elimination in Penghu prison in Penghu prison\nOne missing data.\nOne transferred; One missing data.\nTwo transferred; One released.\nFour released.\nOne relapser.\nFour transferred; Five released. HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \nDuring DAA treatment period, all of patients in sporadic HCV control group completed DAA therapy, while 3 patients in HCV micro-elimination group lost-to-follow (2 transferred; 1 released). During the post-treatment follow-up period, 4 patients in sporadic HCV control group lost-to-follow (4 released), while 6 patients in HCV micro-elimination group lost-to-follow (2 transferred; 4 released). In PP analysis, the overall SVR12 rate was 99.7% (289/290) with comparable SVR12 rates between sporadic HCV control group (98.9%, 86/87) and HCV micro-elimination group (100%, 203/203, P = 0.126, Table 2). Only one patient experienced virological failure (54 years old male, treatment-naïve, HCV-GT3 infection with baseline viral loads of 62,883 IU/mL and FIB-4 of 2.37; relapsed from a 12 wk regimen of glecaprevir/pibrentasvir).\nSafety profiles The safety profiles of both groups were shown in Table 3. None of patients had treatment discontinuation other than released or transferred. None experienced serious adverse event. The frequency of adverse events was 4.3% (4/91) and 1.4% (3/212), respectively, among patients in sporadic control group and HCV micro-elimination group. The most reported adverse events were rash in 3 of 13 (23.1%) patients treated with glecaprevir/pibrentasvir and pruritus in 2 of 212 (0.9%) patients treated with sofosbuvir/velpatasvir. None of patients experienced grade 3 or 4 Laboratory abnormality.\nSafety profiles of hepatitis C virus-infected patients receiving direct-acting antivirals therapy in Penghu prison\nDAA: Directly-acting antivirals; HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \nThe safety profiles of both groups were shown in Table 3. None of patients had treatment discontinuation other than released or transferred. None experienced serious adverse event. The frequency of adverse events was 4.3% (4/91) and 1.4% (3/212), respectively, among patients in sporadic control group and HCV micro-elimination group. The most reported adverse events were rash in 3 of 13 (23.1%) patients treated with glecaprevir/pibrentasvir and pruritus in 2 of 212 (0.9%) patients treated with sofosbuvir/velpatasvir. None of patients experienced grade 3 or 4 Laboratory abnormality.\nSafety profiles of hepatitis C virus-infected patients receiving direct-acting antivirals therapy in Penghu prison\nDAA: Directly-acting antivirals; HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. ","The patient flowchart of HCV mass screen, assessment and treatment was shown in Figure 1. A total of 234 HCV-viremic patients, 208 from mass screening and 26 from outpatient clinics in Penghu Prison were assessed for eligibility of group therapy with sofosbuvir/velpatasvir in December 2019. Twenty-two patients were excluded from anti-HCV therapy due to scheduled to be released from jail (n = 16) or transferred to other jails (n = 3) within 6 mo, unwilling to receive therapy (n = 2) and prior glecaprevir/pibrentasvir treatment failure (n = 1). Finally, 212 patients were recruited for sofosbuvir/velpatasvir therapy initiated in January-February 2020.","The baseline characteristics of 303 HCV-viremic patients, including 212 in HCV micro-elimination campaign and 91 sporadic controls from outpatient clinics before micro-elimination campaign were listed in Table 1. They mean age was 48.4 years with male dominant (99.7%). Thirty (9.9%) had HBV coinfection. The mean FIB-4 was 1.3, with 20 (6.6%) had advanced fibrosis (FIB-4 > 3.25). Only one patient (0.3%) had liver cirrhosis. The mean HCV RNA levels was 6.5 Logs IU/mL, dominant with HCV genotype 1 (HCV-GT1, 42.2%), followed by HCV-GT6 (35.3%), HCV-GT3 (11.6%) and HCV-GT2 (10.6%). Three (1%) patients were prior IFN-experienced. The two groups had comparable characteristics in terms of age, gender, HBV co-infection, liver and renal function tests, FIB-4 score, HCV genotype distribution, and prior history of IFN-based therapy. However, the sporadic patients identified in outpatient clinics had significantly higher proportion of comorbidities, including diabetes, hypertension, hyperlipidemia and an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2, but significantly lower HCV viral loads. None of patient had decompensated cirrhosis nor liver cancer.\nBaseline characteristics of hepatitis C virus-infected patients receiving directly-acting antivirals therapy between sporadic hepatitis C virus therapy in outpatient clinics and campaign of hepatitis C virus micro-elimination in Penghu prison\n56 patients did not have body mass index information (12 patients before campaign of hepatitis C virus (HCV) micro-elimination; 44 patients in campaign of HCV micro-elimination).\n\nP < 0.05. DAA: Directly-acting antivirals; HCV: Hepatitis C virus; BMI: Body mass index; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; LC: Liver cirrhosis; FIB-4: Fibrosis-4 index; HBsAg: Hepatitis B surface antigen; eGFR: Estimated glomerular filtration rate (mL/min/1.73 m2); SOF: Sofosbuvir; VEL: Velpatasvir; LDV: Ledipasvir; GLE: Glecaprevir; PIB: Pibrentasvir; IFN: Interferon.","All of 212 patients in HCV micro-elimination campaign received sofosbuvir/ velpatasvir treatment; while among 91 sporadic patients with DAA therapy before HCV micro-elimination campaign, 78 (85.7%) received 12 wk of sofosbuvir/Ledipasvir and 13 (14.3%) received 8-12 wk of glecaprevir/pibrentasvir according to the Taiwan HCV guideline[12,13].\nIn ITT analysis, the overall SVR12 rate was 95.4% (289/303) with comparable SVR12 rates between sporadic HCV control group (94.5%, 86/91) and HCV micro-elimination group (95.8%, 203/212, P = 0.126, Table 2).\nVirological responses of hepatitis C virus-infected patients receiving directly-acting antivirals therapy before and during campaign of hepatitis C virus micro-elimination in Penghu prison in Penghu prison\nOne missing data.\nOne transferred; One missing data.\nTwo transferred; One released.\nFour released.\nOne relapser.\nFour transferred; Five released. HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \nDuring DAA treatment period, all of patients in sporadic HCV control group completed DAA therapy, while 3 patients in HCV micro-elimination group lost-to-follow (2 transferred; 1 released). During the post-treatment follow-up period, 4 patients in sporadic HCV control group lost-to-follow (4 released), while 6 patients in HCV micro-elimination group lost-to-follow (2 transferred; 4 released). In PP analysis, the overall SVR12 rate was 99.7% (289/290) with comparable SVR12 rates between sporadic HCV control group (98.9%, 86/87) and HCV micro-elimination group (100%, 203/203, P = 0.126, Table 2). Only one patient experienced virological failure (54 years old male, treatment-naïve, HCV-GT3 infection with baseline viral loads of 62,883 IU/mL and FIB-4 of 2.37; relapsed from a 12 wk regimen of glecaprevir/pibrentasvir).","The safety profiles of both groups were shown in Table 3. None of patients had treatment discontinuation other than released or transferred. None experienced serious adverse event. The frequency of adverse events was 4.3% (4/91) and 1.4% (3/212), respectively, among patients in sporadic control group and HCV micro-elimination group. The most reported adverse events were rash in 3 of 13 (23.1%) patients treated with glecaprevir/pibrentasvir and pruritus in 2 of 212 (0.9%) patients treated with sofosbuvir/velpatasvir. None of patients experienced grade 3 or 4 Laboratory abnormality.\nSafety profiles of hepatitis C virus-infected patients receiving direct-acting antivirals therapy in Penghu prison\nDAA: Directly-acting antivirals; HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. ","In the current study, we demonstrated that mass screening combined with onsite group therapy by using a simplified pan-genotypic DAA regimen, 12 wk of sofosbuvir/velpatasvir, provides an “one-size fits all” solution toward the achievement of HCV micro-elimination in prisoners. The SVR rate was 95.6% in ITT population and 100% in PP population after excluding the inmates released or transferred before end-of-follow-up. The high SVR rate was observed in this PWID-dominant population, which HCV genotype distribution was diverse, including genotypes 1a, 1b, 2, 3 and 6. \nRecent advance in the development of IFN-free pan-genotypic DAA regimens has remarkably improved the treatment efficacy with an overall SVR rates of > 90%. Therefore, WHO set the global of HCV elimination by 2030, through the achievement of > 90% diagnosis rate and > 80% treatment rate for eligible patients[16]. Nevertheless, there are many barriers in each HCV care cascade toward HCV elimination at the population level[11,23]. To overcome the barriers, combining the concept of micro-elimination and an outreach strategy with immediate onsite treatment would be a more efficient and practical approach to achieve that goal[18,24]. The current study compared the HCV-infected inmates identified sporadically in outpatient clinics of Penghu Prison from 2017 to 2019 before mass screening and the patients identified by mass screening. We found that mass screening identified 208 HCV-viremic patients in a 5 d screening program from 1137 inmates (encountered around two-third of total inmates in Penghu Prison), compared to 91 HCV-viremic inmates treated in outpatient clinics from 2017 to September 2019. Our results demonstrated that mass screening with immediate onsite treatment provide much more efficient and practical solution to overcome the gaps of disease awareness and link-to-care in the HCV care cascades toward HCV micro-elimination in prisoners. In addition, we implemented “HCV reflex testing” in the mass screening program to scale-up and speed-up the diagnosis and link-to-care for treatment uptake of HCV infections[25].\nPWID is known as the major risk factor of HCV infection and transmission. Although the anti-HCV prevalence in PWID prisoners decreased from 91% in 2014 to 34.8% in 2019 by the strategy of safe injection in Taiwan[21], almost all (97.6%) of HCV-infected prisoners were PWID. Given the lack of vaccine available and high risk of transmission, the strategy of universal screening and concept of “treatment as prevention” are the keys to HCV elimination in prison as well as PWID.\nWe observed that the sporadic HCV-infected prisoners identified in outpatient clinics had significantly higher proportion of comorbidities, including diabetes, hypertension, hyperlipidemia and eGFR, than those participating in the HCV micro-elimination campaign. It implicated that a great proportion of identified sporadically in outpatient clinics were due to concomitant morbidities; by contrast, many HCV-infected patients were unaware to their HCV diseases. In our mass screening, only 36.6% (145/396) of HCV-infected prisoners were aware of HCV infection before screening[21]. It indicates that the implementation of an outreach strategy with universal mass screen is necessary for HCV micro-elimination in prison.\nDespite of the advances in the management of HCV infections, DAA therapy in incarcerated HCV-infected people remains many obstacles to be resolved, including disease unawareness, lack of updated information about the benefits of new DAA treatment, uncertainty of treatment right[26], poor accessibility due to of onsite treatment facilities or HCV treaters. Another difficulty for HCV treatment in prisoners is the unexpected or scheduled releasing from prison or transferring to other prisons, which frequently leads to the interruption of treatment or lost-to- follow up[20,27]. We are lucky that the Taiwan Health Insurance covered all incarcerated people, including all of the laboratory tests and ultrasound sonography and the cost of DAA regimens. Each prison has a contracted hospital providing point-of-care facility. Before initiating DAA therapy, we excluded the patients with expected release or transfer within 24 wk, and negotiated with the authority to avoid unnecessary transferring to other prisons during the period of HCV treatment and follow-up once the inmates entering the DAA course. Eventually we achieved a high treatment rate of 90.6% (212/234) and a high treatment complete rate of 95.8% (203/212), with a high cure rate at 100% (212/212).\nBefore the IFN-free DAA available, the lower SVR rate, much longer treatment duration and frequent adverse events of IFN-based treatment discouraged HCV-infected prisoners from receiving treatment[10]. IFN-free DAA regimens revolutionized HCV treatment which has largely extended the indication for various HCV-infected patients. Nevertheless, the application of typical DAA regimens are based on HCV genotype, presence of decompensated cirrhosis, renal function, and prior treatment experience. The two pangenotypic DAA regimens, sofosbuvir/velpatasvir, and glecaprevir/pibrentasvir, have achieved very high SVR rates of > 95%, regardless of HCV GTs, except for treatment-experienced cirrhotic HCV GT3 patients or GT3b patients[8,12,13]. Recently, to improve the access to anti-HCV therapy, reduce the cost of laboratory tests and the relative complexity of genotype-based treatment strategies, simplified treatment without many information needed for treatment decision are recommended to facilitate the care cascade among populations who are historically less engaged in healthcare, such as PWIDs and prisoners[8]. EASL recommends simplified, genotyping/subtyping-free regimens for IFN-free DAA treatment-naïve (except sofosbuvir plus ribavirin), HCV-infected or HCV-HIV coinfected adolescent and adult patients without cirrhosis or with compensated cirrhosis, regardless of HCV genotypes[8]. These recommendations are a universal 12 wk regimen of sofosbuvir/velpatasvir for all patients or glecaprevir/pibrentasvir, 8 wk for non-cirrhotic, 12 wk for compensated cirrhotic, and 16 wk for HCV GT3 patients, respectively. There are only four information needed before treatment, including the presence of HCV viremia, potential drug-drug interactions, and prior treatment experience, and presence of cirrhosis. The advantages of glecaprevir/pibrentasvir is a shorter 8 wk regimen for treatment-naïve HCV patients and IFN-experienced non-cirrhotic patients with compensated liver diseases, which would be benefit for prisoners who are expected to be released or transferred in a short term. However, glecaprevir, a protease inhibitor, is contraindicated for patients with hepatic decompensation and at risk for rare occurrence of serious drug-induced liver injury[28]. Also, glecaprevir/ pibrentasvir has higher pill burden, three tablets a d. The advantages of sofosbuvir/ velpatasvir include a universal fixed 12 wk regimen, one tablet a d, for all HCV patients with compensated liver diseases, less frequency of potential drug-drug interactions[29], and safety for those with hepatic decompensation. However, a 12 wk regimen with sofosbuvir/velpatasvir needs one more visit and monitoring when compared to an 8 wk regimen with glecaprevir/pibrentasvir. Therefore, we select sofosbuvir/velpatasvir as the antiviral regimen for our outreach onsite treatment. In our study, all HCV-viremic prisoners fit the criteria of simplified, genotyping/ subtyping-free regimens, except one who failed to prior glecaprevir/pibrentasvir therapy and was not enrolled for sofosbuvir/velpatasvir treatment. In our PP analysis, the overall SVR12 rate was comparable between HCV patient group (98.9%, 86/87) and HCV micro-elimination group (100%, 203/203). Our study provided evidence for the concept that simplified, genotyping/subtyping-free regimens can achieve high SVR12 rate in HCV-infected PWID-dominant prisoners.\nIn our study, none of prisoners had DAA treatment discontinuation due to adverse events. None experienced serious adverse event. These data indicated that the simplified, genotyping/subtyping-free regimen, sofosbuvir/velpatasvir, was safe and well tolerated for HCV-infected PWID-dominant prisoners. Very few adverse events were reported in both groups, whatever using sofosbuvir/Ledipasvir, glecaprevir/ pibrentasvir and sofosbuvir/velpatasvir, when compared to the data from clinical trials[30,31]. It might be due to that current population was younger and less patients with advanced fibrosis or chronic kidney diseases.\nThere were some limitations in our study. First, not all inmates in Penghu Prison participated our mass screening. Strategies and policy to encourage inmates to receive HCV screening is mandatory to achieve the goal of WHO. Second, unexpected prisoners’ transferral and release could not be completely avoided, which caused incomplete treatment and follow-up. Successfully linking the released or transferred people to another HCV treaters could help completing HCV treatment and follow-up. Third, there was no reimbursement for the retreatment of prior DAA failed patients in Taiwan at the time of the current study.","Well-designed strategies for mass screening and treatment for HCV-infected prisoners can be implemented successfully by the collaboration between physicians and prison authorities. We demonstrated that mass screening followed by immediate onsite treatment with a simplified pangenotypic DAA regimen, sofosbuvir/velpatasvir, provides successful strategies toward HCV micro-elimination among prisoners."],"string":"[\n \"Hepatitis C virus (HCV) infection is a progressive and blood-borne infectious disease that can lead to end stage liver diseases, such as hepatic decompensation, liver cirrhosis, and hepatocellular carcinoma[1,2]. Iatrogenic transmission of HCV, such as blood transfusion and surgery, has decreased in developed countries. Whereas people who inject drugs (PWID) has become the major population of HCV transmission, which could consist of approximately 80% of HCV-infected patients[3]. Given that lack of vaccine available, “treatment as prevention” for HCV transmission in PWID is very important for HCV elimination.\\nPrisoners are at high risk of HCV infection, with prevalence rates ranging from 3.1% to 38%[4,5]. The high prevalence of HCV infection in prisoners is resulted from unsafe lifestyles, psychiatric disorders, and social problems before they are incarcerated. Recently, PWID has been the most important risk factor of HCV infection in prisoners[6]. The anti-HCV prevalence rate could be as high as 91% among PWID prisoners[7]. Screening and eliminating HCV infection in prisoners is therefore an important social health issue.\\nAccording to the American Association for the Study of Liver Diseases and European Association for the Study of the Liver (EASL) guidelines, all HCV viremic patients should be treated if life span is expected more than one year[8,9]. HCV therapeutic strategies have been revolutionized significantly because of the availability of direct-acting antivirals (DAA)[10]. Interferon (IFN)-based regimens for HCV infection have serious side effects, long therapeutic duration, and contraindications, leading to the huge gaps in HCV care cascade[11]. The current IFN-free DAA regimens provide shorter treatment duration, very high treatment efficacy and safety profiles, not only for general population[12], but also for special populations[13], such as HCV/human immunodeficiency virus (HIV) coinfected patients, hepatitis B virus (HBV)/HCV coinfected patients and patients with chronic kidney diseases in real-world clinical settings[14,15].\\nWorld Health Organization (WHO) set a global goal of HCV elimination by 2030[16], and Taiwan authority is even ambitious by 2025[17]. To achieve the goal, implementation of the concept of HCV micro-elimination is regarding as an efficient and practical strategy[18]. We have proved that “universal mass screening plus outreach onsite treatment” is the key to achieve HCV micro-elimination among patients under maintenance hemodialysis[19].\\nRecently, the latest EASL HCV guideline recommended simplified, genotyping/ subtyping-free, pangenotypic anti-HCV treatment, either sofosbuvir/ velpatasvir or glecaprevir/pibrentasvir, to increase the accessibility and global cure rates among patients with > 12 years, chronic hepatitis C without cirrhosis or with compensated cirrhosis, with or without HIV co-infection, whatever treatment-naïve or IFN-experienced[8].\\nSince HCV treatment is not frequently administered to prisoners due to unawareness of HCV infection, difficultly management, easily loss to follow-up, and lack of hepatologist in prison[20], collaboration between hepatologists and prison authorities to carry out strategies for HCV diagnosis and treatment in prisoners in highly demanded. Herein, we implemented an outreach strategy in combination with universal mass screen and onsite treatment with a simplified pan-genotypic DAA regimen, 12 wk of sofosbuvir/velpatasvir, toward HCV micro-elimination in a PWID-dominant prison in Taiwan.\",\n \"HCV-viremic patients were recruited from two cohorts in Penghu Prison (Agency of Corrections, Ministry of Justice, Taiwan), a PWID-dominant prison (Figure 1).\\n\\nPatient flowchart of hepatitis C virus treatment with a simplified pan-genotypic directly-acting antivirals regimen in Penghu Prison. HCV: Hepatitis C virus; DAA: Directly-acting antivirals; SOL/VEL: Sofosbuvir/velpatasvir; GEL/PIB: Glecaprevir/pibrentasvir; EOTVR: Virological response at end-of-treatment; SVR12: Sustained viral response at post-treatment wk 12.\",\n \"In September 2019, we conducted a 5 d universal mass screening of viral hepatitis in Penghu Prison. These inclusion criteria were prisoners, who were at least 20 years old, being willing to enter the study for screening of viral hepatitis. The study of mass screening was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (IRB: KMUHIRB-SV(I)-20190033). All participants provided written informed consents. A total of 1137 subjects from 1697 inmates participated the mass screening[21]. Among them, 396 (34.8%) subjects had anti-HCV seropositivity; 208 (52.5%) of the 396 subjects were seropositive for HCV ribonucleic acid (RNA) and linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\",\n \"Another 26 HCV-viremic subjects identified in outpatient clinics of Penghu Prison between August to December 2019 were also linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\\nAll patients received pretreatment evaluation in December 2019, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions. A 12 wk, oral pan-genotypic regimen of sofosbuvir/velpatasvir 400/100 mg fixed-dose combination once daily was initiated in January-February 2020. \",\n \"A total of 91 HCV-viremic patients identified in outpatient clinics of Penghu Prison and treated with DAA before mass screening from 2017 to 2019 were enrolled as a control. The selection of DAA regimens were based on physician’s discretion according to the viral genotype and criteria of reimbursement of National Health Insurance Administration, Taiwan. All patients received pretreatment evaluation, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions.\\nAll participants signed informed consent forms. These enrolled inmates of our study were protected according to the guidelines of the Declaration of Helsinki. The current study of DAA therapy was approved by the Institutional Review Board of Tri-Service General Hospital (IRB: TSGHIRB 2-107-05-080).\",\n \"Anti-HCV antibody was determined by the third generation, commercially available immunoassay (Ax SYM HCV III; Abbott Laboratories, North Chicago, IL). HCV RNA viral loads and genotype were determined by real-time PCR assays [RealTime HCV; Abbott Molecular, Des Plaines IL, United States; detection limit: 12 IU/mL])[22]. Liver cirrhosis was defined by the presence of clinical, radiological, endoscopic or laboratory evidence of cirrhosis and/or portal hypertension or fibrosis-4 index (FIB-4) (> 6.5). Laboratory data monitoring and assessment of side effects were performed at treatment wk 2, 4, 8 and end-of-treatment (EOT), and 12 wk after EOT.\\nThe primary endpoint was sustained virological response (SVR12, defined as undetectable HCV RNA throughout 12 wk of the post-treatment follow-up period).\",\n \"The efficacy of all DAA regimens was determined in a intent-to-treat (ITT) population (all enrolled patients with at least one dose of DAA) and a per-protocol (PP) population (subjects receiving at least one dose of DAA and retained in Penghu Prison throughout the DAA treatment and follow-up period). Safety assessments reported adverse event (AE), serious adverse event (SAE) and laboratory abnormalities in the ITT population. Continuous variables are expressed as means ± standard deviation (SD), and categorical variables are expressed as percentages. The differences of continuous variables are estimated by the Student’s t test. The differences in categorical variables are analyzed using the Chi-square test. The on-treatment and off-treatment virological response rates were analyzed in number and percentages with 95% confidence interval (CI). All data analyses were performed using the SPSS software version 18.0 (SPSS Inc., Chicago, Illinois, United States).\",\n \"Patient flowchart of HCV micro-elimination campaign The patient flowchart of HCV mass screen, assessment and treatment was shown in Figure 1. A total of 234 HCV-viremic patients, 208 from mass screening and 26 from outpatient clinics in Penghu Prison were assessed for eligibility of group therapy with sofosbuvir/velpatasvir in December 2019. Twenty-two patients were excluded from anti-HCV therapy due to scheduled to be released from jail (n = 16) or transferred to other jails (n = 3) within 6 mo, unwilling to receive therapy (n = 2) and prior glecaprevir/pibrentasvir treatment failure (n = 1). Finally, 212 patients were recruited for sofosbuvir/velpatasvir therapy initiated in January-February 2020.\\nThe patient flowchart of HCV mass screen, assessment and treatment was shown in Figure 1. A total of 234 HCV-viremic patients, 208 from mass screening and 26 from outpatient clinics in Penghu Prison were assessed for eligibility of group therapy with sofosbuvir/velpatasvir in December 2019. Twenty-two patients were excluded from anti-HCV therapy due to scheduled to be released from jail (n = 16) or transferred to other jails (n = 3) within 6 mo, unwilling to receive therapy (n = 2) and prior glecaprevir/pibrentasvir treatment failure (n = 1). Finally, 212 patients were recruited for sofosbuvir/velpatasvir therapy initiated in January-February 2020.\\nPatient characteristics The baseline characteristics of 303 HCV-viremic patients, including 212 in HCV micro-elimination campaign and 91 sporadic controls from outpatient clinics before micro-elimination campaign were listed in Table 1. They mean age was 48.4 years with male dominant (99.7%). Thirty (9.9%) had HBV coinfection. The mean FIB-4 was 1.3, with 20 (6.6%) had advanced fibrosis (FIB-4 > 3.25). Only one patient (0.3%) had liver cirrhosis. The mean HCV RNA levels was 6.5 Logs IU/mL, dominant with HCV genotype 1 (HCV-GT1, 42.2%), followed by HCV-GT6 (35.3%), HCV-GT3 (11.6%) and HCV-GT2 (10.6%). Three (1%) patients were prior IFN-experienced. The two groups had comparable characteristics in terms of age, gender, HBV co-infection, liver and renal function tests, FIB-4 score, HCV genotype distribution, and prior history of IFN-based therapy. However, the sporadic patients identified in outpatient clinics had significantly higher proportion of comorbidities, including diabetes, hypertension, hyperlipidemia and an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2, but significantly lower HCV viral loads. None of patient had decompensated cirrhosis nor liver cancer.\\nBaseline characteristics of hepatitis C virus-infected patients receiving directly-acting antivirals therapy between sporadic hepatitis C virus therapy in outpatient clinics and campaign of hepatitis C virus micro-elimination in Penghu prison\\n56 patients did not have body mass index information (12 patients before campaign of hepatitis C virus (HCV) micro-elimination; 44 patients in campaign of HCV micro-elimination).\\n\\nP < 0.05. DAA: Directly-acting antivirals; HCV: Hepatitis C virus; BMI: Body mass index; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; LC: Liver cirrhosis; FIB-4: Fibrosis-4 index; HBsAg: Hepatitis B surface antigen; eGFR: Estimated glomerular filtration rate (mL/min/1.73 m2); SOF: Sofosbuvir; VEL: Velpatasvir; LDV: Ledipasvir; GLE: Glecaprevir; PIB: Pibrentasvir; IFN: Interferon.\\nThe baseline characteristics of 303 HCV-viremic patients, including 212 in HCV micro-elimination campaign and 91 sporadic controls from outpatient clinics before micro-elimination campaign were listed in Table 1. They mean age was 48.4 years with male dominant (99.7%). Thirty (9.9%) had HBV coinfection. The mean FIB-4 was 1.3, with 20 (6.6%) had advanced fibrosis (FIB-4 > 3.25). Only one patient (0.3%) had liver cirrhosis. The mean HCV RNA levels was 6.5 Logs IU/mL, dominant with HCV genotype 1 (HCV-GT1, 42.2%), followed by HCV-GT6 (35.3%), HCV-GT3 (11.6%) and HCV-GT2 (10.6%). Three (1%) patients were prior IFN-experienced. The two groups had comparable characteristics in terms of age, gender, HBV co-infection, liver and renal function tests, FIB-4 score, HCV genotype distribution, and prior history of IFN-based therapy. However, the sporadic patients identified in outpatient clinics had significantly higher proportion of comorbidities, including diabetes, hypertension, hyperlipidemia and an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2, but significantly lower HCV viral loads. None of patient had decompensated cirrhosis nor liver cancer.\\nBaseline characteristics of hepatitis C virus-infected patients receiving directly-acting antivirals therapy between sporadic hepatitis C virus therapy in outpatient clinics and campaign of hepatitis C virus micro-elimination in Penghu prison\\n56 patients did not have body mass index information (12 patients before campaign of hepatitis C virus (HCV) micro-elimination; 44 patients in campaign of HCV micro-elimination).\\n\\nP < 0.05. DAA: Directly-acting antivirals; HCV: Hepatitis C virus; BMI: Body mass index; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; LC: Liver cirrhosis; FIB-4: Fibrosis-4 index; HBsAg: Hepatitis B surface antigen; eGFR: Estimated glomerular filtration rate (mL/min/1.73 m2); SOF: Sofosbuvir; VEL: Velpatasvir; LDV: Ledipasvir; GLE: Glecaprevir; PIB: Pibrentasvir; IFN: Interferon.\\nTreatment efficacy All of 212 patients in HCV micro-elimination campaign received sofosbuvir/ velpatasvir treatment; while among 91 sporadic patients with DAA therapy before HCV micro-elimination campaign, 78 (85.7%) received 12 wk of sofosbuvir/Ledipasvir and 13 (14.3%) received 8-12 wk of glecaprevir/pibrentasvir according to the Taiwan HCV guideline[12,13].\\nIn ITT analysis, the overall SVR12 rate was 95.4% (289/303) with comparable SVR12 rates between sporadic HCV control group (94.5%, 86/91) and HCV micro-elimination group (95.8%, 203/212, P = 0.126, Table 2).\\nVirological responses of hepatitis C virus-infected patients receiving directly-acting antivirals therapy before and during campaign of hepatitis C virus micro-elimination in Penghu prison in Penghu prison\\nOne missing data.\\nOne transferred; One missing data.\\nTwo transferred; One released.\\nFour released.\\nOne relapser.\\nFour transferred; Five released. HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \\nDuring DAA treatment period, all of patients in sporadic HCV control group completed DAA therapy, while 3 patients in HCV micro-elimination group lost-to-follow (2 transferred; 1 released). During the post-treatment follow-up period, 4 patients in sporadic HCV control group lost-to-follow (4 released), while 6 patients in HCV micro-elimination group lost-to-follow (2 transferred; 4 released). In PP analysis, the overall SVR12 rate was 99.7% (289/290) with comparable SVR12 rates between sporadic HCV control group (98.9%, 86/87) and HCV micro-elimination group (100%, 203/203, P = 0.126, Table 2). Only one patient experienced virological failure (54 years old male, treatment-naïve, HCV-GT3 infection with baseline viral loads of 62,883 IU/mL and FIB-4 of 2.37; relapsed from a 12 wk regimen of glecaprevir/pibrentasvir).\\nAll of 212 patients in HCV micro-elimination campaign received sofosbuvir/ velpatasvir treatment; while among 91 sporadic patients with DAA therapy before HCV micro-elimination campaign, 78 (85.7%) received 12 wk of sofosbuvir/Ledipasvir and 13 (14.3%) received 8-12 wk of glecaprevir/pibrentasvir according to the Taiwan HCV guideline[12,13].\\nIn ITT analysis, the overall SVR12 rate was 95.4% (289/303) with comparable SVR12 rates between sporadic HCV control group (94.5%, 86/91) and HCV micro-elimination group (95.8%, 203/212, P = 0.126, Table 2).\\nVirological responses of hepatitis C virus-infected patients receiving directly-acting antivirals therapy before and during campaign of hepatitis C virus micro-elimination in Penghu prison in Penghu prison\\nOne missing data.\\nOne transferred; One missing data.\\nTwo transferred; One released.\\nFour released.\\nOne relapser.\\nFour transferred; Five released. HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \\nDuring DAA treatment period, all of patients in sporadic HCV control group completed DAA therapy, while 3 patients in HCV micro-elimination group lost-to-follow (2 transferred; 1 released). During the post-treatment follow-up period, 4 patients in sporadic HCV control group lost-to-follow (4 released), while 6 patients in HCV micro-elimination group lost-to-follow (2 transferred; 4 released). In PP analysis, the overall SVR12 rate was 99.7% (289/290) with comparable SVR12 rates between sporadic HCV control group (98.9%, 86/87) and HCV micro-elimination group (100%, 203/203, P = 0.126, Table 2). Only one patient experienced virological failure (54 years old male, treatment-naïve, HCV-GT3 infection with baseline viral loads of 62,883 IU/mL and FIB-4 of 2.37; relapsed from a 12 wk regimen of glecaprevir/pibrentasvir).\\nSafety profiles The safety profiles of both groups were shown in Table 3. None of patients had treatment discontinuation other than released or transferred. None experienced serious adverse event. The frequency of adverse events was 4.3% (4/91) and 1.4% (3/212), respectively, among patients in sporadic control group and HCV micro-elimination group. The most reported adverse events were rash in 3 of 13 (23.1%) patients treated with glecaprevir/pibrentasvir and pruritus in 2 of 212 (0.9%) patients treated with sofosbuvir/velpatasvir. None of patients experienced grade 3 or 4 Laboratory abnormality.\\nSafety profiles of hepatitis C virus-infected patients receiving direct-acting antivirals therapy in Penghu prison\\nDAA: Directly-acting antivirals; HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \\nThe safety profiles of both groups were shown in Table 3. None of patients had treatment discontinuation other than released or transferred. None experienced serious adverse event. The frequency of adverse events was 4.3% (4/91) and 1.4% (3/212), respectively, among patients in sporadic control group and HCV micro-elimination group. The most reported adverse events were rash in 3 of 13 (23.1%) patients treated with glecaprevir/pibrentasvir and pruritus in 2 of 212 (0.9%) patients treated with sofosbuvir/velpatasvir. None of patients experienced grade 3 or 4 Laboratory abnormality.\\nSafety profiles of hepatitis C virus-infected patients receiving direct-acting antivirals therapy in Penghu prison\\nDAA: Directly-acting antivirals; HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \",\n \"The patient flowchart of HCV mass screen, assessment and treatment was shown in Figure 1. A total of 234 HCV-viremic patients, 208 from mass screening and 26 from outpatient clinics in Penghu Prison were assessed for eligibility of group therapy with sofosbuvir/velpatasvir in December 2019. Twenty-two patients were excluded from anti-HCV therapy due to scheduled to be released from jail (n = 16) or transferred to other jails (n = 3) within 6 mo, unwilling to receive therapy (n = 2) and prior glecaprevir/pibrentasvir treatment failure (n = 1). Finally, 212 patients were recruited for sofosbuvir/velpatasvir therapy initiated in January-February 2020.\",\n \"The baseline characteristics of 303 HCV-viremic patients, including 212 in HCV micro-elimination campaign and 91 sporadic controls from outpatient clinics before micro-elimination campaign were listed in Table 1. They mean age was 48.4 years with male dominant (99.7%). Thirty (9.9%) had HBV coinfection. The mean FIB-4 was 1.3, with 20 (6.6%) had advanced fibrosis (FIB-4 > 3.25). Only one patient (0.3%) had liver cirrhosis. The mean HCV RNA levels was 6.5 Logs IU/mL, dominant with HCV genotype 1 (HCV-GT1, 42.2%), followed by HCV-GT6 (35.3%), HCV-GT3 (11.6%) and HCV-GT2 (10.6%). Three (1%) patients were prior IFN-experienced. The two groups had comparable characteristics in terms of age, gender, HBV co-infection, liver and renal function tests, FIB-4 score, HCV genotype distribution, and prior history of IFN-based therapy. However, the sporadic patients identified in outpatient clinics had significantly higher proportion of comorbidities, including diabetes, hypertension, hyperlipidemia and an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2, but significantly lower HCV viral loads. None of patient had decompensated cirrhosis nor liver cancer.\\nBaseline characteristics of hepatitis C virus-infected patients receiving directly-acting antivirals therapy between sporadic hepatitis C virus therapy in outpatient clinics and campaign of hepatitis C virus micro-elimination in Penghu prison\\n56 patients did not have body mass index information (12 patients before campaign of hepatitis C virus (HCV) micro-elimination; 44 patients in campaign of HCV micro-elimination).\\n\\nP < 0.05. DAA: Directly-acting antivirals; HCV: Hepatitis C virus; BMI: Body mass index; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; LC: Liver cirrhosis; FIB-4: Fibrosis-4 index; HBsAg: Hepatitis B surface antigen; eGFR: Estimated glomerular filtration rate (mL/min/1.73 m2); SOF: Sofosbuvir; VEL: Velpatasvir; LDV: Ledipasvir; GLE: Glecaprevir; PIB: Pibrentasvir; IFN: Interferon.\",\n \"All of 212 patients in HCV micro-elimination campaign received sofosbuvir/ velpatasvir treatment; while among 91 sporadic patients with DAA therapy before HCV micro-elimination campaign, 78 (85.7%) received 12 wk of sofosbuvir/Ledipasvir and 13 (14.3%) received 8-12 wk of glecaprevir/pibrentasvir according to the Taiwan HCV guideline[12,13].\\nIn ITT analysis, the overall SVR12 rate was 95.4% (289/303) with comparable SVR12 rates between sporadic HCV control group (94.5%, 86/91) and HCV micro-elimination group (95.8%, 203/212, P = 0.126, Table 2).\\nVirological responses of hepatitis C virus-infected patients receiving directly-acting antivirals therapy before and during campaign of hepatitis C virus micro-elimination in Penghu prison in Penghu prison\\nOne missing data.\\nOne transferred; One missing data.\\nTwo transferred; One released.\\nFour released.\\nOne relapser.\\nFour transferred; Five released. HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \\nDuring DAA treatment period, all of patients in sporadic HCV control group completed DAA therapy, while 3 patients in HCV micro-elimination group lost-to-follow (2 transferred; 1 released). During the post-treatment follow-up period, 4 patients in sporadic HCV control group lost-to-follow (4 released), while 6 patients in HCV micro-elimination group lost-to-follow (2 transferred; 4 released). In PP analysis, the overall SVR12 rate was 99.7% (289/290) with comparable SVR12 rates between sporadic HCV control group (98.9%, 86/87) and HCV micro-elimination group (100%, 203/203, P = 0.126, Table 2). Only one patient experienced virological failure (54 years old male, treatment-naïve, HCV-GT3 infection with baseline viral loads of 62,883 IU/mL and FIB-4 of 2.37; relapsed from a 12 wk regimen of glecaprevir/pibrentasvir).\",\n \"The safety profiles of both groups were shown in Table 3. None of patients had treatment discontinuation other than released or transferred. None experienced serious adverse event. The frequency of adverse events was 4.3% (4/91) and 1.4% (3/212), respectively, among patients in sporadic control group and HCV micro-elimination group. The most reported adverse events were rash in 3 of 13 (23.1%) patients treated with glecaprevir/pibrentasvir and pruritus in 2 of 212 (0.9%) patients treated with sofosbuvir/velpatasvir. None of patients experienced grade 3 or 4 Laboratory abnormality.\\nSafety profiles of hepatitis C virus-infected patients receiving direct-acting antivirals therapy in Penghu prison\\nDAA: Directly-acting antivirals; HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \",\n \"In the current study, we demonstrated that mass screening combined with onsite group therapy by using a simplified pan-genotypic DAA regimen, 12 wk of sofosbuvir/velpatasvir, provides an “one-size fits all” solution toward the achievement of HCV micro-elimination in prisoners. The SVR rate was 95.6% in ITT population and 100% in PP population after excluding the inmates released or transferred before end-of-follow-up. The high SVR rate was observed in this PWID-dominant population, which HCV genotype distribution was diverse, including genotypes 1a, 1b, 2, 3 and 6. \\nRecent advance in the development of IFN-free pan-genotypic DAA regimens has remarkably improved the treatment efficacy with an overall SVR rates of > 90%. Therefore, WHO set the global of HCV elimination by 2030, through the achievement of > 90% diagnosis rate and > 80% treatment rate for eligible patients[16]. Nevertheless, there are many barriers in each HCV care cascade toward HCV elimination at the population level[11,23]. To overcome the barriers, combining the concept of micro-elimination and an outreach strategy with immediate onsite treatment would be a more efficient and practical approach to achieve that goal[18,24]. The current study compared the HCV-infected inmates identified sporadically in outpatient clinics of Penghu Prison from 2017 to 2019 before mass screening and the patients identified by mass screening. We found that mass screening identified 208 HCV-viremic patients in a 5 d screening program from 1137 inmates (encountered around two-third of total inmates in Penghu Prison), compared to 91 HCV-viremic inmates treated in outpatient clinics from 2017 to September 2019. Our results demonstrated that mass screening with immediate onsite treatment provide much more efficient and practical solution to overcome the gaps of disease awareness and link-to-care in the HCV care cascades toward HCV micro-elimination in prisoners. In addition, we implemented “HCV reflex testing” in the mass screening program to scale-up and speed-up the diagnosis and link-to-care for treatment uptake of HCV infections[25].\\nPWID is known as the major risk factor of HCV infection and transmission. Although the anti-HCV prevalence in PWID prisoners decreased from 91% in 2014 to 34.8% in 2019 by the strategy of safe injection in Taiwan[21], almost all (97.6%) of HCV-infected prisoners were PWID. Given the lack of vaccine available and high risk of transmission, the strategy of universal screening and concept of “treatment as prevention” are the keys to HCV elimination in prison as well as PWID.\\nWe observed that the sporadic HCV-infected prisoners identified in outpatient clinics had significantly higher proportion of comorbidities, including diabetes, hypertension, hyperlipidemia and eGFR, than those participating in the HCV micro-elimination campaign. It implicated that a great proportion of identified sporadically in outpatient clinics were due to concomitant morbidities; by contrast, many HCV-infected patients were unaware to their HCV diseases. In our mass screening, only 36.6% (145/396) of HCV-infected prisoners were aware of HCV infection before screening[21]. It indicates that the implementation of an outreach strategy with universal mass screen is necessary for HCV micro-elimination in prison.\\nDespite of the advances in the management of HCV infections, DAA therapy in incarcerated HCV-infected people remains many obstacles to be resolved, including disease unawareness, lack of updated information about the benefits of new DAA treatment, uncertainty of treatment right[26], poor accessibility due to of onsite treatment facilities or HCV treaters. Another difficulty for HCV treatment in prisoners is the unexpected or scheduled releasing from prison or transferring to other prisons, which frequently leads to the interruption of treatment or lost-to- follow up[20,27]. We are lucky that the Taiwan Health Insurance covered all incarcerated people, including all of the laboratory tests and ultrasound sonography and the cost of DAA regimens. Each prison has a contracted hospital providing point-of-care facility. Before initiating DAA therapy, we excluded the patients with expected release or transfer within 24 wk, and negotiated with the authority to avoid unnecessary transferring to other prisons during the period of HCV treatment and follow-up once the inmates entering the DAA course. Eventually we achieved a high treatment rate of 90.6% (212/234) and a high treatment complete rate of 95.8% (203/212), with a high cure rate at 100% (212/212).\\nBefore the IFN-free DAA available, the lower SVR rate, much longer treatment duration and frequent adverse events of IFN-based treatment discouraged HCV-infected prisoners from receiving treatment[10]. IFN-free DAA regimens revolutionized HCV treatment which has largely extended the indication for various HCV-infected patients. Nevertheless, the application of typical DAA regimens are based on HCV genotype, presence of decompensated cirrhosis, renal function, and prior treatment experience. The two pangenotypic DAA regimens, sofosbuvir/velpatasvir, and glecaprevir/pibrentasvir, have achieved very high SVR rates of > 95%, regardless of HCV GTs, except for treatment-experienced cirrhotic HCV GT3 patients or GT3b patients[8,12,13]. Recently, to improve the access to anti-HCV therapy, reduce the cost of laboratory tests and the relative complexity of genotype-based treatment strategies, simplified treatment without many information needed for treatment decision are recommended to facilitate the care cascade among populations who are historically less engaged in healthcare, such as PWIDs and prisoners[8]. EASL recommends simplified, genotyping/subtyping-free regimens for IFN-free DAA treatment-naïve (except sofosbuvir plus ribavirin), HCV-infected or HCV-HIV coinfected adolescent and adult patients without cirrhosis or with compensated cirrhosis, regardless of HCV genotypes[8]. These recommendations are a universal 12 wk regimen of sofosbuvir/velpatasvir for all patients or glecaprevir/pibrentasvir, 8 wk for non-cirrhotic, 12 wk for compensated cirrhotic, and 16 wk for HCV GT3 patients, respectively. There are only four information needed before treatment, including the presence of HCV viremia, potential drug-drug interactions, and prior treatment experience, and presence of cirrhosis. The advantages of glecaprevir/pibrentasvir is a shorter 8 wk regimen for treatment-naïve HCV patients and IFN-experienced non-cirrhotic patients with compensated liver diseases, which would be benefit for prisoners who are expected to be released or transferred in a short term. However, glecaprevir, a protease inhibitor, is contraindicated for patients with hepatic decompensation and at risk for rare occurrence of serious drug-induced liver injury[28]. Also, glecaprevir/ pibrentasvir has higher pill burden, three tablets a d. The advantages of sofosbuvir/ velpatasvir include a universal fixed 12 wk regimen, one tablet a d, for all HCV patients with compensated liver diseases, less frequency of potential drug-drug interactions[29], and safety for those with hepatic decompensation. However, a 12 wk regimen with sofosbuvir/velpatasvir needs one more visit and monitoring when compared to an 8 wk regimen with glecaprevir/pibrentasvir. Therefore, we select sofosbuvir/velpatasvir as the antiviral regimen for our outreach onsite treatment. In our study, all HCV-viremic prisoners fit the criteria of simplified, genotyping/ subtyping-free regimens, except one who failed to prior glecaprevir/pibrentasvir therapy and was not enrolled for sofosbuvir/velpatasvir treatment. In our PP analysis, the overall SVR12 rate was comparable between HCV patient group (98.9%, 86/87) and HCV micro-elimination group (100%, 203/203). Our study provided evidence for the concept that simplified, genotyping/subtyping-free regimens can achieve high SVR12 rate in HCV-infected PWID-dominant prisoners.\\nIn our study, none of prisoners had DAA treatment discontinuation due to adverse events. None experienced serious adverse event. These data indicated that the simplified, genotyping/subtyping-free regimen, sofosbuvir/velpatasvir, was safe and well tolerated for HCV-infected PWID-dominant prisoners. Very few adverse events were reported in both groups, whatever using sofosbuvir/Ledipasvir, glecaprevir/ pibrentasvir and sofosbuvir/velpatasvir, when compared to the data from clinical trials[30,31]. It might be due to that current population was younger and less patients with advanced fibrosis or chronic kidney diseases.\\nThere were some limitations in our study. First, not all inmates in Penghu Prison participated our mass screening. Strategies and policy to encourage inmates to receive HCV screening is mandatory to achieve the goal of WHO. Second, unexpected prisoners’ transferral and release could not be completely avoided, which caused incomplete treatment and follow-up. Successfully linking the released or transferred people to another HCV treaters could help completing HCV treatment and follow-up. Third, there was no reimbursement for the retreatment of prior DAA failed patients in Taiwan at the time of the current study.\",\n \"Well-designed strategies for mass screening and treatment for HCV-infected prisoners can be implemented successfully by the collaboration between physicians and prison authorities. We demonstrated that mass screening followed by immediate onsite treatment with a simplified pangenotypic DAA regimen, sofosbuvir/velpatasvir, provides successful strategies toward HCV micro-elimination among prisoners.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","MATERIALS AND METHODS","Patients linked to onsite treatment program for HCV micro-elimination","HCV-viremic patients identified by a universal mass screening","HCV-viremic patients identified in outpatient clinics during the period of HCV mass screening","Patients identified and treated by DAAs in outpatient clinics before mass screening","Assessment, monitoring and endpoints","Statistical analyses.","RESULTS","Patient flowchart of HCV micro-elimination campaign","Patient characteristics ","Treatment efficacy","Safety profiles","DISCUSSION","CONCLUSION"],"string":"[\n \"INTRODUCTION\",\n \"MATERIALS AND METHODS\",\n \"Patients linked to onsite treatment program for HCV micro-elimination\",\n \"HCV-viremic patients identified by a universal mass screening\",\n \"HCV-viremic patients identified in outpatient clinics during the period of HCV mass screening\",\n \"Patients identified and treated by DAAs in outpatient clinics before mass screening\",\n \"Assessment, monitoring and endpoints\",\n \"Statistical analyses.\",\n \"RESULTS\",\n \"Patient flowchart of HCV micro-elimination campaign\",\n \"Patient characteristics \",\n \"Treatment efficacy\",\n \"Safety profiles\",\n \"DISCUSSION\",\n \"CONCLUSION\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Hepatitis C virus (HCV) infection is a progressive and blood-borne infectious disease that can lead to end stage liver diseases, such as hepatic decompensation, liver cirrhosis, and hepatocellular carcinoma[1,2]. Iatrogenic transmission of HCV, such as blood transfusion and surgery, has decreased in developed countries. Whereas people who inject drugs (PWID) has become the major population of HCV transmission, which could consist of approximately 80% of HCV-infected patients[3]. Given that lack of vaccine available, “treatment as prevention” for HCV transmission in PWID is very important for HCV elimination.\nPrisoners are at high risk of HCV infection, with prevalence rates ranging from 3.1% to 38%[4,5]. The high prevalence of HCV infection in prisoners is resulted from unsafe lifestyles, psychiatric disorders, and social problems before they are incarcerated. Recently, PWID has been the most important risk factor of HCV infection in prisoners[6]. The anti-HCV prevalence rate could be as high as 91% among PWID prisoners[7]. Screening and eliminating HCV infection in prisoners is therefore an important social health issue.\nAccording to the American Association for the Study of Liver Diseases and European Association for the Study of the Liver (EASL) guidelines, all HCV viremic patients should be treated if life span is expected more than one year[8,9]. HCV therapeutic strategies have been revolutionized significantly because of the availability of direct-acting antivirals (DAA)[10]. Interferon (IFN)-based regimens for HCV infection have serious side effects, long therapeutic duration, and contraindications, leading to the huge gaps in HCV care cascade[11]. The current IFN-free DAA regimens provide shorter treatment duration, very high treatment efficacy and safety profiles, not only for general population[12], but also for special populations[13], such as HCV/human immunodeficiency virus (HIV) coinfected patients, hepatitis B virus (HBV)/HCV coinfected patients and patients with chronic kidney diseases in real-world clinical settings[14,15].\nWorld Health Organization (WHO) set a global goal of HCV elimination by 2030[16], and Taiwan authority is even ambitious by 2025[17]. To achieve the goal, implementation of the concept of HCV micro-elimination is regarding as an efficient and practical strategy[18]. We have proved that “universal mass screening plus outreach onsite treatment” is the key to achieve HCV micro-elimination among patients under maintenance hemodialysis[19].\nRecently, the latest EASL HCV guideline recommended simplified, genotyping/ subtyping-free, pangenotypic anti-HCV treatment, either sofosbuvir/ velpatasvir or glecaprevir/pibrentasvir, to increase the accessibility and global cure rates among patients with > 12 years, chronic hepatitis C without cirrhosis or with compensated cirrhosis, with or without HIV co-infection, whatever treatment-naïve or IFN-experienced[8].\nSince HCV treatment is not frequently administered to prisoners due to unawareness of HCV infection, difficultly management, easily loss to follow-up, and lack of hepatologist in prison[20], collaboration between hepatologists and prison authorities to carry out strategies for HCV diagnosis and treatment in prisoners in highly demanded. Herein, we implemented an outreach strategy in combination with universal mass screen and onsite treatment with a simplified pan-genotypic DAA regimen, 12 wk of sofosbuvir/velpatasvir, toward HCV micro-elimination in a PWID-dominant prison in Taiwan.","Patients linked to onsite treatment program for HCV micro-elimination HCV-viremic patients were recruited from two cohorts in Penghu Prison (Agency of Corrections, Ministry of Justice, Taiwan), a PWID-dominant prison (Figure 1).\n\nPatient flowchart of hepatitis C virus treatment with a simplified pan-genotypic directly-acting antivirals regimen in Penghu Prison. HCV: Hepatitis C virus; DAA: Directly-acting antivirals; SOL/VEL: Sofosbuvir/velpatasvir; GEL/PIB: Glecaprevir/pibrentasvir; EOTVR: Virological response at end-of-treatment; SVR12: Sustained viral response at post-treatment wk 12.\nHCV-viremic patients were recruited from two cohorts in Penghu Prison (Agency of Corrections, Ministry of Justice, Taiwan), a PWID-dominant prison (Figure 1).\n\nPatient flowchart of hepatitis C virus treatment with a simplified pan-genotypic directly-acting antivirals regimen in Penghu Prison. HCV: Hepatitis C virus; DAA: Directly-acting antivirals; SOL/VEL: Sofosbuvir/velpatasvir; GEL/PIB: Glecaprevir/pibrentasvir; EOTVR: Virological response at end-of-treatment; SVR12: Sustained viral response at post-treatment wk 12.\nHCV-viremic patients identified by a universal mass screening In September 2019, we conducted a 5 d universal mass screening of viral hepatitis in Penghu Prison. These inclusion criteria were prisoners, who were at least 20 years old, being willing to enter the study for screening of viral hepatitis. The study of mass screening was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (IRB: KMUHIRB-SV(I)-20190033). All participants provided written informed consents. A total of 1137 subjects from 1697 inmates participated the mass screening[21]. Among them, 396 (34.8%) subjects had anti-HCV seropositivity; 208 (52.5%) of the 396 subjects were seropositive for HCV ribonucleic acid (RNA) and linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\nIn September 2019, we conducted a 5 d universal mass screening of viral hepatitis in Penghu Prison. These inclusion criteria were prisoners, who were at least 20 years old, being willing to enter the study for screening of viral hepatitis. The study of mass screening was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (IRB: KMUHIRB-SV(I)-20190033). All participants provided written informed consents. A total of 1137 subjects from 1697 inmates participated the mass screening[21]. Among them, 396 (34.8%) subjects had anti-HCV seropositivity; 208 (52.5%) of the 396 subjects were seropositive for HCV ribonucleic acid (RNA) and linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\nHCV-viremic patients identified in outpatient clinics during the period of HCV mass screening Another 26 HCV-viremic subjects identified in outpatient clinics of Penghu Prison between August to December 2019 were also linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\nAll patients received pretreatment evaluation in December 2019, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions. A 12 wk, oral pan-genotypic regimen of sofosbuvir/velpatasvir 400/100 mg fixed-dose combination once daily was initiated in January-February 2020. \nAnother 26 HCV-viremic subjects identified in outpatient clinics of Penghu Prison between August to December 2019 were also linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\nAll patients received pretreatment evaluation in December 2019, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions. A 12 wk, oral pan-genotypic regimen of sofosbuvir/velpatasvir 400/100 mg fixed-dose combination once daily was initiated in January-February 2020. \nPatients identified and treated by DAAs in outpatient clinics before mass screening A total of 91 HCV-viremic patients identified in outpatient clinics of Penghu Prison and treated with DAA before mass screening from 2017 to 2019 were enrolled as a control. The selection of DAA regimens were based on physician’s discretion according to the viral genotype and criteria of reimbursement of National Health Insurance Administration, Taiwan. All patients received pretreatment evaluation, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions.\nAll participants signed informed consent forms. These enrolled inmates of our study were protected according to the guidelines of the Declaration of Helsinki. The current study of DAA therapy was approved by the Institutional Review Board of Tri-Service General Hospital (IRB: TSGHIRB 2-107-05-080).\nA total of 91 HCV-viremic patients identified in outpatient clinics of Penghu Prison and treated with DAA before mass screening from 2017 to 2019 were enrolled as a control. The selection of DAA regimens were based on physician’s discretion according to the viral genotype and criteria of reimbursement of National Health Insurance Administration, Taiwan. All patients received pretreatment evaluation, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions.\nAll participants signed informed consent forms. These enrolled inmates of our study were protected according to the guidelines of the Declaration of Helsinki. The current study of DAA therapy was approved by the Institutional Review Board of Tri-Service General Hospital (IRB: TSGHIRB 2-107-05-080).\nAssessment, monitoring and endpoints Anti-HCV antibody was determined by the third generation, commercially available immunoassay (Ax SYM HCV III; Abbott Laboratories, North Chicago, IL). HCV RNA viral loads and genotype were determined by real-time PCR assays [RealTime HCV; Abbott Molecular, Des Plaines IL, United States; detection limit: 12 IU/mL])[22]. Liver cirrhosis was defined by the presence of clinical, radiological, endoscopic or laboratory evidence of cirrhosis and/or portal hypertension or fibrosis-4 index (FIB-4) (> 6.5). Laboratory data monitoring and assessment of side effects were performed at treatment wk 2, 4, 8 and end-of-treatment (EOT), and 12 wk after EOT.\nThe primary endpoint was sustained virological response (SVR12, defined as undetectable HCV RNA throughout 12 wk of the post-treatment follow-up period).\nAnti-HCV antibody was determined by the third generation, commercially available immunoassay (Ax SYM HCV III; Abbott Laboratories, North Chicago, IL). HCV RNA viral loads and genotype were determined by real-time PCR assays [RealTime HCV; Abbott Molecular, Des Plaines IL, United States; detection limit: 12 IU/mL])[22]. Liver cirrhosis was defined by the presence of clinical, radiological, endoscopic or laboratory evidence of cirrhosis and/or portal hypertension or fibrosis-4 index (FIB-4) (> 6.5). Laboratory data monitoring and assessment of side effects were performed at treatment wk 2, 4, 8 and end-of-treatment (EOT), and 12 wk after EOT.\nThe primary endpoint was sustained virological response (SVR12, defined as undetectable HCV RNA throughout 12 wk of the post-treatment follow-up period).\nStatistical analyses. The efficacy of all DAA regimens was determined in a intent-to-treat (ITT) population (all enrolled patients with at least one dose of DAA) and a per-protocol (PP) population (subjects receiving at least one dose of DAA and retained in Penghu Prison throughout the DAA treatment and follow-up period). Safety assessments reported adverse event (AE), serious adverse event (SAE) and laboratory abnormalities in the ITT population. Continuous variables are expressed as means ± standard deviation (SD), and categorical variables are expressed as percentages. The differences of continuous variables are estimated by the Student’s t test. The differences in categorical variables are analyzed using the Chi-square test. The on-treatment and off-treatment virological response rates were analyzed in number and percentages with 95% confidence interval (CI). All data analyses were performed using the SPSS software version 18.0 (SPSS Inc., Chicago, Illinois, United States).\nThe efficacy of all DAA regimens was determined in a intent-to-treat (ITT) population (all enrolled patients with at least one dose of DAA) and a per-protocol (PP) population (subjects receiving at least one dose of DAA and retained in Penghu Prison throughout the DAA treatment and follow-up period). Safety assessments reported adverse event (AE), serious adverse event (SAE) and laboratory abnormalities in the ITT population. Continuous variables are expressed as means ± standard deviation (SD), and categorical variables are expressed as percentages. The differences of continuous variables are estimated by the Student’s t test. The differences in categorical variables are analyzed using the Chi-square test. The on-treatment and off-treatment virological response rates were analyzed in number and percentages with 95% confidence interval (CI). All data analyses were performed using the SPSS software version 18.0 (SPSS Inc., Chicago, Illinois, United States).","HCV-viremic patients were recruited from two cohorts in Penghu Prison (Agency of Corrections, Ministry of Justice, Taiwan), a PWID-dominant prison (Figure 1).\n\nPatient flowchart of hepatitis C virus treatment with a simplified pan-genotypic directly-acting antivirals regimen in Penghu Prison. HCV: Hepatitis C virus; DAA: Directly-acting antivirals; SOL/VEL: Sofosbuvir/velpatasvir; GEL/PIB: Glecaprevir/pibrentasvir; EOTVR: Virological response at end-of-treatment; SVR12: Sustained viral response at post-treatment wk 12.","In September 2019, we conducted a 5 d universal mass screening of viral hepatitis in Penghu Prison. These inclusion criteria were prisoners, who were at least 20 years old, being willing to enter the study for screening of viral hepatitis. The study of mass screening was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (IRB: KMUHIRB-SV(I)-20190033). All participants provided written informed consents. A total of 1137 subjects from 1697 inmates participated the mass screening[21]. Among them, 396 (34.8%) subjects had anti-HCV seropositivity; 208 (52.5%) of the 396 subjects were seropositive for HCV ribonucleic acid (RNA) and linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.","Another 26 HCV-viremic subjects identified in outpatient clinics of Penghu Prison between August to December 2019 were also linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\nAll patients received pretreatment evaluation in December 2019, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions. A 12 wk, oral pan-genotypic regimen of sofosbuvir/velpatasvir 400/100 mg fixed-dose combination once daily was initiated in January-February 2020. ","A total of 91 HCV-viremic patients identified in outpatient clinics of Penghu Prison and treated with DAA before mass screening from 2017 to 2019 were enrolled as a control. The selection of DAA regimens were based on physician’s discretion according to the viral genotype and criteria of reimbursement of National Health Insurance Administration, Taiwan. All patients received pretreatment evaluation, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions.\nAll participants signed informed consent forms. These enrolled inmates of our study were protected according to the guidelines of the Declaration of Helsinki. The current study of DAA therapy was approved by the Institutional Review Board of Tri-Service General Hospital (IRB: TSGHIRB 2-107-05-080).","Anti-HCV antibody was determined by the third generation, commercially available immunoassay (Ax SYM HCV III; Abbott Laboratories, North Chicago, IL). HCV RNA viral loads and genotype were determined by real-time PCR assays [RealTime HCV; Abbott Molecular, Des Plaines IL, United States; detection limit: 12 IU/mL])[22]. Liver cirrhosis was defined by the presence of clinical, radiological, endoscopic or laboratory evidence of cirrhosis and/or portal hypertension or fibrosis-4 index (FIB-4) (> 6.5). Laboratory data monitoring and assessment of side effects were performed at treatment wk 2, 4, 8 and end-of-treatment (EOT), and 12 wk after EOT.\nThe primary endpoint was sustained virological response (SVR12, defined as undetectable HCV RNA throughout 12 wk of the post-treatment follow-up period).","The efficacy of all DAA regimens was determined in a intent-to-treat (ITT) population (all enrolled patients with at least one dose of DAA) and a per-protocol (PP) population (subjects receiving at least one dose of DAA and retained in Penghu Prison throughout the DAA treatment and follow-up period). Safety assessments reported adverse event (AE), serious adverse event (SAE) and laboratory abnormalities in the ITT population. Continuous variables are expressed as means ± standard deviation (SD), and categorical variables are expressed as percentages. The differences of continuous variables are estimated by the Student’s t test. The differences in categorical variables are analyzed using the Chi-square test. The on-treatment and off-treatment virological response rates were analyzed in number and percentages with 95% confidence interval (CI). All data analyses were performed using the SPSS software version 18.0 (SPSS Inc., Chicago, Illinois, United States).","Patient flowchart of HCV micro-elimination campaign The patient flowchart of HCV mass screen, assessment and treatment was shown in Figure 1. A total of 234 HCV-viremic patients, 208 from mass screening and 26 from outpatient clinics in Penghu Prison were assessed for eligibility of group therapy with sofosbuvir/velpatasvir in December 2019. Twenty-two patients were excluded from anti-HCV therapy due to scheduled to be released from jail (n = 16) or transferred to other jails (n = 3) within 6 mo, unwilling to receive therapy (n = 2) and prior glecaprevir/pibrentasvir treatment failure (n = 1). Finally, 212 patients were recruited for sofosbuvir/velpatasvir therapy initiated in January-February 2020.\nThe patient flowchart of HCV mass screen, assessment and treatment was shown in Figure 1. A total of 234 HCV-viremic patients, 208 from mass screening and 26 from outpatient clinics in Penghu Prison were assessed for eligibility of group therapy with sofosbuvir/velpatasvir in December 2019. Twenty-two patients were excluded from anti-HCV therapy due to scheduled to be released from jail (n = 16) or transferred to other jails (n = 3) within 6 mo, unwilling to receive therapy (n = 2) and prior glecaprevir/pibrentasvir treatment failure (n = 1). Finally, 212 patients were recruited for sofosbuvir/velpatasvir therapy initiated in January-February 2020.\nPatient characteristics The baseline characteristics of 303 HCV-viremic patients, including 212 in HCV micro-elimination campaign and 91 sporadic controls from outpatient clinics before micro-elimination campaign were listed in Table 1. They mean age was 48.4 years with male dominant (99.7%). Thirty (9.9%) had HBV coinfection. The mean FIB-4 was 1.3, with 20 (6.6%) had advanced fibrosis (FIB-4 > 3.25). Only one patient (0.3%) had liver cirrhosis. The mean HCV RNA levels was 6.5 Logs IU/mL, dominant with HCV genotype 1 (HCV-GT1, 42.2%), followed by HCV-GT6 (35.3%), HCV-GT3 (11.6%) and HCV-GT2 (10.6%). Three (1%) patients were prior IFN-experienced. The two groups had comparable characteristics in terms of age, gender, HBV co-infection, liver and renal function tests, FIB-4 score, HCV genotype distribution, and prior history of IFN-based therapy. However, the sporadic patients identified in outpatient clinics had significantly higher proportion of comorbidities, including diabetes, hypertension, hyperlipidemia and an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2, but significantly lower HCV viral loads. None of patient had decompensated cirrhosis nor liver cancer.\nBaseline characteristics of hepatitis C virus-infected patients receiving directly-acting antivirals therapy between sporadic hepatitis C virus therapy in outpatient clinics and campaign of hepatitis C virus micro-elimination in Penghu prison\n56 patients did not have body mass index information (12 patients before campaign of hepatitis C virus (HCV) micro-elimination; 44 patients in campaign of HCV micro-elimination).\n\nP < 0.05. DAA: Directly-acting antivirals; HCV: Hepatitis C virus; BMI: Body mass index; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; LC: Liver cirrhosis; FIB-4: Fibrosis-4 index; HBsAg: Hepatitis B surface antigen; eGFR: Estimated glomerular filtration rate (mL/min/1.73 m2); SOF: Sofosbuvir; VEL: Velpatasvir; LDV: Ledipasvir; GLE: Glecaprevir; PIB: Pibrentasvir; IFN: Interferon.\nThe baseline characteristics of 303 HCV-viremic patients, including 212 in HCV micro-elimination campaign and 91 sporadic controls from outpatient clinics before micro-elimination campaign were listed in Table 1. They mean age was 48.4 years with male dominant (99.7%). Thirty (9.9%) had HBV coinfection. The mean FIB-4 was 1.3, with 20 (6.6%) had advanced fibrosis (FIB-4 > 3.25). Only one patient (0.3%) had liver cirrhosis. The mean HCV RNA levels was 6.5 Logs IU/mL, dominant with HCV genotype 1 (HCV-GT1, 42.2%), followed by HCV-GT6 (35.3%), HCV-GT3 (11.6%) and HCV-GT2 (10.6%). Three (1%) patients were prior IFN-experienced. The two groups had comparable characteristics in terms of age, gender, HBV co-infection, liver and renal function tests, FIB-4 score, HCV genotype distribution, and prior history of IFN-based therapy. However, the sporadic patients identified in outpatient clinics had significantly higher proportion of comorbidities, including diabetes, hypertension, hyperlipidemia and an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2, but significantly lower HCV viral loads. None of patient had decompensated cirrhosis nor liver cancer.\nBaseline characteristics of hepatitis C virus-infected patients receiving directly-acting antivirals therapy between sporadic hepatitis C virus therapy in outpatient clinics and campaign of hepatitis C virus micro-elimination in Penghu prison\n56 patients did not have body mass index information (12 patients before campaign of hepatitis C virus (HCV) micro-elimination; 44 patients in campaign of HCV micro-elimination).\n\nP < 0.05. DAA: Directly-acting antivirals; HCV: Hepatitis C virus; BMI: Body mass index; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; LC: Liver cirrhosis; FIB-4: Fibrosis-4 index; HBsAg: Hepatitis B surface antigen; eGFR: Estimated glomerular filtration rate (mL/min/1.73 m2); SOF: Sofosbuvir; VEL: Velpatasvir; LDV: Ledipasvir; GLE: Glecaprevir; PIB: Pibrentasvir; IFN: Interferon.\nTreatment efficacy All of 212 patients in HCV micro-elimination campaign received sofosbuvir/ velpatasvir treatment; while among 91 sporadic patients with DAA therapy before HCV micro-elimination campaign, 78 (85.7%) received 12 wk of sofosbuvir/Ledipasvir and 13 (14.3%) received 8-12 wk of glecaprevir/pibrentasvir according to the Taiwan HCV guideline[12,13].\nIn ITT analysis, the overall SVR12 rate was 95.4% (289/303) with comparable SVR12 rates between sporadic HCV control group (94.5%, 86/91) and HCV micro-elimination group (95.8%, 203/212, P = 0.126, Table 2).\nVirological responses of hepatitis C virus-infected patients receiving directly-acting antivirals therapy before and during campaign of hepatitis C virus micro-elimination in Penghu prison in Penghu prison\nOne missing data.\nOne transferred; One missing data.\nTwo transferred; One released.\nFour released.\nOne relapser.\nFour transferred; Five released. HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \nDuring DAA treatment period, all of patients in sporadic HCV control group completed DAA therapy, while 3 patients in HCV micro-elimination group lost-to-follow (2 transferred; 1 released). During the post-treatment follow-up period, 4 patients in sporadic HCV control group lost-to-follow (4 released), while 6 patients in HCV micro-elimination group lost-to-follow (2 transferred; 4 released). In PP analysis, the overall SVR12 rate was 99.7% (289/290) with comparable SVR12 rates between sporadic HCV control group (98.9%, 86/87) and HCV micro-elimination group (100%, 203/203, P = 0.126, Table 2). Only one patient experienced virological failure (54 years old male, treatment-naïve, HCV-GT3 infection with baseline viral loads of 62,883 IU/mL and FIB-4 of 2.37; relapsed from a 12 wk regimen of glecaprevir/pibrentasvir).\nAll of 212 patients in HCV micro-elimination campaign received sofosbuvir/ velpatasvir treatment; while among 91 sporadic patients with DAA therapy before HCV micro-elimination campaign, 78 (85.7%) received 12 wk of sofosbuvir/Ledipasvir and 13 (14.3%) received 8-12 wk of glecaprevir/pibrentasvir according to the Taiwan HCV guideline[12,13].\nIn ITT analysis, the overall SVR12 rate was 95.4% (289/303) with comparable SVR12 rates between sporadic HCV control group (94.5%, 86/91) and HCV micro-elimination group (95.8%, 203/212, P = 0.126, Table 2).\nVirological responses of hepatitis C virus-infected patients receiving directly-acting antivirals therapy before and during campaign of hepatitis C virus micro-elimination in Penghu prison in Penghu prison\nOne missing data.\nOne transferred; One missing data.\nTwo transferred; One released.\nFour released.\nOne relapser.\nFour transferred; Five released. HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \nDuring DAA treatment period, all of patients in sporadic HCV control group completed DAA therapy, while 3 patients in HCV micro-elimination group lost-to-follow (2 transferred; 1 released). During the post-treatment follow-up period, 4 patients in sporadic HCV control group lost-to-follow (4 released), while 6 patients in HCV micro-elimination group lost-to-follow (2 transferred; 4 released). In PP analysis, the overall SVR12 rate was 99.7% (289/290) with comparable SVR12 rates between sporadic HCV control group (98.9%, 86/87) and HCV micro-elimination group (100%, 203/203, P = 0.126, Table 2). Only one patient experienced virological failure (54 years old male, treatment-naïve, HCV-GT3 infection with baseline viral loads of 62,883 IU/mL and FIB-4 of 2.37; relapsed from a 12 wk regimen of glecaprevir/pibrentasvir).\nSafety profiles The safety profiles of both groups were shown in Table 3. None of patients had treatment discontinuation other than released or transferred. None experienced serious adverse event. The frequency of adverse events was 4.3% (4/91) and 1.4% (3/212), respectively, among patients in sporadic control group and HCV micro-elimination group. The most reported adverse events were rash in 3 of 13 (23.1%) patients treated with glecaprevir/pibrentasvir and pruritus in 2 of 212 (0.9%) patients treated with sofosbuvir/velpatasvir. None of patients experienced grade 3 or 4 Laboratory abnormality.\nSafety profiles of hepatitis C virus-infected patients receiving direct-acting antivirals therapy in Penghu prison\nDAA: Directly-acting antivirals; HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \nThe safety profiles of both groups were shown in Table 3. None of patients had treatment discontinuation other than released or transferred. None experienced serious adverse event. The frequency of adverse events was 4.3% (4/91) and 1.4% (3/212), respectively, among patients in sporadic control group and HCV micro-elimination group. The most reported adverse events were rash in 3 of 13 (23.1%) patients treated with glecaprevir/pibrentasvir and pruritus in 2 of 212 (0.9%) patients treated with sofosbuvir/velpatasvir. None of patients experienced grade 3 or 4 Laboratory abnormality.\nSafety profiles of hepatitis C virus-infected patients receiving direct-acting antivirals therapy in Penghu prison\nDAA: Directly-acting antivirals; HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. ","The patient flowchart of HCV mass screen, assessment and treatment was shown in Figure 1. A total of 234 HCV-viremic patients, 208 from mass screening and 26 from outpatient clinics in Penghu Prison were assessed for eligibility of group therapy with sofosbuvir/velpatasvir in December 2019. Twenty-two patients were excluded from anti-HCV therapy due to scheduled to be released from jail (n = 16) or transferred to other jails (n = 3) within 6 mo, unwilling to receive therapy (n = 2) and prior glecaprevir/pibrentasvir treatment failure (n = 1). Finally, 212 patients were recruited for sofosbuvir/velpatasvir therapy initiated in January-February 2020.","The baseline characteristics of 303 HCV-viremic patients, including 212 in HCV micro-elimination campaign and 91 sporadic controls from outpatient clinics before micro-elimination campaign were listed in Table 1. They mean age was 48.4 years with male dominant (99.7%). Thirty (9.9%) had HBV coinfection. The mean FIB-4 was 1.3, with 20 (6.6%) had advanced fibrosis (FIB-4 > 3.25). Only one patient (0.3%) had liver cirrhosis. The mean HCV RNA levels was 6.5 Logs IU/mL, dominant with HCV genotype 1 (HCV-GT1, 42.2%), followed by HCV-GT6 (35.3%), HCV-GT3 (11.6%) and HCV-GT2 (10.6%). Three (1%) patients were prior IFN-experienced. The two groups had comparable characteristics in terms of age, gender, HBV co-infection, liver and renal function tests, FIB-4 score, HCV genotype distribution, and prior history of IFN-based therapy. However, the sporadic patients identified in outpatient clinics had significantly higher proportion of comorbidities, including diabetes, hypertension, hyperlipidemia and an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2, but significantly lower HCV viral loads. None of patient had decompensated cirrhosis nor liver cancer.\nBaseline characteristics of hepatitis C virus-infected patients receiving directly-acting antivirals therapy between sporadic hepatitis C virus therapy in outpatient clinics and campaign of hepatitis C virus micro-elimination in Penghu prison\n56 patients did not have body mass index information (12 patients before campaign of hepatitis C virus (HCV) micro-elimination; 44 patients in campaign of HCV micro-elimination).\n\nP < 0.05. DAA: Directly-acting antivirals; HCV: Hepatitis C virus; BMI: Body mass index; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; LC: Liver cirrhosis; FIB-4: Fibrosis-4 index; HBsAg: Hepatitis B surface antigen; eGFR: Estimated glomerular filtration rate (mL/min/1.73 m2); SOF: Sofosbuvir; VEL: Velpatasvir; LDV: Ledipasvir; GLE: Glecaprevir; PIB: Pibrentasvir; IFN: Interferon.","All of 212 patients in HCV micro-elimination campaign received sofosbuvir/ velpatasvir treatment; while among 91 sporadic patients with DAA therapy before HCV micro-elimination campaign, 78 (85.7%) received 12 wk of sofosbuvir/Ledipasvir and 13 (14.3%) received 8-12 wk of glecaprevir/pibrentasvir according to the Taiwan HCV guideline[12,13].\nIn ITT analysis, the overall SVR12 rate was 95.4% (289/303) with comparable SVR12 rates between sporadic HCV control group (94.5%, 86/91) and HCV micro-elimination group (95.8%, 203/212, P = 0.126, Table 2).\nVirological responses of hepatitis C virus-infected patients receiving directly-acting antivirals therapy before and during campaign of hepatitis C virus micro-elimination in Penghu prison in Penghu prison\nOne missing data.\nOne transferred; One missing data.\nTwo transferred; One released.\nFour released.\nOne relapser.\nFour transferred; Five released. HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \nDuring DAA treatment period, all of patients in sporadic HCV control group completed DAA therapy, while 3 patients in HCV micro-elimination group lost-to-follow (2 transferred; 1 released). During the post-treatment follow-up period, 4 patients in sporadic HCV control group lost-to-follow (4 released), while 6 patients in HCV micro-elimination group lost-to-follow (2 transferred; 4 released). In PP analysis, the overall SVR12 rate was 99.7% (289/290) with comparable SVR12 rates between sporadic HCV control group (98.9%, 86/87) and HCV micro-elimination group (100%, 203/203, P = 0.126, Table 2). Only one patient experienced virological failure (54 years old male, treatment-naïve, HCV-GT3 infection with baseline viral loads of 62,883 IU/mL and FIB-4 of 2.37; relapsed from a 12 wk regimen of glecaprevir/pibrentasvir).","The safety profiles of both groups were shown in Table 3. None of patients had treatment discontinuation other than released or transferred. None experienced serious adverse event. The frequency of adverse events was 4.3% (4/91) and 1.4% (3/212), respectively, among patients in sporadic control group and HCV micro-elimination group. The most reported adverse events were rash in 3 of 13 (23.1%) patients treated with glecaprevir/pibrentasvir and pruritus in 2 of 212 (0.9%) patients treated with sofosbuvir/velpatasvir. None of patients experienced grade 3 or 4 Laboratory abnormality.\nSafety profiles of hepatitis C virus-infected patients receiving direct-acting antivirals therapy in Penghu prison\nDAA: Directly-acting antivirals; HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. ","In the current study, we demonstrated that mass screening combined with onsite group therapy by using a simplified pan-genotypic DAA regimen, 12 wk of sofosbuvir/velpatasvir, provides an “one-size fits all” solution toward the achievement of HCV micro-elimination in prisoners. The SVR rate was 95.6% in ITT population and 100% in PP population after excluding the inmates released or transferred before end-of-follow-up. The high SVR rate was observed in this PWID-dominant population, which HCV genotype distribution was diverse, including genotypes 1a, 1b, 2, 3 and 6. \nRecent advance in the development of IFN-free pan-genotypic DAA regimens has remarkably improved the treatment efficacy with an overall SVR rates of > 90%. Therefore, WHO set the global of HCV elimination by 2030, through the achievement of > 90% diagnosis rate and > 80% treatment rate for eligible patients[16]. Nevertheless, there are many barriers in each HCV care cascade toward HCV elimination at the population level[11,23]. To overcome the barriers, combining the concept of micro-elimination and an outreach strategy with immediate onsite treatment would be a more efficient and practical approach to achieve that goal[18,24]. The current study compared the HCV-infected inmates identified sporadically in outpatient clinics of Penghu Prison from 2017 to 2019 before mass screening and the patients identified by mass screening. We found that mass screening identified 208 HCV-viremic patients in a 5 d screening program from 1137 inmates (encountered around two-third of total inmates in Penghu Prison), compared to 91 HCV-viremic inmates treated in outpatient clinics from 2017 to September 2019. Our results demonstrated that mass screening with immediate onsite treatment provide much more efficient and practical solution to overcome the gaps of disease awareness and link-to-care in the HCV care cascades toward HCV micro-elimination in prisoners. In addition, we implemented “HCV reflex testing” in the mass screening program to scale-up and speed-up the diagnosis and link-to-care for treatment uptake of HCV infections[25].\nPWID is known as the major risk factor of HCV infection and transmission. Although the anti-HCV prevalence in PWID prisoners decreased from 91% in 2014 to 34.8% in 2019 by the strategy of safe injection in Taiwan[21], almost all (97.6%) of HCV-infected prisoners were PWID. Given the lack of vaccine available and high risk of transmission, the strategy of universal screening and concept of “treatment as prevention” are the keys to HCV elimination in prison as well as PWID.\nWe observed that the sporadic HCV-infected prisoners identified in outpatient clinics had significantly higher proportion of comorbidities, including diabetes, hypertension, hyperlipidemia and eGFR, than those participating in the HCV micro-elimination campaign. It implicated that a great proportion of identified sporadically in outpatient clinics were due to concomitant morbidities; by contrast, many HCV-infected patients were unaware to their HCV diseases. In our mass screening, only 36.6% (145/396) of HCV-infected prisoners were aware of HCV infection before screening[21]. It indicates that the implementation of an outreach strategy with universal mass screen is necessary for HCV micro-elimination in prison.\nDespite of the advances in the management of HCV infections, DAA therapy in incarcerated HCV-infected people remains many obstacles to be resolved, including disease unawareness, lack of updated information about the benefits of new DAA treatment, uncertainty of treatment right[26], poor accessibility due to of onsite treatment facilities or HCV treaters. Another difficulty for HCV treatment in prisoners is the unexpected or scheduled releasing from prison or transferring to other prisons, which frequently leads to the interruption of treatment or lost-to- follow up[20,27]. We are lucky that the Taiwan Health Insurance covered all incarcerated people, including all of the laboratory tests and ultrasound sonography and the cost of DAA regimens. Each prison has a contracted hospital providing point-of-care facility. Before initiating DAA therapy, we excluded the patients with expected release or transfer within 24 wk, and negotiated with the authority to avoid unnecessary transferring to other prisons during the period of HCV treatment and follow-up once the inmates entering the DAA course. Eventually we achieved a high treatment rate of 90.6% (212/234) and a high treatment complete rate of 95.8% (203/212), with a high cure rate at 100% (212/212).\nBefore the IFN-free DAA available, the lower SVR rate, much longer treatment duration and frequent adverse events of IFN-based treatment discouraged HCV-infected prisoners from receiving treatment[10]. IFN-free DAA regimens revolutionized HCV treatment which has largely extended the indication for various HCV-infected patients. Nevertheless, the application of typical DAA regimens are based on HCV genotype, presence of decompensated cirrhosis, renal function, and prior treatment experience. The two pangenotypic DAA regimens, sofosbuvir/velpatasvir, and glecaprevir/pibrentasvir, have achieved very high SVR rates of > 95%, regardless of HCV GTs, except for treatment-experienced cirrhotic HCV GT3 patients or GT3b patients[8,12,13]. Recently, to improve the access to anti-HCV therapy, reduce the cost of laboratory tests and the relative complexity of genotype-based treatment strategies, simplified treatment without many information needed for treatment decision are recommended to facilitate the care cascade among populations who are historically less engaged in healthcare, such as PWIDs and prisoners[8]. EASL recommends simplified, genotyping/subtyping-free regimens for IFN-free DAA treatment-naïve (except sofosbuvir plus ribavirin), HCV-infected or HCV-HIV coinfected adolescent and adult patients without cirrhosis or with compensated cirrhosis, regardless of HCV genotypes[8]. These recommendations are a universal 12 wk regimen of sofosbuvir/velpatasvir for all patients or glecaprevir/pibrentasvir, 8 wk for non-cirrhotic, 12 wk for compensated cirrhotic, and 16 wk for HCV GT3 patients, respectively. There are only four information needed before treatment, including the presence of HCV viremia, potential drug-drug interactions, and prior treatment experience, and presence of cirrhosis. The advantages of glecaprevir/pibrentasvir is a shorter 8 wk regimen for treatment-naïve HCV patients and IFN-experienced non-cirrhotic patients with compensated liver diseases, which would be benefit for prisoners who are expected to be released or transferred in a short term. However, glecaprevir, a protease inhibitor, is contraindicated for patients with hepatic decompensation and at risk for rare occurrence of serious drug-induced liver injury[28]. Also, glecaprevir/ pibrentasvir has higher pill burden, three tablets a d. The advantages of sofosbuvir/ velpatasvir include a universal fixed 12 wk regimen, one tablet a d, for all HCV patients with compensated liver diseases, less frequency of potential drug-drug interactions[29], and safety for those with hepatic decompensation. However, a 12 wk regimen with sofosbuvir/velpatasvir needs one more visit and monitoring when compared to an 8 wk regimen with glecaprevir/pibrentasvir. Therefore, we select sofosbuvir/velpatasvir as the antiviral regimen for our outreach onsite treatment. In our study, all HCV-viremic prisoners fit the criteria of simplified, genotyping/ subtyping-free regimens, except one who failed to prior glecaprevir/pibrentasvir therapy and was not enrolled for sofosbuvir/velpatasvir treatment. In our PP analysis, the overall SVR12 rate was comparable between HCV patient group (98.9%, 86/87) and HCV micro-elimination group (100%, 203/203). Our study provided evidence for the concept that simplified, genotyping/subtyping-free regimens can achieve high SVR12 rate in HCV-infected PWID-dominant prisoners.\nIn our study, none of prisoners had DAA treatment discontinuation due to adverse events. None experienced serious adverse event. These data indicated that the simplified, genotyping/subtyping-free regimen, sofosbuvir/velpatasvir, was safe and well tolerated for HCV-infected PWID-dominant prisoners. Very few adverse events were reported in both groups, whatever using sofosbuvir/Ledipasvir, glecaprevir/ pibrentasvir and sofosbuvir/velpatasvir, when compared to the data from clinical trials[30,31]. It might be due to that current population was younger and less patients with advanced fibrosis or chronic kidney diseases.\nThere were some limitations in our study. First, not all inmates in Penghu Prison participated our mass screening. Strategies and policy to encourage inmates to receive HCV screening is mandatory to achieve the goal of WHO. Second, unexpected prisoners’ transferral and release could not be completely avoided, which caused incomplete treatment and follow-up. Successfully linking the released or transferred people to another HCV treaters could help completing HCV treatment and follow-up. Third, there was no reimbursement for the retreatment of prior DAA failed patients in Taiwan at the time of the current study.","Well-designed strategies for mass screening and treatment for HCV-infected prisoners can be implemented successfully by the collaboration between physicians and prison authorities. We demonstrated that mass screening followed by immediate onsite treatment with a simplified pangenotypic DAA regimen, sofosbuvir/velpatasvir, provides successful strategies toward HCV micro-elimination among prisoners."],"string":"[\n \"Hepatitis C virus (HCV) infection is a progressive and blood-borne infectious disease that can lead to end stage liver diseases, such as hepatic decompensation, liver cirrhosis, and hepatocellular carcinoma[1,2]. Iatrogenic transmission of HCV, such as blood transfusion and surgery, has decreased in developed countries. Whereas people who inject drugs (PWID) has become the major population of HCV transmission, which could consist of approximately 80% of HCV-infected patients[3]. Given that lack of vaccine available, “treatment as prevention” for HCV transmission in PWID is very important for HCV elimination.\\nPrisoners are at high risk of HCV infection, with prevalence rates ranging from 3.1% to 38%[4,5]. The high prevalence of HCV infection in prisoners is resulted from unsafe lifestyles, psychiatric disorders, and social problems before they are incarcerated. Recently, PWID has been the most important risk factor of HCV infection in prisoners[6]. The anti-HCV prevalence rate could be as high as 91% among PWID prisoners[7]. Screening and eliminating HCV infection in prisoners is therefore an important social health issue.\\nAccording to the American Association for the Study of Liver Diseases and European Association for the Study of the Liver (EASL) guidelines, all HCV viremic patients should be treated if life span is expected more than one year[8,9]. HCV therapeutic strategies have been revolutionized significantly because of the availability of direct-acting antivirals (DAA)[10]. Interferon (IFN)-based regimens for HCV infection have serious side effects, long therapeutic duration, and contraindications, leading to the huge gaps in HCV care cascade[11]. The current IFN-free DAA regimens provide shorter treatment duration, very high treatment efficacy and safety profiles, not only for general population[12], but also for special populations[13], such as HCV/human immunodeficiency virus (HIV) coinfected patients, hepatitis B virus (HBV)/HCV coinfected patients and patients with chronic kidney diseases in real-world clinical settings[14,15].\\nWorld Health Organization (WHO) set a global goal of HCV elimination by 2030[16], and Taiwan authority is even ambitious by 2025[17]. To achieve the goal, implementation of the concept of HCV micro-elimination is regarding as an efficient and practical strategy[18]. We have proved that “universal mass screening plus outreach onsite treatment” is the key to achieve HCV micro-elimination among patients under maintenance hemodialysis[19].\\nRecently, the latest EASL HCV guideline recommended simplified, genotyping/ subtyping-free, pangenotypic anti-HCV treatment, either sofosbuvir/ velpatasvir or glecaprevir/pibrentasvir, to increase the accessibility and global cure rates among patients with > 12 years, chronic hepatitis C without cirrhosis or with compensated cirrhosis, with or without HIV co-infection, whatever treatment-naïve or IFN-experienced[8].\\nSince HCV treatment is not frequently administered to prisoners due to unawareness of HCV infection, difficultly management, easily loss to follow-up, and lack of hepatologist in prison[20], collaboration between hepatologists and prison authorities to carry out strategies for HCV diagnosis and treatment in prisoners in highly demanded. Herein, we implemented an outreach strategy in combination with universal mass screen and onsite treatment with a simplified pan-genotypic DAA regimen, 12 wk of sofosbuvir/velpatasvir, toward HCV micro-elimination in a PWID-dominant prison in Taiwan.\",\n \"Patients linked to onsite treatment program for HCV micro-elimination HCV-viremic patients were recruited from two cohorts in Penghu Prison (Agency of Corrections, Ministry of Justice, Taiwan), a PWID-dominant prison (Figure 1).\\n\\nPatient flowchart of hepatitis C virus treatment with a simplified pan-genotypic directly-acting antivirals regimen in Penghu Prison. HCV: Hepatitis C virus; DAA: Directly-acting antivirals; SOL/VEL: Sofosbuvir/velpatasvir; GEL/PIB: Glecaprevir/pibrentasvir; EOTVR: Virological response at end-of-treatment; SVR12: Sustained viral response at post-treatment wk 12.\\nHCV-viremic patients were recruited from two cohorts in Penghu Prison (Agency of Corrections, Ministry of Justice, Taiwan), a PWID-dominant prison (Figure 1).\\n\\nPatient flowchart of hepatitis C virus treatment with a simplified pan-genotypic directly-acting antivirals regimen in Penghu Prison. HCV: Hepatitis C virus; DAA: Directly-acting antivirals; SOL/VEL: Sofosbuvir/velpatasvir; GEL/PIB: Glecaprevir/pibrentasvir; EOTVR: Virological response at end-of-treatment; SVR12: Sustained viral response at post-treatment wk 12.\\nHCV-viremic patients identified by a universal mass screening In September 2019, we conducted a 5 d universal mass screening of viral hepatitis in Penghu Prison. These inclusion criteria were prisoners, who were at least 20 years old, being willing to enter the study for screening of viral hepatitis. The study of mass screening was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (IRB: KMUHIRB-SV(I)-20190033). All participants provided written informed consents. A total of 1137 subjects from 1697 inmates participated the mass screening[21]. Among them, 396 (34.8%) subjects had anti-HCV seropositivity; 208 (52.5%) of the 396 subjects were seropositive for HCV ribonucleic acid (RNA) and linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\\nIn September 2019, we conducted a 5 d universal mass screening of viral hepatitis in Penghu Prison. These inclusion criteria were prisoners, who were at least 20 years old, being willing to enter the study for screening of viral hepatitis. The study of mass screening was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (IRB: KMUHIRB-SV(I)-20190033). All participants provided written informed consents. A total of 1137 subjects from 1697 inmates participated the mass screening[21]. Among them, 396 (34.8%) subjects had anti-HCV seropositivity; 208 (52.5%) of the 396 subjects were seropositive for HCV ribonucleic acid (RNA) and linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\\nHCV-viremic patients identified in outpatient clinics during the period of HCV mass screening Another 26 HCV-viremic subjects identified in outpatient clinics of Penghu Prison between August to December 2019 were also linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\\nAll patients received pretreatment evaluation in December 2019, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions. A 12 wk, oral pan-genotypic regimen of sofosbuvir/velpatasvir 400/100 mg fixed-dose combination once daily was initiated in January-February 2020. \\nAnother 26 HCV-viremic subjects identified in outpatient clinics of Penghu Prison between August to December 2019 were also linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\\nAll patients received pretreatment evaluation in December 2019, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions. A 12 wk, oral pan-genotypic regimen of sofosbuvir/velpatasvir 400/100 mg fixed-dose combination once daily was initiated in January-February 2020. \\nPatients identified and treated by DAAs in outpatient clinics before mass screening A total of 91 HCV-viremic patients identified in outpatient clinics of Penghu Prison and treated with DAA before mass screening from 2017 to 2019 were enrolled as a control. The selection of DAA regimens were based on physician’s discretion according to the viral genotype and criteria of reimbursement of National Health Insurance Administration, Taiwan. All patients received pretreatment evaluation, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions.\\nAll participants signed informed consent forms. These enrolled inmates of our study were protected according to the guidelines of the Declaration of Helsinki. The current study of DAA therapy was approved by the Institutional Review Board of Tri-Service General Hospital (IRB: TSGHIRB 2-107-05-080).\\nA total of 91 HCV-viremic patients identified in outpatient clinics of Penghu Prison and treated with DAA before mass screening from 2017 to 2019 were enrolled as a control. The selection of DAA regimens were based on physician’s discretion according to the viral genotype and criteria of reimbursement of National Health Insurance Administration, Taiwan. All patients received pretreatment evaluation, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions.\\nAll participants signed informed consent forms. These enrolled inmates of our study were protected according to the guidelines of the Declaration of Helsinki. The current study of DAA therapy was approved by the Institutional Review Board of Tri-Service General Hospital (IRB: TSGHIRB 2-107-05-080).\\nAssessment, monitoring and endpoints Anti-HCV antibody was determined by the third generation, commercially available immunoassay (Ax SYM HCV III; Abbott Laboratories, North Chicago, IL). HCV RNA viral loads and genotype were determined by real-time PCR assays [RealTime HCV; Abbott Molecular, Des Plaines IL, United States; detection limit: 12 IU/mL])[22]. Liver cirrhosis was defined by the presence of clinical, radiological, endoscopic or laboratory evidence of cirrhosis and/or portal hypertension or fibrosis-4 index (FIB-4) (> 6.5). Laboratory data monitoring and assessment of side effects were performed at treatment wk 2, 4, 8 and end-of-treatment (EOT), and 12 wk after EOT.\\nThe primary endpoint was sustained virological response (SVR12, defined as undetectable HCV RNA throughout 12 wk of the post-treatment follow-up period).\\nAnti-HCV antibody was determined by the third generation, commercially available immunoassay (Ax SYM HCV III; Abbott Laboratories, North Chicago, IL). HCV RNA viral loads and genotype were determined by real-time PCR assays [RealTime HCV; Abbott Molecular, Des Plaines IL, United States; detection limit: 12 IU/mL])[22]. Liver cirrhosis was defined by the presence of clinical, radiological, endoscopic or laboratory evidence of cirrhosis and/or portal hypertension or fibrosis-4 index (FIB-4) (> 6.5). Laboratory data monitoring and assessment of side effects were performed at treatment wk 2, 4, 8 and end-of-treatment (EOT), and 12 wk after EOT.\\nThe primary endpoint was sustained virological response (SVR12, defined as undetectable HCV RNA throughout 12 wk of the post-treatment follow-up period).\\nStatistical analyses. The efficacy of all DAA regimens was determined in a intent-to-treat (ITT) population (all enrolled patients with at least one dose of DAA) and a per-protocol (PP) population (subjects receiving at least one dose of DAA and retained in Penghu Prison throughout the DAA treatment and follow-up period). Safety assessments reported adverse event (AE), serious adverse event (SAE) and laboratory abnormalities in the ITT population. Continuous variables are expressed as means ± standard deviation (SD), and categorical variables are expressed as percentages. The differences of continuous variables are estimated by the Student’s t test. The differences in categorical variables are analyzed using the Chi-square test. The on-treatment and off-treatment virological response rates were analyzed in number and percentages with 95% confidence interval (CI). All data analyses were performed using the SPSS software version 18.0 (SPSS Inc., Chicago, Illinois, United States).\\nThe efficacy of all DAA regimens was determined in a intent-to-treat (ITT) population (all enrolled patients with at least one dose of DAA) and a per-protocol (PP) population (subjects receiving at least one dose of DAA and retained in Penghu Prison throughout the DAA treatment and follow-up period). Safety assessments reported adverse event (AE), serious adverse event (SAE) and laboratory abnormalities in the ITT population. Continuous variables are expressed as means ± standard deviation (SD), and categorical variables are expressed as percentages. The differences of continuous variables are estimated by the Student’s t test. The differences in categorical variables are analyzed using the Chi-square test. The on-treatment and off-treatment virological response rates were analyzed in number and percentages with 95% confidence interval (CI). All data analyses were performed using the SPSS software version 18.0 (SPSS Inc., Chicago, Illinois, United States).\",\n \"HCV-viremic patients were recruited from two cohorts in Penghu Prison (Agency of Corrections, Ministry of Justice, Taiwan), a PWID-dominant prison (Figure 1).\\n\\nPatient flowchart of hepatitis C virus treatment with a simplified pan-genotypic directly-acting antivirals regimen in Penghu Prison. HCV: Hepatitis C virus; DAA: Directly-acting antivirals; SOL/VEL: Sofosbuvir/velpatasvir; GEL/PIB: Glecaprevir/pibrentasvir; EOTVR: Virological response at end-of-treatment; SVR12: Sustained viral response at post-treatment wk 12.\",\n \"In September 2019, we conducted a 5 d universal mass screening of viral hepatitis in Penghu Prison. These inclusion criteria were prisoners, who were at least 20 years old, being willing to enter the study for screening of viral hepatitis. The study of mass screening was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (IRB: KMUHIRB-SV(I)-20190033). All participants provided written informed consents. A total of 1137 subjects from 1697 inmates participated the mass screening[21]. Among them, 396 (34.8%) subjects had anti-HCV seropositivity; 208 (52.5%) of the 396 subjects were seropositive for HCV ribonucleic acid (RNA) and linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\",\n \"Another 26 HCV-viremic subjects identified in outpatient clinics of Penghu Prison between August to December 2019 were also linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\\nAll patients received pretreatment evaluation in December 2019, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions. A 12 wk, oral pan-genotypic regimen of sofosbuvir/velpatasvir 400/100 mg fixed-dose combination once daily was initiated in January-February 2020. \",\n \"A total of 91 HCV-viremic patients identified in outpatient clinics of Penghu Prison and treated with DAA before mass screening from 2017 to 2019 were enrolled as a control. The selection of DAA regimens were based on physician’s discretion according to the viral genotype and criteria of reimbursement of National Health Insurance Administration, Taiwan. All patients received pretreatment evaluation, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions.\\nAll participants signed informed consent forms. These enrolled inmates of our study were protected according to the guidelines of the Declaration of Helsinki. The current study of DAA therapy was approved by the Institutional Review Board of Tri-Service General Hospital (IRB: TSGHIRB 2-107-05-080).\",\n \"Anti-HCV antibody was determined by the third generation, commercially available immunoassay (Ax SYM HCV III; Abbott Laboratories, North Chicago, IL). HCV RNA viral loads and genotype were determined by real-time PCR assays [RealTime HCV; Abbott Molecular, Des Plaines IL, United States; detection limit: 12 IU/mL])[22]. Liver cirrhosis was defined by the presence of clinical, radiological, endoscopic or laboratory evidence of cirrhosis and/or portal hypertension or fibrosis-4 index (FIB-4) (> 6.5). Laboratory data monitoring and assessment of side effects were performed at treatment wk 2, 4, 8 and end-of-treatment (EOT), and 12 wk after EOT.\\nThe primary endpoint was sustained virological response (SVR12, defined as undetectable HCV RNA throughout 12 wk of the post-treatment follow-up period).\",\n \"The efficacy of all DAA regimens was determined in a intent-to-treat (ITT) population (all enrolled patients with at least one dose of DAA) and a per-protocol (PP) population (subjects receiving at least one dose of DAA and retained in Penghu Prison throughout the DAA treatment and follow-up period). Safety assessments reported adverse event (AE), serious adverse event (SAE) and laboratory abnormalities in the ITT population. Continuous variables are expressed as means ± standard deviation (SD), and categorical variables are expressed as percentages. The differences of continuous variables are estimated by the Student’s t test. The differences in categorical variables are analyzed using the Chi-square test. The on-treatment and off-treatment virological response rates were analyzed in number and percentages with 95% confidence interval (CI). All data analyses were performed using the SPSS software version 18.0 (SPSS Inc., Chicago, Illinois, United States).\",\n \"Patient flowchart of HCV micro-elimination campaign The patient flowchart of HCV mass screen, assessment and treatment was shown in Figure 1. A total of 234 HCV-viremic patients, 208 from mass screening and 26 from outpatient clinics in Penghu Prison were assessed for eligibility of group therapy with sofosbuvir/velpatasvir in December 2019. Twenty-two patients were excluded from anti-HCV therapy due to scheduled to be released from jail (n = 16) or transferred to other jails (n = 3) within 6 mo, unwilling to receive therapy (n = 2) and prior glecaprevir/pibrentasvir treatment failure (n = 1). Finally, 212 patients were recruited for sofosbuvir/velpatasvir therapy initiated in January-February 2020.\\nThe patient flowchart of HCV mass screen, assessment and treatment was shown in Figure 1. A total of 234 HCV-viremic patients, 208 from mass screening and 26 from outpatient clinics in Penghu Prison were assessed for eligibility of group therapy with sofosbuvir/velpatasvir in December 2019. Twenty-two patients were excluded from anti-HCV therapy due to scheduled to be released from jail (n = 16) or transferred to other jails (n = 3) within 6 mo, unwilling to receive therapy (n = 2) and prior glecaprevir/pibrentasvir treatment failure (n = 1). Finally, 212 patients were recruited for sofosbuvir/velpatasvir therapy initiated in January-February 2020.\\nPatient characteristics The baseline characteristics of 303 HCV-viremic patients, including 212 in HCV micro-elimination campaign and 91 sporadic controls from outpatient clinics before micro-elimination campaign were listed in Table 1. They mean age was 48.4 years with male dominant (99.7%). Thirty (9.9%) had HBV coinfection. The mean FIB-4 was 1.3, with 20 (6.6%) had advanced fibrosis (FIB-4 > 3.25). Only one patient (0.3%) had liver cirrhosis. The mean HCV RNA levels was 6.5 Logs IU/mL, dominant with HCV genotype 1 (HCV-GT1, 42.2%), followed by HCV-GT6 (35.3%), HCV-GT3 (11.6%) and HCV-GT2 (10.6%). Three (1%) patients were prior IFN-experienced. The two groups had comparable characteristics in terms of age, gender, HBV co-infection, liver and renal function tests, FIB-4 score, HCV genotype distribution, and prior history of IFN-based therapy. However, the sporadic patients identified in outpatient clinics had significantly higher proportion of comorbidities, including diabetes, hypertension, hyperlipidemia and an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2, but significantly lower HCV viral loads. None of patient had decompensated cirrhosis nor liver cancer.\\nBaseline characteristics of hepatitis C virus-infected patients receiving directly-acting antivirals therapy between sporadic hepatitis C virus therapy in outpatient clinics and campaign of hepatitis C virus micro-elimination in Penghu prison\\n56 patients did not have body mass index information (12 patients before campaign of hepatitis C virus (HCV) micro-elimination; 44 patients in campaign of HCV micro-elimination).\\n\\nP < 0.05. DAA: Directly-acting antivirals; HCV: Hepatitis C virus; BMI: Body mass index; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; LC: Liver cirrhosis; FIB-4: Fibrosis-4 index; HBsAg: Hepatitis B surface antigen; eGFR: Estimated glomerular filtration rate (mL/min/1.73 m2); SOF: Sofosbuvir; VEL: Velpatasvir; LDV: Ledipasvir; GLE: Glecaprevir; PIB: Pibrentasvir; IFN: Interferon.\\nThe baseline characteristics of 303 HCV-viremic patients, including 212 in HCV micro-elimination campaign and 91 sporadic controls from outpatient clinics before micro-elimination campaign were listed in Table 1. They mean age was 48.4 years with male dominant (99.7%). Thirty (9.9%) had HBV coinfection. The mean FIB-4 was 1.3, with 20 (6.6%) had advanced fibrosis (FIB-4 > 3.25). Only one patient (0.3%) had liver cirrhosis. The mean HCV RNA levels was 6.5 Logs IU/mL, dominant with HCV genotype 1 (HCV-GT1, 42.2%), followed by HCV-GT6 (35.3%), HCV-GT3 (11.6%) and HCV-GT2 (10.6%). Three (1%) patients were prior IFN-experienced. The two groups had comparable characteristics in terms of age, gender, HBV co-infection, liver and renal function tests, FIB-4 score, HCV genotype distribution, and prior history of IFN-based therapy. However, the sporadic patients identified in outpatient clinics had significantly higher proportion of comorbidities, including diabetes, hypertension, hyperlipidemia and an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2, but significantly lower HCV viral loads. None of patient had decompensated cirrhosis nor liver cancer.\\nBaseline characteristics of hepatitis C virus-infected patients receiving directly-acting antivirals therapy between sporadic hepatitis C virus therapy in outpatient clinics and campaign of hepatitis C virus micro-elimination in Penghu prison\\n56 patients did not have body mass index information (12 patients before campaign of hepatitis C virus (HCV) micro-elimination; 44 patients in campaign of HCV micro-elimination).\\n\\nP < 0.05. DAA: Directly-acting antivirals; HCV: Hepatitis C virus; BMI: Body mass index; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; LC: Liver cirrhosis; FIB-4: Fibrosis-4 index; HBsAg: Hepatitis B surface antigen; eGFR: Estimated glomerular filtration rate (mL/min/1.73 m2); SOF: Sofosbuvir; VEL: Velpatasvir; LDV: Ledipasvir; GLE: Glecaprevir; PIB: Pibrentasvir; IFN: Interferon.\\nTreatment efficacy All of 212 patients in HCV micro-elimination campaign received sofosbuvir/ velpatasvir treatment; while among 91 sporadic patients with DAA therapy before HCV micro-elimination campaign, 78 (85.7%) received 12 wk of sofosbuvir/Ledipasvir and 13 (14.3%) received 8-12 wk of glecaprevir/pibrentasvir according to the Taiwan HCV guideline[12,13].\\nIn ITT analysis, the overall SVR12 rate was 95.4% (289/303) with comparable SVR12 rates between sporadic HCV control group (94.5%, 86/91) and HCV micro-elimination group (95.8%, 203/212, P = 0.126, Table 2).\\nVirological responses of hepatitis C virus-infected patients receiving directly-acting antivirals therapy before and during campaign of hepatitis C virus micro-elimination in Penghu prison in Penghu prison\\nOne missing data.\\nOne transferred; One missing data.\\nTwo transferred; One released.\\nFour released.\\nOne relapser.\\nFour transferred; Five released. HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \\nDuring DAA treatment period, all of patients in sporadic HCV control group completed DAA therapy, while 3 patients in HCV micro-elimination group lost-to-follow (2 transferred; 1 released). During the post-treatment follow-up period, 4 patients in sporadic HCV control group lost-to-follow (4 released), while 6 patients in HCV micro-elimination group lost-to-follow (2 transferred; 4 released). In PP analysis, the overall SVR12 rate was 99.7% (289/290) with comparable SVR12 rates between sporadic HCV control group (98.9%, 86/87) and HCV micro-elimination group (100%, 203/203, P = 0.126, Table 2). Only one patient experienced virological failure (54 years old male, treatment-naïve, HCV-GT3 infection with baseline viral loads of 62,883 IU/mL and FIB-4 of 2.37; relapsed from a 12 wk regimen of glecaprevir/pibrentasvir).\\nAll of 212 patients in HCV micro-elimination campaign received sofosbuvir/ velpatasvir treatment; while among 91 sporadic patients with DAA therapy before HCV micro-elimination campaign, 78 (85.7%) received 12 wk of sofosbuvir/Ledipasvir and 13 (14.3%) received 8-12 wk of glecaprevir/pibrentasvir according to the Taiwan HCV guideline[12,13].\\nIn ITT analysis, the overall SVR12 rate was 95.4% (289/303) with comparable SVR12 rates between sporadic HCV control group (94.5%, 86/91) and HCV micro-elimination group (95.8%, 203/212, P = 0.126, Table 2).\\nVirological responses of hepatitis C virus-infected patients receiving directly-acting antivirals therapy before and during campaign of hepatitis C virus micro-elimination in Penghu prison in Penghu prison\\nOne missing data.\\nOne transferred; One missing data.\\nTwo transferred; One released.\\nFour released.\\nOne relapser.\\nFour transferred; Five released. HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \\nDuring DAA treatment period, all of patients in sporadic HCV control group completed DAA therapy, while 3 patients in HCV micro-elimination group lost-to-follow (2 transferred; 1 released). During the post-treatment follow-up period, 4 patients in sporadic HCV control group lost-to-follow (4 released), while 6 patients in HCV micro-elimination group lost-to-follow (2 transferred; 4 released). In PP analysis, the overall SVR12 rate was 99.7% (289/290) with comparable SVR12 rates between sporadic HCV control group (98.9%, 86/87) and HCV micro-elimination group (100%, 203/203, P = 0.126, Table 2). Only one patient experienced virological failure (54 years old male, treatment-naïve, HCV-GT3 infection with baseline viral loads of 62,883 IU/mL and FIB-4 of 2.37; relapsed from a 12 wk regimen of glecaprevir/pibrentasvir).\\nSafety profiles The safety profiles of both groups were shown in Table 3. None of patients had treatment discontinuation other than released or transferred. None experienced serious adverse event. The frequency of adverse events was 4.3% (4/91) and 1.4% (3/212), respectively, among patients in sporadic control group and HCV micro-elimination group. The most reported adverse events were rash in 3 of 13 (23.1%) patients treated with glecaprevir/pibrentasvir and pruritus in 2 of 212 (0.9%) patients treated with sofosbuvir/velpatasvir. None of patients experienced grade 3 or 4 Laboratory abnormality.\\nSafety profiles of hepatitis C virus-infected patients receiving direct-acting antivirals therapy in Penghu prison\\nDAA: Directly-acting antivirals; HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \\nThe safety profiles of both groups were shown in Table 3. None of patients had treatment discontinuation other than released or transferred. None experienced serious adverse event. The frequency of adverse events was 4.3% (4/91) and 1.4% (3/212), respectively, among patients in sporadic control group and HCV micro-elimination group. The most reported adverse events were rash in 3 of 13 (23.1%) patients treated with glecaprevir/pibrentasvir and pruritus in 2 of 212 (0.9%) patients treated with sofosbuvir/velpatasvir. None of patients experienced grade 3 or 4 Laboratory abnormality.\\nSafety profiles of hepatitis C virus-infected patients receiving direct-acting antivirals therapy in Penghu prison\\nDAA: Directly-acting antivirals; HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \",\n \"The patient flowchart of HCV mass screen, assessment and treatment was shown in Figure 1. A total of 234 HCV-viremic patients, 208 from mass screening and 26 from outpatient clinics in Penghu Prison were assessed for eligibility of group therapy with sofosbuvir/velpatasvir in December 2019. Twenty-two patients were excluded from anti-HCV therapy due to scheduled to be released from jail (n = 16) or transferred to other jails (n = 3) within 6 mo, unwilling to receive therapy (n = 2) and prior glecaprevir/pibrentasvir treatment failure (n = 1). Finally, 212 patients were recruited for sofosbuvir/velpatasvir therapy initiated in January-February 2020.\",\n \"The baseline characteristics of 303 HCV-viremic patients, including 212 in HCV micro-elimination campaign and 91 sporadic controls from outpatient clinics before micro-elimination campaign were listed in Table 1. They mean age was 48.4 years with male dominant (99.7%). Thirty (9.9%) had HBV coinfection. The mean FIB-4 was 1.3, with 20 (6.6%) had advanced fibrosis (FIB-4 > 3.25). Only one patient (0.3%) had liver cirrhosis. The mean HCV RNA levels was 6.5 Logs IU/mL, dominant with HCV genotype 1 (HCV-GT1, 42.2%), followed by HCV-GT6 (35.3%), HCV-GT3 (11.6%) and HCV-GT2 (10.6%). Three (1%) patients were prior IFN-experienced. The two groups had comparable characteristics in terms of age, gender, HBV co-infection, liver and renal function tests, FIB-4 score, HCV genotype distribution, and prior history of IFN-based therapy. However, the sporadic patients identified in outpatient clinics had significantly higher proportion of comorbidities, including diabetes, hypertension, hyperlipidemia and an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2, but significantly lower HCV viral loads. None of patient had decompensated cirrhosis nor liver cancer.\\nBaseline characteristics of hepatitis C virus-infected patients receiving directly-acting antivirals therapy between sporadic hepatitis C virus therapy in outpatient clinics and campaign of hepatitis C virus micro-elimination in Penghu prison\\n56 patients did not have body mass index information (12 patients before campaign of hepatitis C virus (HCV) micro-elimination; 44 patients in campaign of HCV micro-elimination).\\n\\nP < 0.05. DAA: Directly-acting antivirals; HCV: Hepatitis C virus; BMI: Body mass index; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; LC: Liver cirrhosis; FIB-4: Fibrosis-4 index; HBsAg: Hepatitis B surface antigen; eGFR: Estimated glomerular filtration rate (mL/min/1.73 m2); SOF: Sofosbuvir; VEL: Velpatasvir; LDV: Ledipasvir; GLE: Glecaprevir; PIB: Pibrentasvir; IFN: Interferon.\",\n \"All of 212 patients in HCV micro-elimination campaign received sofosbuvir/ velpatasvir treatment; while among 91 sporadic patients with DAA therapy before HCV micro-elimination campaign, 78 (85.7%) received 12 wk of sofosbuvir/Ledipasvir and 13 (14.3%) received 8-12 wk of glecaprevir/pibrentasvir according to the Taiwan HCV guideline[12,13].\\nIn ITT analysis, the overall SVR12 rate was 95.4% (289/303) with comparable SVR12 rates between sporadic HCV control group (94.5%, 86/91) and HCV micro-elimination group (95.8%, 203/212, P = 0.126, Table 2).\\nVirological responses of hepatitis C virus-infected patients receiving directly-acting antivirals therapy before and during campaign of hepatitis C virus micro-elimination in Penghu prison in Penghu prison\\nOne missing data.\\nOne transferred; One missing data.\\nTwo transferred; One released.\\nFour released.\\nOne relapser.\\nFour transferred; Five released. HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \\nDuring DAA treatment period, all of patients in sporadic HCV control group completed DAA therapy, while 3 patients in HCV micro-elimination group lost-to-follow (2 transferred; 1 released). During the post-treatment follow-up period, 4 patients in sporadic HCV control group lost-to-follow (4 released), while 6 patients in HCV micro-elimination group lost-to-follow (2 transferred; 4 released). In PP analysis, the overall SVR12 rate was 99.7% (289/290) with comparable SVR12 rates between sporadic HCV control group (98.9%, 86/87) and HCV micro-elimination group (100%, 203/203, P = 0.126, Table 2). Only one patient experienced virological failure (54 years old male, treatment-naïve, HCV-GT3 infection with baseline viral loads of 62,883 IU/mL and FIB-4 of 2.37; relapsed from a 12 wk regimen of glecaprevir/pibrentasvir).\",\n \"The safety profiles of both groups were shown in Table 3. None of patients had treatment discontinuation other than released or transferred. None experienced serious adverse event. The frequency of adverse events was 4.3% (4/91) and 1.4% (3/212), respectively, among patients in sporadic control group and HCV micro-elimination group. The most reported adverse events were rash in 3 of 13 (23.1%) patients treated with glecaprevir/pibrentasvir and pruritus in 2 of 212 (0.9%) patients treated with sofosbuvir/velpatasvir. None of patients experienced grade 3 or 4 Laboratory abnormality.\\nSafety profiles of hepatitis C virus-infected patients receiving direct-acting antivirals therapy in Penghu prison\\nDAA: Directly-acting antivirals; HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \",\n \"In the current study, we demonstrated that mass screening combined with onsite group therapy by using a simplified pan-genotypic DAA regimen, 12 wk of sofosbuvir/velpatasvir, provides an “one-size fits all” solution toward the achievement of HCV micro-elimination in prisoners. The SVR rate was 95.6% in ITT population and 100% in PP population after excluding the inmates released or transferred before end-of-follow-up. The high SVR rate was observed in this PWID-dominant population, which HCV genotype distribution was diverse, including genotypes 1a, 1b, 2, 3 and 6. \\nRecent advance in the development of IFN-free pan-genotypic DAA regimens has remarkably improved the treatment efficacy with an overall SVR rates of > 90%. Therefore, WHO set the global of HCV elimination by 2030, through the achievement of > 90% diagnosis rate and > 80% treatment rate for eligible patients[16]. Nevertheless, there are many barriers in each HCV care cascade toward HCV elimination at the population level[11,23]. To overcome the barriers, combining the concept of micro-elimination and an outreach strategy with immediate onsite treatment would be a more efficient and practical approach to achieve that goal[18,24]. The current study compared the HCV-infected inmates identified sporadically in outpatient clinics of Penghu Prison from 2017 to 2019 before mass screening and the patients identified by mass screening. We found that mass screening identified 208 HCV-viremic patients in a 5 d screening program from 1137 inmates (encountered around two-third of total inmates in Penghu Prison), compared to 91 HCV-viremic inmates treated in outpatient clinics from 2017 to September 2019. Our results demonstrated that mass screening with immediate onsite treatment provide much more efficient and practical solution to overcome the gaps of disease awareness and link-to-care in the HCV care cascades toward HCV micro-elimination in prisoners. In addition, we implemented “HCV reflex testing” in the mass screening program to scale-up and speed-up the diagnosis and link-to-care for treatment uptake of HCV infections[25].\\nPWID is known as the major risk factor of HCV infection and transmission. Although the anti-HCV prevalence in PWID prisoners decreased from 91% in 2014 to 34.8% in 2019 by the strategy of safe injection in Taiwan[21], almost all (97.6%) of HCV-infected prisoners were PWID. Given the lack of vaccine available and high risk of transmission, the strategy of universal screening and concept of “treatment as prevention” are the keys to HCV elimination in prison as well as PWID.\\nWe observed that the sporadic HCV-infected prisoners identified in outpatient clinics had significantly higher proportion of comorbidities, including diabetes, hypertension, hyperlipidemia and eGFR, than those participating in the HCV micro-elimination campaign. It implicated that a great proportion of identified sporadically in outpatient clinics were due to concomitant morbidities; by contrast, many HCV-infected patients were unaware to their HCV diseases. In our mass screening, only 36.6% (145/396) of HCV-infected prisoners were aware of HCV infection before screening[21]. It indicates that the implementation of an outreach strategy with universal mass screen is necessary for HCV micro-elimination in prison.\\nDespite of the advances in the management of HCV infections, DAA therapy in incarcerated HCV-infected people remains many obstacles to be resolved, including disease unawareness, lack of updated information about the benefits of new DAA treatment, uncertainty of treatment right[26], poor accessibility due to of onsite treatment facilities or HCV treaters. Another difficulty for HCV treatment in prisoners is the unexpected or scheduled releasing from prison or transferring to other prisons, which frequently leads to the interruption of treatment or lost-to- follow up[20,27]. We are lucky that the Taiwan Health Insurance covered all incarcerated people, including all of the laboratory tests and ultrasound sonography and the cost of DAA regimens. Each prison has a contracted hospital providing point-of-care facility. Before initiating DAA therapy, we excluded the patients with expected release or transfer within 24 wk, and negotiated with the authority to avoid unnecessary transferring to other prisons during the period of HCV treatment and follow-up once the inmates entering the DAA course. Eventually we achieved a high treatment rate of 90.6% (212/234) and a high treatment complete rate of 95.8% (203/212), with a high cure rate at 100% (212/212).\\nBefore the IFN-free DAA available, the lower SVR rate, much longer treatment duration and frequent adverse events of IFN-based treatment discouraged HCV-infected prisoners from receiving treatment[10]. IFN-free DAA regimens revolutionized HCV treatment which has largely extended the indication for various HCV-infected patients. Nevertheless, the application of typical DAA regimens are based on HCV genotype, presence of decompensated cirrhosis, renal function, and prior treatment experience. The two pangenotypic DAA regimens, sofosbuvir/velpatasvir, and glecaprevir/pibrentasvir, have achieved very high SVR rates of > 95%, regardless of HCV GTs, except for treatment-experienced cirrhotic HCV GT3 patients or GT3b patients[8,12,13]. Recently, to improve the access to anti-HCV therapy, reduce the cost of laboratory tests and the relative complexity of genotype-based treatment strategies, simplified treatment without many information needed for treatment decision are recommended to facilitate the care cascade among populations who are historically less engaged in healthcare, such as PWIDs and prisoners[8]. EASL recommends simplified, genotyping/subtyping-free regimens for IFN-free DAA treatment-naïve (except sofosbuvir plus ribavirin), HCV-infected or HCV-HIV coinfected adolescent and adult patients without cirrhosis or with compensated cirrhosis, regardless of HCV genotypes[8]. These recommendations are a universal 12 wk regimen of sofosbuvir/velpatasvir for all patients or glecaprevir/pibrentasvir, 8 wk for non-cirrhotic, 12 wk for compensated cirrhotic, and 16 wk for HCV GT3 patients, respectively. There are only four information needed before treatment, including the presence of HCV viremia, potential drug-drug interactions, and prior treatment experience, and presence of cirrhosis. The advantages of glecaprevir/pibrentasvir is a shorter 8 wk regimen for treatment-naïve HCV patients and IFN-experienced non-cirrhotic patients with compensated liver diseases, which would be benefit for prisoners who are expected to be released or transferred in a short term. However, glecaprevir, a protease inhibitor, is contraindicated for patients with hepatic decompensation and at risk for rare occurrence of serious drug-induced liver injury[28]. Also, glecaprevir/ pibrentasvir has higher pill burden, three tablets a d. The advantages of sofosbuvir/ velpatasvir include a universal fixed 12 wk regimen, one tablet a d, for all HCV patients with compensated liver diseases, less frequency of potential drug-drug interactions[29], and safety for those with hepatic decompensation. However, a 12 wk regimen with sofosbuvir/velpatasvir needs one more visit and monitoring when compared to an 8 wk regimen with glecaprevir/pibrentasvir. Therefore, we select sofosbuvir/velpatasvir as the antiviral regimen for our outreach onsite treatment. In our study, all HCV-viremic prisoners fit the criteria of simplified, genotyping/ subtyping-free regimens, except one who failed to prior glecaprevir/pibrentasvir therapy and was not enrolled for sofosbuvir/velpatasvir treatment. In our PP analysis, the overall SVR12 rate was comparable between HCV patient group (98.9%, 86/87) and HCV micro-elimination group (100%, 203/203). Our study provided evidence for the concept that simplified, genotyping/subtyping-free regimens can achieve high SVR12 rate in HCV-infected PWID-dominant prisoners.\\nIn our study, none of prisoners had DAA treatment discontinuation due to adverse events. None experienced serious adverse event. These data indicated that the simplified, genotyping/subtyping-free regimen, sofosbuvir/velpatasvir, was safe and well tolerated for HCV-infected PWID-dominant prisoners. Very few adverse events were reported in both groups, whatever using sofosbuvir/Ledipasvir, glecaprevir/ pibrentasvir and sofosbuvir/velpatasvir, when compared to the data from clinical trials[30,31]. It might be due to that current population was younger and less patients with advanced fibrosis or chronic kidney diseases.\\nThere were some limitations in our study. First, not all inmates in Penghu Prison participated our mass screening. Strategies and policy to encourage inmates to receive HCV screening is mandatory to achieve the goal of WHO. Second, unexpected prisoners’ transferral and release could not be completely avoided, which caused incomplete treatment and follow-up. Successfully linking the released or transferred people to another HCV treaters could help completing HCV treatment and follow-up. Third, there was no reimbursement for the retreatment of prior DAA failed patients in Taiwan at the time of the current study.\",\n \"Well-designed strategies for mass screening and treatment for HCV-infected prisoners can be implemented successfully by the collaboration between physicians and prison authorities. We demonstrated that mass screening followed by immediate onsite treatment with a simplified pangenotypic DAA regimen, sofosbuvir/velpatasvir, provides successful strategies toward HCV micro-elimination among prisoners.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"keywords":{"kind":"list like","value":["Direct-acting antivirals","Sofosbuvir","Velpatasvir","People who inject drugs","Universal screen"],"string":"[\n \"Direct-acting antivirals\",\n \"Sofosbuvir\",\n \"Velpatasvir\",\n \"People who inject drugs\",\n \"Universal screen\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nHepatitis C virus (HCV) infection is a progressive and blood-borne infectious disease that can lead to end stage liver diseases, such as hepatic decompensation, liver cirrhosis, and hepatocellular carcinoma[1,2]. Iatrogenic transmission of HCV, such as blood transfusion and surgery, has decreased in developed countries. Whereas people who inject drugs (PWID) has become the major population of HCV transmission, which could consist of approximately 80% of HCV-infected patients[3]. Given that lack of vaccine available, “treatment as prevention” for HCV transmission in PWID is very important for HCV elimination.\nPrisoners are at high risk of HCV infection, with prevalence rates ranging from 3.1% to 38%[4,5]. The high prevalence of HCV infection in prisoners is resulted from unsafe lifestyles, psychiatric disorders, and social problems before they are incarcerated. Recently, PWID has been the most important risk factor of HCV infection in prisoners[6]. The anti-HCV prevalence rate could be as high as 91% among PWID prisoners[7]. Screening and eliminating HCV infection in prisoners is therefore an important social health issue.\nAccording to the American Association for the Study of Liver Diseases and European Association for the Study of the Liver (EASL) guidelines, all HCV viremic patients should be treated if life span is expected more than one year[8,9]. HCV therapeutic strategies have been revolutionized significantly because of the availability of direct-acting antivirals (DAA)[10]. Interferon (IFN)-based regimens for HCV infection have serious side effects, long therapeutic duration, and contraindications, leading to the huge gaps in HCV care cascade[11]. The current IFN-free DAA regimens provide shorter treatment duration, very high treatment efficacy and safety profiles, not only for general population[12], but also for special populations[13], such as HCV/human immunodeficiency virus (HIV) coinfected patients, hepatitis B virus (HBV)/HCV coinfected patients and patients with chronic kidney diseases in real-world clinical settings[14,15].\nWorld Health Organization (WHO) set a global goal of HCV elimination by 2030[16], and Taiwan authority is even ambitious by 2025[17]. To achieve the goal, implementation of the concept of HCV micro-elimination is regarding as an efficient and practical strategy[18]. We have proved that “universal mass screening plus outreach onsite treatment” is the key to achieve HCV micro-elimination among patients under maintenance hemodialysis[19].\nRecently, the latest EASL HCV guideline recommended simplified, genotyping/ subtyping-free, pangenotypic anti-HCV treatment, either sofosbuvir/ velpatasvir or glecaprevir/pibrentasvir, to increase the accessibility and global cure rates among patients with > 12 years, chronic hepatitis C without cirrhosis or with compensated cirrhosis, with or without HIV co-infection, whatever treatment-naïve or IFN-experienced[8].\nSince HCV treatment is not frequently administered to prisoners due to unawareness of HCV infection, difficultly management, easily loss to follow-up, and lack of hepatologist in prison[20], collaboration between hepatologists and prison authorities to carry out strategies for HCV diagnosis and treatment in prisoners in highly demanded. Herein, we implemented an outreach strategy in combination with universal mass screen and onsite treatment with a simplified pan-genotypic DAA regimen, 12 wk of sofosbuvir/velpatasvir, toward HCV micro-elimination in a PWID-dominant prison in Taiwan.\n\nMATERIALS AND METHODS:\nPatients linked to onsite treatment program for HCV micro-elimination HCV-viremic patients were recruited from two cohorts in Penghu Prison (Agency of Corrections, Ministry of Justice, Taiwan), a PWID-dominant prison (Figure 1).\n\nPatient flowchart of hepatitis C virus treatment with a simplified pan-genotypic directly-acting antivirals regimen in Penghu Prison. HCV: Hepatitis C virus; DAA: Directly-acting antivirals; SOL/VEL: Sofosbuvir/velpatasvir; GEL/PIB: Glecaprevir/pibrentasvir; EOTVR: Virological response at end-of-treatment; SVR12: Sustained viral response at post-treatment wk 12.\nHCV-viremic patients were recruited from two cohorts in Penghu Prison (Agency of Corrections, Ministry of Justice, Taiwan), a PWID-dominant prison (Figure 1).\n\nPatient flowchart of hepatitis C virus treatment with a simplified pan-genotypic directly-acting antivirals regimen in Penghu Prison. HCV: Hepatitis C virus; DAA: Directly-acting antivirals; SOL/VEL: Sofosbuvir/velpatasvir; GEL/PIB: Glecaprevir/pibrentasvir; EOTVR: Virological response at end-of-treatment; SVR12: Sustained viral response at post-treatment wk 12.\nHCV-viremic patients identified by a universal mass screening In September 2019, we conducted a 5 d universal mass screening of viral hepatitis in Penghu Prison. These inclusion criteria were prisoners, who were at least 20 years old, being willing to enter the study for screening of viral hepatitis. The study of mass screening was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (IRB: KMUHIRB-SV(I)-20190033). All participants provided written informed consents. A total of 1137 subjects from 1697 inmates participated the mass screening[21]. Among them, 396 (34.8%) subjects had anti-HCV seropositivity; 208 (52.5%) of the 396 subjects were seropositive for HCV ribonucleic acid (RNA) and linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\nIn September 2019, we conducted a 5 d universal mass screening of viral hepatitis in Penghu Prison. These inclusion criteria were prisoners, who were at least 20 years old, being willing to enter the study for screening of viral hepatitis. The study of mass screening was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (IRB: KMUHIRB-SV(I)-20190033). All participants provided written informed consents. A total of 1137 subjects from 1697 inmates participated the mass screening[21]. Among them, 396 (34.8%) subjects had anti-HCV seropositivity; 208 (52.5%) of the 396 subjects were seropositive for HCV ribonucleic acid (RNA) and linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\nHCV-viremic patients identified in outpatient clinics during the period of HCV mass screening Another 26 HCV-viremic subjects identified in outpatient clinics of Penghu Prison between August to December 2019 were also linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\nAll patients received pretreatment evaluation in December 2019, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions. A 12 wk, oral pan-genotypic regimen of sofosbuvir/velpatasvir 400/100 mg fixed-dose combination once daily was initiated in January-February 2020. \nAnother 26 HCV-viremic subjects identified in outpatient clinics of Penghu Prison between August to December 2019 were also linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\nAll patients received pretreatment evaluation in December 2019, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions. A 12 wk, oral pan-genotypic regimen of sofosbuvir/velpatasvir 400/100 mg fixed-dose combination once daily was initiated in January-February 2020. \nPatients identified and treated by DAAs in outpatient clinics before mass screening A total of 91 HCV-viremic patients identified in outpatient clinics of Penghu Prison and treated with DAA before mass screening from 2017 to 2019 were enrolled as a control. The selection of DAA regimens were based on physician’s discretion according to the viral genotype and criteria of reimbursement of National Health Insurance Administration, Taiwan. All patients received pretreatment evaluation, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions.\nAll participants signed informed consent forms. These enrolled inmates of our study were protected according to the guidelines of the Declaration of Helsinki. The current study of DAA therapy was approved by the Institutional Review Board of Tri-Service General Hospital (IRB: TSGHIRB 2-107-05-080).\nA total of 91 HCV-viremic patients identified in outpatient clinics of Penghu Prison and treated with DAA before mass screening from 2017 to 2019 were enrolled as a control. The selection of DAA regimens were based on physician’s discretion according to the viral genotype and criteria of reimbursement of National Health Insurance Administration, Taiwan. All patients received pretreatment evaluation, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions.\nAll participants signed informed consent forms. These enrolled inmates of our study were protected according to the guidelines of the Declaration of Helsinki. The current study of DAA therapy was approved by the Institutional Review Board of Tri-Service General Hospital (IRB: TSGHIRB 2-107-05-080).\nAssessment, monitoring and endpoints Anti-HCV antibody was determined by the third generation, commercially available immunoassay (Ax SYM HCV III; Abbott Laboratories, North Chicago, IL). HCV RNA viral loads and genotype were determined by real-time PCR assays [RealTime HCV; Abbott Molecular, Des Plaines IL, United States; detection limit: 12 IU/mL])[22]. Liver cirrhosis was defined by the presence of clinical, radiological, endoscopic or laboratory evidence of cirrhosis and/or portal hypertension or fibrosis-4 index (FIB-4) (> 6.5). Laboratory data monitoring and assessment of side effects were performed at treatment wk 2, 4, 8 and end-of-treatment (EOT), and 12 wk after EOT.\nThe primary endpoint was sustained virological response (SVR12, defined as undetectable HCV RNA throughout 12 wk of the post-treatment follow-up period).\nAnti-HCV antibody was determined by the third generation, commercially available immunoassay (Ax SYM HCV III; Abbott Laboratories, North Chicago, IL). HCV RNA viral loads and genotype were determined by real-time PCR assays [RealTime HCV; Abbott Molecular, Des Plaines IL, United States; detection limit: 12 IU/mL])[22]. Liver cirrhosis was defined by the presence of clinical, radiological, endoscopic or laboratory evidence of cirrhosis and/or portal hypertension or fibrosis-4 index (FIB-4) (> 6.5). Laboratory data monitoring and assessment of side effects were performed at treatment wk 2, 4, 8 and end-of-treatment (EOT), and 12 wk after EOT.\nThe primary endpoint was sustained virological response (SVR12, defined as undetectable HCV RNA throughout 12 wk of the post-treatment follow-up period).\nStatistical analyses. The efficacy of all DAA regimens was determined in a intent-to-treat (ITT) population (all enrolled patients with at least one dose of DAA) and a per-protocol (PP) population (subjects receiving at least one dose of DAA and retained in Penghu Prison throughout the DAA treatment and follow-up period). Safety assessments reported adverse event (AE), serious adverse event (SAE) and laboratory abnormalities in the ITT population. Continuous variables are expressed as means ± standard deviation (SD), and categorical variables are expressed as percentages. The differences of continuous variables are estimated by the Student’s t test. The differences in categorical variables are analyzed using the Chi-square test. The on-treatment and off-treatment virological response rates were analyzed in number and percentages with 95% confidence interval (CI). All data analyses were performed using the SPSS software version 18.0 (SPSS Inc., Chicago, Illinois, United States).\nThe efficacy of all DAA regimens was determined in a intent-to-treat (ITT) population (all enrolled patients with at least one dose of DAA) and a per-protocol (PP) population (subjects receiving at least one dose of DAA and retained in Penghu Prison throughout the DAA treatment and follow-up period). Safety assessments reported adverse event (AE), serious adverse event (SAE) and laboratory abnormalities in the ITT population. Continuous variables are expressed as means ± standard deviation (SD), and categorical variables are expressed as percentages. The differences of continuous variables are estimated by the Student’s t test. The differences in categorical variables are analyzed using the Chi-square test. The on-treatment and off-treatment virological response rates were analyzed in number and percentages with 95% confidence interval (CI). All data analyses were performed using the SPSS software version 18.0 (SPSS Inc., Chicago, Illinois, United States).\n\nPatients linked to onsite treatment program for HCV micro-elimination:\nHCV-viremic patients were recruited from two cohorts in Penghu Prison (Agency of Corrections, Ministry of Justice, Taiwan), a PWID-dominant prison (Figure 1).\n\nPatient flowchart of hepatitis C virus treatment with a simplified pan-genotypic directly-acting antivirals regimen in Penghu Prison. HCV: Hepatitis C virus; DAA: Directly-acting antivirals; SOL/VEL: Sofosbuvir/velpatasvir; GEL/PIB: Glecaprevir/pibrentasvir; EOTVR: Virological response at end-of-treatment; SVR12: Sustained viral response at post-treatment wk 12.\n\nHCV-viremic patients identified by a universal mass screening:\nIn September 2019, we conducted a 5 d universal mass screening of viral hepatitis in Penghu Prison. These inclusion criteria were prisoners, who were at least 20 years old, being willing to enter the study for screening of viral hepatitis. The study of mass screening was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (IRB: KMUHIRB-SV(I)-20190033). All participants provided written informed consents. A total of 1137 subjects from 1697 inmates participated the mass screening[21]. Among them, 396 (34.8%) subjects had anti-HCV seropositivity; 208 (52.5%) of the 396 subjects were seropositive for HCV ribonucleic acid (RNA) and linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\n\nHCV-viremic patients identified in outpatient clinics during the period of HCV mass screening:\nAnother 26 HCV-viremic subjects identified in outpatient clinics of Penghu Prison between August to December 2019 were also linked to the onsite HCV treatment program with universal sofosbuvir/velpatasvir regimen.\nAll patients received pretreatment evaluation in December 2019, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions. A 12 wk, oral pan-genotypic regimen of sofosbuvir/velpatasvir 400/100 mg fixed-dose combination once daily was initiated in January-February 2020. \n\nPatients identified and treated by DAAs in outpatient clinics before mass screening:\nA total of 91 HCV-viremic patients identified in outpatient clinics of Penghu Prison and treated with DAA before mass screening from 2017 to 2019 were enrolled as a control. The selection of DAA regimens were based on physician’s discretion according to the viral genotype and criteria of reimbursement of National Health Insurance Administration, Taiwan. All patients received pretreatment evaluation, including medical history, liver and renal function tests, complete blood cell counts, HCV viral loads and genotyping, abdominal sonography and assessment of potential drug–drug interactions.\nAll participants signed informed consent forms. These enrolled inmates of our study were protected according to the guidelines of the Declaration of Helsinki. The current study of DAA therapy was approved by the Institutional Review Board of Tri-Service General Hospital (IRB: TSGHIRB 2-107-05-080).\n\nAssessment, monitoring and endpoints:\nAnti-HCV antibody was determined by the third generation, commercially available immunoassay (Ax SYM HCV III; Abbott Laboratories, North Chicago, IL). HCV RNA viral loads and genotype were determined by real-time PCR assays [RealTime HCV; Abbott Molecular, Des Plaines IL, United States; detection limit: 12 IU/mL])[22]. Liver cirrhosis was defined by the presence of clinical, radiological, endoscopic or laboratory evidence of cirrhosis and/or portal hypertension or fibrosis-4 index (FIB-4) (> 6.5). Laboratory data monitoring and assessment of side effects were performed at treatment wk 2, 4, 8 and end-of-treatment (EOT), and 12 wk after EOT.\nThe primary endpoint was sustained virological response (SVR12, defined as undetectable HCV RNA throughout 12 wk of the post-treatment follow-up period).\n\nStatistical analyses.:\nThe efficacy of all DAA regimens was determined in a intent-to-treat (ITT) population (all enrolled patients with at least one dose of DAA) and a per-protocol (PP) population (subjects receiving at least one dose of DAA and retained in Penghu Prison throughout the DAA treatment and follow-up period). Safety assessments reported adverse event (AE), serious adverse event (SAE) and laboratory abnormalities in the ITT population. Continuous variables are expressed as means ± standard deviation (SD), and categorical variables are expressed as percentages. The differences of continuous variables are estimated by the Student’s t test. The differences in categorical variables are analyzed using the Chi-square test. The on-treatment and off-treatment virological response rates were analyzed in number and percentages with 95% confidence interval (CI). All data analyses were performed using the SPSS software version 18.0 (SPSS Inc., Chicago, Illinois, United States).\n\nRESULTS:\nPatient flowchart of HCV micro-elimination campaign The patient flowchart of HCV mass screen, assessment and treatment was shown in Figure 1. A total of 234 HCV-viremic patients, 208 from mass screening and 26 from outpatient clinics in Penghu Prison were assessed for eligibility of group therapy with sofosbuvir/velpatasvir in December 2019. Twenty-two patients were excluded from anti-HCV therapy due to scheduled to be released from jail (n = 16) or transferred to other jails (n = 3) within 6 mo, unwilling to receive therapy (n = 2) and prior glecaprevir/pibrentasvir treatment failure (n = 1). Finally, 212 patients were recruited for sofosbuvir/velpatasvir therapy initiated in January-February 2020.\nThe patient flowchart of HCV mass screen, assessment and treatment was shown in Figure 1. A total of 234 HCV-viremic patients, 208 from mass screening and 26 from outpatient clinics in Penghu Prison were assessed for eligibility of group therapy with sofosbuvir/velpatasvir in December 2019. Twenty-two patients were excluded from anti-HCV therapy due to scheduled to be released from jail (n = 16) or transferred to other jails (n = 3) within 6 mo, unwilling to receive therapy (n = 2) and prior glecaprevir/pibrentasvir treatment failure (n = 1). Finally, 212 patients were recruited for sofosbuvir/velpatasvir therapy initiated in January-February 2020.\nPatient characteristics The baseline characteristics of 303 HCV-viremic patients, including 212 in HCV micro-elimination campaign and 91 sporadic controls from outpatient clinics before micro-elimination campaign were listed in Table 1. They mean age was 48.4 years with male dominant (99.7%). Thirty (9.9%) had HBV coinfection. The mean FIB-4 was 1.3, with 20 (6.6%) had advanced fibrosis (FIB-4 > 3.25). Only one patient (0.3%) had liver cirrhosis. The mean HCV RNA levels was 6.5 Logs IU/mL, dominant with HCV genotype 1 (HCV-GT1, 42.2%), followed by HCV-GT6 (35.3%), HCV-GT3 (11.6%) and HCV-GT2 (10.6%). Three (1%) patients were prior IFN-experienced. The two groups had comparable characteristics in terms of age, gender, HBV co-infection, liver and renal function tests, FIB-4 score, HCV genotype distribution, and prior history of IFN-based therapy. However, the sporadic patients identified in outpatient clinics had significantly higher proportion of comorbidities, including diabetes, hypertension, hyperlipidemia and an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2, but significantly lower HCV viral loads. None of patient had decompensated cirrhosis nor liver cancer.\nBaseline characteristics of hepatitis C virus-infected patients receiving directly-acting antivirals therapy between sporadic hepatitis C virus therapy in outpatient clinics and campaign of hepatitis C virus micro-elimination in Penghu prison\n56 patients did not have body mass index information (12 patients before campaign of hepatitis C virus (HCV) micro-elimination; 44 patients in campaign of HCV micro-elimination).\n\nP < 0.05. DAA: Directly-acting antivirals; HCV: Hepatitis C virus; BMI: Body mass index; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; LC: Liver cirrhosis; FIB-4: Fibrosis-4 index; HBsAg: Hepatitis B surface antigen; eGFR: Estimated glomerular filtration rate (mL/min/1.73 m2); SOF: Sofosbuvir; VEL: Velpatasvir; LDV: Ledipasvir; GLE: Glecaprevir; PIB: Pibrentasvir; IFN: Interferon.\nThe baseline characteristics of 303 HCV-viremic patients, including 212 in HCV micro-elimination campaign and 91 sporadic controls from outpatient clinics before micro-elimination campaign were listed in Table 1. They mean age was 48.4 years with male dominant (99.7%). Thirty (9.9%) had HBV coinfection. The mean FIB-4 was 1.3, with 20 (6.6%) had advanced fibrosis (FIB-4 > 3.25). Only one patient (0.3%) had liver cirrhosis. The mean HCV RNA levels was 6.5 Logs IU/mL, dominant with HCV genotype 1 (HCV-GT1, 42.2%), followed by HCV-GT6 (35.3%), HCV-GT3 (11.6%) and HCV-GT2 (10.6%). Three (1%) patients were prior IFN-experienced. The two groups had comparable characteristics in terms of age, gender, HBV co-infection, liver and renal function tests, FIB-4 score, HCV genotype distribution, and prior history of IFN-based therapy. However, the sporadic patients identified in outpatient clinics had significantly higher proportion of comorbidities, including diabetes, hypertension, hyperlipidemia and an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2, but significantly lower HCV viral loads. None of patient had decompensated cirrhosis nor liver cancer.\nBaseline characteristics of hepatitis C virus-infected patients receiving directly-acting antivirals therapy between sporadic hepatitis C virus therapy in outpatient clinics and campaign of hepatitis C virus micro-elimination in Penghu prison\n56 patients did not have body mass index information (12 patients before campaign of hepatitis C virus (HCV) micro-elimination; 44 patients in campaign of HCV micro-elimination).\n\nP < 0.05. DAA: Directly-acting antivirals; HCV: Hepatitis C virus; BMI: Body mass index; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; LC: Liver cirrhosis; FIB-4: Fibrosis-4 index; HBsAg: Hepatitis B surface antigen; eGFR: Estimated glomerular filtration rate (mL/min/1.73 m2); SOF: Sofosbuvir; VEL: Velpatasvir; LDV: Ledipasvir; GLE: Glecaprevir; PIB: Pibrentasvir; IFN: Interferon.\nTreatment efficacy All of 212 patients in HCV micro-elimination campaign received sofosbuvir/ velpatasvir treatment; while among 91 sporadic patients with DAA therapy before HCV micro-elimination campaign, 78 (85.7%) received 12 wk of sofosbuvir/Ledipasvir and 13 (14.3%) received 8-12 wk of glecaprevir/pibrentasvir according to the Taiwan HCV guideline[12,13].\nIn ITT analysis, the overall SVR12 rate was 95.4% (289/303) with comparable SVR12 rates between sporadic HCV control group (94.5%, 86/91) and HCV micro-elimination group (95.8%, 203/212, P = 0.126, Table 2).\nVirological responses of hepatitis C virus-infected patients receiving directly-acting antivirals therapy before and during campaign of hepatitis C virus micro-elimination in Penghu prison in Penghu prison\nOne missing data.\nOne transferred; One missing data.\nTwo transferred; One released.\nFour released.\nOne relapser.\nFour transferred; Five released. HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \nDuring DAA treatment period, all of patients in sporadic HCV control group completed DAA therapy, while 3 patients in HCV micro-elimination group lost-to-follow (2 transferred; 1 released). During the post-treatment follow-up period, 4 patients in sporadic HCV control group lost-to-follow (4 released), while 6 patients in HCV micro-elimination group lost-to-follow (2 transferred; 4 released). In PP analysis, the overall SVR12 rate was 99.7% (289/290) with comparable SVR12 rates between sporadic HCV control group (98.9%, 86/87) and HCV micro-elimination group (100%, 203/203, P = 0.126, Table 2). Only one patient experienced virological failure (54 years old male, treatment-naïve, HCV-GT3 infection with baseline viral loads of 62,883 IU/mL and FIB-4 of 2.37; relapsed from a 12 wk regimen of glecaprevir/pibrentasvir).\nAll of 212 patients in HCV micro-elimination campaign received sofosbuvir/ velpatasvir treatment; while among 91 sporadic patients with DAA therapy before HCV micro-elimination campaign, 78 (85.7%) received 12 wk of sofosbuvir/Ledipasvir and 13 (14.3%) received 8-12 wk of glecaprevir/pibrentasvir according to the Taiwan HCV guideline[12,13].\nIn ITT analysis, the overall SVR12 rate was 95.4% (289/303) with comparable SVR12 rates between sporadic HCV control group (94.5%, 86/91) and HCV micro-elimination group (95.8%, 203/212, P = 0.126, Table 2).\nVirological responses of hepatitis C virus-infected patients receiving directly-acting antivirals therapy before and during campaign of hepatitis C virus micro-elimination in Penghu prison in Penghu prison\nOne missing data.\nOne transferred; One missing data.\nTwo transferred; One released.\nFour released.\nOne relapser.\nFour transferred; Five released. HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \nDuring DAA treatment period, all of patients in sporadic HCV control group completed DAA therapy, while 3 patients in HCV micro-elimination group lost-to-follow (2 transferred; 1 released). During the post-treatment follow-up period, 4 patients in sporadic HCV control group lost-to-follow (4 released), while 6 patients in HCV micro-elimination group lost-to-follow (2 transferred; 4 released). In PP analysis, the overall SVR12 rate was 99.7% (289/290) with comparable SVR12 rates between sporadic HCV control group (98.9%, 86/87) and HCV micro-elimination group (100%, 203/203, P = 0.126, Table 2). Only one patient experienced virological failure (54 years old male, treatment-naïve, HCV-GT3 infection with baseline viral loads of 62,883 IU/mL and FIB-4 of 2.37; relapsed from a 12 wk regimen of glecaprevir/pibrentasvir).\nSafety profiles The safety profiles of both groups were shown in Table 3. None of patients had treatment discontinuation other than released or transferred. None experienced serious adverse event. The frequency of adverse events was 4.3% (4/91) and 1.4% (3/212), respectively, among patients in sporadic control group and HCV micro-elimination group. The most reported adverse events were rash in 3 of 13 (23.1%) patients treated with glecaprevir/pibrentasvir and pruritus in 2 of 212 (0.9%) patients treated with sofosbuvir/velpatasvir. None of patients experienced grade 3 or 4 Laboratory abnormality.\nSafety profiles of hepatitis C virus-infected patients receiving direct-acting antivirals therapy in Penghu prison\nDAA: Directly-acting antivirals; HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \nThe safety profiles of both groups were shown in Table 3. None of patients had treatment discontinuation other than released or transferred. None experienced serious adverse event. The frequency of adverse events was 4.3% (4/91) and 1.4% (3/212), respectively, among patients in sporadic control group and HCV micro-elimination group. The most reported adverse events were rash in 3 of 13 (23.1%) patients treated with glecaprevir/pibrentasvir and pruritus in 2 of 212 (0.9%) patients treated with sofosbuvir/velpatasvir. None of patients experienced grade 3 or 4 Laboratory abnormality.\nSafety profiles of hepatitis C virus-infected patients receiving direct-acting antivirals therapy in Penghu prison\nDAA: Directly-acting antivirals; HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \n\nPatient flowchart of HCV micro-elimination campaign:\nThe patient flowchart of HCV mass screen, assessment and treatment was shown in Figure 1. A total of 234 HCV-viremic patients, 208 from mass screening and 26 from outpatient clinics in Penghu Prison were assessed for eligibility of group therapy with sofosbuvir/velpatasvir in December 2019. Twenty-two patients were excluded from anti-HCV therapy due to scheduled to be released from jail (n = 16) or transferred to other jails (n = 3) within 6 mo, unwilling to receive therapy (n = 2) and prior glecaprevir/pibrentasvir treatment failure (n = 1). Finally, 212 patients were recruited for sofosbuvir/velpatasvir therapy initiated in January-February 2020.\n\nPatient characteristics :\nThe baseline characteristics of 303 HCV-viremic patients, including 212 in HCV micro-elimination campaign and 91 sporadic controls from outpatient clinics before micro-elimination campaign were listed in Table 1. They mean age was 48.4 years with male dominant (99.7%). Thirty (9.9%) had HBV coinfection. The mean FIB-4 was 1.3, with 20 (6.6%) had advanced fibrosis (FIB-4 > 3.25). Only one patient (0.3%) had liver cirrhosis. The mean HCV RNA levels was 6.5 Logs IU/mL, dominant with HCV genotype 1 (HCV-GT1, 42.2%), followed by HCV-GT6 (35.3%), HCV-GT3 (11.6%) and HCV-GT2 (10.6%). Three (1%) patients were prior IFN-experienced. The two groups had comparable characteristics in terms of age, gender, HBV co-infection, liver and renal function tests, FIB-4 score, HCV genotype distribution, and prior history of IFN-based therapy. However, the sporadic patients identified in outpatient clinics had significantly higher proportion of comorbidities, including diabetes, hypertension, hyperlipidemia and an estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2, but significantly lower HCV viral loads. None of patient had decompensated cirrhosis nor liver cancer.\nBaseline characteristics of hepatitis C virus-infected patients receiving directly-acting antivirals therapy between sporadic hepatitis C virus therapy in outpatient clinics and campaign of hepatitis C virus micro-elimination in Penghu prison\n56 patients did not have body mass index information (12 patients before campaign of hepatitis C virus (HCV) micro-elimination; 44 patients in campaign of HCV micro-elimination).\n\nP < 0.05. DAA: Directly-acting antivirals; HCV: Hepatitis C virus; BMI: Body mass index; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; LC: Liver cirrhosis; FIB-4: Fibrosis-4 index; HBsAg: Hepatitis B surface antigen; eGFR: Estimated glomerular filtration rate (mL/min/1.73 m2); SOF: Sofosbuvir; VEL: Velpatasvir; LDV: Ledipasvir; GLE: Glecaprevir; PIB: Pibrentasvir; IFN: Interferon.\n\nTreatment efficacy:\nAll of 212 patients in HCV micro-elimination campaign received sofosbuvir/ velpatasvir treatment; while among 91 sporadic patients with DAA therapy before HCV micro-elimination campaign, 78 (85.7%) received 12 wk of sofosbuvir/Ledipasvir and 13 (14.3%) received 8-12 wk of glecaprevir/pibrentasvir according to the Taiwan HCV guideline[12,13].\nIn ITT analysis, the overall SVR12 rate was 95.4% (289/303) with comparable SVR12 rates between sporadic HCV control group (94.5%, 86/91) and HCV micro-elimination group (95.8%, 203/212, P = 0.126, Table 2).\nVirological responses of hepatitis C virus-infected patients receiving directly-acting antivirals therapy before and during campaign of hepatitis C virus micro-elimination in Penghu prison in Penghu prison\nOne missing data.\nOne transferred; One missing data.\nTwo transferred; One released.\nFour released.\nOne relapser.\nFour transferred; Five released. HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \nDuring DAA treatment period, all of patients in sporadic HCV control group completed DAA therapy, while 3 patients in HCV micro-elimination group lost-to-follow (2 transferred; 1 released). During the post-treatment follow-up period, 4 patients in sporadic HCV control group lost-to-follow (4 released), while 6 patients in HCV micro-elimination group lost-to-follow (2 transferred; 4 released). In PP analysis, the overall SVR12 rate was 99.7% (289/290) with comparable SVR12 rates between sporadic HCV control group (98.9%, 86/87) and HCV micro-elimination group (100%, 203/203, P = 0.126, Table 2). Only one patient experienced virological failure (54 years old male, treatment-naïve, HCV-GT3 infection with baseline viral loads of 62,883 IU/mL and FIB-4 of 2.37; relapsed from a 12 wk regimen of glecaprevir/pibrentasvir).\n\nSafety profiles:\nThe safety profiles of both groups were shown in Table 3. None of patients had treatment discontinuation other than released or transferred. None experienced serious adverse event. The frequency of adverse events was 4.3% (4/91) and 1.4% (3/212), respectively, among patients in sporadic control group and HCV micro-elimination group. The most reported adverse events were rash in 3 of 13 (23.1%) patients treated with glecaprevir/pibrentasvir and pruritus in 2 of 212 (0.9%) patients treated with sofosbuvir/velpatasvir. None of patients experienced grade 3 or 4 Laboratory abnormality.\nSafety profiles of hepatitis C virus-infected patients receiving direct-acting antivirals therapy in Penghu prison\nDAA: Directly-acting antivirals; HCV: Hepatitis C virus; VEL: Velpatasvir; SOF: Sofosbuvir. \n\nDISCUSSION:\nIn the current study, we demonstrated that mass screening combined with onsite group therapy by using a simplified pan-genotypic DAA regimen, 12 wk of sofosbuvir/velpatasvir, provides an “one-size fits all” solution toward the achievement of HCV micro-elimination in prisoners. The SVR rate was 95.6% in ITT population and 100% in PP population after excluding the inmates released or transferred before end-of-follow-up. The high SVR rate was observed in this PWID-dominant population, which HCV genotype distribution was diverse, including genotypes 1a, 1b, 2, 3 and 6. \nRecent advance in the development of IFN-free pan-genotypic DAA regimens has remarkably improved the treatment efficacy with an overall SVR rates of > 90%. Therefore, WHO set the global of HCV elimination by 2030, through the achievement of > 90% diagnosis rate and > 80% treatment rate for eligible patients[16]. Nevertheless, there are many barriers in each HCV care cascade toward HCV elimination at the population level[11,23]. To overcome the barriers, combining the concept of micro-elimination and an outreach strategy with immediate onsite treatment would be a more efficient and practical approach to achieve that goal[18,24]. The current study compared the HCV-infected inmates identified sporadically in outpatient clinics of Penghu Prison from 2017 to 2019 before mass screening and the patients identified by mass screening. We found that mass screening identified 208 HCV-viremic patients in a 5 d screening program from 1137 inmates (encountered around two-third of total inmates in Penghu Prison), compared to 91 HCV-viremic inmates treated in outpatient clinics from 2017 to September 2019. Our results demonstrated that mass screening with immediate onsite treatment provide much more efficient and practical solution to overcome the gaps of disease awareness and link-to-care in the HCV care cascades toward HCV micro-elimination in prisoners. In addition, we implemented “HCV reflex testing” in the mass screening program to scale-up and speed-up the diagnosis and link-to-care for treatment uptake of HCV infections[25].\nPWID is known as the major risk factor of HCV infection and transmission. Although the anti-HCV prevalence in PWID prisoners decreased from 91% in 2014 to 34.8% in 2019 by the strategy of safe injection in Taiwan[21], almost all (97.6%) of HCV-infected prisoners were PWID. Given the lack of vaccine available and high risk of transmission, the strategy of universal screening and concept of “treatment as prevention” are the keys to HCV elimination in prison as well as PWID.\nWe observed that the sporadic HCV-infected prisoners identified in outpatient clinics had significantly higher proportion of comorbidities, including diabetes, hypertension, hyperlipidemia and eGFR, than those participating in the HCV micro-elimination campaign. It implicated that a great proportion of identified sporadically in outpatient clinics were due to concomitant morbidities; by contrast, many HCV-infected patients were unaware to their HCV diseases. In our mass screening, only 36.6% (145/396) of HCV-infected prisoners were aware of HCV infection before screening[21]. It indicates that the implementation of an outreach strategy with universal mass screen is necessary for HCV micro-elimination in prison.\nDespite of the advances in the management of HCV infections, DAA therapy in incarcerated HCV-infected people remains many obstacles to be resolved, including disease unawareness, lack of updated information about the benefits of new DAA treatment, uncertainty of treatment right[26], poor accessibility due to of onsite treatment facilities or HCV treaters. Another difficulty for HCV treatment in prisoners is the unexpected or scheduled releasing from prison or transferring to other prisons, which frequently leads to the interruption of treatment or lost-to- follow up[20,27]. We are lucky that the Taiwan Health Insurance covered all incarcerated people, including all of the laboratory tests and ultrasound sonography and the cost of DAA regimens. Each prison has a contracted hospital providing point-of-care facility. Before initiating DAA therapy, we excluded the patients with expected release or transfer within 24 wk, and negotiated with the authority to avoid unnecessary transferring to other prisons during the period of HCV treatment and follow-up once the inmates entering the DAA course. Eventually we achieved a high treatment rate of 90.6% (212/234) and a high treatment complete rate of 95.8% (203/212), with a high cure rate at 100% (212/212).\nBefore the IFN-free DAA available, the lower SVR rate, much longer treatment duration and frequent adverse events of IFN-based treatment discouraged HCV-infected prisoners from receiving treatment[10]. IFN-free DAA regimens revolutionized HCV treatment which has largely extended the indication for various HCV-infected patients. Nevertheless, the application of typical DAA regimens are based on HCV genotype, presence of decompensated cirrhosis, renal function, and prior treatment experience. The two pangenotypic DAA regimens, sofosbuvir/velpatasvir, and glecaprevir/pibrentasvir, have achieved very high SVR rates of > 95%, regardless of HCV GTs, except for treatment-experienced cirrhotic HCV GT3 patients or GT3b patients[8,12,13]. Recently, to improve the access to anti-HCV therapy, reduce the cost of laboratory tests and the relative complexity of genotype-based treatment strategies, simplified treatment without many information needed for treatment decision are recommended to facilitate the care cascade among populations who are historically less engaged in healthcare, such as PWIDs and prisoners[8]. EASL recommends simplified, genotyping/subtyping-free regimens for IFN-free DAA treatment-naïve (except sofosbuvir plus ribavirin), HCV-infected or HCV-HIV coinfected adolescent and adult patients without cirrhosis or with compensated cirrhosis, regardless of HCV genotypes[8]. These recommendations are a universal 12 wk regimen of sofosbuvir/velpatasvir for all patients or glecaprevir/pibrentasvir, 8 wk for non-cirrhotic, 12 wk for compensated cirrhotic, and 16 wk for HCV GT3 patients, respectively. There are only four information needed before treatment, including the presence of HCV viremia, potential drug-drug interactions, and prior treatment experience, and presence of cirrhosis. The advantages of glecaprevir/pibrentasvir is a shorter 8 wk regimen for treatment-naïve HCV patients and IFN-experienced non-cirrhotic patients with compensated liver diseases, which would be benefit for prisoners who are expected to be released or transferred in a short term. However, glecaprevir, a protease inhibitor, is contraindicated for patients with hepatic decompensation and at risk for rare occurrence of serious drug-induced liver injury[28]. Also, glecaprevir/ pibrentasvir has higher pill burden, three tablets a d. The advantages of sofosbuvir/ velpatasvir include a universal fixed 12 wk regimen, one tablet a d, for all HCV patients with compensated liver diseases, less frequency of potential drug-drug interactions[29], and safety for those with hepatic decompensation. However, a 12 wk regimen with sofosbuvir/velpatasvir needs one more visit and monitoring when compared to an 8 wk regimen with glecaprevir/pibrentasvir. Therefore, we select sofosbuvir/velpatasvir as the antiviral regimen for our outreach onsite treatment. In our study, all HCV-viremic prisoners fit the criteria of simplified, genotyping/ subtyping-free regimens, except one who failed to prior glecaprevir/pibrentasvir therapy and was not enrolled for sofosbuvir/velpatasvir treatment. In our PP analysis, the overall SVR12 rate was comparable between HCV patient group (98.9%, 86/87) and HCV micro-elimination group (100%, 203/203). Our study provided evidence for the concept that simplified, genotyping/subtyping-free regimens can achieve high SVR12 rate in HCV-infected PWID-dominant prisoners.\nIn our study, none of prisoners had DAA treatment discontinuation due to adverse events. None experienced serious adverse event. These data indicated that the simplified, genotyping/subtyping-free regimen, sofosbuvir/velpatasvir, was safe and well tolerated for HCV-infected PWID-dominant prisoners. Very few adverse events were reported in both groups, whatever using sofosbuvir/Ledipasvir, glecaprevir/ pibrentasvir and sofosbuvir/velpatasvir, when compared to the data from clinical trials[30,31]. It might be due to that current population was younger and less patients with advanced fibrosis or chronic kidney diseases.\nThere were some limitations in our study. First, not all inmates in Penghu Prison participated our mass screening. Strategies and policy to encourage inmates to receive HCV screening is mandatory to achieve the goal of WHO. Second, unexpected prisoners’ transferral and release could not be completely avoided, which caused incomplete treatment and follow-up. Successfully linking the released or transferred people to another HCV treaters could help completing HCV treatment and follow-up. Third, there was no reimbursement for the retreatment of prior DAA failed patients in Taiwan at the time of the current study.\n\nCONCLUSION:\nWell-designed strategies for mass screening and treatment for HCV-infected prisoners can be implemented successfully by the collaboration between physicians and prison authorities. We demonstrated that mass screening followed by immediate onsite treatment with a simplified pangenotypic DAA regimen, sofosbuvir/velpatasvir, provides successful strategies toward HCV micro-elimination among prisoners.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nPrisoners are at risk of hepatitis C virus (HCV) infection, especially among the people who inject drugs (PWID). We implemented an outreach strategy in combination with universal mass screening and immediate onsite treatment with a simplified pan-genotypic direct-acting antivirals (DAA) regimen, 12 wk of sofosbuvir/velpatasvir, in a PWID-dominant prison in Taiwan.\n\nMethods:\nHCV-viremic patients were recruited for onsite treatment program for HCV micro-elimination with a pangenotypic DAA regimen, 12 wk of sofosbuvir/ velpatasvir, from two cohorts in Penghu Prison, either identified by mass screen or in outpatient clinics, in September 2019. Another group of HCV-viremic patients identified sporadically in outpatient clinics before mass screening were enrolled as a control group. The primary endpoint was sustained virological response (SVR12, defined as undetectable HCV ribonucleic acid (RNA) 12 wk after end-of-treatment).\n\nResults:\nA total of 212 HCV-viremic subjects were recruited for HCV micro-elimination campaign; 91 patients treated with sofosbuvir/Ledipasvir or glecaprevir/ pibrentasvir before mass screening were enrolled as a control. The HCV micro-elimination group had significantly lower proportion of diabetes, hypertension, hyperlipidemia, advanced fibrosis and chronic kidney diseases, but higher levels of HCV RNA. The SVR12 rate was comparable between the HCV micro-elimination and control groups, 95.8% (203/212) vs 94.5% (86/91), respectively, in intent-to-treat analysis, and 100% (203/203) vs 98.9% (86/87), respectively, in per-protocol analysis. There was no virological failure, treatment discontinuation, and serious adverse event among sofosbuvir/velpatasvir-treated patients in the HCV micro-elimination group.\n\nConclusions:\nOutreach mass screening followed by immediate onsite treatment with a simplified pangenotypic DAA regimen, sofosbuvir/velpatasvir, provides successful strategies toward HCV micro-elimination among prisoners."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nHepatitis C virus (HCV) infection is a progressive and blood-borne infectious disease that can lead to end stage liver diseases, such as hepatic decompensation, liver cirrhosis, and hepatocellular carcinoma[1,2]. Iatrogenic transmission of HCV, such as blood transfusion and surgery, has decreased in developed countries. Whereas people who inject drugs (PWID) has become the major population of HCV transmission, which could consist of approximately 80% of HCV-infected patients[3]. Given that lack of vaccine available, “treatment as prevention” for HCV transmission in PWID is very important for HCV elimination.\nPrisoners are at high risk of HCV infection, with prevalence rates ranging from 3.1% to 38%[4,5]. The high prevalence of HCV infection in prisoners is resulted from unsafe lifestyles, psychiatric disorders, and social problems before they are incarcerated. Recently, PWID has been the most important risk factor of HCV infection in prisoners[6]. The anti-HCV prevalence rate could be as high as 91% among PWID prisoners[7]. Screening and eliminating HCV infection in prisoners is therefore an important social health issue.\nAccording to the American Association for the Study of Liver Diseases and European Association for the Study of the Liver (EASL) guidelines, all HCV viremic patients should be treated if life span is expected more than one year[8,9]. HCV therapeutic strategies have been revolutionized significantly because of the availability of direct-acting antivirals (DAA)[10]. Interferon (IFN)-based regimens for HCV infection have serious side effects, long therapeutic duration, and contraindications, leading to the huge gaps in HCV care cascade[11]. The current IFN-free DAA regimens provide shorter treatment duration, very high treatment efficacy and safety profiles, not only for general population[12], but also for special populations[13], such as HCV/human immunodeficiency virus (HIV) coinfected patients, hepatitis B virus (HBV)/HCV coinfected patients and patients with chronic kidney diseases in real-world clinical settings[14,15].\nWorld Health Organization (WHO) set a global goal of HCV elimination by 2030[16], and Taiwan authority is even ambitious by 2025[17]. To achieve the goal, implementation of the concept of HCV micro-elimination is regarding as an efficient and practical strategy[18]. We have proved that “universal mass screening plus outreach onsite treatment” is the key to achieve HCV micro-elimination among patients under maintenance hemodialysis[19].\nRecently, the latest EASL HCV guideline recommended simplified, genotyping/ subtyping-free, pangenotypic anti-HCV treatment, either sofosbuvir/ velpatasvir or glecaprevir/pibrentasvir, to increase the accessibility and global cure rates among patients with > 12 years, chronic hepatitis C without cirrhosis or with compensated cirrhosis, with or without HIV co-infection, whatever treatment-naïve or IFN-experienced[8].\nSince HCV treatment is not frequently administered to prisoners due to unawareness of HCV infection, difficultly management, easily loss to follow-up, and lack of hepatologist in prison[20], collaboration between hepatologists and prison authorities to carry out strategies for HCV diagnosis and treatment in prisoners in highly demanded. Herein, we implemented an outreach strategy in combination with universal mass screen and onsite treatment with a simplified pan-genotypic DAA regimen, 12 wk of sofosbuvir/velpatasvir, toward HCV micro-elimination in a PWID-dominant prison in Taiwan.\n\nCONCLUSION:\nOur study provided evidence for the concept that simplified, genotyping/subtyping-free regimens can achieve high SVR12 rate in HCV-infected prisoners. In the future, it is possible to implement the strategy to all prisoners in our country."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nPrisoners are at risk of hepatitis C virus (HCV) infection, especially among the people who inject drugs (PWID). We implemented an outreach strategy in combination with universal mass screening and immediate onsite treatment with a simplified pan-genotypic direct-acting antivirals (DAA) regimen, 12 wk of sofosbuvir/velpatasvir, in a PWID-dominant prison in Taiwan.\n\nMethods:\nHCV-viremic patients were recruited for onsite treatment program for HCV micro-elimination with a pangenotypic DAA regimen, 12 wk of sofosbuvir/ velpatasvir, from two cohorts in Penghu Prison, either identified by mass screen or in outpatient clinics, in September 2019. Another group of HCV-viremic patients identified sporadically in outpatient clinics before mass screening were enrolled as a control group. The primary endpoint was sustained virological response (SVR12, defined as undetectable HCV ribonucleic acid (RNA) 12 wk after end-of-treatment).\n\nResults:\nA total of 212 HCV-viremic subjects were recruited for HCV micro-elimination campaign; 91 patients treated with sofosbuvir/Ledipasvir or glecaprevir/ pibrentasvir before mass screening were enrolled as a control. The HCV micro-elimination group had significantly lower proportion of diabetes, hypertension, hyperlipidemia, advanced fibrosis and chronic kidney diseases, but higher levels of HCV RNA. The SVR12 rate was comparable between the HCV micro-elimination and control groups, 95.8% (203/212) vs 94.5% (86/91), respectively, in intent-to-treat analysis, and 100% (203/203) vs 98.9% (86/87), respectively, in per-protocol analysis. There was no virological failure, treatment discontinuation, and serious adverse event among sofosbuvir/velpatasvir-treated patients in the HCV micro-elimination group.\n\nConclusions:\nOutreach mass screening followed by immediate onsite treatment with a simplified pangenotypic DAA regimen, sofosbuvir/velpatasvir, provides successful strategies toward HCV micro-elimination among prisoners."},"whole_article_text_length":{"kind":"number","value":8364,"string":"8,364"},"whole_article_abstract_length":{"kind":"number","value":367,"string":"367"},"other_sections_lengths":{"kind":"list like","value":[625,109,139,105,156,162,186,2176,131,412,381,153,1667,59],"string":"[\n 625,\n 109,\n 139,\n 105,\n 156,\n 162,\n 186,\n 2176,\n 131,\n 412,\n 381,\n 153,\n 1667,\n 59\n]","isConversational":false},"num_sections":{"kind":"number","value":15,"string":"15"},"most_frequent_words":{"kind":"list like","value":["hcv","patients","treatment","elimination","daa","micro","micro 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| Prisons [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"null"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Antiviral Agents | Hepacivirus | Hepatitis C | Hepatitis C, Chronic | Humans | Prisons [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Antiviral Agents | Hepacivirus | Hepatitis C | Hepatitis C, Chronic | Humans | Prisons [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Antiviral Agents | Hepacivirus | Hepatitis C | Hepatitis C, Chronic | Humans | Prisons [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hcv elimination prisoners | pwid important hcv | hcv infected pwid | inmates receive hcv | prison hcv hepatitis [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] hcv elimination prisoners | pwid important hcv | hcv infected pwid | inmates 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elimination | mass | screening [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] hcv | patients | treatment | hepatitis | daa | elimination | micro | micro elimination | mass | screening [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] PWID ||| 12 | PWID | Taiwan [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] HCV micro-elimination | DAA | 12 | two | Penghu Prison | September 2019 ||| ||| RNA | 12 [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"null"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] HCV micro-elimination [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] PWID ||| 12 | PWID | Taiwan ||| HCV micro-elimination | DAA | 12 | two | Penghu Prison | September 2019 ||| ||| RNA | 12 ||| 212 | HCV micro-elimination | 91 ||| HCV | HCV RNA ||| 95.8% | 94.5% | 100% | 203/203 | 98.9% ||| ||| HCV micro-elimination [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] PWID ||| 12 | PWID | Taiwan ||| HCV micro-elimination | DAA | 12 | two | Penghu Prison | September 2019 ||| ||| RNA | 12 ||| 212 | HCV micro-elimination | 91 ||| HCV | HCV RNA ||| 95.8% | 94.5% | 100% | 203/203 | 98.9% ||| ||| HCV micro-elimination [SUMMARY]"}}},{"rowIdx":54,"cells":{"title":{"kind":"string","value":"Prevalence of body-focused repetitive behaviors in three large medical colleges of Karachi: a cross-sectional study."},"pmid":{"kind":"string","value":"23116460"},"background_abstract":{"kind":"string","value":"Body-focused repetitive behaviors (BFRBs) that include skin picking (dermatillomania), hair pulling (trichotillomania) and nail biting (onychophagia), lead to harmful physical and psychological sequelae.The objective was to determine the prevalence of BFRBs among students attending three large medical colleges of Karachi. It is imperative to come up with frequency to design strategies to decrease the burden and adverse effects associated with BFRBs among medical students."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"A cross-sectional study was conducted among 210 students attending Aga Khan University, Dow Medical College and Sind Medical College, Karachi, in equal proportion. Data were collected using a pre tested tool, \"Habit Questionnaire\". Diagnoses were made on the criteria that a student must be involved in an activity 5 times or more per day for 4 weeks or more. Convenience sampling was done to recruit the participants aged 18 years and above after getting written informed consent."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The overall prevalence of BFRBs was found to be 46 (22%). For those positive for BFRBs, gender distribution was as follows: females 29 (13.9%) and males 17 (8.1%). Among these students, 19 (9.0%) were engaged in dermatillomania, 28 (13.3%) in trichotillomania and 13 (6.2%) in onychophagia."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"High proportions of BFRBs are reported among medical students of Karachi. Key health messages and interventions to reduce stress and anxiety among students may help in curtailing the burden of this disease which has serious adverse consequences."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adolescent","Adult","Cross-Sectional Studies","Female","Humans","Male","Obsessive-Compulsive Disorder","Pakistan","Schools, Medical","Young Adult"],"string":"[\n \"Adolescent\",\n \"Adult\",\n \"Cross-Sectional Studies\",\n \"Female\",\n \"Humans\",\n \"Male\",\n \"Obsessive-Compulsive Disorder\",\n \"Pakistan\",\n \"Schools, Medical\",\n \"Young Adult\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"3508914"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Body-focused repetitive behaviors (BFRBs) refer to a group of behaviors that include skin picking (dermatillomania), hair pulling (trichotillomania) and nail biting (onychophagia), which result in physical and psychological difficulties\n[1]. These behaviors for some individuals are simply referred to as nervous habits\n[2]. However, these nervous habits become problematic when they interfere with the person’s everyday functioning. When these BFRBs cross this line, then they are classified as Impulse Control Disorders.\nBFRBs most often begin in late childhood or adolescence. They are among the most poorly understood, misdiagnosed, and under treated groups of disorders. The key factor underlying BFRBs is difficulty resisting the urge or impulse to perform a certain behavior that causes a degree of relief. The behavior continues because the BFRB results in a more pleasant state therefore, it is negatively reinforced.\nPrevalence estimates indicates that such behaviors are quite common among students. Nail biting of 2 times or more per week was reported among 63.6% students in United States of America\n[2]. Another study from USA, using the stringent criteria of 5 times or more per week stated 21.8% students engaged in mouth, lips or cheeks chewing and 10.1% engaged in habitual nail biting\n[3]. A recent survey of college students found that 13.7% of the sample endorsed in at least one repetitive behavior that occurred more than five times per day for at least 4 weeks and produced some type of psychological or physical disruption of functioning\n[4]. Based on questionnaire screenings, a lifetime ICD rate of 3.5% was found in college students of Germany\n[5]. Very limited literature and no prevalence estimates regarding BFRBs could be found from Asian countries including Pakistan.\nThe treatment for BFRBs may include a combination of psychotropic medications and cognitive behavioral therapy (CBT). CBT often involves Habit Reversal Training and Exposure and Response Prevention (aka Exposure and Ritual Prevention).\nSince limited literature from our part of the world is available on body-focused repetitive behaviors and its impact on medical students and their lifestyles, it is imperative to spread awareness regarding them and find out ways and means to prevent them. It is therefore essential to come up with frequency to design strategies to decrease the prevalence of BFRBs and thus prevent their adverse effects among medical students.\nWe thus aimed to determine the prevalence of BFRBs among students attending three large medical colleges of Karachi."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"The study was conducted in three large medical colleges of Karachi: The Aga Khan University, Dow Medical College and Sindh Medical College. Students from Karachi as well as interior and foreign attend these institutes; hence provide better representation from different socio economic classes and cultures. The combined strength of student of the three colleges was approximately 3000. The study was conducted in July 2010.\nThe investigation was a cross-sectional study. Students were recruited in equal proportion from each college. A self-administered pre tested questionnaire was used for data collection. Data was collected by trained medical students. The study was approved by the Ethical Review Committee of Aga Khan University and Dow Medical College. Written informed consent was obtained from each student and an explanation of the purposes of the research was provided to them.\nEligibility criteria to participate in the study comprised of registered medical students aged 18 and above. However students less than 18 years and not willing to consent to participate in this study were excluded.\nA d “Habit Questionnaire” was used for gathering information on repetitive behaviors. Habit is a brief five items, self-report questionnaire that provides a standardized assessment of the frequency and duration of BFRBs.\nThe Habit Questionnaire has been found to possess moderate test–retest reliability of 0.69 of diagnosis of BFRB, p<0.001\n[4].\nParticipants were asked to indicate if they engage in the following repetitive behaviors: hair pulling, hair manipulation, nail biting, skin biting and mouth chewing. For each repetitive behavior, participants who acknowledged engaging in the specified behavior were asked about the frequency of the behavior (i.e., fewer or greater than five times per day) and the duration of the behavior (i.e., less than 4 weeks or longer than 4 weeks). Participants were also asked to specify if the behavior caused impairment, which was defined as (1) interference with daily functioning, (2) resultant injuries with possible permanent scarring or damage, (3) medical attention as a result of the behavior or (4) recommendation to cease the behavior by a medical professional. To rule out conditions that commonly co-occur with BFRBs, the final section of the Habit Questionnaire asked participants to indicate if they had ever been diagnosed with obsessive–compulsive disorder, Tourette’s syndrome, autism, Asperger’s syndrome or a developmental disability.\nTo meet the criteria for a BFRB, the participants needed to answer “yes” to engaging in at least one repetitive behavior five or more times per day, and the behavior had to be present for at least 4 weeks. Participants were also needed to report that the particular behavior interferes with functioning, caused an injury, caused him or her to seek medical attention or elicited a recommendation to stop the behavior by a medical professional.\nOur focus was on three behaviors: Trichotillomania (including ‘hair pulling’ and ‘hair manipulation’), Onychophagia (including ‘nail biting’) and Dermatillomania (including ‘chew mouth lips or cheek’ and ‘bite on any other areas’).\n Statistical analysis A sample size of at least 210 was required to estimate the prevalence of BFRBs among medical students assuming 14% prevalence figure of the Pakistani population, along with 80 percent power, 0.05 significance level, 5 percent bond on error and 10% adjustment for non-response rate. Convenience sampling was used by getting the questionnaires filled during regular college hours from students at four locations in the medical college: the lecture halls, laboratories, library and canteen. Questionnaires were filled on consecutive days until the required sample size was achieved.\nData was entered and analyzed using Statistical Package for the Social Sciences (SPSS) version 13.0. Initially descriptive statistics, frequencies and proportions were generated. Continuous variables were analyzed by t – test and categorical by chi-square or Fisher exact, where appropriate.\nA sample size of at least 210 was required to estimate the prevalence of BFRBs among medical students assuming 14% prevalence figure of the Pakistani population, along with 80 percent power, 0.05 significance level, 5 percent bond on error and 10% adjustment for non-response rate. Convenience sampling was used by getting the questionnaires filled during regular college hours from students at four locations in the medical college: the lecture halls, laboratories, library and canteen. Questionnaires were filled on consecutive days until the required sample size was achieved.\nData was entered and analyzed using Statistical Package for the Social Sciences (SPSS) version 13.0. Initially descriptive statistics, frequencies and proportions were generated. Continuous variables were analyzed by t – test and categorical by chi-square or Fisher exact, where appropriate."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"The prevalence of BFRBs among medical students of Karachi was found to be 46 (22%). For those positive for BFRBs, gender distribution was as follows: females 29 (13.9%) and males 17 (8.1%). The average age of participants was 21.5 ranging from 18–27 (refer Table\n1). Among these students, 19 (9.0%) were engaged in dermatillomania, 28 (13.3%) in trichotillomania and 13 (6.2%) in onychophagia (refer Table\n2).\nBaseline characteristics of participants\nBFRBs characteristics among medical students in Karachi\nAs discussed earlier, to meet criteria for BFRBs, student must report engaging in an activity more than five times a day for at least 4 weeks or more. Table\n3 clearly shows that majority of the students that were involved in an activity repeated it less than five times per day, rendering them ineligible to be labeled as being involved in BFRBs. Also, far less number of students engaging in a BFRB said to have been involved in it for 4 weeks to 12 months while majority fell into 12 months or more category: 7 out of 9 males and 18 out of 19 females for hair pulling; 20 out of 31 males and 23 out of 34 females for hair manipulation; 11 out of 11 males 25 out of 36 females for nail biting; 17 out of 23 males and 38 out of 51 females for chewing mouth, lips or cheeks and 4 out 5 males and 6 out of 9 females for biting other areas.\nPrevalence and gender wise distributions of BFRBs\nMany of the students being engaged in an activity also reported that that activity resulted in noticeable hair loss, interfered with day to day activity, caused injury, permanent scar or damage or made them seek medical attention. Few of them reported that a medical professional suggested them to stop the behavior while very few said that the behavior occurred under the influence of alcohol or other drugs (for details refer Table\n3). Besides this, as shown in the previous studies, we did obtain the same test retest reliability."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"The above facts clearly indicate a comparatively higher prevalence of BFRBs among medical students of Karachi than other populations, supporting the fact that these students are a victim of great stress and anxiety in their daily lives. High occurrence of BFRBs among them can be greatly detrimental and thus there is an utter need to spread awareness regarding these behaviors to save the students from its negative impacts. Key health messages and interventions to reduce stress and anxiety among students may help in curtailing the burden of this disease which has serious adverse consequences."},"other_sections_titles":{"kind":"list like","value":["Background","Statistical analysis","Abbreviations","Competing interests","Authors’ contributions","Authors’ information"],"string":"[\n \"Background\",\n \"Statistical analysis\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Authors’ information\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Body-focused repetitive behaviors (BFRBs) refer to a group of behaviors that include skin picking (dermatillomania), hair pulling (trichotillomania) and nail biting (onychophagia), which result in physical and psychological difficulties\n[1]. These behaviors for some individuals are simply referred to as nervous habits\n[2]. However, these nervous habits become problematic when they interfere with the person’s everyday functioning. When these BFRBs cross this line, then they are classified as Impulse Control Disorders.\nBFRBs most often begin in late childhood or adolescence. They are among the most poorly understood, misdiagnosed, and under treated groups of disorders. The key factor underlying BFRBs is difficulty resisting the urge or impulse to perform a certain behavior that causes a degree of relief. The behavior continues because the BFRB results in a more pleasant state therefore, it is negatively reinforced.\nPrevalence estimates indicates that such behaviors are quite common among students. Nail biting of 2 times or more per week was reported among 63.6% students in United States of America\n[2]. Another study from USA, using the stringent criteria of 5 times or more per week stated 21.8% students engaged in mouth, lips or cheeks chewing and 10.1% engaged in habitual nail biting\n[3]. A recent survey of college students found that 13.7% of the sample endorsed in at least one repetitive behavior that occurred more than five times per day for at least 4 weeks and produced some type of psychological or physical disruption of functioning\n[4]. Based on questionnaire screenings, a lifetime ICD rate of 3.5% was found in college students of Germany\n[5]. Very limited literature and no prevalence estimates regarding BFRBs could be found from Asian countries including Pakistan.\nThe treatment for BFRBs may include a combination of psychotropic medications and cognitive behavioral therapy (CBT). CBT often involves Habit Reversal Training and Exposure and Response Prevention (aka Exposure and Ritual Prevention).\nSince limited literature from our part of the world is available on body-focused repetitive behaviors and its impact on medical students and their lifestyles, it is imperative to spread awareness regarding them and find out ways and means to prevent them. It is therefore essential to come up with frequency to design strategies to decrease the prevalence of BFRBs and thus prevent their adverse effects among medical students.\nWe thus aimed to determine the prevalence of BFRBs among students attending three large medical colleges of Karachi.","A sample size of at least 210 was required to estimate the prevalence of BFRBs among medical students assuming 14% prevalence figure of the Pakistani population, along with 80 percent power, 0.05 significance level, 5 percent bond on error and 10% adjustment for non-response rate. Convenience sampling was used by getting the questionnaires filled during regular college hours from students at four locations in the medical college: the lecture halls, laboratories, library and canteen. Questionnaires were filled on consecutive days until the required sample size was achieved.\nData was entered and analyzed using Statistical Package for the Social Sciences (SPSS) version 13.0. Initially descriptive statistics, frequencies and proportions were generated. Continuous variables were analyzed by t – test and categorical by chi-square or Fisher exact, where appropriate.","BFRBs: Body Focused Repetitive Behaviors; CBT: Cognitive Behavioral Therapy; ICD: Impulse Control Disorder; SPSS: Statistical Package for the Social Sciences.","We do not have any competing interests to declare.","EUS came up with the idea, did literature review, made the proposal, did data collection, entered data into Statistical Package for the Social Sciences, analyzed it and wrote the paper. SSN helped with literature review, did data collection, entered data into Statistical Package for the Social Sciences and analyzed it. HN’s role was as a consultant psychiatrist, BA supervised the overall conduction of study. All authors read and approved the final manuscript.","EUS and SSN are final year medical students at Dow Medical College, Dow University of Health Sciences, Karachi, Pakistan. HN = MBBS, FCPS, BA MSc."],"string":"[\n \"Body-focused repetitive behaviors (BFRBs) refer to a group of behaviors that include skin picking (dermatillomania), hair pulling (trichotillomania) and nail biting (onychophagia), which result in physical and psychological difficulties\\n[1]. These behaviors for some individuals are simply referred to as nervous habits\\n[2]. However, these nervous habits become problematic when they interfere with the person’s everyday functioning. When these BFRBs cross this line, then they are classified as Impulse Control Disorders.\\nBFRBs most often begin in late childhood or adolescence. They are among the most poorly understood, misdiagnosed, and under treated groups of disorders. The key factor underlying BFRBs is difficulty resisting the urge or impulse to perform a certain behavior that causes a degree of relief. The behavior continues because the BFRB results in a more pleasant state therefore, it is negatively reinforced.\\nPrevalence estimates indicates that such behaviors are quite common among students. Nail biting of 2 times or more per week was reported among 63.6% students in United States of America\\n[2]. Another study from USA, using the stringent criteria of 5 times or more per week stated 21.8% students engaged in mouth, lips or cheeks chewing and 10.1% engaged in habitual nail biting\\n[3]. A recent survey of college students found that 13.7% of the sample endorsed in at least one repetitive behavior that occurred more than five times per day for at least 4 weeks and produced some type of psychological or physical disruption of functioning\\n[4]. Based on questionnaire screenings, a lifetime ICD rate of 3.5% was found in college students of Germany\\n[5]. Very limited literature and no prevalence estimates regarding BFRBs could be found from Asian countries including Pakistan.\\nThe treatment for BFRBs may include a combination of psychotropic medications and cognitive behavioral therapy (CBT). CBT often involves Habit Reversal Training and Exposure and Response Prevention (aka Exposure and Ritual Prevention).\\nSince limited literature from our part of the world is available on body-focused repetitive behaviors and its impact on medical students and their lifestyles, it is imperative to spread awareness regarding them and find out ways and means to prevent them. It is therefore essential to come up with frequency to design strategies to decrease the prevalence of BFRBs and thus prevent their adverse effects among medical students.\\nWe thus aimed to determine the prevalence of BFRBs among students attending three large medical colleges of Karachi.\",\n \"A sample size of at least 210 was required to estimate the prevalence of BFRBs among medical students assuming 14% prevalence figure of the Pakistani population, along with 80 percent power, 0.05 significance level, 5 percent bond on error and 10% adjustment for non-response rate. Convenience sampling was used by getting the questionnaires filled during regular college hours from students at four locations in the medical college: the lecture halls, laboratories, library and canteen. Questionnaires were filled on consecutive days until the required sample size was achieved.\\nData was entered and analyzed using Statistical Package for the Social Sciences (SPSS) version 13.0. Initially descriptive statistics, frequencies and proportions were generated. Continuous variables were analyzed by t – test and categorical by chi-square or Fisher exact, where appropriate.\",\n \"BFRBs: Body Focused Repetitive Behaviors; CBT: Cognitive Behavioral Therapy; ICD: Impulse Control Disorder; SPSS: Statistical Package for the Social Sciences.\",\n \"We do not have any competing interests to declare.\",\n \"EUS came up with the idea, did literature review, made the proposal, did data collection, entered data into Statistical Package for the Social Sciences, analyzed it and wrote the paper. SSN helped with literature review, did data collection, entered data into Statistical Package for the Social Sciences and analyzed it. HN’s role was as a consultant psychiatrist, BA supervised the overall conduction of study. All authors read and approved the final manuscript.\",\n \"EUS and SSN are final year medical students at Dow Medical College, Dow University of Health Sciences, Karachi, Pakistan. HN = MBBS, FCPS, BA MSc.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Statistical analysis","Results","Discussion","Conclusions","Abbreviations","Competing interests","Authors’ contributions","Authors’ information"],"string":"[\n \"Background\",\n \"Methods\",\n \"Statistical analysis\",\n \"Results\",\n \"Discussion\",\n \"Conclusions\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Authors’ information\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Body-focused repetitive behaviors (BFRBs) refer to a group of behaviors that include skin picking (dermatillomania), hair pulling (trichotillomania) and nail biting (onychophagia), which result in physical and psychological difficulties\n[1]. These behaviors for some individuals are simply referred to as nervous habits\n[2]. However, these nervous habits become problematic when they interfere with the person’s everyday functioning. When these BFRBs cross this line, then they are classified as Impulse Control Disorders.\nBFRBs most often begin in late childhood or adolescence. They are among the most poorly understood, misdiagnosed, and under treated groups of disorders. The key factor underlying BFRBs is difficulty resisting the urge or impulse to perform a certain behavior that causes a degree of relief. The behavior continues because the BFRB results in a more pleasant state therefore, it is negatively reinforced.\nPrevalence estimates indicates that such behaviors are quite common among students. Nail biting of 2 times or more per week was reported among 63.6% students in United States of America\n[2]. Another study from USA, using the stringent criteria of 5 times or more per week stated 21.8% students engaged in mouth, lips or cheeks chewing and 10.1% engaged in habitual nail biting\n[3]. A recent survey of college students found that 13.7% of the sample endorsed in at least one repetitive behavior that occurred more than five times per day for at least 4 weeks and produced some type of psychological or physical disruption of functioning\n[4]. Based on questionnaire screenings, a lifetime ICD rate of 3.5% was found in college students of Germany\n[5]. Very limited literature and no prevalence estimates regarding BFRBs could be found from Asian countries including Pakistan.\nThe treatment for BFRBs may include a combination of psychotropic medications and cognitive behavioral therapy (CBT). CBT often involves Habit Reversal Training and Exposure and Response Prevention (aka Exposure and Ritual Prevention).\nSince limited literature from our part of the world is available on body-focused repetitive behaviors and its impact on medical students and their lifestyles, it is imperative to spread awareness regarding them and find out ways and means to prevent them. It is therefore essential to come up with frequency to design strategies to decrease the prevalence of BFRBs and thus prevent their adverse effects among medical students.\nWe thus aimed to determine the prevalence of BFRBs among students attending three large medical colleges of Karachi.","The study was conducted in three large medical colleges of Karachi: The Aga Khan University, Dow Medical College and Sindh Medical College. Students from Karachi as well as interior and foreign attend these institutes; hence provide better representation from different socio economic classes and cultures. The combined strength of student of the three colleges was approximately 3000. The study was conducted in July 2010.\nThe investigation was a cross-sectional study. Students were recruited in equal proportion from each college. A self-administered pre tested questionnaire was used for data collection. Data was collected by trained medical students. The study was approved by the Ethical Review Committee of Aga Khan University and Dow Medical College. Written informed consent was obtained from each student and an explanation of the purposes of the research was provided to them.\nEligibility criteria to participate in the study comprised of registered medical students aged 18 and above. However students less than 18 years and not willing to consent to participate in this study were excluded.\nA d “Habit Questionnaire” was used for gathering information on repetitive behaviors. Habit is a brief five items, self-report questionnaire that provides a standardized assessment of the frequency and duration of BFRBs.\nThe Habit Questionnaire has been found to possess moderate test–retest reliability of 0.69 of diagnosis of BFRB, p<0.001\n[4].\nParticipants were asked to indicate if they engage in the following repetitive behaviors: hair pulling, hair manipulation, nail biting, skin biting and mouth chewing. For each repetitive behavior, participants who acknowledged engaging in the specified behavior were asked about the frequency of the behavior (i.e., fewer or greater than five times per day) and the duration of the behavior (i.e., less than 4 weeks or longer than 4 weeks). Participants were also asked to specify if the behavior caused impairment, which was defined as (1) interference with daily functioning, (2) resultant injuries with possible permanent scarring or damage, (3) medical attention as a result of the behavior or (4) recommendation to cease the behavior by a medical professional. To rule out conditions that commonly co-occur with BFRBs, the final section of the Habit Questionnaire asked participants to indicate if they had ever been diagnosed with obsessive–compulsive disorder, Tourette’s syndrome, autism, Asperger’s syndrome or a developmental disability.\nTo meet the criteria for a BFRB, the participants needed to answer “yes” to engaging in at least one repetitive behavior five or more times per day, and the behavior had to be present for at least 4 weeks. Participants were also needed to report that the particular behavior interferes with functioning, caused an injury, caused him or her to seek medical attention or elicited a recommendation to stop the behavior by a medical professional.\nOur focus was on three behaviors: Trichotillomania (including ‘hair pulling’ and ‘hair manipulation’), Onychophagia (including ‘nail biting’) and Dermatillomania (including ‘chew mouth lips or cheek’ and ‘bite on any other areas’).\n Statistical analysis A sample size of at least 210 was required to estimate the prevalence of BFRBs among medical students assuming 14% prevalence figure of the Pakistani population, along with 80 percent power, 0.05 significance level, 5 percent bond on error and 10% adjustment for non-response rate. Convenience sampling was used by getting the questionnaires filled during regular college hours from students at four locations in the medical college: the lecture halls, laboratories, library and canteen. Questionnaires were filled on consecutive days until the required sample size was achieved.\nData was entered and analyzed using Statistical Package for the Social Sciences (SPSS) version 13.0. Initially descriptive statistics, frequencies and proportions were generated. Continuous variables were analyzed by t – test and categorical by chi-square or Fisher exact, where appropriate.\nA sample size of at least 210 was required to estimate the prevalence of BFRBs among medical students assuming 14% prevalence figure of the Pakistani population, along with 80 percent power, 0.05 significance level, 5 percent bond on error and 10% adjustment for non-response rate. Convenience sampling was used by getting the questionnaires filled during regular college hours from students at four locations in the medical college: the lecture halls, laboratories, library and canteen. Questionnaires were filled on consecutive days until the required sample size was achieved.\nData was entered and analyzed using Statistical Package for the Social Sciences (SPSS) version 13.0. Initially descriptive statistics, frequencies and proportions were generated. Continuous variables were analyzed by t – test and categorical by chi-square or Fisher exact, where appropriate.","A sample size of at least 210 was required to estimate the prevalence of BFRBs among medical students assuming 14% prevalence figure of the Pakistani population, along with 80 percent power, 0.05 significance level, 5 percent bond on error and 10% adjustment for non-response rate. Convenience sampling was used by getting the questionnaires filled during regular college hours from students at four locations in the medical college: the lecture halls, laboratories, library and canteen. Questionnaires were filled on consecutive days until the required sample size was achieved.\nData was entered and analyzed using Statistical Package for the Social Sciences (SPSS) version 13.0. Initially descriptive statistics, frequencies and proportions were generated. Continuous variables were analyzed by t – test and categorical by chi-square or Fisher exact, where appropriate.","The prevalence of BFRBs among medical students of Karachi was found to be 46 (22%). For those positive for BFRBs, gender distribution was as follows: females 29 (13.9%) and males 17 (8.1%). The average age of participants was 21.5 ranging from 18–27 (refer Table\n1). Among these students, 19 (9.0%) were engaged in dermatillomania, 28 (13.3%) in trichotillomania and 13 (6.2%) in onychophagia (refer Table\n2).\nBaseline characteristics of participants\nBFRBs characteristics among medical students in Karachi\nAs discussed earlier, to meet criteria for BFRBs, student must report engaging in an activity more than five times a day for at least 4 weeks or more. Table\n3 clearly shows that majority of the students that were involved in an activity repeated it less than five times per day, rendering them ineligible to be labeled as being involved in BFRBs. Also, far less number of students engaging in a BFRB said to have been involved in it for 4 weeks to 12 months while majority fell into 12 months or more category: 7 out of 9 males and 18 out of 19 females for hair pulling; 20 out of 31 males and 23 out of 34 females for hair manipulation; 11 out of 11 males 25 out of 36 females for nail biting; 17 out of 23 males and 38 out of 51 females for chewing mouth, lips or cheeks and 4 out 5 males and 6 out of 9 females for biting other areas.\nPrevalence and gender wise distributions of BFRBs\nMany of the students being engaged in an activity also reported that that activity resulted in noticeable hair loss, interfered with day to day activity, caused injury, permanent scar or damage or made them seek medical attention. Few of them reported that a medical professional suggested them to stop the behavior while very few said that the behavior occurred under the influence of alcohol or other drugs (for details refer Table\n3). Besides this, as shown in the previous studies, we did obtain the same test retest reliability.","BFRBs are not uncommon among medical students of Karachi. It is therefore imperative to identify the prevalence associated with BFRBs to design interventions to curtail the burden.\nDisorder-specific rates in our study ranged from 6.2%-13.3%, which is in agreement with the rates of 12.8% and 10.0% of skin picking and nail biting respectively according to a study conducted in the U.S.\n[6]. Our study also found greater occurrence among females than males, a finding consistent with previous studies\n[1,7,8]. Literature shows that stressful conditions tend to trigger BFRBs but may not be necessarily associated. Thus this higher prevalence may indicate that females are more prone to stressful conditions and thus tend to get engaged more frequently in BFRBs than males.\nAs mentioned above, stress may trigger BFRBs. Besides that, other reasons such as socioeconomic factors may also be the cause of stress and anxiety among different sets of students. But since 1) no literature is available proving any such facts, 2) our sample is limited and has no control population and 3) ours is a cross-sectional study which cannot establish cause and effect relationship, therefore we can’t come up with a valid conclusion in this regard.\nOverall prevalence of BFRBs in our study was found to be much higher than previous surveys for e.g., according to a study, 13.7% of college students endorsed at least one repetitive behavior that occurred more than five times per day for at least 4 weeks and produced some type of psychological or physical disruption of functioning\n[4]. Another source gives a lifetime ICD rate of 3.5% in college students\n[5] while according to a study conducted in United States, 17.1% subjects met criteria for a current ICD\n[6]. Disorder-specific rates are far greater than other studies, such as 2.7-6.4% of skin picking, mouth chewing and nail biting\n[4] and disorder-specific rates of 0.4-1.2%, except for trichotillomania, which was 0%\n[5].\nResearch suggests that skin picking occurs in people with a mean age of onset of around 15 years\n[9] but our study comprised of medical students aged 18 and above, with those indulging in skin picking ranging from 19–26 years with a mean of 22.5 years. Previous studies also identified prevalence of dermatillomania to be approximately 3.8-4.6% of college students\n[8,10] 5.4% of a community sample, 4% of college students and 2% of patients seen in a dermatology clinic\n[8,11-14], and 1.4%, 4.6% and 3.8% by Nancy J. Keuthen on various occasions\n[8,9]. It should be noted here that all these figures are far lower than the prevalence of dermatillomania in our study population that is 9.0%, possibly depicting a greater influence of stress and anxiety on medical students\n[15].\nExpression of repetitive behaviors has been well documented in the trichotillomania literature. Prevalence estimates indicate a frequency of 2.5% of young adults\n[16], being quite lower than the prevalence of trichotillomania found in our study, that is 13.3%. This might be due to inclusion of ‘hair manipulation’ in addition to ‘hair pulling’ in our study.\nIt is interesting to note that medical students of Karachi were found to indulge the most into trichotillomania, followed by dermatillomania and the least prevalent behavior was onychophagia which is contrary to literature present. This might be due to the fact that in our study, Trichotillomania and Dermatillomania both comprised of two behaviors whereas Onychophagia comprised of nail biting alone, as has been mentioned above.\nIn one of the few studies to address the issue of BFRBs, college students were categorized as having a repetitive behavior (habit) if the student reported engaging in a behavior two or more times per week\n[2]. Using this relatively lenient criterion, Hansen et al. found that nail biting occurred in 63.6% of the sample. A subsequent study used more stringent criteria for identifying repetitive behaviors in college students. Stating that the repetitive behavior had to occur at least five times per day to be classified as a habit, it was found that 21.8% of the sample engaged in habitual chewing on mouth, lips, or cheeks and 10.1% engaged in habitual nail biting\n[3].\nAs discussed earlier, BFRBs may produce a variety of physical sequelae. Thus, to accurately ascertain the extent to which BFRBs are an actual diagnosable problem, not only must data be collected on how frequently these behaviors occur in an individual but also and more importantly the extent to which these behaviors produce some type of impairment be considered. Unfortunately, the Hansen et al. (1990) and Woods et al. (1996) did not incorporate this variable into their operational definitions when determining the prevalence of BFRBs. The tool that we have used for our study, the ‘Habit Questionnaire’ follows criteria that cover all factors and variables associated with BFRBs and thus give a better estimate of its prevalence with greater accuracy. Even after following such strict criteria, prevalence in our study was quite high, pointing towards the fact that medical students of Karachi are greatly prone to such negative behaviors.\nThere are a number of aspects of our study which limit conclusions. Due to excessive workload on medical students, the specifications of questionnaires might not have been accurately filled or due to extra stress on students having their exam season and vice versa, their BFRBs might have been affected which might have affected the overall results. Also, since ours is a cross-sectional study, cause and effect relationship for the identified associated factors could not be established. Because we have carried out this study among medical students only, we cannot give any absolute predictions of prevalence among the general population. Besides that, we cannot comment on the age and gender distribution of BFRBs since medical students fall into a particular age group only and also because the majority of students at the above mentioned study setting were females, it may have affected gender distribution of such behaviors. Also, we have not considered co-morbidities of different behaviors which limit conclusions.\nThis study offers a number of avenues for future research. Additional research should be conducted to establish prevalence rates among different populations including children, adolescents and elderly, and among populations with cultural differences. It may also be useful to examine the general phenomenology of BFRBs among typically developing persons, including possible co-occurring psychological symptoms such as anxiety or negative affective states. Studies should also be conducted to find out the factors associated with BFRBs and its various possible consequences. Doing so may elucidate important components of treatment and methods to avoid engaging into such behaviors that are so physically and socially disadvantageous.","The above facts clearly indicate a comparatively higher prevalence of BFRBs among medical students of Karachi than other populations, supporting the fact that these students are a victim of great stress and anxiety in their daily lives. High occurrence of BFRBs among them can be greatly detrimental and thus there is an utter need to spread awareness regarding these behaviors to save the students from its negative impacts. Key health messages and interventions to reduce stress and anxiety among students may help in curtailing the burden of this disease which has serious adverse consequences.","BFRBs: Body Focused Repetitive Behaviors; CBT: Cognitive Behavioral Therapy; ICD: Impulse Control Disorder; SPSS: Statistical Package for the Social Sciences.","We do not have any competing interests to declare.","EUS came up with the idea, did literature review, made the proposal, did data collection, entered data into Statistical Package for the Social Sciences, analyzed it and wrote the paper. SSN helped with literature review, did data collection, entered data into Statistical Package for the Social Sciences and analyzed it. HN’s role was as a consultant psychiatrist, BA supervised the overall conduction of study. All authors read and approved the final manuscript.","EUS and SSN are final year medical students at Dow Medical College, Dow University of Health Sciences, Karachi, Pakistan. HN = MBBS, FCPS, BA MSc."],"string":"[\n \"Body-focused repetitive behaviors (BFRBs) refer to a group of behaviors that include skin picking (dermatillomania), hair pulling (trichotillomania) and nail biting (onychophagia), which result in physical and psychological difficulties\\n[1]. These behaviors for some individuals are simply referred to as nervous habits\\n[2]. However, these nervous habits become problematic when they interfere with the person’s everyday functioning. When these BFRBs cross this line, then they are classified as Impulse Control Disorders.\\nBFRBs most often begin in late childhood or adolescence. They are among the most poorly understood, misdiagnosed, and under treated groups of disorders. The key factor underlying BFRBs is difficulty resisting the urge or impulse to perform a certain behavior that causes a degree of relief. The behavior continues because the BFRB results in a more pleasant state therefore, it is negatively reinforced.\\nPrevalence estimates indicates that such behaviors are quite common among students. Nail biting of 2 times or more per week was reported among 63.6% students in United States of America\\n[2]. Another study from USA, using the stringent criteria of 5 times or more per week stated 21.8% students engaged in mouth, lips or cheeks chewing and 10.1% engaged in habitual nail biting\\n[3]. A recent survey of college students found that 13.7% of the sample endorsed in at least one repetitive behavior that occurred more than five times per day for at least 4 weeks and produced some type of psychological or physical disruption of functioning\\n[4]. Based on questionnaire screenings, a lifetime ICD rate of 3.5% was found in college students of Germany\\n[5]. Very limited literature and no prevalence estimates regarding BFRBs could be found from Asian countries including Pakistan.\\nThe treatment for BFRBs may include a combination of psychotropic medications and cognitive behavioral therapy (CBT). CBT often involves Habit Reversal Training and Exposure and Response Prevention (aka Exposure and Ritual Prevention).\\nSince limited literature from our part of the world is available on body-focused repetitive behaviors and its impact on medical students and their lifestyles, it is imperative to spread awareness regarding them and find out ways and means to prevent them. It is therefore essential to come up with frequency to design strategies to decrease the prevalence of BFRBs and thus prevent their adverse effects among medical students.\\nWe thus aimed to determine the prevalence of BFRBs among students attending three large medical colleges of Karachi.\",\n \"The study was conducted in three large medical colleges of Karachi: The Aga Khan University, Dow Medical College and Sindh Medical College. Students from Karachi as well as interior and foreign attend these institutes; hence provide better representation from different socio economic classes and cultures. The combined strength of student of the three colleges was approximately 3000. The study was conducted in July 2010.\\nThe investigation was a cross-sectional study. Students were recruited in equal proportion from each college. A self-administered pre tested questionnaire was used for data collection. Data was collected by trained medical students. The study was approved by the Ethical Review Committee of Aga Khan University and Dow Medical College. Written informed consent was obtained from each student and an explanation of the purposes of the research was provided to them.\\nEligibility criteria to participate in the study comprised of registered medical students aged 18 and above. However students less than 18 years and not willing to consent to participate in this study were excluded.\\nA d “Habit Questionnaire” was used for gathering information on repetitive behaviors. Habit is a brief five items, self-report questionnaire that provides a standardized assessment of the frequency and duration of BFRBs.\\nThe Habit Questionnaire has been found to possess moderate test–retest reliability of 0.69 of diagnosis of BFRB, p<0.001\\n[4].\\nParticipants were asked to indicate if they engage in the following repetitive behaviors: hair pulling, hair manipulation, nail biting, skin biting and mouth chewing. For each repetitive behavior, participants who acknowledged engaging in the specified behavior were asked about the frequency of the behavior (i.e., fewer or greater than five times per day) and the duration of the behavior (i.e., less than 4 weeks or longer than 4 weeks). Participants were also asked to specify if the behavior caused impairment, which was defined as (1) interference with daily functioning, (2) resultant injuries with possible permanent scarring or damage, (3) medical attention as a result of the behavior or (4) recommendation to cease the behavior by a medical professional. To rule out conditions that commonly co-occur with BFRBs, the final section of the Habit Questionnaire asked participants to indicate if they had ever been diagnosed with obsessive–compulsive disorder, Tourette’s syndrome, autism, Asperger’s syndrome or a developmental disability.\\nTo meet the criteria for a BFRB, the participants needed to answer “yes” to engaging in at least one repetitive behavior five or more times per day, and the behavior had to be present for at least 4 weeks. Participants were also needed to report that the particular behavior interferes with functioning, caused an injury, caused him or her to seek medical attention or elicited a recommendation to stop the behavior by a medical professional.\\nOur focus was on three behaviors: Trichotillomania (including ‘hair pulling’ and ‘hair manipulation’), Onychophagia (including ‘nail biting’) and Dermatillomania (including ‘chew mouth lips or cheek’ and ‘bite on any other areas’).\\n Statistical analysis A sample size of at least 210 was required to estimate the prevalence of BFRBs among medical students assuming 14% prevalence figure of the Pakistani population, along with 80 percent power, 0.05 significance level, 5 percent bond on error and 10% adjustment for non-response rate. Convenience sampling was used by getting the questionnaires filled during regular college hours from students at four locations in the medical college: the lecture halls, laboratories, library and canteen. Questionnaires were filled on consecutive days until the required sample size was achieved.\\nData was entered and analyzed using Statistical Package for the Social Sciences (SPSS) version 13.0. Initially descriptive statistics, frequencies and proportions were generated. Continuous variables were analyzed by t – test and categorical by chi-square or Fisher exact, where appropriate.\\nA sample size of at least 210 was required to estimate the prevalence of BFRBs among medical students assuming 14% prevalence figure of the Pakistani population, along with 80 percent power, 0.05 significance level, 5 percent bond on error and 10% adjustment for non-response rate. Convenience sampling was used by getting the questionnaires filled during regular college hours from students at four locations in the medical college: the lecture halls, laboratories, library and canteen. Questionnaires were filled on consecutive days until the required sample size was achieved.\\nData was entered and analyzed using Statistical Package for the Social Sciences (SPSS) version 13.0. Initially descriptive statistics, frequencies and proportions were generated. Continuous variables were analyzed by t – test and categorical by chi-square or Fisher exact, where appropriate.\",\n \"A sample size of at least 210 was required to estimate the prevalence of BFRBs among medical students assuming 14% prevalence figure of the Pakistani population, along with 80 percent power, 0.05 significance level, 5 percent bond on error and 10% adjustment for non-response rate. Convenience sampling was used by getting the questionnaires filled during regular college hours from students at four locations in the medical college: the lecture halls, laboratories, library and canteen. Questionnaires were filled on consecutive days until the required sample size was achieved.\\nData was entered and analyzed using Statistical Package for the Social Sciences (SPSS) version 13.0. Initially descriptive statistics, frequencies and proportions were generated. Continuous variables were analyzed by t – test and categorical by chi-square or Fisher exact, where appropriate.\",\n \"The prevalence of BFRBs among medical students of Karachi was found to be 46 (22%). For those positive for BFRBs, gender distribution was as follows: females 29 (13.9%) and males 17 (8.1%). The average age of participants was 21.5 ranging from 18–27 (refer Table\\n1). Among these students, 19 (9.0%) were engaged in dermatillomania, 28 (13.3%) in trichotillomania and 13 (6.2%) in onychophagia (refer Table\\n2).\\nBaseline characteristics of participants\\nBFRBs characteristics among medical students in Karachi\\nAs discussed earlier, to meet criteria for BFRBs, student must report engaging in an activity more than five times a day for at least 4 weeks or more. Table\\n3 clearly shows that majority of the students that were involved in an activity repeated it less than five times per day, rendering them ineligible to be labeled as being involved in BFRBs. Also, far less number of students engaging in a BFRB said to have been involved in it for 4 weeks to 12 months while majority fell into 12 months or more category: 7 out of 9 males and 18 out of 19 females for hair pulling; 20 out of 31 males and 23 out of 34 females for hair manipulation; 11 out of 11 males 25 out of 36 females for nail biting; 17 out of 23 males and 38 out of 51 females for chewing mouth, lips or cheeks and 4 out 5 males and 6 out of 9 females for biting other areas.\\nPrevalence and gender wise distributions of BFRBs\\nMany of the students being engaged in an activity also reported that that activity resulted in noticeable hair loss, interfered with day to day activity, caused injury, permanent scar or damage or made them seek medical attention. Few of them reported that a medical professional suggested them to stop the behavior while very few said that the behavior occurred under the influence of alcohol or other drugs (for details refer Table\\n3). Besides this, as shown in the previous studies, we did obtain the same test retest reliability.\",\n \"BFRBs are not uncommon among medical students of Karachi. It is therefore imperative to identify the prevalence associated with BFRBs to design interventions to curtail the burden.\\nDisorder-specific rates in our study ranged from 6.2%-13.3%, which is in agreement with the rates of 12.8% and 10.0% of skin picking and nail biting respectively according to a study conducted in the U.S.\\n[6]. Our study also found greater occurrence among females than males, a finding consistent with previous studies\\n[1,7,8]. Literature shows that stressful conditions tend to trigger BFRBs but may not be necessarily associated. Thus this higher prevalence may indicate that females are more prone to stressful conditions and thus tend to get engaged more frequently in BFRBs than males.\\nAs mentioned above, stress may trigger BFRBs. Besides that, other reasons such as socioeconomic factors may also be the cause of stress and anxiety among different sets of students. But since 1) no literature is available proving any such facts, 2) our sample is limited and has no control population and 3) ours is a cross-sectional study which cannot establish cause and effect relationship, therefore we can’t come up with a valid conclusion in this regard.\\nOverall prevalence of BFRBs in our study was found to be much higher than previous surveys for e.g., according to a study, 13.7% of college students endorsed at least one repetitive behavior that occurred more than five times per day for at least 4 weeks and produced some type of psychological or physical disruption of functioning\\n[4]. Another source gives a lifetime ICD rate of 3.5% in college students\\n[5] while according to a study conducted in United States, 17.1% subjects met criteria for a current ICD\\n[6]. Disorder-specific rates are far greater than other studies, such as 2.7-6.4% of skin picking, mouth chewing and nail biting\\n[4] and disorder-specific rates of 0.4-1.2%, except for trichotillomania, which was 0%\\n[5].\\nResearch suggests that skin picking occurs in people with a mean age of onset of around 15 years\\n[9] but our study comprised of medical students aged 18 and above, with those indulging in skin picking ranging from 19–26 years with a mean of 22.5 years. Previous studies also identified prevalence of dermatillomania to be approximately 3.8-4.6% of college students\\n[8,10] 5.4% of a community sample, 4% of college students and 2% of patients seen in a dermatology clinic\\n[8,11-14], and 1.4%, 4.6% and 3.8% by Nancy J. Keuthen on various occasions\\n[8,9]. It should be noted here that all these figures are far lower than the prevalence of dermatillomania in our study population that is 9.0%, possibly depicting a greater influence of stress and anxiety on medical students\\n[15].\\nExpression of repetitive behaviors has been well documented in the trichotillomania literature. Prevalence estimates indicate a frequency of 2.5% of young adults\\n[16], being quite lower than the prevalence of trichotillomania found in our study, that is 13.3%. This might be due to inclusion of ‘hair manipulation’ in addition to ‘hair pulling’ in our study.\\nIt is interesting to note that medical students of Karachi were found to indulge the most into trichotillomania, followed by dermatillomania and the least prevalent behavior was onychophagia which is contrary to literature present. This might be due to the fact that in our study, Trichotillomania and Dermatillomania both comprised of two behaviors whereas Onychophagia comprised of nail biting alone, as has been mentioned above.\\nIn one of the few studies to address the issue of BFRBs, college students were categorized as having a repetitive behavior (habit) if the student reported engaging in a behavior two or more times per week\\n[2]. Using this relatively lenient criterion, Hansen et al. found that nail biting occurred in 63.6% of the sample. A subsequent study used more stringent criteria for identifying repetitive behaviors in college students. Stating that the repetitive behavior had to occur at least five times per day to be classified as a habit, it was found that 21.8% of the sample engaged in habitual chewing on mouth, lips, or cheeks and 10.1% engaged in habitual nail biting\\n[3].\\nAs discussed earlier, BFRBs may produce a variety of physical sequelae. Thus, to accurately ascertain the extent to which BFRBs are an actual diagnosable problem, not only must data be collected on how frequently these behaviors occur in an individual but also and more importantly the extent to which these behaviors produce some type of impairment be considered. Unfortunately, the Hansen et al. (1990) and Woods et al. (1996) did not incorporate this variable into their operational definitions when determining the prevalence of BFRBs. The tool that we have used for our study, the ‘Habit Questionnaire’ follows criteria that cover all factors and variables associated with BFRBs and thus give a better estimate of its prevalence with greater accuracy. Even after following such strict criteria, prevalence in our study was quite high, pointing towards the fact that medical students of Karachi are greatly prone to such negative behaviors.\\nThere are a number of aspects of our study which limit conclusions. Due to excessive workload on medical students, the specifications of questionnaires might not have been accurately filled or due to extra stress on students having their exam season and vice versa, their BFRBs might have been affected which might have affected the overall results. Also, since ours is a cross-sectional study, cause and effect relationship for the identified associated factors could not be established. Because we have carried out this study among medical students only, we cannot give any absolute predictions of prevalence among the general population. Besides that, we cannot comment on the age and gender distribution of BFRBs since medical students fall into a particular age group only and also because the majority of students at the above mentioned study setting were females, it may have affected gender distribution of such behaviors. Also, we have not considered co-morbidities of different behaviors which limit conclusions.\\nThis study offers a number of avenues for future research. Additional research should be conducted to establish prevalence rates among different populations including children, adolescents and elderly, and among populations with cultural differences. It may also be useful to examine the general phenomenology of BFRBs among typically developing persons, including possible co-occurring psychological symptoms such as anxiety or negative affective states. Studies should also be conducted to find out the factors associated with BFRBs and its various possible consequences. Doing so may elucidate important components of treatment and methods to avoid engaging into such behaviors that are so physically and socially disadvantageous.\",\n \"The above facts clearly indicate a comparatively higher prevalence of BFRBs among medical students of Karachi than other populations, supporting the fact that these students are a victim of great stress and anxiety in their daily lives. High occurrence of BFRBs among them can be greatly detrimental and thus there is an utter need to spread awareness regarding these behaviors to save the students from its negative impacts. Key health messages and interventions to reduce stress and anxiety among students may help in curtailing the burden of this disease which has serious adverse consequences.\",\n \"BFRBs: Body Focused Repetitive Behaviors; CBT: Cognitive Behavioral Therapy; ICD: Impulse Control Disorder; SPSS: Statistical Package for the Social Sciences.\",\n \"We do not have any competing interests to declare.\",\n \"EUS came up with the idea, did literature review, made the proposal, did data collection, entered data into Statistical Package for the Social Sciences, analyzed it and wrote the paper. SSN helped with literature review, did data collection, entered data into Statistical Package for the Social Sciences and analyzed it. HN’s role was as a consultant psychiatrist, BA supervised the overall conduction of study. All authors read and approved the final manuscript.\",\n \"EUS and SSN are final year medical students at Dow Medical College, Dow University of Health Sciences, Karachi, Pakistan. HN = MBBS, FCPS, BA MSc.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,"results","discussion","conclusions",null,null,null,null],"string":"[\n null,\n \"methods\",\n null,\n \"results\",\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null,\n null\n]","isConversational":false},"keywords":{"kind":"list like","value":["BFRBs","Dermatillomania","Trichotillomania","Onychophagia"],"string":"[\n \"BFRBs\",\n \"Dermatillomania\",\n \"Trichotillomania\",\n \"Onychophagia\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Background:\nBody-focused repetitive behaviors (BFRBs) refer to a group of behaviors that include skin picking (dermatillomania), hair pulling (trichotillomania) and nail biting (onychophagia), which result in physical and psychological difficulties\n[1]. These behaviors for some individuals are simply referred to as nervous habits\n[2]. However, these nervous habits become problematic when they interfere with the person’s everyday functioning. When these BFRBs cross this line, then they are classified as Impulse Control Disorders.\nBFRBs most often begin in late childhood or adolescence. They are among the most poorly understood, misdiagnosed, and under treated groups of disorders. The key factor underlying BFRBs is difficulty resisting the urge or impulse to perform a certain behavior that causes a degree of relief. The behavior continues because the BFRB results in a more pleasant state therefore, it is negatively reinforced.\nPrevalence estimates indicates that such behaviors are quite common among students. Nail biting of 2 times or more per week was reported among 63.6% students in United States of America\n[2]. Another study from USA, using the stringent criteria of 5 times or more per week stated 21.8% students engaged in mouth, lips or cheeks chewing and 10.1% engaged in habitual nail biting\n[3]. A recent survey of college students found that 13.7% of the sample endorsed in at least one repetitive behavior that occurred more than five times per day for at least 4 weeks and produced some type of psychological or physical disruption of functioning\n[4]. Based on questionnaire screenings, a lifetime ICD rate of 3.5% was found in college students of Germany\n[5]. Very limited literature and no prevalence estimates regarding BFRBs could be found from Asian countries including Pakistan.\nThe treatment for BFRBs may include a combination of psychotropic medications and cognitive behavioral therapy (CBT). CBT often involves Habit Reversal Training and Exposure and Response Prevention (aka Exposure and Ritual Prevention).\nSince limited literature from our part of the world is available on body-focused repetitive behaviors and its impact on medical students and their lifestyles, it is imperative to spread awareness regarding them and find out ways and means to prevent them. It is therefore essential to come up with frequency to design strategies to decrease the prevalence of BFRBs and thus prevent their adverse effects among medical students.\nWe thus aimed to determine the prevalence of BFRBs among students attending three large medical colleges of Karachi.\n\nMethods:\nThe study was conducted in three large medical colleges of Karachi: The Aga Khan University, Dow Medical College and Sindh Medical College. Students from Karachi as well as interior and foreign attend these institutes; hence provide better representation from different socio economic classes and cultures. The combined strength of student of the three colleges was approximately 3000. The study was conducted in July 2010.\nThe investigation was a cross-sectional study. Students were recruited in equal proportion from each college. A self-administered pre tested questionnaire was used for data collection. Data was collected by trained medical students. The study was approved by the Ethical Review Committee of Aga Khan University and Dow Medical College. Written informed consent was obtained from each student and an explanation of the purposes of the research was provided to them.\nEligibility criteria to participate in the study comprised of registered medical students aged 18 and above. However students less than 18 years and not willing to consent to participate in this study were excluded.\nA d “Habit Questionnaire” was used for gathering information on repetitive behaviors. Habit is a brief five items, self-report questionnaire that provides a standardized assessment of the frequency and duration of BFRBs.\nThe Habit Questionnaire has been found to possess moderate test–retest reliability of 0.69 of diagnosis of BFRB, p<0.001\n[4].\nParticipants were asked to indicate if they engage in the following repetitive behaviors: hair pulling, hair manipulation, nail biting, skin biting and mouth chewing. For each repetitive behavior, participants who acknowledged engaging in the specified behavior were asked about the frequency of the behavior (i.e., fewer or greater than five times per day) and the duration of the behavior (i.e., less than 4 weeks or longer than 4 weeks). Participants were also asked to specify if the behavior caused impairment, which was defined as (1) interference with daily functioning, (2) resultant injuries with possible permanent scarring or damage, (3) medical attention as a result of the behavior or (4) recommendation to cease the behavior by a medical professional. To rule out conditions that commonly co-occur with BFRBs, the final section of the Habit Questionnaire asked participants to indicate if they had ever been diagnosed with obsessive–compulsive disorder, Tourette’s syndrome, autism, Asperger’s syndrome or a developmental disability.\nTo meet the criteria for a BFRB, the participants needed to answer “yes” to engaging in at least one repetitive behavior five or more times per day, and the behavior had to be present for at least 4 weeks. Participants were also needed to report that the particular behavior interferes with functioning, caused an injury, caused him or her to seek medical attention or elicited a recommendation to stop the behavior by a medical professional.\nOur focus was on three behaviors: Trichotillomania (including ‘hair pulling’ and ‘hair manipulation’), Onychophagia (including ‘nail biting’) and Dermatillomania (including ‘chew mouth lips or cheek’ and ‘bite on any other areas’).\n Statistical analysis A sample size of at least 210 was required to estimate the prevalence of BFRBs among medical students assuming 14% prevalence figure of the Pakistani population, along with 80 percent power, 0.05 significance level, 5 percent bond on error and 10% adjustment for non-response rate. Convenience sampling was used by getting the questionnaires filled during regular college hours from students at four locations in the medical college: the lecture halls, laboratories, library and canteen. Questionnaires were filled on consecutive days until the required sample size was achieved.\nData was entered and analyzed using Statistical Package for the Social Sciences (SPSS) version 13.0. Initially descriptive statistics, frequencies and proportions were generated. Continuous variables were analyzed by t – test and categorical by chi-square or Fisher exact, where appropriate.\nA sample size of at least 210 was required to estimate the prevalence of BFRBs among medical students assuming 14% prevalence figure of the Pakistani population, along with 80 percent power, 0.05 significance level, 5 percent bond on error and 10% adjustment for non-response rate. Convenience sampling was used by getting the questionnaires filled during regular college hours from students at four locations in the medical college: the lecture halls, laboratories, library and canteen. Questionnaires were filled on consecutive days until the required sample size was achieved.\nData was entered and analyzed using Statistical Package for the Social Sciences (SPSS) version 13.0. Initially descriptive statistics, frequencies and proportions were generated. Continuous variables were analyzed by t – test and categorical by chi-square or Fisher exact, where appropriate.\n\nStatistical analysis:\nA sample size of at least 210 was required to estimate the prevalence of BFRBs among medical students assuming 14% prevalence figure of the Pakistani population, along with 80 percent power, 0.05 significance level, 5 percent bond on error and 10% adjustment for non-response rate. Convenience sampling was used by getting the questionnaires filled during regular college hours from students at four locations in the medical college: the lecture halls, laboratories, library and canteen. Questionnaires were filled on consecutive days until the required sample size was achieved.\nData was entered and analyzed using Statistical Package for the Social Sciences (SPSS) version 13.0. Initially descriptive statistics, frequencies and proportions were generated. Continuous variables were analyzed by t – test and categorical by chi-square or Fisher exact, where appropriate.\n\nResults:\nThe prevalence of BFRBs among medical students of Karachi was found to be 46 (22%). For those positive for BFRBs, gender distribution was as follows: females 29 (13.9%) and males 17 (8.1%). The average age of participants was 21.5 ranging from 18–27 (refer Table\n1). Among these students, 19 (9.0%) were engaged in dermatillomania, 28 (13.3%) in trichotillomania and 13 (6.2%) in onychophagia (refer Table\n2).\nBaseline characteristics of participants\nBFRBs characteristics among medical students in Karachi\nAs discussed earlier, to meet criteria for BFRBs, student must report engaging in an activity more than five times a day for at least 4 weeks or more. Table\n3 clearly shows that majority of the students that were involved in an activity repeated it less than five times per day, rendering them ineligible to be labeled as being involved in BFRBs. Also, far less number of students engaging in a BFRB said to have been involved in it for 4 weeks to 12 months while majority fell into 12 months or more category: 7 out of 9 males and 18 out of 19 females for hair pulling; 20 out of 31 males and 23 out of 34 females for hair manipulation; 11 out of 11 males 25 out of 36 females for nail biting; 17 out of 23 males and 38 out of 51 females for chewing mouth, lips or cheeks and 4 out 5 males and 6 out of 9 females for biting other areas.\nPrevalence and gender wise distributions of BFRBs\nMany of the students being engaged in an activity also reported that that activity resulted in noticeable hair loss, interfered with day to day activity, caused injury, permanent scar or damage or made them seek medical attention. Few of them reported that a medical professional suggested them to stop the behavior while very few said that the behavior occurred under the influence of alcohol or other drugs (for details refer Table\n3). Besides this, as shown in the previous studies, we did obtain the same test retest reliability.\n\nDiscussion:\nBFRBs are not uncommon among medical students of Karachi. It is therefore imperative to identify the prevalence associated with BFRBs to design interventions to curtail the burden.\nDisorder-specific rates in our study ranged from 6.2%-13.3%, which is in agreement with the rates of 12.8% and 10.0% of skin picking and nail biting respectively according to a study conducted in the U.S.\n[6]. Our study also found greater occurrence among females than males, a finding consistent with previous studies\n[1,7,8]. Literature shows that stressful conditions tend to trigger BFRBs but may not be necessarily associated. Thus this higher prevalence may indicate that females are more prone to stressful conditions and thus tend to get engaged more frequently in BFRBs than males.\nAs mentioned above, stress may trigger BFRBs. Besides that, other reasons such as socioeconomic factors may also be the cause of stress and anxiety among different sets of students. But since 1) no literature is available proving any such facts, 2) our sample is limited and has no control population and 3) ours is a cross-sectional study which cannot establish cause and effect relationship, therefore we can’t come up with a valid conclusion in this regard.\nOverall prevalence of BFRBs in our study was found to be much higher than previous surveys for e.g., according to a study, 13.7% of college students endorsed at least one repetitive behavior that occurred more than five times per day for at least 4 weeks and produced some type of psychological or physical disruption of functioning\n[4]. Another source gives a lifetime ICD rate of 3.5% in college students\n[5] while according to a study conducted in United States, 17.1% subjects met criteria for a current ICD\n[6]. Disorder-specific rates are far greater than other studies, such as 2.7-6.4% of skin picking, mouth chewing and nail biting\n[4] and disorder-specific rates of 0.4-1.2%, except for trichotillomania, which was 0%\n[5].\nResearch suggests that skin picking occurs in people with a mean age of onset of around 15 years\n[9] but our study comprised of medical students aged 18 and above, with those indulging in skin picking ranging from 19–26 years with a mean of 22.5 years. Previous studies also identified prevalence of dermatillomania to be approximately 3.8-4.6% of college students\n[8,10] 5.4% of a community sample, 4% of college students and 2% of patients seen in a dermatology clinic\n[8,11-14], and 1.4%, 4.6% and 3.8% by Nancy J. Keuthen on various occasions\n[8,9]. It should be noted here that all these figures are far lower than the prevalence of dermatillomania in our study population that is 9.0%, possibly depicting a greater influence of stress and anxiety on medical students\n[15].\nExpression of repetitive behaviors has been well documented in the trichotillomania literature. Prevalence estimates indicate a frequency of 2.5% of young adults\n[16], being quite lower than the prevalence of trichotillomania found in our study, that is 13.3%. This might be due to inclusion of ‘hair manipulation’ in addition to ‘hair pulling’ in our study.\nIt is interesting to note that medical students of Karachi were found to indulge the most into trichotillomania, followed by dermatillomania and the least prevalent behavior was onychophagia which is contrary to literature present. This might be due to the fact that in our study, Trichotillomania and Dermatillomania both comprised of two behaviors whereas Onychophagia comprised of nail biting alone, as has been mentioned above.\nIn one of the few studies to address the issue of BFRBs, college students were categorized as having a repetitive behavior (habit) if the student reported engaging in a behavior two or more times per week\n[2]. Using this relatively lenient criterion, Hansen et al. found that nail biting occurred in 63.6% of the sample. A subsequent study used more stringent criteria for identifying repetitive behaviors in college students. Stating that the repetitive behavior had to occur at least five times per day to be classified as a habit, it was found that 21.8% of the sample engaged in habitual chewing on mouth, lips, or cheeks and 10.1% engaged in habitual nail biting\n[3].\nAs discussed earlier, BFRBs may produce a variety of physical sequelae. Thus, to accurately ascertain the extent to which BFRBs are an actual diagnosable problem, not only must data be collected on how frequently these behaviors occur in an individual but also and more importantly the extent to which these behaviors produce some type of impairment be considered. Unfortunately, the Hansen et al. (1990) and Woods et al. (1996) did not incorporate this variable into their operational definitions when determining the prevalence of BFRBs. The tool that we have used for our study, the ‘Habit Questionnaire’ follows criteria that cover all factors and variables associated with BFRBs and thus give a better estimate of its prevalence with greater accuracy. Even after following such strict criteria, prevalence in our study was quite high, pointing towards the fact that medical students of Karachi are greatly prone to such negative behaviors.\nThere are a number of aspects of our study which limit conclusions. Due to excessive workload on medical students, the specifications of questionnaires might not have been accurately filled or due to extra stress on students having their exam season and vice versa, their BFRBs might have been affected which might have affected the overall results. Also, since ours is a cross-sectional study, cause and effect relationship for the identified associated factors could not be established. Because we have carried out this study among medical students only, we cannot give any absolute predictions of prevalence among the general population. Besides that, we cannot comment on the age and gender distribution of BFRBs since medical students fall into a particular age group only and also because the majority of students at the above mentioned study setting were females, it may have affected gender distribution of such behaviors. Also, we have not considered co-morbidities of different behaviors which limit conclusions.\nThis study offers a number of avenues for future research. Additional research should be conducted to establish prevalence rates among different populations including children, adolescents and elderly, and among populations with cultural differences. It may also be useful to examine the general phenomenology of BFRBs among typically developing persons, including possible co-occurring psychological symptoms such as anxiety or negative affective states. Studies should also be conducted to find out the factors associated with BFRBs and its various possible consequences. Doing so may elucidate important components of treatment and methods to avoid engaging into such behaviors that are so physically and socially disadvantageous.\n\nConclusions:\nThe above facts clearly indicate a comparatively higher prevalence of BFRBs among medical students of Karachi than other populations, supporting the fact that these students are a victim of great stress and anxiety in their daily lives. High occurrence of BFRBs among them can be greatly detrimental and thus there is an utter need to spread awareness regarding these behaviors to save the students from its negative impacts. Key health messages and interventions to reduce stress and anxiety among students may help in curtailing the burden of this disease which has serious adverse consequences.\n\nAbbreviations:\nBFRBs: Body Focused Repetitive Behaviors; CBT: Cognitive Behavioral Therapy; ICD: Impulse Control Disorder; SPSS: Statistical Package for the Social Sciences.\n\nCompeting interests:\nWe do not have any competing interests to declare.\n\nAuthors’ contributions:\nEUS came up with the idea, did literature review, made the proposal, did data collection, entered data into Statistical Package for the Social Sciences, analyzed it and wrote the paper. SSN helped with literature review, did data collection, entered data into Statistical Package for the Social Sciences and analyzed it. HN’s role was as a consultant psychiatrist, BA supervised the overall conduction of study. All authors read and approved the final manuscript.\n\nAuthors’ information:\nEUS and SSN are final year medical students at Dow Medical College, Dow University of Health Sciences, Karachi, Pakistan. HN = MBBS, FCPS, BA MSc.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nBody-focused repetitive behaviors (BFRBs) that include skin picking (dermatillomania), hair pulling (trichotillomania) and nail biting (onychophagia), lead to harmful physical and psychological sequelae.The objective was to determine the prevalence of BFRBs among students attending three large medical colleges of Karachi. It is imperative to come up with frequency to design strategies to decrease the burden and adverse effects associated with BFRBs among medical students.\n\nMethods:\nA cross-sectional study was conducted among 210 students attending Aga Khan University, Dow Medical College and Sind Medical College, Karachi, in equal proportion. Data were collected using a pre tested tool, \"Habit Questionnaire\". Diagnoses were made on the criteria that a student must be involved in an activity 5 times or more per day for 4 weeks or more. Convenience sampling was done to recruit the participants aged 18 years and above after getting written informed consent.\n\nResults:\nThe overall prevalence of BFRBs was found to be 46 (22%). For those positive for BFRBs, gender distribution was as follows: females 29 (13.9%) and males 17 (8.1%). Among these students, 19 (9.0%) were engaged in dermatillomania, 28 (13.3%) in trichotillomania and 13 (6.2%) in onychophagia.\n\nConclusions:\nHigh proportions of BFRBs are reported among medical students of Karachi. Key health messages and interventions to reduce stress and anxiety among students may help in curtailing the burden of this disease which has serious adverse consequences."},"background_conclusion_text":{"kind":"string","value":"Background:\nBody-focused repetitive behaviors (BFRBs) refer to a group of behaviors that include skin picking (dermatillomania), hair pulling (trichotillomania) and nail biting (onychophagia), which result in physical and psychological difficulties\n[1]. These behaviors for some individuals are simply referred to as nervous habits\n[2]. However, these nervous habits become problematic when they interfere with the person’s everyday functioning. When these BFRBs cross this line, then they are classified as Impulse Control Disorders.\nBFRBs most often begin in late childhood or adolescence. They are among the most poorly understood, misdiagnosed, and under treated groups of disorders. The key factor underlying BFRBs is difficulty resisting the urge or impulse to perform a certain behavior that causes a degree of relief. The behavior continues because the BFRB results in a more pleasant state therefore, it is negatively reinforced.\nPrevalence estimates indicates that such behaviors are quite common among students. Nail biting of 2 times or more per week was reported among 63.6% students in United States of America\n[2]. Another study from USA, using the stringent criteria of 5 times or more per week stated 21.8% students engaged in mouth, lips or cheeks chewing and 10.1% engaged in habitual nail biting\n[3]. A recent survey of college students found that 13.7% of the sample endorsed in at least one repetitive behavior that occurred more than five times per day for at least 4 weeks and produced some type of psychological or physical disruption of functioning\n[4]. Based on questionnaire screenings, a lifetime ICD rate of 3.5% was found in college students of Germany\n[5]. Very limited literature and no prevalence estimates regarding BFRBs could be found from Asian countries including Pakistan.\nThe treatment for BFRBs may include a combination of psychotropic medications and cognitive behavioral therapy (CBT). CBT often involves Habit Reversal Training and Exposure and Response Prevention (aka Exposure and Ritual Prevention).\nSince limited literature from our part of the world is available on body-focused repetitive behaviors and its impact on medical students and their lifestyles, it is imperative to spread awareness regarding them and find out ways and means to prevent them. It is therefore essential to come up with frequency to design strategies to decrease the prevalence of BFRBs and thus prevent their adverse effects among medical students.\nWe thus aimed to determine the prevalence of BFRBs among students attending three large medical colleges of Karachi.\n\nConclusions:\nThe above facts clearly indicate a comparatively higher prevalence of BFRBs among medical students of Karachi than other populations, supporting the fact that these students are a victim of great stress and anxiety in their daily lives. High occurrence of BFRBs among them can be greatly detrimental and thus there is an utter need to spread awareness regarding these behaviors to save the students from its negative impacts. Key health messages and interventions to reduce stress and anxiety among students may help in curtailing the burden of this disease which has serious adverse consequences."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nBody-focused repetitive behaviors (BFRBs) that include skin picking (dermatillomania), hair pulling (trichotillomania) and nail biting (onychophagia), lead to harmful physical and psychological sequelae.The objective was to determine the prevalence of BFRBs among students attending three large medical colleges of Karachi. It is imperative to come up with frequency to design strategies to decrease the burden and adverse effects associated with BFRBs among medical students.\n\nMethods:\nA cross-sectional study was conducted among 210 students attending Aga Khan University, Dow Medical College and Sind Medical College, Karachi, in equal proportion. Data were collected using a pre tested tool, \"Habit Questionnaire\". Diagnoses were made on the criteria that a student must be involved in an activity 5 times or more per day for 4 weeks or more. Convenience sampling was done to recruit the participants aged 18 years and above after getting written informed consent.\n\nResults:\nThe overall prevalence of BFRBs was found to be 46 (22%). For those positive for BFRBs, gender distribution was as follows: females 29 (13.9%) and males 17 (8.1%). Among these students, 19 (9.0%) were engaged in dermatillomania, 28 (13.3%) in trichotillomania and 13 (6.2%) in onychophagia.\n\nConclusions:\nHigh proportions of BFRBs are reported among medical students of Karachi. Key health messages and interventions to reduce stress and anxiety among students may help in curtailing the burden of this disease which has serious adverse consequences."},"whole_article_text_length":{"kind":"number","value":3503,"string":"3,503"},"whole_article_abstract_length":{"kind":"number","value":294,"string":"294"},"other_sections_lengths":{"kind":"list like","value":[470,150,28,10,85,32],"string":"[\n 470,\n 150,\n 28,\n 10,\n 85,\n 32\n]","isConversational":false},"num_sections":{"kind":"number","value":10,"string":"10"},"most_frequent_words":{"kind":"list like","value":["students","bfrbs","medical","study","prevalence","behavior","college","behaviors","medical students","repetitive"],"string":"[\n \"students\",\n \"bfrbs\",\n \"medical\",\n \"study\",\n \"prevalence\",\n \"behavior\",\n \"college\",\n \"behaviors\",\n \"medical students\",\n \"repetitive\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["nail biting mentioned","behaviors 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[SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] students | bfrbs | medical | interests declare | competing interests declare | competing interests | declare | competing | interests | study [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] students | bfrbs | medical | interests declare | competing interests declare | competing interests | declare | competing | interests | study [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| BFRBs | three | Karachi ||| BFRBs [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 210 | Aga Khan University | Dow Medical College | Sind Medical College | Karachi ||| ||| 5 | 4 weeks ||| 18 years [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] BFRBs | 46 | 22% ||| BFRBs | 29 | 13.9% | 17 | 8.1% ||| 19 | 9.0% | dermatillomania | 28 | 13.3% | trichotillomania | 13 | 6.2% | onychophagia [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Karachi ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| BFRBs | three | Karachi ||| BFRBs ||| 210 | Aga Khan University | Dow Medical College | Sind Medical College | Karachi ||| ||| 5 | 4 weeks ||| 18 years ||| ||| BFRBs | 46 | 22% ||| BFRBs | 29 | 13.9% | 17 | 8.1% ||| 19 | 9.0% | dermatillomania | 28 | 13.3% | trichotillomania | 13 | 6.2% | onychophagia ||| Karachi ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| BFRBs | three | Karachi ||| BFRBs ||| 210 | Aga Khan University | Dow Medical College | Sind Medical College | Karachi ||| ||| 5 | 4 weeks ||| 18 years ||| ||| BFRBs | 46 | 22% ||| BFRBs | 29 | 13.9% | 17 | 8.1% ||| 19 | 9.0% | dermatillomania | 28 | 13.3% | trichotillomania | 13 | 6.2% | onychophagia ||| Karachi ||| [SUMMARY]"}}},{"rowIdx":55,"cells":{"title":{"kind":"string","value":"Thoracotomy is better than thoracoscopic lobectomy in the lymph node dissection of lung cancer: a systematic review and meta-analysis."},"pmid":{"kind":"string","value":"27855709"},"background_abstract":{"kind":"string","value":"The aim of this study was to investigate which surgical method is better in lymph node (LN) dissection of lung cancer."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"A comprehensive search of PubMed, Ovid MEDLINE, EMBASE, Web of Science, ScienceDirect, the Cochrane Library, Scopus, and Google Scholar was performed to identify studies comparing thoracoscopic lobectomy (video-assisted thoracic surgery (VATS) group) and thoracotomy (open group) in LN dissection."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Twenty-nine articles met the inclusion criteria and involved 2763 patients in the VATS group and 3484 patients in the open group. The meta-analysis showed that fewer total LNs (95% confidence interval [CI] -1.52 to -0.73, p < 0.0001) and N2 LNs (95% CI -1.25 to -0.10, p = 0.02) were dissected in the VATS group. A similar number of total LN stations, N2 LN stations, and N1 LNs were harvested in both groups. Only one study reported that fewer N1 LN stations were dissected in the VATS group (1.4 ± 0.5 vs. 1.6 ± 0.6, p = 0.04)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Open lobectomy could achieve better LN dissection efficacy than thoracoscopic lobectomy in the treatment of lung cancer, especially in the N2 LNs dissection. These findings require validation by high-quality, large-scale randomized controlled trials."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Carcinoma, Non-Small-Cell Lung","Humans","Lung Neoplasms","Lymph Node Excision","Lymphatic Metastasis","Mediastinum","Neoplasm Staging","Pneumonectomy","Thoracic Surgery, Video-Assisted","Thoracotomy"],"string":"[\n \"Carcinoma, Non-Small-Cell Lung\",\n \"Humans\",\n \"Lung Neoplasms\",\n \"Lymph Node Excision\",\n \"Lymphatic Metastasis\",\n \"Mediastinum\",\n \"Neoplasm Staging\",\n \"Pneumonectomy\",\n \"Thoracic Surgery, Video-Assisted\",\n \"Thoracotomy\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"5114806"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Lung cancer is the leading cause of cancer deaths in many countries [1, 2]. Surgical treatment is the preferred treatment for early-stage non-small cell lung cancer (NSCLC). D’Cunha’s research showed that N1 and N2 lymph nodes (LNs) were positive in 27.5% of patients with lung cancer under lobectomy [3]. However, non-invasive examinations, such as computed tomography (CT) and positron emission tomography-computed tomography (PET-CT), are not sensitive and specific for the clinical staging of lung cancer. Video-assisted thoracic surgery (VATS) is the preferred surgical procedure, with fewer incidences of postoperative complications and a higher survival rate compared with thoracotomy [4–7]. However, whether VATS can achieve the same LN dissection efficacy is controversial, and there remains a lack of high-quality, large-scale clinical research.\nTo determine whether VATS can achieve the same LN dissection efficacy as thoracotomy in lung cancer, we performed a systemic review and meta-analysis."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Search results and quality assessment of the included studies We initially identified 2341 publications from the database and reference list searches and reviewed 29 articles for final analysis (Fig. 1). The articles involved a total of 6247 patients, of whom 2763 underwent VATS and 3484 underwent thoracotomy. Of these 29 publications, three studies were RCTs and 26 were retrospective studies. According to the NOS and Jadad scales assessment scores, 23 articles were of good quality and the remaining six were medium quality. The baseline characteristics of these articles are listed in Table 1.Fig. 1Flow diagram of screened and included papers\nTable 1Summary of the 29 trials included in the present meta-analysisStudyInstitutionEnrolled yearNo. of patientsClinical stageOutcomesDesignQualityVATSOpenVATSOpen1995Kirby [28] USASingle1991.10–1993.121991.10–1993.122530I①RCT31998Morikawa [29] JPNSingle1996.04–1996.121995.01–1996.033941I–II③Retrospective92000Luketich [30] USASingleNot mentionedNot mentioned3131I①Retrospective72000Sugi [31] JPNSingle1993.01–1994.061993.01–1994.064852Ia③, ⑤RCT32001Nomori [32] JPNSingle1999.08–2000.121998.04–1999.073333I①,⑦,⑧Retrospective72005Watanabe [33] JPNSingle1997–20041997–2004221190I①, ③, ⑦, ⑧Retrospective82006Petersen [34] USASingle2001–20051996–20051285I–IV②Retrospective82006Shigemura [35] JPNMulti1999.01–2004.011999.01–2004.015055Ia①Retrospective92008Shiraishi [36] JPNSingle1994.11–2005.101994.11–2005.102055I③, ⑤Retrospective82008Watanabe [37] JPNSingle1997–20061997–20063732I①, ②, ③, ④Retrospective82008Whitson [38] USASingleNot mentionedNot mentioned67Not mentioned①Retrospective62009Nakanishi [39] JPNSingle2000.04–2007.012000.04–2007.011314I–IV③Retrospective82009Okur [40] TRSingle2007.01–2007.112007.01–2007.112028I②Retrospective82010Denlinger [21] USASingle2000.01–2008.082000.01–2008.0879464I①, ③, ⑤Retrospective82011D’Amico [41] USASingle2007.01–2010.092007.01–2010.09199189I–III②,④Retrospective72012Bu [42] CHNSingle2001.05–2011.042001.05–2011.044687Not mentioned①, ②Retrospective72012Li [43] CHNSingle2006.09–2009.122006.09–2009.122947I③, ④Retrospective82012Licht [44] DNKMulti2007.01–2011.122007.01–2011.12717796I②Retrospective82013Fan [45] CHNSingle2005.01–2010.122005.01–2010.127977I–II①, ②Retrospective82013Lee [22] USASingle1990.05–2011.121990.05–2011.12141115Not mentioned①, ②, ③, ④, ⑤, ⑥Retrospective82013Palade [46] GERSingle2008.05–2011.122008.05–2011.123232I①, ⑦, ⑧RCT32013Zhong [47] CHNSingle2006.03–2011.082006.03–2011.086790I①, ②, ③, ④Retrospective82014Li [48] CHNSingle2011.02–2013.022011.02–2013.022132I–II①Retrospective82014Stephens [4] USASingle2002.01–2011.122002.01–2011.12307307I②Retrospective82015Cai [49] CHNSingle2010.01–2012.052010.01–2012.057167I–II①, ②Retrospective92015Kuritzky [50] USASingle2007–20122007–201274224I②Retrospective82015Murakawa [51] JPNSingle2001–20102001–2010101101I①, ②Retrospective82015Nwogu [6] USAMulti2004.10–2010.062004.10–2010.06175175I–II①, ②Retrospective72015Zhang [52] CHNSingle2012.10–2013.112012.10–2013.117028I①Retrospective9① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\n\nFlow diagram of screened and included papers\nSummary of the 29 trials included in the present meta-analysis\n① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\n\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\nWe initially identified 2341 publications from the database and reference list searches and reviewed 29 articles for final analysis (Fig. 1). The articles involved a total of 6247 patients, of whom 2763 underwent VATS and 3484 underwent thoracotomy. Of these 29 publications, three studies were RCTs and 26 were retrospective studies. According to the NOS and Jadad scales assessment scores, 23 articles were of good quality and the remaining six were medium quality. The baseline characteristics of these articles are listed in Table 1.Fig. 1Flow diagram of screened and included papers\nTable 1Summary of the 29 trials included in the present meta-analysisStudyInstitutionEnrolled yearNo. of patientsClinical stageOutcomesDesignQualityVATSOpenVATSOpen1995Kirby [28] USASingle1991.10–1993.121991.10–1993.122530I①RCT31998Morikawa [29] JPNSingle1996.04–1996.121995.01–1996.033941I–II③Retrospective92000Luketich [30] USASingleNot mentionedNot mentioned3131I①Retrospective72000Sugi [31] JPNSingle1993.01–1994.061993.01–1994.064852Ia③, ⑤RCT32001Nomori [32] JPNSingle1999.08–2000.121998.04–1999.073333I①,⑦,⑧Retrospective72005Watanabe [33] JPNSingle1997–20041997–2004221190I①, ③, ⑦, ⑧Retrospective82006Petersen [34] USASingle2001–20051996–20051285I–IV②Retrospective82006Shigemura [35] JPNMulti1999.01–2004.011999.01–2004.015055Ia①Retrospective92008Shiraishi [36] JPNSingle1994.11–2005.101994.11–2005.102055I③, ⑤Retrospective82008Watanabe [37] JPNSingle1997–20061997–20063732I①, ②, ③, ④Retrospective82008Whitson [38] USASingleNot mentionedNot mentioned67Not mentioned①Retrospective62009Nakanishi [39] JPNSingle2000.04–2007.012000.04–2007.011314I–IV③Retrospective82009Okur [40] TRSingle2007.01–2007.112007.01–2007.112028I②Retrospective82010Denlinger [21] USASingle2000.01–2008.082000.01–2008.0879464I①, ③, ⑤Retrospective82011D’Amico [41] USASingle2007.01–2010.092007.01–2010.09199189I–III②,④Retrospective72012Bu [42] CHNSingle2001.05–2011.042001.05–2011.044687Not mentioned①, ②Retrospective72012Li [43] CHNSingle2006.09–2009.122006.09–2009.122947I③, ④Retrospective82012Licht [44] DNKMulti2007.01–2011.122007.01–2011.12717796I②Retrospective82013Fan [45] CHNSingle2005.01–2010.122005.01–2010.127977I–II①, ②Retrospective82013Lee [22] USASingle1990.05–2011.121990.05–2011.12141115Not mentioned①, ②, ③, ④, ⑤, ⑥Retrospective82013Palade [46] GERSingle2008.05–2011.122008.05–2011.123232I①, ⑦, ⑧RCT32013Zhong [47] CHNSingle2006.03–2011.082006.03–2011.086790I①, ②, ③, ④Retrospective82014Li [48] CHNSingle2011.02–2013.022011.02–2013.022132I–II①Retrospective82014Stephens [4] USASingle2002.01–2011.122002.01–2011.12307307I②Retrospective82015Cai [49] CHNSingle2010.01–2012.052010.01–2012.057167I–II①, ②Retrospective92015Kuritzky [50] USASingle2007–20122007–201274224I②Retrospective82015Murakawa [51] JPNSingle2001–20102001–2010101101I①, ②Retrospective82015Nwogu [6] USAMulti2004.10–2010.062004.10–2010.06175175I–II①, ②Retrospective72015Zhang [52] CHNSingle2012.10–2013.112012.10–2013.117028I①Retrospective9① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\n\nFlow diagram of screened and included papers\nSummary of the 29 trials included in the present meta-analysis\n① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\n\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\n Comparison of total LNN and LNS We identified 19 articles for total LNN comparison. They involved 1297 patients in the VATS group and 1731 patients in the open group (thoracotomy). The heterogeneity between these studies was acceptable (p = 0.02, I\n2 = 44%). Fewer total LNs were dissected in the VATS group as compared with the open group (95% CI −1.52 to −0.73, p < 0.00001, Fig. 2a).Fig. 2Forest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\n\nForest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\nFourteen articles were identified for total LNS comparison. They involved 2046 patients in the VATS group and 2373 patients in the open group. The mean difference in total LNS between the two groups was not significant (95% CI −0.28 to 0.06, p = 0.20), with significant heterogeneity across studies (p < 0.00001, I\n2 = 73%, Fig. 2b).\nWe identified 19 articles for total LNN comparison. They involved 1297 patients in the VATS group and 1731 patients in the open group (thoracotomy). The heterogeneity between these studies was acceptable (p = 0.02, I\n2 = 44%). Fewer total LNs were dissected in the VATS group as compared with the open group (95% CI −1.52 to −0.73, p < 0.00001, Fig. 2a).Fig. 2Forest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\n\nForest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\nFourteen articles were identified for total LNS comparison. They involved 2046 patients in the VATS group and 2373 patients in the open group. The mean difference in total LNS between the two groups was not significant (95% CI −0.28 to 0.06, p = 0.20), with significant heterogeneity across studies (p < 0.00001, I\n2 = 73%, Fig. 2b).\n Comparison of N2 LNN and LNS Eleven articles were identified for N2 LNN comparison. They involved 726 patients in the VATS group and 1132 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.08, I\n2 = 41%). Fewer N2 LNs were dissected in the VATS group as compared with the open group (95% CI −1.38 to −0.49, p < 0.0001, Fig. 3a).Fig. 3Forest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\n\nForest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\nFive articles were identified for N2 LNS comparison. They involved 473 patients in the VATS group and 473 patients in the open group. The mean difference in N2 LNS between the two groups was not significant (95% CI −0.46 to 0.23, p = 0.50), with significant heterogeneity across studies (p = 0.002, I\n2 = 76%, Fig. 3b).\nEleven articles were identified for N2 LNN comparison. They involved 726 patients in the VATS group and 1132 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.08, I\n2 = 41%). Fewer N2 LNs were dissected in the VATS group as compared with the open group (95% CI −1.38 to −0.49, p < 0.0001, Fig. 3a).Fig. 3Forest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\n\nForest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\nFive articles were identified for N2 LNS comparison. They involved 473 patients in the VATS group and 473 patients in the open group. The mean difference in N2 LNS between the two groups was not significant (95% CI −0.46 to 0.23, p = 0.50), with significant heterogeneity across studies (p = 0.002, I\n2 = 76%, Fig. 3b).\n Comparison of N1 LNN and LNS Four articles were identified for N1 LNN comparison. They involved 288 patients in the VATS group and 686 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.44, I\n2 = 60%). The mean difference in N1 LNN between the two groups was not significant (95% CI −0.71 to 0.08, p = 0.11, Fig. 4).Fig. 4Forest plot of the mean difference in N1 LNN in the VATS group vs. the open group\n\nForest plot of the mean difference in N1 LNN in the VATS group vs. the open group\nOnly one article was identified for N1 LNS comparison. They involved 141 patients in the VATS group and 115 patients in the open group. The result showed that fewer N1 LN stations were dissected in the VATS group (1.4 ± 0.5 vs. 1.6 ± 0.6, p = 0.04).\nFour articles were identified for N1 LNN comparison. They involved 288 patients in the VATS group and 686 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.44, I\n2 = 60%). The mean difference in N1 LNN between the two groups was not significant (95% CI −0.71 to 0.08, p = 0.11, Fig. 4).Fig. 4Forest plot of the mean difference in N1 LNN in the VATS group vs. the open group\n\nForest plot of the mean difference in N1 LNN in the VATS group vs. the open group\nOnly one article was identified for N1 LNS comparison. They involved 141 patients in the VATS group and 115 patients in the open group. The result showed that fewer N1 LN stations were dissected in the VATS group (1.4 ± 0.5 vs. 1.6 ± 0.6, p = 0.04).\n Publication bias The funnel plot for publication bias (standard error by total LNN comparison) demonstrated marked evidence of symmetry (Fig. 5), indicating no publication bias. The combined effect size yielded a Z value of 5.64, with a corresponding p < 0.00001. This result indicates that the fail-safe N value was relevant.Fig. 5Funnel plot of the mean difference in total LNN in the VATS group vs. the open group\n\nFunnel plot of the mean difference in total LNN in the VATS group vs. the open group\nThe funnel plot for publication bias (standard error by total LNN comparison) demonstrated marked evidence of symmetry (Fig. 5), indicating no publication bias. The combined effect size yielded a Z value of 5.64, with a corresponding p < 0.00001. This result indicates that the fail-safe N value was relevant.Fig. 5Funnel plot of the mean difference in total LNN in the VATS group vs. the open group\n\nFunnel plot of the mean difference in total LNN in the VATS group vs. the open group"},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"Less total and mediastinal LNs were evaluated with VATS than with thoracotomy in the present study. Both approaches harvested a similar number of total LN stations, mediastinal LN stations, and N1 LNs. However, owing to the possible existing bias in the original studies, inter-study heterogeneity, and the inherent limitations of our meta-analysis, the findings require validation in high-quality, large-scale RCTs."},"other_sections_titles":{"kind":"list like","value":["Search strategy","Inclusion criteria and exclusion criteria","Data extraction","Quality assessment for included studies","Statistical analysis","Search results and quality assessment of the included studies","Comparison of total LNN and LNS","Comparison of N2 LNN and LNS","Comparison of N1 LNN and LNS","Publication bias"],"string":"[\n \"Search strategy\",\n \"Inclusion criteria and exclusion criteria\",\n \"Data extraction\",\n \"Quality assessment for included studies\",\n \"Statistical analysis\",\n \"Search results and quality assessment of the included studies\",\n \"Comparison of total LNN and LNS\",\n \"Comparison of N2 LNN and LNS\",\n \"Comparison of N1 LNN and LNS\",\n \"Publication bias\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["MEDLINE and manual searches were performed by two investigators independently and in duplicate to identify all relevant scientific articles published from January 1990 to May 2016. The MEDLINE search was performed using PubMed, Ovid MEDLINE, EMBASE, Web of Science, ScienceDirect, The Cochrane Library, Scopus, and Google Scholar. The MeSH terms “lung cancer or lung neoplasm”, “thoracotomy or open surgery”, and “video-assisted thoracic surgery or VATS” and comparative study were used.","The following inclusion criteria were applied: (1) published in English, (2) compared the LN dissection of thoracoscopic lobectomy with thoracotomy in treating patients with lung cancer, and (3) the most recent study was chosen when duplication of data is in more than one article.\nReviews without original data, case reports, meta-analyses, letters, expert opinions, and animal studies were excluded. Studies on robotic-assisted VATS were also excluded.","Two investigators independently extracted data from the eligible studies. The extracted data included first author, year of publication, geographical area, study design, duration of enrollment, information on preoperative staging, number of patients per group, LN number (LNN), and LN station number (LNS).","Two investigators independently assessed the quality of each included study using the Newcastle-Ottawa Scale (NOS) for non-randomized studies and the Jadad scale for randomized controlled trials (RCTs).\nThe NOS evaluates the quality of studies by analyzing three items: selection, comparability, and exposure. The scale assigns a maximum of nine points to each study: a maximum of four points for selection, two points for comparability, and three points for exposure. Therefore, the highest quality study would score nine points. In our analysis, high-quality studies were defined as those that scored nine or eight points; medium-quality studies were those that scored seven or six points [8].\nThe Jadad scale (five points) contained questions for three main parts: randomization, masking, and accountability of all patients (withdrawals and dropouts). Studies scored ≥3 points were considered as high quality [9].","Meta-analysis was conducted by Review Manager 5.3 and SPSS 18.0, p value < 0.05 suggested statistically significant. The differences were compared between the two groups using analysis of variance for continuous variables and pooled relative risk (RR) with 95% confidence interval (CI) for categorical variables. We used I\n2 and Cochran Q to evaluate the between-study heterogeneity. A random-effects model was adopted when the heterogeneity was significant (p ≤ 0.10 and I\n2 > 50%); otherwise, a fixed-effects model was used. Rank correlation test of funnel plot asymmetry was used to assess the potential publication bias.","We initially identified 2341 publications from the database and reference list searches and reviewed 29 articles for final analysis (Fig. 1). The articles involved a total of 6247 patients, of whom 2763 underwent VATS and 3484 underwent thoracotomy. Of these 29 publications, three studies were RCTs and 26 were retrospective studies. According to the NOS and Jadad scales assessment scores, 23 articles were of good quality and the remaining six were medium quality. The baseline characteristics of these articles are listed in Table 1.Fig. 1Flow diagram of screened and included papers\nTable 1Summary of the 29 trials included in the present meta-analysisStudyInstitutionEnrolled yearNo. of patientsClinical stageOutcomesDesignQualityVATSOpenVATSOpen1995Kirby [28] USASingle1991.10–1993.121991.10–1993.122530I①RCT31998Morikawa [29] JPNSingle1996.04–1996.121995.01–1996.033941I–II③Retrospective92000Luketich [30] USASingleNot mentionedNot mentioned3131I①Retrospective72000Sugi [31] JPNSingle1993.01–1994.061993.01–1994.064852Ia③, ⑤RCT32001Nomori [32] JPNSingle1999.08–2000.121998.04–1999.073333I①,⑦,⑧Retrospective72005Watanabe [33] JPNSingle1997–20041997–2004221190I①, ③, ⑦, ⑧Retrospective82006Petersen [34] USASingle2001–20051996–20051285I–IV②Retrospective82006Shigemura [35] JPNMulti1999.01–2004.011999.01–2004.015055Ia①Retrospective92008Shiraishi [36] JPNSingle1994.11–2005.101994.11–2005.102055I③, ⑤Retrospective82008Watanabe [37] JPNSingle1997–20061997–20063732I①, ②, ③, ④Retrospective82008Whitson [38] USASingleNot mentionedNot mentioned67Not mentioned①Retrospective62009Nakanishi [39] JPNSingle2000.04–2007.012000.04–2007.011314I–IV③Retrospective82009Okur [40] TRSingle2007.01–2007.112007.01–2007.112028I②Retrospective82010Denlinger [21] USASingle2000.01–2008.082000.01–2008.0879464I①, ③, ⑤Retrospective82011D’Amico [41] USASingle2007.01–2010.092007.01–2010.09199189I–III②,④Retrospective72012Bu [42] CHNSingle2001.05–2011.042001.05–2011.044687Not mentioned①, ②Retrospective72012Li [43] CHNSingle2006.09–2009.122006.09–2009.122947I③, ④Retrospective82012Licht [44] DNKMulti2007.01–2011.122007.01–2011.12717796I②Retrospective82013Fan [45] CHNSingle2005.01–2010.122005.01–2010.127977I–II①, ②Retrospective82013Lee [22] USASingle1990.05–2011.121990.05–2011.12141115Not mentioned①, ②, ③, ④, ⑤, ⑥Retrospective82013Palade [46] GERSingle2008.05–2011.122008.05–2011.123232I①, ⑦, ⑧RCT32013Zhong [47] CHNSingle2006.03–2011.082006.03–2011.086790I①, ②, ③, ④Retrospective82014Li [48] CHNSingle2011.02–2013.022011.02–2013.022132I–II①Retrospective82014Stephens [4] USASingle2002.01–2011.122002.01–2011.12307307I②Retrospective82015Cai [49] CHNSingle2010.01–2012.052010.01–2012.057167I–II①, ②Retrospective92015Kuritzky [50] USASingle2007–20122007–201274224I②Retrospective82015Murakawa [51] JPNSingle2001–20102001–2010101101I①, ②Retrospective82015Nwogu [6] USAMulti2004.10–2010.062004.10–2010.06175175I–II①, ②Retrospective72015Zhang [52] CHNSingle2012.10–2013.112012.10–2013.117028I①Retrospective9① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\n\nFlow diagram of screened and included papers\nSummary of the 29 trials included in the present meta-analysis\n① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\n\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial","We identified 19 articles for total LNN comparison. They involved 1297 patients in the VATS group and 1731 patients in the open group (thoracotomy). The heterogeneity between these studies was acceptable (p = 0.02, I\n2 = 44%). Fewer total LNs were dissected in the VATS group as compared with the open group (95% CI −1.52 to −0.73, p < 0.00001, Fig. 2a).Fig. 2Forest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\n\nForest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\nFourteen articles were identified for total LNS comparison. They involved 2046 patients in the VATS group and 2373 patients in the open group. The mean difference in total LNS between the two groups was not significant (95% CI −0.28 to 0.06, p = 0.20), with significant heterogeneity across studies (p < 0.00001, I\n2 = 73%, Fig. 2b).","Eleven articles were identified for N2 LNN comparison. They involved 726 patients in the VATS group and 1132 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.08, I\n2 = 41%). Fewer N2 LNs were dissected in the VATS group as compared with the open group (95% CI −1.38 to −0.49, p < 0.0001, Fig. 3a).Fig. 3Forest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\n\nForest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\nFive articles were identified for N2 LNS comparison. They involved 473 patients in the VATS group and 473 patients in the open group. The mean difference in N2 LNS between the two groups was not significant (95% CI −0.46 to 0.23, p = 0.50), with significant heterogeneity across studies (p = 0.002, I\n2 = 76%, Fig. 3b).","Four articles were identified for N1 LNN comparison. They involved 288 patients in the VATS group and 686 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.44, I\n2 = 60%). The mean difference in N1 LNN between the two groups was not significant (95% CI −0.71 to 0.08, p = 0.11, Fig. 4).Fig. 4Forest plot of the mean difference in N1 LNN in the VATS group vs. the open group\n\nForest plot of the mean difference in N1 LNN in the VATS group vs. the open group\nOnly one article was identified for N1 LNS comparison. They involved 141 patients in the VATS group and 115 patients in the open group. The result showed that fewer N1 LN stations were dissected in the VATS group (1.4 ± 0.5 vs. 1.6 ± 0.6, p = 0.04).","The funnel plot for publication bias (standard error by total LNN comparison) demonstrated marked evidence of symmetry (Fig. 5), indicating no publication bias. The combined effect size yielded a Z value of 5.64, with a corresponding p < 0.00001. This result indicates that the fail-safe N value was relevant.Fig. 5Funnel plot of the mean difference in total LNN in the VATS group vs. the open group\n\nFunnel plot of the mean difference in total LNN in the VATS group vs. the open group"],"string":"[\n \"MEDLINE and manual searches were performed by two investigators independently and in duplicate to identify all relevant scientific articles published from January 1990 to May 2016. The MEDLINE search was performed using PubMed, Ovid MEDLINE, EMBASE, Web of Science, ScienceDirect, The Cochrane Library, Scopus, and Google Scholar. The MeSH terms “lung cancer or lung neoplasm”, “thoracotomy or open surgery”, and “video-assisted thoracic surgery or VATS” and comparative study were used.\",\n \"The following inclusion criteria were applied: (1) published in English, (2) compared the LN dissection of thoracoscopic lobectomy with thoracotomy in treating patients with lung cancer, and (3) the most recent study was chosen when duplication of data is in more than one article.\\nReviews without original data, case reports, meta-analyses, letters, expert opinions, and animal studies were excluded. Studies on robotic-assisted VATS were also excluded.\",\n \"Two investigators independently extracted data from the eligible studies. The extracted data included first author, year of publication, geographical area, study design, duration of enrollment, information on preoperative staging, number of patients per group, LN number (LNN), and LN station number (LNS).\",\n \"Two investigators independently assessed the quality of each included study using the Newcastle-Ottawa Scale (NOS) for non-randomized studies and the Jadad scale for randomized controlled trials (RCTs).\\nThe NOS evaluates the quality of studies by analyzing three items: selection, comparability, and exposure. The scale assigns a maximum of nine points to each study: a maximum of four points for selection, two points for comparability, and three points for exposure. Therefore, the highest quality study would score nine points. In our analysis, high-quality studies were defined as those that scored nine or eight points; medium-quality studies were those that scored seven or six points [8].\\nThe Jadad scale (five points) contained questions for three main parts: randomization, masking, and accountability of all patients (withdrawals and dropouts). Studies scored ≥3 points were considered as high quality [9].\",\n \"Meta-analysis was conducted by Review Manager 5.3 and SPSS 18.0, p value < 0.05 suggested statistically significant. The differences were compared between the two groups using analysis of variance for continuous variables and pooled relative risk (RR) with 95% confidence interval (CI) for categorical variables. We used I\\n2 and Cochran Q to evaluate the between-study heterogeneity. A random-effects model was adopted when the heterogeneity was significant (p ≤ 0.10 and I\\n2 > 50%); otherwise, a fixed-effects model was used. Rank correlation test of funnel plot asymmetry was used to assess the potential publication bias.\",\n \"We initially identified 2341 publications from the database and reference list searches and reviewed 29 articles for final analysis (Fig. 1). The articles involved a total of 6247 patients, of whom 2763 underwent VATS and 3484 underwent thoracotomy. Of these 29 publications, three studies were RCTs and 26 were retrospective studies. According to the NOS and Jadad scales assessment scores, 23 articles were of good quality and the remaining six were medium quality. The baseline characteristics of these articles are listed in Table 1.Fig. 1Flow diagram of screened and included papers\\nTable 1Summary of the 29 trials included in the present meta-analysisStudyInstitutionEnrolled yearNo. of patientsClinical stageOutcomesDesignQualityVATSOpenVATSOpen1995Kirby [28] USASingle1991.10–1993.121991.10–1993.122530I①RCT31998Morikawa [29] JPNSingle1996.04–1996.121995.01–1996.033941I–II③Retrospective92000Luketich [30] USASingleNot mentionedNot mentioned3131I①Retrospective72000Sugi [31] JPNSingle1993.01–1994.061993.01–1994.064852Ia③, ⑤RCT32001Nomori [32] JPNSingle1999.08–2000.121998.04–1999.073333I①,⑦,⑧Retrospective72005Watanabe [33] JPNSingle1997–20041997–2004221190I①, ③, ⑦, ⑧Retrospective82006Petersen [34] USASingle2001–20051996–20051285I–IV②Retrospective82006Shigemura [35] JPNMulti1999.01–2004.011999.01–2004.015055Ia①Retrospective92008Shiraishi [36] JPNSingle1994.11–2005.101994.11–2005.102055I③, ⑤Retrospective82008Watanabe [37] JPNSingle1997–20061997–20063732I①, ②, ③, ④Retrospective82008Whitson [38] USASingleNot mentionedNot mentioned67Not mentioned①Retrospective62009Nakanishi [39] JPNSingle2000.04–2007.012000.04–2007.011314I–IV③Retrospective82009Okur [40] TRSingle2007.01–2007.112007.01–2007.112028I②Retrospective82010Denlinger [21] USASingle2000.01–2008.082000.01–2008.0879464I①, ③, ⑤Retrospective82011D’Amico [41] USASingle2007.01–2010.092007.01–2010.09199189I–III②,④Retrospective72012Bu [42] CHNSingle2001.05–2011.042001.05–2011.044687Not mentioned①, ②Retrospective72012Li [43] CHNSingle2006.09–2009.122006.09–2009.122947I③, ④Retrospective82012Licht [44] DNKMulti2007.01–2011.122007.01–2011.12717796I②Retrospective82013Fan [45] CHNSingle2005.01–2010.122005.01–2010.127977I–II①, ②Retrospective82013Lee [22] USASingle1990.05–2011.121990.05–2011.12141115Not mentioned①, ②, ③, ④, ⑤, ⑥Retrospective82013Palade [46] GERSingle2008.05–2011.122008.05–2011.123232I①, ⑦, ⑧RCT32013Zhong [47] CHNSingle2006.03–2011.082006.03–2011.086790I①, ②, ③, ④Retrospective82014Li [48] CHNSingle2011.02–2013.022011.02–2013.022132I–II①Retrospective82014Stephens [4] USASingle2002.01–2011.122002.01–2011.12307307I②Retrospective82015Cai [49] CHNSingle2010.01–2012.052010.01–2012.057167I–II①, ②Retrospective92015Kuritzky [50] USASingle2007–20122007–201274224I②Retrospective82015Murakawa [51] JPNSingle2001–20102001–2010101101I①, ②Retrospective82015Nwogu [6] USAMulti2004.10–2010.062004.10–2010.06175175I–II①, ②Retrospective72015Zhang [52] CHNSingle2012.10–2013.112012.10–2013.117028I①Retrospective9① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\\n\\nFlow diagram of screened and included papers\\nSummary of the 29 trials included in the present meta-analysis\\n① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\\n\\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\",\n \"We identified 19 articles for total LNN comparison. They involved 1297 patients in the VATS group and 1731 patients in the open group (thoracotomy). The heterogeneity between these studies was acceptable (p = 0.02, I\\n2 = 44%). Fewer total LNs were dissected in the VATS group as compared with the open group (95% CI −1.52 to −0.73, p < 0.00001, Fig. 2a).Fig. 2Forest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\\n\\nForest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\\nFourteen articles were identified for total LNS comparison. They involved 2046 patients in the VATS group and 2373 patients in the open group. The mean difference in total LNS between the two groups was not significant (95% CI −0.28 to 0.06, p = 0.20), with significant heterogeneity across studies (p < 0.00001, I\\n2 = 73%, Fig. 2b).\",\n \"Eleven articles were identified for N2 LNN comparison. They involved 726 patients in the VATS group and 1132 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.08, I\\n2 = 41%). Fewer N2 LNs were dissected in the VATS group as compared with the open group (95% CI −1.38 to −0.49, p < 0.0001, Fig. 3a).Fig. 3Forest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\\n\\nForest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\\nFive articles were identified for N2 LNS comparison. They involved 473 patients in the VATS group and 473 patients in the open group. The mean difference in N2 LNS between the two groups was not significant (95% CI −0.46 to 0.23, p = 0.50), with significant heterogeneity across studies (p = 0.002, I\\n2 = 76%, Fig. 3b).\",\n \"Four articles were identified for N1 LNN comparison. They involved 288 patients in the VATS group and 686 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.44, I\\n2 = 60%). The mean difference in N1 LNN between the two groups was not significant (95% CI −0.71 to 0.08, p = 0.11, Fig. 4).Fig. 4Forest plot of the mean difference in N1 LNN in the VATS group vs. the open group\\n\\nForest plot of the mean difference in N1 LNN in the VATS group vs. the open group\\nOnly one article was identified for N1 LNS comparison. They involved 141 patients in the VATS group and 115 patients in the open group. The result showed that fewer N1 LN stations were dissected in the VATS group (1.4 ± 0.5 vs. 1.6 ± 0.6, p = 0.04).\",\n \"The funnel plot for publication bias (standard error by total LNN comparison) demonstrated marked evidence of symmetry (Fig. 5), indicating no publication bias. The combined effect size yielded a Z value of 5.64, with a corresponding p < 0.00001. This result indicates that the fail-safe N value was relevant.Fig. 5Funnel plot of the mean difference in total LNN in the VATS group vs. the open group\\n\\nFunnel plot of the mean difference in total LNN in the VATS group vs. the open group\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Search strategy","Inclusion criteria and exclusion criteria","Data extraction","Quality assessment for included studies","Statistical analysis","Results","Search results and quality assessment of the included studies","Comparison of total LNN and LNS","Comparison of N2 LNN and LNS","Comparison of N1 LNN and LNS","Publication bias","Discussion","Conclusions"],"string":"[\n \"Background\",\n \"Methods\",\n \"Search strategy\",\n \"Inclusion criteria and exclusion criteria\",\n \"Data extraction\",\n \"Quality assessment for included studies\",\n \"Statistical analysis\",\n \"Results\",\n \"Search results and quality assessment of the included studies\",\n \"Comparison of total LNN and LNS\",\n \"Comparison of N2 LNN and LNS\",\n \"Comparison of N1 LNN and LNS\",\n \"Publication bias\",\n \"Discussion\",\n \"Conclusions\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Lung cancer is the leading cause of cancer deaths in many countries [1, 2]. Surgical treatment is the preferred treatment for early-stage non-small cell lung cancer (NSCLC). D’Cunha’s research showed that N1 and N2 lymph nodes (LNs) were positive in 27.5% of patients with lung cancer under lobectomy [3]. However, non-invasive examinations, such as computed tomography (CT) and positron emission tomography-computed tomography (PET-CT), are not sensitive and specific for the clinical staging of lung cancer. Video-assisted thoracic surgery (VATS) is the preferred surgical procedure, with fewer incidences of postoperative complications and a higher survival rate compared with thoracotomy [4–7]. However, whether VATS can achieve the same LN dissection efficacy is controversial, and there remains a lack of high-quality, large-scale clinical research.\nTo determine whether VATS can achieve the same LN dissection efficacy as thoracotomy in lung cancer, we performed a systemic review and meta-analysis."," Search strategy MEDLINE and manual searches were performed by two investigators independently and in duplicate to identify all relevant scientific articles published from January 1990 to May 2016. The MEDLINE search was performed using PubMed, Ovid MEDLINE, EMBASE, Web of Science, ScienceDirect, The Cochrane Library, Scopus, and Google Scholar. The MeSH terms “lung cancer or lung neoplasm”, “thoracotomy or open surgery”, and “video-assisted thoracic surgery or VATS” and comparative study were used.\nMEDLINE and manual searches were performed by two investigators independently and in duplicate to identify all relevant scientific articles published from January 1990 to May 2016. The MEDLINE search was performed using PubMed, Ovid MEDLINE, EMBASE, Web of Science, ScienceDirect, The Cochrane Library, Scopus, and Google Scholar. The MeSH terms “lung cancer or lung neoplasm”, “thoracotomy or open surgery”, and “video-assisted thoracic surgery or VATS” and comparative study were used.\n Inclusion criteria and exclusion criteria The following inclusion criteria were applied: (1) published in English, (2) compared the LN dissection of thoracoscopic lobectomy with thoracotomy in treating patients with lung cancer, and (3) the most recent study was chosen when duplication of data is in more than one article.\nReviews without original data, case reports, meta-analyses, letters, expert opinions, and animal studies were excluded. Studies on robotic-assisted VATS were also excluded.\nThe following inclusion criteria were applied: (1) published in English, (2) compared the LN dissection of thoracoscopic lobectomy with thoracotomy in treating patients with lung cancer, and (3) the most recent study was chosen when duplication of data is in more than one article.\nReviews without original data, case reports, meta-analyses, letters, expert opinions, and animal studies were excluded. Studies on robotic-assisted VATS were also excluded.\n Data extraction Two investigators independently extracted data from the eligible studies. The extracted data included first author, year of publication, geographical area, study design, duration of enrollment, information on preoperative staging, number of patients per group, LN number (LNN), and LN station number (LNS).\nTwo investigators independently extracted data from the eligible studies. The extracted data included first author, year of publication, geographical area, study design, duration of enrollment, information on preoperative staging, number of patients per group, LN number (LNN), and LN station number (LNS).\n Quality assessment for included studies Two investigators independently assessed the quality of each included study using the Newcastle-Ottawa Scale (NOS) for non-randomized studies and the Jadad scale for randomized controlled trials (RCTs).\nThe NOS evaluates the quality of studies by analyzing three items: selection, comparability, and exposure. The scale assigns a maximum of nine points to each study: a maximum of four points for selection, two points for comparability, and three points for exposure. Therefore, the highest quality study would score nine points. In our analysis, high-quality studies were defined as those that scored nine or eight points; medium-quality studies were those that scored seven or six points [8].\nThe Jadad scale (five points) contained questions for three main parts: randomization, masking, and accountability of all patients (withdrawals and dropouts). Studies scored ≥3 points were considered as high quality [9].\nTwo investigators independently assessed the quality of each included study using the Newcastle-Ottawa Scale (NOS) for non-randomized studies and the Jadad scale for randomized controlled trials (RCTs).\nThe NOS evaluates the quality of studies by analyzing three items: selection, comparability, and exposure. The scale assigns a maximum of nine points to each study: a maximum of four points for selection, two points for comparability, and three points for exposure. Therefore, the highest quality study would score nine points. In our analysis, high-quality studies were defined as those that scored nine or eight points; medium-quality studies were those that scored seven or six points [8].\nThe Jadad scale (five points) contained questions for three main parts: randomization, masking, and accountability of all patients (withdrawals and dropouts). Studies scored ≥3 points were considered as high quality [9].\n Statistical analysis Meta-analysis was conducted by Review Manager 5.3 and SPSS 18.0, p value < 0.05 suggested statistically significant. The differences were compared between the two groups using analysis of variance for continuous variables and pooled relative risk (RR) with 95% confidence interval (CI) for categorical variables. We used I\n2 and Cochran Q to evaluate the between-study heterogeneity. A random-effects model was adopted when the heterogeneity was significant (p ≤ 0.10 and I\n2 > 50%); otherwise, a fixed-effects model was used. Rank correlation test of funnel plot asymmetry was used to assess the potential publication bias.\nMeta-analysis was conducted by Review Manager 5.3 and SPSS 18.0, p value < 0.05 suggested statistically significant. The differences were compared between the two groups using analysis of variance for continuous variables and pooled relative risk (RR) with 95% confidence interval (CI) for categorical variables. We used I\n2 and Cochran Q to evaluate the between-study heterogeneity. A random-effects model was adopted when the heterogeneity was significant (p ≤ 0.10 and I\n2 > 50%); otherwise, a fixed-effects model was used. Rank correlation test of funnel plot asymmetry was used to assess the potential publication bias.","MEDLINE and manual searches were performed by two investigators independently and in duplicate to identify all relevant scientific articles published from January 1990 to May 2016. The MEDLINE search was performed using PubMed, Ovid MEDLINE, EMBASE, Web of Science, ScienceDirect, The Cochrane Library, Scopus, and Google Scholar. The MeSH terms “lung cancer or lung neoplasm”, “thoracotomy or open surgery”, and “video-assisted thoracic surgery or VATS” and comparative study were used.","The following inclusion criteria were applied: (1) published in English, (2) compared the LN dissection of thoracoscopic lobectomy with thoracotomy in treating patients with lung cancer, and (3) the most recent study was chosen when duplication of data is in more than one article.\nReviews without original data, case reports, meta-analyses, letters, expert opinions, and animal studies were excluded. Studies on robotic-assisted VATS were also excluded.","Two investigators independently extracted data from the eligible studies. The extracted data included first author, year of publication, geographical area, study design, duration of enrollment, information on preoperative staging, number of patients per group, LN number (LNN), and LN station number (LNS).","Two investigators independently assessed the quality of each included study using the Newcastle-Ottawa Scale (NOS) for non-randomized studies and the Jadad scale for randomized controlled trials (RCTs).\nThe NOS evaluates the quality of studies by analyzing three items: selection, comparability, and exposure. The scale assigns a maximum of nine points to each study: a maximum of four points for selection, two points for comparability, and three points for exposure. Therefore, the highest quality study would score nine points. In our analysis, high-quality studies were defined as those that scored nine or eight points; medium-quality studies were those that scored seven or six points [8].\nThe Jadad scale (five points) contained questions for three main parts: randomization, masking, and accountability of all patients (withdrawals and dropouts). Studies scored ≥3 points were considered as high quality [9].","Meta-analysis was conducted by Review Manager 5.3 and SPSS 18.0, p value < 0.05 suggested statistically significant. The differences were compared between the two groups using analysis of variance for continuous variables and pooled relative risk (RR) with 95% confidence interval (CI) for categorical variables. We used I\n2 and Cochran Q to evaluate the between-study heterogeneity. A random-effects model was adopted when the heterogeneity was significant (p ≤ 0.10 and I\n2 > 50%); otherwise, a fixed-effects model was used. Rank correlation test of funnel plot asymmetry was used to assess the potential publication bias."," Search results and quality assessment of the included studies We initially identified 2341 publications from the database and reference list searches and reviewed 29 articles for final analysis (Fig. 1). The articles involved a total of 6247 patients, of whom 2763 underwent VATS and 3484 underwent thoracotomy. Of these 29 publications, three studies were RCTs and 26 were retrospective studies. According to the NOS and Jadad scales assessment scores, 23 articles were of good quality and the remaining six were medium quality. The baseline characteristics of these articles are listed in Table 1.Fig. 1Flow diagram of screened and included papers\nTable 1Summary of the 29 trials included in the present meta-analysisStudyInstitutionEnrolled yearNo. of patientsClinical stageOutcomesDesignQualityVATSOpenVATSOpen1995Kirby [28] USASingle1991.10–1993.121991.10–1993.122530I①RCT31998Morikawa [29] JPNSingle1996.04–1996.121995.01–1996.033941I–II③Retrospective92000Luketich [30] USASingleNot mentionedNot mentioned3131I①Retrospective72000Sugi [31] JPNSingle1993.01–1994.061993.01–1994.064852Ia③, ⑤RCT32001Nomori [32] JPNSingle1999.08–2000.121998.04–1999.073333I①,⑦,⑧Retrospective72005Watanabe [33] JPNSingle1997–20041997–2004221190I①, ③, ⑦, ⑧Retrospective82006Petersen [34] USASingle2001–20051996–20051285I–IV②Retrospective82006Shigemura [35] JPNMulti1999.01–2004.011999.01–2004.015055Ia①Retrospective92008Shiraishi [36] JPNSingle1994.11–2005.101994.11–2005.102055I③, ⑤Retrospective82008Watanabe [37] JPNSingle1997–20061997–20063732I①, ②, ③, ④Retrospective82008Whitson [38] USASingleNot mentionedNot mentioned67Not mentioned①Retrospective62009Nakanishi [39] JPNSingle2000.04–2007.012000.04–2007.011314I–IV③Retrospective82009Okur [40] TRSingle2007.01–2007.112007.01–2007.112028I②Retrospective82010Denlinger [21] USASingle2000.01–2008.082000.01–2008.0879464I①, ③, ⑤Retrospective82011D’Amico [41] USASingle2007.01–2010.092007.01–2010.09199189I–III②,④Retrospective72012Bu [42] CHNSingle2001.05–2011.042001.05–2011.044687Not mentioned①, ②Retrospective72012Li [43] CHNSingle2006.09–2009.122006.09–2009.122947I③, ④Retrospective82012Licht [44] DNKMulti2007.01–2011.122007.01–2011.12717796I②Retrospective82013Fan [45] CHNSingle2005.01–2010.122005.01–2010.127977I–II①, ②Retrospective82013Lee [22] USASingle1990.05–2011.121990.05–2011.12141115Not mentioned①, ②, ③, ④, ⑤, ⑥Retrospective82013Palade [46] GERSingle2008.05–2011.122008.05–2011.123232I①, ⑦, ⑧RCT32013Zhong [47] CHNSingle2006.03–2011.082006.03–2011.086790I①, ②, ③, ④Retrospective82014Li [48] CHNSingle2011.02–2013.022011.02–2013.022132I–II①Retrospective82014Stephens [4] USASingle2002.01–2011.122002.01–2011.12307307I②Retrospective82015Cai [49] CHNSingle2010.01–2012.052010.01–2012.057167I–II①, ②Retrospective92015Kuritzky [50] USASingle2007–20122007–201274224I②Retrospective82015Murakawa [51] JPNSingle2001–20102001–2010101101I①, ②Retrospective82015Nwogu [6] USAMulti2004.10–2010.062004.10–2010.06175175I–II①, ②Retrospective72015Zhang [52] CHNSingle2012.10–2013.112012.10–2013.117028I①Retrospective9① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\n\nFlow diagram of screened and included papers\nSummary of the 29 trials included in the present meta-analysis\n① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\n\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\nWe initially identified 2341 publications from the database and reference list searches and reviewed 29 articles for final analysis (Fig. 1). The articles involved a total of 6247 patients, of whom 2763 underwent VATS and 3484 underwent thoracotomy. Of these 29 publications, three studies were RCTs and 26 were retrospective studies. According to the NOS and Jadad scales assessment scores, 23 articles were of good quality and the remaining six were medium quality. The baseline characteristics of these articles are listed in Table 1.Fig. 1Flow diagram of screened and included papers\nTable 1Summary of the 29 trials included in the present meta-analysisStudyInstitutionEnrolled yearNo. of patientsClinical stageOutcomesDesignQualityVATSOpenVATSOpen1995Kirby [28] USASingle1991.10–1993.121991.10–1993.122530I①RCT31998Morikawa [29] JPNSingle1996.04–1996.121995.01–1996.033941I–II③Retrospective92000Luketich [30] USASingleNot mentionedNot mentioned3131I①Retrospective72000Sugi [31] JPNSingle1993.01–1994.061993.01–1994.064852Ia③, ⑤RCT32001Nomori [32] JPNSingle1999.08–2000.121998.04–1999.073333I①,⑦,⑧Retrospective72005Watanabe [33] JPNSingle1997–20041997–2004221190I①, ③, ⑦, ⑧Retrospective82006Petersen [34] USASingle2001–20051996–20051285I–IV②Retrospective82006Shigemura [35] JPNMulti1999.01–2004.011999.01–2004.015055Ia①Retrospective92008Shiraishi [36] JPNSingle1994.11–2005.101994.11–2005.102055I③, ⑤Retrospective82008Watanabe [37] JPNSingle1997–20061997–20063732I①, ②, ③, ④Retrospective82008Whitson [38] USASingleNot mentionedNot mentioned67Not mentioned①Retrospective62009Nakanishi [39] JPNSingle2000.04–2007.012000.04–2007.011314I–IV③Retrospective82009Okur [40] TRSingle2007.01–2007.112007.01–2007.112028I②Retrospective82010Denlinger [21] USASingle2000.01–2008.082000.01–2008.0879464I①, ③, ⑤Retrospective82011D’Amico [41] USASingle2007.01–2010.092007.01–2010.09199189I–III②,④Retrospective72012Bu [42] CHNSingle2001.05–2011.042001.05–2011.044687Not mentioned①, ②Retrospective72012Li [43] CHNSingle2006.09–2009.122006.09–2009.122947I③, ④Retrospective82012Licht [44] DNKMulti2007.01–2011.122007.01–2011.12717796I②Retrospective82013Fan [45] CHNSingle2005.01–2010.122005.01–2010.127977I–II①, ②Retrospective82013Lee [22] USASingle1990.05–2011.121990.05–2011.12141115Not mentioned①, ②, ③, ④, ⑤, ⑥Retrospective82013Palade [46] GERSingle2008.05–2011.122008.05–2011.123232I①, ⑦, ⑧RCT32013Zhong [47] CHNSingle2006.03–2011.082006.03–2011.086790I①, ②, ③, ④Retrospective82014Li [48] CHNSingle2011.02–2013.022011.02–2013.022132I–II①Retrospective82014Stephens [4] USASingle2002.01–2011.122002.01–2011.12307307I②Retrospective82015Cai [49] CHNSingle2010.01–2012.052010.01–2012.057167I–II①, ②Retrospective92015Kuritzky [50] USASingle2007–20122007–201274224I②Retrospective82015Murakawa [51] JPNSingle2001–20102001–2010101101I①, ②Retrospective82015Nwogu [6] USAMulti2004.10–2010.062004.10–2010.06175175I–II①, ②Retrospective72015Zhang [52] CHNSingle2012.10–2013.112012.10–2013.117028I①Retrospective9① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\n\nFlow diagram of screened and included papers\nSummary of the 29 trials included in the present meta-analysis\n① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\n\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\n Comparison of total LNN and LNS We identified 19 articles for total LNN comparison. They involved 1297 patients in the VATS group and 1731 patients in the open group (thoracotomy). The heterogeneity between these studies was acceptable (p = 0.02, I\n2 = 44%). Fewer total LNs were dissected in the VATS group as compared with the open group (95% CI −1.52 to −0.73, p < 0.00001, Fig. 2a).Fig. 2Forest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\n\nForest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\nFourteen articles were identified for total LNS comparison. They involved 2046 patients in the VATS group and 2373 patients in the open group. The mean difference in total LNS between the two groups was not significant (95% CI −0.28 to 0.06, p = 0.20), with significant heterogeneity across studies (p < 0.00001, I\n2 = 73%, Fig. 2b).\nWe identified 19 articles for total LNN comparison. They involved 1297 patients in the VATS group and 1731 patients in the open group (thoracotomy). The heterogeneity between these studies was acceptable (p = 0.02, I\n2 = 44%). Fewer total LNs were dissected in the VATS group as compared with the open group (95% CI −1.52 to −0.73, p < 0.00001, Fig. 2a).Fig. 2Forest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\n\nForest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\nFourteen articles were identified for total LNS comparison. They involved 2046 patients in the VATS group and 2373 patients in the open group. The mean difference in total LNS between the two groups was not significant (95% CI −0.28 to 0.06, p = 0.20), with significant heterogeneity across studies (p < 0.00001, I\n2 = 73%, Fig. 2b).\n Comparison of N2 LNN and LNS Eleven articles were identified for N2 LNN comparison. They involved 726 patients in the VATS group and 1132 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.08, I\n2 = 41%). Fewer N2 LNs were dissected in the VATS group as compared with the open group (95% CI −1.38 to −0.49, p < 0.0001, Fig. 3a).Fig. 3Forest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\n\nForest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\nFive articles were identified for N2 LNS comparison. They involved 473 patients in the VATS group and 473 patients in the open group. The mean difference in N2 LNS between the two groups was not significant (95% CI −0.46 to 0.23, p = 0.50), with significant heterogeneity across studies (p = 0.002, I\n2 = 76%, Fig. 3b).\nEleven articles were identified for N2 LNN comparison. They involved 726 patients in the VATS group and 1132 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.08, I\n2 = 41%). Fewer N2 LNs were dissected in the VATS group as compared with the open group (95% CI −1.38 to −0.49, p < 0.0001, Fig. 3a).Fig. 3Forest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\n\nForest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\nFive articles were identified for N2 LNS comparison. They involved 473 patients in the VATS group and 473 patients in the open group. The mean difference in N2 LNS between the two groups was not significant (95% CI −0.46 to 0.23, p = 0.50), with significant heterogeneity across studies (p = 0.002, I\n2 = 76%, Fig. 3b).\n Comparison of N1 LNN and LNS Four articles were identified for N1 LNN comparison. They involved 288 patients in the VATS group and 686 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.44, I\n2 = 60%). The mean difference in N1 LNN between the two groups was not significant (95% CI −0.71 to 0.08, p = 0.11, Fig. 4).Fig. 4Forest plot of the mean difference in N1 LNN in the VATS group vs. the open group\n\nForest plot of the mean difference in N1 LNN in the VATS group vs. the open group\nOnly one article was identified for N1 LNS comparison. They involved 141 patients in the VATS group and 115 patients in the open group. The result showed that fewer N1 LN stations were dissected in the VATS group (1.4 ± 0.5 vs. 1.6 ± 0.6, p = 0.04).\nFour articles were identified for N1 LNN comparison. They involved 288 patients in the VATS group and 686 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.44, I\n2 = 60%). The mean difference in N1 LNN between the two groups was not significant (95% CI −0.71 to 0.08, p = 0.11, Fig. 4).Fig. 4Forest plot of the mean difference in N1 LNN in the VATS group vs. the open group\n\nForest plot of the mean difference in N1 LNN in the VATS group vs. the open group\nOnly one article was identified for N1 LNS comparison. They involved 141 patients in the VATS group and 115 patients in the open group. The result showed that fewer N1 LN stations were dissected in the VATS group (1.4 ± 0.5 vs. 1.6 ± 0.6, p = 0.04).\n Publication bias The funnel plot for publication bias (standard error by total LNN comparison) demonstrated marked evidence of symmetry (Fig. 5), indicating no publication bias. The combined effect size yielded a Z value of 5.64, with a corresponding p < 0.00001. This result indicates that the fail-safe N value was relevant.Fig. 5Funnel plot of the mean difference in total LNN in the VATS group vs. the open group\n\nFunnel plot of the mean difference in total LNN in the VATS group vs. the open group\nThe funnel plot for publication bias (standard error by total LNN comparison) demonstrated marked evidence of symmetry (Fig. 5), indicating no publication bias. The combined effect size yielded a Z value of 5.64, with a corresponding p < 0.00001. This result indicates that the fail-safe N value was relevant.Fig. 5Funnel plot of the mean difference in total LNN in the VATS group vs. the open group\n\nFunnel plot of the mean difference in total LNN in the VATS group vs. the open group","We initially identified 2341 publications from the database and reference list searches and reviewed 29 articles for final analysis (Fig. 1). The articles involved a total of 6247 patients, of whom 2763 underwent VATS and 3484 underwent thoracotomy. Of these 29 publications, three studies were RCTs and 26 were retrospective studies. According to the NOS and Jadad scales assessment scores, 23 articles were of good quality and the remaining six were medium quality. The baseline characteristics of these articles are listed in Table 1.Fig. 1Flow diagram of screened and included papers\nTable 1Summary of the 29 trials included in the present meta-analysisStudyInstitutionEnrolled yearNo. of patientsClinical stageOutcomesDesignQualityVATSOpenVATSOpen1995Kirby [28] USASingle1991.10–1993.121991.10–1993.122530I①RCT31998Morikawa [29] JPNSingle1996.04–1996.121995.01–1996.033941I–II③Retrospective92000Luketich [30] USASingleNot mentionedNot mentioned3131I①Retrospective72000Sugi [31] JPNSingle1993.01–1994.061993.01–1994.064852Ia③, ⑤RCT32001Nomori [32] JPNSingle1999.08–2000.121998.04–1999.073333I①,⑦,⑧Retrospective72005Watanabe [33] JPNSingle1997–20041997–2004221190I①, ③, ⑦, ⑧Retrospective82006Petersen [34] USASingle2001–20051996–20051285I–IV②Retrospective82006Shigemura [35] JPNMulti1999.01–2004.011999.01–2004.015055Ia①Retrospective92008Shiraishi [36] JPNSingle1994.11–2005.101994.11–2005.102055I③, ⑤Retrospective82008Watanabe [37] JPNSingle1997–20061997–20063732I①, ②, ③, ④Retrospective82008Whitson [38] USASingleNot mentionedNot mentioned67Not mentioned①Retrospective62009Nakanishi [39] JPNSingle2000.04–2007.012000.04–2007.011314I–IV③Retrospective82009Okur [40] TRSingle2007.01–2007.112007.01–2007.112028I②Retrospective82010Denlinger [21] USASingle2000.01–2008.082000.01–2008.0879464I①, ③, ⑤Retrospective82011D’Amico [41] USASingle2007.01–2010.092007.01–2010.09199189I–III②,④Retrospective72012Bu [42] CHNSingle2001.05–2011.042001.05–2011.044687Not mentioned①, ②Retrospective72012Li [43] CHNSingle2006.09–2009.122006.09–2009.122947I③, ④Retrospective82012Licht [44] DNKMulti2007.01–2011.122007.01–2011.12717796I②Retrospective82013Fan [45] CHNSingle2005.01–2010.122005.01–2010.127977I–II①, ②Retrospective82013Lee [22] USASingle1990.05–2011.121990.05–2011.12141115Not mentioned①, ②, ③, ④, ⑤, ⑥Retrospective82013Palade [46] GERSingle2008.05–2011.122008.05–2011.123232I①, ⑦, ⑧RCT32013Zhong [47] CHNSingle2006.03–2011.082006.03–2011.086790I①, ②, ③, ④Retrospective82014Li [48] CHNSingle2011.02–2013.022011.02–2013.022132I–II①Retrospective82014Stephens [4] USASingle2002.01–2011.122002.01–2011.12307307I②Retrospective82015Cai [49] CHNSingle2010.01–2012.052010.01–2012.057167I–II①, ②Retrospective92015Kuritzky [50] USASingle2007–20122007–201274224I②Retrospective82015Murakawa [51] JPNSingle2001–20102001–2010101101I①, ②Retrospective82015Nwogu [6] USAMulti2004.10–2010.062004.10–2010.06175175I–II①, ②Retrospective72015Zhang [52] CHNSingle2012.10–2013.112012.10–2013.117028I①Retrospective9① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\n\nFlow diagram of screened and included papers\nSummary of the 29 trials included in the present meta-analysis\n① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\n\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial","We identified 19 articles for total LNN comparison. They involved 1297 patients in the VATS group and 1731 patients in the open group (thoracotomy). The heterogeneity between these studies was acceptable (p = 0.02, I\n2 = 44%). Fewer total LNs were dissected in the VATS group as compared with the open group (95% CI −1.52 to −0.73, p < 0.00001, Fig. 2a).Fig. 2Forest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\n\nForest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\nFourteen articles were identified for total LNS comparison. They involved 2046 patients in the VATS group and 2373 patients in the open group. The mean difference in total LNS between the two groups was not significant (95% CI −0.28 to 0.06, p = 0.20), with significant heterogeneity across studies (p < 0.00001, I\n2 = 73%, Fig. 2b).","Eleven articles were identified for N2 LNN comparison. They involved 726 patients in the VATS group and 1132 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.08, I\n2 = 41%). Fewer N2 LNs were dissected in the VATS group as compared with the open group (95% CI −1.38 to −0.49, p < 0.0001, Fig. 3a).Fig. 3Forest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\n\nForest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\nFive articles were identified for N2 LNS comparison. They involved 473 patients in the VATS group and 473 patients in the open group. The mean difference in N2 LNS between the two groups was not significant (95% CI −0.46 to 0.23, p = 0.50), with significant heterogeneity across studies (p = 0.002, I\n2 = 76%, Fig. 3b).","Four articles were identified for N1 LNN comparison. They involved 288 patients in the VATS group and 686 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.44, I\n2 = 60%). The mean difference in N1 LNN between the two groups was not significant (95% CI −0.71 to 0.08, p = 0.11, Fig. 4).Fig. 4Forest plot of the mean difference in N1 LNN in the VATS group vs. the open group\n\nForest plot of the mean difference in N1 LNN in the VATS group vs. the open group\nOnly one article was identified for N1 LNS comparison. They involved 141 patients in the VATS group and 115 patients in the open group. The result showed that fewer N1 LN stations were dissected in the VATS group (1.4 ± 0.5 vs. 1.6 ± 0.6, p = 0.04).","The funnel plot for publication bias (standard error by total LNN comparison) demonstrated marked evidence of symmetry (Fig. 5), indicating no publication bias. The combined effect size yielded a Z value of 5.64, with a corresponding p < 0.00001. This result indicates that the fail-safe N value was relevant.Fig. 5Funnel plot of the mean difference in total LNN in the VATS group vs. the open group\n\nFunnel plot of the mean difference in total LNN in the VATS group vs. the open group","The presence or absence of mediastinal LN metastases is a critical component to accurate staging and therefore a key component of the surgical management of NSCLC [10]. Both overall and disease-free survival have been associated with the number of LNs dissected [11, 12]. Lardinois compared LNs dissection versus sampling; the results showed a longer disease-free survival and better local tumor control in dissection groups [13]. Focusing on stage IA lung cancer, Xu reported a similar result and suggested that the number of N2 stations served as a more significant prognostic factor [14]. As a result, current guidelines from the National Comprehensive Cancer Network and European Society of Thoracic Surgeons recommend that all patients with resectable NSCLC should have a complete systematic nodal dissection, with at least three N2 stations dissected [15, 16].\nAlthough VATS is associated with many benefits in comparison to thoracotomy, whether VATS can achieve the same LN dissection efficacy in patients with lung cancer remains controversial [7, 17, 18]. Flores reported that the surgical field in VATS facilitated the dissection of LNs adjacent to the blood vessels and the trachea and found that smaller nodes achieved better LN dissection [19]. Ramos showed that VATS dissected more total LNS (5.1 ± 1.1 vs. 4.5 ± 1.2, p < 0.001) and mediastinal LNS (3.4 ± 0.9 vs. 3.2 ± 0.9, p = 0.022) when compared with thoracotomy [20]. By contrast, Denlinger reported that significantly more overall LNs were dissected in the open group than in the VATS group (8.9 ± 5.2 vs. 7.1 ± 5.2, p = 0.0006) [21]. Similar results were reported by Lee and colleagues in their retrospective study of LN evaluation achieved by VATS lobectomy compared with that by open lobectomy [22]. A secondary analysis including 752 original participants of the American College of Surgeons Oncology Group Z0030 trial compared patients who underwent lobectomy by VATS with patients who underwent thoracotomy. The results showed that there was no significant difference in the overall number of LNs retrieved between the two groups (15 vs. 19, p = 0.147) [23].\nThe present study included a total of 7568 patients from nine countries, providing the most comprehensive evidence for LN dissection efficacy by VATS to date. The meta-analysis showed that fewer total LNN and similar total LNS were dissected in the VATS group as compared with that in the open group. If we only included studies focusing on clinical stage I lung cancer, the results were similar (total LNN, 95% CI −1.67 to −0.61, p < 0.0001; total LNS, 95% CI −0.62 to 0.02, p = 0.07). It suggests that surgeons may not have the ability to perform systematic lymphadenectomy in VATS or ignore the importance of systematic lymphadenectomy for various reasons (earlier tumor stage, worrying about damage to vital organs, and so on).\nIn the comparison of N1 and N2 LN dissection, our results showed that similar number of N1 LNN and N2 LNS could be harvested by VATS, while fewer N2 LNN were harvested by VATS as compared with thoracotomy. Only one article reported on N1 LNS comparison between the two groups and showed better efficiency in the open group (1.4 ± 0.5 vs. 1.6 ± 0.6, p = 0.04) [22].\nIt was controversial that removing more N2 LNs could increase the accuracy of clinical staging of NSCLC. Boffa et al. compared the completeness of surgical LN evaluation during anatomic resection of primary lung cancer by open and VATS approaches in 11,531 patients from The Society of Thoracic Surgeons-General Thoracic Database. The results showed nodal upstaging in 14.3% (1024 patients) of the open group and in 11.6% (508 patients) of the VATS group (p < 0.001). The study suggested that surgeons should be encouraged to apply a systematic approach to hilar and peribronchial LN dissection during VATS lobectomy for lung cancer, particularly as they were adopting this approach [24].\nSome surgeons might also worry about the complications caused by the systematic LN dissection. As with open thoracotomy, systematic mediastinal LN dissection under VATS may increase the risk of intraoperative bleeding (bronchial arteries, etc.), tracheobronchial injury, recurrent nerve injury, prolonged air leak, atrial fibrillation, and pulmonary edema [25]. In other papers, Watanabe et al. reported similar mortality and morbidity of mediastinal LN dissection by VATS vs. open lobectomy, indicating that systematic mediastinal LN dissection by VATS is a safe procedure [26]. Zhang et al. compared complications such as chylothorax and nerve injury between VATS and open thoracotomy in a meta-analysis. The results showed that these events were similar in both groups [27].\nThe possible limitations of our study must be considered when interpreting the findings described herein. First, including only English papers might have resulted in language bias. Second, including 7568 participants from 36 studies with only three RCTs might have weakened the quality of the results. Third, the number of dissected LNs varied significantly between the included studies. Different doctors have different understanding of LN dissection and might be at different stages of the learning curve. Some data did not meet the National Comprehensive Cancer Network guide requirements for lung cancer surgery treatment of systematic LN dissection, which may have affected the reliability of the results. Fourth, there is great potential for LN fragmentation during dissection. Different pathologists and different counting procedures might lead to false LNN counts, which might increase the heterogeneity between studies but would not alter the overall results. Finally, we did not analyze the survival difference between VATS and open thoracotomy. Our analysis compared LN harvest capability between two evaluation procedures only from a surgical point of view and tried to give further proof of satisfied oncologic efficacy by VATS.","Less total and mediastinal LNs were evaluated with VATS than with thoracotomy in the present study. Both approaches harvested a similar number of total LN stations, mediastinal LN stations, and N1 LNs. However, owing to the possible existing bias in the original studies, inter-study heterogeneity, and the inherent limitations of our meta-analysis, the findings require validation in high-quality, large-scale RCTs."],"string":"[\n \"Lung cancer is the leading cause of cancer deaths in many countries [1, 2]. Surgical treatment is the preferred treatment for early-stage non-small cell lung cancer (NSCLC). D’Cunha’s research showed that N1 and N2 lymph nodes (LNs) were positive in 27.5% of patients with lung cancer under lobectomy [3]. However, non-invasive examinations, such as computed tomography (CT) and positron emission tomography-computed tomography (PET-CT), are not sensitive and specific for the clinical staging of lung cancer. Video-assisted thoracic surgery (VATS) is the preferred surgical procedure, with fewer incidences of postoperative complications and a higher survival rate compared with thoracotomy [4–7]. However, whether VATS can achieve the same LN dissection efficacy is controversial, and there remains a lack of high-quality, large-scale clinical research.\\nTo determine whether VATS can achieve the same LN dissection efficacy as thoracotomy in lung cancer, we performed a systemic review and meta-analysis.\",\n \" Search strategy MEDLINE and manual searches were performed by two investigators independently and in duplicate to identify all relevant scientific articles published from January 1990 to May 2016. The MEDLINE search was performed using PubMed, Ovid MEDLINE, EMBASE, Web of Science, ScienceDirect, The Cochrane Library, Scopus, and Google Scholar. The MeSH terms “lung cancer or lung neoplasm”, “thoracotomy or open surgery”, and “video-assisted thoracic surgery or VATS” and comparative study were used.\\nMEDLINE and manual searches were performed by two investigators independently and in duplicate to identify all relevant scientific articles published from January 1990 to May 2016. The MEDLINE search was performed using PubMed, Ovid MEDLINE, EMBASE, Web of Science, ScienceDirect, The Cochrane Library, Scopus, and Google Scholar. The MeSH terms “lung cancer or lung neoplasm”, “thoracotomy or open surgery”, and “video-assisted thoracic surgery or VATS” and comparative study were used.\\n Inclusion criteria and exclusion criteria The following inclusion criteria were applied: (1) published in English, (2) compared the LN dissection of thoracoscopic lobectomy with thoracotomy in treating patients with lung cancer, and (3) the most recent study was chosen when duplication of data is in more than one article.\\nReviews without original data, case reports, meta-analyses, letters, expert opinions, and animal studies were excluded. Studies on robotic-assisted VATS were also excluded.\\nThe following inclusion criteria were applied: (1) published in English, (2) compared the LN dissection of thoracoscopic lobectomy with thoracotomy in treating patients with lung cancer, and (3) the most recent study was chosen when duplication of data is in more than one article.\\nReviews without original data, case reports, meta-analyses, letters, expert opinions, and animal studies were excluded. Studies on robotic-assisted VATS were also excluded.\\n Data extraction Two investigators independently extracted data from the eligible studies. The extracted data included first author, year of publication, geographical area, study design, duration of enrollment, information on preoperative staging, number of patients per group, LN number (LNN), and LN station number (LNS).\\nTwo investigators independently extracted data from the eligible studies. The extracted data included first author, year of publication, geographical area, study design, duration of enrollment, information on preoperative staging, number of patients per group, LN number (LNN), and LN station number (LNS).\\n Quality assessment for included studies Two investigators independently assessed the quality of each included study using the Newcastle-Ottawa Scale (NOS) for non-randomized studies and the Jadad scale for randomized controlled trials (RCTs).\\nThe NOS evaluates the quality of studies by analyzing three items: selection, comparability, and exposure. The scale assigns a maximum of nine points to each study: a maximum of four points for selection, two points for comparability, and three points for exposure. Therefore, the highest quality study would score nine points. In our analysis, high-quality studies were defined as those that scored nine or eight points; medium-quality studies were those that scored seven or six points [8].\\nThe Jadad scale (five points) contained questions for three main parts: randomization, masking, and accountability of all patients (withdrawals and dropouts). Studies scored ≥3 points were considered as high quality [9].\\nTwo investigators independently assessed the quality of each included study using the Newcastle-Ottawa Scale (NOS) for non-randomized studies and the Jadad scale for randomized controlled trials (RCTs).\\nThe NOS evaluates the quality of studies by analyzing three items: selection, comparability, and exposure. The scale assigns a maximum of nine points to each study: a maximum of four points for selection, two points for comparability, and three points for exposure. Therefore, the highest quality study would score nine points. In our analysis, high-quality studies were defined as those that scored nine or eight points; medium-quality studies were those that scored seven or six points [8].\\nThe Jadad scale (five points) contained questions for three main parts: randomization, masking, and accountability of all patients (withdrawals and dropouts). Studies scored ≥3 points were considered as high quality [9].\\n Statistical analysis Meta-analysis was conducted by Review Manager 5.3 and SPSS 18.0, p value < 0.05 suggested statistically significant. The differences were compared between the two groups using analysis of variance for continuous variables and pooled relative risk (RR) with 95% confidence interval (CI) for categorical variables. We used I\\n2 and Cochran Q to evaluate the between-study heterogeneity. A random-effects model was adopted when the heterogeneity was significant (p ≤ 0.10 and I\\n2 > 50%); otherwise, a fixed-effects model was used. Rank correlation test of funnel plot asymmetry was used to assess the potential publication bias.\\nMeta-analysis was conducted by Review Manager 5.3 and SPSS 18.0, p value < 0.05 suggested statistically significant. The differences were compared between the two groups using analysis of variance for continuous variables and pooled relative risk (RR) with 95% confidence interval (CI) for categorical variables. We used I\\n2 and Cochran Q to evaluate the between-study heterogeneity. A random-effects model was adopted when the heterogeneity was significant (p ≤ 0.10 and I\\n2 > 50%); otherwise, a fixed-effects model was used. Rank correlation test of funnel plot asymmetry was used to assess the potential publication bias.\",\n \"MEDLINE and manual searches were performed by two investigators independently and in duplicate to identify all relevant scientific articles published from January 1990 to May 2016. The MEDLINE search was performed using PubMed, Ovid MEDLINE, EMBASE, Web of Science, ScienceDirect, The Cochrane Library, Scopus, and Google Scholar. The MeSH terms “lung cancer or lung neoplasm”, “thoracotomy or open surgery”, and “video-assisted thoracic surgery or VATS” and comparative study were used.\",\n \"The following inclusion criteria were applied: (1) published in English, (2) compared the LN dissection of thoracoscopic lobectomy with thoracotomy in treating patients with lung cancer, and (3) the most recent study was chosen when duplication of data is in more than one article.\\nReviews without original data, case reports, meta-analyses, letters, expert opinions, and animal studies were excluded. Studies on robotic-assisted VATS were also excluded.\",\n \"Two investigators independently extracted data from the eligible studies. The extracted data included first author, year of publication, geographical area, study design, duration of enrollment, information on preoperative staging, number of patients per group, LN number (LNN), and LN station number (LNS).\",\n \"Two investigators independently assessed the quality of each included study using the Newcastle-Ottawa Scale (NOS) for non-randomized studies and the Jadad scale for randomized controlled trials (RCTs).\\nThe NOS evaluates the quality of studies by analyzing three items: selection, comparability, and exposure. The scale assigns a maximum of nine points to each study: a maximum of four points for selection, two points for comparability, and three points for exposure. Therefore, the highest quality study would score nine points. In our analysis, high-quality studies were defined as those that scored nine or eight points; medium-quality studies were those that scored seven or six points [8].\\nThe Jadad scale (five points) contained questions for three main parts: randomization, masking, and accountability of all patients (withdrawals and dropouts). Studies scored ≥3 points were considered as high quality [9].\",\n \"Meta-analysis was conducted by Review Manager 5.3 and SPSS 18.0, p value < 0.05 suggested statistically significant. The differences were compared between the two groups using analysis of variance for continuous variables and pooled relative risk (RR) with 95% confidence interval (CI) for categorical variables. We used I\\n2 and Cochran Q to evaluate the between-study heterogeneity. A random-effects model was adopted when the heterogeneity was significant (p ≤ 0.10 and I\\n2 > 50%); otherwise, a fixed-effects model was used. Rank correlation test of funnel plot asymmetry was used to assess the potential publication bias.\",\n \" Search results and quality assessment of the included studies We initially identified 2341 publications from the database and reference list searches and reviewed 29 articles for final analysis (Fig. 1). The articles involved a total of 6247 patients, of whom 2763 underwent VATS and 3484 underwent thoracotomy. Of these 29 publications, three studies were RCTs and 26 were retrospective studies. According to the NOS and Jadad scales assessment scores, 23 articles were of good quality and the remaining six were medium quality. The baseline characteristics of these articles are listed in Table 1.Fig. 1Flow diagram of screened and included papers\\nTable 1Summary of the 29 trials included in the present meta-analysisStudyInstitutionEnrolled yearNo. of patientsClinical stageOutcomesDesignQualityVATSOpenVATSOpen1995Kirby [28] USASingle1991.10–1993.121991.10–1993.122530I①RCT31998Morikawa [29] JPNSingle1996.04–1996.121995.01–1996.033941I–II③Retrospective92000Luketich [30] USASingleNot mentionedNot mentioned3131I①Retrospective72000Sugi [31] JPNSingle1993.01–1994.061993.01–1994.064852Ia③, ⑤RCT32001Nomori [32] JPNSingle1999.08–2000.121998.04–1999.073333I①,⑦,⑧Retrospective72005Watanabe [33] JPNSingle1997–20041997–2004221190I①, ③, ⑦, ⑧Retrospective82006Petersen [34] USASingle2001–20051996–20051285I–IV②Retrospective82006Shigemura [35] JPNMulti1999.01–2004.011999.01–2004.015055Ia①Retrospective92008Shiraishi [36] JPNSingle1994.11–2005.101994.11–2005.102055I③, ⑤Retrospective82008Watanabe [37] JPNSingle1997–20061997–20063732I①, ②, ③, ④Retrospective82008Whitson [38] USASingleNot mentionedNot mentioned67Not mentioned①Retrospective62009Nakanishi [39] JPNSingle2000.04–2007.012000.04–2007.011314I–IV③Retrospective82009Okur [40] TRSingle2007.01–2007.112007.01–2007.112028I②Retrospective82010Denlinger [21] USASingle2000.01–2008.082000.01–2008.0879464I①, ③, ⑤Retrospective82011D’Amico [41] USASingle2007.01–2010.092007.01–2010.09199189I–III②,④Retrospective72012Bu [42] CHNSingle2001.05–2011.042001.05–2011.044687Not mentioned①, ②Retrospective72012Li [43] CHNSingle2006.09–2009.122006.09–2009.122947I③, ④Retrospective82012Licht [44] DNKMulti2007.01–2011.122007.01–2011.12717796I②Retrospective82013Fan [45] CHNSingle2005.01–2010.122005.01–2010.127977I–II①, ②Retrospective82013Lee [22] USASingle1990.05–2011.121990.05–2011.12141115Not mentioned①, ②, ③, ④, ⑤, ⑥Retrospective82013Palade [46] GERSingle2008.05–2011.122008.05–2011.123232I①, ⑦, ⑧RCT32013Zhong [47] CHNSingle2006.03–2011.082006.03–2011.086790I①, ②, ③, ④Retrospective82014Li [48] CHNSingle2011.02–2013.022011.02–2013.022132I–II①Retrospective82014Stephens [4] USASingle2002.01–2011.122002.01–2011.12307307I②Retrospective82015Cai [49] CHNSingle2010.01–2012.052010.01–2012.057167I–II①, ②Retrospective92015Kuritzky [50] USASingle2007–20122007–201274224I②Retrospective82015Murakawa [51] JPNSingle2001–20102001–2010101101I①, ②Retrospective82015Nwogu [6] USAMulti2004.10–2010.062004.10–2010.06175175I–II①, ②Retrospective72015Zhang [52] CHNSingle2012.10–2013.112012.10–2013.117028I①Retrospective9① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\\n\\nFlow diagram of screened and included papers\\nSummary of the 29 trials included in the present meta-analysis\\n① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\\n\\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\\nWe initially identified 2341 publications from the database and reference list searches and reviewed 29 articles for final analysis (Fig. 1). The articles involved a total of 6247 patients, of whom 2763 underwent VATS and 3484 underwent thoracotomy. Of these 29 publications, three studies were RCTs and 26 were retrospective studies. According to the NOS and Jadad scales assessment scores, 23 articles were of good quality and the remaining six were medium quality. The baseline characteristics of these articles are listed in Table 1.Fig. 1Flow diagram of screened and included papers\\nTable 1Summary of the 29 trials included in the present meta-analysisStudyInstitutionEnrolled yearNo. of patientsClinical stageOutcomesDesignQualityVATSOpenVATSOpen1995Kirby [28] USASingle1991.10–1993.121991.10–1993.122530I①RCT31998Morikawa [29] JPNSingle1996.04–1996.121995.01–1996.033941I–II③Retrospective92000Luketich [30] USASingleNot mentionedNot mentioned3131I①Retrospective72000Sugi [31] JPNSingle1993.01–1994.061993.01–1994.064852Ia③, ⑤RCT32001Nomori [32] JPNSingle1999.08–2000.121998.04–1999.073333I①,⑦,⑧Retrospective72005Watanabe [33] JPNSingle1997–20041997–2004221190I①, ③, ⑦, ⑧Retrospective82006Petersen [34] USASingle2001–20051996–20051285I–IV②Retrospective82006Shigemura [35] JPNMulti1999.01–2004.011999.01–2004.015055Ia①Retrospective92008Shiraishi [36] JPNSingle1994.11–2005.101994.11–2005.102055I③, ⑤Retrospective82008Watanabe [37] JPNSingle1997–20061997–20063732I①, ②, ③, ④Retrospective82008Whitson [38] USASingleNot mentionedNot mentioned67Not mentioned①Retrospective62009Nakanishi [39] JPNSingle2000.04–2007.012000.04–2007.011314I–IV③Retrospective82009Okur [40] TRSingle2007.01–2007.112007.01–2007.112028I②Retrospective82010Denlinger [21] USASingle2000.01–2008.082000.01–2008.0879464I①, ③, ⑤Retrospective82011D’Amico [41] USASingle2007.01–2010.092007.01–2010.09199189I–III②,④Retrospective72012Bu [42] CHNSingle2001.05–2011.042001.05–2011.044687Not mentioned①, ②Retrospective72012Li [43] CHNSingle2006.09–2009.122006.09–2009.122947I③, ④Retrospective82012Licht [44] DNKMulti2007.01–2011.122007.01–2011.12717796I②Retrospective82013Fan [45] CHNSingle2005.01–2010.122005.01–2010.127977I–II①, ②Retrospective82013Lee [22] USASingle1990.05–2011.121990.05–2011.12141115Not mentioned①, ②, ③, ④, ⑤, ⑥Retrospective82013Palade [46] GERSingle2008.05–2011.122008.05–2011.123232I①, ⑦, ⑧RCT32013Zhong [47] CHNSingle2006.03–2011.082006.03–2011.086790I①, ②, ③, ④Retrospective82014Li [48] CHNSingle2011.02–2013.022011.02–2013.022132I–II①Retrospective82014Stephens [4] USASingle2002.01–2011.122002.01–2011.12307307I②Retrospective82015Cai [49] CHNSingle2010.01–2012.052010.01–2012.057167I–II①, ②Retrospective92015Kuritzky [50] USASingle2007–20122007–201274224I②Retrospective82015Murakawa [51] JPNSingle2001–20102001–2010101101I①, ②Retrospective82015Nwogu [6] USAMulti2004.10–2010.062004.10–2010.06175175I–II①, ②Retrospective72015Zhang [52] CHNSingle2012.10–2013.112012.10–2013.117028I①Retrospective9① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\\n\\nFlow diagram of screened and included papers\\nSummary of the 29 trials included in the present meta-analysis\\n① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\\n\\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\\n Comparison of total LNN and LNS We identified 19 articles for total LNN comparison. They involved 1297 patients in the VATS group and 1731 patients in the open group (thoracotomy). The heterogeneity between these studies was acceptable (p = 0.02, I\\n2 = 44%). Fewer total LNs were dissected in the VATS group as compared with the open group (95% CI −1.52 to −0.73, p < 0.00001, Fig. 2a).Fig. 2Forest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\\n\\nForest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\\nFourteen articles were identified for total LNS comparison. They involved 2046 patients in the VATS group and 2373 patients in the open group. The mean difference in total LNS between the two groups was not significant (95% CI −0.28 to 0.06, p = 0.20), with significant heterogeneity across studies (p < 0.00001, I\\n2 = 73%, Fig. 2b).\\nWe identified 19 articles for total LNN comparison. They involved 1297 patients in the VATS group and 1731 patients in the open group (thoracotomy). The heterogeneity between these studies was acceptable (p = 0.02, I\\n2 = 44%). Fewer total LNs were dissected in the VATS group as compared with the open group (95% CI −1.52 to −0.73, p < 0.00001, Fig. 2a).Fig. 2Forest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\\n\\nForest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\\nFourteen articles were identified for total LNS comparison. They involved 2046 patients in the VATS group and 2373 patients in the open group. The mean difference in total LNS between the two groups was not significant (95% CI −0.28 to 0.06, p = 0.20), with significant heterogeneity across studies (p < 0.00001, I\\n2 = 73%, Fig. 2b).\\n Comparison of N2 LNN and LNS Eleven articles were identified for N2 LNN comparison. They involved 726 patients in the VATS group and 1132 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.08, I\\n2 = 41%). Fewer N2 LNs were dissected in the VATS group as compared with the open group (95% CI −1.38 to −0.49, p < 0.0001, Fig. 3a).Fig. 3Forest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\\n\\nForest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\\nFive articles were identified for N2 LNS comparison. They involved 473 patients in the VATS group and 473 patients in the open group. The mean difference in N2 LNS between the two groups was not significant (95% CI −0.46 to 0.23, p = 0.50), with significant heterogeneity across studies (p = 0.002, I\\n2 = 76%, Fig. 3b).\\nEleven articles were identified for N2 LNN comparison. They involved 726 patients in the VATS group and 1132 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.08, I\\n2 = 41%). Fewer N2 LNs were dissected in the VATS group as compared with the open group (95% CI −1.38 to −0.49, p < 0.0001, Fig. 3a).Fig. 3Forest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\\n\\nForest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\\nFive articles were identified for N2 LNS comparison. They involved 473 patients in the VATS group and 473 patients in the open group. The mean difference in N2 LNS between the two groups was not significant (95% CI −0.46 to 0.23, p = 0.50), with significant heterogeneity across studies (p = 0.002, I\\n2 = 76%, Fig. 3b).\\n Comparison of N1 LNN and LNS Four articles were identified for N1 LNN comparison. They involved 288 patients in the VATS group and 686 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.44, I\\n2 = 60%). The mean difference in N1 LNN between the two groups was not significant (95% CI −0.71 to 0.08, p = 0.11, Fig. 4).Fig. 4Forest plot of the mean difference in N1 LNN in the VATS group vs. the open group\\n\\nForest plot of the mean difference in N1 LNN in the VATS group vs. the open group\\nOnly one article was identified for N1 LNS comparison. They involved 141 patients in the VATS group and 115 patients in the open group. The result showed that fewer N1 LN stations were dissected in the VATS group (1.4 ± 0.5 vs. 1.6 ± 0.6, p = 0.04).\\nFour articles were identified for N1 LNN comparison. They involved 288 patients in the VATS group and 686 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.44, I\\n2 = 60%). The mean difference in N1 LNN between the two groups was not significant (95% CI −0.71 to 0.08, p = 0.11, Fig. 4).Fig. 4Forest plot of the mean difference in N1 LNN in the VATS group vs. the open group\\n\\nForest plot of the mean difference in N1 LNN in the VATS group vs. the open group\\nOnly one article was identified for N1 LNS comparison. They involved 141 patients in the VATS group and 115 patients in the open group. The result showed that fewer N1 LN stations were dissected in the VATS group (1.4 ± 0.5 vs. 1.6 ± 0.6, p = 0.04).\\n Publication bias The funnel plot for publication bias (standard error by total LNN comparison) demonstrated marked evidence of symmetry (Fig. 5), indicating no publication bias. The combined effect size yielded a Z value of 5.64, with a corresponding p < 0.00001. This result indicates that the fail-safe N value was relevant.Fig. 5Funnel plot of the mean difference in total LNN in the VATS group vs. the open group\\n\\nFunnel plot of the mean difference in total LNN in the VATS group vs. the open group\\nThe funnel plot for publication bias (standard error by total LNN comparison) demonstrated marked evidence of symmetry (Fig. 5), indicating no publication bias. The combined effect size yielded a Z value of 5.64, with a corresponding p < 0.00001. This result indicates that the fail-safe N value was relevant.Fig. 5Funnel plot of the mean difference in total LNN in the VATS group vs. the open group\\n\\nFunnel plot of the mean difference in total LNN in the VATS group vs. the open group\",\n \"We initially identified 2341 publications from the database and reference list searches and reviewed 29 articles for final analysis (Fig. 1). The articles involved a total of 6247 patients, of whom 2763 underwent VATS and 3484 underwent thoracotomy. Of these 29 publications, three studies were RCTs and 26 were retrospective studies. According to the NOS and Jadad scales assessment scores, 23 articles were of good quality and the remaining six were medium quality. The baseline characteristics of these articles are listed in Table 1.Fig. 1Flow diagram of screened and included papers\\nTable 1Summary of the 29 trials included in the present meta-analysisStudyInstitutionEnrolled yearNo. of patientsClinical stageOutcomesDesignQualityVATSOpenVATSOpen1995Kirby [28] USASingle1991.10–1993.121991.10–1993.122530I①RCT31998Morikawa [29] JPNSingle1996.04–1996.121995.01–1996.033941I–II③Retrospective92000Luketich [30] USASingleNot mentionedNot mentioned3131I①Retrospective72000Sugi [31] JPNSingle1993.01–1994.061993.01–1994.064852Ia③, ⑤RCT32001Nomori [32] JPNSingle1999.08–2000.121998.04–1999.073333I①,⑦,⑧Retrospective72005Watanabe [33] JPNSingle1997–20041997–2004221190I①, ③, ⑦, ⑧Retrospective82006Petersen [34] USASingle2001–20051996–20051285I–IV②Retrospective82006Shigemura [35] JPNMulti1999.01–2004.011999.01–2004.015055Ia①Retrospective92008Shiraishi [36] JPNSingle1994.11–2005.101994.11–2005.102055I③, ⑤Retrospective82008Watanabe [37] JPNSingle1997–20061997–20063732I①, ②, ③, ④Retrospective82008Whitson [38] USASingleNot mentionedNot mentioned67Not mentioned①Retrospective62009Nakanishi [39] JPNSingle2000.04–2007.012000.04–2007.011314I–IV③Retrospective82009Okur [40] TRSingle2007.01–2007.112007.01–2007.112028I②Retrospective82010Denlinger [21] USASingle2000.01–2008.082000.01–2008.0879464I①, ③, ⑤Retrospective82011D’Amico [41] USASingle2007.01–2010.092007.01–2010.09199189I–III②,④Retrospective72012Bu [42] CHNSingle2001.05–2011.042001.05–2011.044687Not mentioned①, ②Retrospective72012Li [43] CHNSingle2006.09–2009.122006.09–2009.122947I③, ④Retrospective82012Licht [44] DNKMulti2007.01–2011.122007.01–2011.12717796I②Retrospective82013Fan [45] CHNSingle2005.01–2010.122005.01–2010.127977I–II①, ②Retrospective82013Lee [22] USASingle1990.05–2011.121990.05–2011.12141115Not mentioned①, ②, ③, ④, ⑤, ⑥Retrospective82013Palade [46] GERSingle2008.05–2011.122008.05–2011.123232I①, ⑦, ⑧RCT32013Zhong [47] CHNSingle2006.03–2011.082006.03–2011.086790I①, ②, ③, ④Retrospective82014Li [48] CHNSingle2011.02–2013.022011.02–2013.022132I–II①Retrospective82014Stephens [4] USASingle2002.01–2011.122002.01–2011.12307307I②Retrospective82015Cai [49] CHNSingle2010.01–2012.052010.01–2012.057167I–II①, ②Retrospective92015Kuritzky [50] USASingle2007–20122007–201274224I②Retrospective82015Murakawa [51] JPNSingle2001–20102001–2010101101I①, ②Retrospective82015Nwogu [6] USAMulti2004.10–2010.062004.10–2010.06175175I–II①, ②Retrospective72015Zhang [52] CHNSingle2012.10–2013.112012.10–2013.117028I①Retrospective9① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\\n\\nFlow diagram of screened and included papers\\nSummary of the 29 trials included in the present meta-analysis\\n① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\\n\\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\",\n \"We identified 19 articles for total LNN comparison. They involved 1297 patients in the VATS group and 1731 patients in the open group (thoracotomy). The heterogeneity between these studies was acceptable (p = 0.02, I\\n2 = 44%). Fewer total LNs were dissected in the VATS group as compared with the open group (95% CI −1.52 to −0.73, p < 0.00001, Fig. 2a).Fig. 2Forest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\\n\\nForest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\\nFourteen articles were identified for total LNS comparison. They involved 2046 patients in the VATS group and 2373 patients in the open group. The mean difference in total LNS between the two groups was not significant (95% CI −0.28 to 0.06, p = 0.20), with significant heterogeneity across studies (p < 0.00001, I\\n2 = 73%, Fig. 2b).\",\n \"Eleven articles were identified for N2 LNN comparison. They involved 726 patients in the VATS group and 1132 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.08, I\\n2 = 41%). Fewer N2 LNs were dissected in the VATS group as compared with the open group (95% CI −1.38 to −0.49, p < 0.0001, Fig. 3a).Fig. 3Forest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\\n\\nForest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\\nFive articles were identified for N2 LNS comparison. They involved 473 patients in the VATS group and 473 patients in the open group. The mean difference in N2 LNS between the two groups was not significant (95% CI −0.46 to 0.23, p = 0.50), with significant heterogeneity across studies (p = 0.002, I\\n2 = 76%, Fig. 3b).\",\n \"Four articles were identified for N1 LNN comparison. They involved 288 patients in the VATS group and 686 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.44, I\\n2 = 60%). The mean difference in N1 LNN between the two groups was not significant (95% CI −0.71 to 0.08, p = 0.11, Fig. 4).Fig. 4Forest plot of the mean difference in N1 LNN in the VATS group vs. the open group\\n\\nForest plot of the mean difference in N1 LNN in the VATS group vs. the open group\\nOnly one article was identified for N1 LNS comparison. They involved 141 patients in the VATS group and 115 patients in the open group. The result showed that fewer N1 LN stations were dissected in the VATS group (1.4 ± 0.5 vs. 1.6 ± 0.6, p = 0.04).\",\n \"The funnel plot for publication bias (standard error by total LNN comparison) demonstrated marked evidence of symmetry (Fig. 5), indicating no publication bias. The combined effect size yielded a Z value of 5.64, with a corresponding p < 0.00001. This result indicates that the fail-safe N value was relevant.Fig. 5Funnel plot of the mean difference in total LNN in the VATS group vs. the open group\\n\\nFunnel plot of the mean difference in total LNN in the VATS group vs. the open group\",\n \"The presence or absence of mediastinal LN metastases is a critical component to accurate staging and therefore a key component of the surgical management of NSCLC [10]. Both overall and disease-free survival have been associated with the number of LNs dissected [11, 12]. Lardinois compared LNs dissection versus sampling; the results showed a longer disease-free survival and better local tumor control in dissection groups [13]. Focusing on stage IA lung cancer, Xu reported a similar result and suggested that the number of N2 stations served as a more significant prognostic factor [14]. As a result, current guidelines from the National Comprehensive Cancer Network and European Society of Thoracic Surgeons recommend that all patients with resectable NSCLC should have a complete systematic nodal dissection, with at least three N2 stations dissected [15, 16].\\nAlthough VATS is associated with many benefits in comparison to thoracotomy, whether VATS can achieve the same LN dissection efficacy in patients with lung cancer remains controversial [7, 17, 18]. Flores reported that the surgical field in VATS facilitated the dissection of LNs adjacent to the blood vessels and the trachea and found that smaller nodes achieved better LN dissection [19]. Ramos showed that VATS dissected more total LNS (5.1 ± 1.1 vs. 4.5 ± 1.2, p < 0.001) and mediastinal LNS (3.4 ± 0.9 vs. 3.2 ± 0.9, p = 0.022) when compared with thoracotomy [20]. By contrast, Denlinger reported that significantly more overall LNs were dissected in the open group than in the VATS group (8.9 ± 5.2 vs. 7.1 ± 5.2, p = 0.0006) [21]. Similar results were reported by Lee and colleagues in their retrospective study of LN evaluation achieved by VATS lobectomy compared with that by open lobectomy [22]. A secondary analysis including 752 original participants of the American College of Surgeons Oncology Group Z0030 trial compared patients who underwent lobectomy by VATS with patients who underwent thoracotomy. The results showed that there was no significant difference in the overall number of LNs retrieved between the two groups (15 vs. 19, p = 0.147) [23].\\nThe present study included a total of 7568 patients from nine countries, providing the most comprehensive evidence for LN dissection efficacy by VATS to date. The meta-analysis showed that fewer total LNN and similar total LNS were dissected in the VATS group as compared with that in the open group. If we only included studies focusing on clinical stage I lung cancer, the results were similar (total LNN, 95% CI −1.67 to −0.61, p < 0.0001; total LNS, 95% CI −0.62 to 0.02, p = 0.07). It suggests that surgeons may not have the ability to perform systematic lymphadenectomy in VATS or ignore the importance of systematic lymphadenectomy for various reasons (earlier tumor stage, worrying about damage to vital organs, and so on).\\nIn the comparison of N1 and N2 LN dissection, our results showed that similar number of N1 LNN and N2 LNS could be harvested by VATS, while fewer N2 LNN were harvested by VATS as compared with thoracotomy. Only one article reported on N1 LNS comparison between the two groups and showed better efficiency in the open group (1.4 ± 0.5 vs. 1.6 ± 0.6, p = 0.04) [22].\\nIt was controversial that removing more N2 LNs could increase the accuracy of clinical staging of NSCLC. Boffa et al. compared the completeness of surgical LN evaluation during anatomic resection of primary lung cancer by open and VATS approaches in 11,531 patients from The Society of Thoracic Surgeons-General Thoracic Database. The results showed nodal upstaging in 14.3% (1024 patients) of the open group and in 11.6% (508 patients) of the VATS group (p < 0.001). The study suggested that surgeons should be encouraged to apply a systematic approach to hilar and peribronchial LN dissection during VATS lobectomy for lung cancer, particularly as they were adopting this approach [24].\\nSome surgeons might also worry about the complications caused by the systematic LN dissection. As with open thoracotomy, systematic mediastinal LN dissection under VATS may increase the risk of intraoperative bleeding (bronchial arteries, etc.), tracheobronchial injury, recurrent nerve injury, prolonged air leak, atrial fibrillation, and pulmonary edema [25]. In other papers, Watanabe et al. reported similar mortality and morbidity of mediastinal LN dissection by VATS vs. open lobectomy, indicating that systematic mediastinal LN dissection by VATS is a safe procedure [26]. Zhang et al. compared complications such as chylothorax and nerve injury between VATS and open thoracotomy in a meta-analysis. The results showed that these events were similar in both groups [27].\\nThe possible limitations of our study must be considered when interpreting the findings described herein. First, including only English papers might have resulted in language bias. Second, including 7568 participants from 36 studies with only three RCTs might have weakened the quality of the results. Third, the number of dissected LNs varied significantly between the included studies. Different doctors have different understanding of LN dissection and might be at different stages of the learning curve. Some data did not meet the National Comprehensive Cancer Network guide requirements for lung cancer surgery treatment of systematic LN dissection, which may have affected the reliability of the results. Fourth, there is great potential for LN fragmentation during dissection. Different pathologists and different counting procedures might lead to false LNN counts, which might increase the heterogeneity between studies but would not alter the overall results. Finally, we did not analyze the survival difference between VATS and open thoracotomy. Our analysis compared LN harvest capability between two evaluation procedures only from a surgical point of view and tried to give further proof of satisfied oncologic efficacy by VATS.\",\n \"Less total and mediastinal LNs were evaluated with VATS than with thoracotomy in the present study. Both approaches harvested a similar number of total LN stations, mediastinal LN stations, and N1 LNs. However, owing to the possible existing bias in the original studies, inter-study heterogeneity, and the inherent limitations of our meta-analysis, the findings require validation in high-quality, large-scale RCTs.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["introduction","materials|methods",null,null,null,null,null,"results",null,null,null,null,null,"discussion","conclusion"],"string":"[\n \"introduction\",\n \"materials|methods\",\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusion\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["Video-assisted thoracic surgery","Thoracotomy","Meta-analysis","Lung cancer","Lymph node dissection"],"string":"[\n \"Video-assisted thoracic surgery\",\n \"Thoracotomy\",\n \"Meta-analysis\",\n \"Lung cancer\",\n \"Lymph node dissection\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Background:\nLung cancer is the leading cause of cancer deaths in many countries [1, 2]. Surgical treatment is the preferred treatment for early-stage non-small cell lung cancer (NSCLC). D’Cunha’s research showed that N1 and N2 lymph nodes (LNs) were positive in 27.5% of patients with lung cancer under lobectomy [3]. However, non-invasive examinations, such as computed tomography (CT) and positron emission tomography-computed tomography (PET-CT), are not sensitive and specific for the clinical staging of lung cancer. Video-assisted thoracic surgery (VATS) is the preferred surgical procedure, with fewer incidences of postoperative complications and a higher survival rate compared with thoracotomy [4–7]. However, whether VATS can achieve the same LN dissection efficacy is controversial, and there remains a lack of high-quality, large-scale clinical research.\nTo determine whether VATS can achieve the same LN dissection efficacy as thoracotomy in lung cancer, we performed a systemic review and meta-analysis.\n\nMethods:\n Search strategy MEDLINE and manual searches were performed by two investigators independently and in duplicate to identify all relevant scientific articles published from January 1990 to May 2016. The MEDLINE search was performed using PubMed, Ovid MEDLINE, EMBASE, Web of Science, ScienceDirect, The Cochrane Library, Scopus, and Google Scholar. The MeSH terms “lung cancer or lung neoplasm”, “thoracotomy or open surgery”, and “video-assisted thoracic surgery or VATS” and comparative study were used.\nMEDLINE and manual searches were performed by two investigators independently and in duplicate to identify all relevant scientific articles published from January 1990 to May 2016. The MEDLINE search was performed using PubMed, Ovid MEDLINE, EMBASE, Web of Science, ScienceDirect, The Cochrane Library, Scopus, and Google Scholar. The MeSH terms “lung cancer or lung neoplasm”, “thoracotomy or open surgery”, and “video-assisted thoracic surgery or VATS” and comparative study were used.\n Inclusion criteria and exclusion criteria The following inclusion criteria were applied: (1) published in English, (2) compared the LN dissection of thoracoscopic lobectomy with thoracotomy in treating patients with lung cancer, and (3) the most recent study was chosen when duplication of data is in more than one article.\nReviews without original data, case reports, meta-analyses, letters, expert opinions, and animal studies were excluded. Studies on robotic-assisted VATS were also excluded.\nThe following inclusion criteria were applied: (1) published in English, (2) compared the LN dissection of thoracoscopic lobectomy with thoracotomy in treating patients with lung cancer, and (3) the most recent study was chosen when duplication of data is in more than one article.\nReviews without original data, case reports, meta-analyses, letters, expert opinions, and animal studies were excluded. Studies on robotic-assisted VATS were also excluded.\n Data extraction Two investigators independently extracted data from the eligible studies. The extracted data included first author, year of publication, geographical area, study design, duration of enrollment, information on preoperative staging, number of patients per group, LN number (LNN), and LN station number (LNS).\nTwo investigators independently extracted data from the eligible studies. The extracted data included first author, year of publication, geographical area, study design, duration of enrollment, information on preoperative staging, number of patients per group, LN number (LNN), and LN station number (LNS).\n Quality assessment for included studies Two investigators independently assessed the quality of each included study using the Newcastle-Ottawa Scale (NOS) for non-randomized studies and the Jadad scale for randomized controlled trials (RCTs).\nThe NOS evaluates the quality of studies by analyzing three items: selection, comparability, and exposure. The scale assigns a maximum of nine points to each study: a maximum of four points for selection, two points for comparability, and three points for exposure. Therefore, the highest quality study would score nine points. In our analysis, high-quality studies were defined as those that scored nine or eight points; medium-quality studies were those that scored seven or six points [8].\nThe Jadad scale (five points) contained questions for three main parts: randomization, masking, and accountability of all patients (withdrawals and dropouts). Studies scored ≥3 points were considered as high quality [9].\nTwo investigators independently assessed the quality of each included study using the Newcastle-Ottawa Scale (NOS) for non-randomized studies and the Jadad scale for randomized controlled trials (RCTs).\nThe NOS evaluates the quality of studies by analyzing three items: selection, comparability, and exposure. The scale assigns a maximum of nine points to each study: a maximum of four points for selection, two points for comparability, and three points for exposure. Therefore, the highest quality study would score nine points. In our analysis, high-quality studies were defined as those that scored nine or eight points; medium-quality studies were those that scored seven or six points [8].\nThe Jadad scale (five points) contained questions for three main parts: randomization, masking, and accountability of all patients (withdrawals and dropouts). Studies scored ≥3 points were considered as high quality [9].\n Statistical analysis Meta-analysis was conducted by Review Manager 5.3 and SPSS 18.0, p value < 0.05 suggested statistically significant. The differences were compared between the two groups using analysis of variance for continuous variables and pooled relative risk (RR) with 95% confidence interval (CI) for categorical variables. We used I\n2 and Cochran Q to evaluate the between-study heterogeneity. A random-effects model was adopted when the heterogeneity was significant (p ≤ 0.10 and I\n2 > 50%); otherwise, a fixed-effects model was used. Rank correlation test of funnel plot asymmetry was used to assess the potential publication bias.\nMeta-analysis was conducted by Review Manager 5.3 and SPSS 18.0, p value < 0.05 suggested statistically significant. The differences were compared between the two groups using analysis of variance for continuous variables and pooled relative risk (RR) with 95% confidence interval (CI) for categorical variables. We used I\n2 and Cochran Q to evaluate the between-study heterogeneity. A random-effects model was adopted when the heterogeneity was significant (p ≤ 0.10 and I\n2 > 50%); otherwise, a fixed-effects model was used. Rank correlation test of funnel plot asymmetry was used to assess the potential publication bias.\n\nSearch strategy:\nMEDLINE and manual searches were performed by two investigators independently and in duplicate to identify all relevant scientific articles published from January 1990 to May 2016. The MEDLINE search was performed using PubMed, Ovid MEDLINE, EMBASE, Web of Science, ScienceDirect, The Cochrane Library, Scopus, and Google Scholar. The MeSH terms “lung cancer or lung neoplasm”, “thoracotomy or open surgery”, and “video-assisted thoracic surgery or VATS” and comparative study were used.\n\nInclusion criteria and exclusion criteria:\nThe following inclusion criteria were applied: (1) published in English, (2) compared the LN dissection of thoracoscopic lobectomy with thoracotomy in treating patients with lung cancer, and (3) the most recent study was chosen when duplication of data is in more than one article.\nReviews without original data, case reports, meta-analyses, letters, expert opinions, and animal studies were excluded. Studies on robotic-assisted VATS were also excluded.\n\nData extraction:\nTwo investigators independently extracted data from the eligible studies. The extracted data included first author, year of publication, geographical area, study design, duration of enrollment, information on preoperative staging, number of patients per group, LN number (LNN), and LN station number (LNS).\n\nQuality assessment for included studies:\nTwo investigators independently assessed the quality of each included study using the Newcastle-Ottawa Scale (NOS) for non-randomized studies and the Jadad scale for randomized controlled trials (RCTs).\nThe NOS evaluates the quality of studies by analyzing three items: selection, comparability, and exposure. The scale assigns a maximum of nine points to each study: a maximum of four points for selection, two points for comparability, and three points for exposure. Therefore, the highest quality study would score nine points. In our analysis, high-quality studies were defined as those that scored nine or eight points; medium-quality studies were those that scored seven or six points [8].\nThe Jadad scale (five points) contained questions for three main parts: randomization, masking, and accountability of all patients (withdrawals and dropouts). Studies scored ≥3 points were considered as high quality [9].\n\nStatistical analysis:\nMeta-analysis was conducted by Review Manager 5.3 and SPSS 18.0, p value < 0.05 suggested statistically significant. The differences were compared between the two groups using analysis of variance for continuous variables and pooled relative risk (RR) with 95% confidence interval (CI) for categorical variables. We used I\n2 and Cochran Q to evaluate the between-study heterogeneity. A random-effects model was adopted when the heterogeneity was significant (p ≤ 0.10 and I\n2 > 50%); otherwise, a fixed-effects model was used. Rank correlation test of funnel plot asymmetry was used to assess the potential publication bias.\n\nResults:\n Search results and quality assessment of the included studies We initially identified 2341 publications from the database and reference list searches and reviewed 29 articles for final analysis (Fig. 1). The articles involved a total of 6247 patients, of whom 2763 underwent VATS and 3484 underwent thoracotomy. Of these 29 publications, three studies were RCTs and 26 were retrospective studies. According to the NOS and Jadad scales assessment scores, 23 articles were of good quality and the remaining six were medium quality. The baseline characteristics of these articles are listed in Table 1.Fig. 1Flow diagram of screened and included papers\nTable 1Summary of the 29 trials included in the present meta-analysisStudyInstitutionEnrolled yearNo. of patientsClinical stageOutcomesDesignQualityVATSOpenVATSOpen1995Kirby [28] USASingle1991.10–1993.121991.10–1993.122530I①RCT31998Morikawa [29] JPNSingle1996.04–1996.121995.01–1996.033941I–II③Retrospective92000Luketich [30] USASingleNot mentionedNot mentioned3131I①Retrospective72000Sugi [31] JPNSingle1993.01–1994.061993.01–1994.064852Ia③, ⑤RCT32001Nomori [32] JPNSingle1999.08–2000.121998.04–1999.073333I①,⑦,⑧Retrospective72005Watanabe [33] JPNSingle1997–20041997–2004221190I①, ③, ⑦, ⑧Retrospective82006Petersen [34] USASingle2001–20051996–20051285I–IV②Retrospective82006Shigemura [35] JPNMulti1999.01–2004.011999.01–2004.015055Ia①Retrospective92008Shiraishi [36] JPNSingle1994.11–2005.101994.11–2005.102055I③, ⑤Retrospective82008Watanabe [37] JPNSingle1997–20061997–20063732I①, ②, ③, ④Retrospective82008Whitson [38] USASingleNot mentionedNot mentioned67Not mentioned①Retrospective62009Nakanishi [39] JPNSingle2000.04–2007.012000.04–2007.011314I–IV③Retrospective82009Okur [40] TRSingle2007.01–2007.112007.01–2007.112028I②Retrospective82010Denlinger [21] USASingle2000.01–2008.082000.01–2008.0879464I①, ③, ⑤Retrospective82011D’Amico [41] USASingle2007.01–2010.092007.01–2010.09199189I–III②,④Retrospective72012Bu [42] CHNSingle2001.05–2011.042001.05–2011.044687Not mentioned①, ②Retrospective72012Li [43] CHNSingle2006.09–2009.122006.09–2009.122947I③, ④Retrospective82012Licht [44] DNKMulti2007.01–2011.122007.01–2011.12717796I②Retrospective82013Fan [45] CHNSingle2005.01–2010.122005.01–2010.127977I–II①, ②Retrospective82013Lee [22] USASingle1990.05–2011.121990.05–2011.12141115Not mentioned①, ②, ③, ④, ⑤, ⑥Retrospective82013Palade [46] GERSingle2008.05–2011.122008.05–2011.123232I①, ⑦, ⑧RCT32013Zhong [47] CHNSingle2006.03–2011.082006.03–2011.086790I①, ②, ③, ④Retrospective82014Li [48] CHNSingle2011.02–2013.022011.02–2013.022132I–II①Retrospective82014Stephens [4] USASingle2002.01–2011.122002.01–2011.12307307I②Retrospective82015Cai [49] CHNSingle2010.01–2012.052010.01–2012.057167I–II①, ②Retrospective92015Kuritzky [50] USASingle2007–20122007–201274224I②Retrospective82015Murakawa [51] JPNSingle2001–20102001–2010101101I①, ②Retrospective82015Nwogu [6] USAMulti2004.10–2010.062004.10–2010.06175175I–II①, ②Retrospective72015Zhang [52] CHNSingle2012.10–2013.112012.10–2013.117028I①Retrospective9① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\n\nFlow diagram of screened and included papers\nSummary of the 29 trials included in the present meta-analysis\n① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\n\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\nWe initially identified 2341 publications from the database and reference list searches and reviewed 29 articles for final analysis (Fig. 1). The articles involved a total of 6247 patients, of whom 2763 underwent VATS and 3484 underwent thoracotomy. Of these 29 publications, three studies were RCTs and 26 were retrospective studies. According to the NOS and Jadad scales assessment scores, 23 articles were of good quality and the remaining six were medium quality. The baseline characteristics of these articles are listed in Table 1.Fig. 1Flow diagram of screened and included papers\nTable 1Summary of the 29 trials included in the present meta-analysisStudyInstitutionEnrolled yearNo. of patientsClinical stageOutcomesDesignQualityVATSOpenVATSOpen1995Kirby [28] USASingle1991.10–1993.121991.10–1993.122530I①RCT31998Morikawa [29] JPNSingle1996.04–1996.121995.01–1996.033941I–II③Retrospective92000Luketich [30] USASingleNot mentionedNot mentioned3131I①Retrospective72000Sugi [31] JPNSingle1993.01–1994.061993.01–1994.064852Ia③, ⑤RCT32001Nomori [32] JPNSingle1999.08–2000.121998.04–1999.073333I①,⑦,⑧Retrospective72005Watanabe [33] JPNSingle1997–20041997–2004221190I①, ③, ⑦, ⑧Retrospective82006Petersen [34] USASingle2001–20051996–20051285I–IV②Retrospective82006Shigemura [35] JPNMulti1999.01–2004.011999.01–2004.015055Ia①Retrospective92008Shiraishi [36] JPNSingle1994.11–2005.101994.11–2005.102055I③, ⑤Retrospective82008Watanabe [37] JPNSingle1997–20061997–20063732I①, ②, ③, ④Retrospective82008Whitson [38] USASingleNot mentionedNot mentioned67Not mentioned①Retrospective62009Nakanishi [39] JPNSingle2000.04–2007.012000.04–2007.011314I–IV③Retrospective82009Okur [40] TRSingle2007.01–2007.112007.01–2007.112028I②Retrospective82010Denlinger [21] USASingle2000.01–2008.082000.01–2008.0879464I①, ③, ⑤Retrospective82011D’Amico [41] USASingle2007.01–2010.092007.01–2010.09199189I–III②,④Retrospective72012Bu [42] CHNSingle2001.05–2011.042001.05–2011.044687Not mentioned①, ②Retrospective72012Li [43] CHNSingle2006.09–2009.122006.09–2009.122947I③, ④Retrospective82012Licht [44] DNKMulti2007.01–2011.122007.01–2011.12717796I②Retrospective82013Fan [45] CHNSingle2005.01–2010.122005.01–2010.127977I–II①, ②Retrospective82013Lee [22] USASingle1990.05–2011.121990.05–2011.12141115Not mentioned①, ②, ③, ④, ⑤, ⑥Retrospective82013Palade [46] GERSingle2008.05–2011.122008.05–2011.123232I①, ⑦, ⑧RCT32013Zhong [47] CHNSingle2006.03–2011.082006.03–2011.086790I①, ②, ③, ④Retrospective82014Li [48] CHNSingle2011.02–2013.022011.02–2013.022132I–II①Retrospective82014Stephens [4] USASingle2002.01–2011.122002.01–2011.12307307I②Retrospective82015Cai [49] CHNSingle2010.01–2012.052010.01–2012.057167I–II①, ②Retrospective92015Kuritzky [50] USASingle2007–20122007–201274224I②Retrospective82015Murakawa [51] JPNSingle2001–20102001–2010101101I①, ②Retrospective82015Nwogu [6] USAMulti2004.10–2010.062004.10–2010.06175175I–II①, ②Retrospective72015Zhang [52] CHNSingle2012.10–2013.112012.10–2013.117028I①Retrospective9① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\n\nFlow diagram of screened and included papers\nSummary of the 29 trials included in the present meta-analysis\n① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\n\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\n Comparison of total LNN and LNS We identified 19 articles for total LNN comparison. They involved 1297 patients in the VATS group and 1731 patients in the open group (thoracotomy). The heterogeneity between these studies was acceptable (p = 0.02, I\n2 = 44%). Fewer total LNs were dissected in the VATS group as compared with the open group (95% CI −1.52 to −0.73, p < 0.00001, Fig. 2a).Fig. 2Forest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\n\nForest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\nFourteen articles were identified for total LNS comparison. They involved 2046 patients in the VATS group and 2373 patients in the open group. The mean difference in total LNS between the two groups was not significant (95% CI −0.28 to 0.06, p = 0.20), with significant heterogeneity across studies (p < 0.00001, I\n2 = 73%, Fig. 2b).\nWe identified 19 articles for total LNN comparison. They involved 1297 patients in the VATS group and 1731 patients in the open group (thoracotomy). The heterogeneity between these studies was acceptable (p = 0.02, I\n2 = 44%). Fewer total LNs were dissected in the VATS group as compared with the open group (95% CI −1.52 to −0.73, p < 0.00001, Fig. 2a).Fig. 2Forest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\n\nForest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\nFourteen articles were identified for total LNS comparison. They involved 2046 patients in the VATS group and 2373 patients in the open group. The mean difference in total LNS between the two groups was not significant (95% CI −0.28 to 0.06, p = 0.20), with significant heterogeneity across studies (p < 0.00001, I\n2 = 73%, Fig. 2b).\n Comparison of N2 LNN and LNS Eleven articles were identified for N2 LNN comparison. They involved 726 patients in the VATS group and 1132 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.08, I\n2 = 41%). Fewer N2 LNs were dissected in the VATS group as compared with the open group (95% CI −1.38 to −0.49, p < 0.0001, Fig. 3a).Fig. 3Forest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\n\nForest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\nFive articles were identified for N2 LNS comparison. They involved 473 patients in the VATS group and 473 patients in the open group. The mean difference in N2 LNS between the two groups was not significant (95% CI −0.46 to 0.23, p = 0.50), with significant heterogeneity across studies (p = 0.002, I\n2 = 76%, Fig. 3b).\nEleven articles were identified for N2 LNN comparison. They involved 726 patients in the VATS group and 1132 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.08, I\n2 = 41%). Fewer N2 LNs were dissected in the VATS group as compared with the open group (95% CI −1.38 to −0.49, p < 0.0001, Fig. 3a).Fig. 3Forest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\n\nForest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\nFive articles were identified for N2 LNS comparison. They involved 473 patients in the VATS group and 473 patients in the open group. The mean difference in N2 LNS between the two groups was not significant (95% CI −0.46 to 0.23, p = 0.50), with significant heterogeneity across studies (p = 0.002, I\n2 = 76%, Fig. 3b).\n Comparison of N1 LNN and LNS Four articles were identified for N1 LNN comparison. They involved 288 patients in the VATS group and 686 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.44, I\n2 = 60%). The mean difference in N1 LNN between the two groups was not significant (95% CI −0.71 to 0.08, p = 0.11, Fig. 4).Fig. 4Forest plot of the mean difference in N1 LNN in the VATS group vs. the open group\n\nForest plot of the mean difference in N1 LNN in the VATS group vs. the open group\nOnly one article was identified for N1 LNS comparison. They involved 141 patients in the VATS group and 115 patients in the open group. The result showed that fewer N1 LN stations were dissected in the VATS group (1.4 ± 0.5 vs. 1.6 ± 0.6, p = 0.04).\nFour articles were identified for N1 LNN comparison. They involved 288 patients in the VATS group and 686 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.44, I\n2 = 60%). The mean difference in N1 LNN between the two groups was not significant (95% CI −0.71 to 0.08, p = 0.11, Fig. 4).Fig. 4Forest plot of the mean difference in N1 LNN in the VATS group vs. the open group\n\nForest plot of the mean difference in N1 LNN in the VATS group vs. the open group\nOnly one article was identified for N1 LNS comparison. They involved 141 patients in the VATS group and 115 patients in the open group. The result showed that fewer N1 LN stations were dissected in the VATS group (1.4 ± 0.5 vs. 1.6 ± 0.6, p = 0.04).\n Publication bias The funnel plot for publication bias (standard error by total LNN comparison) demonstrated marked evidence of symmetry (Fig. 5), indicating no publication bias. The combined effect size yielded a Z value of 5.64, with a corresponding p < 0.00001. This result indicates that the fail-safe N value was relevant.Fig. 5Funnel plot of the mean difference in total LNN in the VATS group vs. the open group\n\nFunnel plot of the mean difference in total LNN in the VATS group vs. the open group\nThe funnel plot for publication bias (standard error by total LNN comparison) demonstrated marked evidence of symmetry (Fig. 5), indicating no publication bias. The combined effect size yielded a Z value of 5.64, with a corresponding p < 0.00001. This result indicates that the fail-safe N value was relevant.Fig. 5Funnel plot of the mean difference in total LNN in the VATS group vs. the open group\n\nFunnel plot of the mean difference in total LNN in the VATS group vs. the open group\n\nSearch results and quality assessment of the included studies:\nWe initially identified 2341 publications from the database and reference list searches and reviewed 29 articles for final analysis (Fig. 1). The articles involved a total of 6247 patients, of whom 2763 underwent VATS and 3484 underwent thoracotomy. Of these 29 publications, three studies were RCTs and 26 were retrospective studies. According to the NOS and Jadad scales assessment scores, 23 articles were of good quality and the remaining six were medium quality. The baseline characteristics of these articles are listed in Table 1.Fig. 1Flow diagram of screened and included papers\nTable 1Summary of the 29 trials included in the present meta-analysisStudyInstitutionEnrolled yearNo. of patientsClinical stageOutcomesDesignQualityVATSOpenVATSOpen1995Kirby [28] USASingle1991.10–1993.121991.10–1993.122530I①RCT31998Morikawa [29] JPNSingle1996.04–1996.121995.01–1996.033941I–II③Retrospective92000Luketich [30] USASingleNot mentionedNot mentioned3131I①Retrospective72000Sugi [31] JPNSingle1993.01–1994.061993.01–1994.064852Ia③, ⑤RCT32001Nomori [32] JPNSingle1999.08–2000.121998.04–1999.073333I①,⑦,⑧Retrospective72005Watanabe [33] JPNSingle1997–20041997–2004221190I①, ③, ⑦, ⑧Retrospective82006Petersen [34] USASingle2001–20051996–20051285I–IV②Retrospective82006Shigemura [35] JPNMulti1999.01–2004.011999.01–2004.015055Ia①Retrospective92008Shiraishi [36] JPNSingle1994.11–2005.101994.11–2005.102055I③, ⑤Retrospective82008Watanabe [37] JPNSingle1997–20061997–20063732I①, ②, ③, ④Retrospective82008Whitson [38] USASingleNot mentionedNot mentioned67Not mentioned①Retrospective62009Nakanishi [39] JPNSingle2000.04–2007.012000.04–2007.011314I–IV③Retrospective82009Okur [40] TRSingle2007.01–2007.112007.01–2007.112028I②Retrospective82010Denlinger [21] USASingle2000.01–2008.082000.01–2008.0879464I①, ③, ⑤Retrospective82011D’Amico [41] USASingle2007.01–2010.092007.01–2010.09199189I–III②,④Retrospective72012Bu [42] CHNSingle2001.05–2011.042001.05–2011.044687Not mentioned①, ②Retrospective72012Li [43] CHNSingle2006.09–2009.122006.09–2009.122947I③, ④Retrospective82012Licht [44] DNKMulti2007.01–2011.122007.01–2011.12717796I②Retrospective82013Fan [45] CHNSingle2005.01–2010.122005.01–2010.127977I–II①, ②Retrospective82013Lee [22] USASingle1990.05–2011.121990.05–2011.12141115Not mentioned①, ②, ③, ④, ⑤, ⑥Retrospective82013Palade [46] GERSingle2008.05–2011.122008.05–2011.123232I①, ⑦, ⑧RCT32013Zhong [47] CHNSingle2006.03–2011.082006.03–2011.086790I①, ②, ③, ④Retrospective82014Li [48] CHNSingle2011.02–2013.022011.02–2013.022132I–II①Retrospective82014Stephens [4] USASingle2002.01–2011.122002.01–2011.12307307I②Retrospective82015Cai [49] CHNSingle2010.01–2012.052010.01–2012.057167I–II①, ②Retrospective92015Kuritzky [50] USASingle2007–20122007–201274224I②Retrospective82015Murakawa [51] JPNSingle2001–20102001–2010101101I①, ②Retrospective82015Nwogu [6] USAMulti2004.10–2010.062004.10–2010.06175175I–II①, ②Retrospective72015Zhang [52] CHNSingle2012.10–2013.112012.10–2013.117028I①Retrospective9① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\n\nFlow diagram of screened and included papers\nSummary of the 29 trials included in the present meta-analysis\n① total lymph node number, ② total lymph node station number, ③ N2 LNN, ④ N2 LNS, ⑤ N1 LNN, ⑥ N1 LNS, ⑦ left-side LNN, ⑧ right-side LNN\n\nCHN China, DNK Denmark, JPN Japan, GER Germany, TR Turkey, USA United States of America, RCT randomized controlled trial\n\nComparison of total LNN and LNS:\nWe identified 19 articles for total LNN comparison. They involved 1297 patients in the VATS group and 1731 patients in the open group (thoracotomy). The heterogeneity between these studies was acceptable (p = 0.02, I\n2 = 44%). Fewer total LNs were dissected in the VATS group as compared with the open group (95% CI −1.52 to −0.73, p < 0.00001, Fig. 2a).Fig. 2Forest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\n\nForest plot of the mean difference in total LNN (a) and LNS (b) in the VATS group vs. the open group\nFourteen articles were identified for total LNS comparison. They involved 2046 patients in the VATS group and 2373 patients in the open group. The mean difference in total LNS between the two groups was not significant (95% CI −0.28 to 0.06, p = 0.20), with significant heterogeneity across studies (p < 0.00001, I\n2 = 73%, Fig. 2b).\n\nComparison of N2 LNN and LNS:\nEleven articles were identified for N2 LNN comparison. They involved 726 patients in the VATS group and 1132 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.08, I\n2 = 41%). Fewer N2 LNs were dissected in the VATS group as compared with the open group (95% CI −1.38 to −0.49, p < 0.0001, Fig. 3a).Fig. 3Forest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\n\nForest plot of the mean difference in N2 LNN (a) and LNS (b) in the VATS group vs. the open group\nFive articles were identified for N2 LNS comparison. They involved 473 patients in the VATS group and 473 patients in the open group. The mean difference in N2 LNS between the two groups was not significant (95% CI −0.46 to 0.23, p = 0.50), with significant heterogeneity across studies (p = 0.002, I\n2 = 76%, Fig. 3b).\n\nComparison of N1 LNN and LNS:\nFour articles were identified for N1 LNN comparison. They involved 288 patients in the VATS group and 686 patients in the open group. The heterogeneity between these studies was acceptable (p = 0.44, I\n2 = 60%). The mean difference in N1 LNN between the two groups was not significant (95% CI −0.71 to 0.08, p = 0.11, Fig. 4).Fig. 4Forest plot of the mean difference in N1 LNN in the VATS group vs. the open group\n\nForest plot of the mean difference in N1 LNN in the VATS group vs. the open group\nOnly one article was identified for N1 LNS comparison. They involved 141 patients in the VATS group and 115 patients in the open group. The result showed that fewer N1 LN stations were dissected in the VATS group (1.4 ± 0.5 vs. 1.6 ± 0.6, p = 0.04).\n\nPublication bias:\nThe funnel plot for publication bias (standard error by total LNN comparison) demonstrated marked evidence of symmetry (Fig. 5), indicating no publication bias. The combined effect size yielded a Z value of 5.64, with a corresponding p < 0.00001. This result indicates that the fail-safe N value was relevant.Fig. 5Funnel plot of the mean difference in total LNN in the VATS group vs. the open group\n\nFunnel plot of the mean difference in total LNN in the VATS group vs. the open group\n\nDiscussion:\nThe presence or absence of mediastinal LN metastases is a critical component to accurate staging and therefore a key component of the surgical management of NSCLC [10]. Both overall and disease-free survival have been associated with the number of LNs dissected [11, 12]. Lardinois compared LNs dissection versus sampling; the results showed a longer disease-free survival and better local tumor control in dissection groups [13]. Focusing on stage IA lung cancer, Xu reported a similar result and suggested that the number of N2 stations served as a more significant prognostic factor [14]. As a result, current guidelines from the National Comprehensive Cancer Network and European Society of Thoracic Surgeons recommend that all patients with resectable NSCLC should have a complete systematic nodal dissection, with at least three N2 stations dissected [15, 16].\nAlthough VATS is associated with many benefits in comparison to thoracotomy, whether VATS can achieve the same LN dissection efficacy in patients with lung cancer remains controversial [7, 17, 18]. Flores reported that the surgical field in VATS facilitated the dissection of LNs adjacent to the blood vessels and the trachea and found that smaller nodes achieved better LN dissection [19]. Ramos showed that VATS dissected more total LNS (5.1 ± 1.1 vs. 4.5 ± 1.2, p < 0.001) and mediastinal LNS (3.4 ± 0.9 vs. 3.2 ± 0.9, p = 0.022) when compared with thoracotomy [20]. By contrast, Denlinger reported that significantly more overall LNs were dissected in the open group than in the VATS group (8.9 ± 5.2 vs. 7.1 ± 5.2, p = 0.0006) [21]. Similar results were reported by Lee and colleagues in their retrospective study of LN evaluation achieved by VATS lobectomy compared with that by open lobectomy [22]. A secondary analysis including 752 original participants of the American College of Surgeons Oncology Group Z0030 trial compared patients who underwent lobectomy by VATS with patients who underwent thoracotomy. The results showed that there was no significant difference in the overall number of LNs retrieved between the two groups (15 vs. 19, p = 0.147) [23].\nThe present study included a total of 7568 patients from nine countries, providing the most comprehensive evidence for LN dissection efficacy by VATS to date. The meta-analysis showed that fewer total LNN and similar total LNS were dissected in the VATS group as compared with that in the open group. If we only included studies focusing on clinical stage I lung cancer, the results were similar (total LNN, 95% CI −1.67 to −0.61, p < 0.0001; total LNS, 95% CI −0.62 to 0.02, p = 0.07). It suggests that surgeons may not have the ability to perform systematic lymphadenectomy in VATS or ignore the importance of systematic lymphadenectomy for various reasons (earlier tumor stage, worrying about damage to vital organs, and so on).\nIn the comparison of N1 and N2 LN dissection, our results showed that similar number of N1 LNN and N2 LNS could be harvested by VATS, while fewer N2 LNN were harvested by VATS as compared with thoracotomy. Only one article reported on N1 LNS comparison between the two groups and showed better efficiency in the open group (1.4 ± 0.5 vs. 1.6 ± 0.6, p = 0.04) [22].\nIt was controversial that removing more N2 LNs could increase the accuracy of clinical staging of NSCLC. Boffa et al. compared the completeness of surgical LN evaluation during anatomic resection of primary lung cancer by open and VATS approaches in 11,531 patients from The Society of Thoracic Surgeons-General Thoracic Database. The results showed nodal upstaging in 14.3% (1024 patients) of the open group and in 11.6% (508 patients) of the VATS group (p < 0.001). The study suggested that surgeons should be encouraged to apply a systematic approach to hilar and peribronchial LN dissection during VATS lobectomy for lung cancer, particularly as they were adopting this approach [24].\nSome surgeons might also worry about the complications caused by the systematic LN dissection. As with open thoracotomy, systematic mediastinal LN dissection under VATS may increase the risk of intraoperative bleeding (bronchial arteries, etc.), tracheobronchial injury, recurrent nerve injury, prolonged air leak, atrial fibrillation, and pulmonary edema [25]. In other papers, Watanabe et al. reported similar mortality and morbidity of mediastinal LN dissection by VATS vs. open lobectomy, indicating that systematic mediastinal LN dissection by VATS is a safe procedure [26]. Zhang et al. compared complications such as chylothorax and nerve injury between VATS and open thoracotomy in a meta-analysis. The results showed that these events were similar in both groups [27].\nThe possible limitations of our study must be considered when interpreting the findings described herein. First, including only English papers might have resulted in language bias. Second, including 7568 participants from 36 studies with only three RCTs might have weakened the quality of the results. Third, the number of dissected LNs varied significantly between the included studies. Different doctors have different understanding of LN dissection and might be at different stages of the learning curve. Some data did not meet the National Comprehensive Cancer Network guide requirements for lung cancer surgery treatment of systematic LN dissection, which may have affected the reliability of the results. Fourth, there is great potential for LN fragmentation during dissection. Different pathologists and different counting procedures might lead to false LNN counts, which might increase the heterogeneity between studies but would not alter the overall results. Finally, we did not analyze the survival difference between VATS and open thoracotomy. Our analysis compared LN harvest capability between two evaluation procedures only from a surgical point of view and tried to give further proof of satisfied oncologic efficacy by VATS.\n\nConclusions:\nLess total and mediastinal LNs were evaluated with VATS than with thoracotomy in the present study. Both approaches harvested a similar number of total LN stations, mediastinal LN stations, and N1 LNs. However, owing to the possible existing bias in the original studies, inter-study heterogeneity, and the inherent limitations of our meta-analysis, the findings require validation in high-quality, large-scale RCTs.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe aim of this study was to investigate which surgical method is better in lymph node (LN) dissection of lung cancer.\n\nMethods:\nA comprehensive search of PubMed, Ovid MEDLINE, EMBASE, Web of Science, ScienceDirect, the Cochrane Library, Scopus, and Google Scholar was performed to identify studies comparing thoracoscopic lobectomy (video-assisted thoracic surgery (VATS) group) and thoracotomy (open group) in LN dissection.\n\nResults:\nTwenty-nine articles met the inclusion criteria and involved 2763 patients in the VATS group and 3484 patients in the open group. The meta-analysis showed that fewer total LNs (95% confidence interval [CI] -1.52 to -0.73, p < 0.0001) and N2 LNs (95% CI -1.25 to -0.10, p = 0.02) were dissected in the VATS group. A similar number of total LN stations, N2 LN stations, and N1 LNs were harvested in both groups. Only one study reported that fewer N1 LN stations were dissected in the VATS group (1.4 ± 0.5 vs. 1.6 ± 0.6, p = 0.04).\n\nConclusions:\nOpen lobectomy could achieve better LN dissection efficacy than thoracoscopic lobectomy in the treatment of lung cancer, especially in the N2 LNs dissection. These findings require validation by high-quality, large-scale randomized controlled trials."},"background_conclusion_text":{"kind":"string","value":"Background:\nLung cancer is the leading cause of cancer deaths in many countries [1, 2]. Surgical treatment is the preferred treatment for early-stage non-small cell lung cancer (NSCLC). D’Cunha’s research showed that N1 and N2 lymph nodes (LNs) were positive in 27.5% of patients with lung cancer under lobectomy [3]. However, non-invasive examinations, such as computed tomography (CT) and positron emission tomography-computed tomography (PET-CT), are not sensitive and specific for the clinical staging of lung cancer. Video-assisted thoracic surgery (VATS) is the preferred surgical procedure, with fewer incidences of postoperative complications and a higher survival rate compared with thoracotomy [4–7]. However, whether VATS can achieve the same LN dissection efficacy is controversial, and there remains a lack of high-quality, large-scale clinical research.\nTo determine whether VATS can achieve the same LN dissection efficacy as thoracotomy in lung cancer, we performed a systemic review and meta-analysis.\n\nConclusions:\nLess total and mediastinal LNs were evaluated with VATS than with thoracotomy in the present study. Both approaches harvested a similar number of total LN stations, mediastinal LN stations, and N1 LNs. However, owing to the possible existing bias in the original studies, inter-study heterogeneity, and the inherent limitations of our meta-analysis, the findings require validation in high-quality, large-scale RCTs."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nThe aim of this study was to investigate which surgical method is better in lymph node (LN) dissection of lung cancer.\n\nMethods:\nA comprehensive search of PubMed, Ovid MEDLINE, EMBASE, Web of Science, ScienceDirect, the Cochrane Library, Scopus, and Google Scholar was performed to identify studies comparing thoracoscopic lobectomy (video-assisted thoracic surgery (VATS) group) and thoracotomy (open group) in LN dissection.\n\nResults:\nTwenty-nine articles met the inclusion criteria and involved 2763 patients in the VATS group and 3484 patients in the open group. The meta-analysis showed that fewer total LNs (95% confidence interval [CI] -1.52 to -0.73, p < 0.0001) and N2 LNs (95% CI -1.25 to -0.10, p = 0.02) were dissected in the VATS group. A similar number of total LN stations, N2 LN stations, and N1 LNs were harvested in both groups. Only one study reported that fewer N1 LN stations were dissected in the VATS group (1.4 ± 0.5 vs. 1.6 ± 0.6, p = 0.04).\n\nConclusions:\nOpen lobectomy could achieve better LN dissection efficacy than thoracoscopic lobectomy in the treatment of lung cancer, especially in the N2 LNs dissection. These findings require validation by high-quality, large-scale randomized controlled trials."},"whole_article_text_length":{"kind":"number","value":6718,"string":"6,718"},"whole_article_abstract_length":{"kind":"number","value":266,"string":"266"},"other_sections_lengths":{"kind":"list like","value":[90,88,56,176,128,467,218,215,177,101],"string":"[\n 90,\n 88,\n 56,\n 176,\n 128,\n 467,\n 218,\n 215,\n 177,\n 101\n]","isConversational":false},"num_sections":{"kind":"number","value":15,"string":"15"},"most_frequent_words":{"kind":"list like","value":["group","vats","lnn","lns","open","01","patients","studies","vats group","open group"],"string":"[\n \"group\",\n \"vats\",\n \"lnn\",\n \"lns\",\n \"open\",\n \"01\",\n \"patients\",\n \"studies\",\n \"vats group\",\n \"open group\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["dissection thoracoscopic lobectomy","vats lobectomy lung","dissection efficacy thoracotomy","staging lung cancer","efficacy thoracotomy lung"],"string":"[\n \"dissection thoracoscopic lobectomy\",\n \"vats lobectomy lung\",\n \"dissection efficacy thoracotomy\",\n \"staging lung cancer\",\n \"efficacy thoracotomy lung\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Video-assisted thoracic surgery | Thoracotomy | Meta-analysis | Lung cancer | Lymph node dissection [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Video-assisted thoracic surgery | Thoracotomy | Meta-analysis | Lung cancer | Lymph node dissection [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Video-assisted thoracic surgery | Thoracotomy | Meta-analysis | Lung cancer | Lymph node dissection [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Video-assisted thoracic surgery | Thoracotomy | Meta-analysis | Lung cancer | Lymph node dissection [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Video-assisted thoracic surgery | Thoracotomy | Meta-analysis | Lung cancer | Lymph node dissection [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Carcinoma, Non-Small-Cell Lung | Humans | Lung Neoplasms | Lymph Node Excision | Lymphatic Metastasis | Mediastinum | Neoplasm Staging | Pneumonectomy | Thoracic Surgery, Video-Assisted | Thoracotomy [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Carcinoma, Non-Small-Cell Lung | Humans | Lung Neoplasms | Lymph Node Excision | Lymphatic Metastasis | Mediastinum | Neoplasm Staging | Pneumonectomy | Thoracic Surgery, Video-Assisted | Thoracotomy [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Carcinoma, Non-Small-Cell Lung | Humans | Lung Neoplasms | Lymph Node Excision | Lymphatic Metastasis | Mediastinum | Neoplasm Staging | Pneumonectomy | Thoracic Surgery, Video-Assisted | Thoracotomy [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Carcinoma, Non-Small-Cell Lung | Humans | Lung Neoplasms | Lymph Node Excision | Lymphatic Metastasis | Mediastinum | Neoplasm Staging | Pneumonectomy | Thoracic Surgery, Video-Assisted | Thoracotomy [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Carcinoma, Non-Small-Cell Lung | Humans | Lung Neoplasms | Lymph Node Excision | Lymphatic Metastasis | Mediastinum | Neoplasm Staging | Pneumonectomy | Thoracic Surgery, Video-Assisted | Thoracotomy [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] dissection thoracoscopic lobectomy | vats lobectomy lung | dissection efficacy thoracotomy | staging lung cancer | efficacy thoracotomy lung [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] dissection thoracoscopic lobectomy | vats lobectomy lung | dissection efficacy thoracotomy | staging lung cancer | efficacy thoracotomy lung [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] dissection thoracoscopic lobectomy | vats lobectomy lung | dissection efficacy thoracotomy | staging lung cancer | efficacy thoracotomy lung [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] dissection thoracoscopic lobectomy | vats lobectomy lung | dissection efficacy thoracotomy | staging lung cancer | efficacy thoracotomy lung [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] dissection thoracoscopic lobectomy | vats lobectomy lung | dissection efficacy thoracotomy | staging lung cancer | efficacy thoracotomy lung [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] group | vats | lnn | lns | open | 01 | patients | studies | vats group | open group [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] group | vats | lnn | lns | open | 01 | patients | studies | vats group | open group [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] group | vats | lnn | lns | open | 01 | patients | studies | vats group | open group [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] group | vats | lnn | lns | open | 01 | patients | studies | vats group | open group [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] group | vats | lnn | lns | open | 01 | patients | studies | vats group | open group [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cancer | lung | lung cancer | tomography | preferred | computed | ct | computed tomography | research | efficacy [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] group | 01 | lnn | 2011 | vats group | open group | total | open | lns | fig [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] mediastinal | ln stations | stations | total | study | ln | total ln stations mediastinal | n1 lns owing | n1 lns owing possible | approaches harvested similar number [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] group | vats | vats group | open | open group | lnn | points | total | lns | 01 [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] group | vats | vats group | open | open group | lnn | points | total | lns | 01 [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] LN [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Twenty-nine | 2763 | 3484 ||| 95% | CI] -1.52 | 0.0001 | 95% | CI | -0.10 | 0.02 | VATS ||| LN | N2 LN | N1 ||| Only one | N1 LN | 1.4 ± 0.5 | 1.6 ± | 0.6 | 0.04 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] LN ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] LN ||| PubMed | Ovid MEDLINE | EMBASE | ScienceDirect | the Cochrane Library | Google Scholar | VATS | LN ||| Twenty-nine | 2763 | 3484 ||| 95% | CI] -1.52 | 0.0001 | 95% | CI | -0.10 | 0.02 | VATS ||| LN | N2 LN | N1 ||| Only one | N1 LN | 1.4 ± 0.5 | 1.6 ± | 0.6 | 0.04 ||| LN ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] LN ||| PubMed | Ovid MEDLINE | EMBASE | ScienceDirect | the Cochrane Library | Google Scholar | VATS | LN ||| Twenty-nine | 2763 | 3484 ||| 95% | CI] -1.52 | 0.0001 | 95% | CI | -0.10 | 0.02 | VATS ||| LN | N2 LN | N1 ||| Only one | N1 LN | 1.4 ± 0.5 | 1.6 ± | 0.6 | 0.04 ||| LN ||| [SUMMARY]"}}},{"rowIdx":56,"cells":{"title":{"kind":"string","value":"Expression of PH Domain Leucine-rich Repeat Protein Phosphatase, Forkhead Homeobox Type O 3a and RAD51, and their Relationships with Clinicopathologic Features and Prognosis in Ovarian Serous Adenocarcinoma."},"pmid":{"kind":"string","value":"28139510"},"background_abstract":{"kind":"string","value":"Ovarian serous adenocarcinoma can be divided into low- and high-grade tumors, which exhibit substantial differences in pathogenesis, clinicopathology, and prognosis. This study aimed to investigate the differences in the PH domain leucine-rich repeat protein phosphatase (PHLPP), forkhead homeobox type O 3a (FoxO3a), and RAD51 protein expressions, and their associations with prognosis in patients with low- and high-grade ovarian serous adenocarcinomas."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"The PHLPP, FoxO3a, and RAD51 protein expressions were examined in 94 high- and 26 low-grade ovarian serous adenocarcinomas by immunohistochemistry. The differences in expression and their relationships with pathological features and prognosis were analyzed."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"In high-grade serous adenocarcinomas, the positive rates of PHLPP and FoxO3a were 24.5% and 26.6%, while in low-grade tumors, they were 23.1% and 26.9%, respectively (P < 0.05 vs. the control specimens; low- vs. high-grade: P > 0.05). The positive rates of RAD51 were 70.2% and 65.4% in high- and low-grade serous adenocarcinomas, respectively (P < 0.05 vs. the control specimens; low- vs. high-grade: P > 0.05). Meanwhile, in high-grade tumors, Stage III/IV tumors and lymph node and omental metastases were significantly associated with lower PHLPP and FoxO3a and higher RAD51 expression. The 5-year survival rates of patients with PHLPP- and FoxO3a-positive high-grade tumors (43.5% and 36.0%) were significantly higher than in patients with PHLPP-negative tumors (5.6% and 7.2%, respectively; P< 0.05). Similarly, the 5-year survival rate of RAD51-positive patients (3.0%) was significantly lower than in negative patients (42.9%; P< 0.05). In low-grade tumors, the PHLPP, FoxO3a, and RAD51 expressions were not significantly correlated with lymph node metastasis, omental metastasis, Federation of Gynecology and Obstetrics stage, or prognosis."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Abnormal PHLPP, FoxO3a, and RAD51 protein expressions may be involved in the development of high- and low-grade ovarian serous adenocarcinomas, suggesting common molecular pathways. Decreased PHLPP and FoxO3a and increased RAD51 protein expression may be important molecular markers for poor prognosis, and RAD51 may be an independent prognosis factor, of high-grade, but not low-grade, ovarian serous adenocarcinomas."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Aged","Biomarkers, Tumor","Cystadenocarcinoma, Serous","Female","Forkhead Box Protein O3","Humans","Immunohistochemistry","Lymphatic Metastasis","Middle Aged","Neoplasm Staging","Nuclear Proteins","Ovarian Neoplasms","Phosphoprotein Phosphatases","Prognosis","Rad51 Recombinase"],"string":"[\n \"Adult\",\n \"Aged\",\n \"Biomarkers, Tumor\",\n \"Cystadenocarcinoma, Serous\",\n \"Female\",\n \"Forkhead Box Protein O3\",\n \"Humans\",\n \"Immunohistochemistry\",\n \"Lymphatic Metastasis\",\n \"Middle Aged\",\n \"Neoplasm Staging\",\n \"Nuclear Proteins\",\n \"Ovarian Neoplasms\",\n \"Phosphoprotein Phosphatases\",\n \"Prognosis\",\n \"Rad51 Recombinase\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"5308009"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Ovarian serous adenocarcinoma is the most common ovarian epithelial malignant tumor. It can be divided into low- and high-grade tumors, which exhibit substantial differences in the pathogenesis, clinicopathological characteristics, and prognosis.[1] Low-grade serous carcinomas undergo a progressive, gradual developmental process from benign serous cystadenoma to borderline serous cystadenoma to malignant serous adenocarcinoma, while high-grade serous adenocarcinomas develop directly from the oviduct epithelium or ovarian inclusion cysts.\nHowever, it remains unclear whether the two tumor types have completely different molecular mechanisms, whether there is a common molecular basis, and whether the genes involved in the development of these tumors affect the patients’ prognosis. Recent studies[23] have indicated that the PI3K/Akt signaling pathway is involved in the initiation and development of ovarian serous adenocarcinoma. For example, PH domain leucine-rich repeat protein phosphatase (PHLPP) is known to negatively regulate Akt and its downstream kinase by dephosphorylating the hydrophobic core of Akt (Akt1 Ser473), thereby antagonizing PI3K/Akt signaling and inhibiting tumor growth. Forkhead homeobox type O 3a (FoxO3a), an important downstream signaling molecule of Akt, is involved in the initiation and development of tumor cells and regulation of cell proliferation after being phosphorylated by Akt.[45] Further, RAD51, a DNA double-strand break repair gene,[6] has also been implicated in the PI3K/Akt signaling pathway: phosphorylated FoxO3a binds to the promoter of the downstream gene RAD51, thereby regulating RAD51 and promoting tumor development and metastasis.\nThe present study aimed to (1) determine the differences in the protein expressions of PHLPP, FoxO3a, and RAD51 between high- and low-grade ovarian serous adenocarcinomas, (2) explore the roles of these three genes in the initiation of the different grades of ovarian serous adenocarcinoma, and (3) analyze the relationships between their protein expressions and prognosis."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Ethics The study was approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University and was conducted in accordance with the Declaration of Helsinki 1975, as revised in 2000.\nThe study was approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University and was conducted in accordance with the Declaration of Helsinki 1975, as revised in 2000.\n Participant selection and description Patients with ovarian serous adenocarcinoma hospitalized and treated from January 2006 to January 2011 in our hospital were included in the ovarian serous adenocarcinoma groups. No patients received chemotherapy and radiotherapy preoperatively, and their diagnosis was confirmed by pathological examinations postoperatively. All patients had complete clinical data and were graded according to the World Health Organization histological classification for ovarian tumors (2014 edition). Ninety-four and twenty-six cases of high- and low-grade ovarian serous adenocarcinomas were included in this study. Among the high-grade group (mean age, 55 years; range, 36–78 years), there were 26 and 68 patients with International Federation of Gynecology and Obstetrics (FIGO) Stage (2014 edition) I–II and III–IV disease, respectively. There were thirty and 64 patients with and without lymph node metastasis (LNM), and 41 and 53 patients with and without omental metastasis, respectively. Among the low-grade group (mean age, 44 years; range, 26–70 years), there were 18 and eight patients with FIGO Stage I–II and III–IV disease, respectively. There were 4 and 22 patients with and without LNM, and 5 and 21 patients with and without omental metastasis, respectively. Moreover, 21 cases of borderline serous cystadenoma (mean age, 38 years; range, 18–59 years) and 35 cases of benign serous cystadenoma (mean age, 45 years; range, 22–69 years) were included, along with thirty normal ovaries and thirty oviducts (mean age, 48 years; range, 36–62 years) removed from patients undergoing surgery for uterine fibroids, which were used as controls for the low- and high-grade tumor groups, respectively.\nExpression of TP53 protein in high- and low-grade serous carcinoma: Among the 94 cases of high-grade, 92 cases (97.9%) showed strong staining of TP53, while among the 26 cases of low-grade serous adenocarcinoma, there was no case of strong staining.\nPatients with ovarian serous adenocarcinoma hospitalized and treated from January 2006 to January 2011 in our hospital were included in the ovarian serous adenocarcinoma groups. No patients received chemotherapy and radiotherapy preoperatively, and their diagnosis was confirmed by pathological examinations postoperatively. All patients had complete clinical data and were graded according to the World Health Organization histological classification for ovarian tumors (2014 edition). Ninety-four and twenty-six cases of high- and low-grade ovarian serous adenocarcinomas were included in this study. Among the high-grade group (mean age, 55 years; range, 36–78 years), there were 26 and 68 patients with International Federation of Gynecology and Obstetrics (FIGO) Stage (2014 edition) I–II and III–IV disease, respectively. There were thirty and 64 patients with and without lymph node metastasis (LNM), and 41 and 53 patients with and without omental metastasis, respectively. Among the low-grade group (mean age, 44 years; range, 26–70 years), there were 18 and eight patients with FIGO Stage I–II and III–IV disease, respectively. There were 4 and 22 patients with and without LNM, and 5 and 21 patients with and without omental metastasis, respectively. Moreover, 21 cases of borderline serous cystadenoma (mean age, 38 years; range, 18–59 years) and 35 cases of benign serous cystadenoma (mean age, 45 years; range, 22–69 years) were included, along with thirty normal ovaries and thirty oviducts (mean age, 48 years; range, 36–62 years) removed from patients undergoing surgery for uterine fibroids, which were used as controls for the low- and high-grade tumor groups, respectively.\nExpression of TP53 protein in high- and low-grade serous carcinoma: Among the 94 cases of high-grade, 92 cases (97.9%) showed strong staining of TP53, while among the 26 cases of low-grade serous adenocarcinoma, there was no case of strong staining.\n Technical information \nExperimental procedures\n\nThe operative specimens were examined, and the patients were diagnosed by experienced pathologists. Paraffin blocks of the specimens were cut, and immunohistochemical staining of the sections was conducted as follows: the samples were sliced, deparaffinized, dehydrated, and subjected to antigen retrieval before blocking with goat serum. Subsequently, the slides were placed in a humidifier chamber and incubated at room temperature for 45 min. The tissues were incubated with primary antibodies (working concentrations of anti-PHLPP, RAD51, and FoxO3a of 1:100, 1:100, and 1:150, respectively, Biogot Biotechnology Co., Ltd., Nanjing, China) in the sealed humidifier chamber at 4°C overnight. After washing of the slides, goat anti-rabbit secondary antibodies were added and the slides were incubated in the humidifier at 37°C for 30 min. Next, horseradish peroxidase-labeled streptavidin was added and the slides were incubated in the humidifier at 37°C for 30 min, after which the tissues were stained by 3,3’-diaminobenzidine, followed by counterstaining, destaining, dehydration, clearing, and mounting. Finally, the slides were air-dried and observed under a microscope.\n\nEvaluation standard for staining\n\nThe slides were scored by pathologists according to the below criteria for PHLPP, FoxO3a, and RAD51 under a microscope. Ten high-magnification fields were counted, and the average cell numbers were used.\n PH domain leucine-rich repeat protein phosphatase-positive criteria Cells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\nCells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\n Forkhead homeobox type O 3a-positive criteria Cells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\nCells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\n RAD51-positive criteria Cells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\nCells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\n\nExperimental procedures\n\nThe operative specimens were examined, and the patients were diagnosed by experienced pathologists. Paraffin blocks of the specimens were cut, and immunohistochemical staining of the sections was conducted as follows: the samples were sliced, deparaffinized, dehydrated, and subjected to antigen retrieval before blocking with goat serum. Subsequently, the slides were placed in a humidifier chamber and incubated at room temperature for 45 min. The tissues were incubated with primary antibodies (working concentrations of anti-PHLPP, RAD51, and FoxO3a of 1:100, 1:100, and 1:150, respectively, Biogot Biotechnology Co., Ltd., Nanjing, China) in the sealed humidifier chamber at 4°C overnight. After washing of the slides, goat anti-rabbit secondary antibodies were added and the slides were incubated in the humidifier at 37°C for 30 min. Next, horseradish peroxidase-labeled streptavidin was added and the slides were incubated in the humidifier at 37°C for 30 min, after which the tissues were stained by 3,3’-diaminobenzidine, followed by counterstaining, destaining, dehydration, clearing, and mounting. Finally, the slides were air-dried and observed under a microscope.\n\nEvaluation standard for staining\n\nThe slides were scored by pathologists according to the below criteria for PHLPP, FoxO3a, and RAD51 under a microscope. Ten high-magnification fields were counted, and the average cell numbers were used.\n PH domain leucine-rich repeat protein phosphatase-positive criteria Cells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\nCells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\n Forkhead homeobox type O 3a-positive criteria Cells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\nCells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\n RAD51-positive criteria Cells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\nCells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\n Statistical analyses SPSS 16.0 software (SPSS Inc., USA) was used for the statistical analysis. Categorical data were analyzed by the Chi-square test, and the survival rates were calculated by Kaplan-Meier univariate analysis. Differences in the survival rates between the different groups were determined by the log-rank test. Cox multivariate survival analysis was used for analysis of independent prognostic factors. The statistical significance level was set as P < 0.05.\nSPSS 16.0 software (SPSS Inc., USA) was used for the statistical analysis. Categorical data were analyzed by the Chi-square test, and the survival rates were calculated by Kaplan-Meier univariate analysis. Differences in the survival rates between the different groups were determined by the log-rank test. Cox multivariate survival analysis was used for analysis of independent prognostic factors. The statistical significance level was set as P < 0.05."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Expression of PH domain leucine-rich repeat protein phosphatase in ovarian serous adenocarcinomas and its association with clinicopathological features and prognosis Among 94 cases of high-grade serous adenocarcinoma, 23 cases (24.5%) were PHLPP-positive, which was significantly lower than the positive rate of 86.7% in the normal oviduct group (P < 0.05). Of 26 low-grade serous adenocarcinoma cases, 6 (23.1%) were positive, which was significantly lower than that in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, there was no difference in the positive expression rates between high- and low-grade tumors [P > 0.05; Figure 1 and Table 1].\nProtein expression of PH domain leucine-rich repeat protein phosphatase in high- and low-grade serous ovarian adenocarcinoma by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\nProtein expression of PHLPP in high- and low-grade ovarian serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. PHLPP: PH domain leucine-rich repeat protein phosphatase.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower PHLPP protein expression levels [P < 0.05; Table 2]. On the other hand, there were no significant correlations between the clinicopathological features and PHLPP protein expression in low-grade tumors [P > 0.05].\nRelationships between PHLPP, FoxO3a, and RAD51 protein expressions and clinicopathological features of high-grade serous ovarian adenocarcinoma\nLNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a.\nAmong the 94 patients with high-grade serous adenocarcinoma, the 5-year survival rates of PHLPP-negative and positive patients were 5.6% and 43.5%, respectively [P < 0.05, Figure 2]. Among the 26 cases of low-grade serous adenocarcinoma, however, the expression of PHLPP did not significantly associate with the 5-year survival rate of the patients [P > 0.05].\nFive-year survival curves of PH domain leucine-rich repeat protein phosphatase-positive and negative patients with high-grade ovarian serous adenocarcinomas.\nAmong 94 cases of high-grade serous adenocarcinoma, 23 cases (24.5%) were PHLPP-positive, which was significantly lower than the positive rate of 86.7% in the normal oviduct group (P < 0.05). Of 26 low-grade serous adenocarcinoma cases, 6 (23.1%) were positive, which was significantly lower than that in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, there was no difference in the positive expression rates between high- and low-grade tumors [P > 0.05; Figure 1 and Table 1].\nProtein expression of PH domain leucine-rich repeat protein phosphatase in high- and low-grade serous ovarian adenocarcinoma by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\nProtein expression of PHLPP in high- and low-grade ovarian serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. PHLPP: PH domain leucine-rich repeat protein phosphatase.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower PHLPP protein expression levels [P < 0.05; Table 2]. On the other hand, there were no significant correlations between the clinicopathological features and PHLPP protein expression in low-grade tumors [P > 0.05].\nRelationships between PHLPP, FoxO3a, and RAD51 protein expressions and clinicopathological features of high-grade serous ovarian adenocarcinoma\nLNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a.\nAmong the 94 patients with high-grade serous adenocarcinoma, the 5-year survival rates of PHLPP-negative and positive patients were 5.6% and 43.5%, respectively [P < 0.05, Figure 2]. Among the 26 cases of low-grade serous adenocarcinoma, however, the expression of PHLPP did not significantly associate with the 5-year survival rate of the patients [P > 0.05].\nFive-year survival curves of PH domain leucine-rich repeat protein phosphatase-positive and negative patients with high-grade ovarian serous adenocarcinomas.\n Expression of forkhead homeobox type O 3a in ovarian serous adenocarcinoma and its association with clinicopathological features and prognosis Among the 94 cases of high-grade serous adenocarcinoma, 25 (26.6%) were positive for FoxO3a protein expression, which was significantly lower than the positive rate of 90.0% in the normal oviduct group (P < 0.05). Among the 26 cases of low-grade serous carcinoma, 7 (26.9%) were positive, which was significantly lower than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, the difference in the positive expression rates between the high- and low-grade groups was not significant [P > 0.05; Figure 3 and Table 3].\nProtein expression of forkhead homeobox type O 3a in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\nProtein expression of FoxO3a in high- and low-grade serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. FoxO3a: Forkhead homeobox type O 3a.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower FoxO3a protein expression [P < 0.05, Table 2]. On the other hand, in low-grade tumors, the FoxO3a protein expression showed no association with clinicopathological features (P > 0.05).\nAmong the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of FoxO3a-negative and positive patients were 7.2% and 36.0%, respectively [P < 0.05, Figure 4]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of FoxO3a showed no significant association with the 5-year survival rate (P > 0.05).\nFive-year survival curves of forkhead homeobox type O 3a-positive and negative patients with high-grade ovarian serous adenocarcinomas.\nAmong the 94 cases of high-grade serous adenocarcinoma, 25 (26.6%) were positive for FoxO3a protein expression, which was significantly lower than the positive rate of 90.0% in the normal oviduct group (P < 0.05). Among the 26 cases of low-grade serous carcinoma, 7 (26.9%) were positive, which was significantly lower than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, the difference in the positive expression rates between the high- and low-grade groups was not significant [P > 0.05; Figure 3 and Table 3].\nProtein expression of forkhead homeobox type O 3a in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\nProtein expression of FoxO3a in high- and low-grade serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. FoxO3a: Forkhead homeobox type O 3a.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower FoxO3a protein expression [P < 0.05, Table 2]. On the other hand, in low-grade tumors, the FoxO3a protein expression showed no association with clinicopathological features (P > 0.05).\nAmong the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of FoxO3a-negative and positive patients were 7.2% and 36.0%, respectively [P < 0.05, Figure 4]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of FoxO3a showed no significant association with the 5-year survival rate (P > 0.05).\nFive-year survival curves of forkhead homeobox type O 3a-positive and negative patients with high-grade ovarian serous adenocarcinomas.\n Expression of RAD51 in ovarian serous adenocarcinoma and its association with clinicopathological features and prognosis Among the 94 cases of high-grade serous adenocarcinoma, 66 (70.2%) were positive for RAD51 protein, which was significantly higher than that in the normal oviduct group [P < 0.05; Figure 5 and Table 4]. Among the 26 cases of low-grade serous carcinoma, 17 (65.4%) were positive, which was significantly higher than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups [P < 0.05; Figure 5 and Table 4]. However, the difference in the positive expression rates between the high- and low-grade groups was not significant (P > 0.05).\nProtein expression of RAD51 in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Positive high-grade adenocarcinoma, (b) negative normal oviduct, (c) positive low-grade adenocarcinoma, (d) negative borderline cystadenoma, (e) negative cystadenoma, (f) negative normal ovary.\nProtein expression of RAD51 in high- and low-grade serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with higher RAD51 protein expression levels [P < 0.05; Table 2]. Conversely, there was no significant correlation between clinicopathological features and RAD51 expression in low-grade tumors (P > 0.05).\nFinally, among the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of RAD51-positive and negative patients were 3.0% and 42.9%, respectively [P < 0.05; Figure 6]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of RAD51 showed no significant association with the 5-year survival rate (P > 0.05).\nFive-year survival curves of RAD51-positive and negative patients with high-grade ovarian serous adenocarcinomas.\nAmong the 94 cases of high-grade serous adenocarcinoma, 66 (70.2%) were positive for RAD51 protein, which was significantly higher than that in the normal oviduct group [P < 0.05; Figure 5 and Table 4]. Among the 26 cases of low-grade serous carcinoma, 17 (65.4%) were positive, which was significantly higher than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups [P < 0.05; Figure 5 and Table 4]. However, the difference in the positive expression rates between the high- and low-grade groups was not significant (P > 0.05).\nProtein expression of RAD51 in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Positive high-grade adenocarcinoma, (b) negative normal oviduct, (c) positive low-grade adenocarcinoma, (d) negative borderline cystadenoma, (e) negative cystadenoma, (f) negative normal ovary.\nProtein expression of RAD51 in high- and low-grade serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with higher RAD51 protein expression levels [P < 0.05; Table 2]. Conversely, there was no significant correlation between clinicopathological features and RAD51 expression in low-grade tumors (P > 0.05).\nFinally, among the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of RAD51-positive and negative patients were 3.0% and 42.9%, respectively [P < 0.05; Figure 6]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of RAD51 showed no significant association with the 5-year survival rate (P > 0.05).\nFive-year survival curves of RAD51-positive and negative patients with high-grade ovarian serous adenocarcinomas.\n Prognostic multivariate survival analyses of high-grade serous ovarian adenocarcinoma The Cox multivariate analysis results showed that FIGO staging, LNM, and RAD51 protein expression were independent prognostic factors of patients with high-grade serous ovarian adenocarcinoma [P < 0.05; Table 5].\nMultivariate analysis of independent factors associated with the 5-year overall survival rate in patients with high-grade serous ovarian adenocarcinoma\nRR: Risk ratio; CI: Confidence interval; LNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a; PHLPP: PH domain leucine-rich repeat protein phosphatase.\nThe Cox multivariate analysis results showed that FIGO staging, LNM, and RAD51 protein expression were independent prognostic factors of patients with high-grade serous ovarian adenocarcinoma [P < 0.05; Table 5].\nMultivariate analysis of independent factors associated with the 5-year overall survival rate in patients with high-grade serous ovarian adenocarcinoma\nRR: Risk ratio; CI: Confidence interval; LNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a; PHLPP: PH domain leucine-rich repeat protein phosphatase."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Ethics","Participant selection and description","Technical information","PH domain leucine-rich repeat protein phosphatase-positive criteria","Forkhead homeobox type O 3a-positive criteria","RAD51-positive criteria","Statistical analyses","Expression of PH domain leucine-rich repeat protein phosphatase in ovarian serous adenocarcinomas and its association with clinicopathological features and prognosis","Expression of forkhead homeobox type O 3a in ovarian serous adenocarcinoma and its association with clinicopathological features and prognosis","Expression of RAD51 in ovarian serous adenocarcinoma and its association with clinicopathological features and prognosis","Prognostic multivariate survival analyses of high-grade serous ovarian adenocarcinoma","Role of the tumor suppressor PH domain leucine-rich repeat protein phosphatase in the development of ovarian serous adenocarcinoma and its association with patient prognosis","Role of the tumor suppressor forkhead homeobox type O 3a in the development of ovarian serous adenocarcinoma and its association with patient prognosis","Role of RAD51 in the development of ovarian serous adenocarcinoma and its association with patient prognosis","Financial support and sponsorship"],"string":"[\n \"Ethics\",\n \"Participant selection and description\",\n \"Technical information\",\n \"PH domain leucine-rich repeat protein phosphatase-positive criteria\",\n \"Forkhead homeobox type O 3a-positive criteria\",\n \"RAD51-positive criteria\",\n \"Statistical analyses\",\n \"Expression of PH domain leucine-rich repeat protein phosphatase in ovarian serous adenocarcinomas and its association with clinicopathological features and prognosis\",\n \"Expression of forkhead homeobox type O 3a in ovarian serous adenocarcinoma and its association with clinicopathological features and prognosis\",\n \"Expression of RAD51 in ovarian serous adenocarcinoma and its association with clinicopathological features and prognosis\",\n \"Prognostic multivariate survival analyses of high-grade serous ovarian adenocarcinoma\",\n \"Role of the tumor suppressor PH domain leucine-rich repeat protein phosphatase in the development of ovarian serous adenocarcinoma and its association with patient prognosis\",\n \"Role of the tumor suppressor forkhead homeobox type O 3a in the development of ovarian serous adenocarcinoma and its association with patient prognosis\",\n \"Role of RAD51 in the development of ovarian serous adenocarcinoma and its association with patient prognosis\",\n \"Financial support and sponsorship\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["The study was approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University and was conducted in accordance with the Declaration of Helsinki 1975, as revised in 2000.","Patients with ovarian serous adenocarcinoma hospitalized and treated from January 2006 to January 2011 in our hospital were included in the ovarian serous adenocarcinoma groups. No patients received chemotherapy and radiotherapy preoperatively, and their diagnosis was confirmed by pathological examinations postoperatively. All patients had complete clinical data and were graded according to the World Health Organization histological classification for ovarian tumors (2014 edition). Ninety-four and twenty-six cases of high- and low-grade ovarian serous adenocarcinomas were included in this study. Among the high-grade group (mean age, 55 years; range, 36–78 years), there were 26 and 68 patients with International Federation of Gynecology and Obstetrics (FIGO) Stage (2014 edition) I–II and III–IV disease, respectively. There were thirty and 64 patients with and without lymph node metastasis (LNM), and 41 and 53 patients with and without omental metastasis, respectively. Among the low-grade group (mean age, 44 years; range, 26–70 years), there were 18 and eight patients with FIGO Stage I–II and III–IV disease, respectively. There were 4 and 22 patients with and without LNM, and 5 and 21 patients with and without omental metastasis, respectively. Moreover, 21 cases of borderline serous cystadenoma (mean age, 38 years; range, 18–59 years) and 35 cases of benign serous cystadenoma (mean age, 45 years; range, 22–69 years) were included, along with thirty normal ovaries and thirty oviducts (mean age, 48 years; range, 36–62 years) removed from patients undergoing surgery for uterine fibroids, which were used as controls for the low- and high-grade tumor groups, respectively.\nExpression of TP53 protein in high- and low-grade serous carcinoma: Among the 94 cases of high-grade, 92 cases (97.9%) showed strong staining of TP53, while among the 26 cases of low-grade serous adenocarcinoma, there was no case of strong staining.","\nExperimental procedures\n\nThe operative specimens were examined, and the patients were diagnosed by experienced pathologists. Paraffin blocks of the specimens were cut, and immunohistochemical staining of the sections was conducted as follows: the samples were sliced, deparaffinized, dehydrated, and subjected to antigen retrieval before blocking with goat serum. Subsequently, the slides were placed in a humidifier chamber and incubated at room temperature for 45 min. The tissues were incubated with primary antibodies (working concentrations of anti-PHLPP, RAD51, and FoxO3a of 1:100, 1:100, and 1:150, respectively, Biogot Biotechnology Co., Ltd., Nanjing, China) in the sealed humidifier chamber at 4°C overnight. After washing of the slides, goat anti-rabbit secondary antibodies were added and the slides were incubated in the humidifier at 37°C for 30 min. Next, horseradish peroxidase-labeled streptavidin was added and the slides were incubated in the humidifier at 37°C for 30 min, after which the tissues were stained by 3,3’-diaminobenzidine, followed by counterstaining, destaining, dehydration, clearing, and mounting. Finally, the slides were air-dried and observed under a microscope.\n\nEvaluation standard for staining\n\nThe slides were scored by pathologists according to the below criteria for PHLPP, FoxO3a, and RAD51 under a microscope. Ten high-magnification fields were counted, and the average cell numbers were used.\n PH domain leucine-rich repeat protein phosphatase-positive criteria Cells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\nCells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\n Forkhead homeobox type O 3a-positive criteria Cells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\nCells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\n RAD51-positive criteria Cells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\nCells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]","Cells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]","Cells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]","Cells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]","SPSS 16.0 software (SPSS Inc., USA) was used for the statistical analysis. Categorical data were analyzed by the Chi-square test, and the survival rates were calculated by Kaplan-Meier univariate analysis. Differences in the survival rates between the different groups were determined by the log-rank test. Cox multivariate survival analysis was used for analysis of independent prognostic factors. The statistical significance level was set as P < 0.05.","Among 94 cases of high-grade serous adenocarcinoma, 23 cases (24.5%) were PHLPP-positive, which was significantly lower than the positive rate of 86.7% in the normal oviduct group (P < 0.05). Of 26 low-grade serous adenocarcinoma cases, 6 (23.1%) were positive, which was significantly lower than that in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, there was no difference in the positive expression rates between high- and low-grade tumors [P > 0.05; Figure 1 and Table 1].\nProtein expression of PH domain leucine-rich repeat protein phosphatase in high- and low-grade serous ovarian adenocarcinoma by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\nProtein expression of PHLPP in high- and low-grade ovarian serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. PHLPP: PH domain leucine-rich repeat protein phosphatase.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower PHLPP protein expression levels [P < 0.05; Table 2]. On the other hand, there were no significant correlations between the clinicopathological features and PHLPP protein expression in low-grade tumors [P > 0.05].\nRelationships between PHLPP, FoxO3a, and RAD51 protein expressions and clinicopathological features of high-grade serous ovarian adenocarcinoma\nLNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a.\nAmong the 94 patients with high-grade serous adenocarcinoma, the 5-year survival rates of PHLPP-negative and positive patients were 5.6% and 43.5%, respectively [P < 0.05, Figure 2]. Among the 26 cases of low-grade serous adenocarcinoma, however, the expression of PHLPP did not significantly associate with the 5-year survival rate of the patients [P > 0.05].\nFive-year survival curves of PH domain leucine-rich repeat protein phosphatase-positive and negative patients with high-grade ovarian serous adenocarcinomas.","Among the 94 cases of high-grade serous adenocarcinoma, 25 (26.6%) were positive for FoxO3a protein expression, which was significantly lower than the positive rate of 90.0% in the normal oviduct group (P < 0.05). Among the 26 cases of low-grade serous carcinoma, 7 (26.9%) were positive, which was significantly lower than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, the difference in the positive expression rates between the high- and low-grade groups was not significant [P > 0.05; Figure 3 and Table 3].\nProtein expression of forkhead homeobox type O 3a in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\nProtein expression of FoxO3a in high- and low-grade serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. FoxO3a: Forkhead homeobox type O 3a.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower FoxO3a protein expression [P < 0.05, Table 2]. On the other hand, in low-grade tumors, the FoxO3a protein expression showed no association with clinicopathological features (P > 0.05).\nAmong the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of FoxO3a-negative and positive patients were 7.2% and 36.0%, respectively [P < 0.05, Figure 4]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of FoxO3a showed no significant association with the 5-year survival rate (P > 0.05).\nFive-year survival curves of forkhead homeobox type O 3a-positive and negative patients with high-grade ovarian serous adenocarcinomas.","Among the 94 cases of high-grade serous adenocarcinoma, 66 (70.2%) were positive for RAD51 protein, which was significantly higher than that in the normal oviduct group [P < 0.05; Figure 5 and Table 4]. Among the 26 cases of low-grade serous carcinoma, 17 (65.4%) were positive, which was significantly higher than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups [P < 0.05; Figure 5 and Table 4]. However, the difference in the positive expression rates between the high- and low-grade groups was not significant (P > 0.05).\nProtein expression of RAD51 in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Positive high-grade adenocarcinoma, (b) negative normal oviduct, (c) positive low-grade adenocarcinoma, (d) negative borderline cystadenoma, (e) negative cystadenoma, (f) negative normal ovary.\nProtein expression of RAD51 in high- and low-grade serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with higher RAD51 protein expression levels [P < 0.05; Table 2]. Conversely, there was no significant correlation between clinicopathological features and RAD51 expression in low-grade tumors (P > 0.05).\nFinally, among the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of RAD51-positive and negative patients were 3.0% and 42.9%, respectively [P < 0.05; Figure 6]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of RAD51 showed no significant association with the 5-year survival rate (P > 0.05).\nFive-year survival curves of RAD51-positive and negative patients with high-grade ovarian serous adenocarcinomas.","The Cox multivariate analysis results showed that FIGO staging, LNM, and RAD51 protein expression were independent prognostic factors of patients with high-grade serous ovarian adenocarcinoma [P < 0.05; Table 5].\nMultivariate analysis of independent factors associated with the 5-year overall survival rate in patients with high-grade serous ovarian adenocarcinoma\nRR: Risk ratio; CI: Confidence interval; LNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a; PHLPP: PH domain leucine-rich repeat protein phosphatase.","The tumor suppressor PHLPP, located at 18q21.33, can directly dephosphorylate ser473 in the hydrophobic group of Akt, thus negatively regulating Akt and its downstream targets. It has been reported that PHLPP can act synergistically with PTEN, another well-known tumor suppressor gene, thereby significantly inhibiting the proliferation of colorectal cancer cells, and negatively regulating PI3K/Akt signaling.[11] Accordingly, studies have shown that the PHLPP expression is significantly decreased in colon, breast, prostate, and lung cancers.[5712]\nThe results of the present study showed that the positive rate of PHLPP protein expression in the high-grade group was significantly lower than that in the oviduct group. The PHLPP-positive expression rate was decreased gradually from normal ovarian tissue to benign tumors to borderline tumors to low-grade adenocarcinomas, indicating that loss of PHLPP expression may be associated with the initiation of ovarian serous adenocarcinoma development, whereas no significant difference was observed between high-grade and low-grade tumors. Further, the PHLPP expression level showed significant associations with LNM, FIGO stages, and omental metastasis in high-grade tumors, but not in low-grade tumors. Similarly, the 5-year survival rates of PHLPP-positive and negative high-grade tumors significant differed, whereas no difference was seen in the low-grade tumors. These findings suggest that PHLPP may be a predictor of prognosis of high-grade ovarian serous adenocarcinoma.","FoxO3a is an important member of the FOXO family, located on at 6q21, and encodes a 673-amino acid long protein.[13] FoxO3a, as an important signaling molecule downstream of Akt in the PI3K-Akt signaling pathway, binds to the 14-3-3 chaperone protein after being phosphorylated by Akt, and subsequently translocates from the nucleus to the cytoplasm, thereby activating the apoptotic genes Bim, FasL, and tumor necrosis factor and its related apoptosis-inducing ligand TRAIL, consequently promoting apoptosis.[14] In addition to its role in apoptosis, FoxO3a plays critical roles in regulating cell proliferation, metabolism, the stress response, and the life span of cancer cells.[15] Particularly, another target molecule of FoxO3a is the cell cycle-regulating gene P27; overexpression of FoxO3a can arrest the cell cycle in the G0/G1 phase by upregulating P27. In addition, overexpression of FoxO3a can significantly inhibit the proliferation of tumor cells and result in tumor cell G2 arrest, further promoting tumor cell apoptosis.[1617]\nThe results of our analyses showed that the FoxO3a-positive rate in the high-grade tumors was significantly lower than that in the normal oviducts. In the low-grade adenocarcinomas, borderline cystadenomas, cystadenomas, and normal ovaries, the positive expression rates of FoxO3a protein were gradually reduced from normal ovaries to the low-grade adenocarcinomas, and the FoxO3 positive rate of the low-grade tumors was significantly lower than those of the borderline cystadenomas, cystadenomas, and normal ovaries, while the positive rate of borderline cystadenomas was significantly lower than those of cystadenomas and normal ovaries. Moreover, the FoxO3a expression level showed significant associations with LNM, FIGO stage, and omental metastasis in high-grade, but not low-grade tumors. These results suggest that FoxO3a may play a role in the oncogenesis and development of ovarian serous adenocarcinomas. In the high-grade tumors, the 5-year survival rate of FoxO3a-positive patients was significantly higher than that of the negative patients, whereas no difference in survival was seen in low-grade tumors, indicating that positive FoxO3a expression may have a long-term protective effect on cancer patients, and FoxO3a may hence have certain significance in the prediction of the patients’ long-term prognosis. Interestingly, it has been reported that tumor cells with low FoxO3a expression can develop drug resistance to paclitaxel and that inhibiting FoxO3a expression with RNAi can reduce E1A-mediated sensitivity to paclitaxel.[18] Therefore, FoxO3a protein may represent a potential prognostic indicator for high-grade ovarian serous adenocarcinoma.","RAD51 is a human homologous recombination repair gene. Too strong or too weak homologous recombination will result in genome instability and DNA damage, and the consequent gene loss, loss of heterozygosity, and/or genetic translocation are important causes of tumorigenesis and tumor progression.[19] Studies have found that RAD51 protein is overexpression in the breast, prostate, pancreatic, and colon cancers. Such overexpression of RAD51 can provide certain advantages for the growth of cancer cells by promoting the initiation and progression of cancer. However, to date, studies on the expression of RAD51 in ovarian cancers are few.\nThe results of this study showed that the RAD51-positive rate in high-grade tumors was significantly higher than that of normal oviducts, suggesting that RAD51 may play a role in the initiation of high-grade ovarian serous carcinomas. The RAD51-positive rate gradually increased from normal ovaries to cystadenomas to borderline cystadenomas to low-grade serous adenocarcinomas. Particularly, the positive rate of low-grade tumors was significantly higher than that in benign tumors and normal oviducts. The gradual change in the RAD51 expression suggests that the initiation of low-grade serous adenocarcinoma is a gradual procedure from benign to borderline to low-grade adenocarcinoma. However, no significant difference in the expression of RAD51 was found between high- and low-grade tumors. The expression level of RAD51 showed significant associations with LNM, FIGO stage, and omental metastasis in high-grade, but not low-grade tumors.\nHerein, the 5-year survival rate of patients with RAD51-positive high-grade adenocarcinomas was significantly lower than that of patients with RAD51-negative tumors, while no difference in survival was found in low-grade tumors. In other words, we found that high-grade tumor patients with high expression of RAD51 had a lower survival rate and poor prognosis, indicating that RAD51 may be a prognostic factor in high-grade ovarian serous adenocarcinoma. Furthermore, multivariate survival analyses showed that RAD51 was an independent prognostic factor, which may be of clinical significance in terms of the development of molecular targeted therapy, in addition to FIGO stage, and LNM. Further investigations on RAD51 are required to confirm these findings and to hopefully be able to improve the quality of life and prognosis of patients using targeted therapy.\nIn conclusion, the results of the present study indicate that PHLPP, FoxO3a, and RAD51 may play certain roles in the pathogenesis of the different grades of ovarian serous adenocarcinomas. Meanwhile, they also suggest the possible presence of common molecular mechanisms in the pathogenesis of the different grades. Increased protein expression of RAD51 may be an important molecular marker for poor prognosis of high-grade ovarian serous adenocarcinomas. However, considering the limited number of cases selected, which may have affected the significance of the results, further studies are required to improve and supplement these findings.","This work were supported by grants from the Key Scientific Research program in Medical Science of Hebei Province, China (No. 20150318) and Science and Technology support program of Hebei Province, China (No. 16277794D)."],"string":"[\n \"The study was approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University and was conducted in accordance with the Declaration of Helsinki 1975, as revised in 2000.\",\n \"Patients with ovarian serous adenocarcinoma hospitalized and treated from January 2006 to January 2011 in our hospital were included in the ovarian serous adenocarcinoma groups. No patients received chemotherapy and radiotherapy preoperatively, and their diagnosis was confirmed by pathological examinations postoperatively. All patients had complete clinical data and were graded according to the World Health Organization histological classification for ovarian tumors (2014 edition). Ninety-four and twenty-six cases of high- and low-grade ovarian serous adenocarcinomas were included in this study. Among the high-grade group (mean age, 55 years; range, 36–78 years), there were 26 and 68 patients with International Federation of Gynecology and Obstetrics (FIGO) Stage (2014 edition) I–II and III–IV disease, respectively. There were thirty and 64 patients with and without lymph node metastasis (LNM), and 41 and 53 patients with and without omental metastasis, respectively. Among the low-grade group (mean age, 44 years; range, 26–70 years), there were 18 and eight patients with FIGO Stage I–II and III–IV disease, respectively. There were 4 and 22 patients with and without LNM, and 5 and 21 patients with and without omental metastasis, respectively. Moreover, 21 cases of borderline serous cystadenoma (mean age, 38 years; range, 18–59 years) and 35 cases of benign serous cystadenoma (mean age, 45 years; range, 22–69 years) were included, along with thirty normal ovaries and thirty oviducts (mean age, 48 years; range, 36–62 years) removed from patients undergoing surgery for uterine fibroids, which were used as controls for the low- and high-grade tumor groups, respectively.\\nExpression of TP53 protein in high- and low-grade serous carcinoma: Among the 94 cases of high-grade, 92 cases (97.9%) showed strong staining of TP53, while among the 26 cases of low-grade serous adenocarcinoma, there was no case of strong staining.\",\n \"\\nExperimental procedures\\n\\nThe operative specimens were examined, and the patients were diagnosed by experienced pathologists. Paraffin blocks of the specimens were cut, and immunohistochemical staining of the sections was conducted as follows: the samples were sliced, deparaffinized, dehydrated, and subjected to antigen retrieval before blocking with goat serum. Subsequently, the slides were placed in a humidifier chamber and incubated at room temperature for 45 min. The tissues were incubated with primary antibodies (working concentrations of anti-PHLPP, RAD51, and FoxO3a of 1:100, 1:100, and 1:150, respectively, Biogot Biotechnology Co., Ltd., Nanjing, China) in the sealed humidifier chamber at 4°C overnight. After washing of the slides, goat anti-rabbit secondary antibodies were added and the slides were incubated in the humidifier at 37°C for 30 min. Next, horseradish peroxidase-labeled streptavidin was added and the slides were incubated in the humidifier at 37°C for 30 min, after which the tissues were stained by 3,3’-diaminobenzidine, followed by counterstaining, destaining, dehydration, clearing, and mounting. Finally, the slides were air-dried and observed under a microscope.\\n\\nEvaluation standard for staining\\n\\nThe slides were scored by pathologists according to the below criteria for PHLPP, FoxO3a, and RAD51 under a microscope. Ten high-magnification fields were counted, and the average cell numbers were used.\\n PH domain leucine-rich repeat protein phosphatase-positive criteria Cells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\\nCells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\\n Forkhead homeobox type O 3a-positive criteria Cells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\\nCells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\\n RAD51-positive criteria Cells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\\nCells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\",\n \"Cells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\",\n \"Cells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\",\n \"Cells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\",\n \"SPSS 16.0 software (SPSS Inc., USA) was used for the statistical analysis. Categorical data were analyzed by the Chi-square test, and the survival rates were calculated by Kaplan-Meier univariate analysis. Differences in the survival rates between the different groups were determined by the log-rank test. Cox multivariate survival analysis was used for analysis of independent prognostic factors. The statistical significance level was set as P < 0.05.\",\n \"Among 94 cases of high-grade serous adenocarcinoma, 23 cases (24.5%) were PHLPP-positive, which was significantly lower than the positive rate of 86.7% in the normal oviduct group (P < 0.05). Of 26 low-grade serous adenocarcinoma cases, 6 (23.1%) were positive, which was significantly lower than that in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, there was no difference in the positive expression rates between high- and low-grade tumors [P > 0.05; Figure 1 and Table 1].\\nProtein expression of PH domain leucine-rich repeat protein phosphatase in high- and low-grade serous ovarian adenocarcinoma by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\\nProtein expression of PHLPP in high- and low-grade ovarian serous adenocarcinomas\\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. PHLPP: PH domain leucine-rich repeat protein phosphatase.\\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower PHLPP protein expression levels [P < 0.05; Table 2]. On the other hand, there were no significant correlations between the clinicopathological features and PHLPP protein expression in low-grade tumors [P > 0.05].\\nRelationships between PHLPP, FoxO3a, and RAD51 protein expressions and clinicopathological features of high-grade serous ovarian adenocarcinoma\\nLNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a.\\nAmong the 94 patients with high-grade serous adenocarcinoma, the 5-year survival rates of PHLPP-negative and positive patients were 5.6% and 43.5%, respectively [P < 0.05, Figure 2]. Among the 26 cases of low-grade serous adenocarcinoma, however, the expression of PHLPP did not significantly associate with the 5-year survival rate of the patients [P > 0.05].\\nFive-year survival curves of PH domain leucine-rich repeat protein phosphatase-positive and negative patients with high-grade ovarian serous adenocarcinomas.\",\n \"Among the 94 cases of high-grade serous adenocarcinoma, 25 (26.6%) were positive for FoxO3a protein expression, which was significantly lower than the positive rate of 90.0% in the normal oviduct group (P < 0.05). Among the 26 cases of low-grade serous carcinoma, 7 (26.9%) were positive, which was significantly lower than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, the difference in the positive expression rates between the high- and low-grade groups was not significant [P > 0.05; Figure 3 and Table 3].\\nProtein expression of forkhead homeobox type O 3a in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\\nProtein expression of FoxO3a in high- and low-grade serous adenocarcinomas\\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. FoxO3a: Forkhead homeobox type O 3a.\\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower FoxO3a protein expression [P < 0.05, Table 2]. On the other hand, in low-grade tumors, the FoxO3a protein expression showed no association with clinicopathological features (P > 0.05).\\nAmong the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of FoxO3a-negative and positive patients were 7.2% and 36.0%, respectively [P < 0.05, Figure 4]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of FoxO3a showed no significant association with the 5-year survival rate (P > 0.05).\\nFive-year survival curves of forkhead homeobox type O 3a-positive and negative patients with high-grade ovarian serous adenocarcinomas.\",\n \"Among the 94 cases of high-grade serous adenocarcinoma, 66 (70.2%) were positive for RAD51 protein, which was significantly higher than that in the normal oviduct group [P < 0.05; Figure 5 and Table 4]. Among the 26 cases of low-grade serous carcinoma, 17 (65.4%) were positive, which was significantly higher than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups [P < 0.05; Figure 5 and Table 4]. However, the difference in the positive expression rates between the high- and low-grade groups was not significant (P > 0.05).\\nProtein expression of RAD51 in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Positive high-grade adenocarcinoma, (b) negative normal oviduct, (c) positive low-grade adenocarcinoma, (d) negative borderline cystadenoma, (e) negative cystadenoma, (f) negative normal ovary.\\nProtein expression of RAD51 in high- and low-grade serous adenocarcinomas\\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas.\\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with higher RAD51 protein expression levels [P < 0.05; Table 2]. Conversely, there was no significant correlation between clinicopathological features and RAD51 expression in low-grade tumors (P > 0.05).\\nFinally, among the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of RAD51-positive and negative patients were 3.0% and 42.9%, respectively [P < 0.05; Figure 6]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of RAD51 showed no significant association with the 5-year survival rate (P > 0.05).\\nFive-year survival curves of RAD51-positive and negative patients with high-grade ovarian serous adenocarcinomas.\",\n \"The Cox multivariate analysis results showed that FIGO staging, LNM, and RAD51 protein expression were independent prognostic factors of patients with high-grade serous ovarian adenocarcinoma [P < 0.05; Table 5].\\nMultivariate analysis of independent factors associated with the 5-year overall survival rate in patients with high-grade serous ovarian adenocarcinoma\\nRR: Risk ratio; CI: Confidence interval; LNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a; PHLPP: PH domain leucine-rich repeat protein phosphatase.\",\n \"The tumor suppressor PHLPP, located at 18q21.33, can directly dephosphorylate ser473 in the hydrophobic group of Akt, thus negatively regulating Akt and its downstream targets. It has been reported that PHLPP can act synergistically with PTEN, another well-known tumor suppressor gene, thereby significantly inhibiting the proliferation of colorectal cancer cells, and negatively regulating PI3K/Akt signaling.[11] Accordingly, studies have shown that the PHLPP expression is significantly decreased in colon, breast, prostate, and lung cancers.[5712]\\nThe results of the present study showed that the positive rate of PHLPP protein expression in the high-grade group was significantly lower than that in the oviduct group. The PHLPP-positive expression rate was decreased gradually from normal ovarian tissue to benign tumors to borderline tumors to low-grade adenocarcinomas, indicating that loss of PHLPP expression may be associated with the initiation of ovarian serous adenocarcinoma development, whereas no significant difference was observed between high-grade and low-grade tumors. Further, the PHLPP expression level showed significant associations with LNM, FIGO stages, and omental metastasis in high-grade tumors, but not in low-grade tumors. Similarly, the 5-year survival rates of PHLPP-positive and negative high-grade tumors significant differed, whereas no difference was seen in the low-grade tumors. These findings suggest that PHLPP may be a predictor of prognosis of high-grade ovarian serous adenocarcinoma.\",\n \"FoxO3a is an important member of the FOXO family, located on at 6q21, and encodes a 673-amino acid long protein.[13] FoxO3a, as an important signaling molecule downstream of Akt in the PI3K-Akt signaling pathway, binds to the 14-3-3 chaperone protein after being phosphorylated by Akt, and subsequently translocates from the nucleus to the cytoplasm, thereby activating the apoptotic genes Bim, FasL, and tumor necrosis factor and its related apoptosis-inducing ligand TRAIL, consequently promoting apoptosis.[14] In addition to its role in apoptosis, FoxO3a plays critical roles in regulating cell proliferation, metabolism, the stress response, and the life span of cancer cells.[15] Particularly, another target molecule of FoxO3a is the cell cycle-regulating gene P27; overexpression of FoxO3a can arrest the cell cycle in the G0/G1 phase by upregulating P27. In addition, overexpression of FoxO3a can significantly inhibit the proliferation of tumor cells and result in tumor cell G2 arrest, further promoting tumor cell apoptosis.[1617]\\nThe results of our analyses showed that the FoxO3a-positive rate in the high-grade tumors was significantly lower than that in the normal oviducts. In the low-grade adenocarcinomas, borderline cystadenomas, cystadenomas, and normal ovaries, the positive expression rates of FoxO3a protein were gradually reduced from normal ovaries to the low-grade adenocarcinomas, and the FoxO3 positive rate of the low-grade tumors was significantly lower than those of the borderline cystadenomas, cystadenomas, and normal ovaries, while the positive rate of borderline cystadenomas was significantly lower than those of cystadenomas and normal ovaries. Moreover, the FoxO3a expression level showed significant associations with LNM, FIGO stage, and omental metastasis in high-grade, but not low-grade tumors. These results suggest that FoxO3a may play a role in the oncogenesis and development of ovarian serous adenocarcinomas. In the high-grade tumors, the 5-year survival rate of FoxO3a-positive patients was significantly higher than that of the negative patients, whereas no difference in survival was seen in low-grade tumors, indicating that positive FoxO3a expression may have a long-term protective effect on cancer patients, and FoxO3a may hence have certain significance in the prediction of the patients’ long-term prognosis. Interestingly, it has been reported that tumor cells with low FoxO3a expression can develop drug resistance to paclitaxel and that inhibiting FoxO3a expression with RNAi can reduce E1A-mediated sensitivity to paclitaxel.[18] Therefore, FoxO3a protein may represent a potential prognostic indicator for high-grade ovarian serous adenocarcinoma.\",\n \"RAD51 is a human homologous recombination repair gene. Too strong or too weak homologous recombination will result in genome instability and DNA damage, and the consequent gene loss, loss of heterozygosity, and/or genetic translocation are important causes of tumorigenesis and tumor progression.[19] Studies have found that RAD51 protein is overexpression in the breast, prostate, pancreatic, and colon cancers. Such overexpression of RAD51 can provide certain advantages for the growth of cancer cells by promoting the initiation and progression of cancer. However, to date, studies on the expression of RAD51 in ovarian cancers are few.\\nThe results of this study showed that the RAD51-positive rate in high-grade tumors was significantly higher than that of normal oviducts, suggesting that RAD51 may play a role in the initiation of high-grade ovarian serous carcinomas. The RAD51-positive rate gradually increased from normal ovaries to cystadenomas to borderline cystadenomas to low-grade serous adenocarcinomas. Particularly, the positive rate of low-grade tumors was significantly higher than that in benign tumors and normal oviducts. The gradual change in the RAD51 expression suggests that the initiation of low-grade serous adenocarcinoma is a gradual procedure from benign to borderline to low-grade adenocarcinoma. However, no significant difference in the expression of RAD51 was found between high- and low-grade tumors. The expression level of RAD51 showed significant associations with LNM, FIGO stage, and omental metastasis in high-grade, but not low-grade tumors.\\nHerein, the 5-year survival rate of patients with RAD51-positive high-grade adenocarcinomas was significantly lower than that of patients with RAD51-negative tumors, while no difference in survival was found in low-grade tumors. In other words, we found that high-grade tumor patients with high expression of RAD51 had a lower survival rate and poor prognosis, indicating that RAD51 may be a prognostic factor in high-grade ovarian serous adenocarcinoma. Furthermore, multivariate survival analyses showed that RAD51 was an independent prognostic factor, which may be of clinical significance in terms of the development of molecular targeted therapy, in addition to FIGO stage, and LNM. Further investigations on RAD51 are required to confirm these findings and to hopefully be able to improve the quality of life and prognosis of patients using targeted therapy.\\nIn conclusion, the results of the present study indicate that PHLPP, FoxO3a, and RAD51 may play certain roles in the pathogenesis of the different grades of ovarian serous adenocarcinomas. Meanwhile, they also suggest the possible presence of common molecular mechanisms in the pathogenesis of the different grades. Increased protein expression of RAD51 may be an important molecular marker for poor prognosis of high-grade ovarian serous adenocarcinomas. However, considering the limited number of cases selected, which may have affected the significance of the results, further studies are required to improve and supplement these findings.\",\n \"This work were supported by grants from the Key Scientific Research program in Medical Science of Hebei Province, China (No. 20150318) and Science and Technology support program of Hebei Province, China (No. 16277794D).\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Introduction","Methods","Ethics","Participant selection and description","Technical information","PH domain leucine-rich repeat protein phosphatase-positive criteria","Forkhead homeobox type O 3a-positive criteria","RAD51-positive criteria","Statistical analyses","Results","Expression of PH domain leucine-rich repeat protein phosphatase in ovarian serous adenocarcinomas and its association with clinicopathological features and prognosis","Expression of forkhead homeobox type O 3a in ovarian serous adenocarcinoma and its association with clinicopathological features and prognosis","Expression of RAD51 in ovarian serous adenocarcinoma and its association with clinicopathological features and prognosis","Prognostic multivariate survival analyses of high-grade serous ovarian adenocarcinoma","Discussion","Role of the tumor suppressor PH domain leucine-rich repeat protein phosphatase in the development of ovarian serous adenocarcinoma and its association with patient prognosis","Role of the tumor suppressor forkhead homeobox type O 3a in the development of ovarian serous adenocarcinoma and its association with patient prognosis","Role of RAD51 in the development of ovarian serous adenocarcinoma and its association with patient prognosis","Financial support and sponsorship","Conflicts of interest"],"string":"[\n \"Introduction\",\n \"Methods\",\n \"Ethics\",\n \"Participant selection and description\",\n \"Technical information\",\n \"PH domain leucine-rich repeat protein phosphatase-positive criteria\",\n \"Forkhead homeobox type O 3a-positive criteria\",\n \"RAD51-positive criteria\",\n \"Statistical analyses\",\n \"Results\",\n \"Expression of PH domain leucine-rich repeat protein phosphatase in ovarian serous adenocarcinomas and its association with clinicopathological features and prognosis\",\n \"Expression of forkhead homeobox type O 3a in ovarian serous adenocarcinoma and its association with clinicopathological features and prognosis\",\n \"Expression of RAD51 in ovarian serous adenocarcinoma and its association with clinicopathological features and prognosis\",\n \"Prognostic multivariate survival analyses of high-grade serous ovarian adenocarcinoma\",\n \"Discussion\",\n \"Role of the tumor suppressor PH domain leucine-rich repeat protein phosphatase in the development of ovarian serous adenocarcinoma and its association with patient prognosis\",\n \"Role of the tumor suppressor forkhead homeobox type O 3a in the development of ovarian serous adenocarcinoma and its association with patient prognosis\",\n \"Role of RAD51 in the development of ovarian serous adenocarcinoma and its association with patient prognosis\",\n \"Financial support and sponsorship\",\n \"Conflicts of interest\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Ovarian serous adenocarcinoma is the most common ovarian epithelial malignant tumor. It can be divided into low- and high-grade tumors, which exhibit substantial differences in the pathogenesis, clinicopathological characteristics, and prognosis.[1] Low-grade serous carcinomas undergo a progressive, gradual developmental process from benign serous cystadenoma to borderline serous cystadenoma to malignant serous adenocarcinoma, while high-grade serous adenocarcinomas develop directly from the oviduct epithelium or ovarian inclusion cysts.\nHowever, it remains unclear whether the two tumor types have completely different molecular mechanisms, whether there is a common molecular basis, and whether the genes involved in the development of these tumors affect the patients’ prognosis. Recent studies[23] have indicated that the PI3K/Akt signaling pathway is involved in the initiation and development of ovarian serous adenocarcinoma. For example, PH domain leucine-rich repeat protein phosphatase (PHLPP) is known to negatively regulate Akt and its downstream kinase by dephosphorylating the hydrophobic core of Akt (Akt1 Ser473), thereby antagonizing PI3K/Akt signaling and inhibiting tumor growth. Forkhead homeobox type O 3a (FoxO3a), an important downstream signaling molecule of Akt, is involved in the initiation and development of tumor cells and regulation of cell proliferation after being phosphorylated by Akt.[45] Further, RAD51, a DNA double-strand break repair gene,[6] has also been implicated in the PI3K/Akt signaling pathway: phosphorylated FoxO3a binds to the promoter of the downstream gene RAD51, thereby regulating RAD51 and promoting tumor development and metastasis.\nThe present study aimed to (1) determine the differences in the protein expressions of PHLPP, FoxO3a, and RAD51 between high- and low-grade ovarian serous adenocarcinomas, (2) explore the roles of these three genes in the initiation of the different grades of ovarian serous adenocarcinoma, and (3) analyze the relationships between their protein expressions and prognosis."," Ethics The study was approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University and was conducted in accordance with the Declaration of Helsinki 1975, as revised in 2000.\nThe study was approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University and was conducted in accordance with the Declaration of Helsinki 1975, as revised in 2000.\n Participant selection and description Patients with ovarian serous adenocarcinoma hospitalized and treated from January 2006 to January 2011 in our hospital were included in the ovarian serous adenocarcinoma groups. No patients received chemotherapy and radiotherapy preoperatively, and their diagnosis was confirmed by pathological examinations postoperatively. All patients had complete clinical data and were graded according to the World Health Organization histological classification for ovarian tumors (2014 edition). Ninety-four and twenty-six cases of high- and low-grade ovarian serous adenocarcinomas were included in this study. Among the high-grade group (mean age, 55 years; range, 36–78 years), there were 26 and 68 patients with International Federation of Gynecology and Obstetrics (FIGO) Stage (2014 edition) I–II and III–IV disease, respectively. There were thirty and 64 patients with and without lymph node metastasis (LNM), and 41 and 53 patients with and without omental metastasis, respectively. Among the low-grade group (mean age, 44 years; range, 26–70 years), there were 18 and eight patients with FIGO Stage I–II and III–IV disease, respectively. There were 4 and 22 patients with and without LNM, and 5 and 21 patients with and without omental metastasis, respectively. Moreover, 21 cases of borderline serous cystadenoma (mean age, 38 years; range, 18–59 years) and 35 cases of benign serous cystadenoma (mean age, 45 years; range, 22–69 years) were included, along with thirty normal ovaries and thirty oviducts (mean age, 48 years; range, 36–62 years) removed from patients undergoing surgery for uterine fibroids, which were used as controls for the low- and high-grade tumor groups, respectively.\nExpression of TP53 protein in high- and low-grade serous carcinoma: Among the 94 cases of high-grade, 92 cases (97.9%) showed strong staining of TP53, while among the 26 cases of low-grade serous adenocarcinoma, there was no case of strong staining.\nPatients with ovarian serous adenocarcinoma hospitalized and treated from January 2006 to January 2011 in our hospital were included in the ovarian serous adenocarcinoma groups. No patients received chemotherapy and radiotherapy preoperatively, and their diagnosis was confirmed by pathological examinations postoperatively. All patients had complete clinical data and were graded according to the World Health Organization histological classification for ovarian tumors (2014 edition). Ninety-four and twenty-six cases of high- and low-grade ovarian serous adenocarcinomas were included in this study. Among the high-grade group (mean age, 55 years; range, 36–78 years), there were 26 and 68 patients with International Federation of Gynecology and Obstetrics (FIGO) Stage (2014 edition) I–II and III–IV disease, respectively. There were thirty and 64 patients with and without lymph node metastasis (LNM), and 41 and 53 patients with and without omental metastasis, respectively. Among the low-grade group (mean age, 44 years; range, 26–70 years), there were 18 and eight patients with FIGO Stage I–II and III–IV disease, respectively. There were 4 and 22 patients with and without LNM, and 5 and 21 patients with and without omental metastasis, respectively. Moreover, 21 cases of borderline serous cystadenoma (mean age, 38 years; range, 18–59 years) and 35 cases of benign serous cystadenoma (mean age, 45 years; range, 22–69 years) were included, along with thirty normal ovaries and thirty oviducts (mean age, 48 years; range, 36–62 years) removed from patients undergoing surgery for uterine fibroids, which were used as controls for the low- and high-grade tumor groups, respectively.\nExpression of TP53 protein in high- and low-grade serous carcinoma: Among the 94 cases of high-grade, 92 cases (97.9%) showed strong staining of TP53, while among the 26 cases of low-grade serous adenocarcinoma, there was no case of strong staining.\n Technical information \nExperimental procedures\n\nThe operative specimens were examined, and the patients were diagnosed by experienced pathologists. Paraffin blocks of the specimens were cut, and immunohistochemical staining of the sections was conducted as follows: the samples were sliced, deparaffinized, dehydrated, and subjected to antigen retrieval before blocking with goat serum. Subsequently, the slides were placed in a humidifier chamber and incubated at room temperature for 45 min. The tissues were incubated with primary antibodies (working concentrations of anti-PHLPP, RAD51, and FoxO3a of 1:100, 1:100, and 1:150, respectively, Biogot Biotechnology Co., Ltd., Nanjing, China) in the sealed humidifier chamber at 4°C overnight. After washing of the slides, goat anti-rabbit secondary antibodies were added and the slides were incubated in the humidifier at 37°C for 30 min. Next, horseradish peroxidase-labeled streptavidin was added and the slides were incubated in the humidifier at 37°C for 30 min, after which the tissues were stained by 3,3’-diaminobenzidine, followed by counterstaining, destaining, dehydration, clearing, and mounting. Finally, the slides were air-dried and observed under a microscope.\n\nEvaluation standard for staining\n\nThe slides were scored by pathologists according to the below criteria for PHLPP, FoxO3a, and RAD51 under a microscope. Ten high-magnification fields were counted, and the average cell numbers were used.\n PH domain leucine-rich repeat protein phosphatase-positive criteria Cells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\nCells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\n Forkhead homeobox type O 3a-positive criteria Cells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\nCells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\n RAD51-positive criteria Cells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\nCells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\n\nExperimental procedures\n\nThe operative specimens were examined, and the patients were diagnosed by experienced pathologists. Paraffin blocks of the specimens were cut, and immunohistochemical staining of the sections was conducted as follows: the samples were sliced, deparaffinized, dehydrated, and subjected to antigen retrieval before blocking with goat serum. Subsequently, the slides were placed in a humidifier chamber and incubated at room temperature for 45 min. The tissues were incubated with primary antibodies (working concentrations of anti-PHLPP, RAD51, and FoxO3a of 1:100, 1:100, and 1:150, respectively, Biogot Biotechnology Co., Ltd., Nanjing, China) in the sealed humidifier chamber at 4°C overnight. After washing of the slides, goat anti-rabbit secondary antibodies were added and the slides were incubated in the humidifier at 37°C for 30 min. Next, horseradish peroxidase-labeled streptavidin was added and the slides were incubated in the humidifier at 37°C for 30 min, after which the tissues were stained by 3,3’-diaminobenzidine, followed by counterstaining, destaining, dehydration, clearing, and mounting. Finally, the slides were air-dried and observed under a microscope.\n\nEvaluation standard for staining\n\nThe slides were scored by pathologists according to the below criteria for PHLPP, FoxO3a, and RAD51 under a microscope. Ten high-magnification fields were counted, and the average cell numbers were used.\n PH domain leucine-rich repeat protein phosphatase-positive criteria Cells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\nCells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\n Forkhead homeobox type O 3a-positive criteria Cells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\nCells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\n RAD51-positive criteria Cells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\nCells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\n Statistical analyses SPSS 16.0 software (SPSS Inc., USA) was used for the statistical analysis. Categorical data were analyzed by the Chi-square test, and the survival rates were calculated by Kaplan-Meier univariate analysis. Differences in the survival rates between the different groups were determined by the log-rank test. Cox multivariate survival analysis was used for analysis of independent prognostic factors. The statistical significance level was set as P < 0.05.\nSPSS 16.0 software (SPSS Inc., USA) was used for the statistical analysis. Categorical data were analyzed by the Chi-square test, and the survival rates were calculated by Kaplan-Meier univariate analysis. Differences in the survival rates between the different groups were determined by the log-rank test. Cox multivariate survival analysis was used for analysis of independent prognostic factors. The statistical significance level was set as P < 0.05.","The study was approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University and was conducted in accordance with the Declaration of Helsinki 1975, as revised in 2000.","Patients with ovarian serous adenocarcinoma hospitalized and treated from January 2006 to January 2011 in our hospital were included in the ovarian serous adenocarcinoma groups. No patients received chemotherapy and radiotherapy preoperatively, and their diagnosis was confirmed by pathological examinations postoperatively. All patients had complete clinical data and were graded according to the World Health Organization histological classification for ovarian tumors (2014 edition). Ninety-four and twenty-six cases of high- and low-grade ovarian serous adenocarcinomas were included in this study. Among the high-grade group (mean age, 55 years; range, 36–78 years), there were 26 and 68 patients with International Federation of Gynecology and Obstetrics (FIGO) Stage (2014 edition) I–II and III–IV disease, respectively. There were thirty and 64 patients with and without lymph node metastasis (LNM), and 41 and 53 patients with and without omental metastasis, respectively. Among the low-grade group (mean age, 44 years; range, 26–70 years), there were 18 and eight patients with FIGO Stage I–II and III–IV disease, respectively. There were 4 and 22 patients with and without LNM, and 5 and 21 patients with and without omental metastasis, respectively. Moreover, 21 cases of borderline serous cystadenoma (mean age, 38 years; range, 18–59 years) and 35 cases of benign serous cystadenoma (mean age, 45 years; range, 22–69 years) were included, along with thirty normal ovaries and thirty oviducts (mean age, 48 years; range, 36–62 years) removed from patients undergoing surgery for uterine fibroids, which were used as controls for the low- and high-grade tumor groups, respectively.\nExpression of TP53 protein in high- and low-grade serous carcinoma: Among the 94 cases of high-grade, 92 cases (97.9%) showed strong staining of TP53, while among the 26 cases of low-grade serous adenocarcinoma, there was no case of strong staining.","\nExperimental procedures\n\nThe operative specimens were examined, and the patients were diagnosed by experienced pathologists. Paraffin blocks of the specimens were cut, and immunohistochemical staining of the sections was conducted as follows: the samples were sliced, deparaffinized, dehydrated, and subjected to antigen retrieval before blocking with goat serum. Subsequently, the slides were placed in a humidifier chamber and incubated at room temperature for 45 min. The tissues were incubated with primary antibodies (working concentrations of anti-PHLPP, RAD51, and FoxO3a of 1:100, 1:100, and 1:150, respectively, Biogot Biotechnology Co., Ltd., Nanjing, China) in the sealed humidifier chamber at 4°C overnight. After washing of the slides, goat anti-rabbit secondary antibodies were added and the slides were incubated in the humidifier at 37°C for 30 min. Next, horseradish peroxidase-labeled streptavidin was added and the slides were incubated in the humidifier at 37°C for 30 min, after which the tissues were stained by 3,3’-diaminobenzidine, followed by counterstaining, destaining, dehydration, clearing, and mounting. Finally, the slides were air-dried and observed under a microscope.\n\nEvaluation standard for staining\n\nThe slides were scored by pathologists according to the below criteria for PHLPP, FoxO3a, and RAD51 under a microscope. Ten high-magnification fields were counted, and the average cell numbers were used.\n PH domain leucine-rich repeat protein phosphatase-positive criteria Cells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\nCells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\n Forkhead homeobox type O 3a-positive criteria Cells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\nCells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\n RAD51-positive criteria Cells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\nCells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]","Cells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]","Cells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]","Cells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]","SPSS 16.0 software (SPSS Inc., USA) was used for the statistical analysis. Categorical data were analyzed by the Chi-square test, and the survival rates were calculated by Kaplan-Meier univariate analysis. Differences in the survival rates between the different groups were determined by the log-rank test. Cox multivariate survival analysis was used for analysis of independent prognostic factors. The statistical significance level was set as P < 0.05."," Expression of PH domain leucine-rich repeat protein phosphatase in ovarian serous adenocarcinomas and its association with clinicopathological features and prognosis Among 94 cases of high-grade serous adenocarcinoma, 23 cases (24.5%) were PHLPP-positive, which was significantly lower than the positive rate of 86.7% in the normal oviduct group (P < 0.05). Of 26 low-grade serous adenocarcinoma cases, 6 (23.1%) were positive, which was significantly lower than that in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, there was no difference in the positive expression rates between high- and low-grade tumors [P > 0.05; Figure 1 and Table 1].\nProtein expression of PH domain leucine-rich repeat protein phosphatase in high- and low-grade serous ovarian adenocarcinoma by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\nProtein expression of PHLPP in high- and low-grade ovarian serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. PHLPP: PH domain leucine-rich repeat protein phosphatase.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower PHLPP protein expression levels [P < 0.05; Table 2]. On the other hand, there were no significant correlations between the clinicopathological features and PHLPP protein expression in low-grade tumors [P > 0.05].\nRelationships between PHLPP, FoxO3a, and RAD51 protein expressions and clinicopathological features of high-grade serous ovarian adenocarcinoma\nLNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a.\nAmong the 94 patients with high-grade serous adenocarcinoma, the 5-year survival rates of PHLPP-negative and positive patients were 5.6% and 43.5%, respectively [P < 0.05, Figure 2]. Among the 26 cases of low-grade serous adenocarcinoma, however, the expression of PHLPP did not significantly associate with the 5-year survival rate of the patients [P > 0.05].\nFive-year survival curves of PH domain leucine-rich repeat protein phosphatase-positive and negative patients with high-grade ovarian serous adenocarcinomas.\nAmong 94 cases of high-grade serous adenocarcinoma, 23 cases (24.5%) were PHLPP-positive, which was significantly lower than the positive rate of 86.7% in the normal oviduct group (P < 0.05). Of 26 low-grade serous adenocarcinoma cases, 6 (23.1%) were positive, which was significantly lower than that in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, there was no difference in the positive expression rates between high- and low-grade tumors [P > 0.05; Figure 1 and Table 1].\nProtein expression of PH domain leucine-rich repeat protein phosphatase in high- and low-grade serous ovarian adenocarcinoma by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\nProtein expression of PHLPP in high- and low-grade ovarian serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. PHLPP: PH domain leucine-rich repeat protein phosphatase.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower PHLPP protein expression levels [P < 0.05; Table 2]. On the other hand, there were no significant correlations between the clinicopathological features and PHLPP protein expression in low-grade tumors [P > 0.05].\nRelationships between PHLPP, FoxO3a, and RAD51 protein expressions and clinicopathological features of high-grade serous ovarian adenocarcinoma\nLNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a.\nAmong the 94 patients with high-grade serous adenocarcinoma, the 5-year survival rates of PHLPP-negative and positive patients were 5.6% and 43.5%, respectively [P < 0.05, Figure 2]. Among the 26 cases of low-grade serous adenocarcinoma, however, the expression of PHLPP did not significantly associate with the 5-year survival rate of the patients [P > 0.05].\nFive-year survival curves of PH domain leucine-rich repeat protein phosphatase-positive and negative patients with high-grade ovarian serous adenocarcinomas.\n Expression of forkhead homeobox type O 3a in ovarian serous adenocarcinoma and its association with clinicopathological features and prognosis Among the 94 cases of high-grade serous adenocarcinoma, 25 (26.6%) were positive for FoxO3a protein expression, which was significantly lower than the positive rate of 90.0% in the normal oviduct group (P < 0.05). Among the 26 cases of low-grade serous carcinoma, 7 (26.9%) were positive, which was significantly lower than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, the difference in the positive expression rates between the high- and low-grade groups was not significant [P > 0.05; Figure 3 and Table 3].\nProtein expression of forkhead homeobox type O 3a in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\nProtein expression of FoxO3a in high- and low-grade serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. FoxO3a: Forkhead homeobox type O 3a.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower FoxO3a protein expression [P < 0.05, Table 2]. On the other hand, in low-grade tumors, the FoxO3a protein expression showed no association with clinicopathological features (P > 0.05).\nAmong the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of FoxO3a-negative and positive patients were 7.2% and 36.0%, respectively [P < 0.05, Figure 4]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of FoxO3a showed no significant association with the 5-year survival rate (P > 0.05).\nFive-year survival curves of forkhead homeobox type O 3a-positive and negative patients with high-grade ovarian serous adenocarcinomas.\nAmong the 94 cases of high-grade serous adenocarcinoma, 25 (26.6%) were positive for FoxO3a protein expression, which was significantly lower than the positive rate of 90.0% in the normal oviduct group (P < 0.05). Among the 26 cases of low-grade serous carcinoma, 7 (26.9%) were positive, which was significantly lower than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, the difference in the positive expression rates between the high- and low-grade groups was not significant [P > 0.05; Figure 3 and Table 3].\nProtein expression of forkhead homeobox type O 3a in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\nProtein expression of FoxO3a in high- and low-grade serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. FoxO3a: Forkhead homeobox type O 3a.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower FoxO3a protein expression [P < 0.05, Table 2]. On the other hand, in low-grade tumors, the FoxO3a protein expression showed no association with clinicopathological features (P > 0.05).\nAmong the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of FoxO3a-negative and positive patients were 7.2% and 36.0%, respectively [P < 0.05, Figure 4]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of FoxO3a showed no significant association with the 5-year survival rate (P > 0.05).\nFive-year survival curves of forkhead homeobox type O 3a-positive and negative patients with high-grade ovarian serous adenocarcinomas.\n Expression of RAD51 in ovarian serous adenocarcinoma and its association with clinicopathological features and prognosis Among the 94 cases of high-grade serous adenocarcinoma, 66 (70.2%) were positive for RAD51 protein, which was significantly higher than that in the normal oviduct group [P < 0.05; Figure 5 and Table 4]. Among the 26 cases of low-grade serous carcinoma, 17 (65.4%) were positive, which was significantly higher than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups [P < 0.05; Figure 5 and Table 4]. However, the difference in the positive expression rates between the high- and low-grade groups was not significant (P > 0.05).\nProtein expression of RAD51 in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Positive high-grade adenocarcinoma, (b) negative normal oviduct, (c) positive low-grade adenocarcinoma, (d) negative borderline cystadenoma, (e) negative cystadenoma, (f) negative normal ovary.\nProtein expression of RAD51 in high- and low-grade serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with higher RAD51 protein expression levels [P < 0.05; Table 2]. Conversely, there was no significant correlation between clinicopathological features and RAD51 expression in low-grade tumors (P > 0.05).\nFinally, among the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of RAD51-positive and negative patients were 3.0% and 42.9%, respectively [P < 0.05; Figure 6]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of RAD51 showed no significant association with the 5-year survival rate (P > 0.05).\nFive-year survival curves of RAD51-positive and negative patients with high-grade ovarian serous adenocarcinomas.\nAmong the 94 cases of high-grade serous adenocarcinoma, 66 (70.2%) were positive for RAD51 protein, which was significantly higher than that in the normal oviduct group [P < 0.05; Figure 5 and Table 4]. Among the 26 cases of low-grade serous carcinoma, 17 (65.4%) were positive, which was significantly higher than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups [P < 0.05; Figure 5 and Table 4]. However, the difference in the positive expression rates between the high- and low-grade groups was not significant (P > 0.05).\nProtein expression of RAD51 in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Positive high-grade adenocarcinoma, (b) negative normal oviduct, (c) positive low-grade adenocarcinoma, (d) negative borderline cystadenoma, (e) negative cystadenoma, (f) negative normal ovary.\nProtein expression of RAD51 in high- and low-grade serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with higher RAD51 protein expression levels [P < 0.05; Table 2]. Conversely, there was no significant correlation between clinicopathological features and RAD51 expression in low-grade tumors (P > 0.05).\nFinally, among the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of RAD51-positive and negative patients were 3.0% and 42.9%, respectively [P < 0.05; Figure 6]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of RAD51 showed no significant association with the 5-year survival rate (P > 0.05).\nFive-year survival curves of RAD51-positive and negative patients with high-grade ovarian serous adenocarcinomas.\n Prognostic multivariate survival analyses of high-grade serous ovarian adenocarcinoma The Cox multivariate analysis results showed that FIGO staging, LNM, and RAD51 protein expression were independent prognostic factors of patients with high-grade serous ovarian adenocarcinoma [P < 0.05; Table 5].\nMultivariate analysis of independent factors associated with the 5-year overall survival rate in patients with high-grade serous ovarian adenocarcinoma\nRR: Risk ratio; CI: Confidence interval; LNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a; PHLPP: PH domain leucine-rich repeat protein phosphatase.\nThe Cox multivariate analysis results showed that FIGO staging, LNM, and RAD51 protein expression were independent prognostic factors of patients with high-grade serous ovarian adenocarcinoma [P < 0.05; Table 5].\nMultivariate analysis of independent factors associated with the 5-year overall survival rate in patients with high-grade serous ovarian adenocarcinoma\nRR: Risk ratio; CI: Confidence interval; LNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a; PHLPP: PH domain leucine-rich repeat protein phosphatase.","Among 94 cases of high-grade serous adenocarcinoma, 23 cases (24.5%) were PHLPP-positive, which was significantly lower than the positive rate of 86.7% in the normal oviduct group (P < 0.05). Of 26 low-grade serous adenocarcinoma cases, 6 (23.1%) were positive, which was significantly lower than that in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, there was no difference in the positive expression rates between high- and low-grade tumors [P > 0.05; Figure 1 and Table 1].\nProtein expression of PH domain leucine-rich repeat protein phosphatase in high- and low-grade serous ovarian adenocarcinoma by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\nProtein expression of PHLPP in high- and low-grade ovarian serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. PHLPP: PH domain leucine-rich repeat protein phosphatase.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower PHLPP protein expression levels [P < 0.05; Table 2]. On the other hand, there were no significant correlations between the clinicopathological features and PHLPP protein expression in low-grade tumors [P > 0.05].\nRelationships between PHLPP, FoxO3a, and RAD51 protein expressions and clinicopathological features of high-grade serous ovarian adenocarcinoma\nLNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a.\nAmong the 94 patients with high-grade serous adenocarcinoma, the 5-year survival rates of PHLPP-negative and positive patients were 5.6% and 43.5%, respectively [P < 0.05, Figure 2]. Among the 26 cases of low-grade serous adenocarcinoma, however, the expression of PHLPP did not significantly associate with the 5-year survival rate of the patients [P > 0.05].\nFive-year survival curves of PH domain leucine-rich repeat protein phosphatase-positive and negative patients with high-grade ovarian serous adenocarcinomas.","Among the 94 cases of high-grade serous adenocarcinoma, 25 (26.6%) were positive for FoxO3a protein expression, which was significantly lower than the positive rate of 90.0% in the normal oviduct group (P < 0.05). Among the 26 cases of low-grade serous carcinoma, 7 (26.9%) were positive, which was significantly lower than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, the difference in the positive expression rates between the high- and low-grade groups was not significant [P > 0.05; Figure 3 and Table 3].\nProtein expression of forkhead homeobox type O 3a in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\nProtein expression of FoxO3a in high- and low-grade serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. FoxO3a: Forkhead homeobox type O 3a.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower FoxO3a protein expression [P < 0.05, Table 2]. On the other hand, in low-grade tumors, the FoxO3a protein expression showed no association with clinicopathological features (P > 0.05).\nAmong the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of FoxO3a-negative and positive patients were 7.2% and 36.0%, respectively [P < 0.05, Figure 4]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of FoxO3a showed no significant association with the 5-year survival rate (P > 0.05).\nFive-year survival curves of forkhead homeobox type O 3a-positive and negative patients with high-grade ovarian serous adenocarcinomas.","Among the 94 cases of high-grade serous adenocarcinoma, 66 (70.2%) were positive for RAD51 protein, which was significantly higher than that in the normal oviduct group [P < 0.05; Figure 5 and Table 4]. Among the 26 cases of low-grade serous carcinoma, 17 (65.4%) were positive, which was significantly higher than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups [P < 0.05; Figure 5 and Table 4]. However, the difference in the positive expression rates between the high- and low-grade groups was not significant (P > 0.05).\nProtein expression of RAD51 in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Positive high-grade adenocarcinoma, (b) negative normal oviduct, (c) positive low-grade adenocarcinoma, (d) negative borderline cystadenoma, (e) negative cystadenoma, (f) negative normal ovary.\nProtein expression of RAD51 in high- and low-grade serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with higher RAD51 protein expression levels [P < 0.05; Table 2]. Conversely, there was no significant correlation between clinicopathological features and RAD51 expression in low-grade tumors (P > 0.05).\nFinally, among the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of RAD51-positive and negative patients were 3.0% and 42.9%, respectively [P < 0.05; Figure 6]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of RAD51 showed no significant association with the 5-year survival rate (P > 0.05).\nFive-year survival curves of RAD51-positive and negative patients with high-grade ovarian serous adenocarcinomas.","The Cox multivariate analysis results showed that FIGO staging, LNM, and RAD51 protein expression were independent prognostic factors of patients with high-grade serous ovarian adenocarcinoma [P < 0.05; Table 5].\nMultivariate analysis of independent factors associated with the 5-year overall survival rate in patients with high-grade serous ovarian adenocarcinoma\nRR: Risk ratio; CI: Confidence interval; LNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a; PHLPP: PH domain leucine-rich repeat protein phosphatase.","Ovarian cancer is one of the most common malignancies of the female reproductive system, and has the highest mortality among all gynecologic malignant tumors, thus seriously affecting women's health and lives. Recent studies have suggested that ovarian serous adenocarcinomas can be divided into high- and low-grade tumors and that the initiation mechanisms, clinical pathological features, and prognoses of the two types greatly differ.[110] Thus, ovarian serous adenocarcinomas have complex biological behaviors. However, although the pathogeneses of the two types involve different molecular pathways, there is some common basis. Recent studies have found that the PI3K/Akt signaling pathway is closely associated with the initiation and progression of ovarian cancer. PHLPP, a known tumor suppressor, can negatively regulate Akt and its downstream kinases, thereby antagonizing the PI3K/Akt signaling pathway and inhibit tumor growth. Moreover, as an important downstream signaling molecule of Akt, after being phosphorylated by Akt, FoxO3a is involved in the initiation and development of tumor cells and regulates cell proliferation. RAD51, a downstream target of FoxO3a, also plays an important role in PI3K/Akt signaling; FoxO3a binds to the promoter of RAD51, consequently regulating RAD51 and promoting tumor development and metastasis. In this study, by examining the differences in the protein expression levels of PHLPP, FoxO3a, and RAD51 between high- and low-grade ovarian serous adenocarcinomas, the roles of the three genes in the development of the different grades of ovarian serous adenocarcinoma were explored, and the relationships between the protein expression levels and the patient prognosis were analyzed.\n Role of the tumor suppressor PH domain leucine-rich repeat protein phosphatase in the development of ovarian serous adenocarcinoma and its association with patient prognosis The tumor suppressor PHLPP, located at 18q21.33, can directly dephosphorylate ser473 in the hydrophobic group of Akt, thus negatively regulating Akt and its downstream targets. It has been reported that PHLPP can act synergistically with PTEN, another well-known tumor suppressor gene, thereby significantly inhibiting the proliferation of colorectal cancer cells, and negatively regulating PI3K/Akt signaling.[11] Accordingly, studies have shown that the PHLPP expression is significantly decreased in colon, breast, prostate, and lung cancers.[5712]\nThe results of the present study showed that the positive rate of PHLPP protein expression in the high-grade group was significantly lower than that in the oviduct group. The PHLPP-positive expression rate was decreased gradually from normal ovarian tissue to benign tumors to borderline tumors to low-grade adenocarcinomas, indicating that loss of PHLPP expression may be associated with the initiation of ovarian serous adenocarcinoma development, whereas no significant difference was observed between high-grade and low-grade tumors. Further, the PHLPP expression level showed significant associations with LNM, FIGO stages, and omental metastasis in high-grade tumors, but not in low-grade tumors. Similarly, the 5-year survival rates of PHLPP-positive and negative high-grade tumors significant differed, whereas no difference was seen in the low-grade tumors. These findings suggest that PHLPP may be a predictor of prognosis of high-grade ovarian serous adenocarcinoma.\nThe tumor suppressor PHLPP, located at 18q21.33, can directly dephosphorylate ser473 in the hydrophobic group of Akt, thus negatively regulating Akt and its downstream targets. It has been reported that PHLPP can act synergistically with PTEN, another well-known tumor suppressor gene, thereby significantly inhibiting the proliferation of colorectal cancer cells, and negatively regulating PI3K/Akt signaling.[11] Accordingly, studies have shown that the PHLPP expression is significantly decreased in colon, breast, prostate, and lung cancers.[5712]\nThe results of the present study showed that the positive rate of PHLPP protein expression in the high-grade group was significantly lower than that in the oviduct group. The PHLPP-positive expression rate was decreased gradually from normal ovarian tissue to benign tumors to borderline tumors to low-grade adenocarcinomas, indicating that loss of PHLPP expression may be associated with the initiation of ovarian serous adenocarcinoma development, whereas no significant difference was observed between high-grade and low-grade tumors. Further, the PHLPP expression level showed significant associations with LNM, FIGO stages, and omental metastasis in high-grade tumors, but not in low-grade tumors. Similarly, the 5-year survival rates of PHLPP-positive and negative high-grade tumors significant differed, whereas no difference was seen in the low-grade tumors. These findings suggest that PHLPP may be a predictor of prognosis of high-grade ovarian serous adenocarcinoma.\n Role of the tumor suppressor forkhead homeobox type O 3a in the development of ovarian serous adenocarcinoma and its association with patient prognosis FoxO3a is an important member of the FOXO family, located on at 6q21, and encodes a 673-amino acid long protein.[13] FoxO3a, as an important signaling molecule downstream of Akt in the PI3K-Akt signaling pathway, binds to the 14-3-3 chaperone protein after being phosphorylated by Akt, and subsequently translocates from the nucleus to the cytoplasm, thereby activating the apoptotic genes Bim, FasL, and tumor necrosis factor and its related apoptosis-inducing ligand TRAIL, consequently promoting apoptosis.[14] In addition to its role in apoptosis, FoxO3a plays critical roles in regulating cell proliferation, metabolism, the stress response, and the life span of cancer cells.[15] Particularly, another target molecule of FoxO3a is the cell cycle-regulating gene P27; overexpression of FoxO3a can arrest the cell cycle in the G0/G1 phase by upregulating P27. In addition, overexpression of FoxO3a can significantly inhibit the proliferation of tumor cells and result in tumor cell G2 arrest, further promoting tumor cell apoptosis.[1617]\nThe results of our analyses showed that the FoxO3a-positive rate in the high-grade tumors was significantly lower than that in the normal oviducts. In the low-grade adenocarcinomas, borderline cystadenomas, cystadenomas, and normal ovaries, the positive expression rates of FoxO3a protein were gradually reduced from normal ovaries to the low-grade adenocarcinomas, and the FoxO3 positive rate of the low-grade tumors was significantly lower than those of the borderline cystadenomas, cystadenomas, and normal ovaries, while the positive rate of borderline cystadenomas was significantly lower than those of cystadenomas and normal ovaries. Moreover, the FoxO3a expression level showed significant associations with LNM, FIGO stage, and omental metastasis in high-grade, but not low-grade tumors. These results suggest that FoxO3a may play a role in the oncogenesis and development of ovarian serous adenocarcinomas. In the high-grade tumors, the 5-year survival rate of FoxO3a-positive patients was significantly higher than that of the negative patients, whereas no difference in survival was seen in low-grade tumors, indicating that positive FoxO3a expression may have a long-term protective effect on cancer patients, and FoxO3a may hence have certain significance in the prediction of the patients’ long-term prognosis. Interestingly, it has been reported that tumor cells with low FoxO3a expression can develop drug resistance to paclitaxel and that inhibiting FoxO3a expression with RNAi can reduce E1A-mediated sensitivity to paclitaxel.[18] Therefore, FoxO3a protein may represent a potential prognostic indicator for high-grade ovarian serous adenocarcinoma.\nFoxO3a is an important member of the FOXO family, located on at 6q21, and encodes a 673-amino acid long protein.[13] FoxO3a, as an important signaling molecule downstream of Akt in the PI3K-Akt signaling pathway, binds to the 14-3-3 chaperone protein after being phosphorylated by Akt, and subsequently translocates from the nucleus to the cytoplasm, thereby activating the apoptotic genes Bim, FasL, and tumor necrosis factor and its related apoptosis-inducing ligand TRAIL, consequently promoting apoptosis.[14] In addition to its role in apoptosis, FoxO3a plays critical roles in regulating cell proliferation, metabolism, the stress response, and the life span of cancer cells.[15] Particularly, another target molecule of FoxO3a is the cell cycle-regulating gene P27; overexpression of FoxO3a can arrest the cell cycle in the G0/G1 phase by upregulating P27. In addition, overexpression of FoxO3a can significantly inhibit the proliferation of tumor cells and result in tumor cell G2 arrest, further promoting tumor cell apoptosis.[1617]\nThe results of our analyses showed that the FoxO3a-positive rate in the high-grade tumors was significantly lower than that in the normal oviducts. In the low-grade adenocarcinomas, borderline cystadenomas, cystadenomas, and normal ovaries, the positive expression rates of FoxO3a protein were gradually reduced from normal ovaries to the low-grade adenocarcinomas, and the FoxO3 positive rate of the low-grade tumors was significantly lower than those of the borderline cystadenomas, cystadenomas, and normal ovaries, while the positive rate of borderline cystadenomas was significantly lower than those of cystadenomas and normal ovaries. Moreover, the FoxO3a expression level showed significant associations with LNM, FIGO stage, and omental metastasis in high-grade, but not low-grade tumors. These results suggest that FoxO3a may play a role in the oncogenesis and development of ovarian serous adenocarcinomas. In the high-grade tumors, the 5-year survival rate of FoxO3a-positive patients was significantly higher than that of the negative patients, whereas no difference in survival was seen in low-grade tumors, indicating that positive FoxO3a expression may have a long-term protective effect on cancer patients, and FoxO3a may hence have certain significance in the prediction of the patients’ long-term prognosis. Interestingly, it has been reported that tumor cells with low FoxO3a expression can develop drug resistance to paclitaxel and that inhibiting FoxO3a expression with RNAi can reduce E1A-mediated sensitivity to paclitaxel.[18] Therefore, FoxO3a protein may represent a potential prognostic indicator for high-grade ovarian serous adenocarcinoma.\n Role of RAD51 in the development of ovarian serous adenocarcinoma and its association with patient prognosis RAD51 is a human homologous recombination repair gene. Too strong or too weak homologous recombination will result in genome instability and DNA damage, and the consequent gene loss, loss of heterozygosity, and/or genetic translocation are important causes of tumorigenesis and tumor progression.[19] Studies have found that RAD51 protein is overexpression in the breast, prostate, pancreatic, and colon cancers. Such overexpression of RAD51 can provide certain advantages for the growth of cancer cells by promoting the initiation and progression of cancer. However, to date, studies on the expression of RAD51 in ovarian cancers are few.\nThe results of this study showed that the RAD51-positive rate in high-grade tumors was significantly higher than that of normal oviducts, suggesting that RAD51 may play a role in the initiation of high-grade ovarian serous carcinomas. The RAD51-positive rate gradually increased from normal ovaries to cystadenomas to borderline cystadenomas to low-grade serous adenocarcinomas. Particularly, the positive rate of low-grade tumors was significantly higher than that in benign tumors and normal oviducts. The gradual change in the RAD51 expression suggests that the initiation of low-grade serous adenocarcinoma is a gradual procedure from benign to borderline to low-grade adenocarcinoma. However, no significant difference in the expression of RAD51 was found between high- and low-grade tumors. The expression level of RAD51 showed significant associations with LNM, FIGO stage, and omental metastasis in high-grade, but not low-grade tumors.\nHerein, the 5-year survival rate of patients with RAD51-positive high-grade adenocarcinomas was significantly lower than that of patients with RAD51-negative tumors, while no difference in survival was found in low-grade tumors. In other words, we found that high-grade tumor patients with high expression of RAD51 had a lower survival rate and poor prognosis, indicating that RAD51 may be a prognostic factor in high-grade ovarian serous adenocarcinoma. Furthermore, multivariate survival analyses showed that RAD51 was an independent prognostic factor, which may be of clinical significance in terms of the development of molecular targeted therapy, in addition to FIGO stage, and LNM. Further investigations on RAD51 are required to confirm these findings and to hopefully be able to improve the quality of life and prognosis of patients using targeted therapy.\nIn conclusion, the results of the present study indicate that PHLPP, FoxO3a, and RAD51 may play certain roles in the pathogenesis of the different grades of ovarian serous adenocarcinomas. Meanwhile, they also suggest the possible presence of common molecular mechanisms in the pathogenesis of the different grades. Increased protein expression of RAD51 may be an important molecular marker for poor prognosis of high-grade ovarian serous adenocarcinomas. However, considering the limited number of cases selected, which may have affected the significance of the results, further studies are required to improve and supplement these findings.\nRAD51 is a human homologous recombination repair gene. Too strong or too weak homologous recombination will result in genome instability and DNA damage, and the consequent gene loss, loss of heterozygosity, and/or genetic translocation are important causes of tumorigenesis and tumor progression.[19] Studies have found that RAD51 protein is overexpression in the breast, prostate, pancreatic, and colon cancers. Such overexpression of RAD51 can provide certain advantages for the growth of cancer cells by promoting the initiation and progression of cancer. However, to date, studies on the expression of RAD51 in ovarian cancers are few.\nThe results of this study showed that the RAD51-positive rate in high-grade tumors was significantly higher than that of normal oviducts, suggesting that RAD51 may play a role in the initiation of high-grade ovarian serous carcinomas. The RAD51-positive rate gradually increased from normal ovaries to cystadenomas to borderline cystadenomas to low-grade serous adenocarcinomas. Particularly, the positive rate of low-grade tumors was significantly higher than that in benign tumors and normal oviducts. The gradual change in the RAD51 expression suggests that the initiation of low-grade serous adenocarcinoma is a gradual procedure from benign to borderline to low-grade adenocarcinoma. However, no significant difference in the expression of RAD51 was found between high- and low-grade tumors. The expression level of RAD51 showed significant associations with LNM, FIGO stage, and omental metastasis in high-grade, but not low-grade tumors.\nHerein, the 5-year survival rate of patients with RAD51-positive high-grade adenocarcinomas was significantly lower than that of patients with RAD51-negative tumors, while no difference in survival was found in low-grade tumors. In other words, we found that high-grade tumor patients with high expression of RAD51 had a lower survival rate and poor prognosis, indicating that RAD51 may be a prognostic factor in high-grade ovarian serous adenocarcinoma. Furthermore, multivariate survival analyses showed that RAD51 was an independent prognostic factor, which may be of clinical significance in terms of the development of molecular targeted therapy, in addition to FIGO stage, and LNM. Further investigations on RAD51 are required to confirm these findings and to hopefully be able to improve the quality of life and prognosis of patients using targeted therapy.\nIn conclusion, the results of the present study indicate that PHLPP, FoxO3a, and RAD51 may play certain roles in the pathogenesis of the different grades of ovarian serous adenocarcinomas. Meanwhile, they also suggest the possible presence of common molecular mechanisms in the pathogenesis of the different grades. Increased protein expression of RAD51 may be an important molecular marker for poor prognosis of high-grade ovarian serous adenocarcinomas. However, considering the limited number of cases selected, which may have affected the significance of the results, further studies are required to improve and supplement these findings.\n Financial support and sponsorship This work were supported by grants from the Key Scientific Research program in Medical Science of Hebei Province, China (No. 20150318) and Science and Technology support program of Hebei Province, China (No. 16277794D).\nThis work were supported by grants from the Key Scientific Research program in Medical Science of Hebei Province, China (No. 20150318) and Science and Technology support program of Hebei Province, China (No. 16277794D).\n Conflicts of interest There are no conflicts of interest.\nThere are no conflicts of interest.","The tumor suppressor PHLPP, located at 18q21.33, can directly dephosphorylate ser473 in the hydrophobic group of Akt, thus negatively regulating Akt and its downstream targets. It has been reported that PHLPP can act synergistically with PTEN, another well-known tumor suppressor gene, thereby significantly inhibiting the proliferation of colorectal cancer cells, and negatively regulating PI3K/Akt signaling.[11] Accordingly, studies have shown that the PHLPP expression is significantly decreased in colon, breast, prostate, and lung cancers.[5712]\nThe results of the present study showed that the positive rate of PHLPP protein expression in the high-grade group was significantly lower than that in the oviduct group. The PHLPP-positive expression rate was decreased gradually from normal ovarian tissue to benign tumors to borderline tumors to low-grade adenocarcinomas, indicating that loss of PHLPP expression may be associated with the initiation of ovarian serous adenocarcinoma development, whereas no significant difference was observed between high-grade and low-grade tumors. Further, the PHLPP expression level showed significant associations with LNM, FIGO stages, and omental metastasis in high-grade tumors, but not in low-grade tumors. Similarly, the 5-year survival rates of PHLPP-positive and negative high-grade tumors significant differed, whereas no difference was seen in the low-grade tumors. These findings suggest that PHLPP may be a predictor of prognosis of high-grade ovarian serous adenocarcinoma.","FoxO3a is an important member of the FOXO family, located on at 6q21, and encodes a 673-amino acid long protein.[13] FoxO3a, as an important signaling molecule downstream of Akt in the PI3K-Akt signaling pathway, binds to the 14-3-3 chaperone protein after being phosphorylated by Akt, and subsequently translocates from the nucleus to the cytoplasm, thereby activating the apoptotic genes Bim, FasL, and tumor necrosis factor and its related apoptosis-inducing ligand TRAIL, consequently promoting apoptosis.[14] In addition to its role in apoptosis, FoxO3a plays critical roles in regulating cell proliferation, metabolism, the stress response, and the life span of cancer cells.[15] Particularly, another target molecule of FoxO3a is the cell cycle-regulating gene P27; overexpression of FoxO3a can arrest the cell cycle in the G0/G1 phase by upregulating P27. In addition, overexpression of FoxO3a can significantly inhibit the proliferation of tumor cells and result in tumor cell G2 arrest, further promoting tumor cell apoptosis.[1617]\nThe results of our analyses showed that the FoxO3a-positive rate in the high-grade tumors was significantly lower than that in the normal oviducts. In the low-grade adenocarcinomas, borderline cystadenomas, cystadenomas, and normal ovaries, the positive expression rates of FoxO3a protein were gradually reduced from normal ovaries to the low-grade adenocarcinomas, and the FoxO3 positive rate of the low-grade tumors was significantly lower than those of the borderline cystadenomas, cystadenomas, and normal ovaries, while the positive rate of borderline cystadenomas was significantly lower than those of cystadenomas and normal ovaries. Moreover, the FoxO3a expression level showed significant associations with LNM, FIGO stage, and omental metastasis in high-grade, but not low-grade tumors. These results suggest that FoxO3a may play a role in the oncogenesis and development of ovarian serous adenocarcinomas. In the high-grade tumors, the 5-year survival rate of FoxO3a-positive patients was significantly higher than that of the negative patients, whereas no difference in survival was seen in low-grade tumors, indicating that positive FoxO3a expression may have a long-term protective effect on cancer patients, and FoxO3a may hence have certain significance in the prediction of the patients’ long-term prognosis. Interestingly, it has been reported that tumor cells with low FoxO3a expression can develop drug resistance to paclitaxel and that inhibiting FoxO3a expression with RNAi can reduce E1A-mediated sensitivity to paclitaxel.[18] Therefore, FoxO3a protein may represent a potential prognostic indicator for high-grade ovarian serous adenocarcinoma.","RAD51 is a human homologous recombination repair gene. Too strong or too weak homologous recombination will result in genome instability and DNA damage, and the consequent gene loss, loss of heterozygosity, and/or genetic translocation are important causes of tumorigenesis and tumor progression.[19] Studies have found that RAD51 protein is overexpression in the breast, prostate, pancreatic, and colon cancers. Such overexpression of RAD51 can provide certain advantages for the growth of cancer cells by promoting the initiation and progression of cancer. However, to date, studies on the expression of RAD51 in ovarian cancers are few.\nThe results of this study showed that the RAD51-positive rate in high-grade tumors was significantly higher than that of normal oviducts, suggesting that RAD51 may play a role in the initiation of high-grade ovarian serous carcinomas. The RAD51-positive rate gradually increased from normal ovaries to cystadenomas to borderline cystadenomas to low-grade serous adenocarcinomas. Particularly, the positive rate of low-grade tumors was significantly higher than that in benign tumors and normal oviducts. The gradual change in the RAD51 expression suggests that the initiation of low-grade serous adenocarcinoma is a gradual procedure from benign to borderline to low-grade adenocarcinoma. However, no significant difference in the expression of RAD51 was found between high- and low-grade tumors. The expression level of RAD51 showed significant associations with LNM, FIGO stage, and omental metastasis in high-grade, but not low-grade tumors.\nHerein, the 5-year survival rate of patients with RAD51-positive high-grade adenocarcinomas was significantly lower than that of patients with RAD51-negative tumors, while no difference in survival was found in low-grade tumors. In other words, we found that high-grade tumor patients with high expression of RAD51 had a lower survival rate and poor prognosis, indicating that RAD51 may be a prognostic factor in high-grade ovarian serous adenocarcinoma. Furthermore, multivariate survival analyses showed that RAD51 was an independent prognostic factor, which may be of clinical significance in terms of the development of molecular targeted therapy, in addition to FIGO stage, and LNM. Further investigations on RAD51 are required to confirm these findings and to hopefully be able to improve the quality of life and prognosis of patients using targeted therapy.\nIn conclusion, the results of the present study indicate that PHLPP, FoxO3a, and RAD51 may play certain roles in the pathogenesis of the different grades of ovarian serous adenocarcinomas. Meanwhile, they also suggest the possible presence of common molecular mechanisms in the pathogenesis of the different grades. Increased protein expression of RAD51 may be an important molecular marker for poor prognosis of high-grade ovarian serous adenocarcinomas. However, considering the limited number of cases selected, which may have affected the significance of the results, further studies are required to improve and supplement these findings.","This work were supported by grants from the Key Scientific Research program in Medical Science of Hebei Province, China (No. 20150318) and Science and Technology support program of Hebei Province, China (No. 16277794D).","There are no conflicts of interest."],"string":"[\n \"Ovarian serous adenocarcinoma is the most common ovarian epithelial malignant tumor. It can be divided into low- and high-grade tumors, which exhibit substantial differences in the pathogenesis, clinicopathological characteristics, and prognosis.[1] Low-grade serous carcinomas undergo a progressive, gradual developmental process from benign serous cystadenoma to borderline serous cystadenoma to malignant serous adenocarcinoma, while high-grade serous adenocarcinomas develop directly from the oviduct epithelium or ovarian inclusion cysts.\\nHowever, it remains unclear whether the two tumor types have completely different molecular mechanisms, whether there is a common molecular basis, and whether the genes involved in the development of these tumors affect the patients’ prognosis. Recent studies[23] have indicated that the PI3K/Akt signaling pathway is involved in the initiation and development of ovarian serous adenocarcinoma. For example, PH domain leucine-rich repeat protein phosphatase (PHLPP) is known to negatively regulate Akt and its downstream kinase by dephosphorylating the hydrophobic core of Akt (Akt1 Ser473), thereby antagonizing PI3K/Akt signaling and inhibiting tumor growth. Forkhead homeobox type O 3a (FoxO3a), an important downstream signaling molecule of Akt, is involved in the initiation and development of tumor cells and regulation of cell proliferation after being phosphorylated by Akt.[45] Further, RAD51, a DNA double-strand break repair gene,[6] has also been implicated in the PI3K/Akt signaling pathway: phosphorylated FoxO3a binds to the promoter of the downstream gene RAD51, thereby regulating RAD51 and promoting tumor development and metastasis.\\nThe present study aimed to (1) determine the differences in the protein expressions of PHLPP, FoxO3a, and RAD51 between high- and low-grade ovarian serous adenocarcinomas, (2) explore the roles of these three genes in the initiation of the different grades of ovarian serous adenocarcinoma, and (3) analyze the relationships between their protein expressions and prognosis.\",\n \" Ethics The study was approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University and was conducted in accordance with the Declaration of Helsinki 1975, as revised in 2000.\\nThe study was approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University and was conducted in accordance with the Declaration of Helsinki 1975, as revised in 2000.\\n Participant selection and description Patients with ovarian serous adenocarcinoma hospitalized and treated from January 2006 to January 2011 in our hospital were included in the ovarian serous adenocarcinoma groups. No patients received chemotherapy and radiotherapy preoperatively, and their diagnosis was confirmed by pathological examinations postoperatively. All patients had complete clinical data and were graded according to the World Health Organization histological classification for ovarian tumors (2014 edition). Ninety-four and twenty-six cases of high- and low-grade ovarian serous adenocarcinomas were included in this study. Among the high-grade group (mean age, 55 years; range, 36–78 years), there were 26 and 68 patients with International Federation of Gynecology and Obstetrics (FIGO) Stage (2014 edition) I–II and III–IV disease, respectively. There were thirty and 64 patients with and without lymph node metastasis (LNM), and 41 and 53 patients with and without omental metastasis, respectively. Among the low-grade group (mean age, 44 years; range, 26–70 years), there were 18 and eight patients with FIGO Stage I–II and III–IV disease, respectively. There were 4 and 22 patients with and without LNM, and 5 and 21 patients with and without omental metastasis, respectively. Moreover, 21 cases of borderline serous cystadenoma (mean age, 38 years; range, 18–59 years) and 35 cases of benign serous cystadenoma (mean age, 45 years; range, 22–69 years) were included, along with thirty normal ovaries and thirty oviducts (mean age, 48 years; range, 36–62 years) removed from patients undergoing surgery for uterine fibroids, which were used as controls for the low- and high-grade tumor groups, respectively.\\nExpression of TP53 protein in high- and low-grade serous carcinoma: Among the 94 cases of high-grade, 92 cases (97.9%) showed strong staining of TP53, while among the 26 cases of low-grade serous adenocarcinoma, there was no case of strong staining.\\nPatients with ovarian serous adenocarcinoma hospitalized and treated from January 2006 to January 2011 in our hospital were included in the ovarian serous adenocarcinoma groups. No patients received chemotherapy and radiotherapy preoperatively, and their diagnosis was confirmed by pathological examinations postoperatively. All patients had complete clinical data and were graded according to the World Health Organization histological classification for ovarian tumors (2014 edition). Ninety-four and twenty-six cases of high- and low-grade ovarian serous adenocarcinomas were included in this study. Among the high-grade group (mean age, 55 years; range, 36–78 years), there were 26 and 68 patients with International Federation of Gynecology and Obstetrics (FIGO) Stage (2014 edition) I–II and III–IV disease, respectively. There were thirty and 64 patients with and without lymph node metastasis (LNM), and 41 and 53 patients with and without omental metastasis, respectively. Among the low-grade group (mean age, 44 years; range, 26–70 years), there were 18 and eight patients with FIGO Stage I–II and III–IV disease, respectively. There were 4 and 22 patients with and without LNM, and 5 and 21 patients with and without omental metastasis, respectively. Moreover, 21 cases of borderline serous cystadenoma (mean age, 38 years; range, 18–59 years) and 35 cases of benign serous cystadenoma (mean age, 45 years; range, 22–69 years) were included, along with thirty normal ovaries and thirty oviducts (mean age, 48 years; range, 36–62 years) removed from patients undergoing surgery for uterine fibroids, which were used as controls for the low- and high-grade tumor groups, respectively.\\nExpression of TP53 protein in high- and low-grade serous carcinoma: Among the 94 cases of high-grade, 92 cases (97.9%) showed strong staining of TP53, while among the 26 cases of low-grade serous adenocarcinoma, there was no case of strong staining.\\n Technical information \\nExperimental procedures\\n\\nThe operative specimens were examined, and the patients were diagnosed by experienced pathologists. Paraffin blocks of the specimens were cut, and immunohistochemical staining of the sections was conducted as follows: the samples were sliced, deparaffinized, dehydrated, and subjected to antigen retrieval before blocking with goat serum. Subsequently, the slides were placed in a humidifier chamber and incubated at room temperature for 45 min. The tissues were incubated with primary antibodies (working concentrations of anti-PHLPP, RAD51, and FoxO3a of 1:100, 1:100, and 1:150, respectively, Biogot Biotechnology Co., Ltd., Nanjing, China) in the sealed humidifier chamber at 4°C overnight. After washing of the slides, goat anti-rabbit secondary antibodies were added and the slides were incubated in the humidifier at 37°C for 30 min. Next, horseradish peroxidase-labeled streptavidin was added and the slides were incubated in the humidifier at 37°C for 30 min, after which the tissues were stained by 3,3’-diaminobenzidine, followed by counterstaining, destaining, dehydration, clearing, and mounting. Finally, the slides were air-dried and observed under a microscope.\\n\\nEvaluation standard for staining\\n\\nThe slides were scored by pathologists according to the below criteria for PHLPP, FoxO3a, and RAD51 under a microscope. Ten high-magnification fields were counted, and the average cell numbers were used.\\n PH domain leucine-rich repeat protein phosphatase-positive criteria Cells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\\nCells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\\n Forkhead homeobox type O 3a-positive criteria Cells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\\nCells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\\n RAD51-positive criteria Cells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\\nCells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\\n\\nExperimental procedures\\n\\nThe operative specimens were examined, and the patients were diagnosed by experienced pathologists. Paraffin blocks of the specimens were cut, and immunohistochemical staining of the sections was conducted as follows: the samples were sliced, deparaffinized, dehydrated, and subjected to antigen retrieval before blocking with goat serum. Subsequently, the slides were placed in a humidifier chamber and incubated at room temperature for 45 min. The tissues were incubated with primary antibodies (working concentrations of anti-PHLPP, RAD51, and FoxO3a of 1:100, 1:100, and 1:150, respectively, Biogot Biotechnology Co., Ltd., Nanjing, China) in the sealed humidifier chamber at 4°C overnight. After washing of the slides, goat anti-rabbit secondary antibodies were added and the slides were incubated in the humidifier at 37°C for 30 min. Next, horseradish peroxidase-labeled streptavidin was added and the slides were incubated in the humidifier at 37°C for 30 min, after which the tissues were stained by 3,3’-diaminobenzidine, followed by counterstaining, destaining, dehydration, clearing, and mounting. Finally, the slides were air-dried and observed under a microscope.\\n\\nEvaluation standard for staining\\n\\nThe slides were scored by pathologists according to the below criteria for PHLPP, FoxO3a, and RAD51 under a microscope. Ten high-magnification fields were counted, and the average cell numbers were used.\\n PH domain leucine-rich repeat protein phosphatase-positive criteria Cells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\\nCells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\\n Forkhead homeobox type O 3a-positive criteria Cells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\\nCells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\\n RAD51-positive criteria Cells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\\nCells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\\n Statistical analyses SPSS 16.0 software (SPSS Inc., USA) was used for the statistical analysis. Categorical data were analyzed by the Chi-square test, and the survival rates were calculated by Kaplan-Meier univariate analysis. Differences in the survival rates between the different groups were determined by the log-rank test. Cox multivariate survival analysis was used for analysis of independent prognostic factors. The statistical significance level was set as P < 0.05.\\nSPSS 16.0 software (SPSS Inc., USA) was used for the statistical analysis. Categorical data were analyzed by the Chi-square test, and the survival rates were calculated by Kaplan-Meier univariate analysis. Differences in the survival rates between the different groups were determined by the log-rank test. Cox multivariate survival analysis was used for analysis of independent prognostic factors. The statistical significance level was set as P < 0.05.\",\n \"The study was approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University and was conducted in accordance with the Declaration of Helsinki 1975, as revised in 2000.\",\n \"Patients with ovarian serous adenocarcinoma hospitalized and treated from January 2006 to January 2011 in our hospital were included in the ovarian serous adenocarcinoma groups. No patients received chemotherapy and radiotherapy preoperatively, and their diagnosis was confirmed by pathological examinations postoperatively. All patients had complete clinical data and were graded according to the World Health Organization histological classification for ovarian tumors (2014 edition). Ninety-four and twenty-six cases of high- and low-grade ovarian serous adenocarcinomas were included in this study. Among the high-grade group (mean age, 55 years; range, 36–78 years), there were 26 and 68 patients with International Federation of Gynecology and Obstetrics (FIGO) Stage (2014 edition) I–II and III–IV disease, respectively. There were thirty and 64 patients with and without lymph node metastasis (LNM), and 41 and 53 patients with and without omental metastasis, respectively. Among the low-grade group (mean age, 44 years; range, 26–70 years), there were 18 and eight patients with FIGO Stage I–II and III–IV disease, respectively. There were 4 and 22 patients with and without LNM, and 5 and 21 patients with and without omental metastasis, respectively. Moreover, 21 cases of borderline serous cystadenoma (mean age, 38 years; range, 18–59 years) and 35 cases of benign serous cystadenoma (mean age, 45 years; range, 22–69 years) were included, along with thirty normal ovaries and thirty oviducts (mean age, 48 years; range, 36–62 years) removed from patients undergoing surgery for uterine fibroids, which were used as controls for the low- and high-grade tumor groups, respectively.\\nExpression of TP53 protein in high- and low-grade serous carcinoma: Among the 94 cases of high-grade, 92 cases (97.9%) showed strong staining of TP53, while among the 26 cases of low-grade serous adenocarcinoma, there was no case of strong staining.\",\n \"\\nExperimental procedures\\n\\nThe operative specimens were examined, and the patients were diagnosed by experienced pathologists. Paraffin blocks of the specimens were cut, and immunohistochemical staining of the sections was conducted as follows: the samples were sliced, deparaffinized, dehydrated, and subjected to antigen retrieval before blocking with goat serum. Subsequently, the slides were placed in a humidifier chamber and incubated at room temperature for 45 min. The tissues were incubated with primary antibodies (working concentrations of anti-PHLPP, RAD51, and FoxO3a of 1:100, 1:100, and 1:150, respectively, Biogot Biotechnology Co., Ltd., Nanjing, China) in the sealed humidifier chamber at 4°C overnight. After washing of the slides, goat anti-rabbit secondary antibodies were added and the slides were incubated in the humidifier at 37°C for 30 min. Next, horseradish peroxidase-labeled streptavidin was added and the slides were incubated in the humidifier at 37°C for 30 min, after which the tissues were stained by 3,3’-diaminobenzidine, followed by counterstaining, destaining, dehydration, clearing, and mounting. Finally, the slides were air-dried and observed under a microscope.\\n\\nEvaluation standard for staining\\n\\nThe slides were scored by pathologists according to the below criteria for PHLPP, FoxO3a, and RAD51 under a microscope. Ten high-magnification fields were counted, and the average cell numbers were used.\\n PH domain leucine-rich repeat protein phosphatase-positive criteria Cells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\\nCells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\\n Forkhead homeobox type O 3a-positive criteria Cells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\\nCells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\\n RAD51-positive criteria Cells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\\nCells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\",\n \"Cells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\",\n \"Cells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\",\n \"Cells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\",\n \"SPSS 16.0 software (SPSS Inc., USA) was used for the statistical analysis. Categorical data were analyzed by the Chi-square test, and the survival rates were calculated by Kaplan-Meier univariate analysis. Differences in the survival rates between the different groups were determined by the log-rank test. Cox multivariate survival analysis was used for analysis of independent prognostic factors. The statistical significance level was set as P < 0.05.\",\n \" Expression of PH domain leucine-rich repeat protein phosphatase in ovarian serous adenocarcinomas and its association with clinicopathological features and prognosis Among 94 cases of high-grade serous adenocarcinoma, 23 cases (24.5%) were PHLPP-positive, which was significantly lower than the positive rate of 86.7% in the normal oviduct group (P < 0.05). Of 26 low-grade serous adenocarcinoma cases, 6 (23.1%) were positive, which was significantly lower than that in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, there was no difference in the positive expression rates between high- and low-grade tumors [P > 0.05; Figure 1 and Table 1].\\nProtein expression of PH domain leucine-rich repeat protein phosphatase in high- and low-grade serous ovarian adenocarcinoma by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\\nProtein expression of PHLPP in high- and low-grade ovarian serous adenocarcinomas\\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. PHLPP: PH domain leucine-rich repeat protein phosphatase.\\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower PHLPP protein expression levels [P < 0.05; Table 2]. On the other hand, there were no significant correlations between the clinicopathological features and PHLPP protein expression in low-grade tumors [P > 0.05].\\nRelationships between PHLPP, FoxO3a, and RAD51 protein expressions and clinicopathological features of high-grade serous ovarian adenocarcinoma\\nLNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a.\\nAmong the 94 patients with high-grade serous adenocarcinoma, the 5-year survival rates of PHLPP-negative and positive patients were 5.6% and 43.5%, respectively [P < 0.05, Figure 2]. Among the 26 cases of low-grade serous adenocarcinoma, however, the expression of PHLPP did not significantly associate with the 5-year survival rate of the patients [P > 0.05].\\nFive-year survival curves of PH domain leucine-rich repeat protein phosphatase-positive and negative patients with high-grade ovarian serous adenocarcinomas.\\nAmong 94 cases of high-grade serous adenocarcinoma, 23 cases (24.5%) were PHLPP-positive, which was significantly lower than the positive rate of 86.7% in the normal oviduct group (P < 0.05). Of 26 low-grade serous adenocarcinoma cases, 6 (23.1%) were positive, which was significantly lower than that in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, there was no difference in the positive expression rates between high- and low-grade tumors [P > 0.05; Figure 1 and Table 1].\\nProtein expression of PH domain leucine-rich repeat protein phosphatase in high- and low-grade serous ovarian adenocarcinoma by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\\nProtein expression of PHLPP in high- and low-grade ovarian serous adenocarcinomas\\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. PHLPP: PH domain leucine-rich repeat protein phosphatase.\\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower PHLPP protein expression levels [P < 0.05; Table 2]. On the other hand, there were no significant correlations between the clinicopathological features and PHLPP protein expression in low-grade tumors [P > 0.05].\\nRelationships between PHLPP, FoxO3a, and RAD51 protein expressions and clinicopathological features of high-grade serous ovarian adenocarcinoma\\nLNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a.\\nAmong the 94 patients with high-grade serous adenocarcinoma, the 5-year survival rates of PHLPP-negative and positive patients were 5.6% and 43.5%, respectively [P < 0.05, Figure 2]. Among the 26 cases of low-grade serous adenocarcinoma, however, the expression of PHLPP did not significantly associate with the 5-year survival rate of the patients [P > 0.05].\\nFive-year survival curves of PH domain leucine-rich repeat protein phosphatase-positive and negative patients with high-grade ovarian serous adenocarcinomas.\\n Expression of forkhead homeobox type O 3a in ovarian serous adenocarcinoma and its association with clinicopathological features and prognosis Among the 94 cases of high-grade serous adenocarcinoma, 25 (26.6%) were positive for FoxO3a protein expression, which was significantly lower than the positive rate of 90.0% in the normal oviduct group (P < 0.05). Among the 26 cases of low-grade serous carcinoma, 7 (26.9%) were positive, which was significantly lower than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, the difference in the positive expression rates between the high- and low-grade groups was not significant [P > 0.05; Figure 3 and Table 3].\\nProtein expression of forkhead homeobox type O 3a in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\\nProtein expression of FoxO3a in high- and low-grade serous adenocarcinomas\\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. FoxO3a: Forkhead homeobox type O 3a.\\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower FoxO3a protein expression [P < 0.05, Table 2]. On the other hand, in low-grade tumors, the FoxO3a protein expression showed no association with clinicopathological features (P > 0.05).\\nAmong the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of FoxO3a-negative and positive patients were 7.2% and 36.0%, respectively [P < 0.05, Figure 4]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of FoxO3a showed no significant association with the 5-year survival rate (P > 0.05).\\nFive-year survival curves of forkhead homeobox type O 3a-positive and negative patients with high-grade ovarian serous adenocarcinomas.\\nAmong the 94 cases of high-grade serous adenocarcinoma, 25 (26.6%) were positive for FoxO3a protein expression, which was significantly lower than the positive rate of 90.0% in the normal oviduct group (P < 0.05). Among the 26 cases of low-grade serous carcinoma, 7 (26.9%) were positive, which was significantly lower than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, the difference in the positive expression rates between the high- and low-grade groups was not significant [P > 0.05; Figure 3 and Table 3].\\nProtein expression of forkhead homeobox type O 3a in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\\nProtein expression of FoxO3a in high- and low-grade serous adenocarcinomas\\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. FoxO3a: Forkhead homeobox type O 3a.\\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower FoxO3a protein expression [P < 0.05, Table 2]. On the other hand, in low-grade tumors, the FoxO3a protein expression showed no association with clinicopathological features (P > 0.05).\\nAmong the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of FoxO3a-negative and positive patients were 7.2% and 36.0%, respectively [P < 0.05, Figure 4]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of FoxO3a showed no significant association with the 5-year survival rate (P > 0.05).\\nFive-year survival curves of forkhead homeobox type O 3a-positive and negative patients with high-grade ovarian serous adenocarcinomas.\\n Expression of RAD51 in ovarian serous adenocarcinoma and its association with clinicopathological features and prognosis Among the 94 cases of high-grade serous adenocarcinoma, 66 (70.2%) were positive for RAD51 protein, which was significantly higher than that in the normal oviduct group [P < 0.05; Figure 5 and Table 4]. Among the 26 cases of low-grade serous carcinoma, 17 (65.4%) were positive, which was significantly higher than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups [P < 0.05; Figure 5 and Table 4]. However, the difference in the positive expression rates between the high- and low-grade groups was not significant (P > 0.05).\\nProtein expression of RAD51 in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Positive high-grade adenocarcinoma, (b) negative normal oviduct, (c) positive low-grade adenocarcinoma, (d) negative borderline cystadenoma, (e) negative cystadenoma, (f) negative normal ovary.\\nProtein expression of RAD51 in high- and low-grade serous adenocarcinomas\\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas.\\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with higher RAD51 protein expression levels [P < 0.05; Table 2]. Conversely, there was no significant correlation between clinicopathological features and RAD51 expression in low-grade tumors (P > 0.05).\\nFinally, among the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of RAD51-positive and negative patients were 3.0% and 42.9%, respectively [P < 0.05; Figure 6]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of RAD51 showed no significant association with the 5-year survival rate (P > 0.05).\\nFive-year survival curves of RAD51-positive and negative patients with high-grade ovarian serous adenocarcinomas.\\nAmong the 94 cases of high-grade serous adenocarcinoma, 66 (70.2%) were positive for RAD51 protein, which was significantly higher than that in the normal oviduct group [P < 0.05; Figure 5 and Table 4]. Among the 26 cases of low-grade serous carcinoma, 17 (65.4%) were positive, which was significantly higher than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups [P < 0.05; Figure 5 and Table 4]. However, the difference in the positive expression rates between the high- and low-grade groups was not significant (P > 0.05).\\nProtein expression of RAD51 in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Positive high-grade adenocarcinoma, (b) negative normal oviduct, (c) positive low-grade adenocarcinoma, (d) negative borderline cystadenoma, (e) negative cystadenoma, (f) negative normal ovary.\\nProtein expression of RAD51 in high- and low-grade serous adenocarcinomas\\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas.\\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with higher RAD51 protein expression levels [P < 0.05; Table 2]. Conversely, there was no significant correlation between clinicopathological features and RAD51 expression in low-grade tumors (P > 0.05).\\nFinally, among the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of RAD51-positive and negative patients were 3.0% and 42.9%, respectively [P < 0.05; Figure 6]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of RAD51 showed no significant association with the 5-year survival rate (P > 0.05).\\nFive-year survival curves of RAD51-positive and negative patients with high-grade ovarian serous adenocarcinomas.\\n Prognostic multivariate survival analyses of high-grade serous ovarian adenocarcinoma The Cox multivariate analysis results showed that FIGO staging, LNM, and RAD51 protein expression were independent prognostic factors of patients with high-grade serous ovarian adenocarcinoma [P < 0.05; Table 5].\\nMultivariate analysis of independent factors associated with the 5-year overall survival rate in patients with high-grade serous ovarian adenocarcinoma\\nRR: Risk ratio; CI: Confidence interval; LNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a; PHLPP: PH domain leucine-rich repeat protein phosphatase.\\nThe Cox multivariate analysis results showed that FIGO staging, LNM, and RAD51 protein expression were independent prognostic factors of patients with high-grade serous ovarian adenocarcinoma [P < 0.05; Table 5].\\nMultivariate analysis of independent factors associated with the 5-year overall survival rate in patients with high-grade serous ovarian adenocarcinoma\\nRR: Risk ratio; CI: Confidence interval; LNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a; PHLPP: PH domain leucine-rich repeat protein phosphatase.\",\n \"Among 94 cases of high-grade serous adenocarcinoma, 23 cases (24.5%) were PHLPP-positive, which was significantly lower than the positive rate of 86.7% in the normal oviduct group (P < 0.05). Of 26 low-grade serous adenocarcinoma cases, 6 (23.1%) were positive, which was significantly lower than that in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, there was no difference in the positive expression rates between high- and low-grade tumors [P > 0.05; Figure 1 and Table 1].\\nProtein expression of PH domain leucine-rich repeat protein phosphatase in high- and low-grade serous ovarian adenocarcinoma by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\\nProtein expression of PHLPP in high- and low-grade ovarian serous adenocarcinomas\\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. PHLPP: PH domain leucine-rich repeat protein phosphatase.\\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower PHLPP protein expression levels [P < 0.05; Table 2]. On the other hand, there were no significant correlations between the clinicopathological features and PHLPP protein expression in low-grade tumors [P > 0.05].\\nRelationships between PHLPP, FoxO3a, and RAD51 protein expressions and clinicopathological features of high-grade serous ovarian adenocarcinoma\\nLNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a.\\nAmong the 94 patients with high-grade serous adenocarcinoma, the 5-year survival rates of PHLPP-negative and positive patients were 5.6% and 43.5%, respectively [P < 0.05, Figure 2]. Among the 26 cases of low-grade serous adenocarcinoma, however, the expression of PHLPP did not significantly associate with the 5-year survival rate of the patients [P > 0.05].\\nFive-year survival curves of PH domain leucine-rich repeat protein phosphatase-positive and negative patients with high-grade ovarian serous adenocarcinomas.\",\n \"Among the 94 cases of high-grade serous adenocarcinoma, 25 (26.6%) were positive for FoxO3a protein expression, which was significantly lower than the positive rate of 90.0% in the normal oviduct group (P < 0.05). Among the 26 cases of low-grade serous carcinoma, 7 (26.9%) were positive, which was significantly lower than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, the difference in the positive expression rates between the high- and low-grade groups was not significant [P > 0.05; Figure 3 and Table 3].\\nProtein expression of forkhead homeobox type O 3a in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\\nProtein expression of FoxO3a in high- and low-grade serous adenocarcinomas\\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. FoxO3a: Forkhead homeobox type O 3a.\\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower FoxO3a protein expression [P < 0.05, Table 2]. On the other hand, in low-grade tumors, the FoxO3a protein expression showed no association with clinicopathological features (P > 0.05).\\nAmong the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of FoxO3a-negative and positive patients were 7.2% and 36.0%, respectively [P < 0.05, Figure 4]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of FoxO3a showed no significant association with the 5-year survival rate (P > 0.05).\\nFive-year survival curves of forkhead homeobox type O 3a-positive and negative patients with high-grade ovarian serous adenocarcinomas.\",\n \"Among the 94 cases of high-grade serous adenocarcinoma, 66 (70.2%) were positive for RAD51 protein, which was significantly higher than that in the normal oviduct group [P < 0.05; Figure 5 and Table 4]. Among the 26 cases of low-grade serous carcinoma, 17 (65.4%) were positive, which was significantly higher than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups [P < 0.05; Figure 5 and Table 4]. However, the difference in the positive expression rates between the high- and low-grade groups was not significant (P > 0.05).\\nProtein expression of RAD51 in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Positive high-grade adenocarcinoma, (b) negative normal oviduct, (c) positive low-grade adenocarcinoma, (d) negative borderline cystadenoma, (e) negative cystadenoma, (f) negative normal ovary.\\nProtein expression of RAD51 in high- and low-grade serous adenocarcinomas\\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas.\\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with higher RAD51 protein expression levels [P < 0.05; Table 2]. Conversely, there was no significant correlation between clinicopathological features and RAD51 expression in low-grade tumors (P > 0.05).\\nFinally, among the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of RAD51-positive and negative patients were 3.0% and 42.9%, respectively [P < 0.05; Figure 6]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of RAD51 showed no significant association with the 5-year survival rate (P > 0.05).\\nFive-year survival curves of RAD51-positive and negative patients with high-grade ovarian serous adenocarcinomas.\",\n \"The Cox multivariate analysis results showed that FIGO staging, LNM, and RAD51 protein expression were independent prognostic factors of patients with high-grade serous ovarian adenocarcinoma [P < 0.05; Table 5].\\nMultivariate analysis of independent factors associated with the 5-year overall survival rate in patients with high-grade serous ovarian adenocarcinoma\\nRR: Risk ratio; CI: Confidence interval; LNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a; PHLPP: PH domain leucine-rich repeat protein phosphatase.\",\n \"Ovarian cancer is one of the most common malignancies of the female reproductive system, and has the highest mortality among all gynecologic malignant tumors, thus seriously affecting women's health and lives. Recent studies have suggested that ovarian serous adenocarcinomas can be divided into high- and low-grade tumors and that the initiation mechanisms, clinical pathological features, and prognoses of the two types greatly differ.[110] Thus, ovarian serous adenocarcinomas have complex biological behaviors. However, although the pathogeneses of the two types involve different molecular pathways, there is some common basis. Recent studies have found that the PI3K/Akt signaling pathway is closely associated with the initiation and progression of ovarian cancer. PHLPP, a known tumor suppressor, can negatively regulate Akt and its downstream kinases, thereby antagonizing the PI3K/Akt signaling pathway and inhibit tumor growth. Moreover, as an important downstream signaling molecule of Akt, after being phosphorylated by Akt, FoxO3a is involved in the initiation and development of tumor cells and regulates cell proliferation. RAD51, a downstream target of FoxO3a, also plays an important role in PI3K/Akt signaling; FoxO3a binds to the promoter of RAD51, consequently regulating RAD51 and promoting tumor development and metastasis. In this study, by examining the differences in the protein expression levels of PHLPP, FoxO3a, and RAD51 between high- and low-grade ovarian serous adenocarcinomas, the roles of the three genes in the development of the different grades of ovarian serous adenocarcinoma were explored, and the relationships between the protein expression levels and the patient prognosis were analyzed.\\n Role of the tumor suppressor PH domain leucine-rich repeat protein phosphatase in the development of ovarian serous adenocarcinoma and its association with patient prognosis The tumor suppressor PHLPP, located at 18q21.33, can directly dephosphorylate ser473 in the hydrophobic group of Akt, thus negatively regulating Akt and its downstream targets. It has been reported that PHLPP can act synergistically with PTEN, another well-known tumor suppressor gene, thereby significantly inhibiting the proliferation of colorectal cancer cells, and negatively regulating PI3K/Akt signaling.[11] Accordingly, studies have shown that the PHLPP expression is significantly decreased in colon, breast, prostate, and lung cancers.[5712]\\nThe results of the present study showed that the positive rate of PHLPP protein expression in the high-grade group was significantly lower than that in the oviduct group. The PHLPP-positive expression rate was decreased gradually from normal ovarian tissue to benign tumors to borderline tumors to low-grade adenocarcinomas, indicating that loss of PHLPP expression may be associated with the initiation of ovarian serous adenocarcinoma development, whereas no significant difference was observed between high-grade and low-grade tumors. Further, the PHLPP expression level showed significant associations with LNM, FIGO stages, and omental metastasis in high-grade tumors, but not in low-grade tumors. Similarly, the 5-year survival rates of PHLPP-positive and negative high-grade tumors significant differed, whereas no difference was seen in the low-grade tumors. These findings suggest that PHLPP may be a predictor of prognosis of high-grade ovarian serous adenocarcinoma.\\nThe tumor suppressor PHLPP, located at 18q21.33, can directly dephosphorylate ser473 in the hydrophobic group of Akt, thus negatively regulating Akt and its downstream targets. It has been reported that PHLPP can act synergistically with PTEN, another well-known tumor suppressor gene, thereby significantly inhibiting the proliferation of colorectal cancer cells, and negatively regulating PI3K/Akt signaling.[11] Accordingly, studies have shown that the PHLPP expression is significantly decreased in colon, breast, prostate, and lung cancers.[5712]\\nThe results of the present study showed that the positive rate of PHLPP protein expression in the high-grade group was significantly lower than that in the oviduct group. The PHLPP-positive expression rate was decreased gradually from normal ovarian tissue to benign tumors to borderline tumors to low-grade adenocarcinomas, indicating that loss of PHLPP expression may be associated with the initiation of ovarian serous adenocarcinoma development, whereas no significant difference was observed between high-grade and low-grade tumors. Further, the PHLPP expression level showed significant associations with LNM, FIGO stages, and omental metastasis in high-grade tumors, but not in low-grade tumors. Similarly, the 5-year survival rates of PHLPP-positive and negative high-grade tumors significant differed, whereas no difference was seen in the low-grade tumors. These findings suggest that PHLPP may be a predictor of prognosis of high-grade ovarian serous adenocarcinoma.\\n Role of the tumor suppressor forkhead homeobox type O 3a in the development of ovarian serous adenocarcinoma and its association with patient prognosis FoxO3a is an important member of the FOXO family, located on at 6q21, and encodes a 673-amino acid long protein.[13] FoxO3a, as an important signaling molecule downstream of Akt in the PI3K-Akt signaling pathway, binds to the 14-3-3 chaperone protein after being phosphorylated by Akt, and subsequently translocates from the nucleus to the cytoplasm, thereby activating the apoptotic genes Bim, FasL, and tumor necrosis factor and its related apoptosis-inducing ligand TRAIL, consequently promoting apoptosis.[14] In addition to its role in apoptosis, FoxO3a plays critical roles in regulating cell proliferation, metabolism, the stress response, and the life span of cancer cells.[15] Particularly, another target molecule of FoxO3a is the cell cycle-regulating gene P27; overexpression of FoxO3a can arrest the cell cycle in the G0/G1 phase by upregulating P27. In addition, overexpression of FoxO3a can significantly inhibit the proliferation of tumor cells and result in tumor cell G2 arrest, further promoting tumor cell apoptosis.[1617]\\nThe results of our analyses showed that the FoxO3a-positive rate in the high-grade tumors was significantly lower than that in the normal oviducts. In the low-grade adenocarcinomas, borderline cystadenomas, cystadenomas, and normal ovaries, the positive expression rates of FoxO3a protein were gradually reduced from normal ovaries to the low-grade adenocarcinomas, and the FoxO3 positive rate of the low-grade tumors was significantly lower than those of the borderline cystadenomas, cystadenomas, and normal ovaries, while the positive rate of borderline cystadenomas was significantly lower than those of cystadenomas and normal ovaries. Moreover, the FoxO3a expression level showed significant associations with LNM, FIGO stage, and omental metastasis in high-grade, but not low-grade tumors. These results suggest that FoxO3a may play a role in the oncogenesis and development of ovarian serous adenocarcinomas. In the high-grade tumors, the 5-year survival rate of FoxO3a-positive patients was significantly higher than that of the negative patients, whereas no difference in survival was seen in low-grade tumors, indicating that positive FoxO3a expression may have a long-term protective effect on cancer patients, and FoxO3a may hence have certain significance in the prediction of the patients’ long-term prognosis. Interestingly, it has been reported that tumor cells with low FoxO3a expression can develop drug resistance to paclitaxel and that inhibiting FoxO3a expression with RNAi can reduce E1A-mediated sensitivity to paclitaxel.[18] Therefore, FoxO3a protein may represent a potential prognostic indicator for high-grade ovarian serous adenocarcinoma.\\nFoxO3a is an important member of the FOXO family, located on at 6q21, and encodes a 673-amino acid long protein.[13] FoxO3a, as an important signaling molecule downstream of Akt in the PI3K-Akt signaling pathway, binds to the 14-3-3 chaperone protein after being phosphorylated by Akt, and subsequently translocates from the nucleus to the cytoplasm, thereby activating the apoptotic genes Bim, FasL, and tumor necrosis factor and its related apoptosis-inducing ligand TRAIL, consequently promoting apoptosis.[14] In addition to its role in apoptosis, FoxO3a plays critical roles in regulating cell proliferation, metabolism, the stress response, and the life span of cancer cells.[15] Particularly, another target molecule of FoxO3a is the cell cycle-regulating gene P27; overexpression of FoxO3a can arrest the cell cycle in the G0/G1 phase by upregulating P27. In addition, overexpression of FoxO3a can significantly inhibit the proliferation of tumor cells and result in tumor cell G2 arrest, further promoting tumor cell apoptosis.[1617]\\nThe results of our analyses showed that the FoxO3a-positive rate in the high-grade tumors was significantly lower than that in the normal oviducts. In the low-grade adenocarcinomas, borderline cystadenomas, cystadenomas, and normal ovaries, the positive expression rates of FoxO3a protein were gradually reduced from normal ovaries to the low-grade adenocarcinomas, and the FoxO3 positive rate of the low-grade tumors was significantly lower than those of the borderline cystadenomas, cystadenomas, and normal ovaries, while the positive rate of borderline cystadenomas was significantly lower than those of cystadenomas and normal ovaries. Moreover, the FoxO3a expression level showed significant associations with LNM, FIGO stage, and omental metastasis in high-grade, but not low-grade tumors. These results suggest that FoxO3a may play a role in the oncogenesis and development of ovarian serous adenocarcinomas. In the high-grade tumors, the 5-year survival rate of FoxO3a-positive patients was significantly higher than that of the negative patients, whereas no difference in survival was seen in low-grade tumors, indicating that positive FoxO3a expression may have a long-term protective effect on cancer patients, and FoxO3a may hence have certain significance in the prediction of the patients’ long-term prognosis. Interestingly, it has been reported that tumor cells with low FoxO3a expression can develop drug resistance to paclitaxel and that inhibiting FoxO3a expression with RNAi can reduce E1A-mediated sensitivity to paclitaxel.[18] Therefore, FoxO3a protein may represent a potential prognostic indicator for high-grade ovarian serous adenocarcinoma.\\n Role of RAD51 in the development of ovarian serous adenocarcinoma and its association with patient prognosis RAD51 is a human homologous recombination repair gene. Too strong or too weak homologous recombination will result in genome instability and DNA damage, and the consequent gene loss, loss of heterozygosity, and/or genetic translocation are important causes of tumorigenesis and tumor progression.[19] Studies have found that RAD51 protein is overexpression in the breast, prostate, pancreatic, and colon cancers. Such overexpression of RAD51 can provide certain advantages for the growth of cancer cells by promoting the initiation and progression of cancer. However, to date, studies on the expression of RAD51 in ovarian cancers are few.\\nThe results of this study showed that the RAD51-positive rate in high-grade tumors was significantly higher than that of normal oviducts, suggesting that RAD51 may play a role in the initiation of high-grade ovarian serous carcinomas. The RAD51-positive rate gradually increased from normal ovaries to cystadenomas to borderline cystadenomas to low-grade serous adenocarcinomas. Particularly, the positive rate of low-grade tumors was significantly higher than that in benign tumors and normal oviducts. The gradual change in the RAD51 expression suggests that the initiation of low-grade serous adenocarcinoma is a gradual procedure from benign to borderline to low-grade adenocarcinoma. However, no significant difference in the expression of RAD51 was found between high- and low-grade tumors. The expression level of RAD51 showed significant associations with LNM, FIGO stage, and omental metastasis in high-grade, but not low-grade tumors.\\nHerein, the 5-year survival rate of patients with RAD51-positive high-grade adenocarcinomas was significantly lower than that of patients with RAD51-negative tumors, while no difference in survival was found in low-grade tumors. In other words, we found that high-grade tumor patients with high expression of RAD51 had a lower survival rate and poor prognosis, indicating that RAD51 may be a prognostic factor in high-grade ovarian serous adenocarcinoma. Furthermore, multivariate survival analyses showed that RAD51 was an independent prognostic factor, which may be of clinical significance in terms of the development of molecular targeted therapy, in addition to FIGO stage, and LNM. Further investigations on RAD51 are required to confirm these findings and to hopefully be able to improve the quality of life and prognosis of patients using targeted therapy.\\nIn conclusion, the results of the present study indicate that PHLPP, FoxO3a, and RAD51 may play certain roles in the pathogenesis of the different grades of ovarian serous adenocarcinomas. Meanwhile, they also suggest the possible presence of common molecular mechanisms in the pathogenesis of the different grades. Increased protein expression of RAD51 may be an important molecular marker for poor prognosis of high-grade ovarian serous adenocarcinomas. However, considering the limited number of cases selected, which may have affected the significance of the results, further studies are required to improve and supplement these findings.\\nRAD51 is a human homologous recombination repair gene. Too strong or too weak homologous recombination will result in genome instability and DNA damage, and the consequent gene loss, loss of heterozygosity, and/or genetic translocation are important causes of tumorigenesis and tumor progression.[19] Studies have found that RAD51 protein is overexpression in the breast, prostate, pancreatic, and colon cancers. Such overexpression of RAD51 can provide certain advantages for the growth of cancer cells by promoting the initiation and progression of cancer. However, to date, studies on the expression of RAD51 in ovarian cancers are few.\\nThe results of this study showed that the RAD51-positive rate in high-grade tumors was significantly higher than that of normal oviducts, suggesting that RAD51 may play a role in the initiation of high-grade ovarian serous carcinomas. The RAD51-positive rate gradually increased from normal ovaries to cystadenomas to borderline cystadenomas to low-grade serous adenocarcinomas. Particularly, the positive rate of low-grade tumors was significantly higher than that in benign tumors and normal oviducts. The gradual change in the RAD51 expression suggests that the initiation of low-grade serous adenocarcinoma is a gradual procedure from benign to borderline to low-grade adenocarcinoma. However, no significant difference in the expression of RAD51 was found between high- and low-grade tumors. The expression level of RAD51 showed significant associations with LNM, FIGO stage, and omental metastasis in high-grade, but not low-grade tumors.\\nHerein, the 5-year survival rate of patients with RAD51-positive high-grade adenocarcinomas was significantly lower than that of patients with RAD51-negative tumors, while no difference in survival was found in low-grade tumors. In other words, we found that high-grade tumor patients with high expression of RAD51 had a lower survival rate and poor prognosis, indicating that RAD51 may be a prognostic factor in high-grade ovarian serous adenocarcinoma. Furthermore, multivariate survival analyses showed that RAD51 was an independent prognostic factor, which may be of clinical significance in terms of the development of molecular targeted therapy, in addition to FIGO stage, and LNM. Further investigations on RAD51 are required to confirm these findings and to hopefully be able to improve the quality of life and prognosis of patients using targeted therapy.\\nIn conclusion, the results of the present study indicate that PHLPP, FoxO3a, and RAD51 may play certain roles in the pathogenesis of the different grades of ovarian serous adenocarcinomas. Meanwhile, they also suggest the possible presence of common molecular mechanisms in the pathogenesis of the different grades. Increased protein expression of RAD51 may be an important molecular marker for poor prognosis of high-grade ovarian serous adenocarcinomas. However, considering the limited number of cases selected, which may have affected the significance of the results, further studies are required to improve and supplement these findings.\\n Financial support and sponsorship This work were supported by grants from the Key Scientific Research program in Medical Science of Hebei Province, China (No. 20150318) and Science and Technology support program of Hebei Province, China (No. 16277794D).\\nThis work were supported by grants from the Key Scientific Research program in Medical Science of Hebei Province, China (No. 20150318) and Science and Technology support program of Hebei Province, China (No. 16277794D).\\n Conflicts of interest There are no conflicts of interest.\\nThere are no conflicts of interest.\",\n \"The tumor suppressor PHLPP, located at 18q21.33, can directly dephosphorylate ser473 in the hydrophobic group of Akt, thus negatively regulating Akt and its downstream targets. It has been reported that PHLPP can act synergistically with PTEN, another well-known tumor suppressor gene, thereby significantly inhibiting the proliferation of colorectal cancer cells, and negatively regulating PI3K/Akt signaling.[11] Accordingly, studies have shown that the PHLPP expression is significantly decreased in colon, breast, prostate, and lung cancers.[5712]\\nThe results of the present study showed that the positive rate of PHLPP protein expression in the high-grade group was significantly lower than that in the oviduct group. The PHLPP-positive expression rate was decreased gradually from normal ovarian tissue to benign tumors to borderline tumors to low-grade adenocarcinomas, indicating that loss of PHLPP expression may be associated with the initiation of ovarian serous adenocarcinoma development, whereas no significant difference was observed between high-grade and low-grade tumors. Further, the PHLPP expression level showed significant associations with LNM, FIGO stages, and omental metastasis in high-grade tumors, but not in low-grade tumors. Similarly, the 5-year survival rates of PHLPP-positive and negative high-grade tumors significant differed, whereas no difference was seen in the low-grade tumors. These findings suggest that PHLPP may be a predictor of prognosis of high-grade ovarian serous adenocarcinoma.\",\n \"FoxO3a is an important member of the FOXO family, located on at 6q21, and encodes a 673-amino acid long protein.[13] FoxO3a, as an important signaling molecule downstream of Akt in the PI3K-Akt signaling pathway, binds to the 14-3-3 chaperone protein after being phosphorylated by Akt, and subsequently translocates from the nucleus to the cytoplasm, thereby activating the apoptotic genes Bim, FasL, and tumor necrosis factor and its related apoptosis-inducing ligand TRAIL, consequently promoting apoptosis.[14] In addition to its role in apoptosis, FoxO3a plays critical roles in regulating cell proliferation, metabolism, the stress response, and the life span of cancer cells.[15] Particularly, another target molecule of FoxO3a is the cell cycle-regulating gene P27; overexpression of FoxO3a can arrest the cell cycle in the G0/G1 phase by upregulating P27. In addition, overexpression of FoxO3a can significantly inhibit the proliferation of tumor cells and result in tumor cell G2 arrest, further promoting tumor cell apoptosis.[1617]\\nThe results of our analyses showed that the FoxO3a-positive rate in the high-grade tumors was significantly lower than that in the normal oviducts. In the low-grade adenocarcinomas, borderline cystadenomas, cystadenomas, and normal ovaries, the positive expression rates of FoxO3a protein were gradually reduced from normal ovaries to the low-grade adenocarcinomas, and the FoxO3 positive rate of the low-grade tumors was significantly lower than those of the borderline cystadenomas, cystadenomas, and normal ovaries, while the positive rate of borderline cystadenomas was significantly lower than those of cystadenomas and normal ovaries. Moreover, the FoxO3a expression level showed significant associations with LNM, FIGO stage, and omental metastasis in high-grade, but not low-grade tumors. These results suggest that FoxO3a may play a role in the oncogenesis and development of ovarian serous adenocarcinomas. In the high-grade tumors, the 5-year survival rate of FoxO3a-positive patients was significantly higher than that of the negative patients, whereas no difference in survival was seen in low-grade tumors, indicating that positive FoxO3a expression may have a long-term protective effect on cancer patients, and FoxO3a may hence have certain significance in the prediction of the patients’ long-term prognosis. Interestingly, it has been reported that tumor cells with low FoxO3a expression can develop drug resistance to paclitaxel and that inhibiting FoxO3a expression with RNAi can reduce E1A-mediated sensitivity to paclitaxel.[18] Therefore, FoxO3a protein may represent a potential prognostic indicator for high-grade ovarian serous adenocarcinoma.\",\n \"RAD51 is a human homologous recombination repair gene. Too strong or too weak homologous recombination will result in genome instability and DNA damage, and the consequent gene loss, loss of heterozygosity, and/or genetic translocation are important causes of tumorigenesis and tumor progression.[19] Studies have found that RAD51 protein is overexpression in the breast, prostate, pancreatic, and colon cancers. Such overexpression of RAD51 can provide certain advantages for the growth of cancer cells by promoting the initiation and progression of cancer. However, to date, studies on the expression of RAD51 in ovarian cancers are few.\\nThe results of this study showed that the RAD51-positive rate in high-grade tumors was significantly higher than that of normal oviducts, suggesting that RAD51 may play a role in the initiation of high-grade ovarian serous carcinomas. The RAD51-positive rate gradually increased from normal ovaries to cystadenomas to borderline cystadenomas to low-grade serous adenocarcinomas. Particularly, the positive rate of low-grade tumors was significantly higher than that in benign tumors and normal oviducts. The gradual change in the RAD51 expression suggests that the initiation of low-grade serous adenocarcinoma is a gradual procedure from benign to borderline to low-grade adenocarcinoma. However, no significant difference in the expression of RAD51 was found between high- and low-grade tumors. The expression level of RAD51 showed significant associations with LNM, FIGO stage, and omental metastasis in high-grade, but not low-grade tumors.\\nHerein, the 5-year survival rate of patients with RAD51-positive high-grade adenocarcinomas was significantly lower than that of patients with RAD51-negative tumors, while no difference in survival was found in low-grade tumors. In other words, we found that high-grade tumor patients with high expression of RAD51 had a lower survival rate and poor prognosis, indicating that RAD51 may be a prognostic factor in high-grade ovarian serous adenocarcinoma. Furthermore, multivariate survival analyses showed that RAD51 was an independent prognostic factor, which may be of clinical significance in terms of the development of molecular targeted therapy, in addition to FIGO stage, and LNM. Further investigations on RAD51 are required to confirm these findings and to hopefully be able to improve the quality of life and prognosis of patients using targeted therapy.\\nIn conclusion, the results of the present study indicate that PHLPP, FoxO3a, and RAD51 may play certain roles in the pathogenesis of the different grades of ovarian serous adenocarcinomas. Meanwhile, they also suggest the possible presence of common molecular mechanisms in the pathogenesis of the different grades. Increased protein expression of RAD51 may be an important molecular marker for poor prognosis of high-grade ovarian serous adenocarcinomas. However, considering the limited number of cases selected, which may have affected the significance of the results, further studies are required to improve and supplement these findings.\",\n \"This work were supported by grants from the Key Scientific Research program in Medical Science of Hebei Province, China (No. 20150318) and Science and Technology support program of Hebei Province, China (No. 16277794D).\",\n \"There are no conflicts of interest.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,null,null,null,null,null,null,"results",null,null,null,null,"discussion",null,null,null,null,"COI-statement"],"string":"[\n \"intro\",\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"discussion\",\n null,\n null,\n null,\n null,\n \"COI-statement\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["Forkhead Homeobox Type O 3a","Immunohistochemistry","Ovarian Serous Adenocarcinomas","PH Domain Leucine-rich Repeat Protein Phosphatase","Prognosis","RAD51"],"string":"[\n \"Forkhead Homeobox Type O 3a\",\n \"Immunohistochemistry\",\n \"Ovarian Serous Adenocarcinomas\",\n \"PH Domain Leucine-rich Repeat Protein Phosphatase\",\n \"Prognosis\",\n \"RAD51\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Introduction:\nOvarian serous adenocarcinoma is the most common ovarian epithelial malignant tumor. It can be divided into low- and high-grade tumors, which exhibit substantial differences in the pathogenesis, clinicopathological characteristics, and prognosis.[1] Low-grade serous carcinomas undergo a progressive, gradual developmental process from benign serous cystadenoma to borderline serous cystadenoma to malignant serous adenocarcinoma, while high-grade serous adenocarcinomas develop directly from the oviduct epithelium or ovarian inclusion cysts.\nHowever, it remains unclear whether the two tumor types have completely different molecular mechanisms, whether there is a common molecular basis, and whether the genes involved in the development of these tumors affect the patients’ prognosis. Recent studies[23] have indicated that the PI3K/Akt signaling pathway is involved in the initiation and development of ovarian serous adenocarcinoma. For example, PH domain leucine-rich repeat protein phosphatase (PHLPP) is known to negatively regulate Akt and its downstream kinase by dephosphorylating the hydrophobic core of Akt (Akt1 Ser473), thereby antagonizing PI3K/Akt signaling and inhibiting tumor growth. Forkhead homeobox type O 3a (FoxO3a), an important downstream signaling molecule of Akt, is involved in the initiation and development of tumor cells and regulation of cell proliferation after being phosphorylated by Akt.[45] Further, RAD51, a DNA double-strand break repair gene,[6] has also been implicated in the PI3K/Akt signaling pathway: phosphorylated FoxO3a binds to the promoter of the downstream gene RAD51, thereby regulating RAD51 and promoting tumor development and metastasis.\nThe present study aimed to (1) determine the differences in the protein expressions of PHLPP, FoxO3a, and RAD51 between high- and low-grade ovarian serous adenocarcinomas, (2) explore the roles of these three genes in the initiation of the different grades of ovarian serous adenocarcinoma, and (3) analyze the relationships between their protein expressions and prognosis.\n\nMethods:\n Ethics The study was approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University and was conducted in accordance with the Declaration of Helsinki 1975, as revised in 2000.\nThe study was approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University and was conducted in accordance with the Declaration of Helsinki 1975, as revised in 2000.\n Participant selection and description Patients with ovarian serous adenocarcinoma hospitalized and treated from January 2006 to January 2011 in our hospital were included in the ovarian serous adenocarcinoma groups. No patients received chemotherapy and radiotherapy preoperatively, and their diagnosis was confirmed by pathological examinations postoperatively. All patients had complete clinical data and were graded according to the World Health Organization histological classification for ovarian tumors (2014 edition). Ninety-four and twenty-six cases of high- and low-grade ovarian serous adenocarcinomas were included in this study. Among the high-grade group (mean age, 55 years; range, 36–78 years), there were 26 and 68 patients with International Federation of Gynecology and Obstetrics (FIGO) Stage (2014 edition) I–II and III–IV disease, respectively. There were thirty and 64 patients with and without lymph node metastasis (LNM), and 41 and 53 patients with and without omental metastasis, respectively. Among the low-grade group (mean age, 44 years; range, 26–70 years), there were 18 and eight patients with FIGO Stage I–II and III–IV disease, respectively. There were 4 and 22 patients with and without LNM, and 5 and 21 patients with and without omental metastasis, respectively. Moreover, 21 cases of borderline serous cystadenoma (mean age, 38 years; range, 18–59 years) and 35 cases of benign serous cystadenoma (mean age, 45 years; range, 22–69 years) were included, along with thirty normal ovaries and thirty oviducts (mean age, 48 years; range, 36–62 years) removed from patients undergoing surgery for uterine fibroids, which were used as controls for the low- and high-grade tumor groups, respectively.\nExpression of TP53 protein in high- and low-grade serous carcinoma: Among the 94 cases of high-grade, 92 cases (97.9%) showed strong staining of TP53, while among the 26 cases of low-grade serous adenocarcinoma, there was no case of strong staining.\nPatients with ovarian serous adenocarcinoma hospitalized and treated from January 2006 to January 2011 in our hospital were included in the ovarian serous adenocarcinoma groups. No patients received chemotherapy and radiotherapy preoperatively, and their diagnosis was confirmed by pathological examinations postoperatively. All patients had complete clinical data and were graded according to the World Health Organization histological classification for ovarian tumors (2014 edition). Ninety-four and twenty-six cases of high- and low-grade ovarian serous adenocarcinomas were included in this study. Among the high-grade group (mean age, 55 years; range, 36–78 years), there were 26 and 68 patients with International Federation of Gynecology and Obstetrics (FIGO) Stage (2014 edition) I–II and III–IV disease, respectively. There were thirty and 64 patients with and without lymph node metastasis (LNM), and 41 and 53 patients with and without omental metastasis, respectively. Among the low-grade group (mean age, 44 years; range, 26–70 years), there were 18 and eight patients with FIGO Stage I–II and III–IV disease, respectively. There were 4 and 22 patients with and without LNM, and 5 and 21 patients with and without omental metastasis, respectively. Moreover, 21 cases of borderline serous cystadenoma (mean age, 38 years; range, 18–59 years) and 35 cases of benign serous cystadenoma (mean age, 45 years; range, 22–69 years) were included, along with thirty normal ovaries and thirty oviducts (mean age, 48 years; range, 36–62 years) removed from patients undergoing surgery for uterine fibroids, which were used as controls for the low- and high-grade tumor groups, respectively.\nExpression of TP53 protein in high- and low-grade serous carcinoma: Among the 94 cases of high-grade, 92 cases (97.9%) showed strong staining of TP53, while among the 26 cases of low-grade serous adenocarcinoma, there was no case of strong staining.\n Technical information \nExperimental procedures\n\nThe operative specimens were examined, and the patients were diagnosed by experienced pathologists. Paraffin blocks of the specimens were cut, and immunohistochemical staining of the sections was conducted as follows: the samples were sliced, deparaffinized, dehydrated, and subjected to antigen retrieval before blocking with goat serum. Subsequently, the slides were placed in a humidifier chamber and incubated at room temperature for 45 min. The tissues were incubated with primary antibodies (working concentrations of anti-PHLPP, RAD51, and FoxO3a of 1:100, 1:100, and 1:150, respectively, Biogot Biotechnology Co., Ltd., Nanjing, China) in the sealed humidifier chamber at 4°C overnight. After washing of the slides, goat anti-rabbit secondary antibodies were added and the slides were incubated in the humidifier at 37°C for 30 min. Next, horseradish peroxidase-labeled streptavidin was added and the slides were incubated in the humidifier at 37°C for 30 min, after which the tissues were stained by 3,3’-diaminobenzidine, followed by counterstaining, destaining, dehydration, clearing, and mounting. Finally, the slides were air-dried and observed under a microscope.\n\nEvaluation standard for staining\n\nThe slides were scored by pathologists according to the below criteria for PHLPP, FoxO3a, and RAD51 under a microscope. Ten high-magnification fields were counted, and the average cell numbers were used.\n PH domain leucine-rich repeat protein phosphatase-positive criteria Cells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\nCells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\n Forkhead homeobox type O 3a-positive criteria Cells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\nCells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\n RAD51-positive criteria Cells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\nCells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\n\nExperimental procedures\n\nThe operative specimens were examined, and the patients were diagnosed by experienced pathologists. Paraffin blocks of the specimens were cut, and immunohistochemical staining of the sections was conducted as follows: the samples were sliced, deparaffinized, dehydrated, and subjected to antigen retrieval before blocking with goat serum. Subsequently, the slides were placed in a humidifier chamber and incubated at room temperature for 45 min. The tissues were incubated with primary antibodies (working concentrations of anti-PHLPP, RAD51, and FoxO3a of 1:100, 1:100, and 1:150, respectively, Biogot Biotechnology Co., Ltd., Nanjing, China) in the sealed humidifier chamber at 4°C overnight. After washing of the slides, goat anti-rabbit secondary antibodies were added and the slides were incubated in the humidifier at 37°C for 30 min. Next, horseradish peroxidase-labeled streptavidin was added and the slides were incubated in the humidifier at 37°C for 30 min, after which the tissues were stained by 3,3’-diaminobenzidine, followed by counterstaining, destaining, dehydration, clearing, and mounting. Finally, the slides were air-dried and observed under a microscope.\n\nEvaluation standard for staining\n\nThe slides were scored by pathologists according to the below criteria for PHLPP, FoxO3a, and RAD51 under a microscope. Ten high-magnification fields were counted, and the average cell numbers were used.\n PH domain leucine-rich repeat protein phosphatase-positive criteria Cells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\nCells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\n Forkhead homeobox type O 3a-positive criteria Cells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\nCells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\n RAD51-positive criteria Cells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\nCells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\n Statistical analyses SPSS 16.0 software (SPSS Inc., USA) was used for the statistical analysis. Categorical data were analyzed by the Chi-square test, and the survival rates were calculated by Kaplan-Meier univariate analysis. Differences in the survival rates between the different groups were determined by the log-rank test. Cox multivariate survival analysis was used for analysis of independent prognostic factors. The statistical significance level was set as P < 0.05.\nSPSS 16.0 software (SPSS Inc., USA) was used for the statistical analysis. Categorical data were analyzed by the Chi-square test, and the survival rates were calculated by Kaplan-Meier univariate analysis. Differences in the survival rates between the different groups were determined by the log-rank test. Cox multivariate survival analysis was used for analysis of independent prognostic factors. The statistical significance level was set as P < 0.05.\n\nEthics:\nThe study was approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University and was conducted in accordance with the Declaration of Helsinki 1975, as revised in 2000.\n\nParticipant selection and description:\nPatients with ovarian serous adenocarcinoma hospitalized and treated from January 2006 to January 2011 in our hospital were included in the ovarian serous adenocarcinoma groups. No patients received chemotherapy and radiotherapy preoperatively, and their diagnosis was confirmed by pathological examinations postoperatively. All patients had complete clinical data and were graded according to the World Health Organization histological classification for ovarian tumors (2014 edition). Ninety-four and twenty-six cases of high- and low-grade ovarian serous adenocarcinomas were included in this study. Among the high-grade group (mean age, 55 years; range, 36–78 years), there were 26 and 68 patients with International Federation of Gynecology and Obstetrics (FIGO) Stage (2014 edition) I–II and III–IV disease, respectively. There were thirty and 64 patients with and without lymph node metastasis (LNM), and 41 and 53 patients with and without omental metastasis, respectively. Among the low-grade group (mean age, 44 years; range, 26–70 years), there were 18 and eight patients with FIGO Stage I–II and III–IV disease, respectively. There were 4 and 22 patients with and without LNM, and 5 and 21 patients with and without omental metastasis, respectively. Moreover, 21 cases of borderline serous cystadenoma (mean age, 38 years; range, 18–59 years) and 35 cases of benign serous cystadenoma (mean age, 45 years; range, 22–69 years) were included, along with thirty normal ovaries and thirty oviducts (mean age, 48 years; range, 36–62 years) removed from patients undergoing surgery for uterine fibroids, which were used as controls for the low- and high-grade tumor groups, respectively.\nExpression of TP53 protein in high- and low-grade serous carcinoma: Among the 94 cases of high-grade, 92 cases (97.9%) showed strong staining of TP53, while among the 26 cases of low-grade serous adenocarcinoma, there was no case of strong staining.\n\nTechnical information:\n\nExperimental procedures\n\nThe operative specimens were examined, and the patients were diagnosed by experienced pathologists. Paraffin blocks of the specimens were cut, and immunohistochemical staining of the sections was conducted as follows: the samples were sliced, deparaffinized, dehydrated, and subjected to antigen retrieval before blocking with goat serum. Subsequently, the slides were placed in a humidifier chamber and incubated at room temperature for 45 min. The tissues were incubated with primary antibodies (working concentrations of anti-PHLPP, RAD51, and FoxO3a of 1:100, 1:100, and 1:150, respectively, Biogot Biotechnology Co., Ltd., Nanjing, China) in the sealed humidifier chamber at 4°C overnight. After washing of the slides, goat anti-rabbit secondary antibodies were added and the slides were incubated in the humidifier at 37°C for 30 min. Next, horseradish peroxidase-labeled streptavidin was added and the slides were incubated in the humidifier at 37°C for 30 min, after which the tissues were stained by 3,3’-diaminobenzidine, followed by counterstaining, destaining, dehydration, clearing, and mounting. Finally, the slides were air-dried and observed under a microscope.\n\nEvaluation standard for staining\n\nThe slides were scored by pathologists according to the below criteria for PHLPP, FoxO3a, and RAD51 under a microscope. Ten high-magnification fields were counted, and the average cell numbers were used.\n PH domain leucine-rich repeat protein phosphatase-positive criteria Cells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\nCells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\n Forkhead homeobox type O 3a-positive criteria Cells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\nCells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\n RAD51-positive criteria Cells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\nCells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\n\nPH domain leucine-rich repeat protein phosphatase-positive criteria:\nCells with cytoplasm stained brown were determined as positive. The staining intensity was scored as follows: uncolored (0), pale yellow (1 point), brown (2 points), and tan (3 points). The percentages of positive cells were scored as 0, 1, 2, 3, and 4 points for <5%, 5–25%, 26–50%, 51–75%, and >75%, respectively. According to the combined results of the two scores, the staining was divided into four levels: 0–1 point was defined as negative (−), ≥2 was defined as positive.[7]\n\nForkhead homeobox type O 3a-positive criteria:\nCells with nuclei staining brown were defined as positive. No staining was scored as 0, positive cells <10% as 1 point, 11–25% as 2 points, and >26% as 3 points. A total score of >2 points was defined as positive.[8]\n\nRAD51-positive criteria:\nCells with cytoplasm or nuclei staining brown were defined as positive. Positive cells <10% and ≥10% were defined as negative and positive expressions, respectively.[9]\n\nStatistical analyses:\nSPSS 16.0 software (SPSS Inc., USA) was used for the statistical analysis. Categorical data were analyzed by the Chi-square test, and the survival rates were calculated by Kaplan-Meier univariate analysis. Differences in the survival rates between the different groups were determined by the log-rank test. Cox multivariate survival analysis was used for analysis of independent prognostic factors. The statistical significance level was set as P < 0.05.\n\nResults:\n Expression of PH domain leucine-rich repeat protein phosphatase in ovarian serous adenocarcinomas and its association with clinicopathological features and prognosis Among 94 cases of high-grade serous adenocarcinoma, 23 cases (24.5%) were PHLPP-positive, which was significantly lower than the positive rate of 86.7% in the normal oviduct group (P < 0.05). Of 26 low-grade serous adenocarcinoma cases, 6 (23.1%) were positive, which was significantly lower than that in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, there was no difference in the positive expression rates between high- and low-grade tumors [P > 0.05; Figure 1 and Table 1].\nProtein expression of PH domain leucine-rich repeat protein phosphatase in high- and low-grade serous ovarian adenocarcinoma by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\nProtein expression of PHLPP in high- and low-grade ovarian serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. PHLPP: PH domain leucine-rich repeat protein phosphatase.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower PHLPP protein expression levels [P < 0.05; Table 2]. On the other hand, there were no significant correlations between the clinicopathological features and PHLPP protein expression in low-grade tumors [P > 0.05].\nRelationships between PHLPP, FoxO3a, and RAD51 protein expressions and clinicopathological features of high-grade serous ovarian adenocarcinoma\nLNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a.\nAmong the 94 patients with high-grade serous adenocarcinoma, the 5-year survival rates of PHLPP-negative and positive patients were 5.6% and 43.5%, respectively [P < 0.05, Figure 2]. Among the 26 cases of low-grade serous adenocarcinoma, however, the expression of PHLPP did not significantly associate with the 5-year survival rate of the patients [P > 0.05].\nFive-year survival curves of PH domain leucine-rich repeat protein phosphatase-positive and negative patients with high-grade ovarian serous adenocarcinomas.\nAmong 94 cases of high-grade serous adenocarcinoma, 23 cases (24.5%) were PHLPP-positive, which was significantly lower than the positive rate of 86.7% in the normal oviduct group (P < 0.05). Of 26 low-grade serous adenocarcinoma cases, 6 (23.1%) were positive, which was significantly lower than that in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, there was no difference in the positive expression rates between high- and low-grade tumors [P > 0.05; Figure 1 and Table 1].\nProtein expression of PH domain leucine-rich repeat protein phosphatase in high- and low-grade serous ovarian adenocarcinoma by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\nProtein expression of PHLPP in high- and low-grade ovarian serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. PHLPP: PH domain leucine-rich repeat protein phosphatase.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower PHLPP protein expression levels [P < 0.05; Table 2]. On the other hand, there were no significant correlations between the clinicopathological features and PHLPP protein expression in low-grade tumors [P > 0.05].\nRelationships between PHLPP, FoxO3a, and RAD51 protein expressions and clinicopathological features of high-grade serous ovarian adenocarcinoma\nLNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a.\nAmong the 94 patients with high-grade serous adenocarcinoma, the 5-year survival rates of PHLPP-negative and positive patients were 5.6% and 43.5%, respectively [P < 0.05, Figure 2]. Among the 26 cases of low-grade serous adenocarcinoma, however, the expression of PHLPP did not significantly associate with the 5-year survival rate of the patients [P > 0.05].\nFive-year survival curves of PH domain leucine-rich repeat protein phosphatase-positive and negative patients with high-grade ovarian serous adenocarcinomas.\n Expression of forkhead homeobox type O 3a in ovarian serous adenocarcinoma and its association with clinicopathological features and prognosis Among the 94 cases of high-grade serous adenocarcinoma, 25 (26.6%) were positive for FoxO3a protein expression, which was significantly lower than the positive rate of 90.0% in the normal oviduct group (P < 0.05). Among the 26 cases of low-grade serous carcinoma, 7 (26.9%) were positive, which was significantly lower than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, the difference in the positive expression rates between the high- and low-grade groups was not significant [P > 0.05; Figure 3 and Table 3].\nProtein expression of forkhead homeobox type O 3a in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\nProtein expression of FoxO3a in high- and low-grade serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. FoxO3a: Forkhead homeobox type O 3a.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower FoxO3a protein expression [P < 0.05, Table 2]. On the other hand, in low-grade tumors, the FoxO3a protein expression showed no association with clinicopathological features (P > 0.05).\nAmong the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of FoxO3a-negative and positive patients were 7.2% and 36.0%, respectively [P < 0.05, Figure 4]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of FoxO3a showed no significant association with the 5-year survival rate (P > 0.05).\nFive-year survival curves of forkhead homeobox type O 3a-positive and negative patients with high-grade ovarian serous adenocarcinomas.\nAmong the 94 cases of high-grade serous adenocarcinoma, 25 (26.6%) were positive for FoxO3a protein expression, which was significantly lower than the positive rate of 90.0% in the normal oviduct group (P < 0.05). Among the 26 cases of low-grade serous carcinoma, 7 (26.9%) were positive, which was significantly lower than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, the difference in the positive expression rates between the high- and low-grade groups was not significant [P > 0.05; Figure 3 and Table 3].\nProtein expression of forkhead homeobox type O 3a in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\nProtein expression of FoxO3a in high- and low-grade serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. FoxO3a: Forkhead homeobox type O 3a.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower FoxO3a protein expression [P < 0.05, Table 2]. On the other hand, in low-grade tumors, the FoxO3a protein expression showed no association with clinicopathological features (P > 0.05).\nAmong the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of FoxO3a-negative and positive patients were 7.2% and 36.0%, respectively [P < 0.05, Figure 4]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of FoxO3a showed no significant association with the 5-year survival rate (P > 0.05).\nFive-year survival curves of forkhead homeobox type O 3a-positive and negative patients with high-grade ovarian serous adenocarcinomas.\n Expression of RAD51 in ovarian serous adenocarcinoma and its association with clinicopathological features and prognosis Among the 94 cases of high-grade serous adenocarcinoma, 66 (70.2%) were positive for RAD51 protein, which was significantly higher than that in the normal oviduct group [P < 0.05; Figure 5 and Table 4]. Among the 26 cases of low-grade serous carcinoma, 17 (65.4%) were positive, which was significantly higher than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups [P < 0.05; Figure 5 and Table 4]. However, the difference in the positive expression rates between the high- and low-grade groups was not significant (P > 0.05).\nProtein expression of RAD51 in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Positive high-grade adenocarcinoma, (b) negative normal oviduct, (c) positive low-grade adenocarcinoma, (d) negative borderline cystadenoma, (e) negative cystadenoma, (f) negative normal ovary.\nProtein expression of RAD51 in high- and low-grade serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with higher RAD51 protein expression levels [P < 0.05; Table 2]. Conversely, there was no significant correlation between clinicopathological features and RAD51 expression in low-grade tumors (P > 0.05).\nFinally, among the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of RAD51-positive and negative patients were 3.0% and 42.9%, respectively [P < 0.05; Figure 6]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of RAD51 showed no significant association with the 5-year survival rate (P > 0.05).\nFive-year survival curves of RAD51-positive and negative patients with high-grade ovarian serous adenocarcinomas.\nAmong the 94 cases of high-grade serous adenocarcinoma, 66 (70.2%) were positive for RAD51 protein, which was significantly higher than that in the normal oviduct group [P < 0.05; Figure 5 and Table 4]. Among the 26 cases of low-grade serous carcinoma, 17 (65.4%) were positive, which was significantly higher than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups [P < 0.05; Figure 5 and Table 4]. However, the difference in the positive expression rates between the high- and low-grade groups was not significant (P > 0.05).\nProtein expression of RAD51 in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Positive high-grade adenocarcinoma, (b) negative normal oviduct, (c) positive low-grade adenocarcinoma, (d) negative borderline cystadenoma, (e) negative cystadenoma, (f) negative normal ovary.\nProtein expression of RAD51 in high- and low-grade serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with higher RAD51 protein expression levels [P < 0.05; Table 2]. Conversely, there was no significant correlation between clinicopathological features and RAD51 expression in low-grade tumors (P > 0.05).\nFinally, among the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of RAD51-positive and negative patients were 3.0% and 42.9%, respectively [P < 0.05; Figure 6]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of RAD51 showed no significant association with the 5-year survival rate (P > 0.05).\nFive-year survival curves of RAD51-positive and negative patients with high-grade ovarian serous adenocarcinomas.\n Prognostic multivariate survival analyses of high-grade serous ovarian adenocarcinoma The Cox multivariate analysis results showed that FIGO staging, LNM, and RAD51 protein expression were independent prognostic factors of patients with high-grade serous ovarian adenocarcinoma [P < 0.05; Table 5].\nMultivariate analysis of independent factors associated with the 5-year overall survival rate in patients with high-grade serous ovarian adenocarcinoma\nRR: Risk ratio; CI: Confidence interval; LNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a; PHLPP: PH domain leucine-rich repeat protein phosphatase.\nThe Cox multivariate analysis results showed that FIGO staging, LNM, and RAD51 protein expression were independent prognostic factors of patients with high-grade serous ovarian adenocarcinoma [P < 0.05; Table 5].\nMultivariate analysis of independent factors associated with the 5-year overall survival rate in patients with high-grade serous ovarian adenocarcinoma\nRR: Risk ratio; CI: Confidence interval; LNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a; PHLPP: PH domain leucine-rich repeat protein phosphatase.\n\nExpression of PH domain leucine-rich repeat protein phosphatase in ovarian serous adenocarcinomas and its association with clinicopathological features and prognosis:\nAmong 94 cases of high-grade serous adenocarcinoma, 23 cases (24.5%) were PHLPP-positive, which was significantly lower than the positive rate of 86.7% in the normal oviduct group (P < 0.05). Of 26 low-grade serous adenocarcinoma cases, 6 (23.1%) were positive, which was significantly lower than that in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, there was no difference in the positive expression rates between high- and low-grade tumors [P > 0.05; Figure 1 and Table 1].\nProtein expression of PH domain leucine-rich repeat protein phosphatase in high- and low-grade serous ovarian adenocarcinoma by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\nProtein expression of PHLPP in high- and low-grade ovarian serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. PHLPP: PH domain leucine-rich repeat protein phosphatase.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower PHLPP protein expression levels [P < 0.05; Table 2]. On the other hand, there were no significant correlations between the clinicopathological features and PHLPP protein expression in low-grade tumors [P > 0.05].\nRelationships between PHLPP, FoxO3a, and RAD51 protein expressions and clinicopathological features of high-grade serous ovarian adenocarcinoma\nLNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a.\nAmong the 94 patients with high-grade serous adenocarcinoma, the 5-year survival rates of PHLPP-negative and positive patients were 5.6% and 43.5%, respectively [P < 0.05, Figure 2]. Among the 26 cases of low-grade serous adenocarcinoma, however, the expression of PHLPP did not significantly associate with the 5-year survival rate of the patients [P > 0.05].\nFive-year survival curves of PH domain leucine-rich repeat protein phosphatase-positive and negative patients with high-grade ovarian serous adenocarcinomas.\n\nExpression of forkhead homeobox type O 3a in ovarian serous adenocarcinoma and its association with clinicopathological features and prognosis:\nAmong the 94 cases of high-grade serous adenocarcinoma, 25 (26.6%) were positive for FoxO3a protein expression, which was significantly lower than the positive rate of 90.0% in the normal oviduct group (P < 0.05). Among the 26 cases of low-grade serous carcinoma, 7 (26.9%) were positive, which was significantly lower than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups (P < 0.05). However, the difference in the positive expression rates between the high- and low-grade groups was not significant [P > 0.05; Figure 3 and Table 3].\nProtein expression of forkhead homeobox type O 3a in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Negative high-grade adenocarcinoma, (b) positive normal oviduct, (c) negative low-grade adenocarcinoma, (d) positive borderline cystadenoma, (e) positive cystadenoma, (f) positive normal ovary.\nProtein expression of FoxO3a in high- and low-grade serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas. FoxO3a: Forkhead homeobox type O 3a.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with lower FoxO3a protein expression [P < 0.05, Table 2]. On the other hand, in low-grade tumors, the FoxO3a protein expression showed no association with clinicopathological features (P > 0.05).\nAmong the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of FoxO3a-negative and positive patients were 7.2% and 36.0%, respectively [P < 0.05, Figure 4]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of FoxO3a showed no significant association with the 5-year survival rate (P > 0.05).\nFive-year survival curves of forkhead homeobox type O 3a-positive and negative patients with high-grade ovarian serous adenocarcinomas.\n\nExpression of RAD51 in ovarian serous adenocarcinoma and its association with clinicopathological features and prognosis:\nAmong the 94 cases of high-grade serous adenocarcinoma, 66 (70.2%) were positive for RAD51 protein, which was significantly higher than that in the normal oviduct group [P < 0.05; Figure 5 and Table 4]. Among the 26 cases of low-grade serous carcinoma, 17 (65.4%) were positive, which was significantly higher than in the normal ovary, borderline serous cystadenoma, and serous cystadenoma groups [P < 0.05; Figure 5 and Table 4]. However, the difference in the positive expression rates between the high- and low-grade groups was not significant (P > 0.05).\nProtein expression of RAD51 in high- and low-grade serous ovarian adenocarcinomas by immunohistochemical analysis (original magnification ×200). (a) Positive high-grade adenocarcinoma, (b) negative normal oviduct, (c) positive low-grade adenocarcinoma, (d) negative borderline cystadenoma, (e) negative cystadenoma, (f) negative normal ovary.\nProtein expression of RAD51 in high- and low-grade serous adenocarcinomas\n*P<0.05 compared with normal oviducts; †P<0.05 compared with normal ovaries, cystadenomas, and serous cystadenomas.\nIn high-grade tumors, LNM, FIGO Stage III/IV tumors, and omental metastasis were significantly associated with higher RAD51 protein expression levels [P < 0.05; Table 2]. Conversely, there was no significant correlation between clinicopathological features and RAD51 expression in low-grade tumors (P > 0.05).\nFinally, among the 94 cases of high-grade serous adenocarcinoma, the 5-year survival rates of RAD51-positive and negative patients were 3.0% and 42.9%, respectively [P < 0.05; Figure 6]. Among the 26 cases of low-grade serous adenocarcinoma, the expression of RAD51 showed no significant association with the 5-year survival rate (P > 0.05).\nFive-year survival curves of RAD51-positive and negative patients with high-grade ovarian serous adenocarcinomas.\n\nPrognostic multivariate survival analyses of high-grade serous ovarian adenocarcinoma:\nThe Cox multivariate analysis results showed that FIGO staging, LNM, and RAD51 protein expression were independent prognostic factors of patients with high-grade serous ovarian adenocarcinoma [P < 0.05; Table 5].\nMultivariate analysis of independent factors associated with the 5-year overall survival rate in patients with high-grade serous ovarian adenocarcinoma\nRR: Risk ratio; CI: Confidence interval; LNM: Lymph node metastasis; FIGO: International Federation of Gynecology and Obstetrics; OM: Omental metastasis; FoxO3a: Forkhead homeobox type O 3a; PHLPP: PH domain leucine-rich repeat protein phosphatase.\n\nDiscussion:\nOvarian cancer is one of the most common malignancies of the female reproductive system, and has the highest mortality among all gynecologic malignant tumors, thus seriously affecting women's health and lives. Recent studies have suggested that ovarian serous adenocarcinomas can be divided into high- and low-grade tumors and that the initiation mechanisms, clinical pathological features, and prognoses of the two types greatly differ.[110] Thus, ovarian serous adenocarcinomas have complex biological behaviors. However, although the pathogeneses of the two types involve different molecular pathways, there is some common basis. Recent studies have found that the PI3K/Akt signaling pathway is closely associated with the initiation and progression of ovarian cancer. PHLPP, a known tumor suppressor, can negatively regulate Akt and its downstream kinases, thereby antagonizing the PI3K/Akt signaling pathway and inhibit tumor growth. Moreover, as an important downstream signaling molecule of Akt, after being phosphorylated by Akt, FoxO3a is involved in the initiation and development of tumor cells and regulates cell proliferation. RAD51, a downstream target of FoxO3a, also plays an important role in PI3K/Akt signaling; FoxO3a binds to the promoter of RAD51, consequently regulating RAD51 and promoting tumor development and metastasis. In this study, by examining the differences in the protein expression levels of PHLPP, FoxO3a, and RAD51 between high- and low-grade ovarian serous adenocarcinomas, the roles of the three genes in the development of the different grades of ovarian serous adenocarcinoma were explored, and the relationships between the protein expression levels and the patient prognosis were analyzed.\n Role of the tumor suppressor PH domain leucine-rich repeat protein phosphatase in the development of ovarian serous adenocarcinoma and its association with patient prognosis The tumor suppressor PHLPP, located at 18q21.33, can directly dephosphorylate ser473 in the hydrophobic group of Akt, thus negatively regulating Akt and its downstream targets. It has been reported that PHLPP can act synergistically with PTEN, another well-known tumor suppressor gene, thereby significantly inhibiting the proliferation of colorectal cancer cells, and negatively regulating PI3K/Akt signaling.[11] Accordingly, studies have shown that the PHLPP expression is significantly decreased in colon, breast, prostate, and lung cancers.[5712]\nThe results of the present study showed that the positive rate of PHLPP protein expression in the high-grade group was significantly lower than that in the oviduct group. The PHLPP-positive expression rate was decreased gradually from normal ovarian tissue to benign tumors to borderline tumors to low-grade adenocarcinomas, indicating that loss of PHLPP expression may be associated with the initiation of ovarian serous adenocarcinoma development, whereas no significant difference was observed between high-grade and low-grade tumors. Further, the PHLPP expression level showed significant associations with LNM, FIGO stages, and omental metastasis in high-grade tumors, but not in low-grade tumors. Similarly, the 5-year survival rates of PHLPP-positive and negative high-grade tumors significant differed, whereas no difference was seen in the low-grade tumors. These findings suggest that PHLPP may be a predictor of prognosis of high-grade ovarian serous adenocarcinoma.\nThe tumor suppressor PHLPP, located at 18q21.33, can directly dephosphorylate ser473 in the hydrophobic group of Akt, thus negatively regulating Akt and its downstream targets. It has been reported that PHLPP can act synergistically with PTEN, another well-known tumor suppressor gene, thereby significantly inhibiting the proliferation of colorectal cancer cells, and negatively regulating PI3K/Akt signaling.[11] Accordingly, studies have shown that the PHLPP expression is significantly decreased in colon, breast, prostate, and lung cancers.[5712]\nThe results of the present study showed that the positive rate of PHLPP protein expression in the high-grade group was significantly lower than that in the oviduct group. The PHLPP-positive expression rate was decreased gradually from normal ovarian tissue to benign tumors to borderline tumors to low-grade adenocarcinomas, indicating that loss of PHLPP expression may be associated with the initiation of ovarian serous adenocarcinoma development, whereas no significant difference was observed between high-grade and low-grade tumors. Further, the PHLPP expression level showed significant associations with LNM, FIGO stages, and omental metastasis in high-grade tumors, but not in low-grade tumors. Similarly, the 5-year survival rates of PHLPP-positive and negative high-grade tumors significant differed, whereas no difference was seen in the low-grade tumors. These findings suggest that PHLPP may be a predictor of prognosis of high-grade ovarian serous adenocarcinoma.\n Role of the tumor suppressor forkhead homeobox type O 3a in the development of ovarian serous adenocarcinoma and its association with patient prognosis FoxO3a is an important member of the FOXO family, located on at 6q21, and encodes a 673-amino acid long protein.[13] FoxO3a, as an important signaling molecule downstream of Akt in the PI3K-Akt signaling pathway, binds to the 14-3-3 chaperone protein after being phosphorylated by Akt, and subsequently translocates from the nucleus to the cytoplasm, thereby activating the apoptotic genes Bim, FasL, and tumor necrosis factor and its related apoptosis-inducing ligand TRAIL, consequently promoting apoptosis.[14] In addition to its role in apoptosis, FoxO3a plays critical roles in regulating cell proliferation, metabolism, the stress response, and the life span of cancer cells.[15] Particularly, another target molecule of FoxO3a is the cell cycle-regulating gene P27; overexpression of FoxO3a can arrest the cell cycle in the G0/G1 phase by upregulating P27. In addition, overexpression of FoxO3a can significantly inhibit the proliferation of tumor cells and result in tumor cell G2 arrest, further promoting tumor cell apoptosis.[1617]\nThe results of our analyses showed that the FoxO3a-positive rate in the high-grade tumors was significantly lower than that in the normal oviducts. In the low-grade adenocarcinomas, borderline cystadenomas, cystadenomas, and normal ovaries, the positive expression rates of FoxO3a protein were gradually reduced from normal ovaries to the low-grade adenocarcinomas, and the FoxO3 positive rate of the low-grade tumors was significantly lower than those of the borderline cystadenomas, cystadenomas, and normal ovaries, while the positive rate of borderline cystadenomas was significantly lower than those of cystadenomas and normal ovaries. Moreover, the FoxO3a expression level showed significant associations with LNM, FIGO stage, and omental metastasis in high-grade, but not low-grade tumors. These results suggest that FoxO3a may play a role in the oncogenesis and development of ovarian serous adenocarcinomas. In the high-grade tumors, the 5-year survival rate of FoxO3a-positive patients was significantly higher than that of the negative patients, whereas no difference in survival was seen in low-grade tumors, indicating that positive FoxO3a expression may have a long-term protective effect on cancer patients, and FoxO3a may hence have certain significance in the prediction of the patients’ long-term prognosis. Interestingly, it has been reported that tumor cells with low FoxO3a expression can develop drug resistance to paclitaxel and that inhibiting FoxO3a expression with RNAi can reduce E1A-mediated sensitivity to paclitaxel.[18] Therefore, FoxO3a protein may represent a potential prognostic indicator for high-grade ovarian serous adenocarcinoma.\nFoxO3a is an important member of the FOXO family, located on at 6q21, and encodes a 673-amino acid long protein.[13] FoxO3a, as an important signaling molecule downstream of Akt in the PI3K-Akt signaling pathway, binds to the 14-3-3 chaperone protein after being phosphorylated by Akt, and subsequently translocates from the nucleus to the cytoplasm, thereby activating the apoptotic genes Bim, FasL, and tumor necrosis factor and its related apoptosis-inducing ligand TRAIL, consequently promoting apoptosis.[14] In addition to its role in apoptosis, FoxO3a plays critical roles in regulating cell proliferation, metabolism, the stress response, and the life span of cancer cells.[15] Particularly, another target molecule of FoxO3a is the cell cycle-regulating gene P27; overexpression of FoxO3a can arrest the cell cycle in the G0/G1 phase by upregulating P27. In addition, overexpression of FoxO3a can significantly inhibit the proliferation of tumor cells and result in tumor cell G2 arrest, further promoting tumor cell apoptosis.[1617]\nThe results of our analyses showed that the FoxO3a-positive rate in the high-grade tumors was significantly lower than that in the normal oviducts. In the low-grade adenocarcinomas, borderline cystadenomas, cystadenomas, and normal ovaries, the positive expression rates of FoxO3a protein were gradually reduced from normal ovaries to the low-grade adenocarcinomas, and the FoxO3 positive rate of the low-grade tumors was significantly lower than those of the borderline cystadenomas, cystadenomas, and normal ovaries, while the positive rate of borderline cystadenomas was significantly lower than those of cystadenomas and normal ovaries. Moreover, the FoxO3a expression level showed significant associations with LNM, FIGO stage, and omental metastasis in high-grade, but not low-grade tumors. These results suggest that FoxO3a may play a role in the oncogenesis and development of ovarian serous adenocarcinomas. In the high-grade tumors, the 5-year survival rate of FoxO3a-positive patients was significantly higher than that of the negative patients, whereas no difference in survival was seen in low-grade tumors, indicating that positive FoxO3a expression may have a long-term protective effect on cancer patients, and FoxO3a may hence have certain significance in the prediction of the patients’ long-term prognosis. Interestingly, it has been reported that tumor cells with low FoxO3a expression can develop drug resistance to paclitaxel and that inhibiting FoxO3a expression with RNAi can reduce E1A-mediated sensitivity to paclitaxel.[18] Therefore, FoxO3a protein may represent a potential prognostic indicator for high-grade ovarian serous adenocarcinoma.\n Role of RAD51 in the development of ovarian serous adenocarcinoma and its association with patient prognosis RAD51 is a human homologous recombination repair gene. Too strong or too weak homologous recombination will result in genome instability and DNA damage, and the consequent gene loss, loss of heterozygosity, and/or genetic translocation are important causes of tumorigenesis and tumor progression.[19] Studies have found that RAD51 protein is overexpression in the breast, prostate, pancreatic, and colon cancers. Such overexpression of RAD51 can provide certain advantages for the growth of cancer cells by promoting the initiation and progression of cancer. However, to date, studies on the expression of RAD51 in ovarian cancers are few.\nThe results of this study showed that the RAD51-positive rate in high-grade tumors was significantly higher than that of normal oviducts, suggesting that RAD51 may play a role in the initiation of high-grade ovarian serous carcinomas. The RAD51-positive rate gradually increased from normal ovaries to cystadenomas to borderline cystadenomas to low-grade serous adenocarcinomas. Particularly, the positive rate of low-grade tumors was significantly higher than that in benign tumors and normal oviducts. The gradual change in the RAD51 expression suggests that the initiation of low-grade serous adenocarcinoma is a gradual procedure from benign to borderline to low-grade adenocarcinoma. However, no significant difference in the expression of RAD51 was found between high- and low-grade tumors. The expression level of RAD51 showed significant associations with LNM, FIGO stage, and omental metastasis in high-grade, but not low-grade tumors.\nHerein, the 5-year survival rate of patients with RAD51-positive high-grade adenocarcinomas was significantly lower than that of patients with RAD51-negative tumors, while no difference in survival was found in low-grade tumors. In other words, we found that high-grade tumor patients with high expression of RAD51 had a lower survival rate and poor prognosis, indicating that RAD51 may be a prognostic factor in high-grade ovarian serous adenocarcinoma. Furthermore, multivariate survival analyses showed that RAD51 was an independent prognostic factor, which may be of clinical significance in terms of the development of molecular targeted therapy, in addition to FIGO stage, and LNM. Further investigations on RAD51 are required to confirm these findings and to hopefully be able to improve the quality of life and prognosis of patients using targeted therapy.\nIn conclusion, the results of the present study indicate that PHLPP, FoxO3a, and RAD51 may play certain roles in the pathogenesis of the different grades of ovarian serous adenocarcinomas. Meanwhile, they also suggest the possible presence of common molecular mechanisms in the pathogenesis of the different grades. Increased protein expression of RAD51 may be an important molecular marker for poor prognosis of high-grade ovarian serous adenocarcinomas. However, considering the limited number of cases selected, which may have affected the significance of the results, further studies are required to improve and supplement these findings.\nRAD51 is a human homologous recombination repair gene. Too strong or too weak homologous recombination will result in genome instability and DNA damage, and the consequent gene loss, loss of heterozygosity, and/or genetic translocation are important causes of tumorigenesis and tumor progression.[19] Studies have found that RAD51 protein is overexpression in the breast, prostate, pancreatic, and colon cancers. Such overexpression of RAD51 can provide certain advantages for the growth of cancer cells by promoting the initiation and progression of cancer. However, to date, studies on the expression of RAD51 in ovarian cancers are few.\nThe results of this study showed that the RAD51-positive rate in high-grade tumors was significantly higher than that of normal oviducts, suggesting that RAD51 may play a role in the initiation of high-grade ovarian serous carcinomas. The RAD51-positive rate gradually increased from normal ovaries to cystadenomas to borderline cystadenomas to low-grade serous adenocarcinomas. Particularly, the positive rate of low-grade tumors was significantly higher than that in benign tumors and normal oviducts. The gradual change in the RAD51 expression suggests that the initiation of low-grade serous adenocarcinoma is a gradual procedure from benign to borderline to low-grade adenocarcinoma. However, no significant difference in the expression of RAD51 was found between high- and low-grade tumors. The expression level of RAD51 showed significant associations with LNM, FIGO stage, and omental metastasis in high-grade, but not low-grade tumors.\nHerein, the 5-year survival rate of patients with RAD51-positive high-grade adenocarcinomas was significantly lower than that of patients with RAD51-negative tumors, while no difference in survival was found in low-grade tumors. In other words, we found that high-grade tumor patients with high expression of RAD51 had a lower survival rate and poor prognosis, indicating that RAD51 may be a prognostic factor in high-grade ovarian serous adenocarcinoma. Furthermore, multivariate survival analyses showed that RAD51 was an independent prognostic factor, which may be of clinical significance in terms of the development of molecular targeted therapy, in addition to FIGO stage, and LNM. Further investigations on RAD51 are required to confirm these findings and to hopefully be able to improve the quality of life and prognosis of patients using targeted therapy.\nIn conclusion, the results of the present study indicate that PHLPP, FoxO3a, and RAD51 may play certain roles in the pathogenesis of the different grades of ovarian serous adenocarcinomas. Meanwhile, they also suggest the possible presence of common molecular mechanisms in the pathogenesis of the different grades. Increased protein expression of RAD51 may be an important molecular marker for poor prognosis of high-grade ovarian serous adenocarcinomas. However, considering the limited number of cases selected, which may have affected the significance of the results, further studies are required to improve and supplement these findings.\n Financial support and sponsorship This work were supported by grants from the Key Scientific Research program in Medical Science of Hebei Province, China (No. 20150318) and Science and Technology support program of Hebei Province, China (No. 16277794D).\nThis work were supported by grants from the Key Scientific Research program in Medical Science of Hebei Province, China (No. 20150318) and Science and Technology support program of Hebei Province, China (No. 16277794D).\n Conflicts of interest There are no conflicts of interest.\nThere are no conflicts of interest.\n\nRole of the tumor suppressor PH domain leucine-rich repeat protein phosphatase in the development of ovarian serous adenocarcinoma and its association with patient prognosis:\nThe tumor suppressor PHLPP, located at 18q21.33, can directly dephosphorylate ser473 in the hydrophobic group of Akt, thus negatively regulating Akt and its downstream targets. It has been reported that PHLPP can act synergistically with PTEN, another well-known tumor suppressor gene, thereby significantly inhibiting the proliferation of colorectal cancer cells, and negatively regulating PI3K/Akt signaling.[11] Accordingly, studies have shown that the PHLPP expression is significantly decreased in colon, breast, prostate, and lung cancers.[5712]\nThe results of the present study showed that the positive rate of PHLPP protein expression in the high-grade group was significantly lower than that in the oviduct group. The PHLPP-positive expression rate was decreased gradually from normal ovarian tissue to benign tumors to borderline tumors to low-grade adenocarcinomas, indicating that loss of PHLPP expression may be associated with the initiation of ovarian serous adenocarcinoma development, whereas no significant difference was observed between high-grade and low-grade tumors. Further, the PHLPP expression level showed significant associations with LNM, FIGO stages, and omental metastasis in high-grade tumors, but not in low-grade tumors. Similarly, the 5-year survival rates of PHLPP-positive and negative high-grade tumors significant differed, whereas no difference was seen in the low-grade tumors. These findings suggest that PHLPP may be a predictor of prognosis of high-grade ovarian serous adenocarcinoma.\n\nRole of the tumor suppressor forkhead homeobox type O 3a in the development of ovarian serous adenocarcinoma and its association with patient prognosis:\nFoxO3a is an important member of the FOXO family, located on at 6q21, and encodes a 673-amino acid long protein.[13] FoxO3a, as an important signaling molecule downstream of Akt in the PI3K-Akt signaling pathway, binds to the 14-3-3 chaperone protein after being phosphorylated by Akt, and subsequently translocates from the nucleus to the cytoplasm, thereby activating the apoptotic genes Bim, FasL, and tumor necrosis factor and its related apoptosis-inducing ligand TRAIL, consequently promoting apoptosis.[14] In addition to its role in apoptosis, FoxO3a plays critical roles in regulating cell proliferation, metabolism, the stress response, and the life span of cancer cells.[15] Particularly, another target molecule of FoxO3a is the cell cycle-regulating gene P27; overexpression of FoxO3a can arrest the cell cycle in the G0/G1 phase by upregulating P27. In addition, overexpression of FoxO3a can significantly inhibit the proliferation of tumor cells and result in tumor cell G2 arrest, further promoting tumor cell apoptosis.[1617]\nThe results of our analyses showed that the FoxO3a-positive rate in the high-grade tumors was significantly lower than that in the normal oviducts. In the low-grade adenocarcinomas, borderline cystadenomas, cystadenomas, and normal ovaries, the positive expression rates of FoxO3a protein were gradually reduced from normal ovaries to the low-grade adenocarcinomas, and the FoxO3 positive rate of the low-grade tumors was significantly lower than those of the borderline cystadenomas, cystadenomas, and normal ovaries, while the positive rate of borderline cystadenomas was significantly lower than those of cystadenomas and normal ovaries. Moreover, the FoxO3a expression level showed significant associations with LNM, FIGO stage, and omental metastasis in high-grade, but not low-grade tumors. These results suggest that FoxO3a may play a role in the oncogenesis and development of ovarian serous adenocarcinomas. In the high-grade tumors, the 5-year survival rate of FoxO3a-positive patients was significantly higher than that of the negative patients, whereas no difference in survival was seen in low-grade tumors, indicating that positive FoxO3a expression may have a long-term protective effect on cancer patients, and FoxO3a may hence have certain significance in the prediction of the patients’ long-term prognosis. Interestingly, it has been reported that tumor cells with low FoxO3a expression can develop drug resistance to paclitaxel and that inhibiting FoxO3a expression with RNAi can reduce E1A-mediated sensitivity to paclitaxel.[18] Therefore, FoxO3a protein may represent a potential prognostic indicator for high-grade ovarian serous adenocarcinoma.\n\nRole of RAD51 in the development of ovarian serous adenocarcinoma and its association with patient prognosis:\nRAD51 is a human homologous recombination repair gene. Too strong or too weak homologous recombination will result in genome instability and DNA damage, and the consequent gene loss, loss of heterozygosity, and/or genetic translocation are important causes of tumorigenesis and tumor progression.[19] Studies have found that RAD51 protein is overexpression in the breast, prostate, pancreatic, and colon cancers. Such overexpression of RAD51 can provide certain advantages for the growth of cancer cells by promoting the initiation and progression of cancer. However, to date, studies on the expression of RAD51 in ovarian cancers are few.\nThe results of this study showed that the RAD51-positive rate in high-grade tumors was significantly higher than that of normal oviducts, suggesting that RAD51 may play a role in the initiation of high-grade ovarian serous carcinomas. The RAD51-positive rate gradually increased from normal ovaries to cystadenomas to borderline cystadenomas to low-grade serous adenocarcinomas. Particularly, the positive rate of low-grade tumors was significantly higher than that in benign tumors and normal oviducts. The gradual change in the RAD51 expression suggests that the initiation of low-grade serous adenocarcinoma is a gradual procedure from benign to borderline to low-grade adenocarcinoma. However, no significant difference in the expression of RAD51 was found between high- and low-grade tumors. The expression level of RAD51 showed significant associations with LNM, FIGO stage, and omental metastasis in high-grade, but not low-grade tumors.\nHerein, the 5-year survival rate of patients with RAD51-positive high-grade adenocarcinomas was significantly lower than that of patients with RAD51-negative tumors, while no difference in survival was found in low-grade tumors. In other words, we found that high-grade tumor patients with high expression of RAD51 had a lower survival rate and poor prognosis, indicating that RAD51 may be a prognostic factor in high-grade ovarian serous adenocarcinoma. Furthermore, multivariate survival analyses showed that RAD51 was an independent prognostic factor, which may be of clinical significance in terms of the development of molecular targeted therapy, in addition to FIGO stage, and LNM. Further investigations on RAD51 are required to confirm these findings and to hopefully be able to improve the quality of life and prognosis of patients using targeted therapy.\nIn conclusion, the results of the present study indicate that PHLPP, FoxO3a, and RAD51 may play certain roles in the pathogenesis of the different grades of ovarian serous adenocarcinomas. Meanwhile, they also suggest the possible presence of common molecular mechanisms in the pathogenesis of the different grades. Increased protein expression of RAD51 may be an important molecular marker for poor prognosis of high-grade ovarian serous adenocarcinomas. However, considering the limited number of cases selected, which may have affected the significance of the results, further studies are required to improve and supplement these findings.\n\nFinancial support and sponsorship:\nThis work were supported by grants from the Key Scientific Research program in Medical Science of Hebei Province, China (No. 20150318) and Science and Technology support program of Hebei Province, China (No. 16277794D).\n\nConflicts of interest:\nThere are no conflicts of interest.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nOvarian serous adenocarcinoma can be divided into low- and high-grade tumors, which exhibit substantial differences in pathogenesis, clinicopathology, and prognosis. This study aimed to investigate the differences in the PH domain leucine-rich repeat protein phosphatase (PHLPP), forkhead homeobox type O 3a (FoxO3a), and RAD51 protein expressions, and their associations with prognosis in patients with low- and high-grade ovarian serous adenocarcinomas.\n\nMethods:\nThe PHLPP, FoxO3a, and RAD51 protein expressions were examined in 94 high- and 26 low-grade ovarian serous adenocarcinomas by immunohistochemistry. The differences in expression and their relationships with pathological features and prognosis were analyzed.\n\nResults:\nIn high-grade serous adenocarcinomas, the positive rates of PHLPP and FoxO3a were 24.5% and 26.6%, while in low-grade tumors, they were 23.1% and 26.9%, respectively (P < 0.05 vs. the control specimens; low- vs. high-grade: P > 0.05). The positive rates of RAD51 were 70.2% and 65.4% in high- and low-grade serous adenocarcinomas, respectively (P < 0.05 vs. the control specimens; low- vs. high-grade: P > 0.05). Meanwhile, in high-grade tumors, Stage III/IV tumors and lymph node and omental metastases were significantly associated with lower PHLPP and FoxO3a and higher RAD51 expression. The 5-year survival rates of patients with PHLPP- and FoxO3a-positive high-grade tumors (43.5% and 36.0%) were significantly higher than in patients with PHLPP-negative tumors (5.6% and 7.2%, respectively; P< 0.05). Similarly, the 5-year survival rate of RAD51-positive patients (3.0%) was significantly lower than in negative patients (42.9%; P< 0.05). In low-grade tumors, the PHLPP, FoxO3a, and RAD51 expressions were not significantly correlated with lymph node metastasis, omental metastasis, Federation of Gynecology and Obstetrics stage, or prognosis.\n\nConclusions:\nAbnormal PHLPP, FoxO3a, and RAD51 protein expressions may be involved in the development of high- and low-grade ovarian serous adenocarcinomas, suggesting common molecular pathways. Decreased PHLPP and FoxO3a and increased RAD51 protein expression may be important molecular markers for poor prognosis, and RAD51 may be an independent prognosis factor, of high-grade, but not low-grade, ovarian serous adenocarcinomas."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":12826,"string":"12,826"},"whole_article_abstract_length":{"kind":"number","value":454,"string":"454"},"other_sections_lengths":{"kind":"list like","value":[33,378,692,117,52,30,82,458,395,379,112,265,477,538,42],"string":"[\n 33,\n 378,\n 692,\n 117,\n 52,\n 30,\n 82,\n 458,\n 395,\n 379,\n 112,\n 265,\n 477,\n 538,\n 42\n]","isConversational":false},"num_sections":{"kind":"number","value":20,"string":"20"},"most_frequent_words":{"kind":"list like","value":["grade","positive","serous","high","low","low grade","expression","high grade","rad51","adenocarcinoma"],"string":"[\n \"grade\",\n \"positive\",\n \"serous\",\n \"high\",\n \"low\",\n \"low grade\",\n \"expression\",\n \"high grade\",\n \"rad51\",\n \"adenocarcinoma\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["ovarian cancer phlpp","ovarian adenocarcinoma rr","ovarian epithelial malignant","ovarian serous carcinomas","ovarian serous adenocarcinoma"],"string":"[\n \"ovarian cancer phlpp\",\n \"ovarian adenocarcinoma rr\",\n \"ovarian epithelial malignant\",\n \"ovarian serous carcinomas\",\n \"ovarian serous adenocarcinoma\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Forkhead Homeobox Type O 3a | Immunohistochemistry | Ovarian Serous Adenocarcinomas | PH Domain Leucine-rich Repeat Protein Phosphatase | Prognosis | RAD51 [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Forkhead Homeobox Type O 3a | Immunohistochemistry | Ovarian Serous Adenocarcinomas | PH Domain Leucine-rich Repeat Protein Phosphatase | Prognosis | RAD51 [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Forkhead Homeobox Type O 3a | Immunohistochemistry | Ovarian Serous Adenocarcinomas | PH Domain Leucine-rich Repeat Protein Phosphatase | Prognosis | RAD51 [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Forkhead Homeobox Type O 3a | Immunohistochemistry | Ovarian Serous Adenocarcinomas | PH Domain Leucine-rich Repeat Protein Phosphatase | Prognosis | RAD51 [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Biomarkers, Tumor | Cystadenocarcinoma, Serous | Female | Forkhead Box Protein O3 | Humans | Immunohistochemistry | Lymphatic Metastasis | Middle Aged | Neoplasm Staging | Nuclear Proteins | Ovarian Neoplasms | Phosphoprotein Phosphatases | Prognosis | Rad51 Recombinase [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Biomarkers, Tumor | Cystadenocarcinoma, Serous | Female | Forkhead Box Protein O3 | Humans | Immunohistochemistry | Lymphatic Metastasis | Middle Aged | Neoplasm Staging | Nuclear Proteins | Ovarian Neoplasms | Phosphoprotein Phosphatases | Prognosis | Rad51 Recombinase [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Biomarkers, Tumor | Cystadenocarcinoma, Serous | Female | Forkhead Box Protein O3 | Humans | Immunohistochemistry | Lymphatic Metastasis | Middle Aged | Neoplasm Staging | Nuclear Proteins | Ovarian Neoplasms | Phosphoprotein Phosphatases | Prognosis | Rad51 Recombinase [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Biomarkers, Tumor | Cystadenocarcinoma, Serous | Female | Forkhead Box Protein O3 | Humans | Immunohistochemistry | Lymphatic Metastasis | Middle Aged | Neoplasm Staging | Nuclear Proteins | Ovarian Neoplasms | Phosphoprotein Phosphatases | Prognosis | Rad51 Recombinase [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ovarian cancer phlpp | ovarian adenocarcinoma rr | ovarian epithelial malignant | ovarian serous carcinomas | ovarian serous adenocarcinoma [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] ovarian cancer phlpp | ovarian adenocarcinoma rr | ovarian epithelial malignant | ovarian serous carcinomas | ovarian serous adenocarcinoma [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ovarian cancer phlpp | ovarian adenocarcinoma rr | ovarian epithelial malignant | ovarian serous carcinomas | ovarian serous adenocarcinoma [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ovarian cancer phlpp | ovarian adenocarcinoma rr | ovarian epithelial malignant | ovarian serous carcinomas | ovarian serous adenocarcinoma [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] grade | positive | serous | high | low | low grade | expression | high grade | rad51 | adenocarcinoma [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] grade | positive | serous | high | low | low grade | expression | high grade | rad51 | adenocarcinoma [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] grade | positive | serous | high | low | low grade | expression | high grade | rad51 | adenocarcinoma [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] grade | positive | serous | high | low | low grade | expression | high grade | rad51 | adenocarcinoma [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] akt | serous | tumor | signaling | development | ovarian | involved | pi3k | akt signaling | pi3k akt [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] positive | years | staining | points | defined | cells | positive cells | defined positive | brown | slides [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] grade | 05 | serous | high | positive | grade serous | expression | low | low grade | adenocarcinoma [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] grade | positive | serous | high | low | low grade | 05 | expression | high grade | rad51 [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Ovarian ||| O 3a [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] PHLPP | FoxO3a | 94 | 26 ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] PHLPP | FoxO3a | 24.5% | 26.6% | 23.1% | 26.9% | 0.05 ||| 70.2% | 65.4% | 0.05 ||| FoxO3a ||| 5-year | 43.5% | 36.0% | 5.6% and | 7.2% | P< 0.05 ||| 5-year | 3.0% | 42.9% | P< 0.05 ||| PHLPP | FoxO3a | Federation of Gynecology and Obstetrics [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Ovarian ||| O 3a | adenocarcinomas ||| PHLPP | FoxO3a | 94 | 26 ||| ||| PHLPP | FoxO3a | 24.5% | 26.6% | 23.1% | 26.9% | 0.05 ||| 70.2% | 65.4% | 0.05 ||| FoxO3a ||| 5-year | 43.5% | 36.0% | 5.6% and | 7.2% | P< 0.05 ||| 5-year | 3.0% | 42.9% | P< 0.05 ||| PHLPP | FoxO3a | Federation of Gynecology and Obstetrics ||| FoxO3a ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":57,"cells":{"title":{"kind":"string","value":"Novel variants of the newly emerged Anaplasma capra from Korean water deer (Hydropotes inermis argyropus) in South Korea."},"pmid":{"kind":"string","value":"31345253"},"background_abstract":{"kind":"string","value":"Anaplasma spp. are tick-borne Gram-negative obligate intracellular bacteria that infect humans and a wide range of animals. Anaplasma capra has emerged as a human pathogen; however, little is known about the occurrence and genetic identity of this agent in wildlife. The present study aimed to determine the infection rate and genetic profile of this pathogen in wild animals in the Republic of Korea."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"A total of 253 blood samples [198 from Korean water deer (Hydropotes inermis argyropus), 53 from raccoon dogs (Nyctereutes procyonoides) and one sample each from a leopard cat (Prionailurus bengalensis) and a roe deer (Capreolus pygargus)] were collected at Chungbuk Wildlife Center during the period 2015-2018. Genomic DNA was extracted from the samples and screened for presence of Anaplasma species by PCR/sequence analysis of 429 bp of the 16S rRNA gene marker. Anaplasma capra-positive isolates were genetically profiled by amplification of a longer fragment of 16S rRNA (rrs) as well as partial sequences of citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4). Generated sequences of each gene marker were aligned with homologous sequences in the database and phylogenetically analyzed."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Anaplasma capra was detected in blood samples derived from Korean water deer, whereas samples from other animal species were negative. The overall infection rate in tested samples was 13.8% (35/253) and in the water deer the rate was 17.8% (35/198), distributed along the study period from 2015 to 2018. Genetic profiling and a phylogenetic analysis based on analyzed gene markers revealed the occurrence of two distinct strains, clustered in a single clade with counterpart sequences of A. capra in the database."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Anaplasma capra infection were detected in Korean water deer in the Republic of Korea, providing insight into the role of wildlife as a potential reservoir for animal and human anaplasmosis. However, further work is needed in order to evaluate the role of Korean water deer as a host/reservoir host of A. capra."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Anaplasma","Anaplasmosis","Animals","DNA, Bacterial","Deer","Disease Reservoirs","Genetic Variation","Phylogeny","RNA, Ribosomal, 16S","Republic of Korea"],"string":"[\n \"Anaplasma\",\n \"Anaplasmosis\",\n \"Animals\",\n \"DNA, Bacterial\",\n \"Deer\",\n \"Disease Reservoirs\",\n \"Genetic Variation\",\n \"Phylogeny\",\n \"RNA, Ribosomal, 16S\",\n \"Republic of Korea\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"6659236"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"The cosmopolitan genus Anaplasma includes six species of Gram-negative obligate intracellular bacteria that are transmitted by ticks to a wide range of animals, including humans [1–5], resulting in considerable economic losses in the livestock industry and serious public health concerns [6, 7]. Anaplasma phagocytophilum, A. ovis and recently reported A. capra are human pathogens [8–12], whereas other species in the genus have no known zoonotic potential. However, A. platys may have zoonotic potential after frequent reports of human infection [13, 14].\nThe provisional name Anaplasma capra was assigned after its initial characterization in goats (Capra aegagrus hircus) in China [12]. Later, it was isolated from sheep, goats and cattle in different geographical regions [15–19] as well as from various tick species (Haemaphysalis qinghaiensis, H. longicornis, Ixodes persulcatus) [12, 20–23]. Infection of A. capra was also reported in six wild animals in China including three takins (Budorcas taxicolor), two Reeves’s muntjacs (Muntiacus reevesi) and one forest musk deer (Moschus berezovskii) [24].\nAnaplasma species usually parasitize bone marrow-derived elements, including neutrophils (A. phagocytophilum), erythrocytes (A. marginale, A. centrale and A. ovis), monocytes (A. bovis) and platelets (A. platys) [7, 9, 10, 12]. However, A. capra seems to infect endothelial cells, rendering its microscopic detection in blood smears unreliable [12, 15]. In humans, the disease caused by A. capra is generally characterized by undifferentiated fever, headache, malaise, dizziness, myalgia and chills, with potential progression to CNS involvement and cerebrospinal fluid pleocytosis [12].\nAlthough different Anaplasma species have been detected in wildlife [23–29], little is known about the prevalence and genetic identity of A. capra in these animals in Korea. Using molecular tools, the present study aimed at investigating the occurrence and characterizing the genetic profile of this pathogen in wildlife in the Republic of Korea."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"The overall infection rate of A. capra in tested animals was 13.8% (35/253); however, samples from raccoon dogs (n = 53), leopard cat (n = 1) and roe deer (n = 1) were negative. The infection rate in KWD was 17.7% (35/198), distributed as follows: 24.6% (14/57) in 2015; 13.2% (5/38) in 2016; 17.3% (14/81) in 2017; and 9.1% (2/22) in 2018 (Table 2).Table 2Distribution of samples and prevalence of A. capra in animal speciesYearKorean water deerRaccoon dogOther animalsNumber infectedTotal numberInfection rate (%)InfectedNot infectedInfectedNot infectedNot infected201514430210147818.0201653305054311.620171467018LC (1)1410014.0201822009RD (1)2326.3Total number3516305323525313.8aInfection rate/species (%)17.700aOverall infection rateAbbreviations: LC, leopard cat (Prionailurus bengalensis); RD, roe deer (Capreolus pygargus)\n\nDistribution of samples and prevalence of A. capra in animal species\naOverall infection rate\nAbbreviations: LC, leopard cat (Prionailurus bengalensis); RD, roe deer (Capreolus pygargus)\nMolecular and phylogenetic analyses indicated to occurrence of two genetically distinct strains [named Cheongju (23 isolates) and Chungbuk (12 isolates)] of this pathogen. Sequences obtained from both strains were similar to those derived from A. capra from goats, sheep, cattle, ticks and humans; however, they had striking genetic differences, suggesting that they are novel strains. Sequences of the rrs gene fragment of both strains showed an identity of ~ 99.5% with counterparts in database and clustered in the clade of A. capra from different hosts (Fig. 1). Both strains had single nucleotide polymorphisms (SNPs), resulting in four genotypes at this gene locus. Phylogenetic analysis revealed that three sequences designated A. centrale (GenBank: AB211164, AF283007 and GU064903) and two sequences designated Anaplasma spp. (GenBank: AB454075 and AB509223) clustered within the A. capra clade, even though other A. centrale sequences from different hosts and geographical regions formed separate clusters in the ML tree.Fig. 1Maximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of 16S rRNA gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\n\nMaximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of 16S rRNA gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\nThe gltA gene of the Cheongju strain shared a similarity of 99.5% (with two substitutions, A/G at position 456 and T/C at position 533) with gltA sequences KM206274, KJ700628 and MH029895 isolated from a human, goat and tick, respectively [12, 23]. Sequences of the Chungbuk strain showed a similarity of 98–99% with KX685885, KX685886 and MF071308 of A. capra from ticks and sheep [13, 19]. Both strains clustered with their homologous sequences in the A. capra clade (Fig. 2). groEL gene sequences derived from the Cheongju strain shared a similarity of 99% (one substitution) with their counterparts from humans (GenBank: KM206275), goats (GenBank: KJ700629), sheep (GenBank: KX417356) and ticks (GenBank: KR261633 and KR261635), whereas sequences from the Chungbuk strain shared a similarity of 91% with the reference sequences (Fig. 3). The msp2 sequences showed extensive intra- and inter-sequence variations, including multiple InDels and single nucleotide substitutions; however, all sequences remained clustered in the A. capra clade (Fig. 4). A hypervariable stretch was detected between positions 285 and 414 of the generated sequences (corresponding to positions 550 and 679 in the reference sequence KM206276 of A. capra from humans). The msp4 sequences were identical in the two strains and showed an identity of 100% with those from humans (GenBank: KM206277) and ticks (GenBank: KR261637 and KR261640) (Fig. 5).Fig. 2Maximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of gltA gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\nFig. 3Maximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of groEL gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\nFig. 4Maximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of msp2 gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\nFig. 5Maximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of msp4 gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\n\nMaximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of gltA gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\nMaximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of groEL gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\nMaximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of msp2 gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\nMaximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of msp4 gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site"},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"To our knowledge, the results presented herein provide the first evidence for the presence of A. capra in Korean water deer in Korea. As an emerging human pathogen, the detection of A. capra in deer provides insight into the role of wildlife as a potential reservoir for human anaplasmosis. Furthermore, the obtained results expand the known geographical and host range of the Anaplasma capra."},"other_sections_titles":{"kind":"list like","value":["Background","Collection of samples","DNA extraction and PCR amplification","DNA sequence analysis","Phylogenetic analysis"],"string":"[\n \"Background\",\n \"Collection of samples\",\n \"DNA extraction and PCR amplification\",\n \"DNA sequence analysis\",\n \"Phylogenetic analysis\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["The cosmopolitan genus Anaplasma includes six species of Gram-negative obligate intracellular bacteria that are transmitted by ticks to a wide range of animals, including humans [1–5], resulting in considerable economic losses in the livestock industry and serious public health concerns [6, 7]. Anaplasma phagocytophilum, A. ovis and recently reported A. capra are human pathogens [8–12], whereas other species in the genus have no known zoonotic potential. However, A. platys may have zoonotic potential after frequent reports of human infection [13, 14].\nThe provisional name Anaplasma capra was assigned after its initial characterization in goats (Capra aegagrus hircus) in China [12]. Later, it was isolated from sheep, goats and cattle in different geographical regions [15–19] as well as from various tick species (Haemaphysalis qinghaiensis, H. longicornis, Ixodes persulcatus) [12, 20–23]. Infection of A. capra was also reported in six wild animals in China including three takins (Budorcas taxicolor), two Reeves’s muntjacs (Muntiacus reevesi) and one forest musk deer (Moschus berezovskii) [24].\nAnaplasma species usually parasitize bone marrow-derived elements, including neutrophils (A. phagocytophilum), erythrocytes (A. marginale, A. centrale and A. ovis), monocytes (A. bovis) and platelets (A. platys) [7, 9, 10, 12]. However, A. capra seems to infect endothelial cells, rendering its microscopic detection in blood smears unreliable [12, 15]. In humans, the disease caused by A. capra is generally characterized by undifferentiated fever, headache, malaise, dizziness, myalgia and chills, with potential progression to CNS involvement and cerebrospinal fluid pleocytosis [12].\nAlthough different Anaplasma species have been detected in wildlife [23–29], little is known about the prevalence and genetic identity of A. capra in these animals in Korea. Using molecular tools, the present study aimed at investigating the occurrence and characterizing the genetic profile of this pathogen in wildlife in the Republic of Korea.","Chungbuk Wildlife Center is located in Cheongju city, Chungcheongbuk-do province in the Republic of Korea (36°38′13.99ʺN, 127°29′22.99ʺE). The center receives terrestrial and avian wild animals for purposes of treatment from sickness/injuries and/or rehabilitation. Blood samples are collected for diagnosis and treatment of wildlife referred to the Chungbuk Wildlife Center. Blood samples are archived in EDTA-treated tubes and stored at – 80 °C. A total of 253 blood samples including 198 from Korean water deer (Hydropotes inermis argyropus), 53 from raccoon dogs (Nyctereutes procyonoides) and one sample each from a leopard cat (Prionailurus bengalensis) and a roe deer (Capreolus pygargus), collected from January 2015 to June 2018, were used.","Frozen blood samples were thawed at room temperature and genomic DNA was extracted from 200 µl of blood using a Magpurix® Blood Kit and Magpurix® 12s automated nucleic acid purification system (Zinexts Life Science Corp., Taipei, Taiwan), according to the manufacturer’s recommendations. DNA preparations were tested for the presence of Anaplasma species by PCR/sequence analysis of 429 bp of the 16S rRNA gene as described previously [30]. Anaplasma capra-positive isolates were genetically profiled by the amplification of a longer fragment of 16S rRNA (rrs) gene as well as partial sequences of citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4) genes as described previously (Table 1). Amplified fragments were electrophoresed on 1.2% gel loaded with EcoDye™ stain (BIOFACT, Daejeon, Korea) and visualized using UV light.Table 1PCR primers and conditions used in this studyTarget genePrimer namePrimer sequence (5′-3′)Annealing T (°C)Target size (bp)ReferencerrsForwardTTGAGAGTTTGATCCTGGCTCAGAACG571499[12]ReverseWAAGGWGGTAATCCAGCgltAOuter FGCGATTTTAGAGTGYGGAGATTG551031[12]Outer RTACAATACCGGAGTAAAAGTCAAInner FTCATCTCCTGTTGCACGGTGCCC60594[21]Inner RCTCTGAATGAACATGCCCACCCTgroELForwardGCGAGGCGTTAGACAAGTCCATT581129[12]ReverseTCCAGAGATGCAAGCGTGTATAGmsp2Outer FGCGTGTTGATGGCTCTGGT521089[12]Outer RACCAGTATCCTTATTTTTACCInner FGAGTGCACCAGAGCCTAGAA56801This studyInner RTCACCATCACCAAGCACTCTmsp4Outer FCAGTCTGCGCCTGCTCCCTAC55757[12]Outer RAGGAATCTTGCTCCAAGGTTAInner FGGGTTCTGATATGGCATCTTC56656[15]Inner RGGGAAATGTCCTTATAGGATTCGAbbreviations: rrs, 16S rRNA; gltA, citrate synthase; groEL, heat-shock protein; msp2, major surface protein 2; msp4, major surface protein 4; T, temperature\n\nPCR primers and conditions used in this study\nAbbreviations: rrs, 16S rRNA; gltA, citrate synthase; groEL, heat-shock protein; msp2, major surface protein 2; msp4, major surface protein 4; T, temperature","The PCR products (rrs and groEL) or secondary PCR product (for other gene markers) were purified and sequenced, either directly or after cloning in the pGEM-T vector (Promega, Madison, WI, USA), in both directions. Generated sequences were assembled using ChromasPro v.2.1.8 (https://technelysium.com.au/wp/chromaspro/).","The obtained sequences from each genetic locus were aligned with each other and reference sequences, available in GenBank (https://www.ncbi.nlm.nih.gov/), using ClustalX (http://www.clustal.org/) to determine the identity of Anaplasma spp. Evolutionary relationships were inferred based on partial sequences of 16S rRNA, citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4) genes using the maximum likelihood (ML) method implemented in MEGA7 (http://www.megasoftware.net/). The ML phylogenetic analysis was conducted using the Kimura 2-parameter model and 1000 bootstrap replicates. The ML tree was rooted against the nucleotide sequences L36221 (Rickettsia typhi), KY124257 (Rickettsia parkeri), U96733 (Rickettsia rickettsii) and BDDN01000175 (Ehrlichia ruminantium) for 16S rRNA, gltA, groEL and msp4 gene markers, respectively."],"string":"[\n \"The cosmopolitan genus Anaplasma includes six species of Gram-negative obligate intracellular bacteria that are transmitted by ticks to a wide range of animals, including humans [1–5], resulting in considerable economic losses in the livestock industry and serious public health concerns [6, 7]. Anaplasma phagocytophilum, A. ovis and recently reported A. capra are human pathogens [8–12], whereas other species in the genus have no known zoonotic potential. However, A. platys may have zoonotic potential after frequent reports of human infection [13, 14].\\nThe provisional name Anaplasma capra was assigned after its initial characterization in goats (Capra aegagrus hircus) in China [12]. Later, it was isolated from sheep, goats and cattle in different geographical regions [15–19] as well as from various tick species (Haemaphysalis qinghaiensis, H. longicornis, Ixodes persulcatus) [12, 20–23]. Infection of A. capra was also reported in six wild animals in China including three takins (Budorcas taxicolor), two Reeves’s muntjacs (Muntiacus reevesi) and one forest musk deer (Moschus berezovskii) [24].\\nAnaplasma species usually parasitize bone marrow-derived elements, including neutrophils (A. phagocytophilum), erythrocytes (A. marginale, A. centrale and A. ovis), monocytes (A. bovis) and platelets (A. platys) [7, 9, 10, 12]. However, A. capra seems to infect endothelial cells, rendering its microscopic detection in blood smears unreliable [12, 15]. In humans, the disease caused by A. capra is generally characterized by undifferentiated fever, headache, malaise, dizziness, myalgia and chills, with potential progression to CNS involvement and cerebrospinal fluid pleocytosis [12].\\nAlthough different Anaplasma species have been detected in wildlife [23–29], little is known about the prevalence and genetic identity of A. capra in these animals in Korea. Using molecular tools, the present study aimed at investigating the occurrence and characterizing the genetic profile of this pathogen in wildlife in the Republic of Korea.\",\n \"Chungbuk Wildlife Center is located in Cheongju city, Chungcheongbuk-do province in the Republic of Korea (36°38′13.99ʺN, 127°29′22.99ʺE). The center receives terrestrial and avian wild animals for purposes of treatment from sickness/injuries and/or rehabilitation. Blood samples are collected for diagnosis and treatment of wildlife referred to the Chungbuk Wildlife Center. Blood samples are archived in EDTA-treated tubes and stored at – 80 °C. A total of 253 blood samples including 198 from Korean water deer (Hydropotes inermis argyropus), 53 from raccoon dogs (Nyctereutes procyonoides) and one sample each from a leopard cat (Prionailurus bengalensis) and a roe deer (Capreolus pygargus), collected from January 2015 to June 2018, were used.\",\n \"Frozen blood samples were thawed at room temperature and genomic DNA was extracted from 200 µl of blood using a Magpurix® Blood Kit and Magpurix® 12s automated nucleic acid purification system (Zinexts Life Science Corp., Taipei, Taiwan), according to the manufacturer’s recommendations. DNA preparations were tested for the presence of Anaplasma species by PCR/sequence analysis of 429 bp of the 16S rRNA gene as described previously [30]. Anaplasma capra-positive isolates were genetically profiled by the amplification of a longer fragment of 16S rRNA (rrs) gene as well as partial sequences of citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4) genes as described previously (Table 1). Amplified fragments were electrophoresed on 1.2% gel loaded with EcoDye™ stain (BIOFACT, Daejeon, Korea) and visualized using UV light.Table 1PCR primers and conditions used in this studyTarget genePrimer namePrimer sequence (5′-3′)Annealing T (°C)Target size (bp)ReferencerrsForwardTTGAGAGTTTGATCCTGGCTCAGAACG571499[12]ReverseWAAGGWGGTAATCCAGCgltAOuter FGCGATTTTAGAGTGYGGAGATTG551031[12]Outer RTACAATACCGGAGTAAAAGTCAAInner FTCATCTCCTGTTGCACGGTGCCC60594[21]Inner RCTCTGAATGAACATGCCCACCCTgroELForwardGCGAGGCGTTAGACAAGTCCATT581129[12]ReverseTCCAGAGATGCAAGCGTGTATAGmsp2Outer FGCGTGTTGATGGCTCTGGT521089[12]Outer RACCAGTATCCTTATTTTTACCInner FGAGTGCACCAGAGCCTAGAA56801This studyInner RTCACCATCACCAAGCACTCTmsp4Outer FCAGTCTGCGCCTGCTCCCTAC55757[12]Outer RAGGAATCTTGCTCCAAGGTTAInner FGGGTTCTGATATGGCATCTTC56656[15]Inner RGGGAAATGTCCTTATAGGATTCGAbbreviations: rrs, 16S rRNA; gltA, citrate synthase; groEL, heat-shock protein; msp2, major surface protein 2; msp4, major surface protein 4; T, temperature\\n\\nPCR primers and conditions used in this study\\nAbbreviations: rrs, 16S rRNA; gltA, citrate synthase; groEL, heat-shock protein; msp2, major surface protein 2; msp4, major surface protein 4; T, temperature\",\n \"The PCR products (rrs and groEL) or secondary PCR product (for other gene markers) were purified and sequenced, either directly or after cloning in the pGEM-T vector (Promega, Madison, WI, USA), in both directions. Generated sequences were assembled using ChromasPro v.2.1.8 (https://technelysium.com.au/wp/chromaspro/).\",\n \"The obtained sequences from each genetic locus were aligned with each other and reference sequences, available in GenBank (https://www.ncbi.nlm.nih.gov/), using ClustalX (http://www.clustal.org/) to determine the identity of Anaplasma spp. Evolutionary relationships were inferred based on partial sequences of 16S rRNA, citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4) genes using the maximum likelihood (ML) method implemented in MEGA7 (http://www.megasoftware.net/). The ML phylogenetic analysis was conducted using the Kimura 2-parameter model and 1000 bootstrap replicates. The ML tree was rooted against the nucleotide sequences L36221 (Rickettsia typhi), KY124257 (Rickettsia parkeri), U96733 (Rickettsia rickettsii) and BDDN01000175 (Ehrlichia ruminantium) for 16S rRNA, gltA, groEL and msp4 gene markers, respectively.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Collection of samples","DNA extraction and PCR amplification","DNA sequence analysis","Phylogenetic analysis","Results","Discussion","Conclusions"],"string":"[\n \"Background\",\n \"Methods\",\n \"Collection of samples\",\n \"DNA extraction and PCR amplification\",\n \"DNA sequence analysis\",\n \"Phylogenetic analysis\",\n \"Results\",\n \"Discussion\",\n \"Conclusions\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["The cosmopolitan genus Anaplasma includes six species of Gram-negative obligate intracellular bacteria that are transmitted by ticks to a wide range of animals, including humans [1–5], resulting in considerable economic losses in the livestock industry and serious public health concerns [6, 7]. Anaplasma phagocytophilum, A. ovis and recently reported A. capra are human pathogens [8–12], whereas other species in the genus have no known zoonotic potential. However, A. platys may have zoonotic potential after frequent reports of human infection [13, 14].\nThe provisional name Anaplasma capra was assigned after its initial characterization in goats (Capra aegagrus hircus) in China [12]. Later, it was isolated from sheep, goats and cattle in different geographical regions [15–19] as well as from various tick species (Haemaphysalis qinghaiensis, H. longicornis, Ixodes persulcatus) [12, 20–23]. Infection of A. capra was also reported in six wild animals in China including three takins (Budorcas taxicolor), two Reeves’s muntjacs (Muntiacus reevesi) and one forest musk deer (Moschus berezovskii) [24].\nAnaplasma species usually parasitize bone marrow-derived elements, including neutrophils (A. phagocytophilum), erythrocytes (A. marginale, A. centrale and A. ovis), monocytes (A. bovis) and platelets (A. platys) [7, 9, 10, 12]. However, A. capra seems to infect endothelial cells, rendering its microscopic detection in blood smears unreliable [12, 15]. In humans, the disease caused by A. capra is generally characterized by undifferentiated fever, headache, malaise, dizziness, myalgia and chills, with potential progression to CNS involvement and cerebrospinal fluid pleocytosis [12].\nAlthough different Anaplasma species have been detected in wildlife [23–29], little is known about the prevalence and genetic identity of A. capra in these animals in Korea. Using molecular tools, the present study aimed at investigating the occurrence and characterizing the genetic profile of this pathogen in wildlife in the Republic of Korea."," Collection of samples Chungbuk Wildlife Center is located in Cheongju city, Chungcheongbuk-do province in the Republic of Korea (36°38′13.99ʺN, 127°29′22.99ʺE). The center receives terrestrial and avian wild animals for purposes of treatment from sickness/injuries and/or rehabilitation. Blood samples are collected for diagnosis and treatment of wildlife referred to the Chungbuk Wildlife Center. Blood samples are archived in EDTA-treated tubes and stored at – 80 °C. A total of 253 blood samples including 198 from Korean water deer (Hydropotes inermis argyropus), 53 from raccoon dogs (Nyctereutes procyonoides) and one sample each from a leopard cat (Prionailurus bengalensis) and a roe deer (Capreolus pygargus), collected from January 2015 to June 2018, were used.\nChungbuk Wildlife Center is located in Cheongju city, Chungcheongbuk-do province in the Republic of Korea (36°38′13.99ʺN, 127°29′22.99ʺE). The center receives terrestrial and avian wild animals for purposes of treatment from sickness/injuries and/or rehabilitation. Blood samples are collected for diagnosis and treatment of wildlife referred to the Chungbuk Wildlife Center. Blood samples are archived in EDTA-treated tubes and stored at – 80 °C. A total of 253 blood samples including 198 from Korean water deer (Hydropotes inermis argyropus), 53 from raccoon dogs (Nyctereutes procyonoides) and one sample each from a leopard cat (Prionailurus bengalensis) and a roe deer (Capreolus pygargus), collected from January 2015 to June 2018, were used.\n DNA extraction and PCR amplification Frozen blood samples were thawed at room temperature and genomic DNA was extracted from 200 µl of blood using a Magpurix® Blood Kit and Magpurix® 12s automated nucleic acid purification system (Zinexts Life Science Corp., Taipei, Taiwan), according to the manufacturer’s recommendations. DNA preparations were tested for the presence of Anaplasma species by PCR/sequence analysis of 429 bp of the 16S rRNA gene as described previously [30]. Anaplasma capra-positive isolates were genetically profiled by the amplification of a longer fragment of 16S rRNA (rrs) gene as well as partial sequences of citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4) genes as described previously (Table 1). Amplified fragments were electrophoresed on 1.2% gel loaded with EcoDye™ stain (BIOFACT, Daejeon, Korea) and visualized using UV light.Table 1PCR primers and conditions used in this studyTarget genePrimer namePrimer sequence (5′-3′)Annealing T (°C)Target size (bp)ReferencerrsForwardTTGAGAGTTTGATCCTGGCTCAGAACG571499[12]ReverseWAAGGWGGTAATCCAGCgltAOuter FGCGATTTTAGAGTGYGGAGATTG551031[12]Outer RTACAATACCGGAGTAAAAGTCAAInner FTCATCTCCTGTTGCACGGTGCCC60594[21]Inner RCTCTGAATGAACATGCCCACCCTgroELForwardGCGAGGCGTTAGACAAGTCCATT581129[12]ReverseTCCAGAGATGCAAGCGTGTATAGmsp2Outer FGCGTGTTGATGGCTCTGGT521089[12]Outer RACCAGTATCCTTATTTTTACCInner FGAGTGCACCAGAGCCTAGAA56801This studyInner RTCACCATCACCAAGCACTCTmsp4Outer FCAGTCTGCGCCTGCTCCCTAC55757[12]Outer RAGGAATCTTGCTCCAAGGTTAInner FGGGTTCTGATATGGCATCTTC56656[15]Inner RGGGAAATGTCCTTATAGGATTCGAbbreviations: rrs, 16S rRNA; gltA, citrate synthase; groEL, heat-shock protein; msp2, major surface protein 2; msp4, major surface protein 4; T, temperature\n\nPCR primers and conditions used in this study\nAbbreviations: rrs, 16S rRNA; gltA, citrate synthase; groEL, heat-shock protein; msp2, major surface protein 2; msp4, major surface protein 4; T, temperature\nFrozen blood samples were thawed at room temperature and genomic DNA was extracted from 200 µl of blood using a Magpurix® Blood Kit and Magpurix® 12s automated nucleic acid purification system (Zinexts Life Science Corp., Taipei, Taiwan), according to the manufacturer’s recommendations. DNA preparations were tested for the presence of Anaplasma species by PCR/sequence analysis of 429 bp of the 16S rRNA gene as described previously [30]. Anaplasma capra-positive isolates were genetically profiled by the amplification of a longer fragment of 16S rRNA (rrs) gene as well as partial sequences of citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4) genes as described previously (Table 1). Amplified fragments were electrophoresed on 1.2% gel loaded with EcoDye™ stain (BIOFACT, Daejeon, Korea) and visualized using UV light.Table 1PCR primers and conditions used in this studyTarget genePrimer namePrimer sequence (5′-3′)Annealing T (°C)Target size (bp)ReferencerrsForwardTTGAGAGTTTGATCCTGGCTCAGAACG571499[12]ReverseWAAGGWGGTAATCCAGCgltAOuter FGCGATTTTAGAGTGYGGAGATTG551031[12]Outer RTACAATACCGGAGTAAAAGTCAAInner FTCATCTCCTGTTGCACGGTGCCC60594[21]Inner RCTCTGAATGAACATGCCCACCCTgroELForwardGCGAGGCGTTAGACAAGTCCATT581129[12]ReverseTCCAGAGATGCAAGCGTGTATAGmsp2Outer FGCGTGTTGATGGCTCTGGT521089[12]Outer RACCAGTATCCTTATTTTTACCInner FGAGTGCACCAGAGCCTAGAA56801This studyInner RTCACCATCACCAAGCACTCTmsp4Outer FCAGTCTGCGCCTGCTCCCTAC55757[12]Outer RAGGAATCTTGCTCCAAGGTTAInner FGGGTTCTGATATGGCATCTTC56656[15]Inner RGGGAAATGTCCTTATAGGATTCGAbbreviations: rrs, 16S rRNA; gltA, citrate synthase; groEL, heat-shock protein; msp2, major surface protein 2; msp4, major surface protein 4; T, temperature\n\nPCR primers and conditions used in this study\nAbbreviations: rrs, 16S rRNA; gltA, citrate synthase; groEL, heat-shock protein; msp2, major surface protein 2; msp4, major surface protein 4; T, temperature\n DNA sequence analysis The PCR products (rrs and groEL) or secondary PCR product (for other gene markers) were purified and sequenced, either directly or after cloning in the pGEM-T vector (Promega, Madison, WI, USA), in both directions. Generated sequences were assembled using ChromasPro v.2.1.8 (https://technelysium.com.au/wp/chromaspro/).\nThe PCR products (rrs and groEL) or secondary PCR product (for other gene markers) were purified and sequenced, either directly or after cloning in the pGEM-T vector (Promega, Madison, WI, USA), in both directions. Generated sequences were assembled using ChromasPro v.2.1.8 (https://technelysium.com.au/wp/chromaspro/).\n Phylogenetic analysis The obtained sequences from each genetic locus were aligned with each other and reference sequences, available in GenBank (https://www.ncbi.nlm.nih.gov/), using ClustalX (http://www.clustal.org/) to determine the identity of Anaplasma spp. Evolutionary relationships were inferred based on partial sequences of 16S rRNA, citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4) genes using the maximum likelihood (ML) method implemented in MEGA7 (http://www.megasoftware.net/). The ML phylogenetic analysis was conducted using the Kimura 2-parameter model and 1000 bootstrap replicates. The ML tree was rooted against the nucleotide sequences L36221 (Rickettsia typhi), KY124257 (Rickettsia parkeri), U96733 (Rickettsia rickettsii) and BDDN01000175 (Ehrlichia ruminantium) for 16S rRNA, gltA, groEL and msp4 gene markers, respectively.\nThe obtained sequences from each genetic locus were aligned with each other and reference sequences, available in GenBank (https://www.ncbi.nlm.nih.gov/), using ClustalX (http://www.clustal.org/) to determine the identity of Anaplasma spp. Evolutionary relationships were inferred based on partial sequences of 16S rRNA, citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4) genes using the maximum likelihood (ML) method implemented in MEGA7 (http://www.megasoftware.net/). The ML phylogenetic analysis was conducted using the Kimura 2-parameter model and 1000 bootstrap replicates. The ML tree was rooted against the nucleotide sequences L36221 (Rickettsia typhi), KY124257 (Rickettsia parkeri), U96733 (Rickettsia rickettsii) and BDDN01000175 (Ehrlichia ruminantium) for 16S rRNA, gltA, groEL and msp4 gene markers, respectively.","Chungbuk Wildlife Center is located in Cheongju city, Chungcheongbuk-do province in the Republic of Korea (36°38′13.99ʺN, 127°29′22.99ʺE). The center receives terrestrial and avian wild animals for purposes of treatment from sickness/injuries and/or rehabilitation. Blood samples are collected for diagnosis and treatment of wildlife referred to the Chungbuk Wildlife Center. Blood samples are archived in EDTA-treated tubes and stored at – 80 °C. A total of 253 blood samples including 198 from Korean water deer (Hydropotes inermis argyropus), 53 from raccoon dogs (Nyctereutes procyonoides) and one sample each from a leopard cat (Prionailurus bengalensis) and a roe deer (Capreolus pygargus), collected from January 2015 to June 2018, were used.","Frozen blood samples were thawed at room temperature and genomic DNA was extracted from 200 µl of blood using a Magpurix® Blood Kit and Magpurix® 12s automated nucleic acid purification system (Zinexts Life Science Corp., Taipei, Taiwan), according to the manufacturer’s recommendations. DNA preparations were tested for the presence of Anaplasma species by PCR/sequence analysis of 429 bp of the 16S rRNA gene as described previously [30]. Anaplasma capra-positive isolates were genetically profiled by the amplification of a longer fragment of 16S rRNA (rrs) gene as well as partial sequences of citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4) genes as described previously (Table 1). Amplified fragments were electrophoresed on 1.2% gel loaded with EcoDye™ stain (BIOFACT, Daejeon, Korea) and visualized using UV light.Table 1PCR primers and conditions used in this studyTarget genePrimer namePrimer sequence (5′-3′)Annealing T (°C)Target size (bp)ReferencerrsForwardTTGAGAGTTTGATCCTGGCTCAGAACG571499[12]ReverseWAAGGWGGTAATCCAGCgltAOuter FGCGATTTTAGAGTGYGGAGATTG551031[12]Outer RTACAATACCGGAGTAAAAGTCAAInner FTCATCTCCTGTTGCACGGTGCCC60594[21]Inner RCTCTGAATGAACATGCCCACCCTgroELForwardGCGAGGCGTTAGACAAGTCCATT581129[12]ReverseTCCAGAGATGCAAGCGTGTATAGmsp2Outer FGCGTGTTGATGGCTCTGGT521089[12]Outer RACCAGTATCCTTATTTTTACCInner FGAGTGCACCAGAGCCTAGAA56801This studyInner RTCACCATCACCAAGCACTCTmsp4Outer FCAGTCTGCGCCTGCTCCCTAC55757[12]Outer RAGGAATCTTGCTCCAAGGTTAInner FGGGTTCTGATATGGCATCTTC56656[15]Inner RGGGAAATGTCCTTATAGGATTCGAbbreviations: rrs, 16S rRNA; gltA, citrate synthase; groEL, heat-shock protein; msp2, major surface protein 2; msp4, major surface protein 4; T, temperature\n\nPCR primers and conditions used in this study\nAbbreviations: rrs, 16S rRNA; gltA, citrate synthase; groEL, heat-shock protein; msp2, major surface protein 2; msp4, major surface protein 4; T, temperature","The PCR products (rrs and groEL) or secondary PCR product (for other gene markers) were purified and sequenced, either directly or after cloning in the pGEM-T vector (Promega, Madison, WI, USA), in both directions. Generated sequences were assembled using ChromasPro v.2.1.8 (https://technelysium.com.au/wp/chromaspro/).","The obtained sequences from each genetic locus were aligned with each other and reference sequences, available in GenBank (https://www.ncbi.nlm.nih.gov/), using ClustalX (http://www.clustal.org/) to determine the identity of Anaplasma spp. Evolutionary relationships were inferred based on partial sequences of 16S rRNA, citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4) genes using the maximum likelihood (ML) method implemented in MEGA7 (http://www.megasoftware.net/). The ML phylogenetic analysis was conducted using the Kimura 2-parameter model and 1000 bootstrap replicates. The ML tree was rooted against the nucleotide sequences L36221 (Rickettsia typhi), KY124257 (Rickettsia parkeri), U96733 (Rickettsia rickettsii) and BDDN01000175 (Ehrlichia ruminantium) for 16S rRNA, gltA, groEL and msp4 gene markers, respectively.","The overall infection rate of A. capra in tested animals was 13.8% (35/253); however, samples from raccoon dogs (n = 53), leopard cat (n = 1) and roe deer (n = 1) were negative. The infection rate in KWD was 17.7% (35/198), distributed as follows: 24.6% (14/57) in 2015; 13.2% (5/38) in 2016; 17.3% (14/81) in 2017; and 9.1% (2/22) in 2018 (Table 2).Table 2Distribution of samples and prevalence of A. capra in animal speciesYearKorean water deerRaccoon dogOther animalsNumber infectedTotal numberInfection rate (%)InfectedNot infectedInfectedNot infectedNot infected201514430210147818.0201653305054311.620171467018LC (1)1410014.0201822009RD (1)2326.3Total number3516305323525313.8aInfection rate/species (%)17.700aOverall infection rateAbbreviations: LC, leopard cat (Prionailurus bengalensis); RD, roe deer (Capreolus pygargus)\n\nDistribution of samples and prevalence of A. capra in animal species\naOverall infection rate\nAbbreviations: LC, leopard cat (Prionailurus bengalensis); RD, roe deer (Capreolus pygargus)\nMolecular and phylogenetic analyses indicated to occurrence of two genetically distinct strains [named Cheongju (23 isolates) and Chungbuk (12 isolates)] of this pathogen. Sequences obtained from both strains were similar to those derived from A. capra from goats, sheep, cattle, ticks and humans; however, they had striking genetic differences, suggesting that they are novel strains. Sequences of the rrs gene fragment of both strains showed an identity of ~ 99.5% with counterparts in database and clustered in the clade of A. capra from different hosts (Fig. 1). Both strains had single nucleotide polymorphisms (SNPs), resulting in four genotypes at this gene locus. Phylogenetic analysis revealed that three sequences designated A. centrale (GenBank: AB211164, AF283007 and GU064903) and two sequences designated Anaplasma spp. (GenBank: AB454075 and AB509223) clustered within the A. capra clade, even though other A. centrale sequences from different hosts and geographical regions formed separate clusters in the ML tree.Fig. 1Maximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of 16S rRNA gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\n\nMaximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of 16S rRNA gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\nThe gltA gene of the Cheongju strain shared a similarity of 99.5% (with two substitutions, A/G at position 456 and T/C at position 533) with gltA sequences KM206274, KJ700628 and MH029895 isolated from a human, goat and tick, respectively [12, 23]. Sequences of the Chungbuk strain showed a similarity of 98–99% with KX685885, KX685886 and MF071308 of A. capra from ticks and sheep [13, 19]. Both strains clustered with their homologous sequences in the A. capra clade (Fig. 2). groEL gene sequences derived from the Cheongju strain shared a similarity of 99% (one substitution) with their counterparts from humans (GenBank: KM206275), goats (GenBank: KJ700629), sheep (GenBank: KX417356) and ticks (GenBank: KR261633 and KR261635), whereas sequences from the Chungbuk strain shared a similarity of 91% with the reference sequences (Fig. 3). The msp2 sequences showed extensive intra- and inter-sequence variations, including multiple InDels and single nucleotide substitutions; however, all sequences remained clustered in the A. capra clade (Fig. 4). A hypervariable stretch was detected between positions 285 and 414 of the generated sequences (corresponding to positions 550 and 679 in the reference sequence KM206276 of A. capra from humans). The msp4 sequences were identical in the two strains and showed an identity of 100% with those from humans (GenBank: KM206277) and ticks (GenBank: KR261637 and KR261640) (Fig. 5).Fig. 2Maximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of gltA gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\nFig. 3Maximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of groEL gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\nFig. 4Maximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of msp2 gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\nFig. 5Maximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of msp4 gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\n\nMaximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of gltA gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\nMaximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of groEL gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\nMaximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of msp2 gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\nMaximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of msp4 gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site","Wild animals act as reservoirs for a wide range of pathogens [31–33]. The emergence of infectious disease agents of wildlife origin is a prominent challenge to public health and the livestock industry [34–36]. Anaplasma capra has recently been isolated from human patients in China with non-specific clinical manifestations, with potential progression to CNS complications, suggesting that this species could pose a substantial threat to public health [12, 37]. We detected A. capra DNA in blood samples of 35 out of 198 KWD (17.7% infection rate) at the Chungbuk Wildlife Center, Korea. Epidemiological data for this pathogen in wildlife are lacking in Korea; however, our findings are similar to those obtained from wildlife (five takins, three Himalayan gorals, three Reeves’s muntjacs, one forest musk deer and one wild boar) in China [24]. In addition, a low percentage of infection rate was reported cattle, sheep and goats in China, Sweden and Korea [15–19], indicating that A. capra has a broad host range. Occurrence of infection during the study period from 2015 to 2018 indicates the persistence of the infection in KWD, suggesting that the species may act as a reservoir for this pathogen. However, it is difficult to explain the negative results from raccoon dogs in the present study. This may be attributed to persistent infection making the pathogen below detectable level in the blood of these animals. In support of this view, A. capra has been reported to infect endothelial cells [12, 15], making its detection in the blood possible in case of considerable bacteremia or released endothelial cells, resembling Rickettsia species [12]. Furthermore, the sample size and species and/or the age of animals may play a role in these findings. Further investigations are needed to clarify these points.\nOur genetic profiling results indicated that the newly generated 16S rRNA gene sequences shared a homology of > 99.5% with sequences of A. capra strains from humans, sheep, goats, cattle and ticks [12, 15, 16, 18, 20, 22], suggesting that they likely are within the same species of bacteria [38, 39]. Clustering of sequences named A. centrale from deer (Cervus nippon nippon) (GenBank: AB211164) and cattle (GenBank: AF283007) in Japan and from ticks (Haemaphysalis longicornis) in Korea (GenBank: GU064903), as well as Anaplasma spp. from deer (GenBank: AB454075; direct submission) and Japanese serow (Capricornis crispus) (GenBank: AB509223) in the same clade of A. capra indicate a close phylogenetic relationship. The clustering pattern of these sequences in the A. capra clade does not support their assignment as sister taxa and suggests that these isolates are in fact A. capra [15, 25] and may need re-description, since these sequences were more related to Chungbuk strain in the A. capra clade.\nThe results obtained using different gene markers showed considerable sequence variation, suggesting that A. capra has a high degree of genetic diversity. Notably, extensive genetic variation was detected in msp2. Consistent with our results, sequence variation at the studied gene markers has been reported previously among isolates from ticks, sheep and goats [12, 15–18, 20–25]. Similarly, genetic variation is common in other Anaplasma species [40–45]. Although genetic diversity is reportedly related to infectivity, virulence, pathogenicity, niche preference, immune evasion, and/or host adaptability [46–49], this has not been established in A. capra and further studies are needed to evaluate these relationships.\nDue to the extinction of natural predators, the KWD is thriving in Korea and has been designated as “harmful wildlife” by the Ministry of Environment in 1994 owing to harmful interactions with humans and their properties. This close interaction poses substantial threats to domestic animals and human health in Korea. This study was limited by analyzing samples from one geographical area and few animal species, which my lead to biases in the results. A large-scale study is underway to fully elucidate the host range of wildlife, vector ticks, pathogenicity and geographical distribution of this organism in Korea.","To our knowledge, the results presented herein provide the first evidence for the presence of A. capra in Korean water deer in Korea. As an emerging human pathogen, the detection of A. capra in deer provides insight into the role of wildlife as a potential reservoir for human anaplasmosis. Furthermore, the obtained results expand the known geographical and host range of the Anaplasma capra."],"string":"[\n \"The cosmopolitan genus Anaplasma includes six species of Gram-negative obligate intracellular bacteria that are transmitted by ticks to a wide range of animals, including humans [1–5], resulting in considerable economic losses in the livestock industry and serious public health concerns [6, 7]. Anaplasma phagocytophilum, A. ovis and recently reported A. capra are human pathogens [8–12], whereas other species in the genus have no known zoonotic potential. However, A. platys may have zoonotic potential after frequent reports of human infection [13, 14].\\nThe provisional name Anaplasma capra was assigned after its initial characterization in goats (Capra aegagrus hircus) in China [12]. Later, it was isolated from sheep, goats and cattle in different geographical regions [15–19] as well as from various tick species (Haemaphysalis qinghaiensis, H. longicornis, Ixodes persulcatus) [12, 20–23]. Infection of A. capra was also reported in six wild animals in China including three takins (Budorcas taxicolor), two Reeves’s muntjacs (Muntiacus reevesi) and one forest musk deer (Moschus berezovskii) [24].\\nAnaplasma species usually parasitize bone marrow-derived elements, including neutrophils (A. phagocytophilum), erythrocytes (A. marginale, A. centrale and A. ovis), monocytes (A. bovis) and platelets (A. platys) [7, 9, 10, 12]. However, A. capra seems to infect endothelial cells, rendering its microscopic detection in blood smears unreliable [12, 15]. In humans, the disease caused by A. capra is generally characterized by undifferentiated fever, headache, malaise, dizziness, myalgia and chills, with potential progression to CNS involvement and cerebrospinal fluid pleocytosis [12].\\nAlthough different Anaplasma species have been detected in wildlife [23–29], little is known about the prevalence and genetic identity of A. capra in these animals in Korea. Using molecular tools, the present study aimed at investigating the occurrence and characterizing the genetic profile of this pathogen in wildlife in the Republic of Korea.\",\n \" Collection of samples Chungbuk Wildlife Center is located in Cheongju city, Chungcheongbuk-do province in the Republic of Korea (36°38′13.99ʺN, 127°29′22.99ʺE). The center receives terrestrial and avian wild animals for purposes of treatment from sickness/injuries and/or rehabilitation. Blood samples are collected for diagnosis and treatment of wildlife referred to the Chungbuk Wildlife Center. Blood samples are archived in EDTA-treated tubes and stored at – 80 °C. A total of 253 blood samples including 198 from Korean water deer (Hydropotes inermis argyropus), 53 from raccoon dogs (Nyctereutes procyonoides) and one sample each from a leopard cat (Prionailurus bengalensis) and a roe deer (Capreolus pygargus), collected from January 2015 to June 2018, were used.\\nChungbuk Wildlife Center is located in Cheongju city, Chungcheongbuk-do province in the Republic of Korea (36°38′13.99ʺN, 127°29′22.99ʺE). The center receives terrestrial and avian wild animals for purposes of treatment from sickness/injuries and/or rehabilitation. Blood samples are collected for diagnosis and treatment of wildlife referred to the Chungbuk Wildlife Center. Blood samples are archived in EDTA-treated tubes and stored at – 80 °C. A total of 253 blood samples including 198 from Korean water deer (Hydropotes inermis argyropus), 53 from raccoon dogs (Nyctereutes procyonoides) and one sample each from a leopard cat (Prionailurus bengalensis) and a roe deer (Capreolus pygargus), collected from January 2015 to June 2018, were used.\\n DNA extraction and PCR amplification Frozen blood samples were thawed at room temperature and genomic DNA was extracted from 200 µl of blood using a Magpurix® Blood Kit and Magpurix® 12s automated nucleic acid purification system (Zinexts Life Science Corp., Taipei, Taiwan), according to the manufacturer’s recommendations. DNA preparations were tested for the presence of Anaplasma species by PCR/sequence analysis of 429 bp of the 16S rRNA gene as described previously [30]. Anaplasma capra-positive isolates were genetically profiled by the amplification of a longer fragment of 16S rRNA (rrs) gene as well as partial sequences of citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4) genes as described previously (Table 1). Amplified fragments were electrophoresed on 1.2% gel loaded with EcoDye™ stain (BIOFACT, Daejeon, Korea) and visualized using UV light.Table 1PCR primers and conditions used in this studyTarget genePrimer namePrimer sequence (5′-3′)Annealing T (°C)Target size (bp)ReferencerrsForwardTTGAGAGTTTGATCCTGGCTCAGAACG571499[12]ReverseWAAGGWGGTAATCCAGCgltAOuter FGCGATTTTAGAGTGYGGAGATTG551031[12]Outer RTACAATACCGGAGTAAAAGTCAAInner FTCATCTCCTGTTGCACGGTGCCC60594[21]Inner RCTCTGAATGAACATGCCCACCCTgroELForwardGCGAGGCGTTAGACAAGTCCATT581129[12]ReverseTCCAGAGATGCAAGCGTGTATAGmsp2Outer FGCGTGTTGATGGCTCTGGT521089[12]Outer RACCAGTATCCTTATTTTTACCInner FGAGTGCACCAGAGCCTAGAA56801This studyInner RTCACCATCACCAAGCACTCTmsp4Outer FCAGTCTGCGCCTGCTCCCTAC55757[12]Outer RAGGAATCTTGCTCCAAGGTTAInner FGGGTTCTGATATGGCATCTTC56656[15]Inner RGGGAAATGTCCTTATAGGATTCGAbbreviations: rrs, 16S rRNA; gltA, citrate synthase; groEL, heat-shock protein; msp2, major surface protein 2; msp4, major surface protein 4; T, temperature\\n\\nPCR primers and conditions used in this study\\nAbbreviations: rrs, 16S rRNA; gltA, citrate synthase; groEL, heat-shock protein; msp2, major surface protein 2; msp4, major surface protein 4; T, temperature\\nFrozen blood samples were thawed at room temperature and genomic DNA was extracted from 200 µl of blood using a Magpurix® Blood Kit and Magpurix® 12s automated nucleic acid purification system (Zinexts Life Science Corp., Taipei, Taiwan), according to the manufacturer’s recommendations. DNA preparations were tested for the presence of Anaplasma species by PCR/sequence analysis of 429 bp of the 16S rRNA gene as described previously [30]. Anaplasma capra-positive isolates were genetically profiled by the amplification of a longer fragment of 16S rRNA (rrs) gene as well as partial sequences of citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4) genes as described previously (Table 1). Amplified fragments were electrophoresed on 1.2% gel loaded with EcoDye™ stain (BIOFACT, Daejeon, Korea) and visualized using UV light.Table 1PCR primers and conditions used in this studyTarget genePrimer namePrimer sequence (5′-3′)Annealing T (°C)Target size (bp)ReferencerrsForwardTTGAGAGTTTGATCCTGGCTCAGAACG571499[12]ReverseWAAGGWGGTAATCCAGCgltAOuter FGCGATTTTAGAGTGYGGAGATTG551031[12]Outer RTACAATACCGGAGTAAAAGTCAAInner FTCATCTCCTGTTGCACGGTGCCC60594[21]Inner RCTCTGAATGAACATGCCCACCCTgroELForwardGCGAGGCGTTAGACAAGTCCATT581129[12]ReverseTCCAGAGATGCAAGCGTGTATAGmsp2Outer FGCGTGTTGATGGCTCTGGT521089[12]Outer RACCAGTATCCTTATTTTTACCInner FGAGTGCACCAGAGCCTAGAA56801This studyInner RTCACCATCACCAAGCACTCTmsp4Outer FCAGTCTGCGCCTGCTCCCTAC55757[12]Outer RAGGAATCTTGCTCCAAGGTTAInner FGGGTTCTGATATGGCATCTTC56656[15]Inner RGGGAAATGTCCTTATAGGATTCGAbbreviations: rrs, 16S rRNA; gltA, citrate synthase; groEL, heat-shock protein; msp2, major surface protein 2; msp4, major surface protein 4; T, temperature\\n\\nPCR primers and conditions used in this study\\nAbbreviations: rrs, 16S rRNA; gltA, citrate synthase; groEL, heat-shock protein; msp2, major surface protein 2; msp4, major surface protein 4; T, temperature\\n DNA sequence analysis The PCR products (rrs and groEL) or secondary PCR product (for other gene markers) were purified and sequenced, either directly or after cloning in the pGEM-T vector (Promega, Madison, WI, USA), in both directions. Generated sequences were assembled using ChromasPro v.2.1.8 (https://technelysium.com.au/wp/chromaspro/).\\nThe PCR products (rrs and groEL) or secondary PCR product (for other gene markers) were purified and sequenced, either directly or after cloning in the pGEM-T vector (Promega, Madison, WI, USA), in both directions. Generated sequences were assembled using ChromasPro v.2.1.8 (https://technelysium.com.au/wp/chromaspro/).\\n Phylogenetic analysis The obtained sequences from each genetic locus were aligned with each other and reference sequences, available in GenBank (https://www.ncbi.nlm.nih.gov/), using ClustalX (http://www.clustal.org/) to determine the identity of Anaplasma spp. Evolutionary relationships were inferred based on partial sequences of 16S rRNA, citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4) genes using the maximum likelihood (ML) method implemented in MEGA7 (http://www.megasoftware.net/). The ML phylogenetic analysis was conducted using the Kimura 2-parameter model and 1000 bootstrap replicates. The ML tree was rooted against the nucleotide sequences L36221 (Rickettsia typhi), KY124257 (Rickettsia parkeri), U96733 (Rickettsia rickettsii) and BDDN01000175 (Ehrlichia ruminantium) for 16S rRNA, gltA, groEL and msp4 gene markers, respectively.\\nThe obtained sequences from each genetic locus were aligned with each other and reference sequences, available in GenBank (https://www.ncbi.nlm.nih.gov/), using ClustalX (http://www.clustal.org/) to determine the identity of Anaplasma spp. Evolutionary relationships were inferred based on partial sequences of 16S rRNA, citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4) genes using the maximum likelihood (ML) method implemented in MEGA7 (http://www.megasoftware.net/). The ML phylogenetic analysis was conducted using the Kimura 2-parameter model and 1000 bootstrap replicates. The ML tree was rooted against the nucleotide sequences L36221 (Rickettsia typhi), KY124257 (Rickettsia parkeri), U96733 (Rickettsia rickettsii) and BDDN01000175 (Ehrlichia ruminantium) for 16S rRNA, gltA, groEL and msp4 gene markers, respectively.\",\n \"Chungbuk Wildlife Center is located in Cheongju city, Chungcheongbuk-do province in the Republic of Korea (36°38′13.99ʺN, 127°29′22.99ʺE). The center receives terrestrial and avian wild animals for purposes of treatment from sickness/injuries and/or rehabilitation. Blood samples are collected for diagnosis and treatment of wildlife referred to the Chungbuk Wildlife Center. Blood samples are archived in EDTA-treated tubes and stored at – 80 °C. A total of 253 blood samples including 198 from Korean water deer (Hydropotes inermis argyropus), 53 from raccoon dogs (Nyctereutes procyonoides) and one sample each from a leopard cat (Prionailurus bengalensis) and a roe deer (Capreolus pygargus), collected from January 2015 to June 2018, were used.\",\n \"Frozen blood samples were thawed at room temperature and genomic DNA was extracted from 200 µl of blood using a Magpurix® Blood Kit and Magpurix® 12s automated nucleic acid purification system (Zinexts Life Science Corp., Taipei, Taiwan), according to the manufacturer’s recommendations. DNA preparations were tested for the presence of Anaplasma species by PCR/sequence analysis of 429 bp of the 16S rRNA gene as described previously [30]. Anaplasma capra-positive isolates were genetically profiled by the amplification of a longer fragment of 16S rRNA (rrs) gene as well as partial sequences of citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4) genes as described previously (Table 1). Amplified fragments were electrophoresed on 1.2% gel loaded with EcoDye™ stain (BIOFACT, Daejeon, Korea) and visualized using UV light.Table 1PCR primers and conditions used in this studyTarget genePrimer namePrimer sequence (5′-3′)Annealing T (°C)Target size (bp)ReferencerrsForwardTTGAGAGTTTGATCCTGGCTCAGAACG571499[12]ReverseWAAGGWGGTAATCCAGCgltAOuter FGCGATTTTAGAGTGYGGAGATTG551031[12]Outer RTACAATACCGGAGTAAAAGTCAAInner FTCATCTCCTGTTGCACGGTGCCC60594[21]Inner RCTCTGAATGAACATGCCCACCCTgroELForwardGCGAGGCGTTAGACAAGTCCATT581129[12]ReverseTCCAGAGATGCAAGCGTGTATAGmsp2Outer FGCGTGTTGATGGCTCTGGT521089[12]Outer RACCAGTATCCTTATTTTTACCInner FGAGTGCACCAGAGCCTAGAA56801This studyInner RTCACCATCACCAAGCACTCTmsp4Outer FCAGTCTGCGCCTGCTCCCTAC55757[12]Outer RAGGAATCTTGCTCCAAGGTTAInner FGGGTTCTGATATGGCATCTTC56656[15]Inner RGGGAAATGTCCTTATAGGATTCGAbbreviations: rrs, 16S rRNA; gltA, citrate synthase; groEL, heat-shock protein; msp2, major surface protein 2; msp4, major surface protein 4; T, temperature\\n\\nPCR primers and conditions used in this study\\nAbbreviations: rrs, 16S rRNA; gltA, citrate synthase; groEL, heat-shock protein; msp2, major surface protein 2; msp4, major surface protein 4; T, temperature\",\n \"The PCR products (rrs and groEL) or secondary PCR product (for other gene markers) were purified and sequenced, either directly or after cloning in the pGEM-T vector (Promega, Madison, WI, USA), in both directions. Generated sequences were assembled using ChromasPro v.2.1.8 (https://technelysium.com.au/wp/chromaspro/).\",\n \"The obtained sequences from each genetic locus were aligned with each other and reference sequences, available in GenBank (https://www.ncbi.nlm.nih.gov/), using ClustalX (http://www.clustal.org/) to determine the identity of Anaplasma spp. Evolutionary relationships were inferred based on partial sequences of 16S rRNA, citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4) genes using the maximum likelihood (ML) method implemented in MEGA7 (http://www.megasoftware.net/). The ML phylogenetic analysis was conducted using the Kimura 2-parameter model and 1000 bootstrap replicates. The ML tree was rooted against the nucleotide sequences L36221 (Rickettsia typhi), KY124257 (Rickettsia parkeri), U96733 (Rickettsia rickettsii) and BDDN01000175 (Ehrlichia ruminantium) for 16S rRNA, gltA, groEL and msp4 gene markers, respectively.\",\n \"The overall infection rate of A. capra in tested animals was 13.8% (35/253); however, samples from raccoon dogs (n = 53), leopard cat (n = 1) and roe deer (n = 1) were negative. The infection rate in KWD was 17.7% (35/198), distributed as follows: 24.6% (14/57) in 2015; 13.2% (5/38) in 2016; 17.3% (14/81) in 2017; and 9.1% (2/22) in 2018 (Table 2).Table 2Distribution of samples and prevalence of A. capra in animal speciesYearKorean water deerRaccoon dogOther animalsNumber infectedTotal numberInfection rate (%)InfectedNot infectedInfectedNot infectedNot infected201514430210147818.0201653305054311.620171467018LC (1)1410014.0201822009RD (1)2326.3Total number3516305323525313.8aInfection rate/species (%)17.700aOverall infection rateAbbreviations: LC, leopard cat (Prionailurus bengalensis); RD, roe deer (Capreolus pygargus)\\n\\nDistribution of samples and prevalence of A. capra in animal species\\naOverall infection rate\\nAbbreviations: LC, leopard cat (Prionailurus bengalensis); RD, roe deer (Capreolus pygargus)\\nMolecular and phylogenetic analyses indicated to occurrence of two genetically distinct strains [named Cheongju (23 isolates) and Chungbuk (12 isolates)] of this pathogen. Sequences obtained from both strains were similar to those derived from A. capra from goats, sheep, cattle, ticks and humans; however, they had striking genetic differences, suggesting that they are novel strains. Sequences of the rrs gene fragment of both strains showed an identity of ~ 99.5% with counterparts in database and clustered in the clade of A. capra from different hosts (Fig. 1). Both strains had single nucleotide polymorphisms (SNPs), resulting in four genotypes at this gene locus. Phylogenetic analysis revealed that three sequences designated A. centrale (GenBank: AB211164, AF283007 and GU064903) and two sequences designated Anaplasma spp. (GenBank: AB454075 and AB509223) clustered within the A. capra clade, even though other A. centrale sequences from different hosts and geographical regions formed separate clusters in the ML tree.Fig. 1Maximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of 16S rRNA gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\\n\\nMaximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of 16S rRNA gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\\nThe gltA gene of the Cheongju strain shared a similarity of 99.5% (with two substitutions, A/G at position 456 and T/C at position 533) with gltA sequences KM206274, KJ700628 and MH029895 isolated from a human, goat and tick, respectively [12, 23]. Sequences of the Chungbuk strain showed a similarity of 98–99% with KX685885, KX685886 and MF071308 of A. capra from ticks and sheep [13, 19]. Both strains clustered with their homologous sequences in the A. capra clade (Fig. 2). groEL gene sequences derived from the Cheongju strain shared a similarity of 99% (one substitution) with their counterparts from humans (GenBank: KM206275), goats (GenBank: KJ700629), sheep (GenBank: KX417356) and ticks (GenBank: KR261633 and KR261635), whereas sequences from the Chungbuk strain shared a similarity of 91% with the reference sequences (Fig. 3). The msp2 sequences showed extensive intra- and inter-sequence variations, including multiple InDels and single nucleotide substitutions; however, all sequences remained clustered in the A. capra clade (Fig. 4). A hypervariable stretch was detected between positions 285 and 414 of the generated sequences (corresponding to positions 550 and 679 in the reference sequence KM206276 of A. capra from humans). The msp4 sequences were identical in the two strains and showed an identity of 100% with those from humans (GenBank: KM206277) and ticks (GenBank: KR261637 and KR261640) (Fig. 5).Fig. 2Maximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of gltA gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\\nFig. 3Maximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of groEL gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\\nFig. 4Maximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of msp2 gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\\nFig. 5Maximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of msp4 gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\\n\\nMaximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of gltA gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\\nMaximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of groEL gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\\nMaximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of msp2 gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\\nMaximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of msp4 gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\",\n \"Wild animals act as reservoirs for a wide range of pathogens [31–33]. The emergence of infectious disease agents of wildlife origin is a prominent challenge to public health and the livestock industry [34–36]. Anaplasma capra has recently been isolated from human patients in China with non-specific clinical manifestations, with potential progression to CNS complications, suggesting that this species could pose a substantial threat to public health [12, 37]. We detected A. capra DNA in blood samples of 35 out of 198 KWD (17.7% infection rate) at the Chungbuk Wildlife Center, Korea. Epidemiological data for this pathogen in wildlife are lacking in Korea; however, our findings are similar to those obtained from wildlife (five takins, three Himalayan gorals, three Reeves’s muntjacs, one forest musk deer and one wild boar) in China [24]. In addition, a low percentage of infection rate was reported cattle, sheep and goats in China, Sweden and Korea [15–19], indicating that A. capra has a broad host range. Occurrence of infection during the study period from 2015 to 2018 indicates the persistence of the infection in KWD, suggesting that the species may act as a reservoir for this pathogen. However, it is difficult to explain the negative results from raccoon dogs in the present study. This may be attributed to persistent infection making the pathogen below detectable level in the blood of these animals. In support of this view, A. capra has been reported to infect endothelial cells [12, 15], making its detection in the blood possible in case of considerable bacteremia or released endothelial cells, resembling Rickettsia species [12]. Furthermore, the sample size and species and/or the age of animals may play a role in these findings. Further investigations are needed to clarify these points.\\nOur genetic profiling results indicated that the newly generated 16S rRNA gene sequences shared a homology of > 99.5% with sequences of A. capra strains from humans, sheep, goats, cattle and ticks [12, 15, 16, 18, 20, 22], suggesting that they likely are within the same species of bacteria [38, 39]. Clustering of sequences named A. centrale from deer (Cervus nippon nippon) (GenBank: AB211164) and cattle (GenBank: AF283007) in Japan and from ticks (Haemaphysalis longicornis) in Korea (GenBank: GU064903), as well as Anaplasma spp. from deer (GenBank: AB454075; direct submission) and Japanese serow (Capricornis crispus) (GenBank: AB509223) in the same clade of A. capra indicate a close phylogenetic relationship. The clustering pattern of these sequences in the A. capra clade does not support their assignment as sister taxa and suggests that these isolates are in fact A. capra [15, 25] and may need re-description, since these sequences were more related to Chungbuk strain in the A. capra clade.\\nThe results obtained using different gene markers showed considerable sequence variation, suggesting that A. capra has a high degree of genetic diversity. Notably, extensive genetic variation was detected in msp2. Consistent with our results, sequence variation at the studied gene markers has been reported previously among isolates from ticks, sheep and goats [12, 15–18, 20–25]. Similarly, genetic variation is common in other Anaplasma species [40–45]. Although genetic diversity is reportedly related to infectivity, virulence, pathogenicity, niche preference, immune evasion, and/or host adaptability [46–49], this has not been established in A. capra and further studies are needed to evaluate these relationships.\\nDue to the extinction of natural predators, the KWD is thriving in Korea and has been designated as “harmful wildlife” by the Ministry of Environment in 1994 owing to harmful interactions with humans and their properties. This close interaction poses substantial threats to domestic animals and human health in Korea. This study was limited by analyzing samples from one geographical area and few animal species, which my lead to biases in the results. A large-scale study is underway to fully elucidate the host range of wildlife, vector ticks, pathogenicity and geographical distribution of this organism in Korea.\",\n \"To our knowledge, the results presented herein provide the first evidence for the presence of A. capra in Korean water deer in Korea. As an emerging human pathogen, the detection of A. capra in deer provides insight into the role of wildlife as a potential reservoir for human anaplasmosis. Furthermore, the obtained results expand the known geographical and host range of the Anaplasma capra.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":[null,"materials|methods",null,null,null,null,"results","discussion","conclusion"],"string":"[\n null,\n \"materials|methods\",\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\",\n \"conclusion\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["\nAnaplasma capra\n","Korean water deer (Hydropotes inermis argyropus)","South Korea"],"string":"[\n \"\\nAnaplasma capra\\n\",\n \"Korean water deer (Hydropotes inermis argyropus)\",\n \"South Korea\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Background:\nThe cosmopolitan genus Anaplasma includes six species of Gram-negative obligate intracellular bacteria that are transmitted by ticks to a wide range of animals, including humans [1–5], resulting in considerable economic losses in the livestock industry and serious public health concerns [6, 7]. Anaplasma phagocytophilum, A. ovis and recently reported A. capra are human pathogens [8–12], whereas other species in the genus have no known zoonotic potential. However, A. platys may have zoonotic potential after frequent reports of human infection [13, 14].\nThe provisional name Anaplasma capra was assigned after its initial characterization in goats (Capra aegagrus hircus) in China [12]. Later, it was isolated from sheep, goats and cattle in different geographical regions [15–19] as well as from various tick species (Haemaphysalis qinghaiensis, H. longicornis, Ixodes persulcatus) [12, 20–23]. Infection of A. capra was also reported in six wild animals in China including three takins (Budorcas taxicolor), two Reeves’s muntjacs (Muntiacus reevesi) and one forest musk deer (Moschus berezovskii) [24].\nAnaplasma species usually parasitize bone marrow-derived elements, including neutrophils (A. phagocytophilum), erythrocytes (A. marginale, A. centrale and A. ovis), monocytes (A. bovis) and platelets (A. platys) [7, 9, 10, 12]. However, A. capra seems to infect endothelial cells, rendering its microscopic detection in blood smears unreliable [12, 15]. In humans, the disease caused by A. capra is generally characterized by undifferentiated fever, headache, malaise, dizziness, myalgia and chills, with potential progression to CNS involvement and cerebrospinal fluid pleocytosis [12].\nAlthough different Anaplasma species have been detected in wildlife [23–29], little is known about the prevalence and genetic identity of A. capra in these animals in Korea. Using molecular tools, the present study aimed at investigating the occurrence and characterizing the genetic profile of this pathogen in wildlife in the Republic of Korea.\n\nMethods:\n Collection of samples Chungbuk Wildlife Center is located in Cheongju city, Chungcheongbuk-do province in the Republic of Korea (36°38′13.99ʺN, 127°29′22.99ʺE). The center receives terrestrial and avian wild animals for purposes of treatment from sickness/injuries and/or rehabilitation. Blood samples are collected for diagnosis and treatment of wildlife referred to the Chungbuk Wildlife Center. Blood samples are archived in EDTA-treated tubes and stored at – 80 °C. A total of 253 blood samples including 198 from Korean water deer (Hydropotes inermis argyropus), 53 from raccoon dogs (Nyctereutes procyonoides) and one sample each from a leopard cat (Prionailurus bengalensis) and a roe deer (Capreolus pygargus), collected from January 2015 to June 2018, were used.\nChungbuk Wildlife Center is located in Cheongju city, Chungcheongbuk-do province in the Republic of Korea (36°38′13.99ʺN, 127°29′22.99ʺE). The center receives terrestrial and avian wild animals for purposes of treatment from sickness/injuries and/or rehabilitation. Blood samples are collected for diagnosis and treatment of wildlife referred to the Chungbuk Wildlife Center. Blood samples are archived in EDTA-treated tubes and stored at – 80 °C. A total of 253 blood samples including 198 from Korean water deer (Hydropotes inermis argyropus), 53 from raccoon dogs (Nyctereutes procyonoides) and one sample each from a leopard cat (Prionailurus bengalensis) and a roe deer (Capreolus pygargus), collected from January 2015 to June 2018, were used.\n DNA extraction and PCR amplification Frozen blood samples were thawed at room temperature and genomic DNA was extracted from 200 µl of blood using a Magpurix® Blood Kit and Magpurix® 12s automated nucleic acid purification system (Zinexts Life Science Corp., Taipei, Taiwan), according to the manufacturer’s recommendations. DNA preparations were tested for the presence of Anaplasma species by PCR/sequence analysis of 429 bp of the 16S rRNA gene as described previously [30]. Anaplasma capra-positive isolates were genetically profiled by the amplification of a longer fragment of 16S rRNA (rrs) gene as well as partial sequences of citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4) genes as described previously (Table 1). Amplified fragments were electrophoresed on 1.2% gel loaded with EcoDye™ stain (BIOFACT, Daejeon, Korea) and visualized using UV light.Table 1PCR primers and conditions used in this studyTarget genePrimer namePrimer sequence (5′-3′)Annealing T (°C)Target size (bp)ReferencerrsForwardTTGAGAGTTTGATCCTGGCTCAGAACG571499[12]ReverseWAAGGWGGTAATCCAGCgltAOuter FGCGATTTTAGAGTGYGGAGATTG551031[12]Outer RTACAATACCGGAGTAAAAGTCAAInner FTCATCTCCTGTTGCACGGTGCCC60594[21]Inner RCTCTGAATGAACATGCCCACCCTgroELForwardGCGAGGCGTTAGACAAGTCCATT581129[12]ReverseTCCAGAGATGCAAGCGTGTATAGmsp2Outer FGCGTGTTGATGGCTCTGGT521089[12]Outer RACCAGTATCCTTATTTTTACCInner FGAGTGCACCAGAGCCTAGAA56801This studyInner RTCACCATCACCAAGCACTCTmsp4Outer FCAGTCTGCGCCTGCTCCCTAC55757[12]Outer RAGGAATCTTGCTCCAAGGTTAInner FGGGTTCTGATATGGCATCTTC56656[15]Inner RGGGAAATGTCCTTATAGGATTCGAbbreviations: rrs, 16S rRNA; gltA, citrate synthase; groEL, heat-shock protein; msp2, major surface protein 2; msp4, major surface protein 4; T, temperature\n\nPCR primers and conditions used in this study\nAbbreviations: rrs, 16S rRNA; gltA, citrate synthase; groEL, heat-shock protein; msp2, major surface protein 2; msp4, major surface protein 4; T, temperature\nFrozen blood samples were thawed at room temperature and genomic DNA was extracted from 200 µl of blood using a Magpurix® Blood Kit and Magpurix® 12s automated nucleic acid purification system (Zinexts Life Science Corp., Taipei, Taiwan), according to the manufacturer’s recommendations. DNA preparations were tested for the presence of Anaplasma species by PCR/sequence analysis of 429 bp of the 16S rRNA gene as described previously [30]. Anaplasma capra-positive isolates were genetically profiled by the amplification of a longer fragment of 16S rRNA (rrs) gene as well as partial sequences of citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4) genes as described previously (Table 1). Amplified fragments were electrophoresed on 1.2% gel loaded with EcoDye™ stain (BIOFACT, Daejeon, Korea) and visualized using UV light.Table 1PCR primers and conditions used in this studyTarget genePrimer namePrimer sequence (5′-3′)Annealing T (°C)Target size (bp)ReferencerrsForwardTTGAGAGTTTGATCCTGGCTCAGAACG571499[12]ReverseWAAGGWGGTAATCCAGCgltAOuter FGCGATTTTAGAGTGYGGAGATTG551031[12]Outer RTACAATACCGGAGTAAAAGTCAAInner FTCATCTCCTGTTGCACGGTGCCC60594[21]Inner RCTCTGAATGAACATGCCCACCCTgroELForwardGCGAGGCGTTAGACAAGTCCATT581129[12]ReverseTCCAGAGATGCAAGCGTGTATAGmsp2Outer FGCGTGTTGATGGCTCTGGT521089[12]Outer RACCAGTATCCTTATTTTTACCInner FGAGTGCACCAGAGCCTAGAA56801This studyInner RTCACCATCACCAAGCACTCTmsp4Outer FCAGTCTGCGCCTGCTCCCTAC55757[12]Outer RAGGAATCTTGCTCCAAGGTTAInner FGGGTTCTGATATGGCATCTTC56656[15]Inner RGGGAAATGTCCTTATAGGATTCGAbbreviations: rrs, 16S rRNA; gltA, citrate synthase; groEL, heat-shock protein; msp2, major surface protein 2; msp4, major surface protein 4; T, temperature\n\nPCR primers and conditions used in this study\nAbbreviations: rrs, 16S rRNA; gltA, citrate synthase; groEL, heat-shock protein; msp2, major surface protein 2; msp4, major surface protein 4; T, temperature\n DNA sequence analysis The PCR products (rrs and groEL) or secondary PCR product (for other gene markers) were purified and sequenced, either directly or after cloning in the pGEM-T vector (Promega, Madison, WI, USA), in both directions. Generated sequences were assembled using ChromasPro v.2.1.8 (https://technelysium.com.au/wp/chromaspro/).\nThe PCR products (rrs and groEL) or secondary PCR product (for other gene markers) were purified and sequenced, either directly or after cloning in the pGEM-T vector (Promega, Madison, WI, USA), in both directions. Generated sequences were assembled using ChromasPro v.2.1.8 (https://technelysium.com.au/wp/chromaspro/).\n Phylogenetic analysis The obtained sequences from each genetic locus were aligned with each other and reference sequences, available in GenBank (https://www.ncbi.nlm.nih.gov/), using ClustalX (http://www.clustal.org/) to determine the identity of Anaplasma spp. Evolutionary relationships were inferred based on partial sequences of 16S rRNA, citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4) genes using the maximum likelihood (ML) method implemented in MEGA7 (http://www.megasoftware.net/). The ML phylogenetic analysis was conducted using the Kimura 2-parameter model and 1000 bootstrap replicates. The ML tree was rooted against the nucleotide sequences L36221 (Rickettsia typhi), KY124257 (Rickettsia parkeri), U96733 (Rickettsia rickettsii) and BDDN01000175 (Ehrlichia ruminantium) for 16S rRNA, gltA, groEL and msp4 gene markers, respectively.\nThe obtained sequences from each genetic locus were aligned with each other and reference sequences, available in GenBank (https://www.ncbi.nlm.nih.gov/), using ClustalX (http://www.clustal.org/) to determine the identity of Anaplasma spp. Evolutionary relationships were inferred based on partial sequences of 16S rRNA, citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4) genes using the maximum likelihood (ML) method implemented in MEGA7 (http://www.megasoftware.net/). The ML phylogenetic analysis was conducted using the Kimura 2-parameter model and 1000 bootstrap replicates. The ML tree was rooted against the nucleotide sequences L36221 (Rickettsia typhi), KY124257 (Rickettsia parkeri), U96733 (Rickettsia rickettsii) and BDDN01000175 (Ehrlichia ruminantium) for 16S rRNA, gltA, groEL and msp4 gene markers, respectively.\n\nCollection of samples:\nChungbuk Wildlife Center is located in Cheongju city, Chungcheongbuk-do province in the Republic of Korea (36°38′13.99ʺN, 127°29′22.99ʺE). The center receives terrestrial and avian wild animals for purposes of treatment from sickness/injuries and/or rehabilitation. Blood samples are collected for diagnosis and treatment of wildlife referred to the Chungbuk Wildlife Center. Blood samples are archived in EDTA-treated tubes and stored at – 80 °C. A total of 253 blood samples including 198 from Korean water deer (Hydropotes inermis argyropus), 53 from raccoon dogs (Nyctereutes procyonoides) and one sample each from a leopard cat (Prionailurus bengalensis) and a roe deer (Capreolus pygargus), collected from January 2015 to June 2018, were used.\n\nDNA extraction and PCR amplification:\nFrozen blood samples were thawed at room temperature and genomic DNA was extracted from 200 µl of blood using a Magpurix® Blood Kit and Magpurix® 12s automated nucleic acid purification system (Zinexts Life Science Corp., Taipei, Taiwan), according to the manufacturer’s recommendations. DNA preparations were tested for the presence of Anaplasma species by PCR/sequence analysis of 429 bp of the 16S rRNA gene as described previously [30]. Anaplasma capra-positive isolates were genetically profiled by the amplification of a longer fragment of 16S rRNA (rrs) gene as well as partial sequences of citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4) genes as described previously (Table 1). Amplified fragments were electrophoresed on 1.2% gel loaded with EcoDye™ stain (BIOFACT, Daejeon, Korea) and visualized using UV light.Table 1PCR primers and conditions used in this studyTarget genePrimer namePrimer sequence (5′-3′)Annealing T (°C)Target size (bp)ReferencerrsForwardTTGAGAGTTTGATCCTGGCTCAGAACG571499[12]ReverseWAAGGWGGTAATCCAGCgltAOuter FGCGATTTTAGAGTGYGGAGATTG551031[12]Outer RTACAATACCGGAGTAAAAGTCAAInner FTCATCTCCTGTTGCACGGTGCCC60594[21]Inner RCTCTGAATGAACATGCCCACCCTgroELForwardGCGAGGCGTTAGACAAGTCCATT581129[12]ReverseTCCAGAGATGCAAGCGTGTATAGmsp2Outer FGCGTGTTGATGGCTCTGGT521089[12]Outer RACCAGTATCCTTATTTTTACCInner FGAGTGCACCAGAGCCTAGAA56801This studyInner RTCACCATCACCAAGCACTCTmsp4Outer FCAGTCTGCGCCTGCTCCCTAC55757[12]Outer RAGGAATCTTGCTCCAAGGTTAInner FGGGTTCTGATATGGCATCTTC56656[15]Inner RGGGAAATGTCCTTATAGGATTCGAbbreviations: rrs, 16S rRNA; gltA, citrate synthase; groEL, heat-shock protein; msp2, major surface protein 2; msp4, major surface protein 4; T, temperature\n\nPCR primers and conditions used in this study\nAbbreviations: rrs, 16S rRNA; gltA, citrate synthase; groEL, heat-shock protein; msp2, major surface protein 2; msp4, major surface protein 4; T, temperature\n\nDNA sequence analysis:\nThe PCR products (rrs and groEL) or secondary PCR product (for other gene markers) were purified and sequenced, either directly or after cloning in the pGEM-T vector (Promega, Madison, WI, USA), in both directions. Generated sequences were assembled using ChromasPro v.2.1.8 (https://technelysium.com.au/wp/chromaspro/).\n\nPhylogenetic analysis:\nThe obtained sequences from each genetic locus were aligned with each other and reference sequences, available in GenBank (https://www.ncbi.nlm.nih.gov/), using ClustalX (http://www.clustal.org/) to determine the identity of Anaplasma spp. Evolutionary relationships were inferred based on partial sequences of 16S rRNA, citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4) genes using the maximum likelihood (ML) method implemented in MEGA7 (http://www.megasoftware.net/). The ML phylogenetic analysis was conducted using the Kimura 2-parameter model and 1000 bootstrap replicates. The ML tree was rooted against the nucleotide sequences L36221 (Rickettsia typhi), KY124257 (Rickettsia parkeri), U96733 (Rickettsia rickettsii) and BDDN01000175 (Ehrlichia ruminantium) for 16S rRNA, gltA, groEL and msp4 gene markers, respectively.\n\nResults:\nThe overall infection rate of A. capra in tested animals was 13.8% (35/253); however, samples from raccoon dogs (n = 53), leopard cat (n = 1) and roe deer (n = 1) were negative. The infection rate in KWD was 17.7% (35/198), distributed as follows: 24.6% (14/57) in 2015; 13.2% (5/38) in 2016; 17.3% (14/81) in 2017; and 9.1% (2/22) in 2018 (Table 2).Table 2Distribution of samples and prevalence of A. capra in animal speciesYearKorean water deerRaccoon dogOther animalsNumber infectedTotal numberInfection rate (%)InfectedNot infectedInfectedNot infectedNot infected201514430210147818.0201653305054311.620171467018LC (1)1410014.0201822009RD (1)2326.3Total number3516305323525313.8aInfection rate/species (%)17.700aOverall infection rateAbbreviations: LC, leopard cat (Prionailurus bengalensis); RD, roe deer (Capreolus pygargus)\n\nDistribution of samples and prevalence of A. capra in animal species\naOverall infection rate\nAbbreviations: LC, leopard cat (Prionailurus bengalensis); RD, roe deer (Capreolus pygargus)\nMolecular and phylogenetic analyses indicated to occurrence of two genetically distinct strains [named Cheongju (23 isolates) and Chungbuk (12 isolates)] of this pathogen. Sequences obtained from both strains were similar to those derived from A. capra from goats, sheep, cattle, ticks and humans; however, they had striking genetic differences, suggesting that they are novel strains. Sequences of the rrs gene fragment of both strains showed an identity of ~ 99.5% with counterparts in database and clustered in the clade of A. capra from different hosts (Fig. 1). Both strains had single nucleotide polymorphisms (SNPs), resulting in four genotypes at this gene locus. Phylogenetic analysis revealed that three sequences designated A. centrale (GenBank: AB211164, AF283007 and GU064903) and two sequences designated Anaplasma spp. (GenBank: AB454075 and AB509223) clustered within the A. capra clade, even though other A. centrale sequences from different hosts and geographical regions formed separate clusters in the ML tree.Fig. 1Maximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of 16S rRNA gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\n\nMaximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of 16S rRNA gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\nThe gltA gene of the Cheongju strain shared a similarity of 99.5% (with two substitutions, A/G at position 456 and T/C at position 533) with gltA sequences KM206274, KJ700628 and MH029895 isolated from a human, goat and tick, respectively [12, 23]. Sequences of the Chungbuk strain showed a similarity of 98–99% with KX685885, KX685886 and MF071308 of A. capra from ticks and sheep [13, 19]. Both strains clustered with their homologous sequences in the A. capra clade (Fig. 2). groEL gene sequences derived from the Cheongju strain shared a similarity of 99% (one substitution) with their counterparts from humans (GenBank: KM206275), goats (GenBank: KJ700629), sheep (GenBank: KX417356) and ticks (GenBank: KR261633 and KR261635), whereas sequences from the Chungbuk strain shared a similarity of 91% with the reference sequences (Fig. 3). The msp2 sequences showed extensive intra- and inter-sequence variations, including multiple InDels and single nucleotide substitutions; however, all sequences remained clustered in the A. capra clade (Fig. 4). A hypervariable stretch was detected between positions 285 and 414 of the generated sequences (corresponding to positions 550 and 679 in the reference sequence KM206276 of A. capra from humans). The msp4 sequences were identical in the two strains and showed an identity of 100% with those from humans (GenBank: KM206277) and ticks (GenBank: KR261637 and KR261640) (Fig. 5).Fig. 2Maximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of gltA gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\nFig. 3Maximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of groEL gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\nFig. 4Maximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of msp2 gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\nFig. 5Maximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of msp4 gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\n\nMaximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of gltA gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\nMaximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of groEL gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\nMaximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of msp2 gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\nMaximum-likelihood phylogenetic trees of Anaplasma species based on partial sequences of msp4 gene. The tree was constructed using MEGA7 with the Kimura 2-parameter model. The newly generated sequences are indicated by diamonds. The numbers at nodes represent bootstrap values. The scale-bar represents the number of nucleotide substitutions per site\n\nDiscussion:\nWild animals act as reservoirs for a wide range of pathogens [31–33]. The emergence of infectious disease agents of wildlife origin is a prominent challenge to public health and the livestock industry [34–36]. Anaplasma capra has recently been isolated from human patients in China with non-specific clinical manifestations, with potential progression to CNS complications, suggesting that this species could pose a substantial threat to public health [12, 37]. We detected A. capra DNA in blood samples of 35 out of 198 KWD (17.7% infection rate) at the Chungbuk Wildlife Center, Korea. Epidemiological data for this pathogen in wildlife are lacking in Korea; however, our findings are similar to those obtained from wildlife (five takins, three Himalayan gorals, three Reeves’s muntjacs, one forest musk deer and one wild boar) in China [24]. In addition, a low percentage of infection rate was reported cattle, sheep and goats in China, Sweden and Korea [15–19], indicating that A. capra has a broad host range. Occurrence of infection during the study period from 2015 to 2018 indicates the persistence of the infection in KWD, suggesting that the species may act as a reservoir for this pathogen. However, it is difficult to explain the negative results from raccoon dogs in the present study. This may be attributed to persistent infection making the pathogen below detectable level in the blood of these animals. In support of this view, A. capra has been reported to infect endothelial cells [12, 15], making its detection in the blood possible in case of considerable bacteremia or released endothelial cells, resembling Rickettsia species [12]. Furthermore, the sample size and species and/or the age of animals may play a role in these findings. Further investigations are needed to clarify these points.\nOur genetic profiling results indicated that the newly generated 16S rRNA gene sequences shared a homology of > 99.5% with sequences of A. capra strains from humans, sheep, goats, cattle and ticks [12, 15, 16, 18, 20, 22], suggesting that they likely are within the same species of bacteria [38, 39]. Clustering of sequences named A. centrale from deer (Cervus nippon nippon) (GenBank: AB211164) and cattle (GenBank: AF283007) in Japan and from ticks (Haemaphysalis longicornis) in Korea (GenBank: GU064903), as well as Anaplasma spp. from deer (GenBank: AB454075; direct submission) and Japanese serow (Capricornis crispus) (GenBank: AB509223) in the same clade of A. capra indicate a close phylogenetic relationship. The clustering pattern of these sequences in the A. capra clade does not support their assignment as sister taxa and suggests that these isolates are in fact A. capra [15, 25] and may need re-description, since these sequences were more related to Chungbuk strain in the A. capra clade.\nThe results obtained using different gene markers showed considerable sequence variation, suggesting that A. capra has a high degree of genetic diversity. Notably, extensive genetic variation was detected in msp2. Consistent with our results, sequence variation at the studied gene markers has been reported previously among isolates from ticks, sheep and goats [12, 15–18, 20–25]. Similarly, genetic variation is common in other Anaplasma species [40–45]. Although genetic diversity is reportedly related to infectivity, virulence, pathogenicity, niche preference, immune evasion, and/or host adaptability [46–49], this has not been established in A. capra and further studies are needed to evaluate these relationships.\nDue to the extinction of natural predators, the KWD is thriving in Korea and has been designated as “harmful wildlife” by the Ministry of Environment in 1994 owing to harmful interactions with humans and their properties. This close interaction poses substantial threats to domestic animals and human health in Korea. This study was limited by analyzing samples from one geographical area and few animal species, which my lead to biases in the results. A large-scale study is underway to fully elucidate the host range of wildlife, vector ticks, pathogenicity and geographical distribution of this organism in Korea.\n\nConclusions:\nTo our knowledge, the results presented herein provide the first evidence for the presence of A. capra in Korean water deer in Korea. As an emerging human pathogen, the detection of A. capra in deer provides insight into the role of wildlife as a potential reservoir for human anaplasmosis. Furthermore, the obtained results expand the known geographical and host range of the Anaplasma capra.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nAnaplasma spp. are tick-borne Gram-negative obligate intracellular bacteria that infect humans and a wide range of animals. Anaplasma capra has emerged as a human pathogen; however, little is known about the occurrence and genetic identity of this agent in wildlife. The present study aimed to determine the infection rate and genetic profile of this pathogen in wild animals in the Republic of Korea.\n\nMethods:\nA total of 253 blood samples [198 from Korean water deer (Hydropotes inermis argyropus), 53 from raccoon dogs (Nyctereutes procyonoides) and one sample each from a leopard cat (Prionailurus bengalensis) and a roe deer (Capreolus pygargus)] were collected at Chungbuk Wildlife Center during the period 2015-2018. Genomic DNA was extracted from the samples and screened for presence of Anaplasma species by PCR/sequence analysis of 429 bp of the 16S rRNA gene marker. Anaplasma capra-positive isolates were genetically profiled by amplification of a longer fragment of 16S rRNA (rrs) as well as partial sequences of citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4). Generated sequences of each gene marker were aligned with homologous sequences in the database and phylogenetically analyzed.\n\nResults:\nAnaplasma capra was detected in blood samples derived from Korean water deer, whereas samples from other animal species were negative. The overall infection rate in tested samples was 13.8% (35/253) and in the water deer the rate was 17.8% (35/198), distributed along the study period from 2015 to 2018. Genetic profiling and a phylogenetic analysis based on analyzed gene markers revealed the occurrence of two distinct strains, clustered in a single clade with counterpart sequences of A. capra in the database.\n\nConclusions:\nAnaplasma capra infection were detected in Korean water deer in the Republic of Korea, providing insight into the role of wildlife as a potential reservoir for animal and human anaplasmosis. However, further work is needed in order to evaluate the role of Korean water deer as a host/reservoir host of A. capra."},"background_conclusion_text":{"kind":"string","value":"Background:\nThe cosmopolitan genus Anaplasma includes six species of Gram-negative obligate intracellular bacteria that are transmitted by ticks to a wide range of animals, including humans [1–5], resulting in considerable economic losses in the livestock industry and serious public health concerns [6, 7]. Anaplasma phagocytophilum, A. ovis and recently reported A. capra are human pathogens [8–12], whereas other species in the genus have no known zoonotic potential. However, A. platys may have zoonotic potential after frequent reports of human infection [13, 14].\nThe provisional name Anaplasma capra was assigned after its initial characterization in goats (Capra aegagrus hircus) in China [12]. Later, it was isolated from sheep, goats and cattle in different geographical regions [15–19] as well as from various tick species (Haemaphysalis qinghaiensis, H. longicornis, Ixodes persulcatus) [12, 20–23]. Infection of A. capra was also reported in six wild animals in China including three takins (Budorcas taxicolor), two Reeves’s muntjacs (Muntiacus reevesi) and one forest musk deer (Moschus berezovskii) [24].\nAnaplasma species usually parasitize bone marrow-derived elements, including neutrophils (A. phagocytophilum), erythrocytes (A. marginale, A. centrale and A. ovis), monocytes (A. bovis) and platelets (A. platys) [7, 9, 10, 12]. However, A. capra seems to infect endothelial cells, rendering its microscopic detection in blood smears unreliable [12, 15]. In humans, the disease caused by A. capra is generally characterized by undifferentiated fever, headache, malaise, dizziness, myalgia and chills, with potential progression to CNS involvement and cerebrospinal fluid pleocytosis [12].\nAlthough different Anaplasma species have been detected in wildlife [23–29], little is known about the prevalence and genetic identity of A. capra in these animals in Korea. Using molecular tools, the present study aimed at investigating the occurrence and characterizing the genetic profile of this pathogen in wildlife in the Republic of Korea.\n\nConclusions:\nTo our knowledge, the results presented herein provide the first evidence for the presence of A. capra in Korean water deer in Korea. As an emerging human pathogen, the detection of A. capra in deer provides insight into the role of wildlife as a potential reservoir for human anaplasmosis. Furthermore, the obtained results expand the known geographical and host range of the Anaplasma capra."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nAnaplasma spp. are tick-borne Gram-negative obligate intracellular bacteria that infect humans and a wide range of animals. Anaplasma capra has emerged as a human pathogen; however, little is known about the occurrence and genetic identity of this agent in wildlife. The present study aimed to determine the infection rate and genetic profile of this pathogen in wild animals in the Republic of Korea.\n\nMethods:\nA total of 253 blood samples [198 from Korean water deer (Hydropotes inermis argyropus), 53 from raccoon dogs (Nyctereutes procyonoides) and one sample each from a leopard cat (Prionailurus bengalensis) and a roe deer (Capreolus pygargus)] were collected at Chungbuk Wildlife Center during the period 2015-2018. Genomic DNA was extracted from the samples and screened for presence of Anaplasma species by PCR/sequence analysis of 429 bp of the 16S rRNA gene marker. Anaplasma capra-positive isolates were genetically profiled by amplification of a longer fragment of 16S rRNA (rrs) as well as partial sequences of citrate synthase (gltA), heat-shock protein (groEL), major surface protein 2 (msp2) and major surface protein 4 (msp4). Generated sequences of each gene marker were aligned with homologous sequences in the database and phylogenetically analyzed.\n\nResults:\nAnaplasma capra was detected in blood samples derived from Korean water deer, whereas samples from other animal species were negative. The overall infection rate in tested samples was 13.8% (35/253) and in the water deer the rate was 17.8% (35/198), distributed along the study period from 2015 to 2018. Genetic profiling and a phylogenetic analysis based on analyzed gene markers revealed the occurrence of two distinct strains, clustered in a single clade with counterpart sequences of A. capra in the database.\n\nConclusions:\nAnaplasma capra infection were detected in Korean water deer in the Republic of Korea, providing insight into the role of wildlife as a potential reservoir for animal and human anaplasmosis. However, further work is needed in order to evaluate the role of Korean water deer as a host/reservoir host of A. capra."},"whole_article_text_length":{"kind":"number","value":4543,"string":"4,543"},"whole_article_abstract_length":{"kind":"number","value":402,"string":"402"},"other_sections_lengths":{"kind":"list like","value":[384,141,291,59,157],"string":"[\n 384,\n 141,\n 291,\n 59,\n 157\n]","isConversational":false},"num_sections":{"kind":"number","value":9,"string":"9"},"most_frequent_words":{"kind":"list like","value":["sequences","protein","capra","anaplasma","gene","12","species","surface protein","surface","major surface protein"],"string":"[\n \"sequences\",\n \"protein\",\n \"capra\",\n \"anaplasma\",\n \"gene\",\n \"12\",\n \"species\",\n \"surface protein\",\n \"surface\",\n \"major surface protein\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["different anaplasma species","anaplasma species detected","ticks pathogenicity","anaplasma spp deer","capra human pathogens"],"string":"[\n \"different anaplasma species\",\n \"anaplasma species detected\",\n \"ticks pathogenicity\",\n \"anaplasma spp deer\",\n \"capra human pathogens\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] \nAnaplasma capra\n | Korean water deer (Hydropotes inermis argyropus) | South Korea [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] \nAnaplasma capra\n | Korean water deer (Hydropotes inermis argyropus) | South Korea [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] \nAnaplasma capra\n | Korean water deer (Hydropotes inermis argyropus) | South Korea [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] \nAnaplasma capra\n | Korean water deer (Hydropotes inermis argyropus) | South Korea [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] \nAnaplasma capra\n | Korean water deer (Hydropotes inermis argyropus) | South Korea [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Anaplasma | Anaplasmosis | Animals | DNA, Bacterial | Deer | Disease Reservoirs | Genetic Variation | Phylogeny | RNA, Ribosomal, 16S | Republic of Korea [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Anaplasma | Anaplasmosis | Animals | DNA, Bacterial | Deer | Disease Reservoirs | Genetic Variation | Phylogeny | RNA, Ribosomal, 16S | Republic of Korea [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Anaplasma | Anaplasmosis | Animals | DNA, Bacterial | Deer | Disease Reservoirs | Genetic Variation | Phylogeny | RNA, Ribosomal, 16S | Republic of Korea [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Anaplasma | Anaplasmosis | Animals | DNA, Bacterial | Deer | Disease Reservoirs | Genetic Variation | Phylogeny | RNA, Ribosomal, 16S | Republic of Korea [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Anaplasma | Anaplasmosis | Animals | DNA, Bacterial | Deer | Disease Reservoirs | Genetic Variation | Phylogeny | RNA, Ribosomal, 16S | Republic of Korea [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] different anaplasma species | anaplasma species detected | ticks pathogenicity | anaplasma spp deer | capra human pathogens [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] different anaplasma species | anaplasma species detected | ticks pathogenicity | anaplasma spp deer | capra human pathogens [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] different anaplasma species | anaplasma species detected | ticks pathogenicity | anaplasma spp deer | capra human pathogens [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] different anaplasma species | anaplasma species detected | ticks pathogenicity | anaplasma spp deer | capra human pathogens [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] different anaplasma species | anaplasma species detected | ticks pathogenicity | anaplasma spp deer | capra human pathogens [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] sequences | protein | capra | anaplasma | gene | 12 | species | surface protein | surface | major surface protein [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] sequences | protein | capra | anaplasma | gene | 12 | species | surface protein | surface | major surface protein [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] sequences | protein | capra | anaplasma | gene | 12 | species | surface protein | surface | major surface protein 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[SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] results | capra | human | deer | deer korea emerging | capra korean water | capra korean | known geographical host range | emerging | results expand known [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] protein | sequences | capra | surface | major | major surface | major surface protein | surface protein | 12 | wildlife [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] protein | sequences | capra | surface | major | major surface | major surface protein | surface protein | 12 | wildlife [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Gram-negative ||| ||| the Republic of Korea [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Korean ||| 13.8% | 17.8% | 2015 | 2018 ||| two | A. [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Korean | the Republic of Korea ||| Korean | A. [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Gram-negative ||| ||| the Republic of Korea ||| 253 | 198 | Korean | 53 | Nyctereutes | one | Chungbuk Wildlife Center | 2015-2018 ||| Anaplasma | PCR | 429 bp | 16S ||| 16S | 2 | 4 ||| ||| ||| Korean ||| 13.8% | 17.8% | 2015 | 2018 ||| two | A. ||| Korean | the Republic of Korea ||| Korean | A. [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Gram-negative ||| ||| the Republic of Korea ||| 253 | 198 | Korean | 53 | Nyctereutes | one | Chungbuk Wildlife Center | 2015-2018 ||| Anaplasma | PCR | 429 bp | 16S ||| 16S | 2 | 4 ||| ||| ||| Korean ||| 13.8% | 17.8% | 2015 | 2018 ||| two | A. ||| Korean | the Republic of Korea ||| Korean | A. [SUMMARY]"}}},{"rowIdx":58,"cells":{"title":{"kind":"string","value":"Well-Being, Physical Activity, and Social Support in Octogenarians with Heart Failure during COVID-19 Confinement: A Mixed-Methods Study."},"pmid":{"kind":"string","value":"36430033"},"background_abstract":{"kind":"string","value":"This study aimed to compare well-being and physical activity (PA) before and during COVID-19 confinement in older adults with heart failure (HF), to compare well-being and PA during COVID-19 confinement in octogenarians and non-octogenarians, and to explore well-being, social support, attention to symptoms, and assistance needs during confinement in this population."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"A mixed-methods design was performed. Well-being (Cantril Ladder of Life) and PA (International Physical Activity Questionnaire) were assessed. Semi-structured interviews were performed to assess the rest of the variables."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"120 participants were evaluated (74.16 ± 12.90 years; octogenarians = 44.16%, non-octogenarians = 55.83%). Both groups showed lower well-being and performed less PA during confinement than before (p < 0.001). Octogenarians reported lower well-being (p = 0.02), higher sedentary time (p = 0.03), and lower levels of moderate PA (p = 0.04) during confinement. Most individuals in the sample considered their well-being to have decreased during confinement, 30% reported decreased social support, 50% increased their attention to symptoms, and 60% were not satisfied with the assistance received. Octogenarians were more severely impacted during confinement than non-octogenarians in terms of well-being, attention to symptoms, and assistance needs."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Well-being and PA decreased during confinement, although octogenarians were more affected than non-octogenarians. Remote monitoring strategies are needed in elders with HF to control health outcomes in critical periods, especially in octogenarians."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Humans","Aged","Aged, 80 and over","COVID-19","Social Support","Heart Failure","Exercise","Sedentary Behavior"],"string":"[\n \"Humans\",\n \"Aged\",\n \"Aged, 80 and over\",\n \"COVID-19\",\n \"Social Support\",\n \"Heart Failure\",\n \"Exercise\",\n \"Sedentary Behavior\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"9690854"},"background_title":{"kind":"string","value":"1. Introduction"},"background_text":{"kind":"string","value":"Heart failure (HF) is a major clinical and public health problem [1]. HF constitutes a complex debilitating syndrome with significant health consequences that trigger high burdens of mortality and hospitalization, particularly among those aged 65 and older [2].\nThe COVID-19 pandemic has caused an alteration and restructuration of healthcare systems. The routine of care of HF patients has abruptly changed compared to pre-COVID-19 in terms of an increase in in-person outpatient visits and an increase in telehealth-based programs [3]. Moreover, because government-approved nationwide confinement may have led to a decrease in physical activity (PA), which could also lower well-being, there may be serious consequences for cardiometabolic morbimortality in HF patients. Therefore, COVID-19 represents a serious threat for HF patients, even more in elders with HF [4,5,6]. On the other hand, both confinement and fear of contagion may have reduced social support and assistance for elder patients with HF during COVID-19 confinement [7].\nIn order to mitigate these consequences, promotion of self-management and optimization of clinical assistance of patients with HF, as well as the use of measures such as telehealth, telerehabilitation, mobile applications, and electronic heart rate devices, have been employed [8,9]. Previous trials have found several limitations such as the lack of physical examination, depersonalization of healthcare, and lack of familiarity with the platform [10]. Moreover, there are groups of population that seem not to be ready to use these telematic measures, due to very old age, poor hearing, cognitive dysfunction, language barriers, or limited education, which may require the assistance of a family member or a caregiver [5].\nPrevious quantitative studies have analyzed how confinement affected patients with HF, in terms of fear to visit the hospital [3], differences in physical activity [11,12], mental health [13], safety of telehealth programs [14], etc. Few qualitative studies have been performed in order to characterize the value of technology in supporting caregiving for individuals with HF [15], examine the caregiving experiences and coping strategies of older adults with HF during the ongoing pandemic [16], and explore patients’ and clinicians’ experiences of managing HF during COVID-19 pandemic [17]. However, to our knowledge, no mixed-methods studies have been conducted in this regard. Mix-methods designs offer the possibility for participants to give their opinion about issues that may not have been considered in pre-set quantitative questionnaires. In addition, the literature regarding delivery of health care has highlighted the role of patient-reported outcomes and also of experience measures [10].\nEven though previous studies have been performed in patients with HF, most of them take into account the middle-aged population or, when assessing the elderly, ages range from 60 to 80 years, thus information regarding adults older than 80 years old is scarce [11,13,14,16,18,19]. Therefore, further research is needed to focus on the clinical conditions of the octogenarian population and to analyze the differences between octogenarians and non-octogenarians.\nThis study aimed to (1) compare well-being and PA before and during COVID-19 confinement in older patients with HF; (2) compare well-being and PA during COVID-19 confinement in octogenarians and non-octogenarians with HF; and (3) explore well-being, social support, attention to HF symptoms, assistance needs, and suggestions to improve care during COVID-19 confinement in this population."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"3. Results"},"results_text":{"kind":"string","value":"A total of 120 HF patients were assessed before and during COVID-19 confinement, of whom 60.80% were male and 39.20% were women. Mean (SD) age was 74.16 ± 12.90 years old. The sample consisted of non-octogenarians (<80 years old) (55.83% of the sample) and octogenarians (≥80 years) (44.16 % of the sample). All subjects had a diagnosis of HF, with a mean (SD) time of evolution of 78.73 ± 94.21 months. Table 2 shows the sociodemographic characteristics of the sample.\nTable 3 shows the results of well-being and physical activity before and after confinement. The sample reported significantly lower well-being (p < 0.001) during confinement than before confinement. Similarly, lower levels of light PA (p < 0.001), moderate PA (p < 0.001), and total PA (p < 0.001) were observed during confinement when compared to before confinement. In addition, sedentary time was higher during confinement than before confinement (p < 0.001). However, no significant differences were found in vigorous PA (p > 0.05).\nTwo-factor repeated measures MANOVA revealed significant interaction effects on well-being and PA variables (F(5,114) = 3.33, p = 0.01, η2p = 0.89). Five univariate variables showed non-significant interaction effects for “group by age” and “time measurements”, including light PA, moderate PA, vigorous PA, sedentary time, and total PA (p > 0.05). However, the well-being univariate variable was found to have significant interaction effects for only “group by age” and “time measurements” (F(1,118 = 14.56, p < 0.001, η2p = 0.97). Table 4 shows the comparisons of well-being and physical activity in octogenarians vs. non-octogenarians before and after confinement. Post-hoc analysis showed that before confinement, the levels of light, moderate, vigorous, and total PA, as well as sedentary time were similar between both groups (p > 0.05). However, during confinement, octogenarians showed significantly lower values of well-being (p = 0.02) and moderate PA (p = 0.04), as well as higher sedentary time (p = 0.03) than non-octogenarians. Regarding between-time measurements comparisons, there were significant differences before and during confinement in well-being (p < 0.001, respectively), light PA (p < 0.001, respectively), moderate PA (p = 0.001 and p = 0.02, respectively), sedentary time (p < 0.001, respectively), and total PA (p < 0.001, respectively) in both non-octogenarians and octogenarians. However, there were no significant differences by time measurements in vigorous PA (p > 0.05) in any of the two groups.\nRegarding the qualitative data analysis, five categories were obtained: (i) alterations in well-being; (ii) changes in social support; (iii) attention to HF symptoms; (iv) assistance needs; and (v) suggestions to improve care. The identified categories were divided into 32 subcategories. The identified categories and subcategories are described as follows:(i)Alterations in well-being. This category defines the main areas of well-being affected by COVID-19. It is composed of the following six subcategories: (1) cognitive; (2) the confinement has been positive on his/her well-being; (3) the confinement has not changed his/her routines; (4) emotional; (5) physical; (6) social.(ii)Changes in social support. This category shows the main changes in social support for people with HF during COVID-19. It is composed of six subcategories: (1) receives care from institutions; (2) receives care from his/her relatives; (3) difficulty in communication; (4) no family visits; (5) receives outside assistance; (6) reduced social contact.(iii)Attention to HF symptoms. This category highlights the main characteristics related to the attention to HF symptoms during confinement. It is composed of ten subcategories: (1) self-diagnosis of his/her health condition; (2) avoids social contact; (3) maintains healthy habits; (4) increased dependence; (5) fear of COVID-19; (6) no changes due to COVID-19; (7) does not follow doctor’s treatment; (8) concern for the health of his/her relatives; (9) concern for his/her own health; (10) health problems.(iv)Assistance needs. This category defines the needs of care and highlights the main needs or care requirements of HF patients during confinement. This category is composed of two subcategories and four contexts that define assistance needs: (1) no assistance needs; (2) if had assistance needs: (2a) assistance from family members; (2b) outside assistance (ambulance, telephone assistance, telecare button, caregiver, person for household chores and shopping, cardiac rehabilitation, neighbors); (2c) needs more assistance than received; (2d) total dependency.(v)Suggestions to improve care. This category highlights the suggestions of HF patients in order to improve their care during COVID-19 confinement. It is composed of the following eight subcategories: (1) help from politicians and from institutions; (2) demonstrate care to the patient; (3) increase the availability of physicians; (4) increase efficiency and patient care; (5) no suggestions; (6) speed in caring for patients; (7) receive written feedback from telephone consultations; (8) satisfied with the assistance received.\nAlterations in well-being. This category defines the main areas of well-being affected by COVID-19. It is composed of the following six subcategories: (1) cognitive; (2) the confinement has been positive on his/her well-being; (3) the confinement has not changed his/her routines; (4) emotional; (5) physical; (6) social.\nChanges in social support. This category shows the main changes in social support for people with HF during COVID-19. It is composed of six subcategories: (1) receives care from institutions; (2) receives care from his/her relatives; (3) difficulty in communication; (4) no family visits; (5) receives outside assistance; (6) reduced social contact.\nAttention to HF symptoms. This category highlights the main characteristics related to the attention to HF symptoms during confinement. It is composed of ten subcategories: (1) self-diagnosis of his/her health condition; (2) avoids social contact; (3) maintains healthy habits; (4) increased dependence; (5) fear of COVID-19; (6) no changes due to COVID-19; (7) does not follow doctor’s treatment; (8) concern for the health of his/her relatives; (9) concern for his/her own health; (10) health problems.\nAssistance needs. This category defines the needs of care and highlights the main needs or care requirements of HF patients during confinement. This category is composed of two subcategories and four contexts that define assistance needs: (1) no assistance needs; (2) if had assistance needs: (2a) assistance from family members; (2b) outside assistance (ambulance, telephone assistance, telecare button, caregiver, person for household chores and shopping, cardiac rehabilitation, neighbors); (2c) needs more assistance than received; (2d) total dependency.\nSuggestions to improve care. This category highlights the suggestions of HF patients in order to improve their care during COVID-19 confinement. It is composed of the following eight subcategories: (1) help from politicians and from institutions; (2) demonstrate care to the patient; (3) increase the availability of physicians; (4) increase efficiency and patient care; (5) no suggestions; (6) speed in caring for patients; (7) receive written feedback from telephone consultations; (8) satisfied with the assistance received.\nIn the qualitative results, the most reported subcategories in each analyzed category are described. Findings are shown by verbatim excerpts from the interviews.\nRegarding well-being, the 52.50% of the sample considered that COVID-19 altered their well-being to a great extent. Figure 1 shows differences between octogenarians vs. non-octogenarians in alterations in well-being caused by COVID-19 confinement. Alterations in emotional factors stand out in both groups, according to participant 45: “because it is very scary that people dye because of COVID-19. Our life has changed because we can’t go calmed down the street, nor when we meet family or friends” and participant 5: “I started teleworking and it has been overwhelming because we had more work than ever”. Moreover, physical alterations were highlighted, as participant 3 stated: “regarding physical level, I get more tired when making any effort”. It should be noted that non-octogenarians reported that confinement had less impact on their habits, as indicated by participant 164: “the confinement seemed very good to me because I don´t usually go out and I am with my husband and my daughter, so it is fine for me” and participant 133: “regarding confinement, I could climb stairs as I did before”.\nOn the other hand, 30% of the individuals reported that social support decreased during confinement. Figure 2 shows differences between octogenarians vs. non-octogenarians in changes in social support during COVID-19 confinement. In this regard, the most important change in octogenarians was the increase of care provided by their relatives, as shown by participant 1: “my family members are much more concerned about me and about the fact that I comply with the treatments” and participant 63: “I am living with my daughter, because due to confinement, I needed her help and care and I did not want to be alone”. In contrast, non-octogenarians perceived they did not received care during confinement from physicians, or they did not receive enough information in this regard; according to participant 187: “No doctor has come to my house to give information to me regarding suggestions of care and aspects to take into account related to confinement“ and participant 156: “I have not received any support”.\nIn addition, 50% of the individuals reported that their attention to HF symptoms increased during confinement. Figure 3 shows differences between octogenarians vs. non-octogenarians in attention to HF symptoms during COVID-19 confinement. There was a greater concern for health in non-octogenarians, as shown by participant 101: “I worry more about my health and I don’t want to get worse” and participant 196: “a lot of concern for me and for my wife. We don’t want to get sick, so we take more care of ourselves and pay more attention to our symptoms”. Similarly, there was a greater fear of COVID-19 in non-octogenarians, as indicated by participant 28: “I am scared, I am very afraid of COVID-19 [...] I am a patient at risk” and participant 87: “I pay more attention to my symptoms because of fear and because I have more time”. Although only slightly, octogenarians showed a greater concern for avoiding social contact, as expressed by participant 98: “I avoid contact with friends, even with relatives” and participant 178: “I only go out to the street one day a week and by car”.\nRegarding assistance needs of HF patients, 60% of individuals in the sample were not satisfied with the assistance received. As shown in Figure 4, both groups needed assistance, with a greater predominance in octogenarians, as reported by participant 1: “I have a 24-h caregiver, she runs all my errands, goes shopping, helps me in the bath, checks my pills” and participant 23: “another caregiver to clean the house and go shopping”. On the other hand, non-octogenarians considered that they needed more help than they received, as reported by participant 19: “I need more attention because my legs that are swelling more, and I don’t understand why” and participant 229: “the doctor is more available. The general practitioner does not take care of me, he only gives medication to me, we cannot go to the outpatient clinic”.\nRegarding suggestions to improve care (Figure 5), half of the individuals had no suggestions in this regard. Non-octogenarians suggested the need of offering a greater efficiency and attention to HF patients, as participant 39 assured: “I need to continue with the rehabilitation sessions that were cancelled due to the confinement. The suggestion I make is that those of us who are more dependent and who need more attention should get it. They leave us alone at home as if nothing happened, and that is bad for us. I suggest more attention, that the nurse or physiotherapist comes to check that I am doing well. Self-care starts with good information and making sure that I am doing well, then I will be able to do it alone” and participant 114: “they should call us to follow up our situation, it is hard for me to follow the recommendations”. However, those aged <80 suggested a lower availability of doctors, as suggested by participant 189: “now that I have pain and doctors do not attend me” and participant 98: “maybe if doctors were more available they could solve my doubts”, as well as reporting satisfaction with the assistance they were offered, as indicated by participant 23: “I am happy with the assistance received, and I send encouragement to the health workers” and participant 101: “very competent people, I am very happy with the treatment”.\nFinally, the key points of the qualitative data analysis were that most of the individuals sampled considered that well-being decreased during confinement, 30% reported that social support decreased, 50% increased their attention to symptoms, and 60% were not satisfied with the assistance received. Octogenarians were more affected during lockdown than non-octogenarians in terms of well-being, attention to symptoms, and support needs."},"conclusions_title":{"kind":"string","value":"5. Conclusions"},"conclusions_text":{"kind":"string","value":"Well-being and PA levels decreased during COVID-19 confinement in older adults with HF. Moreover, octogenarians were more severely impacted during confinement than non-octogenarians in terms of lower well-being, lower attention to their HF symptoms, and higher assistance needs, although non-octogenarians reported lower social support than non-octogenarians. Thus, the development of remote monitoring strategies is needed in older adults with HF in order to maintain an adequate level of PA and control health outcomes in critical periods, especially for octogenarians with HF."},"other_sections_titles":{"kind":"list like","value":["2. Materials and Methods","2.2. Design","2.3. Data Collection","2.4. Sample Size Calculation","2.5. Data Analysis"],"string":"[\n \"2. Materials and Methods\",\n \"2.2. Design\",\n \"2.3. Data Collection\",\n \"2.4. Sample Size Calculation\",\n \"2.5. Data Analysis\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":[" 2.1. Participants and Setting The sample consisted of 120 participants. Participants were recruited at an outpatient clinic and were assessed between November 2019 and April 2020. Inclusion criteria included: (1) diagnosis of HF, (2) age ≥ 60 years, and (3) being cognitively capable of completing the assessments. Written informed consent was obtained before participation. The principles of voluntariness and confidentiality were respected. All participants were informed about the objectives and procedures of the study. All procedures were conducted in accordance with the principles of the Declaration of Helsinki. The study was approved by the Institutional Review Board Ethics Committee (2020-440-1).\nThe sample consisted of 120 participants. Participants were recruited at an outpatient clinic and were assessed between November 2019 and April 2020. Inclusion criteria included: (1) diagnosis of HF, (2) age ≥ 60 years, and (3) being cognitively capable of completing the assessments. Written informed consent was obtained before participation. The principles of voluntariness and confidentiality were respected. All participants were informed about the objectives and procedures of the study. All procedures were conducted in accordance with the principles of the Declaration of Helsinki. The study was approved by the Institutional Review Board Ethics Committee (2020-440-1).\n 2.2. Design A mixed concurrent triangulation design (dItrIaC) was carried out according to Creswell et al., Hernández et al., and García et al. [20,21,22]. These authors indicated that this design aims to confirm results, cross-validate results between quantitative and qualitative data, and apply the advantages of each method. Both quantitative and qualitative phases have equal importance in the research. In addition, both methods were applied at the same time, thus data collection and analysis were performed at the same time. Regarding the quantitative phase, a descriptive and cross-sectional design was carried out. Regarding the qualitative phase, a phenomenological design was used, in which the immediate subjective experience was analyzed as the basis of knowledge, whilst phenomena were studied from the perspective of the participants, and their referential framework was preserved. In addition, in the qualitative phase, interest was maintained in knowing how people experience and interpret the social world, which they construct in interaction through language [23].\nA mixed concurrent triangulation design (dItrIaC) was carried out according to Creswell et al., Hernández et al., and García et al. [20,21,22]. These authors indicated that this design aims to confirm results, cross-validate results between quantitative and qualitative data, and apply the advantages of each method. Both quantitative and qualitative phases have equal importance in the research. In addition, both methods were applied at the same time, thus data collection and analysis were performed at the same time. Regarding the quantitative phase, a descriptive and cross-sectional design was carried out. Regarding the qualitative phase, a phenomenological design was used, in which the immediate subjective experience was analyzed as the basis of knowledge, whilst phenomena were studied from the perspective of the participants, and their referential framework was preserved. In addition, in the qualitative phase, interest was maintained in knowing how people experience and interpret the social world, which they construct in interaction through language [23].\n 2.3. Data Collection All participants were included in the quantitative and in the qualitative analysis. Sociodemographic data (age, sex, time since HF diagnosis, education, marital status) were obtained by clinical interview through a trained researcher.\nIn the quantitative phase, the following outcomes were evaluated before and during COVID-19 confinement:(1)Well-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].(2)Physical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\nWell-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].\nPhysical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\nRegarding the qualitative phase, semi-structured interviews were performed. Participants were invited to share information before and during COVID-19 confinement, as well as examples of situations they experienced. Table 1 shows the questions of the semi-structured interview performed.\nAll participants were included in the quantitative and in the qualitative analysis. Sociodemographic data (age, sex, time since HF diagnosis, education, marital status) were obtained by clinical interview through a trained researcher.\nIn the quantitative phase, the following outcomes were evaluated before and during COVID-19 confinement:(1)Well-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].(2)Physical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\nWell-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].\nPhysical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\nRegarding the qualitative phase, semi-structured interviews were performed. Participants were invited to share information before and during COVID-19 confinement, as well as examples of situations they experienced. Table 1 shows the questions of the semi-structured interview performed.\n 2.4. Sample Size Calculation Anticipating medium-sized differences in well-being and PA assessed before and during COVID-19 confinement in older adults with HF, the a priori power analysis (within-between interaction ANOVA) with two independent groups (octogenarians vs. non-octogenarians) and two measurement times (before vs. during COVID-19 confinement) yielded a needed total sample size of 98 participants using the following settings: f = 0.25, alpha = 0.05 (p-value), 1 − beta = 0.80 (power), correlation among repeated measures = 0.50.\nAnticipating medium-sized differences in well-being and PA assessed before and during COVID-19 confinement in older adults with HF, the a priori power analysis (within-between interaction ANOVA) with two independent groups (octogenarians vs. non-octogenarians) and two measurement times (before vs. during COVID-19 confinement) yielded a needed total sample size of 98 participants using the following settings: f = 0.25, alpha = 0.05 (p-value), 1 − beta = 0.80 (power), correlation among repeated measures = 0.50.\n 2.5. Data Analysis The statistical analysis in the quantitative phase was performed using SPSS version 26.0 (IBM SPSS, Inc., Chicago, IL., USA). An ANOVA test was used to explore differences between time measurements (i.e., before and during COVID-19 confinement) in the well-being and PA variables in the entire sample. Additionally, a two-factor mixed multivariate analysis of variance (MANOVA) with a between-subjects factor “groups by age” with two categories (i.e., non-octogenarians (<80 years) and octogenarians (≥80 years)) and a within-subject factor “time measurements” with two categories (i.e., before and during COVID-19 confinement) was performed in the abovementioned variables. Post-hoc analyses were requested using the Bonferroni correction. We evaluated the assumption of homoscedasticity using Levene’s test and the sphericity using Mauchly’s test. Partial eta-squared (η2p) values within the repeated measures ANOVA were used to express the effect size (i.e., small ≥ 0.01, medium ≥ 0.06, large ≥ 0.14). The α level was set equal to or less than 0.05 for all tests.\nThe data analysis in the qualitative phase was supported by computer-assisted qualitative data analysis software (CAQDAS) [33], specifically with the use of NVivo software version 12.0 (QSR International, Inc., Burlington, MA, USA). The treatment of the data followed the classical qualitative data analysis system [34,35]. This model involved the following steps [36]:(1)Data reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].(2)Layout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.(3)Obtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\nData reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].\nLayout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.\nObtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\nThe statistical analysis in the quantitative phase was performed using SPSS version 26.0 (IBM SPSS, Inc., Chicago, IL., USA). An ANOVA test was used to explore differences between time measurements (i.e., before and during COVID-19 confinement) in the well-being and PA variables in the entire sample. Additionally, a two-factor mixed multivariate analysis of variance (MANOVA) with a between-subjects factor “groups by age” with two categories (i.e., non-octogenarians (<80 years) and octogenarians (≥80 years)) and a within-subject factor “time measurements” with two categories (i.e., before and during COVID-19 confinement) was performed in the abovementioned variables. Post-hoc analyses were requested using the Bonferroni correction. We evaluated the assumption of homoscedasticity using Levene’s test and the sphericity using Mauchly’s test. Partial eta-squared (η2p) values within the repeated measures ANOVA were used to express the effect size (i.e., small ≥ 0.01, medium ≥ 0.06, large ≥ 0.14). The α level was set equal to or less than 0.05 for all tests.\nThe data analysis in the qualitative phase was supported by computer-assisted qualitative data analysis software (CAQDAS) [33], specifically with the use of NVivo software version 12.0 (QSR International, Inc., Burlington, MA, USA). The treatment of the data followed the classical qualitative data analysis system [34,35]. This model involved the following steps [36]:(1)Data reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].(2)Layout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.(3)Obtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\nData reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].\nLayout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.\nObtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.","A mixed concurrent triangulation design (dItrIaC) was carried out according to Creswell et al., Hernández et al., and García et al. [20,21,22]. These authors indicated that this design aims to confirm results, cross-validate results between quantitative and qualitative data, and apply the advantages of each method. Both quantitative and qualitative phases have equal importance in the research. In addition, both methods were applied at the same time, thus data collection and analysis were performed at the same time. Regarding the quantitative phase, a descriptive and cross-sectional design was carried out. Regarding the qualitative phase, a phenomenological design was used, in which the immediate subjective experience was analyzed as the basis of knowledge, whilst phenomena were studied from the perspective of the participants, and their referential framework was preserved. In addition, in the qualitative phase, interest was maintained in knowing how people experience and interpret the social world, which they construct in interaction through language [23].","All participants were included in the quantitative and in the qualitative analysis. Sociodemographic data (age, sex, time since HF diagnosis, education, marital status) were obtained by clinical interview through a trained researcher.\nIn the quantitative phase, the following outcomes were evaluated before and during COVID-19 confinement:(1)Well-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].(2)Physical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\nWell-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].\nPhysical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\nRegarding the qualitative phase, semi-structured interviews were performed. Participants were invited to share information before and during COVID-19 confinement, as well as examples of situations they experienced. Table 1 shows the questions of the semi-structured interview performed.","Anticipating medium-sized differences in well-being and PA assessed before and during COVID-19 confinement in older adults with HF, the a priori power analysis (within-between interaction ANOVA) with two independent groups (octogenarians vs. non-octogenarians) and two measurement times (before vs. during COVID-19 confinement) yielded a needed total sample size of 98 participants using the following settings: f = 0.25, alpha = 0.05 (p-value), 1 − beta = 0.80 (power), correlation among repeated measures = 0.50.","The statistical analysis in the quantitative phase was performed using SPSS version 26.0 (IBM SPSS, Inc., Chicago, IL., USA). An ANOVA test was used to explore differences between time measurements (i.e., before and during COVID-19 confinement) in the well-being and PA variables in the entire sample. Additionally, a two-factor mixed multivariate analysis of variance (MANOVA) with a between-subjects factor “groups by age” with two categories (i.e., non-octogenarians (<80 years) and octogenarians (≥80 years)) and a within-subject factor “time measurements” with two categories (i.e., before and during COVID-19 confinement) was performed in the abovementioned variables. Post-hoc analyses were requested using the Bonferroni correction. We evaluated the assumption of homoscedasticity using Levene’s test and the sphericity using Mauchly’s test. Partial eta-squared (η2p) values within the repeated measures ANOVA were used to express the effect size (i.e., small ≥ 0.01, medium ≥ 0.06, large ≥ 0.14). The α level was set equal to or less than 0.05 for all tests.\nThe data analysis in the qualitative phase was supported by computer-assisted qualitative data analysis software (CAQDAS) [33], specifically with the use of NVivo software version 12.0 (QSR International, Inc., Burlington, MA, USA). The treatment of the data followed the classical qualitative data analysis system [34,35]. This model involved the following steps [36]:(1)Data reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].(2)Layout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.(3)Obtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\nData reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].\nLayout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.\nObtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed."],"string":"[\n \" 2.1. Participants and Setting The sample consisted of 120 participants. Participants were recruited at an outpatient clinic and were assessed between November 2019 and April 2020. Inclusion criteria included: (1) diagnosis of HF, (2) age ≥ 60 years, and (3) being cognitively capable of completing the assessments. Written informed consent was obtained before participation. The principles of voluntariness and confidentiality were respected. All participants were informed about the objectives and procedures of the study. All procedures were conducted in accordance with the principles of the Declaration of Helsinki. The study was approved by the Institutional Review Board Ethics Committee (2020-440-1).\\nThe sample consisted of 120 participants. Participants were recruited at an outpatient clinic and were assessed between November 2019 and April 2020. Inclusion criteria included: (1) diagnosis of HF, (2) age ≥ 60 years, and (3) being cognitively capable of completing the assessments. Written informed consent was obtained before participation. The principles of voluntariness and confidentiality were respected. All participants were informed about the objectives and procedures of the study. All procedures were conducted in accordance with the principles of the Declaration of Helsinki. The study was approved by the Institutional Review Board Ethics Committee (2020-440-1).\\n 2.2. Design A mixed concurrent triangulation design (dItrIaC) was carried out according to Creswell et al., Hernández et al., and García et al. [20,21,22]. These authors indicated that this design aims to confirm results, cross-validate results between quantitative and qualitative data, and apply the advantages of each method. Both quantitative and qualitative phases have equal importance in the research. In addition, both methods were applied at the same time, thus data collection and analysis were performed at the same time. Regarding the quantitative phase, a descriptive and cross-sectional design was carried out. Regarding the qualitative phase, a phenomenological design was used, in which the immediate subjective experience was analyzed as the basis of knowledge, whilst phenomena were studied from the perspective of the participants, and their referential framework was preserved. In addition, in the qualitative phase, interest was maintained in knowing how people experience and interpret the social world, which they construct in interaction through language [23].\\nA mixed concurrent triangulation design (dItrIaC) was carried out according to Creswell et al., Hernández et al., and García et al. [20,21,22]. These authors indicated that this design aims to confirm results, cross-validate results between quantitative and qualitative data, and apply the advantages of each method. Both quantitative and qualitative phases have equal importance in the research. In addition, both methods were applied at the same time, thus data collection and analysis were performed at the same time. Regarding the quantitative phase, a descriptive and cross-sectional design was carried out. Regarding the qualitative phase, a phenomenological design was used, in which the immediate subjective experience was analyzed as the basis of knowledge, whilst phenomena were studied from the perspective of the participants, and their referential framework was preserved. In addition, in the qualitative phase, interest was maintained in knowing how people experience and interpret the social world, which they construct in interaction through language [23].\\n 2.3. Data Collection All participants were included in the quantitative and in the qualitative analysis. Sociodemographic data (age, sex, time since HF diagnosis, education, marital status) were obtained by clinical interview through a trained researcher.\\nIn the quantitative phase, the following outcomes were evaluated before and during COVID-19 confinement:(1)Well-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].(2)Physical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\\nWell-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].\\nPhysical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\\nRegarding the qualitative phase, semi-structured interviews were performed. Participants were invited to share information before and during COVID-19 confinement, as well as examples of situations they experienced. Table 1 shows the questions of the semi-structured interview performed.\\nAll participants were included in the quantitative and in the qualitative analysis. Sociodemographic data (age, sex, time since HF diagnosis, education, marital status) were obtained by clinical interview through a trained researcher.\\nIn the quantitative phase, the following outcomes were evaluated before and during COVID-19 confinement:(1)Well-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].(2)Physical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\\nWell-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].\\nPhysical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\\nRegarding the qualitative phase, semi-structured interviews were performed. Participants were invited to share information before and during COVID-19 confinement, as well as examples of situations they experienced. Table 1 shows the questions of the semi-structured interview performed.\\n 2.4. Sample Size Calculation Anticipating medium-sized differences in well-being and PA assessed before and during COVID-19 confinement in older adults with HF, the a priori power analysis (within-between interaction ANOVA) with two independent groups (octogenarians vs. non-octogenarians) and two measurement times (before vs. during COVID-19 confinement) yielded a needed total sample size of 98 participants using the following settings: f = 0.25, alpha = 0.05 (p-value), 1 − beta = 0.80 (power), correlation among repeated measures = 0.50.\\nAnticipating medium-sized differences in well-being and PA assessed before and during COVID-19 confinement in older adults with HF, the a priori power analysis (within-between interaction ANOVA) with two independent groups (octogenarians vs. non-octogenarians) and two measurement times (before vs. during COVID-19 confinement) yielded a needed total sample size of 98 participants using the following settings: f = 0.25, alpha = 0.05 (p-value), 1 − beta = 0.80 (power), correlation among repeated measures = 0.50.\\n 2.5. Data Analysis The statistical analysis in the quantitative phase was performed using SPSS version 26.0 (IBM SPSS, Inc., Chicago, IL., USA). An ANOVA test was used to explore differences between time measurements (i.e., before and during COVID-19 confinement) in the well-being and PA variables in the entire sample. Additionally, a two-factor mixed multivariate analysis of variance (MANOVA) with a between-subjects factor “groups by age” with two categories (i.e., non-octogenarians (<80 years) and octogenarians (≥80 years)) and a within-subject factor “time measurements” with two categories (i.e., before and during COVID-19 confinement) was performed in the abovementioned variables. Post-hoc analyses were requested using the Bonferroni correction. We evaluated the assumption of homoscedasticity using Levene’s test and the sphericity using Mauchly’s test. Partial eta-squared (η2p) values within the repeated measures ANOVA were used to express the effect size (i.e., small ≥ 0.01, medium ≥ 0.06, large ≥ 0.14). The α level was set equal to or less than 0.05 for all tests.\\nThe data analysis in the qualitative phase was supported by computer-assisted qualitative data analysis software (CAQDAS) [33], specifically with the use of NVivo software version 12.0 (QSR International, Inc., Burlington, MA, USA). The treatment of the data followed the classical qualitative data analysis system [34,35]. This model involved the following steps [36]:(1)Data reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].(2)Layout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.(3)Obtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\\nData reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].\\nLayout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.\\nObtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\\nThe statistical analysis in the quantitative phase was performed using SPSS version 26.0 (IBM SPSS, Inc., Chicago, IL., USA). An ANOVA test was used to explore differences between time measurements (i.e., before and during COVID-19 confinement) in the well-being and PA variables in the entire sample. Additionally, a two-factor mixed multivariate analysis of variance (MANOVA) with a between-subjects factor “groups by age” with two categories (i.e., non-octogenarians (<80 years) and octogenarians (≥80 years)) and a within-subject factor “time measurements” with two categories (i.e., before and during COVID-19 confinement) was performed in the abovementioned variables. Post-hoc analyses were requested using the Bonferroni correction. We evaluated the assumption of homoscedasticity using Levene’s test and the sphericity using Mauchly’s test. Partial eta-squared (η2p) values within the repeated measures ANOVA were used to express the effect size (i.e., small ≥ 0.01, medium ≥ 0.06, large ≥ 0.14). The α level was set equal to or less than 0.05 for all tests.\\nThe data analysis in the qualitative phase was supported by computer-assisted qualitative data analysis software (CAQDAS) [33], specifically with the use of NVivo software version 12.0 (QSR International, Inc., Burlington, MA, USA). The treatment of the data followed the classical qualitative data analysis system [34,35]. This model involved the following steps [36]:(1)Data reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].(2)Layout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.(3)Obtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\\nData reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].\\nLayout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.\\nObtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\",\n \"A mixed concurrent triangulation design (dItrIaC) was carried out according to Creswell et al., Hernández et al., and García et al. [20,21,22]. These authors indicated that this design aims to confirm results, cross-validate results between quantitative and qualitative data, and apply the advantages of each method. Both quantitative and qualitative phases have equal importance in the research. In addition, both methods were applied at the same time, thus data collection and analysis were performed at the same time. Regarding the quantitative phase, a descriptive and cross-sectional design was carried out. Regarding the qualitative phase, a phenomenological design was used, in which the immediate subjective experience was analyzed as the basis of knowledge, whilst phenomena were studied from the perspective of the participants, and their referential framework was preserved. In addition, in the qualitative phase, interest was maintained in knowing how people experience and interpret the social world, which they construct in interaction through language [23].\",\n \"All participants were included in the quantitative and in the qualitative analysis. Sociodemographic data (age, sex, time since HF diagnosis, education, marital status) were obtained by clinical interview through a trained researcher.\\nIn the quantitative phase, the following outcomes were evaluated before and during COVID-19 confinement:(1)Well-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].(2)Physical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\\nWell-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].\\nPhysical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\\nRegarding the qualitative phase, semi-structured interviews were performed. Participants were invited to share information before and during COVID-19 confinement, as well as examples of situations they experienced. Table 1 shows the questions of the semi-structured interview performed.\",\n \"Anticipating medium-sized differences in well-being and PA assessed before and during COVID-19 confinement in older adults with HF, the a priori power analysis (within-between interaction ANOVA) with two independent groups (octogenarians vs. non-octogenarians) and two measurement times (before vs. during COVID-19 confinement) yielded a needed total sample size of 98 participants using the following settings: f = 0.25, alpha = 0.05 (p-value), 1 − beta = 0.80 (power), correlation among repeated measures = 0.50.\",\n \"The statistical analysis in the quantitative phase was performed using SPSS version 26.0 (IBM SPSS, Inc., Chicago, IL., USA). An ANOVA test was used to explore differences between time measurements (i.e., before and during COVID-19 confinement) in the well-being and PA variables in the entire sample. Additionally, a two-factor mixed multivariate analysis of variance (MANOVA) with a between-subjects factor “groups by age” with two categories (i.e., non-octogenarians (<80 years) and octogenarians (≥80 years)) and a within-subject factor “time measurements” with two categories (i.e., before and during COVID-19 confinement) was performed in the abovementioned variables. Post-hoc analyses were requested using the Bonferroni correction. We evaluated the assumption of homoscedasticity using Levene’s test and the sphericity using Mauchly’s test. Partial eta-squared (η2p) values within the repeated measures ANOVA were used to express the effect size (i.e., small ≥ 0.01, medium ≥ 0.06, large ≥ 0.14). The α level was set equal to or less than 0.05 for all tests.\\nThe data analysis in the qualitative phase was supported by computer-assisted qualitative data analysis software (CAQDAS) [33], specifically with the use of NVivo software version 12.0 (QSR International, Inc., Burlington, MA, USA). The treatment of the data followed the classical qualitative data analysis system [34,35]. This model involved the following steps [36]:(1)Data reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].(2)Layout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.(3)Obtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\\nData reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].\\nLayout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.\\nObtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["1. Introduction","2. Materials and Methods","2.1. Participants and Setting","2.2. Design","2.3. Data Collection","2.4. Sample Size Calculation","2.5. Data Analysis","3. Results","4. Discussion","5. Conclusions"],"string":"[\n \"1. Introduction\",\n \"2. Materials and Methods\",\n \"2.1. Participants and Setting\",\n \"2.2. Design\",\n \"2.3. Data Collection\",\n \"2.4. Sample Size Calculation\",\n \"2.5. Data Analysis\",\n \"3. Results\",\n \"4. Discussion\",\n \"5. Conclusions\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Heart failure (HF) is a major clinical and public health problem [1]. HF constitutes a complex debilitating syndrome with significant health consequences that trigger high burdens of mortality and hospitalization, particularly among those aged 65 and older [2].\nThe COVID-19 pandemic has caused an alteration and restructuration of healthcare systems. The routine of care of HF patients has abruptly changed compared to pre-COVID-19 in terms of an increase in in-person outpatient visits and an increase in telehealth-based programs [3]. Moreover, because government-approved nationwide confinement may have led to a decrease in physical activity (PA), which could also lower well-being, there may be serious consequences for cardiometabolic morbimortality in HF patients. Therefore, COVID-19 represents a serious threat for HF patients, even more in elders with HF [4,5,6]. On the other hand, both confinement and fear of contagion may have reduced social support and assistance for elder patients with HF during COVID-19 confinement [7].\nIn order to mitigate these consequences, promotion of self-management and optimization of clinical assistance of patients with HF, as well as the use of measures such as telehealth, telerehabilitation, mobile applications, and electronic heart rate devices, have been employed [8,9]. Previous trials have found several limitations such as the lack of physical examination, depersonalization of healthcare, and lack of familiarity with the platform [10]. Moreover, there are groups of population that seem not to be ready to use these telematic measures, due to very old age, poor hearing, cognitive dysfunction, language barriers, or limited education, which may require the assistance of a family member or a caregiver [5].\nPrevious quantitative studies have analyzed how confinement affected patients with HF, in terms of fear to visit the hospital [3], differences in physical activity [11,12], mental health [13], safety of telehealth programs [14], etc. Few qualitative studies have been performed in order to characterize the value of technology in supporting caregiving for individuals with HF [15], examine the caregiving experiences and coping strategies of older adults with HF during the ongoing pandemic [16], and explore patients’ and clinicians’ experiences of managing HF during COVID-19 pandemic [17]. However, to our knowledge, no mixed-methods studies have been conducted in this regard. Mix-methods designs offer the possibility for participants to give their opinion about issues that may not have been considered in pre-set quantitative questionnaires. In addition, the literature regarding delivery of health care has highlighted the role of patient-reported outcomes and also of experience measures [10].\nEven though previous studies have been performed in patients with HF, most of them take into account the middle-aged population or, when assessing the elderly, ages range from 60 to 80 years, thus information regarding adults older than 80 years old is scarce [11,13,14,16,18,19]. Therefore, further research is needed to focus on the clinical conditions of the octogenarian population and to analyze the differences between octogenarians and non-octogenarians.\nThis study aimed to (1) compare well-being and PA before and during COVID-19 confinement in older patients with HF; (2) compare well-being and PA during COVID-19 confinement in octogenarians and non-octogenarians with HF; and (3) explore well-being, social support, attention to HF symptoms, assistance needs, and suggestions to improve care during COVID-19 confinement in this population."," 2.1. Participants and Setting The sample consisted of 120 participants. Participants were recruited at an outpatient clinic and were assessed between November 2019 and April 2020. Inclusion criteria included: (1) diagnosis of HF, (2) age ≥ 60 years, and (3) being cognitively capable of completing the assessments. Written informed consent was obtained before participation. The principles of voluntariness and confidentiality were respected. All participants were informed about the objectives and procedures of the study. All procedures were conducted in accordance with the principles of the Declaration of Helsinki. The study was approved by the Institutional Review Board Ethics Committee (2020-440-1).\nThe sample consisted of 120 participants. Participants were recruited at an outpatient clinic and were assessed between November 2019 and April 2020. Inclusion criteria included: (1) diagnosis of HF, (2) age ≥ 60 years, and (3) being cognitively capable of completing the assessments. Written informed consent was obtained before participation. The principles of voluntariness and confidentiality were respected. All participants were informed about the objectives and procedures of the study. All procedures were conducted in accordance with the principles of the Declaration of Helsinki. The study was approved by the Institutional Review Board Ethics Committee (2020-440-1).\n 2.2. Design A mixed concurrent triangulation design (dItrIaC) was carried out according to Creswell et al., Hernández et al., and García et al. [20,21,22]. These authors indicated that this design aims to confirm results, cross-validate results between quantitative and qualitative data, and apply the advantages of each method. Both quantitative and qualitative phases have equal importance in the research. In addition, both methods were applied at the same time, thus data collection and analysis were performed at the same time. Regarding the quantitative phase, a descriptive and cross-sectional design was carried out. Regarding the qualitative phase, a phenomenological design was used, in which the immediate subjective experience was analyzed as the basis of knowledge, whilst phenomena were studied from the perspective of the participants, and their referential framework was preserved. In addition, in the qualitative phase, interest was maintained in knowing how people experience and interpret the social world, which they construct in interaction through language [23].\nA mixed concurrent triangulation design (dItrIaC) was carried out according to Creswell et al., Hernández et al., and García et al. [20,21,22]. These authors indicated that this design aims to confirm results, cross-validate results between quantitative and qualitative data, and apply the advantages of each method. Both quantitative and qualitative phases have equal importance in the research. In addition, both methods were applied at the same time, thus data collection and analysis were performed at the same time. Regarding the quantitative phase, a descriptive and cross-sectional design was carried out. Regarding the qualitative phase, a phenomenological design was used, in which the immediate subjective experience was analyzed as the basis of knowledge, whilst phenomena were studied from the perspective of the participants, and their referential framework was preserved. In addition, in the qualitative phase, interest was maintained in knowing how people experience and interpret the social world, which they construct in interaction through language [23].\n 2.3. Data Collection All participants were included in the quantitative and in the qualitative analysis. Sociodemographic data (age, sex, time since HF diagnosis, education, marital status) were obtained by clinical interview through a trained researcher.\nIn the quantitative phase, the following outcomes were evaluated before and during COVID-19 confinement:(1)Well-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].(2)Physical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\nWell-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].\nPhysical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\nRegarding the qualitative phase, semi-structured interviews were performed. Participants were invited to share information before and during COVID-19 confinement, as well as examples of situations they experienced. Table 1 shows the questions of the semi-structured interview performed.\nAll participants were included in the quantitative and in the qualitative analysis. Sociodemographic data (age, sex, time since HF diagnosis, education, marital status) were obtained by clinical interview through a trained researcher.\nIn the quantitative phase, the following outcomes were evaluated before and during COVID-19 confinement:(1)Well-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].(2)Physical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\nWell-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].\nPhysical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\nRegarding the qualitative phase, semi-structured interviews were performed. Participants were invited to share information before and during COVID-19 confinement, as well as examples of situations they experienced. Table 1 shows the questions of the semi-structured interview performed.\n 2.4. Sample Size Calculation Anticipating medium-sized differences in well-being and PA assessed before and during COVID-19 confinement in older adults with HF, the a priori power analysis (within-between interaction ANOVA) with two independent groups (octogenarians vs. non-octogenarians) and two measurement times (before vs. during COVID-19 confinement) yielded a needed total sample size of 98 participants using the following settings: f = 0.25, alpha = 0.05 (p-value), 1 − beta = 0.80 (power), correlation among repeated measures = 0.50.\nAnticipating medium-sized differences in well-being and PA assessed before and during COVID-19 confinement in older adults with HF, the a priori power analysis (within-between interaction ANOVA) with two independent groups (octogenarians vs. non-octogenarians) and two measurement times (before vs. during COVID-19 confinement) yielded a needed total sample size of 98 participants using the following settings: f = 0.25, alpha = 0.05 (p-value), 1 − beta = 0.80 (power), correlation among repeated measures = 0.50.\n 2.5. Data Analysis The statistical analysis in the quantitative phase was performed using SPSS version 26.0 (IBM SPSS, Inc., Chicago, IL., USA). An ANOVA test was used to explore differences between time measurements (i.e., before and during COVID-19 confinement) in the well-being and PA variables in the entire sample. Additionally, a two-factor mixed multivariate analysis of variance (MANOVA) with a between-subjects factor “groups by age” with two categories (i.e., non-octogenarians (<80 years) and octogenarians (≥80 years)) and a within-subject factor “time measurements” with two categories (i.e., before and during COVID-19 confinement) was performed in the abovementioned variables. Post-hoc analyses were requested using the Bonferroni correction. We evaluated the assumption of homoscedasticity using Levene’s test and the sphericity using Mauchly’s test. Partial eta-squared (η2p) values within the repeated measures ANOVA were used to express the effect size (i.e., small ≥ 0.01, medium ≥ 0.06, large ≥ 0.14). The α level was set equal to or less than 0.05 for all tests.\nThe data analysis in the qualitative phase was supported by computer-assisted qualitative data analysis software (CAQDAS) [33], specifically with the use of NVivo software version 12.0 (QSR International, Inc., Burlington, MA, USA). The treatment of the data followed the classical qualitative data analysis system [34,35]. This model involved the following steps [36]:(1)Data reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].(2)Layout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.(3)Obtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\nData reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].\nLayout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.\nObtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\nThe statistical analysis in the quantitative phase was performed using SPSS version 26.0 (IBM SPSS, Inc., Chicago, IL., USA). An ANOVA test was used to explore differences between time measurements (i.e., before and during COVID-19 confinement) in the well-being and PA variables in the entire sample. Additionally, a two-factor mixed multivariate analysis of variance (MANOVA) with a between-subjects factor “groups by age” with two categories (i.e., non-octogenarians (<80 years) and octogenarians (≥80 years)) and a within-subject factor “time measurements” with two categories (i.e., before and during COVID-19 confinement) was performed in the abovementioned variables. Post-hoc analyses were requested using the Bonferroni correction. We evaluated the assumption of homoscedasticity using Levene’s test and the sphericity using Mauchly’s test. Partial eta-squared (η2p) values within the repeated measures ANOVA were used to express the effect size (i.e., small ≥ 0.01, medium ≥ 0.06, large ≥ 0.14). The α level was set equal to or less than 0.05 for all tests.\nThe data analysis in the qualitative phase was supported by computer-assisted qualitative data analysis software (CAQDAS) [33], specifically with the use of NVivo software version 12.0 (QSR International, Inc., Burlington, MA, USA). The treatment of the data followed the classical qualitative data analysis system [34,35]. This model involved the following steps [36]:(1)Data reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].(2)Layout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.(3)Obtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\nData reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].\nLayout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.\nObtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.","The sample consisted of 120 participants. Participants were recruited at an outpatient clinic and were assessed between November 2019 and April 2020. Inclusion criteria included: (1) diagnosis of HF, (2) age ≥ 60 years, and (3) being cognitively capable of completing the assessments. Written informed consent was obtained before participation. The principles of voluntariness and confidentiality were respected. All participants were informed about the objectives and procedures of the study. All procedures were conducted in accordance with the principles of the Declaration of Helsinki. The study was approved by the Institutional Review Board Ethics Committee (2020-440-1).","A mixed concurrent triangulation design (dItrIaC) was carried out according to Creswell et al., Hernández et al., and García et al. [20,21,22]. These authors indicated that this design aims to confirm results, cross-validate results between quantitative and qualitative data, and apply the advantages of each method. Both quantitative and qualitative phases have equal importance in the research. In addition, both methods were applied at the same time, thus data collection and analysis were performed at the same time. Regarding the quantitative phase, a descriptive and cross-sectional design was carried out. Regarding the qualitative phase, a phenomenological design was used, in which the immediate subjective experience was analyzed as the basis of knowledge, whilst phenomena were studied from the perspective of the participants, and their referential framework was preserved. In addition, in the qualitative phase, interest was maintained in knowing how people experience and interpret the social world, which they construct in interaction through language [23].","All participants were included in the quantitative and in the qualitative analysis. Sociodemographic data (age, sex, time since HF diagnosis, education, marital status) were obtained by clinical interview through a trained researcher.\nIn the quantitative phase, the following outcomes were evaluated before and during COVID-19 confinement:(1)Well-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].(2)Physical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\nWell-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].\nPhysical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\nRegarding the qualitative phase, semi-structured interviews were performed. Participants were invited to share information before and during COVID-19 confinement, as well as examples of situations they experienced. Table 1 shows the questions of the semi-structured interview performed.","Anticipating medium-sized differences in well-being and PA assessed before and during COVID-19 confinement in older adults with HF, the a priori power analysis (within-between interaction ANOVA) with two independent groups (octogenarians vs. non-octogenarians) and two measurement times (before vs. during COVID-19 confinement) yielded a needed total sample size of 98 participants using the following settings: f = 0.25, alpha = 0.05 (p-value), 1 − beta = 0.80 (power), correlation among repeated measures = 0.50.","The statistical analysis in the quantitative phase was performed using SPSS version 26.0 (IBM SPSS, Inc., Chicago, IL., USA). An ANOVA test was used to explore differences between time measurements (i.e., before and during COVID-19 confinement) in the well-being and PA variables in the entire sample. Additionally, a two-factor mixed multivariate analysis of variance (MANOVA) with a between-subjects factor “groups by age” with two categories (i.e., non-octogenarians (<80 years) and octogenarians (≥80 years)) and a within-subject factor “time measurements” with two categories (i.e., before and during COVID-19 confinement) was performed in the abovementioned variables. Post-hoc analyses were requested using the Bonferroni correction. We evaluated the assumption of homoscedasticity using Levene’s test and the sphericity using Mauchly’s test. Partial eta-squared (η2p) values within the repeated measures ANOVA were used to express the effect size (i.e., small ≥ 0.01, medium ≥ 0.06, large ≥ 0.14). The α level was set equal to or less than 0.05 for all tests.\nThe data analysis in the qualitative phase was supported by computer-assisted qualitative data analysis software (CAQDAS) [33], specifically with the use of NVivo software version 12.0 (QSR International, Inc., Burlington, MA, USA). The treatment of the data followed the classical qualitative data analysis system [34,35]. This model involved the following steps [36]:(1)Data reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].(2)Layout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.(3)Obtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\nData reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].\nLayout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.\nObtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.","A total of 120 HF patients were assessed before and during COVID-19 confinement, of whom 60.80% were male and 39.20% were women. Mean (SD) age was 74.16 ± 12.90 years old. The sample consisted of non-octogenarians (<80 years old) (55.83% of the sample) and octogenarians (≥80 years) (44.16 % of the sample). All subjects had a diagnosis of HF, with a mean (SD) time of evolution of 78.73 ± 94.21 months. Table 2 shows the sociodemographic characteristics of the sample.\nTable 3 shows the results of well-being and physical activity before and after confinement. The sample reported significantly lower well-being (p < 0.001) during confinement than before confinement. Similarly, lower levels of light PA (p < 0.001), moderate PA (p < 0.001), and total PA (p < 0.001) were observed during confinement when compared to before confinement. In addition, sedentary time was higher during confinement than before confinement (p < 0.001). However, no significant differences were found in vigorous PA (p > 0.05).\nTwo-factor repeated measures MANOVA revealed significant interaction effects on well-being and PA variables (F(5,114) = 3.33, p = 0.01, η2p = 0.89). Five univariate variables showed non-significant interaction effects for “group by age” and “time measurements”, including light PA, moderate PA, vigorous PA, sedentary time, and total PA (p > 0.05). However, the well-being univariate variable was found to have significant interaction effects for only “group by age” and “time measurements” (F(1,118 = 14.56, p < 0.001, η2p = 0.97). Table 4 shows the comparisons of well-being and physical activity in octogenarians vs. non-octogenarians before and after confinement. Post-hoc analysis showed that before confinement, the levels of light, moderate, vigorous, and total PA, as well as sedentary time were similar between both groups (p > 0.05). However, during confinement, octogenarians showed significantly lower values of well-being (p = 0.02) and moderate PA (p = 0.04), as well as higher sedentary time (p = 0.03) than non-octogenarians. Regarding between-time measurements comparisons, there were significant differences before and during confinement in well-being (p < 0.001, respectively), light PA (p < 0.001, respectively), moderate PA (p = 0.001 and p = 0.02, respectively), sedentary time (p < 0.001, respectively), and total PA (p < 0.001, respectively) in both non-octogenarians and octogenarians. However, there were no significant differences by time measurements in vigorous PA (p > 0.05) in any of the two groups.\nRegarding the qualitative data analysis, five categories were obtained: (i) alterations in well-being; (ii) changes in social support; (iii) attention to HF symptoms; (iv) assistance needs; and (v) suggestions to improve care. The identified categories were divided into 32 subcategories. The identified categories and subcategories are described as follows:(i)Alterations in well-being. This category defines the main areas of well-being affected by COVID-19. It is composed of the following six subcategories: (1) cognitive; (2) the confinement has been positive on his/her well-being; (3) the confinement has not changed his/her routines; (4) emotional; (5) physical; (6) social.(ii)Changes in social support. This category shows the main changes in social support for people with HF during COVID-19. It is composed of six subcategories: (1) receives care from institutions; (2) receives care from his/her relatives; (3) difficulty in communication; (4) no family visits; (5) receives outside assistance; (6) reduced social contact.(iii)Attention to HF symptoms. This category highlights the main characteristics related to the attention to HF symptoms during confinement. It is composed of ten subcategories: (1) self-diagnosis of his/her health condition; (2) avoids social contact; (3) maintains healthy habits; (4) increased dependence; (5) fear of COVID-19; (6) no changes due to COVID-19; (7) does not follow doctor’s treatment; (8) concern for the health of his/her relatives; (9) concern for his/her own health; (10) health problems.(iv)Assistance needs. This category defines the needs of care and highlights the main needs or care requirements of HF patients during confinement. This category is composed of two subcategories and four contexts that define assistance needs: (1) no assistance needs; (2) if had assistance needs: (2a) assistance from family members; (2b) outside assistance (ambulance, telephone assistance, telecare button, caregiver, person for household chores and shopping, cardiac rehabilitation, neighbors); (2c) needs more assistance than received; (2d) total dependency.(v)Suggestions to improve care. This category highlights the suggestions of HF patients in order to improve their care during COVID-19 confinement. It is composed of the following eight subcategories: (1) help from politicians and from institutions; (2) demonstrate care to the patient; (3) increase the availability of physicians; (4) increase efficiency and patient care; (5) no suggestions; (6) speed in caring for patients; (7) receive written feedback from telephone consultations; (8) satisfied with the assistance received.\nAlterations in well-being. This category defines the main areas of well-being affected by COVID-19. It is composed of the following six subcategories: (1) cognitive; (2) the confinement has been positive on his/her well-being; (3) the confinement has not changed his/her routines; (4) emotional; (5) physical; (6) social.\nChanges in social support. This category shows the main changes in social support for people with HF during COVID-19. It is composed of six subcategories: (1) receives care from institutions; (2) receives care from his/her relatives; (3) difficulty in communication; (4) no family visits; (5) receives outside assistance; (6) reduced social contact.\nAttention to HF symptoms. This category highlights the main characteristics related to the attention to HF symptoms during confinement. It is composed of ten subcategories: (1) self-diagnosis of his/her health condition; (2) avoids social contact; (3) maintains healthy habits; (4) increased dependence; (5) fear of COVID-19; (6) no changes due to COVID-19; (7) does not follow doctor’s treatment; (8) concern for the health of his/her relatives; (9) concern for his/her own health; (10) health problems.\nAssistance needs. This category defines the needs of care and highlights the main needs or care requirements of HF patients during confinement. This category is composed of two subcategories and four contexts that define assistance needs: (1) no assistance needs; (2) if had assistance needs: (2a) assistance from family members; (2b) outside assistance (ambulance, telephone assistance, telecare button, caregiver, person for household chores and shopping, cardiac rehabilitation, neighbors); (2c) needs more assistance than received; (2d) total dependency.\nSuggestions to improve care. This category highlights the suggestions of HF patients in order to improve their care during COVID-19 confinement. It is composed of the following eight subcategories: (1) help from politicians and from institutions; (2) demonstrate care to the patient; (3) increase the availability of physicians; (4) increase efficiency and patient care; (5) no suggestions; (6) speed in caring for patients; (7) receive written feedback from telephone consultations; (8) satisfied with the assistance received.\nIn the qualitative results, the most reported subcategories in each analyzed category are described. Findings are shown by verbatim excerpts from the interviews.\nRegarding well-being, the 52.50% of the sample considered that COVID-19 altered their well-being to a great extent. Figure 1 shows differences between octogenarians vs. non-octogenarians in alterations in well-being caused by COVID-19 confinement. Alterations in emotional factors stand out in both groups, according to participant 45: “because it is very scary that people dye because of COVID-19. Our life has changed because we can’t go calmed down the street, nor when we meet family or friends” and participant 5: “I started teleworking and it has been overwhelming because we had more work than ever”. Moreover, physical alterations were highlighted, as participant 3 stated: “regarding physical level, I get more tired when making any effort”. It should be noted that non-octogenarians reported that confinement had less impact on their habits, as indicated by participant 164: “the confinement seemed very good to me because I don´t usually go out and I am with my husband and my daughter, so it is fine for me” and participant 133: “regarding confinement, I could climb stairs as I did before”.\nOn the other hand, 30% of the individuals reported that social support decreased during confinement. Figure 2 shows differences between octogenarians vs. non-octogenarians in changes in social support during COVID-19 confinement. In this regard, the most important change in octogenarians was the increase of care provided by their relatives, as shown by participant 1: “my family members are much more concerned about me and about the fact that I comply with the treatments” and participant 63: “I am living with my daughter, because due to confinement, I needed her help and care and I did not want to be alone”. In contrast, non-octogenarians perceived they did not received care during confinement from physicians, or they did not receive enough information in this regard; according to participant 187: “No doctor has come to my house to give information to me regarding suggestions of care and aspects to take into account related to confinement“ and participant 156: “I have not received any support”.\nIn addition, 50% of the individuals reported that their attention to HF symptoms increased during confinement. Figure 3 shows differences between octogenarians vs. non-octogenarians in attention to HF symptoms during COVID-19 confinement. There was a greater concern for health in non-octogenarians, as shown by participant 101: “I worry more about my health and I don’t want to get worse” and participant 196: “a lot of concern for me and for my wife. We don’t want to get sick, so we take more care of ourselves and pay more attention to our symptoms”. Similarly, there was a greater fear of COVID-19 in non-octogenarians, as indicated by participant 28: “I am scared, I am very afraid of COVID-19 [...] I am a patient at risk” and participant 87: “I pay more attention to my symptoms because of fear and because I have more time”. Although only slightly, octogenarians showed a greater concern for avoiding social contact, as expressed by participant 98: “I avoid contact with friends, even with relatives” and participant 178: “I only go out to the street one day a week and by car”.\nRegarding assistance needs of HF patients, 60% of individuals in the sample were not satisfied with the assistance received. As shown in Figure 4, both groups needed assistance, with a greater predominance in octogenarians, as reported by participant 1: “I have a 24-h caregiver, she runs all my errands, goes shopping, helps me in the bath, checks my pills” and participant 23: “another caregiver to clean the house and go shopping”. On the other hand, non-octogenarians considered that they needed more help than they received, as reported by participant 19: “I need more attention because my legs that are swelling more, and I don’t understand why” and participant 229: “the doctor is more available. The general practitioner does not take care of me, he only gives medication to me, we cannot go to the outpatient clinic”.\nRegarding suggestions to improve care (Figure 5), half of the individuals had no suggestions in this regard. Non-octogenarians suggested the need of offering a greater efficiency and attention to HF patients, as participant 39 assured: “I need to continue with the rehabilitation sessions that were cancelled due to the confinement. The suggestion I make is that those of us who are more dependent and who need more attention should get it. They leave us alone at home as if nothing happened, and that is bad for us. I suggest more attention, that the nurse or physiotherapist comes to check that I am doing well. Self-care starts with good information and making sure that I am doing well, then I will be able to do it alone” and participant 114: “they should call us to follow up our situation, it is hard for me to follow the recommendations”. However, those aged <80 suggested a lower availability of doctors, as suggested by participant 189: “now that I have pain and doctors do not attend me” and participant 98: “maybe if doctors were more available they could solve my doubts”, as well as reporting satisfaction with the assistance they were offered, as indicated by participant 23: “I am happy with the assistance received, and I send encouragement to the health workers” and participant 101: “very competent people, I am very happy with the treatment”.\nFinally, the key points of the qualitative data analysis were that most of the individuals sampled considered that well-being decreased during confinement, 30% reported that social support decreased, 50% increased their attention to symptoms, and 60% were not satisfied with the assistance received. Octogenarians were more affected during lockdown than non-octogenarians in terms of well-being, attention to symptoms, and support needs.","The findings of this study showed that well-being and PA levels of older adults with HF decreased during COVID-19 confinement. Regarding the comparison between octogenarians and non-octogenarians, octogenarians reported lower well-being, higher sedentary time, and lower levels of moderate PA than non-octogenarians during confinement. Moreover, octogenarians were more severely impacted during confinement than non-octogenarians in terms of exhibiting lower well-being. These results are in accordance with the patient-reported experiences, where octogenarians and non-octogenarians explained that confinement affected their well-being, especially regarding emotional status and physical fitness. Furthermore, octogenarian patients of this study reported that confinement affected several aspects of their daily lives. These results are especially important because previous studies have found that patients with HF are at a higher risk of developing mental health problems and seem to have a lower capacity to enjoy daily activities compared with people without HF symptoms [13]. In addition, in the study of Rantanen et al. [40], people more than 85 years old, both men and women, reported significantly lower quality of life during COVID-19 confinement. In addition, those with lower cognitive functioning, lower emotional stability, and living alone may be at risk of poorer self-reported mental and physical health [41]. Therefore, it is important to stress that nurses and other cardiac providers should identify vulnerabilities in sustained HF self-care behaviors and well-being among older adults, especially among octogenarians, to improve their well-being and physical activity and to improve satisfaction with the care they receive [19].\nFurthermore, the decrease in PA and the increase in sedentary time that we found in older adults with HF during confinement, are in line with the results reported by Vetrovsky et al. [11], Angelo-Brasca et al. [42], and Caraballo et al. [12], who also found a reduction of PA levels in HF patients. It is important to highlight the decrease of PA levels since although physical condition was not evaluated, previous studies suggest that the decrease of PA levels could lead to an important deterioration of physical fitness, which is an important predictor of HF morbidity and mortality in this population [43,44]. In turn, a higher functional status is a predictor of an enhanced quality of life in octogenarians [45]. Therefore, the promotion of active aging seems to be a key point in order to improve or sustain quality of life, especially during social distancing [40].\nA lower attention to their HF symptoms and higher assistance needs were found in octogenarians. However, non-octogenarians reported lower social support than octogenarians. In our sample, it seems that octogenarians reported an increase of care provided by their relatives, which may have influenced their social support; thus, octogenarians presented higher social support than non-octogenarians. In this regard, our results are not in line with those reported by Golden et al. [46], who found that loneliness increased with age. In addition, fear of contagion implied that patients preferred telephone visits, although both groups, especially octogenarians, reported that they needed assistance. These results are similar to those obtained by Mcilvennan et al. [3], who reported patients’ expressions of fear, reluctance to visit the hospital, and lower early symptom reporting. However, our results evidenced that participants were mostly satisfied with the care they received. In contrast, Raman and Vyselaar [10] reported that patients preferred in-person visits since telehealth visits were considered as presenting inferior quality due to the lack of physical examination, emotional detachment from care providers, and general unfamiliarity. Independently of participants´ opinions, telehealth models for outpatients with HF have been demonstrated to be safe and seem not to increase mortality, suggesting that telehealth outpatient visits in patients with HF could be safely incorporated into clinical practice [14,17].\nGiven the lack of studies that use a mixed-methods methodology to investigate well-being, PA, social support, attention to symptoms, assistance needs, and suggestions to improve care in older patients with HF during COVID-19 confinement, this study aimed to fill this gap for a better understanding of this condition. The comparison by age in octogenarians vs. non-octogenarians and the qualitative approach, which involved in-depth interviews with HF patients, led to the collection of relevant data not normally discussed or shared in healthcare research.\nHowever, there are several limitations in this study. First, the study was performed in a single center; therefore, results may not be generalizable to the entire older population with HF. Additionally, the findings may not be representative of all patients, since our results can only be generalized to countries with similar restrictions to those established in Spain, such as Italy, France, or the Czech Republic. Second, the small sample size, although not unusual in qualitative research that requires extensive and detailed analysis of each patient, may not be representative of people with HF in Spain. Nevertheless, further studies should be performed with larger sample sizes. Future studies should also investigate the consequences of this reduction of PA and implement appropriate protocols to ensure good health outcomes in older adults, considering well-being, social support, attention to HF symptoms, and assistance needs during critical periods.","Well-being and PA levels decreased during COVID-19 confinement in older adults with HF. Moreover, octogenarians were more severely impacted during confinement than non-octogenarians in terms of lower well-being, lower attention to their HF symptoms, and higher assistance needs, although non-octogenarians reported lower social support than non-octogenarians. Thus, the development of remote monitoring strategies is needed in older adults with HF in order to maintain an adequate level of PA and control health outcomes in critical periods, especially for octogenarians with HF."],"string":"[\n \"Heart failure (HF) is a major clinical and public health problem [1]. HF constitutes a complex debilitating syndrome with significant health consequences that trigger high burdens of mortality and hospitalization, particularly among those aged 65 and older [2].\\nThe COVID-19 pandemic has caused an alteration and restructuration of healthcare systems. The routine of care of HF patients has abruptly changed compared to pre-COVID-19 in terms of an increase in in-person outpatient visits and an increase in telehealth-based programs [3]. Moreover, because government-approved nationwide confinement may have led to a decrease in physical activity (PA), which could also lower well-being, there may be serious consequences for cardiometabolic morbimortality in HF patients. Therefore, COVID-19 represents a serious threat for HF patients, even more in elders with HF [4,5,6]. On the other hand, both confinement and fear of contagion may have reduced social support and assistance for elder patients with HF during COVID-19 confinement [7].\\nIn order to mitigate these consequences, promotion of self-management and optimization of clinical assistance of patients with HF, as well as the use of measures such as telehealth, telerehabilitation, mobile applications, and electronic heart rate devices, have been employed [8,9]. Previous trials have found several limitations such as the lack of physical examination, depersonalization of healthcare, and lack of familiarity with the platform [10]. Moreover, there are groups of population that seem not to be ready to use these telematic measures, due to very old age, poor hearing, cognitive dysfunction, language barriers, or limited education, which may require the assistance of a family member or a caregiver [5].\\nPrevious quantitative studies have analyzed how confinement affected patients with HF, in terms of fear to visit the hospital [3], differences in physical activity [11,12], mental health [13], safety of telehealth programs [14], etc. Few qualitative studies have been performed in order to characterize the value of technology in supporting caregiving for individuals with HF [15], examine the caregiving experiences and coping strategies of older adults with HF during the ongoing pandemic [16], and explore patients’ and clinicians’ experiences of managing HF during COVID-19 pandemic [17]. However, to our knowledge, no mixed-methods studies have been conducted in this regard. Mix-methods designs offer the possibility for participants to give their opinion about issues that may not have been considered in pre-set quantitative questionnaires. In addition, the literature regarding delivery of health care has highlighted the role of patient-reported outcomes and also of experience measures [10].\\nEven though previous studies have been performed in patients with HF, most of them take into account the middle-aged population or, when assessing the elderly, ages range from 60 to 80 years, thus information regarding adults older than 80 years old is scarce [11,13,14,16,18,19]. Therefore, further research is needed to focus on the clinical conditions of the octogenarian population and to analyze the differences between octogenarians and non-octogenarians.\\nThis study aimed to (1) compare well-being and PA before and during COVID-19 confinement in older patients with HF; (2) compare well-being and PA during COVID-19 confinement in octogenarians and non-octogenarians with HF; and (3) explore well-being, social support, attention to HF symptoms, assistance needs, and suggestions to improve care during COVID-19 confinement in this population.\",\n \" 2.1. Participants and Setting The sample consisted of 120 participants. Participants were recruited at an outpatient clinic and were assessed between November 2019 and April 2020. Inclusion criteria included: (1) diagnosis of HF, (2) age ≥ 60 years, and (3) being cognitively capable of completing the assessments. Written informed consent was obtained before participation. The principles of voluntariness and confidentiality were respected. All participants were informed about the objectives and procedures of the study. All procedures were conducted in accordance with the principles of the Declaration of Helsinki. The study was approved by the Institutional Review Board Ethics Committee (2020-440-1).\\nThe sample consisted of 120 participants. Participants were recruited at an outpatient clinic and were assessed between November 2019 and April 2020. Inclusion criteria included: (1) diagnosis of HF, (2) age ≥ 60 years, and (3) being cognitively capable of completing the assessments. Written informed consent was obtained before participation. The principles of voluntariness and confidentiality were respected. All participants were informed about the objectives and procedures of the study. All procedures were conducted in accordance with the principles of the Declaration of Helsinki. The study was approved by the Institutional Review Board Ethics Committee (2020-440-1).\\n 2.2. Design A mixed concurrent triangulation design (dItrIaC) was carried out according to Creswell et al., Hernández et al., and García et al. [20,21,22]. These authors indicated that this design aims to confirm results, cross-validate results between quantitative and qualitative data, and apply the advantages of each method. Both quantitative and qualitative phases have equal importance in the research. In addition, both methods were applied at the same time, thus data collection and analysis were performed at the same time. Regarding the quantitative phase, a descriptive and cross-sectional design was carried out. Regarding the qualitative phase, a phenomenological design was used, in which the immediate subjective experience was analyzed as the basis of knowledge, whilst phenomena were studied from the perspective of the participants, and their referential framework was preserved. In addition, in the qualitative phase, interest was maintained in knowing how people experience and interpret the social world, which they construct in interaction through language [23].\\nA mixed concurrent triangulation design (dItrIaC) was carried out according to Creswell et al., Hernández et al., and García et al. [20,21,22]. These authors indicated that this design aims to confirm results, cross-validate results between quantitative and qualitative data, and apply the advantages of each method. Both quantitative and qualitative phases have equal importance in the research. In addition, both methods were applied at the same time, thus data collection and analysis were performed at the same time. Regarding the quantitative phase, a descriptive and cross-sectional design was carried out. Regarding the qualitative phase, a phenomenological design was used, in which the immediate subjective experience was analyzed as the basis of knowledge, whilst phenomena were studied from the perspective of the participants, and their referential framework was preserved. In addition, in the qualitative phase, interest was maintained in knowing how people experience and interpret the social world, which they construct in interaction through language [23].\\n 2.3. Data Collection All participants were included in the quantitative and in the qualitative analysis. Sociodemographic data (age, sex, time since HF diagnosis, education, marital status) were obtained by clinical interview through a trained researcher.\\nIn the quantitative phase, the following outcomes were evaluated before and during COVID-19 confinement:(1)Well-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].(2)Physical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\\nWell-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].\\nPhysical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\\nRegarding the qualitative phase, semi-structured interviews were performed. Participants were invited to share information before and during COVID-19 confinement, as well as examples of situations they experienced. Table 1 shows the questions of the semi-structured interview performed.\\nAll participants were included in the quantitative and in the qualitative analysis. Sociodemographic data (age, sex, time since HF diagnosis, education, marital status) were obtained by clinical interview through a trained researcher.\\nIn the quantitative phase, the following outcomes were evaluated before and during COVID-19 confinement:(1)Well-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].(2)Physical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\\nWell-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].\\nPhysical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\\nRegarding the qualitative phase, semi-structured interviews were performed. Participants were invited to share information before and during COVID-19 confinement, as well as examples of situations they experienced. Table 1 shows the questions of the semi-structured interview performed.\\n 2.4. Sample Size Calculation Anticipating medium-sized differences in well-being and PA assessed before and during COVID-19 confinement in older adults with HF, the a priori power analysis (within-between interaction ANOVA) with two independent groups (octogenarians vs. non-octogenarians) and two measurement times (before vs. during COVID-19 confinement) yielded a needed total sample size of 98 participants using the following settings: f = 0.25, alpha = 0.05 (p-value), 1 − beta = 0.80 (power), correlation among repeated measures = 0.50.\\nAnticipating medium-sized differences in well-being and PA assessed before and during COVID-19 confinement in older adults with HF, the a priori power analysis (within-between interaction ANOVA) with two independent groups (octogenarians vs. non-octogenarians) and two measurement times (before vs. during COVID-19 confinement) yielded a needed total sample size of 98 participants using the following settings: f = 0.25, alpha = 0.05 (p-value), 1 − beta = 0.80 (power), correlation among repeated measures = 0.50.\\n 2.5. Data Analysis The statistical analysis in the quantitative phase was performed using SPSS version 26.0 (IBM SPSS, Inc., Chicago, IL., USA). An ANOVA test was used to explore differences between time measurements (i.e., before and during COVID-19 confinement) in the well-being and PA variables in the entire sample. Additionally, a two-factor mixed multivariate analysis of variance (MANOVA) with a between-subjects factor “groups by age” with two categories (i.e., non-octogenarians (<80 years) and octogenarians (≥80 years)) and a within-subject factor “time measurements” with two categories (i.e., before and during COVID-19 confinement) was performed in the abovementioned variables. Post-hoc analyses were requested using the Bonferroni correction. We evaluated the assumption of homoscedasticity using Levene’s test and the sphericity using Mauchly’s test. Partial eta-squared (η2p) values within the repeated measures ANOVA were used to express the effect size (i.e., small ≥ 0.01, medium ≥ 0.06, large ≥ 0.14). The α level was set equal to or less than 0.05 for all tests.\\nThe data analysis in the qualitative phase was supported by computer-assisted qualitative data analysis software (CAQDAS) [33], specifically with the use of NVivo software version 12.0 (QSR International, Inc., Burlington, MA, USA). The treatment of the data followed the classical qualitative data analysis system [34,35]. This model involved the following steps [36]:(1)Data reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].(2)Layout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.(3)Obtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\\nData reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].\\nLayout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.\\nObtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\\nThe statistical analysis in the quantitative phase was performed using SPSS version 26.0 (IBM SPSS, Inc., Chicago, IL., USA). An ANOVA test was used to explore differences between time measurements (i.e., before and during COVID-19 confinement) in the well-being and PA variables in the entire sample. Additionally, a two-factor mixed multivariate analysis of variance (MANOVA) with a between-subjects factor “groups by age” with two categories (i.e., non-octogenarians (<80 years) and octogenarians (≥80 years)) and a within-subject factor “time measurements” with two categories (i.e., before and during COVID-19 confinement) was performed in the abovementioned variables. Post-hoc analyses were requested using the Bonferroni correction. We evaluated the assumption of homoscedasticity using Levene’s test and the sphericity using Mauchly’s test. Partial eta-squared (η2p) values within the repeated measures ANOVA were used to express the effect size (i.e., small ≥ 0.01, medium ≥ 0.06, large ≥ 0.14). The α level was set equal to or less than 0.05 for all tests.\\nThe data analysis in the qualitative phase was supported by computer-assisted qualitative data analysis software (CAQDAS) [33], specifically with the use of NVivo software version 12.0 (QSR International, Inc., Burlington, MA, USA). The treatment of the data followed the classical qualitative data analysis system [34,35]. This model involved the following steps [36]:(1)Data reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].(2)Layout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.(3)Obtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\\nData reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].\\nLayout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.\\nObtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\",\n \"The sample consisted of 120 participants. Participants were recruited at an outpatient clinic and were assessed between November 2019 and April 2020. Inclusion criteria included: (1) diagnosis of HF, (2) age ≥ 60 years, and (3) being cognitively capable of completing the assessments. Written informed consent was obtained before participation. The principles of voluntariness and confidentiality were respected. All participants were informed about the objectives and procedures of the study. All procedures were conducted in accordance with the principles of the Declaration of Helsinki. The study was approved by the Institutional Review Board Ethics Committee (2020-440-1).\",\n \"A mixed concurrent triangulation design (dItrIaC) was carried out according to Creswell et al., Hernández et al., and García et al. [20,21,22]. These authors indicated that this design aims to confirm results, cross-validate results between quantitative and qualitative data, and apply the advantages of each method. Both quantitative and qualitative phases have equal importance in the research. In addition, both methods were applied at the same time, thus data collection and analysis were performed at the same time. Regarding the quantitative phase, a descriptive and cross-sectional design was carried out. Regarding the qualitative phase, a phenomenological design was used, in which the immediate subjective experience was analyzed as the basis of knowledge, whilst phenomena were studied from the perspective of the participants, and their referential framework was preserved. In addition, in the qualitative phase, interest was maintained in knowing how people experience and interpret the social world, which they construct in interaction through language [23].\",\n \"All participants were included in the quantitative and in the qualitative analysis. Sociodemographic data (age, sex, time since HF diagnosis, education, marital status) were obtained by clinical interview through a trained researcher.\\nIn the quantitative phase, the following outcomes were evaluated before and during COVID-19 confinement:(1)Well-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].(2)Physical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\\nWell-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].\\nPhysical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\\nRegarding the qualitative phase, semi-structured interviews were performed. Participants were invited to share information before and during COVID-19 confinement, as well as examples of situations they experienced. Table 1 shows the questions of the semi-structured interview performed.\",\n \"Anticipating medium-sized differences in well-being and PA assessed before and during COVID-19 confinement in older adults with HF, the a priori power analysis (within-between interaction ANOVA) with two independent groups (octogenarians vs. non-octogenarians) and two measurement times (before vs. during COVID-19 confinement) yielded a needed total sample size of 98 participants using the following settings: f = 0.25, alpha = 0.05 (p-value), 1 − beta = 0.80 (power), correlation among repeated measures = 0.50.\",\n \"The statistical analysis in the quantitative phase was performed using SPSS version 26.0 (IBM SPSS, Inc., Chicago, IL., USA). An ANOVA test was used to explore differences between time measurements (i.e., before and during COVID-19 confinement) in the well-being and PA variables in the entire sample. Additionally, a two-factor mixed multivariate analysis of variance (MANOVA) with a between-subjects factor “groups by age” with two categories (i.e., non-octogenarians (<80 years) and octogenarians (≥80 years)) and a within-subject factor “time measurements” with two categories (i.e., before and during COVID-19 confinement) was performed in the abovementioned variables. Post-hoc analyses were requested using the Bonferroni correction. We evaluated the assumption of homoscedasticity using Levene’s test and the sphericity using Mauchly’s test. Partial eta-squared (η2p) values within the repeated measures ANOVA were used to express the effect size (i.e., small ≥ 0.01, medium ≥ 0.06, large ≥ 0.14). The α level was set equal to or less than 0.05 for all tests.\\nThe data analysis in the qualitative phase was supported by computer-assisted qualitative data analysis software (CAQDAS) [33], specifically with the use of NVivo software version 12.0 (QSR International, Inc., Burlington, MA, USA). The treatment of the data followed the classical qualitative data analysis system [34,35]. This model involved the following steps [36]:(1)Data reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].(2)Layout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.(3)Obtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\\nData reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].\\nLayout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.\\nObtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\",\n \"A total of 120 HF patients were assessed before and during COVID-19 confinement, of whom 60.80% were male and 39.20% were women. Mean (SD) age was 74.16 ± 12.90 years old. The sample consisted of non-octogenarians (<80 years old) (55.83% of the sample) and octogenarians (≥80 years) (44.16 % of the sample). All subjects had a diagnosis of HF, with a mean (SD) time of evolution of 78.73 ± 94.21 months. Table 2 shows the sociodemographic characteristics of the sample.\\nTable 3 shows the results of well-being and physical activity before and after confinement. The sample reported significantly lower well-being (p < 0.001) during confinement than before confinement. Similarly, lower levels of light PA (p < 0.001), moderate PA (p < 0.001), and total PA (p < 0.001) were observed during confinement when compared to before confinement. In addition, sedentary time was higher during confinement than before confinement (p < 0.001). However, no significant differences were found in vigorous PA (p > 0.05).\\nTwo-factor repeated measures MANOVA revealed significant interaction effects on well-being and PA variables (F(5,114) = 3.33, p = 0.01, η2p = 0.89). Five univariate variables showed non-significant interaction effects for “group by age” and “time measurements”, including light PA, moderate PA, vigorous PA, sedentary time, and total PA (p > 0.05). However, the well-being univariate variable was found to have significant interaction effects for only “group by age” and “time measurements” (F(1,118 = 14.56, p < 0.001, η2p = 0.97). Table 4 shows the comparisons of well-being and physical activity in octogenarians vs. non-octogenarians before and after confinement. Post-hoc analysis showed that before confinement, the levels of light, moderate, vigorous, and total PA, as well as sedentary time were similar between both groups (p > 0.05). However, during confinement, octogenarians showed significantly lower values of well-being (p = 0.02) and moderate PA (p = 0.04), as well as higher sedentary time (p = 0.03) than non-octogenarians. Regarding between-time measurements comparisons, there were significant differences before and during confinement in well-being (p < 0.001, respectively), light PA (p < 0.001, respectively), moderate PA (p = 0.001 and p = 0.02, respectively), sedentary time (p < 0.001, respectively), and total PA (p < 0.001, respectively) in both non-octogenarians and octogenarians. However, there were no significant differences by time measurements in vigorous PA (p > 0.05) in any of the two groups.\\nRegarding the qualitative data analysis, five categories were obtained: (i) alterations in well-being; (ii) changes in social support; (iii) attention to HF symptoms; (iv) assistance needs; and (v) suggestions to improve care. The identified categories were divided into 32 subcategories. The identified categories and subcategories are described as follows:(i)Alterations in well-being. This category defines the main areas of well-being affected by COVID-19. It is composed of the following six subcategories: (1) cognitive; (2) the confinement has been positive on his/her well-being; (3) the confinement has not changed his/her routines; (4) emotional; (5) physical; (6) social.(ii)Changes in social support. This category shows the main changes in social support for people with HF during COVID-19. It is composed of six subcategories: (1) receives care from institutions; (2) receives care from his/her relatives; (3) difficulty in communication; (4) no family visits; (5) receives outside assistance; (6) reduced social contact.(iii)Attention to HF symptoms. This category highlights the main characteristics related to the attention to HF symptoms during confinement. It is composed of ten subcategories: (1) self-diagnosis of his/her health condition; (2) avoids social contact; (3) maintains healthy habits; (4) increased dependence; (5) fear of COVID-19; (6) no changes due to COVID-19; (7) does not follow doctor’s treatment; (8) concern for the health of his/her relatives; (9) concern for his/her own health; (10) health problems.(iv)Assistance needs. This category defines the needs of care and highlights the main needs or care requirements of HF patients during confinement. This category is composed of two subcategories and four contexts that define assistance needs: (1) no assistance needs; (2) if had assistance needs: (2a) assistance from family members; (2b) outside assistance (ambulance, telephone assistance, telecare button, caregiver, person for household chores and shopping, cardiac rehabilitation, neighbors); (2c) needs more assistance than received; (2d) total dependency.(v)Suggestions to improve care. This category highlights the suggestions of HF patients in order to improve their care during COVID-19 confinement. It is composed of the following eight subcategories: (1) help from politicians and from institutions; (2) demonstrate care to the patient; (3) increase the availability of physicians; (4) increase efficiency and patient care; (5) no suggestions; (6) speed in caring for patients; (7) receive written feedback from telephone consultations; (8) satisfied with the assistance received.\\nAlterations in well-being. This category defines the main areas of well-being affected by COVID-19. It is composed of the following six subcategories: (1) cognitive; (2) the confinement has been positive on his/her well-being; (3) the confinement has not changed his/her routines; (4) emotional; (5) physical; (6) social.\\nChanges in social support. This category shows the main changes in social support for people with HF during COVID-19. It is composed of six subcategories: (1) receives care from institutions; (2) receives care from his/her relatives; (3) difficulty in communication; (4) no family visits; (5) receives outside assistance; (6) reduced social contact.\\nAttention to HF symptoms. This category highlights the main characteristics related to the attention to HF symptoms during confinement. It is composed of ten subcategories: (1) self-diagnosis of his/her health condition; (2) avoids social contact; (3) maintains healthy habits; (4) increased dependence; (5) fear of COVID-19; (6) no changes due to COVID-19; (7) does not follow doctor’s treatment; (8) concern for the health of his/her relatives; (9) concern for his/her own health; (10) health problems.\\nAssistance needs. This category defines the needs of care and highlights the main needs or care requirements of HF patients during confinement. This category is composed of two subcategories and four contexts that define assistance needs: (1) no assistance needs; (2) if had assistance needs: (2a) assistance from family members; (2b) outside assistance (ambulance, telephone assistance, telecare button, caregiver, person for household chores and shopping, cardiac rehabilitation, neighbors); (2c) needs more assistance than received; (2d) total dependency.\\nSuggestions to improve care. This category highlights the suggestions of HF patients in order to improve their care during COVID-19 confinement. It is composed of the following eight subcategories: (1) help from politicians and from institutions; (2) demonstrate care to the patient; (3) increase the availability of physicians; (4) increase efficiency and patient care; (5) no suggestions; (6) speed in caring for patients; (7) receive written feedback from telephone consultations; (8) satisfied with the assistance received.\\nIn the qualitative results, the most reported subcategories in each analyzed category are described. Findings are shown by verbatim excerpts from the interviews.\\nRegarding well-being, the 52.50% of the sample considered that COVID-19 altered their well-being to a great extent. Figure 1 shows differences between octogenarians vs. non-octogenarians in alterations in well-being caused by COVID-19 confinement. Alterations in emotional factors stand out in both groups, according to participant 45: “because it is very scary that people dye because of COVID-19. Our life has changed because we can’t go calmed down the street, nor when we meet family or friends” and participant 5: “I started teleworking and it has been overwhelming because we had more work than ever”. Moreover, physical alterations were highlighted, as participant 3 stated: “regarding physical level, I get more tired when making any effort”. It should be noted that non-octogenarians reported that confinement had less impact on their habits, as indicated by participant 164: “the confinement seemed very good to me because I don´t usually go out and I am with my husband and my daughter, so it is fine for me” and participant 133: “regarding confinement, I could climb stairs as I did before”.\\nOn the other hand, 30% of the individuals reported that social support decreased during confinement. Figure 2 shows differences between octogenarians vs. non-octogenarians in changes in social support during COVID-19 confinement. In this regard, the most important change in octogenarians was the increase of care provided by their relatives, as shown by participant 1: “my family members are much more concerned about me and about the fact that I comply with the treatments” and participant 63: “I am living with my daughter, because due to confinement, I needed her help and care and I did not want to be alone”. In contrast, non-octogenarians perceived they did not received care during confinement from physicians, or they did not receive enough information in this regard; according to participant 187: “No doctor has come to my house to give information to me regarding suggestions of care and aspects to take into account related to confinement“ and participant 156: “I have not received any support”.\\nIn addition, 50% of the individuals reported that their attention to HF symptoms increased during confinement. Figure 3 shows differences between octogenarians vs. non-octogenarians in attention to HF symptoms during COVID-19 confinement. There was a greater concern for health in non-octogenarians, as shown by participant 101: “I worry more about my health and I don’t want to get worse” and participant 196: “a lot of concern for me and for my wife. We don’t want to get sick, so we take more care of ourselves and pay more attention to our symptoms”. Similarly, there was a greater fear of COVID-19 in non-octogenarians, as indicated by participant 28: “I am scared, I am very afraid of COVID-19 [...] I am a patient at risk” and participant 87: “I pay more attention to my symptoms because of fear and because I have more time”. Although only slightly, octogenarians showed a greater concern for avoiding social contact, as expressed by participant 98: “I avoid contact with friends, even with relatives” and participant 178: “I only go out to the street one day a week and by car”.\\nRegarding assistance needs of HF patients, 60% of individuals in the sample were not satisfied with the assistance received. As shown in Figure 4, both groups needed assistance, with a greater predominance in octogenarians, as reported by participant 1: “I have a 24-h caregiver, she runs all my errands, goes shopping, helps me in the bath, checks my pills” and participant 23: “another caregiver to clean the house and go shopping”. On the other hand, non-octogenarians considered that they needed more help than they received, as reported by participant 19: “I need more attention because my legs that are swelling more, and I don’t understand why” and participant 229: “the doctor is more available. The general practitioner does not take care of me, he only gives medication to me, we cannot go to the outpatient clinic”.\\nRegarding suggestions to improve care (Figure 5), half of the individuals had no suggestions in this regard. Non-octogenarians suggested the need of offering a greater efficiency and attention to HF patients, as participant 39 assured: “I need to continue with the rehabilitation sessions that were cancelled due to the confinement. The suggestion I make is that those of us who are more dependent and who need more attention should get it. They leave us alone at home as if nothing happened, and that is bad for us. I suggest more attention, that the nurse or physiotherapist comes to check that I am doing well. Self-care starts with good information and making sure that I am doing well, then I will be able to do it alone” and participant 114: “they should call us to follow up our situation, it is hard for me to follow the recommendations”. However, those aged <80 suggested a lower availability of doctors, as suggested by participant 189: “now that I have pain and doctors do not attend me” and participant 98: “maybe if doctors were more available they could solve my doubts”, as well as reporting satisfaction with the assistance they were offered, as indicated by participant 23: “I am happy with the assistance received, and I send encouragement to the health workers” and participant 101: “very competent people, I am very happy with the treatment”.\\nFinally, the key points of the qualitative data analysis were that most of the individuals sampled considered that well-being decreased during confinement, 30% reported that social support decreased, 50% increased their attention to symptoms, and 60% were not satisfied with the assistance received. Octogenarians were more affected during lockdown than non-octogenarians in terms of well-being, attention to symptoms, and support needs.\",\n \"The findings of this study showed that well-being and PA levels of older adults with HF decreased during COVID-19 confinement. Regarding the comparison between octogenarians and non-octogenarians, octogenarians reported lower well-being, higher sedentary time, and lower levels of moderate PA than non-octogenarians during confinement. Moreover, octogenarians were more severely impacted during confinement than non-octogenarians in terms of exhibiting lower well-being. These results are in accordance with the patient-reported experiences, where octogenarians and non-octogenarians explained that confinement affected their well-being, especially regarding emotional status and physical fitness. Furthermore, octogenarian patients of this study reported that confinement affected several aspects of their daily lives. These results are especially important because previous studies have found that patients with HF are at a higher risk of developing mental health problems and seem to have a lower capacity to enjoy daily activities compared with people without HF symptoms [13]. In addition, in the study of Rantanen et al. [40], people more than 85 years old, both men and women, reported significantly lower quality of life during COVID-19 confinement. In addition, those with lower cognitive functioning, lower emotional stability, and living alone may be at risk of poorer self-reported mental and physical health [41]. Therefore, it is important to stress that nurses and other cardiac providers should identify vulnerabilities in sustained HF self-care behaviors and well-being among older adults, especially among octogenarians, to improve their well-being and physical activity and to improve satisfaction with the care they receive [19].\\nFurthermore, the decrease in PA and the increase in sedentary time that we found in older adults with HF during confinement, are in line with the results reported by Vetrovsky et al. [11], Angelo-Brasca et al. [42], and Caraballo et al. [12], who also found a reduction of PA levels in HF patients. It is important to highlight the decrease of PA levels since although physical condition was not evaluated, previous studies suggest that the decrease of PA levels could lead to an important deterioration of physical fitness, which is an important predictor of HF morbidity and mortality in this population [43,44]. In turn, a higher functional status is a predictor of an enhanced quality of life in octogenarians [45]. Therefore, the promotion of active aging seems to be a key point in order to improve or sustain quality of life, especially during social distancing [40].\\nA lower attention to their HF symptoms and higher assistance needs were found in octogenarians. However, non-octogenarians reported lower social support than octogenarians. In our sample, it seems that octogenarians reported an increase of care provided by their relatives, which may have influenced their social support; thus, octogenarians presented higher social support than non-octogenarians. In this regard, our results are not in line with those reported by Golden et al. [46], who found that loneliness increased with age. In addition, fear of contagion implied that patients preferred telephone visits, although both groups, especially octogenarians, reported that they needed assistance. These results are similar to those obtained by Mcilvennan et al. [3], who reported patients’ expressions of fear, reluctance to visit the hospital, and lower early symptom reporting. However, our results evidenced that participants were mostly satisfied with the care they received. In contrast, Raman and Vyselaar [10] reported that patients preferred in-person visits since telehealth visits were considered as presenting inferior quality due to the lack of physical examination, emotional detachment from care providers, and general unfamiliarity. Independently of participants´ opinions, telehealth models for outpatients with HF have been demonstrated to be safe and seem not to increase mortality, suggesting that telehealth outpatient visits in patients with HF could be safely incorporated into clinical practice [14,17].\\nGiven the lack of studies that use a mixed-methods methodology to investigate well-being, PA, social support, attention to symptoms, assistance needs, and suggestions to improve care in older patients with HF during COVID-19 confinement, this study aimed to fill this gap for a better understanding of this condition. The comparison by age in octogenarians vs. non-octogenarians and the qualitative approach, which involved in-depth interviews with HF patients, led to the collection of relevant data not normally discussed or shared in healthcare research.\\nHowever, there are several limitations in this study. First, the study was performed in a single center; therefore, results may not be generalizable to the entire older population with HF. Additionally, the findings may not be representative of all patients, since our results can only be generalized to countries with similar restrictions to those established in Spain, such as Italy, France, or the Czech Republic. Second, the small sample size, although not unusual in qualitative research that requires extensive and detailed analysis of each patient, may not be representative of people with HF in Spain. Nevertheless, further studies should be performed with larger sample sizes. Future studies should also investigate the consequences of this reduction of PA and implement appropriate protocols to ensure good health outcomes in older adults, considering well-being, social support, attention to HF symptoms, and assistance needs during critical periods.\",\n \"Well-being and PA levels decreased during COVID-19 confinement in older adults with HF. Moreover, octogenarians were more severely impacted during confinement than non-octogenarians in terms of lower well-being, lower attention to their HF symptoms, and higher assistance needs, although non-octogenarians reported lower social support than non-octogenarians. Thus, the development of remote monitoring strategies is needed in older adults with HF in order to maintain an adequate level of PA and control health outcomes in critical periods, especially for octogenarians with HF.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["intro",null,"subjects",null,null,null,null,"results","discussion","conclusions"],"string":"[\n \"intro\",\n null,\n \"subjects\",\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\",\n \"conclusions\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["heart failure","COVID-19","confinement","well-being","physical activity","mixed-methods study"],"string":"[\n \"heart failure\",\n \"COVID-19\",\n \"confinement\",\n \"well-being\",\n \"physical activity\",\n \"mixed-methods study\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"1. Introduction:\nHeart failure (HF) is a major clinical and public health problem [1]. HF constitutes a complex debilitating syndrome with significant health consequences that trigger high burdens of mortality and hospitalization, particularly among those aged 65 and older [2].\nThe COVID-19 pandemic has caused an alteration and restructuration of healthcare systems. The routine of care of HF patients has abruptly changed compared to pre-COVID-19 in terms of an increase in in-person outpatient visits and an increase in telehealth-based programs [3]. Moreover, because government-approved nationwide confinement may have led to a decrease in physical activity (PA), which could also lower well-being, there may be serious consequences for cardiometabolic morbimortality in HF patients. Therefore, COVID-19 represents a serious threat for HF patients, even more in elders with HF [4,5,6]. On the other hand, both confinement and fear of contagion may have reduced social support and assistance for elder patients with HF during COVID-19 confinement [7].\nIn order to mitigate these consequences, promotion of self-management and optimization of clinical assistance of patients with HF, as well as the use of measures such as telehealth, telerehabilitation, mobile applications, and electronic heart rate devices, have been employed [8,9]. Previous trials have found several limitations such as the lack of physical examination, depersonalization of healthcare, and lack of familiarity with the platform [10]. Moreover, there are groups of population that seem not to be ready to use these telematic measures, due to very old age, poor hearing, cognitive dysfunction, language barriers, or limited education, which may require the assistance of a family member or a caregiver [5].\nPrevious quantitative studies have analyzed how confinement affected patients with HF, in terms of fear to visit the hospital [3], differences in physical activity [11,12], mental health [13], safety of telehealth programs [14], etc. Few qualitative studies have been performed in order to characterize the value of technology in supporting caregiving for individuals with HF [15], examine the caregiving experiences and coping strategies of older adults with HF during the ongoing pandemic [16], and explore patients’ and clinicians’ experiences of managing HF during COVID-19 pandemic [17]. However, to our knowledge, no mixed-methods studies have been conducted in this regard. Mix-methods designs offer the possibility for participants to give their opinion about issues that may not have been considered in pre-set quantitative questionnaires. In addition, the literature regarding delivery of health care has highlighted the role of patient-reported outcomes and also of experience measures [10].\nEven though previous studies have been performed in patients with HF, most of them take into account the middle-aged population or, when assessing the elderly, ages range from 60 to 80 years, thus information regarding adults older than 80 years old is scarce [11,13,14,16,18,19]. Therefore, further research is needed to focus on the clinical conditions of the octogenarian population and to analyze the differences between octogenarians and non-octogenarians.\nThis study aimed to (1) compare well-being and PA before and during COVID-19 confinement in older patients with HF; (2) compare well-being and PA during COVID-19 confinement in octogenarians and non-octogenarians with HF; and (3) explore well-being, social support, attention to HF symptoms, assistance needs, and suggestions to improve care during COVID-19 confinement in this population.\n\n2. Materials and Methods:\n 2.1. Participants and Setting The sample consisted of 120 participants. Participants were recruited at an outpatient clinic and were assessed between November 2019 and April 2020. Inclusion criteria included: (1) diagnosis of HF, (2) age ≥ 60 years, and (3) being cognitively capable of completing the assessments. Written informed consent was obtained before participation. The principles of voluntariness and confidentiality were respected. All participants were informed about the objectives and procedures of the study. All procedures were conducted in accordance with the principles of the Declaration of Helsinki. The study was approved by the Institutional Review Board Ethics Committee (2020-440-1).\nThe sample consisted of 120 participants. Participants were recruited at an outpatient clinic and were assessed between November 2019 and April 2020. Inclusion criteria included: (1) diagnosis of HF, (2) age ≥ 60 years, and (3) being cognitively capable of completing the assessments. Written informed consent was obtained before participation. The principles of voluntariness and confidentiality were respected. All participants were informed about the objectives and procedures of the study. All procedures were conducted in accordance with the principles of the Declaration of Helsinki. The study was approved by the Institutional Review Board Ethics Committee (2020-440-1).\n 2.2. Design A mixed concurrent triangulation design (dItrIaC) was carried out according to Creswell et al., Hernández et al., and García et al. [20,21,22]. These authors indicated that this design aims to confirm results, cross-validate results between quantitative and qualitative data, and apply the advantages of each method. Both quantitative and qualitative phases have equal importance in the research. In addition, both methods were applied at the same time, thus data collection and analysis were performed at the same time. Regarding the quantitative phase, a descriptive and cross-sectional design was carried out. Regarding the qualitative phase, a phenomenological design was used, in which the immediate subjective experience was analyzed as the basis of knowledge, whilst phenomena were studied from the perspective of the participants, and their referential framework was preserved. In addition, in the qualitative phase, interest was maintained in knowing how people experience and interpret the social world, which they construct in interaction through language [23].\nA mixed concurrent triangulation design (dItrIaC) was carried out according to Creswell et al., Hernández et al., and García et al. [20,21,22]. These authors indicated that this design aims to confirm results, cross-validate results between quantitative and qualitative data, and apply the advantages of each method. Both quantitative and qualitative phases have equal importance in the research. In addition, both methods were applied at the same time, thus data collection and analysis were performed at the same time. Regarding the quantitative phase, a descriptive and cross-sectional design was carried out. Regarding the qualitative phase, a phenomenological design was used, in which the immediate subjective experience was analyzed as the basis of knowledge, whilst phenomena were studied from the perspective of the participants, and their referential framework was preserved. In addition, in the qualitative phase, interest was maintained in knowing how people experience and interpret the social world, which they construct in interaction through language [23].\n 2.3. Data Collection All participants were included in the quantitative and in the qualitative analysis. Sociodemographic data (age, sex, time since HF diagnosis, education, marital status) were obtained by clinical interview through a trained researcher.\nIn the quantitative phase, the following outcomes were evaluated before and during COVID-19 confinement:(1)Well-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].(2)Physical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\nWell-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].\nPhysical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\nRegarding the qualitative phase, semi-structured interviews were performed. Participants were invited to share information before and during COVID-19 confinement, as well as examples of situations they experienced. Table 1 shows the questions of the semi-structured interview performed.\nAll participants were included in the quantitative and in the qualitative analysis. Sociodemographic data (age, sex, time since HF diagnosis, education, marital status) were obtained by clinical interview through a trained researcher.\nIn the quantitative phase, the following outcomes were evaluated before and during COVID-19 confinement:(1)Well-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].(2)Physical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\nWell-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].\nPhysical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\nRegarding the qualitative phase, semi-structured interviews were performed. Participants were invited to share information before and during COVID-19 confinement, as well as examples of situations they experienced. Table 1 shows the questions of the semi-structured interview performed.\n 2.4. Sample Size Calculation Anticipating medium-sized differences in well-being and PA assessed before and during COVID-19 confinement in older adults with HF, the a priori power analysis (within-between interaction ANOVA) with two independent groups (octogenarians vs. non-octogenarians) and two measurement times (before vs. during COVID-19 confinement) yielded a needed total sample size of 98 participants using the following settings: f = 0.25, alpha = 0.05 (p-value), 1 − beta = 0.80 (power), correlation among repeated measures = 0.50.\nAnticipating medium-sized differences in well-being and PA assessed before and during COVID-19 confinement in older adults with HF, the a priori power analysis (within-between interaction ANOVA) with two independent groups (octogenarians vs. non-octogenarians) and two measurement times (before vs. during COVID-19 confinement) yielded a needed total sample size of 98 participants using the following settings: f = 0.25, alpha = 0.05 (p-value), 1 − beta = 0.80 (power), correlation among repeated measures = 0.50.\n 2.5. Data Analysis The statistical analysis in the quantitative phase was performed using SPSS version 26.0 (IBM SPSS, Inc., Chicago, IL., USA). An ANOVA test was used to explore differences between time measurements (i.e., before and during COVID-19 confinement) in the well-being and PA variables in the entire sample. Additionally, a two-factor mixed multivariate analysis of variance (MANOVA) with a between-subjects factor “groups by age” with two categories (i.e., non-octogenarians (<80 years) and octogenarians (≥80 years)) and a within-subject factor “time measurements” with two categories (i.e., before and during COVID-19 confinement) was performed in the abovementioned variables. Post-hoc analyses were requested using the Bonferroni correction. We evaluated the assumption of homoscedasticity using Levene’s test and the sphericity using Mauchly’s test. Partial eta-squared (η2p) values within the repeated measures ANOVA were used to express the effect size (i.e., small ≥ 0.01, medium ≥ 0.06, large ≥ 0.14). The α level was set equal to or less than 0.05 for all tests.\nThe data analysis in the qualitative phase was supported by computer-assisted qualitative data analysis software (CAQDAS) [33], specifically with the use of NVivo software version 12.0 (QSR International, Inc., Burlington, MA, USA). The treatment of the data followed the classical qualitative data analysis system [34,35]. This model involved the following steps [36]:(1)Data reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].(2)Layout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.(3)Obtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\nData reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].\nLayout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.\nObtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\nThe statistical analysis in the quantitative phase was performed using SPSS version 26.0 (IBM SPSS, Inc., Chicago, IL., USA). An ANOVA test was used to explore differences between time measurements (i.e., before and during COVID-19 confinement) in the well-being and PA variables in the entire sample. Additionally, a two-factor mixed multivariate analysis of variance (MANOVA) with a between-subjects factor “groups by age” with two categories (i.e., non-octogenarians (<80 years) and octogenarians (≥80 years)) and a within-subject factor “time measurements” with two categories (i.e., before and during COVID-19 confinement) was performed in the abovementioned variables. Post-hoc analyses were requested using the Bonferroni correction. We evaluated the assumption of homoscedasticity using Levene’s test and the sphericity using Mauchly’s test. Partial eta-squared (η2p) values within the repeated measures ANOVA were used to express the effect size (i.e., small ≥ 0.01, medium ≥ 0.06, large ≥ 0.14). The α level was set equal to or less than 0.05 for all tests.\nThe data analysis in the qualitative phase was supported by computer-assisted qualitative data analysis software (CAQDAS) [33], specifically with the use of NVivo software version 12.0 (QSR International, Inc., Burlington, MA, USA). The treatment of the data followed the classical qualitative data analysis system [34,35]. This model involved the following steps [36]:(1)Data reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].(2)Layout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.(3)Obtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\nData reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].\nLayout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.\nObtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\n\n2.1. Participants and Setting:\nThe sample consisted of 120 participants. Participants were recruited at an outpatient clinic and were assessed between November 2019 and April 2020. Inclusion criteria included: (1) diagnosis of HF, (2) age ≥ 60 years, and (3) being cognitively capable of completing the assessments. Written informed consent was obtained before participation. The principles of voluntariness and confidentiality were respected. All participants were informed about the objectives and procedures of the study. All procedures were conducted in accordance with the principles of the Declaration of Helsinki. The study was approved by the Institutional Review Board Ethics Committee (2020-440-1).\n\n2.2. Design:\nA mixed concurrent triangulation design (dItrIaC) was carried out according to Creswell et al., Hernández et al., and García et al. [20,21,22]. These authors indicated that this design aims to confirm results, cross-validate results between quantitative and qualitative data, and apply the advantages of each method. Both quantitative and qualitative phases have equal importance in the research. In addition, both methods were applied at the same time, thus data collection and analysis were performed at the same time. Regarding the quantitative phase, a descriptive and cross-sectional design was carried out. Regarding the qualitative phase, a phenomenological design was used, in which the immediate subjective experience was analyzed as the basis of knowledge, whilst phenomena were studied from the perspective of the participants, and their referential framework was preserved. In addition, in the qualitative phase, interest was maintained in knowing how people experience and interpret the social world, which they construct in interaction through language [23].\n\n2.3. Data Collection:\nAll participants were included in the quantitative and in the qualitative analysis. Sociodemographic data (age, sex, time since HF diagnosis, education, marital status) were obtained by clinical interview through a trained researcher.\nIn the quantitative phase, the following outcomes were evaluated before and during COVID-19 confinement:(1)Well-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].(2)Physical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\nWell-being was assessed using the Cantril Ladder of Life [19], a single-item indicator with a ladder of steps numbered from 0 to 10 (0 = the worst possible life, 10 = the best possible life). Participants answered on which step they stand at present. Cantril Ladder of Life validity and test-retest coefficients of 0.70 have been reported in previous studies in patients with acute coronary events. Several studies have previously used this scale in HF patients [24,25,26,27,28].\nPhysical activity was evaluated using the International Physical Activity Questionnaire (IPAQ). It contains seven items for identifying frequency and duration of low, moderate, and vigorous PA as well as inactivity during the past week. Frequency is measured in days and duration in hours and minutes. The answers to the questions were transformed into metabolic equivalent of task (MET-minutes). The total PA score is the sum of vigorous, moderate, and walking PA scores. Typical IPAQ correlations with an accelerometer were 0.80 for reliability [29]. Several studies have previously used this questionnaire in HF patients [30,31,32].\nRegarding the qualitative phase, semi-structured interviews were performed. Participants were invited to share information before and during COVID-19 confinement, as well as examples of situations they experienced. Table 1 shows the questions of the semi-structured interview performed.\n\n2.4. Sample Size Calculation:\nAnticipating medium-sized differences in well-being and PA assessed before and during COVID-19 confinement in older adults with HF, the a priori power analysis (within-between interaction ANOVA) with two independent groups (octogenarians vs. non-octogenarians) and two measurement times (before vs. during COVID-19 confinement) yielded a needed total sample size of 98 participants using the following settings: f = 0.25, alpha = 0.05 (p-value), 1 − beta = 0.80 (power), correlation among repeated measures = 0.50.\n\n2.5. Data Analysis:\nThe statistical analysis in the quantitative phase was performed using SPSS version 26.0 (IBM SPSS, Inc., Chicago, IL., USA). An ANOVA test was used to explore differences between time measurements (i.e., before and during COVID-19 confinement) in the well-being and PA variables in the entire sample. Additionally, a two-factor mixed multivariate analysis of variance (MANOVA) with a between-subjects factor “groups by age” with two categories (i.e., non-octogenarians (<80 years) and octogenarians (≥80 years)) and a within-subject factor “time measurements” with two categories (i.e., before and during COVID-19 confinement) was performed in the abovementioned variables. Post-hoc analyses were requested using the Bonferroni correction. We evaluated the assumption of homoscedasticity using Levene’s test and the sphericity using Mauchly’s test. Partial eta-squared (η2p) values within the repeated measures ANOVA were used to express the effect size (i.e., small ≥ 0.01, medium ≥ 0.06, large ≥ 0.14). The α level was set equal to or less than 0.05 for all tests.\nThe data analysis in the qualitative phase was supported by computer-assisted qualitative data analysis software (CAQDAS) [33], specifically with the use of NVivo software version 12.0 (QSR International, Inc., Burlington, MA, USA). The treatment of the data followed the classical qualitative data analysis system [34,35]. This model involved the following steps [36]:(1)Data reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].(2)Layout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.(3)Obtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\nData reduction. Information was divided into grammatical content units (paragraphs and sentences). Inductive content analysis (elaborating categories from the reading and analysis of the collected material without taking into consideration the initial categories) and deductive content analysis (categories are established a priori whilst the researcher adapts each unit to an already existing category) were performed. The assessment of content belonging to the corresponding category/subcategory was performed based on two levels, intracoder and intercoder, until agreement was reached among the members of the research team [37].\nLayout and grouping. Different graphic resources and information were obtained using CAQDAS as follows: relationships and deep structure of the text [38], graphic representations or visual images of the relationships between concepts [39], and matrices/double-entry tables in which verbal information was included according to the aspects specified by rows and columns [34]. For the calculation of the analysis of the frequency of concurrence of the categories and subcategories, the NVivo coding matrix tool was been used. A matrix was made for each category, taking into account that the subcategories were placed in the rows, whilst the classification of octogenarian/non-octogenarian was placed in the columns. The percentages of each row were calculated based on the total cell references of each subcategory.\nObtention of results and verification of conclusions. This phase involved the use of metaphors and analogies, as well as the inclusion of vignettes and narrative fragments, culminating with the aforementioned triangulation strategies. For textual data, description, interpretation, code counting, concurrence, comparison, and contextualization were performed. For data transformed into numerical values, statistical techniques, comparison, and contextualization were performed.\n\n3. Results:\nA total of 120 HF patients were assessed before and during COVID-19 confinement, of whom 60.80% were male and 39.20% were women. Mean (SD) age was 74.16 ± 12.90 years old. The sample consisted of non-octogenarians (<80 years old) (55.83% of the sample) and octogenarians (≥80 years) (44.16 % of the sample). All subjects had a diagnosis of HF, with a mean (SD) time of evolution of 78.73 ± 94.21 months. Table 2 shows the sociodemographic characteristics of the sample.\nTable 3 shows the results of well-being and physical activity before and after confinement. The sample reported significantly lower well-being (p < 0.001) during confinement than before confinement. Similarly, lower levels of light PA (p < 0.001), moderate PA (p < 0.001), and total PA (p < 0.001) were observed during confinement when compared to before confinement. In addition, sedentary time was higher during confinement than before confinement (p < 0.001). However, no significant differences were found in vigorous PA (p > 0.05).\nTwo-factor repeated measures MANOVA revealed significant interaction effects on well-being and PA variables (F(5,114) = 3.33, p = 0.01, η2p = 0.89). Five univariate variables showed non-significant interaction effects for “group by age” and “time measurements”, including light PA, moderate PA, vigorous PA, sedentary time, and total PA (p > 0.05). However, the well-being univariate variable was found to have significant interaction effects for only “group by age” and “time measurements” (F(1,118 = 14.56, p < 0.001, η2p = 0.97). Table 4 shows the comparisons of well-being and physical activity in octogenarians vs. non-octogenarians before and after confinement. Post-hoc analysis showed that before confinement, the levels of light, moderate, vigorous, and total PA, as well as sedentary time were similar between both groups (p > 0.05). However, during confinement, octogenarians showed significantly lower values of well-being (p = 0.02) and moderate PA (p = 0.04), as well as higher sedentary time (p = 0.03) than non-octogenarians. Regarding between-time measurements comparisons, there were significant differences before and during confinement in well-being (p < 0.001, respectively), light PA (p < 0.001, respectively), moderate PA (p = 0.001 and p = 0.02, respectively), sedentary time (p < 0.001, respectively), and total PA (p < 0.001, respectively) in both non-octogenarians and octogenarians. However, there were no significant differences by time measurements in vigorous PA (p > 0.05) in any of the two groups.\nRegarding the qualitative data analysis, five categories were obtained: (i) alterations in well-being; (ii) changes in social support; (iii) attention to HF symptoms; (iv) assistance needs; and (v) suggestions to improve care. The identified categories were divided into 32 subcategories. The identified categories and subcategories are described as follows:(i)Alterations in well-being. This category defines the main areas of well-being affected by COVID-19. It is composed of the following six subcategories: (1) cognitive; (2) the confinement has been positive on his/her well-being; (3) the confinement has not changed his/her routines; (4) emotional; (5) physical; (6) social.(ii)Changes in social support. This category shows the main changes in social support for people with HF during COVID-19. It is composed of six subcategories: (1) receives care from institutions; (2) receives care from his/her relatives; (3) difficulty in communication; (4) no family visits; (5) receives outside assistance; (6) reduced social contact.(iii)Attention to HF symptoms. This category highlights the main characteristics related to the attention to HF symptoms during confinement. It is composed of ten subcategories: (1) self-diagnosis of his/her health condition; (2) avoids social contact; (3) maintains healthy habits; (4) increased dependence; (5) fear of COVID-19; (6) no changes due to COVID-19; (7) does not follow doctor’s treatment; (8) concern for the health of his/her relatives; (9) concern for his/her own health; (10) health problems.(iv)Assistance needs. This category defines the needs of care and highlights the main needs or care requirements of HF patients during confinement. This category is composed of two subcategories and four contexts that define assistance needs: (1) no assistance needs; (2) if had assistance needs: (2a) assistance from family members; (2b) outside assistance (ambulance, telephone assistance, telecare button, caregiver, person for household chores and shopping, cardiac rehabilitation, neighbors); (2c) needs more assistance than received; (2d) total dependency.(v)Suggestions to improve care. This category highlights the suggestions of HF patients in order to improve their care during COVID-19 confinement. It is composed of the following eight subcategories: (1) help from politicians and from institutions; (2) demonstrate care to the patient; (3) increase the availability of physicians; (4) increase efficiency and patient care; (5) no suggestions; (6) speed in caring for patients; (7) receive written feedback from telephone consultations; (8) satisfied with the assistance received.\nAlterations in well-being. This category defines the main areas of well-being affected by COVID-19. It is composed of the following six subcategories: (1) cognitive; (2) the confinement has been positive on his/her well-being; (3) the confinement has not changed his/her routines; (4) emotional; (5) physical; (6) social.\nChanges in social support. This category shows the main changes in social support for people with HF during COVID-19. It is composed of six subcategories: (1) receives care from institutions; (2) receives care from his/her relatives; (3) difficulty in communication; (4) no family visits; (5) receives outside assistance; (6) reduced social contact.\nAttention to HF symptoms. This category highlights the main characteristics related to the attention to HF symptoms during confinement. It is composed of ten subcategories: (1) self-diagnosis of his/her health condition; (2) avoids social contact; (3) maintains healthy habits; (4) increased dependence; (5) fear of COVID-19; (6) no changes due to COVID-19; (7) does not follow doctor’s treatment; (8) concern for the health of his/her relatives; (9) concern for his/her own health; (10) health problems.\nAssistance needs. This category defines the needs of care and highlights the main needs or care requirements of HF patients during confinement. This category is composed of two subcategories and four contexts that define assistance needs: (1) no assistance needs; (2) if had assistance needs: (2a) assistance from family members; (2b) outside assistance (ambulance, telephone assistance, telecare button, caregiver, person for household chores and shopping, cardiac rehabilitation, neighbors); (2c) needs more assistance than received; (2d) total dependency.\nSuggestions to improve care. This category highlights the suggestions of HF patients in order to improve their care during COVID-19 confinement. It is composed of the following eight subcategories: (1) help from politicians and from institutions; (2) demonstrate care to the patient; (3) increase the availability of physicians; (4) increase efficiency and patient care; (5) no suggestions; (6) speed in caring for patients; (7) receive written feedback from telephone consultations; (8) satisfied with the assistance received.\nIn the qualitative results, the most reported subcategories in each analyzed category are described. Findings are shown by verbatim excerpts from the interviews.\nRegarding well-being, the 52.50% of the sample considered that COVID-19 altered their well-being to a great extent. Figure 1 shows differences between octogenarians vs. non-octogenarians in alterations in well-being caused by COVID-19 confinement. Alterations in emotional factors stand out in both groups, according to participant 45: “because it is very scary that people dye because of COVID-19. Our life has changed because we can’t go calmed down the street, nor when we meet family or friends” and participant 5: “I started teleworking and it has been overwhelming because we had more work than ever”. Moreover, physical alterations were highlighted, as participant 3 stated: “regarding physical level, I get more tired when making any effort”. It should be noted that non-octogenarians reported that confinement had less impact on their habits, as indicated by participant 164: “the confinement seemed very good to me because I don´t usually go out and I am with my husband and my daughter, so it is fine for me” and participant 133: “regarding confinement, I could climb stairs as I did before”.\nOn the other hand, 30% of the individuals reported that social support decreased during confinement. Figure 2 shows differences between octogenarians vs. non-octogenarians in changes in social support during COVID-19 confinement. In this regard, the most important change in octogenarians was the increase of care provided by their relatives, as shown by participant 1: “my family members are much more concerned about me and about the fact that I comply with the treatments” and participant 63: “I am living with my daughter, because due to confinement, I needed her help and care and I did not want to be alone”. In contrast, non-octogenarians perceived they did not received care during confinement from physicians, or they did not receive enough information in this regard; according to participant 187: “No doctor has come to my house to give information to me regarding suggestions of care and aspects to take into account related to confinement“ and participant 156: “I have not received any support”.\nIn addition, 50% of the individuals reported that their attention to HF symptoms increased during confinement. Figure 3 shows differences between octogenarians vs. non-octogenarians in attention to HF symptoms during COVID-19 confinement. There was a greater concern for health in non-octogenarians, as shown by participant 101: “I worry more about my health and I don’t want to get worse” and participant 196: “a lot of concern for me and for my wife. We don’t want to get sick, so we take more care of ourselves and pay more attention to our symptoms”. Similarly, there was a greater fear of COVID-19 in non-octogenarians, as indicated by participant 28: “I am scared, I am very afraid of COVID-19 [...] I am a patient at risk” and participant 87: “I pay more attention to my symptoms because of fear and because I have more time”. Although only slightly, octogenarians showed a greater concern for avoiding social contact, as expressed by participant 98: “I avoid contact with friends, even with relatives” and participant 178: “I only go out to the street one day a week and by car”.\nRegarding assistance needs of HF patients, 60% of individuals in the sample were not satisfied with the assistance received. As shown in Figure 4, both groups needed assistance, with a greater predominance in octogenarians, as reported by participant 1: “I have a 24-h caregiver, she runs all my errands, goes shopping, helps me in the bath, checks my pills” and participant 23: “another caregiver to clean the house and go shopping”. On the other hand, non-octogenarians considered that they needed more help than they received, as reported by participant 19: “I need more attention because my legs that are swelling more, and I don’t understand why” and participant 229: “the doctor is more available. The general practitioner does not take care of me, he only gives medication to me, we cannot go to the outpatient clinic”.\nRegarding suggestions to improve care (Figure 5), half of the individuals had no suggestions in this regard. Non-octogenarians suggested the need of offering a greater efficiency and attention to HF patients, as participant 39 assured: “I need to continue with the rehabilitation sessions that were cancelled due to the confinement. The suggestion I make is that those of us who are more dependent and who need more attention should get it. They leave us alone at home as if nothing happened, and that is bad for us. I suggest more attention, that the nurse or physiotherapist comes to check that I am doing well. Self-care starts with good information and making sure that I am doing well, then I will be able to do it alone” and participant 114: “they should call us to follow up our situation, it is hard for me to follow the recommendations”. However, those aged <80 suggested a lower availability of doctors, as suggested by participant 189: “now that I have pain and doctors do not attend me” and participant 98: “maybe if doctors were more available they could solve my doubts”, as well as reporting satisfaction with the assistance they were offered, as indicated by participant 23: “I am happy with the assistance received, and I send encouragement to the health workers” and participant 101: “very competent people, I am very happy with the treatment”.\nFinally, the key points of the qualitative data analysis were that most of the individuals sampled considered that well-being decreased during confinement, 30% reported that social support decreased, 50% increased their attention to symptoms, and 60% were not satisfied with the assistance received. Octogenarians were more affected during lockdown than non-octogenarians in terms of well-being, attention to symptoms, and support needs.\n\n4. Discussion:\nThe findings of this study showed that well-being and PA levels of older adults with HF decreased during COVID-19 confinement. Regarding the comparison between octogenarians and non-octogenarians, octogenarians reported lower well-being, higher sedentary time, and lower levels of moderate PA than non-octogenarians during confinement. Moreover, octogenarians were more severely impacted during confinement than non-octogenarians in terms of exhibiting lower well-being. These results are in accordance with the patient-reported experiences, where octogenarians and non-octogenarians explained that confinement affected their well-being, especially regarding emotional status and physical fitness. Furthermore, octogenarian patients of this study reported that confinement affected several aspects of their daily lives. These results are especially important because previous studies have found that patients with HF are at a higher risk of developing mental health problems and seem to have a lower capacity to enjoy daily activities compared with people without HF symptoms [13]. In addition, in the study of Rantanen et al. [40], people more than 85 years old, both men and women, reported significantly lower quality of life during COVID-19 confinement. In addition, those with lower cognitive functioning, lower emotional stability, and living alone may be at risk of poorer self-reported mental and physical health [41]. Therefore, it is important to stress that nurses and other cardiac providers should identify vulnerabilities in sustained HF self-care behaviors and well-being among older adults, especially among octogenarians, to improve their well-being and physical activity and to improve satisfaction with the care they receive [19].\nFurthermore, the decrease in PA and the increase in sedentary time that we found in older adults with HF during confinement, are in line with the results reported by Vetrovsky et al. [11], Angelo-Brasca et al. [42], and Caraballo et al. [12], who also found a reduction of PA levels in HF patients. It is important to highlight the decrease of PA levels since although physical condition was not evaluated, previous studies suggest that the decrease of PA levels could lead to an important deterioration of physical fitness, which is an important predictor of HF morbidity and mortality in this population [43,44]. In turn, a higher functional status is a predictor of an enhanced quality of life in octogenarians [45]. Therefore, the promotion of active aging seems to be a key point in order to improve or sustain quality of life, especially during social distancing [40].\nA lower attention to their HF symptoms and higher assistance needs were found in octogenarians. However, non-octogenarians reported lower social support than octogenarians. In our sample, it seems that octogenarians reported an increase of care provided by their relatives, which may have influenced their social support; thus, octogenarians presented higher social support than non-octogenarians. In this regard, our results are not in line with those reported by Golden et al. [46], who found that loneliness increased with age. In addition, fear of contagion implied that patients preferred telephone visits, although both groups, especially octogenarians, reported that they needed assistance. These results are similar to those obtained by Mcilvennan et al. [3], who reported patients’ expressions of fear, reluctance to visit the hospital, and lower early symptom reporting. However, our results evidenced that participants were mostly satisfied with the care they received. In contrast, Raman and Vyselaar [10] reported that patients preferred in-person visits since telehealth visits were considered as presenting inferior quality due to the lack of physical examination, emotional detachment from care providers, and general unfamiliarity. Independently of participants´ opinions, telehealth models for outpatients with HF have been demonstrated to be safe and seem not to increase mortality, suggesting that telehealth outpatient visits in patients with HF could be safely incorporated into clinical practice [14,17].\nGiven the lack of studies that use a mixed-methods methodology to investigate well-being, PA, social support, attention to symptoms, assistance needs, and suggestions to improve care in older patients with HF during COVID-19 confinement, this study aimed to fill this gap for a better understanding of this condition. The comparison by age in octogenarians vs. non-octogenarians and the qualitative approach, which involved in-depth interviews with HF patients, led to the collection of relevant data not normally discussed or shared in healthcare research.\nHowever, there are several limitations in this study. First, the study was performed in a single center; therefore, results may not be generalizable to the entire older population with HF. Additionally, the findings may not be representative of all patients, since our results can only be generalized to countries with similar restrictions to those established in Spain, such as Italy, France, or the Czech Republic. Second, the small sample size, although not unusual in qualitative research that requires extensive and detailed analysis of each patient, may not be representative of people with HF in Spain. Nevertheless, further studies should be performed with larger sample sizes. Future studies should also investigate the consequences of this reduction of PA and implement appropriate protocols to ensure good health outcomes in older adults, considering well-being, social support, attention to HF symptoms, and assistance needs during critical periods.\n\n5. Conclusions:\nWell-being and PA levels decreased during COVID-19 confinement in older adults with HF. Moreover, octogenarians were more severely impacted during confinement than non-octogenarians in terms of lower well-being, lower attention to their HF symptoms, and higher assistance needs, although non-octogenarians reported lower social support than non-octogenarians. Thus, the development of remote monitoring strategies is needed in older adults with HF in order to maintain an adequate level of PA and control health outcomes in critical periods, especially for octogenarians with HF.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThis study aimed to compare well-being and physical activity (PA) before and during COVID-19 confinement in older adults with heart failure (HF), to compare well-being and PA during COVID-19 confinement in octogenarians and non-octogenarians, and to explore well-being, social support, attention to symptoms, and assistance needs during confinement in this population.\n\nMethods:\nA mixed-methods design was performed. Well-being (Cantril Ladder of Life) and PA (International Physical Activity Questionnaire) were assessed. Semi-structured interviews were performed to assess the rest of the variables.\n\nResults:\n120 participants were evaluated (74.16 ± 12.90 years; octogenarians = 44.16%, non-octogenarians = 55.83%). Both groups showed lower well-being and performed less PA during confinement than before (p < 0.001). Octogenarians reported lower well-being (p = 0.02), higher sedentary time (p = 0.03), and lower levels of moderate PA (p = 0.04) during confinement. Most individuals in the sample considered their well-being to have decreased during confinement, 30% reported decreased social support, 50% increased their attention to symptoms, and 60% were not satisfied with the assistance received. Octogenarians were more severely impacted during confinement than non-octogenarians in terms of well-being, attention to symptoms, and assistance needs.\n\nConclusions:\nWell-being and PA decreased during confinement, although octogenarians were more affected than non-octogenarians. Remote monitoring strategies are needed in elders with HF to control health outcomes in critical periods, especially in octogenarians."},"background_conclusion_text":{"kind":"string","value":"1. Introduction:\nHeart failure (HF) is a major clinical and public health problem [1]. HF constitutes a complex debilitating syndrome with significant health consequences that trigger high burdens of mortality and hospitalization, particularly among those aged 65 and older [2].\nThe COVID-19 pandemic has caused an alteration and restructuration of healthcare systems. The routine of care of HF patients has abruptly changed compared to pre-COVID-19 in terms of an increase in in-person outpatient visits and an increase in telehealth-based programs [3]. Moreover, because government-approved nationwide confinement may have led to a decrease in physical activity (PA), which could also lower well-being, there may be serious consequences for cardiometabolic morbimortality in HF patients. Therefore, COVID-19 represents a serious threat for HF patients, even more in elders with HF [4,5,6]. On the other hand, both confinement and fear of contagion may have reduced social support and assistance for elder patients with HF during COVID-19 confinement [7].\nIn order to mitigate these consequences, promotion of self-management and optimization of clinical assistance of patients with HF, as well as the use of measures such as telehealth, telerehabilitation, mobile applications, and electronic heart rate devices, have been employed [8,9]. Previous trials have found several limitations such as the lack of physical examination, depersonalization of healthcare, and lack of familiarity with the platform [10]. Moreover, there are groups of population that seem not to be ready to use these telematic measures, due to very old age, poor hearing, cognitive dysfunction, language barriers, or limited education, which may require the assistance of a family member or a caregiver [5].\nPrevious quantitative studies have analyzed how confinement affected patients with HF, in terms of fear to visit the hospital [3], differences in physical activity [11,12], mental health [13], safety of telehealth programs [14], etc. Few qualitative studies have been performed in order to characterize the value of technology in supporting caregiving for individuals with HF [15], examine the caregiving experiences and coping strategies of older adults with HF during the ongoing pandemic [16], and explore patients’ and clinicians’ experiences of managing HF during COVID-19 pandemic [17]. However, to our knowledge, no mixed-methods studies have been conducted in this regard. Mix-methods designs offer the possibility for participants to give their opinion about issues that may not have been considered in pre-set quantitative questionnaires. In addition, the literature regarding delivery of health care has highlighted the role of patient-reported outcomes and also of experience measures [10].\nEven though previous studies have been performed in patients with HF, most of them take into account the middle-aged population or, when assessing the elderly, ages range from 60 to 80 years, thus information regarding adults older than 80 years old is scarce [11,13,14,16,18,19]. Therefore, further research is needed to focus on the clinical conditions of the octogenarian population and to analyze the differences between octogenarians and non-octogenarians.\nThis study aimed to (1) compare well-being and PA before and during COVID-19 confinement in older patients with HF; (2) compare well-being and PA during COVID-19 confinement in octogenarians and non-octogenarians with HF; and (3) explore well-being, social support, attention to HF symptoms, assistance needs, and suggestions to improve care during COVID-19 confinement in this population.\n\n5. Conclusions:\nWell-being and PA levels decreased during COVID-19 confinement in older adults with HF. Moreover, octogenarians were more severely impacted during confinement than non-octogenarians in terms of lower well-being, lower attention to their HF symptoms, and higher assistance needs, although non-octogenarians reported lower social support than non-octogenarians. Thus, the development of remote monitoring strategies is needed in older adults with HF in order to maintain an adequate level of PA and control health outcomes in critical periods, especially for octogenarians with HF."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nThis study aimed to compare well-being and physical activity (PA) before and during COVID-19 confinement in older adults with heart failure (HF), to compare well-being and PA during COVID-19 confinement in octogenarians and non-octogenarians, and to explore well-being, social support, attention to symptoms, and assistance needs during confinement in this population.\n\nMethods:\nA mixed-methods design was performed. Well-being (Cantril Ladder of Life) and PA (International Physical Activity Questionnaire) were assessed. Semi-structured interviews were performed to assess the rest of the variables.\n\nResults:\n120 participants were evaluated (74.16 ± 12.90 years; octogenarians = 44.16%, non-octogenarians = 55.83%). Both groups showed lower well-being and performed less PA during confinement than before (p < 0.001). Octogenarians reported lower well-being (p = 0.02), higher sedentary time (p = 0.03), and lower levels of moderate PA (p = 0.04) during confinement. Most individuals in the sample considered their well-being to have decreased during confinement, 30% reported decreased social support, 50% increased their attention to symptoms, and 60% were not satisfied with the assistance received. Octogenarians were more severely impacted during confinement than non-octogenarians in terms of well-being, attention to symptoms, and assistance needs.\n\nConclusions:\nWell-being and PA decreased during confinement, although octogenarians were more affected than non-octogenarians. Remote monitoring strategies are needed in elders with HF to control health outcomes in critical periods, especially in octogenarians."},"whole_article_text_length":{"kind":"number","value":10195,"string":"10,195"},"whole_article_abstract_length":{"kind":"number","value":315,"string":"315"},"other_sections_lengths":{"kind":"list like","value":[3736,189,521,100,921],"string":"[\n 3736,\n 189,\n 521,\n 100,\n 921\n]","isConversational":false},"num_sections":{"kind":"number","value":10,"string":"10"},"most_frequent_words":{"kind":"list like","value":["hf","confinement","octogenarians","19","analysis","pa","covid","covid 19","patients","data"],"string":"[\n \"hf\",\n \"confinement\",\n \"octogenarians\",\n \"19\",\n \"analysis\",\n \"pa\",\n \"covid\",\n \"covid 19\",\n \"patients\",\n \"data\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["covid 19 pandemic","covid 19 life","elder patients","older patients hf","confinement older patients"],"string":"[\n \"covid 19 pandemic\",\n \"covid 19 life\",\n \"elder patients\",\n \"older patients hf\",\n \"confinement older patients\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] heart failure | COVID-19 | confinement | well-being | physical activity | mixed-methods study [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] heart failure | COVID-19 | confinement | well-being | physical activity | mixed-methods study [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] heart failure | COVID-19 | confinement | well-being | physical activity | mixed-methods study [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] heart failure | COVID-19 | confinement | well-being | physical activity | mixed-methods study [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] heart failure | COVID-19 | confinement | well-being | physical activity | mixed-methods study [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Aged | Aged, 80 and over | COVID-19 | Social Support | Heart Failure | Exercise | Sedentary Behavior [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Aged | Aged, 80 and over | COVID-19 | Social Support | Heart Failure | Exercise | Sedentary Behavior [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Aged | Aged, 80 and over | COVID-19 | Social Support | Heart Failure | Exercise | Sedentary Behavior [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Aged | Aged, 80 and over | COVID-19 | Social Support | Heart Failure | Exercise | Sedentary Behavior [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Aged | Aged, 80 and over | COVID-19 | Social Support | Heart Failure | Exercise | Sedentary Behavior [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] covid 19 pandemic | covid 19 life | elder patients | older patients hf | confinement older patients [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] covid 19 pandemic | covid 19 life | elder patients | older patients hf | confinement older patients [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] covid 19 pandemic | covid 19 life | elder patients | older patients hf | confinement older patients [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] covid 19 pandemic | covid 19 life | elder patients | older patients hf | confinement older patients [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] covid 19 pandemic | covid 19 life | elder patients | older patients hf | confinement older patients [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hf | confinement | octogenarians | 19 | analysis | pa | covid | covid 19 | patients | data [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] hf | confinement | octogenarians | 19 | analysis | pa | covid | covid 19 | patients | data [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] hf | confinement | octogenarians | 19 | analysis | pa | covid | covid 19 | patients | data [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hf | confinement | octogenarians | 19 | analysis | pa | covid | covid 19 | patients | data [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] hf | confinement | octogenarians | 19 | analysis | pa | covid | covid 19 | patients | data [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hf | patients | patients hf | 19 | covid 19 | covid | population | confinement | pandemic | assistance [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] participant | care | assistance | confinement | octogenarians | needs | 001 | composed | attention | subcategories [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] octogenarians | lower | hf | non octogenarians | non | adults hf | adults | older | older adults | older adults hf [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hf | octogenarians | confinement | patients | 19 | pa | covid | covid 19 | non | assistance [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] hf | octogenarians | confinement | patients | 19 | pa | covid | covid 19 | non | assistance [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 | COVID-19 | octogenarians [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 120 | 74.16 | 12.90 years | 44.16% | 55.83% ||| p < | 0.001 ||| Octogenarians | 0.02 | 0.03 | PA | 0.04 ||| 30% | 50% | 60% ||| Octogenarians [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| HF [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 | COVID-19 | octogenarians ||| ||| ||| ||| 120 | 74.16 | 12.90 years | 44.16% | 55.83% ||| p < | 0.001 ||| Octogenarians | 0.02 | 0.03 | PA | 0.04 ||| 30% | 50% | 60% ||| Octogenarians ||| ||| HF [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] COVID-19 | COVID-19 | octogenarians ||| ||| ||| ||| 120 | 74.16 | 12.90 years | 44.16% | 55.83% ||| p < | 0.001 ||| Octogenarians | 0.02 | 0.03 | PA | 0.04 ||| 30% | 50% | 60% ||| Octogenarians ||| ||| HF [SUMMARY]"}}},{"rowIdx":59,"cells":{"title":{"kind":"string","value":"Pressure Overload-induced Cardiac Hypertrophy Varies According to Different Ligation Needle Sizes and Body Weights in Mice."},"pmid":{"kind":"string","value":"30226916"},"background_abstract":{"kind":"string","value":"The cardiac hypertrophy (CH) model for mice has been widely used, thereby providing an effective research foundation for CH exploration."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Four needles with different external diameters (0.35, 0.40, 0.45, and 0.50 mm) were used for AAC. 150 male C57BL/6 mice were selected according to body weight (BW) and divided into 3 weight levels: 18 g, 22 g, and 26 g (n = 50 in each group). All weight levels were divided into 5 groups: a sham group (n = 10) and 4 AAC groups using 4 ligation intensities (n = 10 per group). After surgery, survival rates were recorded, echocardiography was performed, hearts were dissected and used for histological detection, and data were statistically analyzed, P < 0.05 was considered statistically significant."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"All mice died in the following AAC groups: 18g/0.35 mm, 22 g/0.35 mm, 26 g/0.35 mm, 22 g/0.40 mm, and 26 g/0.40 mm. All mice with AAC, those ligated with a 0.50-mm needle, and those that underwent sham operation survived. Different death rates occurred in the following AAC groups: 18 g/0.40 mm, 18 g/0.45 mm, 18 g/0.50 mm, 22 g/45 mm, 22 g/0.50 mm, 26 g/0.45 mm, and 26 g/0.50 mm. The heart weight/body weight ratios (5.39 ± 0.85, 6.41 ± 0.68, 4.67 ± 0.37, 5.22 ± 0.42, 4.23 ± 0.28, 5.41 ± 0.14, and 4.02 ± 0.13) were significantly increased compared with those of the sham groups for mice with the same weight levels."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"A 0.45-mm needle led to more obvious CH than did 0.40-mm and 0.50-mm needles and caused extraordinary CH in 18-g mice."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Animals","Aorta, Abdominal","Body Weight","Cardiomegaly","Constriction","Disease Models, Animal","Echocardiography","Ligation","Male","Mice, Inbred C57BL","Needles","Random Allocation","Reference Values","Reproducibility of Results","Time Factors"],"string":"[\n \"Animals\",\n \"Aorta, Abdominal\",\n \"Body Weight\",\n \"Cardiomegaly\",\n \"Constriction\",\n \"Disease Models, Animal\",\n \"Echocardiography\",\n \"Ligation\",\n \"Male\",\n \"Mice, Inbred C57BL\",\n \"Needles\",\n \"Random Allocation\",\n \"Reference Values\",\n \"Reproducibility of Results\",\n \"Time Factors\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"6023638"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Cardiac hypertrophy (CH) is a compensatory pathological change that is usually\ninduced by pressure overload (PO), neurohumoral abnormality, and the effects of\ncytokines. It is characterized by cardiomyocyte hypertrophy and interstitial\nhyperplasia, and it results in an enlarged heart and thickening of the heart walls.\nClinically, CH is involved in the development of many diseases, such as valvular\ndisease, hypertension, arterial stenosis, and primary myocardial hypertrophy. If\nthese diseases develop at their own pace, then cardiac function (CF) will gradually\ndecompensate, leading to heart failure (HF), which severely lowers the quality of\nlife and increases the mortality rate. Therefore, CH is a widespread concern and has\nbeen explored at the molecular level by researchers. Due to the high genomic\nhomology between mice and humans, an established CH model for mice has been widely\nused in animal experiments, thereby providing an effective research foundation for\nCH exploration.\nCurrently, PO-induced CH is a common way to establish the model. Abdominal aortic\nconstriction (AAC) is highly recommended by researchers because of the high success\nrate and the ability to perform surgery without the need for thoracotomy or a\nventilator. However, the modeling effects with different ligating intensities for\ncertain body weights (BWs) have not yet been reported. Therefore, we used 3\nfrequently used mice BWs (18 g, 22 g, and 26 g) and 4 different needle sizes (0.35,\n0.40, 0.45, and 0.50 mm) to establish the CH model for each weight level for AAC,\nsummarized the survival rates, and evaluated the CH effects."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Animal groups and handling One-hundred fifty male C57BL/6 wild-type mice were obtained from the Shanghai\nSLAC Laboratory Animal Co. Ltd (Shanghai, China). All animals were treated and\ncared for in accordance with the Guide for the Care and Use of\nLaboratory Animals (National Institutes of Health, Washington, DC,\n1996). Experimental protocols were approved by our Institutional Animal Care and\nUse Committee of Zhejiang University (Hangzhou, China). Mice were selected\naccording to weights of approximately 18 g (range, 17.3-18.7 g), 22 g (range,\n20.8-23.0 g), and 26 g (range, 25.1-27.0 g), and they were divided into the\nfollowing 3 weight levels: 18 g (18.0 ± 0.3 g; n = 50), 22 g (22.0\n± 0.6 g; n = 50), and 26 g (26.1 ± 0.5 g; n = 50). All weight\nlevels were divided using sortition randomization method to create a sham group\n(n = 10) and 4 AAC groups according to ligating intensities (0.35, 0.40, 0.45,\nand 0.50mm; n = 10 per group). Regarding BW, no significant differences were\nfound among the 5 groups for each weight level (Table S1), and the preoperative BWs of mice that died and those that\nsurvived were not significant (Table\nS2).\nMice were anesthetized with 4% chloralhydrate (0.1ml/1g BW, intraperitoneal\ninjection). When the mice did not respond when their toe was pinched, the limbs\nwere fixed on the operating board in the supine position and the skin was\nprepared by shaving and disinfection with alcohol. Sterile gauze was placed on\nthe right side of the abdomen and a ventrimesal incision approximately 1.5 cm\nwas created starting from the xiphoid. The skin was fixed with a spreader and\nthe viscera was pulled out gently with a swab and placed on the gauze. Then, the\nabdominal aorta was isolated using a blunt dissection technique with curved\nmicroforceps under a microscope. A 6-0 silk suture was snared and pulled back\naround the aorta 1mm above the superior mesenteric artery. A 2-mm blunt\nacupuncture needle (external diameters: 0.35 mm, 0.40 mm, 0.45 mm, and 0.50 mm;\nHuatuo; Suzhou Medical Appliance Factory, Suzhou, China; criterion number\nGB2024-1994) was then placed next to the aorta. The suture was tied snugly\naround the needle and the aorta. The needle was removed immediately after\nligation, the viscera were replaced, the peritoneum and skin were sutured, and\nthe mice were allowed to recover. Aortic ligation was omitted only for the sham\ngroup. After surgery, the ears were cut to differentiate the mice. Then, mice\nwere placed in an incubator at 30ºC until they woke, and they were\nreturned to their cages. Survival status was recorded daily. To observe the\nphysical development of mice under different conditions, BW differences before\nsurgery and at week 8 post-surgery were calculated as the change in BW.\nOne-hundred fifty male C57BL/6 wild-type mice were obtained from the Shanghai\nSLAC Laboratory Animal Co. Ltd (Shanghai, China). All animals were treated and\ncared for in accordance with the Guide for the Care and Use of\nLaboratory Animals (National Institutes of Health, Washington, DC,\n1996). Experimental protocols were approved by our Institutional Animal Care and\nUse Committee of Zhejiang University (Hangzhou, China). Mice were selected\naccording to weights of approximately 18 g (range, 17.3-18.7 g), 22 g (range,\n20.8-23.0 g), and 26 g (range, 25.1-27.0 g), and they were divided into the\nfollowing 3 weight levels: 18 g (18.0 ± 0.3 g; n = 50), 22 g (22.0\n± 0.6 g; n = 50), and 26 g (26.1 ± 0.5 g; n = 50). All weight\nlevels were divided using sortition randomization method to create a sham group\n(n = 10) and 4 AAC groups according to ligating intensities (0.35, 0.40, 0.45,\nand 0.50mm; n = 10 per group). Regarding BW, no significant differences were\nfound among the 5 groups for each weight level (Table S1), and the preoperative BWs of mice that died and those that\nsurvived were not significant (Table\nS2).\nMice were anesthetized with 4% chloralhydrate (0.1ml/1g BW, intraperitoneal\ninjection). When the mice did not respond when their toe was pinched, the limbs\nwere fixed on the operating board in the supine position and the skin was\nprepared by shaving and disinfection with alcohol. Sterile gauze was placed on\nthe right side of the abdomen and a ventrimesal incision approximately 1.5 cm\nwas created starting from the xiphoid. The skin was fixed with a spreader and\nthe viscera was pulled out gently with a swab and placed on the gauze. Then, the\nabdominal aorta was isolated using a blunt dissection technique with curved\nmicroforceps under a microscope. A 6-0 silk suture was snared and pulled back\naround the aorta 1mm above the superior mesenteric artery. A 2-mm blunt\nacupuncture needle (external diameters: 0.35 mm, 0.40 mm, 0.45 mm, and 0.50 mm;\nHuatuo; Suzhou Medical Appliance Factory, Suzhou, China; criterion number\nGB2024-1994) was then placed next to the aorta. The suture was tied snugly\naround the needle and the aorta. The needle was removed immediately after\nligation, the viscera were replaced, the peritoneum and skin were sutured, and\nthe mice were allowed to recover. Aortic ligation was omitted only for the sham\ngroup. After surgery, the ears were cut to differentiate the mice. Then, mice\nwere placed in an incubator at 30ºC until they woke, and they were\nreturned to their cages. Survival status was recorded daily. To observe the\nphysical development of mice under different conditions, BW differences before\nsurgery and at week 8 post-surgery were calculated as the change in BW.\n Echocardiography imaging After post-surgery weeks 4 and 8, mice were weighed and anesthetized with 4%\nchloralhydrate and placed on a warming pad after skin preparation. Transthoracic\n2-dimensional (2D) echocardiography was performed using the GE Vivid E9\nUltrasound echocardiographic system (General Electric Company, Fairfield, CT,\nUSA) with the GE 9L probe (8-MHz linear array transducer; General Electric\nCompany). M-mode parasternal long-axis scans of the left ventricle at the mitral\nchordae level were used to quantify the interventricular septum thickness at\nend-diastole (IVSd), interventricular septum thickness at end-systole (IVSs),\nleft ventricular internal dimension at end-diastole (LVIDd), left ventricular\ninternal dimension at end-systole (LVIDs), left ventricular posterior wall\nthickness at end-diastole (LVPWd), left ventricular posterior wall thickness at\nend-systole (LVPWs), ejection fraction (EF), and fractional shortening (FS). All\nmice were tested using the same parameters.\nAfter post-surgery weeks 4 and 8, mice were weighed and anesthetized with 4%\nchloralhydrate and placed on a warming pad after skin preparation. Transthoracic\n2-dimensional (2D) echocardiography was performed using the GE Vivid E9\nUltrasound echocardiographic system (General Electric Company, Fairfield, CT,\nUSA) with the GE 9L probe (8-MHz linear array transducer; General Electric\nCompany). M-mode parasternal long-axis scans of the left ventricle at the mitral\nchordae level were used to quantify the interventricular septum thickness at\nend-diastole (IVSd), interventricular septum thickness at end-systole (IVSs),\nleft ventricular internal dimension at end-diastole (LVIDd), left ventricular\ninternal dimension at end-systole (LVIDs), left ventricular posterior wall\nthickness at end-diastole (LVPWd), left ventricular posterior wall thickness at\nend-systole (LVPWs), ejection fraction (EF), and fractional shortening (FS). All\nmice were tested using the same parameters.\n Heart weight, heart weight/body weight, and heart weight/tibial\nlength After echocardiographic analysis at 8 weeks post-surgery, mice were sacrificed by\ncervical dislocation and the hearts were dissected. Then, atrial and vascular\ntissues were snipped carefully, leaving the ventricles. The hearts were rinsed\nwith phosphate-buffered saline (PBS), drained by gently squeezing on absorbent\npaper, weighed, photographed under natural light, and fixed in 4%\nparaformaldehyde. The tibial lengths (TLs; mean value of the bilateral tibia)\nwere recorded. Heart weight (HW), BW, and TL were measured, and the HW/BW ratio\nand HW/TL ratio were calculated to evaluate the hypertrophic response to PO.\nAfter echocardiographic analysis at 8 weeks post-surgery, mice were sacrificed by\ncervical dislocation and the hearts were dissected. Then, atrial and vascular\ntissues were snipped carefully, leaving the ventricles. The hearts were rinsed\nwith phosphate-buffered saline (PBS), drained by gently squeezing on absorbent\npaper, weighed, photographed under natural light, and fixed in 4%\nparaformaldehyde. The tibial lengths (TLs; mean value of the bilateral tibia)\nwere recorded. Heart weight (HW), BW, and TL were measured, and the HW/BW ratio\nand HW/TL ratio were calculated to evaluate the hypertrophic response to PO.\n Histological examination of the heart Extracted hearts were fixed in 4% paraformaldehyde for 24h and dehydrated. After\nroutine histologic procedures, the hearts were embedded in paraffin and cut into\n4-µm sections. Sections were stained with hematoxylin and eosin (HE) and\npicrosirius red (PSR). Cardiac cross-sections were captured at 20 ×\nmicroscopic views from HE sections, and 5 thicknesses of the left ventricle in\neach view were selected in systematic sampling, and measured using Image-Pro\nPlus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Then, the mean values\nwere calculated. Cardiomyocyte morphological changes were captured at\n400×microscopic views from HE sections. Interstitial and/or perivascular\ncollagen depositions were captured at 200×microscopic views under\nstandard lights. Collagen was stained red using PSR, thereby indicating\nfibrosis. At least 6 views were selected in a blinded manner, and each\nphotograph was analyzed to reveal the ratio of red collagen to the entire tissue\narea using Image-Pro Plus 6.0. Then, the mean values were calculated.\nExtracted hearts were fixed in 4% paraformaldehyde for 24h and dehydrated. After\nroutine histologic procedures, the hearts were embedded in paraffin and cut into\n4-µm sections. Sections were stained with hematoxylin and eosin (HE) and\npicrosirius red (PSR). Cardiac cross-sections were captured at 20 ×\nmicroscopic views from HE sections, and 5 thicknesses of the left ventricle in\neach view were selected in systematic sampling, and measured using Image-Pro\nPlus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Then, the mean values\nwere calculated. Cardiomyocyte morphological changes were captured at\n400×microscopic views from HE sections. Interstitial and/or perivascular\ncollagen depositions were captured at 200×microscopic views under\nstandard lights. Collagen was stained red using PSR, thereby indicating\nfibrosis. At least 6 views were selected in a blinded manner, and each\nphotograph was analyzed to reveal the ratio of red collagen to the entire tissue\narea using Image-Pro Plus 6.0. Then, the mean values were calculated.\n Statistical Analysis SPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA) was used for all\nstatistical analyses. The Kolmogorov-Smirnov (K-S) test was used to verify the\nnormality of the quantitative variables as appropriate. Data are presented as\nmean ± standard deviation (SD). One-way ANOVA and post-hoc Tukey tests\nwere used to evaluate differences between groups. p < 0.05 was considered\nstatistically significant.\nSPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA) was used for all\nstatistical analyses. The Kolmogorov-Smirnov (K-S) test was used to verify the\nnormality of the quantitative variables as appropriate. Data are presented as\nmean ± standard deviation (SD). One-way ANOVA and post-hoc Tukey tests\nwere used to evaluate differences between groups. p < 0.05 was considered\nstatistically significant."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Excessive AAC may lead to death We monitored mice deaths after surgery according to acute heart failure (AHF)\ncriteria. Data (Table 1) showed that all\ndeaths occurred within 5 days, and a high incidence of death occurred during the\ninitial 24h post-surgery.\nMice deaths after surgery\nThere were no mice deaths in the AAC0.50-mm group or the sham group.\nDeaths were recorded during 3 time periods (0-24h, 24h-3d, and 3-5\nd); 54 deaths occurred within 0-24h post-surgery. The total number\nof deaths was 65.\nWe monitored mice deaths after surgery according to acute heart failure (AHF)\ncriteria. Data (Table 1) showed that all\ndeaths occurred within 5 days, and a high incidence of death occurred during the\ninitial 24h post-surgery.\nMice deaths after surgery\nThere were no mice deaths in the AAC0.50-mm group or the sham group.\nDeaths were recorded during 3 time periods (0-24h, 24h-3d, and 3-5\nd); 54 deaths occurred within 0-24h post-surgery. The total number\nof deaths was 65.\n AAC increases cardiac dimensions and reduces cardiac function Echocardiography was performed at the end of post-operative weeks 4 and 8. At\nweek 4 post-surgery, data (Table 2)\nshowed a trend of heart enlargement for mice with AAC, including thickening of\nthe ventricular wall and an increase in chamber dilation; however, differences\nin EF and FS were not significant, indicating that changes in the heart\nstructure did not have a pronounced effect on cardiac function at that time\npoint. At week 8 post-surgery, the trend of heart enlargement continued;\nhowever, the EF and FS values for the AAC groups decreased significantly. This\nchange in cardiac function from week 4 to week 8 was consistent with systolic\nfunction beginning to be markedly affected at week 4 after PO surgery.\nEchocardiographic outcomes of 18-g, 22-g, and 26-g mice\nIVSd: interventricular septum thickness at end-diastole; IVSs:\ninterventricular septum thickness at end-systole; LVIDd: left\nventricular internal dimension at end-diastole; LVIDs: left\nventricular internal dimension at end-systole; LVPWd: left\nventricular posterior wall thickness at end-diastole; LVPWs: left\nventricular posterior wall thickness at end-systole; EF: ejection\nfraction; FS: fractional shortening. The cardiac dimensions\n(inclding IVSd, IVSs, LVIDd, LVIDs, LVPWd, and LVPWs) (mm) and\nfunctional indices (inclding EF and FS) changes measured by\nechocardiography. Data were statistically analyzed and presented as\nthe mean ± SD. At week 4, cardiac dimensions for the AAC\ngroups significantly increased compared with the sham groups (*p\n< 0.05); at week 8 , more cardiac dimensions significantly\nincreased, and that EF and FS values for the AAC groups all\ndecreased significantly compared to those of the sham groups for 3\nweight levels (*p < 0.05).\nEchocardiography was performed at the end of post-operative weeks 4 and 8. At\nweek 4 post-surgery, data (Table 2)\nshowed a trend of heart enlargement for mice with AAC, including thickening of\nthe ventricular wall and an increase in chamber dilation; however, differences\nin EF and FS were not significant, indicating that changes in the heart\nstructure did not have a pronounced effect on cardiac function at that time\npoint. At week 8 post-surgery, the trend of heart enlargement continued;\nhowever, the EF and FS values for the AAC groups decreased significantly. This\nchange in cardiac function from week 4 to week 8 was consistent with systolic\nfunction beginning to be markedly affected at week 4 after PO surgery.\nEchocardiographic outcomes of 18-g, 22-g, and 26-g mice\nIVSd: interventricular septum thickness at end-diastole; IVSs:\ninterventricular septum thickness at end-systole; LVIDd: left\nventricular internal dimension at end-diastole; LVIDs: left\nventricular internal dimension at end-systole; LVPWd: left\nventricular posterior wall thickness at end-diastole; LVPWs: left\nventricular posterior wall thickness at end-systole; EF: ejection\nfraction; FS: fractional shortening. The cardiac dimensions\n(inclding IVSd, IVSs, LVIDd, LVIDs, LVPWd, and LVPWs) (mm) and\nfunctional indices (inclding EF and FS) changes measured by\nechocardiography. Data were statistically analyzed and presented as\nthe mean ± SD. At week 4, cardiac dimensions for the AAC\ngroups significantly increased compared with the sham groups (*p\n< 0.05); at week 8 , more cardiac dimensions significantly\nincreased, and that EF and FS values for the AAC groups all\ndecreased significantly compared to those of the sham groups for 3\nweight levels (*p < 0.05).\n AAC increases HW, HW/BW, and HW/TL ratio Generally, the increased HW, HW/BW, and HW/TL ratio are the three main indicators\nof CH. In our study, as shown in Table 3,\nwe found that AAC significantly increased HW and caused a significantly higher\nHW/BW ratio and HW/TL ratio compared to the sham groups for all weight levels.\nThe HW, HW/BW, and HW/TL values for the AAC0.45 mm groups were significantly\nhigher than those for the AAC0.50 mm groups. These HW-related indices for the 18\ng/0.45 mm groups were even significantly higher than those for the 18 g/0.40 mm\ngroups.\nHeart weight–related indices of 18-g, 22-g, and 26-g mice\nHW: heart weight; BW: body weight; TL: tibial length. HW(mg),\nHW/BW(mg/g), and HW/TL(mg/cm) were measured and calculated from AAC\nand sham groups of 3 BW levels. Data are presented as the mean\n± SD.\nCompared to the sham group in the same BW level, the heart\nweight–related indices of AAC groups increased significantly (p <\n0.05).\nCompared to the rest groups in the same BW level, the heart\nweight–related indices of the AAC 0.45-mm group increased\nsignificantly (p < 0.05).\nGenerally, the increased HW, HW/BW, and HW/TL ratio are the three main indicators\nof CH. In our study, as shown in Table 3,\nwe found that AAC significantly increased HW and caused a significantly higher\nHW/BW ratio and HW/TL ratio compared to the sham groups for all weight levels.\nThe HW, HW/BW, and HW/TL values for the AAC0.45 mm groups were significantly\nhigher than those for the AAC0.50 mm groups. These HW-related indices for the 18\ng/0.45 mm groups were even significantly higher than those for the 18 g/0.40 mm\ngroups.\nHeart weight–related indices of 18-g, 22-g, and 26-g mice\nHW: heart weight; BW: body weight; TL: tibial length. HW(mg),\nHW/BW(mg/g), and HW/TL(mg/cm) were measured and calculated from AAC\nand sham groups of 3 BW levels. Data are presented as the mean\n± SD.\nCompared to the sham group in the same BW level, the heart\nweight–related indices of AAC groups increased significantly (p <\n0.05).\nCompared to the rest groups in the same BW level, the heart\nweight–related indices of the AAC 0.45-mm group increased\nsignificantly (p < 0.05).\n AAC leads to cardiomyocyte hypertrophy and increases collagen\ndepositions For mice undergoing AAC surgery, the hearts demonstrated different degrees of\nenlargement (Figure 1A), enlargement of the\npapillary muscles, and thickening of the ventricular walls (Figure 1B). Wall thickening increased significantly compared\nwith that of the sham group (Table 4).\nThe sham groups showed normal architecture of the cardiomyocytes compared with\nthe AAC groups. Pathological changes including enlarged, disarrayed, and\neosinophilic cardiomyocytes and cardiomyocytes rich in cytoplasm and\ntrachychromatic and pantomorphic nuclei were observed in each of the AAC groups\n(Figure 1C). Scattered collagen\ndepositions in the interstitial and perivascular spaces were observed in the\nsham groups. In comparison, in some AAC groups, a larger quantity and wider\nrange of red deposits were observed in the interstitial space (Figure 1D), and thickened collagen was\nobserved in the perivascular space, especially in the external vascular wall\n(Figure 1E). Statistical analysis\nindicated that the AAC group had a significantly greater collagen area than the\nsham group (Table 5). These results imply\nthat AAC is capable of inducing PO-induced CH and fibrosis.\n\nFigure 1Cardiomyocyte hypertrophy and collagen deposition histological\nexamination. (A) Gross hearts under natural light. (B) The 20\n× microscopic views of HE sections. (C) The 400 ×\nmicroscopic views of HE sections. (D) Representative\n200×microscopic views under standard lights of PSR sections\nin the interstitial space. (E) Representative 200 ×\nmicroscopic views under standard lights of PSR sections in the\nperivascular space. Fibrosis is presented as red in the PSR\nsections.\n\nCardiomyocyte hypertrophy and collagen deposition histological\nexamination. (A) Gross hearts under natural light. (B) The 20\n× microscopic views of HE sections. (C) The 400 ×\nmicroscopic views of HE sections. (D) Representative\n200×microscopic views under standard lights of PSR sections\nin the interstitial space. (E) Representative 200 ×\nmicroscopic views under standard lights of PSR sections in the\nperivascular space. Fibrosis is presented as red in the PSR\nsections.\nThickness of the left ventricle (mm) based on weight and needle size\nData are presented as the mean ± SD (n = 5).\np < 0.05 represents a significant difference between the abdominal\naortic constriction (AAC) and sham groups.\nPercentage of collagen deposition in the left ventricle based on weight\nand needle size\nData are presented as the mean ± SD (n = 6).\np < 0.05 represents a significant difference between the abdominal\naortic constriction (AAC) and sham groups.\nFor mice undergoing AAC surgery, the hearts demonstrated different degrees of\nenlargement (Figure 1A), enlargement of the\npapillary muscles, and thickening of the ventricular walls (Figure 1B). Wall thickening increased significantly compared\nwith that of the sham group (Table 4).\nThe sham groups showed normal architecture of the cardiomyocytes compared with\nthe AAC groups. Pathological changes including enlarged, disarrayed, and\neosinophilic cardiomyocytes and cardiomyocytes rich in cytoplasm and\ntrachychromatic and pantomorphic nuclei were observed in each of the AAC groups\n(Figure 1C). Scattered collagen\ndepositions in the interstitial and perivascular spaces were observed in the\nsham groups. In comparison, in some AAC groups, a larger quantity and wider\nrange of red deposits were observed in the interstitial space (Figure 1D), and thickened collagen was\nobserved in the perivascular space, especially in the external vascular wall\n(Figure 1E). Statistical analysis\nindicated that the AAC group had a significantly greater collagen area than the\nsham group (Table 5). These results imply\nthat AAC is capable of inducing PO-induced CH and fibrosis.\n\nFigure 1Cardiomyocyte hypertrophy and collagen deposition histological\nexamination. (A) Gross hearts under natural light. (B) The 20\n× microscopic views of HE sections. (C) The 400 ×\nmicroscopic views of HE sections. (D) Representative\n200×microscopic views under standard lights of PSR sections\nin the interstitial space. (E) Representative 200 ×\nmicroscopic views under standard lights of PSR sections in the\nperivascular space. Fibrosis is presented as red in the PSR\nsections.\n\nCardiomyocyte hypertrophy and collagen deposition histological\nexamination. (A) Gross hearts under natural light. (B) The 20\n× microscopic views of HE sections. (C) The 400 ×\nmicroscopic views of HE sections. (D) Representative\n200×microscopic views under standard lights of PSR sections\nin the interstitial space. (E) Representative 200 ×\nmicroscopic views under standard lights of PSR sections in the\nperivascular space. Fibrosis is presented as red in the PSR\nsections.\nThickness of the left ventricle (mm) based on weight and needle size\nData are presented as the mean ± SD (n = 5).\np < 0.05 represents a significant difference between the abdominal\naortic constriction (AAC) and sham groups.\nPercentage of collagen deposition in the left ventricle based on weight\nand needle size\nData are presented as the mean ± SD (n = 6).\np < 0.05 represents a significant difference between the abdominal\naortic constriction (AAC) and sham groups.\n AAC may restrict physical development Analysis showed that with AAC 0.45 mm, BW significantly increased in 18-g mice\ncompared to 22-g and 26-g mice (Table 6),\nindicating that the 18-g groups had higher development potential. In the 18-g\nmice groups, data showed that the value of 18 g/0.40 mm was significantly lower\nthan that of the 18 g/0.45 mm and 18 g/sham groups, and that there were no\nsignificant differences between the 18 g/0.45 mm and 18 g/sham groups (Table 7), indicating that the 18 g/0.45 mm\ngroup had nearly normal physical development. Development of the 18 g/0.40 mm\ngroup was limited.\nBody weight changes with AAC under 0.45 mm needle\nBW: body weight; AAC: abdominal aortic constriction. Data are\npresented as the mean ± SD.\np < 0.05 represents a significant difference between the 18 g/0.45\nmm group and the 22 g/0.45 mm and 26 g/0.45 mm groups.\nBody weight and BW changes in 18-g mice\nBW: body weight. BW changes of 18-g mice before and after surgery for\n8 weeks. Data are presented as the mean ± SD.\np < 0.05 represents a significant difference between the 18 g/0.40\nmm group and the rest groups after surgery.\nAnalysis showed that with AAC 0.45 mm, BW significantly increased in 18-g mice\ncompared to 22-g and 26-g mice (Table 6),\nindicating that the 18-g groups had higher development potential. In the 18-g\nmice groups, data showed that the value of 18 g/0.40 mm was significantly lower\nthan that of the 18 g/0.45 mm and 18 g/sham groups, and that there were no\nsignificant differences between the 18 g/0.45 mm and 18 g/sham groups (Table 7), indicating that the 18 g/0.45 mm\ngroup had nearly normal physical development. Development of the 18 g/0.40 mm\ngroup was limited.\nBody weight changes with AAC under 0.45 mm needle\nBW: body weight; AAC: abdominal aortic constriction. Data are\npresented as the mean ± SD.\np < 0.05 represents a significant difference between the 18 g/0.45\nmm group and the 22 g/0.45 mm and 26 g/0.45 mm groups.\nBody weight and BW changes in 18-g mice\nBW: body weight. BW changes of 18-g mice before and after surgery for\n8 weeks. Data are presented as the mean ± SD.\np < 0.05 represents a significant difference between the 18 g/0.40\nmm group and the rest groups after surgery."},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"We established CH models using 4 ligation needle sizes and 3 weights for mice. Data\nshowed that both of 0.45-mm and 0.50-mm needles lead to CH. However, 0.45mm needle\nbrings more effective model and causes obvious CH in 18-g mice."},"other_sections_titles":{"kind":"list like","value":["Animal groups and handling","Echocardiography imaging","Heart weight, heart weight/body weight, and heart weight/tibial\nlength","Histological examination of the heart","Statistical Analysis","Excessive AAC may lead to death","AAC increases cardiac dimensions and reduces cardiac function","AAC increases HW, HW/BW, and HW/TL ratio","AAC leads to cardiomyocyte hypertrophy and increases collagen\ndepositions","AAC may restrict physical development"],"string":"[\n \"Animal groups and handling\",\n \"Echocardiography imaging\",\n \"Heart weight, heart weight/body weight, and heart weight/tibial\\nlength\",\n \"Histological examination of the heart\",\n \"Statistical Analysis\",\n \"Excessive AAC may lead to death\",\n \"AAC increases cardiac dimensions and reduces cardiac function\",\n \"AAC increases HW, HW/BW, and HW/TL ratio\",\n \"AAC leads to cardiomyocyte hypertrophy and increases collagen\\ndepositions\",\n \"AAC may restrict physical development\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["One-hundred fifty male C57BL/6 wild-type mice were obtained from the Shanghai\nSLAC Laboratory Animal Co. Ltd (Shanghai, China). All animals were treated and\ncared for in accordance with the Guide for the Care and Use of\nLaboratory Animals (National Institutes of Health, Washington, DC,\n1996). Experimental protocols were approved by our Institutional Animal Care and\nUse Committee of Zhejiang University (Hangzhou, China). Mice were selected\naccording to weights of approximately 18 g (range, 17.3-18.7 g), 22 g (range,\n20.8-23.0 g), and 26 g (range, 25.1-27.0 g), and they were divided into the\nfollowing 3 weight levels: 18 g (18.0 ± 0.3 g; n = 50), 22 g (22.0\n± 0.6 g; n = 50), and 26 g (26.1 ± 0.5 g; n = 50). All weight\nlevels were divided using sortition randomization method to create a sham group\n(n = 10) and 4 AAC groups according to ligating intensities (0.35, 0.40, 0.45,\nand 0.50mm; n = 10 per group). Regarding BW, no significant differences were\nfound among the 5 groups for each weight level (Table S1), and the preoperative BWs of mice that died and those that\nsurvived were not significant (Table\nS2).\nMice were anesthetized with 4% chloralhydrate (0.1ml/1g BW, intraperitoneal\ninjection). When the mice did not respond when their toe was pinched, the limbs\nwere fixed on the operating board in the supine position and the skin was\nprepared by shaving and disinfection with alcohol. Sterile gauze was placed on\nthe right side of the abdomen and a ventrimesal incision approximately 1.5 cm\nwas created starting from the xiphoid. The skin was fixed with a spreader and\nthe viscera was pulled out gently with a swab and placed on the gauze. Then, the\nabdominal aorta was isolated using a blunt dissection technique with curved\nmicroforceps under a microscope. A 6-0 silk suture was snared and pulled back\naround the aorta 1mm above the superior mesenteric artery. A 2-mm blunt\nacupuncture needle (external diameters: 0.35 mm, 0.40 mm, 0.45 mm, and 0.50 mm;\nHuatuo; Suzhou Medical Appliance Factory, Suzhou, China; criterion number\nGB2024-1994) was then placed next to the aorta. The suture was tied snugly\naround the needle and the aorta. The needle was removed immediately after\nligation, the viscera were replaced, the peritoneum and skin were sutured, and\nthe mice were allowed to recover. Aortic ligation was omitted only for the sham\ngroup. After surgery, the ears were cut to differentiate the mice. Then, mice\nwere placed in an incubator at 30ºC until they woke, and they were\nreturned to their cages. Survival status was recorded daily. To observe the\nphysical development of mice under different conditions, BW differences before\nsurgery and at week 8 post-surgery were calculated as the change in BW.","After post-surgery weeks 4 and 8, mice were weighed and anesthetized with 4%\nchloralhydrate and placed on a warming pad after skin preparation. Transthoracic\n2-dimensional (2D) echocardiography was performed using the GE Vivid E9\nUltrasound echocardiographic system (General Electric Company, Fairfield, CT,\nUSA) with the GE 9L probe (8-MHz linear array transducer; General Electric\nCompany). M-mode parasternal long-axis scans of the left ventricle at the mitral\nchordae level were used to quantify the interventricular septum thickness at\nend-diastole (IVSd), interventricular septum thickness at end-systole (IVSs),\nleft ventricular internal dimension at end-diastole (LVIDd), left ventricular\ninternal dimension at end-systole (LVIDs), left ventricular posterior wall\nthickness at end-diastole (LVPWd), left ventricular posterior wall thickness at\nend-systole (LVPWs), ejection fraction (EF), and fractional shortening (FS). All\nmice were tested using the same parameters.","After echocardiographic analysis at 8 weeks post-surgery, mice were sacrificed by\ncervical dislocation and the hearts were dissected. Then, atrial and vascular\ntissues were snipped carefully, leaving the ventricles. The hearts were rinsed\nwith phosphate-buffered saline (PBS), drained by gently squeezing on absorbent\npaper, weighed, photographed under natural light, and fixed in 4%\nparaformaldehyde. The tibial lengths (TLs; mean value of the bilateral tibia)\nwere recorded. Heart weight (HW), BW, and TL were measured, and the HW/BW ratio\nand HW/TL ratio were calculated to evaluate the hypertrophic response to PO.","Extracted hearts were fixed in 4% paraformaldehyde for 24h and dehydrated. After\nroutine histologic procedures, the hearts were embedded in paraffin and cut into\n4-µm sections. Sections were stained with hematoxylin and eosin (HE) and\npicrosirius red (PSR). Cardiac cross-sections were captured at 20 ×\nmicroscopic views from HE sections, and 5 thicknesses of the left ventricle in\neach view were selected in systematic sampling, and measured using Image-Pro\nPlus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Then, the mean values\nwere calculated. Cardiomyocyte morphological changes were captured at\n400×microscopic views from HE sections. Interstitial and/or perivascular\ncollagen depositions were captured at 200×microscopic views under\nstandard lights. Collagen was stained red using PSR, thereby indicating\nfibrosis. At least 6 views were selected in a blinded manner, and each\nphotograph was analyzed to reveal the ratio of red collagen to the entire tissue\narea using Image-Pro Plus 6.0. Then, the mean values were calculated.","SPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA) was used for all\nstatistical analyses. The Kolmogorov-Smirnov (K-S) test was used to verify the\nnormality of the quantitative variables as appropriate. Data are presented as\nmean ± standard deviation (SD). One-way ANOVA and post-hoc Tukey tests\nwere used to evaluate differences between groups. p < 0.05 was considered\nstatistically significant.","We monitored mice deaths after surgery according to acute heart failure (AHF)\ncriteria. Data (Table 1) showed that all\ndeaths occurred within 5 days, and a high incidence of death occurred during the\ninitial 24h post-surgery.\nMice deaths after surgery\nThere were no mice deaths in the AAC0.50-mm group or the sham group.\nDeaths were recorded during 3 time periods (0-24h, 24h-3d, and 3-5\nd); 54 deaths occurred within 0-24h post-surgery. The total number\nof deaths was 65.","Echocardiography was performed at the end of post-operative weeks 4 and 8. At\nweek 4 post-surgery, data (Table 2)\nshowed a trend of heart enlargement for mice with AAC, including thickening of\nthe ventricular wall and an increase in chamber dilation; however, differences\nin EF and FS were not significant, indicating that changes in the heart\nstructure did not have a pronounced effect on cardiac function at that time\npoint. At week 8 post-surgery, the trend of heart enlargement continued;\nhowever, the EF and FS values for the AAC groups decreased significantly. This\nchange in cardiac function from week 4 to week 8 was consistent with systolic\nfunction beginning to be markedly affected at week 4 after PO surgery.\nEchocardiographic outcomes of 18-g, 22-g, and 26-g mice\nIVSd: interventricular septum thickness at end-diastole; IVSs:\ninterventricular septum thickness at end-systole; LVIDd: left\nventricular internal dimension at end-diastole; LVIDs: left\nventricular internal dimension at end-systole; LVPWd: left\nventricular posterior wall thickness at end-diastole; LVPWs: left\nventricular posterior wall thickness at end-systole; EF: ejection\nfraction; FS: fractional shortening. The cardiac dimensions\n(inclding IVSd, IVSs, LVIDd, LVIDs, LVPWd, and LVPWs) (mm) and\nfunctional indices (inclding EF and FS) changes measured by\nechocardiography. Data were statistically analyzed and presented as\nthe mean ± SD. At week 4, cardiac dimensions for the AAC\ngroups significantly increased compared with the sham groups (*p\n< 0.05); at week 8 , more cardiac dimensions significantly\nincreased, and that EF and FS values for the AAC groups all\ndecreased significantly compared to those of the sham groups for 3\nweight levels (*p < 0.05).","Generally, the increased HW, HW/BW, and HW/TL ratio are the three main indicators\nof CH. In our study, as shown in Table 3,\nwe found that AAC significantly increased HW and caused a significantly higher\nHW/BW ratio and HW/TL ratio compared to the sham groups for all weight levels.\nThe HW, HW/BW, and HW/TL values for the AAC0.45 mm groups were significantly\nhigher than those for the AAC0.50 mm groups. These HW-related indices for the 18\ng/0.45 mm groups were even significantly higher than those for the 18 g/0.40 mm\ngroups.\nHeart weight–related indices of 18-g, 22-g, and 26-g mice\nHW: heart weight; BW: body weight; TL: tibial length. HW(mg),\nHW/BW(mg/g), and HW/TL(mg/cm) were measured and calculated from AAC\nand sham groups of 3 BW levels. Data are presented as the mean\n± SD.\nCompared to the sham group in the same BW level, the heart\nweight–related indices of AAC groups increased significantly (p <\n0.05).\nCompared to the rest groups in the same BW level, the heart\nweight–related indices of the AAC 0.45-mm group increased\nsignificantly (p < 0.05).","For mice undergoing AAC surgery, the hearts demonstrated different degrees of\nenlargement (Figure 1A), enlargement of the\npapillary muscles, and thickening of the ventricular walls (Figure 1B). Wall thickening increased significantly compared\nwith that of the sham group (Table 4).\nThe sham groups showed normal architecture of the cardiomyocytes compared with\nthe AAC groups. Pathological changes including enlarged, disarrayed, and\neosinophilic cardiomyocytes and cardiomyocytes rich in cytoplasm and\ntrachychromatic and pantomorphic nuclei were observed in each of the AAC groups\n(Figure 1C). Scattered collagen\ndepositions in the interstitial and perivascular spaces were observed in the\nsham groups. In comparison, in some AAC groups, a larger quantity and wider\nrange of red deposits were observed in the interstitial space (Figure 1D), and thickened collagen was\nobserved in the perivascular space, especially in the external vascular wall\n(Figure 1E). Statistical analysis\nindicated that the AAC group had a significantly greater collagen area than the\nsham group (Table 5). These results imply\nthat AAC is capable of inducing PO-induced CH and fibrosis.\n\nFigure 1Cardiomyocyte hypertrophy and collagen deposition histological\nexamination. (A) Gross hearts under natural light. (B) The 20\n× microscopic views of HE sections. (C) The 400 ×\nmicroscopic views of HE sections. (D) Representative\n200×microscopic views under standard lights of PSR sections\nin the interstitial space. (E) Representative 200 ×\nmicroscopic views under standard lights of PSR sections in the\nperivascular space. Fibrosis is presented as red in the PSR\nsections.\n\nCardiomyocyte hypertrophy and collagen deposition histological\nexamination. (A) Gross hearts under natural light. (B) The 20\n× microscopic views of HE sections. (C) The 400 ×\nmicroscopic views of HE sections. (D) Representative\n200×microscopic views under standard lights of PSR sections\nin the interstitial space. (E) Representative 200 ×\nmicroscopic views under standard lights of PSR sections in the\nperivascular space. Fibrosis is presented as red in the PSR\nsections.\nThickness of the left ventricle (mm) based on weight and needle size\nData are presented as the mean ± SD (n = 5).\np < 0.05 represents a significant difference between the abdominal\naortic constriction (AAC) and sham groups.\nPercentage of collagen deposition in the left ventricle based on weight\nand needle size\nData are presented as the mean ± SD (n = 6).\np < 0.05 represents a significant difference between the abdominal\naortic constriction (AAC) and sham groups.","Analysis showed that with AAC 0.45 mm, BW significantly increased in 18-g mice\ncompared to 22-g and 26-g mice (Table 6),\nindicating that the 18-g groups had higher development potential. In the 18-g\nmice groups, data showed that the value of 18 g/0.40 mm was significantly lower\nthan that of the 18 g/0.45 mm and 18 g/sham groups, and that there were no\nsignificant differences between the 18 g/0.45 mm and 18 g/sham groups (Table 7), indicating that the 18 g/0.45 mm\ngroup had nearly normal physical development. Development of the 18 g/0.40 mm\ngroup was limited.\nBody weight changes with AAC under 0.45 mm needle\nBW: body weight; AAC: abdominal aortic constriction. Data are\npresented as the mean ± SD.\np < 0.05 represents a significant difference between the 18 g/0.45\nmm group and the 22 g/0.45 mm and 26 g/0.45 mm groups.\nBody weight and BW changes in 18-g mice\nBW: body weight. BW changes of 18-g mice before and after surgery for\n8 weeks. Data are presented as the mean ± SD.\np < 0.05 represents a significant difference between the 18 g/0.40\nmm group and the rest groups after surgery."],"string":"[\n \"One-hundred fifty male C57BL/6 wild-type mice were obtained from the Shanghai\\nSLAC Laboratory Animal Co. Ltd (Shanghai, China). All animals were treated and\\ncared for in accordance with the Guide for the Care and Use of\\nLaboratory Animals (National Institutes of Health, Washington, DC,\\n1996). Experimental protocols were approved by our Institutional Animal Care and\\nUse Committee of Zhejiang University (Hangzhou, China). Mice were selected\\naccording to weights of approximately 18 g (range, 17.3-18.7 g), 22 g (range,\\n20.8-23.0 g), and 26 g (range, 25.1-27.0 g), and they were divided into the\\nfollowing 3 weight levels: 18 g (18.0 ± 0.3 g; n = 50), 22 g (22.0\\n± 0.6 g; n = 50), and 26 g (26.1 ± 0.5 g; n = 50). All weight\\nlevels were divided using sortition randomization method to create a sham group\\n(n = 10) and 4 AAC groups according to ligating intensities (0.35, 0.40, 0.45,\\nand 0.50mm; n = 10 per group). Regarding BW, no significant differences were\\nfound among the 5 groups for each weight level (Table S1), and the preoperative BWs of mice that died and those that\\nsurvived were not significant (Table\\nS2).\\nMice were anesthetized with 4% chloralhydrate (0.1ml/1g BW, intraperitoneal\\ninjection). When the mice did not respond when their toe was pinched, the limbs\\nwere fixed on the operating board in the supine position and the skin was\\nprepared by shaving and disinfection with alcohol. Sterile gauze was placed on\\nthe right side of the abdomen and a ventrimesal incision approximately 1.5 cm\\nwas created starting from the xiphoid. The skin was fixed with a spreader and\\nthe viscera was pulled out gently with a swab and placed on the gauze. Then, the\\nabdominal aorta was isolated using a blunt dissection technique with curved\\nmicroforceps under a microscope. A 6-0 silk suture was snared and pulled back\\naround the aorta 1mm above the superior mesenteric artery. A 2-mm blunt\\nacupuncture needle (external diameters: 0.35 mm, 0.40 mm, 0.45 mm, and 0.50 mm;\\nHuatuo; Suzhou Medical Appliance Factory, Suzhou, China; criterion number\\nGB2024-1994) was then placed next to the aorta. The suture was tied snugly\\naround the needle and the aorta. The needle was removed immediately after\\nligation, the viscera were replaced, the peritoneum and skin were sutured, and\\nthe mice were allowed to recover. Aortic ligation was omitted only for the sham\\ngroup. After surgery, the ears were cut to differentiate the mice. Then, mice\\nwere placed in an incubator at 30ºC until they woke, and they were\\nreturned to their cages. Survival status was recorded daily. To observe the\\nphysical development of mice under different conditions, BW differences before\\nsurgery and at week 8 post-surgery were calculated as the change in BW.\",\n \"After post-surgery weeks 4 and 8, mice were weighed and anesthetized with 4%\\nchloralhydrate and placed on a warming pad after skin preparation. Transthoracic\\n2-dimensional (2D) echocardiography was performed using the GE Vivid E9\\nUltrasound echocardiographic system (General Electric Company, Fairfield, CT,\\nUSA) with the GE 9L probe (8-MHz linear array transducer; General Electric\\nCompany). M-mode parasternal long-axis scans of the left ventricle at the mitral\\nchordae level were used to quantify the interventricular septum thickness at\\nend-diastole (IVSd), interventricular septum thickness at end-systole (IVSs),\\nleft ventricular internal dimension at end-diastole (LVIDd), left ventricular\\ninternal dimension at end-systole (LVIDs), left ventricular posterior wall\\nthickness at end-diastole (LVPWd), left ventricular posterior wall thickness at\\nend-systole (LVPWs), ejection fraction (EF), and fractional shortening (FS). All\\nmice were tested using the same parameters.\",\n \"After echocardiographic analysis at 8 weeks post-surgery, mice were sacrificed by\\ncervical dislocation and the hearts were dissected. Then, atrial and vascular\\ntissues were snipped carefully, leaving the ventricles. The hearts were rinsed\\nwith phosphate-buffered saline (PBS), drained by gently squeezing on absorbent\\npaper, weighed, photographed under natural light, and fixed in 4%\\nparaformaldehyde. The tibial lengths (TLs; mean value of the bilateral tibia)\\nwere recorded. Heart weight (HW), BW, and TL were measured, and the HW/BW ratio\\nand HW/TL ratio were calculated to evaluate the hypertrophic response to PO.\",\n \"Extracted hearts were fixed in 4% paraformaldehyde for 24h and dehydrated. After\\nroutine histologic procedures, the hearts were embedded in paraffin and cut into\\n4-µm sections. Sections were stained with hematoxylin and eosin (HE) and\\npicrosirius red (PSR). Cardiac cross-sections were captured at 20 ×\\nmicroscopic views from HE sections, and 5 thicknesses of the left ventricle in\\neach view were selected in systematic sampling, and measured using Image-Pro\\nPlus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Then, the mean values\\nwere calculated. Cardiomyocyte morphological changes were captured at\\n400×microscopic views from HE sections. Interstitial and/or perivascular\\ncollagen depositions were captured at 200×microscopic views under\\nstandard lights. Collagen was stained red using PSR, thereby indicating\\nfibrosis. At least 6 views were selected in a blinded manner, and each\\nphotograph was analyzed to reveal the ratio of red collagen to the entire tissue\\narea using Image-Pro Plus 6.0. Then, the mean values were calculated.\",\n \"SPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA) was used for all\\nstatistical analyses. The Kolmogorov-Smirnov (K-S) test was used to verify the\\nnormality of the quantitative variables as appropriate. Data are presented as\\nmean ± standard deviation (SD). One-way ANOVA and post-hoc Tukey tests\\nwere used to evaluate differences between groups. p < 0.05 was considered\\nstatistically significant.\",\n \"We monitored mice deaths after surgery according to acute heart failure (AHF)\\ncriteria. Data (Table 1) showed that all\\ndeaths occurred within 5 days, and a high incidence of death occurred during the\\ninitial 24h post-surgery.\\nMice deaths after surgery\\nThere were no mice deaths in the AAC0.50-mm group or the sham group.\\nDeaths were recorded during 3 time periods (0-24h, 24h-3d, and 3-5\\nd); 54 deaths occurred within 0-24h post-surgery. The total number\\nof deaths was 65.\",\n \"Echocardiography was performed at the end of post-operative weeks 4 and 8. At\\nweek 4 post-surgery, data (Table 2)\\nshowed a trend of heart enlargement for mice with AAC, including thickening of\\nthe ventricular wall and an increase in chamber dilation; however, differences\\nin EF and FS were not significant, indicating that changes in the heart\\nstructure did not have a pronounced effect on cardiac function at that time\\npoint. At week 8 post-surgery, the trend of heart enlargement continued;\\nhowever, the EF and FS values for the AAC groups decreased significantly. This\\nchange in cardiac function from week 4 to week 8 was consistent with systolic\\nfunction beginning to be markedly affected at week 4 after PO surgery.\\nEchocardiographic outcomes of 18-g, 22-g, and 26-g mice\\nIVSd: interventricular septum thickness at end-diastole; IVSs:\\ninterventricular septum thickness at end-systole; LVIDd: left\\nventricular internal dimension at end-diastole; LVIDs: left\\nventricular internal dimension at end-systole; LVPWd: left\\nventricular posterior wall thickness at end-diastole; LVPWs: left\\nventricular posterior wall thickness at end-systole; EF: ejection\\nfraction; FS: fractional shortening. The cardiac dimensions\\n(inclding IVSd, IVSs, LVIDd, LVIDs, LVPWd, and LVPWs) (mm) and\\nfunctional indices (inclding EF and FS) changes measured by\\nechocardiography. Data were statistically analyzed and presented as\\nthe mean ± SD. At week 4, cardiac dimensions for the AAC\\ngroups significantly increased compared with the sham groups (*p\\n< 0.05); at week 8 , more cardiac dimensions significantly\\nincreased, and that EF and FS values for the AAC groups all\\ndecreased significantly compared to those of the sham groups for 3\\nweight levels (*p < 0.05).\",\n \"Generally, the increased HW, HW/BW, and HW/TL ratio are the three main indicators\\nof CH. In our study, as shown in Table 3,\\nwe found that AAC significantly increased HW and caused a significantly higher\\nHW/BW ratio and HW/TL ratio compared to the sham groups for all weight levels.\\nThe HW, HW/BW, and HW/TL values for the AAC0.45 mm groups were significantly\\nhigher than those for the AAC0.50 mm groups. These HW-related indices for the 18\\ng/0.45 mm groups were even significantly higher than those for the 18 g/0.40 mm\\ngroups.\\nHeart weight–related indices of 18-g, 22-g, and 26-g mice\\nHW: heart weight; BW: body weight; TL: tibial length. HW(mg),\\nHW/BW(mg/g), and HW/TL(mg/cm) were measured and calculated from AAC\\nand sham groups of 3 BW levels. Data are presented as the mean\\n± SD.\\nCompared to the sham group in the same BW level, the heart\\nweight–related indices of AAC groups increased significantly (p <\\n0.05).\\nCompared to the rest groups in the same BW level, the heart\\nweight–related indices of the AAC 0.45-mm group increased\\nsignificantly (p < 0.05).\",\n \"For mice undergoing AAC surgery, the hearts demonstrated different degrees of\\nenlargement (Figure 1A), enlargement of the\\npapillary muscles, and thickening of the ventricular walls (Figure 1B). Wall thickening increased significantly compared\\nwith that of the sham group (Table 4).\\nThe sham groups showed normal architecture of the cardiomyocytes compared with\\nthe AAC groups. Pathological changes including enlarged, disarrayed, and\\neosinophilic cardiomyocytes and cardiomyocytes rich in cytoplasm and\\ntrachychromatic and pantomorphic nuclei were observed in each of the AAC groups\\n(Figure 1C). Scattered collagen\\ndepositions in the interstitial and perivascular spaces were observed in the\\nsham groups. In comparison, in some AAC groups, a larger quantity and wider\\nrange of red deposits were observed in the interstitial space (Figure 1D), and thickened collagen was\\nobserved in the perivascular space, especially in the external vascular wall\\n(Figure 1E). Statistical analysis\\nindicated that the AAC group had a significantly greater collagen area than the\\nsham group (Table 5). These results imply\\nthat AAC is capable of inducing PO-induced CH and fibrosis.\\n\\nFigure 1Cardiomyocyte hypertrophy and collagen deposition histological\\nexamination. (A) Gross hearts under natural light. (B) The 20\\n× microscopic views of HE sections. (C) The 400 ×\\nmicroscopic views of HE sections. (D) Representative\\n200×microscopic views under standard lights of PSR sections\\nin the interstitial space. (E) Representative 200 ×\\nmicroscopic views under standard lights of PSR sections in the\\nperivascular space. Fibrosis is presented as red in the PSR\\nsections.\\n\\nCardiomyocyte hypertrophy and collagen deposition histological\\nexamination. (A) Gross hearts under natural light. (B) The 20\\n× microscopic views of HE sections. (C) The 400 ×\\nmicroscopic views of HE sections. (D) Representative\\n200×microscopic views under standard lights of PSR sections\\nin the interstitial space. (E) Representative 200 ×\\nmicroscopic views under standard lights of PSR sections in the\\nperivascular space. Fibrosis is presented as red in the PSR\\nsections.\\nThickness of the left ventricle (mm) based on weight and needle size\\nData are presented as the mean ± SD (n = 5).\\np < 0.05 represents a significant difference between the abdominal\\naortic constriction (AAC) and sham groups.\\nPercentage of collagen deposition in the left ventricle based on weight\\nand needle size\\nData are presented as the mean ± SD (n = 6).\\np < 0.05 represents a significant difference between the abdominal\\naortic constriction (AAC) and sham groups.\",\n \"Analysis showed that with AAC 0.45 mm, BW significantly increased in 18-g mice\\ncompared to 22-g and 26-g mice (Table 6),\\nindicating that the 18-g groups had higher development potential. In the 18-g\\nmice groups, data showed that the value of 18 g/0.40 mm was significantly lower\\nthan that of the 18 g/0.45 mm and 18 g/sham groups, and that there were no\\nsignificant differences between the 18 g/0.45 mm and 18 g/sham groups (Table 7), indicating that the 18 g/0.45 mm\\ngroup had nearly normal physical development. Development of the 18 g/0.40 mm\\ngroup was limited.\\nBody weight changes with AAC under 0.45 mm needle\\nBW: body weight; AAC: abdominal aortic constriction. Data are\\npresented as the mean ± SD.\\np < 0.05 represents a significant difference between the 18 g/0.45\\nmm group and the 22 g/0.45 mm and 26 g/0.45 mm groups.\\nBody weight and BW changes in 18-g mice\\nBW: body weight. BW changes of 18-g mice before and after surgery for\\n8 weeks. Data are presented as the mean ± SD.\\np < 0.05 represents a significant difference between the 18 g/0.40\\nmm group and the rest groups after surgery.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Introduction","Methods","Animal groups and handling","Echocardiography imaging","Heart weight, heart weight/body weight, and heart weight/tibial\nlength","Histological examination of the heart","Statistical Analysis","Results","Excessive AAC may lead to death","AAC increases cardiac dimensions and reduces cardiac function","AAC increases HW, HW/BW, and HW/TL ratio","AAC leads to cardiomyocyte hypertrophy and increases collagen\ndepositions","AAC may restrict physical development","Discussion","Conclusion"],"string":"[\n \"Introduction\",\n \"Methods\",\n \"Animal groups and handling\",\n \"Echocardiography imaging\",\n \"Heart weight, heart weight/body weight, and heart weight/tibial\\nlength\",\n \"Histological examination of the heart\",\n \"Statistical Analysis\",\n \"Results\",\n \"Excessive AAC may lead to death\",\n \"AAC increases cardiac dimensions and reduces cardiac function\",\n \"AAC increases HW, HW/BW, and HW/TL ratio\",\n \"AAC leads to cardiomyocyte hypertrophy and increases collagen\\ndepositions\",\n \"AAC may restrict physical development\",\n \"Discussion\",\n \"Conclusion\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Cardiac hypertrophy (CH) is a compensatory pathological change that is usually\ninduced by pressure overload (PO), neurohumoral abnormality, and the effects of\ncytokines. It is characterized by cardiomyocyte hypertrophy and interstitial\nhyperplasia, and it results in an enlarged heart and thickening of the heart walls.\nClinically, CH is involved in the development of many diseases, such as valvular\ndisease, hypertension, arterial stenosis, and primary myocardial hypertrophy. If\nthese diseases develop at their own pace, then cardiac function (CF) will gradually\ndecompensate, leading to heart failure (HF), which severely lowers the quality of\nlife and increases the mortality rate. Therefore, CH is a widespread concern and has\nbeen explored at the molecular level by researchers. Due to the high genomic\nhomology between mice and humans, an established CH model for mice has been widely\nused in animal experiments, thereby providing an effective research foundation for\nCH exploration.\nCurrently, PO-induced CH is a common way to establish the model. Abdominal aortic\nconstriction (AAC) is highly recommended by researchers because of the high success\nrate and the ability to perform surgery without the need for thoracotomy or a\nventilator. However, the modeling effects with different ligating intensities for\ncertain body weights (BWs) have not yet been reported. Therefore, we used 3\nfrequently used mice BWs (18 g, 22 g, and 26 g) and 4 different needle sizes (0.35,\n0.40, 0.45, and 0.50 mm) to establish the CH model for each weight level for AAC,\nsummarized the survival rates, and evaluated the CH effects."," Animal groups and handling One-hundred fifty male C57BL/6 wild-type mice were obtained from the Shanghai\nSLAC Laboratory Animal Co. Ltd (Shanghai, China). All animals were treated and\ncared for in accordance with the Guide for the Care and Use of\nLaboratory Animals (National Institutes of Health, Washington, DC,\n1996). Experimental protocols were approved by our Institutional Animal Care and\nUse Committee of Zhejiang University (Hangzhou, China). Mice were selected\naccording to weights of approximately 18 g (range, 17.3-18.7 g), 22 g (range,\n20.8-23.0 g), and 26 g (range, 25.1-27.0 g), and they were divided into the\nfollowing 3 weight levels: 18 g (18.0 ± 0.3 g; n = 50), 22 g (22.0\n± 0.6 g; n = 50), and 26 g (26.1 ± 0.5 g; n = 50). All weight\nlevels were divided using sortition randomization method to create a sham group\n(n = 10) and 4 AAC groups according to ligating intensities (0.35, 0.40, 0.45,\nand 0.50mm; n = 10 per group). Regarding BW, no significant differences were\nfound among the 5 groups for each weight level (Table S1), and the preoperative BWs of mice that died and those that\nsurvived were not significant (Table\nS2).\nMice were anesthetized with 4% chloralhydrate (0.1ml/1g BW, intraperitoneal\ninjection). When the mice did not respond when their toe was pinched, the limbs\nwere fixed on the operating board in the supine position and the skin was\nprepared by shaving and disinfection with alcohol. Sterile gauze was placed on\nthe right side of the abdomen and a ventrimesal incision approximately 1.5 cm\nwas created starting from the xiphoid. The skin was fixed with a spreader and\nthe viscera was pulled out gently with a swab and placed on the gauze. Then, the\nabdominal aorta was isolated using a blunt dissection technique with curved\nmicroforceps under a microscope. A 6-0 silk suture was snared and pulled back\naround the aorta 1mm above the superior mesenteric artery. A 2-mm blunt\nacupuncture needle (external diameters: 0.35 mm, 0.40 mm, 0.45 mm, and 0.50 mm;\nHuatuo; Suzhou Medical Appliance Factory, Suzhou, China; criterion number\nGB2024-1994) was then placed next to the aorta. The suture was tied snugly\naround the needle and the aorta. The needle was removed immediately after\nligation, the viscera were replaced, the peritoneum and skin were sutured, and\nthe mice were allowed to recover. Aortic ligation was omitted only for the sham\ngroup. After surgery, the ears were cut to differentiate the mice. Then, mice\nwere placed in an incubator at 30ºC until they woke, and they were\nreturned to their cages. Survival status was recorded daily. To observe the\nphysical development of mice under different conditions, BW differences before\nsurgery and at week 8 post-surgery were calculated as the change in BW.\nOne-hundred fifty male C57BL/6 wild-type mice were obtained from the Shanghai\nSLAC Laboratory Animal Co. Ltd (Shanghai, China). All animals were treated and\ncared for in accordance with the Guide for the Care and Use of\nLaboratory Animals (National Institutes of Health, Washington, DC,\n1996). Experimental protocols were approved by our Institutional Animal Care and\nUse Committee of Zhejiang University (Hangzhou, China). Mice were selected\naccording to weights of approximately 18 g (range, 17.3-18.7 g), 22 g (range,\n20.8-23.0 g), and 26 g (range, 25.1-27.0 g), and they were divided into the\nfollowing 3 weight levels: 18 g (18.0 ± 0.3 g; n = 50), 22 g (22.0\n± 0.6 g; n = 50), and 26 g (26.1 ± 0.5 g; n = 50). All weight\nlevels were divided using sortition randomization method to create a sham group\n(n = 10) and 4 AAC groups according to ligating intensities (0.35, 0.40, 0.45,\nand 0.50mm; n = 10 per group). Regarding BW, no significant differences were\nfound among the 5 groups for each weight level (Table S1), and the preoperative BWs of mice that died and those that\nsurvived were not significant (Table\nS2).\nMice were anesthetized with 4% chloralhydrate (0.1ml/1g BW, intraperitoneal\ninjection). When the mice did not respond when their toe was pinched, the limbs\nwere fixed on the operating board in the supine position and the skin was\nprepared by shaving and disinfection with alcohol. Sterile gauze was placed on\nthe right side of the abdomen and a ventrimesal incision approximately 1.5 cm\nwas created starting from the xiphoid. The skin was fixed with a spreader and\nthe viscera was pulled out gently with a swab and placed on the gauze. Then, the\nabdominal aorta was isolated using a blunt dissection technique with curved\nmicroforceps under a microscope. A 6-0 silk suture was snared and pulled back\naround the aorta 1mm above the superior mesenteric artery. A 2-mm blunt\nacupuncture needle (external diameters: 0.35 mm, 0.40 mm, 0.45 mm, and 0.50 mm;\nHuatuo; Suzhou Medical Appliance Factory, Suzhou, China; criterion number\nGB2024-1994) was then placed next to the aorta. The suture was tied snugly\naround the needle and the aorta. The needle was removed immediately after\nligation, the viscera were replaced, the peritoneum and skin were sutured, and\nthe mice were allowed to recover. Aortic ligation was omitted only for the sham\ngroup. After surgery, the ears were cut to differentiate the mice. Then, mice\nwere placed in an incubator at 30ºC until they woke, and they were\nreturned to their cages. Survival status was recorded daily. To observe the\nphysical development of mice under different conditions, BW differences before\nsurgery and at week 8 post-surgery were calculated as the change in BW.\n Echocardiography imaging After post-surgery weeks 4 and 8, mice were weighed and anesthetized with 4%\nchloralhydrate and placed on a warming pad after skin preparation. Transthoracic\n2-dimensional (2D) echocardiography was performed using the GE Vivid E9\nUltrasound echocardiographic system (General Electric Company, Fairfield, CT,\nUSA) with the GE 9L probe (8-MHz linear array transducer; General Electric\nCompany). M-mode parasternal long-axis scans of the left ventricle at the mitral\nchordae level were used to quantify the interventricular septum thickness at\nend-diastole (IVSd), interventricular septum thickness at end-systole (IVSs),\nleft ventricular internal dimension at end-diastole (LVIDd), left ventricular\ninternal dimension at end-systole (LVIDs), left ventricular posterior wall\nthickness at end-diastole (LVPWd), left ventricular posterior wall thickness at\nend-systole (LVPWs), ejection fraction (EF), and fractional shortening (FS). All\nmice were tested using the same parameters.\nAfter post-surgery weeks 4 and 8, mice were weighed and anesthetized with 4%\nchloralhydrate and placed on a warming pad after skin preparation. Transthoracic\n2-dimensional (2D) echocardiography was performed using the GE Vivid E9\nUltrasound echocardiographic system (General Electric Company, Fairfield, CT,\nUSA) with the GE 9L probe (8-MHz linear array transducer; General Electric\nCompany). M-mode parasternal long-axis scans of the left ventricle at the mitral\nchordae level were used to quantify the interventricular septum thickness at\nend-diastole (IVSd), interventricular septum thickness at end-systole (IVSs),\nleft ventricular internal dimension at end-diastole (LVIDd), left ventricular\ninternal dimension at end-systole (LVIDs), left ventricular posterior wall\nthickness at end-diastole (LVPWd), left ventricular posterior wall thickness at\nend-systole (LVPWs), ejection fraction (EF), and fractional shortening (FS). All\nmice were tested using the same parameters.\n Heart weight, heart weight/body weight, and heart weight/tibial\nlength After echocardiographic analysis at 8 weeks post-surgery, mice were sacrificed by\ncervical dislocation and the hearts were dissected. Then, atrial and vascular\ntissues were snipped carefully, leaving the ventricles. The hearts were rinsed\nwith phosphate-buffered saline (PBS), drained by gently squeezing on absorbent\npaper, weighed, photographed under natural light, and fixed in 4%\nparaformaldehyde. The tibial lengths (TLs; mean value of the bilateral tibia)\nwere recorded. Heart weight (HW), BW, and TL were measured, and the HW/BW ratio\nand HW/TL ratio were calculated to evaluate the hypertrophic response to PO.\nAfter echocardiographic analysis at 8 weeks post-surgery, mice were sacrificed by\ncervical dislocation and the hearts were dissected. Then, atrial and vascular\ntissues were snipped carefully, leaving the ventricles. The hearts were rinsed\nwith phosphate-buffered saline (PBS), drained by gently squeezing on absorbent\npaper, weighed, photographed under natural light, and fixed in 4%\nparaformaldehyde. The tibial lengths (TLs; mean value of the bilateral tibia)\nwere recorded. Heart weight (HW), BW, and TL were measured, and the HW/BW ratio\nand HW/TL ratio were calculated to evaluate the hypertrophic response to PO.\n Histological examination of the heart Extracted hearts were fixed in 4% paraformaldehyde for 24h and dehydrated. After\nroutine histologic procedures, the hearts were embedded in paraffin and cut into\n4-µm sections. Sections were stained with hematoxylin and eosin (HE) and\npicrosirius red (PSR). Cardiac cross-sections were captured at 20 ×\nmicroscopic views from HE sections, and 5 thicknesses of the left ventricle in\neach view were selected in systematic sampling, and measured using Image-Pro\nPlus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Then, the mean values\nwere calculated. Cardiomyocyte morphological changes were captured at\n400×microscopic views from HE sections. Interstitial and/or perivascular\ncollagen depositions were captured at 200×microscopic views under\nstandard lights. Collagen was stained red using PSR, thereby indicating\nfibrosis. At least 6 views were selected in a blinded manner, and each\nphotograph was analyzed to reveal the ratio of red collagen to the entire tissue\narea using Image-Pro Plus 6.0. Then, the mean values were calculated.\nExtracted hearts were fixed in 4% paraformaldehyde for 24h and dehydrated. After\nroutine histologic procedures, the hearts were embedded in paraffin and cut into\n4-µm sections. Sections were stained with hematoxylin and eosin (HE) and\npicrosirius red (PSR). Cardiac cross-sections were captured at 20 ×\nmicroscopic views from HE sections, and 5 thicknesses of the left ventricle in\neach view were selected in systematic sampling, and measured using Image-Pro\nPlus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Then, the mean values\nwere calculated. Cardiomyocyte morphological changes were captured at\n400×microscopic views from HE sections. Interstitial and/or perivascular\ncollagen depositions were captured at 200×microscopic views under\nstandard lights. Collagen was stained red using PSR, thereby indicating\nfibrosis. At least 6 views were selected in a blinded manner, and each\nphotograph was analyzed to reveal the ratio of red collagen to the entire tissue\narea using Image-Pro Plus 6.0. Then, the mean values were calculated.\n Statistical Analysis SPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA) was used for all\nstatistical analyses. The Kolmogorov-Smirnov (K-S) test was used to verify the\nnormality of the quantitative variables as appropriate. Data are presented as\nmean ± standard deviation (SD). One-way ANOVA and post-hoc Tukey tests\nwere used to evaluate differences between groups. p < 0.05 was considered\nstatistically significant.\nSPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA) was used for all\nstatistical analyses. The Kolmogorov-Smirnov (K-S) test was used to verify the\nnormality of the quantitative variables as appropriate. Data are presented as\nmean ± standard deviation (SD). One-way ANOVA and post-hoc Tukey tests\nwere used to evaluate differences between groups. p < 0.05 was considered\nstatistically significant.","One-hundred fifty male C57BL/6 wild-type mice were obtained from the Shanghai\nSLAC Laboratory Animal Co. Ltd (Shanghai, China). All animals were treated and\ncared for in accordance with the Guide for the Care and Use of\nLaboratory Animals (National Institutes of Health, Washington, DC,\n1996). Experimental protocols were approved by our Institutional Animal Care and\nUse Committee of Zhejiang University (Hangzhou, China). Mice were selected\naccording to weights of approximately 18 g (range, 17.3-18.7 g), 22 g (range,\n20.8-23.0 g), and 26 g (range, 25.1-27.0 g), and they were divided into the\nfollowing 3 weight levels: 18 g (18.0 ± 0.3 g; n = 50), 22 g (22.0\n± 0.6 g; n = 50), and 26 g (26.1 ± 0.5 g; n = 50). All weight\nlevels were divided using sortition randomization method to create a sham group\n(n = 10) and 4 AAC groups according to ligating intensities (0.35, 0.40, 0.45,\nand 0.50mm; n = 10 per group). Regarding BW, no significant differences were\nfound among the 5 groups for each weight level (Table S1), and the preoperative BWs of mice that died and those that\nsurvived were not significant (Table\nS2).\nMice were anesthetized with 4% chloralhydrate (0.1ml/1g BW, intraperitoneal\ninjection). When the mice did not respond when their toe was pinched, the limbs\nwere fixed on the operating board in the supine position and the skin was\nprepared by shaving and disinfection with alcohol. Sterile gauze was placed on\nthe right side of the abdomen and a ventrimesal incision approximately 1.5 cm\nwas created starting from the xiphoid. The skin was fixed with a spreader and\nthe viscera was pulled out gently with a swab and placed on the gauze. Then, the\nabdominal aorta was isolated using a blunt dissection technique with curved\nmicroforceps under a microscope. A 6-0 silk suture was snared and pulled back\naround the aorta 1mm above the superior mesenteric artery. A 2-mm blunt\nacupuncture needle (external diameters: 0.35 mm, 0.40 mm, 0.45 mm, and 0.50 mm;\nHuatuo; Suzhou Medical Appliance Factory, Suzhou, China; criterion number\nGB2024-1994) was then placed next to the aorta. The suture was tied snugly\naround the needle and the aorta. The needle was removed immediately after\nligation, the viscera were replaced, the peritoneum and skin were sutured, and\nthe mice were allowed to recover. Aortic ligation was omitted only for the sham\ngroup. After surgery, the ears were cut to differentiate the mice. Then, mice\nwere placed in an incubator at 30ºC until they woke, and they were\nreturned to their cages. Survival status was recorded daily. To observe the\nphysical development of mice under different conditions, BW differences before\nsurgery and at week 8 post-surgery were calculated as the change in BW.","After post-surgery weeks 4 and 8, mice were weighed and anesthetized with 4%\nchloralhydrate and placed on a warming pad after skin preparation. Transthoracic\n2-dimensional (2D) echocardiography was performed using the GE Vivid E9\nUltrasound echocardiographic system (General Electric Company, Fairfield, CT,\nUSA) with the GE 9L probe (8-MHz linear array transducer; General Electric\nCompany). M-mode parasternal long-axis scans of the left ventricle at the mitral\nchordae level were used to quantify the interventricular septum thickness at\nend-diastole (IVSd), interventricular septum thickness at end-systole (IVSs),\nleft ventricular internal dimension at end-diastole (LVIDd), left ventricular\ninternal dimension at end-systole (LVIDs), left ventricular posterior wall\nthickness at end-diastole (LVPWd), left ventricular posterior wall thickness at\nend-systole (LVPWs), ejection fraction (EF), and fractional shortening (FS). All\nmice were tested using the same parameters.","After echocardiographic analysis at 8 weeks post-surgery, mice were sacrificed by\ncervical dislocation and the hearts were dissected. Then, atrial and vascular\ntissues were snipped carefully, leaving the ventricles. The hearts were rinsed\nwith phosphate-buffered saline (PBS), drained by gently squeezing on absorbent\npaper, weighed, photographed under natural light, and fixed in 4%\nparaformaldehyde. The tibial lengths (TLs; mean value of the bilateral tibia)\nwere recorded. Heart weight (HW), BW, and TL were measured, and the HW/BW ratio\nand HW/TL ratio were calculated to evaluate the hypertrophic response to PO.","Extracted hearts were fixed in 4% paraformaldehyde for 24h and dehydrated. After\nroutine histologic procedures, the hearts were embedded in paraffin and cut into\n4-µm sections. Sections were stained with hematoxylin and eosin (HE) and\npicrosirius red (PSR). Cardiac cross-sections were captured at 20 ×\nmicroscopic views from HE sections, and 5 thicknesses of the left ventricle in\neach view were selected in systematic sampling, and measured using Image-Pro\nPlus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Then, the mean values\nwere calculated. Cardiomyocyte morphological changes were captured at\n400×microscopic views from HE sections. Interstitial and/or perivascular\ncollagen depositions were captured at 200×microscopic views under\nstandard lights. Collagen was stained red using PSR, thereby indicating\nfibrosis. At least 6 views were selected in a blinded manner, and each\nphotograph was analyzed to reveal the ratio of red collagen to the entire tissue\narea using Image-Pro Plus 6.0. Then, the mean values were calculated.","SPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA) was used for all\nstatistical analyses. The Kolmogorov-Smirnov (K-S) test was used to verify the\nnormality of the quantitative variables as appropriate. Data are presented as\nmean ± standard deviation (SD). One-way ANOVA and post-hoc Tukey tests\nwere used to evaluate differences between groups. p < 0.05 was considered\nstatistically significant."," Excessive AAC may lead to death We monitored mice deaths after surgery according to acute heart failure (AHF)\ncriteria. Data (Table 1) showed that all\ndeaths occurred within 5 days, and a high incidence of death occurred during the\ninitial 24h post-surgery.\nMice deaths after surgery\nThere were no mice deaths in the AAC0.50-mm group or the sham group.\nDeaths were recorded during 3 time periods (0-24h, 24h-3d, and 3-5\nd); 54 deaths occurred within 0-24h post-surgery. The total number\nof deaths was 65.\nWe monitored mice deaths after surgery according to acute heart failure (AHF)\ncriteria. Data (Table 1) showed that all\ndeaths occurred within 5 days, and a high incidence of death occurred during the\ninitial 24h post-surgery.\nMice deaths after surgery\nThere were no mice deaths in the AAC0.50-mm group or the sham group.\nDeaths were recorded during 3 time periods (0-24h, 24h-3d, and 3-5\nd); 54 deaths occurred within 0-24h post-surgery. The total number\nof deaths was 65.\n AAC increases cardiac dimensions and reduces cardiac function Echocardiography was performed at the end of post-operative weeks 4 and 8. At\nweek 4 post-surgery, data (Table 2)\nshowed a trend of heart enlargement for mice with AAC, including thickening of\nthe ventricular wall and an increase in chamber dilation; however, differences\nin EF and FS were not significant, indicating that changes in the heart\nstructure did not have a pronounced effect on cardiac function at that time\npoint. At week 8 post-surgery, the trend of heart enlargement continued;\nhowever, the EF and FS values for the AAC groups decreased significantly. This\nchange in cardiac function from week 4 to week 8 was consistent with systolic\nfunction beginning to be markedly affected at week 4 after PO surgery.\nEchocardiographic outcomes of 18-g, 22-g, and 26-g mice\nIVSd: interventricular septum thickness at end-diastole; IVSs:\ninterventricular septum thickness at end-systole; LVIDd: left\nventricular internal dimension at end-diastole; LVIDs: left\nventricular internal dimension at end-systole; LVPWd: left\nventricular posterior wall thickness at end-diastole; LVPWs: left\nventricular posterior wall thickness at end-systole; EF: ejection\nfraction; FS: fractional shortening. The cardiac dimensions\n(inclding IVSd, IVSs, LVIDd, LVIDs, LVPWd, and LVPWs) (mm) and\nfunctional indices (inclding EF and FS) changes measured by\nechocardiography. Data were statistically analyzed and presented as\nthe mean ± SD. At week 4, cardiac dimensions for the AAC\ngroups significantly increased compared with the sham groups (*p\n< 0.05); at week 8 , more cardiac dimensions significantly\nincreased, and that EF and FS values for the AAC groups all\ndecreased significantly compared to those of the sham groups for 3\nweight levels (*p < 0.05).\nEchocardiography was performed at the end of post-operative weeks 4 and 8. At\nweek 4 post-surgery, data (Table 2)\nshowed a trend of heart enlargement for mice with AAC, including thickening of\nthe ventricular wall and an increase in chamber dilation; however, differences\nin EF and FS were not significant, indicating that changes in the heart\nstructure did not have a pronounced effect on cardiac function at that time\npoint. At week 8 post-surgery, the trend of heart enlargement continued;\nhowever, the EF and FS values for the AAC groups decreased significantly. This\nchange in cardiac function from week 4 to week 8 was consistent with systolic\nfunction beginning to be markedly affected at week 4 after PO surgery.\nEchocardiographic outcomes of 18-g, 22-g, and 26-g mice\nIVSd: interventricular septum thickness at end-diastole; IVSs:\ninterventricular septum thickness at end-systole; LVIDd: left\nventricular internal dimension at end-diastole; LVIDs: left\nventricular internal dimension at end-systole; LVPWd: left\nventricular posterior wall thickness at end-diastole; LVPWs: left\nventricular posterior wall thickness at end-systole; EF: ejection\nfraction; FS: fractional shortening. The cardiac dimensions\n(inclding IVSd, IVSs, LVIDd, LVIDs, LVPWd, and LVPWs) (mm) and\nfunctional indices (inclding EF and FS) changes measured by\nechocardiography. Data were statistically analyzed and presented as\nthe mean ± SD. At week 4, cardiac dimensions for the AAC\ngroups significantly increased compared with the sham groups (*p\n< 0.05); at week 8 , more cardiac dimensions significantly\nincreased, and that EF and FS values for the AAC groups all\ndecreased significantly compared to those of the sham groups for 3\nweight levels (*p < 0.05).\n AAC increases HW, HW/BW, and HW/TL ratio Generally, the increased HW, HW/BW, and HW/TL ratio are the three main indicators\nof CH. In our study, as shown in Table 3,\nwe found that AAC significantly increased HW and caused a significantly higher\nHW/BW ratio and HW/TL ratio compared to the sham groups for all weight levels.\nThe HW, HW/BW, and HW/TL values for the AAC0.45 mm groups were significantly\nhigher than those for the AAC0.50 mm groups. These HW-related indices for the 18\ng/0.45 mm groups were even significantly higher than those for the 18 g/0.40 mm\ngroups.\nHeart weight–related indices of 18-g, 22-g, and 26-g mice\nHW: heart weight; BW: body weight; TL: tibial length. HW(mg),\nHW/BW(mg/g), and HW/TL(mg/cm) were measured and calculated from AAC\nand sham groups of 3 BW levels. Data are presented as the mean\n± SD.\nCompared to the sham group in the same BW level, the heart\nweight–related indices of AAC groups increased significantly (p <\n0.05).\nCompared to the rest groups in the same BW level, the heart\nweight–related indices of the AAC 0.45-mm group increased\nsignificantly (p < 0.05).\nGenerally, the increased HW, HW/BW, and HW/TL ratio are the three main indicators\nof CH. In our study, as shown in Table 3,\nwe found that AAC significantly increased HW and caused a significantly higher\nHW/BW ratio and HW/TL ratio compared to the sham groups for all weight levels.\nThe HW, HW/BW, and HW/TL values for the AAC0.45 mm groups were significantly\nhigher than those for the AAC0.50 mm groups. These HW-related indices for the 18\ng/0.45 mm groups were even significantly higher than those for the 18 g/0.40 mm\ngroups.\nHeart weight–related indices of 18-g, 22-g, and 26-g mice\nHW: heart weight; BW: body weight; TL: tibial length. HW(mg),\nHW/BW(mg/g), and HW/TL(mg/cm) were measured and calculated from AAC\nand sham groups of 3 BW levels. Data are presented as the mean\n± SD.\nCompared to the sham group in the same BW level, the heart\nweight–related indices of AAC groups increased significantly (p <\n0.05).\nCompared to the rest groups in the same BW level, the heart\nweight–related indices of the AAC 0.45-mm group increased\nsignificantly (p < 0.05).\n AAC leads to cardiomyocyte hypertrophy and increases collagen\ndepositions For mice undergoing AAC surgery, the hearts demonstrated different degrees of\nenlargement (Figure 1A), enlargement of the\npapillary muscles, and thickening of the ventricular walls (Figure 1B). Wall thickening increased significantly compared\nwith that of the sham group (Table 4).\nThe sham groups showed normal architecture of the cardiomyocytes compared with\nthe AAC groups. Pathological changes including enlarged, disarrayed, and\neosinophilic cardiomyocytes and cardiomyocytes rich in cytoplasm and\ntrachychromatic and pantomorphic nuclei were observed in each of the AAC groups\n(Figure 1C). Scattered collagen\ndepositions in the interstitial and perivascular spaces were observed in the\nsham groups. In comparison, in some AAC groups, a larger quantity and wider\nrange of red deposits were observed in the interstitial space (Figure 1D), and thickened collagen was\nobserved in the perivascular space, especially in the external vascular wall\n(Figure 1E). Statistical analysis\nindicated that the AAC group had a significantly greater collagen area than the\nsham group (Table 5). These results imply\nthat AAC is capable of inducing PO-induced CH and fibrosis.\n\nFigure 1Cardiomyocyte hypertrophy and collagen deposition histological\nexamination. (A) Gross hearts under natural light. (B) The 20\n× microscopic views of HE sections. (C) The 400 ×\nmicroscopic views of HE sections. (D) Representative\n200×microscopic views under standard lights of PSR sections\nin the interstitial space. (E) Representative 200 ×\nmicroscopic views under standard lights of PSR sections in the\nperivascular space. Fibrosis is presented as red in the PSR\nsections.\n\nCardiomyocyte hypertrophy and collagen deposition histological\nexamination. (A) Gross hearts under natural light. (B) The 20\n× microscopic views of HE sections. (C) The 400 ×\nmicroscopic views of HE sections. (D) Representative\n200×microscopic views under standard lights of PSR sections\nin the interstitial space. (E) Representative 200 ×\nmicroscopic views under standard lights of PSR sections in the\nperivascular space. Fibrosis is presented as red in the PSR\nsections.\nThickness of the left ventricle (mm) based on weight and needle size\nData are presented as the mean ± SD (n = 5).\np < 0.05 represents a significant difference between the abdominal\naortic constriction (AAC) and sham groups.\nPercentage of collagen deposition in the left ventricle based on weight\nand needle size\nData are presented as the mean ± SD (n = 6).\np < 0.05 represents a significant difference between the abdominal\naortic constriction (AAC) and sham groups.\nFor mice undergoing AAC surgery, the hearts demonstrated different degrees of\nenlargement (Figure 1A), enlargement of the\npapillary muscles, and thickening of the ventricular walls (Figure 1B). Wall thickening increased significantly compared\nwith that of the sham group (Table 4).\nThe sham groups showed normal architecture of the cardiomyocytes compared with\nthe AAC groups. Pathological changes including enlarged, disarrayed, and\neosinophilic cardiomyocytes and cardiomyocytes rich in cytoplasm and\ntrachychromatic and pantomorphic nuclei were observed in each of the AAC groups\n(Figure 1C). Scattered collagen\ndepositions in the interstitial and perivascular spaces were observed in the\nsham groups. In comparison, in some AAC groups, a larger quantity and wider\nrange of red deposits were observed in the interstitial space (Figure 1D), and thickened collagen was\nobserved in the perivascular space, especially in the external vascular wall\n(Figure 1E). Statistical analysis\nindicated that the AAC group had a significantly greater collagen area than the\nsham group (Table 5). These results imply\nthat AAC is capable of inducing PO-induced CH and fibrosis.\n\nFigure 1Cardiomyocyte hypertrophy and collagen deposition histological\nexamination. (A) Gross hearts under natural light. (B) The 20\n× microscopic views of HE sections. (C) The 400 ×\nmicroscopic views of HE sections. (D) Representative\n200×microscopic views under standard lights of PSR sections\nin the interstitial space. (E) Representative 200 ×\nmicroscopic views under standard lights of PSR sections in the\nperivascular space. Fibrosis is presented as red in the PSR\nsections.\n\nCardiomyocyte hypertrophy and collagen deposition histological\nexamination. (A) Gross hearts under natural light. (B) The 20\n× microscopic views of HE sections. (C) The 400 ×\nmicroscopic views of HE sections. (D) Representative\n200×microscopic views under standard lights of PSR sections\nin the interstitial space. (E) Representative 200 ×\nmicroscopic views under standard lights of PSR sections in the\nperivascular space. Fibrosis is presented as red in the PSR\nsections.\nThickness of the left ventricle (mm) based on weight and needle size\nData are presented as the mean ± SD (n = 5).\np < 0.05 represents a significant difference between the abdominal\naortic constriction (AAC) and sham groups.\nPercentage of collagen deposition in the left ventricle based on weight\nand needle size\nData are presented as the mean ± SD (n = 6).\np < 0.05 represents a significant difference between the abdominal\naortic constriction (AAC) and sham groups.\n AAC may restrict physical development Analysis showed that with AAC 0.45 mm, BW significantly increased in 18-g mice\ncompared to 22-g and 26-g mice (Table 6),\nindicating that the 18-g groups had higher development potential. In the 18-g\nmice groups, data showed that the value of 18 g/0.40 mm was significantly lower\nthan that of the 18 g/0.45 mm and 18 g/sham groups, and that there were no\nsignificant differences between the 18 g/0.45 mm and 18 g/sham groups (Table 7), indicating that the 18 g/0.45 mm\ngroup had nearly normal physical development. Development of the 18 g/0.40 mm\ngroup was limited.\nBody weight changes with AAC under 0.45 mm needle\nBW: body weight; AAC: abdominal aortic constriction. Data are\npresented as the mean ± SD.\np < 0.05 represents a significant difference between the 18 g/0.45\nmm group and the 22 g/0.45 mm and 26 g/0.45 mm groups.\nBody weight and BW changes in 18-g mice\nBW: body weight. BW changes of 18-g mice before and after surgery for\n8 weeks. Data are presented as the mean ± SD.\np < 0.05 represents a significant difference between the 18 g/0.40\nmm group and the rest groups after surgery.\nAnalysis showed that with AAC 0.45 mm, BW significantly increased in 18-g mice\ncompared to 22-g and 26-g mice (Table 6),\nindicating that the 18-g groups had higher development potential. In the 18-g\nmice groups, data showed that the value of 18 g/0.40 mm was significantly lower\nthan that of the 18 g/0.45 mm and 18 g/sham groups, and that there were no\nsignificant differences between the 18 g/0.45 mm and 18 g/sham groups (Table 7), indicating that the 18 g/0.45 mm\ngroup had nearly normal physical development. Development of the 18 g/0.40 mm\ngroup was limited.\nBody weight changes with AAC under 0.45 mm needle\nBW: body weight; AAC: abdominal aortic constriction. Data are\npresented as the mean ± SD.\np < 0.05 represents a significant difference between the 18 g/0.45\nmm group and the 22 g/0.45 mm and 26 g/0.45 mm groups.\nBody weight and BW changes in 18-g mice\nBW: body weight. BW changes of 18-g mice before and after surgery for\n8 weeks. Data are presented as the mean ± SD.\np < 0.05 represents a significant difference between the 18 g/0.40\nmm group and the rest groups after surgery.","We monitored mice deaths after surgery according to acute heart failure (AHF)\ncriteria. Data (Table 1) showed that all\ndeaths occurred within 5 days, and a high incidence of death occurred during the\ninitial 24h post-surgery.\nMice deaths after surgery\nThere were no mice deaths in the AAC0.50-mm group or the sham group.\nDeaths were recorded during 3 time periods (0-24h, 24h-3d, and 3-5\nd); 54 deaths occurred within 0-24h post-surgery. The total number\nof deaths was 65.","Echocardiography was performed at the end of post-operative weeks 4 and 8. At\nweek 4 post-surgery, data (Table 2)\nshowed a trend of heart enlargement for mice with AAC, including thickening of\nthe ventricular wall and an increase in chamber dilation; however, differences\nin EF and FS were not significant, indicating that changes in the heart\nstructure did not have a pronounced effect on cardiac function at that time\npoint. At week 8 post-surgery, the trend of heart enlargement continued;\nhowever, the EF and FS values for the AAC groups decreased significantly. This\nchange in cardiac function from week 4 to week 8 was consistent with systolic\nfunction beginning to be markedly affected at week 4 after PO surgery.\nEchocardiographic outcomes of 18-g, 22-g, and 26-g mice\nIVSd: interventricular septum thickness at end-diastole; IVSs:\ninterventricular septum thickness at end-systole; LVIDd: left\nventricular internal dimension at end-diastole; LVIDs: left\nventricular internal dimension at end-systole; LVPWd: left\nventricular posterior wall thickness at end-diastole; LVPWs: left\nventricular posterior wall thickness at end-systole; EF: ejection\nfraction; FS: fractional shortening. The cardiac dimensions\n(inclding IVSd, IVSs, LVIDd, LVIDs, LVPWd, and LVPWs) (mm) and\nfunctional indices (inclding EF and FS) changes measured by\nechocardiography. Data were statistically analyzed and presented as\nthe mean ± SD. At week 4, cardiac dimensions for the AAC\ngroups significantly increased compared with the sham groups (*p\n< 0.05); at week 8 , more cardiac dimensions significantly\nincreased, and that EF and FS values for the AAC groups all\ndecreased significantly compared to those of the sham groups for 3\nweight levels (*p < 0.05).","Generally, the increased HW, HW/BW, and HW/TL ratio are the three main indicators\nof CH. In our study, as shown in Table 3,\nwe found that AAC significantly increased HW and caused a significantly higher\nHW/BW ratio and HW/TL ratio compared to the sham groups for all weight levels.\nThe HW, HW/BW, and HW/TL values for the AAC0.45 mm groups were significantly\nhigher than those for the AAC0.50 mm groups. These HW-related indices for the 18\ng/0.45 mm groups were even significantly higher than those for the 18 g/0.40 mm\ngroups.\nHeart weight–related indices of 18-g, 22-g, and 26-g mice\nHW: heart weight; BW: body weight; TL: tibial length. HW(mg),\nHW/BW(mg/g), and HW/TL(mg/cm) were measured and calculated from AAC\nand sham groups of 3 BW levels. Data are presented as the mean\n± SD.\nCompared to the sham group in the same BW level, the heart\nweight–related indices of AAC groups increased significantly (p <\n0.05).\nCompared to the rest groups in the same BW level, the heart\nweight–related indices of the AAC 0.45-mm group increased\nsignificantly (p < 0.05).","For mice undergoing AAC surgery, the hearts demonstrated different degrees of\nenlargement (Figure 1A), enlargement of the\npapillary muscles, and thickening of the ventricular walls (Figure 1B). Wall thickening increased significantly compared\nwith that of the sham group (Table 4).\nThe sham groups showed normal architecture of the cardiomyocytes compared with\nthe AAC groups. Pathological changes including enlarged, disarrayed, and\neosinophilic cardiomyocytes and cardiomyocytes rich in cytoplasm and\ntrachychromatic and pantomorphic nuclei were observed in each of the AAC groups\n(Figure 1C). Scattered collagen\ndepositions in the interstitial and perivascular spaces were observed in the\nsham groups. In comparison, in some AAC groups, a larger quantity and wider\nrange of red deposits were observed in the interstitial space (Figure 1D), and thickened collagen was\nobserved in the perivascular space, especially in the external vascular wall\n(Figure 1E). Statistical analysis\nindicated that the AAC group had a significantly greater collagen area than the\nsham group (Table 5). These results imply\nthat AAC is capable of inducing PO-induced CH and fibrosis.\n\nFigure 1Cardiomyocyte hypertrophy and collagen deposition histological\nexamination. (A) Gross hearts under natural light. (B) The 20\n× microscopic views of HE sections. (C) The 400 ×\nmicroscopic views of HE sections. (D) Representative\n200×microscopic views under standard lights of PSR sections\nin the interstitial space. (E) Representative 200 ×\nmicroscopic views under standard lights of PSR sections in the\nperivascular space. Fibrosis is presented as red in the PSR\nsections.\n\nCardiomyocyte hypertrophy and collagen deposition histological\nexamination. (A) Gross hearts under natural light. (B) The 20\n× microscopic views of HE sections. (C) The 400 ×\nmicroscopic views of HE sections. (D) Representative\n200×microscopic views under standard lights of PSR sections\nin the interstitial space. (E) Representative 200 ×\nmicroscopic views under standard lights of PSR sections in the\nperivascular space. Fibrosis is presented as red in the PSR\nsections.\nThickness of the left ventricle (mm) based on weight and needle size\nData are presented as the mean ± SD (n = 5).\np < 0.05 represents a significant difference between the abdominal\naortic constriction (AAC) and sham groups.\nPercentage of collagen deposition in the left ventricle based on weight\nand needle size\nData are presented as the mean ± SD (n = 6).\np < 0.05 represents a significant difference between the abdominal\naortic constriction (AAC) and sham groups.","Analysis showed that with AAC 0.45 mm, BW significantly increased in 18-g mice\ncompared to 22-g and 26-g mice (Table 6),\nindicating that the 18-g groups had higher development potential. In the 18-g\nmice groups, data showed that the value of 18 g/0.40 mm was significantly lower\nthan that of the 18 g/0.45 mm and 18 g/sham groups, and that there were no\nsignificant differences between the 18 g/0.45 mm and 18 g/sham groups (Table 7), indicating that the 18 g/0.45 mm\ngroup had nearly normal physical development. Development of the 18 g/0.40 mm\ngroup was limited.\nBody weight changes with AAC under 0.45 mm needle\nBW: body weight; AAC: abdominal aortic constriction. Data are\npresented as the mean ± SD.\np < 0.05 represents a significant difference between the 18 g/0.45\nmm group and the 22 g/0.45 mm and 26 g/0.45 mm groups.\nBody weight and BW changes in 18-g mice\nBW: body weight. BW changes of 18-g mice before and after surgery for\n8 weeks. Data are presented as the mean ± SD.\np < 0.05 represents a significant difference between the 18 g/0.40\nmm group and the rest groups after surgery.","In this study, we performed AAC according to 4 different ligating intensities for\nmice of 3 different weight levels to evaluate the survival rates of mice and CH\ninduced by PO under different conditions. This is the first study showing that CH\ndiversities exist among groups under different ligations and BW.\nAAC is widely used in the modeling of CH induced by PO in mice. Needle ligation is\nusually used, and the efficiency of modeling is highly dependent on ligation\nintensity. Nevertheless, excessive constriction will lead to death,1 and our research findings (Table 1) demonstrated this point. In this\nstudy, a 0.35-mm needle caused the death of all mice in the 3 weight levels, and the\n0.40-mm needle caused the death of all mice in 22-g and 26-g groups. Contrarily, all\nmice with AAC that underwent surgery with a 0.50-mm needle or sham operation\nsurvived. Mice in the other groups had different mortality rates. Regarding the\nselection of needles for the BW ranges of this study, a needle smaller than 0.35mm\nin diameter caused stronger constriction and death. However, a needle larger than\n0.50 mm in diameter did not alternatively affect the survival rate, but it did\nreduce the efficiency of CH because of the reduced PO from weaker constriction. This\nis why we chose needles between 0.35 mm and 0.50 mm.\nDeath can occur after AAC. Undoubtedly, AAC increases cardiac afterloading. To cope\nwith the additional biodynamics, the heart exerts a series of adaptive changes,\nincluding activation and hypertrophy of cardiomyocytes and hyperplasia of the\nextracellular matrix.2 This\ncompensational mechanism maintains cardiac output (CO) effectively for a period of\ntime while maintaining the survival of the organism; it is also the basis for the\nestablishment of the CH model. However, when the sudden afterloading is out of the\nrange of cardiomyocyte adjustment, the bloodstream will be limited and cause\nconstriction, resulting in AHF. AHF is typically characterized by rapid changes in\nheart failure (HF) symptoms.3 Sato\net al.4 considered the incidence of\ndeath within 5 days as an assessment criterion of AHF. AHF could moderately or\nmarkedly improve by the second day if effectively controlled. AHF leads to high\nventricular pressure, and high ventricular pressure leads to high pulmonary blood\npressure, thus leading to pulmonary congestion, which is one of the causes of death\nafter AAC.5 Liao et al.6 suggested that cardiogenic\npneumo-edema is the main cause of postoperative death for PO mice. Additionally,\narrhythmia may occur as part of the electrophysiological changes,7 and cardiomyocyte sarcomeres may be\ndisordered during the pathological changes.8 These are all severe threats to the survival rate after AAC.\nOur record of mice death times (Table 1)\nshowed the phenomenon of all deaths occurring within 5 days. A high incidence of\ndeath occurred during the initial 24h, which is in accordance with the\naforementioned AHF criteria. In addition, there is a positive correlation between CO\nand BW;9,10 therefore, compared with the low-weight mice, high-weight\nmice require more CO and will have cardiac afterloading that is more increased than\nthat of low-weight mice with the same aortic constriction. Results of the current\nstudy (Table 1) indicate that higher-weight\nmice had poorer tolerance for AAC, which is reflected in their mortality rates.\nRegarding mice with AAC that underwent ligation with a 0.40-mm needle, all mice in\nthe 22-g and 26-g groups died. However, 6 out of 10 mice survived in the 18-g\ngroup.\nThe diagnosis of CH usually depends on changes in cardiac function and\nmorphology.11\nEchocardiography can be performed in vitro noninvasively during the first assessment\nof CH, and it is especially used to monitor changes in cardiac function.12 We performed echocardiographic\nexaminations of mice at the end of week 4 and week 8 post-surgery. Data (week 4 data\nin Table 2) showed that at the end of week 4,\nthe phenomena of thickened ventricular walls, enlarged ventricular chambers, and\ndecreased cardiac functions were emerging in each AAC group compared with the sham\ngroups, and this diversity was consistent with the characteristic cardiac changes\nthat occur with chronic pressure overload.13,14 These trends\nbecame more pronounced at the end of week 8 (week 8 data in Table 2), when EF and FS, which represent cardiac function, were\nsignificantly lower compared with the sham groups. CH also increased HW. In our\nstudy, the HW, HW/BW ratio, and HW/TL ratio for the AAC groups were significantly\nincreased (Table 3). Cardiac remodeling is\nthe most typical pathological change of CH, including cardiomyocyte hypertrophy and\nthe extracellular matrix increases.15 Our histological results showed increased external diameters\nand ventricular thickness in gross hearts and cross-sections under AAC (Figures 1A and B). HE staining of the AAC groups displayed the hypertrophic pathology\nof cardiomyocytes and nuclei (Figure 1C). PSR\nstaining of the AAC groups displayed extensive collagen depositions (Figure 1D), particularly in the perivascular\nspace (Figure 1E). Statistical analysis showed\nthat the thickness of the left ventricle (Table\n4) and the percentage of collagen deposition (Table 5) were significantly increased in the AAC groups compared\nto the sham group. Regarding the formation of collagen, Kuwahara et al.16 indicated that cardiac fibroblasts\nare activated on day 3 after PO, and that the neoformative fibrous tissues mainly\naffect the diastolic function rather than the systolic function during the initial 4\nweeks. Then, excessive myocardial fibrosis is implicated in systolic dysfunction\nbecause of its more intensive traction, and cardiac function begins to deteriorate\nsignificantly. Regarding EF and FS values for the AAC groups (Table 2), the downward trends from week 4 to week 8 conform to\nthis theory.\nChoosing the proper needle is critical for establishing the CH model. Based on these\nresults, we found that all mice with AAC died when a 0.35-mm needle was used for\nligation for all 3 weight levels and when a 0.40-mm needle was used for ligation for\nthe 22-g and 26-g groups; therefore, these 5 groups of weight-needle pairings were\nclearly unsuitable for use. The 18g/0.40mm group had obvious CH compared with the\nsham group, and its survival rate was acceptable (6 out of 10). However, it should\nstill be excluded because the 18 g/0.45 mm group showed more obvious CH and higher\nsurvival rates (8 out of 10) (Table 1, Table 3). The 0.45-mm and 0.50-mm needles are\navailable for all 3 weight levels, but both can result in definite myocardial\nhypertrophy. However, the values of the HW, HW/BW ratio, and HW/TL ratio for the AAC\nmice when using the 0.45-mm needle were significantly higher than those when using a\n0.50-mm needle for each weight level (Table\n3). Therefore, for all 3 weight levels of our study, a CH model can be\nestablished using a 0.50-mm needle and the survival rate of the mice will not be\nthreatened. However, a 0.45-mm needle leads to more effective CH model, and higher\nmortality than the 0.50-mm needle.\nNormally, with the PO-induced CH model, thinner needles creates more severe aortic\nstenosis and lead to more pronounced CH, and vice versa. However, we observed an\ninteresting phenomenon: the CH level of the 18 g/0.45 mm group was abnormally\nsignificantly higher than that of the 18 g/0.40 mm group (18-g mice in Table 3). Regarding the analysis of BW data\nwith AAC (Table 6), the changes in BW in 18-g\nmice during weeks 0 to 8 were significantly higher than those for the 22-g and 26-g\nmice, indicating that 18-g mice have greater potential for physical development\nafter surgery and that physical development is often accompanied by organ\ndevelopment.17 Therefore,\nthe heart of 18-g mice also has greater development potential. For the same weight\nlevel, the BW change of the 18 g/0.45 mm group during weeks 0 to 8 was significantly\nhigher than that of the 18 g/0.40 mm group (BW change in Table 7). As mentioned, BW is positively related to CO;\ntherefore, perhaps the greater ligation limited CO in the 18 g/0.40 mm group, which\nalso limited physical development and organ development, including development of\nthe heart. At the end of week 8, there was no significant difference in BW for the\n18g/0.45mm group and 18g/sham groups; both had significantly higher BW than the 18\ng/0.40 mm group (BW at week 8 in Table 7).\nThe 0.45-mm needle had no obvious limits in18-g mice, but the BW advantage for the\n18 g/0.45 mm group compared to the 18 g/0.40 mm group depends on greater CO and\nrequires more hypertrophic myocardium for support. So, to establish CH models for\nAAC in mice that have developmental potential, such as 18-g mice, there may be a\nspecial ligating intensity region that can cause more obvious CH than the two\nadjacent regions. However, this phenomenon must comprise multiple factors and is\nworth further study.","We established CH models using 4 ligation needle sizes and 3 weights for mice. Data\nshowed that both of 0.45-mm and 0.50-mm needles lead to CH. However, 0.45mm needle\nbrings more effective model and causes obvious CH in 18-g mice."],"string":"[\n \"Cardiac hypertrophy (CH) is a compensatory pathological change that is usually\\ninduced by pressure overload (PO), neurohumoral abnormality, and the effects of\\ncytokines. It is characterized by cardiomyocyte hypertrophy and interstitial\\nhyperplasia, and it results in an enlarged heart and thickening of the heart walls.\\nClinically, CH is involved in the development of many diseases, such as valvular\\ndisease, hypertension, arterial stenosis, and primary myocardial hypertrophy. If\\nthese diseases develop at their own pace, then cardiac function (CF) will gradually\\ndecompensate, leading to heart failure (HF), which severely lowers the quality of\\nlife and increases the mortality rate. Therefore, CH is a widespread concern and has\\nbeen explored at the molecular level by researchers. Due to the high genomic\\nhomology between mice and humans, an established CH model for mice has been widely\\nused in animal experiments, thereby providing an effective research foundation for\\nCH exploration.\\nCurrently, PO-induced CH is a common way to establish the model. Abdominal aortic\\nconstriction (AAC) is highly recommended by researchers because of the high success\\nrate and the ability to perform surgery without the need for thoracotomy or a\\nventilator. However, the modeling effects with different ligating intensities for\\ncertain body weights (BWs) have not yet been reported. Therefore, we used 3\\nfrequently used mice BWs (18 g, 22 g, and 26 g) and 4 different needle sizes (0.35,\\n0.40, 0.45, and 0.50 mm) to establish the CH model for each weight level for AAC,\\nsummarized the survival rates, and evaluated the CH effects.\",\n \" Animal groups and handling One-hundred fifty male C57BL/6 wild-type mice were obtained from the Shanghai\\nSLAC Laboratory Animal Co. Ltd (Shanghai, China). All animals were treated and\\ncared for in accordance with the Guide for the Care and Use of\\nLaboratory Animals (National Institutes of Health, Washington, DC,\\n1996). Experimental protocols were approved by our Institutional Animal Care and\\nUse Committee of Zhejiang University (Hangzhou, China). Mice were selected\\naccording to weights of approximately 18 g (range, 17.3-18.7 g), 22 g (range,\\n20.8-23.0 g), and 26 g (range, 25.1-27.0 g), and they were divided into the\\nfollowing 3 weight levels: 18 g (18.0 ± 0.3 g; n = 50), 22 g (22.0\\n± 0.6 g; n = 50), and 26 g (26.1 ± 0.5 g; n = 50). All weight\\nlevels were divided using sortition randomization method to create a sham group\\n(n = 10) and 4 AAC groups according to ligating intensities (0.35, 0.40, 0.45,\\nand 0.50mm; n = 10 per group). Regarding BW, no significant differences were\\nfound among the 5 groups for each weight level (Table S1), and the preoperative BWs of mice that died and those that\\nsurvived were not significant (Table\\nS2).\\nMice were anesthetized with 4% chloralhydrate (0.1ml/1g BW, intraperitoneal\\ninjection). When the mice did not respond when their toe was pinched, the limbs\\nwere fixed on the operating board in the supine position and the skin was\\nprepared by shaving and disinfection with alcohol. Sterile gauze was placed on\\nthe right side of the abdomen and a ventrimesal incision approximately 1.5 cm\\nwas created starting from the xiphoid. The skin was fixed with a spreader and\\nthe viscera was pulled out gently with a swab and placed on the gauze. Then, the\\nabdominal aorta was isolated using a blunt dissection technique with curved\\nmicroforceps under a microscope. A 6-0 silk suture was snared and pulled back\\naround the aorta 1mm above the superior mesenteric artery. A 2-mm blunt\\nacupuncture needle (external diameters: 0.35 mm, 0.40 mm, 0.45 mm, and 0.50 mm;\\nHuatuo; Suzhou Medical Appliance Factory, Suzhou, China; criterion number\\nGB2024-1994) was then placed next to the aorta. The suture was tied snugly\\naround the needle and the aorta. The needle was removed immediately after\\nligation, the viscera were replaced, the peritoneum and skin were sutured, and\\nthe mice were allowed to recover. Aortic ligation was omitted only for the sham\\ngroup. After surgery, the ears were cut to differentiate the mice. Then, mice\\nwere placed in an incubator at 30ºC until they woke, and they were\\nreturned to their cages. Survival status was recorded daily. To observe the\\nphysical development of mice under different conditions, BW differences before\\nsurgery and at week 8 post-surgery were calculated as the change in BW.\\nOne-hundred fifty male C57BL/6 wild-type mice were obtained from the Shanghai\\nSLAC Laboratory Animal Co. Ltd (Shanghai, China). All animals were treated and\\ncared for in accordance with the Guide for the Care and Use of\\nLaboratory Animals (National Institutes of Health, Washington, DC,\\n1996). Experimental protocols were approved by our Institutional Animal Care and\\nUse Committee of Zhejiang University (Hangzhou, China). Mice were selected\\naccording to weights of approximately 18 g (range, 17.3-18.7 g), 22 g (range,\\n20.8-23.0 g), and 26 g (range, 25.1-27.0 g), and they were divided into the\\nfollowing 3 weight levels: 18 g (18.0 ± 0.3 g; n = 50), 22 g (22.0\\n± 0.6 g; n = 50), and 26 g (26.1 ± 0.5 g; n = 50). All weight\\nlevels were divided using sortition randomization method to create a sham group\\n(n = 10) and 4 AAC groups according to ligating intensities (0.35, 0.40, 0.45,\\nand 0.50mm; n = 10 per group). Regarding BW, no significant differences were\\nfound among the 5 groups for each weight level (Table S1), and the preoperative BWs of mice that died and those that\\nsurvived were not significant (Table\\nS2).\\nMice were anesthetized with 4% chloralhydrate (0.1ml/1g BW, intraperitoneal\\ninjection). When the mice did not respond when their toe was pinched, the limbs\\nwere fixed on the operating board in the supine position and the skin was\\nprepared by shaving and disinfection with alcohol. Sterile gauze was placed on\\nthe right side of the abdomen and a ventrimesal incision approximately 1.5 cm\\nwas created starting from the xiphoid. The skin was fixed with a spreader and\\nthe viscera was pulled out gently with a swab and placed on the gauze. Then, the\\nabdominal aorta was isolated using a blunt dissection technique with curved\\nmicroforceps under a microscope. A 6-0 silk suture was snared and pulled back\\naround the aorta 1mm above the superior mesenteric artery. A 2-mm blunt\\nacupuncture needle (external diameters: 0.35 mm, 0.40 mm, 0.45 mm, and 0.50 mm;\\nHuatuo; Suzhou Medical Appliance Factory, Suzhou, China; criterion number\\nGB2024-1994) was then placed next to the aorta. The suture was tied snugly\\naround the needle and the aorta. The needle was removed immediately after\\nligation, the viscera were replaced, the peritoneum and skin were sutured, and\\nthe mice were allowed to recover. Aortic ligation was omitted only for the sham\\ngroup. After surgery, the ears were cut to differentiate the mice. Then, mice\\nwere placed in an incubator at 30ºC until they woke, and they were\\nreturned to their cages. Survival status was recorded daily. To observe the\\nphysical development of mice under different conditions, BW differences before\\nsurgery and at week 8 post-surgery were calculated as the change in BW.\\n Echocardiography imaging After post-surgery weeks 4 and 8, mice were weighed and anesthetized with 4%\\nchloralhydrate and placed on a warming pad after skin preparation. Transthoracic\\n2-dimensional (2D) echocardiography was performed using the GE Vivid E9\\nUltrasound echocardiographic system (General Electric Company, Fairfield, CT,\\nUSA) with the GE 9L probe (8-MHz linear array transducer; General Electric\\nCompany). M-mode parasternal long-axis scans of the left ventricle at the mitral\\nchordae level were used to quantify the interventricular septum thickness at\\nend-diastole (IVSd), interventricular septum thickness at end-systole (IVSs),\\nleft ventricular internal dimension at end-diastole (LVIDd), left ventricular\\ninternal dimension at end-systole (LVIDs), left ventricular posterior wall\\nthickness at end-diastole (LVPWd), left ventricular posterior wall thickness at\\nend-systole (LVPWs), ejection fraction (EF), and fractional shortening (FS). All\\nmice were tested using the same parameters.\\nAfter post-surgery weeks 4 and 8, mice were weighed and anesthetized with 4%\\nchloralhydrate and placed on a warming pad after skin preparation. Transthoracic\\n2-dimensional (2D) echocardiography was performed using the GE Vivid E9\\nUltrasound echocardiographic system (General Electric Company, Fairfield, CT,\\nUSA) with the GE 9L probe (8-MHz linear array transducer; General Electric\\nCompany). M-mode parasternal long-axis scans of the left ventricle at the mitral\\nchordae level were used to quantify the interventricular septum thickness at\\nend-diastole (IVSd), interventricular septum thickness at end-systole (IVSs),\\nleft ventricular internal dimension at end-diastole (LVIDd), left ventricular\\ninternal dimension at end-systole (LVIDs), left ventricular posterior wall\\nthickness at end-diastole (LVPWd), left ventricular posterior wall thickness at\\nend-systole (LVPWs), ejection fraction (EF), and fractional shortening (FS). All\\nmice were tested using the same parameters.\\n Heart weight, heart weight/body weight, and heart weight/tibial\\nlength After echocardiographic analysis at 8 weeks post-surgery, mice were sacrificed by\\ncervical dislocation and the hearts were dissected. Then, atrial and vascular\\ntissues were snipped carefully, leaving the ventricles. The hearts were rinsed\\nwith phosphate-buffered saline (PBS), drained by gently squeezing on absorbent\\npaper, weighed, photographed under natural light, and fixed in 4%\\nparaformaldehyde. The tibial lengths (TLs; mean value of the bilateral tibia)\\nwere recorded. Heart weight (HW), BW, and TL were measured, and the HW/BW ratio\\nand HW/TL ratio were calculated to evaluate the hypertrophic response to PO.\\nAfter echocardiographic analysis at 8 weeks post-surgery, mice were sacrificed by\\ncervical dislocation and the hearts were dissected. Then, atrial and vascular\\ntissues were snipped carefully, leaving the ventricles. The hearts were rinsed\\nwith phosphate-buffered saline (PBS), drained by gently squeezing on absorbent\\npaper, weighed, photographed under natural light, and fixed in 4%\\nparaformaldehyde. The tibial lengths (TLs; mean value of the bilateral tibia)\\nwere recorded. Heart weight (HW), BW, and TL were measured, and the HW/BW ratio\\nand HW/TL ratio were calculated to evaluate the hypertrophic response to PO.\\n Histological examination of the heart Extracted hearts were fixed in 4% paraformaldehyde for 24h and dehydrated. After\\nroutine histologic procedures, the hearts were embedded in paraffin and cut into\\n4-µm sections. Sections were stained with hematoxylin and eosin (HE) and\\npicrosirius red (PSR). Cardiac cross-sections were captured at 20 ×\\nmicroscopic views from HE sections, and 5 thicknesses of the left ventricle in\\neach view were selected in systematic sampling, and measured using Image-Pro\\nPlus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Then, the mean values\\nwere calculated. Cardiomyocyte morphological changes were captured at\\n400×microscopic views from HE sections. Interstitial and/or perivascular\\ncollagen depositions were captured at 200×microscopic views under\\nstandard lights. Collagen was stained red using PSR, thereby indicating\\nfibrosis. At least 6 views were selected in a blinded manner, and each\\nphotograph was analyzed to reveal the ratio of red collagen to the entire tissue\\narea using Image-Pro Plus 6.0. Then, the mean values were calculated.\\nExtracted hearts were fixed in 4% paraformaldehyde for 24h and dehydrated. After\\nroutine histologic procedures, the hearts were embedded in paraffin and cut into\\n4-µm sections. Sections were stained with hematoxylin and eosin (HE) and\\npicrosirius red (PSR). Cardiac cross-sections were captured at 20 ×\\nmicroscopic views from HE sections, and 5 thicknesses of the left ventricle in\\neach view were selected in systematic sampling, and measured using Image-Pro\\nPlus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Then, the mean values\\nwere calculated. Cardiomyocyte morphological changes were captured at\\n400×microscopic views from HE sections. Interstitial and/or perivascular\\ncollagen depositions were captured at 200×microscopic views under\\nstandard lights. Collagen was stained red using PSR, thereby indicating\\nfibrosis. At least 6 views were selected in a blinded manner, and each\\nphotograph was analyzed to reveal the ratio of red collagen to the entire tissue\\narea using Image-Pro Plus 6.0. Then, the mean values were calculated.\\n Statistical Analysis SPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA) was used for all\\nstatistical analyses. The Kolmogorov-Smirnov (K-S) test was used to verify the\\nnormality of the quantitative variables as appropriate. Data are presented as\\nmean ± standard deviation (SD). One-way ANOVA and post-hoc Tukey tests\\nwere used to evaluate differences between groups. p < 0.05 was considered\\nstatistically significant.\\nSPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA) was used for all\\nstatistical analyses. The Kolmogorov-Smirnov (K-S) test was used to verify the\\nnormality of the quantitative variables as appropriate. Data are presented as\\nmean ± standard deviation (SD). One-way ANOVA and post-hoc Tukey tests\\nwere used to evaluate differences between groups. p < 0.05 was considered\\nstatistically significant.\",\n \"One-hundred fifty male C57BL/6 wild-type mice were obtained from the Shanghai\\nSLAC Laboratory Animal Co. Ltd (Shanghai, China). All animals were treated and\\ncared for in accordance with the Guide for the Care and Use of\\nLaboratory Animals (National Institutes of Health, Washington, DC,\\n1996). Experimental protocols were approved by our Institutional Animal Care and\\nUse Committee of Zhejiang University (Hangzhou, China). Mice were selected\\naccording to weights of approximately 18 g (range, 17.3-18.7 g), 22 g (range,\\n20.8-23.0 g), and 26 g (range, 25.1-27.0 g), and they were divided into the\\nfollowing 3 weight levels: 18 g (18.0 ± 0.3 g; n = 50), 22 g (22.0\\n± 0.6 g; n = 50), and 26 g (26.1 ± 0.5 g; n = 50). All weight\\nlevels were divided using sortition randomization method to create a sham group\\n(n = 10) and 4 AAC groups according to ligating intensities (0.35, 0.40, 0.45,\\nand 0.50mm; n = 10 per group). Regarding BW, no significant differences were\\nfound among the 5 groups for each weight level (Table S1), and the preoperative BWs of mice that died and those that\\nsurvived were not significant (Table\\nS2).\\nMice were anesthetized with 4% chloralhydrate (0.1ml/1g BW, intraperitoneal\\ninjection). When the mice did not respond when their toe was pinched, the limbs\\nwere fixed on the operating board in the supine position and the skin was\\nprepared by shaving and disinfection with alcohol. Sterile gauze was placed on\\nthe right side of the abdomen and a ventrimesal incision approximately 1.5 cm\\nwas created starting from the xiphoid. The skin was fixed with a spreader and\\nthe viscera was pulled out gently with a swab and placed on the gauze. Then, the\\nabdominal aorta was isolated using a blunt dissection technique with curved\\nmicroforceps under a microscope. A 6-0 silk suture was snared and pulled back\\naround the aorta 1mm above the superior mesenteric artery. A 2-mm blunt\\nacupuncture needle (external diameters: 0.35 mm, 0.40 mm, 0.45 mm, and 0.50 mm;\\nHuatuo; Suzhou Medical Appliance Factory, Suzhou, China; criterion number\\nGB2024-1994) was then placed next to the aorta. The suture was tied snugly\\naround the needle and the aorta. The needle was removed immediately after\\nligation, the viscera were replaced, the peritoneum and skin were sutured, and\\nthe mice were allowed to recover. Aortic ligation was omitted only for the sham\\ngroup. After surgery, the ears were cut to differentiate the mice. Then, mice\\nwere placed in an incubator at 30ºC until they woke, and they were\\nreturned to their cages. Survival status was recorded daily. To observe the\\nphysical development of mice under different conditions, BW differences before\\nsurgery and at week 8 post-surgery were calculated as the change in BW.\",\n \"After post-surgery weeks 4 and 8, mice were weighed and anesthetized with 4%\\nchloralhydrate and placed on a warming pad after skin preparation. Transthoracic\\n2-dimensional (2D) echocardiography was performed using the GE Vivid E9\\nUltrasound echocardiographic system (General Electric Company, Fairfield, CT,\\nUSA) with the GE 9L probe (8-MHz linear array transducer; General Electric\\nCompany). M-mode parasternal long-axis scans of the left ventricle at the mitral\\nchordae level were used to quantify the interventricular septum thickness at\\nend-diastole (IVSd), interventricular septum thickness at end-systole (IVSs),\\nleft ventricular internal dimension at end-diastole (LVIDd), left ventricular\\ninternal dimension at end-systole (LVIDs), left ventricular posterior wall\\nthickness at end-diastole (LVPWd), left ventricular posterior wall thickness at\\nend-systole (LVPWs), ejection fraction (EF), and fractional shortening (FS). All\\nmice were tested using the same parameters.\",\n \"After echocardiographic analysis at 8 weeks post-surgery, mice were sacrificed by\\ncervical dislocation and the hearts were dissected. Then, atrial and vascular\\ntissues were snipped carefully, leaving the ventricles. The hearts were rinsed\\nwith phosphate-buffered saline (PBS), drained by gently squeezing on absorbent\\npaper, weighed, photographed under natural light, and fixed in 4%\\nparaformaldehyde. The tibial lengths (TLs; mean value of the bilateral tibia)\\nwere recorded. Heart weight (HW), BW, and TL were measured, and the HW/BW ratio\\nand HW/TL ratio were calculated to evaluate the hypertrophic response to PO.\",\n \"Extracted hearts were fixed in 4% paraformaldehyde for 24h and dehydrated. After\\nroutine histologic procedures, the hearts were embedded in paraffin and cut into\\n4-µm sections. Sections were stained with hematoxylin and eosin (HE) and\\npicrosirius red (PSR). Cardiac cross-sections were captured at 20 ×\\nmicroscopic views from HE sections, and 5 thicknesses of the left ventricle in\\neach view were selected in systematic sampling, and measured using Image-Pro\\nPlus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Then, the mean values\\nwere calculated. Cardiomyocyte morphological changes were captured at\\n400×microscopic views from HE sections. Interstitial and/or perivascular\\ncollagen depositions were captured at 200×microscopic views under\\nstandard lights. Collagen was stained red using PSR, thereby indicating\\nfibrosis. At least 6 views were selected in a blinded manner, and each\\nphotograph was analyzed to reveal the ratio of red collagen to the entire tissue\\narea using Image-Pro Plus 6.0. Then, the mean values were calculated.\",\n \"SPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA) was used for all\\nstatistical analyses. The Kolmogorov-Smirnov (K-S) test was used to verify the\\nnormality of the quantitative variables as appropriate. Data are presented as\\nmean ± standard deviation (SD). One-way ANOVA and post-hoc Tukey tests\\nwere used to evaluate differences between groups. p < 0.05 was considered\\nstatistically significant.\",\n \" Excessive AAC may lead to death We monitored mice deaths after surgery according to acute heart failure (AHF)\\ncriteria. Data (Table 1) showed that all\\ndeaths occurred within 5 days, and a high incidence of death occurred during the\\ninitial 24h post-surgery.\\nMice deaths after surgery\\nThere were no mice deaths in the AAC0.50-mm group or the sham group.\\nDeaths were recorded during 3 time periods (0-24h, 24h-3d, and 3-5\\nd); 54 deaths occurred within 0-24h post-surgery. The total number\\nof deaths was 65.\\nWe monitored mice deaths after surgery according to acute heart failure (AHF)\\ncriteria. Data (Table 1) showed that all\\ndeaths occurred within 5 days, and a high incidence of death occurred during the\\ninitial 24h post-surgery.\\nMice deaths after surgery\\nThere were no mice deaths in the AAC0.50-mm group or the sham group.\\nDeaths were recorded during 3 time periods (0-24h, 24h-3d, and 3-5\\nd); 54 deaths occurred within 0-24h post-surgery. The total number\\nof deaths was 65.\\n AAC increases cardiac dimensions and reduces cardiac function Echocardiography was performed at the end of post-operative weeks 4 and 8. At\\nweek 4 post-surgery, data (Table 2)\\nshowed a trend of heart enlargement for mice with AAC, including thickening of\\nthe ventricular wall and an increase in chamber dilation; however, differences\\nin EF and FS were not significant, indicating that changes in the heart\\nstructure did not have a pronounced effect on cardiac function at that time\\npoint. At week 8 post-surgery, the trend of heart enlargement continued;\\nhowever, the EF and FS values for the AAC groups decreased significantly. This\\nchange in cardiac function from week 4 to week 8 was consistent with systolic\\nfunction beginning to be markedly affected at week 4 after PO surgery.\\nEchocardiographic outcomes of 18-g, 22-g, and 26-g mice\\nIVSd: interventricular septum thickness at end-diastole; IVSs:\\ninterventricular septum thickness at end-systole; LVIDd: left\\nventricular internal dimension at end-diastole; LVIDs: left\\nventricular internal dimension at end-systole; LVPWd: left\\nventricular posterior wall thickness at end-diastole; LVPWs: left\\nventricular posterior wall thickness at end-systole; EF: ejection\\nfraction; FS: fractional shortening. The cardiac dimensions\\n(inclding IVSd, IVSs, LVIDd, LVIDs, LVPWd, and LVPWs) (mm) and\\nfunctional indices (inclding EF and FS) changes measured by\\nechocardiography. Data were statistically analyzed and presented as\\nthe mean ± SD. At week 4, cardiac dimensions for the AAC\\ngroups significantly increased compared with the sham groups (*p\\n< 0.05); at week 8 , more cardiac dimensions significantly\\nincreased, and that EF and FS values for the AAC groups all\\ndecreased significantly compared to those of the sham groups for 3\\nweight levels (*p < 0.05).\\nEchocardiography was performed at the end of post-operative weeks 4 and 8. At\\nweek 4 post-surgery, data (Table 2)\\nshowed a trend of heart enlargement for mice with AAC, including thickening of\\nthe ventricular wall and an increase in chamber dilation; however, differences\\nin EF and FS were not significant, indicating that changes in the heart\\nstructure did not have a pronounced effect on cardiac function at that time\\npoint. At week 8 post-surgery, the trend of heart enlargement continued;\\nhowever, the EF and FS values for the AAC groups decreased significantly. This\\nchange in cardiac function from week 4 to week 8 was consistent with systolic\\nfunction beginning to be markedly affected at week 4 after PO surgery.\\nEchocardiographic outcomes of 18-g, 22-g, and 26-g mice\\nIVSd: interventricular septum thickness at end-diastole; IVSs:\\ninterventricular septum thickness at end-systole; LVIDd: left\\nventricular internal dimension at end-diastole; LVIDs: left\\nventricular internal dimension at end-systole; LVPWd: left\\nventricular posterior wall thickness at end-diastole; LVPWs: left\\nventricular posterior wall thickness at end-systole; EF: ejection\\nfraction; FS: fractional shortening. The cardiac dimensions\\n(inclding IVSd, IVSs, LVIDd, LVIDs, LVPWd, and LVPWs) (mm) and\\nfunctional indices (inclding EF and FS) changes measured by\\nechocardiography. Data were statistically analyzed and presented as\\nthe mean ± SD. At week 4, cardiac dimensions for the AAC\\ngroups significantly increased compared with the sham groups (*p\\n< 0.05); at week 8 , more cardiac dimensions significantly\\nincreased, and that EF and FS values for the AAC groups all\\ndecreased significantly compared to those of the sham groups for 3\\nweight levels (*p < 0.05).\\n AAC increases HW, HW/BW, and HW/TL ratio Generally, the increased HW, HW/BW, and HW/TL ratio are the three main indicators\\nof CH. In our study, as shown in Table 3,\\nwe found that AAC significantly increased HW and caused a significantly higher\\nHW/BW ratio and HW/TL ratio compared to the sham groups for all weight levels.\\nThe HW, HW/BW, and HW/TL values for the AAC0.45 mm groups were significantly\\nhigher than those for the AAC0.50 mm groups. These HW-related indices for the 18\\ng/0.45 mm groups were even significantly higher than those for the 18 g/0.40 mm\\ngroups.\\nHeart weight–related indices of 18-g, 22-g, and 26-g mice\\nHW: heart weight; BW: body weight; TL: tibial length. HW(mg),\\nHW/BW(mg/g), and HW/TL(mg/cm) were measured and calculated from AAC\\nand sham groups of 3 BW levels. Data are presented as the mean\\n± SD.\\nCompared to the sham group in the same BW level, the heart\\nweight–related indices of AAC groups increased significantly (p <\\n0.05).\\nCompared to the rest groups in the same BW level, the heart\\nweight–related indices of the AAC 0.45-mm group increased\\nsignificantly (p < 0.05).\\nGenerally, the increased HW, HW/BW, and HW/TL ratio are the three main indicators\\nof CH. In our study, as shown in Table 3,\\nwe found that AAC significantly increased HW and caused a significantly higher\\nHW/BW ratio and HW/TL ratio compared to the sham groups for all weight levels.\\nThe HW, HW/BW, and HW/TL values for the AAC0.45 mm groups were significantly\\nhigher than those for the AAC0.50 mm groups. These HW-related indices for the 18\\ng/0.45 mm groups were even significantly higher than those for the 18 g/0.40 mm\\ngroups.\\nHeart weight–related indices of 18-g, 22-g, and 26-g mice\\nHW: heart weight; BW: body weight; TL: tibial length. HW(mg),\\nHW/BW(mg/g), and HW/TL(mg/cm) were measured and calculated from AAC\\nand sham groups of 3 BW levels. Data are presented as the mean\\n± SD.\\nCompared to the sham group in the same BW level, the heart\\nweight–related indices of AAC groups increased significantly (p <\\n0.05).\\nCompared to the rest groups in the same BW level, the heart\\nweight–related indices of the AAC 0.45-mm group increased\\nsignificantly (p < 0.05).\\n AAC leads to cardiomyocyte hypertrophy and increases collagen\\ndepositions For mice undergoing AAC surgery, the hearts demonstrated different degrees of\\nenlargement (Figure 1A), enlargement of the\\npapillary muscles, and thickening of the ventricular walls (Figure 1B). Wall thickening increased significantly compared\\nwith that of the sham group (Table 4).\\nThe sham groups showed normal architecture of the cardiomyocytes compared with\\nthe AAC groups. Pathological changes including enlarged, disarrayed, and\\neosinophilic cardiomyocytes and cardiomyocytes rich in cytoplasm and\\ntrachychromatic and pantomorphic nuclei were observed in each of the AAC groups\\n(Figure 1C). Scattered collagen\\ndepositions in the interstitial and perivascular spaces were observed in the\\nsham groups. In comparison, in some AAC groups, a larger quantity and wider\\nrange of red deposits were observed in the interstitial space (Figure 1D), and thickened collagen was\\nobserved in the perivascular space, especially in the external vascular wall\\n(Figure 1E). Statistical analysis\\nindicated that the AAC group had a significantly greater collagen area than the\\nsham group (Table 5). These results imply\\nthat AAC is capable of inducing PO-induced CH and fibrosis.\\n\\nFigure 1Cardiomyocyte hypertrophy and collagen deposition histological\\nexamination. (A) Gross hearts under natural light. (B) The 20\\n× microscopic views of HE sections. (C) The 400 ×\\nmicroscopic views of HE sections. (D) Representative\\n200×microscopic views under standard lights of PSR sections\\nin the interstitial space. (E) Representative 200 ×\\nmicroscopic views under standard lights of PSR sections in the\\nperivascular space. Fibrosis is presented as red in the PSR\\nsections.\\n\\nCardiomyocyte hypertrophy and collagen deposition histological\\nexamination. (A) Gross hearts under natural light. (B) The 20\\n× microscopic views of HE sections. (C) The 400 ×\\nmicroscopic views of HE sections. (D) Representative\\n200×microscopic views under standard lights of PSR sections\\nin the interstitial space. (E) Representative 200 ×\\nmicroscopic views under standard lights of PSR sections in the\\nperivascular space. Fibrosis is presented as red in the PSR\\nsections.\\nThickness of the left ventricle (mm) based on weight and needle size\\nData are presented as the mean ± SD (n = 5).\\np < 0.05 represents a significant difference between the abdominal\\naortic constriction (AAC) and sham groups.\\nPercentage of collagen deposition in the left ventricle based on weight\\nand needle size\\nData are presented as the mean ± SD (n = 6).\\np < 0.05 represents a significant difference between the abdominal\\naortic constriction (AAC) and sham groups.\\nFor mice undergoing AAC surgery, the hearts demonstrated different degrees of\\nenlargement (Figure 1A), enlargement of the\\npapillary muscles, and thickening of the ventricular walls (Figure 1B). Wall thickening increased significantly compared\\nwith that of the sham group (Table 4).\\nThe sham groups showed normal architecture of the cardiomyocytes compared with\\nthe AAC groups. Pathological changes including enlarged, disarrayed, and\\neosinophilic cardiomyocytes and cardiomyocytes rich in cytoplasm and\\ntrachychromatic and pantomorphic nuclei were observed in each of the AAC groups\\n(Figure 1C). Scattered collagen\\ndepositions in the interstitial and perivascular spaces were observed in the\\nsham groups. In comparison, in some AAC groups, a larger quantity and wider\\nrange of red deposits were observed in the interstitial space (Figure 1D), and thickened collagen was\\nobserved in the perivascular space, especially in the external vascular wall\\n(Figure 1E). Statistical analysis\\nindicated that the AAC group had a significantly greater collagen area than the\\nsham group (Table 5). These results imply\\nthat AAC is capable of inducing PO-induced CH and fibrosis.\\n\\nFigure 1Cardiomyocyte hypertrophy and collagen deposition histological\\nexamination. (A) Gross hearts under natural light. (B) The 20\\n× microscopic views of HE sections. (C) The 400 ×\\nmicroscopic views of HE sections. (D) Representative\\n200×microscopic views under standard lights of PSR sections\\nin the interstitial space. (E) Representative 200 ×\\nmicroscopic views under standard lights of PSR sections in the\\nperivascular space. Fibrosis is presented as red in the PSR\\nsections.\\n\\nCardiomyocyte hypertrophy and collagen deposition histological\\nexamination. (A) Gross hearts under natural light. (B) The 20\\n× microscopic views of HE sections. (C) The 400 ×\\nmicroscopic views of HE sections. (D) Representative\\n200×microscopic views under standard lights of PSR sections\\nin the interstitial space. (E) Representative 200 ×\\nmicroscopic views under standard lights of PSR sections in the\\nperivascular space. Fibrosis is presented as red in the PSR\\nsections.\\nThickness of the left ventricle (mm) based on weight and needle size\\nData are presented as the mean ± SD (n = 5).\\np < 0.05 represents a significant difference between the abdominal\\naortic constriction (AAC) and sham groups.\\nPercentage of collagen deposition in the left ventricle based on weight\\nand needle size\\nData are presented as the mean ± SD (n = 6).\\np < 0.05 represents a significant difference between the abdominal\\naortic constriction (AAC) and sham groups.\\n AAC may restrict physical development Analysis showed that with AAC 0.45 mm, BW significantly increased in 18-g mice\\ncompared to 22-g and 26-g mice (Table 6),\\nindicating that the 18-g groups had higher development potential. In the 18-g\\nmice groups, data showed that the value of 18 g/0.40 mm was significantly lower\\nthan that of the 18 g/0.45 mm and 18 g/sham groups, and that there were no\\nsignificant differences between the 18 g/0.45 mm and 18 g/sham groups (Table 7), indicating that the 18 g/0.45 mm\\ngroup had nearly normal physical development. Development of the 18 g/0.40 mm\\ngroup was limited.\\nBody weight changes with AAC under 0.45 mm needle\\nBW: body weight; AAC: abdominal aortic constriction. Data are\\npresented as the mean ± SD.\\np < 0.05 represents a significant difference between the 18 g/0.45\\nmm group and the 22 g/0.45 mm and 26 g/0.45 mm groups.\\nBody weight and BW changes in 18-g mice\\nBW: body weight. BW changes of 18-g mice before and after surgery for\\n8 weeks. Data are presented as the mean ± SD.\\np < 0.05 represents a significant difference between the 18 g/0.40\\nmm group and the rest groups after surgery.\\nAnalysis showed that with AAC 0.45 mm, BW significantly increased in 18-g mice\\ncompared to 22-g and 26-g mice (Table 6),\\nindicating that the 18-g groups had higher development potential. In the 18-g\\nmice groups, data showed that the value of 18 g/0.40 mm was significantly lower\\nthan that of the 18 g/0.45 mm and 18 g/sham groups, and that there were no\\nsignificant differences between the 18 g/0.45 mm and 18 g/sham groups (Table 7), indicating that the 18 g/0.45 mm\\ngroup had nearly normal physical development. Development of the 18 g/0.40 mm\\ngroup was limited.\\nBody weight changes with AAC under 0.45 mm needle\\nBW: body weight; AAC: abdominal aortic constriction. Data are\\npresented as the mean ± SD.\\np < 0.05 represents a significant difference between the 18 g/0.45\\nmm group and the 22 g/0.45 mm and 26 g/0.45 mm groups.\\nBody weight and BW changes in 18-g mice\\nBW: body weight. BW changes of 18-g mice before and after surgery for\\n8 weeks. Data are presented as the mean ± SD.\\np < 0.05 represents a significant difference between the 18 g/0.40\\nmm group and the rest groups after surgery.\",\n \"We monitored mice deaths after surgery according to acute heart failure (AHF)\\ncriteria. Data (Table 1) showed that all\\ndeaths occurred within 5 days, and a high incidence of death occurred during the\\ninitial 24h post-surgery.\\nMice deaths after surgery\\nThere were no mice deaths in the AAC0.50-mm group or the sham group.\\nDeaths were recorded during 3 time periods (0-24h, 24h-3d, and 3-5\\nd); 54 deaths occurred within 0-24h post-surgery. The total number\\nof deaths was 65.\",\n \"Echocardiography was performed at the end of post-operative weeks 4 and 8. At\\nweek 4 post-surgery, data (Table 2)\\nshowed a trend of heart enlargement for mice with AAC, including thickening of\\nthe ventricular wall and an increase in chamber dilation; however, differences\\nin EF and FS were not significant, indicating that changes in the heart\\nstructure did not have a pronounced effect on cardiac function at that time\\npoint. At week 8 post-surgery, the trend of heart enlargement continued;\\nhowever, the EF and FS values for the AAC groups decreased significantly. This\\nchange in cardiac function from week 4 to week 8 was consistent with systolic\\nfunction beginning to be markedly affected at week 4 after PO surgery.\\nEchocardiographic outcomes of 18-g, 22-g, and 26-g mice\\nIVSd: interventricular septum thickness at end-diastole; IVSs:\\ninterventricular septum thickness at end-systole; LVIDd: left\\nventricular internal dimension at end-diastole; LVIDs: left\\nventricular internal dimension at end-systole; LVPWd: left\\nventricular posterior wall thickness at end-diastole; LVPWs: left\\nventricular posterior wall thickness at end-systole; EF: ejection\\nfraction; FS: fractional shortening. The cardiac dimensions\\n(inclding IVSd, IVSs, LVIDd, LVIDs, LVPWd, and LVPWs) (mm) and\\nfunctional indices (inclding EF and FS) changes measured by\\nechocardiography. Data were statistically analyzed and presented as\\nthe mean ± SD. At week 4, cardiac dimensions for the AAC\\ngroups significantly increased compared with the sham groups (*p\\n< 0.05); at week 8 , more cardiac dimensions significantly\\nincreased, and that EF and FS values for the AAC groups all\\ndecreased significantly compared to those of the sham groups for 3\\nweight levels (*p < 0.05).\",\n \"Generally, the increased HW, HW/BW, and HW/TL ratio are the three main indicators\\nof CH. In our study, as shown in Table 3,\\nwe found that AAC significantly increased HW and caused a significantly higher\\nHW/BW ratio and HW/TL ratio compared to the sham groups for all weight levels.\\nThe HW, HW/BW, and HW/TL values for the AAC0.45 mm groups were significantly\\nhigher than those for the AAC0.50 mm groups. These HW-related indices for the 18\\ng/0.45 mm groups were even significantly higher than those for the 18 g/0.40 mm\\ngroups.\\nHeart weight–related indices of 18-g, 22-g, and 26-g mice\\nHW: heart weight; BW: body weight; TL: tibial length. HW(mg),\\nHW/BW(mg/g), and HW/TL(mg/cm) were measured and calculated from AAC\\nand sham groups of 3 BW levels. Data are presented as the mean\\n± SD.\\nCompared to the sham group in the same BW level, the heart\\nweight–related indices of AAC groups increased significantly (p <\\n0.05).\\nCompared to the rest groups in the same BW level, the heart\\nweight–related indices of the AAC 0.45-mm group increased\\nsignificantly (p < 0.05).\",\n \"For mice undergoing AAC surgery, the hearts demonstrated different degrees of\\nenlargement (Figure 1A), enlargement of the\\npapillary muscles, and thickening of the ventricular walls (Figure 1B). Wall thickening increased significantly compared\\nwith that of the sham group (Table 4).\\nThe sham groups showed normal architecture of the cardiomyocytes compared with\\nthe AAC groups. Pathological changes including enlarged, disarrayed, and\\neosinophilic cardiomyocytes and cardiomyocytes rich in cytoplasm and\\ntrachychromatic and pantomorphic nuclei were observed in each of the AAC groups\\n(Figure 1C). Scattered collagen\\ndepositions in the interstitial and perivascular spaces were observed in the\\nsham groups. In comparison, in some AAC groups, a larger quantity and wider\\nrange of red deposits were observed in the interstitial space (Figure 1D), and thickened collagen was\\nobserved in the perivascular space, especially in the external vascular wall\\n(Figure 1E). Statistical analysis\\nindicated that the AAC group had a significantly greater collagen area than the\\nsham group (Table 5). These results imply\\nthat AAC is capable of inducing PO-induced CH and fibrosis.\\n\\nFigure 1Cardiomyocyte hypertrophy and collagen deposition histological\\nexamination. (A) Gross hearts under natural light. (B) The 20\\n× microscopic views of HE sections. (C) The 400 ×\\nmicroscopic views of HE sections. (D) Representative\\n200×microscopic views under standard lights of PSR sections\\nin the interstitial space. (E) Representative 200 ×\\nmicroscopic views under standard lights of PSR sections in the\\nperivascular space. Fibrosis is presented as red in the PSR\\nsections.\\n\\nCardiomyocyte hypertrophy and collagen deposition histological\\nexamination. (A) Gross hearts under natural light. (B) The 20\\n× microscopic views of HE sections. (C) The 400 ×\\nmicroscopic views of HE sections. (D) Representative\\n200×microscopic views under standard lights of PSR sections\\nin the interstitial space. (E) Representative 200 ×\\nmicroscopic views under standard lights of PSR sections in the\\nperivascular space. Fibrosis is presented as red in the PSR\\nsections.\\nThickness of the left ventricle (mm) based on weight and needle size\\nData are presented as the mean ± SD (n = 5).\\np < 0.05 represents a significant difference between the abdominal\\naortic constriction (AAC) and sham groups.\\nPercentage of collagen deposition in the left ventricle based on weight\\nand needle size\\nData are presented as the mean ± SD (n = 6).\\np < 0.05 represents a significant difference between the abdominal\\naortic constriction (AAC) and sham groups.\",\n \"Analysis showed that with AAC 0.45 mm, BW significantly increased in 18-g mice\\ncompared to 22-g and 26-g mice (Table 6),\\nindicating that the 18-g groups had higher development potential. In the 18-g\\nmice groups, data showed that the value of 18 g/0.40 mm was significantly lower\\nthan that of the 18 g/0.45 mm and 18 g/sham groups, and that there were no\\nsignificant differences between the 18 g/0.45 mm and 18 g/sham groups (Table 7), indicating that the 18 g/0.45 mm\\ngroup had nearly normal physical development. Development of the 18 g/0.40 mm\\ngroup was limited.\\nBody weight changes with AAC under 0.45 mm needle\\nBW: body weight; AAC: abdominal aortic constriction. Data are\\npresented as the mean ± SD.\\np < 0.05 represents a significant difference between the 18 g/0.45\\nmm group and the 22 g/0.45 mm and 26 g/0.45 mm groups.\\nBody weight and BW changes in 18-g mice\\nBW: body weight. BW changes of 18-g mice before and after surgery for\\n8 weeks. Data are presented as the mean ± SD.\\np < 0.05 represents a significant difference between the 18 g/0.40\\nmm group and the rest groups after surgery.\",\n \"In this study, we performed AAC according to 4 different ligating intensities for\\nmice of 3 different weight levels to evaluate the survival rates of mice and CH\\ninduced by PO under different conditions. This is the first study showing that CH\\ndiversities exist among groups under different ligations and BW.\\nAAC is widely used in the modeling of CH induced by PO in mice. Needle ligation is\\nusually used, and the efficiency of modeling is highly dependent on ligation\\nintensity. Nevertheless, excessive constriction will lead to death,1 and our research findings (Table 1) demonstrated this point. In this\\nstudy, a 0.35-mm needle caused the death of all mice in the 3 weight levels, and the\\n0.40-mm needle caused the death of all mice in 22-g and 26-g groups. Contrarily, all\\nmice with AAC that underwent surgery with a 0.50-mm needle or sham operation\\nsurvived. Mice in the other groups had different mortality rates. Regarding the\\nselection of needles for the BW ranges of this study, a needle smaller than 0.35mm\\nin diameter caused stronger constriction and death. However, a needle larger than\\n0.50 mm in diameter did not alternatively affect the survival rate, but it did\\nreduce the efficiency of CH because of the reduced PO from weaker constriction. This\\nis why we chose needles between 0.35 mm and 0.50 mm.\\nDeath can occur after AAC. Undoubtedly, AAC increases cardiac afterloading. To cope\\nwith the additional biodynamics, the heart exerts a series of adaptive changes,\\nincluding activation and hypertrophy of cardiomyocytes and hyperplasia of the\\nextracellular matrix.2 This\\ncompensational mechanism maintains cardiac output (CO) effectively for a period of\\ntime while maintaining the survival of the organism; it is also the basis for the\\nestablishment of the CH model. However, when the sudden afterloading is out of the\\nrange of cardiomyocyte adjustment, the bloodstream will be limited and cause\\nconstriction, resulting in AHF. AHF is typically characterized by rapid changes in\\nheart failure (HF) symptoms.3 Sato\\net al.4 considered the incidence of\\ndeath within 5 days as an assessment criterion of AHF. AHF could moderately or\\nmarkedly improve by the second day if effectively controlled. AHF leads to high\\nventricular pressure, and high ventricular pressure leads to high pulmonary blood\\npressure, thus leading to pulmonary congestion, which is one of the causes of death\\nafter AAC.5 Liao et al.6 suggested that cardiogenic\\npneumo-edema is the main cause of postoperative death for PO mice. Additionally,\\narrhythmia may occur as part of the electrophysiological changes,7 and cardiomyocyte sarcomeres may be\\ndisordered during the pathological changes.8 These are all severe threats to the survival rate after AAC.\\nOur record of mice death times (Table 1)\\nshowed the phenomenon of all deaths occurring within 5 days. A high incidence of\\ndeath occurred during the initial 24h, which is in accordance with the\\naforementioned AHF criteria. In addition, there is a positive correlation between CO\\nand BW;9,10 therefore, compared with the low-weight mice, high-weight\\nmice require more CO and will have cardiac afterloading that is more increased than\\nthat of low-weight mice with the same aortic constriction. Results of the current\\nstudy (Table 1) indicate that higher-weight\\nmice had poorer tolerance for AAC, which is reflected in their mortality rates.\\nRegarding mice with AAC that underwent ligation with a 0.40-mm needle, all mice in\\nthe 22-g and 26-g groups died. However, 6 out of 10 mice survived in the 18-g\\ngroup.\\nThe diagnosis of CH usually depends on changes in cardiac function and\\nmorphology.11\\nEchocardiography can be performed in vitro noninvasively during the first assessment\\nof CH, and it is especially used to monitor changes in cardiac function.12 We performed echocardiographic\\nexaminations of mice at the end of week 4 and week 8 post-surgery. Data (week 4 data\\nin Table 2) showed that at the end of week 4,\\nthe phenomena of thickened ventricular walls, enlarged ventricular chambers, and\\ndecreased cardiac functions were emerging in each AAC group compared with the sham\\ngroups, and this diversity was consistent with the characteristic cardiac changes\\nthat occur with chronic pressure overload.13,14 These trends\\nbecame more pronounced at the end of week 8 (week 8 data in Table 2), when EF and FS, which represent cardiac function, were\\nsignificantly lower compared with the sham groups. CH also increased HW. In our\\nstudy, the HW, HW/BW ratio, and HW/TL ratio for the AAC groups were significantly\\nincreased (Table 3). Cardiac remodeling is\\nthe most typical pathological change of CH, including cardiomyocyte hypertrophy and\\nthe extracellular matrix increases.15 Our histological results showed increased external diameters\\nand ventricular thickness in gross hearts and cross-sections under AAC (Figures 1A and B). HE staining of the AAC groups displayed the hypertrophic pathology\\nof cardiomyocytes and nuclei (Figure 1C). PSR\\nstaining of the AAC groups displayed extensive collagen depositions (Figure 1D), particularly in the perivascular\\nspace (Figure 1E). Statistical analysis showed\\nthat the thickness of the left ventricle (Table\\n4) and the percentage of collagen deposition (Table 5) were significantly increased in the AAC groups compared\\nto the sham group. Regarding the formation of collagen, Kuwahara et al.16 indicated that cardiac fibroblasts\\nare activated on day 3 after PO, and that the neoformative fibrous tissues mainly\\naffect the diastolic function rather than the systolic function during the initial 4\\nweeks. Then, excessive myocardial fibrosis is implicated in systolic dysfunction\\nbecause of its more intensive traction, and cardiac function begins to deteriorate\\nsignificantly. Regarding EF and FS values for the AAC groups (Table 2), the downward trends from week 4 to week 8 conform to\\nthis theory.\\nChoosing the proper needle is critical for establishing the CH model. Based on these\\nresults, we found that all mice with AAC died when a 0.35-mm needle was used for\\nligation for all 3 weight levels and when a 0.40-mm needle was used for ligation for\\nthe 22-g and 26-g groups; therefore, these 5 groups of weight-needle pairings were\\nclearly unsuitable for use. The 18g/0.40mm group had obvious CH compared with the\\nsham group, and its survival rate was acceptable (6 out of 10). However, it should\\nstill be excluded because the 18 g/0.45 mm group showed more obvious CH and higher\\nsurvival rates (8 out of 10) (Table 1, Table 3). The 0.45-mm and 0.50-mm needles are\\navailable for all 3 weight levels, but both can result in definite myocardial\\nhypertrophy. However, the values of the HW, HW/BW ratio, and HW/TL ratio for the AAC\\nmice when using the 0.45-mm needle were significantly higher than those when using a\\n0.50-mm needle for each weight level (Table\\n3). Therefore, for all 3 weight levels of our study, a CH model can be\\nestablished using a 0.50-mm needle and the survival rate of the mice will not be\\nthreatened. However, a 0.45-mm needle leads to more effective CH model, and higher\\nmortality than the 0.50-mm needle.\\nNormally, with the PO-induced CH model, thinner needles creates more severe aortic\\nstenosis and lead to more pronounced CH, and vice versa. However, we observed an\\ninteresting phenomenon: the CH level of the 18 g/0.45 mm group was abnormally\\nsignificantly higher than that of the 18 g/0.40 mm group (18-g mice in Table 3). Regarding the analysis of BW data\\nwith AAC (Table 6), the changes in BW in 18-g\\nmice during weeks 0 to 8 were significantly higher than those for the 22-g and 26-g\\nmice, indicating that 18-g mice have greater potential for physical development\\nafter surgery and that physical development is often accompanied by organ\\ndevelopment.17 Therefore,\\nthe heart of 18-g mice also has greater development potential. For the same weight\\nlevel, the BW change of the 18 g/0.45 mm group during weeks 0 to 8 was significantly\\nhigher than that of the 18 g/0.40 mm group (BW change in Table 7). As mentioned, BW is positively related to CO;\\ntherefore, perhaps the greater ligation limited CO in the 18 g/0.40 mm group, which\\nalso limited physical development and organ development, including development of\\nthe heart. At the end of week 8, there was no significant difference in BW for the\\n18g/0.45mm group and 18g/sham groups; both had significantly higher BW than the 18\\ng/0.40 mm group (BW at week 8 in Table 7).\\nThe 0.45-mm needle had no obvious limits in18-g mice, but the BW advantage for the\\n18 g/0.45 mm group compared to the 18 g/0.40 mm group depends on greater CO and\\nrequires more hypertrophic myocardium for support. So, to establish CH models for\\nAAC in mice that have developmental potential, such as 18-g mice, there may be a\\nspecial ligating intensity region that can cause more obvious CH than the two\\nadjacent regions. However, this phenomenon must comprise multiple factors and is\\nworth further study.\",\n \"We established CH models using 4 ligation needle sizes and 3 weights for mice. Data\\nshowed that both of 0.45-mm and 0.50-mm needles lead to CH. However, 0.45mm needle\\nbrings more effective model and causes obvious CH in 18-g mice.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,null,null,null,null,"results",null,null,null,null,null,"discussion","conclusions"],"string":"[\n \"intro\",\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["Cardiomegaly","Body Weight","Heart Failure","Needles/utilization","Rats"],"string":"[\n \"Cardiomegaly\",\n \"Body Weight\",\n \"Heart Failure\",\n \"Needles/utilization\",\n \"Rats\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Introduction:\nCardiac hypertrophy (CH) is a compensatory pathological change that is usually\ninduced by pressure overload (PO), neurohumoral abnormality, and the effects of\ncytokines. It is characterized by cardiomyocyte hypertrophy and interstitial\nhyperplasia, and it results in an enlarged heart and thickening of the heart walls.\nClinically, CH is involved in the development of many diseases, such as valvular\ndisease, hypertension, arterial stenosis, and primary myocardial hypertrophy. If\nthese diseases develop at their own pace, then cardiac function (CF) will gradually\ndecompensate, leading to heart failure (HF), which severely lowers the quality of\nlife and increases the mortality rate. Therefore, CH is a widespread concern and has\nbeen explored at the molecular level by researchers. Due to the high genomic\nhomology between mice and humans, an established CH model for mice has been widely\nused in animal experiments, thereby providing an effective research foundation for\nCH exploration.\nCurrently, PO-induced CH is a common way to establish the model. Abdominal aortic\nconstriction (AAC) is highly recommended by researchers because of the high success\nrate and the ability to perform surgery without the need for thoracotomy or a\nventilator. However, the modeling effects with different ligating intensities for\ncertain body weights (BWs) have not yet been reported. Therefore, we used 3\nfrequently used mice BWs (18 g, 22 g, and 26 g) and 4 different needle sizes (0.35,\n0.40, 0.45, and 0.50 mm) to establish the CH model for each weight level for AAC,\nsummarized the survival rates, and evaluated the CH effects.\n\nMethods:\n Animal groups and handling One-hundred fifty male C57BL/6 wild-type mice were obtained from the Shanghai\nSLAC Laboratory Animal Co. Ltd (Shanghai, China). All animals were treated and\ncared for in accordance with the Guide for the Care and Use of\nLaboratory Animals (National Institutes of Health, Washington, DC,\n1996). Experimental protocols were approved by our Institutional Animal Care and\nUse Committee of Zhejiang University (Hangzhou, China). Mice were selected\naccording to weights of approximately 18 g (range, 17.3-18.7 g), 22 g (range,\n20.8-23.0 g), and 26 g (range, 25.1-27.0 g), and they were divided into the\nfollowing 3 weight levels: 18 g (18.0 ± 0.3 g; n = 50), 22 g (22.0\n± 0.6 g; n = 50), and 26 g (26.1 ± 0.5 g; n = 50). All weight\nlevels were divided using sortition randomization method to create a sham group\n(n = 10) and 4 AAC groups according to ligating intensities (0.35, 0.40, 0.45,\nand 0.50mm; n = 10 per group). Regarding BW, no significant differences were\nfound among the 5 groups for each weight level (Table S1), and the preoperative BWs of mice that died and those that\nsurvived were not significant (Table\nS2).\nMice were anesthetized with 4% chloralhydrate (0.1ml/1g BW, intraperitoneal\ninjection). When the mice did not respond when their toe was pinched, the limbs\nwere fixed on the operating board in the supine position and the skin was\nprepared by shaving and disinfection with alcohol. Sterile gauze was placed on\nthe right side of the abdomen and a ventrimesal incision approximately 1.5 cm\nwas created starting from the xiphoid. The skin was fixed with a spreader and\nthe viscera was pulled out gently with a swab and placed on the gauze. Then, the\nabdominal aorta was isolated using a blunt dissection technique with curved\nmicroforceps under a microscope. A 6-0 silk suture was snared and pulled back\naround the aorta 1mm above the superior mesenteric artery. A 2-mm blunt\nacupuncture needle (external diameters: 0.35 mm, 0.40 mm, 0.45 mm, and 0.50 mm;\nHuatuo; Suzhou Medical Appliance Factory, Suzhou, China; criterion number\nGB2024-1994) was then placed next to the aorta. The suture was tied snugly\naround the needle and the aorta. The needle was removed immediately after\nligation, the viscera were replaced, the peritoneum and skin were sutured, and\nthe mice were allowed to recover. Aortic ligation was omitted only for the sham\ngroup. After surgery, the ears were cut to differentiate the mice. Then, mice\nwere placed in an incubator at 30ºC until they woke, and they were\nreturned to their cages. Survival status was recorded daily. To observe the\nphysical development of mice under different conditions, BW differences before\nsurgery and at week 8 post-surgery were calculated as the change in BW.\nOne-hundred fifty male C57BL/6 wild-type mice were obtained from the Shanghai\nSLAC Laboratory Animal Co. Ltd (Shanghai, China). All animals were treated and\ncared for in accordance with the Guide for the Care and Use of\nLaboratory Animals (National Institutes of Health, Washington, DC,\n1996). Experimental protocols were approved by our Institutional Animal Care and\nUse Committee of Zhejiang University (Hangzhou, China). Mice were selected\naccording to weights of approximately 18 g (range, 17.3-18.7 g), 22 g (range,\n20.8-23.0 g), and 26 g (range, 25.1-27.0 g), and they were divided into the\nfollowing 3 weight levels: 18 g (18.0 ± 0.3 g; n = 50), 22 g (22.0\n± 0.6 g; n = 50), and 26 g (26.1 ± 0.5 g; n = 50). All weight\nlevels were divided using sortition randomization method to create a sham group\n(n = 10) and 4 AAC groups according to ligating intensities (0.35, 0.40, 0.45,\nand 0.50mm; n = 10 per group). Regarding BW, no significant differences were\nfound among the 5 groups for each weight level (Table S1), and the preoperative BWs of mice that died and those that\nsurvived were not significant (Table\nS2).\nMice were anesthetized with 4% chloralhydrate (0.1ml/1g BW, intraperitoneal\ninjection). When the mice did not respond when their toe was pinched, the limbs\nwere fixed on the operating board in the supine position and the skin was\nprepared by shaving and disinfection with alcohol. Sterile gauze was placed on\nthe right side of the abdomen and a ventrimesal incision approximately 1.5 cm\nwas created starting from the xiphoid. The skin was fixed with a spreader and\nthe viscera was pulled out gently with a swab and placed on the gauze. Then, the\nabdominal aorta was isolated using a blunt dissection technique with curved\nmicroforceps under a microscope. A 6-0 silk suture was snared and pulled back\naround the aorta 1mm above the superior mesenteric artery. A 2-mm blunt\nacupuncture needle (external diameters: 0.35 mm, 0.40 mm, 0.45 mm, and 0.50 mm;\nHuatuo; Suzhou Medical Appliance Factory, Suzhou, China; criterion number\nGB2024-1994) was then placed next to the aorta. The suture was tied snugly\naround the needle and the aorta. The needle was removed immediately after\nligation, the viscera were replaced, the peritoneum and skin were sutured, and\nthe mice were allowed to recover. Aortic ligation was omitted only for the sham\ngroup. After surgery, the ears were cut to differentiate the mice. Then, mice\nwere placed in an incubator at 30ºC until they woke, and they were\nreturned to their cages. Survival status was recorded daily. To observe the\nphysical development of mice under different conditions, BW differences before\nsurgery and at week 8 post-surgery were calculated as the change in BW.\n Echocardiography imaging After post-surgery weeks 4 and 8, mice were weighed and anesthetized with 4%\nchloralhydrate and placed on a warming pad after skin preparation. Transthoracic\n2-dimensional (2D) echocardiography was performed using the GE Vivid E9\nUltrasound echocardiographic system (General Electric Company, Fairfield, CT,\nUSA) with the GE 9L probe (8-MHz linear array transducer; General Electric\nCompany). M-mode parasternal long-axis scans of the left ventricle at the mitral\nchordae level were used to quantify the interventricular septum thickness at\nend-diastole (IVSd), interventricular septum thickness at end-systole (IVSs),\nleft ventricular internal dimension at end-diastole (LVIDd), left ventricular\ninternal dimension at end-systole (LVIDs), left ventricular posterior wall\nthickness at end-diastole (LVPWd), left ventricular posterior wall thickness at\nend-systole (LVPWs), ejection fraction (EF), and fractional shortening (FS). All\nmice were tested using the same parameters.\nAfter post-surgery weeks 4 and 8, mice were weighed and anesthetized with 4%\nchloralhydrate and placed on a warming pad after skin preparation. Transthoracic\n2-dimensional (2D) echocardiography was performed using the GE Vivid E9\nUltrasound echocardiographic system (General Electric Company, Fairfield, CT,\nUSA) with the GE 9L probe (8-MHz linear array transducer; General Electric\nCompany). M-mode parasternal long-axis scans of the left ventricle at the mitral\nchordae level were used to quantify the interventricular septum thickness at\nend-diastole (IVSd), interventricular septum thickness at end-systole (IVSs),\nleft ventricular internal dimension at end-diastole (LVIDd), left ventricular\ninternal dimension at end-systole (LVIDs), left ventricular posterior wall\nthickness at end-diastole (LVPWd), left ventricular posterior wall thickness at\nend-systole (LVPWs), ejection fraction (EF), and fractional shortening (FS). All\nmice were tested using the same parameters.\n Heart weight, heart weight/body weight, and heart weight/tibial\nlength After echocardiographic analysis at 8 weeks post-surgery, mice were sacrificed by\ncervical dislocation and the hearts were dissected. Then, atrial and vascular\ntissues were snipped carefully, leaving the ventricles. The hearts were rinsed\nwith phosphate-buffered saline (PBS), drained by gently squeezing on absorbent\npaper, weighed, photographed under natural light, and fixed in 4%\nparaformaldehyde. The tibial lengths (TLs; mean value of the bilateral tibia)\nwere recorded. Heart weight (HW), BW, and TL were measured, and the HW/BW ratio\nand HW/TL ratio were calculated to evaluate the hypertrophic response to PO.\nAfter echocardiographic analysis at 8 weeks post-surgery, mice were sacrificed by\ncervical dislocation and the hearts were dissected. Then, atrial and vascular\ntissues were snipped carefully, leaving the ventricles. The hearts were rinsed\nwith phosphate-buffered saline (PBS), drained by gently squeezing on absorbent\npaper, weighed, photographed under natural light, and fixed in 4%\nparaformaldehyde. The tibial lengths (TLs; mean value of the bilateral tibia)\nwere recorded. Heart weight (HW), BW, and TL were measured, and the HW/BW ratio\nand HW/TL ratio were calculated to evaluate the hypertrophic response to PO.\n Histological examination of the heart Extracted hearts were fixed in 4% paraformaldehyde for 24h and dehydrated. After\nroutine histologic procedures, the hearts were embedded in paraffin and cut into\n4-µm sections. Sections were stained with hematoxylin and eosin (HE) and\npicrosirius red (PSR). Cardiac cross-sections were captured at 20 ×\nmicroscopic views from HE sections, and 5 thicknesses of the left ventricle in\neach view were selected in systematic sampling, and measured using Image-Pro\nPlus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Then, the mean values\nwere calculated. Cardiomyocyte morphological changes were captured at\n400×microscopic views from HE sections. Interstitial and/or perivascular\ncollagen depositions were captured at 200×microscopic views under\nstandard lights. Collagen was stained red using PSR, thereby indicating\nfibrosis. At least 6 views were selected in a blinded manner, and each\nphotograph was analyzed to reveal the ratio of red collagen to the entire tissue\narea using Image-Pro Plus 6.0. Then, the mean values were calculated.\nExtracted hearts were fixed in 4% paraformaldehyde for 24h and dehydrated. After\nroutine histologic procedures, the hearts were embedded in paraffin and cut into\n4-µm sections. Sections were stained with hematoxylin and eosin (HE) and\npicrosirius red (PSR). Cardiac cross-sections were captured at 20 ×\nmicroscopic views from HE sections, and 5 thicknesses of the left ventricle in\neach view were selected in systematic sampling, and measured using Image-Pro\nPlus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Then, the mean values\nwere calculated. Cardiomyocyte morphological changes were captured at\n400×microscopic views from HE sections. Interstitial and/or perivascular\ncollagen depositions were captured at 200×microscopic views under\nstandard lights. Collagen was stained red using PSR, thereby indicating\nfibrosis. At least 6 views were selected in a blinded manner, and each\nphotograph was analyzed to reveal the ratio of red collagen to the entire tissue\narea using Image-Pro Plus 6.0. Then, the mean values were calculated.\n Statistical Analysis SPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA) was used for all\nstatistical analyses. The Kolmogorov-Smirnov (K-S) test was used to verify the\nnormality of the quantitative variables as appropriate. Data are presented as\nmean ± standard deviation (SD). One-way ANOVA and post-hoc Tukey tests\nwere used to evaluate differences between groups. p < 0.05 was considered\nstatistically significant.\nSPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA) was used for all\nstatistical analyses. The Kolmogorov-Smirnov (K-S) test was used to verify the\nnormality of the quantitative variables as appropriate. Data are presented as\nmean ± standard deviation (SD). One-way ANOVA and post-hoc Tukey tests\nwere used to evaluate differences between groups. p < 0.05 was considered\nstatistically significant.\n\nAnimal groups and handling:\nOne-hundred fifty male C57BL/6 wild-type mice were obtained from the Shanghai\nSLAC Laboratory Animal Co. Ltd (Shanghai, China). All animals were treated and\ncared for in accordance with the Guide for the Care and Use of\nLaboratory Animals (National Institutes of Health, Washington, DC,\n1996). Experimental protocols were approved by our Institutional Animal Care and\nUse Committee of Zhejiang University (Hangzhou, China). Mice were selected\naccording to weights of approximately 18 g (range, 17.3-18.7 g), 22 g (range,\n20.8-23.0 g), and 26 g (range, 25.1-27.0 g), and they were divided into the\nfollowing 3 weight levels: 18 g (18.0 ± 0.3 g; n = 50), 22 g (22.0\n± 0.6 g; n = 50), and 26 g (26.1 ± 0.5 g; n = 50). All weight\nlevels were divided using sortition randomization method to create a sham group\n(n = 10) and 4 AAC groups according to ligating intensities (0.35, 0.40, 0.45,\nand 0.50mm; n = 10 per group). Regarding BW, no significant differences were\nfound among the 5 groups for each weight level (Table S1), and the preoperative BWs of mice that died and those that\nsurvived were not significant (Table\nS2).\nMice were anesthetized with 4% chloralhydrate (0.1ml/1g BW, intraperitoneal\ninjection). When the mice did not respond when their toe was pinched, the limbs\nwere fixed on the operating board in the supine position and the skin was\nprepared by shaving and disinfection with alcohol. Sterile gauze was placed on\nthe right side of the abdomen and a ventrimesal incision approximately 1.5 cm\nwas created starting from the xiphoid. The skin was fixed with a spreader and\nthe viscera was pulled out gently with a swab and placed on the gauze. Then, the\nabdominal aorta was isolated using a blunt dissection technique with curved\nmicroforceps under a microscope. A 6-0 silk suture was snared and pulled back\naround the aorta 1mm above the superior mesenteric artery. A 2-mm blunt\nacupuncture needle (external diameters: 0.35 mm, 0.40 mm, 0.45 mm, and 0.50 mm;\nHuatuo; Suzhou Medical Appliance Factory, Suzhou, China; criterion number\nGB2024-1994) was then placed next to the aorta. The suture was tied snugly\naround the needle and the aorta. The needle was removed immediately after\nligation, the viscera were replaced, the peritoneum and skin were sutured, and\nthe mice were allowed to recover. Aortic ligation was omitted only for the sham\ngroup. After surgery, the ears were cut to differentiate the mice. Then, mice\nwere placed in an incubator at 30ºC until they woke, and they were\nreturned to their cages. Survival status was recorded daily. To observe the\nphysical development of mice under different conditions, BW differences before\nsurgery and at week 8 post-surgery were calculated as the change in BW.\n\nEchocardiography imaging:\nAfter post-surgery weeks 4 and 8, mice were weighed and anesthetized with 4%\nchloralhydrate and placed on a warming pad after skin preparation. Transthoracic\n2-dimensional (2D) echocardiography was performed using the GE Vivid E9\nUltrasound echocardiographic system (General Electric Company, Fairfield, CT,\nUSA) with the GE 9L probe (8-MHz linear array transducer; General Electric\nCompany). M-mode parasternal long-axis scans of the left ventricle at the mitral\nchordae level were used to quantify the interventricular septum thickness at\nend-diastole (IVSd), interventricular septum thickness at end-systole (IVSs),\nleft ventricular internal dimension at end-diastole (LVIDd), left ventricular\ninternal dimension at end-systole (LVIDs), left ventricular posterior wall\nthickness at end-diastole (LVPWd), left ventricular posterior wall thickness at\nend-systole (LVPWs), ejection fraction (EF), and fractional shortening (FS). All\nmice were tested using the same parameters.\n\nHeart weight, heart weight/body weight, and heart weight/tibial\nlength:\nAfter echocardiographic analysis at 8 weeks post-surgery, mice were sacrificed by\ncervical dislocation and the hearts were dissected. Then, atrial and vascular\ntissues were snipped carefully, leaving the ventricles. The hearts were rinsed\nwith phosphate-buffered saline (PBS), drained by gently squeezing on absorbent\npaper, weighed, photographed under natural light, and fixed in 4%\nparaformaldehyde. The tibial lengths (TLs; mean value of the bilateral tibia)\nwere recorded. Heart weight (HW), BW, and TL were measured, and the HW/BW ratio\nand HW/TL ratio were calculated to evaluate the hypertrophic response to PO.\n\nHistological examination of the heart:\nExtracted hearts were fixed in 4% paraformaldehyde for 24h and dehydrated. After\nroutine histologic procedures, the hearts were embedded in paraffin and cut into\n4-µm sections. Sections were stained with hematoxylin and eosin (HE) and\npicrosirius red (PSR). Cardiac cross-sections were captured at 20 ×\nmicroscopic views from HE sections, and 5 thicknesses of the left ventricle in\neach view were selected in systematic sampling, and measured using Image-Pro\nPlus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Then, the mean values\nwere calculated. Cardiomyocyte morphological changes were captured at\n400×microscopic views from HE sections. Interstitial and/or perivascular\ncollagen depositions were captured at 200×microscopic views under\nstandard lights. Collagen was stained red using PSR, thereby indicating\nfibrosis. At least 6 views were selected in a blinded manner, and each\nphotograph was analyzed to reveal the ratio of red collagen to the entire tissue\narea using Image-Pro Plus 6.0. Then, the mean values were calculated.\n\nStatistical Analysis:\nSPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA) was used for all\nstatistical analyses. The Kolmogorov-Smirnov (K-S) test was used to verify the\nnormality of the quantitative variables as appropriate. Data are presented as\nmean ± standard deviation (SD). One-way ANOVA and post-hoc Tukey tests\nwere used to evaluate differences between groups. p < 0.05 was considered\nstatistically significant.\n\nResults:\n Excessive AAC may lead to death We monitored mice deaths after surgery according to acute heart failure (AHF)\ncriteria. Data (Table 1) showed that all\ndeaths occurred within 5 days, and a high incidence of death occurred during the\ninitial 24h post-surgery.\nMice deaths after surgery\nThere were no mice deaths in the AAC0.50-mm group or the sham group.\nDeaths were recorded during 3 time periods (0-24h, 24h-3d, and 3-5\nd); 54 deaths occurred within 0-24h post-surgery. The total number\nof deaths was 65.\nWe monitored mice deaths after surgery according to acute heart failure (AHF)\ncriteria. Data (Table 1) showed that all\ndeaths occurred within 5 days, and a high incidence of death occurred during the\ninitial 24h post-surgery.\nMice deaths after surgery\nThere were no mice deaths in the AAC0.50-mm group or the sham group.\nDeaths were recorded during 3 time periods (0-24h, 24h-3d, and 3-5\nd); 54 deaths occurred within 0-24h post-surgery. The total number\nof deaths was 65.\n AAC increases cardiac dimensions and reduces cardiac function Echocardiography was performed at the end of post-operative weeks 4 and 8. At\nweek 4 post-surgery, data (Table 2)\nshowed a trend of heart enlargement for mice with AAC, including thickening of\nthe ventricular wall and an increase in chamber dilation; however, differences\nin EF and FS were not significant, indicating that changes in the heart\nstructure did not have a pronounced effect on cardiac function at that time\npoint. At week 8 post-surgery, the trend of heart enlargement continued;\nhowever, the EF and FS values for the AAC groups decreased significantly. This\nchange in cardiac function from week 4 to week 8 was consistent with systolic\nfunction beginning to be markedly affected at week 4 after PO surgery.\nEchocardiographic outcomes of 18-g, 22-g, and 26-g mice\nIVSd: interventricular septum thickness at end-diastole; IVSs:\ninterventricular septum thickness at end-systole; LVIDd: left\nventricular internal dimension at end-diastole; LVIDs: left\nventricular internal dimension at end-systole; LVPWd: left\nventricular posterior wall thickness at end-diastole; LVPWs: left\nventricular posterior wall thickness at end-systole; EF: ejection\nfraction; FS: fractional shortening. The cardiac dimensions\n(inclding IVSd, IVSs, LVIDd, LVIDs, LVPWd, and LVPWs) (mm) and\nfunctional indices (inclding EF and FS) changes measured by\nechocardiography. Data were statistically analyzed and presented as\nthe mean ± SD. At week 4, cardiac dimensions for the AAC\ngroups significantly increased compared with the sham groups (*p\n< 0.05); at week 8 , more cardiac dimensions significantly\nincreased, and that EF and FS values for the AAC groups all\ndecreased significantly compared to those of the sham groups for 3\nweight levels (*p < 0.05).\nEchocardiography was performed at the end of post-operative weeks 4 and 8. At\nweek 4 post-surgery, data (Table 2)\nshowed a trend of heart enlargement for mice with AAC, including thickening of\nthe ventricular wall and an increase in chamber dilation; however, differences\nin EF and FS were not significant, indicating that changes in the heart\nstructure did not have a pronounced effect on cardiac function at that time\npoint. At week 8 post-surgery, the trend of heart enlargement continued;\nhowever, the EF and FS values for the AAC groups decreased significantly. This\nchange in cardiac function from week 4 to week 8 was consistent with systolic\nfunction beginning to be markedly affected at week 4 after PO surgery.\nEchocardiographic outcomes of 18-g, 22-g, and 26-g mice\nIVSd: interventricular septum thickness at end-diastole; IVSs:\ninterventricular septum thickness at end-systole; LVIDd: left\nventricular internal dimension at end-diastole; LVIDs: left\nventricular internal dimension at end-systole; LVPWd: left\nventricular posterior wall thickness at end-diastole; LVPWs: left\nventricular posterior wall thickness at end-systole; EF: ejection\nfraction; FS: fractional shortening. The cardiac dimensions\n(inclding IVSd, IVSs, LVIDd, LVIDs, LVPWd, and LVPWs) (mm) and\nfunctional indices (inclding EF and FS) changes measured by\nechocardiography. Data were statistically analyzed and presented as\nthe mean ± SD. At week 4, cardiac dimensions for the AAC\ngroups significantly increased compared with the sham groups (*p\n< 0.05); at week 8 , more cardiac dimensions significantly\nincreased, and that EF and FS values for the AAC groups all\ndecreased significantly compared to those of the sham groups for 3\nweight levels (*p < 0.05).\n AAC increases HW, HW/BW, and HW/TL ratio Generally, the increased HW, HW/BW, and HW/TL ratio are the three main indicators\nof CH. In our study, as shown in Table 3,\nwe found that AAC significantly increased HW and caused a significantly higher\nHW/BW ratio and HW/TL ratio compared to the sham groups for all weight levels.\nThe HW, HW/BW, and HW/TL values for the AAC0.45 mm groups were significantly\nhigher than those for the AAC0.50 mm groups. These HW-related indices for the 18\ng/0.45 mm groups were even significantly higher than those for the 18 g/0.40 mm\ngroups.\nHeart weight–related indices of 18-g, 22-g, and 26-g mice\nHW: heart weight; BW: body weight; TL: tibial length. HW(mg),\nHW/BW(mg/g), and HW/TL(mg/cm) were measured and calculated from AAC\nand sham groups of 3 BW levels. Data are presented as the mean\n± SD.\nCompared to the sham group in the same BW level, the heart\nweight–related indices of AAC groups increased significantly (p <\n0.05).\nCompared to the rest groups in the same BW level, the heart\nweight–related indices of the AAC 0.45-mm group increased\nsignificantly (p < 0.05).\nGenerally, the increased HW, HW/BW, and HW/TL ratio are the three main indicators\nof CH. In our study, as shown in Table 3,\nwe found that AAC significantly increased HW and caused a significantly higher\nHW/BW ratio and HW/TL ratio compared to the sham groups for all weight levels.\nThe HW, HW/BW, and HW/TL values for the AAC0.45 mm groups were significantly\nhigher than those for the AAC0.50 mm groups. These HW-related indices for the 18\ng/0.45 mm groups were even significantly higher than those for the 18 g/0.40 mm\ngroups.\nHeart weight–related indices of 18-g, 22-g, and 26-g mice\nHW: heart weight; BW: body weight; TL: tibial length. HW(mg),\nHW/BW(mg/g), and HW/TL(mg/cm) were measured and calculated from AAC\nand sham groups of 3 BW levels. Data are presented as the mean\n± SD.\nCompared to the sham group in the same BW level, the heart\nweight–related indices of AAC groups increased significantly (p <\n0.05).\nCompared to the rest groups in the same BW level, the heart\nweight–related indices of the AAC 0.45-mm group increased\nsignificantly (p < 0.05).\n AAC leads to cardiomyocyte hypertrophy and increases collagen\ndepositions For mice undergoing AAC surgery, the hearts demonstrated different degrees of\nenlargement (Figure 1A), enlargement of the\npapillary muscles, and thickening of the ventricular walls (Figure 1B). Wall thickening increased significantly compared\nwith that of the sham group (Table 4).\nThe sham groups showed normal architecture of the cardiomyocytes compared with\nthe AAC groups. Pathological changes including enlarged, disarrayed, and\neosinophilic cardiomyocytes and cardiomyocytes rich in cytoplasm and\ntrachychromatic and pantomorphic nuclei were observed in each of the AAC groups\n(Figure 1C). Scattered collagen\ndepositions in the interstitial and perivascular spaces were observed in the\nsham groups. In comparison, in some AAC groups, a larger quantity and wider\nrange of red deposits were observed in the interstitial space (Figure 1D), and thickened collagen was\nobserved in the perivascular space, especially in the external vascular wall\n(Figure 1E). Statistical analysis\nindicated that the AAC group had a significantly greater collagen area than the\nsham group (Table 5). These results imply\nthat AAC is capable of inducing PO-induced CH and fibrosis.\n\nFigure 1Cardiomyocyte hypertrophy and collagen deposition histological\nexamination. (A) Gross hearts under natural light. (B) The 20\n× microscopic views of HE sections. (C) The 400 ×\nmicroscopic views of HE sections. (D) Representative\n200×microscopic views under standard lights of PSR sections\nin the interstitial space. (E) Representative 200 ×\nmicroscopic views under standard lights of PSR sections in the\nperivascular space. Fibrosis is presented as red in the PSR\nsections.\n\nCardiomyocyte hypertrophy and collagen deposition histological\nexamination. (A) Gross hearts under natural light. (B) The 20\n× microscopic views of HE sections. (C) The 400 ×\nmicroscopic views of HE sections. (D) Representative\n200×microscopic views under standard lights of PSR sections\nin the interstitial space. (E) Representative 200 ×\nmicroscopic views under standard lights of PSR sections in the\nperivascular space. Fibrosis is presented as red in the PSR\nsections.\nThickness of the left ventricle (mm) based on weight and needle size\nData are presented as the mean ± SD (n = 5).\np < 0.05 represents a significant difference between the abdominal\naortic constriction (AAC) and sham groups.\nPercentage of collagen deposition in the left ventricle based on weight\nand needle size\nData are presented as the mean ± SD (n = 6).\np < 0.05 represents a significant difference between the abdominal\naortic constriction (AAC) and sham groups.\nFor mice undergoing AAC surgery, the hearts demonstrated different degrees of\nenlargement (Figure 1A), enlargement of the\npapillary muscles, and thickening of the ventricular walls (Figure 1B). Wall thickening increased significantly compared\nwith that of the sham group (Table 4).\nThe sham groups showed normal architecture of the cardiomyocytes compared with\nthe AAC groups. Pathological changes including enlarged, disarrayed, and\neosinophilic cardiomyocytes and cardiomyocytes rich in cytoplasm and\ntrachychromatic and pantomorphic nuclei were observed in each of the AAC groups\n(Figure 1C). Scattered collagen\ndepositions in the interstitial and perivascular spaces were observed in the\nsham groups. In comparison, in some AAC groups, a larger quantity and wider\nrange of red deposits were observed in the interstitial space (Figure 1D), and thickened collagen was\nobserved in the perivascular space, especially in the external vascular wall\n(Figure 1E). Statistical analysis\nindicated that the AAC group had a significantly greater collagen area than the\nsham group (Table 5). These results imply\nthat AAC is capable of inducing PO-induced CH and fibrosis.\n\nFigure 1Cardiomyocyte hypertrophy and collagen deposition histological\nexamination. (A) Gross hearts under natural light. (B) The 20\n× microscopic views of HE sections. (C) The 400 ×\nmicroscopic views of HE sections. (D) Representative\n200×microscopic views under standard lights of PSR sections\nin the interstitial space. (E) Representative 200 ×\nmicroscopic views under standard lights of PSR sections in the\nperivascular space. Fibrosis is presented as red in the PSR\nsections.\n\nCardiomyocyte hypertrophy and collagen deposition histological\nexamination. (A) Gross hearts under natural light. (B) The 20\n× microscopic views of HE sections. (C) The 400 ×\nmicroscopic views of HE sections. (D) Representative\n200×microscopic views under standard lights of PSR sections\nin the interstitial space. (E) Representative 200 ×\nmicroscopic views under standard lights of PSR sections in the\nperivascular space. Fibrosis is presented as red in the PSR\nsections.\nThickness of the left ventricle (mm) based on weight and needle size\nData are presented as the mean ± SD (n = 5).\np < 0.05 represents a significant difference between the abdominal\naortic constriction (AAC) and sham groups.\nPercentage of collagen deposition in the left ventricle based on weight\nand needle size\nData are presented as the mean ± SD (n = 6).\np < 0.05 represents a significant difference between the abdominal\naortic constriction (AAC) and sham groups.\n AAC may restrict physical development Analysis showed that with AAC 0.45 mm, BW significantly increased in 18-g mice\ncompared to 22-g and 26-g mice (Table 6),\nindicating that the 18-g groups had higher development potential. In the 18-g\nmice groups, data showed that the value of 18 g/0.40 mm was significantly lower\nthan that of the 18 g/0.45 mm and 18 g/sham groups, and that there were no\nsignificant differences between the 18 g/0.45 mm and 18 g/sham groups (Table 7), indicating that the 18 g/0.45 mm\ngroup had nearly normal physical development. Development of the 18 g/0.40 mm\ngroup was limited.\nBody weight changes with AAC under 0.45 mm needle\nBW: body weight; AAC: abdominal aortic constriction. Data are\npresented as the mean ± SD.\np < 0.05 represents a significant difference between the 18 g/0.45\nmm group and the 22 g/0.45 mm and 26 g/0.45 mm groups.\nBody weight and BW changes in 18-g mice\nBW: body weight. BW changes of 18-g mice before and after surgery for\n8 weeks. Data are presented as the mean ± SD.\np < 0.05 represents a significant difference between the 18 g/0.40\nmm group and the rest groups after surgery.\nAnalysis showed that with AAC 0.45 mm, BW significantly increased in 18-g mice\ncompared to 22-g and 26-g mice (Table 6),\nindicating that the 18-g groups had higher development potential. In the 18-g\nmice groups, data showed that the value of 18 g/0.40 mm was significantly lower\nthan that of the 18 g/0.45 mm and 18 g/sham groups, and that there were no\nsignificant differences between the 18 g/0.45 mm and 18 g/sham groups (Table 7), indicating that the 18 g/0.45 mm\ngroup had nearly normal physical development. Development of the 18 g/0.40 mm\ngroup was limited.\nBody weight changes with AAC under 0.45 mm needle\nBW: body weight; AAC: abdominal aortic constriction. Data are\npresented as the mean ± SD.\np < 0.05 represents a significant difference between the 18 g/0.45\nmm group and the 22 g/0.45 mm and 26 g/0.45 mm groups.\nBody weight and BW changes in 18-g mice\nBW: body weight. BW changes of 18-g mice before and after surgery for\n8 weeks. Data are presented as the mean ± SD.\np < 0.05 represents a significant difference between the 18 g/0.40\nmm group and the rest groups after surgery.\n\nExcessive AAC may lead to death:\nWe monitored mice deaths after surgery according to acute heart failure (AHF)\ncriteria. Data (Table 1) showed that all\ndeaths occurred within 5 days, and a high incidence of death occurred during the\ninitial 24h post-surgery.\nMice deaths after surgery\nThere were no mice deaths in the AAC0.50-mm group or the sham group.\nDeaths were recorded during 3 time periods (0-24h, 24h-3d, and 3-5\nd); 54 deaths occurred within 0-24h post-surgery. The total number\nof deaths was 65.\n\nAAC increases cardiac dimensions and reduces cardiac function:\nEchocardiography was performed at the end of post-operative weeks 4 and 8. At\nweek 4 post-surgery, data (Table 2)\nshowed a trend of heart enlargement for mice with AAC, including thickening of\nthe ventricular wall and an increase in chamber dilation; however, differences\nin EF and FS were not significant, indicating that changes in the heart\nstructure did not have a pronounced effect on cardiac function at that time\npoint. At week 8 post-surgery, the trend of heart enlargement continued;\nhowever, the EF and FS values for the AAC groups decreased significantly. This\nchange in cardiac function from week 4 to week 8 was consistent with systolic\nfunction beginning to be markedly affected at week 4 after PO surgery.\nEchocardiographic outcomes of 18-g, 22-g, and 26-g mice\nIVSd: interventricular septum thickness at end-diastole; IVSs:\ninterventricular septum thickness at end-systole; LVIDd: left\nventricular internal dimension at end-diastole; LVIDs: left\nventricular internal dimension at end-systole; LVPWd: left\nventricular posterior wall thickness at end-diastole; LVPWs: left\nventricular posterior wall thickness at end-systole; EF: ejection\nfraction; FS: fractional shortening. The cardiac dimensions\n(inclding IVSd, IVSs, LVIDd, LVIDs, LVPWd, and LVPWs) (mm) and\nfunctional indices (inclding EF and FS) changes measured by\nechocardiography. Data were statistically analyzed and presented as\nthe mean ± SD. At week 4, cardiac dimensions for the AAC\ngroups significantly increased compared with the sham groups (*p\n< 0.05); at week 8 , more cardiac dimensions significantly\nincreased, and that EF and FS values for the AAC groups all\ndecreased significantly compared to those of the sham groups for 3\nweight levels (*p < 0.05).\n\nAAC increases HW, HW/BW, and HW/TL ratio:\nGenerally, the increased HW, HW/BW, and HW/TL ratio are the three main indicators\nof CH. In our study, as shown in Table 3,\nwe found that AAC significantly increased HW and caused a significantly higher\nHW/BW ratio and HW/TL ratio compared to the sham groups for all weight levels.\nThe HW, HW/BW, and HW/TL values for the AAC0.45 mm groups were significantly\nhigher than those for the AAC0.50 mm groups. These HW-related indices for the 18\ng/0.45 mm groups were even significantly higher than those for the 18 g/0.40 mm\ngroups.\nHeart weight–related indices of 18-g, 22-g, and 26-g mice\nHW: heart weight; BW: body weight; TL: tibial length. HW(mg),\nHW/BW(mg/g), and HW/TL(mg/cm) were measured and calculated from AAC\nand sham groups of 3 BW levels. Data are presented as the mean\n± SD.\nCompared to the sham group in the same BW level, the heart\nweight–related indices of AAC groups increased significantly (p <\n0.05).\nCompared to the rest groups in the same BW level, the heart\nweight–related indices of the AAC 0.45-mm group increased\nsignificantly (p < 0.05).\n\nAAC leads to cardiomyocyte hypertrophy and increases collagen\ndepositions:\nFor mice undergoing AAC surgery, the hearts demonstrated different degrees of\nenlargement (Figure 1A), enlargement of the\npapillary muscles, and thickening of the ventricular walls (Figure 1B). Wall thickening increased significantly compared\nwith that of the sham group (Table 4).\nThe sham groups showed normal architecture of the cardiomyocytes compared with\nthe AAC groups. Pathological changes including enlarged, disarrayed, and\neosinophilic cardiomyocytes and cardiomyocytes rich in cytoplasm and\ntrachychromatic and pantomorphic nuclei were observed in each of the AAC groups\n(Figure 1C). Scattered collagen\ndepositions in the interstitial and perivascular spaces were observed in the\nsham groups. In comparison, in some AAC groups, a larger quantity and wider\nrange of red deposits were observed in the interstitial space (Figure 1D), and thickened collagen was\nobserved in the perivascular space, especially in the external vascular wall\n(Figure 1E). Statistical analysis\nindicated that the AAC group had a significantly greater collagen area than the\nsham group (Table 5). These results imply\nthat AAC is capable of inducing PO-induced CH and fibrosis.\n\nFigure 1Cardiomyocyte hypertrophy and collagen deposition histological\nexamination. (A) Gross hearts under natural light. (B) The 20\n× microscopic views of HE sections. (C) The 400 ×\nmicroscopic views of HE sections. (D) Representative\n200×microscopic views under standard lights of PSR sections\nin the interstitial space. (E) Representative 200 ×\nmicroscopic views under standard lights of PSR sections in the\nperivascular space. Fibrosis is presented as red in the PSR\nsections.\n\nCardiomyocyte hypertrophy and collagen deposition histological\nexamination. (A) Gross hearts under natural light. (B) The 20\n× microscopic views of HE sections. (C) The 400 ×\nmicroscopic views of HE sections. (D) Representative\n200×microscopic views under standard lights of PSR sections\nin the interstitial space. (E) Representative 200 ×\nmicroscopic views under standard lights of PSR sections in the\nperivascular space. Fibrosis is presented as red in the PSR\nsections.\nThickness of the left ventricle (mm) based on weight and needle size\nData are presented as the mean ± SD (n = 5).\np < 0.05 represents a significant difference between the abdominal\naortic constriction (AAC) and sham groups.\nPercentage of collagen deposition in the left ventricle based on weight\nand needle size\nData are presented as the mean ± SD (n = 6).\np < 0.05 represents a significant difference between the abdominal\naortic constriction (AAC) and sham groups.\n\nAAC may restrict physical development:\nAnalysis showed that with AAC 0.45 mm, BW significantly increased in 18-g mice\ncompared to 22-g and 26-g mice (Table 6),\nindicating that the 18-g groups had higher development potential. In the 18-g\nmice groups, data showed that the value of 18 g/0.40 mm was significantly lower\nthan that of the 18 g/0.45 mm and 18 g/sham groups, and that there were no\nsignificant differences between the 18 g/0.45 mm and 18 g/sham groups (Table 7), indicating that the 18 g/0.45 mm\ngroup had nearly normal physical development. Development of the 18 g/0.40 mm\ngroup was limited.\nBody weight changes with AAC under 0.45 mm needle\nBW: body weight; AAC: abdominal aortic constriction. Data are\npresented as the mean ± SD.\np < 0.05 represents a significant difference between the 18 g/0.45\nmm group and the 22 g/0.45 mm and 26 g/0.45 mm groups.\nBody weight and BW changes in 18-g mice\nBW: body weight. BW changes of 18-g mice before and after surgery for\n8 weeks. Data are presented as the mean ± SD.\np < 0.05 represents a significant difference between the 18 g/0.40\nmm group and the rest groups after surgery.\n\nDiscussion:\nIn this study, we performed AAC according to 4 different ligating intensities for\nmice of 3 different weight levels to evaluate the survival rates of mice and CH\ninduced by PO under different conditions. This is the first study showing that CH\ndiversities exist among groups under different ligations and BW.\nAAC is widely used in the modeling of CH induced by PO in mice. Needle ligation is\nusually used, and the efficiency of modeling is highly dependent on ligation\nintensity. Nevertheless, excessive constriction will lead to death,1 and our research findings (Table 1) demonstrated this point. In this\nstudy, a 0.35-mm needle caused the death of all mice in the 3 weight levels, and the\n0.40-mm needle caused the death of all mice in 22-g and 26-g groups. Contrarily, all\nmice with AAC that underwent surgery with a 0.50-mm needle or sham operation\nsurvived. Mice in the other groups had different mortality rates. Regarding the\nselection of needles for the BW ranges of this study, a needle smaller than 0.35mm\nin diameter caused stronger constriction and death. However, a needle larger than\n0.50 mm in diameter did not alternatively affect the survival rate, but it did\nreduce the efficiency of CH because of the reduced PO from weaker constriction. This\nis why we chose needles between 0.35 mm and 0.50 mm.\nDeath can occur after AAC. Undoubtedly, AAC increases cardiac afterloading. To cope\nwith the additional biodynamics, the heart exerts a series of adaptive changes,\nincluding activation and hypertrophy of cardiomyocytes and hyperplasia of the\nextracellular matrix.2 This\ncompensational mechanism maintains cardiac output (CO) effectively for a period of\ntime while maintaining the survival of the organism; it is also the basis for the\nestablishment of the CH model. However, when the sudden afterloading is out of the\nrange of cardiomyocyte adjustment, the bloodstream will be limited and cause\nconstriction, resulting in AHF. AHF is typically characterized by rapid changes in\nheart failure (HF) symptoms.3 Sato\net al.4 considered the incidence of\ndeath within 5 days as an assessment criterion of AHF. AHF could moderately or\nmarkedly improve by the second day if effectively controlled. AHF leads to high\nventricular pressure, and high ventricular pressure leads to high pulmonary blood\npressure, thus leading to pulmonary congestion, which is one of the causes of death\nafter AAC.5 Liao et al.6 suggested that cardiogenic\npneumo-edema is the main cause of postoperative death for PO mice. Additionally,\narrhythmia may occur as part of the electrophysiological changes,7 and cardiomyocyte sarcomeres may be\ndisordered during the pathological changes.8 These are all severe threats to the survival rate after AAC.\nOur record of mice death times (Table 1)\nshowed the phenomenon of all deaths occurring within 5 days. A high incidence of\ndeath occurred during the initial 24h, which is in accordance with the\naforementioned AHF criteria. In addition, there is a positive correlation between CO\nand BW;9,10 therefore, compared with the low-weight mice, high-weight\nmice require more CO and will have cardiac afterloading that is more increased than\nthat of low-weight mice with the same aortic constriction. Results of the current\nstudy (Table 1) indicate that higher-weight\nmice had poorer tolerance for AAC, which is reflected in their mortality rates.\nRegarding mice with AAC that underwent ligation with a 0.40-mm needle, all mice in\nthe 22-g and 26-g groups died. However, 6 out of 10 mice survived in the 18-g\ngroup.\nThe diagnosis of CH usually depends on changes in cardiac function and\nmorphology.11\nEchocardiography can be performed in vitro noninvasively during the first assessment\nof CH, and it is especially used to monitor changes in cardiac function.12 We performed echocardiographic\nexaminations of mice at the end of week 4 and week 8 post-surgery. Data (week 4 data\nin Table 2) showed that at the end of week 4,\nthe phenomena of thickened ventricular walls, enlarged ventricular chambers, and\ndecreased cardiac functions were emerging in each AAC group compared with the sham\ngroups, and this diversity was consistent with the characteristic cardiac changes\nthat occur with chronic pressure overload.13,14 These trends\nbecame more pronounced at the end of week 8 (week 8 data in Table 2), when EF and FS, which represent cardiac function, were\nsignificantly lower compared with the sham groups. CH also increased HW. In our\nstudy, the HW, HW/BW ratio, and HW/TL ratio for the AAC groups were significantly\nincreased (Table 3). Cardiac remodeling is\nthe most typical pathological change of CH, including cardiomyocyte hypertrophy and\nthe extracellular matrix increases.15 Our histological results showed increased external diameters\nand ventricular thickness in gross hearts and cross-sections under AAC (Figures 1A and B). HE staining of the AAC groups displayed the hypertrophic pathology\nof cardiomyocytes and nuclei (Figure 1C). PSR\nstaining of the AAC groups displayed extensive collagen depositions (Figure 1D), particularly in the perivascular\nspace (Figure 1E). Statistical analysis showed\nthat the thickness of the left ventricle (Table\n4) and the percentage of collagen deposition (Table 5) were significantly increased in the AAC groups compared\nto the sham group. Regarding the formation of collagen, Kuwahara et al.16 indicated that cardiac fibroblasts\nare activated on day 3 after PO, and that the neoformative fibrous tissues mainly\naffect the diastolic function rather than the systolic function during the initial 4\nweeks. Then, excessive myocardial fibrosis is implicated in systolic dysfunction\nbecause of its more intensive traction, and cardiac function begins to deteriorate\nsignificantly. Regarding EF and FS values for the AAC groups (Table 2), the downward trends from week 4 to week 8 conform to\nthis theory.\nChoosing the proper needle is critical for establishing the CH model. Based on these\nresults, we found that all mice with AAC died when a 0.35-mm needle was used for\nligation for all 3 weight levels and when a 0.40-mm needle was used for ligation for\nthe 22-g and 26-g groups; therefore, these 5 groups of weight-needle pairings were\nclearly unsuitable for use. The 18g/0.40mm group had obvious CH compared with the\nsham group, and its survival rate was acceptable (6 out of 10). However, it should\nstill be excluded because the 18 g/0.45 mm group showed more obvious CH and higher\nsurvival rates (8 out of 10) (Table 1, Table 3). The 0.45-mm and 0.50-mm needles are\navailable for all 3 weight levels, but both can result in definite myocardial\nhypertrophy. However, the values of the HW, HW/BW ratio, and HW/TL ratio for the AAC\nmice when using the 0.45-mm needle were significantly higher than those when using a\n0.50-mm needle for each weight level (Table\n3). Therefore, for all 3 weight levels of our study, a CH model can be\nestablished using a 0.50-mm needle and the survival rate of the mice will not be\nthreatened. However, a 0.45-mm needle leads to more effective CH model, and higher\nmortality than the 0.50-mm needle.\nNormally, with the PO-induced CH model, thinner needles creates more severe aortic\nstenosis and lead to more pronounced CH, and vice versa. However, we observed an\ninteresting phenomenon: the CH level of the 18 g/0.45 mm group was abnormally\nsignificantly higher than that of the 18 g/0.40 mm group (18-g mice in Table 3). Regarding the analysis of BW data\nwith AAC (Table 6), the changes in BW in 18-g\nmice during weeks 0 to 8 were significantly higher than those for the 22-g and 26-g\nmice, indicating that 18-g mice have greater potential for physical development\nafter surgery and that physical development is often accompanied by organ\ndevelopment.17 Therefore,\nthe heart of 18-g mice also has greater development potential. For the same weight\nlevel, the BW change of the 18 g/0.45 mm group during weeks 0 to 8 was significantly\nhigher than that of the 18 g/0.40 mm group (BW change in Table 7). As mentioned, BW is positively related to CO;\ntherefore, perhaps the greater ligation limited CO in the 18 g/0.40 mm group, which\nalso limited physical development and organ development, including development of\nthe heart. At the end of week 8, there was no significant difference in BW for the\n18g/0.45mm group and 18g/sham groups; both had significantly higher BW than the 18\ng/0.40 mm group (BW at week 8 in Table 7).\nThe 0.45-mm needle had no obvious limits in18-g mice, but the BW advantage for the\n18 g/0.45 mm group compared to the 18 g/0.40 mm group depends on greater CO and\nrequires more hypertrophic myocardium for support. So, to establish CH models for\nAAC in mice that have developmental potential, such as 18-g mice, there may be a\nspecial ligating intensity region that can cause more obvious CH than the two\nadjacent regions. However, this phenomenon must comprise multiple factors and is\nworth further study.\n\nConclusion:\nWe established CH models using 4 ligation needle sizes and 3 weights for mice. Data\nshowed that both of 0.45-mm and 0.50-mm needles lead to CH. However, 0.45mm needle\nbrings more effective model and causes obvious CH in 18-g mice.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe cardiac hypertrophy (CH) model for mice has been widely used, thereby providing an effective research foundation for CH exploration.\n\nMethods:\nFour needles with different external diameters (0.35, 0.40, 0.45, and 0.50 mm) were used for AAC. 150 male C57BL/6 mice were selected according to body weight (BW) and divided into 3 weight levels: 18 g, 22 g, and 26 g (n = 50 in each group). All weight levels were divided into 5 groups: a sham group (n = 10) and 4 AAC groups using 4 ligation intensities (n = 10 per group). After surgery, survival rates were recorded, echocardiography was performed, hearts were dissected and used for histological detection, and data were statistically analyzed, P < 0.05 was considered statistically significant.\n\nResults:\nAll mice died in the following AAC groups: 18g/0.35 mm, 22 g/0.35 mm, 26 g/0.35 mm, 22 g/0.40 mm, and 26 g/0.40 mm. All mice with AAC, those ligated with a 0.50-mm needle, and those that underwent sham operation survived. Different death rates occurred in the following AAC groups: 18 g/0.40 mm, 18 g/0.45 mm, 18 g/0.50 mm, 22 g/45 mm, 22 g/0.50 mm, 26 g/0.45 mm, and 26 g/0.50 mm. The heart weight/body weight ratios (5.39 ± 0.85, 6.41 ± 0.68, 4.67 ± 0.37, 5.22 ± 0.42, 4.23 ± 0.28, 5.41 ± 0.14, and 4.02 ± 0.13) were significantly increased compared with those of the sham groups for mice with the same weight levels.\n\nConclusions:\nA 0.45-mm needle led to more obvious CH than did 0.40-mm and 0.50-mm needles and caused extraordinary CH in 18-g mice."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nCardiac hypertrophy (CH) is a compensatory pathological change that is usually\ninduced by pressure overload (PO), neurohumoral abnormality, and the effects of\ncytokines. It is characterized by cardiomyocyte hypertrophy and interstitial\nhyperplasia, and it results in an enlarged heart and thickening of the heart walls.\nClinically, CH is involved in the development of many diseases, such as valvular\ndisease, hypertension, arterial stenosis, and primary myocardial hypertrophy. If\nthese diseases develop at their own pace, then cardiac function (CF) will gradually\ndecompensate, leading to heart failure (HF), which severely lowers the quality of\nlife and increases the mortality rate. Therefore, CH is a widespread concern and has\nbeen explored at the molecular level by researchers. Due to the high genomic\nhomology between mice and humans, an established CH model for mice has been widely\nused in animal experiments, thereby providing an effective research foundation for\nCH exploration.\nCurrently, PO-induced CH is a common way to establish the model. Abdominal aortic\nconstriction (AAC) is highly recommended by researchers because of the high success\nrate and the ability to perform surgery without the need for thoracotomy or a\nventilator. However, the modeling effects with different ligating intensities for\ncertain body weights (BWs) have not yet been reported. Therefore, we used 3\nfrequently used mice BWs (18 g, 22 g, and 26 g) and 4 different needle sizes (0.35,\n0.40, 0.45, and 0.50 mm) to establish the CH model for each weight level for AAC,\nsummarized the survival rates, and evaluated the CH effects.\n\nConclusion:\nWe established CH models using 4 ligation needle sizes and 3 weights for mice. Data\nshowed that both of 0.45-mm and 0.50-mm needles lead to CH. However, 0.45mm needle\nbrings more effective model and causes obvious CH in 18-g mice."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nThe cardiac hypertrophy (CH) model for mice has been widely used, thereby providing an effective research foundation for CH exploration.\n\nMethods:\nFour needles with different external diameters (0.35, 0.40, 0.45, and 0.50 mm) were used for AAC. 150 male C57BL/6 mice were selected according to body weight (BW) and divided into 3 weight levels: 18 g, 22 g, and 26 g (n = 50 in each group). All weight levels were divided into 5 groups: a sham group (n = 10) and 4 AAC groups using 4 ligation intensities (n = 10 per group). After surgery, survival rates were recorded, echocardiography was performed, hearts were dissected and used for histological detection, and data were statistically analyzed, P < 0.05 was considered statistically significant.\n\nResults:\nAll mice died in the following AAC groups: 18g/0.35 mm, 22 g/0.35 mm, 26 g/0.35 mm, 22 g/0.40 mm, and 26 g/0.40 mm. All mice with AAC, those ligated with a 0.50-mm needle, and those that underwent sham operation survived. Different death rates occurred in the following AAC groups: 18 g/0.40 mm, 18 g/0.45 mm, 18 g/0.50 mm, 22 g/45 mm, 22 g/0.50 mm, 26 g/0.45 mm, and 26 g/0.50 mm. The heart weight/body weight ratios (5.39 ± 0.85, 6.41 ± 0.68, 4.67 ± 0.37, 5.22 ± 0.42, 4.23 ± 0.28, 5.41 ± 0.14, and 4.02 ± 0.13) were significantly increased compared with those of the sham groups for mice with the same weight levels.\n\nConclusions:\nA 0.45-mm needle led to more obvious CH than did 0.40-mm and 0.50-mm needles and caused extraordinary CH in 18-g mice."},"whole_article_text_length":{"kind":"number","value":10645,"string":"10,645"},"whole_article_abstract_length":{"kind":"number","value":342,"string":"342"},"other_sections_lengths":{"kind":"list like","value":[605,203,128,200,87,113,369,268,514,252],"string":"[\n 605,\n 203,\n 128,\n 200,\n 87,\n 113,\n 369,\n 268,\n 514,\n 252\n]","isConversational":false},"num_sections":{"kind":"number","value":15,"string":"15"},"most_frequent_words":{"kind":"list like","value":["mice","groups","mm","aac","18","bw","weight","hw","group","sham"],"string":"[\n \"mice\",\n \"groups\",\n \"mm\",\n \"aac\",\n \"18\",\n \"bw\",\n \"weight\",\n \"hw\",\n \"group\",\n \"sham\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["hypertrophy cardiomyocytes hyperplasia","myocardial hypertrophy values","cardiomyocyte hypertrophy increases","activation hypertrophy cardiomyocytes","cardiac hypertrophy ch"],"string":"[\n \"hypertrophy cardiomyocytes hyperplasia\",\n \"myocardial hypertrophy values\",\n \"cardiomyocyte hypertrophy increases\",\n \"activation hypertrophy cardiomyocytes\",\n \"cardiac hypertrophy ch\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Cardiomegaly | Body Weight | Heart Failure | Needles/utilization | Rats [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Cardiomegaly | Body Weight | Heart Failure | Needles/utilization | Rats [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Cardiomegaly | Body Weight | Heart Failure | Needles/utilization | Rats [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Cardiomegaly | Body Weight | Heart Failure | Needles/utilization | Rats [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Cardiomegaly | Body Weight | Heart Failure | Needles/utilization | Rats [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Cardiomegaly | Body Weight | Heart Failure | Needles/utilization | Rats [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Aorta, Abdominal | Body Weight | Cardiomegaly | Constriction | Disease Models, Animal | Echocardiography | Ligation | Male | Mice, Inbred C57BL | Needles | Random Allocation | Reference Values | Reproducibility of Results | Time Factors [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Aorta, Abdominal | Body Weight | Cardiomegaly | Constriction | Disease Models, Animal | Echocardiography | Ligation | Male | Mice, Inbred C57BL | Needles | Random Allocation | Reference Values | Reproducibility of Results | Time Factors [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Aorta, Abdominal | Body Weight | Cardiomegaly | Constriction | Disease Models, Animal | Echocardiography | Ligation | Male | Mice, Inbred C57BL | Needles | Random Allocation | Reference Values | Reproducibility of Results | Time Factors [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Aorta, Abdominal | Body Weight | Cardiomegaly | Constriction | Disease Models, Animal | Echocardiography | Ligation | Male | Mice, Inbred C57BL | Needles | Random Allocation | Reference Values | Reproducibility of Results | Time Factors [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Aorta, Abdominal | Body Weight | Cardiomegaly | Constriction | Disease Models, Animal | Echocardiography | Ligation | Male | Mice, Inbred C57BL | Needles | Random Allocation | Reference Values | Reproducibility of Results | Time Factors [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Aorta, Abdominal | Body Weight | Cardiomegaly | Constriction | Disease Models, Animal | Echocardiography | Ligation | Male | Mice, Inbred C57BL | Needles | Random Allocation | Reference Values | Reproducibility of Results | Time Factors [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hypertrophy cardiomyocytes hyperplasia | myocardial hypertrophy values | cardiomyocyte hypertrophy increases | activation hypertrophy cardiomyocytes | cardiac hypertrophy ch [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] hypertrophy cardiomyocytes hyperplasia | myocardial hypertrophy values | cardiomyocyte hypertrophy increases | activation hypertrophy cardiomyocytes | cardiac hypertrophy ch [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] hypertrophy cardiomyocytes hyperplasia | myocardial hypertrophy values | cardiomyocyte hypertrophy increases | activation hypertrophy cardiomyocytes | cardiac hypertrophy ch [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] hypertrophy cardiomyocytes hyperplasia | myocardial hypertrophy values | cardiomyocyte hypertrophy increases | activation hypertrophy cardiomyocytes | cardiac hypertrophy ch [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hypertrophy cardiomyocytes hyperplasia | myocardial hypertrophy values | cardiomyocyte hypertrophy increases | activation hypertrophy cardiomyocytes | cardiac hypertrophy ch [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] hypertrophy cardiomyocytes hyperplasia | myocardial hypertrophy values | cardiomyocyte hypertrophy increases | activation hypertrophy cardiomyocytes | cardiac hypertrophy ch [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] mice | groups | mm | aac | 18 | bw | weight | hw | group | sham [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] mice | groups | mm | aac | 18 | bw | weight | hw | group | sham [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] mice | groups | mm | aac | 18 | bw | weight | hw | group | sham [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] mice | groups | mm | aac | 18 | bw | weight | hw | group | sham [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] mice | groups | mm | aac | 18 | bw | weight | hw | group | sham [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] mice | groups | mm | aac | 18 | bw | weight | hw | group | sham [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ch | effects | model | hypertrophy | researchers | diseases | researchers high | establish | ch model | rate [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] mice | placed | end | aorta | left | bw | sections | skin | fixed | views [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] groups | aac | hw | 18 | significantly | mm | bw | sham | sections | 45 mm [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ch | needle | lead ch | mice data | needles lead ch | needles lead | mice data showed 45 | 45mm needle | 45mm needle brings | model causes obvious ch [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] groups | mm | mice | 18 | hw | bw | aac | ch | end | weight [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] groups | mm | mice | 18 | hw | bw | aac | ch | end | weight [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Four | 0.35 | 0.40 | 0.45 | 0.50 mm | AAC ||| 150 | 3 | 18 | 22 | 26 | 50 ||| 5 | sham group | 10 | 4 | 4 | 10 ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] AAC | 18g/0.35 mm | 22 g/0.35 mm | 26 g/0.35 mm | 22 | 26 ||| AAC | 0.50-mm ||| AAC | 18 g/0.40 mm | 18 g/0.45 mm | 18 | 22 | g/45 mm | 22 | g/0.50 mm | 26 g/0.45 mm | 26 ||| 5.39 | 0.85 | 6.41 | 0.68 | 4.67 | 0.37 | 5.22 | 0.42 | 4.23 ± 0.28 | 5.41 | 0.14 | 4.02 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] 0.45-mm | 0.40-mm | 0.50-mm | 18 [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Four | 0.35 | 0.40 | 0.45 | 0.50 mm | AAC ||| 150 | 3 | 18 | 22 | 26 | 50 ||| 5 | sham group | 10 | 4 | 4 | 10 ||| ||| ||| AAC | 18g/0.35 mm | 22 g/0.35 mm | 26 g/0.35 mm | 22 | 26 ||| AAC | 0.50-mm ||| AAC | 18 g/0.40 mm | 18 g/0.45 mm | 18 | 22 | g/45 mm | 22 | g/0.50 mm | 26 g/0.45 mm | 26 ||| 5.39 | 0.85 | 6.41 | 0.68 | 4.67 | 0.37 | 5.22 | 0.42 | 4.23 ± 0.28 | 5.41 | 0.14 | 4.02 ||| 0.45-mm | 0.40-mm | 0.50-mm | 18 [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Four | 0.35 | 0.40 | 0.45 | 0.50 mm | AAC ||| 150 | 3 | 18 | 22 | 26 | 50 ||| 5 | sham group | 10 | 4 | 4 | 10 ||| ||| ||| AAC | 18g/0.35 mm | 22 g/0.35 mm | 26 g/0.35 mm | 22 | 26 ||| AAC | 0.50-mm ||| AAC | 18 g/0.40 mm | 18 g/0.45 mm | 18 | 22 | g/45 mm | 22 | g/0.50 mm | 26 g/0.45 mm | 26 ||| 5.39 | 0.85 | 6.41 | 0.68 | 4.67 | 0.37 | 5.22 | 0.42 | 4.23 ± 0.28 | 5.41 | 0.14 | 4.02 ||| 0.45-mm | 0.40-mm | 0.50-mm | 18 [SUMMARY]"}}},{"rowIdx":60,"cells":{"title":{"kind":"string","value":"Cyanidin 3-glucoside modulated cell cycle progression in liver precancerous lesion, "},"pmid":{"kind":"string","value":"33911466"},"background_abstract":{"kind":"string","value":"Cyanidin-3-O-glucoside (cyan) exhibits antioxidant and anticancer properties. The cell cycle proteins and antimitotic drugs might be promising therapeutic targets in hepatocellular carcinoma."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"In vivo, DEN/2-AAF-induced hepatic PCL, rats were treated with three doses of cyan (10, 15, and 20 mg/kg/d, for four consecutive days per week for 16 wk). Blood and liver tissue samples were collected for measurement of the followings; alpha fetoprotein (AFP) liver function and RNA panel differential expression was evaluated via real time polymerase chain reaction. Histopathological examination of liver sections stained with H&E and immunohistochemical study using glutathione S-transferase placental (GSTP) and proliferating cell nuclear antigen (PCNA) antibodies were assessed."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Cyan administration mitigated the effect of DEN/2-AFF induced PCL, decreased AFP levels, and improved liver function. Remarkably, treatment with cyan dose dependently decreased the long non-coding RNA MALAT1 and tubulin gamma 1 mRNA expressions and increased the levels of miR-125b, all of which are involved in cell cycle and mitotic spindle assembly. Of note, cyan decreased GSTP foci percent area and PCNA positively stained nuclei."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Our results indicated that cyan could be used as a potential therapeutic agent to inhibit liver carcinogenesis in rat model via modulation of cell cycle."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Animals","Anthocyanins","Diethylnitrosamine","Female","Glucosides","Glutathione Transferase","Liver","Liver Neoplasms","Liver Neoplasms, Experimental","Precancerous Conditions","Pregnancy","Rats","Rats, Wistar"],"string":"[\n \"Animals\",\n \"Anthocyanins\",\n \"Diethylnitrosamine\",\n \"Female\",\n \"Glucosides\",\n \"Glutathione Transferase\",\n \"Liver\",\n \"Liver Neoplasms\",\n \"Liver Neoplasms, Experimental\",\n \"Precancerous Conditions\",\n \"Pregnancy\",\n \"Rats\",\n \"Rats, Wistar\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"8047539"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Hepatocellular carcinoma (HCC) is the commonest primary malignancy of the liver. Its main risk factors include hepatitis B, hepatitis C, and non-alcoholic steatohepatitis. HCC is associated with high mortality and morbidity. Its incidence has increased from 1.4 to 6.2 per 100000 cases per year within the last 30 years[1]. HCC burden in Egypt is high due to the high prevalence of HCV, where liver cancers account for 11.75% of all gastrointestinal cancers and 1.68% of all malignancies. In addition, over 70% of all liver malignancies in Egypt are HCC[2].\nTo date, HCC is holding its place as an unresolved dilemma; its resistance to systemic chemotherapy and radiation makes the cure approachable only in very early stages according to Milan criteria, that is, where the tumor size is < 5 cm (when only one lesion is present) or < 3 cm (when 2-3 lesions are present)[3].\nGenes involved in cell cycle control are usually mutated in presence of cancer. Dysregulated mitosis results in genomic instability, which leads to tumor aggressiveness[4]. Microtubules are a key component of mitotic spindles, and thus, a crucial part of the process of mitosis. They form an important target in cancer chemotherapy, inducing arrest of mitosis and cell death. On the other hand, while the overexpression of γ-tubulin (γ-TUBG) showed improved survival among small cell lung cancer patients[5], the overexpression of γ-TUBG is found to be characteristic feature of thyroid and breast cancers[6]. Such variation may arise due to differences in gene transcription and check point regulations in different cancer tissues[7].\nCyanidin-3-glucoside (cyan) is a potential chemotherapeutic and chemo-protective agent. Through its antioxidant activity, it scavenges radical oxygen species, which decreases the overall number of tumorous cells (malignant and benign) in in vivo studies[8]. Cyan has been implicated in some beneficial health activities[9], including reducing age-associated oxidative stress[10], improving cognitive brain function, and exhibiting anti-diabetic[11], anti-inflammation[12], anti-atherogenic[13], and anti-obesity activities[14]. Previously, it has been reported that cyan mediates caspase-3 cleavage with DNA fragmentation. Cyan has also been associated with induction of autophagy, a key element involved in cancer elimination, via induction of autophagy-related gene 5 and microtubule-associated protein 1 light chain 3-II[15]. Cyan has drawn increasing attention because of its potential anti-cancer properties. Cyan may offer a novel avenue for treating HCC. a potential antiproliferative effect on HepG2 cells[16].\nThe present study was conducted to evaluate the effect of cyan in three different doses on cell cycle progression and mitotic assembly disorder in hepatic precancerous lesion (PCL) via assessment of levels of long non-coding RNA (lncRNA) MALAT1, miR-125b, and tubulin 1. We also conducted histopathological and immuno-histochemical examination using H&E staining assessment of levels of glutathione S-transferase placental (GSTP) and proliferating cell nuclear antigen (PCNA) antibodies in male Wistar rat model of diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF)-induced hepatic PCL. "},"methods_title":{"kind":"string","value":"MATERIALS AND METHODS"},"methods_text":{"kind":"string","value":"Chemicals and drugs DEN was obtained as 1 g solution in serum bottle diluted in 0.9% NaCl. 2-AAF was obtained as white powder, while cyan was obtained as black powder. Both 2-AAF and cyan were dissolved in 0.9% NaCl. All chemicals were purchased from Sigma-Aldrich, Cairo, Egypt.\nDEN was obtained as 1 g solution in serum bottle diluted in 0.9% NaCl. 2-AAF was obtained as white powder, while cyan was obtained as black powder. Both 2-AAF and cyan were dissolved in 0.9% NaCl. All chemicals were purchased from Sigma-Aldrich, Cairo, Egypt.\nExperimental animals All animal procedures were approved by the Institutional Animal Ethics Committee of Ain Shams University, Faculty of Medicine. Thirty male Wistar rats weighing 200-250 g were purchased from Nile Pharmaceuticals Company (Cairo, Egypt). Animals were housed in an animal room with temperature 25 ± 2 °C and 12 h light/dark cycle controls. An adaptation period of 1 wk was allowed before initiation of the experimental protocol.\nAll animal procedures were approved by the Institutional Animal Ethics Committee of Ain Shams University, Faculty of Medicine. Thirty male Wistar rats weighing 200-250 g were purchased from Nile Pharmaceuticals Company (Cairo, Egypt). Animals were housed in an animal room with temperature 25 ± 2 °C and 12 h light/dark cycle controls. An adaptation period of 1 wk was allowed before initiation of the experimental protocol.\nExperimental procedures \nInduction of hepatic PCL: DEN was injected intraperitoneally (i.p.) at a dose of 100 mg/kg once weekly for three consecutive weeks. One week after the last DEN injection, a single dose (300 mg/kg) of 2-AAF was injected.\n\nAnimal groups: Rats were divided into five groups (6 rats/each group), including: Naïve group: Rats were injected with a vehicle (0.9% NaCl), PCL group: Rats were injected with DEN and 2-AAF, and three cyan groups (cyan-10, cyan-15, and cyan-20 groups): Rats were injected with DEN and 2-AAF, and orally administered through gastric gavage with cyan in doses of 10, 15, and 20 mg/kg respectively, for four consecutive days per week for 16 wk[17].\n\nInduction of hepatic PCL: DEN was injected intraperitoneally (i.p.) at a dose of 100 mg/kg once weekly for three consecutive weeks. One week after the last DEN injection, a single dose (300 mg/kg) of 2-AAF was injected.\n\nAnimal groups: Rats were divided into five groups (6 rats/each group), including: Naïve group: Rats were injected with a vehicle (0.9% NaCl), PCL group: Rats were injected with DEN and 2-AAF, and three cyan groups (cyan-10, cyan-15, and cyan-20 groups): Rats were injected with DEN and 2-AAF, and orally administered through gastric gavage with cyan in doses of 10, 15, and 20 mg/kg respectively, for four consecutive days per week for 16 wk[17].\nAssessment of the effects of treatment \nBiochemical and molecular studies: After scarifying the animals, blood was withdrawn from each rat from the retro orbital vein and incubated for about half an hour for clotting. Then, the blood sample was centrifuged for 20 min at 5000 RPM to get serum samples. Livers were dissected. The right lobe of the livers was removed, cut into longitudinal sections 2-4 mm in thickness and kept in 10% formalin for histopathological and immunohistochemical examination. The other lobe was kept frozen at -80 °C for molecular analyses. The samples were analyzed for the following parameters: (1) alpha fetoprotein (AFP) levels: AFP level was analyzed using quantitative sandwich rat AFP ELISA kit purchased from MyBiosource Inc. (San Diego, United States) with a sensitivity range of 0.625-20 ng/mL; (2) serum alanine aminotransferase (ALT) levels: The serum ALT was measured according to method described by using kits purchased from Diamond Diagnostic (Cairo, Egypt). ALT catalyzes the transfer of amino groups from specific amino acids to ketoglutaric acid, yielding glutamic acid and oxaloacetic or pyruvic acid, respectively. Ketoacids were then determined calorimetrically after their reaction with 2, 4- dinitrophenylhydrazine; and (3) determination of serum total bilirubin and direct bilirubin: Alkaline methanolysis of bilirubin followed by chloroform extraction of bilirubin methyl esters, and later, separation of these esters by chromatography and spectrophotometric determination at 430 nm.\n\nBiochemical and molecular studies: After scarifying the animals, blood was withdrawn from each rat from the retro orbital vein and incubated for about half an hour for clotting. Then, the blood sample was centrifuged for 20 min at 5000 RPM to get serum samples. Livers were dissected. The right lobe of the livers was removed, cut into longitudinal sections 2-4 mm in thickness and kept in 10% formalin for histopathological and immunohistochemical examination. The other lobe was kept frozen at -80 °C for molecular analyses. The samples were analyzed for the following parameters: (1) alpha fetoprotein (AFP) levels: AFP level was analyzed using quantitative sandwich rat AFP ELISA kit purchased from MyBiosource Inc. (San Diego, United States) with a sensitivity range of 0.625-20 ng/mL; (2) serum alanine aminotransferase (ALT) levels: The serum ALT was measured according to method described by using kits purchased from Diamond Diagnostic (Cairo, Egypt). ALT catalyzes the transfer of amino groups from specific amino acids to ketoglutaric acid, yielding glutamic acid and oxaloacetic or pyruvic acid, respectively. Ketoacids were then determined calorimetrically after their reaction with 2, 4- dinitrophenylhydrazine; and (3) determination of serum total bilirubin and direct bilirubin: Alkaline methanolysis of bilirubin followed by chloroform extraction of bilirubin methyl esters, and later, separation of these esters by chromatography and spectrophotometric determination at 430 nm.\nDetermination of serum albumin Quantitative determination of serum albumin level was quantified using rat albumin ELISA kit. The absorbance at 450 nm is a measure of the concentration of albumin in the test sample.\nQuantitative determination of serum albumin level was quantified using rat albumin ELISA kit. The absorbance at 450 nm is a measure of the concentration of albumin in the test sample.\nBioinformatics-based selection of cell cycle-microtubule assembly- and HCC-specific RNA panel The RNA panel was chosen in three steps: (1) Tubulin, gamma 1 (TUBG1) was retrieved as a crucial player in microtubule formation and progression of the cell cycle through gene atlas expression database (available at https://www.ebi.ac.uk/gxa/home) alongside with verification of the chosen gene from protein Atlas database (available at https://www.proteinatlas.org/), followed by analysis of TUBG1 gene ontology to ensure that it is linked to G2-M transition and mitotic spindle assembly, which is closely linked to cancer development through gene card database (available at https://www.genecards.org/); (2) we selected miR-125b-1-3p targeting TUBG1 mRNA though) miRWALK database available at http://mirwalk.umm.uni-heidelberg.de/), followed by pathway enrichment, which revealed that miR-125b-1-3p is linked to MAP kinase and ubiquitin-mediated proteolysis (strongly correlated with mitotic assembly and cell cycle progression) through (Diana database available at http://diana.imis.athena-innovation.gr/DianaTools/index.php); and (3) lncRNA metastasis-associated lung adenocarcinoma transcript 1, lncRNA MALAT1, was obtained as a lncRNA specific to HCC and targeting miR-125b-1-3p using noncode database available at http://www.noncode.org/ and European Bioinformatics institute database available at https://www.ebi.ac.uk.\nThe RNA panel was chosen in three steps: (1) Tubulin, gamma 1 (TUBG1) was retrieved as a crucial player in microtubule formation and progression of the cell cycle through gene atlas expression database (available at https://www.ebi.ac.uk/gxa/home) alongside with verification of the chosen gene from protein Atlas database (available at https://www.proteinatlas.org/), followed by analysis of TUBG1 gene ontology to ensure that it is linked to G2-M transition and mitotic spindle assembly, which is closely linked to cancer development through gene card database (available at https://www.genecards.org/); (2) we selected miR-125b-1-3p targeting TUBG1 mRNA though) miRWALK database available at http://mirwalk.umm.uni-heidelberg.de/), followed by pathway enrichment, which revealed that miR-125b-1-3p is linked to MAP kinase and ubiquitin-mediated proteolysis (strongly correlated with mitotic assembly and cell cycle progression) through (Diana database available at http://diana.imis.athena-innovation.gr/DianaTools/index.php); and (3) lncRNA metastasis-associated lung adenocarcinoma transcript 1, lncRNA MALAT1, was obtained as a lncRNA specific to HCC and targeting miR-125b-1-3p using noncode database available at http://www.noncode.org/ and European Bioinformatics institute database available at https://www.ebi.ac.uk.\nExtraction of total RNA, including lncRNA and miRNA from liver tissue Total RNA was extracted from liver tissues using miRNeasy Mini kit (Cat No. 217004, Qiagen, United States) according to manufacturer’s protocol. The extracted RNA concentration and integrity were assessed using [(NanoDrop Technologies/Thermo Scientific, Wilmington, DE, United States)], with RNA purities were 1.8-2. The extracted total RNA was reverse transcribed into cDNA using a miScript II RT kit (Cat No. 218160, 218161, Qiagen, United States) on thermal cycler (Bio-Rad; Hercules, CA, United States), according to the manufacturer’s protocol. \nTotal RNA was extracted from liver tissues using miRNeasy Mini kit (Cat No. 217004, Qiagen, United States) according to manufacturer’s protocol. The extracted RNA concentration and integrity were assessed using [(NanoDrop Technologies/Thermo Scientific, Wilmington, DE, United States)], with RNA purities were 1.8-2. The extracted total RNA was reverse transcribed into cDNA using a miScript II RT kit (Cat No. 218160, 218161, Qiagen, United States) on thermal cycler (Bio-Rad; Hercules, CA, United States), according to the manufacturer’s protocol. \nReal time-polymerase chain reaction quantification of the RNA panel in liver tissues The relative expression of TUBG1 mRNA and lncRNA-MALAT1 in liver tissue were measured using a Quantitect SYBR Green Master Mix kit and an RT2 SYBR Green ROX real time-polymerase chain reaction (qPCR) Mastermix (Qiagen), respectively, using a 7500 qPCR Systems (Applied Biosystems, Foster City, CA, United States) detection system) and specific primers (Accession: NM_001128148, NM_003234, NR_002819, and ENST00000534336, respectively) supplied by Qiagen. GAPDH (Accession NM_002046.7) was used as a housekeeping gene. MiR-3163 expression in liver tissue was quantified by PCR using a miScript SYBR Green kit (Qiagen), a miScript universal primer, and a miRNA-specific forward primer (mir-125b-1-3p miScript Primer Assay) (Accession: MIMAT0004592 (5'-ACGGGUUAGGCUCUUGGGAGCU). All steps followed the manufacturer’s suggested protocol, and SNORD68 was used as an internal control. Ct values more than 36 were considered as negative. The specificities of the amplicons for the SYBR Green−based PCR amplification were affirmed by the melting curves. The 2−ΔΔCt technique was used to measure the relative expression of the HCC-specific RNA panel.\nThe relative expression of TUBG1 mRNA and lncRNA-MALAT1 in liver tissue were measured using a Quantitect SYBR Green Master Mix kit and an RT2 SYBR Green ROX real time-polymerase chain reaction (qPCR) Mastermix (Qiagen), respectively, using a 7500 qPCR Systems (Applied Biosystems, Foster City, CA, United States) detection system) and specific primers (Accession: NM_001128148, NM_003234, NR_002819, and ENST00000534336, respectively) supplied by Qiagen. GAPDH (Accession NM_002046.7) was used as a housekeeping gene. MiR-3163 expression in liver tissue was quantified by PCR using a miScript SYBR Green kit (Qiagen), a miScript universal primer, and a miRNA-specific forward primer (mir-125b-1-3p miScript Primer Assay) (Accession: MIMAT0004592 (5'-ACGGGUUAGGCUCUUGGGAGCU). All steps followed the manufacturer’s suggested protocol, and SNORD68 was used as an internal control. Ct values more than 36 were considered as negative. The specificities of the amplicons for the SYBR Green−based PCR amplification were affirmed by the melting curves. The 2−ΔΔCt technique was used to measure the relative expression of the HCC-specific RNA panel.\nHistopathological and immunohistochemical studies Liver sections were cut at 5-μm thickness and stained with H&E. Immunohistochemistry S-P method was used to detect GSTP and PCNA levels. Briefly, the protocol was as follows: (1) the tissues were treated with endogenous peroxidase blocking solution at room temperature for 10 min, and then, incubated in normal nonimmune serum at room temperature for 10 min; (2) mouse anti GSTP or PCNA antibody was added to adjacent tissue sections respectively and incubated overnight at 4 °C; (3) biotin-conjugated second antibody was added to the sections and incubated at room temperature for 10 min; and (4) S-P complex was added at room temperature for 10 min, and then, 2,4-diaminobutyric acid was used for the color reaction. The tissue sections were washed with Poly (butylene succinate) (PBS) (0.01 mol/L, pH 7.4) between each step. Positive and negative controls were simultaneously used to ensure specificity and reliability of the staining process. A positive section was taken as positive control. In negative control, PBS was used to replace the first antibody. GSTP-positivity was indicated by brown coloration of the cytoplasm. Morphometric analysis of the foci percent area was carried out using Leica Q win V.3 software after capturing the images using a Leica DM2500 microscope (Leica, Wetzlar, Germany). The PCNA labeling indices are represented as the expression of positively-stained nuclei (10 fields/slide at 400 ×) as shown below: +, positive expression found in 1-3 fields; ++, positive expression found in 4-6 fields; and +++, positive expression found in 7-10 fields. Rabbit monoclonal PCNA and GSTP antibodies were purchased from Abcam (San Francisco, United States).\nLiver sections were cut at 5-μm thickness and stained with H&E. Immunohistochemistry S-P method was used to detect GSTP and PCNA levels. Briefly, the protocol was as follows: (1) the tissues were treated with endogenous peroxidase blocking solution at room temperature for 10 min, and then, incubated in normal nonimmune serum at room temperature for 10 min; (2) mouse anti GSTP or PCNA antibody was added to adjacent tissue sections respectively and incubated overnight at 4 °C; (3) biotin-conjugated second antibody was added to the sections and incubated at room temperature for 10 min; and (4) S-P complex was added at room temperature for 10 min, and then, 2,4-diaminobutyric acid was used for the color reaction. The tissue sections were washed with Poly (butylene succinate) (PBS) (0.01 mol/L, pH 7.4) between each step. Positive and negative controls were simultaneously used to ensure specificity and reliability of the staining process. A positive section was taken as positive control. In negative control, PBS was used to replace the first antibody. GSTP-positivity was indicated by brown coloration of the cytoplasm. Morphometric analysis of the foci percent area was carried out using Leica Q win V.3 software after capturing the images using a Leica DM2500 microscope (Leica, Wetzlar, Germany). The PCNA labeling indices are represented as the expression of positively-stained nuclei (10 fields/slide at 400 ×) as shown below: +, positive expression found in 1-3 fields; ++, positive expression found in 4-6 fields; and +++, positive expression found in 7-10 fields. Rabbit monoclonal PCNA and GSTP antibodies were purchased from Abcam (San Francisco, United States).\nStatistical analysis All results were expressed as mean ± SD. Statistical analysis was carried out using GraphPad Prism version 6.01. Unpaired t-test was done to compare between naïve and PCL groups. One-way ANOVA, followed by Tukey’s test, was carried out to compare between all groups. P values < 0.05 were considered statistically significant. \nAll results were expressed as mean ± SD. Statistical analysis was carried out using GraphPad Prism version 6.01. Unpaired t-test was done to compare between naïve and PCL groups. One-way ANOVA, followed by Tukey’s test, was carried out to compare between all groups. P values < 0.05 were considered statistically significant. "},"results_title":{"kind":"null"},"results_text":{"kind":"null"},"conclusions_title":{"kind":"string","value":"CONCLUSION"},"conclusions_text":{"kind":"string","value":"Further larger in vitro and in vivo studies are needed to elucidate the mechanism of cyanidin cytotoxicity in hepatocellular carcinoma."},"other_sections_titles":{"kind":"list like","value":["INTRODUCTION","Chemicals and drugs ","Experimental animals","Experimental procedures","Assessment of the effects of treatment","Determination of serum albumin","Bioinformatics-based selection of cell cycle-microtubule assembly- and HCC-specific RNA panel ","Extraction of total RNA, including lncRNA and miRNA from liver tissue","Real time-polymerase chain reaction quantification of the RNA panel in liver tissues","Histopathological and immunohistochemical studies","Statistical analysis","RESULTS","Effect on AFP levels and liver function tests","Effect on expression of lncRNA MALAT1, miR-125b, and TUBG1 (G2/M transition of mitotic cell cycle) in the liver tissue","Histopathological and immunohistochemical studies","DISCUSSION","CONCLUSION"],"string":"[\n \"INTRODUCTION\",\n \"Chemicals and drugs \",\n \"Experimental animals\",\n \"Experimental procedures\",\n \"Assessment of the effects of treatment\",\n \"Determination of serum albumin\",\n \"Bioinformatics-based selection of cell cycle-microtubule assembly- and HCC-specific RNA panel \",\n \"Extraction of total RNA, including lncRNA and miRNA from liver tissue\",\n \"Real time-polymerase chain reaction quantification of the RNA panel in liver tissues\",\n \"Histopathological and immunohistochemical studies\",\n \"Statistical analysis\",\n \"RESULTS\",\n \"Effect on AFP levels and liver function tests\",\n \"Effect on expression of lncRNA MALAT1, miR-125b, and TUBG1 (G2/M transition of mitotic cell cycle) in the liver tissue\",\n \"Histopathological and immunohistochemical studies\",\n \"DISCUSSION\",\n \"CONCLUSION\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Hepatocellular carcinoma (HCC) is the commonest primary malignancy of the liver. Its main risk factors include hepatitis B, hepatitis C, and non-alcoholic steatohepatitis. HCC is associated with high mortality and morbidity. Its incidence has increased from 1.4 to 6.2 per 100000 cases per year within the last 30 years[1]. HCC burden in Egypt is high due to the high prevalence of HCV, where liver cancers account for 11.75% of all gastrointestinal cancers and 1.68% of all malignancies. In addition, over 70% of all liver malignancies in Egypt are HCC[2].\nTo date, HCC is holding its place as an unresolved dilemma; its resistance to systemic chemotherapy and radiation makes the cure approachable only in very early stages according to Milan criteria, that is, where the tumor size is < 5 cm (when only one lesion is present) or < 3 cm (when 2-3 lesions are present)[3].\nGenes involved in cell cycle control are usually mutated in presence of cancer. Dysregulated mitosis results in genomic instability, which leads to tumor aggressiveness[4]. Microtubules are a key component of mitotic spindles, and thus, a crucial part of the process of mitosis. They form an important target in cancer chemotherapy, inducing arrest of mitosis and cell death. On the other hand, while the overexpression of γ-tubulin (γ-TUBG) showed improved survival among small cell lung cancer patients[5], the overexpression of γ-TUBG is found to be characteristic feature of thyroid and breast cancers[6]. Such variation may arise due to differences in gene transcription and check point regulations in different cancer tissues[7].\nCyanidin-3-glucoside (cyan) is a potential chemotherapeutic and chemo-protective agent. Through its antioxidant activity, it scavenges radical oxygen species, which decreases the overall number of tumorous cells (malignant and benign) in in vivo studies[8]. Cyan has been implicated in some beneficial health activities[9], including reducing age-associated oxidative stress[10], improving cognitive brain function, and exhibiting anti-diabetic[11], anti-inflammation[12], anti-atherogenic[13], and anti-obesity activities[14]. Previously, it has been reported that cyan mediates caspase-3 cleavage with DNA fragmentation. Cyan has also been associated with induction of autophagy, a key element involved in cancer elimination, via induction of autophagy-related gene 5 and microtubule-associated protein 1 light chain 3-II[15]. Cyan has drawn increasing attention because of its potential anti-cancer properties. Cyan may offer a novel avenue for treating HCC. a potential antiproliferative effect on HepG2 cells[16].\nThe present study was conducted to evaluate the effect of cyan in three different doses on cell cycle progression and mitotic assembly disorder in hepatic precancerous lesion (PCL) via assessment of levels of long non-coding RNA (lncRNA) MALAT1, miR-125b, and tubulin 1. We also conducted histopathological and immuno-histochemical examination using H&E staining assessment of levels of glutathione S-transferase placental (GSTP) and proliferating cell nuclear antigen (PCNA) antibodies in male Wistar rat model of diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF)-induced hepatic PCL. ","DEN was obtained as 1 g solution in serum bottle diluted in 0.9% NaCl. 2-AAF was obtained as white powder, while cyan was obtained as black powder. Both 2-AAF and cyan were dissolved in 0.9% NaCl. All chemicals were purchased from Sigma-Aldrich, Cairo, Egypt.","All animal procedures were approved by the Institutional Animal Ethics Committee of Ain Shams University, Faculty of Medicine. Thirty male Wistar rats weighing 200-250 g were purchased from Nile Pharmaceuticals Company (Cairo, Egypt). Animals were housed in an animal room with temperature 25 ± 2 °C and 12 h light/dark cycle controls. An adaptation period of 1 wk was allowed before initiation of the experimental protocol.","\nInduction of hepatic PCL: DEN was injected intraperitoneally (i.p.) at a dose of 100 mg/kg once weekly for three consecutive weeks. One week after the last DEN injection, a single dose (300 mg/kg) of 2-AAF was injected.\n\nAnimal groups: Rats were divided into five groups (6 rats/each group), including: Naïve group: Rats were injected with a vehicle (0.9% NaCl), PCL group: Rats were injected with DEN and 2-AAF, and three cyan groups (cyan-10, cyan-15, and cyan-20 groups): Rats were injected with DEN and 2-AAF, and orally administered through gastric gavage with cyan in doses of 10, 15, and 20 mg/kg respectively, for four consecutive days per week for 16 wk[17].","\nBiochemical and molecular studies: After scarifying the animals, blood was withdrawn from each rat from the retro orbital vein and incubated for about half an hour for clotting. Then, the blood sample was centrifuged for 20 min at 5000 RPM to get serum samples. Livers were dissected. The right lobe of the livers was removed, cut into longitudinal sections 2-4 mm in thickness and kept in 10% formalin for histopathological and immunohistochemical examination. The other lobe was kept frozen at -80 °C for molecular analyses. The samples were analyzed for the following parameters: (1) alpha fetoprotein (AFP) levels: AFP level was analyzed using quantitative sandwich rat AFP ELISA kit purchased from MyBiosource Inc. (San Diego, United States) with a sensitivity range of 0.625-20 ng/mL; (2) serum alanine aminotransferase (ALT) levels: The serum ALT was measured according to method described by using kits purchased from Diamond Diagnostic (Cairo, Egypt). ALT catalyzes the transfer of amino groups from specific amino acids to ketoglutaric acid, yielding glutamic acid and oxaloacetic or pyruvic acid, respectively. Ketoacids were then determined calorimetrically after their reaction with 2, 4- dinitrophenylhydrazine; and (3) determination of serum total bilirubin and direct bilirubin: Alkaline methanolysis of bilirubin followed by chloroform extraction of bilirubin methyl esters, and later, separation of these esters by chromatography and spectrophotometric determination at 430 nm.","Quantitative determination of serum albumin level was quantified using rat albumin ELISA kit. The absorbance at 450 nm is a measure of the concentration of albumin in the test sample.","The RNA panel was chosen in three steps: (1) Tubulin, gamma 1 (TUBG1) was retrieved as a crucial player in microtubule formation and progression of the cell cycle through gene atlas expression database (available at https://www.ebi.ac.uk/gxa/home) alongside with verification of the chosen gene from protein Atlas database (available at https://www.proteinatlas.org/), followed by analysis of TUBG1 gene ontology to ensure that it is linked to G2-M transition and mitotic spindle assembly, which is closely linked to cancer development through gene card database (available at https://www.genecards.org/); (2) we selected miR-125b-1-3p targeting TUBG1 mRNA though) miRWALK database available at http://mirwalk.umm.uni-heidelberg.de/), followed by pathway enrichment, which revealed that miR-125b-1-3p is linked to MAP kinase and ubiquitin-mediated proteolysis (strongly correlated with mitotic assembly and cell cycle progression) through (Diana database available at http://diana.imis.athena-innovation.gr/DianaTools/index.php); and (3) lncRNA metastasis-associated lung adenocarcinoma transcript 1, lncRNA MALAT1, was obtained as a lncRNA specific to HCC and targeting miR-125b-1-3p using noncode database available at http://www.noncode.org/ and European Bioinformatics institute database available at https://www.ebi.ac.uk.","Total RNA was extracted from liver tissues using miRNeasy Mini kit (Cat No. 217004, Qiagen, United States) according to manufacturer’s protocol. The extracted RNA concentration and integrity were assessed using [(NanoDrop Technologies/Thermo Scientific, Wilmington, DE, United States)], with RNA purities were 1.8-2. The extracted total RNA was reverse transcribed into cDNA using a miScript II RT kit (Cat No. 218160, 218161, Qiagen, United States) on thermal cycler (Bio-Rad; Hercules, CA, United States), according to the manufacturer’s protocol. ","The relative expression of TUBG1 mRNA and lncRNA-MALAT1 in liver tissue were measured using a Quantitect SYBR Green Master Mix kit and an RT2 SYBR Green ROX real time-polymerase chain reaction (qPCR) Mastermix (Qiagen), respectively, using a 7500 qPCR Systems (Applied Biosystems, Foster City, CA, United States) detection system) and specific primers (Accession: NM_001128148, NM_003234, NR_002819, and ENST00000534336, respectively) supplied by Qiagen. GAPDH (Accession NM_002046.7) was used as a housekeeping gene. MiR-3163 expression in liver tissue was quantified by PCR using a miScript SYBR Green kit (Qiagen), a miScript universal primer, and a miRNA-specific forward primer (mir-125b-1-3p miScript Primer Assay) (Accession: MIMAT0004592 (5'-ACGGGUUAGGCUCUUGGGAGCU). All steps followed the manufacturer’s suggested protocol, and SNORD68 was used as an internal control. Ct values more than 36 were considered as negative. The specificities of the amplicons for the SYBR Green−based PCR amplification were affirmed by the melting curves. The 2−ΔΔCt technique was used to measure the relative expression of the HCC-specific RNA panel.","Liver sections were cut at 5-μm thickness and stained with H&E. Immunohistochemistry S-P method was used to detect GSTP and PCNA levels. Briefly, the protocol was as follows: (1) the tissues were treated with endogenous peroxidase blocking solution at room temperature for 10 min, and then, incubated in normal nonimmune serum at room temperature for 10 min; (2) mouse anti GSTP or PCNA antibody was added to adjacent tissue sections respectively and incubated overnight at 4 °C; (3) biotin-conjugated second antibody was added to the sections and incubated at room temperature for 10 min; and (4) S-P complex was added at room temperature for 10 min, and then, 2,4-diaminobutyric acid was used for the color reaction. The tissue sections were washed with Poly (butylene succinate) (PBS) (0.01 mol/L, pH 7.4) between each step. Positive and negative controls were simultaneously used to ensure specificity and reliability of the staining process. A positive section was taken as positive control. In negative control, PBS was used to replace the first antibody. GSTP-positivity was indicated by brown coloration of the cytoplasm. Morphometric analysis of the foci percent area was carried out using Leica Q win V.3 software after capturing the images using a Leica DM2500 microscope (Leica, Wetzlar, Germany). The PCNA labeling indices are represented as the expression of positively-stained nuclei (10 fields/slide at 400 ×) as shown below: +, positive expression found in 1-3 fields; ++, positive expression found in 4-6 fields; and +++, positive expression found in 7-10 fields. Rabbit monoclonal PCNA and GSTP antibodies were purchased from Abcam (San Francisco, United States).","All results were expressed as mean ± SD. Statistical analysis was carried out using GraphPad Prism version 6.01. Unpaired t-test was done to compare between naïve and PCL groups. One-way ANOVA, followed by Tukey’s test, was carried out to compare between all groups. P values < 0.05 were considered statistically significant. ","Effect on AFP levels and liver function tests As shown in Figure 1, following induction of PCL, rats exhibited a significant increase in AFP levels. AFP levels were significantly decreased in cyan-10, cyan-15, and cyan-20 groups compared to PCL group. There was no significant difference between the AFP levels of the three cyan groups. Liver function tests were affected after receiving DEN/2-AAF in which rats exhibited a significant increase in levels of ALT and total and direct bilirubin, and a significant decrease in the serum albumin (Figure 2). As shown in Figure 2A, rats received cyan. in different doses exhibited a significant decrease in serum ALT compared to PCL group. Cyan-15 group showed significant decrease in serum ALT compared to cyan-10 group. Moreover, cyan-20 group showed significant decrease in serum ALT compared to cyan-10 group. In As depicted in Figure 2B, compared to PCL group, the rats in the three cyan groups exhibited a significant decrease in serum total bilirubin. Furthermore, cyan-20 group showed significant decrease in serum total bilirubin compared to cyan-10 group. Figure 2C: Serum direct bilirubin was significantly decreased in the three cyan groups compared to the PCL group. Moreover, cyan-20 group showed significant decrease in serum direct bilirubin compared to cyan-10 group. Administration of cyan in doses 5, 10 and 20 mg/kg/d produced a significant increase in serum albumin compared to PCL group. However, there was no significant difference between the serum albumin levels of the three cyan groups (Figure 2D).\n\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) on alpha fetoprotein in the serum of rats. Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group, unpaired t test; bP < 0.05 significant differences compared to precancerous lesion group. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; AFP: Alpha fetoprotein.\n\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) in rats. A: Alanine aminotransferase; B: Total bilirubin; C: Direct bilirubin; D: Serum albumin. Values are mean ± SEM; number of animals = 6 rats/each group. bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; ALT: Alanine aminotransferase.\nAs shown in Figure 1, following induction of PCL, rats exhibited a significant increase in AFP levels. AFP levels were significantly decreased in cyan-10, cyan-15, and cyan-20 groups compared to PCL group. There was no significant difference between the AFP levels of the three cyan groups. Liver function tests were affected after receiving DEN/2-AAF in which rats exhibited a significant increase in levels of ALT and total and direct bilirubin, and a significant decrease in the serum albumin (Figure 2). As shown in Figure 2A, rats received cyan. in different doses exhibited a significant decrease in serum ALT compared to PCL group. Cyan-15 group showed significant decrease in serum ALT compared to cyan-10 group. Moreover, cyan-20 group showed significant decrease in serum ALT compared to cyan-10 group. In As depicted in Figure 2B, compared to PCL group, the rats in the three cyan groups exhibited a significant decrease in serum total bilirubin. Furthermore, cyan-20 group showed significant decrease in serum total bilirubin compared to cyan-10 group. Figure 2C: Serum direct bilirubin was significantly decreased in the three cyan groups compared to the PCL group. Moreover, cyan-20 group showed significant decrease in serum direct bilirubin compared to cyan-10 group. Administration of cyan in doses 5, 10 and 20 mg/kg/d produced a significant increase in serum albumin compared to PCL group. However, there was no significant difference between the serum albumin levels of the three cyan groups (Figure 2D).\n\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) on alpha fetoprotein in the serum of rats. Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group, unpaired t test; bP < 0.05 significant differences compared to precancerous lesion group. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; AFP: Alpha fetoprotein.\n\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) in rats. A: Alanine aminotransferase; B: Total bilirubin; C: Direct bilirubin; D: Serum albumin. Values are mean ± SEM; number of animals = 6 rats/each group. bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; ALT: Alanine aminotransferase.\nEffect on expression of lncRNA MALAT1, miR-125b, and TUBG1 (G2/M transition of mitotic cell cycle) in the liver tissue As shown in Figure 3, induction of PCL resulted in significant increase of lncRNA MALAT a significant decrease in RQ miR-125b and significant increase in RQ TUBG1 expressions in comparison to naïve group. RQ of lncRNA MALAT1 in the liver tissue was significantly decreased in cyan -10,15 and cyan-20 groups compared to PCL group. Furthermore, cyan-20 group showed significant decrease in RQ of lncRNA MALAT1 in the liver tissue compared to cyan-10 group (Figure 3A). On the contrary, RQ of miR-125b in the liver tissue was significantly increased in cyan -10, cyan-15 and cyan-20 groups compared to PCL group. In addition, cyan-20 group showed significant increase in RQ of miR-125b in the liver tissue compared to cyan-10 group (Figure 3B). Furthermore, RQ of TUBG1 in the liver tissue was significantly decreased in cyan-10, cyan-15 and cyan-20 groups compared to PCL group. Also, cyan-20 group showed significant decrease in RQ of TUBG1 in the liver tissue compared to cyan-10 group (Figure 3C).\n\nEffect on expression of of long non-coding RNA, MALAT1, miR-125b and tubulin 1 (G2/M transition of mitotic cell cycle) in the liver tissue. A: RQ of long non-coding RNA MALAT1; B: RQ miR-125b; C: RQ TUBGI . Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group; bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; TUBG: Tubulin gamma 1; lncRNA: Long non-coding RNA.\nAs shown in Figure 3, induction of PCL resulted in significant increase of lncRNA MALAT a significant decrease in RQ miR-125b and significant increase in RQ TUBG1 expressions in comparison to naïve group. RQ of lncRNA MALAT1 in the liver tissue was significantly decreased in cyan -10,15 and cyan-20 groups compared to PCL group. Furthermore, cyan-20 group showed significant decrease in RQ of lncRNA MALAT1 in the liver tissue compared to cyan-10 group (Figure 3A). On the contrary, RQ of miR-125b in the liver tissue was significantly increased in cyan -10, cyan-15 and cyan-20 groups compared to PCL group. In addition, cyan-20 group showed significant increase in RQ of miR-125b in the liver tissue compared to cyan-10 group (Figure 3B). Furthermore, RQ of TUBG1 in the liver tissue was significantly decreased in cyan-10, cyan-15 and cyan-20 groups compared to PCL group. Also, cyan-20 group showed significant decrease in RQ of TUBG1 in the liver tissue compared to cyan-10 group (Figure 3C).\n\nEffect on expression of of long non-coding RNA, MALAT1, miR-125b and tubulin 1 (G2/M transition of mitotic cell cycle) in the liver tissue. A: RQ of long non-coding RNA MALAT1; B: RQ miR-125b; C: RQ TUBGI . Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group; bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; TUBG: Tubulin gamma 1; lncRNA: Long non-coding RNA.\nHistopathological and immunohistochemical studies Light microscopic examination of H&E-stained sections of naïve rats’ liver showed normal hepatic architecture, cords of hepatocytes radiating from central vein, portal triads present in between, polygonal hepatocytes with centrally rounded vesicular nuclei, and hepatic sinusoid in between (Figure 4A and B). Liver sections obtained from rats received DEN/2-AAF (Figure 4C-E) showed disruption of normal hepatic lobular architecture along with presence of large, discriminated dysplastic nodules compressing the surrounding liver tissue, with high-grade dysplastic cells displaying increased nuclear: Cytoplasmatic ratio (Figure 4E), nuclear hyperchromatosis, and basophilic cytoplasm. Liver sections of rats treated with different doses of cyanidin (10, 15, and 20 mg/kg) respectively, showed small and less discriminated dysplastic nodules (Figure 4F-H).\n\nPhotomicrographs of liver sections stained with H&E staining. A and B: Naïve group liver sections showed normal hepatic architecture, cords of hepatocytes radiating from central vein and portal triads present in-between and polygonal hepatocytes with central rounded vesicular nuclei, and hepatic sinusoid in-between; C-E: Liver sections of rats received diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF) showed larger, discriminated dysplastic nodules (doted shapes) compressing the surrounding liver tissue with disruption of normal hepatic lobular architecture; D: Liver sections of rats received DEN/2-AAF showed eosinophilic foci of cellular alteration consisting of enlarged hepatocytes with increased acidophilic staining and vaculated nuclei (arrow head); E: Liver sections of rats received DEN/2-AAF showed foci of Clear cell formed of hepatocytes showing variable degrees of cytoplasmic vacuolations and ballooning with pyknotic nuclei (arrow); F-H: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) respectively, showing small and less discriminated dysplastic nodules (doted shapes). (Magnification: A, C, F, G, H × 1000; B, D, E × 400). CV: Central vein; PV: Portal vein; Pt: Portal triads; S: Sinusoid; H: Hepatocytes.\nImmunohistochemical examination with GSTP antibody appeared as large hyperplastic nodules were demonstrated by the brown color. Figure 5A and B shows the liver sections of naïve group, while Figure 5C shows large-positively stained GSTP foci demonstrated in PCL group. Liver sections from rats of all cyan-treated groups exhibited small GSTP-positive small hepatic foci of different sizes scattered in-between negatively-stained hepatic parenchyma (Figure 5D-F). The percent area of GSTP foci was significantly increased in the PCL group compared to naïve group, while all cyan-treated groups showed significant decrease in the GSTP percent area. Furthermore, cyan-15 and -20 showed significant decrease in the GSTP percent area over cyan-10 (Table 1). PCNA immunohistochemical analysis showed an elevated expression of PCNA in the group that received DEN/2-AAF as compared to naïve group (Figure 6A and B). Liver sections obtained from rats received cyan. in its three doses showed decrease in PCNA-positively stained nuclei (Figure 6C-E and Table 1).\n\nPhotomicrographs of liver sections of rats immunohistochemical stained with glutathione S-transferase placental antibody. A and B: Naive group; C: Precancerous lesion group showing multiple glutathione S-transferase placental (GSTP)-positive large hepatic nodules (brown stained nodules) occupying most of section; D-F: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) showing GSTP positive small hepatic foci (brown stained cells = arrow) of different size scattered in-between negatively stained hepatic parenchyma. (Magnification: × 40)\n\nPhotomicrographs of liver sections Immunohistochemical stained with proliferating cell nuclear antigen. A: Negative reaction of control group; B: Diethylnitrosamine + 100 mg 2-acetylaminofluorene group showing positive stained nuclei scattered all over the field; C-E: Liver sections of rats treated with different doses of cyamidine (10, 15, 20 mg/kg) respectively, show few positive hepatocytes sporadically distributed over the field (arrow). (Magnification × 100). \nExpression rate of hepatocytes positive for glutathione S-transferase placental and proliferating cell nuclear antigen were calculated as number of positive field expression in 10 fields per rat liver tissue\nValues are mean ± SD; number of animals = 6 rats/each group. Examined field; 10 fields/liver section. \n\nP < 0.05 significant differences compared to naïve group.\n\nP < 0.05 significant differences compared to precancerous lesion group.\n\nP < 0.05 significant compared to cyanidin-3-O-glucoside-10, One-way ANOVA followed by Tukey’s test. +: Positive expression found in 1-3 fields; ++: Positive expression found in 4-6 fields; +++: Positive expression found in 7-10 field; PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; GSTP: Glutathione S-transferase placental; PCNA: Proliferating cell nuclear antigen.\nLight microscopic examination of H&E-stained sections of naïve rats’ liver showed normal hepatic architecture, cords of hepatocytes radiating from central vein, portal triads present in between, polygonal hepatocytes with centrally rounded vesicular nuclei, and hepatic sinusoid in between (Figure 4A and B). Liver sections obtained from rats received DEN/2-AAF (Figure 4C-E) showed disruption of normal hepatic lobular architecture along with presence of large, discriminated dysplastic nodules compressing the surrounding liver tissue, with high-grade dysplastic cells displaying increased nuclear: Cytoplasmatic ratio (Figure 4E), nuclear hyperchromatosis, and basophilic cytoplasm. Liver sections of rats treated with different doses of cyanidin (10, 15, and 20 mg/kg) respectively, showed small and less discriminated dysplastic nodules (Figure 4F-H).\n\nPhotomicrographs of liver sections stained with H&E staining. A and B: Naïve group liver sections showed normal hepatic architecture, cords of hepatocytes radiating from central vein and portal triads present in-between and polygonal hepatocytes with central rounded vesicular nuclei, and hepatic sinusoid in-between; C-E: Liver sections of rats received diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF) showed larger, discriminated dysplastic nodules (doted shapes) compressing the surrounding liver tissue with disruption of normal hepatic lobular architecture; D: Liver sections of rats received DEN/2-AAF showed eosinophilic foci of cellular alteration consisting of enlarged hepatocytes with increased acidophilic staining and vaculated nuclei (arrow head); E: Liver sections of rats received DEN/2-AAF showed foci of Clear cell formed of hepatocytes showing variable degrees of cytoplasmic vacuolations and ballooning with pyknotic nuclei (arrow); F-H: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) respectively, showing small and less discriminated dysplastic nodules (doted shapes). (Magnification: A, C, F, G, H × 1000; B, D, E × 400). CV: Central vein; PV: Portal vein; Pt: Portal triads; S: Sinusoid; H: Hepatocytes.\nImmunohistochemical examination with GSTP antibody appeared as large hyperplastic nodules were demonstrated by the brown color. Figure 5A and B shows the liver sections of naïve group, while Figure 5C shows large-positively stained GSTP foci demonstrated in PCL group. Liver sections from rats of all cyan-treated groups exhibited small GSTP-positive small hepatic foci of different sizes scattered in-between negatively-stained hepatic parenchyma (Figure 5D-F). The percent area of GSTP foci was significantly increased in the PCL group compared to naïve group, while all cyan-treated groups showed significant decrease in the GSTP percent area. Furthermore, cyan-15 and -20 showed significant decrease in the GSTP percent area over cyan-10 (Table 1). PCNA immunohistochemical analysis showed an elevated expression of PCNA in the group that received DEN/2-AAF as compared to naïve group (Figure 6A and B). Liver sections obtained from rats received cyan. in its three doses showed decrease in PCNA-positively stained nuclei (Figure 6C-E and Table 1).\n\nPhotomicrographs of liver sections of rats immunohistochemical stained with glutathione S-transferase placental antibody. A and B: Naive group; C: Precancerous lesion group showing multiple glutathione S-transferase placental (GSTP)-positive large hepatic nodules (brown stained nodules) occupying most of section; D-F: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) showing GSTP positive small hepatic foci (brown stained cells = arrow) of different size scattered in-between negatively stained hepatic parenchyma. (Magnification: × 40)\n\nPhotomicrographs of liver sections Immunohistochemical stained with proliferating cell nuclear antigen. A: Negative reaction of control group; B: Diethylnitrosamine + 100 mg 2-acetylaminofluorene group showing positive stained nuclei scattered all over the field; C-E: Liver sections of rats treated with different doses of cyamidine (10, 15, 20 mg/kg) respectively, show few positive hepatocytes sporadically distributed over the field (arrow). (Magnification × 100). \nExpression rate of hepatocytes positive for glutathione S-transferase placental and proliferating cell nuclear antigen were calculated as number of positive field expression in 10 fields per rat liver tissue\nValues are mean ± SD; number of animals = 6 rats/each group. Examined field; 10 fields/liver section. \n\nP < 0.05 significant differences compared to naïve group.\n\nP < 0.05 significant differences compared to precancerous lesion group.\n\nP < 0.05 significant compared to cyanidin-3-O-glucoside-10, One-way ANOVA followed by Tukey’s test. +: Positive expression found in 1-3 fields; ++: Positive expression found in 4-6 fields; +++: Positive expression found in 7-10 field; PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; GSTP: Glutathione S-transferase placental; PCNA: Proliferating cell nuclear antigen.","As shown in Figure 1, following induction of PCL, rats exhibited a significant increase in AFP levels. AFP levels were significantly decreased in cyan-10, cyan-15, and cyan-20 groups compared to PCL group. There was no significant difference between the AFP levels of the three cyan groups. Liver function tests were affected after receiving DEN/2-AAF in which rats exhibited a significant increase in levels of ALT and total and direct bilirubin, and a significant decrease in the serum albumin (Figure 2). As shown in Figure 2A, rats received cyan. in different doses exhibited a significant decrease in serum ALT compared to PCL group. Cyan-15 group showed significant decrease in serum ALT compared to cyan-10 group. Moreover, cyan-20 group showed significant decrease in serum ALT compared to cyan-10 group. In As depicted in Figure 2B, compared to PCL group, the rats in the three cyan groups exhibited a significant decrease in serum total bilirubin. Furthermore, cyan-20 group showed significant decrease in serum total bilirubin compared to cyan-10 group. Figure 2C: Serum direct bilirubin was significantly decreased in the three cyan groups compared to the PCL group. Moreover, cyan-20 group showed significant decrease in serum direct bilirubin compared to cyan-10 group. Administration of cyan in doses 5, 10 and 20 mg/kg/d produced a significant increase in serum albumin compared to PCL group. However, there was no significant difference between the serum albumin levels of the three cyan groups (Figure 2D).\n\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) on alpha fetoprotein in the serum of rats. Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group, unpaired t test; bP < 0.05 significant differences compared to precancerous lesion group. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; AFP: Alpha fetoprotein.\n\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) in rats. A: Alanine aminotransferase; B: Total bilirubin; C: Direct bilirubin; D: Serum albumin. Values are mean ± SEM; number of animals = 6 rats/each group. bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; ALT: Alanine aminotransferase.","As shown in Figure 3, induction of PCL resulted in significant increase of lncRNA MALAT a significant decrease in RQ miR-125b and significant increase in RQ TUBG1 expressions in comparison to naïve group. RQ of lncRNA MALAT1 in the liver tissue was significantly decreased in cyan -10,15 and cyan-20 groups compared to PCL group. Furthermore, cyan-20 group showed significant decrease in RQ of lncRNA MALAT1 in the liver tissue compared to cyan-10 group (Figure 3A). On the contrary, RQ of miR-125b in the liver tissue was significantly increased in cyan -10, cyan-15 and cyan-20 groups compared to PCL group. In addition, cyan-20 group showed significant increase in RQ of miR-125b in the liver tissue compared to cyan-10 group (Figure 3B). Furthermore, RQ of TUBG1 in the liver tissue was significantly decreased in cyan-10, cyan-15 and cyan-20 groups compared to PCL group. Also, cyan-20 group showed significant decrease in RQ of TUBG1 in the liver tissue compared to cyan-10 group (Figure 3C).\n\nEffect on expression of of long non-coding RNA, MALAT1, miR-125b and tubulin 1 (G2/M transition of mitotic cell cycle) in the liver tissue. A: RQ of long non-coding RNA MALAT1; B: RQ miR-125b; C: RQ TUBGI . Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group; bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; TUBG: Tubulin gamma 1; lncRNA: Long non-coding RNA.","Light microscopic examination of H&E-stained sections of naïve rats’ liver showed normal hepatic architecture, cords of hepatocytes radiating from central vein, portal triads present in between, polygonal hepatocytes with centrally rounded vesicular nuclei, and hepatic sinusoid in between (Figure 4A and B). Liver sections obtained from rats received DEN/2-AAF (Figure 4C-E) showed disruption of normal hepatic lobular architecture along with presence of large, discriminated dysplastic nodules compressing the surrounding liver tissue, with high-grade dysplastic cells displaying increased nuclear: Cytoplasmatic ratio (Figure 4E), nuclear hyperchromatosis, and basophilic cytoplasm. Liver sections of rats treated with different doses of cyanidin (10, 15, and 20 mg/kg) respectively, showed small and less discriminated dysplastic nodules (Figure 4F-H).\n\nPhotomicrographs of liver sections stained with H&E staining. A and B: Naïve group liver sections showed normal hepatic architecture, cords of hepatocytes radiating from central vein and portal triads present in-between and polygonal hepatocytes with central rounded vesicular nuclei, and hepatic sinusoid in-between; C-E: Liver sections of rats received diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF) showed larger, discriminated dysplastic nodules (doted shapes) compressing the surrounding liver tissue with disruption of normal hepatic lobular architecture; D: Liver sections of rats received DEN/2-AAF showed eosinophilic foci of cellular alteration consisting of enlarged hepatocytes with increased acidophilic staining and vaculated nuclei (arrow head); E: Liver sections of rats received DEN/2-AAF showed foci of Clear cell formed of hepatocytes showing variable degrees of cytoplasmic vacuolations and ballooning with pyknotic nuclei (arrow); F-H: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) respectively, showing small and less discriminated dysplastic nodules (doted shapes). (Magnification: A, C, F, G, H × 1000; B, D, E × 400). CV: Central vein; PV: Portal vein; Pt: Portal triads; S: Sinusoid; H: Hepatocytes.\nImmunohistochemical examination with GSTP antibody appeared as large hyperplastic nodules were demonstrated by the brown color. Figure 5A and B shows the liver sections of naïve group, while Figure 5C shows large-positively stained GSTP foci demonstrated in PCL group. Liver sections from rats of all cyan-treated groups exhibited small GSTP-positive small hepatic foci of different sizes scattered in-between negatively-stained hepatic parenchyma (Figure 5D-F). The percent area of GSTP foci was significantly increased in the PCL group compared to naïve group, while all cyan-treated groups showed significant decrease in the GSTP percent area. Furthermore, cyan-15 and -20 showed significant decrease in the GSTP percent area over cyan-10 (Table 1). PCNA immunohistochemical analysis showed an elevated expression of PCNA in the group that received DEN/2-AAF as compared to naïve group (Figure 6A and B). Liver sections obtained from rats received cyan. in its three doses showed decrease in PCNA-positively stained nuclei (Figure 6C-E and Table 1).\n\nPhotomicrographs of liver sections of rats immunohistochemical stained with glutathione S-transferase placental antibody. A and B: Naive group; C: Precancerous lesion group showing multiple glutathione S-transferase placental (GSTP)-positive large hepatic nodules (brown stained nodules) occupying most of section; D-F: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) showing GSTP positive small hepatic foci (brown stained cells = arrow) of different size scattered in-between negatively stained hepatic parenchyma. (Magnification: × 40)\n\nPhotomicrographs of liver sections Immunohistochemical stained with proliferating cell nuclear antigen. A: Negative reaction of control group; B: Diethylnitrosamine + 100 mg 2-acetylaminofluorene group showing positive stained nuclei scattered all over the field; C-E: Liver sections of rats treated with different doses of cyamidine (10, 15, 20 mg/kg) respectively, show few positive hepatocytes sporadically distributed over the field (arrow). (Magnification × 100). \nExpression rate of hepatocytes positive for glutathione S-transferase placental and proliferating cell nuclear antigen were calculated as number of positive field expression in 10 fields per rat liver tissue\nValues are mean ± SD; number of animals = 6 rats/each group. Examined field; 10 fields/liver section. \n\nP < 0.05 significant differences compared to naïve group.\n\nP < 0.05 significant differences compared to precancerous lesion group.\n\nP < 0.05 significant compared to cyanidin-3-O-glucoside-10, One-way ANOVA followed by Tukey’s test. +: Positive expression found in 1-3 fields; ++: Positive expression found in 4-6 fields; +++: Positive expression found in 7-10 field; PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; GSTP: Glutathione S-transferase placental; PCNA: Proliferating cell nuclear antigen.","Altered expression of mitotic spindle assembly genes has been detected in many cancers. For example, MAD2 gene levels are downregulated in breast carcinoma[18], while mitotic checkpoint serine/BUB1 gene expression is dysregulated in colorectal carcinoma[19]. The impairment of spindle assembly genes are frequently detected in HCC[20,21]. Moreover, inhibition of the mitotic assembly genes has been found to be lethal to cancer cells, and has promising therapeutic effect in cancer treatment[22,23]. \nIn this study, we used several public microarray databases for the construction of simple genetic-epigenetic network specific to HCC and linked to cell cycle and mitotic spindle formation. We assessed the antiproliferative effect of cyan in HCC animal model via modulation of lncRNA-MALAT1-miR-125b-TUBG1 mRNA axis expression.\nMicrotubules are involved both in maintenance of cell shape and motility and in mitotic spindle formation[24]. γ-TUBG is a major player that supports microtubule nucleation. γ-TUBG acts as a binding site for the α/β-tubulin dimer and it has two γ-TUBG genes, TUBG1 and TUBG2 in mammals only[25]. The TUBG levels in the centrosome change during cell cycle progression and usually increases at the start of mitosis. At the end of mitosis, TUBG levels are decreased to interphase levels[26]. Overexpression and altered compartmentalization of γ-TUBG may lead to carcinogenesis[27-29]. Interestingly, γ-TUBG associates interact with tumor suppressor protein C53 in mammalian interphase cells of different cellular origins[30]. Interestingly, γ-TUBG interacts with E2F transcription factors, resulting in upregulation of E2F target genes PCNA and CDKB1[31] and glutathione S transferase activity[32]. On the other hand, the mRNA and protein levels of TUBA1C are upregulated in HCC. Clinically, it affects the survival of patients and the level of metastatic burden along with portal vein thrombosis. In addition, it activates cellular proliferation and migration and is a potential therapeutic target for HCC[33]. Recent studies have shed light on the efficacy of microtubule-binding agents in the treatment of HCC, especially in combination with mammalian target of rapamycin inhibitors[34].\nOne of the few miRNAs that regulate Spindle Assembly Checkpoints (SAC) is miR-125b. It suppresses the expression of Mad1 (a core SAC protein) that is responsible for inhibiting entry into anaphase till metaphase defects are corrected[35]. Importantly, miRNA-125b has been found to be associated with colorectal cancer[36], multiple myeloma[37], and breast cancer[38]. Song et al[39] demonstrated that the expression of miR-125b in hepatocytes was protective and reflected better survival in HCC tissue samples due to restoration of SIRT6 expression[39]. miR-125b-5p inhibits HCC proliferation by targeting TXNRD1, the terminal step of biosynthesis of nucleotides[40].\nDeregulation of lncRNAs has been shown to be crucial in cancer progression, especially HCC[41,42]. Hung et al[43] demonstrated the involvement of lncRNAs in regulation of the expression of cell cycle-related genes, p53 gene regulatory pathway, and apoptosis[43,44]. The lncRNA MALAT1 is overexpressed in several solid tumors and is linked with tumor recurrence[45-47]. MALAT1 coordinate RNA polymerase II transcription, pre-mRNA splicing, and mRNA export[48]. Thus, this lncRNA holds great potential in influencing the local concentration of a specific splicing factor during specific stages of the cell cycle. Depletion of MALAT1 in cancer cells inhibits tumorigenicity[49]. MALAT1 regulates E2F1 transcription factor activity, highlighting its pro-proliferative function[50]. MALAT1 is upregulated in many cancer types, including breast cancer[51], cervical cancer, lung cancer[52], and hepatocarcinoma[53-55]. In a previous study, hypoxia enhanced MALAT1 expression, leading to increase in proliferating and invading activity of Hep3B cells due to negative interaction with miR200a[56]. Huang and his group explored the role of specificity protein 1/3 in regulation of MALAT1 expression in HCC cells[57].\nCyanidin or anthocyanin (a polyphenolic pigment found in plants) confers several pharmacological benefits, including anticancer properties[58-62]. Cyan mediates cytotoxicity against human monocytic leukemia cells via G2/M phase arrest and induction of apoptosis[63]. Tsai et al[64] evaluated anthocyanins as promising anticancer or chemopreventive agents to induce G2/M arrest in HAs-induced leukemia cell cycle arrest via ATM-Chk1/2-Cdc25C axis modulation[64]. They also found that anthocyanin inhibits the proliferation of HCC cells[65,66].\nIn our study, the effect of cyan on cancerous tissue was dose-dependent (10, 15 and 20 mg/kg/d), that is, increasing its effect with increasing the concentration of the drug. This was proved also, with the correlated significant decrease in the percent area of GSTP foci, PCNA expression which are the target of TUBG1 mRNA and the molecular epigenetic markers associated with HCC (lncRNA MALAT1 and miR-125b-1-3p) in the liver tissue of rats in our study (Figure 7). Thus, we can hypothesize that administration of cyan in PCL may result in downregulation of lncRNA MALAT1 with subsequent release of free miR-125b-1-3p that binds to TUBG1 mRNA and downregulate its expression[65]. The changes in the epigenetic-genetic network affect their target genes PCNA and GSTP.\n\nConcept map of study design. GSTP: Glutathione S-transferase placental; PCNA: Proliferating cell nuclear antigen; TUBG: Tubulin; lncRNA: Long non-coding RNA.\nLastly, the cyan-mediated modulation of cell cycle and mitotic spindle assembly that regulate HCC proliferation might affect the capability of the HCC cells to overcome oxidative stress[66,67]. Our data confers the rationale for designing antimitotic approach combining the conventional cytotoxic drugs with phytochemical extracts to inhibit cell cycle progression in cancer. More in vitro functional studies are required to explore the functional mechanism of the chosen RNA panel and validate their role as drug target biomarkers. ","Cyanidin is a natural molecule that holds great potential in unraveling the mystery of cytotoxic pharmacy in the future. It can be used to devise novel antimitotic drugs that can target cell cycle in HCC. Our results also indicated great potential of the TUBG1 mRNA- miR-125b-lncRNA MALAT1 and -1-3p axis as potential drug target biomarker for further investigations."],"string":"[\n \"Hepatocellular carcinoma (HCC) is the commonest primary malignancy of the liver. Its main risk factors include hepatitis B, hepatitis C, and non-alcoholic steatohepatitis. HCC is associated with high mortality and morbidity. Its incidence has increased from 1.4 to 6.2 per 100000 cases per year within the last 30 years[1]. HCC burden in Egypt is high due to the high prevalence of HCV, where liver cancers account for 11.75% of all gastrointestinal cancers and 1.68% of all malignancies. In addition, over 70% of all liver malignancies in Egypt are HCC[2].\\nTo date, HCC is holding its place as an unresolved dilemma; its resistance to systemic chemotherapy and radiation makes the cure approachable only in very early stages according to Milan criteria, that is, where the tumor size is < 5 cm (when only one lesion is present) or < 3 cm (when 2-3 lesions are present)[3].\\nGenes involved in cell cycle control are usually mutated in presence of cancer. Dysregulated mitosis results in genomic instability, which leads to tumor aggressiveness[4]. Microtubules are a key component of mitotic spindles, and thus, a crucial part of the process of mitosis. They form an important target in cancer chemotherapy, inducing arrest of mitosis and cell death. On the other hand, while the overexpression of γ-tubulin (γ-TUBG) showed improved survival among small cell lung cancer patients[5], the overexpression of γ-TUBG is found to be characteristic feature of thyroid and breast cancers[6]. Such variation may arise due to differences in gene transcription and check point regulations in different cancer tissues[7].\\nCyanidin-3-glucoside (cyan) is a potential chemotherapeutic and chemo-protective agent. Through its antioxidant activity, it scavenges radical oxygen species, which decreases the overall number of tumorous cells (malignant and benign) in in vivo studies[8]. Cyan has been implicated in some beneficial health activities[9], including reducing age-associated oxidative stress[10], improving cognitive brain function, and exhibiting anti-diabetic[11], anti-inflammation[12], anti-atherogenic[13], and anti-obesity activities[14]. Previously, it has been reported that cyan mediates caspase-3 cleavage with DNA fragmentation. Cyan has also been associated with induction of autophagy, a key element involved in cancer elimination, via induction of autophagy-related gene 5 and microtubule-associated protein 1 light chain 3-II[15]. Cyan has drawn increasing attention because of its potential anti-cancer properties. Cyan may offer a novel avenue for treating HCC. a potential antiproliferative effect on HepG2 cells[16].\\nThe present study was conducted to evaluate the effect of cyan in three different doses on cell cycle progression and mitotic assembly disorder in hepatic precancerous lesion (PCL) via assessment of levels of long non-coding RNA (lncRNA) MALAT1, miR-125b, and tubulin 1. We also conducted histopathological and immuno-histochemical examination using H&E staining assessment of levels of glutathione S-transferase placental (GSTP) and proliferating cell nuclear antigen (PCNA) antibodies in male Wistar rat model of diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF)-induced hepatic PCL. \",\n \"DEN was obtained as 1 g solution in serum bottle diluted in 0.9% NaCl. 2-AAF was obtained as white powder, while cyan was obtained as black powder. Both 2-AAF and cyan were dissolved in 0.9% NaCl. All chemicals were purchased from Sigma-Aldrich, Cairo, Egypt.\",\n \"All animal procedures were approved by the Institutional Animal Ethics Committee of Ain Shams University, Faculty of Medicine. Thirty male Wistar rats weighing 200-250 g were purchased from Nile Pharmaceuticals Company (Cairo, Egypt). Animals were housed in an animal room with temperature 25 ± 2 °C and 12 h light/dark cycle controls. An adaptation period of 1 wk was allowed before initiation of the experimental protocol.\",\n \"\\nInduction of hepatic PCL: DEN was injected intraperitoneally (i.p.) at a dose of 100 mg/kg once weekly for three consecutive weeks. One week after the last DEN injection, a single dose (300 mg/kg) of 2-AAF was injected.\\n\\nAnimal groups: Rats were divided into five groups (6 rats/each group), including: Naïve group: Rats were injected with a vehicle (0.9% NaCl), PCL group: Rats were injected with DEN and 2-AAF, and three cyan groups (cyan-10, cyan-15, and cyan-20 groups): Rats were injected with DEN and 2-AAF, and orally administered through gastric gavage with cyan in doses of 10, 15, and 20 mg/kg respectively, for four consecutive days per week for 16 wk[17].\",\n \"\\nBiochemical and molecular studies: After scarifying the animals, blood was withdrawn from each rat from the retro orbital vein and incubated for about half an hour for clotting. Then, the blood sample was centrifuged for 20 min at 5000 RPM to get serum samples. Livers were dissected. The right lobe of the livers was removed, cut into longitudinal sections 2-4 mm in thickness and kept in 10% formalin for histopathological and immunohistochemical examination. The other lobe was kept frozen at -80 °C for molecular analyses. The samples were analyzed for the following parameters: (1) alpha fetoprotein (AFP) levels: AFP level was analyzed using quantitative sandwich rat AFP ELISA kit purchased from MyBiosource Inc. (San Diego, United States) with a sensitivity range of 0.625-20 ng/mL; (2) serum alanine aminotransferase (ALT) levels: The serum ALT was measured according to method described by using kits purchased from Diamond Diagnostic (Cairo, Egypt). ALT catalyzes the transfer of amino groups from specific amino acids to ketoglutaric acid, yielding glutamic acid and oxaloacetic or pyruvic acid, respectively. Ketoacids were then determined calorimetrically after their reaction with 2, 4- dinitrophenylhydrazine; and (3) determination of serum total bilirubin and direct bilirubin: Alkaline methanolysis of bilirubin followed by chloroform extraction of bilirubin methyl esters, and later, separation of these esters by chromatography and spectrophotometric determination at 430 nm.\",\n \"Quantitative determination of serum albumin level was quantified using rat albumin ELISA kit. The absorbance at 450 nm is a measure of the concentration of albumin in the test sample.\",\n \"The RNA panel was chosen in three steps: (1) Tubulin, gamma 1 (TUBG1) was retrieved as a crucial player in microtubule formation and progression of the cell cycle through gene atlas expression database (available at https://www.ebi.ac.uk/gxa/home) alongside with verification of the chosen gene from protein Atlas database (available at https://www.proteinatlas.org/), followed by analysis of TUBG1 gene ontology to ensure that it is linked to G2-M transition and mitotic spindle assembly, which is closely linked to cancer development through gene card database (available at https://www.genecards.org/); (2) we selected miR-125b-1-3p targeting TUBG1 mRNA though) miRWALK database available at http://mirwalk.umm.uni-heidelberg.de/), followed by pathway enrichment, which revealed that miR-125b-1-3p is linked to MAP kinase and ubiquitin-mediated proteolysis (strongly correlated with mitotic assembly and cell cycle progression) through (Diana database available at http://diana.imis.athena-innovation.gr/DianaTools/index.php); and (3) lncRNA metastasis-associated lung adenocarcinoma transcript 1, lncRNA MALAT1, was obtained as a lncRNA specific to HCC and targeting miR-125b-1-3p using noncode database available at http://www.noncode.org/ and European Bioinformatics institute database available at https://www.ebi.ac.uk.\",\n \"Total RNA was extracted from liver tissues using miRNeasy Mini kit (Cat No. 217004, Qiagen, United States) according to manufacturer’s protocol. The extracted RNA concentration and integrity were assessed using [(NanoDrop Technologies/Thermo Scientific, Wilmington, DE, United States)], with RNA purities were 1.8-2. The extracted total RNA was reverse transcribed into cDNA using a miScript II RT kit (Cat No. 218160, 218161, Qiagen, United States) on thermal cycler (Bio-Rad; Hercules, CA, United States), according to the manufacturer’s protocol. \",\n \"The relative expression of TUBG1 mRNA and lncRNA-MALAT1 in liver tissue were measured using a Quantitect SYBR Green Master Mix kit and an RT2 SYBR Green ROX real time-polymerase chain reaction (qPCR) Mastermix (Qiagen), respectively, using a 7500 qPCR Systems (Applied Biosystems, Foster City, CA, United States) detection system) and specific primers (Accession: NM_001128148, NM_003234, NR_002819, and ENST00000534336, respectively) supplied by Qiagen. GAPDH (Accession NM_002046.7) was used as a housekeeping gene. MiR-3163 expression in liver tissue was quantified by PCR using a miScript SYBR Green kit (Qiagen), a miScript universal primer, and a miRNA-specific forward primer (mir-125b-1-3p miScript Primer Assay) (Accession: MIMAT0004592 (5'-ACGGGUUAGGCUCUUGGGAGCU). All steps followed the manufacturer’s suggested protocol, and SNORD68 was used as an internal control. Ct values more than 36 were considered as negative. The specificities of the amplicons for the SYBR Green−based PCR amplification were affirmed by the melting curves. The 2−ΔΔCt technique was used to measure the relative expression of the HCC-specific RNA panel.\",\n \"Liver sections were cut at 5-μm thickness and stained with H&E. Immunohistochemistry S-P method was used to detect GSTP and PCNA levels. Briefly, the protocol was as follows: (1) the tissues were treated with endogenous peroxidase blocking solution at room temperature for 10 min, and then, incubated in normal nonimmune serum at room temperature for 10 min; (2) mouse anti GSTP or PCNA antibody was added to adjacent tissue sections respectively and incubated overnight at 4 °C; (3) biotin-conjugated second antibody was added to the sections and incubated at room temperature for 10 min; and (4) S-P complex was added at room temperature for 10 min, and then, 2,4-diaminobutyric acid was used for the color reaction. The tissue sections were washed with Poly (butylene succinate) (PBS) (0.01 mol/L, pH 7.4) between each step. Positive and negative controls were simultaneously used to ensure specificity and reliability of the staining process. A positive section was taken as positive control. In negative control, PBS was used to replace the first antibody. GSTP-positivity was indicated by brown coloration of the cytoplasm. Morphometric analysis of the foci percent area was carried out using Leica Q win V.3 software after capturing the images using a Leica DM2500 microscope (Leica, Wetzlar, Germany). The PCNA labeling indices are represented as the expression of positively-stained nuclei (10 fields/slide at 400 ×) as shown below: +, positive expression found in 1-3 fields; ++, positive expression found in 4-6 fields; and +++, positive expression found in 7-10 fields. Rabbit monoclonal PCNA and GSTP antibodies were purchased from Abcam (San Francisco, United States).\",\n \"All results were expressed as mean ± SD. Statistical analysis was carried out using GraphPad Prism version 6.01. Unpaired t-test was done to compare between naïve and PCL groups. One-way ANOVA, followed by Tukey’s test, was carried out to compare between all groups. P values < 0.05 were considered statistically significant. \",\n \"Effect on AFP levels and liver function tests As shown in Figure 1, following induction of PCL, rats exhibited a significant increase in AFP levels. AFP levels were significantly decreased in cyan-10, cyan-15, and cyan-20 groups compared to PCL group. There was no significant difference between the AFP levels of the three cyan groups. Liver function tests were affected after receiving DEN/2-AAF in which rats exhibited a significant increase in levels of ALT and total and direct bilirubin, and a significant decrease in the serum albumin (Figure 2). As shown in Figure 2A, rats received cyan. in different doses exhibited a significant decrease in serum ALT compared to PCL group. Cyan-15 group showed significant decrease in serum ALT compared to cyan-10 group. Moreover, cyan-20 group showed significant decrease in serum ALT compared to cyan-10 group. In As depicted in Figure 2B, compared to PCL group, the rats in the three cyan groups exhibited a significant decrease in serum total bilirubin. Furthermore, cyan-20 group showed significant decrease in serum total bilirubin compared to cyan-10 group. Figure 2C: Serum direct bilirubin was significantly decreased in the three cyan groups compared to the PCL group. Moreover, cyan-20 group showed significant decrease in serum direct bilirubin compared to cyan-10 group. Administration of cyan in doses 5, 10 and 20 mg/kg/d produced a significant increase in serum albumin compared to PCL group. However, there was no significant difference between the serum albumin levels of the three cyan groups (Figure 2D).\\n\\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) on alpha fetoprotein in the serum of rats. Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group, unpaired t test; bP < 0.05 significant differences compared to precancerous lesion group. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; AFP: Alpha fetoprotein.\\n\\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) in rats. A: Alanine aminotransferase; B: Total bilirubin; C: Direct bilirubin; D: Serum albumin. Values are mean ± SEM; number of animals = 6 rats/each group. bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; ALT: Alanine aminotransferase.\\nAs shown in Figure 1, following induction of PCL, rats exhibited a significant increase in AFP levels. AFP levels were significantly decreased in cyan-10, cyan-15, and cyan-20 groups compared to PCL group. There was no significant difference between the AFP levels of the three cyan groups. Liver function tests were affected after receiving DEN/2-AAF in which rats exhibited a significant increase in levels of ALT and total and direct bilirubin, and a significant decrease in the serum albumin (Figure 2). As shown in Figure 2A, rats received cyan. in different doses exhibited a significant decrease in serum ALT compared to PCL group. Cyan-15 group showed significant decrease in serum ALT compared to cyan-10 group. Moreover, cyan-20 group showed significant decrease in serum ALT compared to cyan-10 group. In As depicted in Figure 2B, compared to PCL group, the rats in the three cyan groups exhibited a significant decrease in serum total bilirubin. Furthermore, cyan-20 group showed significant decrease in serum total bilirubin compared to cyan-10 group. Figure 2C: Serum direct bilirubin was significantly decreased in the three cyan groups compared to the PCL group. Moreover, cyan-20 group showed significant decrease in serum direct bilirubin compared to cyan-10 group. Administration of cyan in doses 5, 10 and 20 mg/kg/d produced a significant increase in serum albumin compared to PCL group. However, there was no significant difference between the serum albumin levels of the three cyan groups (Figure 2D).\\n\\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) on alpha fetoprotein in the serum of rats. Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group, unpaired t test; bP < 0.05 significant differences compared to precancerous lesion group. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; AFP: Alpha fetoprotein.\\n\\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) in rats. A: Alanine aminotransferase; B: Total bilirubin; C: Direct bilirubin; D: Serum albumin. Values are mean ± SEM; number of animals = 6 rats/each group. bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; ALT: Alanine aminotransferase.\\nEffect on expression of lncRNA MALAT1, miR-125b, and TUBG1 (G2/M transition of mitotic cell cycle) in the liver tissue As shown in Figure 3, induction of PCL resulted in significant increase of lncRNA MALAT a significant decrease in RQ miR-125b and significant increase in RQ TUBG1 expressions in comparison to naïve group. RQ of lncRNA MALAT1 in the liver tissue was significantly decreased in cyan -10,15 and cyan-20 groups compared to PCL group. Furthermore, cyan-20 group showed significant decrease in RQ of lncRNA MALAT1 in the liver tissue compared to cyan-10 group (Figure 3A). On the contrary, RQ of miR-125b in the liver tissue was significantly increased in cyan -10, cyan-15 and cyan-20 groups compared to PCL group. In addition, cyan-20 group showed significant increase in RQ of miR-125b in the liver tissue compared to cyan-10 group (Figure 3B). Furthermore, RQ of TUBG1 in the liver tissue was significantly decreased in cyan-10, cyan-15 and cyan-20 groups compared to PCL group. Also, cyan-20 group showed significant decrease in RQ of TUBG1 in the liver tissue compared to cyan-10 group (Figure 3C).\\n\\nEffect on expression of of long non-coding RNA, MALAT1, miR-125b and tubulin 1 (G2/M transition of mitotic cell cycle) in the liver tissue. A: RQ of long non-coding RNA MALAT1; B: RQ miR-125b; C: RQ TUBGI . Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group; bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; TUBG: Tubulin gamma 1; lncRNA: Long non-coding RNA.\\nAs shown in Figure 3, induction of PCL resulted in significant increase of lncRNA MALAT a significant decrease in RQ miR-125b and significant increase in RQ TUBG1 expressions in comparison to naïve group. RQ of lncRNA MALAT1 in the liver tissue was significantly decreased in cyan -10,15 and cyan-20 groups compared to PCL group. Furthermore, cyan-20 group showed significant decrease in RQ of lncRNA MALAT1 in the liver tissue compared to cyan-10 group (Figure 3A). On the contrary, RQ of miR-125b in the liver tissue was significantly increased in cyan -10, cyan-15 and cyan-20 groups compared to PCL group. In addition, cyan-20 group showed significant increase in RQ of miR-125b in the liver tissue compared to cyan-10 group (Figure 3B). Furthermore, RQ of TUBG1 in the liver tissue was significantly decreased in cyan-10, cyan-15 and cyan-20 groups compared to PCL group. Also, cyan-20 group showed significant decrease in RQ of TUBG1 in the liver tissue compared to cyan-10 group (Figure 3C).\\n\\nEffect on expression of of long non-coding RNA, MALAT1, miR-125b and tubulin 1 (G2/M transition of mitotic cell cycle) in the liver tissue. A: RQ of long non-coding RNA MALAT1; B: RQ miR-125b; C: RQ TUBGI . Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group; bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; TUBG: Tubulin gamma 1; lncRNA: Long non-coding RNA.\\nHistopathological and immunohistochemical studies Light microscopic examination of H&E-stained sections of naïve rats’ liver showed normal hepatic architecture, cords of hepatocytes radiating from central vein, portal triads present in between, polygonal hepatocytes with centrally rounded vesicular nuclei, and hepatic sinusoid in between (Figure 4A and B). Liver sections obtained from rats received DEN/2-AAF (Figure 4C-E) showed disruption of normal hepatic lobular architecture along with presence of large, discriminated dysplastic nodules compressing the surrounding liver tissue, with high-grade dysplastic cells displaying increased nuclear: Cytoplasmatic ratio (Figure 4E), nuclear hyperchromatosis, and basophilic cytoplasm. Liver sections of rats treated with different doses of cyanidin (10, 15, and 20 mg/kg) respectively, showed small and less discriminated dysplastic nodules (Figure 4F-H).\\n\\nPhotomicrographs of liver sections stained with H&E staining. A and B: Naïve group liver sections showed normal hepatic architecture, cords of hepatocytes radiating from central vein and portal triads present in-between and polygonal hepatocytes with central rounded vesicular nuclei, and hepatic sinusoid in-between; C-E: Liver sections of rats received diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF) showed larger, discriminated dysplastic nodules (doted shapes) compressing the surrounding liver tissue with disruption of normal hepatic lobular architecture; D: Liver sections of rats received DEN/2-AAF showed eosinophilic foci of cellular alteration consisting of enlarged hepatocytes with increased acidophilic staining and vaculated nuclei (arrow head); E: Liver sections of rats received DEN/2-AAF showed foci of Clear cell formed of hepatocytes showing variable degrees of cytoplasmic vacuolations and ballooning with pyknotic nuclei (arrow); F-H: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) respectively, showing small and less discriminated dysplastic nodules (doted shapes). (Magnification: A, C, F, G, H × 1000; B, D, E × 400). CV: Central vein; PV: Portal vein; Pt: Portal triads; S: Sinusoid; H: Hepatocytes.\\nImmunohistochemical examination with GSTP antibody appeared as large hyperplastic nodules were demonstrated by the brown color. Figure 5A and B shows the liver sections of naïve group, while Figure 5C shows large-positively stained GSTP foci demonstrated in PCL group. Liver sections from rats of all cyan-treated groups exhibited small GSTP-positive small hepatic foci of different sizes scattered in-between negatively-stained hepatic parenchyma (Figure 5D-F). The percent area of GSTP foci was significantly increased in the PCL group compared to naïve group, while all cyan-treated groups showed significant decrease in the GSTP percent area. Furthermore, cyan-15 and -20 showed significant decrease in the GSTP percent area over cyan-10 (Table 1). PCNA immunohistochemical analysis showed an elevated expression of PCNA in the group that received DEN/2-AAF as compared to naïve group (Figure 6A and B). Liver sections obtained from rats received cyan. in its three doses showed decrease in PCNA-positively stained nuclei (Figure 6C-E and Table 1).\\n\\nPhotomicrographs of liver sections of rats immunohistochemical stained with glutathione S-transferase placental antibody. A and B: Naive group; C: Precancerous lesion group showing multiple glutathione S-transferase placental (GSTP)-positive large hepatic nodules (brown stained nodules) occupying most of section; D-F: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) showing GSTP positive small hepatic foci (brown stained cells = arrow) of different size scattered in-between negatively stained hepatic parenchyma. (Magnification: × 40)\\n\\nPhotomicrographs of liver sections Immunohistochemical stained with proliferating cell nuclear antigen. A: Negative reaction of control group; B: Diethylnitrosamine + 100 mg 2-acetylaminofluorene group showing positive stained nuclei scattered all over the field; C-E: Liver sections of rats treated with different doses of cyamidine (10, 15, 20 mg/kg) respectively, show few positive hepatocytes sporadically distributed over the field (arrow). (Magnification × 100). \\nExpression rate of hepatocytes positive for glutathione S-transferase placental and proliferating cell nuclear antigen were calculated as number of positive field expression in 10 fields per rat liver tissue\\nValues are mean ± SD; number of animals = 6 rats/each group. Examined field; 10 fields/liver section. \\n\\nP < 0.05 significant differences compared to naïve group.\\n\\nP < 0.05 significant differences compared to precancerous lesion group.\\n\\nP < 0.05 significant compared to cyanidin-3-O-glucoside-10, One-way ANOVA followed by Tukey’s test. +: Positive expression found in 1-3 fields; ++: Positive expression found in 4-6 fields; +++: Positive expression found in 7-10 field; PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; GSTP: Glutathione S-transferase placental; PCNA: Proliferating cell nuclear antigen.\\nLight microscopic examination of H&E-stained sections of naïve rats’ liver showed normal hepatic architecture, cords of hepatocytes radiating from central vein, portal triads present in between, polygonal hepatocytes with centrally rounded vesicular nuclei, and hepatic sinusoid in between (Figure 4A and B). Liver sections obtained from rats received DEN/2-AAF (Figure 4C-E) showed disruption of normal hepatic lobular architecture along with presence of large, discriminated dysplastic nodules compressing the surrounding liver tissue, with high-grade dysplastic cells displaying increased nuclear: Cytoplasmatic ratio (Figure 4E), nuclear hyperchromatosis, and basophilic cytoplasm. Liver sections of rats treated with different doses of cyanidin (10, 15, and 20 mg/kg) respectively, showed small and less discriminated dysplastic nodules (Figure 4F-H).\\n\\nPhotomicrographs of liver sections stained with H&E staining. A and B: Naïve group liver sections showed normal hepatic architecture, cords of hepatocytes radiating from central vein and portal triads present in-between and polygonal hepatocytes with central rounded vesicular nuclei, and hepatic sinusoid in-between; C-E: Liver sections of rats received diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF) showed larger, discriminated dysplastic nodules (doted shapes) compressing the surrounding liver tissue with disruption of normal hepatic lobular architecture; D: Liver sections of rats received DEN/2-AAF showed eosinophilic foci of cellular alteration consisting of enlarged hepatocytes with increased acidophilic staining and vaculated nuclei (arrow head); E: Liver sections of rats received DEN/2-AAF showed foci of Clear cell formed of hepatocytes showing variable degrees of cytoplasmic vacuolations and ballooning with pyknotic nuclei (arrow); F-H: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) respectively, showing small and less discriminated dysplastic nodules (doted shapes). (Magnification: A, C, F, G, H × 1000; B, D, E × 400). CV: Central vein; PV: Portal vein; Pt: Portal triads; S: Sinusoid; H: Hepatocytes.\\nImmunohistochemical examination with GSTP antibody appeared as large hyperplastic nodules were demonstrated by the brown color. Figure 5A and B shows the liver sections of naïve group, while Figure 5C shows large-positively stained GSTP foci demonstrated in PCL group. Liver sections from rats of all cyan-treated groups exhibited small GSTP-positive small hepatic foci of different sizes scattered in-between negatively-stained hepatic parenchyma (Figure 5D-F). The percent area of GSTP foci was significantly increased in the PCL group compared to naïve group, while all cyan-treated groups showed significant decrease in the GSTP percent area. Furthermore, cyan-15 and -20 showed significant decrease in the GSTP percent area over cyan-10 (Table 1). PCNA immunohistochemical analysis showed an elevated expression of PCNA in the group that received DEN/2-AAF as compared to naïve group (Figure 6A and B). Liver sections obtained from rats received cyan. in its three doses showed decrease in PCNA-positively stained nuclei (Figure 6C-E and Table 1).\\n\\nPhotomicrographs of liver sections of rats immunohistochemical stained with glutathione S-transferase placental antibody. A and B: Naive group; C: Precancerous lesion group showing multiple glutathione S-transferase placental (GSTP)-positive large hepatic nodules (brown stained nodules) occupying most of section; D-F: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) showing GSTP positive small hepatic foci (brown stained cells = arrow) of different size scattered in-between negatively stained hepatic parenchyma. (Magnification: × 40)\\n\\nPhotomicrographs of liver sections Immunohistochemical stained with proliferating cell nuclear antigen. A: Negative reaction of control group; B: Diethylnitrosamine + 100 mg 2-acetylaminofluorene group showing positive stained nuclei scattered all over the field; C-E: Liver sections of rats treated with different doses of cyamidine (10, 15, 20 mg/kg) respectively, show few positive hepatocytes sporadically distributed over the field (arrow). (Magnification × 100). \\nExpression rate of hepatocytes positive for glutathione S-transferase placental and proliferating cell nuclear antigen were calculated as number of positive field expression in 10 fields per rat liver tissue\\nValues are mean ± SD; number of animals = 6 rats/each group. Examined field; 10 fields/liver section. \\n\\nP < 0.05 significant differences compared to naïve group.\\n\\nP < 0.05 significant differences compared to precancerous lesion group.\\n\\nP < 0.05 significant compared to cyanidin-3-O-glucoside-10, One-way ANOVA followed by Tukey’s test. +: Positive expression found in 1-3 fields; ++: Positive expression found in 4-6 fields; +++: Positive expression found in 7-10 field; PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; GSTP: Glutathione S-transferase placental; PCNA: Proliferating cell nuclear antigen.\",\n \"As shown in Figure 1, following induction of PCL, rats exhibited a significant increase in AFP levels. AFP levels were significantly decreased in cyan-10, cyan-15, and cyan-20 groups compared to PCL group. There was no significant difference between the AFP levels of the three cyan groups. Liver function tests were affected after receiving DEN/2-AAF in which rats exhibited a significant increase in levels of ALT and total and direct bilirubin, and a significant decrease in the serum albumin (Figure 2). As shown in Figure 2A, rats received cyan. in different doses exhibited a significant decrease in serum ALT compared to PCL group. Cyan-15 group showed significant decrease in serum ALT compared to cyan-10 group. Moreover, cyan-20 group showed significant decrease in serum ALT compared to cyan-10 group. In As depicted in Figure 2B, compared to PCL group, the rats in the three cyan groups exhibited a significant decrease in serum total bilirubin. Furthermore, cyan-20 group showed significant decrease in serum total bilirubin compared to cyan-10 group. Figure 2C: Serum direct bilirubin was significantly decreased in the three cyan groups compared to the PCL group. Moreover, cyan-20 group showed significant decrease in serum direct bilirubin compared to cyan-10 group. Administration of cyan in doses 5, 10 and 20 mg/kg/d produced a significant increase in serum albumin compared to PCL group. However, there was no significant difference between the serum albumin levels of the three cyan groups (Figure 2D).\\n\\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) on alpha fetoprotein in the serum of rats. Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group, unpaired t test; bP < 0.05 significant differences compared to precancerous lesion group. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; AFP: Alpha fetoprotein.\\n\\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) in rats. A: Alanine aminotransferase; B: Total bilirubin; C: Direct bilirubin; D: Serum albumin. Values are mean ± SEM; number of animals = 6 rats/each group. bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; ALT: Alanine aminotransferase.\",\n \"As shown in Figure 3, induction of PCL resulted in significant increase of lncRNA MALAT a significant decrease in RQ miR-125b and significant increase in RQ TUBG1 expressions in comparison to naïve group. RQ of lncRNA MALAT1 in the liver tissue was significantly decreased in cyan -10,15 and cyan-20 groups compared to PCL group. Furthermore, cyan-20 group showed significant decrease in RQ of lncRNA MALAT1 in the liver tissue compared to cyan-10 group (Figure 3A). On the contrary, RQ of miR-125b in the liver tissue was significantly increased in cyan -10, cyan-15 and cyan-20 groups compared to PCL group. In addition, cyan-20 group showed significant increase in RQ of miR-125b in the liver tissue compared to cyan-10 group (Figure 3B). Furthermore, RQ of TUBG1 in the liver tissue was significantly decreased in cyan-10, cyan-15 and cyan-20 groups compared to PCL group. Also, cyan-20 group showed significant decrease in RQ of TUBG1 in the liver tissue compared to cyan-10 group (Figure 3C).\\n\\nEffect on expression of of long non-coding RNA, MALAT1, miR-125b and tubulin 1 (G2/M transition of mitotic cell cycle) in the liver tissue. A: RQ of long non-coding RNA MALAT1; B: RQ miR-125b; C: RQ TUBGI . Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group; bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; TUBG: Tubulin gamma 1; lncRNA: Long non-coding RNA.\",\n \"Light microscopic examination of H&E-stained sections of naïve rats’ liver showed normal hepatic architecture, cords of hepatocytes radiating from central vein, portal triads present in between, polygonal hepatocytes with centrally rounded vesicular nuclei, and hepatic sinusoid in between (Figure 4A and B). Liver sections obtained from rats received DEN/2-AAF (Figure 4C-E) showed disruption of normal hepatic lobular architecture along with presence of large, discriminated dysplastic nodules compressing the surrounding liver tissue, with high-grade dysplastic cells displaying increased nuclear: Cytoplasmatic ratio (Figure 4E), nuclear hyperchromatosis, and basophilic cytoplasm. Liver sections of rats treated with different doses of cyanidin (10, 15, and 20 mg/kg) respectively, showed small and less discriminated dysplastic nodules (Figure 4F-H).\\n\\nPhotomicrographs of liver sections stained with H&E staining. A and B: Naïve group liver sections showed normal hepatic architecture, cords of hepatocytes radiating from central vein and portal triads present in-between and polygonal hepatocytes with central rounded vesicular nuclei, and hepatic sinusoid in-between; C-E: Liver sections of rats received diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF) showed larger, discriminated dysplastic nodules (doted shapes) compressing the surrounding liver tissue with disruption of normal hepatic lobular architecture; D: Liver sections of rats received DEN/2-AAF showed eosinophilic foci of cellular alteration consisting of enlarged hepatocytes with increased acidophilic staining and vaculated nuclei (arrow head); E: Liver sections of rats received DEN/2-AAF showed foci of Clear cell formed of hepatocytes showing variable degrees of cytoplasmic vacuolations and ballooning with pyknotic nuclei (arrow); F-H: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) respectively, showing small and less discriminated dysplastic nodules (doted shapes). (Magnification: A, C, F, G, H × 1000; B, D, E × 400). CV: Central vein; PV: Portal vein; Pt: Portal triads; S: Sinusoid; H: Hepatocytes.\\nImmunohistochemical examination with GSTP antibody appeared as large hyperplastic nodules were demonstrated by the brown color. Figure 5A and B shows the liver sections of naïve group, while Figure 5C shows large-positively stained GSTP foci demonstrated in PCL group. Liver sections from rats of all cyan-treated groups exhibited small GSTP-positive small hepatic foci of different sizes scattered in-between negatively-stained hepatic parenchyma (Figure 5D-F). The percent area of GSTP foci was significantly increased in the PCL group compared to naïve group, while all cyan-treated groups showed significant decrease in the GSTP percent area. Furthermore, cyan-15 and -20 showed significant decrease in the GSTP percent area over cyan-10 (Table 1). PCNA immunohistochemical analysis showed an elevated expression of PCNA in the group that received DEN/2-AAF as compared to naïve group (Figure 6A and B). Liver sections obtained from rats received cyan. in its three doses showed decrease in PCNA-positively stained nuclei (Figure 6C-E and Table 1).\\n\\nPhotomicrographs of liver sections of rats immunohistochemical stained with glutathione S-transferase placental antibody. A and B: Naive group; C: Precancerous lesion group showing multiple glutathione S-transferase placental (GSTP)-positive large hepatic nodules (brown stained nodules) occupying most of section; D-F: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) showing GSTP positive small hepatic foci (brown stained cells = arrow) of different size scattered in-between negatively stained hepatic parenchyma. (Magnification: × 40)\\n\\nPhotomicrographs of liver sections Immunohistochemical stained with proliferating cell nuclear antigen. A: Negative reaction of control group; B: Diethylnitrosamine + 100 mg 2-acetylaminofluorene group showing positive stained nuclei scattered all over the field; C-E: Liver sections of rats treated with different doses of cyamidine (10, 15, 20 mg/kg) respectively, show few positive hepatocytes sporadically distributed over the field (arrow). (Magnification × 100). \\nExpression rate of hepatocytes positive for glutathione S-transferase placental and proliferating cell nuclear antigen were calculated as number of positive field expression in 10 fields per rat liver tissue\\nValues are mean ± SD; number of animals = 6 rats/each group. Examined field; 10 fields/liver section. \\n\\nP < 0.05 significant differences compared to naïve group.\\n\\nP < 0.05 significant differences compared to precancerous lesion group.\\n\\nP < 0.05 significant compared to cyanidin-3-O-glucoside-10, One-way ANOVA followed by Tukey’s test. +: Positive expression found in 1-3 fields; ++: Positive expression found in 4-6 fields; +++: Positive expression found in 7-10 field; PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; GSTP: Glutathione S-transferase placental; PCNA: Proliferating cell nuclear antigen.\",\n \"Altered expression of mitotic spindle assembly genes has been detected in many cancers. For example, MAD2 gene levels are downregulated in breast carcinoma[18], while mitotic checkpoint serine/BUB1 gene expression is dysregulated in colorectal carcinoma[19]. The impairment of spindle assembly genes are frequently detected in HCC[20,21]. Moreover, inhibition of the mitotic assembly genes has been found to be lethal to cancer cells, and has promising therapeutic effect in cancer treatment[22,23]. \\nIn this study, we used several public microarray databases for the construction of simple genetic-epigenetic network specific to HCC and linked to cell cycle and mitotic spindle formation. We assessed the antiproliferative effect of cyan in HCC animal model via modulation of lncRNA-MALAT1-miR-125b-TUBG1 mRNA axis expression.\\nMicrotubules are involved both in maintenance of cell shape and motility and in mitotic spindle formation[24]. γ-TUBG is a major player that supports microtubule nucleation. γ-TUBG acts as a binding site for the α/β-tubulin dimer and it has two γ-TUBG genes, TUBG1 and TUBG2 in mammals only[25]. The TUBG levels in the centrosome change during cell cycle progression and usually increases at the start of mitosis. At the end of mitosis, TUBG levels are decreased to interphase levels[26]. Overexpression and altered compartmentalization of γ-TUBG may lead to carcinogenesis[27-29]. Interestingly, γ-TUBG associates interact with tumor suppressor protein C53 in mammalian interphase cells of different cellular origins[30]. Interestingly, γ-TUBG interacts with E2F transcription factors, resulting in upregulation of E2F target genes PCNA and CDKB1[31] and glutathione S transferase activity[32]. On the other hand, the mRNA and protein levels of TUBA1C are upregulated in HCC. Clinically, it affects the survival of patients and the level of metastatic burden along with portal vein thrombosis. In addition, it activates cellular proliferation and migration and is a potential therapeutic target for HCC[33]. Recent studies have shed light on the efficacy of microtubule-binding agents in the treatment of HCC, especially in combination with mammalian target of rapamycin inhibitors[34].\\nOne of the few miRNAs that regulate Spindle Assembly Checkpoints (SAC) is miR-125b. It suppresses the expression of Mad1 (a core SAC protein) that is responsible for inhibiting entry into anaphase till metaphase defects are corrected[35]. Importantly, miRNA-125b has been found to be associated with colorectal cancer[36], multiple myeloma[37], and breast cancer[38]. Song et al[39] demonstrated that the expression of miR-125b in hepatocytes was protective and reflected better survival in HCC tissue samples due to restoration of SIRT6 expression[39]. miR-125b-5p inhibits HCC proliferation by targeting TXNRD1, the terminal step of biosynthesis of nucleotides[40].\\nDeregulation of lncRNAs has been shown to be crucial in cancer progression, especially HCC[41,42]. Hung et al[43] demonstrated the involvement of lncRNAs in regulation of the expression of cell cycle-related genes, p53 gene regulatory pathway, and apoptosis[43,44]. The lncRNA MALAT1 is overexpressed in several solid tumors and is linked with tumor recurrence[45-47]. MALAT1 coordinate RNA polymerase II transcription, pre-mRNA splicing, and mRNA export[48]. Thus, this lncRNA holds great potential in influencing the local concentration of a specific splicing factor during specific stages of the cell cycle. Depletion of MALAT1 in cancer cells inhibits tumorigenicity[49]. MALAT1 regulates E2F1 transcription factor activity, highlighting its pro-proliferative function[50]. MALAT1 is upregulated in many cancer types, including breast cancer[51], cervical cancer, lung cancer[52], and hepatocarcinoma[53-55]. In a previous study, hypoxia enhanced MALAT1 expression, leading to increase in proliferating and invading activity of Hep3B cells due to negative interaction with miR200a[56]. Huang and his group explored the role of specificity protein 1/3 in regulation of MALAT1 expression in HCC cells[57].\\nCyanidin or anthocyanin (a polyphenolic pigment found in plants) confers several pharmacological benefits, including anticancer properties[58-62]. Cyan mediates cytotoxicity against human monocytic leukemia cells via G2/M phase arrest and induction of apoptosis[63]. Tsai et al[64] evaluated anthocyanins as promising anticancer or chemopreventive agents to induce G2/M arrest in HAs-induced leukemia cell cycle arrest via ATM-Chk1/2-Cdc25C axis modulation[64]. They also found that anthocyanin inhibits the proliferation of HCC cells[65,66].\\nIn our study, the effect of cyan on cancerous tissue was dose-dependent (10, 15 and 20 mg/kg/d), that is, increasing its effect with increasing the concentration of the drug. This was proved also, with the correlated significant decrease in the percent area of GSTP foci, PCNA expression which are the target of TUBG1 mRNA and the molecular epigenetic markers associated with HCC (lncRNA MALAT1 and miR-125b-1-3p) in the liver tissue of rats in our study (Figure 7). Thus, we can hypothesize that administration of cyan in PCL may result in downregulation of lncRNA MALAT1 with subsequent release of free miR-125b-1-3p that binds to TUBG1 mRNA and downregulate its expression[65]. The changes in the epigenetic-genetic network affect their target genes PCNA and GSTP.\\n\\nConcept map of study design. GSTP: Glutathione S-transferase placental; PCNA: Proliferating cell nuclear antigen; TUBG: Tubulin; lncRNA: Long non-coding RNA.\\nLastly, the cyan-mediated modulation of cell cycle and mitotic spindle assembly that regulate HCC proliferation might affect the capability of the HCC cells to overcome oxidative stress[66,67]. Our data confers the rationale for designing antimitotic approach combining the conventional cytotoxic drugs with phytochemical extracts to inhibit cell cycle progression in cancer. More in vitro functional studies are required to explore the functional mechanism of the chosen RNA panel and validate their role as drug target biomarkers. \",\n \"Cyanidin is a natural molecule that holds great potential in unraveling the mystery of cytotoxic pharmacy in the future. It can be used to devise novel antimitotic drugs that can target cell cycle in HCC. Our results also indicated great potential of the TUBG1 mRNA- miR-125b-lncRNA MALAT1 and -1-3p axis as potential drug target biomarker for further investigations.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","MATERIALS AND METHODS","Chemicals and drugs ","Experimental animals","Experimental procedures","Assessment of the effects of treatment","Determination of serum albumin","Bioinformatics-based selection of cell cycle-microtubule assembly- and HCC-specific RNA panel ","Extraction of total RNA, including lncRNA and miRNA from liver tissue","Real time-polymerase chain reaction quantification of the RNA panel in liver tissues","Histopathological and immunohistochemical studies","Statistical analysis","RESULTS","Effect on AFP levels and liver function tests","Effect on expression of lncRNA MALAT1, miR-125b, and TUBG1 (G2/M transition of mitotic cell cycle) in the liver tissue","Histopathological and immunohistochemical studies","DISCUSSION","CONCLUSION"],"string":"[\n \"INTRODUCTION\",\n \"MATERIALS AND METHODS\",\n \"Chemicals and drugs \",\n \"Experimental animals\",\n \"Experimental procedures\",\n \"Assessment of the effects of treatment\",\n \"Determination of serum albumin\",\n \"Bioinformatics-based selection of cell cycle-microtubule assembly- and HCC-specific RNA panel \",\n \"Extraction of total RNA, including lncRNA and miRNA from liver tissue\",\n \"Real time-polymerase chain reaction quantification of the RNA panel in liver tissues\",\n \"Histopathological and immunohistochemical studies\",\n \"Statistical analysis\",\n \"RESULTS\",\n \"Effect on AFP levels and liver function tests\",\n \"Effect on expression of lncRNA MALAT1, miR-125b, and TUBG1 (G2/M transition of mitotic cell cycle) in the liver tissue\",\n \"Histopathological and immunohistochemical studies\",\n \"DISCUSSION\",\n \"CONCLUSION\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Hepatocellular carcinoma (HCC) is the commonest primary malignancy of the liver. Its main risk factors include hepatitis B, hepatitis C, and non-alcoholic steatohepatitis. HCC is associated with high mortality and morbidity. Its incidence has increased from 1.4 to 6.2 per 100000 cases per year within the last 30 years[1]. HCC burden in Egypt is high due to the high prevalence of HCV, where liver cancers account for 11.75% of all gastrointestinal cancers and 1.68% of all malignancies. In addition, over 70% of all liver malignancies in Egypt are HCC[2].\nTo date, HCC is holding its place as an unresolved dilemma; its resistance to systemic chemotherapy and radiation makes the cure approachable only in very early stages according to Milan criteria, that is, where the tumor size is < 5 cm (when only one lesion is present) or < 3 cm (when 2-3 lesions are present)[3].\nGenes involved in cell cycle control are usually mutated in presence of cancer. Dysregulated mitosis results in genomic instability, which leads to tumor aggressiveness[4]. Microtubules are a key component of mitotic spindles, and thus, a crucial part of the process of mitosis. They form an important target in cancer chemotherapy, inducing arrest of mitosis and cell death. On the other hand, while the overexpression of γ-tubulin (γ-TUBG) showed improved survival among small cell lung cancer patients[5], the overexpression of γ-TUBG is found to be characteristic feature of thyroid and breast cancers[6]. Such variation may arise due to differences in gene transcription and check point regulations in different cancer tissues[7].\nCyanidin-3-glucoside (cyan) is a potential chemotherapeutic and chemo-protective agent. Through its antioxidant activity, it scavenges radical oxygen species, which decreases the overall number of tumorous cells (malignant and benign) in in vivo studies[8]. Cyan has been implicated in some beneficial health activities[9], including reducing age-associated oxidative stress[10], improving cognitive brain function, and exhibiting anti-diabetic[11], anti-inflammation[12], anti-atherogenic[13], and anti-obesity activities[14]. Previously, it has been reported that cyan mediates caspase-3 cleavage with DNA fragmentation. Cyan has also been associated with induction of autophagy, a key element involved in cancer elimination, via induction of autophagy-related gene 5 and microtubule-associated protein 1 light chain 3-II[15]. Cyan has drawn increasing attention because of its potential anti-cancer properties. Cyan may offer a novel avenue for treating HCC. a potential antiproliferative effect on HepG2 cells[16].\nThe present study was conducted to evaluate the effect of cyan in three different doses on cell cycle progression and mitotic assembly disorder in hepatic precancerous lesion (PCL) via assessment of levels of long non-coding RNA (lncRNA) MALAT1, miR-125b, and tubulin 1. We also conducted histopathological and immuno-histochemical examination using H&E staining assessment of levels of glutathione S-transferase placental (GSTP) and proliferating cell nuclear antigen (PCNA) antibodies in male Wistar rat model of diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF)-induced hepatic PCL. ","Chemicals and drugs DEN was obtained as 1 g solution in serum bottle diluted in 0.9% NaCl. 2-AAF was obtained as white powder, while cyan was obtained as black powder. Both 2-AAF and cyan were dissolved in 0.9% NaCl. All chemicals were purchased from Sigma-Aldrich, Cairo, Egypt.\nDEN was obtained as 1 g solution in serum bottle diluted in 0.9% NaCl. 2-AAF was obtained as white powder, while cyan was obtained as black powder. Both 2-AAF and cyan were dissolved in 0.9% NaCl. All chemicals were purchased from Sigma-Aldrich, Cairo, Egypt.\nExperimental animals All animal procedures were approved by the Institutional Animal Ethics Committee of Ain Shams University, Faculty of Medicine. Thirty male Wistar rats weighing 200-250 g were purchased from Nile Pharmaceuticals Company (Cairo, Egypt). Animals were housed in an animal room with temperature 25 ± 2 °C and 12 h light/dark cycle controls. An adaptation period of 1 wk was allowed before initiation of the experimental protocol.\nAll animal procedures were approved by the Institutional Animal Ethics Committee of Ain Shams University, Faculty of Medicine. Thirty male Wistar rats weighing 200-250 g were purchased from Nile Pharmaceuticals Company (Cairo, Egypt). Animals were housed in an animal room with temperature 25 ± 2 °C and 12 h light/dark cycle controls. An adaptation period of 1 wk was allowed before initiation of the experimental protocol.\nExperimental procedures \nInduction of hepatic PCL: DEN was injected intraperitoneally (i.p.) at a dose of 100 mg/kg once weekly for three consecutive weeks. One week after the last DEN injection, a single dose (300 mg/kg) of 2-AAF was injected.\n\nAnimal groups: Rats were divided into five groups (6 rats/each group), including: Naïve group: Rats were injected with a vehicle (0.9% NaCl), PCL group: Rats were injected with DEN and 2-AAF, and three cyan groups (cyan-10, cyan-15, and cyan-20 groups): Rats were injected with DEN and 2-AAF, and orally administered through gastric gavage with cyan in doses of 10, 15, and 20 mg/kg respectively, for four consecutive days per week for 16 wk[17].\n\nInduction of hepatic PCL: DEN was injected intraperitoneally (i.p.) at a dose of 100 mg/kg once weekly for three consecutive weeks. One week after the last DEN injection, a single dose (300 mg/kg) of 2-AAF was injected.\n\nAnimal groups: Rats were divided into five groups (6 rats/each group), including: Naïve group: Rats were injected with a vehicle (0.9% NaCl), PCL group: Rats were injected with DEN and 2-AAF, and three cyan groups (cyan-10, cyan-15, and cyan-20 groups): Rats were injected with DEN and 2-AAF, and orally administered through gastric gavage with cyan in doses of 10, 15, and 20 mg/kg respectively, for four consecutive days per week for 16 wk[17].\nAssessment of the effects of treatment \nBiochemical and molecular studies: After scarifying the animals, blood was withdrawn from each rat from the retro orbital vein and incubated for about half an hour for clotting. Then, the blood sample was centrifuged for 20 min at 5000 RPM to get serum samples. Livers were dissected. The right lobe of the livers was removed, cut into longitudinal sections 2-4 mm in thickness and kept in 10% formalin for histopathological and immunohistochemical examination. The other lobe was kept frozen at -80 °C for molecular analyses. The samples were analyzed for the following parameters: (1) alpha fetoprotein (AFP) levels: AFP level was analyzed using quantitative sandwich rat AFP ELISA kit purchased from MyBiosource Inc. (San Diego, United States) with a sensitivity range of 0.625-20 ng/mL; (2) serum alanine aminotransferase (ALT) levels: The serum ALT was measured according to method described by using kits purchased from Diamond Diagnostic (Cairo, Egypt). ALT catalyzes the transfer of amino groups from specific amino acids to ketoglutaric acid, yielding glutamic acid and oxaloacetic or pyruvic acid, respectively. Ketoacids were then determined calorimetrically after their reaction with 2, 4- dinitrophenylhydrazine; and (3) determination of serum total bilirubin and direct bilirubin: Alkaline methanolysis of bilirubin followed by chloroform extraction of bilirubin methyl esters, and later, separation of these esters by chromatography and spectrophotometric determination at 430 nm.\n\nBiochemical and molecular studies: After scarifying the animals, blood was withdrawn from each rat from the retro orbital vein and incubated for about half an hour for clotting. Then, the blood sample was centrifuged for 20 min at 5000 RPM to get serum samples. Livers were dissected. The right lobe of the livers was removed, cut into longitudinal sections 2-4 mm in thickness and kept in 10% formalin for histopathological and immunohistochemical examination. The other lobe was kept frozen at -80 °C for molecular analyses. The samples were analyzed for the following parameters: (1) alpha fetoprotein (AFP) levels: AFP level was analyzed using quantitative sandwich rat AFP ELISA kit purchased from MyBiosource Inc. (San Diego, United States) with a sensitivity range of 0.625-20 ng/mL; (2) serum alanine aminotransferase (ALT) levels: The serum ALT was measured according to method described by using kits purchased from Diamond Diagnostic (Cairo, Egypt). ALT catalyzes the transfer of amino groups from specific amino acids to ketoglutaric acid, yielding glutamic acid and oxaloacetic or pyruvic acid, respectively. Ketoacids were then determined calorimetrically after their reaction with 2, 4- dinitrophenylhydrazine; and (3) determination of serum total bilirubin and direct bilirubin: Alkaline methanolysis of bilirubin followed by chloroform extraction of bilirubin methyl esters, and later, separation of these esters by chromatography and spectrophotometric determination at 430 nm.\nDetermination of serum albumin Quantitative determination of serum albumin level was quantified using rat albumin ELISA kit. The absorbance at 450 nm is a measure of the concentration of albumin in the test sample.\nQuantitative determination of serum albumin level was quantified using rat albumin ELISA kit. The absorbance at 450 nm is a measure of the concentration of albumin in the test sample.\nBioinformatics-based selection of cell cycle-microtubule assembly- and HCC-specific RNA panel The RNA panel was chosen in three steps: (1) Tubulin, gamma 1 (TUBG1) was retrieved as a crucial player in microtubule formation and progression of the cell cycle through gene atlas expression database (available at https://www.ebi.ac.uk/gxa/home) alongside with verification of the chosen gene from protein Atlas database (available at https://www.proteinatlas.org/), followed by analysis of TUBG1 gene ontology to ensure that it is linked to G2-M transition and mitotic spindle assembly, which is closely linked to cancer development through gene card database (available at https://www.genecards.org/); (2) we selected miR-125b-1-3p targeting TUBG1 mRNA though) miRWALK database available at http://mirwalk.umm.uni-heidelberg.de/), followed by pathway enrichment, which revealed that miR-125b-1-3p is linked to MAP kinase and ubiquitin-mediated proteolysis (strongly correlated with mitotic assembly and cell cycle progression) through (Diana database available at http://diana.imis.athena-innovation.gr/DianaTools/index.php); and (3) lncRNA metastasis-associated lung adenocarcinoma transcript 1, lncRNA MALAT1, was obtained as a lncRNA specific to HCC and targeting miR-125b-1-3p using noncode database available at http://www.noncode.org/ and European Bioinformatics institute database available at https://www.ebi.ac.uk.\nThe RNA panel was chosen in three steps: (1) Tubulin, gamma 1 (TUBG1) was retrieved as a crucial player in microtubule formation and progression of the cell cycle through gene atlas expression database (available at https://www.ebi.ac.uk/gxa/home) alongside with verification of the chosen gene from protein Atlas database (available at https://www.proteinatlas.org/), followed by analysis of TUBG1 gene ontology to ensure that it is linked to G2-M transition and mitotic spindle assembly, which is closely linked to cancer development through gene card database (available at https://www.genecards.org/); (2) we selected miR-125b-1-3p targeting TUBG1 mRNA though) miRWALK database available at http://mirwalk.umm.uni-heidelberg.de/), followed by pathway enrichment, which revealed that miR-125b-1-3p is linked to MAP kinase and ubiquitin-mediated proteolysis (strongly correlated with mitotic assembly and cell cycle progression) through (Diana database available at http://diana.imis.athena-innovation.gr/DianaTools/index.php); and (3) lncRNA metastasis-associated lung adenocarcinoma transcript 1, lncRNA MALAT1, was obtained as a lncRNA specific to HCC and targeting miR-125b-1-3p using noncode database available at http://www.noncode.org/ and European Bioinformatics institute database available at https://www.ebi.ac.uk.\nExtraction of total RNA, including lncRNA and miRNA from liver tissue Total RNA was extracted from liver tissues using miRNeasy Mini kit (Cat No. 217004, Qiagen, United States) according to manufacturer’s protocol. The extracted RNA concentration and integrity were assessed using [(NanoDrop Technologies/Thermo Scientific, Wilmington, DE, United States)], with RNA purities were 1.8-2. The extracted total RNA was reverse transcribed into cDNA using a miScript II RT kit (Cat No. 218160, 218161, Qiagen, United States) on thermal cycler (Bio-Rad; Hercules, CA, United States), according to the manufacturer’s protocol. \nTotal RNA was extracted from liver tissues using miRNeasy Mini kit (Cat No. 217004, Qiagen, United States) according to manufacturer’s protocol. The extracted RNA concentration and integrity were assessed using [(NanoDrop Technologies/Thermo Scientific, Wilmington, DE, United States)], with RNA purities were 1.8-2. The extracted total RNA was reverse transcribed into cDNA using a miScript II RT kit (Cat No. 218160, 218161, Qiagen, United States) on thermal cycler (Bio-Rad; Hercules, CA, United States), according to the manufacturer’s protocol. \nReal time-polymerase chain reaction quantification of the RNA panel in liver tissues The relative expression of TUBG1 mRNA and lncRNA-MALAT1 in liver tissue were measured using a Quantitect SYBR Green Master Mix kit and an RT2 SYBR Green ROX real time-polymerase chain reaction (qPCR) Mastermix (Qiagen), respectively, using a 7500 qPCR Systems (Applied Biosystems, Foster City, CA, United States) detection system) and specific primers (Accession: NM_001128148, NM_003234, NR_002819, and ENST00000534336, respectively) supplied by Qiagen. GAPDH (Accession NM_002046.7) was used as a housekeeping gene. MiR-3163 expression in liver tissue was quantified by PCR using a miScript SYBR Green kit (Qiagen), a miScript universal primer, and a miRNA-specific forward primer (mir-125b-1-3p miScript Primer Assay) (Accession: MIMAT0004592 (5'-ACGGGUUAGGCUCUUGGGAGCU). All steps followed the manufacturer’s suggested protocol, and SNORD68 was used as an internal control. Ct values more than 36 were considered as negative. The specificities of the amplicons for the SYBR Green−based PCR amplification were affirmed by the melting curves. The 2−ΔΔCt technique was used to measure the relative expression of the HCC-specific RNA panel.\nThe relative expression of TUBG1 mRNA and lncRNA-MALAT1 in liver tissue were measured using a Quantitect SYBR Green Master Mix kit and an RT2 SYBR Green ROX real time-polymerase chain reaction (qPCR) Mastermix (Qiagen), respectively, using a 7500 qPCR Systems (Applied Biosystems, Foster City, CA, United States) detection system) and specific primers (Accession: NM_001128148, NM_003234, NR_002819, and ENST00000534336, respectively) supplied by Qiagen. GAPDH (Accession NM_002046.7) was used as a housekeeping gene. MiR-3163 expression in liver tissue was quantified by PCR using a miScript SYBR Green kit (Qiagen), a miScript universal primer, and a miRNA-specific forward primer (mir-125b-1-3p miScript Primer Assay) (Accession: MIMAT0004592 (5'-ACGGGUUAGGCUCUUGGGAGCU). All steps followed the manufacturer’s suggested protocol, and SNORD68 was used as an internal control. Ct values more than 36 were considered as negative. The specificities of the amplicons for the SYBR Green−based PCR amplification were affirmed by the melting curves. The 2−ΔΔCt technique was used to measure the relative expression of the HCC-specific RNA panel.\nHistopathological and immunohistochemical studies Liver sections were cut at 5-μm thickness and stained with H&E. Immunohistochemistry S-P method was used to detect GSTP and PCNA levels. Briefly, the protocol was as follows: (1) the tissues were treated with endogenous peroxidase blocking solution at room temperature for 10 min, and then, incubated in normal nonimmune serum at room temperature for 10 min; (2) mouse anti GSTP or PCNA antibody was added to adjacent tissue sections respectively and incubated overnight at 4 °C; (3) biotin-conjugated second antibody was added to the sections and incubated at room temperature for 10 min; and (4) S-P complex was added at room temperature for 10 min, and then, 2,4-diaminobutyric acid was used for the color reaction. The tissue sections were washed with Poly (butylene succinate) (PBS) (0.01 mol/L, pH 7.4) between each step. Positive and negative controls were simultaneously used to ensure specificity and reliability of the staining process. A positive section was taken as positive control. In negative control, PBS was used to replace the first antibody. GSTP-positivity was indicated by brown coloration of the cytoplasm. Morphometric analysis of the foci percent area was carried out using Leica Q win V.3 software after capturing the images using a Leica DM2500 microscope (Leica, Wetzlar, Germany). The PCNA labeling indices are represented as the expression of positively-stained nuclei (10 fields/slide at 400 ×) as shown below: +, positive expression found in 1-3 fields; ++, positive expression found in 4-6 fields; and +++, positive expression found in 7-10 fields. Rabbit monoclonal PCNA and GSTP antibodies were purchased from Abcam (San Francisco, United States).\nLiver sections were cut at 5-μm thickness and stained with H&E. Immunohistochemistry S-P method was used to detect GSTP and PCNA levels. Briefly, the protocol was as follows: (1) the tissues were treated with endogenous peroxidase blocking solution at room temperature for 10 min, and then, incubated in normal nonimmune serum at room temperature for 10 min; (2) mouse anti GSTP or PCNA antibody was added to adjacent tissue sections respectively and incubated overnight at 4 °C; (3) biotin-conjugated second antibody was added to the sections and incubated at room temperature for 10 min; and (4) S-P complex was added at room temperature for 10 min, and then, 2,4-diaminobutyric acid was used for the color reaction. The tissue sections were washed with Poly (butylene succinate) (PBS) (0.01 mol/L, pH 7.4) between each step. Positive and negative controls were simultaneously used to ensure specificity and reliability of the staining process. A positive section was taken as positive control. In negative control, PBS was used to replace the first antibody. GSTP-positivity was indicated by brown coloration of the cytoplasm. Morphometric analysis of the foci percent area was carried out using Leica Q win V.3 software after capturing the images using a Leica DM2500 microscope (Leica, Wetzlar, Germany). The PCNA labeling indices are represented as the expression of positively-stained nuclei (10 fields/slide at 400 ×) as shown below: +, positive expression found in 1-3 fields; ++, positive expression found in 4-6 fields; and +++, positive expression found in 7-10 fields. Rabbit monoclonal PCNA and GSTP antibodies were purchased from Abcam (San Francisco, United States).\nStatistical analysis All results were expressed as mean ± SD. Statistical analysis was carried out using GraphPad Prism version 6.01. Unpaired t-test was done to compare between naïve and PCL groups. One-way ANOVA, followed by Tukey’s test, was carried out to compare between all groups. P values < 0.05 were considered statistically significant. \nAll results were expressed as mean ± SD. Statistical analysis was carried out using GraphPad Prism version 6.01. Unpaired t-test was done to compare between naïve and PCL groups. One-way ANOVA, followed by Tukey’s test, was carried out to compare between all groups. P values < 0.05 were considered statistically significant. ","DEN was obtained as 1 g solution in serum bottle diluted in 0.9% NaCl. 2-AAF was obtained as white powder, while cyan was obtained as black powder. Both 2-AAF and cyan were dissolved in 0.9% NaCl. All chemicals were purchased from Sigma-Aldrich, Cairo, Egypt.","All animal procedures were approved by the Institutional Animal Ethics Committee of Ain Shams University, Faculty of Medicine. Thirty male Wistar rats weighing 200-250 g were purchased from Nile Pharmaceuticals Company (Cairo, Egypt). Animals were housed in an animal room with temperature 25 ± 2 °C and 12 h light/dark cycle controls. An adaptation period of 1 wk was allowed before initiation of the experimental protocol.","\nInduction of hepatic PCL: DEN was injected intraperitoneally (i.p.) at a dose of 100 mg/kg once weekly for three consecutive weeks. One week after the last DEN injection, a single dose (300 mg/kg) of 2-AAF was injected.\n\nAnimal groups: Rats were divided into five groups (6 rats/each group), including: Naïve group: Rats were injected with a vehicle (0.9% NaCl), PCL group: Rats were injected with DEN and 2-AAF, and three cyan groups (cyan-10, cyan-15, and cyan-20 groups): Rats were injected with DEN and 2-AAF, and orally administered through gastric gavage with cyan in doses of 10, 15, and 20 mg/kg respectively, for four consecutive days per week for 16 wk[17].","\nBiochemical and molecular studies: After scarifying the animals, blood was withdrawn from each rat from the retro orbital vein and incubated for about half an hour for clotting. Then, the blood sample was centrifuged for 20 min at 5000 RPM to get serum samples. Livers were dissected. The right lobe of the livers was removed, cut into longitudinal sections 2-4 mm in thickness and kept in 10% formalin for histopathological and immunohistochemical examination. The other lobe was kept frozen at -80 °C for molecular analyses. The samples were analyzed for the following parameters: (1) alpha fetoprotein (AFP) levels: AFP level was analyzed using quantitative sandwich rat AFP ELISA kit purchased from MyBiosource Inc. (San Diego, United States) with a sensitivity range of 0.625-20 ng/mL; (2) serum alanine aminotransferase (ALT) levels: The serum ALT was measured according to method described by using kits purchased from Diamond Diagnostic (Cairo, Egypt). ALT catalyzes the transfer of amino groups from specific amino acids to ketoglutaric acid, yielding glutamic acid and oxaloacetic or pyruvic acid, respectively. Ketoacids were then determined calorimetrically after their reaction with 2, 4- dinitrophenylhydrazine; and (3) determination of serum total bilirubin and direct bilirubin: Alkaline methanolysis of bilirubin followed by chloroform extraction of bilirubin methyl esters, and later, separation of these esters by chromatography and spectrophotometric determination at 430 nm.","Quantitative determination of serum albumin level was quantified using rat albumin ELISA kit. The absorbance at 450 nm is a measure of the concentration of albumin in the test sample.","The RNA panel was chosen in three steps: (1) Tubulin, gamma 1 (TUBG1) was retrieved as a crucial player in microtubule formation and progression of the cell cycle through gene atlas expression database (available at https://www.ebi.ac.uk/gxa/home) alongside with verification of the chosen gene from protein Atlas database (available at https://www.proteinatlas.org/), followed by analysis of TUBG1 gene ontology to ensure that it is linked to G2-M transition and mitotic spindle assembly, which is closely linked to cancer development through gene card database (available at https://www.genecards.org/); (2) we selected miR-125b-1-3p targeting TUBG1 mRNA though) miRWALK database available at http://mirwalk.umm.uni-heidelberg.de/), followed by pathway enrichment, which revealed that miR-125b-1-3p is linked to MAP kinase and ubiquitin-mediated proteolysis (strongly correlated with mitotic assembly and cell cycle progression) through (Diana database available at http://diana.imis.athena-innovation.gr/DianaTools/index.php); and (3) lncRNA metastasis-associated lung adenocarcinoma transcript 1, lncRNA MALAT1, was obtained as a lncRNA specific to HCC and targeting miR-125b-1-3p using noncode database available at http://www.noncode.org/ and European Bioinformatics institute database available at https://www.ebi.ac.uk.","Total RNA was extracted from liver tissues using miRNeasy Mini kit (Cat No. 217004, Qiagen, United States) according to manufacturer’s protocol. The extracted RNA concentration and integrity were assessed using [(NanoDrop Technologies/Thermo Scientific, Wilmington, DE, United States)], with RNA purities were 1.8-2. The extracted total RNA was reverse transcribed into cDNA using a miScript II RT kit (Cat No. 218160, 218161, Qiagen, United States) on thermal cycler (Bio-Rad; Hercules, CA, United States), according to the manufacturer’s protocol. ","The relative expression of TUBG1 mRNA and lncRNA-MALAT1 in liver tissue were measured using a Quantitect SYBR Green Master Mix kit and an RT2 SYBR Green ROX real time-polymerase chain reaction (qPCR) Mastermix (Qiagen), respectively, using a 7500 qPCR Systems (Applied Biosystems, Foster City, CA, United States) detection system) and specific primers (Accession: NM_001128148, NM_003234, NR_002819, and ENST00000534336, respectively) supplied by Qiagen. GAPDH (Accession NM_002046.7) was used as a housekeeping gene. MiR-3163 expression in liver tissue was quantified by PCR using a miScript SYBR Green kit (Qiagen), a miScript universal primer, and a miRNA-specific forward primer (mir-125b-1-3p miScript Primer Assay) (Accession: MIMAT0004592 (5'-ACGGGUUAGGCUCUUGGGAGCU). All steps followed the manufacturer’s suggested protocol, and SNORD68 was used as an internal control. Ct values more than 36 were considered as negative. The specificities of the amplicons for the SYBR Green−based PCR amplification were affirmed by the melting curves. The 2−ΔΔCt technique was used to measure the relative expression of the HCC-specific RNA panel.","Liver sections were cut at 5-μm thickness and stained with H&E. Immunohistochemistry S-P method was used to detect GSTP and PCNA levels. Briefly, the protocol was as follows: (1) the tissues were treated with endogenous peroxidase blocking solution at room temperature for 10 min, and then, incubated in normal nonimmune serum at room temperature for 10 min; (2) mouse anti GSTP or PCNA antibody was added to adjacent tissue sections respectively and incubated overnight at 4 °C; (3) biotin-conjugated second antibody was added to the sections and incubated at room temperature for 10 min; and (4) S-P complex was added at room temperature for 10 min, and then, 2,4-diaminobutyric acid was used for the color reaction. The tissue sections were washed with Poly (butylene succinate) (PBS) (0.01 mol/L, pH 7.4) between each step. Positive and negative controls were simultaneously used to ensure specificity and reliability of the staining process. A positive section was taken as positive control. In negative control, PBS was used to replace the first antibody. GSTP-positivity was indicated by brown coloration of the cytoplasm. Morphometric analysis of the foci percent area was carried out using Leica Q win V.3 software after capturing the images using a Leica DM2500 microscope (Leica, Wetzlar, Germany). The PCNA labeling indices are represented as the expression of positively-stained nuclei (10 fields/slide at 400 ×) as shown below: +, positive expression found in 1-3 fields; ++, positive expression found in 4-6 fields; and +++, positive expression found in 7-10 fields. Rabbit monoclonal PCNA and GSTP antibodies were purchased from Abcam (San Francisco, United States).","All results were expressed as mean ± SD. Statistical analysis was carried out using GraphPad Prism version 6.01. Unpaired t-test was done to compare between naïve and PCL groups. One-way ANOVA, followed by Tukey’s test, was carried out to compare between all groups. P values < 0.05 were considered statistically significant. ","Effect on AFP levels and liver function tests As shown in Figure 1, following induction of PCL, rats exhibited a significant increase in AFP levels. AFP levels were significantly decreased in cyan-10, cyan-15, and cyan-20 groups compared to PCL group. There was no significant difference between the AFP levels of the three cyan groups. Liver function tests were affected after receiving DEN/2-AAF in which rats exhibited a significant increase in levels of ALT and total and direct bilirubin, and a significant decrease in the serum albumin (Figure 2). As shown in Figure 2A, rats received cyan. in different doses exhibited a significant decrease in serum ALT compared to PCL group. Cyan-15 group showed significant decrease in serum ALT compared to cyan-10 group. Moreover, cyan-20 group showed significant decrease in serum ALT compared to cyan-10 group. In As depicted in Figure 2B, compared to PCL group, the rats in the three cyan groups exhibited a significant decrease in serum total bilirubin. Furthermore, cyan-20 group showed significant decrease in serum total bilirubin compared to cyan-10 group. Figure 2C: Serum direct bilirubin was significantly decreased in the three cyan groups compared to the PCL group. Moreover, cyan-20 group showed significant decrease in serum direct bilirubin compared to cyan-10 group. Administration of cyan in doses 5, 10 and 20 mg/kg/d produced a significant increase in serum albumin compared to PCL group. However, there was no significant difference between the serum albumin levels of the three cyan groups (Figure 2D).\n\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) on alpha fetoprotein in the serum of rats. Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group, unpaired t test; bP < 0.05 significant differences compared to precancerous lesion group. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; AFP: Alpha fetoprotein.\n\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) in rats. A: Alanine aminotransferase; B: Total bilirubin; C: Direct bilirubin; D: Serum albumin. Values are mean ± SEM; number of animals = 6 rats/each group. bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; ALT: Alanine aminotransferase.\nAs shown in Figure 1, following induction of PCL, rats exhibited a significant increase in AFP levels. AFP levels were significantly decreased in cyan-10, cyan-15, and cyan-20 groups compared to PCL group. There was no significant difference between the AFP levels of the three cyan groups. Liver function tests were affected after receiving DEN/2-AAF in which rats exhibited a significant increase in levels of ALT and total and direct bilirubin, and a significant decrease in the serum albumin (Figure 2). As shown in Figure 2A, rats received cyan. in different doses exhibited a significant decrease in serum ALT compared to PCL group. Cyan-15 group showed significant decrease in serum ALT compared to cyan-10 group. Moreover, cyan-20 group showed significant decrease in serum ALT compared to cyan-10 group. In As depicted in Figure 2B, compared to PCL group, the rats in the three cyan groups exhibited a significant decrease in serum total bilirubin. Furthermore, cyan-20 group showed significant decrease in serum total bilirubin compared to cyan-10 group. Figure 2C: Serum direct bilirubin was significantly decreased in the three cyan groups compared to the PCL group. Moreover, cyan-20 group showed significant decrease in serum direct bilirubin compared to cyan-10 group. Administration of cyan in doses 5, 10 and 20 mg/kg/d produced a significant increase in serum albumin compared to PCL group. However, there was no significant difference between the serum albumin levels of the three cyan groups (Figure 2D).\n\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) on alpha fetoprotein in the serum of rats. Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group, unpaired t test; bP < 0.05 significant differences compared to precancerous lesion group. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; AFP: Alpha fetoprotein.\n\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) in rats. A: Alanine aminotransferase; B: Total bilirubin; C: Direct bilirubin; D: Serum albumin. Values are mean ± SEM; number of animals = 6 rats/each group. bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; ALT: Alanine aminotransferase.\nEffect on expression of lncRNA MALAT1, miR-125b, and TUBG1 (G2/M transition of mitotic cell cycle) in the liver tissue As shown in Figure 3, induction of PCL resulted in significant increase of lncRNA MALAT a significant decrease in RQ miR-125b and significant increase in RQ TUBG1 expressions in comparison to naïve group. RQ of lncRNA MALAT1 in the liver tissue was significantly decreased in cyan -10,15 and cyan-20 groups compared to PCL group. Furthermore, cyan-20 group showed significant decrease in RQ of lncRNA MALAT1 in the liver tissue compared to cyan-10 group (Figure 3A). On the contrary, RQ of miR-125b in the liver tissue was significantly increased in cyan -10, cyan-15 and cyan-20 groups compared to PCL group. In addition, cyan-20 group showed significant increase in RQ of miR-125b in the liver tissue compared to cyan-10 group (Figure 3B). Furthermore, RQ of TUBG1 in the liver tissue was significantly decreased in cyan-10, cyan-15 and cyan-20 groups compared to PCL group. Also, cyan-20 group showed significant decrease in RQ of TUBG1 in the liver tissue compared to cyan-10 group (Figure 3C).\n\nEffect on expression of of long non-coding RNA, MALAT1, miR-125b and tubulin 1 (G2/M transition of mitotic cell cycle) in the liver tissue. A: RQ of long non-coding RNA MALAT1; B: RQ miR-125b; C: RQ TUBGI . Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group; bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; TUBG: Tubulin gamma 1; lncRNA: Long non-coding RNA.\nAs shown in Figure 3, induction of PCL resulted in significant increase of lncRNA MALAT a significant decrease in RQ miR-125b and significant increase in RQ TUBG1 expressions in comparison to naïve group. RQ of lncRNA MALAT1 in the liver tissue was significantly decreased in cyan -10,15 and cyan-20 groups compared to PCL group. Furthermore, cyan-20 group showed significant decrease in RQ of lncRNA MALAT1 in the liver tissue compared to cyan-10 group (Figure 3A). On the contrary, RQ of miR-125b in the liver tissue was significantly increased in cyan -10, cyan-15 and cyan-20 groups compared to PCL group. In addition, cyan-20 group showed significant increase in RQ of miR-125b in the liver tissue compared to cyan-10 group (Figure 3B). Furthermore, RQ of TUBG1 in the liver tissue was significantly decreased in cyan-10, cyan-15 and cyan-20 groups compared to PCL group. Also, cyan-20 group showed significant decrease in RQ of TUBG1 in the liver tissue compared to cyan-10 group (Figure 3C).\n\nEffect on expression of of long non-coding RNA, MALAT1, miR-125b and tubulin 1 (G2/M transition of mitotic cell cycle) in the liver tissue. A: RQ of long non-coding RNA MALAT1; B: RQ miR-125b; C: RQ TUBGI . Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group; bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; TUBG: Tubulin gamma 1; lncRNA: Long non-coding RNA.\nHistopathological and immunohistochemical studies Light microscopic examination of H&E-stained sections of naïve rats’ liver showed normal hepatic architecture, cords of hepatocytes radiating from central vein, portal triads present in between, polygonal hepatocytes with centrally rounded vesicular nuclei, and hepatic sinusoid in between (Figure 4A and B). Liver sections obtained from rats received DEN/2-AAF (Figure 4C-E) showed disruption of normal hepatic lobular architecture along with presence of large, discriminated dysplastic nodules compressing the surrounding liver tissue, with high-grade dysplastic cells displaying increased nuclear: Cytoplasmatic ratio (Figure 4E), nuclear hyperchromatosis, and basophilic cytoplasm. Liver sections of rats treated with different doses of cyanidin (10, 15, and 20 mg/kg) respectively, showed small and less discriminated dysplastic nodules (Figure 4F-H).\n\nPhotomicrographs of liver sections stained with H&E staining. A and B: Naïve group liver sections showed normal hepatic architecture, cords of hepatocytes radiating from central vein and portal triads present in-between and polygonal hepatocytes with central rounded vesicular nuclei, and hepatic sinusoid in-between; C-E: Liver sections of rats received diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF) showed larger, discriminated dysplastic nodules (doted shapes) compressing the surrounding liver tissue with disruption of normal hepatic lobular architecture; D: Liver sections of rats received DEN/2-AAF showed eosinophilic foci of cellular alteration consisting of enlarged hepatocytes with increased acidophilic staining and vaculated nuclei (arrow head); E: Liver sections of rats received DEN/2-AAF showed foci of Clear cell formed of hepatocytes showing variable degrees of cytoplasmic vacuolations and ballooning with pyknotic nuclei (arrow); F-H: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) respectively, showing small and less discriminated dysplastic nodules (doted shapes). (Magnification: A, C, F, G, H × 1000; B, D, E × 400). CV: Central vein; PV: Portal vein; Pt: Portal triads; S: Sinusoid; H: Hepatocytes.\nImmunohistochemical examination with GSTP antibody appeared as large hyperplastic nodules were demonstrated by the brown color. Figure 5A and B shows the liver sections of naïve group, while Figure 5C shows large-positively stained GSTP foci demonstrated in PCL group. Liver sections from rats of all cyan-treated groups exhibited small GSTP-positive small hepatic foci of different sizes scattered in-between negatively-stained hepatic parenchyma (Figure 5D-F). The percent area of GSTP foci was significantly increased in the PCL group compared to naïve group, while all cyan-treated groups showed significant decrease in the GSTP percent area. Furthermore, cyan-15 and -20 showed significant decrease in the GSTP percent area over cyan-10 (Table 1). PCNA immunohistochemical analysis showed an elevated expression of PCNA in the group that received DEN/2-AAF as compared to naïve group (Figure 6A and B). Liver sections obtained from rats received cyan. in its three doses showed decrease in PCNA-positively stained nuclei (Figure 6C-E and Table 1).\n\nPhotomicrographs of liver sections of rats immunohistochemical stained with glutathione S-transferase placental antibody. A and B: Naive group; C: Precancerous lesion group showing multiple glutathione S-transferase placental (GSTP)-positive large hepatic nodules (brown stained nodules) occupying most of section; D-F: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) showing GSTP positive small hepatic foci (brown stained cells = arrow) of different size scattered in-between negatively stained hepatic parenchyma. (Magnification: × 40)\n\nPhotomicrographs of liver sections Immunohistochemical stained with proliferating cell nuclear antigen. A: Negative reaction of control group; B: Diethylnitrosamine + 100 mg 2-acetylaminofluorene group showing positive stained nuclei scattered all over the field; C-E: Liver sections of rats treated with different doses of cyamidine (10, 15, 20 mg/kg) respectively, show few positive hepatocytes sporadically distributed over the field (arrow). (Magnification × 100). \nExpression rate of hepatocytes positive for glutathione S-transferase placental and proliferating cell nuclear antigen were calculated as number of positive field expression in 10 fields per rat liver tissue\nValues are mean ± SD; number of animals = 6 rats/each group. Examined field; 10 fields/liver section. \n\nP < 0.05 significant differences compared to naïve group.\n\nP < 0.05 significant differences compared to precancerous lesion group.\n\nP < 0.05 significant compared to cyanidin-3-O-glucoside-10, One-way ANOVA followed by Tukey’s test. +: Positive expression found in 1-3 fields; ++: Positive expression found in 4-6 fields; +++: Positive expression found in 7-10 field; PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; GSTP: Glutathione S-transferase placental; PCNA: Proliferating cell nuclear antigen.\nLight microscopic examination of H&E-stained sections of naïve rats’ liver showed normal hepatic architecture, cords of hepatocytes radiating from central vein, portal triads present in between, polygonal hepatocytes with centrally rounded vesicular nuclei, and hepatic sinusoid in between (Figure 4A and B). Liver sections obtained from rats received DEN/2-AAF (Figure 4C-E) showed disruption of normal hepatic lobular architecture along with presence of large, discriminated dysplastic nodules compressing the surrounding liver tissue, with high-grade dysplastic cells displaying increased nuclear: Cytoplasmatic ratio (Figure 4E), nuclear hyperchromatosis, and basophilic cytoplasm. Liver sections of rats treated with different doses of cyanidin (10, 15, and 20 mg/kg) respectively, showed small and less discriminated dysplastic nodules (Figure 4F-H).\n\nPhotomicrographs of liver sections stained with H&E staining. A and B: Naïve group liver sections showed normal hepatic architecture, cords of hepatocytes radiating from central vein and portal triads present in-between and polygonal hepatocytes with central rounded vesicular nuclei, and hepatic sinusoid in-between; C-E: Liver sections of rats received diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF) showed larger, discriminated dysplastic nodules (doted shapes) compressing the surrounding liver tissue with disruption of normal hepatic lobular architecture; D: Liver sections of rats received DEN/2-AAF showed eosinophilic foci of cellular alteration consisting of enlarged hepatocytes with increased acidophilic staining and vaculated nuclei (arrow head); E: Liver sections of rats received DEN/2-AAF showed foci of Clear cell formed of hepatocytes showing variable degrees of cytoplasmic vacuolations and ballooning with pyknotic nuclei (arrow); F-H: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) respectively, showing small and less discriminated dysplastic nodules (doted shapes). (Magnification: A, C, F, G, H × 1000; B, D, E × 400). CV: Central vein; PV: Portal vein; Pt: Portal triads; S: Sinusoid; H: Hepatocytes.\nImmunohistochemical examination with GSTP antibody appeared as large hyperplastic nodules were demonstrated by the brown color. Figure 5A and B shows the liver sections of naïve group, while Figure 5C shows large-positively stained GSTP foci demonstrated in PCL group. Liver sections from rats of all cyan-treated groups exhibited small GSTP-positive small hepatic foci of different sizes scattered in-between negatively-stained hepatic parenchyma (Figure 5D-F). The percent area of GSTP foci was significantly increased in the PCL group compared to naïve group, while all cyan-treated groups showed significant decrease in the GSTP percent area. Furthermore, cyan-15 and -20 showed significant decrease in the GSTP percent area over cyan-10 (Table 1). PCNA immunohistochemical analysis showed an elevated expression of PCNA in the group that received DEN/2-AAF as compared to naïve group (Figure 6A and B). Liver sections obtained from rats received cyan. in its three doses showed decrease in PCNA-positively stained nuclei (Figure 6C-E and Table 1).\n\nPhotomicrographs of liver sections of rats immunohistochemical stained with glutathione S-transferase placental antibody. A and B: Naive group; C: Precancerous lesion group showing multiple glutathione S-transferase placental (GSTP)-positive large hepatic nodules (brown stained nodules) occupying most of section; D-F: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) showing GSTP positive small hepatic foci (brown stained cells = arrow) of different size scattered in-between negatively stained hepatic parenchyma. (Magnification: × 40)\n\nPhotomicrographs of liver sections Immunohistochemical stained with proliferating cell nuclear antigen. A: Negative reaction of control group; B: Diethylnitrosamine + 100 mg 2-acetylaminofluorene group showing positive stained nuclei scattered all over the field; C-E: Liver sections of rats treated with different doses of cyamidine (10, 15, 20 mg/kg) respectively, show few positive hepatocytes sporadically distributed over the field (arrow). (Magnification × 100). \nExpression rate of hepatocytes positive for glutathione S-transferase placental and proliferating cell nuclear antigen were calculated as number of positive field expression in 10 fields per rat liver tissue\nValues are mean ± SD; number of animals = 6 rats/each group. Examined field; 10 fields/liver section. \n\nP < 0.05 significant differences compared to naïve group.\n\nP < 0.05 significant differences compared to precancerous lesion group.\n\nP < 0.05 significant compared to cyanidin-3-O-glucoside-10, One-way ANOVA followed by Tukey’s test. +: Positive expression found in 1-3 fields; ++: Positive expression found in 4-6 fields; +++: Positive expression found in 7-10 field; PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; GSTP: Glutathione S-transferase placental; PCNA: Proliferating cell nuclear antigen.","As shown in Figure 1, following induction of PCL, rats exhibited a significant increase in AFP levels. AFP levels were significantly decreased in cyan-10, cyan-15, and cyan-20 groups compared to PCL group. There was no significant difference between the AFP levels of the three cyan groups. Liver function tests were affected after receiving DEN/2-AAF in which rats exhibited a significant increase in levels of ALT and total and direct bilirubin, and a significant decrease in the serum albumin (Figure 2). As shown in Figure 2A, rats received cyan. in different doses exhibited a significant decrease in serum ALT compared to PCL group. Cyan-15 group showed significant decrease in serum ALT compared to cyan-10 group. Moreover, cyan-20 group showed significant decrease in serum ALT compared to cyan-10 group. In As depicted in Figure 2B, compared to PCL group, the rats in the three cyan groups exhibited a significant decrease in serum total bilirubin. Furthermore, cyan-20 group showed significant decrease in serum total bilirubin compared to cyan-10 group. Figure 2C: Serum direct bilirubin was significantly decreased in the three cyan groups compared to the PCL group. Moreover, cyan-20 group showed significant decrease in serum direct bilirubin compared to cyan-10 group. Administration of cyan in doses 5, 10 and 20 mg/kg/d produced a significant increase in serum albumin compared to PCL group. However, there was no significant difference between the serum albumin levels of the three cyan groups (Figure 2D).\n\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) on alpha fetoprotein in the serum of rats. Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group, unpaired t test; bP < 0.05 significant differences compared to precancerous lesion group. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; AFP: Alpha fetoprotein.\n\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) in rats. A: Alanine aminotransferase; B: Total bilirubin; C: Direct bilirubin; D: Serum albumin. Values are mean ± SEM; number of animals = 6 rats/each group. bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; ALT: Alanine aminotransferase.","As shown in Figure 3, induction of PCL resulted in significant increase of lncRNA MALAT a significant decrease in RQ miR-125b and significant increase in RQ TUBG1 expressions in comparison to naïve group. RQ of lncRNA MALAT1 in the liver tissue was significantly decreased in cyan -10,15 and cyan-20 groups compared to PCL group. Furthermore, cyan-20 group showed significant decrease in RQ of lncRNA MALAT1 in the liver tissue compared to cyan-10 group (Figure 3A). On the contrary, RQ of miR-125b in the liver tissue was significantly increased in cyan -10, cyan-15 and cyan-20 groups compared to PCL group. In addition, cyan-20 group showed significant increase in RQ of miR-125b in the liver tissue compared to cyan-10 group (Figure 3B). Furthermore, RQ of TUBG1 in the liver tissue was significantly decreased in cyan-10, cyan-15 and cyan-20 groups compared to PCL group. Also, cyan-20 group showed significant decrease in RQ of TUBG1 in the liver tissue compared to cyan-10 group (Figure 3C).\n\nEffect on expression of of long non-coding RNA, MALAT1, miR-125b and tubulin 1 (G2/M transition of mitotic cell cycle) in the liver tissue. A: RQ of long non-coding RNA MALAT1; B: RQ miR-125b; C: RQ TUBGI . Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group; bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; TUBG: Tubulin gamma 1; lncRNA: Long non-coding RNA.","Light microscopic examination of H&E-stained sections of naïve rats’ liver showed normal hepatic architecture, cords of hepatocytes radiating from central vein, portal triads present in between, polygonal hepatocytes with centrally rounded vesicular nuclei, and hepatic sinusoid in between (Figure 4A and B). Liver sections obtained from rats received DEN/2-AAF (Figure 4C-E) showed disruption of normal hepatic lobular architecture along with presence of large, discriminated dysplastic nodules compressing the surrounding liver tissue, with high-grade dysplastic cells displaying increased nuclear: Cytoplasmatic ratio (Figure 4E), nuclear hyperchromatosis, and basophilic cytoplasm. Liver sections of rats treated with different doses of cyanidin (10, 15, and 20 mg/kg) respectively, showed small and less discriminated dysplastic nodules (Figure 4F-H).\n\nPhotomicrographs of liver sections stained with H&E staining. A and B: Naïve group liver sections showed normal hepatic architecture, cords of hepatocytes radiating from central vein and portal triads present in-between and polygonal hepatocytes with central rounded vesicular nuclei, and hepatic sinusoid in-between; C-E: Liver sections of rats received diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF) showed larger, discriminated dysplastic nodules (doted shapes) compressing the surrounding liver tissue with disruption of normal hepatic lobular architecture; D: Liver sections of rats received DEN/2-AAF showed eosinophilic foci of cellular alteration consisting of enlarged hepatocytes with increased acidophilic staining and vaculated nuclei (arrow head); E: Liver sections of rats received DEN/2-AAF showed foci of Clear cell formed of hepatocytes showing variable degrees of cytoplasmic vacuolations and ballooning with pyknotic nuclei (arrow); F-H: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) respectively, showing small and less discriminated dysplastic nodules (doted shapes). (Magnification: A, C, F, G, H × 1000; B, D, E × 400). CV: Central vein; PV: Portal vein; Pt: Portal triads; S: Sinusoid; H: Hepatocytes.\nImmunohistochemical examination with GSTP antibody appeared as large hyperplastic nodules were demonstrated by the brown color. Figure 5A and B shows the liver sections of naïve group, while Figure 5C shows large-positively stained GSTP foci demonstrated in PCL group. Liver sections from rats of all cyan-treated groups exhibited small GSTP-positive small hepatic foci of different sizes scattered in-between negatively-stained hepatic parenchyma (Figure 5D-F). The percent area of GSTP foci was significantly increased in the PCL group compared to naïve group, while all cyan-treated groups showed significant decrease in the GSTP percent area. Furthermore, cyan-15 and -20 showed significant decrease in the GSTP percent area over cyan-10 (Table 1). PCNA immunohistochemical analysis showed an elevated expression of PCNA in the group that received DEN/2-AAF as compared to naïve group (Figure 6A and B). Liver sections obtained from rats received cyan. in its three doses showed decrease in PCNA-positively stained nuclei (Figure 6C-E and Table 1).\n\nPhotomicrographs of liver sections of rats immunohistochemical stained with glutathione S-transferase placental antibody. A and B: Naive group; C: Precancerous lesion group showing multiple glutathione S-transferase placental (GSTP)-positive large hepatic nodules (brown stained nodules) occupying most of section; D-F: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) showing GSTP positive small hepatic foci (brown stained cells = arrow) of different size scattered in-between negatively stained hepatic parenchyma. (Magnification: × 40)\n\nPhotomicrographs of liver sections Immunohistochemical stained with proliferating cell nuclear antigen. A: Negative reaction of control group; B: Diethylnitrosamine + 100 mg 2-acetylaminofluorene group showing positive stained nuclei scattered all over the field; C-E: Liver sections of rats treated with different doses of cyamidine (10, 15, 20 mg/kg) respectively, show few positive hepatocytes sporadically distributed over the field (arrow). (Magnification × 100). \nExpression rate of hepatocytes positive for glutathione S-transferase placental and proliferating cell nuclear antigen were calculated as number of positive field expression in 10 fields per rat liver tissue\nValues are mean ± SD; number of animals = 6 rats/each group. Examined field; 10 fields/liver section. \n\nP < 0.05 significant differences compared to naïve group.\n\nP < 0.05 significant differences compared to precancerous lesion group.\n\nP < 0.05 significant compared to cyanidin-3-O-glucoside-10, One-way ANOVA followed by Tukey’s test. +: Positive expression found in 1-3 fields; ++: Positive expression found in 4-6 fields; +++: Positive expression found in 7-10 field; PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; GSTP: Glutathione S-transferase placental; PCNA: Proliferating cell nuclear antigen.","Altered expression of mitotic spindle assembly genes has been detected in many cancers. For example, MAD2 gene levels are downregulated in breast carcinoma[18], while mitotic checkpoint serine/BUB1 gene expression is dysregulated in colorectal carcinoma[19]. The impairment of spindle assembly genes are frequently detected in HCC[20,21]. Moreover, inhibition of the mitotic assembly genes has been found to be lethal to cancer cells, and has promising therapeutic effect in cancer treatment[22,23]. \nIn this study, we used several public microarray databases for the construction of simple genetic-epigenetic network specific to HCC and linked to cell cycle and mitotic spindle formation. We assessed the antiproliferative effect of cyan in HCC animal model via modulation of lncRNA-MALAT1-miR-125b-TUBG1 mRNA axis expression.\nMicrotubules are involved both in maintenance of cell shape and motility and in mitotic spindle formation[24]. γ-TUBG is a major player that supports microtubule nucleation. γ-TUBG acts as a binding site for the α/β-tubulin dimer and it has two γ-TUBG genes, TUBG1 and TUBG2 in mammals only[25]. The TUBG levels in the centrosome change during cell cycle progression and usually increases at the start of mitosis. At the end of mitosis, TUBG levels are decreased to interphase levels[26]. Overexpression and altered compartmentalization of γ-TUBG may lead to carcinogenesis[27-29]. Interestingly, γ-TUBG associates interact with tumor suppressor protein C53 in mammalian interphase cells of different cellular origins[30]. Interestingly, γ-TUBG interacts with E2F transcription factors, resulting in upregulation of E2F target genes PCNA and CDKB1[31] and glutathione S transferase activity[32]. On the other hand, the mRNA and protein levels of TUBA1C are upregulated in HCC. Clinically, it affects the survival of patients and the level of metastatic burden along with portal vein thrombosis. In addition, it activates cellular proliferation and migration and is a potential therapeutic target for HCC[33]. Recent studies have shed light on the efficacy of microtubule-binding agents in the treatment of HCC, especially in combination with mammalian target of rapamycin inhibitors[34].\nOne of the few miRNAs that regulate Spindle Assembly Checkpoints (SAC) is miR-125b. It suppresses the expression of Mad1 (a core SAC protein) that is responsible for inhibiting entry into anaphase till metaphase defects are corrected[35]. Importantly, miRNA-125b has been found to be associated with colorectal cancer[36], multiple myeloma[37], and breast cancer[38]. Song et al[39] demonstrated that the expression of miR-125b in hepatocytes was protective and reflected better survival in HCC tissue samples due to restoration of SIRT6 expression[39]. miR-125b-5p inhibits HCC proliferation by targeting TXNRD1, the terminal step of biosynthesis of nucleotides[40].\nDeregulation of lncRNAs has been shown to be crucial in cancer progression, especially HCC[41,42]. Hung et al[43] demonstrated the involvement of lncRNAs in regulation of the expression of cell cycle-related genes, p53 gene regulatory pathway, and apoptosis[43,44]. The lncRNA MALAT1 is overexpressed in several solid tumors and is linked with tumor recurrence[45-47]. MALAT1 coordinate RNA polymerase II transcription, pre-mRNA splicing, and mRNA export[48]. Thus, this lncRNA holds great potential in influencing the local concentration of a specific splicing factor during specific stages of the cell cycle. Depletion of MALAT1 in cancer cells inhibits tumorigenicity[49]. MALAT1 regulates E2F1 transcription factor activity, highlighting its pro-proliferative function[50]. MALAT1 is upregulated in many cancer types, including breast cancer[51], cervical cancer, lung cancer[52], and hepatocarcinoma[53-55]. In a previous study, hypoxia enhanced MALAT1 expression, leading to increase in proliferating and invading activity of Hep3B cells due to negative interaction with miR200a[56]. Huang and his group explored the role of specificity protein 1/3 in regulation of MALAT1 expression in HCC cells[57].\nCyanidin or anthocyanin (a polyphenolic pigment found in plants) confers several pharmacological benefits, including anticancer properties[58-62]. Cyan mediates cytotoxicity against human monocytic leukemia cells via G2/M phase arrest and induction of apoptosis[63]. Tsai et al[64] evaluated anthocyanins as promising anticancer or chemopreventive agents to induce G2/M arrest in HAs-induced leukemia cell cycle arrest via ATM-Chk1/2-Cdc25C axis modulation[64]. They also found that anthocyanin inhibits the proliferation of HCC cells[65,66].\nIn our study, the effect of cyan on cancerous tissue was dose-dependent (10, 15 and 20 mg/kg/d), that is, increasing its effect with increasing the concentration of the drug. This was proved also, with the correlated significant decrease in the percent area of GSTP foci, PCNA expression which are the target of TUBG1 mRNA and the molecular epigenetic markers associated with HCC (lncRNA MALAT1 and miR-125b-1-3p) in the liver tissue of rats in our study (Figure 7). Thus, we can hypothesize that administration of cyan in PCL may result in downregulation of lncRNA MALAT1 with subsequent release of free miR-125b-1-3p that binds to TUBG1 mRNA and downregulate its expression[65]. The changes in the epigenetic-genetic network affect their target genes PCNA and GSTP.\n\nConcept map of study design. GSTP: Glutathione S-transferase placental; PCNA: Proliferating cell nuclear antigen; TUBG: Tubulin; lncRNA: Long non-coding RNA.\nLastly, the cyan-mediated modulation of cell cycle and mitotic spindle assembly that regulate HCC proliferation might affect the capability of the HCC cells to overcome oxidative stress[66,67]. Our data confers the rationale for designing antimitotic approach combining the conventional cytotoxic drugs with phytochemical extracts to inhibit cell cycle progression in cancer. More in vitro functional studies are required to explore the functional mechanism of the chosen RNA panel and validate their role as drug target biomarkers. ","Cyanidin is a natural molecule that holds great potential in unraveling the mystery of cytotoxic pharmacy in the future. It can be used to devise novel antimitotic drugs that can target cell cycle in HCC. Our results also indicated great potential of the TUBG1 mRNA- miR-125b-lncRNA MALAT1 and -1-3p axis as potential drug target biomarker for further investigations."],"string":"[\n \"Hepatocellular carcinoma (HCC) is the commonest primary malignancy of the liver. Its main risk factors include hepatitis B, hepatitis C, and non-alcoholic steatohepatitis. HCC is associated with high mortality and morbidity. Its incidence has increased from 1.4 to 6.2 per 100000 cases per year within the last 30 years[1]. HCC burden in Egypt is high due to the high prevalence of HCV, where liver cancers account for 11.75% of all gastrointestinal cancers and 1.68% of all malignancies. In addition, over 70% of all liver malignancies in Egypt are HCC[2].\\nTo date, HCC is holding its place as an unresolved dilemma; its resistance to systemic chemotherapy and radiation makes the cure approachable only in very early stages according to Milan criteria, that is, where the tumor size is < 5 cm (when only one lesion is present) or < 3 cm (when 2-3 lesions are present)[3].\\nGenes involved in cell cycle control are usually mutated in presence of cancer. Dysregulated mitosis results in genomic instability, which leads to tumor aggressiveness[4]. Microtubules are a key component of mitotic spindles, and thus, a crucial part of the process of mitosis. They form an important target in cancer chemotherapy, inducing arrest of mitosis and cell death. On the other hand, while the overexpression of γ-tubulin (γ-TUBG) showed improved survival among small cell lung cancer patients[5], the overexpression of γ-TUBG is found to be characteristic feature of thyroid and breast cancers[6]. Such variation may arise due to differences in gene transcription and check point regulations in different cancer tissues[7].\\nCyanidin-3-glucoside (cyan) is a potential chemotherapeutic and chemo-protective agent. Through its antioxidant activity, it scavenges radical oxygen species, which decreases the overall number of tumorous cells (malignant and benign) in in vivo studies[8]. Cyan has been implicated in some beneficial health activities[9], including reducing age-associated oxidative stress[10], improving cognitive brain function, and exhibiting anti-diabetic[11], anti-inflammation[12], anti-atherogenic[13], and anti-obesity activities[14]. Previously, it has been reported that cyan mediates caspase-3 cleavage with DNA fragmentation. Cyan has also been associated with induction of autophagy, a key element involved in cancer elimination, via induction of autophagy-related gene 5 and microtubule-associated protein 1 light chain 3-II[15]. Cyan has drawn increasing attention because of its potential anti-cancer properties. Cyan may offer a novel avenue for treating HCC. a potential antiproliferative effect on HepG2 cells[16].\\nThe present study was conducted to evaluate the effect of cyan in three different doses on cell cycle progression and mitotic assembly disorder in hepatic precancerous lesion (PCL) via assessment of levels of long non-coding RNA (lncRNA) MALAT1, miR-125b, and tubulin 1. We also conducted histopathological and immuno-histochemical examination using H&E staining assessment of levels of glutathione S-transferase placental (GSTP) and proliferating cell nuclear antigen (PCNA) antibodies in male Wistar rat model of diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF)-induced hepatic PCL. \",\n \"Chemicals and drugs DEN was obtained as 1 g solution in serum bottle diluted in 0.9% NaCl. 2-AAF was obtained as white powder, while cyan was obtained as black powder. Both 2-AAF and cyan were dissolved in 0.9% NaCl. All chemicals were purchased from Sigma-Aldrich, Cairo, Egypt.\\nDEN was obtained as 1 g solution in serum bottle diluted in 0.9% NaCl. 2-AAF was obtained as white powder, while cyan was obtained as black powder. Both 2-AAF and cyan were dissolved in 0.9% NaCl. All chemicals were purchased from Sigma-Aldrich, Cairo, Egypt.\\nExperimental animals All animal procedures were approved by the Institutional Animal Ethics Committee of Ain Shams University, Faculty of Medicine. Thirty male Wistar rats weighing 200-250 g were purchased from Nile Pharmaceuticals Company (Cairo, Egypt). Animals were housed in an animal room with temperature 25 ± 2 °C and 12 h light/dark cycle controls. An adaptation period of 1 wk was allowed before initiation of the experimental protocol.\\nAll animal procedures were approved by the Institutional Animal Ethics Committee of Ain Shams University, Faculty of Medicine. Thirty male Wistar rats weighing 200-250 g were purchased from Nile Pharmaceuticals Company (Cairo, Egypt). Animals were housed in an animal room with temperature 25 ± 2 °C and 12 h light/dark cycle controls. An adaptation period of 1 wk was allowed before initiation of the experimental protocol.\\nExperimental procedures \\nInduction of hepatic PCL: DEN was injected intraperitoneally (i.p.) at a dose of 100 mg/kg once weekly for three consecutive weeks. One week after the last DEN injection, a single dose (300 mg/kg) of 2-AAF was injected.\\n\\nAnimal groups: Rats were divided into five groups (6 rats/each group), including: Naïve group: Rats were injected with a vehicle (0.9% NaCl), PCL group: Rats were injected with DEN and 2-AAF, and three cyan groups (cyan-10, cyan-15, and cyan-20 groups): Rats were injected with DEN and 2-AAF, and orally administered through gastric gavage with cyan in doses of 10, 15, and 20 mg/kg respectively, for four consecutive days per week for 16 wk[17].\\n\\nInduction of hepatic PCL: DEN was injected intraperitoneally (i.p.) at a dose of 100 mg/kg once weekly for three consecutive weeks. One week after the last DEN injection, a single dose (300 mg/kg) of 2-AAF was injected.\\n\\nAnimal groups: Rats were divided into five groups (6 rats/each group), including: Naïve group: Rats were injected with a vehicle (0.9% NaCl), PCL group: Rats were injected with DEN and 2-AAF, and three cyan groups (cyan-10, cyan-15, and cyan-20 groups): Rats were injected with DEN and 2-AAF, and orally administered through gastric gavage with cyan in doses of 10, 15, and 20 mg/kg respectively, for four consecutive days per week for 16 wk[17].\\nAssessment of the effects of treatment \\nBiochemical and molecular studies: After scarifying the animals, blood was withdrawn from each rat from the retro orbital vein and incubated for about half an hour for clotting. Then, the blood sample was centrifuged for 20 min at 5000 RPM to get serum samples. Livers were dissected. The right lobe of the livers was removed, cut into longitudinal sections 2-4 mm in thickness and kept in 10% formalin for histopathological and immunohistochemical examination. The other lobe was kept frozen at -80 °C for molecular analyses. The samples were analyzed for the following parameters: (1) alpha fetoprotein (AFP) levels: AFP level was analyzed using quantitative sandwich rat AFP ELISA kit purchased from MyBiosource Inc. (San Diego, United States) with a sensitivity range of 0.625-20 ng/mL; (2) serum alanine aminotransferase (ALT) levels: The serum ALT was measured according to method described by using kits purchased from Diamond Diagnostic (Cairo, Egypt). ALT catalyzes the transfer of amino groups from specific amino acids to ketoglutaric acid, yielding glutamic acid and oxaloacetic or pyruvic acid, respectively. Ketoacids were then determined calorimetrically after their reaction with 2, 4- dinitrophenylhydrazine; and (3) determination of serum total bilirubin and direct bilirubin: Alkaline methanolysis of bilirubin followed by chloroform extraction of bilirubin methyl esters, and later, separation of these esters by chromatography and spectrophotometric determination at 430 nm.\\n\\nBiochemical and molecular studies: After scarifying the animals, blood was withdrawn from each rat from the retro orbital vein and incubated for about half an hour for clotting. Then, the blood sample was centrifuged for 20 min at 5000 RPM to get serum samples. Livers were dissected. The right lobe of the livers was removed, cut into longitudinal sections 2-4 mm in thickness and kept in 10% formalin for histopathological and immunohistochemical examination. The other lobe was kept frozen at -80 °C for molecular analyses. The samples were analyzed for the following parameters: (1) alpha fetoprotein (AFP) levels: AFP level was analyzed using quantitative sandwich rat AFP ELISA kit purchased from MyBiosource Inc. (San Diego, United States) with a sensitivity range of 0.625-20 ng/mL; (2) serum alanine aminotransferase (ALT) levels: The serum ALT was measured according to method described by using kits purchased from Diamond Diagnostic (Cairo, Egypt). ALT catalyzes the transfer of amino groups from specific amino acids to ketoglutaric acid, yielding glutamic acid and oxaloacetic or pyruvic acid, respectively. Ketoacids were then determined calorimetrically after their reaction with 2, 4- dinitrophenylhydrazine; and (3) determination of serum total bilirubin and direct bilirubin: Alkaline methanolysis of bilirubin followed by chloroform extraction of bilirubin methyl esters, and later, separation of these esters by chromatography and spectrophotometric determination at 430 nm.\\nDetermination of serum albumin Quantitative determination of serum albumin level was quantified using rat albumin ELISA kit. The absorbance at 450 nm is a measure of the concentration of albumin in the test sample.\\nQuantitative determination of serum albumin level was quantified using rat albumin ELISA kit. The absorbance at 450 nm is a measure of the concentration of albumin in the test sample.\\nBioinformatics-based selection of cell cycle-microtubule assembly- and HCC-specific RNA panel The RNA panel was chosen in three steps: (1) Tubulin, gamma 1 (TUBG1) was retrieved as a crucial player in microtubule formation and progression of the cell cycle through gene atlas expression database (available at https://www.ebi.ac.uk/gxa/home) alongside with verification of the chosen gene from protein Atlas database (available at https://www.proteinatlas.org/), followed by analysis of TUBG1 gene ontology to ensure that it is linked to G2-M transition and mitotic spindle assembly, which is closely linked to cancer development through gene card database (available at https://www.genecards.org/); (2) we selected miR-125b-1-3p targeting TUBG1 mRNA though) miRWALK database available at http://mirwalk.umm.uni-heidelberg.de/), followed by pathway enrichment, which revealed that miR-125b-1-3p is linked to MAP kinase and ubiquitin-mediated proteolysis (strongly correlated with mitotic assembly and cell cycle progression) through (Diana database available at http://diana.imis.athena-innovation.gr/DianaTools/index.php); and (3) lncRNA metastasis-associated lung adenocarcinoma transcript 1, lncRNA MALAT1, was obtained as a lncRNA specific to HCC and targeting miR-125b-1-3p using noncode database available at http://www.noncode.org/ and European Bioinformatics institute database available at https://www.ebi.ac.uk.\\nThe RNA panel was chosen in three steps: (1) Tubulin, gamma 1 (TUBG1) was retrieved as a crucial player in microtubule formation and progression of the cell cycle through gene atlas expression database (available at https://www.ebi.ac.uk/gxa/home) alongside with verification of the chosen gene from protein Atlas database (available at https://www.proteinatlas.org/), followed by analysis of TUBG1 gene ontology to ensure that it is linked to G2-M transition and mitotic spindle assembly, which is closely linked to cancer development through gene card database (available at https://www.genecards.org/); (2) we selected miR-125b-1-3p targeting TUBG1 mRNA though) miRWALK database available at http://mirwalk.umm.uni-heidelberg.de/), followed by pathway enrichment, which revealed that miR-125b-1-3p is linked to MAP kinase and ubiquitin-mediated proteolysis (strongly correlated with mitotic assembly and cell cycle progression) through (Diana database available at http://diana.imis.athena-innovation.gr/DianaTools/index.php); and (3) lncRNA metastasis-associated lung adenocarcinoma transcript 1, lncRNA MALAT1, was obtained as a lncRNA specific to HCC and targeting miR-125b-1-3p using noncode database available at http://www.noncode.org/ and European Bioinformatics institute database available at https://www.ebi.ac.uk.\\nExtraction of total RNA, including lncRNA and miRNA from liver tissue Total RNA was extracted from liver tissues using miRNeasy Mini kit (Cat No. 217004, Qiagen, United States) according to manufacturer’s protocol. The extracted RNA concentration and integrity were assessed using [(NanoDrop Technologies/Thermo Scientific, Wilmington, DE, United States)], with RNA purities were 1.8-2. The extracted total RNA was reverse transcribed into cDNA using a miScript II RT kit (Cat No. 218160, 218161, Qiagen, United States) on thermal cycler (Bio-Rad; Hercules, CA, United States), according to the manufacturer’s protocol. \\nTotal RNA was extracted from liver tissues using miRNeasy Mini kit (Cat No. 217004, Qiagen, United States) according to manufacturer’s protocol. The extracted RNA concentration and integrity were assessed using [(NanoDrop Technologies/Thermo Scientific, Wilmington, DE, United States)], with RNA purities were 1.8-2. The extracted total RNA was reverse transcribed into cDNA using a miScript II RT kit (Cat No. 218160, 218161, Qiagen, United States) on thermal cycler (Bio-Rad; Hercules, CA, United States), according to the manufacturer’s protocol. \\nReal time-polymerase chain reaction quantification of the RNA panel in liver tissues The relative expression of TUBG1 mRNA and lncRNA-MALAT1 in liver tissue were measured using a Quantitect SYBR Green Master Mix kit and an RT2 SYBR Green ROX real time-polymerase chain reaction (qPCR) Mastermix (Qiagen), respectively, using a 7500 qPCR Systems (Applied Biosystems, Foster City, CA, United States) detection system) and specific primers (Accession: NM_001128148, NM_003234, NR_002819, and ENST00000534336, respectively) supplied by Qiagen. GAPDH (Accession NM_002046.7) was used as a housekeeping gene. MiR-3163 expression in liver tissue was quantified by PCR using a miScript SYBR Green kit (Qiagen), a miScript universal primer, and a miRNA-specific forward primer (mir-125b-1-3p miScript Primer Assay) (Accession: MIMAT0004592 (5'-ACGGGUUAGGCUCUUGGGAGCU). All steps followed the manufacturer’s suggested protocol, and SNORD68 was used as an internal control. Ct values more than 36 were considered as negative. The specificities of the amplicons for the SYBR Green−based PCR amplification were affirmed by the melting curves. The 2−ΔΔCt technique was used to measure the relative expression of the HCC-specific RNA panel.\\nThe relative expression of TUBG1 mRNA and lncRNA-MALAT1 in liver tissue were measured using a Quantitect SYBR Green Master Mix kit and an RT2 SYBR Green ROX real time-polymerase chain reaction (qPCR) Mastermix (Qiagen), respectively, using a 7500 qPCR Systems (Applied Biosystems, Foster City, CA, United States) detection system) and specific primers (Accession: NM_001128148, NM_003234, NR_002819, and ENST00000534336, respectively) supplied by Qiagen. GAPDH (Accession NM_002046.7) was used as a housekeeping gene. MiR-3163 expression in liver tissue was quantified by PCR using a miScript SYBR Green kit (Qiagen), a miScript universal primer, and a miRNA-specific forward primer (mir-125b-1-3p miScript Primer Assay) (Accession: MIMAT0004592 (5'-ACGGGUUAGGCUCUUGGGAGCU). All steps followed the manufacturer’s suggested protocol, and SNORD68 was used as an internal control. Ct values more than 36 were considered as negative. The specificities of the amplicons for the SYBR Green−based PCR amplification were affirmed by the melting curves. The 2−ΔΔCt technique was used to measure the relative expression of the HCC-specific RNA panel.\\nHistopathological and immunohistochemical studies Liver sections were cut at 5-μm thickness and stained with H&E. Immunohistochemistry S-P method was used to detect GSTP and PCNA levels. Briefly, the protocol was as follows: (1) the tissues were treated with endogenous peroxidase blocking solution at room temperature for 10 min, and then, incubated in normal nonimmune serum at room temperature for 10 min; (2) mouse anti GSTP or PCNA antibody was added to adjacent tissue sections respectively and incubated overnight at 4 °C; (3) biotin-conjugated second antibody was added to the sections and incubated at room temperature for 10 min; and (4) S-P complex was added at room temperature for 10 min, and then, 2,4-diaminobutyric acid was used for the color reaction. The tissue sections were washed with Poly (butylene succinate) (PBS) (0.01 mol/L, pH 7.4) between each step. Positive and negative controls were simultaneously used to ensure specificity and reliability of the staining process. A positive section was taken as positive control. In negative control, PBS was used to replace the first antibody. GSTP-positivity was indicated by brown coloration of the cytoplasm. Morphometric analysis of the foci percent area was carried out using Leica Q win V.3 software after capturing the images using a Leica DM2500 microscope (Leica, Wetzlar, Germany). The PCNA labeling indices are represented as the expression of positively-stained nuclei (10 fields/slide at 400 ×) as shown below: +, positive expression found in 1-3 fields; ++, positive expression found in 4-6 fields; and +++, positive expression found in 7-10 fields. Rabbit monoclonal PCNA and GSTP antibodies were purchased from Abcam (San Francisco, United States).\\nLiver sections were cut at 5-μm thickness and stained with H&E. Immunohistochemistry S-P method was used to detect GSTP and PCNA levels. Briefly, the protocol was as follows: (1) the tissues were treated with endogenous peroxidase blocking solution at room temperature for 10 min, and then, incubated in normal nonimmune serum at room temperature for 10 min; (2) mouse anti GSTP or PCNA antibody was added to adjacent tissue sections respectively and incubated overnight at 4 °C; (3) biotin-conjugated second antibody was added to the sections and incubated at room temperature for 10 min; and (4) S-P complex was added at room temperature for 10 min, and then, 2,4-diaminobutyric acid was used for the color reaction. The tissue sections were washed with Poly (butylene succinate) (PBS) (0.01 mol/L, pH 7.4) between each step. Positive and negative controls were simultaneously used to ensure specificity and reliability of the staining process. A positive section was taken as positive control. In negative control, PBS was used to replace the first antibody. GSTP-positivity was indicated by brown coloration of the cytoplasm. Morphometric analysis of the foci percent area was carried out using Leica Q win V.3 software after capturing the images using a Leica DM2500 microscope (Leica, Wetzlar, Germany). The PCNA labeling indices are represented as the expression of positively-stained nuclei (10 fields/slide at 400 ×) as shown below: +, positive expression found in 1-3 fields; ++, positive expression found in 4-6 fields; and +++, positive expression found in 7-10 fields. Rabbit monoclonal PCNA and GSTP antibodies were purchased from Abcam (San Francisco, United States).\\nStatistical analysis All results were expressed as mean ± SD. Statistical analysis was carried out using GraphPad Prism version 6.01. Unpaired t-test was done to compare between naïve and PCL groups. One-way ANOVA, followed by Tukey’s test, was carried out to compare between all groups. P values < 0.05 were considered statistically significant. \\nAll results were expressed as mean ± SD. Statistical analysis was carried out using GraphPad Prism version 6.01. Unpaired t-test was done to compare between naïve and PCL groups. One-way ANOVA, followed by Tukey’s test, was carried out to compare between all groups. P values < 0.05 were considered statistically significant. \",\n \"DEN was obtained as 1 g solution in serum bottle diluted in 0.9% NaCl. 2-AAF was obtained as white powder, while cyan was obtained as black powder. Both 2-AAF and cyan were dissolved in 0.9% NaCl. All chemicals were purchased from Sigma-Aldrich, Cairo, Egypt.\",\n \"All animal procedures were approved by the Institutional Animal Ethics Committee of Ain Shams University, Faculty of Medicine. Thirty male Wistar rats weighing 200-250 g were purchased from Nile Pharmaceuticals Company (Cairo, Egypt). Animals were housed in an animal room with temperature 25 ± 2 °C and 12 h light/dark cycle controls. An adaptation period of 1 wk was allowed before initiation of the experimental protocol.\",\n \"\\nInduction of hepatic PCL: DEN was injected intraperitoneally (i.p.) at a dose of 100 mg/kg once weekly for three consecutive weeks. One week after the last DEN injection, a single dose (300 mg/kg) of 2-AAF was injected.\\n\\nAnimal groups: Rats were divided into five groups (6 rats/each group), including: Naïve group: Rats were injected with a vehicle (0.9% NaCl), PCL group: Rats were injected with DEN and 2-AAF, and three cyan groups (cyan-10, cyan-15, and cyan-20 groups): Rats were injected with DEN and 2-AAF, and orally administered through gastric gavage with cyan in doses of 10, 15, and 20 mg/kg respectively, for four consecutive days per week for 16 wk[17].\",\n \"\\nBiochemical and molecular studies: After scarifying the animals, blood was withdrawn from each rat from the retro orbital vein and incubated for about half an hour for clotting. Then, the blood sample was centrifuged for 20 min at 5000 RPM to get serum samples. Livers were dissected. The right lobe of the livers was removed, cut into longitudinal sections 2-4 mm in thickness and kept in 10% formalin for histopathological and immunohistochemical examination. The other lobe was kept frozen at -80 °C for molecular analyses. The samples were analyzed for the following parameters: (1) alpha fetoprotein (AFP) levels: AFP level was analyzed using quantitative sandwich rat AFP ELISA kit purchased from MyBiosource Inc. (San Diego, United States) with a sensitivity range of 0.625-20 ng/mL; (2) serum alanine aminotransferase (ALT) levels: The serum ALT was measured according to method described by using kits purchased from Diamond Diagnostic (Cairo, Egypt). ALT catalyzes the transfer of amino groups from specific amino acids to ketoglutaric acid, yielding glutamic acid and oxaloacetic or pyruvic acid, respectively. Ketoacids were then determined calorimetrically after their reaction with 2, 4- dinitrophenylhydrazine; and (3) determination of serum total bilirubin and direct bilirubin: Alkaline methanolysis of bilirubin followed by chloroform extraction of bilirubin methyl esters, and later, separation of these esters by chromatography and spectrophotometric determination at 430 nm.\",\n \"Quantitative determination of serum albumin level was quantified using rat albumin ELISA kit. The absorbance at 450 nm is a measure of the concentration of albumin in the test sample.\",\n \"The RNA panel was chosen in three steps: (1) Tubulin, gamma 1 (TUBG1) was retrieved as a crucial player in microtubule formation and progression of the cell cycle through gene atlas expression database (available at https://www.ebi.ac.uk/gxa/home) alongside with verification of the chosen gene from protein Atlas database (available at https://www.proteinatlas.org/), followed by analysis of TUBG1 gene ontology to ensure that it is linked to G2-M transition and mitotic spindle assembly, which is closely linked to cancer development through gene card database (available at https://www.genecards.org/); (2) we selected miR-125b-1-3p targeting TUBG1 mRNA though) miRWALK database available at http://mirwalk.umm.uni-heidelberg.de/), followed by pathway enrichment, which revealed that miR-125b-1-3p is linked to MAP kinase and ubiquitin-mediated proteolysis (strongly correlated with mitotic assembly and cell cycle progression) through (Diana database available at http://diana.imis.athena-innovation.gr/DianaTools/index.php); and (3) lncRNA metastasis-associated lung adenocarcinoma transcript 1, lncRNA MALAT1, was obtained as a lncRNA specific to HCC and targeting miR-125b-1-3p using noncode database available at http://www.noncode.org/ and European Bioinformatics institute database available at https://www.ebi.ac.uk.\",\n \"Total RNA was extracted from liver tissues using miRNeasy Mini kit (Cat No. 217004, Qiagen, United States) according to manufacturer’s protocol. The extracted RNA concentration and integrity were assessed using [(NanoDrop Technologies/Thermo Scientific, Wilmington, DE, United States)], with RNA purities were 1.8-2. The extracted total RNA was reverse transcribed into cDNA using a miScript II RT kit (Cat No. 218160, 218161, Qiagen, United States) on thermal cycler (Bio-Rad; Hercules, CA, United States), according to the manufacturer’s protocol. \",\n \"The relative expression of TUBG1 mRNA and lncRNA-MALAT1 in liver tissue were measured using a Quantitect SYBR Green Master Mix kit and an RT2 SYBR Green ROX real time-polymerase chain reaction (qPCR) Mastermix (Qiagen), respectively, using a 7500 qPCR Systems (Applied Biosystems, Foster City, CA, United States) detection system) and specific primers (Accession: NM_001128148, NM_003234, NR_002819, and ENST00000534336, respectively) supplied by Qiagen. GAPDH (Accession NM_002046.7) was used as a housekeeping gene. MiR-3163 expression in liver tissue was quantified by PCR using a miScript SYBR Green kit (Qiagen), a miScript universal primer, and a miRNA-specific forward primer (mir-125b-1-3p miScript Primer Assay) (Accession: MIMAT0004592 (5'-ACGGGUUAGGCUCUUGGGAGCU). All steps followed the manufacturer’s suggested protocol, and SNORD68 was used as an internal control. Ct values more than 36 were considered as negative. The specificities of the amplicons for the SYBR Green−based PCR amplification were affirmed by the melting curves. The 2−ΔΔCt technique was used to measure the relative expression of the HCC-specific RNA panel.\",\n \"Liver sections were cut at 5-μm thickness and stained with H&E. Immunohistochemistry S-P method was used to detect GSTP and PCNA levels. Briefly, the protocol was as follows: (1) the tissues were treated with endogenous peroxidase blocking solution at room temperature for 10 min, and then, incubated in normal nonimmune serum at room temperature for 10 min; (2) mouse anti GSTP or PCNA antibody was added to adjacent tissue sections respectively and incubated overnight at 4 °C; (3) biotin-conjugated second antibody was added to the sections and incubated at room temperature for 10 min; and (4) S-P complex was added at room temperature for 10 min, and then, 2,4-diaminobutyric acid was used for the color reaction. The tissue sections were washed with Poly (butylene succinate) (PBS) (0.01 mol/L, pH 7.4) between each step. Positive and negative controls were simultaneously used to ensure specificity and reliability of the staining process. A positive section was taken as positive control. In negative control, PBS was used to replace the first antibody. GSTP-positivity was indicated by brown coloration of the cytoplasm. Morphometric analysis of the foci percent area was carried out using Leica Q win V.3 software after capturing the images using a Leica DM2500 microscope (Leica, Wetzlar, Germany). The PCNA labeling indices are represented as the expression of positively-stained nuclei (10 fields/slide at 400 ×) as shown below: +, positive expression found in 1-3 fields; ++, positive expression found in 4-6 fields; and +++, positive expression found in 7-10 fields. Rabbit monoclonal PCNA and GSTP antibodies were purchased from Abcam (San Francisco, United States).\",\n \"All results were expressed as mean ± SD. Statistical analysis was carried out using GraphPad Prism version 6.01. Unpaired t-test was done to compare between naïve and PCL groups. One-way ANOVA, followed by Tukey’s test, was carried out to compare between all groups. P values < 0.05 were considered statistically significant. \",\n \"Effect on AFP levels and liver function tests As shown in Figure 1, following induction of PCL, rats exhibited a significant increase in AFP levels. AFP levels were significantly decreased in cyan-10, cyan-15, and cyan-20 groups compared to PCL group. There was no significant difference between the AFP levels of the three cyan groups. Liver function tests were affected after receiving DEN/2-AAF in which rats exhibited a significant increase in levels of ALT and total and direct bilirubin, and a significant decrease in the serum albumin (Figure 2). As shown in Figure 2A, rats received cyan. in different doses exhibited a significant decrease in serum ALT compared to PCL group. Cyan-15 group showed significant decrease in serum ALT compared to cyan-10 group. Moreover, cyan-20 group showed significant decrease in serum ALT compared to cyan-10 group. In As depicted in Figure 2B, compared to PCL group, the rats in the three cyan groups exhibited a significant decrease in serum total bilirubin. Furthermore, cyan-20 group showed significant decrease in serum total bilirubin compared to cyan-10 group. Figure 2C: Serum direct bilirubin was significantly decreased in the three cyan groups compared to the PCL group. Moreover, cyan-20 group showed significant decrease in serum direct bilirubin compared to cyan-10 group. Administration of cyan in doses 5, 10 and 20 mg/kg/d produced a significant increase in serum albumin compared to PCL group. However, there was no significant difference between the serum albumin levels of the three cyan groups (Figure 2D).\\n\\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) on alpha fetoprotein in the serum of rats. Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group, unpaired t test; bP < 0.05 significant differences compared to precancerous lesion group. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; AFP: Alpha fetoprotein.\\n\\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) in rats. A: Alanine aminotransferase; B: Total bilirubin; C: Direct bilirubin; D: Serum albumin. Values are mean ± SEM; number of animals = 6 rats/each group. bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; ALT: Alanine aminotransferase.\\nAs shown in Figure 1, following induction of PCL, rats exhibited a significant increase in AFP levels. AFP levels were significantly decreased in cyan-10, cyan-15, and cyan-20 groups compared to PCL group. There was no significant difference between the AFP levels of the three cyan groups. Liver function tests were affected after receiving DEN/2-AAF in which rats exhibited a significant increase in levels of ALT and total and direct bilirubin, and a significant decrease in the serum albumin (Figure 2). As shown in Figure 2A, rats received cyan. in different doses exhibited a significant decrease in serum ALT compared to PCL group. Cyan-15 group showed significant decrease in serum ALT compared to cyan-10 group. Moreover, cyan-20 group showed significant decrease in serum ALT compared to cyan-10 group. In As depicted in Figure 2B, compared to PCL group, the rats in the three cyan groups exhibited a significant decrease in serum total bilirubin. Furthermore, cyan-20 group showed significant decrease in serum total bilirubin compared to cyan-10 group. Figure 2C: Serum direct bilirubin was significantly decreased in the three cyan groups compared to the PCL group. Moreover, cyan-20 group showed significant decrease in serum direct bilirubin compared to cyan-10 group. Administration of cyan in doses 5, 10 and 20 mg/kg/d produced a significant increase in serum albumin compared to PCL group. However, there was no significant difference between the serum albumin levels of the three cyan groups (Figure 2D).\\n\\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) on alpha fetoprotein in the serum of rats. Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group, unpaired t test; bP < 0.05 significant differences compared to precancerous lesion group. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; AFP: Alpha fetoprotein.\\n\\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) in rats. A: Alanine aminotransferase; B: Total bilirubin; C: Direct bilirubin; D: Serum albumin. Values are mean ± SEM; number of animals = 6 rats/each group. bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; ALT: Alanine aminotransferase.\\nEffect on expression of lncRNA MALAT1, miR-125b, and TUBG1 (G2/M transition of mitotic cell cycle) in the liver tissue As shown in Figure 3, induction of PCL resulted in significant increase of lncRNA MALAT a significant decrease in RQ miR-125b and significant increase in RQ TUBG1 expressions in comparison to naïve group. RQ of lncRNA MALAT1 in the liver tissue was significantly decreased in cyan -10,15 and cyan-20 groups compared to PCL group. Furthermore, cyan-20 group showed significant decrease in RQ of lncRNA MALAT1 in the liver tissue compared to cyan-10 group (Figure 3A). On the contrary, RQ of miR-125b in the liver tissue was significantly increased in cyan -10, cyan-15 and cyan-20 groups compared to PCL group. In addition, cyan-20 group showed significant increase in RQ of miR-125b in the liver tissue compared to cyan-10 group (Figure 3B). Furthermore, RQ of TUBG1 in the liver tissue was significantly decreased in cyan-10, cyan-15 and cyan-20 groups compared to PCL group. Also, cyan-20 group showed significant decrease in RQ of TUBG1 in the liver tissue compared to cyan-10 group (Figure 3C).\\n\\nEffect on expression of of long non-coding RNA, MALAT1, miR-125b and tubulin 1 (G2/M transition of mitotic cell cycle) in the liver tissue. A: RQ of long non-coding RNA MALAT1; B: RQ miR-125b; C: RQ TUBGI . Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group; bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; TUBG: Tubulin gamma 1; lncRNA: Long non-coding RNA.\\nAs shown in Figure 3, induction of PCL resulted in significant increase of lncRNA MALAT a significant decrease in RQ miR-125b and significant increase in RQ TUBG1 expressions in comparison to naïve group. RQ of lncRNA MALAT1 in the liver tissue was significantly decreased in cyan -10,15 and cyan-20 groups compared to PCL group. Furthermore, cyan-20 group showed significant decrease in RQ of lncRNA MALAT1 in the liver tissue compared to cyan-10 group (Figure 3A). On the contrary, RQ of miR-125b in the liver tissue was significantly increased in cyan -10, cyan-15 and cyan-20 groups compared to PCL group. In addition, cyan-20 group showed significant increase in RQ of miR-125b in the liver tissue compared to cyan-10 group (Figure 3B). Furthermore, RQ of TUBG1 in the liver tissue was significantly decreased in cyan-10, cyan-15 and cyan-20 groups compared to PCL group. Also, cyan-20 group showed significant decrease in RQ of TUBG1 in the liver tissue compared to cyan-10 group (Figure 3C).\\n\\nEffect on expression of of long non-coding RNA, MALAT1, miR-125b and tubulin 1 (G2/M transition of mitotic cell cycle) in the liver tissue. A: RQ of long non-coding RNA MALAT1; B: RQ miR-125b; C: RQ TUBGI . Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group; bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; TUBG: Tubulin gamma 1; lncRNA: Long non-coding RNA.\\nHistopathological and immunohistochemical studies Light microscopic examination of H&E-stained sections of naïve rats’ liver showed normal hepatic architecture, cords of hepatocytes radiating from central vein, portal triads present in between, polygonal hepatocytes with centrally rounded vesicular nuclei, and hepatic sinusoid in between (Figure 4A and B). Liver sections obtained from rats received DEN/2-AAF (Figure 4C-E) showed disruption of normal hepatic lobular architecture along with presence of large, discriminated dysplastic nodules compressing the surrounding liver tissue, with high-grade dysplastic cells displaying increased nuclear: Cytoplasmatic ratio (Figure 4E), nuclear hyperchromatosis, and basophilic cytoplasm. Liver sections of rats treated with different doses of cyanidin (10, 15, and 20 mg/kg) respectively, showed small and less discriminated dysplastic nodules (Figure 4F-H).\\n\\nPhotomicrographs of liver sections stained with H&E staining. A and B: Naïve group liver sections showed normal hepatic architecture, cords of hepatocytes radiating from central vein and portal triads present in-between and polygonal hepatocytes with central rounded vesicular nuclei, and hepatic sinusoid in-between; C-E: Liver sections of rats received diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF) showed larger, discriminated dysplastic nodules (doted shapes) compressing the surrounding liver tissue with disruption of normal hepatic lobular architecture; D: Liver sections of rats received DEN/2-AAF showed eosinophilic foci of cellular alteration consisting of enlarged hepatocytes with increased acidophilic staining and vaculated nuclei (arrow head); E: Liver sections of rats received DEN/2-AAF showed foci of Clear cell formed of hepatocytes showing variable degrees of cytoplasmic vacuolations and ballooning with pyknotic nuclei (arrow); F-H: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) respectively, showing small and less discriminated dysplastic nodules (doted shapes). (Magnification: A, C, F, G, H × 1000; B, D, E × 400). CV: Central vein; PV: Portal vein; Pt: Portal triads; S: Sinusoid; H: Hepatocytes.\\nImmunohistochemical examination with GSTP antibody appeared as large hyperplastic nodules were demonstrated by the brown color. Figure 5A and B shows the liver sections of naïve group, while Figure 5C shows large-positively stained GSTP foci demonstrated in PCL group. Liver sections from rats of all cyan-treated groups exhibited small GSTP-positive small hepatic foci of different sizes scattered in-between negatively-stained hepatic parenchyma (Figure 5D-F). The percent area of GSTP foci was significantly increased in the PCL group compared to naïve group, while all cyan-treated groups showed significant decrease in the GSTP percent area. Furthermore, cyan-15 and -20 showed significant decrease in the GSTP percent area over cyan-10 (Table 1). PCNA immunohistochemical analysis showed an elevated expression of PCNA in the group that received DEN/2-AAF as compared to naïve group (Figure 6A and B). Liver sections obtained from rats received cyan. in its three doses showed decrease in PCNA-positively stained nuclei (Figure 6C-E and Table 1).\\n\\nPhotomicrographs of liver sections of rats immunohistochemical stained with glutathione S-transferase placental antibody. A and B: Naive group; C: Precancerous lesion group showing multiple glutathione S-transferase placental (GSTP)-positive large hepatic nodules (brown stained nodules) occupying most of section; D-F: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) showing GSTP positive small hepatic foci (brown stained cells = arrow) of different size scattered in-between negatively stained hepatic parenchyma. (Magnification: × 40)\\n\\nPhotomicrographs of liver sections Immunohistochemical stained with proliferating cell nuclear antigen. A: Negative reaction of control group; B: Diethylnitrosamine + 100 mg 2-acetylaminofluorene group showing positive stained nuclei scattered all over the field; C-E: Liver sections of rats treated with different doses of cyamidine (10, 15, 20 mg/kg) respectively, show few positive hepatocytes sporadically distributed over the field (arrow). (Magnification × 100). \\nExpression rate of hepatocytes positive for glutathione S-transferase placental and proliferating cell nuclear antigen were calculated as number of positive field expression in 10 fields per rat liver tissue\\nValues are mean ± SD; number of animals = 6 rats/each group. Examined field; 10 fields/liver section. \\n\\nP < 0.05 significant differences compared to naïve group.\\n\\nP < 0.05 significant differences compared to precancerous lesion group.\\n\\nP < 0.05 significant compared to cyanidin-3-O-glucoside-10, One-way ANOVA followed by Tukey’s test. +: Positive expression found in 1-3 fields; ++: Positive expression found in 4-6 fields; +++: Positive expression found in 7-10 field; PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; GSTP: Glutathione S-transferase placental; PCNA: Proliferating cell nuclear antigen.\\nLight microscopic examination of H&E-stained sections of naïve rats’ liver showed normal hepatic architecture, cords of hepatocytes radiating from central vein, portal triads present in between, polygonal hepatocytes with centrally rounded vesicular nuclei, and hepatic sinusoid in between (Figure 4A and B). Liver sections obtained from rats received DEN/2-AAF (Figure 4C-E) showed disruption of normal hepatic lobular architecture along with presence of large, discriminated dysplastic nodules compressing the surrounding liver tissue, with high-grade dysplastic cells displaying increased nuclear: Cytoplasmatic ratio (Figure 4E), nuclear hyperchromatosis, and basophilic cytoplasm. Liver sections of rats treated with different doses of cyanidin (10, 15, and 20 mg/kg) respectively, showed small and less discriminated dysplastic nodules (Figure 4F-H).\\n\\nPhotomicrographs of liver sections stained with H&E staining. A and B: Naïve group liver sections showed normal hepatic architecture, cords of hepatocytes radiating from central vein and portal triads present in-between and polygonal hepatocytes with central rounded vesicular nuclei, and hepatic sinusoid in-between; C-E: Liver sections of rats received diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF) showed larger, discriminated dysplastic nodules (doted shapes) compressing the surrounding liver tissue with disruption of normal hepatic lobular architecture; D: Liver sections of rats received DEN/2-AAF showed eosinophilic foci of cellular alteration consisting of enlarged hepatocytes with increased acidophilic staining and vaculated nuclei (arrow head); E: Liver sections of rats received DEN/2-AAF showed foci of Clear cell formed of hepatocytes showing variable degrees of cytoplasmic vacuolations and ballooning with pyknotic nuclei (arrow); F-H: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) respectively, showing small and less discriminated dysplastic nodules (doted shapes). (Magnification: A, C, F, G, H × 1000; B, D, E × 400). CV: Central vein; PV: Portal vein; Pt: Portal triads; S: Sinusoid; H: Hepatocytes.\\nImmunohistochemical examination with GSTP antibody appeared as large hyperplastic nodules were demonstrated by the brown color. Figure 5A and B shows the liver sections of naïve group, while Figure 5C shows large-positively stained GSTP foci demonstrated in PCL group. Liver sections from rats of all cyan-treated groups exhibited small GSTP-positive small hepatic foci of different sizes scattered in-between negatively-stained hepatic parenchyma (Figure 5D-F). The percent area of GSTP foci was significantly increased in the PCL group compared to naïve group, while all cyan-treated groups showed significant decrease in the GSTP percent area. Furthermore, cyan-15 and -20 showed significant decrease in the GSTP percent area over cyan-10 (Table 1). PCNA immunohistochemical analysis showed an elevated expression of PCNA in the group that received DEN/2-AAF as compared to naïve group (Figure 6A and B). Liver sections obtained from rats received cyan. in its three doses showed decrease in PCNA-positively stained nuclei (Figure 6C-E and Table 1).\\n\\nPhotomicrographs of liver sections of rats immunohistochemical stained with glutathione S-transferase placental antibody. A and B: Naive group; C: Precancerous lesion group showing multiple glutathione S-transferase placental (GSTP)-positive large hepatic nodules (brown stained nodules) occupying most of section; D-F: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) showing GSTP positive small hepatic foci (brown stained cells = arrow) of different size scattered in-between negatively stained hepatic parenchyma. (Magnification: × 40)\\n\\nPhotomicrographs of liver sections Immunohistochemical stained with proliferating cell nuclear antigen. A: Negative reaction of control group; B: Diethylnitrosamine + 100 mg 2-acetylaminofluorene group showing positive stained nuclei scattered all over the field; C-E: Liver sections of rats treated with different doses of cyamidine (10, 15, 20 mg/kg) respectively, show few positive hepatocytes sporadically distributed over the field (arrow). (Magnification × 100). \\nExpression rate of hepatocytes positive for glutathione S-transferase placental and proliferating cell nuclear antigen were calculated as number of positive field expression in 10 fields per rat liver tissue\\nValues are mean ± SD; number of animals = 6 rats/each group. Examined field; 10 fields/liver section. \\n\\nP < 0.05 significant differences compared to naïve group.\\n\\nP < 0.05 significant differences compared to precancerous lesion group.\\n\\nP < 0.05 significant compared to cyanidin-3-O-glucoside-10, One-way ANOVA followed by Tukey’s test. +: Positive expression found in 1-3 fields; ++: Positive expression found in 4-6 fields; +++: Positive expression found in 7-10 field; PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; GSTP: Glutathione S-transferase placental; PCNA: Proliferating cell nuclear antigen.\",\n \"As shown in Figure 1, following induction of PCL, rats exhibited a significant increase in AFP levels. AFP levels were significantly decreased in cyan-10, cyan-15, and cyan-20 groups compared to PCL group. There was no significant difference between the AFP levels of the three cyan groups. Liver function tests were affected after receiving DEN/2-AAF in which rats exhibited a significant increase in levels of ALT and total and direct bilirubin, and a significant decrease in the serum albumin (Figure 2). As shown in Figure 2A, rats received cyan. in different doses exhibited a significant decrease in serum ALT compared to PCL group. Cyan-15 group showed significant decrease in serum ALT compared to cyan-10 group. Moreover, cyan-20 group showed significant decrease in serum ALT compared to cyan-10 group. In As depicted in Figure 2B, compared to PCL group, the rats in the three cyan groups exhibited a significant decrease in serum total bilirubin. Furthermore, cyan-20 group showed significant decrease in serum total bilirubin compared to cyan-10 group. Figure 2C: Serum direct bilirubin was significantly decreased in the three cyan groups compared to the PCL group. Moreover, cyan-20 group showed significant decrease in serum direct bilirubin compared to cyan-10 group. Administration of cyan in doses 5, 10 and 20 mg/kg/d produced a significant increase in serum albumin compared to PCL group. However, there was no significant difference between the serum albumin levels of the three cyan groups (Figure 2D).\\n\\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) on alpha fetoprotein in the serum of rats. Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group, unpaired t test; bP < 0.05 significant differences compared to precancerous lesion group. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; AFP: Alpha fetoprotein.\\n\\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) in rats. A: Alanine aminotransferase; B: Total bilirubin; C: Direct bilirubin; D: Serum albumin. Values are mean ± SEM; number of animals = 6 rats/each group. bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; ALT: Alanine aminotransferase.\",\n \"As shown in Figure 3, induction of PCL resulted in significant increase of lncRNA MALAT a significant decrease in RQ miR-125b and significant increase in RQ TUBG1 expressions in comparison to naïve group. RQ of lncRNA MALAT1 in the liver tissue was significantly decreased in cyan -10,15 and cyan-20 groups compared to PCL group. Furthermore, cyan-20 group showed significant decrease in RQ of lncRNA MALAT1 in the liver tissue compared to cyan-10 group (Figure 3A). On the contrary, RQ of miR-125b in the liver tissue was significantly increased in cyan -10, cyan-15 and cyan-20 groups compared to PCL group. In addition, cyan-20 group showed significant increase in RQ of miR-125b in the liver tissue compared to cyan-10 group (Figure 3B). Furthermore, RQ of TUBG1 in the liver tissue was significantly decreased in cyan-10, cyan-15 and cyan-20 groups compared to PCL group. Also, cyan-20 group showed significant decrease in RQ of TUBG1 in the liver tissue compared to cyan-10 group (Figure 3C).\\n\\nEffect on expression of of long non-coding RNA, MALAT1, miR-125b and tubulin 1 (G2/M transition of mitotic cell cycle) in the liver tissue. A: RQ of long non-coding RNA MALAT1; B: RQ miR-125b; C: RQ TUBGI . Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group; bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; TUBG: Tubulin gamma 1; lncRNA: Long non-coding RNA.\",\n \"Light microscopic examination of H&E-stained sections of naïve rats’ liver showed normal hepatic architecture, cords of hepatocytes radiating from central vein, portal triads present in between, polygonal hepatocytes with centrally rounded vesicular nuclei, and hepatic sinusoid in between (Figure 4A and B). Liver sections obtained from rats received DEN/2-AAF (Figure 4C-E) showed disruption of normal hepatic lobular architecture along with presence of large, discriminated dysplastic nodules compressing the surrounding liver tissue, with high-grade dysplastic cells displaying increased nuclear: Cytoplasmatic ratio (Figure 4E), nuclear hyperchromatosis, and basophilic cytoplasm. Liver sections of rats treated with different doses of cyanidin (10, 15, and 20 mg/kg) respectively, showed small and less discriminated dysplastic nodules (Figure 4F-H).\\n\\nPhotomicrographs of liver sections stained with H&E staining. A and B: Naïve group liver sections showed normal hepatic architecture, cords of hepatocytes radiating from central vein and portal triads present in-between and polygonal hepatocytes with central rounded vesicular nuclei, and hepatic sinusoid in-between; C-E: Liver sections of rats received diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF) showed larger, discriminated dysplastic nodules (doted shapes) compressing the surrounding liver tissue with disruption of normal hepatic lobular architecture; D: Liver sections of rats received DEN/2-AAF showed eosinophilic foci of cellular alteration consisting of enlarged hepatocytes with increased acidophilic staining and vaculated nuclei (arrow head); E: Liver sections of rats received DEN/2-AAF showed foci of Clear cell formed of hepatocytes showing variable degrees of cytoplasmic vacuolations and ballooning with pyknotic nuclei (arrow); F-H: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) respectively, showing small and less discriminated dysplastic nodules (doted shapes). (Magnification: A, C, F, G, H × 1000; B, D, E × 400). CV: Central vein; PV: Portal vein; Pt: Portal triads; S: Sinusoid; H: Hepatocytes.\\nImmunohistochemical examination with GSTP antibody appeared as large hyperplastic nodules were demonstrated by the brown color. Figure 5A and B shows the liver sections of naïve group, while Figure 5C shows large-positively stained GSTP foci demonstrated in PCL group. Liver sections from rats of all cyan-treated groups exhibited small GSTP-positive small hepatic foci of different sizes scattered in-between negatively-stained hepatic parenchyma (Figure 5D-F). The percent area of GSTP foci was significantly increased in the PCL group compared to naïve group, while all cyan-treated groups showed significant decrease in the GSTP percent area. Furthermore, cyan-15 and -20 showed significant decrease in the GSTP percent area over cyan-10 (Table 1). PCNA immunohistochemical analysis showed an elevated expression of PCNA in the group that received DEN/2-AAF as compared to naïve group (Figure 6A and B). Liver sections obtained from rats received cyan. in its three doses showed decrease in PCNA-positively stained nuclei (Figure 6C-E and Table 1).\\n\\nPhotomicrographs of liver sections of rats immunohistochemical stained with glutathione S-transferase placental antibody. A and B: Naive group; C: Precancerous lesion group showing multiple glutathione S-transferase placental (GSTP)-positive large hepatic nodules (brown stained nodules) occupying most of section; D-F: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) showing GSTP positive small hepatic foci (brown stained cells = arrow) of different size scattered in-between negatively stained hepatic parenchyma. (Magnification: × 40)\\n\\nPhotomicrographs of liver sections Immunohistochemical stained with proliferating cell nuclear antigen. A: Negative reaction of control group; B: Diethylnitrosamine + 100 mg 2-acetylaminofluorene group showing positive stained nuclei scattered all over the field; C-E: Liver sections of rats treated with different doses of cyamidine (10, 15, 20 mg/kg) respectively, show few positive hepatocytes sporadically distributed over the field (arrow). (Magnification × 100). \\nExpression rate of hepatocytes positive for glutathione S-transferase placental and proliferating cell nuclear antigen were calculated as number of positive field expression in 10 fields per rat liver tissue\\nValues are mean ± SD; number of animals = 6 rats/each group. Examined field; 10 fields/liver section. \\n\\nP < 0.05 significant differences compared to naïve group.\\n\\nP < 0.05 significant differences compared to precancerous lesion group.\\n\\nP < 0.05 significant compared to cyanidin-3-O-glucoside-10, One-way ANOVA followed by Tukey’s test. +: Positive expression found in 1-3 fields; ++: Positive expression found in 4-6 fields; +++: Positive expression found in 7-10 field; PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; GSTP: Glutathione S-transferase placental; PCNA: Proliferating cell nuclear antigen.\",\n \"Altered expression of mitotic spindle assembly genes has been detected in many cancers. For example, MAD2 gene levels are downregulated in breast carcinoma[18], while mitotic checkpoint serine/BUB1 gene expression is dysregulated in colorectal carcinoma[19]. The impairment of spindle assembly genes are frequently detected in HCC[20,21]. Moreover, inhibition of the mitotic assembly genes has been found to be lethal to cancer cells, and has promising therapeutic effect in cancer treatment[22,23]. \\nIn this study, we used several public microarray databases for the construction of simple genetic-epigenetic network specific to HCC and linked to cell cycle and mitotic spindle formation. We assessed the antiproliferative effect of cyan in HCC animal model via modulation of lncRNA-MALAT1-miR-125b-TUBG1 mRNA axis expression.\\nMicrotubules are involved both in maintenance of cell shape and motility and in mitotic spindle formation[24]. γ-TUBG is a major player that supports microtubule nucleation. γ-TUBG acts as a binding site for the α/β-tubulin dimer and it has two γ-TUBG genes, TUBG1 and TUBG2 in mammals only[25]. The TUBG levels in the centrosome change during cell cycle progression and usually increases at the start of mitosis. At the end of mitosis, TUBG levels are decreased to interphase levels[26]. Overexpression and altered compartmentalization of γ-TUBG may lead to carcinogenesis[27-29]. Interestingly, γ-TUBG associates interact with tumor suppressor protein C53 in mammalian interphase cells of different cellular origins[30]. Interestingly, γ-TUBG interacts with E2F transcription factors, resulting in upregulation of E2F target genes PCNA and CDKB1[31] and glutathione S transferase activity[32]. On the other hand, the mRNA and protein levels of TUBA1C are upregulated in HCC. Clinically, it affects the survival of patients and the level of metastatic burden along with portal vein thrombosis. In addition, it activates cellular proliferation and migration and is a potential therapeutic target for HCC[33]. Recent studies have shed light on the efficacy of microtubule-binding agents in the treatment of HCC, especially in combination with mammalian target of rapamycin inhibitors[34].\\nOne of the few miRNAs that regulate Spindle Assembly Checkpoints (SAC) is miR-125b. It suppresses the expression of Mad1 (a core SAC protein) that is responsible for inhibiting entry into anaphase till metaphase defects are corrected[35]. Importantly, miRNA-125b has been found to be associated with colorectal cancer[36], multiple myeloma[37], and breast cancer[38]. Song et al[39] demonstrated that the expression of miR-125b in hepatocytes was protective and reflected better survival in HCC tissue samples due to restoration of SIRT6 expression[39]. miR-125b-5p inhibits HCC proliferation by targeting TXNRD1, the terminal step of biosynthesis of nucleotides[40].\\nDeregulation of lncRNAs has been shown to be crucial in cancer progression, especially HCC[41,42]. Hung et al[43] demonstrated the involvement of lncRNAs in regulation of the expression of cell cycle-related genes, p53 gene regulatory pathway, and apoptosis[43,44]. The lncRNA MALAT1 is overexpressed in several solid tumors and is linked with tumor recurrence[45-47]. MALAT1 coordinate RNA polymerase II transcription, pre-mRNA splicing, and mRNA export[48]. Thus, this lncRNA holds great potential in influencing the local concentration of a specific splicing factor during specific stages of the cell cycle. Depletion of MALAT1 in cancer cells inhibits tumorigenicity[49]. MALAT1 regulates E2F1 transcription factor activity, highlighting its pro-proliferative function[50]. MALAT1 is upregulated in many cancer types, including breast cancer[51], cervical cancer, lung cancer[52], and hepatocarcinoma[53-55]. In a previous study, hypoxia enhanced MALAT1 expression, leading to increase in proliferating and invading activity of Hep3B cells due to negative interaction with miR200a[56]. Huang and his group explored the role of specificity protein 1/3 in regulation of MALAT1 expression in HCC cells[57].\\nCyanidin or anthocyanin (a polyphenolic pigment found in plants) confers several pharmacological benefits, including anticancer properties[58-62]. Cyan mediates cytotoxicity against human monocytic leukemia cells via G2/M phase arrest and induction of apoptosis[63]. Tsai et al[64] evaluated anthocyanins as promising anticancer or chemopreventive agents to induce G2/M arrest in HAs-induced leukemia cell cycle arrest via ATM-Chk1/2-Cdc25C axis modulation[64]. They also found that anthocyanin inhibits the proliferation of HCC cells[65,66].\\nIn our study, the effect of cyan on cancerous tissue was dose-dependent (10, 15 and 20 mg/kg/d), that is, increasing its effect with increasing the concentration of the drug. This was proved also, with the correlated significant decrease in the percent area of GSTP foci, PCNA expression which are the target of TUBG1 mRNA and the molecular epigenetic markers associated with HCC (lncRNA MALAT1 and miR-125b-1-3p) in the liver tissue of rats in our study (Figure 7). Thus, we can hypothesize that administration of cyan in PCL may result in downregulation of lncRNA MALAT1 with subsequent release of free miR-125b-1-3p that binds to TUBG1 mRNA and downregulate its expression[65]. The changes in the epigenetic-genetic network affect their target genes PCNA and GSTP.\\n\\nConcept map of study design. GSTP: Glutathione S-transferase placental; PCNA: Proliferating cell nuclear antigen; TUBG: Tubulin; lncRNA: Long non-coding RNA.\\nLastly, the cyan-mediated modulation of cell cycle and mitotic spindle assembly that regulate HCC proliferation might affect the capability of the HCC cells to overcome oxidative stress[66,67]. Our data confers the rationale for designing antimitotic approach combining the conventional cytotoxic drugs with phytochemical extracts to inhibit cell cycle progression in cancer. More in vitro functional studies are required to explore the functional mechanism of the chosen RNA panel and validate their role as drug target biomarkers. \",\n \"Cyanidin is a natural molecule that holds great potential in unraveling the mystery of cytotoxic pharmacy in the future. It can be used to devise novel antimitotic drugs that can target cell cycle in HCC. Our results also indicated great potential of the TUBG1 mRNA- miR-125b-lncRNA MALAT1 and -1-3p axis as potential drug target biomarker for further investigations.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"keywords":{"kind":"list like","value":["Hepatocellular carcinoma therapy","Hepatocellular-carcinoma growth","Hepatocellular-carcinoma model","Hepatocellular-carcinoma size"],"string":"[\n \"Hepatocellular carcinoma therapy\",\n \"Hepatocellular-carcinoma growth\",\n \"Hepatocellular-carcinoma model\",\n \"Hepatocellular-carcinoma size\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nHepatocellular carcinoma (HCC) is the commonest primary malignancy of the liver. Its main risk factors include hepatitis B, hepatitis C, and non-alcoholic steatohepatitis. HCC is associated with high mortality and morbidity. Its incidence has increased from 1.4 to 6.2 per 100000 cases per year within the last 30 years[1]. HCC burden in Egypt is high due to the high prevalence of HCV, where liver cancers account for 11.75% of all gastrointestinal cancers and 1.68% of all malignancies. In addition, over 70% of all liver malignancies in Egypt are HCC[2].\nTo date, HCC is holding its place as an unresolved dilemma; its resistance to systemic chemotherapy and radiation makes the cure approachable only in very early stages according to Milan criteria, that is, where the tumor size is < 5 cm (when only one lesion is present) or < 3 cm (when 2-3 lesions are present)[3].\nGenes involved in cell cycle control are usually mutated in presence of cancer. Dysregulated mitosis results in genomic instability, which leads to tumor aggressiveness[4]. Microtubules are a key component of mitotic spindles, and thus, a crucial part of the process of mitosis. They form an important target in cancer chemotherapy, inducing arrest of mitosis and cell death. On the other hand, while the overexpression of γ-tubulin (γ-TUBG) showed improved survival among small cell lung cancer patients[5], the overexpression of γ-TUBG is found to be characteristic feature of thyroid and breast cancers[6]. Such variation may arise due to differences in gene transcription and check point regulations in different cancer tissues[7].\nCyanidin-3-glucoside (cyan) is a potential chemotherapeutic and chemo-protective agent. Through its antioxidant activity, it scavenges radical oxygen species, which decreases the overall number of tumorous cells (malignant and benign) in in vivo studies[8]. Cyan has been implicated in some beneficial health activities[9], including reducing age-associated oxidative stress[10], improving cognitive brain function, and exhibiting anti-diabetic[11], anti-inflammation[12], anti-atherogenic[13], and anti-obesity activities[14]. Previously, it has been reported that cyan mediates caspase-3 cleavage with DNA fragmentation. Cyan has also been associated with induction of autophagy, a key element involved in cancer elimination, via induction of autophagy-related gene 5 and microtubule-associated protein 1 light chain 3-II[15]. Cyan has drawn increasing attention because of its potential anti-cancer properties. Cyan may offer a novel avenue for treating HCC. a potential antiproliferative effect on HepG2 cells[16].\nThe present study was conducted to evaluate the effect of cyan in three different doses on cell cycle progression and mitotic assembly disorder in hepatic precancerous lesion (PCL) via assessment of levels of long non-coding RNA (lncRNA) MALAT1, miR-125b, and tubulin 1. We also conducted histopathological and immuno-histochemical examination using H&E staining assessment of levels of glutathione S-transferase placental (GSTP) and proliferating cell nuclear antigen (PCNA) antibodies in male Wistar rat model of diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF)-induced hepatic PCL. \n\nMATERIALS AND METHODS:\nChemicals and drugs DEN was obtained as 1 g solution in serum bottle diluted in 0.9% NaCl. 2-AAF was obtained as white powder, while cyan was obtained as black powder. Both 2-AAF and cyan were dissolved in 0.9% NaCl. All chemicals were purchased from Sigma-Aldrich, Cairo, Egypt.\nDEN was obtained as 1 g solution in serum bottle diluted in 0.9% NaCl. 2-AAF was obtained as white powder, while cyan was obtained as black powder. Both 2-AAF and cyan were dissolved in 0.9% NaCl. All chemicals were purchased from Sigma-Aldrich, Cairo, Egypt.\nExperimental animals All animal procedures were approved by the Institutional Animal Ethics Committee of Ain Shams University, Faculty of Medicine. Thirty male Wistar rats weighing 200-250 g were purchased from Nile Pharmaceuticals Company (Cairo, Egypt). Animals were housed in an animal room with temperature 25 ± 2 °C and 12 h light/dark cycle controls. An adaptation period of 1 wk was allowed before initiation of the experimental protocol.\nAll animal procedures were approved by the Institutional Animal Ethics Committee of Ain Shams University, Faculty of Medicine. Thirty male Wistar rats weighing 200-250 g were purchased from Nile Pharmaceuticals Company (Cairo, Egypt). Animals were housed in an animal room with temperature 25 ± 2 °C and 12 h light/dark cycle controls. An adaptation period of 1 wk was allowed before initiation of the experimental protocol.\nExperimental procedures \nInduction of hepatic PCL: DEN was injected intraperitoneally (i.p.) at a dose of 100 mg/kg once weekly for three consecutive weeks. One week after the last DEN injection, a single dose (300 mg/kg) of 2-AAF was injected.\n\nAnimal groups: Rats were divided into five groups (6 rats/each group), including: Naïve group: Rats were injected with a vehicle (0.9% NaCl), PCL group: Rats were injected with DEN and 2-AAF, and three cyan groups (cyan-10, cyan-15, and cyan-20 groups): Rats were injected with DEN and 2-AAF, and orally administered through gastric gavage with cyan in doses of 10, 15, and 20 mg/kg respectively, for four consecutive days per week for 16 wk[17].\n\nInduction of hepatic PCL: DEN was injected intraperitoneally (i.p.) at a dose of 100 mg/kg once weekly for three consecutive weeks. One week after the last DEN injection, a single dose (300 mg/kg) of 2-AAF was injected.\n\nAnimal groups: Rats were divided into five groups (6 rats/each group), including: Naïve group: Rats were injected with a vehicle (0.9% NaCl), PCL group: Rats were injected with DEN and 2-AAF, and three cyan groups (cyan-10, cyan-15, and cyan-20 groups): Rats were injected with DEN and 2-AAF, and orally administered through gastric gavage with cyan in doses of 10, 15, and 20 mg/kg respectively, for four consecutive days per week for 16 wk[17].\nAssessment of the effects of treatment \nBiochemical and molecular studies: After scarifying the animals, blood was withdrawn from each rat from the retro orbital vein and incubated for about half an hour for clotting. Then, the blood sample was centrifuged for 20 min at 5000 RPM to get serum samples. Livers were dissected. The right lobe of the livers was removed, cut into longitudinal sections 2-4 mm in thickness and kept in 10% formalin for histopathological and immunohistochemical examination. The other lobe was kept frozen at -80 °C for molecular analyses. The samples were analyzed for the following parameters: (1) alpha fetoprotein (AFP) levels: AFP level was analyzed using quantitative sandwich rat AFP ELISA kit purchased from MyBiosource Inc. (San Diego, United States) with a sensitivity range of 0.625-20 ng/mL; (2) serum alanine aminotransferase (ALT) levels: The serum ALT was measured according to method described by using kits purchased from Diamond Diagnostic (Cairo, Egypt). ALT catalyzes the transfer of amino groups from specific amino acids to ketoglutaric acid, yielding glutamic acid and oxaloacetic or pyruvic acid, respectively. Ketoacids were then determined calorimetrically after their reaction with 2, 4- dinitrophenylhydrazine; and (3) determination of serum total bilirubin and direct bilirubin: Alkaline methanolysis of bilirubin followed by chloroform extraction of bilirubin methyl esters, and later, separation of these esters by chromatography and spectrophotometric determination at 430 nm.\n\nBiochemical and molecular studies: After scarifying the animals, blood was withdrawn from each rat from the retro orbital vein and incubated for about half an hour for clotting. Then, the blood sample was centrifuged for 20 min at 5000 RPM to get serum samples. Livers were dissected. The right lobe of the livers was removed, cut into longitudinal sections 2-4 mm in thickness and kept in 10% formalin for histopathological and immunohistochemical examination. The other lobe was kept frozen at -80 °C for molecular analyses. The samples were analyzed for the following parameters: (1) alpha fetoprotein (AFP) levels: AFP level was analyzed using quantitative sandwich rat AFP ELISA kit purchased from MyBiosource Inc. (San Diego, United States) with a sensitivity range of 0.625-20 ng/mL; (2) serum alanine aminotransferase (ALT) levels: The serum ALT was measured according to method described by using kits purchased from Diamond Diagnostic (Cairo, Egypt). ALT catalyzes the transfer of amino groups from specific amino acids to ketoglutaric acid, yielding glutamic acid and oxaloacetic or pyruvic acid, respectively. Ketoacids were then determined calorimetrically after their reaction with 2, 4- dinitrophenylhydrazine; and (3) determination of serum total bilirubin and direct bilirubin: Alkaline methanolysis of bilirubin followed by chloroform extraction of bilirubin methyl esters, and later, separation of these esters by chromatography and spectrophotometric determination at 430 nm.\nDetermination of serum albumin Quantitative determination of serum albumin level was quantified using rat albumin ELISA kit. The absorbance at 450 nm is a measure of the concentration of albumin in the test sample.\nQuantitative determination of serum albumin level was quantified using rat albumin ELISA kit. The absorbance at 450 nm is a measure of the concentration of albumin in the test sample.\nBioinformatics-based selection of cell cycle-microtubule assembly- and HCC-specific RNA panel The RNA panel was chosen in three steps: (1) Tubulin, gamma 1 (TUBG1) was retrieved as a crucial player in microtubule formation and progression of the cell cycle through gene atlas expression database (available at https://www.ebi.ac.uk/gxa/home) alongside with verification of the chosen gene from protein Atlas database (available at https://www.proteinatlas.org/), followed by analysis of TUBG1 gene ontology to ensure that it is linked to G2-M transition and mitotic spindle assembly, which is closely linked to cancer development through gene card database (available at https://www.genecards.org/); (2) we selected miR-125b-1-3p targeting TUBG1 mRNA though) miRWALK database available at http://mirwalk.umm.uni-heidelberg.de/), followed by pathway enrichment, which revealed that miR-125b-1-3p is linked to MAP kinase and ubiquitin-mediated proteolysis (strongly correlated with mitotic assembly and cell cycle progression) through (Diana database available at http://diana.imis.athena-innovation.gr/DianaTools/index.php); and (3) lncRNA metastasis-associated lung adenocarcinoma transcript 1, lncRNA MALAT1, was obtained as a lncRNA specific to HCC and targeting miR-125b-1-3p using noncode database available at http://www.noncode.org/ and European Bioinformatics institute database available at https://www.ebi.ac.uk.\nThe RNA panel was chosen in three steps: (1) Tubulin, gamma 1 (TUBG1) was retrieved as a crucial player in microtubule formation and progression of the cell cycle through gene atlas expression database (available at https://www.ebi.ac.uk/gxa/home) alongside with verification of the chosen gene from protein Atlas database (available at https://www.proteinatlas.org/), followed by analysis of TUBG1 gene ontology to ensure that it is linked to G2-M transition and mitotic spindle assembly, which is closely linked to cancer development through gene card database (available at https://www.genecards.org/); (2) we selected miR-125b-1-3p targeting TUBG1 mRNA though) miRWALK database available at http://mirwalk.umm.uni-heidelberg.de/), followed by pathway enrichment, which revealed that miR-125b-1-3p is linked to MAP kinase and ubiquitin-mediated proteolysis (strongly correlated with mitotic assembly and cell cycle progression) through (Diana database available at http://diana.imis.athena-innovation.gr/DianaTools/index.php); and (3) lncRNA metastasis-associated lung adenocarcinoma transcript 1, lncRNA MALAT1, was obtained as a lncRNA specific to HCC and targeting miR-125b-1-3p using noncode database available at http://www.noncode.org/ and European Bioinformatics institute database available at https://www.ebi.ac.uk.\nExtraction of total RNA, including lncRNA and miRNA from liver tissue Total RNA was extracted from liver tissues using miRNeasy Mini kit (Cat No. 217004, Qiagen, United States) according to manufacturer’s protocol. The extracted RNA concentration and integrity were assessed using [(NanoDrop Technologies/Thermo Scientific, Wilmington, DE, United States)], with RNA purities were 1.8-2. The extracted total RNA was reverse transcribed into cDNA using a miScript II RT kit (Cat No. 218160, 218161, Qiagen, United States) on thermal cycler (Bio-Rad; Hercules, CA, United States), according to the manufacturer’s protocol. \nTotal RNA was extracted from liver tissues using miRNeasy Mini kit (Cat No. 217004, Qiagen, United States) according to manufacturer’s protocol. The extracted RNA concentration and integrity were assessed using [(NanoDrop Technologies/Thermo Scientific, Wilmington, DE, United States)], with RNA purities were 1.8-2. The extracted total RNA was reverse transcribed into cDNA using a miScript II RT kit (Cat No. 218160, 218161, Qiagen, United States) on thermal cycler (Bio-Rad; Hercules, CA, United States), according to the manufacturer’s protocol. \nReal time-polymerase chain reaction quantification of the RNA panel in liver tissues The relative expression of TUBG1 mRNA and lncRNA-MALAT1 in liver tissue were measured using a Quantitect SYBR Green Master Mix kit and an RT2 SYBR Green ROX real time-polymerase chain reaction (qPCR) Mastermix (Qiagen), respectively, using a 7500 qPCR Systems (Applied Biosystems, Foster City, CA, United States) detection system) and specific primers (Accession: NM_001128148, NM_003234, NR_002819, and ENST00000534336, respectively) supplied by Qiagen. GAPDH (Accession NM_002046.7) was used as a housekeeping gene. MiR-3163 expression in liver tissue was quantified by PCR using a miScript SYBR Green kit (Qiagen), a miScript universal primer, and a miRNA-specific forward primer (mir-125b-1-3p miScript Primer Assay) (Accession: MIMAT0004592 (5'-ACGGGUUAGGCUCUUGGGAGCU). All steps followed the manufacturer’s suggested protocol, and SNORD68 was used as an internal control. Ct values more than 36 were considered as negative. The specificities of the amplicons for the SYBR Green−based PCR amplification were affirmed by the melting curves. The 2−ΔΔCt technique was used to measure the relative expression of the HCC-specific RNA panel.\nThe relative expression of TUBG1 mRNA and lncRNA-MALAT1 in liver tissue were measured using a Quantitect SYBR Green Master Mix kit and an RT2 SYBR Green ROX real time-polymerase chain reaction (qPCR) Mastermix (Qiagen), respectively, using a 7500 qPCR Systems (Applied Biosystems, Foster City, CA, United States) detection system) and specific primers (Accession: NM_001128148, NM_003234, NR_002819, and ENST00000534336, respectively) supplied by Qiagen. GAPDH (Accession NM_002046.7) was used as a housekeeping gene. MiR-3163 expression in liver tissue was quantified by PCR using a miScript SYBR Green kit (Qiagen), a miScript universal primer, and a miRNA-specific forward primer (mir-125b-1-3p miScript Primer Assay) (Accession: MIMAT0004592 (5'-ACGGGUUAGGCUCUUGGGAGCU). All steps followed the manufacturer’s suggested protocol, and SNORD68 was used as an internal control. Ct values more than 36 were considered as negative. The specificities of the amplicons for the SYBR Green−based PCR amplification were affirmed by the melting curves. The 2−ΔΔCt technique was used to measure the relative expression of the HCC-specific RNA panel.\nHistopathological and immunohistochemical studies Liver sections were cut at 5-μm thickness and stained with H&E. Immunohistochemistry S-P method was used to detect GSTP and PCNA levels. Briefly, the protocol was as follows: (1) the tissues were treated with endogenous peroxidase blocking solution at room temperature for 10 min, and then, incubated in normal nonimmune serum at room temperature for 10 min; (2) mouse anti GSTP or PCNA antibody was added to adjacent tissue sections respectively and incubated overnight at 4 °C; (3) biotin-conjugated second antibody was added to the sections and incubated at room temperature for 10 min; and (4) S-P complex was added at room temperature for 10 min, and then, 2,4-diaminobutyric acid was used for the color reaction. The tissue sections were washed with Poly (butylene succinate) (PBS) (0.01 mol/L, pH 7.4) between each step. Positive and negative controls were simultaneously used to ensure specificity and reliability of the staining process. A positive section was taken as positive control. In negative control, PBS was used to replace the first antibody. GSTP-positivity was indicated by brown coloration of the cytoplasm. Morphometric analysis of the foci percent area was carried out using Leica Q win V.3 software after capturing the images using a Leica DM2500 microscope (Leica, Wetzlar, Germany). The PCNA labeling indices are represented as the expression of positively-stained nuclei (10 fields/slide at 400 ×) as shown below: +, positive expression found in 1-3 fields; ++, positive expression found in 4-6 fields; and +++, positive expression found in 7-10 fields. Rabbit monoclonal PCNA and GSTP antibodies were purchased from Abcam (San Francisco, United States).\nLiver sections were cut at 5-μm thickness and stained with H&E. Immunohistochemistry S-P method was used to detect GSTP and PCNA levels. Briefly, the protocol was as follows: (1) the tissues were treated with endogenous peroxidase blocking solution at room temperature for 10 min, and then, incubated in normal nonimmune serum at room temperature for 10 min; (2) mouse anti GSTP or PCNA antibody was added to adjacent tissue sections respectively and incubated overnight at 4 °C; (3) biotin-conjugated second antibody was added to the sections and incubated at room temperature for 10 min; and (4) S-P complex was added at room temperature for 10 min, and then, 2,4-diaminobutyric acid was used for the color reaction. The tissue sections were washed with Poly (butylene succinate) (PBS) (0.01 mol/L, pH 7.4) between each step. Positive and negative controls were simultaneously used to ensure specificity and reliability of the staining process. A positive section was taken as positive control. In negative control, PBS was used to replace the first antibody. GSTP-positivity was indicated by brown coloration of the cytoplasm. Morphometric analysis of the foci percent area was carried out using Leica Q win V.3 software after capturing the images using a Leica DM2500 microscope (Leica, Wetzlar, Germany). The PCNA labeling indices are represented as the expression of positively-stained nuclei (10 fields/slide at 400 ×) as shown below: +, positive expression found in 1-3 fields; ++, positive expression found in 4-6 fields; and +++, positive expression found in 7-10 fields. Rabbit monoclonal PCNA and GSTP antibodies were purchased from Abcam (San Francisco, United States).\nStatistical analysis All results were expressed as mean ± SD. Statistical analysis was carried out using GraphPad Prism version 6.01. Unpaired t-test was done to compare between naïve and PCL groups. One-way ANOVA, followed by Tukey’s test, was carried out to compare between all groups. P values < 0.05 were considered statistically significant. \nAll results were expressed as mean ± SD. Statistical analysis was carried out using GraphPad Prism version 6.01. Unpaired t-test was done to compare between naïve and PCL groups. One-way ANOVA, followed by Tukey’s test, was carried out to compare between all groups. P values < 0.05 were considered statistically significant. \n\nChemicals and drugs :\nDEN was obtained as 1 g solution in serum bottle diluted in 0.9% NaCl. 2-AAF was obtained as white powder, while cyan was obtained as black powder. Both 2-AAF and cyan were dissolved in 0.9% NaCl. All chemicals were purchased from Sigma-Aldrich, Cairo, Egypt.\n\nExperimental animals:\nAll animal procedures were approved by the Institutional Animal Ethics Committee of Ain Shams University, Faculty of Medicine. Thirty male Wistar rats weighing 200-250 g were purchased from Nile Pharmaceuticals Company (Cairo, Egypt). Animals were housed in an animal room with temperature 25 ± 2 °C and 12 h light/dark cycle controls. An adaptation period of 1 wk was allowed before initiation of the experimental protocol.\n\nExperimental procedures:\n\nInduction of hepatic PCL: DEN was injected intraperitoneally (i.p.) at a dose of 100 mg/kg once weekly for three consecutive weeks. One week after the last DEN injection, a single dose (300 mg/kg) of 2-AAF was injected.\n\nAnimal groups: Rats were divided into five groups (6 rats/each group), including: Naïve group: Rats were injected with a vehicle (0.9% NaCl), PCL group: Rats were injected with DEN and 2-AAF, and three cyan groups (cyan-10, cyan-15, and cyan-20 groups): Rats were injected with DEN and 2-AAF, and orally administered through gastric gavage with cyan in doses of 10, 15, and 20 mg/kg respectively, for four consecutive days per week for 16 wk[17].\n\nAssessment of the effects of treatment:\n\nBiochemical and molecular studies: After scarifying the animals, blood was withdrawn from each rat from the retro orbital vein and incubated for about half an hour for clotting. Then, the blood sample was centrifuged for 20 min at 5000 RPM to get serum samples. Livers were dissected. The right lobe of the livers was removed, cut into longitudinal sections 2-4 mm in thickness and kept in 10% formalin for histopathological and immunohistochemical examination. The other lobe was kept frozen at -80 °C for molecular analyses. The samples were analyzed for the following parameters: (1) alpha fetoprotein (AFP) levels: AFP level was analyzed using quantitative sandwich rat AFP ELISA kit purchased from MyBiosource Inc. (San Diego, United States) with a sensitivity range of 0.625-20 ng/mL; (2) serum alanine aminotransferase (ALT) levels: The serum ALT was measured according to method described by using kits purchased from Diamond Diagnostic (Cairo, Egypt). ALT catalyzes the transfer of amino groups from specific amino acids to ketoglutaric acid, yielding glutamic acid and oxaloacetic or pyruvic acid, respectively. Ketoacids were then determined calorimetrically after their reaction with 2, 4- dinitrophenylhydrazine; and (3) determination of serum total bilirubin and direct bilirubin: Alkaline methanolysis of bilirubin followed by chloroform extraction of bilirubin methyl esters, and later, separation of these esters by chromatography and spectrophotometric determination at 430 nm.\n\nDetermination of serum albumin:\nQuantitative determination of serum albumin level was quantified using rat albumin ELISA kit. The absorbance at 450 nm is a measure of the concentration of albumin in the test sample.\n\nBioinformatics-based selection of cell cycle-microtubule assembly- and HCC-specific RNA panel :\nThe RNA panel was chosen in three steps: (1) Tubulin, gamma 1 (TUBG1) was retrieved as a crucial player in microtubule formation and progression of the cell cycle through gene atlas expression database (available at https://www.ebi.ac.uk/gxa/home) alongside with verification of the chosen gene from protein Atlas database (available at https://www.proteinatlas.org/), followed by analysis of TUBG1 gene ontology to ensure that it is linked to G2-M transition and mitotic spindle assembly, which is closely linked to cancer development through gene card database (available at https://www.genecards.org/); (2) we selected miR-125b-1-3p targeting TUBG1 mRNA though) miRWALK database available at http://mirwalk.umm.uni-heidelberg.de/), followed by pathway enrichment, which revealed that miR-125b-1-3p is linked to MAP kinase and ubiquitin-mediated proteolysis (strongly correlated with mitotic assembly and cell cycle progression) through (Diana database available at http://diana.imis.athena-innovation.gr/DianaTools/index.php); and (3) lncRNA metastasis-associated lung adenocarcinoma transcript 1, lncRNA MALAT1, was obtained as a lncRNA specific to HCC and targeting miR-125b-1-3p using noncode database available at http://www.noncode.org/ and European Bioinformatics institute database available at https://www.ebi.ac.uk.\n\nExtraction of total RNA, including lncRNA and miRNA from liver tissue:\nTotal RNA was extracted from liver tissues using miRNeasy Mini kit (Cat No. 217004, Qiagen, United States) according to manufacturer’s protocol. The extracted RNA concentration and integrity were assessed using [(NanoDrop Technologies/Thermo Scientific, Wilmington, DE, United States)], with RNA purities were 1.8-2. The extracted total RNA was reverse transcribed into cDNA using a miScript II RT kit (Cat No. 218160, 218161, Qiagen, United States) on thermal cycler (Bio-Rad; Hercules, CA, United States), according to the manufacturer’s protocol. \n\nReal time-polymerase chain reaction quantification of the RNA panel in liver tissues:\nThe relative expression of TUBG1 mRNA and lncRNA-MALAT1 in liver tissue were measured using a Quantitect SYBR Green Master Mix kit and an RT2 SYBR Green ROX real time-polymerase chain reaction (qPCR) Mastermix (Qiagen), respectively, using a 7500 qPCR Systems (Applied Biosystems, Foster City, CA, United States) detection system) and specific primers (Accession: NM_001128148, NM_003234, NR_002819, and ENST00000534336, respectively) supplied by Qiagen. GAPDH (Accession NM_002046.7) was used as a housekeeping gene. MiR-3163 expression in liver tissue was quantified by PCR using a miScript SYBR Green kit (Qiagen), a miScript universal primer, and a miRNA-specific forward primer (mir-125b-1-3p miScript Primer Assay) (Accession: MIMAT0004592 (5'-ACGGGUUAGGCUCUUGGGAGCU). All steps followed the manufacturer’s suggested protocol, and SNORD68 was used as an internal control. Ct values more than 36 were considered as negative. The specificities of the amplicons for the SYBR Green−based PCR amplification were affirmed by the melting curves. The 2−ΔΔCt technique was used to measure the relative expression of the HCC-specific RNA panel.\n\nHistopathological and immunohistochemical studies:\nLiver sections were cut at 5-μm thickness and stained with H&E. Immunohistochemistry S-P method was used to detect GSTP and PCNA levels. Briefly, the protocol was as follows: (1) the tissues were treated with endogenous peroxidase blocking solution at room temperature for 10 min, and then, incubated in normal nonimmune serum at room temperature for 10 min; (2) mouse anti GSTP or PCNA antibody was added to adjacent tissue sections respectively and incubated overnight at 4 °C; (3) biotin-conjugated second antibody was added to the sections and incubated at room temperature for 10 min; and (4) S-P complex was added at room temperature for 10 min, and then, 2,4-diaminobutyric acid was used for the color reaction. The tissue sections were washed with Poly (butylene succinate) (PBS) (0.01 mol/L, pH 7.4) between each step. Positive and negative controls were simultaneously used to ensure specificity and reliability of the staining process. A positive section was taken as positive control. In negative control, PBS was used to replace the first antibody. GSTP-positivity was indicated by brown coloration of the cytoplasm. Morphometric analysis of the foci percent area was carried out using Leica Q win V.3 software after capturing the images using a Leica DM2500 microscope (Leica, Wetzlar, Germany). The PCNA labeling indices are represented as the expression of positively-stained nuclei (10 fields/slide at 400 ×) as shown below: +, positive expression found in 1-3 fields; ++, positive expression found in 4-6 fields; and +++, positive expression found in 7-10 fields. Rabbit monoclonal PCNA and GSTP antibodies were purchased from Abcam (San Francisco, United States).\n\nStatistical analysis:\nAll results were expressed as mean ± SD. Statistical analysis was carried out using GraphPad Prism version 6.01. Unpaired t-test was done to compare between naïve and PCL groups. One-way ANOVA, followed by Tukey’s test, was carried out to compare between all groups. P values < 0.05 were considered statistically significant. \n\nRESULTS:\nEffect on AFP levels and liver function tests As shown in Figure 1, following induction of PCL, rats exhibited a significant increase in AFP levels. AFP levels were significantly decreased in cyan-10, cyan-15, and cyan-20 groups compared to PCL group. There was no significant difference between the AFP levels of the three cyan groups. Liver function tests were affected after receiving DEN/2-AAF in which rats exhibited a significant increase in levels of ALT and total and direct bilirubin, and a significant decrease in the serum albumin (Figure 2). As shown in Figure 2A, rats received cyan. in different doses exhibited a significant decrease in serum ALT compared to PCL group. Cyan-15 group showed significant decrease in serum ALT compared to cyan-10 group. Moreover, cyan-20 group showed significant decrease in serum ALT compared to cyan-10 group. In As depicted in Figure 2B, compared to PCL group, the rats in the three cyan groups exhibited a significant decrease in serum total bilirubin. Furthermore, cyan-20 group showed significant decrease in serum total bilirubin compared to cyan-10 group. Figure 2C: Serum direct bilirubin was significantly decreased in the three cyan groups compared to the PCL group. Moreover, cyan-20 group showed significant decrease in serum direct bilirubin compared to cyan-10 group. Administration of cyan in doses 5, 10 and 20 mg/kg/d produced a significant increase in serum albumin compared to PCL group. However, there was no significant difference between the serum albumin levels of the three cyan groups (Figure 2D).\n\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) on alpha fetoprotein in the serum of rats. Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group, unpaired t test; bP < 0.05 significant differences compared to precancerous lesion group. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; AFP: Alpha fetoprotein.\n\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) in rats. A: Alanine aminotransferase; B: Total bilirubin; C: Direct bilirubin; D: Serum albumin. Values are mean ± SEM; number of animals = 6 rats/each group. bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; ALT: Alanine aminotransferase.\nAs shown in Figure 1, following induction of PCL, rats exhibited a significant increase in AFP levels. AFP levels were significantly decreased in cyan-10, cyan-15, and cyan-20 groups compared to PCL group. There was no significant difference between the AFP levels of the three cyan groups. Liver function tests were affected after receiving DEN/2-AAF in which rats exhibited a significant increase in levels of ALT and total and direct bilirubin, and a significant decrease in the serum albumin (Figure 2). As shown in Figure 2A, rats received cyan. in different doses exhibited a significant decrease in serum ALT compared to PCL group. Cyan-15 group showed significant decrease in serum ALT compared to cyan-10 group. Moreover, cyan-20 group showed significant decrease in serum ALT compared to cyan-10 group. In As depicted in Figure 2B, compared to PCL group, the rats in the three cyan groups exhibited a significant decrease in serum total bilirubin. Furthermore, cyan-20 group showed significant decrease in serum total bilirubin compared to cyan-10 group. Figure 2C: Serum direct bilirubin was significantly decreased in the three cyan groups compared to the PCL group. Moreover, cyan-20 group showed significant decrease in serum direct bilirubin compared to cyan-10 group. Administration of cyan in doses 5, 10 and 20 mg/kg/d produced a significant increase in serum albumin compared to PCL group. However, there was no significant difference between the serum albumin levels of the three cyan groups (Figure 2D).\n\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) on alpha fetoprotein in the serum of rats. Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group, unpaired t test; bP < 0.05 significant differences compared to precancerous lesion group. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; AFP: Alpha fetoprotein.\n\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) in rats. A: Alanine aminotransferase; B: Total bilirubin; C: Direct bilirubin; D: Serum albumin. Values are mean ± SEM; number of animals = 6 rats/each group. bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; ALT: Alanine aminotransferase.\nEffect on expression of lncRNA MALAT1, miR-125b, and TUBG1 (G2/M transition of mitotic cell cycle) in the liver tissue As shown in Figure 3, induction of PCL resulted in significant increase of lncRNA MALAT a significant decrease in RQ miR-125b and significant increase in RQ TUBG1 expressions in comparison to naïve group. RQ of lncRNA MALAT1 in the liver tissue was significantly decreased in cyan -10,15 and cyan-20 groups compared to PCL group. Furthermore, cyan-20 group showed significant decrease in RQ of lncRNA MALAT1 in the liver tissue compared to cyan-10 group (Figure 3A). On the contrary, RQ of miR-125b in the liver tissue was significantly increased in cyan -10, cyan-15 and cyan-20 groups compared to PCL group. In addition, cyan-20 group showed significant increase in RQ of miR-125b in the liver tissue compared to cyan-10 group (Figure 3B). Furthermore, RQ of TUBG1 in the liver tissue was significantly decreased in cyan-10, cyan-15 and cyan-20 groups compared to PCL group. Also, cyan-20 group showed significant decrease in RQ of TUBG1 in the liver tissue compared to cyan-10 group (Figure 3C).\n\nEffect on expression of of long non-coding RNA, MALAT1, miR-125b and tubulin 1 (G2/M transition of mitotic cell cycle) in the liver tissue. A: RQ of long non-coding RNA MALAT1; B: RQ miR-125b; C: RQ TUBGI . Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group; bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; TUBG: Tubulin gamma 1; lncRNA: Long non-coding RNA.\nAs shown in Figure 3, induction of PCL resulted in significant increase of lncRNA MALAT a significant decrease in RQ miR-125b and significant increase in RQ TUBG1 expressions in comparison to naïve group. RQ of lncRNA MALAT1 in the liver tissue was significantly decreased in cyan -10,15 and cyan-20 groups compared to PCL group. Furthermore, cyan-20 group showed significant decrease in RQ of lncRNA MALAT1 in the liver tissue compared to cyan-10 group (Figure 3A). On the contrary, RQ of miR-125b in the liver tissue was significantly increased in cyan -10, cyan-15 and cyan-20 groups compared to PCL group. In addition, cyan-20 group showed significant increase in RQ of miR-125b in the liver tissue compared to cyan-10 group (Figure 3B). Furthermore, RQ of TUBG1 in the liver tissue was significantly decreased in cyan-10, cyan-15 and cyan-20 groups compared to PCL group. Also, cyan-20 group showed significant decrease in RQ of TUBG1 in the liver tissue compared to cyan-10 group (Figure 3C).\n\nEffect on expression of of long non-coding RNA, MALAT1, miR-125b and tubulin 1 (G2/M transition of mitotic cell cycle) in the liver tissue. A: RQ of long non-coding RNA MALAT1; B: RQ miR-125b; C: RQ TUBGI . Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group; bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; TUBG: Tubulin gamma 1; lncRNA: Long non-coding RNA.\nHistopathological and immunohistochemical studies Light microscopic examination of H&E-stained sections of naïve rats’ liver showed normal hepatic architecture, cords of hepatocytes radiating from central vein, portal triads present in between, polygonal hepatocytes with centrally rounded vesicular nuclei, and hepatic sinusoid in between (Figure 4A and B). Liver sections obtained from rats received DEN/2-AAF (Figure 4C-E) showed disruption of normal hepatic lobular architecture along with presence of large, discriminated dysplastic nodules compressing the surrounding liver tissue, with high-grade dysplastic cells displaying increased nuclear: Cytoplasmatic ratio (Figure 4E), nuclear hyperchromatosis, and basophilic cytoplasm. Liver sections of rats treated with different doses of cyanidin (10, 15, and 20 mg/kg) respectively, showed small and less discriminated dysplastic nodules (Figure 4F-H).\n\nPhotomicrographs of liver sections stained with H&E staining. A and B: Naïve group liver sections showed normal hepatic architecture, cords of hepatocytes radiating from central vein and portal triads present in-between and polygonal hepatocytes with central rounded vesicular nuclei, and hepatic sinusoid in-between; C-E: Liver sections of rats received diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF) showed larger, discriminated dysplastic nodules (doted shapes) compressing the surrounding liver tissue with disruption of normal hepatic lobular architecture; D: Liver sections of rats received DEN/2-AAF showed eosinophilic foci of cellular alteration consisting of enlarged hepatocytes with increased acidophilic staining and vaculated nuclei (arrow head); E: Liver sections of rats received DEN/2-AAF showed foci of Clear cell formed of hepatocytes showing variable degrees of cytoplasmic vacuolations and ballooning with pyknotic nuclei (arrow); F-H: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) respectively, showing small and less discriminated dysplastic nodules (doted shapes). (Magnification: A, C, F, G, H × 1000; B, D, E × 400). CV: Central vein; PV: Portal vein; Pt: Portal triads; S: Sinusoid; H: Hepatocytes.\nImmunohistochemical examination with GSTP antibody appeared as large hyperplastic nodules were demonstrated by the brown color. Figure 5A and B shows the liver sections of naïve group, while Figure 5C shows large-positively stained GSTP foci demonstrated in PCL group. Liver sections from rats of all cyan-treated groups exhibited small GSTP-positive small hepatic foci of different sizes scattered in-between negatively-stained hepatic parenchyma (Figure 5D-F). The percent area of GSTP foci was significantly increased in the PCL group compared to naïve group, while all cyan-treated groups showed significant decrease in the GSTP percent area. Furthermore, cyan-15 and -20 showed significant decrease in the GSTP percent area over cyan-10 (Table 1). PCNA immunohistochemical analysis showed an elevated expression of PCNA in the group that received DEN/2-AAF as compared to naïve group (Figure 6A and B). Liver sections obtained from rats received cyan. in its three doses showed decrease in PCNA-positively stained nuclei (Figure 6C-E and Table 1).\n\nPhotomicrographs of liver sections of rats immunohistochemical stained with glutathione S-transferase placental antibody. A and B: Naive group; C: Precancerous lesion group showing multiple glutathione S-transferase placental (GSTP)-positive large hepatic nodules (brown stained nodules) occupying most of section; D-F: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) showing GSTP positive small hepatic foci (brown stained cells = arrow) of different size scattered in-between negatively stained hepatic parenchyma. (Magnification: × 40)\n\nPhotomicrographs of liver sections Immunohistochemical stained with proliferating cell nuclear antigen. A: Negative reaction of control group; B: Diethylnitrosamine + 100 mg 2-acetylaminofluorene group showing positive stained nuclei scattered all over the field; C-E: Liver sections of rats treated with different doses of cyamidine (10, 15, 20 mg/kg) respectively, show few positive hepatocytes sporadically distributed over the field (arrow). (Magnification × 100). \nExpression rate of hepatocytes positive for glutathione S-transferase placental and proliferating cell nuclear antigen were calculated as number of positive field expression in 10 fields per rat liver tissue\nValues are mean ± SD; number of animals = 6 rats/each group. Examined field; 10 fields/liver section. \n\nP < 0.05 significant differences compared to naïve group.\n\nP < 0.05 significant differences compared to precancerous lesion group.\n\nP < 0.05 significant compared to cyanidin-3-O-glucoside-10, One-way ANOVA followed by Tukey’s test. +: Positive expression found in 1-3 fields; ++: Positive expression found in 4-6 fields; +++: Positive expression found in 7-10 field; PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; GSTP: Glutathione S-transferase placental; PCNA: Proliferating cell nuclear antigen.\nLight microscopic examination of H&E-stained sections of naïve rats’ liver showed normal hepatic architecture, cords of hepatocytes radiating from central vein, portal triads present in between, polygonal hepatocytes with centrally rounded vesicular nuclei, and hepatic sinusoid in between (Figure 4A and B). Liver sections obtained from rats received DEN/2-AAF (Figure 4C-E) showed disruption of normal hepatic lobular architecture along with presence of large, discriminated dysplastic nodules compressing the surrounding liver tissue, with high-grade dysplastic cells displaying increased nuclear: Cytoplasmatic ratio (Figure 4E), nuclear hyperchromatosis, and basophilic cytoplasm. Liver sections of rats treated with different doses of cyanidin (10, 15, and 20 mg/kg) respectively, showed small and less discriminated dysplastic nodules (Figure 4F-H).\n\nPhotomicrographs of liver sections stained with H&E staining. A and B: Naïve group liver sections showed normal hepatic architecture, cords of hepatocytes radiating from central vein and portal triads present in-between and polygonal hepatocytes with central rounded vesicular nuclei, and hepatic sinusoid in-between; C-E: Liver sections of rats received diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF) showed larger, discriminated dysplastic nodules (doted shapes) compressing the surrounding liver tissue with disruption of normal hepatic lobular architecture; D: Liver sections of rats received DEN/2-AAF showed eosinophilic foci of cellular alteration consisting of enlarged hepatocytes with increased acidophilic staining and vaculated nuclei (arrow head); E: Liver sections of rats received DEN/2-AAF showed foci of Clear cell formed of hepatocytes showing variable degrees of cytoplasmic vacuolations and ballooning with pyknotic nuclei (arrow); F-H: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) respectively, showing small and less discriminated dysplastic nodules (doted shapes). (Magnification: A, C, F, G, H × 1000; B, D, E × 400). CV: Central vein; PV: Portal vein; Pt: Portal triads; S: Sinusoid; H: Hepatocytes.\nImmunohistochemical examination with GSTP antibody appeared as large hyperplastic nodules were demonstrated by the brown color. Figure 5A and B shows the liver sections of naïve group, while Figure 5C shows large-positively stained GSTP foci demonstrated in PCL group. Liver sections from rats of all cyan-treated groups exhibited small GSTP-positive small hepatic foci of different sizes scattered in-between negatively-stained hepatic parenchyma (Figure 5D-F). The percent area of GSTP foci was significantly increased in the PCL group compared to naïve group, while all cyan-treated groups showed significant decrease in the GSTP percent area. Furthermore, cyan-15 and -20 showed significant decrease in the GSTP percent area over cyan-10 (Table 1). PCNA immunohistochemical analysis showed an elevated expression of PCNA in the group that received DEN/2-AAF as compared to naïve group (Figure 6A and B). Liver sections obtained from rats received cyan. in its three doses showed decrease in PCNA-positively stained nuclei (Figure 6C-E and Table 1).\n\nPhotomicrographs of liver sections of rats immunohistochemical stained with glutathione S-transferase placental antibody. A and B: Naive group; C: Precancerous lesion group showing multiple glutathione S-transferase placental (GSTP)-positive large hepatic nodules (brown stained nodules) occupying most of section; D-F: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) showing GSTP positive small hepatic foci (brown stained cells = arrow) of different size scattered in-between negatively stained hepatic parenchyma. (Magnification: × 40)\n\nPhotomicrographs of liver sections Immunohistochemical stained with proliferating cell nuclear antigen. A: Negative reaction of control group; B: Diethylnitrosamine + 100 mg 2-acetylaminofluorene group showing positive stained nuclei scattered all over the field; C-E: Liver sections of rats treated with different doses of cyamidine (10, 15, 20 mg/kg) respectively, show few positive hepatocytes sporadically distributed over the field (arrow). (Magnification × 100). \nExpression rate of hepatocytes positive for glutathione S-transferase placental and proliferating cell nuclear antigen were calculated as number of positive field expression in 10 fields per rat liver tissue\nValues are mean ± SD; number of animals = 6 rats/each group. Examined field; 10 fields/liver section. \n\nP < 0.05 significant differences compared to naïve group.\n\nP < 0.05 significant differences compared to precancerous lesion group.\n\nP < 0.05 significant compared to cyanidin-3-O-glucoside-10, One-way ANOVA followed by Tukey’s test. +: Positive expression found in 1-3 fields; ++: Positive expression found in 4-6 fields; +++: Positive expression found in 7-10 field; PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; GSTP: Glutathione S-transferase placental; PCNA: Proliferating cell nuclear antigen.\n\nEffect on AFP levels and liver function tests:\nAs shown in Figure 1, following induction of PCL, rats exhibited a significant increase in AFP levels. AFP levels were significantly decreased in cyan-10, cyan-15, and cyan-20 groups compared to PCL group. There was no significant difference between the AFP levels of the three cyan groups. Liver function tests were affected after receiving DEN/2-AAF in which rats exhibited a significant increase in levels of ALT and total and direct bilirubin, and a significant decrease in the serum albumin (Figure 2). As shown in Figure 2A, rats received cyan. in different doses exhibited a significant decrease in serum ALT compared to PCL group. Cyan-15 group showed significant decrease in serum ALT compared to cyan-10 group. Moreover, cyan-20 group showed significant decrease in serum ALT compared to cyan-10 group. In As depicted in Figure 2B, compared to PCL group, the rats in the three cyan groups exhibited a significant decrease in serum total bilirubin. Furthermore, cyan-20 group showed significant decrease in serum total bilirubin compared to cyan-10 group. Figure 2C: Serum direct bilirubin was significantly decreased in the three cyan groups compared to the PCL group. Moreover, cyan-20 group showed significant decrease in serum direct bilirubin compared to cyan-10 group. Administration of cyan in doses 5, 10 and 20 mg/kg/d produced a significant increase in serum albumin compared to PCL group. However, there was no significant difference between the serum albumin levels of the three cyan groups (Figure 2D).\n\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) on alpha fetoprotein in the serum of rats. Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group, unpaired t test; bP < 0.05 significant differences compared to precancerous lesion group. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; AFP: Alpha fetoprotein.\n\nEffect of cyanidin 3-glucoside at different doses (10, 15 and 20 mg/kg/d) in rats. A: Alanine aminotransferase; B: Total bilirubin; C: Direct bilirubin; D: Serum albumin. Values are mean ± SEM; number of animals = 6 rats/each group. bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; ALT: Alanine aminotransferase.\n\nEffect on expression of lncRNA MALAT1, miR-125b, and TUBG1 (G2/M transition of mitotic cell cycle) in the liver tissue:\nAs shown in Figure 3, induction of PCL resulted in significant increase of lncRNA MALAT a significant decrease in RQ miR-125b and significant increase in RQ TUBG1 expressions in comparison to naïve group. RQ of lncRNA MALAT1 in the liver tissue was significantly decreased in cyan -10,15 and cyan-20 groups compared to PCL group. Furthermore, cyan-20 group showed significant decrease in RQ of lncRNA MALAT1 in the liver tissue compared to cyan-10 group (Figure 3A). On the contrary, RQ of miR-125b in the liver tissue was significantly increased in cyan -10, cyan-15 and cyan-20 groups compared to PCL group. In addition, cyan-20 group showed significant increase in RQ of miR-125b in the liver tissue compared to cyan-10 group (Figure 3B). Furthermore, RQ of TUBG1 in the liver tissue was significantly decreased in cyan-10, cyan-15 and cyan-20 groups compared to PCL group. Also, cyan-20 group showed significant decrease in RQ of TUBG1 in the liver tissue compared to cyan-10 group (Figure 3C).\n\nEffect on expression of of long non-coding RNA, MALAT1, miR-125b and tubulin 1 (G2/M transition of mitotic cell cycle) in the liver tissue. A: RQ of long non-coding RNA MALAT1; B: RQ miR-125b; C: RQ TUBGI . Values are mean ± SEM; number of animals = 6 rats/each group. aP < 0.05 significant differences compared to naïve group; bP < 0.05 significant differences compared to precancerous lesion group; cP < 0.05 significant differences between the 2 selected groups, One-way ANOVA followed by Tukey’s test. PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; TUBG: Tubulin gamma 1; lncRNA: Long non-coding RNA.\n\nHistopathological and immunohistochemical studies:\nLight microscopic examination of H&E-stained sections of naïve rats’ liver showed normal hepatic architecture, cords of hepatocytes radiating from central vein, portal triads present in between, polygonal hepatocytes with centrally rounded vesicular nuclei, and hepatic sinusoid in between (Figure 4A and B). Liver sections obtained from rats received DEN/2-AAF (Figure 4C-E) showed disruption of normal hepatic lobular architecture along with presence of large, discriminated dysplastic nodules compressing the surrounding liver tissue, with high-grade dysplastic cells displaying increased nuclear: Cytoplasmatic ratio (Figure 4E), nuclear hyperchromatosis, and basophilic cytoplasm. Liver sections of rats treated with different doses of cyanidin (10, 15, and 20 mg/kg) respectively, showed small and less discriminated dysplastic nodules (Figure 4F-H).\n\nPhotomicrographs of liver sections stained with H&E staining. A and B: Naïve group liver sections showed normal hepatic architecture, cords of hepatocytes radiating from central vein and portal triads present in-between and polygonal hepatocytes with central rounded vesicular nuclei, and hepatic sinusoid in-between; C-E: Liver sections of rats received diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF) showed larger, discriminated dysplastic nodules (doted shapes) compressing the surrounding liver tissue with disruption of normal hepatic lobular architecture; D: Liver sections of rats received DEN/2-AAF showed eosinophilic foci of cellular alteration consisting of enlarged hepatocytes with increased acidophilic staining and vaculated nuclei (arrow head); E: Liver sections of rats received DEN/2-AAF showed foci of Clear cell formed of hepatocytes showing variable degrees of cytoplasmic vacuolations and ballooning with pyknotic nuclei (arrow); F-H: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) respectively, showing small and less discriminated dysplastic nodules (doted shapes). (Magnification: A, C, F, G, H × 1000; B, D, E × 400). CV: Central vein; PV: Portal vein; Pt: Portal triads; S: Sinusoid; H: Hepatocytes.\nImmunohistochemical examination with GSTP antibody appeared as large hyperplastic nodules were demonstrated by the brown color. Figure 5A and B shows the liver sections of naïve group, while Figure 5C shows large-positively stained GSTP foci demonstrated in PCL group. Liver sections from rats of all cyan-treated groups exhibited small GSTP-positive small hepatic foci of different sizes scattered in-between negatively-stained hepatic parenchyma (Figure 5D-F). The percent area of GSTP foci was significantly increased in the PCL group compared to naïve group, while all cyan-treated groups showed significant decrease in the GSTP percent area. Furthermore, cyan-15 and -20 showed significant decrease in the GSTP percent area over cyan-10 (Table 1). PCNA immunohistochemical analysis showed an elevated expression of PCNA in the group that received DEN/2-AAF as compared to naïve group (Figure 6A and B). Liver sections obtained from rats received cyan. in its three doses showed decrease in PCNA-positively stained nuclei (Figure 6C-E and Table 1).\n\nPhotomicrographs of liver sections of rats immunohistochemical stained with glutathione S-transferase placental antibody. A and B: Naive group; C: Precancerous lesion group showing multiple glutathione S-transferase placental (GSTP)-positive large hepatic nodules (brown stained nodules) occupying most of section; D-F: Liver sections of rats treated with different doses of cyanidin (10, 15, 20 mg/kg) showing GSTP positive small hepatic foci (brown stained cells = arrow) of different size scattered in-between negatively stained hepatic parenchyma. (Magnification: × 40)\n\nPhotomicrographs of liver sections Immunohistochemical stained with proliferating cell nuclear antigen. A: Negative reaction of control group; B: Diethylnitrosamine + 100 mg 2-acetylaminofluorene group showing positive stained nuclei scattered all over the field; C-E: Liver sections of rats treated with different doses of cyamidine (10, 15, 20 mg/kg) respectively, show few positive hepatocytes sporadically distributed over the field (arrow). (Magnification × 100). \nExpression rate of hepatocytes positive for glutathione S-transferase placental and proliferating cell nuclear antigen were calculated as number of positive field expression in 10 fields per rat liver tissue\nValues are mean ± SD; number of animals = 6 rats/each group. Examined field; 10 fields/liver section. \n\nP < 0.05 significant differences compared to naïve group.\n\nP < 0.05 significant differences compared to precancerous lesion group.\n\nP < 0.05 significant compared to cyanidin-3-O-glucoside-10, One-way ANOVA followed by Tukey’s test. +: Positive expression found in 1-3 fields; ++: Positive expression found in 4-6 fields; +++: Positive expression found in 7-10 field; PCL: Precancerous lesion; Cyan: Cyanidin-3-O-glucoside; GSTP: Glutathione S-transferase placental; PCNA: Proliferating cell nuclear antigen.\n\nDISCUSSION:\nAltered expression of mitotic spindle assembly genes has been detected in many cancers. For example, MAD2 gene levels are downregulated in breast carcinoma[18], while mitotic checkpoint serine/BUB1 gene expression is dysregulated in colorectal carcinoma[19]. The impairment of spindle assembly genes are frequently detected in HCC[20,21]. Moreover, inhibition of the mitotic assembly genes has been found to be lethal to cancer cells, and has promising therapeutic effect in cancer treatment[22,23]. \nIn this study, we used several public microarray databases for the construction of simple genetic-epigenetic network specific to HCC and linked to cell cycle and mitotic spindle formation. We assessed the antiproliferative effect of cyan in HCC animal model via modulation of lncRNA-MALAT1-miR-125b-TUBG1 mRNA axis expression.\nMicrotubules are involved both in maintenance of cell shape and motility and in mitotic spindle formation[24]. γ-TUBG is a major player that supports microtubule nucleation. γ-TUBG acts as a binding site for the α/β-tubulin dimer and it has two γ-TUBG genes, TUBG1 and TUBG2 in mammals only[25]. The TUBG levels in the centrosome change during cell cycle progression and usually increases at the start of mitosis. At the end of mitosis, TUBG levels are decreased to interphase levels[26]. Overexpression and altered compartmentalization of γ-TUBG may lead to carcinogenesis[27-29]. Interestingly, γ-TUBG associates interact with tumor suppressor protein C53 in mammalian interphase cells of different cellular origins[30]. Interestingly, γ-TUBG interacts with E2F transcription factors, resulting in upregulation of E2F target genes PCNA and CDKB1[31] and glutathione S transferase activity[32]. On the other hand, the mRNA and protein levels of TUBA1C are upregulated in HCC. Clinically, it affects the survival of patients and the level of metastatic burden along with portal vein thrombosis. In addition, it activates cellular proliferation and migration and is a potential therapeutic target for HCC[33]. Recent studies have shed light on the efficacy of microtubule-binding agents in the treatment of HCC, especially in combination with mammalian target of rapamycin inhibitors[34].\nOne of the few miRNAs that regulate Spindle Assembly Checkpoints (SAC) is miR-125b. It suppresses the expression of Mad1 (a core SAC protein) that is responsible for inhibiting entry into anaphase till metaphase defects are corrected[35]. Importantly, miRNA-125b has been found to be associated with colorectal cancer[36], multiple myeloma[37], and breast cancer[38]. Song et al[39] demonstrated that the expression of miR-125b in hepatocytes was protective and reflected better survival in HCC tissue samples due to restoration of SIRT6 expression[39]. miR-125b-5p inhibits HCC proliferation by targeting TXNRD1, the terminal step of biosynthesis of nucleotides[40].\nDeregulation of lncRNAs has been shown to be crucial in cancer progression, especially HCC[41,42]. Hung et al[43] demonstrated the involvement of lncRNAs in regulation of the expression of cell cycle-related genes, p53 gene regulatory pathway, and apoptosis[43,44]. The lncRNA MALAT1 is overexpressed in several solid tumors and is linked with tumor recurrence[45-47]. MALAT1 coordinate RNA polymerase II transcription, pre-mRNA splicing, and mRNA export[48]. Thus, this lncRNA holds great potential in influencing the local concentration of a specific splicing factor during specific stages of the cell cycle. Depletion of MALAT1 in cancer cells inhibits tumorigenicity[49]. MALAT1 regulates E2F1 transcription factor activity, highlighting its pro-proliferative function[50]. MALAT1 is upregulated in many cancer types, including breast cancer[51], cervical cancer, lung cancer[52], and hepatocarcinoma[53-55]. In a previous study, hypoxia enhanced MALAT1 expression, leading to increase in proliferating and invading activity of Hep3B cells due to negative interaction with miR200a[56]. Huang and his group explored the role of specificity protein 1/3 in regulation of MALAT1 expression in HCC cells[57].\nCyanidin or anthocyanin (a polyphenolic pigment found in plants) confers several pharmacological benefits, including anticancer properties[58-62]. Cyan mediates cytotoxicity against human monocytic leukemia cells via G2/M phase arrest and induction of apoptosis[63]. Tsai et al[64] evaluated anthocyanins as promising anticancer or chemopreventive agents to induce G2/M arrest in HAs-induced leukemia cell cycle arrest via ATM-Chk1/2-Cdc25C axis modulation[64]. They also found that anthocyanin inhibits the proliferation of HCC cells[65,66].\nIn our study, the effect of cyan on cancerous tissue was dose-dependent (10, 15 and 20 mg/kg/d), that is, increasing its effect with increasing the concentration of the drug. This was proved also, with the correlated significant decrease in the percent area of GSTP foci, PCNA expression which are the target of TUBG1 mRNA and the molecular epigenetic markers associated with HCC (lncRNA MALAT1 and miR-125b-1-3p) in the liver tissue of rats in our study (Figure 7). Thus, we can hypothesize that administration of cyan in PCL may result in downregulation of lncRNA MALAT1 with subsequent release of free miR-125b-1-3p that binds to TUBG1 mRNA and downregulate its expression[65]. The changes in the epigenetic-genetic network affect their target genes PCNA and GSTP.\n\nConcept map of study design. GSTP: Glutathione S-transferase placental; PCNA: Proliferating cell nuclear antigen; TUBG: Tubulin; lncRNA: Long non-coding RNA.\nLastly, the cyan-mediated modulation of cell cycle and mitotic spindle assembly that regulate HCC proliferation might affect the capability of the HCC cells to overcome oxidative stress[66,67]. Our data confers the rationale for designing antimitotic approach combining the conventional cytotoxic drugs with phytochemical extracts to inhibit cell cycle progression in cancer. More in vitro functional studies are required to explore the functional mechanism of the chosen RNA panel and validate their role as drug target biomarkers. \n\nCONCLUSION:\nCyanidin is a natural molecule that holds great potential in unraveling the mystery of cytotoxic pharmacy in the future. It can be used to devise novel antimitotic drugs that can target cell cycle in HCC. Our results also indicated great potential of the TUBG1 mRNA- miR-125b-lncRNA MALAT1 and -1-3p axis as potential drug target biomarker for further investigations.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nCyanidin-3-O-glucoside (cyan) exhibits antioxidant and anticancer properties. The cell cycle proteins and antimitotic drugs might be promising therapeutic targets in hepatocellular carcinoma.\n\nMethods:\nIn vivo, DEN/2-AAF-induced hepatic PCL, rats were treated with three doses of cyan (10, 15, and 20 mg/kg/d, for four consecutive days per week for 16 wk). Blood and liver tissue samples were collected for measurement of the followings; alpha fetoprotein (AFP) liver function and RNA panel differential expression was evaluated via real time polymerase chain reaction. Histopathological examination of liver sections stained with H&E and immunohistochemical study using glutathione S-transferase placental (GSTP) and proliferating cell nuclear antigen (PCNA) antibodies were assessed.\n\nResults:\nCyan administration mitigated the effect of DEN/2-AFF induced PCL, decreased AFP levels, and improved liver function. Remarkably, treatment with cyan dose dependently decreased the long non-coding RNA MALAT1 and tubulin gamma 1 mRNA expressions and increased the levels of miR-125b, all of which are involved in cell cycle and mitotic spindle assembly. Of note, cyan decreased GSTP foci percent area and PCNA positively stained nuclei.\n\nConclusions:\nOur results indicated that cyan could be used as a potential therapeutic agent to inhibit liver carcinogenesis in rat model via modulation of cell cycle."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nHepatocellular carcinoma (HCC) is the commonest primary malignancy of the liver. Its main risk factors include hepatitis B, hepatitis C, and non-alcoholic steatohepatitis. HCC is associated with high mortality and morbidity. Its incidence has increased from 1.4 to 6.2 per 100000 cases per year within the last 30 years[1]. HCC burden in Egypt is high due to the high prevalence of HCV, where liver cancers account for 11.75% of all gastrointestinal cancers and 1.68% of all malignancies. In addition, over 70% of all liver malignancies in Egypt are HCC[2].\nTo date, HCC is holding its place as an unresolved dilemma; its resistance to systemic chemotherapy and radiation makes the cure approachable only in very early stages according to Milan criteria, that is, where the tumor size is < 5 cm (when only one lesion is present) or < 3 cm (when 2-3 lesions are present)[3].\nGenes involved in cell cycle control are usually mutated in presence of cancer. Dysregulated mitosis results in genomic instability, which leads to tumor aggressiveness[4]. Microtubules are a key component of mitotic spindles, and thus, a crucial part of the process of mitosis. They form an important target in cancer chemotherapy, inducing arrest of mitosis and cell death. On the other hand, while the overexpression of γ-tubulin (γ-TUBG) showed improved survival among small cell lung cancer patients[5], the overexpression of γ-TUBG is found to be characteristic feature of thyroid and breast cancers[6]. Such variation may arise due to differences in gene transcription and check point regulations in different cancer tissues[7].\nCyanidin-3-glucoside (cyan) is a potential chemotherapeutic and chemo-protective agent. Through its antioxidant activity, it scavenges radical oxygen species, which decreases the overall number of tumorous cells (malignant and benign) in in vivo studies[8]. Cyan has been implicated in some beneficial health activities[9], including reducing age-associated oxidative stress[10], improving cognitive brain function, and exhibiting anti-diabetic[11], anti-inflammation[12], anti-atherogenic[13], and anti-obesity activities[14]. Previously, it has been reported that cyan mediates caspase-3 cleavage with DNA fragmentation. Cyan has also been associated with induction of autophagy, a key element involved in cancer elimination, via induction of autophagy-related gene 5 and microtubule-associated protein 1 light chain 3-II[15]. Cyan has drawn increasing attention because of its potential anti-cancer properties. Cyan may offer a novel avenue for treating HCC. a potential antiproliferative effect on HepG2 cells[16].\nThe present study was conducted to evaluate the effect of cyan in three different doses on cell cycle progression and mitotic assembly disorder in hepatic precancerous lesion (PCL) via assessment of levels of long non-coding RNA (lncRNA) MALAT1, miR-125b, and tubulin 1. We also conducted histopathological and immuno-histochemical examination using H&E staining assessment of levels of glutathione S-transferase placental (GSTP) and proliferating cell nuclear antigen (PCNA) antibodies in male Wistar rat model of diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF)-induced hepatic PCL. \n\nCONCLUSION:\nFurther larger in vitro and in vivo studies are needed to elucidate the mechanism of cyanidin cytotoxicity in hepatocellular carcinoma."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nCyanidin-3-O-glucoside (cyan) exhibits antioxidant and anticancer properties. The cell cycle proteins and antimitotic drugs might be promising therapeutic targets in hepatocellular carcinoma.\n\nMethods:\nIn vivo, DEN/2-AAF-induced hepatic PCL, rats were treated with three doses of cyan (10, 15, and 20 mg/kg/d, for four consecutive days per week for 16 wk). Blood and liver tissue samples were collected for measurement of the followings; alpha fetoprotein (AFP) liver function and RNA panel differential expression was evaluated via real time polymerase chain reaction. Histopathological examination of liver sections stained with H&E and immunohistochemical study using glutathione S-transferase placental (GSTP) and proliferating cell nuclear antigen (PCNA) antibodies were assessed.\n\nResults:\nCyan administration mitigated the effect of DEN/2-AFF induced PCL, decreased AFP levels, and improved liver function. Remarkably, treatment with cyan dose dependently decreased the long non-coding RNA MALAT1 and tubulin gamma 1 mRNA expressions and increased the levels of miR-125b, all of which are involved in cell cycle and mitotic spindle assembly. Of note, cyan decreased GSTP foci percent area and PCNA positively stained nuclei.\n\nConclusions:\nOur results indicated that cyan could be used as a potential therapeutic agent to inhibit liver carcinogenesis in rat model via modulation of cell cycle."},"whole_article_text_length":{"kind":"number","value":11831,"string":"11,831"},"whole_article_abstract_length":{"kind":"number","value":257,"string":"257"},"other_sections_lengths":{"kind":"list like","value":[594,59,79,157,267,32,206,113,209,339,63,3530,476,321,947,1081,65],"string":"[\n 594,\n 59,\n 79,\n 157,\n 267,\n 32,\n 206,\n 113,\n 209,\n 339,\n 63,\n 3530,\n 476,\n 321,\n 947,\n 1081,\n 65\n]","isConversational":false},"num_sections":{"kind":"number","value":18,"string":"18"},"most_frequent_words":{"kind":"list like","value":["cyan","group","liver","10","significant","rats","compared","20","sections","pcl"],"string":"[\n \"cyan\",\n \"group\",\n \"liver\",\n \"10\",\n \"significant\",\n \"rats\",\n \"compared\",\n \"20\",\n \"sections\",\n \"pcl\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["introduction hepatocellular carcinoma","carcinoma hcc commonest","hcv liver cancers","liver malignancies egypt","malignancies egypt hcc"],"string":"[\n \"introduction hepatocellular carcinoma\",\n \"carcinoma hcc commonest\",\n \"hcv liver cancers\",\n \"liver malignancies egypt\",\n \"malignancies egypt hcc\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"null"},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Hepatocellular carcinoma therapy | Hepatocellular-carcinoma growth | Hepatocellular-carcinoma model | Hepatocellular-carcinoma size [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Hepatocellular carcinoma therapy | Hepatocellular-carcinoma growth | Hepatocellular-carcinoma model | Hepatocellular-carcinoma size [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"null"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Hepatocellular carcinoma therapy | Hepatocellular-carcinoma growth | Hepatocellular-carcinoma model | Hepatocellular-carcinoma size [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Hepatocellular carcinoma therapy | Hepatocellular-carcinoma growth | Hepatocellular-carcinoma model | Hepatocellular-carcinoma size [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Hepatocellular carcinoma therapy | Hepatocellular-carcinoma growth | Hepatocellular-carcinoma model | Hepatocellular-carcinoma size [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Anthocyanins | Diethylnitrosamine | Female | Glucosides | Glutathione Transferase | Liver | Liver Neoplasms | Liver Neoplasms, Experimental | Precancerous Conditions | Pregnancy | Rats | Rats, Wistar [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Anthocyanins | Diethylnitrosamine | Female | Glucosides | Glutathione Transferase | Liver | Liver Neoplasms | Liver Neoplasms, Experimental | Precancerous Conditions | Pregnancy | Rats | Rats, Wistar [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"null"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Anthocyanins | Diethylnitrosamine | Female | Glucosides | Glutathione Transferase | Liver | Liver Neoplasms | Liver Neoplasms, Experimental | Precancerous Conditions | Pregnancy | Rats | Rats, Wistar [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Anthocyanins | Diethylnitrosamine | Female | Glucosides | Glutathione Transferase | Liver | Liver Neoplasms | Liver Neoplasms, Experimental | Precancerous Conditions | Pregnancy | Rats | Rats, Wistar [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Anthocyanins | Diethylnitrosamine | Female | Glucosides | Glutathione Transferase | Liver | Liver Neoplasms | Liver Neoplasms, Experimental | Precancerous Conditions | Pregnancy | Rats | Rats, Wistar [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] introduction hepatocellular carcinoma | carcinoma hcc commonest | hcv liver cancers | liver malignancies egypt | malignancies egypt hcc [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] introduction hepatocellular carcinoma | carcinoma hcc commonest | hcv liver cancers | liver malignancies egypt | malignancies egypt hcc [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"null"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] introduction hepatocellular carcinoma | carcinoma hcc commonest | hcv liver cancers | liver malignancies egypt | malignancies egypt hcc [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] introduction hepatocellular carcinoma | carcinoma hcc commonest | hcv liver cancers | liver malignancies egypt | malignancies egypt hcc [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] introduction hepatocellular carcinoma | carcinoma hcc commonest | hcv liver cancers | liver malignancies egypt | malignancies egypt hcc [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cyan | group | liver | 10 | significant | rats | compared | 20 | sections | pcl [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] cyan | group | liver | 10 | significant | rats | compared | 20 | sections | pcl [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"null"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] cyan | group | liver | 10 | significant | rats | compared | 20 | sections | pcl [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cyan | group | liver | 10 | significant | rats | compared | 20 | sections | pcl [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cyan | group | liver | 10 | significant | rats | compared | 20 | sections | pcl [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cancer | anti | hcc | cyan | associated | cell | mitosis | cancers | present | potential [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] database available | database | available | united states | united | states | injected | www | positive | serum [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"null"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] potential | great potential | great | target | pharmacy future devise novel | future devise | future devise novel | future devise novel antimitotic | pharmacy future devise | pharmacy future [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] cyan | group | significant | 10 | rats | compared | liver | serum | groups | 20 [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] cyan | group | significant | 10 | rats | compared | liver | serum | groups | 20 [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] DEN/2-AAF | PCL | three | 10 | 15 | 20 mg/kg | four consecutive days per week | 16 ||| AFP | RNA ||| H&E [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"null"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| DEN/2-AAF | PCL | three | 10 | 15 | 20 mg/kg | four consecutive days per week | 16 ||| AFP | RNA ||| H&E ||| Cyan | PCL | AFP ||| RNA MALAT1 | 1 ||| GSTP foci percent | PCNA ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| DEN/2-AAF | PCL | three | 10 | 15 | 20 mg/kg | four consecutive days per week | 16 ||| AFP | RNA ||| H&E ||| Cyan | PCL | AFP ||| RNA MALAT1 | 1 ||| GSTP foci percent | PCNA ||| [SUMMARY]"}}},{"rowIdx":61,"cells":{"title":{"kind":"string","value":"Lipoteichoic acid induces surfactant protein-A biosynthesis in human alveolar type II epithelial cells through activating the MEK1/2-ERK1/2-NF-κB pathway."},"pmid":{"kind":"string","value":"23031213"},"background_abstract":{"kind":"string","value":"Lipoteichoic acid (LTA), a gram-positive bacterial outer membrane component, can cause septic shock. Our previous studies showed that the gram-negative endotoxin, lipopolysaccharide (LPS), could induce surfactant protein-A (SP-A) production in human alveolar epithelial (A549) cells."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"A549 cells were exposed to LTA. Levels of SP-A, nuclear factor (NF)-κB, extracellular signal-regulated kinase 1/2 (ERK1/2), and mitogen-activated/extracellular signal-regulated kinase kinase (MEK)1 were determined."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Exposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability. Meanwhile, when exposed to 30 μg/ml LTA for 1, 6, and 24 h, the biosynthesis of SP-A mRNA and protein in A549 cells significantly increased. As to the mechanism, LTA enhanced cytosolic and nuclear NF-κB levels in time-dependent manners. Pretreatment with BAY 11-7082, an inhibitor of NF-κB activation, significantly inhibited LTA-induced SP-A mRNA expression. Sequentially, LTA time-dependently augmented phosphorylation of ERK1/2. In addition, levels of phosphorylated MEK1 were augmented following treatment with LTA."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Therefore, this study showed that LTA can increase SP-A synthesis in human alveolar type II epithelial cells through sequentially activating the MEK1-ERK1/2-NF-κB-dependent pathway."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Alveolar Epithelial Cells","Cell Culture Techniques","Cell Survival","Humans","Immunoblotting","Lipopolysaccharides","Mitogen-Activated Protein Kinase 1","Mitogen-Activated Protein Kinase 3","NF-kappa B","Pulmonary Surfactant-Associated Protein A","Real-Time Polymerase Chain Reaction","Signal Transduction","Teichoic Acids"],"string":"[\n \"Alveolar Epithelial Cells\",\n \"Cell Culture Techniques\",\n \"Cell Survival\",\n \"Humans\",\n \"Immunoblotting\",\n \"Lipopolysaccharides\",\n \"Mitogen-Activated Protein Kinase 1\",\n \"Mitogen-Activated Protein Kinase 3\",\n \"NF-kappa B\",\n \"Pulmonary Surfactant-Associated Protein A\",\n \"Real-Time Polymerase Chain Reaction\",\n \"Signal Transduction\",\n \"Teichoic Acids\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"3492077"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Sepsis can lead to multiorgan failure and death and appears to be triggered by bacterial products, such as lipopolysaccharide (LPS) from gram-negative bacteria and lipoteichoic acid (LTA) from gram-positive ones \n[1-3]. Infection of the respiratory tract caused by gram-positive bacteria and pneumonia combined with acute lung injury (ALI) are usually the leading causes of mortality by sepsis \n[4]. In the past few decades, the incidences of sepsis and septic shock have been increasing \n[5]. Although endotoxin-activated events are clearly important in gram-negative infection, gram-positive bacteria also have crucial roles, but less is known about host responses to them \n[6]. The increasing prevalence of sepsis from gram-positive bacterial pathogens necessitates a reevaluation of the basic assumptions about the molecular pathogenesis of ALI.\nAlveolar epithelial type II cells contribute to the maintenance of mucosal integrity by modulating the production of surfactants \n[7]. Pulmonary surfactants play important roles in protecting the lung during endotoxin-induced injury and infection \n[8,9]. Surfactant protein (SP)-A is the most abundant pulmonary surfactant protein. Levels of SP-A in bronchiolar lavage fluid are modulated in gram-negative or -positive bacteria-caused lung diseases, including severe pneumonia, acute respiratory distress syndrome, and cardiogenic lung edema \n[10]. Thus, altering lung SP-A levels can be an effective indicator for pulmonary infection and inflammation. Our previous study showed that LPS selectively induced spa gene expression in human alveolar epithelial A549 cells \n[11].\nLTA, an outer membrane component of gram-positive bacteria, was shown to be one of the critical factors participating in the pathogenesis of sepsis \n[12,13]. LTA can stimulate inflammatory responses in the lung \n[14,15]. Therefore, understanding the mechanisms that regulate LTA-mediated cell activation is crucial for diagnosis, treatment, or prognosis of lung inflammatory diseases. In response to stimuli, LTA can activate macrophages to produce massive amounts of inflammatory factors that exhibit systemic effects in the general circulation \n[16]. LTA can induce the secretion of various cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α \n[17]. These data suggest that LTA can selectively modify gene transcription of various cell types and sequentially augment and possibly initiate tissue inflammation.\nMitogen-activated protein kinases (MAPKs) are serine/threonine kinases. The first MAPK isoforms to be cloned and characterized were the extracellular signal-regulated kinase 1 and 2 (ERK 1/2) \n[18,19]. ERK 1/2 are well documented to be activated by a family of dual-specificity kinases known as the mitogen-activated/ERK kinases (MEKs) \n[16,20]. A previous study demonstrated that LTA can selectively activate the ERK pathway in the cornea \n[21]. Our previous study showed that LTA induced TNF-α and IL-6 expressions by means of stimulating phosphorylation of ERK1/2 in macrophages \n[16]. In addition, LTA also triggered translocation of nuclear factor (NF)-κB from the cytoplasm to nuclei and its transactivation activity. Meanwhile, the mechanisms responsible for LTA-induced spa gene expression in alveolar epithelial cells are still unknown. In this study, we attempted to evaluate the effects of LTA on SP-1 synthesis in human alveolar type II epithelial cells and its possible mechanisms."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Toxicity of LTA to A549 cells Cell morphology and viability were assayed to evaluate the toxicity of LTA to human alveolar epithelial A549 cells. Exposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability (data not shown). When exposed to 30 μg/ml LTA for 1, 6, and 24 h, the viability of A549 cells was not influenced. Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h did not alter cell morphology (data not shown).\nCell morphology and viability were assayed to evaluate the toxicity of LTA to human alveolar epithelial A549 cells. Exposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability (data not shown). When exposed to 30 μg/ml LTA for 1, 6, and 24 h, the viability of A549 cells was not influenced. Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h did not alter cell morphology (data not shown).\n LTA-induced enhancement of SP-A biosynthesis in A549 cells The effects of LTA on SP-A levels in A549 cells were evaluated by an immunoblotting analysis (Figure \n1). In untreated A549 cells, low levels of SP-A were immunodetected (Figure \n1A, top panel, lane 1). After exposure to 30 μg/ml LTA for 1 h, levels of SP-A were found to be augmented (lane 2). When treated for 6 and 24 h, LTA obviously increased amounts of SP-A in A549 cells. β-Actin was immunodetected (Figure \n1A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n1B). Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h respectively caused significant 176%, 230%, and 270% increases in SP-A levels.\nEffects of lipoteichoic acid (LTA) on the production of surfactant protein(SP)-A. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h (A). Cellular proteins were prepared for the immunoblotting analyses. Amounts of SP-A were immunodetected (A, top pane). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\nThe effects of LTA on SP-A levels in A549 cells were evaluated by an immunoblotting analysis (Figure \n1). In untreated A549 cells, low levels of SP-A were immunodetected (Figure \n1A, top panel, lane 1). After exposure to 30 μg/ml LTA for 1 h, levels of SP-A were found to be augmented (lane 2). When treated for 6 and 24 h, LTA obviously increased amounts of SP-A in A549 cells. β-Actin was immunodetected (Figure \n1A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n1B). Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h respectively caused significant 176%, 230%, and 270% increases in SP-A levels.\nEffects of lipoteichoic acid (LTA) on the production of surfactant protein(SP)-A. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h (A). Cellular proteins were prepared for the immunoblotting analyses. Amounts of SP-A were immunodetected (A, top pane). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\n LTA-induced SP-A mRNA expression in A549 cells Induction of SP-A mRNA expression by LTA was quantified using a real-time PCR analysis (Figure \n2). After exposure to LTA for 1 h, the levels of SP-A mRNA in A549 cells were increased by 2.1-fold. Exposure of A549 cells to LTA for 6 and 24 h caused 2.8- and 3.7-fold increases in the levels of SP-A mRNA, respectively (Figure \n2). Pretreatment of A549 cells with BAY 11–7082, an inhibitor of NF-κB activation, for 1 h did not change SP-A mRNA expression (data not shown). However, BAY 11–7082 significantly inhibited LTA-induced SP-A mRNA production by 70% (Figure \n2).\nEffects of lipoteichoic acid (LTA) on induction of surfactant protein(SP)-A mRNA. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. In addition, A549 cells were pretreated with BAY 11–7082 (BAY), an inhibitor of NF-κB activation, for 1 h and then exposed to LTA for another 24 h. mRNA was prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Each value represents the mean ± SEM for n = 3. Symbols * and # indicate that the value significantly (p < 0.05) differed from the respective control and LTA-treated group, respectively.\nInduction of SP-A mRNA expression by LTA was quantified using a real-time PCR analysis (Figure \n2). After exposure to LTA for 1 h, the levels of SP-A mRNA in A549 cells were increased by 2.1-fold. Exposure of A549 cells to LTA for 6 and 24 h caused 2.8- and 3.7-fold increases in the levels of SP-A mRNA, respectively (Figure \n2). Pretreatment of A549 cells with BAY 11–7082, an inhibitor of NF-κB activation, for 1 h did not change SP-A mRNA expression (data not shown). However, BAY 11–7082 significantly inhibited LTA-induced SP-A mRNA production by 70% (Figure \n2).\nEffects of lipoteichoic acid (LTA) on induction of surfactant protein(SP)-A mRNA. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. In addition, A549 cells were pretreated with BAY 11–7082 (BAY), an inhibitor of NF-κB activation, for 1 h and then exposed to LTA for another 24 h. mRNA was prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Each value represents the mean ± SEM for n = 3. Symbols * and # indicate that the value significantly (p < 0.05) differed from the respective control and LTA-treated group, respectively.\n Augmentation of NF-κB expression and translocation by LTA Mechanisms of LTA-induced SP-A augmentation were evaluated by analyses of NF-κB expression and translocation (Figures \n3 and \n4). Exposure of A549 cells to LTA for 1 h enhanced levels of cytosolic NF-κB (Figure \n3A, top panel, lane 1). After treatment for 6 and 24 h, the expression of cytosolic NF-κB was obviously augmented (lanes 3 and 4). β-Actin was immunodetected (Figure \n3A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n3B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased NF-κB production by 181%, 200%, and 230%, respectively.\nEffects of lipoteichoic acid (LTA) on the expression of the transcription factor, nuclear factor (NF)-κB. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Levels of cytosolic NF-κB p65 (cNF-κB) were immunodetected (A, top panel). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\nEffects of lipoteichoic acid (LTA) on translocation of the transcription factor, nuclear factor (NF)-κB, from the cytoplasm to nuclei. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Amounts of nuclear NF-κB p65 (nNF-κB) were immunodetected (A, top panel). Proliferating cell nuclear antigen (PCNA) was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\nTreatment of A549 cells with LTA for 1 h increased levels of nuclear NF-κB (Figure \n4A, top panel, lane 2). When exposed for 6 and 24 h, translocation of NF-κB from the cytoplasm to nuclei notably increased (lanes 3 and 4). Amounts of PCNA in A549 cells were immunodetected (Figure \n4A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n4B). Exposure of A549 cells to LTA for 1, 6, and 24 h respectively caused significant 176%, 340%, 530% enhancements in levels of nuclear NF-κB.\nMechanisms of LTA-induced SP-A augmentation were evaluated by analyses of NF-κB expression and translocation (Figures \n3 and \n4). Exposure of A549 cells to LTA for 1 h enhanced levels of cytosolic NF-κB (Figure \n3A, top panel, lane 1). After treatment for 6 and 24 h, the expression of cytosolic NF-κB was obviously augmented (lanes 3 and 4). β-Actin was immunodetected (Figure \n3A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n3B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased NF-κB production by 181%, 200%, and 230%, respectively.\nEffects of lipoteichoic acid (LTA) on the expression of the transcription factor, nuclear factor (NF)-κB. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Levels of cytosolic NF-κB p65 (cNF-κB) were immunodetected (A, top panel). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\nEffects of lipoteichoic acid (LTA) on translocation of the transcription factor, nuclear factor (NF)-κB, from the cytoplasm to nuclei. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Amounts of nuclear NF-κB p65 (nNF-κB) were immunodetected (A, top panel). Proliferating cell nuclear antigen (PCNA) was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\nTreatment of A549 cells with LTA for 1 h increased levels of nuclear NF-κB (Figure \n4A, top panel, lane 2). When exposed for 6 and 24 h, translocation of NF-κB from the cytoplasm to nuclei notably increased (lanes 3 and 4). Amounts of PCNA in A549 cells were immunodetected (Figure \n4A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n4B). Exposure of A549 cells to LTA for 1, 6, and 24 h respectively caused significant 176%, 340%, 530% enhancements in levels of nuclear NF-κB.\n LTA-enhanced phosphorylation of ERK1/2 The reason why LTA improved NF-κB activation was further investigated by assaying ERK1/2 phosphorylation (Figure \n5). Treatment of A549 cells with LTA for 1 h increased the amounts of phosphorylated ERK1/2 (Figure \n5A, top panel, lane 2). Levels of phosphorylated ERK1/2 in A549 cells were obviously raised after exposure to LTA for 6 and 24 h (lanes 3 and 4). Amounts of β-actin in A549 cells were immunodetected (Figure \n5A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n5B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased ERK1 phosphorylation by 259%, 170%, and 334%, respectively. In comparison, levels of phosphorylated ERK2 were respectively augmented by 8.2-, 6.4-, and 7.8-fold following LTA administration for 1, 6, and 24 h (Figure \n5B).\nEffects of lipoteichoic acid (LTA) on the phosphorylation of extracellular signal-regulated kinase (ERK)1/2. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated ERK1/2 (p-ERK1/2) were immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.\nThe reason why LTA improved NF-κB activation was further investigated by assaying ERK1/2 phosphorylation (Figure \n5). Treatment of A549 cells with LTA for 1 h increased the amounts of phosphorylated ERK1/2 (Figure \n5A, top panel, lane 2). Levels of phosphorylated ERK1/2 in A549 cells were obviously raised after exposure to LTA for 6 and 24 h (lanes 3 and 4). Amounts of β-actin in A549 cells were immunodetected (Figure \n5A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n5B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased ERK1 phosphorylation by 259%, 170%, and 334%, respectively. In comparison, levels of phosphorylated ERK2 were respectively augmented by 8.2-, 6.4-, and 7.8-fold following LTA administration for 1, 6, and 24 h (Figure \n5B).\nEffects of lipoteichoic acid (LTA) on the phosphorylation of extracellular signal-regulated kinase (ERK)1/2. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated ERK1/2 (p-ERK1/2) were immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.\n LTA-induced activation of MEK1 Phosphorylation of MEK1 was assayed to determine the mechanism of LTA-induced ERK1/2 activation (Figure \n6). Low levels of phosphorylated MEK1 were detected in untreated A549 cells (Figure \n6A, top panel, lane 1). However, exposure of A549 cells to LTA for 1 h stimulated MEK1 phosphorylation (lane 2). After exposure for 6 and 24 h, the amounts of phosphorylated MEK1 had obviously increased (lanes 3 and 4). β-actin in A549 cells was immunodetected (Figure \n6A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n6B). Treatment of A549 cells with LTA for 1, 6, and 24 h respectively caused significant 82%, 330%, and 370% increases in levels of phosphorylated MEK1.\nEffects of lipoteichoic acid (LTA) on the phosphorylation of mitogen-activated/extracellular signal-regulated kinase kinase (MEK)1. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated MEK1 (p-MEK1) was immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.\nPhosphorylation of MEK1 was assayed to determine the mechanism of LTA-induced ERK1/2 activation (Figure \n6). Low levels of phosphorylated MEK1 were detected in untreated A549 cells (Figure \n6A, top panel, lane 1). However, exposure of A549 cells to LTA for 1 h stimulated MEK1 phosphorylation (lane 2). After exposure for 6 and 24 h, the amounts of phosphorylated MEK1 had obviously increased (lanes 3 and 4). β-actin in A549 cells was immunodetected (Figure \n6A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n6B). Treatment of A549 cells with LTA for 1, 6, and 24 h respectively caused significant 82%, 330%, and 370% increases in levels of phosphorylated MEK1.\nEffects of lipoteichoic acid (LTA) on the phosphorylation of mitogen-activated/extracellular signal-regulated kinase kinase (MEK)1. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated MEK1 (p-MEK1) was immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"In summary, we used an alveolar epithelial cell model to study the immunomodulatory responses of LTA. Our results revealed that LTA can induce inflammatory responses in alveolar epithelial A549 cells by means of enhancing SP-A mRNA and protein syntheses. Moreover, the signal-transducing mechanisms of LTA-caused regulation of SP-A expression arise through the cascade phosphorylations of MEK1 and ERK1/2. In succession, LTA increased NF-κB expression and translocation. LTA-induced SP-A production in alveolar type II epithelial cells may indicate the status of gram-positive bacteria-caused septic shock and acute lung injury. More molecular pathways should be investigated and proven in the future. However, there are certain limitations of this study, including the use of A549 cells, which are derived from human lung carcinoma. The effects of LTA on A549 cells may differ from those on normal alveolar epithelial cells. Thus, we will perform a translational study to evaluate the effects of LTA on alveolar epithelial cells of animals with acute lung injury."},"other_sections_titles":{"kind":"list like","value":["Background","Cell culture and drug treatment","Assay of cell viability","Immunoblotting analyses of SP-A, NF-κB, and phosphorylated and non-phosphorylated ERK1/2 and MEK1","Extraction of nuclear proteins and immunodetection","Real-time polymerase chain reaction (PCR) assays","Statistical analysis","Toxicity of LTA to A549 cells","LTA-induced enhancement of SP-A biosynthesis in A549 cells","LTA-induced SP-A mRNA expression in A549 cells","Augmentation of NF-κB expression and translocation by LTA","LTA-enhanced phosphorylation of ERK1/2","LTA-induced activation of MEK1","Abbreviations","Competing interests","Authors’ contributions"],"string":"[\n \"Background\",\n \"Cell culture and drug treatment\",\n \"Assay of cell viability\",\n \"Immunoblotting analyses of SP-A, NF-κB, and phosphorylated and non-phosphorylated ERK1/2 and MEK1\",\n \"Extraction of nuclear proteins and immunodetection\",\n \"Real-time polymerase chain reaction (PCR) assays\",\n \"Statistical analysis\",\n \"Toxicity of LTA to A549 cells\",\n \"LTA-induced enhancement of SP-A biosynthesis in A549 cells\",\n \"LTA-induced SP-A mRNA expression in A549 cells\",\n \"Augmentation of NF-κB expression and translocation by LTA\",\n \"LTA-enhanced phosphorylation of ERK1/2\",\n \"LTA-induced activation of MEK1\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Sepsis can lead to multiorgan failure and death and appears to be triggered by bacterial products, such as lipopolysaccharide (LPS) from gram-negative bacteria and lipoteichoic acid (LTA) from gram-positive ones \n[1-3]. Infection of the respiratory tract caused by gram-positive bacteria and pneumonia combined with acute lung injury (ALI) are usually the leading causes of mortality by sepsis \n[4]. In the past few decades, the incidences of sepsis and septic shock have been increasing \n[5]. Although endotoxin-activated events are clearly important in gram-negative infection, gram-positive bacteria also have crucial roles, but less is known about host responses to them \n[6]. The increasing prevalence of sepsis from gram-positive bacterial pathogens necessitates a reevaluation of the basic assumptions about the molecular pathogenesis of ALI.\nAlveolar epithelial type II cells contribute to the maintenance of mucosal integrity by modulating the production of surfactants \n[7]. Pulmonary surfactants play important roles in protecting the lung during endotoxin-induced injury and infection \n[8,9]. Surfactant protein (SP)-A is the most abundant pulmonary surfactant protein. Levels of SP-A in bronchiolar lavage fluid are modulated in gram-negative or -positive bacteria-caused lung diseases, including severe pneumonia, acute respiratory distress syndrome, and cardiogenic lung edema \n[10]. Thus, altering lung SP-A levels can be an effective indicator for pulmonary infection and inflammation. Our previous study showed that LPS selectively induced spa gene expression in human alveolar epithelial A549 cells \n[11].\nLTA, an outer membrane component of gram-positive bacteria, was shown to be one of the critical factors participating in the pathogenesis of sepsis \n[12,13]. LTA can stimulate inflammatory responses in the lung \n[14,15]. Therefore, understanding the mechanisms that regulate LTA-mediated cell activation is crucial for diagnosis, treatment, or prognosis of lung inflammatory diseases. In response to stimuli, LTA can activate macrophages to produce massive amounts of inflammatory factors that exhibit systemic effects in the general circulation \n[16]. LTA can induce the secretion of various cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α \n[17]. These data suggest that LTA can selectively modify gene transcription of various cell types and sequentially augment and possibly initiate tissue inflammation.\nMitogen-activated protein kinases (MAPKs) are serine/threonine kinases. The first MAPK isoforms to be cloned and characterized were the extracellular signal-regulated kinase 1 and 2 (ERK 1/2) \n[18,19]. ERK 1/2 are well documented to be activated by a family of dual-specificity kinases known as the mitogen-activated/ERK kinases (MEKs) \n[16,20]. A previous study demonstrated that LTA can selectively activate the ERK pathway in the cornea \n[21]. Our previous study showed that LTA induced TNF-α and IL-6 expressions by means of stimulating phosphorylation of ERK1/2 in macrophages \n[16]. In addition, LTA also triggered translocation of nuclear factor (NF)-κB from the cytoplasm to nuclei and its transactivation activity. Meanwhile, the mechanisms responsible for LTA-induced spa gene expression in alveolar epithelial cells are still unknown. In this study, we attempted to evaluate the effects of LTA on SP-1 synthesis in human alveolar type II epithelial cells and its possible mechanisms.","A human lung carcinoma type II epithelial cell line (A549) was cultured following a previous method \n[3]. A549 cells were grown in Dulbecco's modified Eagle's medium (DMEM)/Ham’s F12 culture medium (Sigma, St. Louis, MO, USA), supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 U/ml penicillin G, 100 μg/ml streptomycin, and 2 mM l-glutamine. A549 cells were seeded in 75-cm2 culture flasks at 37 °C in a humidified atmosphere of 5% CO2. Cells were grown to confluence before drug treatment. LTA purchased from Sigma was dissolved in phosphate-buffered saline (PBS) (0.14 M NaCl, 2.6 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4). BAY 11–7082, an inhibitor of NF-κB activation, was also purchased from Sigma.","Cell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previously described \n[22]. Briefly, A549 cells (104 cells/well) were seeded in 96-well tissue culture plates overnight. After drug treatment, macrophages were cultured in new medium containing 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide for a further 3 h. The blue formazan products in the macrophages were dissolved in dimethyl sulfoxide and spectrophotometrically measured at a wavelength of 550 nm.","Protein levels were immunodetected according to a previously described method \n[11]. After drug treatment, cell lysates were prepared in ice-cold radioimmunoprecipitation assay buffer (25 mM Tris–HCl (pH 7.2), 0.1% sodium dodecylsulfate (SDS), 1% Triton X-100, 1% sodium deoxycholate, 0.15 M NaCl, and 1 mM EDTA). Protein concentrations were quantified using a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Proteins (50 μg/well) were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Immunodetection of SP-A and NF-κB was carried out using rabbit polyclonal antibodies against human SP-A and NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cellular β-actin protein was immunodetected using a mouse monoclonal antibody (mAb) against mouse β-actin (Sigma) as the internal standard. These protein bands were quantified using a digital imaging system (UVtec, Cambridge, UK). Phosphorylated ERK1/2 and MEK1 were immunodetected using a rabbit polyclonal antibody against phosphorylated residues of ERK1/2 and MEK1 (Cell Signaling, Danvers, MA, USA). Nonphosphorylated ERK2 and MEK1 were immunodetected as the internal controls (Cell Signaling). Intensities of the immunoreactive bands were determined using a digital imaging system (Wallac Victor 1420, PerkinElmer, Melbourne, Australia).","Amounts of nuclear transcription factors were quantified following a previously described method \n[20]. After drug treatment, nuclear extracts of macrophages were prepared. Protein concentrations were quantified by a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Nuclear proteins (50 μg/well) were subjected to SDS-PAGE and transferred to nitrocellulose membranes. After blocking, NF-κB was immunodetected using a rabbit polyclonal antibody against mouse NF-κB p65 (Santa Cruz Biotechnology). A proliferating cell nuclear antigen (PCNA) was detected using a mouse mAb against the rat PCNA protein (Santa Cruz Biotechnology) as the internal standard. Intensities of the immunoreactive bands were determined using a digital imaging system (Wallac Victor 1420, PerkinElmer).","Messenger (m)RNA from A549 cells exposed to LTA were prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Oligonucleotides for the PCR analyses of SP-A mRNA and β-actin mRNA were designed and synthesized by Clontech Laboratories (Palo Alto, CA, USA). The oligonucleotide sequences of the upstream and downstream primers for these mRNA analyses were respectively 5'-TGA AAGGGAGTTCTAGCATCTCACAGA-3' and 5'-ACATATGCCTATGTAGGCCTGACTGAG-3' for SP-A mRNA, and 5'- GTCTACATGTCTCGATCCCACTTA A -3' and 5'-GGTCTTTCTCTCTCATCGCGCTC-5' for β-actin mRNA. A quantitative PCR analysis was carried out using iQSYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad) as described previously \n[11].","Statistical differences were considered significant when the p value of Duncan’s multiple-range test was <0.05. Statistical analysis between groups over time was carried out by a two-way analysis of variance (ANOVA).","Cell morphology and viability were assayed to evaluate the toxicity of LTA to human alveolar epithelial A549 cells. Exposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability (data not shown). When exposed to 30 μg/ml LTA for 1, 6, and 24 h, the viability of A549 cells was not influenced. Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h did not alter cell morphology (data not shown).","The effects of LTA on SP-A levels in A549 cells were evaluated by an immunoblotting analysis (Figure \n1). In untreated A549 cells, low levels of SP-A were immunodetected (Figure \n1A, top panel, lane 1). After exposure to 30 μg/ml LTA for 1 h, levels of SP-A were found to be augmented (lane 2). When treated for 6 and 24 h, LTA obviously increased amounts of SP-A in A549 cells. β-Actin was immunodetected (Figure \n1A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n1B). Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h respectively caused significant 176%, 230%, and 270% increases in SP-A levels.\nEffects of lipoteichoic acid (LTA) on the production of surfactant protein(SP)-A. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h (A). Cellular proteins were prepared for the immunoblotting analyses. Amounts of SP-A were immunodetected (A, top pane). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.","Induction of SP-A mRNA expression by LTA was quantified using a real-time PCR analysis (Figure \n2). After exposure to LTA for 1 h, the levels of SP-A mRNA in A549 cells were increased by 2.1-fold. Exposure of A549 cells to LTA for 6 and 24 h caused 2.8- and 3.7-fold increases in the levels of SP-A mRNA, respectively (Figure \n2). Pretreatment of A549 cells with BAY 11–7082, an inhibitor of NF-κB activation, for 1 h did not change SP-A mRNA expression (data not shown). However, BAY 11–7082 significantly inhibited LTA-induced SP-A mRNA production by 70% (Figure \n2).\nEffects of lipoteichoic acid (LTA) on induction of surfactant protein(SP)-A mRNA. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. In addition, A549 cells were pretreated with BAY 11–7082 (BAY), an inhibitor of NF-κB activation, for 1 h and then exposed to LTA for another 24 h. mRNA was prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Each value represents the mean ± SEM for n = 3. Symbols * and # indicate that the value significantly (p < 0.05) differed from the respective control and LTA-treated group, respectively.","Mechanisms of LTA-induced SP-A augmentation were evaluated by analyses of NF-κB expression and translocation (Figures \n3 and \n4). Exposure of A549 cells to LTA for 1 h enhanced levels of cytosolic NF-κB (Figure \n3A, top panel, lane 1). After treatment for 6 and 24 h, the expression of cytosolic NF-κB was obviously augmented (lanes 3 and 4). β-Actin was immunodetected (Figure \n3A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n3B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased NF-κB production by 181%, 200%, and 230%, respectively.\nEffects of lipoteichoic acid (LTA) on the expression of the transcription factor, nuclear factor (NF)-κB. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Levels of cytosolic NF-κB p65 (cNF-κB) were immunodetected (A, top panel). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\nEffects of lipoteichoic acid (LTA) on translocation of the transcription factor, nuclear factor (NF)-κB, from the cytoplasm to nuclei. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Amounts of nuclear NF-κB p65 (nNF-κB) were immunodetected (A, top panel). Proliferating cell nuclear antigen (PCNA) was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\nTreatment of A549 cells with LTA for 1 h increased levels of nuclear NF-κB (Figure \n4A, top panel, lane 2). When exposed for 6 and 24 h, translocation of NF-κB from the cytoplasm to nuclei notably increased (lanes 3 and 4). Amounts of PCNA in A549 cells were immunodetected (Figure \n4A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n4B). Exposure of A549 cells to LTA for 1, 6, and 24 h respectively caused significant 176%, 340%, 530% enhancements in levels of nuclear NF-κB.","The reason why LTA improved NF-κB activation was further investigated by assaying ERK1/2 phosphorylation (Figure \n5). Treatment of A549 cells with LTA for 1 h increased the amounts of phosphorylated ERK1/2 (Figure \n5A, top panel, lane 2). Levels of phosphorylated ERK1/2 in A549 cells were obviously raised after exposure to LTA for 6 and 24 h (lanes 3 and 4). Amounts of β-actin in A549 cells were immunodetected (Figure \n5A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n5B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased ERK1 phosphorylation by 259%, 170%, and 334%, respectively. In comparison, levels of phosphorylated ERK2 were respectively augmented by 8.2-, 6.4-, and 7.8-fold following LTA administration for 1, 6, and 24 h (Figure \n5B).\nEffects of lipoteichoic acid (LTA) on the phosphorylation of extracellular signal-regulated kinase (ERK)1/2. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated ERK1/2 (p-ERK1/2) were immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.","Phosphorylation of MEK1 was assayed to determine the mechanism of LTA-induced ERK1/2 activation (Figure \n6). Low levels of phosphorylated MEK1 were detected in untreated A549 cells (Figure \n6A, top panel, lane 1). However, exposure of A549 cells to LTA for 1 h stimulated MEK1 phosphorylation (lane 2). After exposure for 6 and 24 h, the amounts of phosphorylated MEK1 had obviously increased (lanes 3 and 4). β-actin in A549 cells was immunodetected (Figure \n6A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n6B). Treatment of A549 cells with LTA for 1, 6, and 24 h respectively caused significant 82%, 330%, and 370% increases in levels of phosphorylated MEK1.\nEffects of lipoteichoic acid (LTA) on the phosphorylation of mitogen-activated/extracellular signal-regulated kinase kinase (MEK)1. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated MEK1 (p-MEK1) was immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.","ALI: Acute lung injury; ERK1/2: Extracellular signal-regulated kinase 1/2; IL: Interleukin; LPS: Lipopolysaccharide; LTA: Lipoteichoic acid; NF-κB: Nuclear factor-κB; MAPKs: Mitogen-activated protein kinases; MEK1: Mitogen-activated/extracellular signal-regulated kinase kinase 1; PCNA: Proliferating cell nuclear antigen; SDS-PAGE: Sodium dodecylsulfate polyacrylamide gel electrophoresis; SP-A: Surfactant protein-A; TLR2: Toll-like receptor 2; TNF-α: Tumor necrosis factor-α.","The authors declare that they have no competing interests.","FLL, CYC, and RMC visualized experimental design. YTT and HLT refined the experimental approach. TGC did the statistical analysis. TLC had significant intellectual input into the development of this work, and added to the Discussion. All authors reviewed data and results, and had significant input into the writing of the final manuscript. All authors read and approved the final manuscript."],"string":"[\n \"Sepsis can lead to multiorgan failure and death and appears to be triggered by bacterial products, such as lipopolysaccharide (LPS) from gram-negative bacteria and lipoteichoic acid (LTA) from gram-positive ones \\n[1-3]. Infection of the respiratory tract caused by gram-positive bacteria and pneumonia combined with acute lung injury (ALI) are usually the leading causes of mortality by sepsis \\n[4]. In the past few decades, the incidences of sepsis and septic shock have been increasing \\n[5]. Although endotoxin-activated events are clearly important in gram-negative infection, gram-positive bacteria also have crucial roles, but less is known about host responses to them \\n[6]. The increasing prevalence of sepsis from gram-positive bacterial pathogens necessitates a reevaluation of the basic assumptions about the molecular pathogenesis of ALI.\\nAlveolar epithelial type II cells contribute to the maintenance of mucosal integrity by modulating the production of surfactants \\n[7]. Pulmonary surfactants play important roles in protecting the lung during endotoxin-induced injury and infection \\n[8,9]. Surfactant protein (SP)-A is the most abundant pulmonary surfactant protein. Levels of SP-A in bronchiolar lavage fluid are modulated in gram-negative or -positive bacteria-caused lung diseases, including severe pneumonia, acute respiratory distress syndrome, and cardiogenic lung edema \\n[10]. Thus, altering lung SP-A levels can be an effective indicator for pulmonary infection and inflammation. Our previous study showed that LPS selectively induced spa gene expression in human alveolar epithelial A549 cells \\n[11].\\nLTA, an outer membrane component of gram-positive bacteria, was shown to be one of the critical factors participating in the pathogenesis of sepsis \\n[12,13]. LTA can stimulate inflammatory responses in the lung \\n[14,15]. Therefore, understanding the mechanisms that regulate LTA-mediated cell activation is crucial for diagnosis, treatment, or prognosis of lung inflammatory diseases. In response to stimuli, LTA can activate macrophages to produce massive amounts of inflammatory factors that exhibit systemic effects in the general circulation \\n[16]. LTA can induce the secretion of various cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α \\n[17]. These data suggest that LTA can selectively modify gene transcription of various cell types and sequentially augment and possibly initiate tissue inflammation.\\nMitogen-activated protein kinases (MAPKs) are serine/threonine kinases. The first MAPK isoforms to be cloned and characterized were the extracellular signal-regulated kinase 1 and 2 (ERK 1/2) \\n[18,19]. ERK 1/2 are well documented to be activated by a family of dual-specificity kinases known as the mitogen-activated/ERK kinases (MEKs) \\n[16,20]. A previous study demonstrated that LTA can selectively activate the ERK pathway in the cornea \\n[21]. Our previous study showed that LTA induced TNF-α and IL-6 expressions by means of stimulating phosphorylation of ERK1/2 in macrophages \\n[16]. In addition, LTA also triggered translocation of nuclear factor (NF)-κB from the cytoplasm to nuclei and its transactivation activity. Meanwhile, the mechanisms responsible for LTA-induced spa gene expression in alveolar epithelial cells are still unknown. In this study, we attempted to evaluate the effects of LTA on SP-1 synthesis in human alveolar type II epithelial cells and its possible mechanisms.\",\n \"A human lung carcinoma type II epithelial cell line (A549) was cultured following a previous method \\n[3]. A549 cells were grown in Dulbecco's modified Eagle's medium (DMEM)/Ham’s F12 culture medium (Sigma, St. Louis, MO, USA), supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 U/ml penicillin G, 100 μg/ml streptomycin, and 2 mM l-glutamine. A549 cells were seeded in 75-cm2 culture flasks at 37 °C in a humidified atmosphere of 5% CO2. Cells were grown to confluence before drug treatment. LTA purchased from Sigma was dissolved in phosphate-buffered saline (PBS) (0.14 M NaCl, 2.6 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4). BAY 11–7082, an inhibitor of NF-κB activation, was also purchased from Sigma.\",\n \"Cell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previously described \\n[22]. Briefly, A549 cells (104 cells/well) were seeded in 96-well tissue culture plates overnight. After drug treatment, macrophages were cultured in new medium containing 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide for a further 3 h. The blue formazan products in the macrophages were dissolved in dimethyl sulfoxide and spectrophotometrically measured at a wavelength of 550 nm.\",\n \"Protein levels were immunodetected according to a previously described method \\n[11]. After drug treatment, cell lysates were prepared in ice-cold radioimmunoprecipitation assay buffer (25 mM Tris–HCl (pH 7.2), 0.1% sodium dodecylsulfate (SDS), 1% Triton X-100, 1% sodium deoxycholate, 0.15 M NaCl, and 1 mM EDTA). Protein concentrations were quantified using a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Proteins (50 μg/well) were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Immunodetection of SP-A and NF-κB was carried out using rabbit polyclonal antibodies against human SP-A and NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cellular β-actin protein was immunodetected using a mouse monoclonal antibody (mAb) against mouse β-actin (Sigma) as the internal standard. These protein bands were quantified using a digital imaging system (UVtec, Cambridge, UK). Phosphorylated ERK1/2 and MEK1 were immunodetected using a rabbit polyclonal antibody against phosphorylated residues of ERK1/2 and MEK1 (Cell Signaling, Danvers, MA, USA). Nonphosphorylated ERK2 and MEK1 were immunodetected as the internal controls (Cell Signaling). Intensities of the immunoreactive bands were determined using a digital imaging system (Wallac Victor 1420, PerkinElmer, Melbourne, Australia).\",\n \"Amounts of nuclear transcription factors were quantified following a previously described method \\n[20]. After drug treatment, nuclear extracts of macrophages were prepared. Protein concentrations were quantified by a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Nuclear proteins (50 μg/well) were subjected to SDS-PAGE and transferred to nitrocellulose membranes. After blocking, NF-κB was immunodetected using a rabbit polyclonal antibody against mouse NF-κB p65 (Santa Cruz Biotechnology). A proliferating cell nuclear antigen (PCNA) was detected using a mouse mAb against the rat PCNA protein (Santa Cruz Biotechnology) as the internal standard. Intensities of the immunoreactive bands were determined using a digital imaging system (Wallac Victor 1420, PerkinElmer).\",\n \"Messenger (m)RNA from A549 cells exposed to LTA were prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Oligonucleotides for the PCR analyses of SP-A mRNA and β-actin mRNA were designed and synthesized by Clontech Laboratories (Palo Alto, CA, USA). The oligonucleotide sequences of the upstream and downstream primers for these mRNA analyses were respectively 5'-TGA AAGGGAGTTCTAGCATCTCACAGA-3' and 5'-ACATATGCCTATGTAGGCCTGACTGAG-3' for SP-A mRNA, and 5'- GTCTACATGTCTCGATCCCACTTA A -3' and 5'-GGTCTTTCTCTCTCATCGCGCTC-5' for β-actin mRNA. A quantitative PCR analysis was carried out using iQSYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad) as described previously \\n[11].\",\n \"Statistical differences were considered significant when the p value of Duncan’s multiple-range test was <0.05. Statistical analysis between groups over time was carried out by a two-way analysis of variance (ANOVA).\",\n \"Cell morphology and viability were assayed to evaluate the toxicity of LTA to human alveolar epithelial A549 cells. Exposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability (data not shown). When exposed to 30 μg/ml LTA for 1, 6, and 24 h, the viability of A549 cells was not influenced. Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h did not alter cell morphology (data not shown).\",\n \"The effects of LTA on SP-A levels in A549 cells were evaluated by an immunoblotting analysis (Figure \\n1). In untreated A549 cells, low levels of SP-A were immunodetected (Figure \\n1A, top panel, lane 1). After exposure to 30 μg/ml LTA for 1 h, levels of SP-A were found to be augmented (lane 2). When treated for 6 and 24 h, LTA obviously increased amounts of SP-A in A549 cells. β-Actin was immunodetected (Figure \\n1A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \\n1B). Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h respectively caused significant 176%, 230%, and 270% increases in SP-A levels.\\nEffects of lipoteichoic acid (LTA) on the production of surfactant protein(SP)-A. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h (A). Cellular proteins were prepared for the immunoblotting analyses. Amounts of SP-A were immunodetected (A, top pane). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\",\n \"Induction of SP-A mRNA expression by LTA was quantified using a real-time PCR analysis (Figure \\n2). After exposure to LTA for 1 h, the levels of SP-A mRNA in A549 cells were increased by 2.1-fold. Exposure of A549 cells to LTA for 6 and 24 h caused 2.8- and 3.7-fold increases in the levels of SP-A mRNA, respectively (Figure \\n2). Pretreatment of A549 cells with BAY 11–7082, an inhibitor of NF-κB activation, for 1 h did not change SP-A mRNA expression (data not shown). However, BAY 11–7082 significantly inhibited LTA-induced SP-A mRNA production by 70% (Figure \\n2).\\nEffects of lipoteichoic acid (LTA) on induction of surfactant protein(SP)-A mRNA. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. In addition, A549 cells were pretreated with BAY 11–7082 (BAY), an inhibitor of NF-κB activation, for 1 h and then exposed to LTA for another 24 h. mRNA was prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Each value represents the mean ± SEM for n = 3. Symbols * and # indicate that the value significantly (p < 0.05) differed from the respective control and LTA-treated group, respectively.\",\n \"Mechanisms of LTA-induced SP-A augmentation were evaluated by analyses of NF-κB expression and translocation (Figures \\n3 and \\n4). Exposure of A549 cells to LTA for 1 h enhanced levels of cytosolic NF-κB (Figure \\n3A, top panel, lane 1). After treatment for 6 and 24 h, the expression of cytosolic NF-κB was obviously augmented (lanes 3 and 4). β-Actin was immunodetected (Figure \\n3A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \\n3B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased NF-κB production by 181%, 200%, and 230%, respectively.\\nEffects of lipoteichoic acid (LTA) on the expression of the transcription factor, nuclear factor (NF)-κB. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Levels of cytosolic NF-κB p65 (cNF-κB) were immunodetected (A, top panel). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\\nEffects of lipoteichoic acid (LTA) on translocation of the transcription factor, nuclear factor (NF)-κB, from the cytoplasm to nuclei. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Amounts of nuclear NF-κB p65 (nNF-κB) were immunodetected (A, top panel). Proliferating cell nuclear antigen (PCNA) was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\\nTreatment of A549 cells with LTA for 1 h increased levels of nuclear NF-κB (Figure \\n4A, top panel, lane 2). When exposed for 6 and 24 h, translocation of NF-κB from the cytoplasm to nuclei notably increased (lanes 3 and 4). Amounts of PCNA in A549 cells were immunodetected (Figure \\n4A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \\n4B). Exposure of A549 cells to LTA for 1, 6, and 24 h respectively caused significant 176%, 340%, 530% enhancements in levels of nuclear NF-κB.\",\n \"The reason why LTA improved NF-κB activation was further investigated by assaying ERK1/2 phosphorylation (Figure \\n5). Treatment of A549 cells with LTA for 1 h increased the amounts of phosphorylated ERK1/2 (Figure \\n5A, top panel, lane 2). Levels of phosphorylated ERK1/2 in A549 cells were obviously raised after exposure to LTA for 6 and 24 h (lanes 3 and 4). Amounts of β-actin in A549 cells were immunodetected (Figure \\n5A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \\n5B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased ERK1 phosphorylation by 259%, 170%, and 334%, respectively. In comparison, levels of phosphorylated ERK2 were respectively augmented by 8.2-, 6.4-, and 7.8-fold following LTA administration for 1, 6, and 24 h (Figure \\n5B).\\nEffects of lipoteichoic acid (LTA) on the phosphorylation of extracellular signal-regulated kinase (ERK)1/2. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated ERK1/2 (p-ERK1/2) were immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.\",\n \"Phosphorylation of MEK1 was assayed to determine the mechanism of LTA-induced ERK1/2 activation (Figure \\n6). Low levels of phosphorylated MEK1 were detected in untreated A549 cells (Figure \\n6A, top panel, lane 1). However, exposure of A549 cells to LTA for 1 h stimulated MEK1 phosphorylation (lane 2). After exposure for 6 and 24 h, the amounts of phosphorylated MEK1 had obviously increased (lanes 3 and 4). β-actin in A549 cells was immunodetected (Figure \\n6A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \\n6B). Treatment of A549 cells with LTA for 1, 6, and 24 h respectively caused significant 82%, 330%, and 370% increases in levels of phosphorylated MEK1.\\nEffects of lipoteichoic acid (LTA) on the phosphorylation of mitogen-activated/extracellular signal-regulated kinase kinase (MEK)1. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated MEK1 (p-MEK1) was immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.\",\n \"ALI: Acute lung injury; ERK1/2: Extracellular signal-regulated kinase 1/2; IL: Interleukin; LPS: Lipopolysaccharide; LTA: Lipoteichoic acid; NF-κB: Nuclear factor-κB; MAPKs: Mitogen-activated protein kinases; MEK1: Mitogen-activated/extracellular signal-regulated kinase kinase 1; PCNA: Proliferating cell nuclear antigen; SDS-PAGE: Sodium dodecylsulfate polyacrylamide gel electrophoresis; SP-A: Surfactant protein-A; TLR2: Toll-like receptor 2; TNF-α: Tumor necrosis factor-α.\",\n \"The authors declare that they have no competing interests.\",\n \"FLL, CYC, and RMC visualized experimental design. YTT and HLT refined the experimental approach. TGC did the statistical analysis. TLC had significant intellectual input into the development of this work, and added to the Discussion. All authors reviewed data and results, and had significant input into the writing of the final manuscript. All authors read and approved the final manuscript.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Background","Materials and methods","Cell culture and drug treatment","Assay of cell viability","Immunoblotting analyses of SP-A, NF-κB, and phosphorylated and non-phosphorylated ERK1/2 and MEK1","Extraction of nuclear proteins and immunodetection","Real-time polymerase chain reaction (PCR) assays","Statistical analysis","Results","Toxicity of LTA to A549 cells","LTA-induced enhancement of SP-A biosynthesis in A549 cells","LTA-induced SP-A mRNA expression in A549 cells","Augmentation of NF-κB expression and translocation by LTA","LTA-enhanced phosphorylation of ERK1/2","LTA-induced activation of MEK1","Discussion","Conclusions","Abbreviations","Competing interests","Authors’ contributions"],"string":"[\n \"Background\",\n \"Materials and methods\",\n \"Cell culture and drug treatment\",\n \"Assay of cell viability\",\n \"Immunoblotting analyses of SP-A, NF-κB, and phosphorylated and non-phosphorylated ERK1/2 and MEK1\",\n \"Extraction of nuclear proteins and immunodetection\",\n \"Real-time polymerase chain reaction (PCR) assays\",\n \"Statistical analysis\",\n \"Results\",\n \"Toxicity of LTA to A549 cells\",\n \"LTA-induced enhancement of SP-A biosynthesis in A549 cells\",\n \"LTA-induced SP-A mRNA expression in A549 cells\",\n \"Augmentation of NF-κB expression and translocation by LTA\",\n \"LTA-enhanced phosphorylation of ERK1/2\",\n \"LTA-induced activation of MEK1\",\n \"Discussion\",\n \"Conclusions\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Sepsis can lead to multiorgan failure and death and appears to be triggered by bacterial products, such as lipopolysaccharide (LPS) from gram-negative bacteria and lipoteichoic acid (LTA) from gram-positive ones \n[1-3]. Infection of the respiratory tract caused by gram-positive bacteria and pneumonia combined with acute lung injury (ALI) are usually the leading causes of mortality by sepsis \n[4]. In the past few decades, the incidences of sepsis and septic shock have been increasing \n[5]. Although endotoxin-activated events are clearly important in gram-negative infection, gram-positive bacteria also have crucial roles, but less is known about host responses to them \n[6]. The increasing prevalence of sepsis from gram-positive bacterial pathogens necessitates a reevaluation of the basic assumptions about the molecular pathogenesis of ALI.\nAlveolar epithelial type II cells contribute to the maintenance of mucosal integrity by modulating the production of surfactants \n[7]. Pulmonary surfactants play important roles in protecting the lung during endotoxin-induced injury and infection \n[8,9]. Surfactant protein (SP)-A is the most abundant pulmonary surfactant protein. Levels of SP-A in bronchiolar lavage fluid are modulated in gram-negative or -positive bacteria-caused lung diseases, including severe pneumonia, acute respiratory distress syndrome, and cardiogenic lung edema \n[10]. Thus, altering lung SP-A levels can be an effective indicator for pulmonary infection and inflammation. Our previous study showed that LPS selectively induced spa gene expression in human alveolar epithelial A549 cells \n[11].\nLTA, an outer membrane component of gram-positive bacteria, was shown to be one of the critical factors participating in the pathogenesis of sepsis \n[12,13]. LTA can stimulate inflammatory responses in the lung \n[14,15]. Therefore, understanding the mechanisms that regulate LTA-mediated cell activation is crucial for diagnosis, treatment, or prognosis of lung inflammatory diseases. In response to stimuli, LTA can activate macrophages to produce massive amounts of inflammatory factors that exhibit systemic effects in the general circulation \n[16]. LTA can induce the secretion of various cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α \n[17]. These data suggest that LTA can selectively modify gene transcription of various cell types and sequentially augment and possibly initiate tissue inflammation.\nMitogen-activated protein kinases (MAPKs) are serine/threonine kinases. The first MAPK isoforms to be cloned and characterized were the extracellular signal-regulated kinase 1 and 2 (ERK 1/2) \n[18,19]. ERK 1/2 are well documented to be activated by a family of dual-specificity kinases known as the mitogen-activated/ERK kinases (MEKs) \n[16,20]. A previous study demonstrated that LTA can selectively activate the ERK pathway in the cornea \n[21]. Our previous study showed that LTA induced TNF-α and IL-6 expressions by means of stimulating phosphorylation of ERK1/2 in macrophages \n[16]. In addition, LTA also triggered translocation of nuclear factor (NF)-κB from the cytoplasm to nuclei and its transactivation activity. Meanwhile, the mechanisms responsible for LTA-induced spa gene expression in alveolar epithelial cells are still unknown. In this study, we attempted to evaluate the effects of LTA on SP-1 synthesis in human alveolar type II epithelial cells and its possible mechanisms."," Cell culture and drug treatment A human lung carcinoma type II epithelial cell line (A549) was cultured following a previous method \n[3]. A549 cells were grown in Dulbecco's modified Eagle's medium (DMEM)/Ham’s F12 culture medium (Sigma, St. Louis, MO, USA), supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 U/ml penicillin G, 100 μg/ml streptomycin, and 2 mM l-glutamine. A549 cells were seeded in 75-cm2 culture flasks at 37 °C in a humidified atmosphere of 5% CO2. Cells were grown to confluence before drug treatment. LTA purchased from Sigma was dissolved in phosphate-buffered saline (PBS) (0.14 M NaCl, 2.6 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4). BAY 11–7082, an inhibitor of NF-κB activation, was also purchased from Sigma.\nA human lung carcinoma type II epithelial cell line (A549) was cultured following a previous method \n[3]. A549 cells were grown in Dulbecco's modified Eagle's medium (DMEM)/Ham’s F12 culture medium (Sigma, St. Louis, MO, USA), supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 U/ml penicillin G, 100 μg/ml streptomycin, and 2 mM l-glutamine. A549 cells were seeded in 75-cm2 culture flasks at 37 °C in a humidified atmosphere of 5% CO2. Cells were grown to confluence before drug treatment. LTA purchased from Sigma was dissolved in phosphate-buffered saline (PBS) (0.14 M NaCl, 2.6 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4). BAY 11–7082, an inhibitor of NF-κB activation, was also purchased from Sigma.\n Assay of cell viability Cell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previously described \n[22]. Briefly, A549 cells (104 cells/well) were seeded in 96-well tissue culture plates overnight. After drug treatment, macrophages were cultured in new medium containing 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide for a further 3 h. The blue formazan products in the macrophages were dissolved in dimethyl sulfoxide and spectrophotometrically measured at a wavelength of 550 nm.\nCell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previously described \n[22]. Briefly, A549 cells (104 cells/well) were seeded in 96-well tissue culture plates overnight. After drug treatment, macrophages were cultured in new medium containing 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide for a further 3 h. The blue formazan products in the macrophages were dissolved in dimethyl sulfoxide and spectrophotometrically measured at a wavelength of 550 nm.\n Immunoblotting analyses of SP-A, NF-κB, and phosphorylated and non-phosphorylated ERK1/2 and MEK1 Protein levels were immunodetected according to a previously described method \n[11]. After drug treatment, cell lysates were prepared in ice-cold radioimmunoprecipitation assay buffer (25 mM Tris–HCl (pH 7.2), 0.1% sodium dodecylsulfate (SDS), 1% Triton X-100, 1% sodium deoxycholate, 0.15 M NaCl, and 1 mM EDTA). Protein concentrations were quantified using a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Proteins (50 μg/well) were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Immunodetection of SP-A and NF-κB was carried out using rabbit polyclonal antibodies against human SP-A and NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cellular β-actin protein was immunodetected using a mouse monoclonal antibody (mAb) against mouse β-actin (Sigma) as the internal standard. These protein bands were quantified using a digital imaging system (UVtec, Cambridge, UK). Phosphorylated ERK1/2 and MEK1 were immunodetected using a rabbit polyclonal antibody against phosphorylated residues of ERK1/2 and MEK1 (Cell Signaling, Danvers, MA, USA). Nonphosphorylated ERK2 and MEK1 were immunodetected as the internal controls (Cell Signaling). Intensities of the immunoreactive bands were determined using a digital imaging system (Wallac Victor 1420, PerkinElmer, Melbourne, Australia).\nProtein levels were immunodetected according to a previously described method \n[11]. After drug treatment, cell lysates were prepared in ice-cold radioimmunoprecipitation assay buffer (25 mM Tris–HCl (pH 7.2), 0.1% sodium dodecylsulfate (SDS), 1% Triton X-100, 1% sodium deoxycholate, 0.15 M NaCl, and 1 mM EDTA). Protein concentrations were quantified using a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Proteins (50 μg/well) were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Immunodetection of SP-A and NF-κB was carried out using rabbit polyclonal antibodies against human SP-A and NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cellular β-actin protein was immunodetected using a mouse monoclonal antibody (mAb) against mouse β-actin (Sigma) as the internal standard. These protein bands were quantified using a digital imaging system (UVtec, Cambridge, UK). Phosphorylated ERK1/2 and MEK1 were immunodetected using a rabbit polyclonal antibody against phosphorylated residues of ERK1/2 and MEK1 (Cell Signaling, Danvers, MA, USA). Nonphosphorylated ERK2 and MEK1 were immunodetected as the internal controls (Cell Signaling). Intensities of the immunoreactive bands were determined using a digital imaging system (Wallac Victor 1420, PerkinElmer, Melbourne, Australia).\n Extraction of nuclear proteins and immunodetection Amounts of nuclear transcription factors were quantified following a previously described method \n[20]. After drug treatment, nuclear extracts of macrophages were prepared. Protein concentrations were quantified by a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Nuclear proteins (50 μg/well) were subjected to SDS-PAGE and transferred to nitrocellulose membranes. After blocking, NF-κB was immunodetected using a rabbit polyclonal antibody against mouse NF-κB p65 (Santa Cruz Biotechnology). A proliferating cell nuclear antigen (PCNA) was detected using a mouse mAb against the rat PCNA protein (Santa Cruz Biotechnology) as the internal standard. Intensities of the immunoreactive bands were determined using a digital imaging system (Wallac Victor 1420, PerkinElmer).\nAmounts of nuclear transcription factors were quantified following a previously described method \n[20]. After drug treatment, nuclear extracts of macrophages were prepared. Protein concentrations were quantified by a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Nuclear proteins (50 μg/well) were subjected to SDS-PAGE and transferred to nitrocellulose membranes. After blocking, NF-κB was immunodetected using a rabbit polyclonal antibody against mouse NF-κB p65 (Santa Cruz Biotechnology). A proliferating cell nuclear antigen (PCNA) was detected using a mouse mAb against the rat PCNA protein (Santa Cruz Biotechnology) as the internal standard. Intensities of the immunoreactive bands were determined using a digital imaging system (Wallac Victor 1420, PerkinElmer).\n Real-time polymerase chain reaction (PCR) assays Messenger (m)RNA from A549 cells exposed to LTA were prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Oligonucleotides for the PCR analyses of SP-A mRNA and β-actin mRNA were designed and synthesized by Clontech Laboratories (Palo Alto, CA, USA). The oligonucleotide sequences of the upstream and downstream primers for these mRNA analyses were respectively 5'-TGA AAGGGAGTTCTAGCATCTCACAGA-3' and 5'-ACATATGCCTATGTAGGCCTGACTGAG-3' for SP-A mRNA, and 5'- GTCTACATGTCTCGATCCCACTTA A -3' and 5'-GGTCTTTCTCTCTCATCGCGCTC-5' for β-actin mRNA. A quantitative PCR analysis was carried out using iQSYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad) as described previously \n[11].\nMessenger (m)RNA from A549 cells exposed to LTA were prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Oligonucleotides for the PCR analyses of SP-A mRNA and β-actin mRNA were designed and synthesized by Clontech Laboratories (Palo Alto, CA, USA). The oligonucleotide sequences of the upstream and downstream primers for these mRNA analyses were respectively 5'-TGA AAGGGAGTTCTAGCATCTCACAGA-3' and 5'-ACATATGCCTATGTAGGCCTGACTGAG-3' for SP-A mRNA, and 5'- GTCTACATGTCTCGATCCCACTTA A -3' and 5'-GGTCTTTCTCTCTCATCGCGCTC-5' for β-actin mRNA. A quantitative PCR analysis was carried out using iQSYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad) as described previously \n[11].\n Statistical analysis Statistical differences were considered significant when the p value of Duncan’s multiple-range test was <0.05. Statistical analysis between groups over time was carried out by a two-way analysis of variance (ANOVA).\nStatistical differences were considered significant when the p value of Duncan’s multiple-range test was <0.05. Statistical analysis between groups over time was carried out by a two-way analysis of variance (ANOVA).","A human lung carcinoma type II epithelial cell line (A549) was cultured following a previous method \n[3]. A549 cells were grown in Dulbecco's modified Eagle's medium (DMEM)/Ham’s F12 culture medium (Sigma, St. Louis, MO, USA), supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 U/ml penicillin G, 100 μg/ml streptomycin, and 2 mM l-glutamine. A549 cells were seeded in 75-cm2 culture flasks at 37 °C in a humidified atmosphere of 5% CO2. Cells were grown to confluence before drug treatment. LTA purchased from Sigma was dissolved in phosphate-buffered saline (PBS) (0.14 M NaCl, 2.6 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4). BAY 11–7082, an inhibitor of NF-κB activation, was also purchased from Sigma.","Cell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previously described \n[22]. Briefly, A549 cells (104 cells/well) were seeded in 96-well tissue culture plates overnight. After drug treatment, macrophages were cultured in new medium containing 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide for a further 3 h. The blue formazan products in the macrophages were dissolved in dimethyl sulfoxide and spectrophotometrically measured at a wavelength of 550 nm.","Protein levels were immunodetected according to a previously described method \n[11]. After drug treatment, cell lysates were prepared in ice-cold radioimmunoprecipitation assay buffer (25 mM Tris–HCl (pH 7.2), 0.1% sodium dodecylsulfate (SDS), 1% Triton X-100, 1% sodium deoxycholate, 0.15 M NaCl, and 1 mM EDTA). Protein concentrations were quantified using a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Proteins (50 μg/well) were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Immunodetection of SP-A and NF-κB was carried out using rabbit polyclonal antibodies against human SP-A and NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cellular β-actin protein was immunodetected using a mouse monoclonal antibody (mAb) against mouse β-actin (Sigma) as the internal standard. These protein bands were quantified using a digital imaging system (UVtec, Cambridge, UK). Phosphorylated ERK1/2 and MEK1 were immunodetected using a rabbit polyclonal antibody against phosphorylated residues of ERK1/2 and MEK1 (Cell Signaling, Danvers, MA, USA). Nonphosphorylated ERK2 and MEK1 were immunodetected as the internal controls (Cell Signaling). Intensities of the immunoreactive bands were determined using a digital imaging system (Wallac Victor 1420, PerkinElmer, Melbourne, Australia).","Amounts of nuclear transcription factors were quantified following a previously described method \n[20]. After drug treatment, nuclear extracts of macrophages were prepared. Protein concentrations were quantified by a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Nuclear proteins (50 μg/well) were subjected to SDS-PAGE and transferred to nitrocellulose membranes. After blocking, NF-κB was immunodetected using a rabbit polyclonal antibody against mouse NF-κB p65 (Santa Cruz Biotechnology). A proliferating cell nuclear antigen (PCNA) was detected using a mouse mAb against the rat PCNA protein (Santa Cruz Biotechnology) as the internal standard. Intensities of the immunoreactive bands were determined using a digital imaging system (Wallac Victor 1420, PerkinElmer).","Messenger (m)RNA from A549 cells exposed to LTA were prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Oligonucleotides for the PCR analyses of SP-A mRNA and β-actin mRNA were designed and synthesized by Clontech Laboratories (Palo Alto, CA, USA). The oligonucleotide sequences of the upstream and downstream primers for these mRNA analyses were respectively 5'-TGA AAGGGAGTTCTAGCATCTCACAGA-3' and 5'-ACATATGCCTATGTAGGCCTGACTGAG-3' for SP-A mRNA, and 5'- GTCTACATGTCTCGATCCCACTTA A -3' and 5'-GGTCTTTCTCTCTCATCGCGCTC-5' for β-actin mRNA. A quantitative PCR analysis was carried out using iQSYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad) as described previously \n[11].","Statistical differences were considered significant when the p value of Duncan’s multiple-range test was <0.05. Statistical analysis between groups over time was carried out by a two-way analysis of variance (ANOVA)."," Toxicity of LTA to A549 cells Cell morphology and viability were assayed to evaluate the toxicity of LTA to human alveolar epithelial A549 cells. Exposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability (data not shown). When exposed to 30 μg/ml LTA for 1, 6, and 24 h, the viability of A549 cells was not influenced. Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h did not alter cell morphology (data not shown).\nCell morphology and viability were assayed to evaluate the toxicity of LTA to human alveolar epithelial A549 cells. Exposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability (data not shown). When exposed to 30 μg/ml LTA for 1, 6, and 24 h, the viability of A549 cells was not influenced. Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h did not alter cell morphology (data not shown).\n LTA-induced enhancement of SP-A biosynthesis in A549 cells The effects of LTA on SP-A levels in A549 cells were evaluated by an immunoblotting analysis (Figure \n1). In untreated A549 cells, low levels of SP-A were immunodetected (Figure \n1A, top panel, lane 1). After exposure to 30 μg/ml LTA for 1 h, levels of SP-A were found to be augmented (lane 2). When treated for 6 and 24 h, LTA obviously increased amounts of SP-A in A549 cells. β-Actin was immunodetected (Figure \n1A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n1B). Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h respectively caused significant 176%, 230%, and 270% increases in SP-A levels.\nEffects of lipoteichoic acid (LTA) on the production of surfactant protein(SP)-A. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h (A). Cellular proteins were prepared for the immunoblotting analyses. Amounts of SP-A were immunodetected (A, top pane). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\nThe effects of LTA on SP-A levels in A549 cells were evaluated by an immunoblotting analysis (Figure \n1). In untreated A549 cells, low levels of SP-A were immunodetected (Figure \n1A, top panel, lane 1). After exposure to 30 μg/ml LTA for 1 h, levels of SP-A were found to be augmented (lane 2). When treated for 6 and 24 h, LTA obviously increased amounts of SP-A in A549 cells. β-Actin was immunodetected (Figure \n1A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n1B). Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h respectively caused significant 176%, 230%, and 270% increases in SP-A levels.\nEffects of lipoteichoic acid (LTA) on the production of surfactant protein(SP)-A. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h (A). Cellular proteins were prepared for the immunoblotting analyses. Amounts of SP-A were immunodetected (A, top pane). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\n LTA-induced SP-A mRNA expression in A549 cells Induction of SP-A mRNA expression by LTA was quantified using a real-time PCR analysis (Figure \n2). After exposure to LTA for 1 h, the levels of SP-A mRNA in A549 cells were increased by 2.1-fold. Exposure of A549 cells to LTA for 6 and 24 h caused 2.8- and 3.7-fold increases in the levels of SP-A mRNA, respectively (Figure \n2). Pretreatment of A549 cells with BAY 11–7082, an inhibitor of NF-κB activation, for 1 h did not change SP-A mRNA expression (data not shown). However, BAY 11–7082 significantly inhibited LTA-induced SP-A mRNA production by 70% (Figure \n2).\nEffects of lipoteichoic acid (LTA) on induction of surfactant protein(SP)-A mRNA. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. In addition, A549 cells were pretreated with BAY 11–7082 (BAY), an inhibitor of NF-κB activation, for 1 h and then exposed to LTA for another 24 h. mRNA was prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Each value represents the mean ± SEM for n = 3. Symbols * and # indicate that the value significantly (p < 0.05) differed from the respective control and LTA-treated group, respectively.\nInduction of SP-A mRNA expression by LTA was quantified using a real-time PCR analysis (Figure \n2). After exposure to LTA for 1 h, the levels of SP-A mRNA in A549 cells were increased by 2.1-fold. Exposure of A549 cells to LTA for 6 and 24 h caused 2.8- and 3.7-fold increases in the levels of SP-A mRNA, respectively (Figure \n2). Pretreatment of A549 cells with BAY 11–7082, an inhibitor of NF-κB activation, for 1 h did not change SP-A mRNA expression (data not shown). However, BAY 11–7082 significantly inhibited LTA-induced SP-A mRNA production by 70% (Figure \n2).\nEffects of lipoteichoic acid (LTA) on induction of surfactant protein(SP)-A mRNA. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. In addition, A549 cells were pretreated with BAY 11–7082 (BAY), an inhibitor of NF-κB activation, for 1 h and then exposed to LTA for another 24 h. mRNA was prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Each value represents the mean ± SEM for n = 3. Symbols * and # indicate that the value significantly (p < 0.05) differed from the respective control and LTA-treated group, respectively.\n Augmentation of NF-κB expression and translocation by LTA Mechanisms of LTA-induced SP-A augmentation were evaluated by analyses of NF-κB expression and translocation (Figures \n3 and \n4). Exposure of A549 cells to LTA for 1 h enhanced levels of cytosolic NF-κB (Figure \n3A, top panel, lane 1). After treatment for 6 and 24 h, the expression of cytosolic NF-κB was obviously augmented (lanes 3 and 4). β-Actin was immunodetected (Figure \n3A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n3B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased NF-κB production by 181%, 200%, and 230%, respectively.\nEffects of lipoteichoic acid (LTA) on the expression of the transcription factor, nuclear factor (NF)-κB. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Levels of cytosolic NF-κB p65 (cNF-κB) were immunodetected (A, top panel). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\nEffects of lipoteichoic acid (LTA) on translocation of the transcription factor, nuclear factor (NF)-κB, from the cytoplasm to nuclei. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Amounts of nuclear NF-κB p65 (nNF-κB) were immunodetected (A, top panel). Proliferating cell nuclear antigen (PCNA) was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\nTreatment of A549 cells with LTA for 1 h increased levels of nuclear NF-κB (Figure \n4A, top panel, lane 2). When exposed for 6 and 24 h, translocation of NF-κB from the cytoplasm to nuclei notably increased (lanes 3 and 4). Amounts of PCNA in A549 cells were immunodetected (Figure \n4A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n4B). Exposure of A549 cells to LTA for 1, 6, and 24 h respectively caused significant 176%, 340%, 530% enhancements in levels of nuclear NF-κB.\nMechanisms of LTA-induced SP-A augmentation were evaluated by analyses of NF-κB expression and translocation (Figures \n3 and \n4). Exposure of A549 cells to LTA for 1 h enhanced levels of cytosolic NF-κB (Figure \n3A, top panel, lane 1). After treatment for 6 and 24 h, the expression of cytosolic NF-κB was obviously augmented (lanes 3 and 4). β-Actin was immunodetected (Figure \n3A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n3B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased NF-κB production by 181%, 200%, and 230%, respectively.\nEffects of lipoteichoic acid (LTA) on the expression of the transcription factor, nuclear factor (NF)-κB. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Levels of cytosolic NF-κB p65 (cNF-κB) were immunodetected (A, top panel). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\nEffects of lipoteichoic acid (LTA) on translocation of the transcription factor, nuclear factor (NF)-κB, from the cytoplasm to nuclei. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Amounts of nuclear NF-κB p65 (nNF-κB) were immunodetected (A, top panel). Proliferating cell nuclear antigen (PCNA) was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\nTreatment of A549 cells with LTA for 1 h increased levels of nuclear NF-κB (Figure \n4A, top panel, lane 2). When exposed for 6 and 24 h, translocation of NF-κB from the cytoplasm to nuclei notably increased (lanes 3 and 4). Amounts of PCNA in A549 cells were immunodetected (Figure \n4A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n4B). Exposure of A549 cells to LTA for 1, 6, and 24 h respectively caused significant 176%, 340%, 530% enhancements in levels of nuclear NF-κB.\n LTA-enhanced phosphorylation of ERK1/2 The reason why LTA improved NF-κB activation was further investigated by assaying ERK1/2 phosphorylation (Figure \n5). Treatment of A549 cells with LTA for 1 h increased the amounts of phosphorylated ERK1/2 (Figure \n5A, top panel, lane 2). Levels of phosphorylated ERK1/2 in A549 cells were obviously raised after exposure to LTA for 6 and 24 h (lanes 3 and 4). Amounts of β-actin in A549 cells were immunodetected (Figure \n5A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n5B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased ERK1 phosphorylation by 259%, 170%, and 334%, respectively. In comparison, levels of phosphorylated ERK2 were respectively augmented by 8.2-, 6.4-, and 7.8-fold following LTA administration for 1, 6, and 24 h (Figure \n5B).\nEffects of lipoteichoic acid (LTA) on the phosphorylation of extracellular signal-regulated kinase (ERK)1/2. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated ERK1/2 (p-ERK1/2) were immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.\nThe reason why LTA improved NF-κB activation was further investigated by assaying ERK1/2 phosphorylation (Figure \n5). Treatment of A549 cells with LTA for 1 h increased the amounts of phosphorylated ERK1/2 (Figure \n5A, top panel, lane 2). Levels of phosphorylated ERK1/2 in A549 cells were obviously raised after exposure to LTA for 6 and 24 h (lanes 3 and 4). Amounts of β-actin in A549 cells were immunodetected (Figure \n5A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n5B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased ERK1 phosphorylation by 259%, 170%, and 334%, respectively. In comparison, levels of phosphorylated ERK2 were respectively augmented by 8.2-, 6.4-, and 7.8-fold following LTA administration for 1, 6, and 24 h (Figure \n5B).\nEffects of lipoteichoic acid (LTA) on the phosphorylation of extracellular signal-regulated kinase (ERK)1/2. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated ERK1/2 (p-ERK1/2) were immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.\n LTA-induced activation of MEK1 Phosphorylation of MEK1 was assayed to determine the mechanism of LTA-induced ERK1/2 activation (Figure \n6). Low levels of phosphorylated MEK1 were detected in untreated A549 cells (Figure \n6A, top panel, lane 1). However, exposure of A549 cells to LTA for 1 h stimulated MEK1 phosphorylation (lane 2). After exposure for 6 and 24 h, the amounts of phosphorylated MEK1 had obviously increased (lanes 3 and 4). β-actin in A549 cells was immunodetected (Figure \n6A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n6B). Treatment of A549 cells with LTA for 1, 6, and 24 h respectively caused significant 82%, 330%, and 370% increases in levels of phosphorylated MEK1.\nEffects of lipoteichoic acid (LTA) on the phosphorylation of mitogen-activated/extracellular signal-regulated kinase kinase (MEK)1. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated MEK1 (p-MEK1) was immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.\nPhosphorylation of MEK1 was assayed to determine the mechanism of LTA-induced ERK1/2 activation (Figure \n6). Low levels of phosphorylated MEK1 were detected in untreated A549 cells (Figure \n6A, top panel, lane 1). However, exposure of A549 cells to LTA for 1 h stimulated MEK1 phosphorylation (lane 2). After exposure for 6 and 24 h, the amounts of phosphorylated MEK1 had obviously increased (lanes 3 and 4). β-actin in A549 cells was immunodetected (Figure \n6A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n6B). Treatment of A549 cells with LTA for 1, 6, and 24 h respectively caused significant 82%, 330%, and 370% increases in levels of phosphorylated MEK1.\nEffects of lipoteichoic acid (LTA) on the phosphorylation of mitogen-activated/extracellular signal-regulated kinase kinase (MEK)1. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated MEK1 (p-MEK1) was immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.","Cell morphology and viability were assayed to evaluate the toxicity of LTA to human alveolar epithelial A549 cells. Exposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability (data not shown). When exposed to 30 μg/ml LTA for 1, 6, and 24 h, the viability of A549 cells was not influenced. Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h did not alter cell morphology (data not shown).","The effects of LTA on SP-A levels in A549 cells were evaluated by an immunoblotting analysis (Figure \n1). In untreated A549 cells, low levels of SP-A were immunodetected (Figure \n1A, top panel, lane 1). After exposure to 30 μg/ml LTA for 1 h, levels of SP-A were found to be augmented (lane 2). When treated for 6 and 24 h, LTA obviously increased amounts of SP-A in A549 cells. β-Actin was immunodetected (Figure \n1A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n1B). Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h respectively caused significant 176%, 230%, and 270% increases in SP-A levels.\nEffects of lipoteichoic acid (LTA) on the production of surfactant protein(SP)-A. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h (A). Cellular proteins were prepared for the immunoblotting analyses. Amounts of SP-A were immunodetected (A, top pane). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.","Induction of SP-A mRNA expression by LTA was quantified using a real-time PCR analysis (Figure \n2). After exposure to LTA for 1 h, the levels of SP-A mRNA in A549 cells were increased by 2.1-fold. Exposure of A549 cells to LTA for 6 and 24 h caused 2.8- and 3.7-fold increases in the levels of SP-A mRNA, respectively (Figure \n2). Pretreatment of A549 cells with BAY 11–7082, an inhibitor of NF-κB activation, for 1 h did not change SP-A mRNA expression (data not shown). However, BAY 11–7082 significantly inhibited LTA-induced SP-A mRNA production by 70% (Figure \n2).\nEffects of lipoteichoic acid (LTA) on induction of surfactant protein(SP)-A mRNA. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. In addition, A549 cells were pretreated with BAY 11–7082 (BAY), an inhibitor of NF-κB activation, for 1 h and then exposed to LTA for another 24 h. mRNA was prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Each value represents the mean ± SEM for n = 3. Symbols * and # indicate that the value significantly (p < 0.05) differed from the respective control and LTA-treated group, respectively.","Mechanisms of LTA-induced SP-A augmentation were evaluated by analyses of NF-κB expression and translocation (Figures \n3 and \n4). Exposure of A549 cells to LTA for 1 h enhanced levels of cytosolic NF-κB (Figure \n3A, top panel, lane 1). After treatment for 6 and 24 h, the expression of cytosolic NF-κB was obviously augmented (lanes 3 and 4). β-Actin was immunodetected (Figure \n3A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n3B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased NF-κB production by 181%, 200%, and 230%, respectively.\nEffects of lipoteichoic acid (LTA) on the expression of the transcription factor, nuclear factor (NF)-κB. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Levels of cytosolic NF-κB p65 (cNF-κB) were immunodetected (A, top panel). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\nEffects of lipoteichoic acid (LTA) on translocation of the transcription factor, nuclear factor (NF)-κB, from the cytoplasm to nuclei. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Amounts of nuclear NF-κB p65 (nNF-κB) were immunodetected (A, top panel). Proliferating cell nuclear antigen (PCNA) was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\nTreatment of A549 cells with LTA for 1 h increased levels of nuclear NF-κB (Figure \n4A, top panel, lane 2). When exposed for 6 and 24 h, translocation of NF-κB from the cytoplasm to nuclei notably increased (lanes 3 and 4). Amounts of PCNA in A549 cells were immunodetected (Figure \n4A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n4B). Exposure of A549 cells to LTA for 1, 6, and 24 h respectively caused significant 176%, 340%, 530% enhancements in levels of nuclear NF-κB.","The reason why LTA improved NF-κB activation was further investigated by assaying ERK1/2 phosphorylation (Figure \n5). Treatment of A549 cells with LTA for 1 h increased the amounts of phosphorylated ERK1/2 (Figure \n5A, top panel, lane 2). Levels of phosphorylated ERK1/2 in A549 cells were obviously raised after exposure to LTA for 6 and 24 h (lanes 3 and 4). Amounts of β-actin in A549 cells were immunodetected (Figure \n5A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n5B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased ERK1 phosphorylation by 259%, 170%, and 334%, respectively. In comparison, levels of phosphorylated ERK2 were respectively augmented by 8.2-, 6.4-, and 7.8-fold following LTA administration for 1, 6, and 24 h (Figure \n5B).\nEffects of lipoteichoic acid (LTA) on the phosphorylation of extracellular signal-regulated kinase (ERK)1/2. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated ERK1/2 (p-ERK1/2) were immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.","Phosphorylation of MEK1 was assayed to determine the mechanism of LTA-induced ERK1/2 activation (Figure \n6). Low levels of phosphorylated MEK1 were detected in untreated A549 cells (Figure \n6A, top panel, lane 1). However, exposure of A549 cells to LTA for 1 h stimulated MEK1 phosphorylation (lane 2). After exposure for 6 and 24 h, the amounts of phosphorylated MEK1 had obviously increased (lanes 3 and 4). β-actin in A549 cells was immunodetected (Figure \n6A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n6B). Treatment of A549 cells with LTA for 1, 6, and 24 h respectively caused significant 82%, 330%, and 370% increases in levels of phosphorylated MEK1.\nEffects of lipoteichoic acid (LTA) on the phosphorylation of mitogen-activated/extracellular signal-regulated kinase kinase (MEK)1. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated MEK1 (p-MEK1) was immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.","LTA represents a class of amphiphilic molecules anchored to the outer face of the cytoplasmic membrane in gram-positive bacteria and is commonly released during cell growth, especially under antibiotic therapy \n[1,2]. It can cause cytokine induction in mononuclear phagocytes \n[17]. In previous studies, LTA concentrations of 0.2~50 μg/ml were detected and stimulated activity of polymononuclear leucocyte functions and release of TNF-α in peripheral blood mononuclear cells \n[23,24]. Meanwhile, LTA levels at the infectious site can reach a high level of 26,694 ng/mL \n[25]. The concentration of LTA used in this study was < 50 μg/ml. Therefore, our results show that LTA at clinically relevant concentrations can activate alveolar type II epithelial cells by stimulating production of surfactants.\nDuring bacterial infection, endotoxins, including LTA and LPS, increase capillary permeability and enhance expressions of cellular adhesion molecules, proinflammatory cytokines, and chemokines \n[1,15]. These endotoxins can lead to most of the clinical manifestations of bacterial infection and are associated with ALI \n[4,5]. In addition, LTA can trigger lung inflammation and causes neutrophil influx into the lungs \n[15,26]. This study showed that in response to LTA stimulation, levels of SP-A mRNA and protein in alveolar A549 cells were time-dependently augmented. SP-A contributes to the pulmonary host defense \n[10,16,27]. A previous study reported that when spa gene expression was knocked-out, susceptibility of the lungs to pathogenic infection was simultaneously raised \n[28]. Our previous study also showed that LPS-mediated toll-like receptor (TLR) 2 signaling in human alveolar epithelial cells might increase SP-A biosynthesis and subsequently lead to an inflammatory response in the lungs \n[3]. As a result, SP-A could be an effective biomarker for detecting pulmonary infection by gram-negative or -positive bacteria.\nThis study showed that LTA increased the expression of NF-κB and its translocation from the cytoplasm to nuclei. NF-κB is a typical transcription factor in response to stimulation by LTA \n[16]. LTA can bind CD14 and then stimulates TLR activation \n[16,29]. After LTA associates with TLR2, NF-κB can be activated by protein kinases and is then translocated to nuclei from the cytoplasm \n[11]. NF-κB regulates certain gene expressions to control cell proliferation, differentiation, and death \n[30,31]. A previous study showed that LTA induced cyclooxygenase-2 expression in epithelial cells via IκB degradation and successive p65 NF-κB translocation \n[32]. LTA could induce SP-A mRNA expression in A549 cells. Our bioinformatic search revealed that NF-κB-DNA-binding motifs were found in the promoter regions of the spa gene. Suppressing NF-κB activation using BAY 11–7082 simultaneously inhibited LTA-induced SP-A mRNA expression. Thus, LTA transcriptionally induces SP-A expression through inducing NF-κB expression and translocation.\nOur present results revealed that the phosphorylation of ERK1/2 was associated with NF-κB activation. Sequentially, ERK1/2-activated IκBα kinase can phosphorylate IκB at two conserved serine residues in the N-terminus, triggering the degradation of this inhibitor and allowing for the rapid translocation of NF-κB into nuclei \n[16,20]. Accordingly, LTA-induced activation of A549 cells is mainly due to the improvement in ERK1/2 phosphorylation. Roles of ERK1 and ERK2 in LTA-induced SP-A expression were not determined in this study but will be validated using RNA interference in our next study. There is growing evidence that the ERK signaling pathway, which contributes to regulating inflammatory events \n[33]. Therefore, LTA regulates SP-A expression in alveolar type II epithelial cells in the course of eliciting ERK1/2 phosphorylation and subsequent activation of the transcription factor, NF-κB.\nERK activation is mediated by at least three different pathways: a Raf/MEK-dependent pathway, a PI3K/Raf-independent pathway that strongly activates MEK, and a third undetermined pathway that directly activates ERK proteins \n[34]. This study showed that LTA time-dependently increased levels of phosphorylated MEK1. Thus, one of the possible reasons explaining why LTA stimulates ERK1/2 activation is the increase in MEK1 phosphorylation. MAPK-regulating signals place this family of protein kinases in an apparently linear signaling cascade downstream of growth factor receptors, adaptor proteins, guanine-nucleotide exchange factors, Ras, Raf, and MEK \n[19]. The present study demonstrates that LTA can induce SP-A expression via MEK-dependent activation of the protein kinase ERK1/2-signaling pathway.","In summary, we used an alveolar epithelial cell model to study the immunomodulatory responses of LTA. Our results revealed that LTA can induce inflammatory responses in alveolar epithelial A549 cells by means of enhancing SP-A mRNA and protein syntheses. Moreover, the signal-transducing mechanisms of LTA-caused regulation of SP-A expression arise through the cascade phosphorylations of MEK1 and ERK1/2. In succession, LTA increased NF-κB expression and translocation. LTA-induced SP-A production in alveolar type II epithelial cells may indicate the status of gram-positive bacteria-caused septic shock and acute lung injury. More molecular pathways should be investigated and proven in the future. However, there are certain limitations of this study, including the use of A549 cells, which are derived from human lung carcinoma. The effects of LTA on A549 cells may differ from those on normal alveolar epithelial cells. Thus, we will perform a translational study to evaluate the effects of LTA on alveolar epithelial cells of animals with acute lung injury.","ALI: Acute lung injury; ERK1/2: Extracellular signal-regulated kinase 1/2; IL: Interleukin; LPS: Lipopolysaccharide; LTA: Lipoteichoic acid; NF-κB: Nuclear factor-κB; MAPKs: Mitogen-activated protein kinases; MEK1: Mitogen-activated/extracellular signal-regulated kinase kinase 1; PCNA: Proliferating cell nuclear antigen; SDS-PAGE: Sodium dodecylsulfate polyacrylamide gel electrophoresis; SP-A: Surfactant protein-A; TLR2: Toll-like receptor 2; TNF-α: Tumor necrosis factor-α.","The authors declare that they have no competing interests.","FLL, CYC, and RMC visualized experimental design. YTT and HLT refined the experimental approach. TGC did the statistical analysis. TLC had significant intellectual input into the development of this work, and added to the Discussion. All authors reviewed data and results, and had significant input into the writing of the final manuscript. All authors read and approved the final manuscript."],"string":"[\n \"Sepsis can lead to multiorgan failure and death and appears to be triggered by bacterial products, such as lipopolysaccharide (LPS) from gram-negative bacteria and lipoteichoic acid (LTA) from gram-positive ones \\n[1-3]. Infection of the respiratory tract caused by gram-positive bacteria and pneumonia combined with acute lung injury (ALI) are usually the leading causes of mortality by sepsis \\n[4]. In the past few decades, the incidences of sepsis and septic shock have been increasing \\n[5]. Although endotoxin-activated events are clearly important in gram-negative infection, gram-positive bacteria also have crucial roles, but less is known about host responses to them \\n[6]. The increasing prevalence of sepsis from gram-positive bacterial pathogens necessitates a reevaluation of the basic assumptions about the molecular pathogenesis of ALI.\\nAlveolar epithelial type II cells contribute to the maintenance of mucosal integrity by modulating the production of surfactants \\n[7]. Pulmonary surfactants play important roles in protecting the lung during endotoxin-induced injury and infection \\n[8,9]. Surfactant protein (SP)-A is the most abundant pulmonary surfactant protein. Levels of SP-A in bronchiolar lavage fluid are modulated in gram-negative or -positive bacteria-caused lung diseases, including severe pneumonia, acute respiratory distress syndrome, and cardiogenic lung edema \\n[10]. Thus, altering lung SP-A levels can be an effective indicator for pulmonary infection and inflammation. Our previous study showed that LPS selectively induced spa gene expression in human alveolar epithelial A549 cells \\n[11].\\nLTA, an outer membrane component of gram-positive bacteria, was shown to be one of the critical factors participating in the pathogenesis of sepsis \\n[12,13]. LTA can stimulate inflammatory responses in the lung \\n[14,15]. Therefore, understanding the mechanisms that regulate LTA-mediated cell activation is crucial for diagnosis, treatment, or prognosis of lung inflammatory diseases. In response to stimuli, LTA can activate macrophages to produce massive amounts of inflammatory factors that exhibit systemic effects in the general circulation \\n[16]. LTA can induce the secretion of various cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α \\n[17]. These data suggest that LTA can selectively modify gene transcription of various cell types and sequentially augment and possibly initiate tissue inflammation.\\nMitogen-activated protein kinases (MAPKs) are serine/threonine kinases. The first MAPK isoforms to be cloned and characterized were the extracellular signal-regulated kinase 1 and 2 (ERK 1/2) \\n[18,19]. ERK 1/2 are well documented to be activated by a family of dual-specificity kinases known as the mitogen-activated/ERK kinases (MEKs) \\n[16,20]. A previous study demonstrated that LTA can selectively activate the ERK pathway in the cornea \\n[21]. Our previous study showed that LTA induced TNF-α and IL-6 expressions by means of stimulating phosphorylation of ERK1/2 in macrophages \\n[16]. In addition, LTA also triggered translocation of nuclear factor (NF)-κB from the cytoplasm to nuclei and its transactivation activity. Meanwhile, the mechanisms responsible for LTA-induced spa gene expression in alveolar epithelial cells are still unknown. In this study, we attempted to evaluate the effects of LTA on SP-1 synthesis in human alveolar type II epithelial cells and its possible mechanisms.\",\n \" Cell culture and drug treatment A human lung carcinoma type II epithelial cell line (A549) was cultured following a previous method \\n[3]. A549 cells were grown in Dulbecco's modified Eagle's medium (DMEM)/Ham’s F12 culture medium (Sigma, St. Louis, MO, USA), supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 U/ml penicillin G, 100 μg/ml streptomycin, and 2 mM l-glutamine. A549 cells were seeded in 75-cm2 culture flasks at 37 °C in a humidified atmosphere of 5% CO2. Cells were grown to confluence before drug treatment. LTA purchased from Sigma was dissolved in phosphate-buffered saline (PBS) (0.14 M NaCl, 2.6 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4). BAY 11–7082, an inhibitor of NF-κB activation, was also purchased from Sigma.\\nA human lung carcinoma type II epithelial cell line (A549) was cultured following a previous method \\n[3]. A549 cells were grown in Dulbecco's modified Eagle's medium (DMEM)/Ham’s F12 culture medium (Sigma, St. Louis, MO, USA), supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 U/ml penicillin G, 100 μg/ml streptomycin, and 2 mM l-glutamine. A549 cells were seeded in 75-cm2 culture flasks at 37 °C in a humidified atmosphere of 5% CO2. Cells were grown to confluence before drug treatment. LTA purchased from Sigma was dissolved in phosphate-buffered saline (PBS) (0.14 M NaCl, 2.6 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4). BAY 11–7082, an inhibitor of NF-κB activation, was also purchased from Sigma.\\n Assay of cell viability Cell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previously described \\n[22]. Briefly, A549 cells (104 cells/well) were seeded in 96-well tissue culture plates overnight. After drug treatment, macrophages were cultured in new medium containing 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide for a further 3 h. The blue formazan products in the macrophages were dissolved in dimethyl sulfoxide and spectrophotometrically measured at a wavelength of 550 nm.\\nCell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previously described \\n[22]. Briefly, A549 cells (104 cells/well) were seeded in 96-well tissue culture plates overnight. After drug treatment, macrophages were cultured in new medium containing 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide for a further 3 h. The blue formazan products in the macrophages were dissolved in dimethyl sulfoxide and spectrophotometrically measured at a wavelength of 550 nm.\\n Immunoblotting analyses of SP-A, NF-κB, and phosphorylated and non-phosphorylated ERK1/2 and MEK1 Protein levels were immunodetected according to a previously described method \\n[11]. After drug treatment, cell lysates were prepared in ice-cold radioimmunoprecipitation assay buffer (25 mM Tris–HCl (pH 7.2), 0.1% sodium dodecylsulfate (SDS), 1% Triton X-100, 1% sodium deoxycholate, 0.15 M NaCl, and 1 mM EDTA). Protein concentrations were quantified using a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Proteins (50 μg/well) were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Immunodetection of SP-A and NF-κB was carried out using rabbit polyclonal antibodies against human SP-A and NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cellular β-actin protein was immunodetected using a mouse monoclonal antibody (mAb) against mouse β-actin (Sigma) as the internal standard. These protein bands were quantified using a digital imaging system (UVtec, Cambridge, UK). Phosphorylated ERK1/2 and MEK1 were immunodetected using a rabbit polyclonal antibody against phosphorylated residues of ERK1/2 and MEK1 (Cell Signaling, Danvers, MA, USA). Nonphosphorylated ERK2 and MEK1 were immunodetected as the internal controls (Cell Signaling). Intensities of the immunoreactive bands were determined using a digital imaging system (Wallac Victor 1420, PerkinElmer, Melbourne, Australia).\\nProtein levels were immunodetected according to a previously described method \\n[11]. After drug treatment, cell lysates were prepared in ice-cold radioimmunoprecipitation assay buffer (25 mM Tris–HCl (pH 7.2), 0.1% sodium dodecylsulfate (SDS), 1% Triton X-100, 1% sodium deoxycholate, 0.15 M NaCl, and 1 mM EDTA). Protein concentrations were quantified using a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Proteins (50 μg/well) were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Immunodetection of SP-A and NF-κB was carried out using rabbit polyclonal antibodies against human SP-A and NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cellular β-actin protein was immunodetected using a mouse monoclonal antibody (mAb) against mouse β-actin (Sigma) as the internal standard. These protein bands were quantified using a digital imaging system (UVtec, Cambridge, UK). Phosphorylated ERK1/2 and MEK1 were immunodetected using a rabbit polyclonal antibody against phosphorylated residues of ERK1/2 and MEK1 (Cell Signaling, Danvers, MA, USA). Nonphosphorylated ERK2 and MEK1 were immunodetected as the internal controls (Cell Signaling). Intensities of the immunoreactive bands were determined using a digital imaging system (Wallac Victor 1420, PerkinElmer, Melbourne, Australia).\\n Extraction of nuclear proteins and immunodetection Amounts of nuclear transcription factors were quantified following a previously described method \\n[20]. After drug treatment, nuclear extracts of macrophages were prepared. Protein concentrations were quantified by a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Nuclear proteins (50 μg/well) were subjected to SDS-PAGE and transferred to nitrocellulose membranes. After blocking, NF-κB was immunodetected using a rabbit polyclonal antibody against mouse NF-κB p65 (Santa Cruz Biotechnology). A proliferating cell nuclear antigen (PCNA) was detected using a mouse mAb against the rat PCNA protein (Santa Cruz Biotechnology) as the internal standard. Intensities of the immunoreactive bands were determined using a digital imaging system (Wallac Victor 1420, PerkinElmer).\\nAmounts of nuclear transcription factors were quantified following a previously described method \\n[20]. After drug treatment, nuclear extracts of macrophages were prepared. Protein concentrations were quantified by a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Nuclear proteins (50 μg/well) were subjected to SDS-PAGE and transferred to nitrocellulose membranes. After blocking, NF-κB was immunodetected using a rabbit polyclonal antibody against mouse NF-κB p65 (Santa Cruz Biotechnology). A proliferating cell nuclear antigen (PCNA) was detected using a mouse mAb against the rat PCNA protein (Santa Cruz Biotechnology) as the internal standard. Intensities of the immunoreactive bands were determined using a digital imaging system (Wallac Victor 1420, PerkinElmer).\\n Real-time polymerase chain reaction (PCR) assays Messenger (m)RNA from A549 cells exposed to LTA were prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Oligonucleotides for the PCR analyses of SP-A mRNA and β-actin mRNA were designed and synthesized by Clontech Laboratories (Palo Alto, CA, USA). The oligonucleotide sequences of the upstream and downstream primers for these mRNA analyses were respectively 5'-TGA AAGGGAGTTCTAGCATCTCACAGA-3' and 5'-ACATATGCCTATGTAGGCCTGACTGAG-3' for SP-A mRNA, and 5'- GTCTACATGTCTCGATCCCACTTA A -3' and 5'-GGTCTTTCTCTCTCATCGCGCTC-5' for β-actin mRNA. A quantitative PCR analysis was carried out using iQSYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad) as described previously \\n[11].\\nMessenger (m)RNA from A549 cells exposed to LTA were prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Oligonucleotides for the PCR analyses of SP-A mRNA and β-actin mRNA were designed and synthesized by Clontech Laboratories (Palo Alto, CA, USA). The oligonucleotide sequences of the upstream and downstream primers for these mRNA analyses were respectively 5'-TGA AAGGGAGTTCTAGCATCTCACAGA-3' and 5'-ACATATGCCTATGTAGGCCTGACTGAG-3' for SP-A mRNA, and 5'- GTCTACATGTCTCGATCCCACTTA A -3' and 5'-GGTCTTTCTCTCTCATCGCGCTC-5' for β-actin mRNA. A quantitative PCR analysis was carried out using iQSYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad) as described previously \\n[11].\\n Statistical analysis Statistical differences were considered significant when the p value of Duncan’s multiple-range test was <0.05. Statistical analysis between groups over time was carried out by a two-way analysis of variance (ANOVA).\\nStatistical differences were considered significant when the p value of Duncan’s multiple-range test was <0.05. Statistical analysis between groups over time was carried out by a two-way analysis of variance (ANOVA).\",\n \"A human lung carcinoma type II epithelial cell line (A549) was cultured following a previous method \\n[3]. A549 cells were grown in Dulbecco's modified Eagle's medium (DMEM)/Ham’s F12 culture medium (Sigma, St. Louis, MO, USA), supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 U/ml penicillin G, 100 μg/ml streptomycin, and 2 mM l-glutamine. A549 cells were seeded in 75-cm2 culture flasks at 37 °C in a humidified atmosphere of 5% CO2. Cells were grown to confluence before drug treatment. LTA purchased from Sigma was dissolved in phosphate-buffered saline (PBS) (0.14 M NaCl, 2.6 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4). BAY 11–7082, an inhibitor of NF-κB activation, was also purchased from Sigma.\",\n \"Cell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previously described \\n[22]. Briefly, A549 cells (104 cells/well) were seeded in 96-well tissue culture plates overnight. After drug treatment, macrophages were cultured in new medium containing 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide for a further 3 h. The blue formazan products in the macrophages were dissolved in dimethyl sulfoxide and spectrophotometrically measured at a wavelength of 550 nm.\",\n \"Protein levels were immunodetected according to a previously described method \\n[11]. After drug treatment, cell lysates were prepared in ice-cold radioimmunoprecipitation assay buffer (25 mM Tris–HCl (pH 7.2), 0.1% sodium dodecylsulfate (SDS), 1% Triton X-100, 1% sodium deoxycholate, 0.15 M NaCl, and 1 mM EDTA). Protein concentrations were quantified using a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Proteins (50 μg/well) were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Immunodetection of SP-A and NF-κB was carried out using rabbit polyclonal antibodies against human SP-A and NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cellular β-actin protein was immunodetected using a mouse monoclonal antibody (mAb) against mouse β-actin (Sigma) as the internal standard. These protein bands were quantified using a digital imaging system (UVtec, Cambridge, UK). Phosphorylated ERK1/2 and MEK1 were immunodetected using a rabbit polyclonal antibody against phosphorylated residues of ERK1/2 and MEK1 (Cell Signaling, Danvers, MA, USA). Nonphosphorylated ERK2 and MEK1 were immunodetected as the internal controls (Cell Signaling). Intensities of the immunoreactive bands were determined using a digital imaging system (Wallac Victor 1420, PerkinElmer, Melbourne, Australia).\",\n \"Amounts of nuclear transcription factors were quantified following a previously described method \\n[20]. After drug treatment, nuclear extracts of macrophages were prepared. Protein concentrations were quantified by a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Nuclear proteins (50 μg/well) were subjected to SDS-PAGE and transferred to nitrocellulose membranes. After blocking, NF-κB was immunodetected using a rabbit polyclonal antibody against mouse NF-κB p65 (Santa Cruz Biotechnology). A proliferating cell nuclear antigen (PCNA) was detected using a mouse mAb against the rat PCNA protein (Santa Cruz Biotechnology) as the internal standard. Intensities of the immunoreactive bands were determined using a digital imaging system (Wallac Victor 1420, PerkinElmer).\",\n \"Messenger (m)RNA from A549 cells exposed to LTA were prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Oligonucleotides for the PCR analyses of SP-A mRNA and β-actin mRNA were designed and synthesized by Clontech Laboratories (Palo Alto, CA, USA). The oligonucleotide sequences of the upstream and downstream primers for these mRNA analyses were respectively 5'-TGA AAGGGAGTTCTAGCATCTCACAGA-3' and 5'-ACATATGCCTATGTAGGCCTGACTGAG-3' for SP-A mRNA, and 5'- GTCTACATGTCTCGATCCCACTTA A -3' and 5'-GGTCTTTCTCTCTCATCGCGCTC-5' for β-actin mRNA. A quantitative PCR analysis was carried out using iQSYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad) as described previously \\n[11].\",\n \"Statistical differences were considered significant when the p value of Duncan’s multiple-range test was <0.05. Statistical analysis between groups over time was carried out by a two-way analysis of variance (ANOVA).\",\n \" Toxicity of LTA to A549 cells Cell morphology and viability were assayed to evaluate the toxicity of LTA to human alveolar epithelial A549 cells. Exposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability (data not shown). When exposed to 30 μg/ml LTA for 1, 6, and 24 h, the viability of A549 cells was not influenced. Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h did not alter cell morphology (data not shown).\\nCell morphology and viability were assayed to evaluate the toxicity of LTA to human alveolar epithelial A549 cells. Exposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability (data not shown). When exposed to 30 μg/ml LTA for 1, 6, and 24 h, the viability of A549 cells was not influenced. Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h did not alter cell morphology (data not shown).\\n LTA-induced enhancement of SP-A biosynthesis in A549 cells The effects of LTA on SP-A levels in A549 cells were evaluated by an immunoblotting analysis (Figure \\n1). In untreated A549 cells, low levels of SP-A were immunodetected (Figure \\n1A, top panel, lane 1). After exposure to 30 μg/ml LTA for 1 h, levels of SP-A were found to be augmented (lane 2). When treated for 6 and 24 h, LTA obviously increased amounts of SP-A in A549 cells. β-Actin was immunodetected (Figure \\n1A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \\n1B). Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h respectively caused significant 176%, 230%, and 270% increases in SP-A levels.\\nEffects of lipoteichoic acid (LTA) on the production of surfactant protein(SP)-A. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h (A). Cellular proteins were prepared for the immunoblotting analyses. Amounts of SP-A were immunodetected (A, top pane). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\\nThe effects of LTA on SP-A levels in A549 cells were evaluated by an immunoblotting analysis (Figure \\n1). In untreated A549 cells, low levels of SP-A were immunodetected (Figure \\n1A, top panel, lane 1). After exposure to 30 μg/ml LTA for 1 h, levels of SP-A were found to be augmented (lane 2). When treated for 6 and 24 h, LTA obviously increased amounts of SP-A in A549 cells. β-Actin was immunodetected (Figure \\n1A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \\n1B). Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h respectively caused significant 176%, 230%, and 270% increases in SP-A levels.\\nEffects of lipoteichoic acid (LTA) on the production of surfactant protein(SP)-A. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h (A). Cellular proteins were prepared for the immunoblotting analyses. Amounts of SP-A were immunodetected (A, top pane). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\\n LTA-induced SP-A mRNA expression in A549 cells Induction of SP-A mRNA expression by LTA was quantified using a real-time PCR analysis (Figure \\n2). After exposure to LTA for 1 h, the levels of SP-A mRNA in A549 cells were increased by 2.1-fold. Exposure of A549 cells to LTA for 6 and 24 h caused 2.8- and 3.7-fold increases in the levels of SP-A mRNA, respectively (Figure \\n2). Pretreatment of A549 cells with BAY 11–7082, an inhibitor of NF-κB activation, for 1 h did not change SP-A mRNA expression (data not shown). However, BAY 11–7082 significantly inhibited LTA-induced SP-A mRNA production by 70% (Figure \\n2).\\nEffects of lipoteichoic acid (LTA) on induction of surfactant protein(SP)-A mRNA. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. In addition, A549 cells were pretreated with BAY 11–7082 (BAY), an inhibitor of NF-κB activation, for 1 h and then exposed to LTA for another 24 h. mRNA was prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Each value represents the mean ± SEM for n = 3. Symbols * and # indicate that the value significantly (p < 0.05) differed from the respective control and LTA-treated group, respectively.\\nInduction of SP-A mRNA expression by LTA was quantified using a real-time PCR analysis (Figure \\n2). After exposure to LTA for 1 h, the levels of SP-A mRNA in A549 cells were increased by 2.1-fold. Exposure of A549 cells to LTA for 6 and 24 h caused 2.8- and 3.7-fold increases in the levels of SP-A mRNA, respectively (Figure \\n2). Pretreatment of A549 cells with BAY 11–7082, an inhibitor of NF-κB activation, for 1 h did not change SP-A mRNA expression (data not shown). However, BAY 11–7082 significantly inhibited LTA-induced SP-A mRNA production by 70% (Figure \\n2).\\nEffects of lipoteichoic acid (LTA) on induction of surfactant protein(SP)-A mRNA. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. In addition, A549 cells were pretreated with BAY 11–7082 (BAY), an inhibitor of NF-κB activation, for 1 h and then exposed to LTA for another 24 h. mRNA was prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Each value represents the mean ± SEM for n = 3. Symbols * and # indicate that the value significantly (p < 0.05) differed from the respective control and LTA-treated group, respectively.\\n Augmentation of NF-κB expression and translocation by LTA Mechanisms of LTA-induced SP-A augmentation were evaluated by analyses of NF-κB expression and translocation (Figures \\n3 and \\n4). Exposure of A549 cells to LTA for 1 h enhanced levels of cytosolic NF-κB (Figure \\n3A, top panel, lane 1). After treatment for 6 and 24 h, the expression of cytosolic NF-κB was obviously augmented (lanes 3 and 4). β-Actin was immunodetected (Figure \\n3A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \\n3B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased NF-κB production by 181%, 200%, and 230%, respectively.\\nEffects of lipoteichoic acid (LTA) on the expression of the transcription factor, nuclear factor (NF)-κB. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Levels of cytosolic NF-κB p65 (cNF-κB) were immunodetected (A, top panel). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\\nEffects of lipoteichoic acid (LTA) on translocation of the transcription factor, nuclear factor (NF)-κB, from the cytoplasm to nuclei. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Amounts of nuclear NF-κB p65 (nNF-κB) were immunodetected (A, top panel). Proliferating cell nuclear antigen (PCNA) was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\\nTreatment of A549 cells with LTA for 1 h increased levels of nuclear NF-κB (Figure \\n4A, top panel, lane 2). When exposed for 6 and 24 h, translocation of NF-κB from the cytoplasm to nuclei notably increased (lanes 3 and 4). Amounts of PCNA in A549 cells were immunodetected (Figure \\n4A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \\n4B). Exposure of A549 cells to LTA for 1, 6, and 24 h respectively caused significant 176%, 340%, 530% enhancements in levels of nuclear NF-κB.\\nMechanisms of LTA-induced SP-A augmentation were evaluated by analyses of NF-κB expression and translocation (Figures \\n3 and \\n4). Exposure of A549 cells to LTA for 1 h enhanced levels of cytosolic NF-κB (Figure \\n3A, top panel, lane 1). After treatment for 6 and 24 h, the expression of cytosolic NF-κB was obviously augmented (lanes 3 and 4). β-Actin was immunodetected (Figure \\n3A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \\n3B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased NF-κB production by 181%, 200%, and 230%, respectively.\\nEffects of lipoteichoic acid (LTA) on the expression of the transcription factor, nuclear factor (NF)-κB. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Levels of cytosolic NF-κB p65 (cNF-κB) were immunodetected (A, top panel). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\\nEffects of lipoteichoic acid (LTA) on translocation of the transcription factor, nuclear factor (NF)-κB, from the cytoplasm to nuclei. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Amounts of nuclear NF-κB p65 (nNF-κB) were immunodetected (A, top panel). Proliferating cell nuclear antigen (PCNA) was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\\nTreatment of A549 cells with LTA for 1 h increased levels of nuclear NF-κB (Figure \\n4A, top panel, lane 2). When exposed for 6 and 24 h, translocation of NF-κB from the cytoplasm to nuclei notably increased (lanes 3 and 4). Amounts of PCNA in A549 cells were immunodetected (Figure \\n4A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \\n4B). Exposure of A549 cells to LTA for 1, 6, and 24 h respectively caused significant 176%, 340%, 530% enhancements in levels of nuclear NF-κB.\\n LTA-enhanced phosphorylation of ERK1/2 The reason why LTA improved NF-κB activation was further investigated by assaying ERK1/2 phosphorylation (Figure \\n5). Treatment of A549 cells with LTA for 1 h increased the amounts of phosphorylated ERK1/2 (Figure \\n5A, top panel, lane 2). Levels of phosphorylated ERK1/2 in A549 cells were obviously raised after exposure to LTA for 6 and 24 h (lanes 3 and 4). Amounts of β-actin in A549 cells were immunodetected (Figure \\n5A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \\n5B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased ERK1 phosphorylation by 259%, 170%, and 334%, respectively. In comparison, levels of phosphorylated ERK2 were respectively augmented by 8.2-, 6.4-, and 7.8-fold following LTA administration for 1, 6, and 24 h (Figure \\n5B).\\nEffects of lipoteichoic acid (LTA) on the phosphorylation of extracellular signal-regulated kinase (ERK)1/2. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated ERK1/2 (p-ERK1/2) were immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.\\nThe reason why LTA improved NF-κB activation was further investigated by assaying ERK1/2 phosphorylation (Figure \\n5). Treatment of A549 cells with LTA for 1 h increased the amounts of phosphorylated ERK1/2 (Figure \\n5A, top panel, lane 2). Levels of phosphorylated ERK1/2 in A549 cells were obviously raised after exposure to LTA for 6 and 24 h (lanes 3 and 4). Amounts of β-actin in A549 cells were immunodetected (Figure \\n5A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \\n5B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased ERK1 phosphorylation by 259%, 170%, and 334%, respectively. In comparison, levels of phosphorylated ERK2 were respectively augmented by 8.2-, 6.4-, and 7.8-fold following LTA administration for 1, 6, and 24 h (Figure \\n5B).\\nEffects of lipoteichoic acid (LTA) on the phosphorylation of extracellular signal-regulated kinase (ERK)1/2. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated ERK1/2 (p-ERK1/2) were immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.\\n LTA-induced activation of MEK1 Phosphorylation of MEK1 was assayed to determine the mechanism of LTA-induced ERK1/2 activation (Figure \\n6). Low levels of phosphorylated MEK1 were detected in untreated A549 cells (Figure \\n6A, top panel, lane 1). However, exposure of A549 cells to LTA for 1 h stimulated MEK1 phosphorylation (lane 2). After exposure for 6 and 24 h, the amounts of phosphorylated MEK1 had obviously increased (lanes 3 and 4). β-actin in A549 cells was immunodetected (Figure \\n6A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \\n6B). Treatment of A549 cells with LTA for 1, 6, and 24 h respectively caused significant 82%, 330%, and 370% increases in levels of phosphorylated MEK1.\\nEffects of lipoteichoic acid (LTA) on the phosphorylation of mitogen-activated/extracellular signal-regulated kinase kinase (MEK)1. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated MEK1 (p-MEK1) was immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.\\nPhosphorylation of MEK1 was assayed to determine the mechanism of LTA-induced ERK1/2 activation (Figure \\n6). Low levels of phosphorylated MEK1 were detected in untreated A549 cells (Figure \\n6A, top panel, lane 1). However, exposure of A549 cells to LTA for 1 h stimulated MEK1 phosphorylation (lane 2). After exposure for 6 and 24 h, the amounts of phosphorylated MEK1 had obviously increased (lanes 3 and 4). β-actin in A549 cells was immunodetected (Figure \\n6A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \\n6B). Treatment of A549 cells with LTA for 1, 6, and 24 h respectively caused significant 82%, 330%, and 370% increases in levels of phosphorylated MEK1.\\nEffects of lipoteichoic acid (LTA) on the phosphorylation of mitogen-activated/extracellular signal-regulated kinase kinase (MEK)1. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated MEK1 (p-MEK1) was immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.\",\n \"Cell morphology and viability were assayed to evaluate the toxicity of LTA to human alveolar epithelial A549 cells. Exposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability (data not shown). When exposed to 30 μg/ml LTA for 1, 6, and 24 h, the viability of A549 cells was not influenced. Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h did not alter cell morphology (data not shown).\",\n \"The effects of LTA on SP-A levels in A549 cells were evaluated by an immunoblotting analysis (Figure \\n1). In untreated A549 cells, low levels of SP-A were immunodetected (Figure \\n1A, top panel, lane 1). After exposure to 30 μg/ml LTA for 1 h, levels of SP-A were found to be augmented (lane 2). When treated for 6 and 24 h, LTA obviously increased amounts of SP-A in A549 cells. β-Actin was immunodetected (Figure \\n1A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \\n1B). Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h respectively caused significant 176%, 230%, and 270% increases in SP-A levels.\\nEffects of lipoteichoic acid (LTA) on the production of surfactant protein(SP)-A. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h (A). Cellular proteins were prepared for the immunoblotting analyses. Amounts of SP-A were immunodetected (A, top pane). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\",\n \"Induction of SP-A mRNA expression by LTA was quantified using a real-time PCR analysis (Figure \\n2). After exposure to LTA for 1 h, the levels of SP-A mRNA in A549 cells were increased by 2.1-fold. Exposure of A549 cells to LTA for 6 and 24 h caused 2.8- and 3.7-fold increases in the levels of SP-A mRNA, respectively (Figure \\n2). Pretreatment of A549 cells with BAY 11–7082, an inhibitor of NF-κB activation, for 1 h did not change SP-A mRNA expression (data not shown). However, BAY 11–7082 significantly inhibited LTA-induced SP-A mRNA production by 70% (Figure \\n2).\\nEffects of lipoteichoic acid (LTA) on induction of surfactant protein(SP)-A mRNA. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. In addition, A549 cells were pretreated with BAY 11–7082 (BAY), an inhibitor of NF-κB activation, for 1 h and then exposed to LTA for another 24 h. mRNA was prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Each value represents the mean ± SEM for n = 3. Symbols * and # indicate that the value significantly (p < 0.05) differed from the respective control and LTA-treated group, respectively.\",\n \"Mechanisms of LTA-induced SP-A augmentation were evaluated by analyses of NF-κB expression and translocation (Figures \\n3 and \\n4). Exposure of A549 cells to LTA for 1 h enhanced levels of cytosolic NF-κB (Figure \\n3A, top panel, lane 1). After treatment for 6 and 24 h, the expression of cytosolic NF-κB was obviously augmented (lanes 3 and 4). β-Actin was immunodetected (Figure \\n3A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \\n3B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased NF-κB production by 181%, 200%, and 230%, respectively.\\nEffects of lipoteichoic acid (LTA) on the expression of the transcription factor, nuclear factor (NF)-κB. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Levels of cytosolic NF-κB p65 (cNF-κB) were immunodetected (A, top panel). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\\nEffects of lipoteichoic acid (LTA) on translocation of the transcription factor, nuclear factor (NF)-κB, from the cytoplasm to nuclei. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Amounts of nuclear NF-κB p65 (nNF-κB) were immunodetected (A, top panel). Proliferating cell nuclear antigen (PCNA) was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\\nTreatment of A549 cells with LTA for 1 h increased levels of nuclear NF-κB (Figure \\n4A, top panel, lane 2). When exposed for 6 and 24 h, translocation of NF-κB from the cytoplasm to nuclei notably increased (lanes 3 and 4). Amounts of PCNA in A549 cells were immunodetected (Figure \\n4A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \\n4B). Exposure of A549 cells to LTA for 1, 6, and 24 h respectively caused significant 176%, 340%, 530% enhancements in levels of nuclear NF-κB.\",\n \"The reason why LTA improved NF-κB activation was further investigated by assaying ERK1/2 phosphorylation (Figure \\n5). Treatment of A549 cells with LTA for 1 h increased the amounts of phosphorylated ERK1/2 (Figure \\n5A, top panel, lane 2). Levels of phosphorylated ERK1/2 in A549 cells were obviously raised after exposure to LTA for 6 and 24 h (lanes 3 and 4). Amounts of β-actin in A549 cells were immunodetected (Figure \\n5A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \\n5B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased ERK1 phosphorylation by 259%, 170%, and 334%, respectively. In comparison, levels of phosphorylated ERK2 were respectively augmented by 8.2-, 6.4-, and 7.8-fold following LTA administration for 1, 6, and 24 h (Figure \\n5B).\\nEffects of lipoteichoic acid (LTA) on the phosphorylation of extracellular signal-regulated kinase (ERK)1/2. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated ERK1/2 (p-ERK1/2) were immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.\",\n \"Phosphorylation of MEK1 was assayed to determine the mechanism of LTA-induced ERK1/2 activation (Figure \\n6). Low levels of phosphorylated MEK1 were detected in untreated A549 cells (Figure \\n6A, top panel, lane 1). However, exposure of A549 cells to LTA for 1 h stimulated MEK1 phosphorylation (lane 2). After exposure for 6 and 24 h, the amounts of phosphorylated MEK1 had obviously increased (lanes 3 and 4). β-actin in A549 cells was immunodetected (Figure \\n6A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \\n6B). Treatment of A549 cells with LTA for 1, 6, and 24 h respectively caused significant 82%, 330%, and 370% increases in levels of phosphorylated MEK1.\\nEffects of lipoteichoic acid (LTA) on the phosphorylation of mitogen-activated/extracellular signal-regulated kinase kinase (MEK)1. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated MEK1 (p-MEK1) was immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.\",\n \"LTA represents a class of amphiphilic molecules anchored to the outer face of the cytoplasmic membrane in gram-positive bacteria and is commonly released during cell growth, especially under antibiotic therapy \\n[1,2]. It can cause cytokine induction in mononuclear phagocytes \\n[17]. In previous studies, LTA concentrations of 0.2~50 μg/ml were detected and stimulated activity of polymononuclear leucocyte functions and release of TNF-α in peripheral blood mononuclear cells \\n[23,24]. Meanwhile, LTA levels at the infectious site can reach a high level of 26,694 ng/mL \\n[25]. The concentration of LTA used in this study was < 50 μg/ml. Therefore, our results show that LTA at clinically relevant concentrations can activate alveolar type II epithelial cells by stimulating production of surfactants.\\nDuring bacterial infection, endotoxins, including LTA and LPS, increase capillary permeability and enhance expressions of cellular adhesion molecules, proinflammatory cytokines, and chemokines \\n[1,15]. These endotoxins can lead to most of the clinical manifestations of bacterial infection and are associated with ALI \\n[4,5]. In addition, LTA can trigger lung inflammation and causes neutrophil influx into the lungs \\n[15,26]. This study showed that in response to LTA stimulation, levels of SP-A mRNA and protein in alveolar A549 cells were time-dependently augmented. SP-A contributes to the pulmonary host defense \\n[10,16,27]. A previous study reported that when spa gene expression was knocked-out, susceptibility of the lungs to pathogenic infection was simultaneously raised \\n[28]. Our previous study also showed that LPS-mediated toll-like receptor (TLR) 2 signaling in human alveolar epithelial cells might increase SP-A biosynthesis and subsequently lead to an inflammatory response in the lungs \\n[3]. As a result, SP-A could be an effective biomarker for detecting pulmonary infection by gram-negative or -positive bacteria.\\nThis study showed that LTA increased the expression of NF-κB and its translocation from the cytoplasm to nuclei. NF-κB is a typical transcription factor in response to stimulation by LTA \\n[16]. LTA can bind CD14 and then stimulates TLR activation \\n[16,29]. After LTA associates with TLR2, NF-κB can be activated by protein kinases and is then translocated to nuclei from the cytoplasm \\n[11]. NF-κB regulates certain gene expressions to control cell proliferation, differentiation, and death \\n[30,31]. A previous study showed that LTA induced cyclooxygenase-2 expression in epithelial cells via IκB degradation and successive p65 NF-κB translocation \\n[32]. LTA could induce SP-A mRNA expression in A549 cells. Our bioinformatic search revealed that NF-κB-DNA-binding motifs were found in the promoter regions of the spa gene. Suppressing NF-κB activation using BAY 11–7082 simultaneously inhibited LTA-induced SP-A mRNA expression. Thus, LTA transcriptionally induces SP-A expression through inducing NF-κB expression and translocation.\\nOur present results revealed that the phosphorylation of ERK1/2 was associated with NF-κB activation. Sequentially, ERK1/2-activated IκBα kinase can phosphorylate IκB at two conserved serine residues in the N-terminus, triggering the degradation of this inhibitor and allowing for the rapid translocation of NF-κB into nuclei \\n[16,20]. Accordingly, LTA-induced activation of A549 cells is mainly due to the improvement in ERK1/2 phosphorylation. Roles of ERK1 and ERK2 in LTA-induced SP-A expression were not determined in this study but will be validated using RNA interference in our next study. There is growing evidence that the ERK signaling pathway, which contributes to regulating inflammatory events \\n[33]. Therefore, LTA regulates SP-A expression in alveolar type II epithelial cells in the course of eliciting ERK1/2 phosphorylation and subsequent activation of the transcription factor, NF-κB.\\nERK activation is mediated by at least three different pathways: a Raf/MEK-dependent pathway, a PI3K/Raf-independent pathway that strongly activates MEK, and a third undetermined pathway that directly activates ERK proteins \\n[34]. This study showed that LTA time-dependently increased levels of phosphorylated MEK1. Thus, one of the possible reasons explaining why LTA stimulates ERK1/2 activation is the increase in MEK1 phosphorylation. MAPK-regulating signals place this family of protein kinases in an apparently linear signaling cascade downstream of growth factor receptors, adaptor proteins, guanine-nucleotide exchange factors, Ras, Raf, and MEK \\n[19]. The present study demonstrates that LTA can induce SP-A expression via MEK-dependent activation of the protein kinase ERK1/2-signaling pathway.\",\n \"In summary, we used an alveolar epithelial cell model to study the immunomodulatory responses of LTA. Our results revealed that LTA can induce inflammatory responses in alveolar epithelial A549 cells by means of enhancing SP-A mRNA and protein syntheses. Moreover, the signal-transducing mechanisms of LTA-caused regulation of SP-A expression arise through the cascade phosphorylations of MEK1 and ERK1/2. In succession, LTA increased NF-κB expression and translocation. LTA-induced SP-A production in alveolar type II epithelial cells may indicate the status of gram-positive bacteria-caused septic shock and acute lung injury. More molecular pathways should be investigated and proven in the future. However, there are certain limitations of this study, including the use of A549 cells, which are derived from human lung carcinoma. The effects of LTA on A549 cells may differ from those on normal alveolar epithelial cells. Thus, we will perform a translational study to evaluate the effects of LTA on alveolar epithelial cells of animals with acute lung injury.\",\n \"ALI: Acute lung injury; ERK1/2: Extracellular signal-regulated kinase 1/2; IL: Interleukin; LPS: Lipopolysaccharide; LTA: Lipoteichoic acid; NF-κB: Nuclear factor-κB; MAPKs: Mitogen-activated protein kinases; MEK1: Mitogen-activated/extracellular signal-regulated kinase kinase 1; PCNA: Proliferating cell nuclear antigen; SDS-PAGE: Sodium dodecylsulfate polyacrylamide gel electrophoresis; SP-A: Surfactant protein-A; TLR2: Toll-like receptor 2; TNF-α: Tumor necrosis factor-α.\",\n \"The authors declare that they have no competing interests.\",\n \"FLL, CYC, and RMC visualized experimental design. YTT and HLT refined the experimental approach. TGC did the statistical analysis. TLC had significant intellectual input into the development of this work, and added to the Discussion. All authors reviewed data and results, and had significant input into the writing of the final manuscript. All authors read and approved the final manuscript.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":[null,"materials|methods",null,null,null,null,null,null,"results",null,null,null,null,null,null,"discussion","conclusions",null,null,null],"string":"[\n null,\n \"materials|methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null\n]","isConversational":false},"keywords":{"kind":"list like","value":["Lipoteichoic acid","Alveolar epithelial cells","Surfactant protein-A","MEK/ERK/NF-κB"],"string":"[\n \"Lipoteichoic acid\",\n \"Alveolar epithelial cells\",\n \"Surfactant protein-A\",\n \"MEK/ERK/NF-κB\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Background:\nSepsis can lead to multiorgan failure and death and appears to be triggered by bacterial products, such as lipopolysaccharide (LPS) from gram-negative bacteria and lipoteichoic acid (LTA) from gram-positive ones \n[1-3]. Infection of the respiratory tract caused by gram-positive bacteria and pneumonia combined with acute lung injury (ALI) are usually the leading causes of mortality by sepsis \n[4]. In the past few decades, the incidences of sepsis and septic shock have been increasing \n[5]. Although endotoxin-activated events are clearly important in gram-negative infection, gram-positive bacteria also have crucial roles, but less is known about host responses to them \n[6]. The increasing prevalence of sepsis from gram-positive bacterial pathogens necessitates a reevaluation of the basic assumptions about the molecular pathogenesis of ALI.\nAlveolar epithelial type II cells contribute to the maintenance of mucosal integrity by modulating the production of surfactants \n[7]. Pulmonary surfactants play important roles in protecting the lung during endotoxin-induced injury and infection \n[8,9]. Surfactant protein (SP)-A is the most abundant pulmonary surfactant protein. Levels of SP-A in bronchiolar lavage fluid are modulated in gram-negative or -positive bacteria-caused lung diseases, including severe pneumonia, acute respiratory distress syndrome, and cardiogenic lung edema \n[10]. Thus, altering lung SP-A levels can be an effective indicator for pulmonary infection and inflammation. Our previous study showed that LPS selectively induced spa gene expression in human alveolar epithelial A549 cells \n[11].\nLTA, an outer membrane component of gram-positive bacteria, was shown to be one of the critical factors participating in the pathogenesis of sepsis \n[12,13]. LTA can stimulate inflammatory responses in the lung \n[14,15]. Therefore, understanding the mechanisms that regulate LTA-mediated cell activation is crucial for diagnosis, treatment, or prognosis of lung inflammatory diseases. In response to stimuli, LTA can activate macrophages to produce massive amounts of inflammatory factors that exhibit systemic effects in the general circulation \n[16]. LTA can induce the secretion of various cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α \n[17]. These data suggest that LTA can selectively modify gene transcription of various cell types and sequentially augment and possibly initiate tissue inflammation.\nMitogen-activated protein kinases (MAPKs) are serine/threonine kinases. The first MAPK isoforms to be cloned and characterized were the extracellular signal-regulated kinase 1 and 2 (ERK 1/2) \n[18,19]. ERK 1/2 are well documented to be activated by a family of dual-specificity kinases known as the mitogen-activated/ERK kinases (MEKs) \n[16,20]. A previous study demonstrated that LTA can selectively activate the ERK pathway in the cornea \n[21]. Our previous study showed that LTA induced TNF-α and IL-6 expressions by means of stimulating phosphorylation of ERK1/2 in macrophages \n[16]. In addition, LTA also triggered translocation of nuclear factor (NF)-κB from the cytoplasm to nuclei and its transactivation activity. Meanwhile, the mechanisms responsible for LTA-induced spa gene expression in alveolar epithelial cells are still unknown. In this study, we attempted to evaluate the effects of LTA on SP-1 synthesis in human alveolar type II epithelial cells and its possible mechanisms.\n\nMaterials and methods:\n Cell culture and drug treatment A human lung carcinoma type II epithelial cell line (A549) was cultured following a previous method \n[3]. A549 cells were grown in Dulbecco's modified Eagle's medium (DMEM)/Ham’s F12 culture medium (Sigma, St. Louis, MO, USA), supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 U/ml penicillin G, 100 μg/ml streptomycin, and 2 mM l-glutamine. A549 cells were seeded in 75-cm2 culture flasks at 37 °C in a humidified atmosphere of 5% CO2. Cells were grown to confluence before drug treatment. LTA purchased from Sigma was dissolved in phosphate-buffered saline (PBS) (0.14 M NaCl, 2.6 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4). BAY 11–7082, an inhibitor of NF-κB activation, was also purchased from Sigma.\nA human lung carcinoma type II epithelial cell line (A549) was cultured following a previous method \n[3]. A549 cells were grown in Dulbecco's modified Eagle's medium (DMEM)/Ham’s F12 culture medium (Sigma, St. Louis, MO, USA), supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 U/ml penicillin G, 100 μg/ml streptomycin, and 2 mM l-glutamine. A549 cells were seeded in 75-cm2 culture flasks at 37 °C in a humidified atmosphere of 5% CO2. Cells were grown to confluence before drug treatment. LTA purchased from Sigma was dissolved in phosphate-buffered saline (PBS) (0.14 M NaCl, 2.6 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4). BAY 11–7082, an inhibitor of NF-κB activation, was also purchased from Sigma.\n Assay of cell viability Cell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previously described \n[22]. Briefly, A549 cells (104 cells/well) were seeded in 96-well tissue culture plates overnight. After drug treatment, macrophages were cultured in new medium containing 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide for a further 3 h. The blue formazan products in the macrophages were dissolved in dimethyl sulfoxide and spectrophotometrically measured at a wavelength of 550 nm.\nCell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previously described \n[22]. Briefly, A549 cells (104 cells/well) were seeded in 96-well tissue culture plates overnight. After drug treatment, macrophages were cultured in new medium containing 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide for a further 3 h. The blue formazan products in the macrophages were dissolved in dimethyl sulfoxide and spectrophotometrically measured at a wavelength of 550 nm.\n Immunoblotting analyses of SP-A, NF-κB, and phosphorylated and non-phosphorylated ERK1/2 and MEK1 Protein levels were immunodetected according to a previously described method \n[11]. After drug treatment, cell lysates were prepared in ice-cold radioimmunoprecipitation assay buffer (25 mM Tris–HCl (pH 7.2), 0.1% sodium dodecylsulfate (SDS), 1% Triton X-100, 1% sodium deoxycholate, 0.15 M NaCl, and 1 mM EDTA). Protein concentrations were quantified using a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Proteins (50 μg/well) were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Immunodetection of SP-A and NF-κB was carried out using rabbit polyclonal antibodies against human SP-A and NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cellular β-actin protein was immunodetected using a mouse monoclonal antibody (mAb) against mouse β-actin (Sigma) as the internal standard. These protein bands were quantified using a digital imaging system (UVtec, Cambridge, UK). Phosphorylated ERK1/2 and MEK1 were immunodetected using a rabbit polyclonal antibody against phosphorylated residues of ERK1/2 and MEK1 (Cell Signaling, Danvers, MA, USA). Nonphosphorylated ERK2 and MEK1 were immunodetected as the internal controls (Cell Signaling). Intensities of the immunoreactive bands were determined using a digital imaging system (Wallac Victor 1420, PerkinElmer, Melbourne, Australia).\nProtein levels were immunodetected according to a previously described method \n[11]. After drug treatment, cell lysates were prepared in ice-cold radioimmunoprecipitation assay buffer (25 mM Tris–HCl (pH 7.2), 0.1% sodium dodecylsulfate (SDS), 1% Triton X-100, 1% sodium deoxycholate, 0.15 M NaCl, and 1 mM EDTA). Protein concentrations were quantified using a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Proteins (50 μg/well) were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Immunodetection of SP-A and NF-κB was carried out using rabbit polyclonal antibodies against human SP-A and NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cellular β-actin protein was immunodetected using a mouse monoclonal antibody (mAb) against mouse β-actin (Sigma) as the internal standard. These protein bands were quantified using a digital imaging system (UVtec, Cambridge, UK). Phosphorylated ERK1/2 and MEK1 were immunodetected using a rabbit polyclonal antibody against phosphorylated residues of ERK1/2 and MEK1 (Cell Signaling, Danvers, MA, USA). Nonphosphorylated ERK2 and MEK1 were immunodetected as the internal controls (Cell Signaling). Intensities of the immunoreactive bands were determined using a digital imaging system (Wallac Victor 1420, PerkinElmer, Melbourne, Australia).\n Extraction of nuclear proteins and immunodetection Amounts of nuclear transcription factors were quantified following a previously described method \n[20]. After drug treatment, nuclear extracts of macrophages were prepared. Protein concentrations were quantified by a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Nuclear proteins (50 μg/well) were subjected to SDS-PAGE and transferred to nitrocellulose membranes. After blocking, NF-κB was immunodetected using a rabbit polyclonal antibody against mouse NF-κB p65 (Santa Cruz Biotechnology). A proliferating cell nuclear antigen (PCNA) was detected using a mouse mAb against the rat PCNA protein (Santa Cruz Biotechnology) as the internal standard. Intensities of the immunoreactive bands were determined using a digital imaging system (Wallac Victor 1420, PerkinElmer).\nAmounts of nuclear transcription factors were quantified following a previously described method \n[20]. After drug treatment, nuclear extracts of macrophages were prepared. Protein concentrations were quantified by a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Nuclear proteins (50 μg/well) were subjected to SDS-PAGE and transferred to nitrocellulose membranes. After blocking, NF-κB was immunodetected using a rabbit polyclonal antibody against mouse NF-κB p65 (Santa Cruz Biotechnology). A proliferating cell nuclear antigen (PCNA) was detected using a mouse mAb against the rat PCNA protein (Santa Cruz Biotechnology) as the internal standard. Intensities of the immunoreactive bands were determined using a digital imaging system (Wallac Victor 1420, PerkinElmer).\n Real-time polymerase chain reaction (PCR) assays Messenger (m)RNA from A549 cells exposed to LTA were prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Oligonucleotides for the PCR analyses of SP-A mRNA and β-actin mRNA were designed and synthesized by Clontech Laboratories (Palo Alto, CA, USA). The oligonucleotide sequences of the upstream and downstream primers for these mRNA analyses were respectively 5'-TGA AAGGGAGTTCTAGCATCTCACAGA-3' and 5'-ACATATGCCTATGTAGGCCTGACTGAG-3' for SP-A mRNA, and 5'- GTCTACATGTCTCGATCCCACTTA A -3' and 5'-GGTCTTTCTCTCTCATCGCGCTC-5' for β-actin mRNA. A quantitative PCR analysis was carried out using iQSYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad) as described previously \n[11].\nMessenger (m)RNA from A549 cells exposed to LTA were prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Oligonucleotides for the PCR analyses of SP-A mRNA and β-actin mRNA were designed and synthesized by Clontech Laboratories (Palo Alto, CA, USA). The oligonucleotide sequences of the upstream and downstream primers for these mRNA analyses were respectively 5'-TGA AAGGGAGTTCTAGCATCTCACAGA-3' and 5'-ACATATGCCTATGTAGGCCTGACTGAG-3' for SP-A mRNA, and 5'- GTCTACATGTCTCGATCCCACTTA A -3' and 5'-GGTCTTTCTCTCTCATCGCGCTC-5' for β-actin mRNA. A quantitative PCR analysis was carried out using iQSYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad) as described previously \n[11].\n Statistical analysis Statistical differences were considered significant when the p value of Duncan’s multiple-range test was <0.05. Statistical analysis between groups over time was carried out by a two-way analysis of variance (ANOVA).\nStatistical differences were considered significant when the p value of Duncan’s multiple-range test was <0.05. Statistical analysis between groups over time was carried out by a two-way analysis of variance (ANOVA).\n\nCell culture and drug treatment:\nA human lung carcinoma type II epithelial cell line (A549) was cultured following a previous method \n[3]. A549 cells were grown in Dulbecco's modified Eagle's medium (DMEM)/Ham’s F12 culture medium (Sigma, St. Louis, MO, USA), supplemented with 10% (v/v) heat-inactivated fetal calf serum, 100 U/ml penicillin G, 100 μg/ml streptomycin, and 2 mM l-glutamine. A549 cells were seeded in 75-cm2 culture flasks at 37 °C in a humidified atmosphere of 5% CO2. Cells were grown to confluence before drug treatment. LTA purchased from Sigma was dissolved in phosphate-buffered saline (PBS) (0.14 M NaCl, 2.6 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4). BAY 11–7082, an inhibitor of NF-κB activation, was also purchased from Sigma.\n\nAssay of cell viability:\nCell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previously described \n[22]. Briefly, A549 cells (104 cells/well) were seeded in 96-well tissue culture plates overnight. After drug treatment, macrophages were cultured in new medium containing 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide for a further 3 h. The blue formazan products in the macrophages were dissolved in dimethyl sulfoxide and spectrophotometrically measured at a wavelength of 550 nm.\n\nImmunoblotting analyses of SP-A, NF-κB, and phosphorylated and non-phosphorylated ERK1/2 and MEK1:\nProtein levels were immunodetected according to a previously described method \n[11]. After drug treatment, cell lysates were prepared in ice-cold radioimmunoprecipitation assay buffer (25 mM Tris–HCl (pH 7.2), 0.1% sodium dodecylsulfate (SDS), 1% Triton X-100, 1% sodium deoxycholate, 0.15 M NaCl, and 1 mM EDTA). Protein concentrations were quantified using a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Proteins (50 μg/well) were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Immunodetection of SP-A and NF-κB was carried out using rabbit polyclonal antibodies against human SP-A and NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cellular β-actin protein was immunodetected using a mouse monoclonal antibody (mAb) against mouse β-actin (Sigma) as the internal standard. These protein bands were quantified using a digital imaging system (UVtec, Cambridge, UK). Phosphorylated ERK1/2 and MEK1 were immunodetected using a rabbit polyclonal antibody against phosphorylated residues of ERK1/2 and MEK1 (Cell Signaling, Danvers, MA, USA). Nonphosphorylated ERK2 and MEK1 were immunodetected as the internal controls (Cell Signaling). Intensities of the immunoreactive bands were determined using a digital imaging system (Wallac Victor 1420, PerkinElmer, Melbourne, Australia).\n\nExtraction of nuclear proteins and immunodetection:\nAmounts of nuclear transcription factors were quantified following a previously described method \n[20]. After drug treatment, nuclear extracts of macrophages were prepared. Protein concentrations were quantified by a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Nuclear proteins (50 μg/well) were subjected to SDS-PAGE and transferred to nitrocellulose membranes. After blocking, NF-κB was immunodetected using a rabbit polyclonal antibody against mouse NF-κB p65 (Santa Cruz Biotechnology). A proliferating cell nuclear antigen (PCNA) was detected using a mouse mAb against the rat PCNA protein (Santa Cruz Biotechnology) as the internal standard. Intensities of the immunoreactive bands were determined using a digital imaging system (Wallac Victor 1420, PerkinElmer).\n\nReal-time polymerase chain reaction (PCR) assays:\nMessenger (m)RNA from A549 cells exposed to LTA were prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Oligonucleotides for the PCR analyses of SP-A mRNA and β-actin mRNA were designed and synthesized by Clontech Laboratories (Palo Alto, CA, USA). The oligonucleotide sequences of the upstream and downstream primers for these mRNA analyses were respectively 5'-TGA AAGGGAGTTCTAGCATCTCACAGA-3' and 5'-ACATATGCCTATGTAGGCCTGACTGAG-3' for SP-A mRNA, and 5'- GTCTACATGTCTCGATCCCACTTA A -3' and 5'-GGTCTTTCTCTCTCATCGCGCTC-5' for β-actin mRNA. A quantitative PCR analysis was carried out using iQSYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad) as described previously \n[11].\n\nStatistical analysis:\nStatistical differences were considered significant when the p value of Duncan’s multiple-range test was <0.05. Statistical analysis between groups over time was carried out by a two-way analysis of variance (ANOVA).\n\nResults:\n Toxicity of LTA to A549 cells Cell morphology and viability were assayed to evaluate the toxicity of LTA to human alveolar epithelial A549 cells. Exposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability (data not shown). When exposed to 30 μg/ml LTA for 1, 6, and 24 h, the viability of A549 cells was not influenced. Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h did not alter cell morphology (data not shown).\nCell morphology and viability were assayed to evaluate the toxicity of LTA to human alveolar epithelial A549 cells. Exposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability (data not shown). When exposed to 30 μg/ml LTA for 1, 6, and 24 h, the viability of A549 cells was not influenced. Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h did not alter cell morphology (data not shown).\n LTA-induced enhancement of SP-A biosynthesis in A549 cells The effects of LTA on SP-A levels in A549 cells were evaluated by an immunoblotting analysis (Figure \n1). In untreated A549 cells, low levels of SP-A were immunodetected (Figure \n1A, top panel, lane 1). After exposure to 30 μg/ml LTA for 1 h, levels of SP-A were found to be augmented (lane 2). When treated for 6 and 24 h, LTA obviously increased amounts of SP-A in A549 cells. β-Actin was immunodetected (Figure \n1A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n1B). Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h respectively caused significant 176%, 230%, and 270% increases in SP-A levels.\nEffects of lipoteichoic acid (LTA) on the production of surfactant protein(SP)-A. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h (A). Cellular proteins were prepared for the immunoblotting analyses. Amounts of SP-A were immunodetected (A, top pane). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\nThe effects of LTA on SP-A levels in A549 cells were evaluated by an immunoblotting analysis (Figure \n1). In untreated A549 cells, low levels of SP-A were immunodetected (Figure \n1A, top panel, lane 1). After exposure to 30 μg/ml LTA for 1 h, levels of SP-A were found to be augmented (lane 2). When treated for 6 and 24 h, LTA obviously increased amounts of SP-A in A549 cells. β-Actin was immunodetected (Figure \n1A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n1B). Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h respectively caused significant 176%, 230%, and 270% increases in SP-A levels.\nEffects of lipoteichoic acid (LTA) on the production of surfactant protein(SP)-A. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h (A). Cellular proteins were prepared for the immunoblotting analyses. Amounts of SP-A were immunodetected (A, top pane). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\n LTA-induced SP-A mRNA expression in A549 cells Induction of SP-A mRNA expression by LTA was quantified using a real-time PCR analysis (Figure \n2). After exposure to LTA for 1 h, the levels of SP-A mRNA in A549 cells were increased by 2.1-fold. Exposure of A549 cells to LTA for 6 and 24 h caused 2.8- and 3.7-fold increases in the levels of SP-A mRNA, respectively (Figure \n2). Pretreatment of A549 cells with BAY 11–7082, an inhibitor of NF-κB activation, for 1 h did not change SP-A mRNA expression (data not shown). However, BAY 11–7082 significantly inhibited LTA-induced SP-A mRNA production by 70% (Figure \n2).\nEffects of lipoteichoic acid (LTA) on induction of surfactant protein(SP)-A mRNA. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. In addition, A549 cells were pretreated with BAY 11–7082 (BAY), an inhibitor of NF-κB activation, for 1 h and then exposed to LTA for another 24 h. mRNA was prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Each value represents the mean ± SEM for n = 3. Symbols * and # indicate that the value significantly (p < 0.05) differed from the respective control and LTA-treated group, respectively.\nInduction of SP-A mRNA expression by LTA was quantified using a real-time PCR analysis (Figure \n2). After exposure to LTA for 1 h, the levels of SP-A mRNA in A549 cells were increased by 2.1-fold. Exposure of A549 cells to LTA for 6 and 24 h caused 2.8- and 3.7-fold increases in the levels of SP-A mRNA, respectively (Figure \n2). Pretreatment of A549 cells with BAY 11–7082, an inhibitor of NF-κB activation, for 1 h did not change SP-A mRNA expression (data not shown). However, BAY 11–7082 significantly inhibited LTA-induced SP-A mRNA production by 70% (Figure \n2).\nEffects of lipoteichoic acid (LTA) on induction of surfactant protein(SP)-A mRNA. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. In addition, A549 cells were pretreated with BAY 11–7082 (BAY), an inhibitor of NF-κB activation, for 1 h and then exposed to LTA for another 24 h. mRNA was prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Each value represents the mean ± SEM for n = 3. Symbols * and # indicate that the value significantly (p < 0.05) differed from the respective control and LTA-treated group, respectively.\n Augmentation of NF-κB expression and translocation by LTA Mechanisms of LTA-induced SP-A augmentation were evaluated by analyses of NF-κB expression and translocation (Figures \n3 and \n4). Exposure of A549 cells to LTA for 1 h enhanced levels of cytosolic NF-κB (Figure \n3A, top panel, lane 1). After treatment for 6 and 24 h, the expression of cytosolic NF-κB was obviously augmented (lanes 3 and 4). β-Actin was immunodetected (Figure \n3A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n3B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased NF-κB production by 181%, 200%, and 230%, respectively.\nEffects of lipoteichoic acid (LTA) on the expression of the transcription factor, nuclear factor (NF)-κB. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Levels of cytosolic NF-κB p65 (cNF-κB) were immunodetected (A, top panel). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\nEffects of lipoteichoic acid (LTA) on translocation of the transcription factor, nuclear factor (NF)-κB, from the cytoplasm to nuclei. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Amounts of nuclear NF-κB p65 (nNF-κB) were immunodetected (A, top panel). Proliferating cell nuclear antigen (PCNA) was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\nTreatment of A549 cells with LTA for 1 h increased levels of nuclear NF-κB (Figure \n4A, top panel, lane 2). When exposed for 6 and 24 h, translocation of NF-κB from the cytoplasm to nuclei notably increased (lanes 3 and 4). Amounts of PCNA in A549 cells were immunodetected (Figure \n4A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n4B). Exposure of A549 cells to LTA for 1, 6, and 24 h respectively caused significant 176%, 340%, 530% enhancements in levels of nuclear NF-κB.\nMechanisms of LTA-induced SP-A augmentation were evaluated by analyses of NF-κB expression and translocation (Figures \n3 and \n4). Exposure of A549 cells to LTA for 1 h enhanced levels of cytosolic NF-κB (Figure \n3A, top panel, lane 1). After treatment for 6 and 24 h, the expression of cytosolic NF-κB was obviously augmented (lanes 3 and 4). β-Actin was immunodetected (Figure \n3A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n3B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased NF-κB production by 181%, 200%, and 230%, respectively.\nEffects of lipoteichoic acid (LTA) on the expression of the transcription factor, nuclear factor (NF)-κB. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Levels of cytosolic NF-κB p65 (cNF-κB) were immunodetected (A, top panel). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\nEffects of lipoteichoic acid (LTA) on translocation of the transcription factor, nuclear factor (NF)-κB, from the cytoplasm to nuclei. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Amounts of nuclear NF-κB p65 (nNF-κB) were immunodetected (A, top panel). Proliferating cell nuclear antigen (PCNA) was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\nTreatment of A549 cells with LTA for 1 h increased levels of nuclear NF-κB (Figure \n4A, top panel, lane 2). When exposed for 6 and 24 h, translocation of NF-κB from the cytoplasm to nuclei notably increased (lanes 3 and 4). Amounts of PCNA in A549 cells were immunodetected (Figure \n4A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n4B). Exposure of A549 cells to LTA for 1, 6, and 24 h respectively caused significant 176%, 340%, 530% enhancements in levels of nuclear NF-κB.\n LTA-enhanced phosphorylation of ERK1/2 The reason why LTA improved NF-κB activation was further investigated by assaying ERK1/2 phosphorylation (Figure \n5). Treatment of A549 cells with LTA for 1 h increased the amounts of phosphorylated ERK1/2 (Figure \n5A, top panel, lane 2). Levels of phosphorylated ERK1/2 in A549 cells were obviously raised after exposure to LTA for 6 and 24 h (lanes 3 and 4). Amounts of β-actin in A549 cells were immunodetected (Figure \n5A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n5B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased ERK1 phosphorylation by 259%, 170%, and 334%, respectively. In comparison, levels of phosphorylated ERK2 were respectively augmented by 8.2-, 6.4-, and 7.8-fold following LTA administration for 1, 6, and 24 h (Figure \n5B).\nEffects of lipoteichoic acid (LTA) on the phosphorylation of extracellular signal-regulated kinase (ERK)1/2. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated ERK1/2 (p-ERK1/2) were immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.\nThe reason why LTA improved NF-κB activation was further investigated by assaying ERK1/2 phosphorylation (Figure \n5). Treatment of A549 cells with LTA for 1 h increased the amounts of phosphorylated ERK1/2 (Figure \n5A, top panel, lane 2). Levels of phosphorylated ERK1/2 in A549 cells were obviously raised after exposure to LTA for 6 and 24 h (lanes 3 and 4). Amounts of β-actin in A549 cells were immunodetected (Figure \n5A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n5B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased ERK1 phosphorylation by 259%, 170%, and 334%, respectively. In comparison, levels of phosphorylated ERK2 were respectively augmented by 8.2-, 6.4-, and 7.8-fold following LTA administration for 1, 6, and 24 h (Figure \n5B).\nEffects of lipoteichoic acid (LTA) on the phosphorylation of extracellular signal-regulated kinase (ERK)1/2. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated ERK1/2 (p-ERK1/2) were immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.\n LTA-induced activation of MEK1 Phosphorylation of MEK1 was assayed to determine the mechanism of LTA-induced ERK1/2 activation (Figure \n6). Low levels of phosphorylated MEK1 were detected in untreated A549 cells (Figure \n6A, top panel, lane 1). However, exposure of A549 cells to LTA for 1 h stimulated MEK1 phosphorylation (lane 2). After exposure for 6 and 24 h, the amounts of phosphorylated MEK1 had obviously increased (lanes 3 and 4). β-actin in A549 cells was immunodetected (Figure \n6A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n6B). Treatment of A549 cells with LTA for 1, 6, and 24 h respectively caused significant 82%, 330%, and 370% increases in levels of phosphorylated MEK1.\nEffects of lipoteichoic acid (LTA) on the phosphorylation of mitogen-activated/extracellular signal-regulated kinase kinase (MEK)1. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated MEK1 (p-MEK1) was immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.\nPhosphorylation of MEK1 was assayed to determine the mechanism of LTA-induced ERK1/2 activation (Figure \n6). Low levels of phosphorylated MEK1 were detected in untreated A549 cells (Figure \n6A, top panel, lane 1). However, exposure of A549 cells to LTA for 1 h stimulated MEK1 phosphorylation (lane 2). After exposure for 6 and 24 h, the amounts of phosphorylated MEK1 had obviously increased (lanes 3 and 4). β-actin in A549 cells was immunodetected (Figure \n6A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n6B). Treatment of A549 cells with LTA for 1, 6, and 24 h respectively caused significant 82%, 330%, and 370% increases in levels of phosphorylated MEK1.\nEffects of lipoteichoic acid (LTA) on the phosphorylation of mitogen-activated/extracellular signal-regulated kinase kinase (MEK)1. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated MEK1 (p-MEK1) was immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.\n\nToxicity of LTA to A549 cells:\nCell morphology and viability were assayed to evaluate the toxicity of LTA to human alveolar epithelial A549 cells. Exposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability (data not shown). When exposed to 30 μg/ml LTA for 1, 6, and 24 h, the viability of A549 cells was not influenced. Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h did not alter cell morphology (data not shown).\n\nLTA-induced enhancement of SP-A biosynthesis in A549 cells:\nThe effects of LTA on SP-A levels in A549 cells were evaluated by an immunoblotting analysis (Figure \n1). In untreated A549 cells, low levels of SP-A were immunodetected (Figure \n1A, top panel, lane 1). After exposure to 30 μg/ml LTA for 1 h, levels of SP-A were found to be augmented (lane 2). When treated for 6 and 24 h, LTA obviously increased amounts of SP-A in A549 cells. β-Actin was immunodetected (Figure \n1A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n1B). Exposure of A549 cells to 30 μg/ml LTA for 1, 6, and 24 h respectively caused significant 176%, 230%, and 270% increases in SP-A levels.\nEffects of lipoteichoic acid (LTA) on the production of surfactant protein(SP)-A. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h (A). Cellular proteins were prepared for the immunoblotting analyses. Amounts of SP-A were immunodetected (A, top pane). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\n\nLTA-induced SP-A mRNA expression in A549 cells:\nInduction of SP-A mRNA expression by LTA was quantified using a real-time PCR analysis (Figure \n2). After exposure to LTA for 1 h, the levels of SP-A mRNA in A549 cells were increased by 2.1-fold. Exposure of A549 cells to LTA for 6 and 24 h caused 2.8- and 3.7-fold increases in the levels of SP-A mRNA, respectively (Figure \n2). Pretreatment of A549 cells with BAY 11–7082, an inhibitor of NF-κB activation, for 1 h did not change SP-A mRNA expression (data not shown). However, BAY 11–7082 significantly inhibited LTA-induced SP-A mRNA production by 70% (Figure \n2).\nEffects of lipoteichoic acid (LTA) on induction of surfactant protein(SP)-A mRNA. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. In addition, A549 cells were pretreated with BAY 11–7082 (BAY), an inhibitor of NF-κB activation, for 1 h and then exposed to LTA for another 24 h. mRNA was prepared for real-time PCR analyses of SP-A mRNA and β-actin mRNA. Each value represents the mean ± SEM for n = 3. Symbols * and # indicate that the value significantly (p < 0.05) differed from the respective control and LTA-treated group, respectively.\n\nAugmentation of NF-κB expression and translocation by LTA:\nMechanisms of LTA-induced SP-A augmentation were evaluated by analyses of NF-κB expression and translocation (Figures \n3 and \n4). Exposure of A549 cells to LTA for 1 h enhanced levels of cytosolic NF-κB (Figure \n3A, top panel, lane 1). After treatment for 6 and 24 h, the expression of cytosolic NF-κB was obviously augmented (lanes 3 and 4). β-Actin was immunodetected (Figure \n3A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n3B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased NF-κB production by 181%, 200%, and 230%, respectively.\nEffects of lipoteichoic acid (LTA) on the expression of the transcription factor, nuclear factor (NF)-κB. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Levels of cytosolic NF-κB p65 (cNF-κB) were immunodetected (A, top panel). β-Actin was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\nEffects of lipoteichoic acid (LTA) on translocation of the transcription factor, nuclear factor (NF)-κB, from the cytoplasm to nuclei. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Amounts of nuclear NF-κB p65 (nNF-κB) were immunodetected (A, top panel). Proliferating cell nuclear antigen (PCNA) was detected as the internal standard (bottom panel). These protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control groups, p < 0.05. AU, arbitrary unit.\nTreatment of A549 cells with LTA for 1 h increased levels of nuclear NF-κB (Figure \n4A, top panel, lane 2). When exposed for 6 and 24 h, translocation of NF-κB from the cytoplasm to nuclei notably increased (lanes 3 and 4). Amounts of PCNA in A549 cells were immunodetected (Figure \n4A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n4B). Exposure of A549 cells to LTA for 1, 6, and 24 h respectively caused significant 176%, 340%, 530% enhancements in levels of nuclear NF-κB.\n\nLTA-enhanced phosphorylation of ERK1/2:\nThe reason why LTA improved NF-κB activation was further investigated by assaying ERK1/2 phosphorylation (Figure \n5). Treatment of A549 cells with LTA for 1 h increased the amounts of phosphorylated ERK1/2 (Figure \n5A, top panel, lane 2). Levels of phosphorylated ERK1/2 in A549 cells were obviously raised after exposure to LTA for 6 and 24 h (lanes 3 and 4). Amounts of β-actin in A549 cells were immunodetected (Figure \n5A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n5B). Exposure of A549 cells to LTA for 1, 6, and 24 h significantly increased ERK1 phosphorylation by 259%, 170%, and 334%, respectively. In comparison, levels of phosphorylated ERK2 were respectively augmented by 8.2-, 6.4-, and 7.8-fold following LTA administration for 1, 6, and 24 h (Figure \n5B).\nEffects of lipoteichoic acid (LTA) on the phosphorylation of extracellular signal-regulated kinase (ERK)1/2. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated ERK1/2 (p-ERK1/2) were immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.\n\nLTA-induced activation of MEK1:\nPhosphorylation of MEK1 was assayed to determine the mechanism of LTA-induced ERK1/2 activation (Figure \n6). Low levels of phosphorylated MEK1 were detected in untreated A549 cells (Figure \n6A, top panel, lane 1). However, exposure of A549 cells to LTA for 1 h stimulated MEK1 phosphorylation (lane 2). After exposure for 6 and 24 h, the amounts of phosphorylated MEK1 had obviously increased (lanes 3 and 4). β-actin in A549 cells was immunodetected (Figure \n6A, bottom panel). These immunorelated protein bands were quantified and analyzed (Figure \n6B). Treatment of A549 cells with LTA for 1, 6, and 24 h respectively caused significant 82%, 330%, and 370% increases in levels of phosphorylated MEK1.\nEffects of lipoteichoic acid (LTA) on the phosphorylation of mitogen-activated/extracellular signal-regulated kinase kinase (MEK)1. A549 cells were exposed to 30 μg/ml LTA for 1, 6, and 24 h. Phosphorylated MEK1 (p-MEK1) was immunodetected (A, top panel). ERK2 was detected as the internal standard (bottom panel). These immunorelated protein bands were quantified and analyzed (B). Each value represents the mean ± SEM for n = 6. An asterisk (*) indicates that a value significantly differed from the control group, p < 0.05. AU, arbitrary unit.\n\nDiscussion:\nLTA represents a class of amphiphilic molecules anchored to the outer face of the cytoplasmic membrane in gram-positive bacteria and is commonly released during cell growth, especially under antibiotic therapy \n[1,2]. It can cause cytokine induction in mononuclear phagocytes \n[17]. In previous studies, LTA concentrations of 0.2~50 μg/ml were detected and stimulated activity of polymononuclear leucocyte functions and release of TNF-α in peripheral blood mononuclear cells \n[23,24]. Meanwhile, LTA levels at the infectious site can reach a high level of 26,694 ng/mL \n[25]. The concentration of LTA used in this study was < 50 μg/ml. Therefore, our results show that LTA at clinically relevant concentrations can activate alveolar type II epithelial cells by stimulating production of surfactants.\nDuring bacterial infection, endotoxins, including LTA and LPS, increase capillary permeability and enhance expressions of cellular adhesion molecules, proinflammatory cytokines, and chemokines \n[1,15]. These endotoxins can lead to most of the clinical manifestations of bacterial infection and are associated with ALI \n[4,5]. In addition, LTA can trigger lung inflammation and causes neutrophil influx into the lungs \n[15,26]. This study showed that in response to LTA stimulation, levels of SP-A mRNA and protein in alveolar A549 cells were time-dependently augmented. SP-A contributes to the pulmonary host defense \n[10,16,27]. A previous study reported that when spa gene expression was knocked-out, susceptibility of the lungs to pathogenic infection was simultaneously raised \n[28]. Our previous study also showed that LPS-mediated toll-like receptor (TLR) 2 signaling in human alveolar epithelial cells might increase SP-A biosynthesis and subsequently lead to an inflammatory response in the lungs \n[3]. As a result, SP-A could be an effective biomarker for detecting pulmonary infection by gram-negative or -positive bacteria.\nThis study showed that LTA increased the expression of NF-κB and its translocation from the cytoplasm to nuclei. NF-κB is a typical transcription factor in response to stimulation by LTA \n[16]. LTA can bind CD14 and then stimulates TLR activation \n[16,29]. After LTA associates with TLR2, NF-κB can be activated by protein kinases and is then translocated to nuclei from the cytoplasm \n[11]. NF-κB regulates certain gene expressions to control cell proliferation, differentiation, and death \n[30,31]. A previous study showed that LTA induced cyclooxygenase-2 expression in epithelial cells via IκB degradation and successive p65 NF-κB translocation \n[32]. LTA could induce SP-A mRNA expression in A549 cells. Our bioinformatic search revealed that NF-κB-DNA-binding motifs were found in the promoter regions of the spa gene. Suppressing NF-κB activation using BAY 11–7082 simultaneously inhibited LTA-induced SP-A mRNA expression. Thus, LTA transcriptionally induces SP-A expression through inducing NF-κB expression and translocation.\nOur present results revealed that the phosphorylation of ERK1/2 was associated with NF-κB activation. Sequentially, ERK1/2-activated IκBα kinase can phosphorylate IκB at two conserved serine residues in the N-terminus, triggering the degradation of this inhibitor and allowing for the rapid translocation of NF-κB into nuclei \n[16,20]. Accordingly, LTA-induced activation of A549 cells is mainly due to the improvement in ERK1/2 phosphorylation. Roles of ERK1 and ERK2 in LTA-induced SP-A expression were not determined in this study but will be validated using RNA interference in our next study. There is growing evidence that the ERK signaling pathway, which contributes to regulating inflammatory events \n[33]. Therefore, LTA regulates SP-A expression in alveolar type II epithelial cells in the course of eliciting ERK1/2 phosphorylation and subsequent activation of the transcription factor, NF-κB.\nERK activation is mediated by at least three different pathways: a Raf/MEK-dependent pathway, a PI3K/Raf-independent pathway that strongly activates MEK, and a third undetermined pathway that directly activates ERK proteins \n[34]. This study showed that LTA time-dependently increased levels of phosphorylated MEK1. Thus, one of the possible reasons explaining why LTA stimulates ERK1/2 activation is the increase in MEK1 phosphorylation. MAPK-regulating signals place this family of protein kinases in an apparently linear signaling cascade downstream of growth factor receptors, adaptor proteins, guanine-nucleotide exchange factors, Ras, Raf, and MEK \n[19]. The present study demonstrates that LTA can induce SP-A expression via MEK-dependent activation of the protein kinase ERK1/2-signaling pathway.\n\nConclusions:\nIn summary, we used an alveolar epithelial cell model to study the immunomodulatory responses of LTA. Our results revealed that LTA can induce inflammatory responses in alveolar epithelial A549 cells by means of enhancing SP-A mRNA and protein syntheses. Moreover, the signal-transducing mechanisms of LTA-caused regulation of SP-A expression arise through the cascade phosphorylations of MEK1 and ERK1/2. In succession, LTA increased NF-κB expression and translocation. LTA-induced SP-A production in alveolar type II epithelial cells may indicate the status of gram-positive bacteria-caused septic shock and acute lung injury. More molecular pathways should be investigated and proven in the future. However, there are certain limitations of this study, including the use of A549 cells, which are derived from human lung carcinoma. The effects of LTA on A549 cells may differ from those on normal alveolar epithelial cells. Thus, we will perform a translational study to evaluate the effects of LTA on alveolar epithelial cells of animals with acute lung injury.\n\nAbbreviations:\nALI: Acute lung injury; ERK1/2: Extracellular signal-regulated kinase 1/2; IL: Interleukin; LPS: Lipopolysaccharide; LTA: Lipoteichoic acid; NF-κB: Nuclear factor-κB; MAPKs: Mitogen-activated protein kinases; MEK1: Mitogen-activated/extracellular signal-regulated kinase kinase 1; PCNA: Proliferating cell nuclear antigen; SDS-PAGE: Sodium dodecylsulfate polyacrylamide gel electrophoresis; SP-A: Surfactant protein-A; TLR2: Toll-like receptor 2; TNF-α: Tumor necrosis factor-α.\n\nCompeting interests:\nThe authors declare that they have no competing interests.\n\nAuthors’ contributions:\nFLL, CYC, and RMC visualized experimental design. YTT and HLT refined the experimental approach. TGC did the statistical analysis. TLC had significant intellectual input into the development of this work, and added to the Discussion. All authors reviewed data and results, and had significant input into the writing of the final manuscript. All authors read and approved the final manuscript.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nLipoteichoic acid (LTA), a gram-positive bacterial outer membrane component, can cause septic shock. Our previous studies showed that the gram-negative endotoxin, lipopolysaccharide (LPS), could induce surfactant protein-A (SP-A) production in human alveolar epithelial (A549) cells.\n\nMethods:\nA549 cells were exposed to LTA. Levels of SP-A, nuclear factor (NF)-κB, extracellular signal-regulated kinase 1/2 (ERK1/2), and mitogen-activated/extracellular signal-regulated kinase kinase (MEK)1 were determined.\n\nResults:\nExposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability. Meanwhile, when exposed to 30 μg/ml LTA for 1, 6, and 24 h, the biosynthesis of SP-A mRNA and protein in A549 cells significantly increased. As to the mechanism, LTA enhanced cytosolic and nuclear NF-κB levels in time-dependent manners. Pretreatment with BAY 11-7082, an inhibitor of NF-κB activation, significantly inhibited LTA-induced SP-A mRNA expression. Sequentially, LTA time-dependently augmented phosphorylation of ERK1/2. In addition, levels of phosphorylated MEK1 were augmented following treatment with LTA.\n\nConclusions:\nTherefore, this study showed that LTA can increase SP-A synthesis in human alveolar type II epithelial cells through sequentially activating the MEK1-ERK1/2-NF-κB-dependent pathway."},"background_conclusion_text":{"kind":"string","value":"Background:\nSepsis can lead to multiorgan failure and death and appears to be triggered by bacterial products, such as lipopolysaccharide (LPS) from gram-negative bacteria and lipoteichoic acid (LTA) from gram-positive ones \n[1-3]. Infection of the respiratory tract caused by gram-positive bacteria and pneumonia combined with acute lung injury (ALI) are usually the leading causes of mortality by sepsis \n[4]. In the past few decades, the incidences of sepsis and septic shock have been increasing \n[5]. Although endotoxin-activated events are clearly important in gram-negative infection, gram-positive bacteria also have crucial roles, but less is known about host responses to them \n[6]. The increasing prevalence of sepsis from gram-positive bacterial pathogens necessitates a reevaluation of the basic assumptions about the molecular pathogenesis of ALI.\nAlveolar epithelial type II cells contribute to the maintenance of mucosal integrity by modulating the production of surfactants \n[7]. Pulmonary surfactants play important roles in protecting the lung during endotoxin-induced injury and infection \n[8,9]. Surfactant protein (SP)-A is the most abundant pulmonary surfactant protein. Levels of SP-A in bronchiolar lavage fluid are modulated in gram-negative or -positive bacteria-caused lung diseases, including severe pneumonia, acute respiratory distress syndrome, and cardiogenic lung edema \n[10]. Thus, altering lung SP-A levels can be an effective indicator for pulmonary infection and inflammation. Our previous study showed that LPS selectively induced spa gene expression in human alveolar epithelial A549 cells \n[11].\nLTA, an outer membrane component of gram-positive bacteria, was shown to be one of the critical factors participating in the pathogenesis of sepsis \n[12,13]. LTA can stimulate inflammatory responses in the lung \n[14,15]. Therefore, understanding the mechanisms that regulate LTA-mediated cell activation is crucial for diagnosis, treatment, or prognosis of lung inflammatory diseases. In response to stimuli, LTA can activate macrophages to produce massive amounts of inflammatory factors that exhibit systemic effects in the general circulation \n[16]. LTA can induce the secretion of various cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α \n[17]. These data suggest that LTA can selectively modify gene transcription of various cell types and sequentially augment and possibly initiate tissue inflammation.\nMitogen-activated protein kinases (MAPKs) are serine/threonine kinases. The first MAPK isoforms to be cloned and characterized were the extracellular signal-regulated kinase 1 and 2 (ERK 1/2) \n[18,19]. ERK 1/2 are well documented to be activated by a family of dual-specificity kinases known as the mitogen-activated/ERK kinases (MEKs) \n[16,20]. A previous study demonstrated that LTA can selectively activate the ERK pathway in the cornea \n[21]. Our previous study showed that LTA induced TNF-α and IL-6 expressions by means of stimulating phosphorylation of ERK1/2 in macrophages \n[16]. In addition, LTA also triggered translocation of nuclear factor (NF)-κB from the cytoplasm to nuclei and its transactivation activity. Meanwhile, the mechanisms responsible for LTA-induced spa gene expression in alveolar epithelial cells are still unknown. In this study, we attempted to evaluate the effects of LTA on SP-1 synthesis in human alveolar type II epithelial cells and its possible mechanisms.\n\nConclusions:\nIn summary, we used an alveolar epithelial cell model to study the immunomodulatory responses of LTA. Our results revealed that LTA can induce inflammatory responses in alveolar epithelial A549 cells by means of enhancing SP-A mRNA and protein syntheses. Moreover, the signal-transducing mechanisms of LTA-caused regulation of SP-A expression arise through the cascade phosphorylations of MEK1 and ERK1/2. In succession, LTA increased NF-κB expression and translocation. LTA-induced SP-A production in alveolar type II epithelial cells may indicate the status of gram-positive bacteria-caused septic shock and acute lung injury. More molecular pathways should be investigated and proven in the future. However, there are certain limitations of this study, including the use of A549 cells, which are derived from human lung carcinoma. The effects of LTA on A549 cells may differ from those on normal alveolar epithelial cells. Thus, we will perform a translational study to evaluate the effects of LTA on alveolar epithelial cells of animals with acute lung injury."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nLipoteichoic acid (LTA), a gram-positive bacterial outer membrane component, can cause septic shock. Our previous studies showed that the gram-negative endotoxin, lipopolysaccharide (LPS), could induce surfactant protein-A (SP-A) production in human alveolar epithelial (A549) cells.\n\nMethods:\nA549 cells were exposed to LTA. Levels of SP-A, nuclear factor (NF)-κB, extracellular signal-regulated kinase 1/2 (ERK1/2), and mitogen-activated/extracellular signal-regulated kinase kinase (MEK)1 were determined.\n\nResults:\nExposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability. Meanwhile, when exposed to 30 μg/ml LTA for 1, 6, and 24 h, the biosynthesis of SP-A mRNA and protein in A549 cells significantly increased. As to the mechanism, LTA enhanced cytosolic and nuclear NF-κB levels in time-dependent manners. Pretreatment with BAY 11-7082, an inhibitor of NF-κB activation, significantly inhibited LTA-induced SP-A mRNA expression. Sequentially, LTA time-dependently augmented phosphorylation of ERK1/2. In addition, levels of phosphorylated MEK1 were augmented following treatment with LTA.\n\nConclusions:\nTherefore, this study showed that LTA can increase SP-A synthesis in human alveolar type II epithelial cells through sequentially activating the MEK1-ERK1/2-NF-κB-dependent pathway."},"whole_article_text_length":{"kind":"number","value":9975,"string":"9,975"},"whole_article_abstract_length":{"kind":"number","value":280,"string":"280"},"other_sections_lengths":{"kind":"list like","value":[646,169,95,268,143,148,41,103,282,265,515,290,270,103,10,70],"string":"[\n 646,\n 169,\n 95,\n 268,\n 143,\n 148,\n 41,\n 103,\n 282,\n 265,\n 515,\n 290,\n 270,\n 103,\n 10,\n 70\n]","isConversational":false},"num_sections":{"kind":"number","value":20,"string":"20"},"most_frequent_words":{"kind":"list like","value":["lta","cells","a549","a549 cells","sp","κb","nf κb","nf","protein","24"],"string":"[\n \"lta\",\n \"cells\",\n \"a549\",\n \"a549 cells\",\n \"sp\",\n \"κb\",\n \"nf κb\",\n \"nf\",\n \"protein\",\n \"24\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["surfactants pulmonary surfactants","sepsis gram positive","participating pathogenesis sepsis","infection surfactant protein","protecting lung endotoxin"],"string":"[\n \"surfactants pulmonary surfactants\",\n \"sepsis gram positive\",\n \"participating pathogenesis sepsis\",\n \"infection surfactant protein\",\n \"protecting lung endotoxin\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Lipoteichoic acid | Alveolar epithelial cells | Surfactant protein-A | MEK/ERK/NF-κB [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Lipoteichoic acid | Alveolar epithelial cells | Surfactant protein-A | MEK/ERK/NF-κB [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Lipoteichoic acid | Alveolar epithelial cells | Surfactant protein-A | MEK/ERK/NF-κB [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Lipoteichoic acid | Alveolar epithelial cells | Surfactant protein-A | MEK/ERK/NF-κB [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Lipoteichoic acid | Alveolar epithelial cells | Surfactant protein-A | MEK/ERK/NF-κB [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Alveolar Epithelial Cells | Cell Culture Techniques | Cell Survival | Humans | Immunoblotting | Lipopolysaccharides | Mitogen-Activated Protein Kinase 1 | Mitogen-Activated Protein Kinase 3 | NF-kappa B | Pulmonary Surfactant-Associated Protein A | Real-Time Polymerase Chain Reaction | Signal Transduction | Teichoic Acids [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Alveolar Epithelial Cells | Cell Culture Techniques | Cell Survival | Humans | Immunoblotting | Lipopolysaccharides | Mitogen-Activated Protein Kinase 1 | Mitogen-Activated Protein Kinase 3 | NF-kappa B | Pulmonary Surfactant-Associated Protein A | Real-Time Polymerase Chain Reaction | Signal Transduction | Teichoic Acids [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Alveolar Epithelial Cells | Cell Culture Techniques | Cell Survival | Humans | Immunoblotting | Lipopolysaccharides | Mitogen-Activated Protein Kinase 1 | Mitogen-Activated Protein Kinase 3 | NF-kappa B | Pulmonary Surfactant-Associated Protein A | Real-Time Polymerase Chain Reaction | Signal Transduction | Teichoic Acids [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Alveolar Epithelial Cells | Cell Culture Techniques | Cell Survival | Humans | Immunoblotting | Lipopolysaccharides | Mitogen-Activated Protein Kinase 1 | Mitogen-Activated Protein Kinase 3 | NF-kappa B | Pulmonary Surfactant-Associated Protein A | Real-Time Polymerase Chain Reaction | Signal Transduction | Teichoic Acids [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Alveolar Epithelial Cells | Cell Culture Techniques | Cell Survival | Humans | Immunoblotting | Lipopolysaccharides | Mitogen-Activated Protein Kinase 1 | Mitogen-Activated Protein Kinase 3 | NF-kappa B | Pulmonary Surfactant-Associated Protein A | Real-Time Polymerase Chain Reaction | Signal Transduction | Teichoic Acids [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] surfactants pulmonary surfactants | sepsis gram positive | participating pathogenesis sepsis | infection surfactant protein | protecting lung endotoxin [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] surfactants pulmonary surfactants | sepsis gram positive | participating pathogenesis sepsis | infection surfactant protein | protecting lung endotoxin [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] surfactants pulmonary surfactants | sepsis gram positive | participating pathogenesis sepsis | infection surfactant protein | protecting lung endotoxin [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] surfactants pulmonary surfactants | sepsis gram positive | participating pathogenesis sepsis | infection surfactant protein | protecting lung endotoxin [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] surfactants pulmonary surfactants | sepsis gram positive | participating pathogenesis sepsis | infection surfactant protein | protecting lung endotoxin [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] lta | cells | a549 | a549 cells | sp | κb | nf κb | nf | protein | 24 [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] lta | cells | a549 | a549 cells | sp | κb | nf κb | nf | protein | 24 [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] lta | cells | a549 | a549 cells | sp | κb | nf κb | nf | protein | 24 [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] lta | cells | a549 | a549 cells | sp | κb | nf κb | nf | protein | 24 [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] lta | cells | a549 | a549 cells | sp | κb | nf κb | nf | protein | 24 [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] gram | sepsis | lta | positive | lung | gram positive | bacteria | infection | study | positive bacteria [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] lta | figure | cells | a549 cells | a549 | panel | 24 | lta 24 | exposure | sp [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] alveolar | epithelial | lta | alveolar epithelial | cells | epithelial cells | study | lung | responses | lung injury [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] lta | cells | a549 | a549 cells | sp | figure | mrna | 24 | κb | panel [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] lta | cells | a549 | a549 cells | sp | figure | mrna | 24 | κb | panel [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| gram | LPS [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 10 | 30 | 50 μg/ml | 24 ||| 30 μg/ml | 1 | 6 | 24 | SP-A mRNA | A549 ||| LTA | NF-κB ||| NF-κB ||| LTA | ERK1/2 ||| LTA [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] LTA | II | MEK1-ERK1/2-NF-κB-dependent [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| gram | LPS ||| LTA ||| 1/2 ||| ||| 10 | 30 | 50 μg/ml | 24 ||| 30 μg/ml | 1 | 6 | 24 | SP-A mRNA | A549 ||| LTA | NF-κB ||| NF-κB ||| LTA | ERK1/2 ||| LTA ||| LTA | II | MEK1-ERK1/2-NF-κB-dependent [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| gram | LPS ||| LTA ||| 1/2 ||| ||| 10 | 30 | 50 μg/ml | 24 ||| 30 μg/ml | 1 | 6 | 24 | SP-A mRNA | A549 ||| LTA | NF-κB ||| NF-κB ||| LTA | ERK1/2 ||| LTA ||| LTA | II | MEK1-ERK1/2-NF-κB-dependent [SUMMARY]"}}},{"rowIdx":62,"cells":{"title":{"kind":"string","value":"Ecological niche modelling of Hemipteran insects in Cameroon; the paradox of a vector-borne transmission for Mycobacterium ulcerans, the causative agent of Buruli ulcer."},"pmid":{"kind":"string","value":"25344052"},"background_abstract":{"kind":"string","value":"The mode of transmission of the emerging neglected disease Buruli ulcer is unknown. Several potential transmission pathways have been proposed, such as amoebae, or transmission through food webs. Several lines of evidence have suggested that biting aquatic insects, Naucoridae and Belostomatidae, may act as vectors, however this proposal remains controversial."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Herein, based on sampling in Cameroon, we construct an ecological niche model of these insects to describe their spatial distribution. We predict their distribution across West Africa, describe important environmental drivers of their abundance, and examine the correlation between their abundance and Buruli ulcer prevalence in the context of the Bradford-Hill guidelines."},"methods_abstract_label":{"kind":"string","value":"MATERIALS AND METHODS"},"results_abstract":{"kind":"string","value":"We find a significant positive correlation between the abundance of the insects and the prevalence of Buruli ulcer. This correlation changes in space and time, it is significant in one Camerounese study region in (Akonolinga) and not other (Bankim). We discuss notable environmental differences between these regions."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"We interpret the presence of, and change in, this correlation as evidence (though not proof) that these insects may be locally important in the environmental persistence, or transmission, of Mycobacterium. ulcerans. This is consistent with the idea of M. ulcerans as a pathogen transmitted by multiple modes of infection, the importance of any one pathway changing from region to region, depending on the local environmental conditions."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Animals","Buruli Ulcer","Cameroon","Ecosystem","Geographic Mapping","Hemiptera","Humans","Insect Vectors","Mycobacterium ulcerans"],"string":"[\n \"Animals\",\n \"Buruli Ulcer\",\n \"Cameroon\",\n \"Ecosystem\",\n \"Geographic Mapping\",\n \"Hemiptera\",\n \"Humans\",\n \"Insect Vectors\",\n \"Mycobacterium ulcerans\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"4213541"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"The Buruli ulcer is an emerging neglected tropical disease affecting more than 5,000 people per year in West and Central Africa, French Guiana, Latin America and Australia [1]. The disease burden is highest in Africa, where it predominantly affects children under the age of 15, and due to damage to the skin, muscle and bone, can result in severe scarring and crippling deformities if left untreated. The disease is caused by the environmental pathogen Mycobacterium ulcerans.\nThe mode of transmission of M. ulcerans, the method by which it infects humans, is unknown. Many routes of transmission have been proposed, such as transmission by aerosol [2], vector transmission by amoebae [3] or through aquatic networks [4]. During a study of the association between M. ulcerans and aquatic plants in Ghana and Benin aquatic insects were accidentally collected during the sampling procedure, and unexpectedly found to test positive for M. ulcerans\n[1]. The authors proposed that, given that these insects occasionally bite humans, they may be implicated in transmission of M. ulcerans. Aquatic insects have been further implicated after a series of laboratory experiments demonstrated the competency of Naucoridae to act as vectors. Naucoridae are able to acquire M. ulcerans from their diet, and then transmit the pathogen to mice resulting in Buruli-like symptoms [5–8]. Buruli ulcer is commonly associated with lowland, stagnant water [9] and human behaviours associated with water bodies appear to be risk factors for Buruli ulcer infection, which would lend support to the idea of infection occurring in an aquatic context.\nHowever, the role of these insects has been disputed for several reasons. In a two year study of Buruli ulcer endemic and non-endemic sites in Ghana [10], found no evidence for a role in transmission. The population of these insects, and the prevalence of M. ulcerans infection in them, was not significantly different between Buruli ulcer endemic and Buruli ulcer non-endemic sites [10]. The authors argued that, if these insects are vectors, we would expect them to have a higher abundance in Buruli ulcer endemic sites, that rates of M. ulcerans infection of the insects should be higher in Buruli ulcer endemic sites, and that the rate of infection of these insects should be higher than other species of invertebrates.\nThese expectations are based on the Bradford-Hill guidelines for associating insect vectors with human vector-borne disease [11, 12]. These guidelines provide a general framework to explore the association between vectors and disease, based on the consistency, specificity, plausibility and coherence of the proposed mode of transmission.\nConsistency refers to the expectation that the rate of infection of the proposed vectors, which should be consistently strongly positively associated, in time and space, with prevalence of human cases. This also implies human cases should not occur in absence of the proposed vector. The proposed vector should have a demonstrated capacity to physically transmit the pathogen, which has been demonstrated for M. ulcerans in the lab [5]. The interaction between the proposed vector and human infection must be specific and alternative explanations of human infection should be ruled out (though see [4] for alternative explanations). That is, human infection must be demonstrated to not have been the result of other potential modes of transmission. We note that this criterion must be applied with care in cases of multi-host transmission.\nAdditionally, the proposed vector must plausibly be able to be a vector of the pathogen. This criterion is often controversial as it is highly dependent upon the experience of the researcher, and their opinion about what is, and is not, plausible as opposed to merely possible [13]. Most authors in Buruli ulcer research would agree with some basic facts; the waterbugs are infected in the environment [14], they bite humans occasionally, and are able to transmit the bacteria to mice in the lab [5]. However, waterbugs are not known to be vectors of other pathogens, and related Mycobacteria (Mycobacterium tuberculosis, M. leprae, M. marnium) are not known to be vector-borne diseases [10, 12]. The plausibility of this proposed route of transmission is still debated. The final criterion, coherence, is based on what we already know about the pathogen, the vector and the host. Does the proposed method of transmission fit well with our current understanding of its biology? As our understanding of the biology of M. ulcerans improves, this criterion will be answered.\nGiven this framework, how likely is it that Naucoridae and/or Belostomatidae are vectors of the Buruli ulcer disease? Herein, we explore the correlation in time and space between the proposed vectors, Naucoridae and Belostomatidae, and the Buruli ulcer prevalence. We discuss the other Bradford-Hill criteria, but do not focus on them specifically, as it was not directly within the scope of this work. Based on sampling in Cameroon, we characterise the set of suitable habitats within which species of the Families Belostomatidae and Naucoridae can maintain a population (their ecological niche) and describe the spatial distribution of these suitable habitats across West Africa. We then explore any correlations between habitat suitability and Buruli ulcer prevalence at multiple spatial scales."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Distribution of suitable habitat, and its relationship to Buruli ulcer In Akonolinga there was a significantly positive correlation between Buruli ulcer prevalence and average habitat suitability, for both Naucoridae and Belostomatidae, in the wet season (Table 2, Figure 4). This relationship was significant at multiple buffer distances. In contrast, in Bankim there was no significant correlation between Buruli ulcer prevalence and Belostomatidae or Naucoridae average habitat suitability, in either wet or dry seasons or at any buffer distance (Table 3).Table 2\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\nSpeciesSeasonBufferBonferroni pvalueBelostomatidaeDry10-5-4-3-2-1-Village-Wet100.00050.01440.0233-2-1-Village-NaucoridaeDry10-5-4-3-2-1-Village-Wet100.00150.01340.0143-2-1-Village-The column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.Figure 4\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\n\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\n\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.\n\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\n\nSpearman rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Bankim, for both seasons and species\n\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. No significant correlations were observed in Bankim.\nIn Akonolinga there was a significantly positive correlation between Buruli ulcer prevalence and average habitat suitability, for both Naucoridae and Belostomatidae, in the wet season (Table 2, Figure 4). This relationship was significant at multiple buffer distances. In contrast, in Bankim there was no significant correlation between Buruli ulcer prevalence and Belostomatidae or Naucoridae average habitat suitability, in either wet or dry seasons or at any buffer distance (Table 3).Table 2\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\nSpeciesSeasonBufferBonferroni pvalueBelostomatidaeDry10-5-4-3-2-1-Village-Wet100.00050.01440.0233-2-1-Village-NaucoridaeDry10-5-4-3-2-1-Village-Wet100.00150.01340.0143-2-1-Village-The column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.Figure 4\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\n\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\n\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.\n\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\n\nSpearman rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Bankim, for both seasons and species\n\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. No significant correlations were observed in Bankim.\n Ecologically important variables in the distribution of the aquatic insect families Variable importance was evaluated using Jackknife variable removal. Jacknife removes a variable and evaluates the effect of variable removal on the model. In the dry season Belostomatidae and Naucoridae responded in broadly similar fashions; the variable whose removal had the largest effect was GLC 5 km (Figure 5). The land cover categories most suitable for both Belostomatidae and Naucoridae are water bodies, artificial areas, rain fed croplands and forest/grassland mosaics (Figure 6). If one of these categories is the dominant category in 5 km radius, in the dry season, the likelihood of encountering the insect is higher. Unsuitable categories were forest and vegetation/cropland mosaic.In the wet season precipitation is more important than land cover. Precipitation suitability peaks at approximately 300 millimeters per month, and diminishes above or below this (Figure 6). For the dry season there is a simple increase in habitat suitability with increasing precipitation.Figure 5\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.Figure 6\nHabitat suitability for Belostomatidae (1\nst\nand 2\nnd\nrows) and Naucoridae (3\nrd\nand 4\nth\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\n\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.\n\nHabitat suitability for Belostomatidae (1\nst\nand 2\nnd\nrows) and Naucoridae (3\nrd\nand 4\nth\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\nFlow accumulation had a negative association with habitat suitability, and wetness index had a positive association, regardless of season, for both Belostomatidae and Naucoridae.\nVariable importance was evaluated using Jackknife variable removal. Jacknife removes a variable and evaluates the effect of variable removal on the model. In the dry season Belostomatidae and Naucoridae responded in broadly similar fashions; the variable whose removal had the largest effect was GLC 5 km (Figure 5). The land cover categories most suitable for both Belostomatidae and Naucoridae are water bodies, artificial areas, rain fed croplands and forest/grassland mosaics (Figure 6). If one of these categories is the dominant category in 5 km radius, in the dry season, the likelihood of encountering the insect is higher. Unsuitable categories were forest and vegetation/cropland mosaic.In the wet season precipitation is more important than land cover. Precipitation suitability peaks at approximately 300 millimeters per month, and diminishes above or below this (Figure 6). For the dry season there is a simple increase in habitat suitability with increasing precipitation.Figure 5\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.Figure 6\nHabitat suitability for Belostomatidae (1\nst\nand 2\nnd\nrows) and Naucoridae (3\nrd\nand 4\nth\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\n\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.\n\nHabitat suitability for Belostomatidae (1\nst\nand 2\nnd\nrows) and Naucoridae (3\nrd\nand 4\nth\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\nFlow accumulation had a negative association with habitat suitability, and wetness index had a positive association, regardless of season, for both Belostomatidae and Naucoridae.\n Model performance AICc for Naucoridae adults (dry season) was 14.6, and 14.2 in the wet season, as in Table 2. For Belostomatidae adults (dry season) the AICc was 12.5, and 12.2 in the wet season. Scores of overfitting are relative; these scores indicate the Belostomatidae model was less prone to overfitting than the Naucoridae model.\nThe AG data set was also used in model validation. In the dry season Naucoridae adults had an AG AUC of 0.83, and 0.80 in the wet season. Belostomatidae adults had an AG AUC of 0.80 in the dry season, and 0.86 in the wet season. These scores indicate that the models are able to describe the distribution of the insects with good accuracy; the model based on SME dataset is able to accurately replicate the independently collected AG dataset.\nAICc for Naucoridae adults (dry season) was 14.6, and 14.2 in the wet season, as in Table 2. For Belostomatidae adults (dry season) the AICc was 12.5, and 12.2 in the wet season. Scores of overfitting are relative; these scores indicate the Belostomatidae model was less prone to overfitting than the Naucoridae model.\nThe AG data set was also used in model validation. In the dry season Naucoridae adults had an AG AUC of 0.83, and 0.80 in the wet season. Belostomatidae adults had an AG AUC of 0.80 in the dry season, and 0.86 in the wet season. These scores indicate that the models are able to describe the distribution of the insects with good accuracy; the model based on SME dataset is able to accurately replicate the independently collected AG dataset."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Background","Distribution of Belostomatidae and Naucoridae aquatic insect families","Environmental parameters used in ecological niche modelling","Data preparation, niche modelling, and prediction of spatial distribution of suitable habitat","Evaluating model performance","Identifying the relationship between habitat suitability and Buruli ulcer prevalence","Distribution of suitable habitat, and its relationship to Buruli ulcer","Ecologically important variables in the distribution of the aquatic insect families","Model performance"],"string":"[\n \"Background\",\n \"Distribution of Belostomatidae and Naucoridae aquatic insect families\",\n \"Environmental parameters used in ecological niche modelling\",\n \"Data preparation, niche modelling, and prediction of spatial distribution of suitable habitat\",\n \"Evaluating model performance\",\n \"Identifying the relationship between habitat suitability and Buruli ulcer prevalence\",\n \"Distribution of suitable habitat, and its relationship to Buruli ulcer\",\n \"Ecologically important variables in the distribution of the aquatic insect families\",\n \"Model performance\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["The Buruli ulcer is an emerging neglected tropical disease affecting more than 5,000 people per year in West and Central Africa, French Guiana, Latin America and Australia [1]. The disease burden is highest in Africa, where it predominantly affects children under the age of 15, and due to damage to the skin, muscle and bone, can result in severe scarring and crippling deformities if left untreated. The disease is caused by the environmental pathogen Mycobacterium ulcerans.\nThe mode of transmission of M. ulcerans, the method by which it infects humans, is unknown. Many routes of transmission have been proposed, such as transmission by aerosol [2], vector transmission by amoebae [3] or through aquatic networks [4]. During a study of the association between M. ulcerans and aquatic plants in Ghana and Benin aquatic insects were accidentally collected during the sampling procedure, and unexpectedly found to test positive for M. ulcerans\n[1]. The authors proposed that, given that these insects occasionally bite humans, they may be implicated in transmission of M. ulcerans. Aquatic insects have been further implicated after a series of laboratory experiments demonstrated the competency of Naucoridae to act as vectors. Naucoridae are able to acquire M. ulcerans from their diet, and then transmit the pathogen to mice resulting in Buruli-like symptoms [5–8]. Buruli ulcer is commonly associated with lowland, stagnant water [9] and human behaviours associated with water bodies appear to be risk factors for Buruli ulcer infection, which would lend support to the idea of infection occurring in an aquatic context.\nHowever, the role of these insects has been disputed for several reasons. In a two year study of Buruli ulcer endemic and non-endemic sites in Ghana [10], found no evidence for a role in transmission. The population of these insects, and the prevalence of M. ulcerans infection in them, was not significantly different between Buruli ulcer endemic and Buruli ulcer non-endemic sites [10]. The authors argued that, if these insects are vectors, we would expect them to have a higher abundance in Buruli ulcer endemic sites, that rates of M. ulcerans infection of the insects should be higher in Buruli ulcer endemic sites, and that the rate of infection of these insects should be higher than other species of invertebrates.\nThese expectations are based on the Bradford-Hill guidelines for associating insect vectors with human vector-borne disease [11, 12]. These guidelines provide a general framework to explore the association between vectors and disease, based on the consistency, specificity, plausibility and coherence of the proposed mode of transmission.\nConsistency refers to the expectation that the rate of infection of the proposed vectors, which should be consistently strongly positively associated, in time and space, with prevalence of human cases. This also implies human cases should not occur in absence of the proposed vector. The proposed vector should have a demonstrated capacity to physically transmit the pathogen, which has been demonstrated for M. ulcerans in the lab [5]. The interaction between the proposed vector and human infection must be specific and alternative explanations of human infection should be ruled out (though see [4] for alternative explanations). That is, human infection must be demonstrated to not have been the result of other potential modes of transmission. We note that this criterion must be applied with care in cases of multi-host transmission.\nAdditionally, the proposed vector must plausibly be able to be a vector of the pathogen. This criterion is often controversial as it is highly dependent upon the experience of the researcher, and their opinion about what is, and is not, plausible as opposed to merely possible [13]. Most authors in Buruli ulcer research would agree with some basic facts; the waterbugs are infected in the environment [14], they bite humans occasionally, and are able to transmit the bacteria to mice in the lab [5]. However, waterbugs are not known to be vectors of other pathogens, and related Mycobacteria (Mycobacterium tuberculosis, M. leprae, M. marnium) are not known to be vector-borne diseases [10, 12]. The plausibility of this proposed route of transmission is still debated. The final criterion, coherence, is based on what we already know about the pathogen, the vector and the host. Does the proposed method of transmission fit well with our current understanding of its biology? As our understanding of the biology of M. ulcerans improves, this criterion will be answered.\nGiven this framework, how likely is it that Naucoridae and/or Belostomatidae are vectors of the Buruli ulcer disease? Herein, we explore the correlation in time and space between the proposed vectors, Naucoridae and Belostomatidae, and the Buruli ulcer prevalence. We discuss the other Bradford-Hill criteria, but do not focus on them specifically, as it was not directly within the scope of this work. Based on sampling in Cameroon, we characterise the set of suitable habitats within which species of the Families Belostomatidae and Naucoridae can maintain a population (their ecological niche) and describe the spatial distribution of these suitable habitats across West Africa. We then explore any correlations between habitat suitability and Buruli ulcer prevalence at multiple spatial scales.","Data were collected as described in [15] hereafter referred to as the SME dataset. In brief, 36 sample sites in Cameroon were visited monthly from September 2012 to February 2013 (Figure 1), a period including both wet and dry seasons. Dip net sampling was conducted at all sites. Due to limitations of current taxonomic keys, the aquatic insects of interest were only identifiable to the phylogenetic division of Family. A second dataset was used in model validation, using data collected separately by A. Garchitorena (Figure 1). This dataset was collected as described in [16], and is hereafter referred to as the AG dataset. Niche models of Belostomatidae and Naucoridae are constructed using the SME dataset, and then tested on their ability to reproduce the independent AG dataset.Figure 1\nStudy sites Cameroon against local land cover. Data from the SME dataset is in red, and AG data set is in blue. The sample sites span the extent of Cameroon, sampling from every major land cover category.\n\nStudy sites Cameroon against local land cover. Data from the SME dataset is in red, and AG data set is in blue. The sample sites span the extent of Cameroon, sampling from every major land cover category.","Five ecological parameters were used to describe the distribution of suitable habitats: rainfall, flow accumulation, wetness index, land cover at the sample site, and land cover within the flight range of adult insects. These variables were selected on the basis of their likelihood to influence the distribution and condition of water, and are summarised in Table 1. Rainfall was highly seasonal, so we divide models by the season of collection. Models constructed using species distribution data from the dry season used the precipitation in the driest season; models constructed using species distribution data from the wet season used the precipitation in the wettest season. These two variables were taken from the Worldclim database, as BIO13 Precipitation of Wettest Month and BIO14 Precipitation of Driest Month [17]. Flow accumulation was derived using elevation data [18]. Flow accumulation is the surface area contributing water to a particular point, and indicates the potential amount of water available, which is then determined by rainfall. Using the SRTM elevation, flow accumulation was derived using the Fill, Flow Direction and Flow Accumulation tools in ArcMap 10.1 [19]. Wetness index has previously been shown to be associated with the Buruli ulcer [9]. In ecological terms it indicates the topological potential for water to accumulate, and was derived according to Equation 1,Table 1\nEnvironmental variables used in ecological niche modelling\nVariableUnitsOriginal resolutionSourceWetness indexm2\n15 arc-sec (approx 450 m2)SRTMFlow accumulationm2\n15 arc-sec (approx 450 m2)SRTMPrecipitation in wettest seasonmillimeter30 arc-sec (approx 1 km2)Bioclim 13Precipitation in driest seasonmillimeter30 arc-sec (approx 1 km2)Bioclim 14GLC-APUnitless300 m2\nGLC 2009GLC-5 KUnitless300 m2\nGLC 2009All data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for use in Maxent. Resampling used resample() in the library ‘raster’ of the software R.\n\nEnvironmental variables used in ecological niche modelling\n\nAll data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for use in Maxent. Resampling used resample() in the library ‘raster’ of the software R.\n\n\n1\n\n\n\nwhere FA was the flow accumulation, 500 was the cell size in meters, and S was the surface slope in degrees. Large flow accumulation values and flat slopes resulted in high wetness index values, and indicate areas where water is likely to stagnate. In areas where the slope is zero wetness index had no value. Land cover was derived from the Global Land Cover Map 2009 [20]. GLC-At Point (hereafter GLC-AP) is the land cover at the sample site. In a 5 km radius around the site the most common (modal) land cover category was described. For example, a sample site may be in savannah, but surrounded by forest. 5 km was selected as the approximate flight radius of the insects [21–23]. This is termed GLC-5 K.","Insect distribution data from the 36 sample sites (Figure 1) were explored to identify normality, homogeneity of variance and correlation in the five environmental parameters used. Two sites were excluded from analysis as apparent outliers in flow accumulation, otherwise the data were normally distributed and homogenous. Correlation was observed between flow accumulation and wetness index and between precipitation in the driest and in the wettest seasons, however this was not significant (p > 0.05) using a Spearman’s correlation test. All data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for modelling using resample() in the library ‘raster’ of the software R [24].\nAcross the scale of West Africa we assume that absence data is not reliable, as it is more likely to indicate failure of detection rather than evidence of absence. For this reason, we chose to conduct presence-only modelling, and the specific method selected was Maximum Entropy [25]. We used the software Maxent (Maxent 3.3.3 k, [25]) to construct these models. Maxent has been used several times in the past to model the ecological niche of disease vectors [26]. Maximum entropy modelling minimises the divergence between the distribution of the environmental parameters and the species distribution, assuming the species is distributed in the most ecologically efficient manner possible. Maxent describes the environmental parameters across the study region, and is sensitive to the size of the study region. Herein, we generated background points in every raster cell within the extent of our study (that is, every environment was described). The models were replicated 100 times and averaged.\nIn order to select an appropriate extent to the area considered for modelling we chose to confine the spatial extent of our study to the region ecologically interpolated by the sample sites (Figure 2). The sample sites of the SME dataset describe a particular range of environmental conditions for the five environmental parameters (Table 1). We excluded conditions that were not studied by the SME regime (much larger or lower values of rainfall, land cover categories not sampled, flow accumulation not observed at the study sites) to avoid extrapolating beyond the range of ecological parameter values studied. To select this interpolative region we constructed a Multivariate Environmental Similarity Surface (MESS map [27]), excluding areas with negative MESS values from our study region. A MESS map was constructed for each model; models with more samples can be interpolated into larger ecological regions. MESS maps were constructed with the function mess() available in the library dismo in R.Figure 2\nDelineation of the study area. We exclude areas of ecological extrapolation. Across West Africa the red-green coloured region is ecologically interpolative within our sites, according to a multivariate environmental similarity surface (MESS). Regions where we extrapolate beyond the ecology of our study sites are identified using MESS values less than 0. Colour values are MESS values, indicating similarity to sample sites; a value of 100 is identical to the median of the sample sites. The spatial projection of this interpolative region is shown against a map of country borders of West Africa, for Belostomatidae adults in the dry season. This projection changes for each family, developmental stage and season, and though it is spatially discontinuous it is ecologically homogenous.\n\nDelineation of the study area. We exclude areas of ecological extrapolation. Across West Africa the red-green coloured region is ecologically interpolative within our sites, according to a multivariate environmental similarity surface (MESS). Regions where we extrapolate beyond the ecology of our study sites are identified using MESS values less than 0. Colour values are MESS values, indicating similarity to sample sites; a value of 100 is identical to the median of the sample sites. The spatial projection of this interpolative region is shown against a map of country borders of West Africa, for Belostomatidae adults in the dry season. This projection changes for each family, developmental stage and season, and though it is spatially discontinuous it is ecologically homogenous.","We evaluated the performance of these models according to their ability to correctly predict the distribution of the insects (with a separate dataset, the AG dataset) as efficiently as possible (avoiding overfitting). We used two methods to evaluate the models. First, the Akaike information criterion, corrected for small sample size (AICc), was used. AICc was generated as in [28], and penalises complex models to avoid over-fitting the data, low AICc scores indicate the model is not over-fitting. Second, we tested the models ability to predict the AG dataset. The Area Under Curve (AUC) is often used to evaluate Maxent models, and has been criticized previously [29]. We used a modified version of the AUC for model evaluation, termed here AG AUC. The model predictions were compared to the known values as collected by AG. AG AUC values range from 0 to 1, values close to 1 indicate good performance, 0.5 is no better than random. Use of the AG dataset allowed us to use true absence data for model validation, avoiding the problem of pseudo-absence data in Maxent.\nThe purpose of these two different metrics is to consider different aspects of performance, neither were without limitations. Use of the AG dataset allowed a degree of validation across methodologies, indicating the extent to which our results were dependent on a particular sampling regime. AICc is traditionally used to indicate over-fitting in models, however it is sensitive to the size of the ecological niche of the species.","Buruli ulcer prevalence data was collected for two endemic regions in Cameroon, Akonolinga [30] and Bankim [31], as shown in Figure 3. Around the centre of each village a buffer was created, and average habitat suitability in this buffer was correlated to the village Buruli ulcer prevalence using Spearman’s rank correlation coefficient. Seven buffers were used to explore the effect of buffer size and shape. Around the centre of the village circular buffers of 1, 2, 3, 4, 5 and 10 km were selected, and average habitat suitability recorded. We also used a buffer defined by the borders of the village (Figure 3). This buffer changes in size for each village, and represents the approximate extent of the village area. 5 km is approximately the flight radius of the insects, and the distance easily walkable by local people on an average day in Akonolinga [32]. To deal with this multiple testing, Bonferonni’s correction of the p-values was used.Figure 3\nSpatial distribution of Buruli ulcer prevalence in Akonolianga and Bankim, two endemic regions within Cameroon.\n\n\nSpatial distribution of Buruli ulcer prevalence in Akonolianga and Bankim, two endemic regions within Cameroon.\n","In Akonolinga there was a significantly positive correlation between Buruli ulcer prevalence and average habitat suitability, for both Naucoridae and Belostomatidae, in the wet season (Table 2, Figure 4). This relationship was significant at multiple buffer distances. In contrast, in Bankim there was no significant correlation between Buruli ulcer prevalence and Belostomatidae or Naucoridae average habitat suitability, in either wet or dry seasons or at any buffer distance (Table 3).Table 2\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\nSpeciesSeasonBufferBonferroni pvalueBelostomatidaeDry10-5-4-3-2-1-Village-Wet100.00050.01440.0233-2-1-Village-NaucoridaeDry10-5-4-3-2-1-Village-Wet100.00150.01340.0143-2-1-Village-The column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.Figure 4\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\n\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\n\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.\n\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\n\nSpearman rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Bankim, for both seasons and species\n\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. No significant correlations were observed in Bankim.","Variable importance was evaluated using Jackknife variable removal. Jacknife removes a variable and evaluates the effect of variable removal on the model. In the dry season Belostomatidae and Naucoridae responded in broadly similar fashions; the variable whose removal had the largest effect was GLC 5 km (Figure 5). The land cover categories most suitable for both Belostomatidae and Naucoridae are water bodies, artificial areas, rain fed croplands and forest/grassland mosaics (Figure 6). If one of these categories is the dominant category in 5 km radius, in the dry season, the likelihood of encountering the insect is higher. Unsuitable categories were forest and vegetation/cropland mosaic.In the wet season precipitation is more important than land cover. Precipitation suitability peaks at approximately 300 millimeters per month, and diminishes above or below this (Figure 6). For the dry season there is a simple increase in habitat suitability with increasing precipitation.Figure 5\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.Figure 6\nHabitat suitability for Belostomatidae (1\nst\nand 2\nnd\nrows) and Naucoridae (3\nrd\nand 4\nth\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\n\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.\n\nHabitat suitability for Belostomatidae (1\nst\nand 2\nnd\nrows) and Naucoridae (3\nrd\nand 4\nth\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\nFlow accumulation had a negative association with habitat suitability, and wetness index had a positive association, regardless of season, for both Belostomatidae and Naucoridae.","AICc for Naucoridae adults (dry season) was 14.6, and 14.2 in the wet season, as in Table 2. For Belostomatidae adults (dry season) the AICc was 12.5, and 12.2 in the wet season. Scores of overfitting are relative; these scores indicate the Belostomatidae model was less prone to overfitting than the Naucoridae model.\nThe AG data set was also used in model validation. In the dry season Naucoridae adults had an AG AUC of 0.83, and 0.80 in the wet season. Belostomatidae adults had an AG AUC of 0.80 in the dry season, and 0.86 in the wet season. These scores indicate that the models are able to describe the distribution of the insects with good accuracy; the model based on SME dataset is able to accurately replicate the independently collected AG dataset."],"string":"[\n \"The Buruli ulcer is an emerging neglected tropical disease affecting more than 5,000 people per year in West and Central Africa, French Guiana, Latin America and Australia [1]. The disease burden is highest in Africa, where it predominantly affects children under the age of 15, and due to damage to the skin, muscle and bone, can result in severe scarring and crippling deformities if left untreated. The disease is caused by the environmental pathogen Mycobacterium ulcerans.\\nThe mode of transmission of M. ulcerans, the method by which it infects humans, is unknown. Many routes of transmission have been proposed, such as transmission by aerosol [2], vector transmission by amoebae [3] or through aquatic networks [4]. During a study of the association between M. ulcerans and aquatic plants in Ghana and Benin aquatic insects were accidentally collected during the sampling procedure, and unexpectedly found to test positive for M. ulcerans\\n[1]. The authors proposed that, given that these insects occasionally bite humans, they may be implicated in transmission of M. ulcerans. Aquatic insects have been further implicated after a series of laboratory experiments demonstrated the competency of Naucoridae to act as vectors. Naucoridae are able to acquire M. ulcerans from their diet, and then transmit the pathogen to mice resulting in Buruli-like symptoms [5–8]. Buruli ulcer is commonly associated with lowland, stagnant water [9] and human behaviours associated with water bodies appear to be risk factors for Buruli ulcer infection, which would lend support to the idea of infection occurring in an aquatic context.\\nHowever, the role of these insects has been disputed for several reasons. In a two year study of Buruli ulcer endemic and non-endemic sites in Ghana [10], found no evidence for a role in transmission. The population of these insects, and the prevalence of M. ulcerans infection in them, was not significantly different between Buruli ulcer endemic and Buruli ulcer non-endemic sites [10]. The authors argued that, if these insects are vectors, we would expect them to have a higher abundance in Buruli ulcer endemic sites, that rates of M. ulcerans infection of the insects should be higher in Buruli ulcer endemic sites, and that the rate of infection of these insects should be higher than other species of invertebrates.\\nThese expectations are based on the Bradford-Hill guidelines for associating insect vectors with human vector-borne disease [11, 12]. These guidelines provide a general framework to explore the association between vectors and disease, based on the consistency, specificity, plausibility and coherence of the proposed mode of transmission.\\nConsistency refers to the expectation that the rate of infection of the proposed vectors, which should be consistently strongly positively associated, in time and space, with prevalence of human cases. This also implies human cases should not occur in absence of the proposed vector. The proposed vector should have a demonstrated capacity to physically transmit the pathogen, which has been demonstrated for M. ulcerans in the lab [5]. The interaction between the proposed vector and human infection must be specific and alternative explanations of human infection should be ruled out (though see [4] for alternative explanations). That is, human infection must be demonstrated to not have been the result of other potential modes of transmission. We note that this criterion must be applied with care in cases of multi-host transmission.\\nAdditionally, the proposed vector must plausibly be able to be a vector of the pathogen. This criterion is often controversial as it is highly dependent upon the experience of the researcher, and their opinion about what is, and is not, plausible as opposed to merely possible [13]. Most authors in Buruli ulcer research would agree with some basic facts; the waterbugs are infected in the environment [14], they bite humans occasionally, and are able to transmit the bacteria to mice in the lab [5]. However, waterbugs are not known to be vectors of other pathogens, and related Mycobacteria (Mycobacterium tuberculosis, M. leprae, M. marnium) are not known to be vector-borne diseases [10, 12]. The plausibility of this proposed route of transmission is still debated. The final criterion, coherence, is based on what we already know about the pathogen, the vector and the host. Does the proposed method of transmission fit well with our current understanding of its biology? As our understanding of the biology of M. ulcerans improves, this criterion will be answered.\\nGiven this framework, how likely is it that Naucoridae and/or Belostomatidae are vectors of the Buruli ulcer disease? Herein, we explore the correlation in time and space between the proposed vectors, Naucoridae and Belostomatidae, and the Buruli ulcer prevalence. We discuss the other Bradford-Hill criteria, but do not focus on them specifically, as it was not directly within the scope of this work. Based on sampling in Cameroon, we characterise the set of suitable habitats within which species of the Families Belostomatidae and Naucoridae can maintain a population (their ecological niche) and describe the spatial distribution of these suitable habitats across West Africa. We then explore any correlations between habitat suitability and Buruli ulcer prevalence at multiple spatial scales.\",\n \"Data were collected as described in [15] hereafter referred to as the SME dataset. In brief, 36 sample sites in Cameroon were visited monthly from September 2012 to February 2013 (Figure 1), a period including both wet and dry seasons. Dip net sampling was conducted at all sites. Due to limitations of current taxonomic keys, the aquatic insects of interest were only identifiable to the phylogenetic division of Family. A second dataset was used in model validation, using data collected separately by A. Garchitorena (Figure 1). This dataset was collected as described in [16], and is hereafter referred to as the AG dataset. Niche models of Belostomatidae and Naucoridae are constructed using the SME dataset, and then tested on their ability to reproduce the independent AG dataset.Figure 1\\nStudy sites Cameroon against local land cover. Data from the SME dataset is in red, and AG data set is in blue. The sample sites span the extent of Cameroon, sampling from every major land cover category.\\n\\nStudy sites Cameroon against local land cover. Data from the SME dataset is in red, and AG data set is in blue. The sample sites span the extent of Cameroon, sampling from every major land cover category.\",\n \"Five ecological parameters were used to describe the distribution of suitable habitats: rainfall, flow accumulation, wetness index, land cover at the sample site, and land cover within the flight range of adult insects. These variables were selected on the basis of their likelihood to influence the distribution and condition of water, and are summarised in Table 1. Rainfall was highly seasonal, so we divide models by the season of collection. Models constructed using species distribution data from the dry season used the precipitation in the driest season; models constructed using species distribution data from the wet season used the precipitation in the wettest season. These two variables were taken from the Worldclim database, as BIO13 Precipitation of Wettest Month and BIO14 Precipitation of Driest Month [17]. Flow accumulation was derived using elevation data [18]. Flow accumulation is the surface area contributing water to a particular point, and indicates the potential amount of water available, which is then determined by rainfall. Using the SRTM elevation, flow accumulation was derived using the Fill, Flow Direction and Flow Accumulation tools in ArcMap 10.1 [19]. Wetness index has previously been shown to be associated with the Buruli ulcer [9]. In ecological terms it indicates the topological potential for water to accumulate, and was derived according to Equation 1,Table 1\\nEnvironmental variables used in ecological niche modelling\\nVariableUnitsOriginal resolutionSourceWetness indexm2\\n15 arc-sec (approx 450 m2)SRTMFlow accumulationm2\\n15 arc-sec (approx 450 m2)SRTMPrecipitation in wettest seasonmillimeter30 arc-sec (approx 1 km2)Bioclim 13Precipitation in driest seasonmillimeter30 arc-sec (approx 1 km2)Bioclim 14GLC-APUnitless300 m2\\nGLC 2009GLC-5 KUnitless300 m2\\nGLC 2009All data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for use in Maxent. Resampling used resample() in the library ‘raster’ of the software R.\\n\\nEnvironmental variables used in ecological niche modelling\\n\\nAll data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for use in Maxent. Resampling used resample() in the library ‘raster’ of the software R.\\n\\n\\n1\\n\\n\\n\\nwhere FA was the flow accumulation, 500 was the cell size in meters, and S was the surface slope in degrees. Large flow accumulation values and flat slopes resulted in high wetness index values, and indicate areas where water is likely to stagnate. In areas where the slope is zero wetness index had no value. Land cover was derived from the Global Land Cover Map 2009 [20]. GLC-At Point (hereafter GLC-AP) is the land cover at the sample site. In a 5 km radius around the site the most common (modal) land cover category was described. For example, a sample site may be in savannah, but surrounded by forest. 5 km was selected as the approximate flight radius of the insects [21–23]. This is termed GLC-5 K.\",\n \"Insect distribution data from the 36 sample sites (Figure 1) were explored to identify normality, homogeneity of variance and correlation in the five environmental parameters used. Two sites were excluded from analysis as apparent outliers in flow accumulation, otherwise the data were normally distributed and homogenous. Correlation was observed between flow accumulation and wetness index and between precipitation in the driest and in the wettest seasons, however this was not significant (p > 0.05) using a Spearman’s correlation test. All data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for modelling using resample() in the library ‘raster’ of the software R [24].\\nAcross the scale of West Africa we assume that absence data is not reliable, as it is more likely to indicate failure of detection rather than evidence of absence. For this reason, we chose to conduct presence-only modelling, and the specific method selected was Maximum Entropy [25]. We used the software Maxent (Maxent 3.3.3 k, [25]) to construct these models. Maxent has been used several times in the past to model the ecological niche of disease vectors [26]. Maximum entropy modelling minimises the divergence between the distribution of the environmental parameters and the species distribution, assuming the species is distributed in the most ecologically efficient manner possible. Maxent describes the environmental parameters across the study region, and is sensitive to the size of the study region. Herein, we generated background points in every raster cell within the extent of our study (that is, every environment was described). The models were replicated 100 times and averaged.\\nIn order to select an appropriate extent to the area considered for modelling we chose to confine the spatial extent of our study to the region ecologically interpolated by the sample sites (Figure 2). The sample sites of the SME dataset describe a particular range of environmental conditions for the five environmental parameters (Table 1). We excluded conditions that were not studied by the SME regime (much larger or lower values of rainfall, land cover categories not sampled, flow accumulation not observed at the study sites) to avoid extrapolating beyond the range of ecological parameter values studied. To select this interpolative region we constructed a Multivariate Environmental Similarity Surface (MESS map [27]), excluding areas with negative MESS values from our study region. A MESS map was constructed for each model; models with more samples can be interpolated into larger ecological regions. MESS maps were constructed with the function mess() available in the library dismo in R.Figure 2\\nDelineation of the study area. We exclude areas of ecological extrapolation. Across West Africa the red-green coloured region is ecologically interpolative within our sites, according to a multivariate environmental similarity surface (MESS). Regions where we extrapolate beyond the ecology of our study sites are identified using MESS values less than 0. Colour values are MESS values, indicating similarity to sample sites; a value of 100 is identical to the median of the sample sites. The spatial projection of this interpolative region is shown against a map of country borders of West Africa, for Belostomatidae adults in the dry season. This projection changes for each family, developmental stage and season, and though it is spatially discontinuous it is ecologically homogenous.\\n\\nDelineation of the study area. We exclude areas of ecological extrapolation. Across West Africa the red-green coloured region is ecologically interpolative within our sites, according to a multivariate environmental similarity surface (MESS). Regions where we extrapolate beyond the ecology of our study sites are identified using MESS values less than 0. Colour values are MESS values, indicating similarity to sample sites; a value of 100 is identical to the median of the sample sites. The spatial projection of this interpolative region is shown against a map of country borders of West Africa, for Belostomatidae adults in the dry season. This projection changes for each family, developmental stage and season, and though it is spatially discontinuous it is ecologically homogenous.\",\n \"We evaluated the performance of these models according to their ability to correctly predict the distribution of the insects (with a separate dataset, the AG dataset) as efficiently as possible (avoiding overfitting). We used two methods to evaluate the models. First, the Akaike information criterion, corrected for small sample size (AICc), was used. AICc was generated as in [28], and penalises complex models to avoid over-fitting the data, low AICc scores indicate the model is not over-fitting. Second, we tested the models ability to predict the AG dataset. The Area Under Curve (AUC) is often used to evaluate Maxent models, and has been criticized previously [29]. We used a modified version of the AUC for model evaluation, termed here AG AUC. The model predictions were compared to the known values as collected by AG. AG AUC values range from 0 to 1, values close to 1 indicate good performance, 0.5 is no better than random. Use of the AG dataset allowed us to use true absence data for model validation, avoiding the problem of pseudo-absence data in Maxent.\\nThe purpose of these two different metrics is to consider different aspects of performance, neither were without limitations. Use of the AG dataset allowed a degree of validation across methodologies, indicating the extent to which our results were dependent on a particular sampling regime. AICc is traditionally used to indicate over-fitting in models, however it is sensitive to the size of the ecological niche of the species.\",\n \"Buruli ulcer prevalence data was collected for two endemic regions in Cameroon, Akonolinga [30] and Bankim [31], as shown in Figure 3. Around the centre of each village a buffer was created, and average habitat suitability in this buffer was correlated to the village Buruli ulcer prevalence using Spearman’s rank correlation coefficient. Seven buffers were used to explore the effect of buffer size and shape. Around the centre of the village circular buffers of 1, 2, 3, 4, 5 and 10 km were selected, and average habitat suitability recorded. We also used a buffer defined by the borders of the village (Figure 3). This buffer changes in size for each village, and represents the approximate extent of the village area. 5 km is approximately the flight radius of the insects, and the distance easily walkable by local people on an average day in Akonolinga [32]. To deal with this multiple testing, Bonferonni’s correction of the p-values was used.Figure 3\\nSpatial distribution of Buruli ulcer prevalence in Akonolianga and Bankim, two endemic regions within Cameroon.\\n\\n\\nSpatial distribution of Buruli ulcer prevalence in Akonolianga and Bankim, two endemic regions within Cameroon.\\n\",\n \"In Akonolinga there was a significantly positive correlation between Buruli ulcer prevalence and average habitat suitability, for both Naucoridae and Belostomatidae, in the wet season (Table 2, Figure 4). This relationship was significant at multiple buffer distances. In contrast, in Bankim there was no significant correlation between Buruli ulcer prevalence and Belostomatidae or Naucoridae average habitat suitability, in either wet or dry seasons or at any buffer distance (Table 3).Table 2\\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\\nSpeciesSeasonBufferBonferroni pvalueBelostomatidaeDry10-5-4-3-2-1-Village-Wet100.00050.01440.0233-2-1-Village-NaucoridaeDry10-5-4-3-2-1-Village-Wet100.00150.01340.0143-2-1-Village-The column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.Figure 4\\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\\n\\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\\n\\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.\\n\\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\\n\\nSpearman rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Bankim, for both seasons and species\\n\\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. No significant correlations were observed in Bankim.\",\n \"Variable importance was evaluated using Jackknife variable removal. Jacknife removes a variable and evaluates the effect of variable removal on the model. In the dry season Belostomatidae and Naucoridae responded in broadly similar fashions; the variable whose removal had the largest effect was GLC 5 km (Figure 5). The land cover categories most suitable for both Belostomatidae and Naucoridae are water bodies, artificial areas, rain fed croplands and forest/grassland mosaics (Figure 6). If one of these categories is the dominant category in 5 km radius, in the dry season, the likelihood of encountering the insect is higher. Unsuitable categories were forest and vegetation/cropland mosaic.In the wet season precipitation is more important than land cover. Precipitation suitability peaks at approximately 300 millimeters per month, and diminishes above or below this (Figure 6). For the dry season there is a simple increase in habitat suitability with increasing precipitation.Figure 5\\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.Figure 6\\nHabitat suitability for Belostomatidae (1\\nst\\nand 2\\nnd\\nrows) and Naucoridae (3\\nrd\\nand 4\\nth\\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\\n\\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.\\n\\nHabitat suitability for Belostomatidae (1\\nst\\nand 2\\nnd\\nrows) and Naucoridae (3\\nrd\\nand 4\\nth\\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\\nFlow accumulation had a negative association with habitat suitability, and wetness index had a positive association, regardless of season, for both Belostomatidae and Naucoridae.\",\n \"AICc for Naucoridae adults (dry season) was 14.6, and 14.2 in the wet season, as in Table 2. For Belostomatidae adults (dry season) the AICc was 12.5, and 12.2 in the wet season. Scores of overfitting are relative; these scores indicate the Belostomatidae model was less prone to overfitting than the Naucoridae model.\\nThe AG data set was also used in model validation. In the dry season Naucoridae adults had an AG AUC of 0.83, and 0.80 in the wet season. Belostomatidae adults had an AG AUC of 0.80 in the dry season, and 0.86 in the wet season. These scores indicate that the models are able to describe the distribution of the insects with good accuracy; the model based on SME dataset is able to accurately replicate the independently collected AG dataset.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Background","Materials and methods","Distribution of Belostomatidae and Naucoridae aquatic insect families","Environmental parameters used in ecological niche modelling","Data preparation, niche modelling, and prediction of spatial distribution of suitable habitat","Evaluating model performance","Identifying the relationship between habitat suitability and Buruli ulcer prevalence","Results","Distribution of suitable habitat, and its relationship to Buruli ulcer","Ecologically important variables in the distribution of the aquatic insect families","Model performance","Discussion"],"string":"[\n \"Background\",\n \"Materials and methods\",\n \"Distribution of Belostomatidae and Naucoridae aquatic insect families\",\n \"Environmental parameters used in ecological niche modelling\",\n \"Data preparation, niche modelling, and prediction of spatial distribution of suitable habitat\",\n \"Evaluating model performance\",\n \"Identifying the relationship between habitat suitability and Buruli ulcer prevalence\",\n \"Results\",\n \"Distribution of suitable habitat, and its relationship to Buruli ulcer\",\n \"Ecologically important variables in the distribution of the aquatic insect families\",\n \"Model performance\",\n \"Discussion\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["The Buruli ulcer is an emerging neglected tropical disease affecting more than 5,000 people per year in West and Central Africa, French Guiana, Latin America and Australia [1]. The disease burden is highest in Africa, where it predominantly affects children under the age of 15, and due to damage to the skin, muscle and bone, can result in severe scarring and crippling deformities if left untreated. The disease is caused by the environmental pathogen Mycobacterium ulcerans.\nThe mode of transmission of M. ulcerans, the method by which it infects humans, is unknown. Many routes of transmission have been proposed, such as transmission by aerosol [2], vector transmission by amoebae [3] or through aquatic networks [4]. During a study of the association between M. ulcerans and aquatic plants in Ghana and Benin aquatic insects were accidentally collected during the sampling procedure, and unexpectedly found to test positive for M. ulcerans\n[1]. The authors proposed that, given that these insects occasionally bite humans, they may be implicated in transmission of M. ulcerans. Aquatic insects have been further implicated after a series of laboratory experiments demonstrated the competency of Naucoridae to act as vectors. Naucoridae are able to acquire M. ulcerans from their diet, and then transmit the pathogen to mice resulting in Buruli-like symptoms [5–8]. Buruli ulcer is commonly associated with lowland, stagnant water [9] and human behaviours associated with water bodies appear to be risk factors for Buruli ulcer infection, which would lend support to the idea of infection occurring in an aquatic context.\nHowever, the role of these insects has been disputed for several reasons. In a two year study of Buruli ulcer endemic and non-endemic sites in Ghana [10], found no evidence for a role in transmission. The population of these insects, and the prevalence of M. ulcerans infection in them, was not significantly different between Buruli ulcer endemic and Buruli ulcer non-endemic sites [10]. The authors argued that, if these insects are vectors, we would expect them to have a higher abundance in Buruli ulcer endemic sites, that rates of M. ulcerans infection of the insects should be higher in Buruli ulcer endemic sites, and that the rate of infection of these insects should be higher than other species of invertebrates.\nThese expectations are based on the Bradford-Hill guidelines for associating insect vectors with human vector-borne disease [11, 12]. These guidelines provide a general framework to explore the association between vectors and disease, based on the consistency, specificity, plausibility and coherence of the proposed mode of transmission.\nConsistency refers to the expectation that the rate of infection of the proposed vectors, which should be consistently strongly positively associated, in time and space, with prevalence of human cases. This also implies human cases should not occur in absence of the proposed vector. The proposed vector should have a demonstrated capacity to physically transmit the pathogen, which has been demonstrated for M. ulcerans in the lab [5]. The interaction between the proposed vector and human infection must be specific and alternative explanations of human infection should be ruled out (though see [4] for alternative explanations). That is, human infection must be demonstrated to not have been the result of other potential modes of transmission. We note that this criterion must be applied with care in cases of multi-host transmission.\nAdditionally, the proposed vector must plausibly be able to be a vector of the pathogen. This criterion is often controversial as it is highly dependent upon the experience of the researcher, and their opinion about what is, and is not, plausible as opposed to merely possible [13]. Most authors in Buruli ulcer research would agree with some basic facts; the waterbugs are infected in the environment [14], they bite humans occasionally, and are able to transmit the bacteria to mice in the lab [5]. However, waterbugs are not known to be vectors of other pathogens, and related Mycobacteria (Mycobacterium tuberculosis, M. leprae, M. marnium) are not known to be vector-borne diseases [10, 12]. The plausibility of this proposed route of transmission is still debated. The final criterion, coherence, is based on what we already know about the pathogen, the vector and the host. Does the proposed method of transmission fit well with our current understanding of its biology? As our understanding of the biology of M. ulcerans improves, this criterion will be answered.\nGiven this framework, how likely is it that Naucoridae and/or Belostomatidae are vectors of the Buruli ulcer disease? Herein, we explore the correlation in time and space between the proposed vectors, Naucoridae and Belostomatidae, and the Buruli ulcer prevalence. We discuss the other Bradford-Hill criteria, but do not focus on them specifically, as it was not directly within the scope of this work. Based on sampling in Cameroon, we characterise the set of suitable habitats within which species of the Families Belostomatidae and Naucoridae can maintain a population (their ecological niche) and describe the spatial distribution of these suitable habitats across West Africa. We then explore any correlations between habitat suitability and Buruli ulcer prevalence at multiple spatial scales."," Distribution of Belostomatidae and Naucoridae aquatic insect families Data were collected as described in [15] hereafter referred to as the SME dataset. In brief, 36 sample sites in Cameroon were visited monthly from September 2012 to February 2013 (Figure 1), a period including both wet and dry seasons. Dip net sampling was conducted at all sites. Due to limitations of current taxonomic keys, the aquatic insects of interest were only identifiable to the phylogenetic division of Family. A second dataset was used in model validation, using data collected separately by A. Garchitorena (Figure 1). This dataset was collected as described in [16], and is hereafter referred to as the AG dataset. Niche models of Belostomatidae and Naucoridae are constructed using the SME dataset, and then tested on their ability to reproduce the independent AG dataset.Figure 1\nStudy sites Cameroon against local land cover. Data from the SME dataset is in red, and AG data set is in blue. The sample sites span the extent of Cameroon, sampling from every major land cover category.\n\nStudy sites Cameroon against local land cover. Data from the SME dataset is in red, and AG data set is in blue. The sample sites span the extent of Cameroon, sampling from every major land cover category.\nData were collected as described in [15] hereafter referred to as the SME dataset. In brief, 36 sample sites in Cameroon were visited monthly from September 2012 to February 2013 (Figure 1), a period including both wet and dry seasons. Dip net sampling was conducted at all sites. Due to limitations of current taxonomic keys, the aquatic insects of interest were only identifiable to the phylogenetic division of Family. A second dataset was used in model validation, using data collected separately by A. Garchitorena (Figure 1). This dataset was collected as described in [16], and is hereafter referred to as the AG dataset. Niche models of Belostomatidae and Naucoridae are constructed using the SME dataset, and then tested on their ability to reproduce the independent AG dataset.Figure 1\nStudy sites Cameroon against local land cover. Data from the SME dataset is in red, and AG data set is in blue. The sample sites span the extent of Cameroon, sampling from every major land cover category.\n\nStudy sites Cameroon against local land cover. Data from the SME dataset is in red, and AG data set is in blue. The sample sites span the extent of Cameroon, sampling from every major land cover category.\n Environmental parameters used in ecological niche modelling Five ecological parameters were used to describe the distribution of suitable habitats: rainfall, flow accumulation, wetness index, land cover at the sample site, and land cover within the flight range of adult insects. These variables were selected on the basis of their likelihood to influence the distribution and condition of water, and are summarised in Table 1. Rainfall was highly seasonal, so we divide models by the season of collection. Models constructed using species distribution data from the dry season used the precipitation in the driest season; models constructed using species distribution data from the wet season used the precipitation in the wettest season. These two variables were taken from the Worldclim database, as BIO13 Precipitation of Wettest Month and BIO14 Precipitation of Driest Month [17]. Flow accumulation was derived using elevation data [18]. Flow accumulation is the surface area contributing water to a particular point, and indicates the potential amount of water available, which is then determined by rainfall. Using the SRTM elevation, flow accumulation was derived using the Fill, Flow Direction and Flow Accumulation tools in ArcMap 10.1 [19]. Wetness index has previously been shown to be associated with the Buruli ulcer [9]. In ecological terms it indicates the topological potential for water to accumulate, and was derived according to Equation 1,Table 1\nEnvironmental variables used in ecological niche modelling\nVariableUnitsOriginal resolutionSourceWetness indexm2\n15 arc-sec (approx 450 m2)SRTMFlow accumulationm2\n15 arc-sec (approx 450 m2)SRTMPrecipitation in wettest seasonmillimeter30 arc-sec (approx 1 km2)Bioclim 13Precipitation in driest seasonmillimeter30 arc-sec (approx 1 km2)Bioclim 14GLC-APUnitless300 m2\nGLC 2009GLC-5 KUnitless300 m2\nGLC 2009All data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for use in Maxent. Resampling used resample() in the library ‘raster’ of the software R.\n\nEnvironmental variables used in ecological niche modelling\n\nAll data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for use in Maxent. Resampling used resample() in the library ‘raster’ of the software R.\n\n\n1\n\n\n\nwhere FA was the flow accumulation, 500 was the cell size in meters, and S was the surface slope in degrees. Large flow accumulation values and flat slopes resulted in high wetness index values, and indicate areas where water is likely to stagnate. In areas where the slope is zero wetness index had no value. Land cover was derived from the Global Land Cover Map 2009 [20]. GLC-At Point (hereafter GLC-AP) is the land cover at the sample site. In a 5 km radius around the site the most common (modal) land cover category was described. For example, a sample site may be in savannah, but surrounded by forest. 5 km was selected as the approximate flight radius of the insects [21–23]. This is termed GLC-5 K.\nFive ecological parameters were used to describe the distribution of suitable habitats: rainfall, flow accumulation, wetness index, land cover at the sample site, and land cover within the flight range of adult insects. These variables were selected on the basis of their likelihood to influence the distribution and condition of water, and are summarised in Table 1. Rainfall was highly seasonal, so we divide models by the season of collection. Models constructed using species distribution data from the dry season used the precipitation in the driest season; models constructed using species distribution data from the wet season used the precipitation in the wettest season. These two variables were taken from the Worldclim database, as BIO13 Precipitation of Wettest Month and BIO14 Precipitation of Driest Month [17]. Flow accumulation was derived using elevation data [18]. Flow accumulation is the surface area contributing water to a particular point, and indicates the potential amount of water available, which is then determined by rainfall. Using the SRTM elevation, flow accumulation was derived using the Fill, Flow Direction and Flow Accumulation tools in ArcMap 10.1 [19]. Wetness index has previously been shown to be associated with the Buruli ulcer [9]. In ecological terms it indicates the topological potential for water to accumulate, and was derived according to Equation 1,Table 1\nEnvironmental variables used in ecological niche modelling\nVariableUnitsOriginal resolutionSourceWetness indexm2\n15 arc-sec (approx 450 m2)SRTMFlow accumulationm2\n15 arc-sec (approx 450 m2)SRTMPrecipitation in wettest seasonmillimeter30 arc-sec (approx 1 km2)Bioclim 13Precipitation in driest seasonmillimeter30 arc-sec (approx 1 km2)Bioclim 14GLC-APUnitless300 m2\nGLC 2009GLC-5 KUnitless300 m2\nGLC 2009All data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for use in Maxent. Resampling used resample() in the library ‘raster’ of the software R.\n\nEnvironmental variables used in ecological niche modelling\n\nAll data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for use in Maxent. Resampling used resample() in the library ‘raster’ of the software R.\n\n\n1\n\n\n\nwhere FA was the flow accumulation, 500 was the cell size in meters, and S was the surface slope in degrees. Large flow accumulation values and flat slopes resulted in high wetness index values, and indicate areas where water is likely to stagnate. In areas where the slope is zero wetness index had no value. Land cover was derived from the Global Land Cover Map 2009 [20]. GLC-At Point (hereafter GLC-AP) is the land cover at the sample site. In a 5 km radius around the site the most common (modal) land cover category was described. For example, a sample site may be in savannah, but surrounded by forest. 5 km was selected as the approximate flight radius of the insects [21–23]. This is termed GLC-5 K.\n Data preparation, niche modelling, and prediction of spatial distribution of suitable habitat Insect distribution data from the 36 sample sites (Figure 1) were explored to identify normality, homogeneity of variance and correlation in the five environmental parameters used. Two sites were excluded from analysis as apparent outliers in flow accumulation, otherwise the data were normally distributed and homogenous. Correlation was observed between flow accumulation and wetness index and between precipitation in the driest and in the wettest seasons, however this was not significant (p > 0.05) using a Spearman’s correlation test. All data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for modelling using resample() in the library ‘raster’ of the software R [24].\nAcross the scale of West Africa we assume that absence data is not reliable, as it is more likely to indicate failure of detection rather than evidence of absence. For this reason, we chose to conduct presence-only modelling, and the specific method selected was Maximum Entropy [25]. We used the software Maxent (Maxent 3.3.3 k, [25]) to construct these models. Maxent has been used several times in the past to model the ecological niche of disease vectors [26]. Maximum entropy modelling minimises the divergence between the distribution of the environmental parameters and the species distribution, assuming the species is distributed in the most ecologically efficient manner possible. Maxent describes the environmental parameters across the study region, and is sensitive to the size of the study region. Herein, we generated background points in every raster cell within the extent of our study (that is, every environment was described). The models were replicated 100 times and averaged.\nIn order to select an appropriate extent to the area considered for modelling we chose to confine the spatial extent of our study to the region ecologically interpolated by the sample sites (Figure 2). The sample sites of the SME dataset describe a particular range of environmental conditions for the five environmental parameters (Table 1). We excluded conditions that were not studied by the SME regime (much larger or lower values of rainfall, land cover categories not sampled, flow accumulation not observed at the study sites) to avoid extrapolating beyond the range of ecological parameter values studied. To select this interpolative region we constructed a Multivariate Environmental Similarity Surface (MESS map [27]), excluding areas with negative MESS values from our study region. A MESS map was constructed for each model; models with more samples can be interpolated into larger ecological regions. MESS maps were constructed with the function mess() available in the library dismo in R.Figure 2\nDelineation of the study area. We exclude areas of ecological extrapolation. Across West Africa the red-green coloured region is ecologically interpolative within our sites, according to a multivariate environmental similarity surface (MESS). Regions where we extrapolate beyond the ecology of our study sites are identified using MESS values less than 0. Colour values are MESS values, indicating similarity to sample sites; a value of 100 is identical to the median of the sample sites. The spatial projection of this interpolative region is shown against a map of country borders of West Africa, for Belostomatidae adults in the dry season. This projection changes for each family, developmental stage and season, and though it is spatially discontinuous it is ecologically homogenous.\n\nDelineation of the study area. We exclude areas of ecological extrapolation. Across West Africa the red-green coloured region is ecologically interpolative within our sites, according to a multivariate environmental similarity surface (MESS). Regions where we extrapolate beyond the ecology of our study sites are identified using MESS values less than 0. Colour values are MESS values, indicating similarity to sample sites; a value of 100 is identical to the median of the sample sites. The spatial projection of this interpolative region is shown against a map of country borders of West Africa, for Belostomatidae adults in the dry season. This projection changes for each family, developmental stage and season, and though it is spatially discontinuous it is ecologically homogenous.\nInsect distribution data from the 36 sample sites (Figure 1) were explored to identify normality, homogeneity of variance and correlation in the five environmental parameters used. Two sites were excluded from analysis as apparent outliers in flow accumulation, otherwise the data were normally distributed and homogenous. Correlation was observed between flow accumulation and wetness index and between precipitation in the driest and in the wettest seasons, however this was not significant (p > 0.05) using a Spearman’s correlation test. All data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for modelling using resample() in the library ‘raster’ of the software R [24].\nAcross the scale of West Africa we assume that absence data is not reliable, as it is more likely to indicate failure of detection rather than evidence of absence. For this reason, we chose to conduct presence-only modelling, and the specific method selected was Maximum Entropy [25]. We used the software Maxent (Maxent 3.3.3 k, [25]) to construct these models. Maxent has been used several times in the past to model the ecological niche of disease vectors [26]. Maximum entropy modelling minimises the divergence between the distribution of the environmental parameters and the species distribution, assuming the species is distributed in the most ecologically efficient manner possible. Maxent describes the environmental parameters across the study region, and is sensitive to the size of the study region. Herein, we generated background points in every raster cell within the extent of our study (that is, every environment was described). The models were replicated 100 times and averaged.\nIn order to select an appropriate extent to the area considered for modelling we chose to confine the spatial extent of our study to the region ecologically interpolated by the sample sites (Figure 2). The sample sites of the SME dataset describe a particular range of environmental conditions for the five environmental parameters (Table 1). We excluded conditions that were not studied by the SME regime (much larger or lower values of rainfall, land cover categories not sampled, flow accumulation not observed at the study sites) to avoid extrapolating beyond the range of ecological parameter values studied. To select this interpolative region we constructed a Multivariate Environmental Similarity Surface (MESS map [27]), excluding areas with negative MESS values from our study region. A MESS map was constructed for each model; models with more samples can be interpolated into larger ecological regions. MESS maps were constructed with the function mess() available in the library dismo in R.Figure 2\nDelineation of the study area. We exclude areas of ecological extrapolation. Across West Africa the red-green coloured region is ecologically interpolative within our sites, according to a multivariate environmental similarity surface (MESS). Regions where we extrapolate beyond the ecology of our study sites are identified using MESS values less than 0. Colour values are MESS values, indicating similarity to sample sites; a value of 100 is identical to the median of the sample sites. The spatial projection of this interpolative region is shown against a map of country borders of West Africa, for Belostomatidae adults in the dry season. This projection changes for each family, developmental stage and season, and though it is spatially discontinuous it is ecologically homogenous.\n\nDelineation of the study area. We exclude areas of ecological extrapolation. Across West Africa the red-green coloured region is ecologically interpolative within our sites, according to a multivariate environmental similarity surface (MESS). Regions where we extrapolate beyond the ecology of our study sites are identified using MESS values less than 0. Colour values are MESS values, indicating similarity to sample sites; a value of 100 is identical to the median of the sample sites. The spatial projection of this interpolative region is shown against a map of country borders of West Africa, for Belostomatidae adults in the dry season. This projection changes for each family, developmental stage and season, and though it is spatially discontinuous it is ecologically homogenous.\n Evaluating model performance We evaluated the performance of these models according to their ability to correctly predict the distribution of the insects (with a separate dataset, the AG dataset) as efficiently as possible (avoiding overfitting). We used two methods to evaluate the models. First, the Akaike information criterion, corrected for small sample size (AICc), was used. AICc was generated as in [28], and penalises complex models to avoid over-fitting the data, low AICc scores indicate the model is not over-fitting. Second, we tested the models ability to predict the AG dataset. The Area Under Curve (AUC) is often used to evaluate Maxent models, and has been criticized previously [29]. We used a modified version of the AUC for model evaluation, termed here AG AUC. The model predictions were compared to the known values as collected by AG. AG AUC values range from 0 to 1, values close to 1 indicate good performance, 0.5 is no better than random. Use of the AG dataset allowed us to use true absence data for model validation, avoiding the problem of pseudo-absence data in Maxent.\nThe purpose of these two different metrics is to consider different aspects of performance, neither were without limitations. Use of the AG dataset allowed a degree of validation across methodologies, indicating the extent to which our results were dependent on a particular sampling regime. AICc is traditionally used to indicate over-fitting in models, however it is sensitive to the size of the ecological niche of the species.\nWe evaluated the performance of these models according to their ability to correctly predict the distribution of the insects (with a separate dataset, the AG dataset) as efficiently as possible (avoiding overfitting). We used two methods to evaluate the models. First, the Akaike information criterion, corrected for small sample size (AICc), was used. AICc was generated as in [28], and penalises complex models to avoid over-fitting the data, low AICc scores indicate the model is not over-fitting. Second, we tested the models ability to predict the AG dataset. The Area Under Curve (AUC) is often used to evaluate Maxent models, and has been criticized previously [29]. We used a modified version of the AUC for model evaluation, termed here AG AUC. The model predictions were compared to the known values as collected by AG. AG AUC values range from 0 to 1, values close to 1 indicate good performance, 0.5 is no better than random. Use of the AG dataset allowed us to use true absence data for model validation, avoiding the problem of pseudo-absence data in Maxent.\nThe purpose of these two different metrics is to consider different aspects of performance, neither were without limitations. Use of the AG dataset allowed a degree of validation across methodologies, indicating the extent to which our results were dependent on a particular sampling regime. AICc is traditionally used to indicate over-fitting in models, however it is sensitive to the size of the ecological niche of the species.\n Identifying the relationship between habitat suitability and Buruli ulcer prevalence Buruli ulcer prevalence data was collected for two endemic regions in Cameroon, Akonolinga [30] and Bankim [31], as shown in Figure 3. Around the centre of each village a buffer was created, and average habitat suitability in this buffer was correlated to the village Buruli ulcer prevalence using Spearman’s rank correlation coefficient. Seven buffers were used to explore the effect of buffer size and shape. Around the centre of the village circular buffers of 1, 2, 3, 4, 5 and 10 km were selected, and average habitat suitability recorded. We also used a buffer defined by the borders of the village (Figure 3). This buffer changes in size for each village, and represents the approximate extent of the village area. 5 km is approximately the flight radius of the insects, and the distance easily walkable by local people on an average day in Akonolinga [32]. To deal with this multiple testing, Bonferonni’s correction of the p-values was used.Figure 3\nSpatial distribution of Buruli ulcer prevalence in Akonolianga and Bankim, two endemic regions within Cameroon.\n\n\nSpatial distribution of Buruli ulcer prevalence in Akonolianga and Bankim, two endemic regions within Cameroon.\n\nBuruli ulcer prevalence data was collected for two endemic regions in Cameroon, Akonolinga [30] and Bankim [31], as shown in Figure 3. Around the centre of each village a buffer was created, and average habitat suitability in this buffer was correlated to the village Buruli ulcer prevalence using Spearman’s rank correlation coefficient. Seven buffers were used to explore the effect of buffer size and shape. Around the centre of the village circular buffers of 1, 2, 3, 4, 5 and 10 km were selected, and average habitat suitability recorded. We also used a buffer defined by the borders of the village (Figure 3). This buffer changes in size for each village, and represents the approximate extent of the village area. 5 km is approximately the flight radius of the insects, and the distance easily walkable by local people on an average day in Akonolinga [32]. To deal with this multiple testing, Bonferonni’s correction of the p-values was used.Figure 3\nSpatial distribution of Buruli ulcer prevalence in Akonolianga and Bankim, two endemic regions within Cameroon.\n\n\nSpatial distribution of Buruli ulcer prevalence in Akonolianga and Bankim, two endemic regions within Cameroon.\n","Data were collected as described in [15] hereafter referred to as the SME dataset. In brief, 36 sample sites in Cameroon were visited monthly from September 2012 to February 2013 (Figure 1), a period including both wet and dry seasons. Dip net sampling was conducted at all sites. Due to limitations of current taxonomic keys, the aquatic insects of interest were only identifiable to the phylogenetic division of Family. A second dataset was used in model validation, using data collected separately by A. Garchitorena (Figure 1). This dataset was collected as described in [16], and is hereafter referred to as the AG dataset. Niche models of Belostomatidae and Naucoridae are constructed using the SME dataset, and then tested on their ability to reproduce the independent AG dataset.Figure 1\nStudy sites Cameroon against local land cover. Data from the SME dataset is in red, and AG data set is in blue. The sample sites span the extent of Cameroon, sampling from every major land cover category.\n\nStudy sites Cameroon against local land cover. Data from the SME dataset is in red, and AG data set is in blue. The sample sites span the extent of Cameroon, sampling from every major land cover category.","Five ecological parameters were used to describe the distribution of suitable habitats: rainfall, flow accumulation, wetness index, land cover at the sample site, and land cover within the flight range of adult insects. These variables were selected on the basis of their likelihood to influence the distribution and condition of water, and are summarised in Table 1. Rainfall was highly seasonal, so we divide models by the season of collection. Models constructed using species distribution data from the dry season used the precipitation in the driest season; models constructed using species distribution data from the wet season used the precipitation in the wettest season. These two variables were taken from the Worldclim database, as BIO13 Precipitation of Wettest Month and BIO14 Precipitation of Driest Month [17]. Flow accumulation was derived using elevation data [18]. Flow accumulation is the surface area contributing water to a particular point, and indicates the potential amount of water available, which is then determined by rainfall. Using the SRTM elevation, flow accumulation was derived using the Fill, Flow Direction and Flow Accumulation tools in ArcMap 10.1 [19]. Wetness index has previously been shown to be associated with the Buruli ulcer [9]. In ecological terms it indicates the topological potential for water to accumulate, and was derived according to Equation 1,Table 1\nEnvironmental variables used in ecological niche modelling\nVariableUnitsOriginal resolutionSourceWetness indexm2\n15 arc-sec (approx 450 m2)SRTMFlow accumulationm2\n15 arc-sec (approx 450 m2)SRTMPrecipitation in wettest seasonmillimeter30 arc-sec (approx 1 km2)Bioclim 13Precipitation in driest seasonmillimeter30 arc-sec (approx 1 km2)Bioclim 14GLC-APUnitless300 m2\nGLC 2009GLC-5 KUnitless300 m2\nGLC 2009All data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for use in Maxent. Resampling used resample() in the library ‘raster’ of the software R.\n\nEnvironmental variables used in ecological niche modelling\n\nAll data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for use in Maxent. Resampling used resample() in the library ‘raster’ of the software R.\n\n\n1\n\n\n\nwhere FA was the flow accumulation, 500 was the cell size in meters, and S was the surface slope in degrees. Large flow accumulation values and flat slopes resulted in high wetness index values, and indicate areas where water is likely to stagnate. In areas where the slope is zero wetness index had no value. Land cover was derived from the Global Land Cover Map 2009 [20]. GLC-At Point (hereafter GLC-AP) is the land cover at the sample site. In a 5 km radius around the site the most common (modal) land cover category was described. For example, a sample site may be in savannah, but surrounded by forest. 5 km was selected as the approximate flight radius of the insects [21–23]. This is termed GLC-5 K.","Insect distribution data from the 36 sample sites (Figure 1) were explored to identify normality, homogeneity of variance and correlation in the five environmental parameters used. Two sites were excluded from analysis as apparent outliers in flow accumulation, otherwise the data were normally distributed and homogenous. Correlation was observed between flow accumulation and wetness index and between precipitation in the driest and in the wettest seasons, however this was not significant (p > 0.05) using a Spearman’s correlation test. All data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for modelling using resample() in the library ‘raster’ of the software R [24].\nAcross the scale of West Africa we assume that absence data is not reliable, as it is more likely to indicate failure of detection rather than evidence of absence. For this reason, we chose to conduct presence-only modelling, and the specific method selected was Maximum Entropy [25]. We used the software Maxent (Maxent 3.3.3 k, [25]) to construct these models. Maxent has been used several times in the past to model the ecological niche of disease vectors [26]. Maximum entropy modelling minimises the divergence between the distribution of the environmental parameters and the species distribution, assuming the species is distributed in the most ecologically efficient manner possible. Maxent describes the environmental parameters across the study region, and is sensitive to the size of the study region. Herein, we generated background points in every raster cell within the extent of our study (that is, every environment was described). The models were replicated 100 times and averaged.\nIn order to select an appropriate extent to the area considered for modelling we chose to confine the spatial extent of our study to the region ecologically interpolated by the sample sites (Figure 2). The sample sites of the SME dataset describe a particular range of environmental conditions for the five environmental parameters (Table 1). We excluded conditions that were not studied by the SME regime (much larger or lower values of rainfall, land cover categories not sampled, flow accumulation not observed at the study sites) to avoid extrapolating beyond the range of ecological parameter values studied. To select this interpolative region we constructed a Multivariate Environmental Similarity Surface (MESS map [27]), excluding areas with negative MESS values from our study region. A MESS map was constructed for each model; models with more samples can be interpolated into larger ecological regions. MESS maps were constructed with the function mess() available in the library dismo in R.Figure 2\nDelineation of the study area. We exclude areas of ecological extrapolation. Across West Africa the red-green coloured region is ecologically interpolative within our sites, according to a multivariate environmental similarity surface (MESS). Regions where we extrapolate beyond the ecology of our study sites are identified using MESS values less than 0. Colour values are MESS values, indicating similarity to sample sites; a value of 100 is identical to the median of the sample sites. The spatial projection of this interpolative region is shown against a map of country borders of West Africa, for Belostomatidae adults in the dry season. This projection changes for each family, developmental stage and season, and though it is spatially discontinuous it is ecologically homogenous.\n\nDelineation of the study area. We exclude areas of ecological extrapolation. Across West Africa the red-green coloured region is ecologically interpolative within our sites, according to a multivariate environmental similarity surface (MESS). Regions where we extrapolate beyond the ecology of our study sites are identified using MESS values less than 0. Colour values are MESS values, indicating similarity to sample sites; a value of 100 is identical to the median of the sample sites. The spatial projection of this interpolative region is shown against a map of country borders of West Africa, for Belostomatidae adults in the dry season. This projection changes for each family, developmental stage and season, and though it is spatially discontinuous it is ecologically homogenous.","We evaluated the performance of these models according to their ability to correctly predict the distribution of the insects (with a separate dataset, the AG dataset) as efficiently as possible (avoiding overfitting). We used two methods to evaluate the models. First, the Akaike information criterion, corrected for small sample size (AICc), was used. AICc was generated as in [28], and penalises complex models to avoid over-fitting the data, low AICc scores indicate the model is not over-fitting. Second, we tested the models ability to predict the AG dataset. The Area Under Curve (AUC) is often used to evaluate Maxent models, and has been criticized previously [29]. We used a modified version of the AUC for model evaluation, termed here AG AUC. The model predictions were compared to the known values as collected by AG. AG AUC values range from 0 to 1, values close to 1 indicate good performance, 0.5 is no better than random. Use of the AG dataset allowed us to use true absence data for model validation, avoiding the problem of pseudo-absence data in Maxent.\nThe purpose of these two different metrics is to consider different aspects of performance, neither were without limitations. Use of the AG dataset allowed a degree of validation across methodologies, indicating the extent to which our results were dependent on a particular sampling regime. AICc is traditionally used to indicate over-fitting in models, however it is sensitive to the size of the ecological niche of the species.","Buruli ulcer prevalence data was collected for two endemic regions in Cameroon, Akonolinga [30] and Bankim [31], as shown in Figure 3. Around the centre of each village a buffer was created, and average habitat suitability in this buffer was correlated to the village Buruli ulcer prevalence using Spearman’s rank correlation coefficient. Seven buffers were used to explore the effect of buffer size and shape. Around the centre of the village circular buffers of 1, 2, 3, 4, 5 and 10 km were selected, and average habitat suitability recorded. We also used a buffer defined by the borders of the village (Figure 3). This buffer changes in size for each village, and represents the approximate extent of the village area. 5 km is approximately the flight radius of the insects, and the distance easily walkable by local people on an average day in Akonolinga [32]. To deal with this multiple testing, Bonferonni’s correction of the p-values was used.Figure 3\nSpatial distribution of Buruli ulcer prevalence in Akonolianga and Bankim, two endemic regions within Cameroon.\n\n\nSpatial distribution of Buruli ulcer prevalence in Akonolianga and Bankim, two endemic regions within Cameroon.\n"," Distribution of suitable habitat, and its relationship to Buruli ulcer In Akonolinga there was a significantly positive correlation between Buruli ulcer prevalence and average habitat suitability, for both Naucoridae and Belostomatidae, in the wet season (Table 2, Figure 4). This relationship was significant at multiple buffer distances. In contrast, in Bankim there was no significant correlation between Buruli ulcer prevalence and Belostomatidae or Naucoridae average habitat suitability, in either wet or dry seasons or at any buffer distance (Table 3).Table 2\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\nSpeciesSeasonBufferBonferroni pvalueBelostomatidaeDry10-5-4-3-2-1-Village-Wet100.00050.01440.0233-2-1-Village-NaucoridaeDry10-5-4-3-2-1-Village-Wet100.00150.01340.0143-2-1-Village-The column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.Figure 4\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\n\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\n\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.\n\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\n\nSpearman rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Bankim, for both seasons and species\n\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. No significant correlations were observed in Bankim.\nIn Akonolinga there was a significantly positive correlation between Buruli ulcer prevalence and average habitat suitability, for both Naucoridae and Belostomatidae, in the wet season (Table 2, Figure 4). This relationship was significant at multiple buffer distances. In contrast, in Bankim there was no significant correlation between Buruli ulcer prevalence and Belostomatidae or Naucoridae average habitat suitability, in either wet or dry seasons or at any buffer distance (Table 3).Table 2\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\nSpeciesSeasonBufferBonferroni pvalueBelostomatidaeDry10-5-4-3-2-1-Village-Wet100.00050.01440.0233-2-1-Village-NaucoridaeDry10-5-4-3-2-1-Village-Wet100.00150.01340.0143-2-1-Village-The column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.Figure 4\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\n\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\n\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.\n\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\n\nSpearman rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Bankim, for both seasons and species\n\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. No significant correlations were observed in Bankim.\n Ecologically important variables in the distribution of the aquatic insect families Variable importance was evaluated using Jackknife variable removal. Jacknife removes a variable and evaluates the effect of variable removal on the model. In the dry season Belostomatidae and Naucoridae responded in broadly similar fashions; the variable whose removal had the largest effect was GLC 5 km (Figure 5). The land cover categories most suitable for both Belostomatidae and Naucoridae are water bodies, artificial areas, rain fed croplands and forest/grassland mosaics (Figure 6). If one of these categories is the dominant category in 5 km radius, in the dry season, the likelihood of encountering the insect is higher. Unsuitable categories were forest and vegetation/cropland mosaic.In the wet season precipitation is more important than land cover. Precipitation suitability peaks at approximately 300 millimeters per month, and diminishes above or below this (Figure 6). For the dry season there is a simple increase in habitat suitability with increasing precipitation.Figure 5\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.Figure 6\nHabitat suitability for Belostomatidae (1\nst\nand 2\nnd\nrows) and Naucoridae (3\nrd\nand 4\nth\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\n\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.\n\nHabitat suitability for Belostomatidae (1\nst\nand 2\nnd\nrows) and Naucoridae (3\nrd\nand 4\nth\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\nFlow accumulation had a negative association with habitat suitability, and wetness index had a positive association, regardless of season, for both Belostomatidae and Naucoridae.\nVariable importance was evaluated using Jackknife variable removal. Jacknife removes a variable and evaluates the effect of variable removal on the model. In the dry season Belostomatidae and Naucoridae responded in broadly similar fashions; the variable whose removal had the largest effect was GLC 5 km (Figure 5). The land cover categories most suitable for both Belostomatidae and Naucoridae are water bodies, artificial areas, rain fed croplands and forest/grassland mosaics (Figure 6). If one of these categories is the dominant category in 5 km radius, in the dry season, the likelihood of encountering the insect is higher. Unsuitable categories were forest and vegetation/cropland mosaic.In the wet season precipitation is more important than land cover. Precipitation suitability peaks at approximately 300 millimeters per month, and diminishes above or below this (Figure 6). For the dry season there is a simple increase in habitat suitability with increasing precipitation.Figure 5\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.Figure 6\nHabitat suitability for Belostomatidae (1\nst\nand 2\nnd\nrows) and Naucoridae (3\nrd\nand 4\nth\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\n\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.\n\nHabitat suitability for Belostomatidae (1\nst\nand 2\nnd\nrows) and Naucoridae (3\nrd\nand 4\nth\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\nFlow accumulation had a negative association with habitat suitability, and wetness index had a positive association, regardless of season, for both Belostomatidae and Naucoridae.\n Model performance AICc for Naucoridae adults (dry season) was 14.6, and 14.2 in the wet season, as in Table 2. For Belostomatidae adults (dry season) the AICc was 12.5, and 12.2 in the wet season. Scores of overfitting are relative; these scores indicate the Belostomatidae model was less prone to overfitting than the Naucoridae model.\nThe AG data set was also used in model validation. In the dry season Naucoridae adults had an AG AUC of 0.83, and 0.80 in the wet season. Belostomatidae adults had an AG AUC of 0.80 in the dry season, and 0.86 in the wet season. These scores indicate that the models are able to describe the distribution of the insects with good accuracy; the model based on SME dataset is able to accurately replicate the independently collected AG dataset.\nAICc for Naucoridae adults (dry season) was 14.6, and 14.2 in the wet season, as in Table 2. For Belostomatidae adults (dry season) the AICc was 12.5, and 12.2 in the wet season. Scores of overfitting are relative; these scores indicate the Belostomatidae model was less prone to overfitting than the Naucoridae model.\nThe AG data set was also used in model validation. In the dry season Naucoridae adults had an AG AUC of 0.83, and 0.80 in the wet season. Belostomatidae adults had an AG AUC of 0.80 in the dry season, and 0.86 in the wet season. These scores indicate that the models are able to describe the distribution of the insects with good accuracy; the model based on SME dataset is able to accurately replicate the independently collected AG dataset.","In Akonolinga there was a significantly positive correlation between Buruli ulcer prevalence and average habitat suitability, for both Naucoridae and Belostomatidae, in the wet season (Table 2, Figure 4). This relationship was significant at multiple buffer distances. In contrast, in Bankim there was no significant correlation between Buruli ulcer prevalence and Belostomatidae or Naucoridae average habitat suitability, in either wet or dry seasons or at any buffer distance (Table 3).Table 2\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\nSpeciesSeasonBufferBonferroni pvalueBelostomatidaeDry10-5-4-3-2-1-Village-Wet100.00050.01440.0233-2-1-Village-NaucoridaeDry10-5-4-3-2-1-Village-Wet100.00150.01340.0143-2-1-Village-The column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.Figure 4\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\n\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\n\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.\n\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\n\nSpearman rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Bankim, for both seasons and species\n\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. No significant correlations were observed in Bankim.","Variable importance was evaluated using Jackknife variable removal. Jacknife removes a variable and evaluates the effect of variable removal on the model. In the dry season Belostomatidae and Naucoridae responded in broadly similar fashions; the variable whose removal had the largest effect was GLC 5 km (Figure 5). The land cover categories most suitable for both Belostomatidae and Naucoridae are water bodies, artificial areas, rain fed croplands and forest/grassland mosaics (Figure 6). If one of these categories is the dominant category in 5 km radius, in the dry season, the likelihood of encountering the insect is higher. Unsuitable categories were forest and vegetation/cropland mosaic.In the wet season precipitation is more important than land cover. Precipitation suitability peaks at approximately 300 millimeters per month, and diminishes above or below this (Figure 6). For the dry season there is a simple increase in habitat suitability with increasing precipitation.Figure 5\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.Figure 6\nHabitat suitability for Belostomatidae (1\nst\nand 2\nnd\nrows) and Naucoridae (3\nrd\nand 4\nth\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\n\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.\n\nHabitat suitability for Belostomatidae (1\nst\nand 2\nnd\nrows) and Naucoridae (3\nrd\nand 4\nth\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\nFlow accumulation had a negative association with habitat suitability, and wetness index had a positive association, regardless of season, for both Belostomatidae and Naucoridae.","AICc for Naucoridae adults (dry season) was 14.6, and 14.2 in the wet season, as in Table 2. For Belostomatidae adults (dry season) the AICc was 12.5, and 12.2 in the wet season. Scores of overfitting are relative; these scores indicate the Belostomatidae model was less prone to overfitting than the Naucoridae model.\nThe AG data set was also used in model validation. In the dry season Naucoridae adults had an AG AUC of 0.83, and 0.80 in the wet season. Belostomatidae adults had an AG AUC of 0.80 in the dry season, and 0.86 in the wet season. These scores indicate that the models are able to describe the distribution of the insects with good accuracy; the model based on SME dataset is able to accurately replicate the independently collected AG dataset.","We explored the correlation between the distribution of Belostomatidae and Naucoridae and the prevalence of Buruli ulcer. We have found a positive gradient between habitat suitability of Naucoridae and Belostomatidae and Buruli ulcer prevalence. Correlation does not imply causation; this result is not proof that the insects are vectors. However, understanding the reasons for the temporal and spatial changes in this correlation will enable a better understanding of the reasons for changes in Buruli ulcer prevalence.\nThere are significant temporal changes in this correlation between habitat suitability of the insects and Buruli ulcer prevalence; in Akonolinga the Buruli ulcer prevalence is correlated to Naucoridae and Belostomatidae distribution in the wet season, but not in the dry season. Buruli ulcer is known to have complex temporal changes in prevalence [30, 33, 34], as is M. ulcerans\n[14, 35]. It is therefore unsurprising that, if these insects are implicated in maintaining M. ulcerans in the environment, or involved in transmission to humans in some way (either as host vectors, or host carriers), the correlation between Buruli ulcer prevalence and their abundance would change in time.\nWe also observe spatial changes in the correlation; water bug habitat suitability is not correlated to Buruli ulcer prevalence in Bankim, 457 km North of Akonolinga. Speculatively, perhaps other routes of transmission may be more important in this region, for example contact with infected plant biofilms, as suggested in Ghana [36].\nHow do we interpret this result in terms of the Bradford-Hill guidelines? Herein we have focused on the correlation between these insects and the prevalence of the disease in both space and time. While there is a significant positive correlation for the predicted abundance of the aquatic insects and the prevalence of the Buruli ulcer, this correlation is not consistent from region to region. Previous research has proposed that M. ulcerans is transmitted within a multi-host transmission network [4]. In such a situation of multiple hosts the relative importance of any given mode of transmission may be expected to vary in time or space, and our results are consistent with, though not proof of, this hypothesis. The lack of a clear signal between water bugs and Buruli ulcer in Bankim would suggest that may not be key vectors or host carriers in that region. Recent studies have found notable changes in community composition relevant to M. ulcerans distribution, in the Greater Accra, Ashanti and Volta regions of Ghana, for both plant [35] and aquatic insects [37]. This would support the importance of changing biotic communities as a key factor in changing Buruli ulcer prevalence, a priority for future work. We have found that the prevalence of Buruli ulcer is correlated to Belostomatidae and Naucoridae abundance in Akonolinga but not Bankim, we do not know if the plant community composition for these regions, and wider aquatic insect community, also correlates to disease prevalence changes on a similar scale.\nThe ecological niches of both Naucorid and Belostomatid water bugs in West Africa are predominantly determined by the distribution of suitable landcover in a 5 km radius, preferring water bodies, artificial areas and rain fed croplands. The specific land cover at the point of the site (GLC-AP) was less informative. The observation that the most suitable regional land cover class is water bodies is not surprising, but the high suitability of urban areas is curious. Ecologically this could have a variety of causes; there may be changes in the chemical composition of water in these habitats, a reduction in predation pressure, or a greater abundance of food. The specific reasons will require further research.\nOur study has been limited in certain points; the low taxonomic resolution of the insects is a current limitation in this study. Secondly, an important limitation is that the distribution of M. ulcerans in these insects in these areas is unknown. The distribution of Naucoridae and Belostomatidae infected by M. ulcerans may differ from the distribution of Naucoridae and Belostomatidae generally. However, the insects are known to host the bacillus on their carapace, in their body [10, 14, 16]) and in their salivary glands [14] in the wild, and the distribution of the insect necessarily sets a limit to the distribution of infected insects. A related limitation is the unknown incubation time of M. ulcerans; the time from infection to presentation at the hospital, remains unknown. Finally, we have only addressed a single criterion of the Bradford-Hill guidelines; correlation. We have not aimed for a full discussion of the other criteria, and our findings should not be interpreted as proof of the role of these insects as vectors or key host carriers. Rather, we have discussed the existence of, and change in, a correlation between these insects and Buruli ulcer. Future work aims to explore spatial variation in the correlation between Buruli ulcer and the entire plant and animal communities, identifying any similarities between regions where the correlation exists, expanding on previous studies [37] which have focused on the community assemblage differences between M. ulcerans endemic and non-endemic regions of Ghana.\nDespite these limitations, these results are consistent with previous research, which has shown that in Akonolinga the Nyong river is a risk factor for Buruli ulcer [30]. Our results agree with this conclusion; the main focus of suitable habitats for the insects in Akonolinga is the Nyong river, where the existence of large plants near the river banks provides appropriate habitat for Naucoridae and Belostomatidae to forage, develop and reproduce. Previous research has also implicated aquatic insects as important vectors in Akonolinga [14], including detection of M. ulcerans in the saliva of the insects.\nIn conclusion, we find a positive correlation between the abundance of Naucoridae and Belostomatidae suitable habitat and Buruli ulcer prevalence. This correlation is not constant, and changes in time and space. We interpret this as evidence consistent with that idea that Naucoridae and Belostomatidae may be locally important host carriers of M. ulcerans in certain conditions, their importance changing as the environmental conditions change, which would be expected in the situation of multi-host transmission."],"string":"[\n \"The Buruli ulcer is an emerging neglected tropical disease affecting more than 5,000 people per year in West and Central Africa, French Guiana, Latin America and Australia [1]. The disease burden is highest in Africa, where it predominantly affects children under the age of 15, and due to damage to the skin, muscle and bone, can result in severe scarring and crippling deformities if left untreated. The disease is caused by the environmental pathogen Mycobacterium ulcerans.\\nThe mode of transmission of M. ulcerans, the method by which it infects humans, is unknown. Many routes of transmission have been proposed, such as transmission by aerosol [2], vector transmission by amoebae [3] or through aquatic networks [4]. During a study of the association between M. ulcerans and aquatic plants in Ghana and Benin aquatic insects were accidentally collected during the sampling procedure, and unexpectedly found to test positive for M. ulcerans\\n[1]. The authors proposed that, given that these insects occasionally bite humans, they may be implicated in transmission of M. ulcerans. Aquatic insects have been further implicated after a series of laboratory experiments demonstrated the competency of Naucoridae to act as vectors. Naucoridae are able to acquire M. ulcerans from their diet, and then transmit the pathogen to mice resulting in Buruli-like symptoms [5–8]. Buruli ulcer is commonly associated with lowland, stagnant water [9] and human behaviours associated with water bodies appear to be risk factors for Buruli ulcer infection, which would lend support to the idea of infection occurring in an aquatic context.\\nHowever, the role of these insects has been disputed for several reasons. In a two year study of Buruli ulcer endemic and non-endemic sites in Ghana [10], found no evidence for a role in transmission. The population of these insects, and the prevalence of M. ulcerans infection in them, was not significantly different between Buruli ulcer endemic and Buruli ulcer non-endemic sites [10]. The authors argued that, if these insects are vectors, we would expect them to have a higher abundance in Buruli ulcer endemic sites, that rates of M. ulcerans infection of the insects should be higher in Buruli ulcer endemic sites, and that the rate of infection of these insects should be higher than other species of invertebrates.\\nThese expectations are based on the Bradford-Hill guidelines for associating insect vectors with human vector-borne disease [11, 12]. These guidelines provide a general framework to explore the association between vectors and disease, based on the consistency, specificity, plausibility and coherence of the proposed mode of transmission.\\nConsistency refers to the expectation that the rate of infection of the proposed vectors, which should be consistently strongly positively associated, in time and space, with prevalence of human cases. This also implies human cases should not occur in absence of the proposed vector. The proposed vector should have a demonstrated capacity to physically transmit the pathogen, which has been demonstrated for M. ulcerans in the lab [5]. The interaction between the proposed vector and human infection must be specific and alternative explanations of human infection should be ruled out (though see [4] for alternative explanations). That is, human infection must be demonstrated to not have been the result of other potential modes of transmission. We note that this criterion must be applied with care in cases of multi-host transmission.\\nAdditionally, the proposed vector must plausibly be able to be a vector of the pathogen. This criterion is often controversial as it is highly dependent upon the experience of the researcher, and their opinion about what is, and is not, plausible as opposed to merely possible [13]. Most authors in Buruli ulcer research would agree with some basic facts; the waterbugs are infected in the environment [14], they bite humans occasionally, and are able to transmit the bacteria to mice in the lab [5]. However, waterbugs are not known to be vectors of other pathogens, and related Mycobacteria (Mycobacterium tuberculosis, M. leprae, M. marnium) are not known to be vector-borne diseases [10, 12]. The plausibility of this proposed route of transmission is still debated. The final criterion, coherence, is based on what we already know about the pathogen, the vector and the host. Does the proposed method of transmission fit well with our current understanding of its biology? As our understanding of the biology of M. ulcerans improves, this criterion will be answered.\\nGiven this framework, how likely is it that Naucoridae and/or Belostomatidae are vectors of the Buruli ulcer disease? Herein, we explore the correlation in time and space between the proposed vectors, Naucoridae and Belostomatidae, and the Buruli ulcer prevalence. We discuss the other Bradford-Hill criteria, but do not focus on them specifically, as it was not directly within the scope of this work. Based on sampling in Cameroon, we characterise the set of suitable habitats within which species of the Families Belostomatidae and Naucoridae can maintain a population (their ecological niche) and describe the spatial distribution of these suitable habitats across West Africa. We then explore any correlations between habitat suitability and Buruli ulcer prevalence at multiple spatial scales.\",\n \" Distribution of Belostomatidae and Naucoridae aquatic insect families Data were collected as described in [15] hereafter referred to as the SME dataset. In brief, 36 sample sites in Cameroon were visited monthly from September 2012 to February 2013 (Figure 1), a period including both wet and dry seasons. Dip net sampling was conducted at all sites. Due to limitations of current taxonomic keys, the aquatic insects of interest were only identifiable to the phylogenetic division of Family. A second dataset was used in model validation, using data collected separately by A. Garchitorena (Figure 1). This dataset was collected as described in [16], and is hereafter referred to as the AG dataset. Niche models of Belostomatidae and Naucoridae are constructed using the SME dataset, and then tested on their ability to reproduce the independent AG dataset.Figure 1\\nStudy sites Cameroon against local land cover. Data from the SME dataset is in red, and AG data set is in blue. The sample sites span the extent of Cameroon, sampling from every major land cover category.\\n\\nStudy sites Cameroon against local land cover. Data from the SME dataset is in red, and AG data set is in blue. The sample sites span the extent of Cameroon, sampling from every major land cover category.\\nData were collected as described in [15] hereafter referred to as the SME dataset. In brief, 36 sample sites in Cameroon were visited monthly from September 2012 to February 2013 (Figure 1), a period including both wet and dry seasons. Dip net sampling was conducted at all sites. Due to limitations of current taxonomic keys, the aquatic insects of interest were only identifiable to the phylogenetic division of Family. A second dataset was used in model validation, using data collected separately by A. Garchitorena (Figure 1). This dataset was collected as described in [16], and is hereafter referred to as the AG dataset. Niche models of Belostomatidae and Naucoridae are constructed using the SME dataset, and then tested on their ability to reproduce the independent AG dataset.Figure 1\\nStudy sites Cameroon against local land cover. Data from the SME dataset is in red, and AG data set is in blue. The sample sites span the extent of Cameroon, sampling from every major land cover category.\\n\\nStudy sites Cameroon against local land cover. Data from the SME dataset is in red, and AG data set is in blue. The sample sites span the extent of Cameroon, sampling from every major land cover category.\\n Environmental parameters used in ecological niche modelling Five ecological parameters were used to describe the distribution of suitable habitats: rainfall, flow accumulation, wetness index, land cover at the sample site, and land cover within the flight range of adult insects. These variables were selected on the basis of their likelihood to influence the distribution and condition of water, and are summarised in Table 1. Rainfall was highly seasonal, so we divide models by the season of collection. Models constructed using species distribution data from the dry season used the precipitation in the driest season; models constructed using species distribution data from the wet season used the precipitation in the wettest season. These two variables were taken from the Worldclim database, as BIO13 Precipitation of Wettest Month and BIO14 Precipitation of Driest Month [17]. Flow accumulation was derived using elevation data [18]. Flow accumulation is the surface area contributing water to a particular point, and indicates the potential amount of water available, which is then determined by rainfall. Using the SRTM elevation, flow accumulation was derived using the Fill, Flow Direction and Flow Accumulation tools in ArcMap 10.1 [19]. Wetness index has previously been shown to be associated with the Buruli ulcer [9]. In ecological terms it indicates the topological potential for water to accumulate, and was derived according to Equation 1,Table 1\\nEnvironmental variables used in ecological niche modelling\\nVariableUnitsOriginal resolutionSourceWetness indexm2\\n15 arc-sec (approx 450 m2)SRTMFlow accumulationm2\\n15 arc-sec (approx 450 m2)SRTMPrecipitation in wettest seasonmillimeter30 arc-sec (approx 1 km2)Bioclim 13Precipitation in driest seasonmillimeter30 arc-sec (approx 1 km2)Bioclim 14GLC-APUnitless300 m2\\nGLC 2009GLC-5 KUnitless300 m2\\nGLC 2009All data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for use in Maxent. Resampling used resample() in the library ‘raster’ of the software R.\\n\\nEnvironmental variables used in ecological niche modelling\\n\\nAll data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for use in Maxent. Resampling used resample() in the library ‘raster’ of the software R.\\n\\n\\n1\\n\\n\\n\\nwhere FA was the flow accumulation, 500 was the cell size in meters, and S was the surface slope in degrees. Large flow accumulation values and flat slopes resulted in high wetness index values, and indicate areas where water is likely to stagnate. In areas where the slope is zero wetness index had no value. Land cover was derived from the Global Land Cover Map 2009 [20]. GLC-At Point (hereafter GLC-AP) is the land cover at the sample site. In a 5 km radius around the site the most common (modal) land cover category was described. For example, a sample site may be in savannah, but surrounded by forest. 5 km was selected as the approximate flight radius of the insects [21–23]. This is termed GLC-5 K.\\nFive ecological parameters were used to describe the distribution of suitable habitats: rainfall, flow accumulation, wetness index, land cover at the sample site, and land cover within the flight range of adult insects. These variables were selected on the basis of their likelihood to influence the distribution and condition of water, and are summarised in Table 1. Rainfall was highly seasonal, so we divide models by the season of collection. Models constructed using species distribution data from the dry season used the precipitation in the driest season; models constructed using species distribution data from the wet season used the precipitation in the wettest season. These two variables were taken from the Worldclim database, as BIO13 Precipitation of Wettest Month and BIO14 Precipitation of Driest Month [17]. Flow accumulation was derived using elevation data [18]. Flow accumulation is the surface area contributing water to a particular point, and indicates the potential amount of water available, which is then determined by rainfall. Using the SRTM elevation, flow accumulation was derived using the Fill, Flow Direction and Flow Accumulation tools in ArcMap 10.1 [19]. Wetness index has previously been shown to be associated with the Buruli ulcer [9]. In ecological terms it indicates the topological potential for water to accumulate, and was derived according to Equation 1,Table 1\\nEnvironmental variables used in ecological niche modelling\\nVariableUnitsOriginal resolutionSourceWetness indexm2\\n15 arc-sec (approx 450 m2)SRTMFlow accumulationm2\\n15 arc-sec (approx 450 m2)SRTMPrecipitation in wettest seasonmillimeter30 arc-sec (approx 1 km2)Bioclim 13Precipitation in driest seasonmillimeter30 arc-sec (approx 1 km2)Bioclim 14GLC-APUnitless300 m2\\nGLC 2009GLC-5 KUnitless300 m2\\nGLC 2009All data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for use in Maxent. Resampling used resample() in the library ‘raster’ of the software R.\\n\\nEnvironmental variables used in ecological niche modelling\\n\\nAll data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for use in Maxent. Resampling used resample() in the library ‘raster’ of the software R.\\n\\n\\n1\\n\\n\\n\\nwhere FA was the flow accumulation, 500 was the cell size in meters, and S was the surface slope in degrees. Large flow accumulation values and flat slopes resulted in high wetness index values, and indicate areas where water is likely to stagnate. In areas where the slope is zero wetness index had no value. Land cover was derived from the Global Land Cover Map 2009 [20]. GLC-At Point (hereafter GLC-AP) is the land cover at the sample site. In a 5 km radius around the site the most common (modal) land cover category was described. For example, a sample site may be in savannah, but surrounded by forest. 5 km was selected as the approximate flight radius of the insects [21–23]. This is termed GLC-5 K.\\n Data preparation, niche modelling, and prediction of spatial distribution of suitable habitat Insect distribution data from the 36 sample sites (Figure 1) were explored to identify normality, homogeneity of variance and correlation in the five environmental parameters used. Two sites were excluded from analysis as apparent outliers in flow accumulation, otherwise the data were normally distributed and homogenous. Correlation was observed between flow accumulation and wetness index and between precipitation in the driest and in the wettest seasons, however this was not significant (p > 0.05) using a Spearman’s correlation test. All data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for modelling using resample() in the library ‘raster’ of the software R [24].\\nAcross the scale of West Africa we assume that absence data is not reliable, as it is more likely to indicate failure of detection rather than evidence of absence. For this reason, we chose to conduct presence-only modelling, and the specific method selected was Maximum Entropy [25]. We used the software Maxent (Maxent 3.3.3 k, [25]) to construct these models. Maxent has been used several times in the past to model the ecological niche of disease vectors [26]. Maximum entropy modelling minimises the divergence between the distribution of the environmental parameters and the species distribution, assuming the species is distributed in the most ecologically efficient manner possible. Maxent describes the environmental parameters across the study region, and is sensitive to the size of the study region. Herein, we generated background points in every raster cell within the extent of our study (that is, every environment was described). The models were replicated 100 times and averaged.\\nIn order to select an appropriate extent to the area considered for modelling we chose to confine the spatial extent of our study to the region ecologically interpolated by the sample sites (Figure 2). The sample sites of the SME dataset describe a particular range of environmental conditions for the five environmental parameters (Table 1). We excluded conditions that were not studied by the SME regime (much larger or lower values of rainfall, land cover categories not sampled, flow accumulation not observed at the study sites) to avoid extrapolating beyond the range of ecological parameter values studied. To select this interpolative region we constructed a Multivariate Environmental Similarity Surface (MESS map [27]), excluding areas with negative MESS values from our study region. A MESS map was constructed for each model; models with more samples can be interpolated into larger ecological regions. MESS maps were constructed with the function mess() available in the library dismo in R.Figure 2\\nDelineation of the study area. We exclude areas of ecological extrapolation. Across West Africa the red-green coloured region is ecologically interpolative within our sites, according to a multivariate environmental similarity surface (MESS). Regions where we extrapolate beyond the ecology of our study sites are identified using MESS values less than 0. Colour values are MESS values, indicating similarity to sample sites; a value of 100 is identical to the median of the sample sites. The spatial projection of this interpolative region is shown against a map of country borders of West Africa, for Belostomatidae adults in the dry season. This projection changes for each family, developmental stage and season, and though it is spatially discontinuous it is ecologically homogenous.\\n\\nDelineation of the study area. We exclude areas of ecological extrapolation. Across West Africa the red-green coloured region is ecologically interpolative within our sites, according to a multivariate environmental similarity surface (MESS). Regions where we extrapolate beyond the ecology of our study sites are identified using MESS values less than 0. Colour values are MESS values, indicating similarity to sample sites; a value of 100 is identical to the median of the sample sites. The spatial projection of this interpolative region is shown against a map of country borders of West Africa, for Belostomatidae adults in the dry season. This projection changes for each family, developmental stage and season, and though it is spatially discontinuous it is ecologically homogenous.\\nInsect distribution data from the 36 sample sites (Figure 1) were explored to identify normality, homogeneity of variance and correlation in the five environmental parameters used. Two sites were excluded from analysis as apparent outliers in flow accumulation, otherwise the data were normally distributed and homogenous. Correlation was observed between flow accumulation and wetness index and between precipitation in the driest and in the wettest seasons, however this was not significant (p > 0.05) using a Spearman’s correlation test. All data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for modelling using resample() in the library ‘raster’ of the software R [24].\\nAcross the scale of West Africa we assume that absence data is not reliable, as it is more likely to indicate failure of detection rather than evidence of absence. For this reason, we chose to conduct presence-only modelling, and the specific method selected was Maximum Entropy [25]. We used the software Maxent (Maxent 3.3.3 k, [25]) to construct these models. Maxent has been used several times in the past to model the ecological niche of disease vectors [26]. Maximum entropy modelling minimises the divergence between the distribution of the environmental parameters and the species distribution, assuming the species is distributed in the most ecologically efficient manner possible. Maxent describes the environmental parameters across the study region, and is sensitive to the size of the study region. Herein, we generated background points in every raster cell within the extent of our study (that is, every environment was described). The models were replicated 100 times and averaged.\\nIn order to select an appropriate extent to the area considered for modelling we chose to confine the spatial extent of our study to the region ecologically interpolated by the sample sites (Figure 2). The sample sites of the SME dataset describe a particular range of environmental conditions for the five environmental parameters (Table 1). We excluded conditions that were not studied by the SME regime (much larger or lower values of rainfall, land cover categories not sampled, flow accumulation not observed at the study sites) to avoid extrapolating beyond the range of ecological parameter values studied. To select this interpolative region we constructed a Multivariate Environmental Similarity Surface (MESS map [27]), excluding areas with negative MESS values from our study region. A MESS map was constructed for each model; models with more samples can be interpolated into larger ecological regions. MESS maps were constructed with the function mess() available in the library dismo in R.Figure 2\\nDelineation of the study area. We exclude areas of ecological extrapolation. Across West Africa the red-green coloured region is ecologically interpolative within our sites, according to a multivariate environmental similarity surface (MESS). Regions where we extrapolate beyond the ecology of our study sites are identified using MESS values less than 0. Colour values are MESS values, indicating similarity to sample sites; a value of 100 is identical to the median of the sample sites. The spatial projection of this interpolative region is shown against a map of country borders of West Africa, for Belostomatidae adults in the dry season. This projection changes for each family, developmental stage and season, and though it is spatially discontinuous it is ecologically homogenous.\\n\\nDelineation of the study area. We exclude areas of ecological extrapolation. Across West Africa the red-green coloured region is ecologically interpolative within our sites, according to a multivariate environmental similarity surface (MESS). Regions where we extrapolate beyond the ecology of our study sites are identified using MESS values less than 0. Colour values are MESS values, indicating similarity to sample sites; a value of 100 is identical to the median of the sample sites. The spatial projection of this interpolative region is shown against a map of country borders of West Africa, for Belostomatidae adults in the dry season. This projection changes for each family, developmental stage and season, and though it is spatially discontinuous it is ecologically homogenous.\\n Evaluating model performance We evaluated the performance of these models according to their ability to correctly predict the distribution of the insects (with a separate dataset, the AG dataset) as efficiently as possible (avoiding overfitting). We used two methods to evaluate the models. First, the Akaike information criterion, corrected for small sample size (AICc), was used. AICc was generated as in [28], and penalises complex models to avoid over-fitting the data, low AICc scores indicate the model is not over-fitting. Second, we tested the models ability to predict the AG dataset. The Area Under Curve (AUC) is often used to evaluate Maxent models, and has been criticized previously [29]. We used a modified version of the AUC for model evaluation, termed here AG AUC. The model predictions were compared to the known values as collected by AG. AG AUC values range from 0 to 1, values close to 1 indicate good performance, 0.5 is no better than random. Use of the AG dataset allowed us to use true absence data for model validation, avoiding the problem of pseudo-absence data in Maxent.\\nThe purpose of these two different metrics is to consider different aspects of performance, neither were without limitations. Use of the AG dataset allowed a degree of validation across methodologies, indicating the extent to which our results were dependent on a particular sampling regime. AICc is traditionally used to indicate over-fitting in models, however it is sensitive to the size of the ecological niche of the species.\\nWe evaluated the performance of these models according to their ability to correctly predict the distribution of the insects (with a separate dataset, the AG dataset) as efficiently as possible (avoiding overfitting). We used two methods to evaluate the models. First, the Akaike information criterion, corrected for small sample size (AICc), was used. AICc was generated as in [28], and penalises complex models to avoid over-fitting the data, low AICc scores indicate the model is not over-fitting. Second, we tested the models ability to predict the AG dataset. The Area Under Curve (AUC) is often used to evaluate Maxent models, and has been criticized previously [29]. We used a modified version of the AUC for model evaluation, termed here AG AUC. The model predictions were compared to the known values as collected by AG. AG AUC values range from 0 to 1, values close to 1 indicate good performance, 0.5 is no better than random. Use of the AG dataset allowed us to use true absence data for model validation, avoiding the problem of pseudo-absence data in Maxent.\\nThe purpose of these two different metrics is to consider different aspects of performance, neither were without limitations. Use of the AG dataset allowed a degree of validation across methodologies, indicating the extent to which our results were dependent on a particular sampling regime. AICc is traditionally used to indicate over-fitting in models, however it is sensitive to the size of the ecological niche of the species.\\n Identifying the relationship between habitat suitability and Buruli ulcer prevalence Buruli ulcer prevalence data was collected for two endemic regions in Cameroon, Akonolinga [30] and Bankim [31], as shown in Figure 3. Around the centre of each village a buffer was created, and average habitat suitability in this buffer was correlated to the village Buruli ulcer prevalence using Spearman’s rank correlation coefficient. Seven buffers were used to explore the effect of buffer size and shape. Around the centre of the village circular buffers of 1, 2, 3, 4, 5 and 10 km were selected, and average habitat suitability recorded. We also used a buffer defined by the borders of the village (Figure 3). This buffer changes in size for each village, and represents the approximate extent of the village area. 5 km is approximately the flight radius of the insects, and the distance easily walkable by local people on an average day in Akonolinga [32]. To deal with this multiple testing, Bonferonni’s correction of the p-values was used.Figure 3\\nSpatial distribution of Buruli ulcer prevalence in Akonolianga and Bankim, two endemic regions within Cameroon.\\n\\n\\nSpatial distribution of Buruli ulcer prevalence in Akonolianga and Bankim, two endemic regions within Cameroon.\\n\\nBuruli ulcer prevalence data was collected for two endemic regions in Cameroon, Akonolinga [30] and Bankim [31], as shown in Figure 3. Around the centre of each village a buffer was created, and average habitat suitability in this buffer was correlated to the village Buruli ulcer prevalence using Spearman’s rank correlation coefficient. Seven buffers were used to explore the effect of buffer size and shape. Around the centre of the village circular buffers of 1, 2, 3, 4, 5 and 10 km were selected, and average habitat suitability recorded. We also used a buffer defined by the borders of the village (Figure 3). This buffer changes in size for each village, and represents the approximate extent of the village area. 5 km is approximately the flight radius of the insects, and the distance easily walkable by local people on an average day in Akonolinga [32]. To deal with this multiple testing, Bonferonni’s correction of the p-values was used.Figure 3\\nSpatial distribution of Buruli ulcer prevalence in Akonolianga and Bankim, two endemic regions within Cameroon.\\n\\n\\nSpatial distribution of Buruli ulcer prevalence in Akonolianga and Bankim, two endemic regions within Cameroon.\\n\",\n \"Data were collected as described in [15] hereafter referred to as the SME dataset. In brief, 36 sample sites in Cameroon were visited monthly from September 2012 to February 2013 (Figure 1), a period including both wet and dry seasons. Dip net sampling was conducted at all sites. Due to limitations of current taxonomic keys, the aquatic insects of interest were only identifiable to the phylogenetic division of Family. A second dataset was used in model validation, using data collected separately by A. Garchitorena (Figure 1). This dataset was collected as described in [16], and is hereafter referred to as the AG dataset. Niche models of Belostomatidae and Naucoridae are constructed using the SME dataset, and then tested on their ability to reproduce the independent AG dataset.Figure 1\\nStudy sites Cameroon against local land cover. Data from the SME dataset is in red, and AG data set is in blue. The sample sites span the extent of Cameroon, sampling from every major land cover category.\\n\\nStudy sites Cameroon against local land cover. Data from the SME dataset is in red, and AG data set is in blue. The sample sites span the extent of Cameroon, sampling from every major land cover category.\",\n \"Five ecological parameters were used to describe the distribution of suitable habitats: rainfall, flow accumulation, wetness index, land cover at the sample site, and land cover within the flight range of adult insects. These variables were selected on the basis of their likelihood to influence the distribution and condition of water, and are summarised in Table 1. Rainfall was highly seasonal, so we divide models by the season of collection. Models constructed using species distribution data from the dry season used the precipitation in the driest season; models constructed using species distribution data from the wet season used the precipitation in the wettest season. These two variables were taken from the Worldclim database, as BIO13 Precipitation of Wettest Month and BIO14 Precipitation of Driest Month [17]. Flow accumulation was derived using elevation data [18]. Flow accumulation is the surface area contributing water to a particular point, and indicates the potential amount of water available, which is then determined by rainfall. Using the SRTM elevation, flow accumulation was derived using the Fill, Flow Direction and Flow Accumulation tools in ArcMap 10.1 [19]. Wetness index has previously been shown to be associated with the Buruli ulcer [9]. In ecological terms it indicates the topological potential for water to accumulate, and was derived according to Equation 1,Table 1\\nEnvironmental variables used in ecological niche modelling\\nVariableUnitsOriginal resolutionSourceWetness indexm2\\n15 arc-sec (approx 450 m2)SRTMFlow accumulationm2\\n15 arc-sec (approx 450 m2)SRTMPrecipitation in wettest seasonmillimeter30 arc-sec (approx 1 km2)Bioclim 13Precipitation in driest seasonmillimeter30 arc-sec (approx 1 km2)Bioclim 14GLC-APUnitless300 m2\\nGLC 2009GLC-5 KUnitless300 m2\\nGLC 2009All data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for use in Maxent. Resampling used resample() in the library ‘raster’ of the software R.\\n\\nEnvironmental variables used in ecological niche modelling\\n\\nAll data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for use in Maxent. Resampling used resample() in the library ‘raster’ of the software R.\\n\\n\\n1\\n\\n\\n\\nwhere FA was the flow accumulation, 500 was the cell size in meters, and S was the surface slope in degrees. Large flow accumulation values and flat slopes resulted in high wetness index values, and indicate areas where water is likely to stagnate. In areas where the slope is zero wetness index had no value. Land cover was derived from the Global Land Cover Map 2009 [20]. GLC-At Point (hereafter GLC-AP) is the land cover at the sample site. In a 5 km radius around the site the most common (modal) land cover category was described. For example, a sample site may be in savannah, but surrounded by forest. 5 km was selected as the approximate flight radius of the insects [21–23]. This is termed GLC-5 K.\",\n \"Insect distribution data from the 36 sample sites (Figure 1) were explored to identify normality, homogeneity of variance and correlation in the five environmental parameters used. Two sites were excluded from analysis as apparent outliers in flow accumulation, otherwise the data were normally distributed and homogenous. Correlation was observed between flow accumulation and wetness index and between precipitation in the driest and in the wettest seasons, however this was not significant (p > 0.05) using a Spearman’s correlation test. All data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for modelling using resample() in the library ‘raster’ of the software R [24].\\nAcross the scale of West Africa we assume that absence data is not reliable, as it is more likely to indicate failure of detection rather than evidence of absence. For this reason, we chose to conduct presence-only modelling, and the specific method selected was Maximum Entropy [25]. We used the software Maxent (Maxent 3.3.3 k, [25]) to construct these models. Maxent has been used several times in the past to model the ecological niche of disease vectors [26]. Maximum entropy modelling minimises the divergence between the distribution of the environmental parameters and the species distribution, assuming the species is distributed in the most ecologically efficient manner possible. Maxent describes the environmental parameters across the study region, and is sensitive to the size of the study region. Herein, we generated background points in every raster cell within the extent of our study (that is, every environment was described). The models were replicated 100 times and averaged.\\nIn order to select an appropriate extent to the area considered for modelling we chose to confine the spatial extent of our study to the region ecologically interpolated by the sample sites (Figure 2). The sample sites of the SME dataset describe a particular range of environmental conditions for the five environmental parameters (Table 1). We excluded conditions that were not studied by the SME regime (much larger or lower values of rainfall, land cover categories not sampled, flow accumulation not observed at the study sites) to avoid extrapolating beyond the range of ecological parameter values studied. To select this interpolative region we constructed a Multivariate Environmental Similarity Surface (MESS map [27]), excluding areas with negative MESS values from our study region. A MESS map was constructed for each model; models with more samples can be interpolated into larger ecological regions. MESS maps were constructed with the function mess() available in the library dismo in R.Figure 2\\nDelineation of the study area. We exclude areas of ecological extrapolation. Across West Africa the red-green coloured region is ecologically interpolative within our sites, according to a multivariate environmental similarity surface (MESS). Regions where we extrapolate beyond the ecology of our study sites are identified using MESS values less than 0. Colour values are MESS values, indicating similarity to sample sites; a value of 100 is identical to the median of the sample sites. The spatial projection of this interpolative region is shown against a map of country borders of West Africa, for Belostomatidae adults in the dry season. This projection changes for each family, developmental stage and season, and though it is spatially discontinuous it is ecologically homogenous.\\n\\nDelineation of the study area. We exclude areas of ecological extrapolation. Across West Africa the red-green coloured region is ecologically interpolative within our sites, according to a multivariate environmental similarity surface (MESS). Regions where we extrapolate beyond the ecology of our study sites are identified using MESS values less than 0. Colour values are MESS values, indicating similarity to sample sites; a value of 100 is identical to the median of the sample sites. The spatial projection of this interpolative region is shown against a map of country borders of West Africa, for Belostomatidae adults in the dry season. This projection changes for each family, developmental stage and season, and though it is spatially discontinuous it is ecologically homogenous.\",\n \"We evaluated the performance of these models according to their ability to correctly predict the distribution of the insects (with a separate dataset, the AG dataset) as efficiently as possible (avoiding overfitting). We used two methods to evaluate the models. First, the Akaike information criterion, corrected for small sample size (AICc), was used. AICc was generated as in [28], and penalises complex models to avoid over-fitting the data, low AICc scores indicate the model is not over-fitting. Second, we tested the models ability to predict the AG dataset. The Area Under Curve (AUC) is often used to evaluate Maxent models, and has been criticized previously [29]. We used a modified version of the AUC for model evaluation, termed here AG AUC. The model predictions were compared to the known values as collected by AG. AG AUC values range from 0 to 1, values close to 1 indicate good performance, 0.5 is no better than random. Use of the AG dataset allowed us to use true absence data for model validation, avoiding the problem of pseudo-absence data in Maxent.\\nThe purpose of these two different metrics is to consider different aspects of performance, neither were without limitations. Use of the AG dataset allowed a degree of validation across methodologies, indicating the extent to which our results were dependent on a particular sampling regime. AICc is traditionally used to indicate over-fitting in models, however it is sensitive to the size of the ecological niche of the species.\",\n \"Buruli ulcer prevalence data was collected for two endemic regions in Cameroon, Akonolinga [30] and Bankim [31], as shown in Figure 3. Around the centre of each village a buffer was created, and average habitat suitability in this buffer was correlated to the village Buruli ulcer prevalence using Spearman’s rank correlation coefficient. Seven buffers were used to explore the effect of buffer size and shape. Around the centre of the village circular buffers of 1, 2, 3, 4, 5 and 10 km were selected, and average habitat suitability recorded. We also used a buffer defined by the borders of the village (Figure 3). This buffer changes in size for each village, and represents the approximate extent of the village area. 5 km is approximately the flight radius of the insects, and the distance easily walkable by local people on an average day in Akonolinga [32]. To deal with this multiple testing, Bonferonni’s correction of the p-values was used.Figure 3\\nSpatial distribution of Buruli ulcer prevalence in Akonolianga and Bankim, two endemic regions within Cameroon.\\n\\n\\nSpatial distribution of Buruli ulcer prevalence in Akonolianga and Bankim, two endemic regions within Cameroon.\\n\",\n \" Distribution of suitable habitat, and its relationship to Buruli ulcer In Akonolinga there was a significantly positive correlation between Buruli ulcer prevalence and average habitat suitability, for both Naucoridae and Belostomatidae, in the wet season (Table 2, Figure 4). This relationship was significant at multiple buffer distances. In contrast, in Bankim there was no significant correlation between Buruli ulcer prevalence and Belostomatidae or Naucoridae average habitat suitability, in either wet or dry seasons or at any buffer distance (Table 3).Table 2\\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\\nSpeciesSeasonBufferBonferroni pvalueBelostomatidaeDry10-5-4-3-2-1-Village-Wet100.00050.01440.0233-2-1-Village-NaucoridaeDry10-5-4-3-2-1-Village-Wet100.00150.01340.0143-2-1-Village-The column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.Figure 4\\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\\n\\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\\n\\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.\\n\\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\\n\\nSpearman rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Bankim, for both seasons and species\\n\\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. No significant correlations were observed in Bankim.\\nIn Akonolinga there was a significantly positive correlation between Buruli ulcer prevalence and average habitat suitability, for both Naucoridae and Belostomatidae, in the wet season (Table 2, Figure 4). This relationship was significant at multiple buffer distances. In contrast, in Bankim there was no significant correlation between Buruli ulcer prevalence and Belostomatidae or Naucoridae average habitat suitability, in either wet or dry seasons or at any buffer distance (Table 3).Table 2\\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\\nSpeciesSeasonBufferBonferroni pvalueBelostomatidaeDry10-5-4-3-2-1-Village-Wet100.00050.01440.0233-2-1-Village-NaucoridaeDry10-5-4-3-2-1-Village-Wet100.00150.01340.0143-2-1-Village-The column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.Figure 4\\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\\n\\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\\n\\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.\\n\\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\\n\\nSpearman rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Bankim, for both seasons and species\\n\\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. No significant correlations were observed in Bankim.\\n Ecologically important variables in the distribution of the aquatic insect families Variable importance was evaluated using Jackknife variable removal. Jacknife removes a variable and evaluates the effect of variable removal on the model. In the dry season Belostomatidae and Naucoridae responded in broadly similar fashions; the variable whose removal had the largest effect was GLC 5 km (Figure 5). The land cover categories most suitable for both Belostomatidae and Naucoridae are water bodies, artificial areas, rain fed croplands and forest/grassland mosaics (Figure 6). If one of these categories is the dominant category in 5 km radius, in the dry season, the likelihood of encountering the insect is higher. Unsuitable categories were forest and vegetation/cropland mosaic.In the wet season precipitation is more important than land cover. Precipitation suitability peaks at approximately 300 millimeters per month, and diminishes above or below this (Figure 6). For the dry season there is a simple increase in habitat suitability with increasing precipitation.Figure 5\\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.Figure 6\\nHabitat suitability for Belostomatidae (1\\nst\\nand 2\\nnd\\nrows) and Naucoridae (3\\nrd\\nand 4\\nth\\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\\n\\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.\\n\\nHabitat suitability for Belostomatidae (1\\nst\\nand 2\\nnd\\nrows) and Naucoridae (3\\nrd\\nand 4\\nth\\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\\nFlow accumulation had a negative association with habitat suitability, and wetness index had a positive association, regardless of season, for both Belostomatidae and Naucoridae.\\nVariable importance was evaluated using Jackknife variable removal. Jacknife removes a variable and evaluates the effect of variable removal on the model. In the dry season Belostomatidae and Naucoridae responded in broadly similar fashions; the variable whose removal had the largest effect was GLC 5 km (Figure 5). The land cover categories most suitable for both Belostomatidae and Naucoridae are water bodies, artificial areas, rain fed croplands and forest/grassland mosaics (Figure 6). If one of these categories is the dominant category in 5 km radius, in the dry season, the likelihood of encountering the insect is higher. Unsuitable categories were forest and vegetation/cropland mosaic.In the wet season precipitation is more important than land cover. Precipitation suitability peaks at approximately 300 millimeters per month, and diminishes above or below this (Figure 6). For the dry season there is a simple increase in habitat suitability with increasing precipitation.Figure 5\\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.Figure 6\\nHabitat suitability for Belostomatidae (1\\nst\\nand 2\\nnd\\nrows) and Naucoridae (3\\nrd\\nand 4\\nth\\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\\n\\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.\\n\\nHabitat suitability for Belostomatidae (1\\nst\\nand 2\\nnd\\nrows) and Naucoridae (3\\nrd\\nand 4\\nth\\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\\nFlow accumulation had a negative association with habitat suitability, and wetness index had a positive association, regardless of season, for both Belostomatidae and Naucoridae.\\n Model performance AICc for Naucoridae adults (dry season) was 14.6, and 14.2 in the wet season, as in Table 2. For Belostomatidae adults (dry season) the AICc was 12.5, and 12.2 in the wet season. Scores of overfitting are relative; these scores indicate the Belostomatidae model was less prone to overfitting than the Naucoridae model.\\nThe AG data set was also used in model validation. In the dry season Naucoridae adults had an AG AUC of 0.83, and 0.80 in the wet season. Belostomatidae adults had an AG AUC of 0.80 in the dry season, and 0.86 in the wet season. These scores indicate that the models are able to describe the distribution of the insects with good accuracy; the model based on SME dataset is able to accurately replicate the independently collected AG dataset.\\nAICc for Naucoridae adults (dry season) was 14.6, and 14.2 in the wet season, as in Table 2. For Belostomatidae adults (dry season) the AICc was 12.5, and 12.2 in the wet season. Scores of overfitting are relative; these scores indicate the Belostomatidae model was less prone to overfitting than the Naucoridae model.\\nThe AG data set was also used in model validation. In the dry season Naucoridae adults had an AG AUC of 0.83, and 0.80 in the wet season. Belostomatidae adults had an AG AUC of 0.80 in the dry season, and 0.86 in the wet season. These scores indicate that the models are able to describe the distribution of the insects with good accuracy; the model based on SME dataset is able to accurately replicate the independently collected AG dataset.\",\n \"In Akonolinga there was a significantly positive correlation between Buruli ulcer prevalence and average habitat suitability, for both Naucoridae and Belostomatidae, in the wet season (Table 2, Figure 4). This relationship was significant at multiple buffer distances. In contrast, in Bankim there was no significant correlation between Buruli ulcer prevalence and Belostomatidae or Naucoridae average habitat suitability, in either wet or dry seasons or at any buffer distance (Table 3).Table 2\\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\\nSpeciesSeasonBufferBonferroni pvalueBelostomatidaeDry10-5-4-3-2-1-Village-Wet100.00050.01440.0233-2-1-Village-NaucoridaeDry10-5-4-3-2-1-Village-Wet100.00150.01340.0143-2-1-Village-The column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.Figure 4\\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\\n\\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\\n\\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.\\n\\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\\n\\nSpearman rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Bankim, for both seasons and species\\n\\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. No significant correlations were observed in Bankim.\",\n \"Variable importance was evaluated using Jackknife variable removal. Jacknife removes a variable and evaluates the effect of variable removal on the model. In the dry season Belostomatidae and Naucoridae responded in broadly similar fashions; the variable whose removal had the largest effect was GLC 5 km (Figure 5). The land cover categories most suitable for both Belostomatidae and Naucoridae are water bodies, artificial areas, rain fed croplands and forest/grassland mosaics (Figure 6). If one of these categories is the dominant category in 5 km radius, in the dry season, the likelihood of encountering the insect is higher. Unsuitable categories were forest and vegetation/cropland mosaic.In the wet season precipitation is more important than land cover. Precipitation suitability peaks at approximately 300 millimeters per month, and diminishes above or below this (Figure 6). For the dry season there is a simple increase in habitat suitability with increasing precipitation.Figure 5\\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.Figure 6\\nHabitat suitability for Belostomatidae (1\\nst\\nand 2\\nnd\\nrows) and Naucoridae (3\\nrd\\nand 4\\nth\\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\\n\\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.\\n\\nHabitat suitability for Belostomatidae (1\\nst\\nand 2\\nnd\\nrows) and Naucoridae (3\\nrd\\nand 4\\nth\\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\\nFlow accumulation had a negative association with habitat suitability, and wetness index had a positive association, regardless of season, for both Belostomatidae and Naucoridae.\",\n \"AICc for Naucoridae adults (dry season) was 14.6, and 14.2 in the wet season, as in Table 2. For Belostomatidae adults (dry season) the AICc was 12.5, and 12.2 in the wet season. Scores of overfitting are relative; these scores indicate the Belostomatidae model was less prone to overfitting than the Naucoridae model.\\nThe AG data set was also used in model validation. In the dry season Naucoridae adults had an AG AUC of 0.83, and 0.80 in the wet season. Belostomatidae adults had an AG AUC of 0.80 in the dry season, and 0.86 in the wet season. These scores indicate that the models are able to describe the distribution of the insects with good accuracy; the model based on SME dataset is able to accurately replicate the independently collected AG dataset.\",\n \"We explored the correlation between the distribution of Belostomatidae and Naucoridae and the prevalence of Buruli ulcer. We have found a positive gradient between habitat suitability of Naucoridae and Belostomatidae and Buruli ulcer prevalence. Correlation does not imply causation; this result is not proof that the insects are vectors. However, understanding the reasons for the temporal and spatial changes in this correlation will enable a better understanding of the reasons for changes in Buruli ulcer prevalence.\\nThere are significant temporal changes in this correlation between habitat suitability of the insects and Buruli ulcer prevalence; in Akonolinga the Buruli ulcer prevalence is correlated to Naucoridae and Belostomatidae distribution in the wet season, but not in the dry season. Buruli ulcer is known to have complex temporal changes in prevalence [30, 33, 34], as is M. ulcerans\\n[14, 35]. It is therefore unsurprising that, if these insects are implicated in maintaining M. ulcerans in the environment, or involved in transmission to humans in some way (either as host vectors, or host carriers), the correlation between Buruli ulcer prevalence and their abundance would change in time.\\nWe also observe spatial changes in the correlation; water bug habitat suitability is not correlated to Buruli ulcer prevalence in Bankim, 457 km North of Akonolinga. Speculatively, perhaps other routes of transmission may be more important in this region, for example contact with infected plant biofilms, as suggested in Ghana [36].\\nHow do we interpret this result in terms of the Bradford-Hill guidelines? Herein we have focused on the correlation between these insects and the prevalence of the disease in both space and time. While there is a significant positive correlation for the predicted abundance of the aquatic insects and the prevalence of the Buruli ulcer, this correlation is not consistent from region to region. Previous research has proposed that M. ulcerans is transmitted within a multi-host transmission network [4]. In such a situation of multiple hosts the relative importance of any given mode of transmission may be expected to vary in time or space, and our results are consistent with, though not proof of, this hypothesis. The lack of a clear signal between water bugs and Buruli ulcer in Bankim would suggest that may not be key vectors or host carriers in that region. Recent studies have found notable changes in community composition relevant to M. ulcerans distribution, in the Greater Accra, Ashanti and Volta regions of Ghana, for both plant [35] and aquatic insects [37]. This would support the importance of changing biotic communities as a key factor in changing Buruli ulcer prevalence, a priority for future work. We have found that the prevalence of Buruli ulcer is correlated to Belostomatidae and Naucoridae abundance in Akonolinga but not Bankim, we do not know if the plant community composition for these regions, and wider aquatic insect community, also correlates to disease prevalence changes on a similar scale.\\nThe ecological niches of both Naucorid and Belostomatid water bugs in West Africa are predominantly determined by the distribution of suitable landcover in a 5 km radius, preferring water bodies, artificial areas and rain fed croplands. The specific land cover at the point of the site (GLC-AP) was less informative. The observation that the most suitable regional land cover class is water bodies is not surprising, but the high suitability of urban areas is curious. Ecologically this could have a variety of causes; there may be changes in the chemical composition of water in these habitats, a reduction in predation pressure, or a greater abundance of food. The specific reasons will require further research.\\nOur study has been limited in certain points; the low taxonomic resolution of the insects is a current limitation in this study. Secondly, an important limitation is that the distribution of M. ulcerans in these insects in these areas is unknown. The distribution of Naucoridae and Belostomatidae infected by M. ulcerans may differ from the distribution of Naucoridae and Belostomatidae generally. However, the insects are known to host the bacillus on their carapace, in their body [10, 14, 16]) and in their salivary glands [14] in the wild, and the distribution of the insect necessarily sets a limit to the distribution of infected insects. A related limitation is the unknown incubation time of M. ulcerans; the time from infection to presentation at the hospital, remains unknown. Finally, we have only addressed a single criterion of the Bradford-Hill guidelines; correlation. We have not aimed for a full discussion of the other criteria, and our findings should not be interpreted as proof of the role of these insects as vectors or key host carriers. Rather, we have discussed the existence of, and change in, a correlation between these insects and Buruli ulcer. Future work aims to explore spatial variation in the correlation between Buruli ulcer and the entire plant and animal communities, identifying any similarities between regions where the correlation exists, expanding on previous studies [37] which have focused on the community assemblage differences between M. ulcerans endemic and non-endemic regions of Ghana.\\nDespite these limitations, these results are consistent with previous research, which has shown that in Akonolinga the Nyong river is a risk factor for Buruli ulcer [30]. Our results agree with this conclusion; the main focus of suitable habitats for the insects in Akonolinga is the Nyong river, where the existence of large plants near the river banks provides appropriate habitat for Naucoridae and Belostomatidae to forage, develop and reproduce. Previous research has also implicated aquatic insects as important vectors in Akonolinga [14], including detection of M. ulcerans in the saliva of the insects.\\nIn conclusion, we find a positive correlation between the abundance of Naucoridae and Belostomatidae suitable habitat and Buruli ulcer prevalence. This correlation is not constant, and changes in time and space. We interpret this as evidence consistent with that idea that Naucoridae and Belostomatidae may be locally important host carriers of M. ulcerans in certain conditions, their importance changing as the environmental conditions change, which would be expected in the situation of multi-host transmission.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":[null,"materials|methods",null,null,null,null,null,"results",null,null,null,"discussion"],"string":"[\n null,\n \"materials|methods\",\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n \"discussion\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["Ecological niche modelling","Naucoridae","Belostomatidae","Spatial distribution","Habitat suitability","Buruli ulcer","\nMycobacterium ulcerans\n","Vector-borne transmission","Environmentally-acquired disease"],"string":"[\n \"Ecological niche modelling\",\n \"Naucoridae\",\n \"Belostomatidae\",\n \"Spatial distribution\",\n \"Habitat suitability\",\n \"Buruli ulcer\",\n \"\\nMycobacterium ulcerans\\n\",\n \"Vector-borne transmission\",\n \"Environmentally-acquired disease\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Background:\nThe Buruli ulcer is an emerging neglected tropical disease affecting more than 5,000 people per year in West and Central Africa, French Guiana, Latin America and Australia [1]. The disease burden is highest in Africa, where it predominantly affects children under the age of 15, and due to damage to the skin, muscle and bone, can result in severe scarring and crippling deformities if left untreated. The disease is caused by the environmental pathogen Mycobacterium ulcerans.\nThe mode of transmission of M. ulcerans, the method by which it infects humans, is unknown. Many routes of transmission have been proposed, such as transmission by aerosol [2], vector transmission by amoebae [3] or through aquatic networks [4]. During a study of the association between M. ulcerans and aquatic plants in Ghana and Benin aquatic insects were accidentally collected during the sampling procedure, and unexpectedly found to test positive for M. ulcerans\n[1]. The authors proposed that, given that these insects occasionally bite humans, they may be implicated in transmission of M. ulcerans. Aquatic insects have been further implicated after a series of laboratory experiments demonstrated the competency of Naucoridae to act as vectors. Naucoridae are able to acquire M. ulcerans from their diet, and then transmit the pathogen to mice resulting in Buruli-like symptoms [5–8]. Buruli ulcer is commonly associated with lowland, stagnant water [9] and human behaviours associated with water bodies appear to be risk factors for Buruli ulcer infection, which would lend support to the idea of infection occurring in an aquatic context.\nHowever, the role of these insects has been disputed for several reasons. In a two year study of Buruli ulcer endemic and non-endemic sites in Ghana [10], found no evidence for a role in transmission. The population of these insects, and the prevalence of M. ulcerans infection in them, was not significantly different between Buruli ulcer endemic and Buruli ulcer non-endemic sites [10]. The authors argued that, if these insects are vectors, we would expect them to have a higher abundance in Buruli ulcer endemic sites, that rates of M. ulcerans infection of the insects should be higher in Buruli ulcer endemic sites, and that the rate of infection of these insects should be higher than other species of invertebrates.\nThese expectations are based on the Bradford-Hill guidelines for associating insect vectors with human vector-borne disease [11, 12]. These guidelines provide a general framework to explore the association between vectors and disease, based on the consistency, specificity, plausibility and coherence of the proposed mode of transmission.\nConsistency refers to the expectation that the rate of infection of the proposed vectors, which should be consistently strongly positively associated, in time and space, with prevalence of human cases. This also implies human cases should not occur in absence of the proposed vector. The proposed vector should have a demonstrated capacity to physically transmit the pathogen, which has been demonstrated for M. ulcerans in the lab [5]. The interaction between the proposed vector and human infection must be specific and alternative explanations of human infection should be ruled out (though see [4] for alternative explanations). That is, human infection must be demonstrated to not have been the result of other potential modes of transmission. We note that this criterion must be applied with care in cases of multi-host transmission.\nAdditionally, the proposed vector must plausibly be able to be a vector of the pathogen. This criterion is often controversial as it is highly dependent upon the experience of the researcher, and their opinion about what is, and is not, plausible as opposed to merely possible [13]. Most authors in Buruli ulcer research would agree with some basic facts; the waterbugs are infected in the environment [14], they bite humans occasionally, and are able to transmit the bacteria to mice in the lab [5]. However, waterbugs are not known to be vectors of other pathogens, and related Mycobacteria (Mycobacterium tuberculosis, M. leprae, M. marnium) are not known to be vector-borne diseases [10, 12]. The plausibility of this proposed route of transmission is still debated. The final criterion, coherence, is based on what we already know about the pathogen, the vector and the host. Does the proposed method of transmission fit well with our current understanding of its biology? As our understanding of the biology of M. ulcerans improves, this criterion will be answered.\nGiven this framework, how likely is it that Naucoridae and/or Belostomatidae are vectors of the Buruli ulcer disease? Herein, we explore the correlation in time and space between the proposed vectors, Naucoridae and Belostomatidae, and the Buruli ulcer prevalence. We discuss the other Bradford-Hill criteria, but do not focus on them specifically, as it was not directly within the scope of this work. Based on sampling in Cameroon, we characterise the set of suitable habitats within which species of the Families Belostomatidae and Naucoridae can maintain a population (their ecological niche) and describe the spatial distribution of these suitable habitats across West Africa. We then explore any correlations between habitat suitability and Buruli ulcer prevalence at multiple spatial scales.\n\nMaterials and methods:\n Distribution of Belostomatidae and Naucoridae aquatic insect families Data were collected as described in [15] hereafter referred to as the SME dataset. In brief, 36 sample sites in Cameroon were visited monthly from September 2012 to February 2013 (Figure 1), a period including both wet and dry seasons. Dip net sampling was conducted at all sites. Due to limitations of current taxonomic keys, the aquatic insects of interest were only identifiable to the phylogenetic division of Family. A second dataset was used in model validation, using data collected separately by A. Garchitorena (Figure 1). This dataset was collected as described in [16], and is hereafter referred to as the AG dataset. Niche models of Belostomatidae and Naucoridae are constructed using the SME dataset, and then tested on their ability to reproduce the independent AG dataset.Figure 1\nStudy sites Cameroon against local land cover. Data from the SME dataset is in red, and AG data set is in blue. The sample sites span the extent of Cameroon, sampling from every major land cover category.\n\nStudy sites Cameroon against local land cover. Data from the SME dataset is in red, and AG data set is in blue. The sample sites span the extent of Cameroon, sampling from every major land cover category.\nData were collected as described in [15] hereafter referred to as the SME dataset. In brief, 36 sample sites in Cameroon were visited monthly from September 2012 to February 2013 (Figure 1), a period including both wet and dry seasons. Dip net sampling was conducted at all sites. Due to limitations of current taxonomic keys, the aquatic insects of interest were only identifiable to the phylogenetic division of Family. A second dataset was used in model validation, using data collected separately by A. Garchitorena (Figure 1). This dataset was collected as described in [16], and is hereafter referred to as the AG dataset. Niche models of Belostomatidae and Naucoridae are constructed using the SME dataset, and then tested on their ability to reproduce the independent AG dataset.Figure 1\nStudy sites Cameroon against local land cover. Data from the SME dataset is in red, and AG data set is in blue. The sample sites span the extent of Cameroon, sampling from every major land cover category.\n\nStudy sites Cameroon against local land cover. Data from the SME dataset is in red, and AG data set is in blue. The sample sites span the extent of Cameroon, sampling from every major land cover category.\n Environmental parameters used in ecological niche modelling Five ecological parameters were used to describe the distribution of suitable habitats: rainfall, flow accumulation, wetness index, land cover at the sample site, and land cover within the flight range of adult insects. These variables were selected on the basis of their likelihood to influence the distribution and condition of water, and are summarised in Table 1. Rainfall was highly seasonal, so we divide models by the season of collection. Models constructed using species distribution data from the dry season used the precipitation in the driest season; models constructed using species distribution data from the wet season used the precipitation in the wettest season. These two variables were taken from the Worldclim database, as BIO13 Precipitation of Wettest Month and BIO14 Precipitation of Driest Month [17]. Flow accumulation was derived using elevation data [18]. Flow accumulation is the surface area contributing water to a particular point, and indicates the potential amount of water available, which is then determined by rainfall. Using the SRTM elevation, flow accumulation was derived using the Fill, Flow Direction and Flow Accumulation tools in ArcMap 10.1 [19]. Wetness index has previously been shown to be associated with the Buruli ulcer [9]. In ecological terms it indicates the topological potential for water to accumulate, and was derived according to Equation 1,Table 1\nEnvironmental variables used in ecological niche modelling\nVariableUnitsOriginal resolutionSourceWetness indexm2\n15 arc-sec (approx 450 m2)SRTMFlow accumulationm2\n15 arc-sec (approx 450 m2)SRTMPrecipitation in wettest seasonmillimeter30 arc-sec (approx 1 km2)Bioclim 13Precipitation in driest seasonmillimeter30 arc-sec (approx 1 km2)Bioclim 14GLC-APUnitless300 m2\nGLC 2009GLC-5 KUnitless300 m2\nGLC 2009All data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for use in Maxent. Resampling used resample() in the library ‘raster’ of the software R.\n\nEnvironmental variables used in ecological niche modelling\n\nAll data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for use in Maxent. Resampling used resample() in the library ‘raster’ of the software R.\n\n\n1\n\n\n\nwhere FA was the flow accumulation, 500 was the cell size in meters, and S was the surface slope in degrees. Large flow accumulation values and flat slopes resulted in high wetness index values, and indicate areas where water is likely to stagnate. In areas where the slope is zero wetness index had no value. Land cover was derived from the Global Land Cover Map 2009 [20]. GLC-At Point (hereafter GLC-AP) is the land cover at the sample site. In a 5 km radius around the site the most common (modal) land cover category was described. For example, a sample site may be in savannah, but surrounded by forest. 5 km was selected as the approximate flight radius of the insects [21–23]. This is termed GLC-5 K.\nFive ecological parameters were used to describe the distribution of suitable habitats: rainfall, flow accumulation, wetness index, land cover at the sample site, and land cover within the flight range of adult insects. These variables were selected on the basis of their likelihood to influence the distribution and condition of water, and are summarised in Table 1. Rainfall was highly seasonal, so we divide models by the season of collection. Models constructed using species distribution data from the dry season used the precipitation in the driest season; models constructed using species distribution data from the wet season used the precipitation in the wettest season. These two variables were taken from the Worldclim database, as BIO13 Precipitation of Wettest Month and BIO14 Precipitation of Driest Month [17]. Flow accumulation was derived using elevation data [18]. Flow accumulation is the surface area contributing water to a particular point, and indicates the potential amount of water available, which is then determined by rainfall. Using the SRTM elevation, flow accumulation was derived using the Fill, Flow Direction and Flow Accumulation tools in ArcMap 10.1 [19]. Wetness index has previously been shown to be associated with the Buruli ulcer [9]. In ecological terms it indicates the topological potential for water to accumulate, and was derived according to Equation 1,Table 1\nEnvironmental variables used in ecological niche modelling\nVariableUnitsOriginal resolutionSourceWetness indexm2\n15 arc-sec (approx 450 m2)SRTMFlow accumulationm2\n15 arc-sec (approx 450 m2)SRTMPrecipitation in wettest seasonmillimeter30 arc-sec (approx 1 km2)Bioclim 13Precipitation in driest seasonmillimeter30 arc-sec (approx 1 km2)Bioclim 14GLC-APUnitless300 m2\nGLC 2009GLC-5 KUnitless300 m2\nGLC 2009All data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for use in Maxent. Resampling used resample() in the library ‘raster’ of the software R.\n\nEnvironmental variables used in ecological niche modelling\n\nAll data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for use in Maxent. Resampling used resample() in the library ‘raster’ of the software R.\n\n\n1\n\n\n\nwhere FA was the flow accumulation, 500 was the cell size in meters, and S was the surface slope in degrees. Large flow accumulation values and flat slopes resulted in high wetness index values, and indicate areas where water is likely to stagnate. In areas where the slope is zero wetness index had no value. Land cover was derived from the Global Land Cover Map 2009 [20]. GLC-At Point (hereafter GLC-AP) is the land cover at the sample site. In a 5 km radius around the site the most common (modal) land cover category was described. For example, a sample site may be in savannah, but surrounded by forest. 5 km was selected as the approximate flight radius of the insects [21–23]. This is termed GLC-5 K.\n Data preparation, niche modelling, and prediction of spatial distribution of suitable habitat Insect distribution data from the 36 sample sites (Figure 1) were explored to identify normality, homogeneity of variance and correlation in the five environmental parameters used. Two sites were excluded from analysis as apparent outliers in flow accumulation, otherwise the data were normally distributed and homogenous. Correlation was observed between flow accumulation and wetness index and between precipitation in the driest and in the wettest seasons, however this was not significant (p > 0.05) using a Spearman’s correlation test. All data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for modelling using resample() in the library ‘raster’ of the software R [24].\nAcross the scale of West Africa we assume that absence data is not reliable, as it is more likely to indicate failure of detection rather than evidence of absence. For this reason, we chose to conduct presence-only modelling, and the specific method selected was Maximum Entropy [25]. We used the software Maxent (Maxent 3.3.3 k, [25]) to construct these models. Maxent has been used several times in the past to model the ecological niche of disease vectors [26]. Maximum entropy modelling minimises the divergence between the distribution of the environmental parameters and the species distribution, assuming the species is distributed in the most ecologically efficient manner possible. Maxent describes the environmental parameters across the study region, and is sensitive to the size of the study region. Herein, we generated background points in every raster cell within the extent of our study (that is, every environment was described). The models were replicated 100 times and averaged.\nIn order to select an appropriate extent to the area considered for modelling we chose to confine the spatial extent of our study to the region ecologically interpolated by the sample sites (Figure 2). The sample sites of the SME dataset describe a particular range of environmental conditions for the five environmental parameters (Table 1). We excluded conditions that were not studied by the SME regime (much larger or lower values of rainfall, land cover categories not sampled, flow accumulation not observed at the study sites) to avoid extrapolating beyond the range of ecological parameter values studied. To select this interpolative region we constructed a Multivariate Environmental Similarity Surface (MESS map [27]), excluding areas with negative MESS values from our study region. A MESS map was constructed for each model; models with more samples can be interpolated into larger ecological regions. MESS maps were constructed with the function mess() available in the library dismo in R.Figure 2\nDelineation of the study area. We exclude areas of ecological extrapolation. Across West Africa the red-green coloured region is ecologically interpolative within our sites, according to a multivariate environmental similarity surface (MESS). Regions where we extrapolate beyond the ecology of our study sites are identified using MESS values less than 0. Colour values are MESS values, indicating similarity to sample sites; a value of 100 is identical to the median of the sample sites. The spatial projection of this interpolative region is shown against a map of country borders of West Africa, for Belostomatidae adults in the dry season. This projection changes for each family, developmental stage and season, and though it is spatially discontinuous it is ecologically homogenous.\n\nDelineation of the study area. We exclude areas of ecological extrapolation. Across West Africa the red-green coloured region is ecologically interpolative within our sites, according to a multivariate environmental similarity surface (MESS). Regions where we extrapolate beyond the ecology of our study sites are identified using MESS values less than 0. Colour values are MESS values, indicating similarity to sample sites; a value of 100 is identical to the median of the sample sites. The spatial projection of this interpolative region is shown against a map of country borders of West Africa, for Belostomatidae adults in the dry season. This projection changes for each family, developmental stage and season, and though it is spatially discontinuous it is ecologically homogenous.\nInsect distribution data from the 36 sample sites (Figure 1) were explored to identify normality, homogeneity of variance and correlation in the five environmental parameters used. Two sites were excluded from analysis as apparent outliers in flow accumulation, otherwise the data were normally distributed and homogenous. Correlation was observed between flow accumulation and wetness index and between precipitation in the driest and in the wettest seasons, however this was not significant (p > 0.05) using a Spearman’s correlation test. All data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for modelling using resample() in the library ‘raster’ of the software R [24].\nAcross the scale of West Africa we assume that absence data is not reliable, as it is more likely to indicate failure of detection rather than evidence of absence. For this reason, we chose to conduct presence-only modelling, and the specific method selected was Maximum Entropy [25]. We used the software Maxent (Maxent 3.3.3 k, [25]) to construct these models. Maxent has been used several times in the past to model the ecological niche of disease vectors [26]. Maximum entropy modelling minimises the divergence between the distribution of the environmental parameters and the species distribution, assuming the species is distributed in the most ecologically efficient manner possible. Maxent describes the environmental parameters across the study region, and is sensitive to the size of the study region. Herein, we generated background points in every raster cell within the extent of our study (that is, every environment was described). The models were replicated 100 times and averaged.\nIn order to select an appropriate extent to the area considered for modelling we chose to confine the spatial extent of our study to the region ecologically interpolated by the sample sites (Figure 2). The sample sites of the SME dataset describe a particular range of environmental conditions for the five environmental parameters (Table 1). We excluded conditions that were not studied by the SME regime (much larger or lower values of rainfall, land cover categories not sampled, flow accumulation not observed at the study sites) to avoid extrapolating beyond the range of ecological parameter values studied. To select this interpolative region we constructed a Multivariate Environmental Similarity Surface (MESS map [27]), excluding areas with negative MESS values from our study region. A MESS map was constructed for each model; models with more samples can be interpolated into larger ecological regions. MESS maps were constructed with the function mess() available in the library dismo in R.Figure 2\nDelineation of the study area. We exclude areas of ecological extrapolation. Across West Africa the red-green coloured region is ecologically interpolative within our sites, according to a multivariate environmental similarity surface (MESS). Regions where we extrapolate beyond the ecology of our study sites are identified using MESS values less than 0. Colour values are MESS values, indicating similarity to sample sites; a value of 100 is identical to the median of the sample sites. The spatial projection of this interpolative region is shown against a map of country borders of West Africa, for Belostomatidae adults in the dry season. This projection changes for each family, developmental stage and season, and though it is spatially discontinuous it is ecologically homogenous.\n\nDelineation of the study area. We exclude areas of ecological extrapolation. Across West Africa the red-green coloured region is ecologically interpolative within our sites, according to a multivariate environmental similarity surface (MESS). Regions where we extrapolate beyond the ecology of our study sites are identified using MESS values less than 0. Colour values are MESS values, indicating similarity to sample sites; a value of 100 is identical to the median of the sample sites. The spatial projection of this interpolative region is shown against a map of country borders of West Africa, for Belostomatidae adults in the dry season. This projection changes for each family, developmental stage and season, and though it is spatially discontinuous it is ecologically homogenous.\n Evaluating model performance We evaluated the performance of these models according to their ability to correctly predict the distribution of the insects (with a separate dataset, the AG dataset) as efficiently as possible (avoiding overfitting). We used two methods to evaluate the models. First, the Akaike information criterion, corrected for small sample size (AICc), was used. AICc was generated as in [28], and penalises complex models to avoid over-fitting the data, low AICc scores indicate the model is not over-fitting. Second, we tested the models ability to predict the AG dataset. The Area Under Curve (AUC) is often used to evaluate Maxent models, and has been criticized previously [29]. We used a modified version of the AUC for model evaluation, termed here AG AUC. The model predictions were compared to the known values as collected by AG. AG AUC values range from 0 to 1, values close to 1 indicate good performance, 0.5 is no better than random. Use of the AG dataset allowed us to use true absence data for model validation, avoiding the problem of pseudo-absence data in Maxent.\nThe purpose of these two different metrics is to consider different aspects of performance, neither were without limitations. Use of the AG dataset allowed a degree of validation across methodologies, indicating the extent to which our results were dependent on a particular sampling regime. AICc is traditionally used to indicate over-fitting in models, however it is sensitive to the size of the ecological niche of the species.\nWe evaluated the performance of these models according to their ability to correctly predict the distribution of the insects (with a separate dataset, the AG dataset) as efficiently as possible (avoiding overfitting). We used two methods to evaluate the models. First, the Akaike information criterion, corrected for small sample size (AICc), was used. AICc was generated as in [28], and penalises complex models to avoid over-fitting the data, low AICc scores indicate the model is not over-fitting. Second, we tested the models ability to predict the AG dataset. The Area Under Curve (AUC) is often used to evaluate Maxent models, and has been criticized previously [29]. We used a modified version of the AUC for model evaluation, termed here AG AUC. The model predictions were compared to the known values as collected by AG. AG AUC values range from 0 to 1, values close to 1 indicate good performance, 0.5 is no better than random. Use of the AG dataset allowed us to use true absence data for model validation, avoiding the problem of pseudo-absence data in Maxent.\nThe purpose of these two different metrics is to consider different aspects of performance, neither were without limitations. Use of the AG dataset allowed a degree of validation across methodologies, indicating the extent to which our results were dependent on a particular sampling regime. AICc is traditionally used to indicate over-fitting in models, however it is sensitive to the size of the ecological niche of the species.\n Identifying the relationship between habitat suitability and Buruli ulcer prevalence Buruli ulcer prevalence data was collected for two endemic regions in Cameroon, Akonolinga [30] and Bankim [31], as shown in Figure 3. Around the centre of each village a buffer was created, and average habitat suitability in this buffer was correlated to the village Buruli ulcer prevalence using Spearman’s rank correlation coefficient. Seven buffers were used to explore the effect of buffer size and shape. Around the centre of the village circular buffers of 1, 2, 3, 4, 5 and 10 km were selected, and average habitat suitability recorded. We also used a buffer defined by the borders of the village (Figure 3). This buffer changes in size for each village, and represents the approximate extent of the village area. 5 km is approximately the flight radius of the insects, and the distance easily walkable by local people on an average day in Akonolinga [32]. To deal with this multiple testing, Bonferonni’s correction of the p-values was used.Figure 3\nSpatial distribution of Buruli ulcer prevalence in Akonolianga and Bankim, two endemic regions within Cameroon.\n\n\nSpatial distribution of Buruli ulcer prevalence in Akonolianga and Bankim, two endemic regions within Cameroon.\n\nBuruli ulcer prevalence data was collected for two endemic regions in Cameroon, Akonolinga [30] and Bankim [31], as shown in Figure 3. Around the centre of each village a buffer was created, and average habitat suitability in this buffer was correlated to the village Buruli ulcer prevalence using Spearman’s rank correlation coefficient. Seven buffers were used to explore the effect of buffer size and shape. Around the centre of the village circular buffers of 1, 2, 3, 4, 5 and 10 km were selected, and average habitat suitability recorded. We also used a buffer defined by the borders of the village (Figure 3). This buffer changes in size for each village, and represents the approximate extent of the village area. 5 km is approximately the flight radius of the insects, and the distance easily walkable by local people on an average day in Akonolinga [32]. To deal with this multiple testing, Bonferonni’s correction of the p-values was used.Figure 3\nSpatial distribution of Buruli ulcer prevalence in Akonolianga and Bankim, two endemic regions within Cameroon.\n\n\nSpatial distribution of Buruli ulcer prevalence in Akonolianga and Bankim, two endemic regions within Cameroon.\n\n\nDistribution of Belostomatidae and Naucoridae aquatic insect families:\nData were collected as described in [15] hereafter referred to as the SME dataset. In brief, 36 sample sites in Cameroon were visited monthly from September 2012 to February 2013 (Figure 1), a period including both wet and dry seasons. Dip net sampling was conducted at all sites. Due to limitations of current taxonomic keys, the aquatic insects of interest were only identifiable to the phylogenetic division of Family. A second dataset was used in model validation, using data collected separately by A. Garchitorena (Figure 1). This dataset was collected as described in [16], and is hereafter referred to as the AG dataset. Niche models of Belostomatidae and Naucoridae are constructed using the SME dataset, and then tested on their ability to reproduce the independent AG dataset.Figure 1\nStudy sites Cameroon against local land cover. Data from the SME dataset is in red, and AG data set is in blue. The sample sites span the extent of Cameroon, sampling from every major land cover category.\n\nStudy sites Cameroon against local land cover. Data from the SME dataset is in red, and AG data set is in blue. The sample sites span the extent of Cameroon, sampling from every major land cover category.\n\nEnvironmental parameters used in ecological niche modelling:\nFive ecological parameters were used to describe the distribution of suitable habitats: rainfall, flow accumulation, wetness index, land cover at the sample site, and land cover within the flight range of adult insects. These variables were selected on the basis of their likelihood to influence the distribution and condition of water, and are summarised in Table 1. Rainfall was highly seasonal, so we divide models by the season of collection. Models constructed using species distribution data from the dry season used the precipitation in the driest season; models constructed using species distribution data from the wet season used the precipitation in the wettest season. These two variables were taken from the Worldclim database, as BIO13 Precipitation of Wettest Month and BIO14 Precipitation of Driest Month [17]. Flow accumulation was derived using elevation data [18]. Flow accumulation is the surface area contributing water to a particular point, and indicates the potential amount of water available, which is then determined by rainfall. Using the SRTM elevation, flow accumulation was derived using the Fill, Flow Direction and Flow Accumulation tools in ArcMap 10.1 [19]. Wetness index has previously been shown to be associated with the Buruli ulcer [9]. In ecological terms it indicates the topological potential for water to accumulate, and was derived according to Equation 1,Table 1\nEnvironmental variables used in ecological niche modelling\nVariableUnitsOriginal resolutionSourceWetness indexm2\n15 arc-sec (approx 450 m2)SRTMFlow accumulationm2\n15 arc-sec (approx 450 m2)SRTMPrecipitation in wettest seasonmillimeter30 arc-sec (approx 1 km2)Bioclim 13Precipitation in driest seasonmillimeter30 arc-sec (approx 1 km2)Bioclim 14GLC-APUnitless300 m2\nGLC 2009GLC-5 KUnitless300 m2\nGLC 2009All data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for use in Maxent. Resampling used resample() in the library ‘raster’ of the software R.\n\nEnvironmental variables used in ecological niche modelling\n\nAll data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for use in Maxent. Resampling used resample() in the library ‘raster’ of the software R.\n\n\n1\n\n\n\nwhere FA was the flow accumulation, 500 was the cell size in meters, and S was the surface slope in degrees. Large flow accumulation values and flat slopes resulted in high wetness index values, and indicate areas where water is likely to stagnate. In areas where the slope is zero wetness index had no value. Land cover was derived from the Global Land Cover Map 2009 [20]. GLC-At Point (hereafter GLC-AP) is the land cover at the sample site. In a 5 km radius around the site the most common (modal) land cover category was described. For example, a sample site may be in savannah, but surrounded by forest. 5 km was selected as the approximate flight radius of the insects [21–23]. This is termed GLC-5 K.\n\nData preparation, niche modelling, and prediction of spatial distribution of suitable habitat:\nInsect distribution data from the 36 sample sites (Figure 1) were explored to identify normality, homogeneity of variance and correlation in the five environmental parameters used. Two sites were excluded from analysis as apparent outliers in flow accumulation, otherwise the data were normally distributed and homogenous. Correlation was observed between flow accumulation and wetness index and between precipitation in the driest and in the wettest seasons, however this was not significant (p > 0.05) using a Spearman’s correlation test. All data were resampled to a spatial resolution of 0.004 decimal degrees (~300 m2) for modelling using resample() in the library ‘raster’ of the software R [24].\nAcross the scale of West Africa we assume that absence data is not reliable, as it is more likely to indicate failure of detection rather than evidence of absence. For this reason, we chose to conduct presence-only modelling, and the specific method selected was Maximum Entropy [25]. We used the software Maxent (Maxent 3.3.3 k, [25]) to construct these models. Maxent has been used several times in the past to model the ecological niche of disease vectors [26]. Maximum entropy modelling minimises the divergence between the distribution of the environmental parameters and the species distribution, assuming the species is distributed in the most ecologically efficient manner possible. Maxent describes the environmental parameters across the study region, and is sensitive to the size of the study region. Herein, we generated background points in every raster cell within the extent of our study (that is, every environment was described). The models were replicated 100 times and averaged.\nIn order to select an appropriate extent to the area considered for modelling we chose to confine the spatial extent of our study to the region ecologically interpolated by the sample sites (Figure 2). The sample sites of the SME dataset describe a particular range of environmental conditions for the five environmental parameters (Table 1). We excluded conditions that were not studied by the SME regime (much larger or lower values of rainfall, land cover categories not sampled, flow accumulation not observed at the study sites) to avoid extrapolating beyond the range of ecological parameter values studied. To select this interpolative region we constructed a Multivariate Environmental Similarity Surface (MESS map [27]), excluding areas with negative MESS values from our study region. A MESS map was constructed for each model; models with more samples can be interpolated into larger ecological regions. MESS maps were constructed with the function mess() available in the library dismo in R.Figure 2\nDelineation of the study area. We exclude areas of ecological extrapolation. Across West Africa the red-green coloured region is ecologically interpolative within our sites, according to a multivariate environmental similarity surface (MESS). Regions where we extrapolate beyond the ecology of our study sites are identified using MESS values less than 0. Colour values are MESS values, indicating similarity to sample sites; a value of 100 is identical to the median of the sample sites. The spatial projection of this interpolative region is shown against a map of country borders of West Africa, for Belostomatidae adults in the dry season. This projection changes for each family, developmental stage and season, and though it is spatially discontinuous it is ecologically homogenous.\n\nDelineation of the study area. We exclude areas of ecological extrapolation. Across West Africa the red-green coloured region is ecologically interpolative within our sites, according to a multivariate environmental similarity surface (MESS). Regions where we extrapolate beyond the ecology of our study sites are identified using MESS values less than 0. Colour values are MESS values, indicating similarity to sample sites; a value of 100 is identical to the median of the sample sites. The spatial projection of this interpolative region is shown against a map of country borders of West Africa, for Belostomatidae adults in the dry season. This projection changes for each family, developmental stage and season, and though it is spatially discontinuous it is ecologically homogenous.\n\nEvaluating model performance:\nWe evaluated the performance of these models according to their ability to correctly predict the distribution of the insects (with a separate dataset, the AG dataset) as efficiently as possible (avoiding overfitting). We used two methods to evaluate the models. First, the Akaike information criterion, corrected for small sample size (AICc), was used. AICc was generated as in [28], and penalises complex models to avoid over-fitting the data, low AICc scores indicate the model is not over-fitting. Second, we tested the models ability to predict the AG dataset. The Area Under Curve (AUC) is often used to evaluate Maxent models, and has been criticized previously [29]. We used a modified version of the AUC for model evaluation, termed here AG AUC. The model predictions were compared to the known values as collected by AG. AG AUC values range from 0 to 1, values close to 1 indicate good performance, 0.5 is no better than random. Use of the AG dataset allowed us to use true absence data for model validation, avoiding the problem of pseudo-absence data in Maxent.\nThe purpose of these two different metrics is to consider different aspects of performance, neither were without limitations. Use of the AG dataset allowed a degree of validation across methodologies, indicating the extent to which our results were dependent on a particular sampling regime. AICc is traditionally used to indicate over-fitting in models, however it is sensitive to the size of the ecological niche of the species.\n\nIdentifying the relationship between habitat suitability and Buruli ulcer prevalence:\nBuruli ulcer prevalence data was collected for two endemic regions in Cameroon, Akonolinga [30] and Bankim [31], as shown in Figure 3. Around the centre of each village a buffer was created, and average habitat suitability in this buffer was correlated to the village Buruli ulcer prevalence using Spearman’s rank correlation coefficient. Seven buffers were used to explore the effect of buffer size and shape. Around the centre of the village circular buffers of 1, 2, 3, 4, 5 and 10 km were selected, and average habitat suitability recorded. We also used a buffer defined by the borders of the village (Figure 3). This buffer changes in size for each village, and represents the approximate extent of the village area. 5 km is approximately the flight radius of the insects, and the distance easily walkable by local people on an average day in Akonolinga [32]. To deal with this multiple testing, Bonferonni’s correction of the p-values was used.Figure 3\nSpatial distribution of Buruli ulcer prevalence in Akonolianga and Bankim, two endemic regions within Cameroon.\n\n\nSpatial distribution of Buruli ulcer prevalence in Akonolianga and Bankim, two endemic regions within Cameroon.\n\n\nResults:\n Distribution of suitable habitat, and its relationship to Buruli ulcer In Akonolinga there was a significantly positive correlation between Buruli ulcer prevalence and average habitat suitability, for both Naucoridae and Belostomatidae, in the wet season (Table 2, Figure 4). This relationship was significant at multiple buffer distances. In contrast, in Bankim there was no significant correlation between Buruli ulcer prevalence and Belostomatidae or Naucoridae average habitat suitability, in either wet or dry seasons or at any buffer distance (Table 3).Table 2\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\nSpeciesSeasonBufferBonferroni pvalueBelostomatidaeDry10-5-4-3-2-1-Village-Wet100.00050.01440.0233-2-1-Village-NaucoridaeDry10-5-4-3-2-1-Village-Wet100.00150.01340.0143-2-1-Village-The column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.Figure 4\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\n\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\n\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.\n\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\n\nSpearman rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Bankim, for both seasons and species\n\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. No significant correlations were observed in Bankim.\nIn Akonolinga there was a significantly positive correlation between Buruli ulcer prevalence and average habitat suitability, for both Naucoridae and Belostomatidae, in the wet season (Table 2, Figure 4). This relationship was significant at multiple buffer distances. In contrast, in Bankim there was no significant correlation between Buruli ulcer prevalence and Belostomatidae or Naucoridae average habitat suitability, in either wet or dry seasons or at any buffer distance (Table 3).Table 2\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\nSpeciesSeasonBufferBonferroni pvalueBelostomatidaeDry10-5-4-3-2-1-Village-Wet100.00050.01440.0233-2-1-Village-NaucoridaeDry10-5-4-3-2-1-Village-Wet100.00150.01340.0143-2-1-Village-The column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.Figure 4\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\n\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\n\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.\n\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\n\nSpearman rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Bankim, for both seasons and species\n\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. No significant correlations were observed in Bankim.\n Ecologically important variables in the distribution of the aquatic insect families Variable importance was evaluated using Jackknife variable removal. Jacknife removes a variable and evaluates the effect of variable removal on the model. In the dry season Belostomatidae and Naucoridae responded in broadly similar fashions; the variable whose removal had the largest effect was GLC 5 km (Figure 5). The land cover categories most suitable for both Belostomatidae and Naucoridae are water bodies, artificial areas, rain fed croplands and forest/grassland mosaics (Figure 6). If one of these categories is the dominant category in 5 km radius, in the dry season, the likelihood of encountering the insect is higher. Unsuitable categories were forest and vegetation/cropland mosaic.In the wet season precipitation is more important than land cover. Precipitation suitability peaks at approximately 300 millimeters per month, and diminishes above or below this (Figure 6). For the dry season there is a simple increase in habitat suitability with increasing precipitation.Figure 5\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.Figure 6\nHabitat suitability for Belostomatidae (1\nst\nand 2\nnd\nrows) and Naucoridae (3\nrd\nand 4\nth\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\n\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.\n\nHabitat suitability for Belostomatidae (1\nst\nand 2\nnd\nrows) and Naucoridae (3\nrd\nand 4\nth\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\nFlow accumulation had a negative association with habitat suitability, and wetness index had a positive association, regardless of season, for both Belostomatidae and Naucoridae.\nVariable importance was evaluated using Jackknife variable removal. Jacknife removes a variable and evaluates the effect of variable removal on the model. In the dry season Belostomatidae and Naucoridae responded in broadly similar fashions; the variable whose removal had the largest effect was GLC 5 km (Figure 5). The land cover categories most suitable for both Belostomatidae and Naucoridae are water bodies, artificial areas, rain fed croplands and forest/grassland mosaics (Figure 6). If one of these categories is the dominant category in 5 km radius, in the dry season, the likelihood of encountering the insect is higher. Unsuitable categories were forest and vegetation/cropland mosaic.In the wet season precipitation is more important than land cover. Precipitation suitability peaks at approximately 300 millimeters per month, and diminishes above or below this (Figure 6). For the dry season there is a simple increase in habitat suitability with increasing precipitation.Figure 5\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.Figure 6\nHabitat suitability for Belostomatidae (1\nst\nand 2\nnd\nrows) and Naucoridae (3\nrd\nand 4\nth\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\n\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.\n\nHabitat suitability for Belostomatidae (1\nst\nand 2\nnd\nrows) and Naucoridae (3\nrd\nand 4\nth\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\nFlow accumulation had a negative association with habitat suitability, and wetness index had a positive association, regardless of season, for both Belostomatidae and Naucoridae.\n Model performance AICc for Naucoridae adults (dry season) was 14.6, and 14.2 in the wet season, as in Table 2. For Belostomatidae adults (dry season) the AICc was 12.5, and 12.2 in the wet season. Scores of overfitting are relative; these scores indicate the Belostomatidae model was less prone to overfitting than the Naucoridae model.\nThe AG data set was also used in model validation. In the dry season Naucoridae adults had an AG AUC of 0.83, and 0.80 in the wet season. Belostomatidae adults had an AG AUC of 0.80 in the dry season, and 0.86 in the wet season. These scores indicate that the models are able to describe the distribution of the insects with good accuracy; the model based on SME dataset is able to accurately replicate the independently collected AG dataset.\nAICc for Naucoridae adults (dry season) was 14.6, and 14.2 in the wet season, as in Table 2. For Belostomatidae adults (dry season) the AICc was 12.5, and 12.2 in the wet season. Scores of overfitting are relative; these scores indicate the Belostomatidae model was less prone to overfitting than the Naucoridae model.\nThe AG data set was also used in model validation. In the dry season Naucoridae adults had an AG AUC of 0.83, and 0.80 in the wet season. Belostomatidae adults had an AG AUC of 0.80 in the dry season, and 0.86 in the wet season. These scores indicate that the models are able to describe the distribution of the insects with good accuracy; the model based on SME dataset is able to accurately replicate the independently collected AG dataset.\n\nDistribution of suitable habitat, and its relationship to Buruli ulcer:\nIn Akonolinga there was a significantly positive correlation between Buruli ulcer prevalence and average habitat suitability, for both Naucoridae and Belostomatidae, in the wet season (Table 2, Figure 4). This relationship was significant at multiple buffer distances. In contrast, in Bankim there was no significant correlation between Buruli ulcer prevalence and Belostomatidae or Naucoridae average habitat suitability, in either wet or dry seasons or at any buffer distance (Table 3).Table 2\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\nSpeciesSeasonBufferBonferroni pvalueBelostomatidaeDry10-5-4-3-2-1-Village-Wet100.00050.01440.0233-2-1-Village-NaucoridaeDry10-5-4-3-2-1-Village-Wet100.00150.01340.0143-2-1-Village-The column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.Figure 4\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\n\nSpearman’s rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Akonolinga, for both seasons and species\n\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. Significant positive correlations were observed between both Belostomatidae and Naucoridae in the wet season and Buruli ulcer prevalence, but not the dry season.\n\nCorrelation between the prevalence of Buruli ulcer and habitat suitability of Belostomatidae (left) and Naucoridae (right) in the wet season in Akonolinga. Colour indicates use of a linear model (Black) or locally weighted scatterplot smoothing (Red), different ways of viewing the correlation. Buruli ulcer was absent from certain villages (grey dots) where habitat suitability for the insects is high. Because this can skew any correlation between habitat suitability and prevalence, we explored the effect of including (thin lines) or excluding (thick lines) these villages. We note that in either case Spearman’s rank correlation coefficient was significant. For Belostomatidae with Buruli ulcer absent villages p = 0.08, without BU absent villages p = 0.03. For Naucoridae with BU absent villages p = 0.04, without BU absent villages p = 0.02.\n\nSpearman rank correlation coefficients for correlation between Buruli ulcer prevalence and habitat suitability in Bankim, for both seasons and species\n\nThe column labelled Buffer is the distance, in km, around the village centre that the habitat suitability is considered, the buffer labelled village uses village boarders as a buffer (Figure 3). Bonferonni’s p value is the significance of the correlation between the insect and the disease, for clarity only significant values (<0.05) are presented, non-significant values are marked “-”. No significant correlations were observed in Bankim.\n\nEcologically important variables in the distribution of the aquatic insect families:\nVariable importance was evaluated using Jackknife variable removal. Jacknife removes a variable and evaluates the effect of variable removal on the model. In the dry season Belostomatidae and Naucoridae responded in broadly similar fashions; the variable whose removal had the largest effect was GLC 5 km (Figure 5). The land cover categories most suitable for both Belostomatidae and Naucoridae are water bodies, artificial areas, rain fed croplands and forest/grassland mosaics (Figure 6). If one of these categories is the dominant category in 5 km radius, in the dry season, the likelihood of encountering the insect is higher. Unsuitable categories were forest and vegetation/cropland mosaic.In the wet season precipitation is more important than land cover. Precipitation suitability peaks at approximately 300 millimeters per month, and diminishes above or below this (Figure 6). For the dry season there is a simple increase in habitat suitability with increasing precipitation.Figure 5\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.Figure 6\nHabitat suitability for Belostomatidae (1\nst\nand 2\nnd\nrows) and Naucoridae (3\nrd\nand 4\nth\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\n\nImportance of each variable according to Jack-knife AUC for wet and dry seasons. A high value indicates the variable is important; however this is sensitive to correlation within the variables. For both insects the most important variable in the dry season was the land cover in the flight radius (GLC 5 km), in the wet season precipitation was the most important variable.\n\nHabitat suitability for Belostomatidae (1\nst\nand 2\nnd\nrows) and Naucoridae (3\nrd\nand 4\nth\nrows) in the wet and dry seasons. Both insects have a negative relationship with flow accumulation, and a positive relationship with wetness index; an unexpected relationship given that increasing flow accumulation normally means increasing wetness index.\nFlow accumulation had a negative association with habitat suitability, and wetness index had a positive association, regardless of season, for both Belostomatidae and Naucoridae.\n\nModel performance:\nAICc for Naucoridae adults (dry season) was 14.6, and 14.2 in the wet season, as in Table 2. For Belostomatidae adults (dry season) the AICc was 12.5, and 12.2 in the wet season. Scores of overfitting are relative; these scores indicate the Belostomatidae model was less prone to overfitting than the Naucoridae model.\nThe AG data set was also used in model validation. In the dry season Naucoridae adults had an AG AUC of 0.83, and 0.80 in the wet season. Belostomatidae adults had an AG AUC of 0.80 in the dry season, and 0.86 in the wet season. These scores indicate that the models are able to describe the distribution of the insects with good accuracy; the model based on SME dataset is able to accurately replicate the independently collected AG dataset.\n\nDiscussion:\nWe explored the correlation between the distribution of Belostomatidae and Naucoridae and the prevalence of Buruli ulcer. We have found a positive gradient between habitat suitability of Naucoridae and Belostomatidae and Buruli ulcer prevalence. Correlation does not imply causation; this result is not proof that the insects are vectors. However, understanding the reasons for the temporal and spatial changes in this correlation will enable a better understanding of the reasons for changes in Buruli ulcer prevalence.\nThere are significant temporal changes in this correlation between habitat suitability of the insects and Buruli ulcer prevalence; in Akonolinga the Buruli ulcer prevalence is correlated to Naucoridae and Belostomatidae distribution in the wet season, but not in the dry season. Buruli ulcer is known to have complex temporal changes in prevalence [30, 33, 34], as is M. ulcerans\n[14, 35]. It is therefore unsurprising that, if these insects are implicated in maintaining M. ulcerans in the environment, or involved in transmission to humans in some way (either as host vectors, or host carriers), the correlation between Buruli ulcer prevalence and their abundance would change in time.\nWe also observe spatial changes in the correlation; water bug habitat suitability is not correlated to Buruli ulcer prevalence in Bankim, 457 km North of Akonolinga. Speculatively, perhaps other routes of transmission may be more important in this region, for example contact with infected plant biofilms, as suggested in Ghana [36].\nHow do we interpret this result in terms of the Bradford-Hill guidelines? Herein we have focused on the correlation between these insects and the prevalence of the disease in both space and time. While there is a significant positive correlation for the predicted abundance of the aquatic insects and the prevalence of the Buruli ulcer, this correlation is not consistent from region to region. Previous research has proposed that M. ulcerans is transmitted within a multi-host transmission network [4]. In such a situation of multiple hosts the relative importance of any given mode of transmission may be expected to vary in time or space, and our results are consistent with, though not proof of, this hypothesis. The lack of a clear signal between water bugs and Buruli ulcer in Bankim would suggest that may not be key vectors or host carriers in that region. Recent studies have found notable changes in community composition relevant to M. ulcerans distribution, in the Greater Accra, Ashanti and Volta regions of Ghana, for both plant [35] and aquatic insects [37]. This would support the importance of changing biotic communities as a key factor in changing Buruli ulcer prevalence, a priority for future work. We have found that the prevalence of Buruli ulcer is correlated to Belostomatidae and Naucoridae abundance in Akonolinga but not Bankim, we do not know if the plant community composition for these regions, and wider aquatic insect community, also correlates to disease prevalence changes on a similar scale.\nThe ecological niches of both Naucorid and Belostomatid water bugs in West Africa are predominantly determined by the distribution of suitable landcover in a 5 km radius, preferring water bodies, artificial areas and rain fed croplands. The specific land cover at the point of the site (GLC-AP) was less informative. The observation that the most suitable regional land cover class is water bodies is not surprising, but the high suitability of urban areas is curious. Ecologically this could have a variety of causes; there may be changes in the chemical composition of water in these habitats, a reduction in predation pressure, or a greater abundance of food. The specific reasons will require further research.\nOur study has been limited in certain points; the low taxonomic resolution of the insects is a current limitation in this study. Secondly, an important limitation is that the distribution of M. ulcerans in these insects in these areas is unknown. The distribution of Naucoridae and Belostomatidae infected by M. ulcerans may differ from the distribution of Naucoridae and Belostomatidae generally. However, the insects are known to host the bacillus on their carapace, in their body [10, 14, 16]) and in their salivary glands [14] in the wild, and the distribution of the insect necessarily sets a limit to the distribution of infected insects. A related limitation is the unknown incubation time of M. ulcerans; the time from infection to presentation at the hospital, remains unknown. Finally, we have only addressed a single criterion of the Bradford-Hill guidelines; correlation. We have not aimed for a full discussion of the other criteria, and our findings should not be interpreted as proof of the role of these insects as vectors or key host carriers. Rather, we have discussed the existence of, and change in, a correlation between these insects and Buruli ulcer. Future work aims to explore spatial variation in the correlation between Buruli ulcer and the entire plant and animal communities, identifying any similarities between regions where the correlation exists, expanding on previous studies [37] which have focused on the community assemblage differences between M. ulcerans endemic and non-endemic regions of Ghana.\nDespite these limitations, these results are consistent with previous research, which has shown that in Akonolinga the Nyong river is a risk factor for Buruli ulcer [30]. Our results agree with this conclusion; the main focus of suitable habitats for the insects in Akonolinga is the Nyong river, where the existence of large plants near the river banks provides appropriate habitat for Naucoridae and Belostomatidae to forage, develop and reproduce. Previous research has also implicated aquatic insects as important vectors in Akonolinga [14], including detection of M. ulcerans in the saliva of the insects.\nIn conclusion, we find a positive correlation between the abundance of Naucoridae and Belostomatidae suitable habitat and Buruli ulcer prevalence. This correlation is not constant, and changes in time and space. We interpret this as evidence consistent with that idea that Naucoridae and Belostomatidae may be locally important host carriers of M. ulcerans in certain conditions, their importance changing as the environmental conditions change, which would be expected in the situation of multi-host transmission.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe mode of transmission of the emerging neglected disease Buruli ulcer is unknown. Several potential transmission pathways have been proposed, such as amoebae, or transmission through food webs. Several lines of evidence have suggested that biting aquatic insects, Naucoridae and Belostomatidae, may act as vectors, however this proposal remains controversial.\n\nMethods:\nHerein, based on sampling in Cameroon, we construct an ecological niche model of these insects to describe their spatial distribution. We predict their distribution across West Africa, describe important environmental drivers of their abundance, and examine the correlation between their abundance and Buruli ulcer prevalence in the context of the Bradford-Hill guidelines.\n\nResults:\nWe find a significant positive correlation between the abundance of the insects and the prevalence of Buruli ulcer. This correlation changes in space and time, it is significant in one Camerounese study region in (Akonolinga) and not other (Bankim). We discuss notable environmental differences between these regions.\n\nConclusions:\nWe interpret the presence of, and change in, this correlation as evidence (though not proof) that these insects may be locally important in the environmental persistence, or transmission, of Mycobacterium. ulcerans. This is consistent with the idea of M. ulcerans as a pathogen transmitted by multiple modes of infection, the importance of any one pathway changing from region to region, depending on the local environmental conditions."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":13021,"string":"13,021"},"whole_article_abstract_length":{"kind":"number","value":265,"string":"265"},"other_sections_lengths":{"kind":"list like","value":[993,238,559,770,294,232,819,496,153],"string":"[\n 993,\n 238,\n 559,\n 770,\n 294,\n 232,\n 819,\n 496,\n 153\n]","isConversational":false},"num_sections":{"kind":"number","value":12,"string":"12"},"most_frequent_words":{"kind":"list like","value":["season","correlation","buruli","ulcer","buruli ulcer","habitat","belostomatidae","suitability","naucoridae","habitat suitability"],"string":"[\n \"season\",\n \"correlation\",\n \"buruli\",\n \"ulcer\",\n \"buruli ulcer\",\n \"habitat\",\n \"belostomatidae\",\n \"suitability\",\n \"naucoridae\",\n \"habitat suitability\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["association ulcerans aquatic","bugs buruli ulcer","pathogen mycobacterium ulcerans","transmission ulcerans aquatic","distribution ulcerans insects"],"string":"[\n \"association ulcerans aquatic\",\n \"bugs buruli ulcer\",\n \"pathogen mycobacterium ulcerans\",\n \"transmission ulcerans aquatic\",\n \"distribution ulcerans insects\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Ecological niche modelling | Naucoridae | Belostomatidae | Spatial distribution | Habitat suitability | Buruli ulcer | \nMycobacterium ulcerans\n | Vector-borne transmission | Environmentally-acquired disease [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Ecological niche modelling | Naucoridae | Belostomatidae | Spatial distribution | Habitat suitability | Buruli ulcer | \nMycobacterium ulcerans\n | Vector-borne transmission | Environmentally-acquired disease [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Ecological niche modelling | Naucoridae | Belostomatidae | Spatial distribution | Habitat suitability | Buruli ulcer | \nMycobacterium ulcerans\n | Vector-borne transmission | Environmentally-acquired disease [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Buruli Ulcer | Cameroon | Ecosystem | Geographic Mapping | Hemiptera | Humans | Insect Vectors | Mycobacterium ulcerans [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Buruli Ulcer | Cameroon | Ecosystem | Geographic Mapping | Hemiptera | Humans | Insect Vectors | Mycobacterium ulcerans [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Buruli Ulcer | Cameroon | Ecosystem | Geographic Mapping | Hemiptera | Humans | Insect Vectors | Mycobacterium ulcerans [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] association ulcerans aquatic | bugs buruli ulcer | pathogen mycobacterium ulcerans | transmission ulcerans aquatic | distribution ulcerans insects [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] association ulcerans aquatic | bugs buruli ulcer | pathogen mycobacterium ulcerans | transmission ulcerans aquatic | distribution ulcerans insects [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] association ulcerans aquatic | bugs buruli ulcer | pathogen mycobacterium ulcerans | transmission ulcerans aquatic | distribution ulcerans insects [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] season | correlation | buruli | ulcer | buruli ulcer | habitat | belostomatidae | suitability | naucoridae | habitat suitability [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] season | correlation | buruli | ulcer | buruli ulcer | habitat | belostomatidae | suitability | naucoridae | habitat suitability [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] season | correlation | buruli | ulcer | buruli ulcer | habitat | belostomatidae | suitability | naucoridae | habitat suitability [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] transmission | proposed | vector | ulcerans | infection | human | buruli | buruli ulcer | ulcer | vectors [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] season | variable | villages | correlation | suitability | habitat | habitat suitability | village | absent | wet [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] season | buruli | buruli ulcer | ulcer | sites | ag | correlation | village | dataset | prevalence [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Buruli ||| amoebae ||| Naucoridae | Belostomatidae [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Buruli ||| one | Camerounese | Akonolinga | Bankim ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Buruli ||| amoebae ||| Naucoridae | Belostomatidae ||| Cameroon ||| West Africa | Buruli | Bradford ||| Buruli ||| one | Camerounese | Akonolinga | Bankim ||| ||| Mycobacterium ||| ||| M. [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":63,"cells":{"title":{"kind":"string","value":"Feature-specific terrain park-injury rates and risk factors in snowboarders: a case-control study."},"pmid":{"kind":"string","value":"24184587"},"background_abstract":{"kind":"string","value":"Snowboarding is a popular albeit risky sport and terrain park (TP) injuries are more severe than regular slope injuries. TPs contain man-made features that facilitate aerial manoeuvres. The objectives of this study were to determine overall and feature-specific injury rates and the potential risk factors for TP injuries."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Case-control study with exposure estimation, conducted in an Alberta TP during two ski seasons. Cases were snowboarders injured in the TP who presented to ski patrol and/or local emergency departments. Controls were uninjured snowboarders in the same TP. κ Statistics were used to measure the reliability of reported risk factor information. Injury rates were calculated and adjusted logistic regression was used to calculate the feature-specific odds of injury."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Overall, 333 cases and 1261 controls were enrolled. Reliability of risk factor information was κ>0.60 for 21/24 variables. The overall injury rate was 0.75/1000 runs. Rates were highest for jumps and half-pipe (both 2.56/1000 runs) and lowest for rails (0.43/1000 runs) and quarter-pipes (0.24/1000 runs). Compared with rails, there were increased odds of injury for half-pipe (OR 9.63; 95% CI 4.80 to 19.32), jumps (OR 4.29; 95% CI 2.72 to 6.76), mushroom (OR 2.30; 95% CI 1.20 to 4.41) and kickers (OR 1.99; 95% CI 1.27 to 3.12)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Higher feature-specific injury rates and increased odds of injury were associated with features that promote aerial manoeuvres or a large drop to the ground. Further research is required to determine ways to increase snowboarder safety in the TP."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adolescent","Adult","Alberta","Case-Control Studies","Child","Female","Humans","Incidence","Injury Severity Score","Male","Risk Factors","Skiing"],"string":"[\n \"Adolescent\",\n \"Adult\",\n \"Alberta\",\n \"Case-Control Studies\",\n \"Child\",\n \"Female\",\n \"Humans\",\n \"Incidence\",\n \"Injury Severity Score\",\n \"Male\",\n \"Risk Factors\",\n \"Skiing\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"3888610"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Snowboarding is a popular sport1\n2 and the risk of injury is higher for snowboarding than for skiing.3\n4 Being a beginner,5–8 poor weather conditions9 and not wearing protective equipment10–12 increase injury risk. Ski areas often include terrain parks (TPs) with man-made features (eg, jumps, rails and half-pipes) for performing tricks and aerial manoeuvres. In November 2007, Resorts of the Canadian Rockies (RCR) removed all man-made jumps from their TPs because they believed jumps increased injury risk.13 Definitions of common features can be found at: http://www.snowboard-coach.com/freestyle-snowboarding-features.html and in appendix 1.\nBetween 5% and 27% of skiing and snowboarding injuries occur in TPs2\n14–19 and are more severe than regular slope injuries.15\n16\n18 At the 2012 Winter Youth Olympic Games, 35% of all snowboard half-pipe and slope-style competitors were injured.20 Those injured in TPs tend to be snowboarders, male, 13–24 years old, fall from higher heights15 or self-perceived experts.16 There is a dearth of research examining injury rates and intrinsic and extrinsic risk factors for snowboarders in TPs in relation to injury mechanism—a comprehensive approach recommended by sport injury prevention research leaders.21\n22 Therefore, the study objectives were to calculate overall and feature-specific TP injury rates, determine potential risk factors for injury in the TP and assess the reliability of the data collection methods."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Definition of cases and controls This unmatched case-control study was conducted in one Alberta TP during the 2008–2009 and 2009–2010 seasons. There were approximately 290 000 skier–snowboarder visits to the resort annually. Except for the half-pipe and mushroom, the overall TP layout and number of features changed once each season, resulting in four configurations containing all seven feature types. The resort did not assign a difficulty rating to individual features. Helmets were mandatory in the TP.\nCases were snowboarders injured in the TP who presented to either the ski patrol and/or one of two nearby emergency departments (EDs), both Level 1 trauma centres (one adult, one paediatric). Controls were uninjured snowboarders using the same TP. Cases were ‘severe’ if they presented to an ED, and ‘minor’ if they presented to ski patrol only or to ski patrol and a non-emergent healthcare provider. Injuries presenting to the ED represent the public health burden and snowboarders injured in the TPs near hospitals may place a strain on EDs.\nThis unmatched case-control study was conducted in one Alberta TP during the 2008–2009 and 2009–2010 seasons. There were approximately 290 000 skier–snowboarder visits to the resort annually. Except for the half-pipe and mushroom, the overall TP layout and number of features changed once each season, resulting in four configurations containing all seven feature types. The resort did not assign a difficulty rating to individual features. Helmets were mandatory in the TP.\nCases were snowboarders injured in the TP who presented to either the ski patrol and/or one of two nearby emergency departments (EDs), both Level 1 trauma centres (one adult, one paediatric). Controls were uninjured snowboarders using the same TP. Cases were ‘severe’ if they presented to an ED, and ‘minor’ if they presented to ski patrol only or to ski patrol and a non-emergent healthcare provider. Injuries presenting to the ED represent the public health burden and snowboarders injured in the TPs near hospitals may place a strain on EDs.\n Data collection Case data were collected from ski patrol Accident Report Forms (ARF) and ED medical records. ARFs are completed by the ski patrol for anyone injured and presented to them. ARFs record demographics (age, sex and five-point self-reported ability), injured body region, injury type (fracture, dislocation, sprain/strain, bruise/abrasion/laceration and concussion), environment (temperature, light and snow (groomed/ungroomed)) and contact information. ARFs have previously been used in research14\n18\n19\n23–25 and were collected from the resort biweekly. Snowboarders who presented to the ED were identified from the Regional Emergency Department Information System. Following verbal consent (parent/guardian if snowboarder was <14 years) and confirmation they were injured at the TP of interest, additional self-reported data were collected by telephone. The previously listed information was captured for snowboarders presenting only to the ED, along with years of snowboarding and TP experience, listening to music, wearing wrist guards, previous snowboarding injury and feature used (jump, kicker, box, rail, quarter-pipe, half-pipe or mushroom) when injured. If the case presented to the ski patrol and could not be contacted or did not consent to the telephone interview, only the ARF data were included.\nTo collect feature use among controls, trained research assistants (RAs) at the bottom of the hill observed snowboarders' TP runs in 3 h time slots, 3–4 times a week at various times during the day, and recorded feature use on a map. Data were collected each week from 1 January until the resort closed (end of March in Season 1 and mid-April in Season 2) and included all four TP layouts. RAs approached the first snowboarder, obtained consent and asked the same risk factor information as asked to cases. Snowboarders indicated feature use on the map for features not fully visible to the RA. After each interview, the next snowboarder closest to the RA was approached. Temperature (smartphone Weather Network application), light and snow conditions were recorded on an hourly basis.\nInjury rate denominator data were collected in the same 3 h slots. Snowboarder runs were counted by an RA at the top of the TP, which was the only run to the right of the chairlift and only serviced by that chairlift. Age-group (<12, 12–17 or >17) and sex were visually assessed. To determine accuracy of observed age-group and sex classifications, RAs independently estimated the age-group and sex of a snowboarder entering the TP and then approached them to confirm. This was repeated in 10 min intervals for 3 h blocks and included 337 snowboarders.\nEthical approval was granted by the University of Calgary Conjoint Health Research Ethics Board.\nCase data were collected from ski patrol Accident Report Forms (ARF) and ED medical records. ARFs are completed by the ski patrol for anyone injured and presented to them. ARFs record demographics (age, sex and five-point self-reported ability), injured body region, injury type (fracture, dislocation, sprain/strain, bruise/abrasion/laceration and concussion), environment (temperature, light and snow (groomed/ungroomed)) and contact information. ARFs have previously been used in research14\n18\n19\n23–25 and were collected from the resort biweekly. Snowboarders who presented to the ED were identified from the Regional Emergency Department Information System. Following verbal consent (parent/guardian if snowboarder was <14 years) and confirmation they were injured at the TP of interest, additional self-reported data were collected by telephone. The previously listed information was captured for snowboarders presenting only to the ED, along with years of snowboarding and TP experience, listening to music, wearing wrist guards, previous snowboarding injury and feature used (jump, kicker, box, rail, quarter-pipe, half-pipe or mushroom) when injured. If the case presented to the ski patrol and could not be contacted or did not consent to the telephone interview, only the ARF data were included.\nTo collect feature use among controls, trained research assistants (RAs) at the bottom of the hill observed snowboarders' TP runs in 3 h time slots, 3–4 times a week at various times during the day, and recorded feature use on a map. Data were collected each week from 1 January until the resort closed (end of March in Season 1 and mid-April in Season 2) and included all four TP layouts. RAs approached the first snowboarder, obtained consent and asked the same risk factor information as asked to cases. Snowboarders indicated feature use on the map for features not fully visible to the RA. After each interview, the next snowboarder closest to the RA was approached. Temperature (smartphone Weather Network application), light and snow conditions were recorded on an hourly basis.\nInjury rate denominator data were collected in the same 3 h slots. Snowboarder runs were counted by an RA at the top of the TP, which was the only run to the right of the chairlift and only serviced by that chairlift. Age-group (<12, 12–17 or >17) and sex were visually assessed. To determine accuracy of observed age-group and sex classifications, RAs independently estimated the age-group and sex of a snowboarder entering the TP and then approached them to confirm. This was repeated in 10 min intervals for 3 h blocks and included 337 snowboarders.\nEthical approval was granted by the University of Calgary Conjoint Health Research Ethics Board.\n Analysis Reliability Three pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\nThree pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\n Rates Injury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\nInjury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\n Risk factors The distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\nThe distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\n Reliability Three pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\nThree pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\n Rates Injury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\nInjury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\n Risk factors The distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\nThe distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"A total of 333 cases (107 ski patrol, 174 ski patrol and ED, 18 ski patrol and healthcare provider and 34 ED only) and 1261 controls were included. The consent rate was 79% for cases and 94% for controls (figure 1).\nRecruitment of included snowboarders.\n Reliability Based on initial RA observation and follow-up confirmation, the RAs correctly classified age-group in 78% (κ=0.71; 95% CI 0.49 to 0.93) and sex in 100% of uninjured snowboarders (κ=1.00; 95% CI 0.69 to 1.00). There was significant agreement for age and sex classification between the RAs (p>0.05 for each of the three pairs). For interview responses, κ=0.72 (95% CI 0.60 to 0.85) for feature use at the time of injury and this improved when follow-up interviews were conducted within 2 weeks (κ=0.86; 95% CI 0.64 to 1.00). The overall κ were ≥0.60 except for body region of the second and third injuries and diagnosis of the second injury.\nBased on initial RA observation and follow-up confirmation, the RAs correctly classified age-group in 78% (κ=0.71; 95% CI 0.49 to 0.93) and sex in 100% of uninjured snowboarders (κ=1.00; 95% CI 0.69 to 1.00). There was significant agreement for age and sex classification between the RAs (p>0.05 for each of the three pairs). For interview responses, κ=0.72 (95% CI 0.60 to 0.85) for feature use at the time of injury and this improved when follow-up interviews were conducted within 2 weeks (κ=0.86; 95% CI 0.64 to 1.00). The overall κ were ≥0.60 except for body region of the second and third injuries and diagnosis of the second injury.\n Injuries Overall, 62.5% went to the ED. The most commonly injured body regions were the wrist (20%), and head (14%), while the most common injury type was fracture (36%). Table 1 describes feature use among injured and uninjured TP snowboarders.\nFeatures used by all injured, severely injured snowboarders, and uninjured snowboarders in the terrain park\n*Weighted for time of day, weekday versus weekend and exposure opportunity.\nFemales aged 12–17 and >17 had higher rates of injuries than males, and higher rates of severe injuries at all ages (table 2).\nOverall, sex-specific and age-specific injury rates (per 1000 runs and 95% CI)\nFeatures that promoted aerial manoeuvres or resulted in a greater drop to the ground typically had higher injury rates than features with a small drop (table 3).\nTable 3All and severe injury rates (per 1000 feature exposures and 95% CI)FeatureAll injuries* rate (95% CI)Severe injuries* rate (95% CI)Aerial manoeuvre or substantial drop to the ground Half-pipe2.6 (1.5 to 4.0)1.8 (0.9 to 3.0) Jumps2.6 (2.1 to 3.2)1.8 (1.4 to 2.3) Kickers0.6 (0.5 to 0.8)0.4 (0.3 to 0.5) Mushroom0.5 (0.3 to 0.8)0.2 (0.1 to 0.5)Small drop to the ground Boxes0.7 (0.5 to 0.8)0.4 (0.3 to 0.5) Rails0.4 (0.3 to 0.6)0.2 (0.1 to 0.4) Quarter-pipes0.2 (0.1 to 0.4)0.1 (0.1 to 0.3)*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed). Jumps and half-pipe had the highest overall and severe injury rates. Compared with rails, rates of all injuries were significantly higher on the half-pipe, jumps and boxes (table 4).\nAll and severe injury rates (per 1000 feature exposures and 95% CI)\n*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed).\nFeature-specific injury rate ratios and 95% CI\nN, number; RR, rate ratio.\n*Four snowboarders could not recall feature used at time of injury and 13 snowboarders were not using a feature at the time of injury but were in the boundaries of the terrain park (TP) for a total of 333 TP injuries.\n†Two snowboarders could not recall feature used at time of injury and nine snowboarders were not using a feature at the time of injury but were in the boundaries of the TP for a total of 208 severe TP injuries. Bold refers to statistically significant results.\nBaseline characteristics are presented in table 5. The odds of injury were significantly higher when it was −10°C to 0°C compared with 0–10°C, and at night versus sunny weather. Beginners/novices had significantly lower odds of injury than intermediates, as did those with a previous snowboarding injury, music use, or when it was above 10°C versus 0–10°C. When comparing severely injured with uninjured snowboarders, the same patterns were observed except that music or temperatures above 10°C were not significant. There were no significant differences between severe versus minor injuries.\nSummary of the characteristics of all injured and uninjured snowboarders (crude OR and 95% CI)\n*Odds of injury increases for every increase in year.\n†Compared with no injuries.\n‡Compared with minor injuries.\nN, numbers; N/A, not applicable.\nThe crude associations between injury and feature use showed significantly greater odds of injury for jumps and half-pipe, compared with rails and significantly lower odds of injury for quarter-pipes (table 6). For severe injury versus no injury, the crude odds of severe injury were significantly greater on jumps, half-pipe and kickers compared with rails.\nAssociation between injury and feature type after controlling for confounders (OR and 95% CI)\n*Adjusted for previous injury, ability and temperature. Age, sex, listening to music, wearing wrist guards and light and snow conditions did not change any of the feature-specific estimates more than 15%.\n†Adjusted for music and light. Age, sex, ability, wrist guard use and temperature and snow conditions did not change any of the feature-specific estimates more than 15%.\nThere were significant increases in the adjusted odds of injury for half-pipe (OR 9.63; 95% CI 4.80 to 19.32), jumps (OR 4.29; 95% CI 2.72 to 6.76), mushroom (OR 2.30; 95% CI 1.20 to 4.41) and kickers (OR 1.99; 95% CI 1.27 to 3.12) versus rails. The adjusted odds of severe injury versus no injury were significantly higher for half-pipe, jumps and kickers compared with rails. After accounting for clustering, the 95% CI width increased marginally but significance did not change.\nUsing the imputed dataset, there was no evidence of effect modification of feature by age or sex (p=0.41) versus rails, significantly increased adjusted odds of injury on half-pipe (OR 5.88; 95% CI 3.25 to 10.63) and jumps (OR 4.78; 95% CI 3.22 to 7.11) and significantly decreased odds of injury on quarter-pipes (OR 0.49; 95% CI 0.25 to 0.97).\nOverall, 62.5% went to the ED. The most commonly injured body regions were the wrist (20%), and head (14%), while the most common injury type was fracture (36%). Table 1 describes feature use among injured and uninjured TP snowboarders.\nFeatures used by all injured, severely injured snowboarders, and uninjured snowboarders in the terrain park\n*Weighted for time of day, weekday versus weekend and exposure opportunity.\nFemales aged 12–17 and >17 had higher rates of injuries than males, and higher rates of severe injuries at all ages (table 2).\nOverall, sex-specific and age-specific injury rates (per 1000 runs and 95% CI)\nFeatures that promoted aerial manoeuvres or resulted in a greater drop to the ground typically had higher injury rates than features with a small drop (table 3).\nTable 3All and severe injury rates (per 1000 feature exposures and 95% CI)FeatureAll injuries* rate (95% CI)Severe injuries* rate (95% CI)Aerial manoeuvre or substantial drop to the ground Half-pipe2.6 (1.5 to 4.0)1.8 (0.9 to 3.0) Jumps2.6 (2.1 to 3.2)1.8 (1.4 to 2.3) Kickers0.6 (0.5 to 0.8)0.4 (0.3 to 0.5) Mushroom0.5 (0.3 to 0.8)0.2 (0.1 to 0.5)Small drop to the ground Boxes0.7 (0.5 to 0.8)0.4 (0.3 to 0.5) Rails0.4 (0.3 to 0.6)0.2 (0.1 to 0.4) Quarter-pipes0.2 (0.1 to 0.4)0.1 (0.1 to 0.3)*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed). Jumps and half-pipe had the highest overall and severe injury rates. Compared with rails, rates of all injuries were significantly higher on the half-pipe, jumps and boxes (table 4).\nAll and severe injury rates (per 1000 feature exposures and 95% CI)\n*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed).\nFeature-specific injury rate ratios and 95% CI\nN, number; RR, rate ratio.\n*Four snowboarders could not recall feature used at time of injury and 13 snowboarders were not using a feature at the time of injury but were in the boundaries of the terrain park (TP) for a total of 333 TP injuries.\n†Two snowboarders could not recall feature used at time of injury and nine snowboarders were not using a feature at the time of injury but were in the boundaries of the TP for a total of 208 severe TP injuries. Bold refers to statistically significant results.\nBaseline characteristics are presented in table 5. The odds of injury were significantly higher when it was −10°C to 0°C compared with 0–10°C, and at night versus sunny weather. Beginners/novices had significantly lower odds of injury than intermediates, as did those with a previous snowboarding injury, music use, or when it was above 10°C versus 0–10°C. When comparing severely injured with uninjured snowboarders, the same patterns were observed except that music or temperatures above 10°C were not significant. There were no significant differences between severe versus minor injuries.\nSummary of the characteristics of all injured and uninjured snowboarders (crude OR and 95% CI)\n*Odds of injury increases for every increase in year.\n†Compared with no injuries.\n‡Compared with minor injuries.\nN, numbers; N/A, not applicable.\nThe crude associations between injury and feature use showed significantly greater odds of injury for jumps and half-pipe, compared with rails and significantly lower odds of injury for quarter-pipes (table 6). For severe injury versus no injury, the crude odds of severe injury were significantly greater on jumps, half-pipe and kickers compared with rails.\nAssociation between injury and feature type after controlling for confounders (OR and 95% CI)\n*Adjusted for previous injury, ability and temperature. Age, sex, listening to music, wearing wrist guards and light and snow conditions did not change any of the feature-specific estimates more than 15%.\n†Adjusted for music and light. Age, sex, ability, wrist guard use and temperature and snow conditions did not change any of the feature-specific estimates more than 15%.\nThere were significant increases in the adjusted odds of injury for half-pipe (OR 9.63; 95% CI 4.80 to 19.32), jumps (OR 4.29; 95% CI 2.72 to 6.76), mushroom (OR 2.30; 95% CI 1.20 to 4.41) and kickers (OR 1.99; 95% CI 1.27 to 3.12) versus rails. The adjusted odds of severe injury versus no injury were significantly higher for half-pipe, jumps and kickers compared with rails. After accounting for clustering, the 95% CI width increased marginally but significance did not change.\nUsing the imputed dataset, there was no evidence of effect modification of feature by age or sex (p=0.41) versus rails, significantly increased adjusted odds of injury on half-pipe (OR 5.88; 95% CI 3.25 to 10.63) and jumps (OR 4.78; 95% CI 3.22 to 7.11) and significantly decreased odds of injury on quarter-pipes (OR 0.49; 95% CI 0.25 to 0.97)."},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"Feature-specific injury rates ranged from 2.56 injuries/1000 runs (jumps and half-pipe) to 0.24 injuries/1000 runs (quarter-pipe). Half-pipe, jumps and kickers were significant risk factors for any injury and severe injury. Recommendations have been made for prevention strategy development to reduce TP injury risk. These strategies will require rigorous evaluation.\nWhat are the new findings?In this study, the overall injury rate for snowboarding terrain park (TP) injuries is 0.75 injuries/1000 runs.The injury rates are highest for jumps (2.56/1000 runs) and half-pipe (2.56/1000 runs) and lowest for rails (0.43/1000 runs) and quarter-pipes (0.24/1000 runs).Compared with rails, the odds of injury is significantly higher on the half-pipe (OR 9.63; 95% CI 4.80 to 19.32) and jumps (OR 4.29; 95% CI 2.72 to 6.76).\nHow might it impact on clinical practice in the near future?Terrain parks (TPs) are popular and the majority of snowboarders injured in the TP present to the emergency department.With the identification of potential risk factors for TP injuries among snowboarders, injury prevention programmes can be tailored to those at greatest risk of injury.Should these programmes be effective, clinicians can expect to treat fewer snowboarders injured in the TP.\nIn this study, the overall injury rate for snowboarding terrain park (TP) injuries is 0.75 injuries/1000 runs.\nThe injury rates are highest for jumps (2.56/1000 runs) and half-pipe (2.56/1000 runs) and lowest for rails (0.43/1000 runs) and quarter-pipes (0.24/1000 runs).\nCompared with rails, the odds of injury is significantly higher on the half-pipe (OR 9.63; 95% CI 4.80 to 19.32) and jumps (OR 4.29; 95% CI 2.72 to 6.76).\nTerrain parks (TPs) are popular and the majority of snowboarders injured in the TP present to the emergency department.\nWith the identification of potential risk factors for TP injuries among snowboarders, injury prevention programmes can be tailored to those at greatest risk of injury.\nShould these programmes be effective, clinicians can expect to treat fewer snowboarders injured in the TP."},"other_sections_titles":{"kind":"list like","value":["Introduction","Definition of cases and controls","Data collection","Analysis","Reliability","Rates","Risk factors","Reliability","Injuries","Limitations"],"string":"[\n \"Introduction\",\n \"Definition of cases and controls\",\n \"Data collection\",\n \"Analysis\",\n \"Reliability\",\n \"Rates\",\n \"Risk factors\",\n \"Reliability\",\n \"Injuries\",\n \"Limitations\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Snowboarding is a popular sport1\n2 and the risk of injury is higher for snowboarding than for skiing.3\n4 Being a beginner,5–8 poor weather conditions9 and not wearing protective equipment10–12 increase injury risk. Ski areas often include terrain parks (TPs) with man-made features (eg, jumps, rails and half-pipes) for performing tricks and aerial manoeuvres. In November 2007, Resorts of the Canadian Rockies (RCR) removed all man-made jumps from their TPs because they believed jumps increased injury risk.13 Definitions of common features can be found at: http://www.snowboard-coach.com/freestyle-snowboarding-features.html and in appendix 1.\nBetween 5% and 27% of skiing and snowboarding injuries occur in TPs2\n14–19 and are more severe than regular slope injuries.15\n16\n18 At the 2012 Winter Youth Olympic Games, 35% of all snowboard half-pipe and slope-style competitors were injured.20 Those injured in TPs tend to be snowboarders, male, 13–24 years old, fall from higher heights15 or self-perceived experts.16 There is a dearth of research examining injury rates and intrinsic and extrinsic risk factors for snowboarders in TPs in relation to injury mechanism—a comprehensive approach recommended by sport injury prevention research leaders.21\n22 Therefore, the study objectives were to calculate overall and feature-specific TP injury rates, determine potential risk factors for injury in the TP and assess the reliability of the data collection methods.","This unmatched case-control study was conducted in one Alberta TP during the 2008–2009 and 2009–2010 seasons. There were approximately 290 000 skier–snowboarder visits to the resort annually. Except for the half-pipe and mushroom, the overall TP layout and number of features changed once each season, resulting in four configurations containing all seven feature types. The resort did not assign a difficulty rating to individual features. Helmets were mandatory in the TP.\nCases were snowboarders injured in the TP who presented to either the ski patrol and/or one of two nearby emergency departments (EDs), both Level 1 trauma centres (one adult, one paediatric). Controls were uninjured snowboarders using the same TP. Cases were ‘severe’ if they presented to an ED, and ‘minor’ if they presented to ski patrol only or to ski patrol and a non-emergent healthcare provider. Injuries presenting to the ED represent the public health burden and snowboarders injured in the TPs near hospitals may place a strain on EDs.","Case data were collected from ski patrol Accident Report Forms (ARF) and ED medical records. ARFs are completed by the ski patrol for anyone injured and presented to them. ARFs record demographics (age, sex and five-point self-reported ability), injured body region, injury type (fracture, dislocation, sprain/strain, bruise/abrasion/laceration and concussion), environment (temperature, light and snow (groomed/ungroomed)) and contact information. ARFs have previously been used in research14\n18\n19\n23–25 and were collected from the resort biweekly. Snowboarders who presented to the ED were identified from the Regional Emergency Department Information System. Following verbal consent (parent/guardian if snowboarder was <14 years) and confirmation they were injured at the TP of interest, additional self-reported data were collected by telephone. The previously listed information was captured for snowboarders presenting only to the ED, along with years of snowboarding and TP experience, listening to music, wearing wrist guards, previous snowboarding injury and feature used (jump, kicker, box, rail, quarter-pipe, half-pipe or mushroom) when injured. If the case presented to the ski patrol and could not be contacted or did not consent to the telephone interview, only the ARF data were included.\nTo collect feature use among controls, trained research assistants (RAs) at the bottom of the hill observed snowboarders' TP runs in 3 h time slots, 3–4 times a week at various times during the day, and recorded feature use on a map. Data were collected each week from 1 January until the resort closed (end of March in Season 1 and mid-April in Season 2) and included all four TP layouts. RAs approached the first snowboarder, obtained consent and asked the same risk factor information as asked to cases. Snowboarders indicated feature use on the map for features not fully visible to the RA. After each interview, the next snowboarder closest to the RA was approached. Temperature (smartphone Weather Network application), light and snow conditions were recorded on an hourly basis.\nInjury rate denominator data were collected in the same 3 h slots. Snowboarder runs were counted by an RA at the top of the TP, which was the only run to the right of the chairlift and only serviced by that chairlift. Age-group (<12, 12–17 or >17) and sex were visually assessed. To determine accuracy of observed age-group and sex classifications, RAs independently estimated the age-group and sex of a snowboarder entering the TP and then approached them to confirm. This was repeated in 10 min intervals for 3 h blocks and included 337 snowboarders.\nEthical approval was granted by the University of Calgary Conjoint Health Research Ethics Board."," Reliability Three pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\nThree pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\n Rates Injury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\nInjury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\n Risk factors The distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\nThe distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33","Three pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27","Injury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.","The distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33","Based on initial RA observation and follow-up confirmation, the RAs correctly classified age-group in 78% (κ=0.71; 95% CI 0.49 to 0.93) and sex in 100% of uninjured snowboarders (κ=1.00; 95% CI 0.69 to 1.00). There was significant agreement for age and sex classification between the RAs (p>0.05 for each of the three pairs). For interview responses, κ=0.72 (95% CI 0.60 to 0.85) for feature use at the time of injury and this improved when follow-up interviews were conducted within 2 weeks (κ=0.86; 95% CI 0.64 to 1.00). The overall κ were ≥0.60 except for body region of the second and third injuries and diagnosis of the second injury.","Overall, 62.5% went to the ED. The most commonly injured body regions were the wrist (20%), and head (14%), while the most common injury type was fracture (36%). Table 1 describes feature use among injured and uninjured TP snowboarders.\nFeatures used by all injured, severely injured snowboarders, and uninjured snowboarders in the terrain park\n*Weighted for time of day, weekday versus weekend and exposure opportunity.\nFemales aged 12–17 and >17 had higher rates of injuries than males, and higher rates of severe injuries at all ages (table 2).\nOverall, sex-specific and age-specific injury rates (per 1000 runs and 95% CI)\nFeatures that promoted aerial manoeuvres or resulted in a greater drop to the ground typically had higher injury rates than features with a small drop (table 3).\nTable 3All and severe injury rates (per 1000 feature exposures and 95% CI)FeatureAll injuries* rate (95% CI)Severe injuries* rate (95% CI)Aerial manoeuvre or substantial drop to the ground Half-pipe2.6 (1.5 to 4.0)1.8 (0.9 to 3.0) Jumps2.6 (2.1 to 3.2)1.8 (1.4 to 2.3) Kickers0.6 (0.5 to 0.8)0.4 (0.3 to 0.5) Mushroom0.5 (0.3 to 0.8)0.2 (0.1 to 0.5)Small drop to the ground Boxes0.7 (0.5 to 0.8)0.4 (0.3 to 0.5) Rails0.4 (0.3 to 0.6)0.2 (0.1 to 0.4) Quarter-pipes0.2 (0.1 to 0.4)0.1 (0.1 to 0.3)*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed). Jumps and half-pipe had the highest overall and severe injury rates. Compared with rails, rates of all injuries were significantly higher on the half-pipe, jumps and boxes (table 4).\nAll and severe injury rates (per 1000 feature exposures and 95% CI)\n*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed).\nFeature-specific injury rate ratios and 95% CI\nN, number; RR, rate ratio.\n*Four snowboarders could not recall feature used at time of injury and 13 snowboarders were not using a feature at the time of injury but were in the boundaries of the terrain park (TP) for a total of 333 TP injuries.\n†Two snowboarders could not recall feature used at time of injury and nine snowboarders were not using a feature at the time of injury but were in the boundaries of the TP for a total of 208 severe TP injuries. Bold refers to statistically significant results.\nBaseline characteristics are presented in table 5. The odds of injury were significantly higher when it was −10°C to 0°C compared with 0–10°C, and at night versus sunny weather. Beginners/novices had significantly lower odds of injury than intermediates, as did those with a previous snowboarding injury, music use, or when it was above 10°C versus 0–10°C. When comparing severely injured with uninjured snowboarders, the same patterns were observed except that music or temperatures above 10°C were not significant. There were no significant differences between severe versus minor injuries.\nSummary of the characteristics of all injured and uninjured snowboarders (crude OR and 95% CI)\n*Odds of injury increases for every increase in year.\n†Compared with no injuries.\n‡Compared with minor injuries.\nN, numbers; N/A, not applicable.\nThe crude associations between injury and feature use showed significantly greater odds of injury for jumps and half-pipe, compared with rails and significantly lower odds of injury for quarter-pipes (table 6). For severe injury versus no injury, the crude odds of severe injury were significantly greater on jumps, half-pipe and kickers compared with rails.\nAssociation between injury and feature type after controlling for confounders (OR and 95% CI)\n*Adjusted for previous injury, ability and temperature. Age, sex, listening to music, wearing wrist guards and light and snow conditions did not change any of the feature-specific estimates more than 15%.\n†Adjusted for music and light. Age, sex, ability, wrist guard use and temperature and snow conditions did not change any of the feature-specific estimates more than 15%.\nThere were significant increases in the adjusted odds of injury for half-pipe (OR 9.63; 95% CI 4.80 to 19.32), jumps (OR 4.29; 95% CI 2.72 to 6.76), mushroom (OR 2.30; 95% CI 1.20 to 4.41) and kickers (OR 1.99; 95% CI 1.27 to 3.12) versus rails. The adjusted odds of severe injury versus no injury were significantly higher for half-pipe, jumps and kickers compared with rails. After accounting for clustering, the 95% CI width increased marginally but significance did not change.\nUsing the imputed dataset, there was no evidence of effect modification of feature by age or sex (p=0.41) versus rails, significantly increased adjusted odds of injury on half-pipe (OR 5.88; 95% CI 3.25 to 10.63) and jumps (OR 4.78; 95% CI 3.22 to 7.11) and significantly decreased odds of injury on quarter-pipes (OR 0.49; 95% CI 0.25 to 0.97).","Snowboarders who climbed up to reattempt features were counted as one run, which would underestimate the number of TP runs. Injured snowboarders who did not present to the ski patrol or either of the two nearest EDs were missed. This selection bias resulted in rate underestimation and observed associations between feature and injury would be overestimated if missed snowboarders were injured on rails. However, only 47 (14%) of the included snowboarders said they saw the ski patrol and a non-participating healthcare provider, indicating that most sought treatment at a participating ED.\nThere was potential misclassification by feature if the injured snowboarder could not recall the feature; however, this occurred only four times. Controls may not have correctly reported their feature use when the RA could not see the entire TP and it was not possible to determine the reliability of controls’ feature use. This could have an unpredictable effect on the ORs if it operated differently by feature type. Only age-group and sex of the uninjured controls could be assessed for accuracy and our observations were found to be valid. There was potential for misclassification of severe injury because factors other than severity could predict presentation to the ED, such as parental fear or anxiety influenced by a recent celebrity skiing death.39 It was unknown if non-consenting injured snowboarders presented to a non-study ED and were incorrectly classified as a minor injury. Fortunately, few who saw the ski patrol went to a non-study ED (8%).\nIt is possible that some important confounders were overlooked, such as first attempt at a new feature, manoeuvre performed, speed, fatigue, height or weight. There may be behavioural confounders, such as peer pressure to attempt difficult features or manoeuvres. However, some of these potential confounders were likely accounted for by other variables such as ability, age and sex. Although this study was conducted at only one resort, the TP layout changed four times during the two seasons and this enhances the generalisability of the results."],"string":"[\n \"Snowboarding is a popular sport1\\n2 and the risk of injury is higher for snowboarding than for skiing.3\\n4 Being a beginner,5–8 poor weather conditions9 and not wearing protective equipment10–12 increase injury risk. Ski areas often include terrain parks (TPs) with man-made features (eg, jumps, rails and half-pipes) for performing tricks and aerial manoeuvres. In November 2007, Resorts of the Canadian Rockies (RCR) removed all man-made jumps from their TPs because they believed jumps increased injury risk.13 Definitions of common features can be found at: http://www.snowboard-coach.com/freestyle-snowboarding-features.html and in appendix 1.\\nBetween 5% and 27% of skiing and snowboarding injuries occur in TPs2\\n14–19 and are more severe than regular slope injuries.15\\n16\\n18 At the 2012 Winter Youth Olympic Games, 35% of all snowboard half-pipe and slope-style competitors were injured.20 Those injured in TPs tend to be snowboarders, male, 13–24 years old, fall from higher heights15 or self-perceived experts.16 There is a dearth of research examining injury rates and intrinsic and extrinsic risk factors for snowboarders in TPs in relation to injury mechanism—a comprehensive approach recommended by sport injury prevention research leaders.21\\n22 Therefore, the study objectives were to calculate overall and feature-specific TP injury rates, determine potential risk factors for injury in the TP and assess the reliability of the data collection methods.\",\n \"This unmatched case-control study was conducted in one Alberta TP during the 2008–2009 and 2009–2010 seasons. There were approximately 290 000 skier–snowboarder visits to the resort annually. Except for the half-pipe and mushroom, the overall TP layout and number of features changed once each season, resulting in four configurations containing all seven feature types. The resort did not assign a difficulty rating to individual features. Helmets were mandatory in the TP.\\nCases were snowboarders injured in the TP who presented to either the ski patrol and/or one of two nearby emergency departments (EDs), both Level 1 trauma centres (one adult, one paediatric). Controls were uninjured snowboarders using the same TP. Cases were ‘severe’ if they presented to an ED, and ‘minor’ if they presented to ski patrol only or to ski patrol and a non-emergent healthcare provider. Injuries presenting to the ED represent the public health burden and snowboarders injured in the TPs near hospitals may place a strain on EDs.\",\n \"Case data were collected from ski patrol Accident Report Forms (ARF) and ED medical records. ARFs are completed by the ski patrol for anyone injured and presented to them. ARFs record demographics (age, sex and five-point self-reported ability), injured body region, injury type (fracture, dislocation, sprain/strain, bruise/abrasion/laceration and concussion), environment (temperature, light and snow (groomed/ungroomed)) and contact information. ARFs have previously been used in research14\\n18\\n19\\n23–25 and were collected from the resort biweekly. Snowboarders who presented to the ED were identified from the Regional Emergency Department Information System. Following verbal consent (parent/guardian if snowboarder was <14 years) and confirmation they were injured at the TP of interest, additional self-reported data were collected by telephone. The previously listed information was captured for snowboarders presenting only to the ED, along with years of snowboarding and TP experience, listening to music, wearing wrist guards, previous snowboarding injury and feature used (jump, kicker, box, rail, quarter-pipe, half-pipe or mushroom) when injured. If the case presented to the ski patrol and could not be contacted or did not consent to the telephone interview, only the ARF data were included.\\nTo collect feature use among controls, trained research assistants (RAs) at the bottom of the hill observed snowboarders' TP runs in 3 h time slots, 3–4 times a week at various times during the day, and recorded feature use on a map. Data were collected each week from 1 January until the resort closed (end of March in Season 1 and mid-April in Season 2) and included all four TP layouts. RAs approached the first snowboarder, obtained consent and asked the same risk factor information as asked to cases. Snowboarders indicated feature use on the map for features not fully visible to the RA. After each interview, the next snowboarder closest to the RA was approached. Temperature (smartphone Weather Network application), light and snow conditions were recorded on an hourly basis.\\nInjury rate denominator data were collected in the same 3 h slots. Snowboarder runs were counted by an RA at the top of the TP, which was the only run to the right of the chairlift and only serviced by that chairlift. Age-group (<12, 12–17 or >17) and sex were visually assessed. To determine accuracy of observed age-group and sex classifications, RAs independently estimated the age-group and sex of a snowboarder entering the TP and then approached them to confirm. This was repeated in 10 min intervals for 3 h blocks and included 337 snowboarders.\\nEthical approval was granted by the University of Calgary Conjoint Health Research Ethics Board.\",\n \" Reliability Three pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\\nThree pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\\n Rates Injury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\\nInjury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\\n Risk factors The distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\\nThe distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\",\n \"Three pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\",\n \"Injury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\",\n \"The distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\",\n \"Based on initial RA observation and follow-up confirmation, the RAs correctly classified age-group in 78% (κ=0.71; 95% CI 0.49 to 0.93) and sex in 100% of uninjured snowboarders (κ=1.00; 95% CI 0.69 to 1.00). There was significant agreement for age and sex classification between the RAs (p>0.05 for each of the three pairs). For interview responses, κ=0.72 (95% CI 0.60 to 0.85) for feature use at the time of injury and this improved when follow-up interviews were conducted within 2 weeks (κ=0.86; 95% CI 0.64 to 1.00). The overall κ were ≥0.60 except for body region of the second and third injuries and diagnosis of the second injury.\",\n \"Overall, 62.5% went to the ED. The most commonly injured body regions were the wrist (20%), and head (14%), while the most common injury type was fracture (36%). Table 1 describes feature use among injured and uninjured TP snowboarders.\\nFeatures used by all injured, severely injured snowboarders, and uninjured snowboarders in the terrain park\\n*Weighted for time of day, weekday versus weekend and exposure opportunity.\\nFemales aged 12–17 and >17 had higher rates of injuries than males, and higher rates of severe injuries at all ages (table 2).\\nOverall, sex-specific and age-specific injury rates (per 1000 runs and 95% CI)\\nFeatures that promoted aerial manoeuvres or resulted in a greater drop to the ground typically had higher injury rates than features with a small drop (table 3).\\nTable 3All and severe injury rates (per 1000 feature exposures and 95% CI)FeatureAll injuries* rate (95% CI)Severe injuries* rate (95% CI)Aerial manoeuvre or substantial drop to the ground Half-pipe2.6 (1.5 to 4.0)1.8 (0.9 to 3.0) Jumps2.6 (2.1 to 3.2)1.8 (1.4 to 2.3) Kickers0.6 (0.5 to 0.8)0.4 (0.3 to 0.5) Mushroom0.5 (0.3 to 0.8)0.2 (0.1 to 0.5)Small drop to the ground Boxes0.7 (0.5 to 0.8)0.4 (0.3 to 0.5) Rails0.4 (0.3 to 0.6)0.2 (0.1 to 0.4) Quarter-pipes0.2 (0.1 to 0.4)0.1 (0.1 to 0.3)*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed). Jumps and half-pipe had the highest overall and severe injury rates. Compared with rails, rates of all injuries were significantly higher on the half-pipe, jumps and boxes (table 4).\\nAll and severe injury rates (per 1000 feature exposures and 95% CI)\\n*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed).\\nFeature-specific injury rate ratios and 95% CI\\nN, number; RR, rate ratio.\\n*Four snowboarders could not recall feature used at time of injury and 13 snowboarders were not using a feature at the time of injury but were in the boundaries of the terrain park (TP) for a total of 333 TP injuries.\\n†Two snowboarders could not recall feature used at time of injury and nine snowboarders were not using a feature at the time of injury but were in the boundaries of the TP for a total of 208 severe TP injuries. Bold refers to statistically significant results.\\nBaseline characteristics are presented in table 5. The odds of injury were significantly higher when it was −10°C to 0°C compared with 0–10°C, and at night versus sunny weather. Beginners/novices had significantly lower odds of injury than intermediates, as did those with a previous snowboarding injury, music use, or when it was above 10°C versus 0–10°C. When comparing severely injured with uninjured snowboarders, the same patterns were observed except that music or temperatures above 10°C were not significant. There were no significant differences between severe versus minor injuries.\\nSummary of the characteristics of all injured and uninjured snowboarders (crude OR and 95% CI)\\n*Odds of injury increases for every increase in year.\\n†Compared with no injuries.\\n‡Compared with minor injuries.\\nN, numbers; N/A, not applicable.\\nThe crude associations between injury and feature use showed significantly greater odds of injury for jumps and half-pipe, compared with rails and significantly lower odds of injury for quarter-pipes (table 6). For severe injury versus no injury, the crude odds of severe injury were significantly greater on jumps, half-pipe and kickers compared with rails.\\nAssociation between injury and feature type after controlling for confounders (OR and 95% CI)\\n*Adjusted for previous injury, ability and temperature. Age, sex, listening to music, wearing wrist guards and light and snow conditions did not change any of the feature-specific estimates more than 15%.\\n†Adjusted for music and light. Age, sex, ability, wrist guard use and temperature and snow conditions did not change any of the feature-specific estimates more than 15%.\\nThere were significant increases in the adjusted odds of injury for half-pipe (OR 9.63; 95% CI 4.80 to 19.32), jumps (OR 4.29; 95% CI 2.72 to 6.76), mushroom (OR 2.30; 95% CI 1.20 to 4.41) and kickers (OR 1.99; 95% CI 1.27 to 3.12) versus rails. The adjusted odds of severe injury versus no injury were significantly higher for half-pipe, jumps and kickers compared with rails. After accounting for clustering, the 95% CI width increased marginally but significance did not change.\\nUsing the imputed dataset, there was no evidence of effect modification of feature by age or sex (p=0.41) versus rails, significantly increased adjusted odds of injury on half-pipe (OR 5.88; 95% CI 3.25 to 10.63) and jumps (OR 4.78; 95% CI 3.22 to 7.11) and significantly decreased odds of injury on quarter-pipes (OR 0.49; 95% CI 0.25 to 0.97).\",\n \"Snowboarders who climbed up to reattempt features were counted as one run, which would underestimate the number of TP runs. Injured snowboarders who did not present to the ski patrol or either of the two nearest EDs were missed. This selection bias resulted in rate underestimation and observed associations between feature and injury would be overestimated if missed snowboarders were injured on rails. However, only 47 (14%) of the included snowboarders said they saw the ski patrol and a non-participating healthcare provider, indicating that most sought treatment at a participating ED.\\nThere was potential misclassification by feature if the injured snowboarder could not recall the feature; however, this occurred only four times. Controls may not have correctly reported their feature use when the RA could not see the entire TP and it was not possible to determine the reliability of controls’ feature use. This could have an unpredictable effect on the ORs if it operated differently by feature type. Only age-group and sex of the uninjured controls could be assessed for accuracy and our observations were found to be valid. There was potential for misclassification of severe injury because factors other than severity could predict presentation to the ED, such as parental fear or anxiety influenced by a recent celebrity skiing death.39 It was unknown if non-consenting injured snowboarders presented to a non-study ED and were incorrectly classified as a minor injury. Fortunately, few who saw the ski patrol went to a non-study ED (8%).\\nIt is possible that some important confounders were overlooked, such as first attempt at a new feature, manoeuvre performed, speed, fatigue, height or weight. There may be behavioural confounders, such as peer pressure to attempt difficult features or manoeuvres. However, some of these potential confounders were likely accounted for by other variables such as ability, age and sex. Although this study was conducted at only one resort, the TP layout changed four times during the two seasons and this enhances the generalisability of the results.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Introduction","Methods","Definition of cases and controls","Data collection","Analysis","Reliability","Rates","Risk factors","Results","Reliability","Injuries","Discussion","Limitations","Conclusion","Supplementary Material"],"string":"[\n \"Introduction\",\n \"Methods\",\n \"Definition of cases and controls\",\n \"Data collection\",\n \"Analysis\",\n \"Reliability\",\n \"Rates\",\n \"Risk factors\",\n \"Results\",\n \"Reliability\",\n \"Injuries\",\n \"Discussion\",\n \"Limitations\",\n \"Conclusion\",\n \"Supplementary Material\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Snowboarding is a popular sport1\n2 and the risk of injury is higher for snowboarding than for skiing.3\n4 Being a beginner,5–8 poor weather conditions9 and not wearing protective equipment10–12 increase injury risk. Ski areas often include terrain parks (TPs) with man-made features (eg, jumps, rails and half-pipes) for performing tricks and aerial manoeuvres. In November 2007, Resorts of the Canadian Rockies (RCR) removed all man-made jumps from their TPs because they believed jumps increased injury risk.13 Definitions of common features can be found at: http://www.snowboard-coach.com/freestyle-snowboarding-features.html and in appendix 1.\nBetween 5% and 27% of skiing and snowboarding injuries occur in TPs2\n14–19 and are more severe than regular slope injuries.15\n16\n18 At the 2012 Winter Youth Olympic Games, 35% of all snowboard half-pipe and slope-style competitors were injured.20 Those injured in TPs tend to be snowboarders, male, 13–24 years old, fall from higher heights15 or self-perceived experts.16 There is a dearth of research examining injury rates and intrinsic and extrinsic risk factors for snowboarders in TPs in relation to injury mechanism—a comprehensive approach recommended by sport injury prevention research leaders.21\n22 Therefore, the study objectives were to calculate overall and feature-specific TP injury rates, determine potential risk factors for injury in the TP and assess the reliability of the data collection methods."," Definition of cases and controls This unmatched case-control study was conducted in one Alberta TP during the 2008–2009 and 2009–2010 seasons. There were approximately 290 000 skier–snowboarder visits to the resort annually. Except for the half-pipe and mushroom, the overall TP layout and number of features changed once each season, resulting in four configurations containing all seven feature types. The resort did not assign a difficulty rating to individual features. Helmets were mandatory in the TP.\nCases were snowboarders injured in the TP who presented to either the ski patrol and/or one of two nearby emergency departments (EDs), both Level 1 trauma centres (one adult, one paediatric). Controls were uninjured snowboarders using the same TP. Cases were ‘severe’ if they presented to an ED, and ‘minor’ if they presented to ski patrol only or to ski patrol and a non-emergent healthcare provider. Injuries presenting to the ED represent the public health burden and snowboarders injured in the TPs near hospitals may place a strain on EDs.\nThis unmatched case-control study was conducted in one Alberta TP during the 2008–2009 and 2009–2010 seasons. There were approximately 290 000 skier–snowboarder visits to the resort annually. Except for the half-pipe and mushroom, the overall TP layout and number of features changed once each season, resulting in four configurations containing all seven feature types. The resort did not assign a difficulty rating to individual features. Helmets were mandatory in the TP.\nCases were snowboarders injured in the TP who presented to either the ski patrol and/or one of two nearby emergency departments (EDs), both Level 1 trauma centres (one adult, one paediatric). Controls were uninjured snowboarders using the same TP. Cases were ‘severe’ if they presented to an ED, and ‘minor’ if they presented to ski patrol only or to ski patrol and a non-emergent healthcare provider. Injuries presenting to the ED represent the public health burden and snowboarders injured in the TPs near hospitals may place a strain on EDs.\n Data collection Case data were collected from ski patrol Accident Report Forms (ARF) and ED medical records. ARFs are completed by the ski patrol for anyone injured and presented to them. ARFs record demographics (age, sex and five-point self-reported ability), injured body region, injury type (fracture, dislocation, sprain/strain, bruise/abrasion/laceration and concussion), environment (temperature, light and snow (groomed/ungroomed)) and contact information. ARFs have previously been used in research14\n18\n19\n23–25 and were collected from the resort biweekly. Snowboarders who presented to the ED were identified from the Regional Emergency Department Information System. Following verbal consent (parent/guardian if snowboarder was <14 years) and confirmation they were injured at the TP of interest, additional self-reported data were collected by telephone. The previously listed information was captured for snowboarders presenting only to the ED, along with years of snowboarding and TP experience, listening to music, wearing wrist guards, previous snowboarding injury and feature used (jump, kicker, box, rail, quarter-pipe, half-pipe or mushroom) when injured. If the case presented to the ski patrol and could not be contacted or did not consent to the telephone interview, only the ARF data were included.\nTo collect feature use among controls, trained research assistants (RAs) at the bottom of the hill observed snowboarders' TP runs in 3 h time slots, 3–4 times a week at various times during the day, and recorded feature use on a map. Data were collected each week from 1 January until the resort closed (end of March in Season 1 and mid-April in Season 2) and included all four TP layouts. RAs approached the first snowboarder, obtained consent and asked the same risk factor information as asked to cases. Snowboarders indicated feature use on the map for features not fully visible to the RA. After each interview, the next snowboarder closest to the RA was approached. Temperature (smartphone Weather Network application), light and snow conditions were recorded on an hourly basis.\nInjury rate denominator data were collected in the same 3 h slots. Snowboarder runs were counted by an RA at the top of the TP, which was the only run to the right of the chairlift and only serviced by that chairlift. Age-group (<12, 12–17 or >17) and sex were visually assessed. To determine accuracy of observed age-group and sex classifications, RAs independently estimated the age-group and sex of a snowboarder entering the TP and then approached them to confirm. This was repeated in 10 min intervals for 3 h blocks and included 337 snowboarders.\nEthical approval was granted by the University of Calgary Conjoint Health Research Ethics Board.\nCase data were collected from ski patrol Accident Report Forms (ARF) and ED medical records. ARFs are completed by the ski patrol for anyone injured and presented to them. ARFs record demographics (age, sex and five-point self-reported ability), injured body region, injury type (fracture, dislocation, sprain/strain, bruise/abrasion/laceration and concussion), environment (temperature, light and snow (groomed/ungroomed)) and contact information. ARFs have previously been used in research14\n18\n19\n23–25 and were collected from the resort biweekly. Snowboarders who presented to the ED were identified from the Regional Emergency Department Information System. Following verbal consent (parent/guardian if snowboarder was <14 years) and confirmation they were injured at the TP of interest, additional self-reported data were collected by telephone. The previously listed information was captured for snowboarders presenting only to the ED, along with years of snowboarding and TP experience, listening to music, wearing wrist guards, previous snowboarding injury and feature used (jump, kicker, box, rail, quarter-pipe, half-pipe or mushroom) when injured. If the case presented to the ski patrol and could not be contacted or did not consent to the telephone interview, only the ARF data were included.\nTo collect feature use among controls, trained research assistants (RAs) at the bottom of the hill observed snowboarders' TP runs in 3 h time slots, 3–4 times a week at various times during the day, and recorded feature use on a map. Data were collected each week from 1 January until the resort closed (end of March in Season 1 and mid-April in Season 2) and included all four TP layouts. RAs approached the first snowboarder, obtained consent and asked the same risk factor information as asked to cases. Snowboarders indicated feature use on the map for features not fully visible to the RA. After each interview, the next snowboarder closest to the RA was approached. Temperature (smartphone Weather Network application), light and snow conditions were recorded on an hourly basis.\nInjury rate denominator data were collected in the same 3 h slots. Snowboarder runs were counted by an RA at the top of the TP, which was the only run to the right of the chairlift and only serviced by that chairlift. Age-group (<12, 12–17 or >17) and sex were visually assessed. To determine accuracy of observed age-group and sex classifications, RAs independently estimated the age-group and sex of a snowboarder entering the TP and then approached them to confirm. This was repeated in 10 min intervals for 3 h blocks and included 337 snowboarders.\nEthical approval was granted by the University of Calgary Conjoint Health Research Ethics Board.\n Analysis Reliability Three pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\nThree pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\n Rates Injury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\nInjury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\n Risk factors The distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\nThe distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\n Reliability Three pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\nThree pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\n Rates Injury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\nInjury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\n Risk factors The distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\nThe distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33","This unmatched case-control study was conducted in one Alberta TP during the 2008–2009 and 2009–2010 seasons. There were approximately 290 000 skier–snowboarder visits to the resort annually. Except for the half-pipe and mushroom, the overall TP layout and number of features changed once each season, resulting in four configurations containing all seven feature types. The resort did not assign a difficulty rating to individual features. Helmets were mandatory in the TP.\nCases were snowboarders injured in the TP who presented to either the ski patrol and/or one of two nearby emergency departments (EDs), both Level 1 trauma centres (one adult, one paediatric). Controls were uninjured snowboarders using the same TP. Cases were ‘severe’ if they presented to an ED, and ‘minor’ if they presented to ski patrol only or to ski patrol and a non-emergent healthcare provider. Injuries presenting to the ED represent the public health burden and snowboarders injured in the TPs near hospitals may place a strain on EDs.","Case data were collected from ski patrol Accident Report Forms (ARF) and ED medical records. ARFs are completed by the ski patrol for anyone injured and presented to them. ARFs record demographics (age, sex and five-point self-reported ability), injured body region, injury type (fracture, dislocation, sprain/strain, bruise/abrasion/laceration and concussion), environment (temperature, light and snow (groomed/ungroomed)) and contact information. ARFs have previously been used in research14\n18\n19\n23–25 and were collected from the resort biweekly. Snowboarders who presented to the ED were identified from the Regional Emergency Department Information System. Following verbal consent (parent/guardian if snowboarder was <14 years) and confirmation they were injured at the TP of interest, additional self-reported data were collected by telephone. The previously listed information was captured for snowboarders presenting only to the ED, along with years of snowboarding and TP experience, listening to music, wearing wrist guards, previous snowboarding injury and feature used (jump, kicker, box, rail, quarter-pipe, half-pipe or mushroom) when injured. If the case presented to the ski patrol and could not be contacted or did not consent to the telephone interview, only the ARF data were included.\nTo collect feature use among controls, trained research assistants (RAs) at the bottom of the hill observed snowboarders' TP runs in 3 h time slots, 3–4 times a week at various times during the day, and recorded feature use on a map. Data were collected each week from 1 January until the resort closed (end of March in Season 1 and mid-April in Season 2) and included all four TP layouts. RAs approached the first snowboarder, obtained consent and asked the same risk factor information as asked to cases. Snowboarders indicated feature use on the map for features not fully visible to the RA. After each interview, the next snowboarder closest to the RA was approached. Temperature (smartphone Weather Network application), light and snow conditions were recorded on an hourly basis.\nInjury rate denominator data were collected in the same 3 h slots. Snowboarder runs were counted by an RA at the top of the TP, which was the only run to the right of the chairlift and only serviced by that chairlift. Age-group (<12, 12–17 or >17) and sex were visually assessed. To determine accuracy of observed age-group and sex classifications, RAs independently estimated the age-group and sex of a snowboarder entering the TP and then approached them to confirm. This was repeated in 10 min intervals for 3 h blocks and included 337 snowboarders.\nEthical approval was granted by the University of Calgary Conjoint Health Research Ethics Board."," Reliability Three pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\nThree pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\n Rates Injury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\nInjury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\n Risk factors The distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\nThe distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33","Three pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27","Injury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.","The distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33","A total of 333 cases (107 ski patrol, 174 ski patrol and ED, 18 ski patrol and healthcare provider and 34 ED only) and 1261 controls were included. The consent rate was 79% for cases and 94% for controls (figure 1).\nRecruitment of included snowboarders.\n Reliability Based on initial RA observation and follow-up confirmation, the RAs correctly classified age-group in 78% (κ=0.71; 95% CI 0.49 to 0.93) and sex in 100% of uninjured snowboarders (κ=1.00; 95% CI 0.69 to 1.00). There was significant agreement for age and sex classification between the RAs (p>0.05 for each of the three pairs). For interview responses, κ=0.72 (95% CI 0.60 to 0.85) for feature use at the time of injury and this improved when follow-up interviews were conducted within 2 weeks (κ=0.86; 95% CI 0.64 to 1.00). The overall κ were ≥0.60 except for body region of the second and third injuries and diagnosis of the second injury.\nBased on initial RA observation and follow-up confirmation, the RAs correctly classified age-group in 78% (κ=0.71; 95% CI 0.49 to 0.93) and sex in 100% of uninjured snowboarders (κ=1.00; 95% CI 0.69 to 1.00). There was significant agreement for age and sex classification between the RAs (p>0.05 for each of the three pairs). For interview responses, κ=0.72 (95% CI 0.60 to 0.85) for feature use at the time of injury and this improved when follow-up interviews were conducted within 2 weeks (κ=0.86; 95% CI 0.64 to 1.00). The overall κ were ≥0.60 except for body region of the second and third injuries and diagnosis of the second injury.\n Injuries Overall, 62.5% went to the ED. The most commonly injured body regions were the wrist (20%), and head (14%), while the most common injury type was fracture (36%). Table 1 describes feature use among injured and uninjured TP snowboarders.\nFeatures used by all injured, severely injured snowboarders, and uninjured snowboarders in the terrain park\n*Weighted for time of day, weekday versus weekend and exposure opportunity.\nFemales aged 12–17 and >17 had higher rates of injuries than males, and higher rates of severe injuries at all ages (table 2).\nOverall, sex-specific and age-specific injury rates (per 1000 runs and 95% CI)\nFeatures that promoted aerial manoeuvres or resulted in a greater drop to the ground typically had higher injury rates than features with a small drop (table 3).\nTable 3All and severe injury rates (per 1000 feature exposures and 95% CI)FeatureAll injuries* rate (95% CI)Severe injuries* rate (95% CI)Aerial manoeuvre or substantial drop to the ground Half-pipe2.6 (1.5 to 4.0)1.8 (0.9 to 3.0) Jumps2.6 (2.1 to 3.2)1.8 (1.4 to 2.3) Kickers0.6 (0.5 to 0.8)0.4 (0.3 to 0.5) Mushroom0.5 (0.3 to 0.8)0.2 (0.1 to 0.5)Small drop to the ground Boxes0.7 (0.5 to 0.8)0.4 (0.3 to 0.5) Rails0.4 (0.3 to 0.6)0.2 (0.1 to 0.4) Quarter-pipes0.2 (0.1 to 0.4)0.1 (0.1 to 0.3)*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed). Jumps and half-pipe had the highest overall and severe injury rates. Compared with rails, rates of all injuries were significantly higher on the half-pipe, jumps and boxes (table 4).\nAll and severe injury rates (per 1000 feature exposures and 95% CI)\n*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed).\nFeature-specific injury rate ratios and 95% CI\nN, number; RR, rate ratio.\n*Four snowboarders could not recall feature used at time of injury and 13 snowboarders were not using a feature at the time of injury but were in the boundaries of the terrain park (TP) for a total of 333 TP injuries.\n†Two snowboarders could not recall feature used at time of injury and nine snowboarders were not using a feature at the time of injury but were in the boundaries of the TP for a total of 208 severe TP injuries. Bold refers to statistically significant results.\nBaseline characteristics are presented in table 5. The odds of injury were significantly higher when it was −10°C to 0°C compared with 0–10°C, and at night versus sunny weather. Beginners/novices had significantly lower odds of injury than intermediates, as did those with a previous snowboarding injury, music use, or when it was above 10°C versus 0–10°C. When comparing severely injured with uninjured snowboarders, the same patterns were observed except that music or temperatures above 10°C were not significant. There were no significant differences between severe versus minor injuries.\nSummary of the characteristics of all injured and uninjured snowboarders (crude OR and 95% CI)\n*Odds of injury increases for every increase in year.\n†Compared with no injuries.\n‡Compared with minor injuries.\nN, numbers; N/A, not applicable.\nThe crude associations between injury and feature use showed significantly greater odds of injury for jumps and half-pipe, compared with rails and significantly lower odds of injury for quarter-pipes (table 6). For severe injury versus no injury, the crude odds of severe injury were significantly greater on jumps, half-pipe and kickers compared with rails.\nAssociation between injury and feature type after controlling for confounders (OR and 95% CI)\n*Adjusted for previous injury, ability and temperature. Age, sex, listening to music, wearing wrist guards and light and snow conditions did not change any of the feature-specific estimates more than 15%.\n†Adjusted for music and light. Age, sex, ability, wrist guard use and temperature and snow conditions did not change any of the feature-specific estimates more than 15%.\nThere were significant increases in the adjusted odds of injury for half-pipe (OR 9.63; 95% CI 4.80 to 19.32), jumps (OR 4.29; 95% CI 2.72 to 6.76), mushroom (OR 2.30; 95% CI 1.20 to 4.41) and kickers (OR 1.99; 95% CI 1.27 to 3.12) versus rails. The adjusted odds of severe injury versus no injury were significantly higher for half-pipe, jumps and kickers compared with rails. After accounting for clustering, the 95% CI width increased marginally but significance did not change.\nUsing the imputed dataset, there was no evidence of effect modification of feature by age or sex (p=0.41) versus rails, significantly increased adjusted odds of injury on half-pipe (OR 5.88; 95% CI 3.25 to 10.63) and jumps (OR 4.78; 95% CI 3.22 to 7.11) and significantly decreased odds of injury on quarter-pipes (OR 0.49; 95% CI 0.25 to 0.97).\nOverall, 62.5% went to the ED. The most commonly injured body regions were the wrist (20%), and head (14%), while the most common injury type was fracture (36%). Table 1 describes feature use among injured and uninjured TP snowboarders.\nFeatures used by all injured, severely injured snowboarders, and uninjured snowboarders in the terrain park\n*Weighted for time of day, weekday versus weekend and exposure opportunity.\nFemales aged 12–17 and >17 had higher rates of injuries than males, and higher rates of severe injuries at all ages (table 2).\nOverall, sex-specific and age-specific injury rates (per 1000 runs and 95% CI)\nFeatures that promoted aerial manoeuvres or resulted in a greater drop to the ground typically had higher injury rates than features with a small drop (table 3).\nTable 3All and severe injury rates (per 1000 feature exposures and 95% CI)FeatureAll injuries* rate (95% CI)Severe injuries* rate (95% CI)Aerial manoeuvre or substantial drop to the ground Half-pipe2.6 (1.5 to 4.0)1.8 (0.9 to 3.0) Jumps2.6 (2.1 to 3.2)1.8 (1.4 to 2.3) Kickers0.6 (0.5 to 0.8)0.4 (0.3 to 0.5) Mushroom0.5 (0.3 to 0.8)0.2 (0.1 to 0.5)Small drop to the ground Boxes0.7 (0.5 to 0.8)0.4 (0.3 to 0.5) Rails0.4 (0.3 to 0.6)0.2 (0.1 to 0.4) Quarter-pipes0.2 (0.1 to 0.4)0.1 (0.1 to 0.3)*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed). Jumps and half-pipe had the highest overall and severe injury rates. Compared with rails, rates of all injuries were significantly higher on the half-pipe, jumps and boxes (table 4).\nAll and severe injury rates (per 1000 feature exposures and 95% CI)\n*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed).\nFeature-specific injury rate ratios and 95% CI\nN, number; RR, rate ratio.\n*Four snowboarders could not recall feature used at time of injury and 13 snowboarders were not using a feature at the time of injury but were in the boundaries of the terrain park (TP) for a total of 333 TP injuries.\n†Two snowboarders could not recall feature used at time of injury and nine snowboarders were not using a feature at the time of injury but were in the boundaries of the TP for a total of 208 severe TP injuries. Bold refers to statistically significant results.\nBaseline characteristics are presented in table 5. The odds of injury were significantly higher when it was −10°C to 0°C compared with 0–10°C, and at night versus sunny weather. Beginners/novices had significantly lower odds of injury than intermediates, as did those with a previous snowboarding injury, music use, or when it was above 10°C versus 0–10°C. When comparing severely injured with uninjured snowboarders, the same patterns were observed except that music or temperatures above 10°C were not significant. There were no significant differences between severe versus minor injuries.\nSummary of the characteristics of all injured and uninjured snowboarders (crude OR and 95% CI)\n*Odds of injury increases for every increase in year.\n†Compared with no injuries.\n‡Compared with minor injuries.\nN, numbers; N/A, not applicable.\nThe crude associations between injury and feature use showed significantly greater odds of injury for jumps and half-pipe, compared with rails and significantly lower odds of injury for quarter-pipes (table 6). For severe injury versus no injury, the crude odds of severe injury were significantly greater on jumps, half-pipe and kickers compared with rails.\nAssociation between injury and feature type after controlling for confounders (OR and 95% CI)\n*Adjusted for previous injury, ability and temperature. Age, sex, listening to music, wearing wrist guards and light and snow conditions did not change any of the feature-specific estimates more than 15%.\n†Adjusted for music and light. Age, sex, ability, wrist guard use and temperature and snow conditions did not change any of the feature-specific estimates more than 15%.\nThere were significant increases in the adjusted odds of injury for half-pipe (OR 9.63; 95% CI 4.80 to 19.32), jumps (OR 4.29; 95% CI 2.72 to 6.76), mushroom (OR 2.30; 95% CI 1.20 to 4.41) and kickers (OR 1.99; 95% CI 1.27 to 3.12) versus rails. The adjusted odds of severe injury versus no injury were significantly higher for half-pipe, jumps and kickers compared with rails. After accounting for clustering, the 95% CI width increased marginally but significance did not change.\nUsing the imputed dataset, there was no evidence of effect modification of feature by age or sex (p=0.41) versus rails, significantly increased adjusted odds of injury on half-pipe (OR 5.88; 95% CI 3.25 to 10.63) and jumps (OR 4.78; 95% CI 3.22 to 7.11) and significantly decreased odds of injury on quarter-pipes (OR 0.49; 95% CI 0.25 to 0.97).","Based on initial RA observation and follow-up confirmation, the RAs correctly classified age-group in 78% (κ=0.71; 95% CI 0.49 to 0.93) and sex in 100% of uninjured snowboarders (κ=1.00; 95% CI 0.69 to 1.00). There was significant agreement for age and sex classification between the RAs (p>0.05 for each of the three pairs). For interview responses, κ=0.72 (95% CI 0.60 to 0.85) for feature use at the time of injury and this improved when follow-up interviews were conducted within 2 weeks (κ=0.86; 95% CI 0.64 to 1.00). The overall κ were ≥0.60 except for body region of the second and third injuries and diagnosis of the second injury.","Overall, 62.5% went to the ED. The most commonly injured body regions were the wrist (20%), and head (14%), while the most common injury type was fracture (36%). Table 1 describes feature use among injured and uninjured TP snowboarders.\nFeatures used by all injured, severely injured snowboarders, and uninjured snowboarders in the terrain park\n*Weighted for time of day, weekday versus weekend and exposure opportunity.\nFemales aged 12–17 and >17 had higher rates of injuries than males, and higher rates of severe injuries at all ages (table 2).\nOverall, sex-specific and age-specific injury rates (per 1000 runs and 95% CI)\nFeatures that promoted aerial manoeuvres or resulted in a greater drop to the ground typically had higher injury rates than features with a small drop (table 3).\nTable 3All and severe injury rates (per 1000 feature exposures and 95% CI)FeatureAll injuries* rate (95% CI)Severe injuries* rate (95% CI)Aerial manoeuvre or substantial drop to the ground Half-pipe2.6 (1.5 to 4.0)1.8 (0.9 to 3.0) Jumps2.6 (2.1 to 3.2)1.8 (1.4 to 2.3) Kickers0.6 (0.5 to 0.8)0.4 (0.3 to 0.5) Mushroom0.5 (0.3 to 0.8)0.2 (0.1 to 0.5)Small drop to the ground Boxes0.7 (0.5 to 0.8)0.4 (0.3 to 0.5) Rails0.4 (0.3 to 0.6)0.2 (0.1 to 0.4) Quarter-pipes0.2 (0.1 to 0.4)0.1 (0.1 to 0.3)*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed). Jumps and half-pipe had the highest overall and severe injury rates. Compared with rails, rates of all injuries were significantly higher on the half-pipe, jumps and boxes (table 4).\nAll and severe injury rates (per 1000 feature exposures and 95% CI)\n*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed).\nFeature-specific injury rate ratios and 95% CI\nN, number; RR, rate ratio.\n*Four snowboarders could not recall feature used at time of injury and 13 snowboarders were not using a feature at the time of injury but were in the boundaries of the terrain park (TP) for a total of 333 TP injuries.\n†Two snowboarders could not recall feature used at time of injury and nine snowboarders were not using a feature at the time of injury but were in the boundaries of the TP for a total of 208 severe TP injuries. Bold refers to statistically significant results.\nBaseline characteristics are presented in table 5. The odds of injury were significantly higher when it was −10°C to 0°C compared with 0–10°C, and at night versus sunny weather. Beginners/novices had significantly lower odds of injury than intermediates, as did those with a previous snowboarding injury, music use, or when it was above 10°C versus 0–10°C. When comparing severely injured with uninjured snowboarders, the same patterns were observed except that music or temperatures above 10°C were not significant. There were no significant differences between severe versus minor injuries.\nSummary of the characteristics of all injured and uninjured snowboarders (crude OR and 95% CI)\n*Odds of injury increases for every increase in year.\n†Compared with no injuries.\n‡Compared with minor injuries.\nN, numbers; N/A, not applicable.\nThe crude associations between injury and feature use showed significantly greater odds of injury for jumps and half-pipe, compared with rails and significantly lower odds of injury for quarter-pipes (table 6). For severe injury versus no injury, the crude odds of severe injury were significantly greater on jumps, half-pipe and kickers compared with rails.\nAssociation between injury and feature type after controlling for confounders (OR and 95% CI)\n*Adjusted for previous injury, ability and temperature. Age, sex, listening to music, wearing wrist guards and light and snow conditions did not change any of the feature-specific estimates more than 15%.\n†Adjusted for music and light. Age, sex, ability, wrist guard use and temperature and snow conditions did not change any of the feature-specific estimates more than 15%.\nThere were significant increases in the adjusted odds of injury for half-pipe (OR 9.63; 95% CI 4.80 to 19.32), jumps (OR 4.29; 95% CI 2.72 to 6.76), mushroom (OR 2.30; 95% CI 1.20 to 4.41) and kickers (OR 1.99; 95% CI 1.27 to 3.12) versus rails. The adjusted odds of severe injury versus no injury were significantly higher for half-pipe, jumps and kickers compared with rails. After accounting for clustering, the 95% CI width increased marginally but significance did not change.\nUsing the imputed dataset, there was no evidence of effect modification of feature by age or sex (p=0.41) versus rails, significantly increased adjusted odds of injury on half-pipe (OR 5.88; 95% CI 3.25 to 10.63) and jumps (OR 4.78; 95% CI 3.22 to 7.11) and significantly decreased odds of injury on quarter-pipes (OR 0.49; 95% CI 0.25 to 0.97).","To our knowledge this is the first study to examine feature-specific injury rates and potential risk factors for TP snowboarding injuries. Data collection methods were found to be accurate and reliable for age and sex information. There was ‘substantial’ to ‘perfect’ agreement for most risk factors for injured snowboarders, including feature use.27 Self-reported risk factors were confirmed with reliable sources where possible. The overall injury rate of TP snowboarders was estimated at 0.75/1000 runs. Feature-specific injury rates were higher for features that supported aerial manoeuvres or a large drop to the ground. Aerial features facilitate more air time and we hypothesise that snowboarders may have more opportunity to lose their sense of body position/orientation and land with more force and this increases the likelihood of injury.\nResearch suggests that TP injuries are more severe than those on regular slopes.15\n16\n18 One study reported an overall TP injury rate of 0.62/1000 ski and snowboard days, including both skiers and snowboarders, but only ski patrol injuries.15 If each skier and snowboarder took only one TP run during their day, this rate is lower than our observed rate. It is unknown if the TPs were similar in size or number of features. A literature review34 reported that ski patrol-reported injuries among snowboarders varied from 2.135 to 7.0/361000 outings; however, a TP-specific injury rate or the precise injury definition was not provided.\nHalf-pipes and jumps significantly predicted injury. Torjussen and Bahr37 found professional snowboarders competing in half-pipe and big air competitions had a significantly higher injury risk compared with giant-slalom, where the snowboarder does not leave the ground.\nOverall 34% of snowboarders listened to music through a personal music player. The unadjusted result indicated that listening to music reduced the odds of injury. In a laboratory setting, sport students listening to music while wearing a helmet with built-in speakers had similar mean reaction time to peripheral stimulus as those who were wearing a helmet but not listening to music.38 Similar to non-TP research, snowboarding in suboptimal environmental (bad weather/visibility) conditions affected the odds of injury.9 In contrast to previous research,5–8 we found beginners had significantly reduced odds of injury; perhaps beginners realise they are in an environment beyond their skill level and choose easier features or do not use features as intended (eg, not leaving the ground when going over a jump).\nFuture research should identify and evaluate ways to reduce injuries without sacrificing participation, motor learning or skill development. Injury prevention should focus on risk mitigation. Possible strategies include marking landings, smaller TPs for progression to larger features, marking feature difficulty, controlling speed and slope for take-offs and landings, enforcing TP etiquette rules or reducing the height of aerial features. Further research is needed to develop guidelines regarding optimal TP design. The efficacy of these strategies should be investigated.\n Limitations Snowboarders who climbed up to reattempt features were counted as one run, which would underestimate the number of TP runs. Injured snowboarders who did not present to the ski patrol or either of the two nearest EDs were missed. This selection bias resulted in rate underestimation and observed associations between feature and injury would be overestimated if missed snowboarders were injured on rails. However, only 47 (14%) of the included snowboarders said they saw the ski patrol and a non-participating healthcare provider, indicating that most sought treatment at a participating ED.\nThere was potential misclassification by feature if the injured snowboarder could not recall the feature; however, this occurred only four times. Controls may not have correctly reported their feature use when the RA could not see the entire TP and it was not possible to determine the reliability of controls’ feature use. This could have an unpredictable effect on the ORs if it operated differently by feature type. Only age-group and sex of the uninjured controls could be assessed for accuracy and our observations were found to be valid. There was potential for misclassification of severe injury because factors other than severity could predict presentation to the ED, such as parental fear or anxiety influenced by a recent celebrity skiing death.39 It was unknown if non-consenting injured snowboarders presented to a non-study ED and were incorrectly classified as a minor injury. Fortunately, few who saw the ski patrol went to a non-study ED (8%).\nIt is possible that some important confounders were overlooked, such as first attempt at a new feature, manoeuvre performed, speed, fatigue, height or weight. There may be behavioural confounders, such as peer pressure to attempt difficult features or manoeuvres. However, some of these potential confounders were likely accounted for by other variables such as ability, age and sex. Although this study was conducted at only one resort, the TP layout changed four times during the two seasons and this enhances the generalisability of the results.\nSnowboarders who climbed up to reattempt features were counted as one run, which would underestimate the number of TP runs. Injured snowboarders who did not present to the ski patrol or either of the two nearest EDs were missed. This selection bias resulted in rate underestimation and observed associations between feature and injury would be overestimated if missed snowboarders were injured on rails. However, only 47 (14%) of the included snowboarders said they saw the ski patrol and a non-participating healthcare provider, indicating that most sought treatment at a participating ED.\nThere was potential misclassification by feature if the injured snowboarder could not recall the feature; however, this occurred only four times. Controls may not have correctly reported their feature use when the RA could not see the entire TP and it was not possible to determine the reliability of controls’ feature use. This could have an unpredictable effect on the ORs if it operated differently by feature type. Only age-group and sex of the uninjured controls could be assessed for accuracy and our observations were found to be valid. There was potential for misclassification of severe injury because factors other than severity could predict presentation to the ED, such as parental fear or anxiety influenced by a recent celebrity skiing death.39 It was unknown if non-consenting injured snowboarders presented to a non-study ED and were incorrectly classified as a minor injury. Fortunately, few who saw the ski patrol went to a non-study ED (8%).\nIt is possible that some important confounders were overlooked, such as first attempt at a new feature, manoeuvre performed, speed, fatigue, height or weight. There may be behavioural confounders, such as peer pressure to attempt difficult features or manoeuvres. However, some of these potential confounders were likely accounted for by other variables such as ability, age and sex. Although this study was conducted at only one resort, the TP layout changed four times during the two seasons and this enhances the generalisability of the results.","Snowboarders who climbed up to reattempt features were counted as one run, which would underestimate the number of TP runs. Injured snowboarders who did not present to the ski patrol or either of the two nearest EDs were missed. This selection bias resulted in rate underestimation and observed associations between feature and injury would be overestimated if missed snowboarders were injured on rails. However, only 47 (14%) of the included snowboarders said they saw the ski patrol and a non-participating healthcare provider, indicating that most sought treatment at a participating ED.\nThere was potential misclassification by feature if the injured snowboarder could not recall the feature; however, this occurred only four times. Controls may not have correctly reported their feature use when the RA could not see the entire TP and it was not possible to determine the reliability of controls’ feature use. This could have an unpredictable effect on the ORs if it operated differently by feature type. Only age-group and sex of the uninjured controls could be assessed for accuracy and our observations were found to be valid. There was potential for misclassification of severe injury because factors other than severity could predict presentation to the ED, such as parental fear or anxiety influenced by a recent celebrity skiing death.39 It was unknown if non-consenting injured snowboarders presented to a non-study ED and were incorrectly classified as a minor injury. Fortunately, few who saw the ski patrol went to a non-study ED (8%).\nIt is possible that some important confounders were overlooked, such as first attempt at a new feature, manoeuvre performed, speed, fatigue, height or weight. There may be behavioural confounders, such as peer pressure to attempt difficult features or manoeuvres. However, some of these potential confounders were likely accounted for by other variables such as ability, age and sex. Although this study was conducted at only one resort, the TP layout changed four times during the two seasons and this enhances the generalisability of the results.","Feature-specific injury rates ranged from 2.56 injuries/1000 runs (jumps and half-pipe) to 0.24 injuries/1000 runs (quarter-pipe). Half-pipe, jumps and kickers were significant risk factors for any injury and severe injury. Recommendations have been made for prevention strategy development to reduce TP injury risk. These strategies will require rigorous evaluation.\nWhat are the new findings?In this study, the overall injury rate for snowboarding terrain park (TP) injuries is 0.75 injuries/1000 runs.The injury rates are highest for jumps (2.56/1000 runs) and half-pipe (2.56/1000 runs) and lowest for rails (0.43/1000 runs) and quarter-pipes (0.24/1000 runs).Compared with rails, the odds of injury is significantly higher on the half-pipe (OR 9.63; 95% CI 4.80 to 19.32) and jumps (OR 4.29; 95% CI 2.72 to 6.76).\nHow might it impact on clinical practice in the near future?Terrain parks (TPs) are popular and the majority of snowboarders injured in the TP present to the emergency department.With the identification of potential risk factors for TP injuries among snowboarders, injury prevention programmes can be tailored to those at greatest risk of injury.Should these programmes be effective, clinicians can expect to treat fewer snowboarders injured in the TP.\nIn this study, the overall injury rate for snowboarding terrain park (TP) injuries is 0.75 injuries/1000 runs.\nThe injury rates are highest for jumps (2.56/1000 runs) and half-pipe (2.56/1000 runs) and lowest for rails (0.43/1000 runs) and quarter-pipes (0.24/1000 runs).\nCompared with rails, the odds of injury is significantly higher on the half-pipe (OR 9.63; 95% CI 4.80 to 19.32) and jumps (OR 4.29; 95% CI 2.72 to 6.76).\nTerrain parks (TPs) are popular and the majority of snowboarders injured in the TP present to the emergency department.\nWith the identification of potential risk factors for TP injuries among snowboarders, injury prevention programmes can be tailored to those at greatest risk of injury.\nShould these programmes be effective, clinicians can expect to treat fewer snowboarders injured in the TP.",""],"string":"[\n \"Snowboarding is a popular sport1\\n2 and the risk of injury is higher for snowboarding than for skiing.3\\n4 Being a beginner,5–8 poor weather conditions9 and not wearing protective equipment10–12 increase injury risk. Ski areas often include terrain parks (TPs) with man-made features (eg, jumps, rails and half-pipes) for performing tricks and aerial manoeuvres. In November 2007, Resorts of the Canadian Rockies (RCR) removed all man-made jumps from their TPs because they believed jumps increased injury risk.13 Definitions of common features can be found at: http://www.snowboard-coach.com/freestyle-snowboarding-features.html and in appendix 1.\\nBetween 5% and 27% of skiing and snowboarding injuries occur in TPs2\\n14–19 and are more severe than regular slope injuries.15\\n16\\n18 At the 2012 Winter Youth Olympic Games, 35% of all snowboard half-pipe and slope-style competitors were injured.20 Those injured in TPs tend to be snowboarders, male, 13–24 years old, fall from higher heights15 or self-perceived experts.16 There is a dearth of research examining injury rates and intrinsic and extrinsic risk factors for snowboarders in TPs in relation to injury mechanism—a comprehensive approach recommended by sport injury prevention research leaders.21\\n22 Therefore, the study objectives were to calculate overall and feature-specific TP injury rates, determine potential risk factors for injury in the TP and assess the reliability of the data collection methods.\",\n \" Definition of cases and controls This unmatched case-control study was conducted in one Alberta TP during the 2008–2009 and 2009–2010 seasons. There were approximately 290 000 skier–snowboarder visits to the resort annually. Except for the half-pipe and mushroom, the overall TP layout and number of features changed once each season, resulting in four configurations containing all seven feature types. The resort did not assign a difficulty rating to individual features. Helmets were mandatory in the TP.\\nCases were snowboarders injured in the TP who presented to either the ski patrol and/or one of two nearby emergency departments (EDs), both Level 1 trauma centres (one adult, one paediatric). Controls were uninjured snowboarders using the same TP. Cases were ‘severe’ if they presented to an ED, and ‘minor’ if they presented to ski patrol only or to ski patrol and a non-emergent healthcare provider. Injuries presenting to the ED represent the public health burden and snowboarders injured in the TPs near hospitals may place a strain on EDs.\\nThis unmatched case-control study was conducted in one Alberta TP during the 2008–2009 and 2009–2010 seasons. There were approximately 290 000 skier–snowboarder visits to the resort annually. Except for the half-pipe and mushroom, the overall TP layout and number of features changed once each season, resulting in four configurations containing all seven feature types. The resort did not assign a difficulty rating to individual features. Helmets were mandatory in the TP.\\nCases were snowboarders injured in the TP who presented to either the ski patrol and/or one of two nearby emergency departments (EDs), both Level 1 trauma centres (one adult, one paediatric). Controls were uninjured snowboarders using the same TP. Cases were ‘severe’ if they presented to an ED, and ‘minor’ if they presented to ski patrol only or to ski patrol and a non-emergent healthcare provider. Injuries presenting to the ED represent the public health burden and snowboarders injured in the TPs near hospitals may place a strain on EDs.\\n Data collection Case data were collected from ski patrol Accident Report Forms (ARF) and ED medical records. ARFs are completed by the ski patrol for anyone injured and presented to them. ARFs record demographics (age, sex and five-point self-reported ability), injured body region, injury type (fracture, dislocation, sprain/strain, bruise/abrasion/laceration and concussion), environment (temperature, light and snow (groomed/ungroomed)) and contact information. ARFs have previously been used in research14\\n18\\n19\\n23–25 and were collected from the resort biweekly. Snowboarders who presented to the ED were identified from the Regional Emergency Department Information System. Following verbal consent (parent/guardian if snowboarder was <14 years) and confirmation they were injured at the TP of interest, additional self-reported data were collected by telephone. The previously listed information was captured for snowboarders presenting only to the ED, along with years of snowboarding and TP experience, listening to music, wearing wrist guards, previous snowboarding injury and feature used (jump, kicker, box, rail, quarter-pipe, half-pipe or mushroom) when injured. If the case presented to the ski patrol and could not be contacted or did not consent to the telephone interview, only the ARF data were included.\\nTo collect feature use among controls, trained research assistants (RAs) at the bottom of the hill observed snowboarders' TP runs in 3 h time slots, 3–4 times a week at various times during the day, and recorded feature use on a map. Data were collected each week from 1 January until the resort closed (end of March in Season 1 and mid-April in Season 2) and included all four TP layouts. RAs approached the first snowboarder, obtained consent and asked the same risk factor information as asked to cases. Snowboarders indicated feature use on the map for features not fully visible to the RA. After each interview, the next snowboarder closest to the RA was approached. Temperature (smartphone Weather Network application), light and snow conditions were recorded on an hourly basis.\\nInjury rate denominator data were collected in the same 3 h slots. Snowboarder runs were counted by an RA at the top of the TP, which was the only run to the right of the chairlift and only serviced by that chairlift. Age-group (<12, 12–17 or >17) and sex were visually assessed. To determine accuracy of observed age-group and sex classifications, RAs independently estimated the age-group and sex of a snowboarder entering the TP and then approached them to confirm. This was repeated in 10 min intervals for 3 h blocks and included 337 snowboarders.\\nEthical approval was granted by the University of Calgary Conjoint Health Research Ethics Board.\\nCase data were collected from ski patrol Accident Report Forms (ARF) and ED medical records. ARFs are completed by the ski patrol for anyone injured and presented to them. ARFs record demographics (age, sex and five-point self-reported ability), injured body region, injury type (fracture, dislocation, sprain/strain, bruise/abrasion/laceration and concussion), environment (temperature, light and snow (groomed/ungroomed)) and contact information. ARFs have previously been used in research14\\n18\\n19\\n23–25 and were collected from the resort biweekly. Snowboarders who presented to the ED were identified from the Regional Emergency Department Information System. Following verbal consent (parent/guardian if snowboarder was <14 years) and confirmation they were injured at the TP of interest, additional self-reported data were collected by telephone. The previously listed information was captured for snowboarders presenting only to the ED, along with years of snowboarding and TP experience, listening to music, wearing wrist guards, previous snowboarding injury and feature used (jump, kicker, box, rail, quarter-pipe, half-pipe or mushroom) when injured. If the case presented to the ski patrol and could not be contacted or did not consent to the telephone interview, only the ARF data were included.\\nTo collect feature use among controls, trained research assistants (RAs) at the bottom of the hill observed snowboarders' TP runs in 3 h time slots, 3–4 times a week at various times during the day, and recorded feature use on a map. Data were collected each week from 1 January until the resort closed (end of March in Season 1 and mid-April in Season 2) and included all four TP layouts. RAs approached the first snowboarder, obtained consent and asked the same risk factor information as asked to cases. Snowboarders indicated feature use on the map for features not fully visible to the RA. After each interview, the next snowboarder closest to the RA was approached. Temperature (smartphone Weather Network application), light and snow conditions were recorded on an hourly basis.\\nInjury rate denominator data were collected in the same 3 h slots. Snowboarder runs were counted by an RA at the top of the TP, which was the only run to the right of the chairlift and only serviced by that chairlift. Age-group (<12, 12–17 or >17) and sex were visually assessed. To determine accuracy of observed age-group and sex classifications, RAs independently estimated the age-group and sex of a snowboarder entering the TP and then approached them to confirm. This was repeated in 10 min intervals for 3 h blocks and included 337 snowboarders.\\nEthical approval was granted by the University of Calgary Conjoint Health Research Ethics Board.\\n Analysis Reliability Three pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\\nThree pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\\n Rates Injury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\\nInjury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\\n Risk factors The distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\\nThe distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\\n Reliability Three pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\\nThree pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\\n Rates Injury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\\nInjury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\\n Risk factors The distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\\nThe distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\",\n \"This unmatched case-control study was conducted in one Alberta TP during the 2008–2009 and 2009–2010 seasons. There were approximately 290 000 skier–snowboarder visits to the resort annually. Except for the half-pipe and mushroom, the overall TP layout and number of features changed once each season, resulting in four configurations containing all seven feature types. The resort did not assign a difficulty rating to individual features. Helmets were mandatory in the TP.\\nCases were snowboarders injured in the TP who presented to either the ski patrol and/or one of two nearby emergency departments (EDs), both Level 1 trauma centres (one adult, one paediatric). Controls were uninjured snowboarders using the same TP. Cases were ‘severe’ if they presented to an ED, and ‘minor’ if they presented to ski patrol only or to ski patrol and a non-emergent healthcare provider. Injuries presenting to the ED represent the public health burden and snowboarders injured in the TPs near hospitals may place a strain on EDs.\",\n \"Case data were collected from ski patrol Accident Report Forms (ARF) and ED medical records. ARFs are completed by the ski patrol for anyone injured and presented to them. ARFs record demographics (age, sex and five-point self-reported ability), injured body region, injury type (fracture, dislocation, sprain/strain, bruise/abrasion/laceration and concussion), environment (temperature, light and snow (groomed/ungroomed)) and contact information. ARFs have previously been used in research14\\n18\\n19\\n23–25 and were collected from the resort biweekly. Snowboarders who presented to the ED were identified from the Regional Emergency Department Information System. Following verbal consent (parent/guardian if snowboarder was <14 years) and confirmation they were injured at the TP of interest, additional self-reported data were collected by telephone. The previously listed information was captured for snowboarders presenting only to the ED, along with years of snowboarding and TP experience, listening to music, wearing wrist guards, previous snowboarding injury and feature used (jump, kicker, box, rail, quarter-pipe, half-pipe or mushroom) when injured. If the case presented to the ski patrol and could not be contacted or did not consent to the telephone interview, only the ARF data were included.\\nTo collect feature use among controls, trained research assistants (RAs) at the bottom of the hill observed snowboarders' TP runs in 3 h time slots, 3–4 times a week at various times during the day, and recorded feature use on a map. Data were collected each week from 1 January until the resort closed (end of March in Season 1 and mid-April in Season 2) and included all four TP layouts. RAs approached the first snowboarder, obtained consent and asked the same risk factor information as asked to cases. Snowboarders indicated feature use on the map for features not fully visible to the RA. After each interview, the next snowboarder closest to the RA was approached. Temperature (smartphone Weather Network application), light and snow conditions were recorded on an hourly basis.\\nInjury rate denominator data were collected in the same 3 h slots. Snowboarder runs were counted by an RA at the top of the TP, which was the only run to the right of the chairlift and only serviced by that chairlift. Age-group (<12, 12–17 or >17) and sex were visually assessed. To determine accuracy of observed age-group and sex classifications, RAs independently estimated the age-group and sex of a snowboarder entering the TP and then approached them to confirm. This was repeated in 10 min intervals for 3 h blocks and included 337 snowboarders.\\nEthical approval was granted by the University of Calgary Conjoint Health Research Ethics Board.\",\n \" Reliability Three pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\\nThree pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\\n Rates Injury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\\nInjury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\\n Risk factors The distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\\nThe distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\",\n \"Three pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\",\n \"Injury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\",\n \"The distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\",\n \"A total of 333 cases (107 ski patrol, 174 ski patrol and ED, 18 ski patrol and healthcare provider and 34 ED only) and 1261 controls were included. The consent rate was 79% for cases and 94% for controls (figure 1).\\nRecruitment of included snowboarders.\\n Reliability Based on initial RA observation and follow-up confirmation, the RAs correctly classified age-group in 78% (κ=0.71; 95% CI 0.49 to 0.93) and sex in 100% of uninjured snowboarders (κ=1.00; 95% CI 0.69 to 1.00). There was significant agreement for age and sex classification between the RAs (p>0.05 for each of the three pairs). For interview responses, κ=0.72 (95% CI 0.60 to 0.85) for feature use at the time of injury and this improved when follow-up interviews were conducted within 2 weeks (κ=0.86; 95% CI 0.64 to 1.00). The overall κ were ≥0.60 except for body region of the second and third injuries and diagnosis of the second injury.\\nBased on initial RA observation and follow-up confirmation, the RAs correctly classified age-group in 78% (κ=0.71; 95% CI 0.49 to 0.93) and sex in 100% of uninjured snowboarders (κ=1.00; 95% CI 0.69 to 1.00). There was significant agreement for age and sex classification between the RAs (p>0.05 for each of the three pairs). For interview responses, κ=0.72 (95% CI 0.60 to 0.85) for feature use at the time of injury and this improved when follow-up interviews were conducted within 2 weeks (κ=0.86; 95% CI 0.64 to 1.00). The overall κ were ≥0.60 except for body region of the second and third injuries and diagnosis of the second injury.\\n Injuries Overall, 62.5% went to the ED. The most commonly injured body regions were the wrist (20%), and head (14%), while the most common injury type was fracture (36%). Table 1 describes feature use among injured and uninjured TP snowboarders.\\nFeatures used by all injured, severely injured snowboarders, and uninjured snowboarders in the terrain park\\n*Weighted for time of day, weekday versus weekend and exposure opportunity.\\nFemales aged 12–17 and >17 had higher rates of injuries than males, and higher rates of severe injuries at all ages (table 2).\\nOverall, sex-specific and age-specific injury rates (per 1000 runs and 95% CI)\\nFeatures that promoted aerial manoeuvres or resulted in a greater drop to the ground typically had higher injury rates than features with a small drop (table 3).\\nTable 3All and severe injury rates (per 1000 feature exposures and 95% CI)FeatureAll injuries* rate (95% CI)Severe injuries* rate (95% CI)Aerial manoeuvre or substantial drop to the ground Half-pipe2.6 (1.5 to 4.0)1.8 (0.9 to 3.0) Jumps2.6 (2.1 to 3.2)1.8 (1.4 to 2.3) Kickers0.6 (0.5 to 0.8)0.4 (0.3 to 0.5) Mushroom0.5 (0.3 to 0.8)0.2 (0.1 to 0.5)Small drop to the ground Boxes0.7 (0.5 to 0.8)0.4 (0.3 to 0.5) Rails0.4 (0.3 to 0.6)0.2 (0.1 to 0.4) Quarter-pipes0.2 (0.1 to 0.4)0.1 (0.1 to 0.3)*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed). Jumps and half-pipe had the highest overall and severe injury rates. Compared with rails, rates of all injuries were significantly higher on the half-pipe, jumps and boxes (table 4).\\nAll and severe injury rates (per 1000 feature exposures and 95% CI)\\n*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed).\\nFeature-specific injury rate ratios and 95% CI\\nN, number; RR, rate ratio.\\n*Four snowboarders could not recall feature used at time of injury and 13 snowboarders were not using a feature at the time of injury but were in the boundaries of the terrain park (TP) for a total of 333 TP injuries.\\n†Two snowboarders could not recall feature used at time of injury and nine snowboarders were not using a feature at the time of injury but were in the boundaries of the TP for a total of 208 severe TP injuries. Bold refers to statistically significant results.\\nBaseline characteristics are presented in table 5. The odds of injury were significantly higher when it was −10°C to 0°C compared with 0–10°C, and at night versus sunny weather. Beginners/novices had significantly lower odds of injury than intermediates, as did those with a previous snowboarding injury, music use, or when it was above 10°C versus 0–10°C. When comparing severely injured with uninjured snowboarders, the same patterns were observed except that music or temperatures above 10°C were not significant. There were no significant differences between severe versus minor injuries.\\nSummary of the characteristics of all injured and uninjured snowboarders (crude OR and 95% CI)\\n*Odds of injury increases for every increase in year.\\n†Compared with no injuries.\\n‡Compared with minor injuries.\\nN, numbers; N/A, not applicable.\\nThe crude associations between injury and feature use showed significantly greater odds of injury for jumps and half-pipe, compared with rails and significantly lower odds of injury for quarter-pipes (table 6). For severe injury versus no injury, the crude odds of severe injury were significantly greater on jumps, half-pipe and kickers compared with rails.\\nAssociation between injury and feature type after controlling for confounders (OR and 95% CI)\\n*Adjusted for previous injury, ability and temperature. Age, sex, listening to music, wearing wrist guards and light and snow conditions did not change any of the feature-specific estimates more than 15%.\\n†Adjusted for music and light. Age, sex, ability, wrist guard use and temperature and snow conditions did not change any of the feature-specific estimates more than 15%.\\nThere were significant increases in the adjusted odds of injury for half-pipe (OR 9.63; 95% CI 4.80 to 19.32), jumps (OR 4.29; 95% CI 2.72 to 6.76), mushroom (OR 2.30; 95% CI 1.20 to 4.41) and kickers (OR 1.99; 95% CI 1.27 to 3.12) versus rails. The adjusted odds of severe injury versus no injury were significantly higher for half-pipe, jumps and kickers compared with rails. After accounting for clustering, the 95% CI width increased marginally but significance did not change.\\nUsing the imputed dataset, there was no evidence of effect modification of feature by age or sex (p=0.41) versus rails, significantly increased adjusted odds of injury on half-pipe (OR 5.88; 95% CI 3.25 to 10.63) and jumps (OR 4.78; 95% CI 3.22 to 7.11) and significantly decreased odds of injury on quarter-pipes (OR 0.49; 95% CI 0.25 to 0.97).\\nOverall, 62.5% went to the ED. The most commonly injured body regions were the wrist (20%), and head (14%), while the most common injury type was fracture (36%). Table 1 describes feature use among injured and uninjured TP snowboarders.\\nFeatures used by all injured, severely injured snowboarders, and uninjured snowboarders in the terrain park\\n*Weighted for time of day, weekday versus weekend and exposure opportunity.\\nFemales aged 12–17 and >17 had higher rates of injuries than males, and higher rates of severe injuries at all ages (table 2).\\nOverall, sex-specific and age-specific injury rates (per 1000 runs and 95% CI)\\nFeatures that promoted aerial manoeuvres or resulted in a greater drop to the ground typically had higher injury rates than features with a small drop (table 3).\\nTable 3All and severe injury rates (per 1000 feature exposures and 95% CI)FeatureAll injuries* rate (95% CI)Severe injuries* rate (95% CI)Aerial manoeuvre or substantial drop to the ground Half-pipe2.6 (1.5 to 4.0)1.8 (0.9 to 3.0) Jumps2.6 (2.1 to 3.2)1.8 (1.4 to 2.3) Kickers0.6 (0.5 to 0.8)0.4 (0.3 to 0.5) Mushroom0.5 (0.3 to 0.8)0.2 (0.1 to 0.5)Small drop to the ground Boxes0.7 (0.5 to 0.8)0.4 (0.3 to 0.5) Rails0.4 (0.3 to 0.6)0.2 (0.1 to 0.4) Quarter-pipes0.2 (0.1 to 0.4)0.1 (0.1 to 0.3)*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed). Jumps and half-pipe had the highest overall and severe injury rates. Compared with rails, rates of all injuries were significantly higher on the half-pipe, jumps and boxes (table 4).\\nAll and severe injury rates (per 1000 feature exposures and 95% CI)\\n*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed).\\nFeature-specific injury rate ratios and 95% CI\\nN, number; RR, rate ratio.\\n*Four snowboarders could not recall feature used at time of injury and 13 snowboarders were not using a feature at the time of injury but were in the boundaries of the terrain park (TP) for a total of 333 TP injuries.\\n†Two snowboarders could not recall feature used at time of injury and nine snowboarders were not using a feature at the time of injury but were in the boundaries of the TP for a total of 208 severe TP injuries. Bold refers to statistically significant results.\\nBaseline characteristics are presented in table 5. The odds of injury were significantly higher when it was −10°C to 0°C compared with 0–10°C, and at night versus sunny weather. Beginners/novices had significantly lower odds of injury than intermediates, as did those with a previous snowboarding injury, music use, or when it was above 10°C versus 0–10°C. When comparing severely injured with uninjured snowboarders, the same patterns were observed except that music or temperatures above 10°C were not significant. There were no significant differences between severe versus minor injuries.\\nSummary of the characteristics of all injured and uninjured snowboarders (crude OR and 95% CI)\\n*Odds of injury increases for every increase in year.\\n†Compared with no injuries.\\n‡Compared with minor injuries.\\nN, numbers; N/A, not applicable.\\nThe crude associations between injury and feature use showed significantly greater odds of injury for jumps and half-pipe, compared with rails and significantly lower odds of injury for quarter-pipes (table 6). For severe injury versus no injury, the crude odds of severe injury were significantly greater on jumps, half-pipe and kickers compared with rails.\\nAssociation between injury and feature type after controlling for confounders (OR and 95% CI)\\n*Adjusted for previous injury, ability and temperature. Age, sex, listening to music, wearing wrist guards and light and snow conditions did not change any of the feature-specific estimates more than 15%.\\n†Adjusted for music and light. Age, sex, ability, wrist guard use and temperature and snow conditions did not change any of the feature-specific estimates more than 15%.\\nThere were significant increases in the adjusted odds of injury for half-pipe (OR 9.63; 95% CI 4.80 to 19.32), jumps (OR 4.29; 95% CI 2.72 to 6.76), mushroom (OR 2.30; 95% CI 1.20 to 4.41) and kickers (OR 1.99; 95% CI 1.27 to 3.12) versus rails. The adjusted odds of severe injury versus no injury were significantly higher for half-pipe, jumps and kickers compared with rails. After accounting for clustering, the 95% CI width increased marginally but significance did not change.\\nUsing the imputed dataset, there was no evidence of effect modification of feature by age or sex (p=0.41) versus rails, significantly increased adjusted odds of injury on half-pipe (OR 5.88; 95% CI 3.25 to 10.63) and jumps (OR 4.78; 95% CI 3.22 to 7.11) and significantly decreased odds of injury on quarter-pipes (OR 0.49; 95% CI 0.25 to 0.97).\",\n \"Based on initial RA observation and follow-up confirmation, the RAs correctly classified age-group in 78% (κ=0.71; 95% CI 0.49 to 0.93) and sex in 100% of uninjured snowboarders (κ=1.00; 95% CI 0.69 to 1.00). There was significant agreement for age and sex classification between the RAs (p>0.05 for each of the three pairs). For interview responses, κ=0.72 (95% CI 0.60 to 0.85) for feature use at the time of injury and this improved when follow-up interviews were conducted within 2 weeks (κ=0.86; 95% CI 0.64 to 1.00). The overall κ were ≥0.60 except for body region of the second and third injuries and diagnosis of the second injury.\",\n \"Overall, 62.5% went to the ED. The most commonly injured body regions were the wrist (20%), and head (14%), while the most common injury type was fracture (36%). Table 1 describes feature use among injured and uninjured TP snowboarders.\\nFeatures used by all injured, severely injured snowboarders, and uninjured snowboarders in the terrain park\\n*Weighted for time of day, weekday versus weekend and exposure opportunity.\\nFemales aged 12–17 and >17 had higher rates of injuries than males, and higher rates of severe injuries at all ages (table 2).\\nOverall, sex-specific and age-specific injury rates (per 1000 runs and 95% CI)\\nFeatures that promoted aerial manoeuvres or resulted in a greater drop to the ground typically had higher injury rates than features with a small drop (table 3).\\nTable 3All and severe injury rates (per 1000 feature exposures and 95% CI)FeatureAll injuries* rate (95% CI)Severe injuries* rate (95% CI)Aerial manoeuvre or substantial drop to the ground Half-pipe2.6 (1.5 to 4.0)1.8 (0.9 to 3.0) Jumps2.6 (2.1 to 3.2)1.8 (1.4 to 2.3) Kickers0.6 (0.5 to 0.8)0.4 (0.3 to 0.5) Mushroom0.5 (0.3 to 0.8)0.2 (0.1 to 0.5)Small drop to the ground Boxes0.7 (0.5 to 0.8)0.4 (0.3 to 0.5) Rails0.4 (0.3 to 0.6)0.2 (0.1 to 0.4) Quarter-pipes0.2 (0.1 to 0.4)0.1 (0.1 to 0.3)*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed). Jumps and half-pipe had the highest overall and severe injury rates. Compared with rails, rates of all injuries were significantly higher on the half-pipe, jumps and boxes (table 4).\\nAll and severe injury rates (per 1000 feature exposures and 95% CI)\\n*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed).\\nFeature-specific injury rate ratios and 95% CI\\nN, number; RR, rate ratio.\\n*Four snowboarders could not recall feature used at time of injury and 13 snowboarders were not using a feature at the time of injury but were in the boundaries of the terrain park (TP) for a total of 333 TP injuries.\\n†Two snowboarders could not recall feature used at time of injury and nine snowboarders were not using a feature at the time of injury but were in the boundaries of the TP for a total of 208 severe TP injuries. Bold refers to statistically significant results.\\nBaseline characteristics are presented in table 5. The odds of injury were significantly higher when it was −10°C to 0°C compared with 0–10°C, and at night versus sunny weather. Beginners/novices had significantly lower odds of injury than intermediates, as did those with a previous snowboarding injury, music use, or when it was above 10°C versus 0–10°C. When comparing severely injured with uninjured snowboarders, the same patterns were observed except that music or temperatures above 10°C were not significant. There were no significant differences between severe versus minor injuries.\\nSummary of the characteristics of all injured and uninjured snowboarders (crude OR and 95% CI)\\n*Odds of injury increases for every increase in year.\\n†Compared with no injuries.\\n‡Compared with minor injuries.\\nN, numbers; N/A, not applicable.\\nThe crude associations between injury and feature use showed significantly greater odds of injury for jumps and half-pipe, compared with rails and significantly lower odds of injury for quarter-pipes (table 6). For severe injury versus no injury, the crude odds of severe injury were significantly greater on jumps, half-pipe and kickers compared with rails.\\nAssociation between injury and feature type after controlling for confounders (OR and 95% CI)\\n*Adjusted for previous injury, ability and temperature. Age, sex, listening to music, wearing wrist guards and light and snow conditions did not change any of the feature-specific estimates more than 15%.\\n†Adjusted for music and light. Age, sex, ability, wrist guard use and temperature and snow conditions did not change any of the feature-specific estimates more than 15%.\\nThere were significant increases in the adjusted odds of injury for half-pipe (OR 9.63; 95% CI 4.80 to 19.32), jumps (OR 4.29; 95% CI 2.72 to 6.76), mushroom (OR 2.30; 95% CI 1.20 to 4.41) and kickers (OR 1.99; 95% CI 1.27 to 3.12) versus rails. The adjusted odds of severe injury versus no injury were significantly higher for half-pipe, jumps and kickers compared with rails. After accounting for clustering, the 95% CI width increased marginally but significance did not change.\\nUsing the imputed dataset, there was no evidence of effect modification of feature by age or sex (p=0.41) versus rails, significantly increased adjusted odds of injury on half-pipe (OR 5.88; 95% CI 3.25 to 10.63) and jumps (OR 4.78; 95% CI 3.22 to 7.11) and significantly decreased odds of injury on quarter-pipes (OR 0.49; 95% CI 0.25 to 0.97).\",\n \"To our knowledge this is the first study to examine feature-specific injury rates and potential risk factors for TP snowboarding injuries. Data collection methods were found to be accurate and reliable for age and sex information. There was ‘substantial’ to ‘perfect’ agreement for most risk factors for injured snowboarders, including feature use.27 Self-reported risk factors were confirmed with reliable sources where possible. The overall injury rate of TP snowboarders was estimated at 0.75/1000 runs. Feature-specific injury rates were higher for features that supported aerial manoeuvres or a large drop to the ground. Aerial features facilitate more air time and we hypothesise that snowboarders may have more opportunity to lose their sense of body position/orientation and land with more force and this increases the likelihood of injury.\\nResearch suggests that TP injuries are more severe than those on regular slopes.15\\n16\\n18 One study reported an overall TP injury rate of 0.62/1000 ski and snowboard days, including both skiers and snowboarders, but only ski patrol injuries.15 If each skier and snowboarder took only one TP run during their day, this rate is lower than our observed rate. It is unknown if the TPs were similar in size or number of features. A literature review34 reported that ski patrol-reported injuries among snowboarders varied from 2.135 to 7.0/361000 outings; however, a TP-specific injury rate or the precise injury definition was not provided.\\nHalf-pipes and jumps significantly predicted injury. Torjussen and Bahr37 found professional snowboarders competing in half-pipe and big air competitions had a significantly higher injury risk compared with giant-slalom, where the snowboarder does not leave the ground.\\nOverall 34% of snowboarders listened to music through a personal music player. The unadjusted result indicated that listening to music reduced the odds of injury. In a laboratory setting, sport students listening to music while wearing a helmet with built-in speakers had similar mean reaction time to peripheral stimulus as those who were wearing a helmet but not listening to music.38 Similar to non-TP research, snowboarding in suboptimal environmental (bad weather/visibility) conditions affected the odds of injury.9 In contrast to previous research,5–8 we found beginners had significantly reduced odds of injury; perhaps beginners realise they are in an environment beyond their skill level and choose easier features or do not use features as intended (eg, not leaving the ground when going over a jump).\\nFuture research should identify and evaluate ways to reduce injuries without sacrificing participation, motor learning or skill development. Injury prevention should focus on risk mitigation. Possible strategies include marking landings, smaller TPs for progression to larger features, marking feature difficulty, controlling speed and slope for take-offs and landings, enforcing TP etiquette rules or reducing the height of aerial features. Further research is needed to develop guidelines regarding optimal TP design. The efficacy of these strategies should be investigated.\\n Limitations Snowboarders who climbed up to reattempt features were counted as one run, which would underestimate the number of TP runs. Injured snowboarders who did not present to the ski patrol or either of the two nearest EDs were missed. This selection bias resulted in rate underestimation and observed associations between feature and injury would be overestimated if missed snowboarders were injured on rails. However, only 47 (14%) of the included snowboarders said they saw the ski patrol and a non-participating healthcare provider, indicating that most sought treatment at a participating ED.\\nThere was potential misclassification by feature if the injured snowboarder could not recall the feature; however, this occurred only four times. Controls may not have correctly reported their feature use when the RA could not see the entire TP and it was not possible to determine the reliability of controls’ feature use. This could have an unpredictable effect on the ORs if it operated differently by feature type. Only age-group and sex of the uninjured controls could be assessed for accuracy and our observations were found to be valid. There was potential for misclassification of severe injury because factors other than severity could predict presentation to the ED, such as parental fear or anxiety influenced by a recent celebrity skiing death.39 It was unknown if non-consenting injured snowboarders presented to a non-study ED and were incorrectly classified as a minor injury. Fortunately, few who saw the ski patrol went to a non-study ED (8%).\\nIt is possible that some important confounders were overlooked, such as first attempt at a new feature, manoeuvre performed, speed, fatigue, height or weight. There may be behavioural confounders, such as peer pressure to attempt difficult features or manoeuvres. However, some of these potential confounders were likely accounted for by other variables such as ability, age and sex. Although this study was conducted at only one resort, the TP layout changed four times during the two seasons and this enhances the generalisability of the results.\\nSnowboarders who climbed up to reattempt features were counted as one run, which would underestimate the number of TP runs. Injured snowboarders who did not present to the ski patrol or either of the two nearest EDs were missed. This selection bias resulted in rate underestimation and observed associations between feature and injury would be overestimated if missed snowboarders were injured on rails. However, only 47 (14%) of the included snowboarders said they saw the ski patrol and a non-participating healthcare provider, indicating that most sought treatment at a participating ED.\\nThere was potential misclassification by feature if the injured snowboarder could not recall the feature; however, this occurred only four times. Controls may not have correctly reported their feature use when the RA could not see the entire TP and it was not possible to determine the reliability of controls’ feature use. This could have an unpredictable effect on the ORs if it operated differently by feature type. Only age-group and sex of the uninjured controls could be assessed for accuracy and our observations were found to be valid. There was potential for misclassification of severe injury because factors other than severity could predict presentation to the ED, such as parental fear or anxiety influenced by a recent celebrity skiing death.39 It was unknown if non-consenting injured snowboarders presented to a non-study ED and were incorrectly classified as a minor injury. Fortunately, few who saw the ski patrol went to a non-study ED (8%).\\nIt is possible that some important confounders were overlooked, such as first attempt at a new feature, manoeuvre performed, speed, fatigue, height or weight. There may be behavioural confounders, such as peer pressure to attempt difficult features or manoeuvres. However, some of these potential confounders were likely accounted for by other variables such as ability, age and sex. Although this study was conducted at only one resort, the TP layout changed four times during the two seasons and this enhances the generalisability of the results.\",\n \"Snowboarders who climbed up to reattempt features were counted as one run, which would underestimate the number of TP runs. Injured snowboarders who did not present to the ski patrol or either of the two nearest EDs were missed. This selection bias resulted in rate underestimation and observed associations between feature and injury would be overestimated if missed snowboarders were injured on rails. However, only 47 (14%) of the included snowboarders said they saw the ski patrol and a non-participating healthcare provider, indicating that most sought treatment at a participating ED.\\nThere was potential misclassification by feature if the injured snowboarder could not recall the feature; however, this occurred only four times. Controls may not have correctly reported their feature use when the RA could not see the entire TP and it was not possible to determine the reliability of controls’ feature use. This could have an unpredictable effect on the ORs if it operated differently by feature type. Only age-group and sex of the uninjured controls could be assessed for accuracy and our observations were found to be valid. There was potential for misclassification of severe injury because factors other than severity could predict presentation to the ED, such as parental fear or anxiety influenced by a recent celebrity skiing death.39 It was unknown if non-consenting injured snowboarders presented to a non-study ED and were incorrectly classified as a minor injury. Fortunately, few who saw the ski patrol went to a non-study ED (8%).\\nIt is possible that some important confounders were overlooked, such as first attempt at a new feature, manoeuvre performed, speed, fatigue, height or weight. There may be behavioural confounders, such as peer pressure to attempt difficult features or manoeuvres. However, some of these potential confounders were likely accounted for by other variables such as ability, age and sex. Although this study was conducted at only one resort, the TP layout changed four times during the two seasons and this enhances the generalisability of the results.\",\n \"Feature-specific injury rates ranged from 2.56 injuries/1000 runs (jumps and half-pipe) to 0.24 injuries/1000 runs (quarter-pipe). Half-pipe, jumps and kickers were significant risk factors for any injury and severe injury. Recommendations have been made for prevention strategy development to reduce TP injury risk. These strategies will require rigorous evaluation.\\nWhat are the new findings?In this study, the overall injury rate for snowboarding terrain park (TP) injuries is 0.75 injuries/1000 runs.The injury rates are highest for jumps (2.56/1000 runs) and half-pipe (2.56/1000 runs) and lowest for rails (0.43/1000 runs) and quarter-pipes (0.24/1000 runs).Compared with rails, the odds of injury is significantly higher on the half-pipe (OR 9.63; 95% CI 4.80 to 19.32) and jumps (OR 4.29; 95% CI 2.72 to 6.76).\\nHow might it impact on clinical practice in the near future?Terrain parks (TPs) are popular and the majority of snowboarders injured in the TP present to the emergency department.With the identification of potential risk factors for TP injuries among snowboarders, injury prevention programmes can be tailored to those at greatest risk of injury.Should these programmes be effective, clinicians can expect to treat fewer snowboarders injured in the TP.\\nIn this study, the overall injury rate for snowboarding terrain park (TP) injuries is 0.75 injuries/1000 runs.\\nThe injury rates are highest for jumps (2.56/1000 runs) and half-pipe (2.56/1000 runs) and lowest for rails (0.43/1000 runs) and quarter-pipes (0.24/1000 runs).\\nCompared with rails, the odds of injury is significantly higher on the half-pipe (OR 9.63; 95% CI 4.80 to 19.32) and jumps (OR 4.29; 95% CI 2.72 to 6.76).\\nTerrain parks (TPs) are popular and the majority of snowboarders injured in the TP present to the emergency department.\\nWith the identification of potential risk factors for TP injuries among snowboarders, injury prevention programmes can be tailored to those at greatest risk of injury.\\nShould these programmes be effective, clinicians can expect to treat fewer snowboarders injured in the TP.\",\n \"\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,null,"results",null,null,"discussion",null,"conclusions","supplementary-material"],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n \"discussion\",\n null,\n \"conclusions\",\n \"supplementary-material\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["Epidemiology","Injury Prevention"],"string":"[\n \"Epidemiology\",\n \"Injury Prevention\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Introduction:\nSnowboarding is a popular sport1\n2 and the risk of injury is higher for snowboarding than for skiing.3\n4 Being a beginner,5–8 poor weather conditions9 and not wearing protective equipment10–12 increase injury risk. Ski areas often include terrain parks (TPs) with man-made features (eg, jumps, rails and half-pipes) for performing tricks and aerial manoeuvres. In November 2007, Resorts of the Canadian Rockies (RCR) removed all man-made jumps from their TPs because they believed jumps increased injury risk.13 Definitions of common features can be found at: http://www.snowboard-coach.com/freestyle-snowboarding-features.html and in appendix 1.\nBetween 5% and 27% of skiing and snowboarding injuries occur in TPs2\n14–19 and are more severe than regular slope injuries.15\n16\n18 At the 2012 Winter Youth Olympic Games, 35% of all snowboard half-pipe and slope-style competitors were injured.20 Those injured in TPs tend to be snowboarders, male, 13–24 years old, fall from higher heights15 or self-perceived experts.16 There is a dearth of research examining injury rates and intrinsic and extrinsic risk factors for snowboarders in TPs in relation to injury mechanism—a comprehensive approach recommended by sport injury prevention research leaders.21\n22 Therefore, the study objectives were to calculate overall and feature-specific TP injury rates, determine potential risk factors for injury in the TP and assess the reliability of the data collection methods.\n\nMethods:\n Definition of cases and controls This unmatched case-control study was conducted in one Alberta TP during the 2008–2009 and 2009–2010 seasons. There were approximately 290 000 skier–snowboarder visits to the resort annually. Except for the half-pipe and mushroom, the overall TP layout and number of features changed once each season, resulting in four configurations containing all seven feature types. The resort did not assign a difficulty rating to individual features. Helmets were mandatory in the TP.\nCases were snowboarders injured in the TP who presented to either the ski patrol and/or one of two nearby emergency departments (EDs), both Level 1 trauma centres (one adult, one paediatric). Controls were uninjured snowboarders using the same TP. Cases were ‘severe’ if they presented to an ED, and ‘minor’ if they presented to ski patrol only or to ski patrol and a non-emergent healthcare provider. Injuries presenting to the ED represent the public health burden and snowboarders injured in the TPs near hospitals may place a strain on EDs.\nThis unmatched case-control study was conducted in one Alberta TP during the 2008–2009 and 2009–2010 seasons. There were approximately 290 000 skier–snowboarder visits to the resort annually. Except for the half-pipe and mushroom, the overall TP layout and number of features changed once each season, resulting in four configurations containing all seven feature types. The resort did not assign a difficulty rating to individual features. Helmets were mandatory in the TP.\nCases were snowboarders injured in the TP who presented to either the ski patrol and/or one of two nearby emergency departments (EDs), both Level 1 trauma centres (one adult, one paediatric). Controls were uninjured snowboarders using the same TP. Cases were ‘severe’ if they presented to an ED, and ‘minor’ if they presented to ski patrol only or to ski patrol and a non-emergent healthcare provider. Injuries presenting to the ED represent the public health burden and snowboarders injured in the TPs near hospitals may place a strain on EDs.\n Data collection Case data were collected from ski patrol Accident Report Forms (ARF) and ED medical records. ARFs are completed by the ski patrol for anyone injured and presented to them. ARFs record demographics (age, sex and five-point self-reported ability), injured body region, injury type (fracture, dislocation, sprain/strain, bruise/abrasion/laceration and concussion), environment (temperature, light and snow (groomed/ungroomed)) and contact information. ARFs have previously been used in research14\n18\n19\n23–25 and were collected from the resort biweekly. Snowboarders who presented to the ED were identified from the Regional Emergency Department Information System. Following verbal consent (parent/guardian if snowboarder was <14 years) and confirmation they were injured at the TP of interest, additional self-reported data were collected by telephone. The previously listed information was captured for snowboarders presenting only to the ED, along with years of snowboarding and TP experience, listening to music, wearing wrist guards, previous snowboarding injury and feature used (jump, kicker, box, rail, quarter-pipe, half-pipe or mushroom) when injured. If the case presented to the ski patrol and could not be contacted or did not consent to the telephone interview, only the ARF data were included.\nTo collect feature use among controls, trained research assistants (RAs) at the bottom of the hill observed snowboarders' TP runs in 3 h time slots, 3–4 times a week at various times during the day, and recorded feature use on a map. Data were collected each week from 1 January until the resort closed (end of March in Season 1 and mid-April in Season 2) and included all four TP layouts. RAs approached the first snowboarder, obtained consent and asked the same risk factor information as asked to cases. Snowboarders indicated feature use on the map for features not fully visible to the RA. After each interview, the next snowboarder closest to the RA was approached. Temperature (smartphone Weather Network application), light and snow conditions were recorded on an hourly basis.\nInjury rate denominator data were collected in the same 3 h slots. Snowboarder runs were counted by an RA at the top of the TP, which was the only run to the right of the chairlift and only serviced by that chairlift. Age-group (<12, 12–17 or >17) and sex were visually assessed. To determine accuracy of observed age-group and sex classifications, RAs independently estimated the age-group and sex of a snowboarder entering the TP and then approached them to confirm. This was repeated in 10 min intervals for 3 h blocks and included 337 snowboarders.\nEthical approval was granted by the University of Calgary Conjoint Health Research Ethics Board.\nCase data were collected from ski patrol Accident Report Forms (ARF) and ED medical records. ARFs are completed by the ski patrol for anyone injured and presented to them. ARFs record demographics (age, sex and five-point self-reported ability), injured body region, injury type (fracture, dislocation, sprain/strain, bruise/abrasion/laceration and concussion), environment (temperature, light and snow (groomed/ungroomed)) and contact information. ARFs have previously been used in research14\n18\n19\n23–25 and were collected from the resort biweekly. Snowboarders who presented to the ED were identified from the Regional Emergency Department Information System. Following verbal consent (parent/guardian if snowboarder was <14 years) and confirmation they were injured at the TP of interest, additional self-reported data were collected by telephone. The previously listed information was captured for snowboarders presenting only to the ED, along with years of snowboarding and TP experience, listening to music, wearing wrist guards, previous snowboarding injury and feature used (jump, kicker, box, rail, quarter-pipe, half-pipe or mushroom) when injured. If the case presented to the ski patrol and could not be contacted or did not consent to the telephone interview, only the ARF data were included.\nTo collect feature use among controls, trained research assistants (RAs) at the bottom of the hill observed snowboarders' TP runs in 3 h time slots, 3–4 times a week at various times during the day, and recorded feature use on a map. Data were collected each week from 1 January until the resort closed (end of March in Season 1 and mid-April in Season 2) and included all four TP layouts. RAs approached the first snowboarder, obtained consent and asked the same risk factor information as asked to cases. Snowboarders indicated feature use on the map for features not fully visible to the RA. After each interview, the next snowboarder closest to the RA was approached. Temperature (smartphone Weather Network application), light and snow conditions were recorded on an hourly basis.\nInjury rate denominator data were collected in the same 3 h slots. Snowboarder runs were counted by an RA at the top of the TP, which was the only run to the right of the chairlift and only serviced by that chairlift. Age-group (<12, 12–17 or >17) and sex were visually assessed. To determine accuracy of observed age-group and sex classifications, RAs independently estimated the age-group and sex of a snowboarder entering the TP and then approached them to confirm. This was repeated in 10 min intervals for 3 h blocks and included 337 snowboarders.\nEthical approval was granted by the University of Calgary Conjoint Health Research Ethics Board.\n Analysis Reliability Three pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\nThree pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\n Rates Injury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\nInjury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\n Risk factors The distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\nThe distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\n Reliability Three pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\nThree pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\n Rates Injury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\nInjury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\n Risk factors The distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\nThe distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\n\nDefinition of cases and controls:\nThis unmatched case-control study was conducted in one Alberta TP during the 2008–2009 and 2009–2010 seasons. There were approximately 290 000 skier–snowboarder visits to the resort annually. Except for the half-pipe and mushroom, the overall TP layout and number of features changed once each season, resulting in four configurations containing all seven feature types. The resort did not assign a difficulty rating to individual features. Helmets were mandatory in the TP.\nCases were snowboarders injured in the TP who presented to either the ski patrol and/or one of two nearby emergency departments (EDs), both Level 1 trauma centres (one adult, one paediatric). Controls were uninjured snowboarders using the same TP. Cases were ‘severe’ if they presented to an ED, and ‘minor’ if they presented to ski patrol only or to ski patrol and a non-emergent healthcare provider. Injuries presenting to the ED represent the public health burden and snowboarders injured in the TPs near hospitals may place a strain on EDs.\n\nData collection:\nCase data were collected from ski patrol Accident Report Forms (ARF) and ED medical records. ARFs are completed by the ski patrol for anyone injured and presented to them. ARFs record demographics (age, sex and five-point self-reported ability), injured body region, injury type (fracture, dislocation, sprain/strain, bruise/abrasion/laceration and concussion), environment (temperature, light and snow (groomed/ungroomed)) and contact information. ARFs have previously been used in research14\n18\n19\n23–25 and were collected from the resort biweekly. Snowboarders who presented to the ED were identified from the Regional Emergency Department Information System. Following verbal consent (parent/guardian if snowboarder was <14 years) and confirmation they were injured at the TP of interest, additional self-reported data were collected by telephone. The previously listed information was captured for snowboarders presenting only to the ED, along with years of snowboarding and TP experience, listening to music, wearing wrist guards, previous snowboarding injury and feature used (jump, kicker, box, rail, quarter-pipe, half-pipe or mushroom) when injured. If the case presented to the ski patrol and could not be contacted or did not consent to the telephone interview, only the ARF data were included.\nTo collect feature use among controls, trained research assistants (RAs) at the bottom of the hill observed snowboarders' TP runs in 3 h time slots, 3–4 times a week at various times during the day, and recorded feature use on a map. Data were collected each week from 1 January until the resort closed (end of March in Season 1 and mid-April in Season 2) and included all four TP layouts. RAs approached the first snowboarder, obtained consent and asked the same risk factor information as asked to cases. Snowboarders indicated feature use on the map for features not fully visible to the RA. After each interview, the next snowboarder closest to the RA was approached. Temperature (smartphone Weather Network application), light and snow conditions were recorded on an hourly basis.\nInjury rate denominator data were collected in the same 3 h slots. Snowboarder runs were counted by an RA at the top of the TP, which was the only run to the right of the chairlift and only serviced by that chairlift. Age-group (<12, 12–17 or >17) and sex were visually assessed. To determine accuracy of observed age-group and sex classifications, RAs independently estimated the age-group and sex of a snowboarder entering the TP and then approached them to confirm. This was repeated in 10 min intervals for 3 h blocks and included 337 snowboarders.\nEthical approval was granted by the University of Calgary Conjoint Health Research Ethics Board.\n\nAnalysis:\n Reliability Three pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\nThree pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\n Rates Injury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\nInjury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\n Risk factors The distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\nThe distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\n\nReliability:\nThree pairs of RAs independently classified uninjured snowboarders entering the TP by age-group and sex. The Stuart-Maxwell test for overall marginal homogeneity assessed the reliability within the three pairings.26 To determine accuracy of observed age-group and sex classifications, unweighted κ statistics and 95% CIs were calculated.\nAt the end of Season 1, cases interviewed within the last month were reinterviewed using the original questions. To measure reliability, unweighted κ with 95% CIs were calculated for variables without ordering and weighted κ (κw) with 95% CIs for ordinal variables.27\n\nRates:\nInjury rates were presented as injuries per 1000 runs; the numerator was the number of injured snowboarders over the two seasons and the denominator was the estimated total runs. The denominator was extrapolated from the observed number of snowboarders entering the TP by multiplying the number of runs per 3 h time slot by the number of time slots the TP was open each season. This resulted in a denominator that was representative of participation at different times of the day during weekends and weekdays. The severe injury rate numerator was snowboarders who presented to the ED. Age-group, sex and age–sex-specific injury rates were calculated.\nFeature-specific injury and severe injury rates were calculated. The denominator was the total number of runs taken on that type of feature, extrapolated in the aforementioned manner to reflect exposure opportunity. For example, there were seven opportunities to go over a box during one run but only one opportunity to use a mushroom. The 95% CIs were calculated using the Poisson distribution.28\nOverall, sex-specific and age-specific rate ratios and 95% CIs were calculated comparing each feature with rails. Rails were the reference feature as they were still permitted in RCR TPs13 and were hypothesised to have a lower injury rate possibly due to their smaller drop to the ground.\n\nRisk factors:\nThe distributions of potential risk factors between cases and controls, severe cases and controls and severe cases and minor cases were compared using proportions for dichotomous/polytomous risk factors and means with SDs for continuous risk factors. Unadjusted ORs with 95% CIs were calculated.\nLogistic regression was used to calculate the association between injury versus no injury and feature use using a backwards elimination.29 Potential confounders were: age (continuous), sex (male/female), previous injury (yes/no), self-reported ability (beginner-novice/intermediate/advanced/expert), wrist guard use (yes/no), music use (yes/no), temperature (>10C, 0–10C, −10–0C, <−10C), light (sunny/cloud/night) and snow (groomed/ungroomed). These were entered into the model containing feature (jumps, kickers, half-pipe, quarter-pipe, box, mushroom, rails). Whichever confounder produced the smallest change in the feature-specific ORs was removed, provided the change was <15% and repeated until all potential confounders were removed.30 The 95% CIs were adjusted for the clustering effect of multiple feature use within controls.\nTo compare severe injuries with no injuries and severe injuries with minor injuries, forward selection was used because of smaller sample size.31 Potential confounders were added one at a time to the crude model; the confounder that produced the greatest percent change in a feature OR was retained. This was repeated until either the addition of another confounder no longer changed any of the TP feature estimates by >15% or there was one confounder for every 10 cases.31 The 95% CIs were adjusted for clustering effect of multiple feature use within uninjured controls.\nFor the injury versus no injury comparison, a sensitivity analysis using multiple imputation by chained equations with five imputed datasets was conducted32 to address missing data and test for effect modification by age or sex and feature. Feature, age, sex, ability, previous injury, music, wrist guards, temperature, light and snow were used for imputation. Effect modification was assessed using an omnibus test (p>0.05 indicating no evidence of effect modification), and backwards elimination multiple logistic regression was conducted on the imputed data. Analyses were conducted in Stata/SE V.11.33\n\nResults:\nA total of 333 cases (107 ski patrol, 174 ski patrol and ED, 18 ski patrol and healthcare provider and 34 ED only) and 1261 controls were included. The consent rate was 79% for cases and 94% for controls (figure 1).\nRecruitment of included snowboarders.\n Reliability Based on initial RA observation and follow-up confirmation, the RAs correctly classified age-group in 78% (κ=0.71; 95% CI 0.49 to 0.93) and sex in 100% of uninjured snowboarders (κ=1.00; 95% CI 0.69 to 1.00). There was significant agreement for age and sex classification between the RAs (p>0.05 for each of the three pairs). For interview responses, κ=0.72 (95% CI 0.60 to 0.85) for feature use at the time of injury and this improved when follow-up interviews were conducted within 2 weeks (κ=0.86; 95% CI 0.64 to 1.00). The overall κ were ≥0.60 except for body region of the second and third injuries and diagnosis of the second injury.\nBased on initial RA observation and follow-up confirmation, the RAs correctly classified age-group in 78% (κ=0.71; 95% CI 0.49 to 0.93) and sex in 100% of uninjured snowboarders (κ=1.00; 95% CI 0.69 to 1.00). There was significant agreement for age and sex classification between the RAs (p>0.05 for each of the three pairs). For interview responses, κ=0.72 (95% CI 0.60 to 0.85) for feature use at the time of injury and this improved when follow-up interviews were conducted within 2 weeks (κ=0.86; 95% CI 0.64 to 1.00). The overall κ were ≥0.60 except for body region of the second and third injuries and diagnosis of the second injury.\n Injuries Overall, 62.5% went to the ED. The most commonly injured body regions were the wrist (20%), and head (14%), while the most common injury type was fracture (36%). Table 1 describes feature use among injured and uninjured TP snowboarders.\nFeatures used by all injured, severely injured snowboarders, and uninjured snowboarders in the terrain park\n*Weighted for time of day, weekday versus weekend and exposure opportunity.\nFemales aged 12–17 and >17 had higher rates of injuries than males, and higher rates of severe injuries at all ages (table 2).\nOverall, sex-specific and age-specific injury rates (per 1000 runs and 95% CI)\nFeatures that promoted aerial manoeuvres or resulted in a greater drop to the ground typically had higher injury rates than features with a small drop (table 3).\nTable 3All and severe injury rates (per 1000 feature exposures and 95% CI)FeatureAll injuries* rate (95% CI)Severe injuries* rate (95% CI)Aerial manoeuvre or substantial drop to the ground Half-pipe2.6 (1.5 to 4.0)1.8 (0.9 to 3.0) Jumps2.6 (2.1 to 3.2)1.8 (1.4 to 2.3) Kickers0.6 (0.5 to 0.8)0.4 (0.3 to 0.5) Mushroom0.5 (0.3 to 0.8)0.2 (0.1 to 0.5)Small drop to the ground Boxes0.7 (0.5 to 0.8)0.4 (0.3 to 0.5) Rails0.4 (0.3 to 0.6)0.2 (0.1 to 0.4) Quarter-pipes0.2 (0.1 to 0.4)0.1 (0.1 to 0.3)*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed). Jumps and half-pipe had the highest overall and severe injury rates. Compared with rails, rates of all injuries were significantly higher on the half-pipe, jumps and boxes (table 4).\nAll and severe injury rates (per 1000 feature exposures and 95% CI)\n*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed).\nFeature-specific injury rate ratios and 95% CI\nN, number; RR, rate ratio.\n*Four snowboarders could not recall feature used at time of injury and 13 snowboarders were not using a feature at the time of injury but were in the boundaries of the terrain park (TP) for a total of 333 TP injuries.\n†Two snowboarders could not recall feature used at time of injury and nine snowboarders were not using a feature at the time of injury but were in the boundaries of the TP for a total of 208 severe TP injuries. Bold refers to statistically significant results.\nBaseline characteristics are presented in table 5. The odds of injury were significantly higher when it was −10°C to 0°C compared with 0–10°C, and at night versus sunny weather. Beginners/novices had significantly lower odds of injury than intermediates, as did those with a previous snowboarding injury, music use, or when it was above 10°C versus 0–10°C. When comparing severely injured with uninjured snowboarders, the same patterns were observed except that music or temperatures above 10°C were not significant. There were no significant differences between severe versus minor injuries.\nSummary of the characteristics of all injured and uninjured snowboarders (crude OR and 95% CI)\n*Odds of injury increases for every increase in year.\n†Compared with no injuries.\n‡Compared with minor injuries.\nN, numbers; N/A, not applicable.\nThe crude associations between injury and feature use showed significantly greater odds of injury for jumps and half-pipe, compared with rails and significantly lower odds of injury for quarter-pipes (table 6). For severe injury versus no injury, the crude odds of severe injury were significantly greater on jumps, half-pipe and kickers compared with rails.\nAssociation between injury and feature type after controlling for confounders (OR and 95% CI)\n*Adjusted for previous injury, ability and temperature. Age, sex, listening to music, wearing wrist guards and light and snow conditions did not change any of the feature-specific estimates more than 15%.\n†Adjusted for music and light. Age, sex, ability, wrist guard use and temperature and snow conditions did not change any of the feature-specific estimates more than 15%.\nThere were significant increases in the adjusted odds of injury for half-pipe (OR 9.63; 95% CI 4.80 to 19.32), jumps (OR 4.29; 95% CI 2.72 to 6.76), mushroom (OR 2.30; 95% CI 1.20 to 4.41) and kickers (OR 1.99; 95% CI 1.27 to 3.12) versus rails. The adjusted odds of severe injury versus no injury were significantly higher for half-pipe, jumps and kickers compared with rails. After accounting for clustering, the 95% CI width increased marginally but significance did not change.\nUsing the imputed dataset, there was no evidence of effect modification of feature by age or sex (p=0.41) versus rails, significantly increased adjusted odds of injury on half-pipe (OR 5.88; 95% CI 3.25 to 10.63) and jumps (OR 4.78; 95% CI 3.22 to 7.11) and significantly decreased odds of injury on quarter-pipes (OR 0.49; 95% CI 0.25 to 0.97).\nOverall, 62.5% went to the ED. The most commonly injured body regions were the wrist (20%), and head (14%), while the most common injury type was fracture (36%). Table 1 describes feature use among injured and uninjured TP snowboarders.\nFeatures used by all injured, severely injured snowboarders, and uninjured snowboarders in the terrain park\n*Weighted for time of day, weekday versus weekend and exposure opportunity.\nFemales aged 12–17 and >17 had higher rates of injuries than males, and higher rates of severe injuries at all ages (table 2).\nOverall, sex-specific and age-specific injury rates (per 1000 runs and 95% CI)\nFeatures that promoted aerial manoeuvres or resulted in a greater drop to the ground typically had higher injury rates than features with a small drop (table 3).\nTable 3All and severe injury rates (per 1000 feature exposures and 95% CI)FeatureAll injuries* rate (95% CI)Severe injuries* rate (95% CI)Aerial manoeuvre or substantial drop to the ground Half-pipe2.6 (1.5 to 4.0)1.8 (0.9 to 3.0) Jumps2.6 (2.1 to 3.2)1.8 (1.4 to 2.3) Kickers0.6 (0.5 to 0.8)0.4 (0.3 to 0.5) Mushroom0.5 (0.3 to 0.8)0.2 (0.1 to 0.5)Small drop to the ground Boxes0.7 (0.5 to 0.8)0.4 (0.3 to 0.5) Rails0.4 (0.3 to 0.6)0.2 (0.1 to 0.4) Quarter-pipes0.2 (0.1 to 0.4)0.1 (0.1 to 0.3)*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed). Jumps and half-pipe had the highest overall and severe injury rates. Compared with rails, rates of all injuries were significantly higher on the half-pipe, jumps and boxes (table 4).\nAll and severe injury rates (per 1000 feature exposures and 95% CI)\n*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed).\nFeature-specific injury rate ratios and 95% CI\nN, number; RR, rate ratio.\n*Four snowboarders could not recall feature used at time of injury and 13 snowboarders were not using a feature at the time of injury but were in the boundaries of the terrain park (TP) for a total of 333 TP injuries.\n†Two snowboarders could not recall feature used at time of injury and nine snowboarders were not using a feature at the time of injury but were in the boundaries of the TP for a total of 208 severe TP injuries. Bold refers to statistically significant results.\nBaseline characteristics are presented in table 5. The odds of injury were significantly higher when it was −10°C to 0°C compared with 0–10°C, and at night versus sunny weather. Beginners/novices had significantly lower odds of injury than intermediates, as did those with a previous snowboarding injury, music use, or when it was above 10°C versus 0–10°C. When comparing severely injured with uninjured snowboarders, the same patterns were observed except that music or temperatures above 10°C were not significant. There were no significant differences between severe versus minor injuries.\nSummary of the characteristics of all injured and uninjured snowboarders (crude OR and 95% CI)\n*Odds of injury increases for every increase in year.\n†Compared with no injuries.\n‡Compared with minor injuries.\nN, numbers; N/A, not applicable.\nThe crude associations between injury and feature use showed significantly greater odds of injury for jumps and half-pipe, compared with rails and significantly lower odds of injury for quarter-pipes (table 6). For severe injury versus no injury, the crude odds of severe injury were significantly greater on jumps, half-pipe and kickers compared with rails.\nAssociation between injury and feature type after controlling for confounders (OR and 95% CI)\n*Adjusted for previous injury, ability and temperature. Age, sex, listening to music, wearing wrist guards and light and snow conditions did not change any of the feature-specific estimates more than 15%.\n†Adjusted for music and light. Age, sex, ability, wrist guard use and temperature and snow conditions did not change any of the feature-specific estimates more than 15%.\nThere were significant increases in the adjusted odds of injury for half-pipe (OR 9.63; 95% CI 4.80 to 19.32), jumps (OR 4.29; 95% CI 2.72 to 6.76), mushroom (OR 2.30; 95% CI 1.20 to 4.41) and kickers (OR 1.99; 95% CI 1.27 to 3.12) versus rails. The adjusted odds of severe injury versus no injury were significantly higher for half-pipe, jumps and kickers compared with rails. After accounting for clustering, the 95% CI width increased marginally but significance did not change.\nUsing the imputed dataset, there was no evidence of effect modification of feature by age or sex (p=0.41) versus rails, significantly increased adjusted odds of injury on half-pipe (OR 5.88; 95% CI 3.25 to 10.63) and jumps (OR 4.78; 95% CI 3.22 to 7.11) and significantly decreased odds of injury on quarter-pipes (OR 0.49; 95% CI 0.25 to 0.97).\n\nReliability:\nBased on initial RA observation and follow-up confirmation, the RAs correctly classified age-group in 78% (κ=0.71; 95% CI 0.49 to 0.93) and sex in 100% of uninjured snowboarders (κ=1.00; 95% CI 0.69 to 1.00). There was significant agreement for age and sex classification between the RAs (p>0.05 for each of the three pairs). For interview responses, κ=0.72 (95% CI 0.60 to 0.85) for feature use at the time of injury and this improved when follow-up interviews were conducted within 2 weeks (κ=0.86; 95% CI 0.64 to 1.00). The overall κ were ≥0.60 except for body region of the second and third injuries and diagnosis of the second injury.\n\nInjuries:\nOverall, 62.5% went to the ED. The most commonly injured body regions were the wrist (20%), and head (14%), while the most common injury type was fracture (36%). Table 1 describes feature use among injured and uninjured TP snowboarders.\nFeatures used by all injured, severely injured snowboarders, and uninjured snowboarders in the terrain park\n*Weighted for time of day, weekday versus weekend and exposure opportunity.\nFemales aged 12–17 and >17 had higher rates of injuries than males, and higher rates of severe injuries at all ages (table 2).\nOverall, sex-specific and age-specific injury rates (per 1000 runs and 95% CI)\nFeatures that promoted aerial manoeuvres or resulted in a greater drop to the ground typically had higher injury rates than features with a small drop (table 3).\nTable 3All and severe injury rates (per 1000 feature exposures and 95% CI)FeatureAll injuries* rate (95% CI)Severe injuries* rate (95% CI)Aerial manoeuvre or substantial drop to the ground Half-pipe2.6 (1.5 to 4.0)1.8 (0.9 to 3.0) Jumps2.6 (2.1 to 3.2)1.8 (1.4 to 2.3) Kickers0.6 (0.5 to 0.8)0.4 (0.3 to 0.5) Mushroom0.5 (0.3 to 0.8)0.2 (0.1 to 0.5)Small drop to the ground Boxes0.7 (0.5 to 0.8)0.4 (0.3 to 0.5) Rails0.4 (0.3 to 0.6)0.2 (0.1 to 0.4) Quarter-pipes0.2 (0.1 to 0.4)0.1 (0.1 to 0.3)*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed). Jumps and half-pipe had the highest overall and severe injury rates. Compared with rails, rates of all injuries were significantly higher on the half-pipe, jumps and boxes (table 4).\nAll and severe injury rates (per 1000 feature exposures and 95% CI)\n*Injury rates were adjusted for weekday versus weekend, time of day and exposure opportunity (ie, incorporates days when the terrain park or certain features were closed).\nFeature-specific injury rate ratios and 95% CI\nN, number; RR, rate ratio.\n*Four snowboarders could not recall feature used at time of injury and 13 snowboarders were not using a feature at the time of injury but were in the boundaries of the terrain park (TP) for a total of 333 TP injuries.\n†Two snowboarders could not recall feature used at time of injury and nine snowboarders were not using a feature at the time of injury but were in the boundaries of the TP for a total of 208 severe TP injuries. Bold refers to statistically significant results.\nBaseline characteristics are presented in table 5. The odds of injury were significantly higher when it was −10°C to 0°C compared with 0–10°C, and at night versus sunny weather. Beginners/novices had significantly lower odds of injury than intermediates, as did those with a previous snowboarding injury, music use, or when it was above 10°C versus 0–10°C. When comparing severely injured with uninjured snowboarders, the same patterns were observed except that music or temperatures above 10°C were not significant. There were no significant differences between severe versus minor injuries.\nSummary of the characteristics of all injured and uninjured snowboarders (crude OR and 95% CI)\n*Odds of injury increases for every increase in year.\n†Compared with no injuries.\n‡Compared with minor injuries.\nN, numbers; N/A, not applicable.\nThe crude associations between injury and feature use showed significantly greater odds of injury for jumps and half-pipe, compared with rails and significantly lower odds of injury for quarter-pipes (table 6). For severe injury versus no injury, the crude odds of severe injury were significantly greater on jumps, half-pipe and kickers compared with rails.\nAssociation between injury and feature type after controlling for confounders (OR and 95% CI)\n*Adjusted for previous injury, ability and temperature. Age, sex, listening to music, wearing wrist guards and light and snow conditions did not change any of the feature-specific estimates more than 15%.\n†Adjusted for music and light. Age, sex, ability, wrist guard use and temperature and snow conditions did not change any of the feature-specific estimates more than 15%.\nThere were significant increases in the adjusted odds of injury for half-pipe (OR 9.63; 95% CI 4.80 to 19.32), jumps (OR 4.29; 95% CI 2.72 to 6.76), mushroom (OR 2.30; 95% CI 1.20 to 4.41) and kickers (OR 1.99; 95% CI 1.27 to 3.12) versus rails. The adjusted odds of severe injury versus no injury were significantly higher for half-pipe, jumps and kickers compared with rails. After accounting for clustering, the 95% CI width increased marginally but significance did not change.\nUsing the imputed dataset, there was no evidence of effect modification of feature by age or sex (p=0.41) versus rails, significantly increased adjusted odds of injury on half-pipe (OR 5.88; 95% CI 3.25 to 10.63) and jumps (OR 4.78; 95% CI 3.22 to 7.11) and significantly decreased odds of injury on quarter-pipes (OR 0.49; 95% CI 0.25 to 0.97).\n\nDiscussion:\nTo our knowledge this is the first study to examine feature-specific injury rates and potential risk factors for TP snowboarding injuries. Data collection methods were found to be accurate and reliable for age and sex information. There was ‘substantial’ to ‘perfect’ agreement for most risk factors for injured snowboarders, including feature use.27 Self-reported risk factors were confirmed with reliable sources where possible. The overall injury rate of TP snowboarders was estimated at 0.75/1000 runs. Feature-specific injury rates were higher for features that supported aerial manoeuvres or a large drop to the ground. Aerial features facilitate more air time and we hypothesise that snowboarders may have more opportunity to lose their sense of body position/orientation and land with more force and this increases the likelihood of injury.\nResearch suggests that TP injuries are more severe than those on regular slopes.15\n16\n18 One study reported an overall TP injury rate of 0.62/1000 ski and snowboard days, including both skiers and snowboarders, but only ski patrol injuries.15 If each skier and snowboarder took only one TP run during their day, this rate is lower than our observed rate. It is unknown if the TPs were similar in size or number of features. A literature review34 reported that ski patrol-reported injuries among snowboarders varied from 2.135 to 7.0/361000 outings; however, a TP-specific injury rate or the precise injury definition was not provided.\nHalf-pipes and jumps significantly predicted injury. Torjussen and Bahr37 found professional snowboarders competing in half-pipe and big air competitions had a significantly higher injury risk compared with giant-slalom, where the snowboarder does not leave the ground.\nOverall 34% of snowboarders listened to music through a personal music player. The unadjusted result indicated that listening to music reduced the odds of injury. In a laboratory setting, sport students listening to music while wearing a helmet with built-in speakers had similar mean reaction time to peripheral stimulus as those who were wearing a helmet but not listening to music.38 Similar to non-TP research, snowboarding in suboptimal environmental (bad weather/visibility) conditions affected the odds of injury.9 In contrast to previous research,5–8 we found beginners had significantly reduced odds of injury; perhaps beginners realise they are in an environment beyond their skill level and choose easier features or do not use features as intended (eg, not leaving the ground when going over a jump).\nFuture research should identify and evaluate ways to reduce injuries without sacrificing participation, motor learning or skill development. Injury prevention should focus on risk mitigation. Possible strategies include marking landings, smaller TPs for progression to larger features, marking feature difficulty, controlling speed and slope for take-offs and landings, enforcing TP etiquette rules or reducing the height of aerial features. Further research is needed to develop guidelines regarding optimal TP design. The efficacy of these strategies should be investigated.\n Limitations Snowboarders who climbed up to reattempt features were counted as one run, which would underestimate the number of TP runs. Injured snowboarders who did not present to the ski patrol or either of the two nearest EDs were missed. This selection bias resulted in rate underestimation and observed associations between feature and injury would be overestimated if missed snowboarders were injured on rails. However, only 47 (14%) of the included snowboarders said they saw the ski patrol and a non-participating healthcare provider, indicating that most sought treatment at a participating ED.\nThere was potential misclassification by feature if the injured snowboarder could not recall the feature; however, this occurred only four times. Controls may not have correctly reported their feature use when the RA could not see the entire TP and it was not possible to determine the reliability of controls’ feature use. This could have an unpredictable effect on the ORs if it operated differently by feature type. Only age-group and sex of the uninjured controls could be assessed for accuracy and our observations were found to be valid. There was potential for misclassification of severe injury because factors other than severity could predict presentation to the ED, such as parental fear or anxiety influenced by a recent celebrity skiing death.39 It was unknown if non-consenting injured snowboarders presented to a non-study ED and were incorrectly classified as a minor injury. Fortunately, few who saw the ski patrol went to a non-study ED (8%).\nIt is possible that some important confounders were overlooked, such as first attempt at a new feature, manoeuvre performed, speed, fatigue, height or weight. There may be behavioural confounders, such as peer pressure to attempt difficult features or manoeuvres. However, some of these potential confounders were likely accounted for by other variables such as ability, age and sex. Although this study was conducted at only one resort, the TP layout changed four times during the two seasons and this enhances the generalisability of the results.\nSnowboarders who climbed up to reattempt features were counted as one run, which would underestimate the number of TP runs. Injured snowboarders who did not present to the ski patrol or either of the two nearest EDs were missed. This selection bias resulted in rate underestimation and observed associations between feature and injury would be overestimated if missed snowboarders were injured on rails. However, only 47 (14%) of the included snowboarders said they saw the ski patrol and a non-participating healthcare provider, indicating that most sought treatment at a participating ED.\nThere was potential misclassification by feature if the injured snowboarder could not recall the feature; however, this occurred only four times. Controls may not have correctly reported their feature use when the RA could not see the entire TP and it was not possible to determine the reliability of controls’ feature use. This could have an unpredictable effect on the ORs if it operated differently by feature type. Only age-group and sex of the uninjured controls could be assessed for accuracy and our observations were found to be valid. There was potential for misclassification of severe injury because factors other than severity could predict presentation to the ED, such as parental fear or anxiety influenced by a recent celebrity skiing death.39 It was unknown if non-consenting injured snowboarders presented to a non-study ED and were incorrectly classified as a minor injury. Fortunately, few who saw the ski patrol went to a non-study ED (8%).\nIt is possible that some important confounders were overlooked, such as first attempt at a new feature, manoeuvre performed, speed, fatigue, height or weight. There may be behavioural confounders, such as peer pressure to attempt difficult features or manoeuvres. However, some of these potential confounders were likely accounted for by other variables such as ability, age and sex. Although this study was conducted at only one resort, the TP layout changed four times during the two seasons and this enhances the generalisability of the results.\n\nLimitations:\nSnowboarders who climbed up to reattempt features were counted as one run, which would underestimate the number of TP runs. Injured snowboarders who did not present to the ski patrol or either of the two nearest EDs were missed. This selection bias resulted in rate underestimation and observed associations between feature and injury would be overestimated if missed snowboarders were injured on rails. However, only 47 (14%) of the included snowboarders said they saw the ski patrol and a non-participating healthcare provider, indicating that most sought treatment at a participating ED.\nThere was potential misclassification by feature if the injured snowboarder could not recall the feature; however, this occurred only four times. Controls may not have correctly reported their feature use when the RA could not see the entire TP and it was not possible to determine the reliability of controls’ feature use. This could have an unpredictable effect on the ORs if it operated differently by feature type. Only age-group and sex of the uninjured controls could be assessed for accuracy and our observations were found to be valid. There was potential for misclassification of severe injury because factors other than severity could predict presentation to the ED, such as parental fear or anxiety influenced by a recent celebrity skiing death.39 It was unknown if non-consenting injured snowboarders presented to a non-study ED and were incorrectly classified as a minor injury. Fortunately, few who saw the ski patrol went to a non-study ED (8%).\nIt is possible that some important confounders were overlooked, such as first attempt at a new feature, manoeuvre performed, speed, fatigue, height or weight. There may be behavioural confounders, such as peer pressure to attempt difficult features or manoeuvres. However, some of these potential confounders were likely accounted for by other variables such as ability, age and sex. Although this study was conducted at only one resort, the TP layout changed four times during the two seasons and this enhances the generalisability of the results.\n\nConclusion:\nFeature-specific injury rates ranged from 2.56 injuries/1000 runs (jumps and half-pipe) to 0.24 injuries/1000 runs (quarter-pipe). Half-pipe, jumps and kickers were significant risk factors for any injury and severe injury. Recommendations have been made for prevention strategy development to reduce TP injury risk. These strategies will require rigorous evaluation.\nWhat are the new findings?In this study, the overall injury rate for snowboarding terrain park (TP) injuries is 0.75 injuries/1000 runs.The injury rates are highest for jumps (2.56/1000 runs) and half-pipe (2.56/1000 runs) and lowest for rails (0.43/1000 runs) and quarter-pipes (0.24/1000 runs).Compared with rails, the odds of injury is significantly higher on the half-pipe (OR 9.63; 95% CI 4.80 to 19.32) and jumps (OR 4.29; 95% CI 2.72 to 6.76).\nHow might it impact on clinical practice in the near future?Terrain parks (TPs) are popular and the majority of snowboarders injured in the TP present to the emergency department.With the identification of potential risk factors for TP injuries among snowboarders, injury prevention programmes can be tailored to those at greatest risk of injury.Should these programmes be effective, clinicians can expect to treat fewer snowboarders injured in the TP.\nIn this study, the overall injury rate for snowboarding terrain park (TP) injuries is 0.75 injuries/1000 runs.\nThe injury rates are highest for jumps (2.56/1000 runs) and half-pipe (2.56/1000 runs) and lowest for rails (0.43/1000 runs) and quarter-pipes (0.24/1000 runs).\nCompared with rails, the odds of injury is significantly higher on the half-pipe (OR 9.63; 95% CI 4.80 to 19.32) and jumps (OR 4.29; 95% CI 2.72 to 6.76).\nTerrain parks (TPs) are popular and the majority of snowboarders injured in the TP present to the emergency department.\nWith the identification of potential risk factors for TP injuries among snowboarders, injury prevention programmes can be tailored to those at greatest risk of injury.\nShould these programmes be effective, clinicians can expect to treat fewer snowboarders injured in the TP.\n\nSupplementary Material:\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nSnowboarding is a popular albeit risky sport and terrain park (TP) injuries are more severe than regular slope injuries. TPs contain man-made features that facilitate aerial manoeuvres. The objectives of this study were to determine overall and feature-specific injury rates and the potential risk factors for TP injuries.\n\nMethods:\nCase-control study with exposure estimation, conducted in an Alberta TP during two ski seasons. Cases were snowboarders injured in the TP who presented to ski patrol and/or local emergency departments. Controls were uninjured snowboarders in the same TP. κ Statistics were used to measure the reliability of reported risk factor information. Injury rates were calculated and adjusted logistic regression was used to calculate the feature-specific odds of injury.\n\nResults:\nOverall, 333 cases and 1261 controls were enrolled. Reliability of risk factor information was κ>0.60 for 21/24 variables. The overall injury rate was 0.75/1000 runs. Rates were highest for jumps and half-pipe (both 2.56/1000 runs) and lowest for rails (0.43/1000 runs) and quarter-pipes (0.24/1000 runs). Compared with rails, there were increased odds of injury for half-pipe (OR 9.63; 95% CI 4.80 to 19.32), jumps (OR 4.29; 95% CI 2.72 to 6.76), mushroom (OR 2.30; 95% CI 1.20 to 4.41) and kickers (OR 1.99; 95% CI 1.27 to 3.12).\n\nConclusions:\nHigher feature-specific injury rates and increased odds of injury were associated with features that promote aerial manoeuvres or a large drop to the ground. Further research is required to determine ways to increase snowboarder safety in the TP."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nSnowboarding is a popular sport1\n2 and the risk of injury is higher for snowboarding than for skiing.3\n4 Being a beginner,5–8 poor weather conditions9 and not wearing protective equipment10–12 increase injury risk. Ski areas often include terrain parks (TPs) with man-made features (eg, jumps, rails and half-pipes) for performing tricks and aerial manoeuvres. In November 2007, Resorts of the Canadian Rockies (RCR) removed all man-made jumps from their TPs because they believed jumps increased injury risk.13 Definitions of common features can be found at: http://www.snowboard-coach.com/freestyle-snowboarding-features.html and in appendix 1.\nBetween 5% and 27% of skiing and snowboarding injuries occur in TPs2\n14–19 and are more severe than regular slope injuries.15\n16\n18 At the 2012 Winter Youth Olympic Games, 35% of all snowboard half-pipe and slope-style competitors were injured.20 Those injured in TPs tend to be snowboarders, male, 13–24 years old, fall from higher heights15 or self-perceived experts.16 There is a dearth of research examining injury rates and intrinsic and extrinsic risk factors for snowboarders in TPs in relation to injury mechanism—a comprehensive approach recommended by sport injury prevention research leaders.21\n22 Therefore, the study objectives were to calculate overall and feature-specific TP injury rates, determine potential risk factors for injury in the TP and assess the reliability of the data collection methods.\n\nConclusion:\nFeature-specific injury rates ranged from 2.56 injuries/1000 runs (jumps and half-pipe) to 0.24 injuries/1000 runs (quarter-pipe). Half-pipe, jumps and kickers were significant risk factors for any injury and severe injury. Recommendations have been made for prevention strategy development to reduce TP injury risk. These strategies will require rigorous evaluation.\nWhat are the new findings?In this study, the overall injury rate for snowboarding terrain park (TP) injuries is 0.75 injuries/1000 runs.The injury rates are highest for jumps (2.56/1000 runs) and half-pipe (2.56/1000 runs) and lowest for rails (0.43/1000 runs) and quarter-pipes (0.24/1000 runs).Compared with rails, the odds of injury is significantly higher on the half-pipe (OR 9.63; 95% CI 4.80 to 19.32) and jumps (OR 4.29; 95% CI 2.72 to 6.76).\nHow might it impact on clinical practice in the near future?Terrain parks (TPs) are popular and the majority of snowboarders injured in the TP present to the emergency department.With the identification of potential risk factors for TP injuries among snowboarders, injury prevention programmes can be tailored to those at greatest risk of injury.Should these programmes be effective, clinicians can expect to treat fewer snowboarders injured in the TP.\nIn this study, the overall injury rate for snowboarding terrain park (TP) injuries is 0.75 injuries/1000 runs.\nThe injury rates are highest for jumps (2.56/1000 runs) and half-pipe (2.56/1000 runs) and lowest for rails (0.43/1000 runs) and quarter-pipes (0.24/1000 runs).\nCompared with rails, the odds of injury is significantly higher on the half-pipe (OR 9.63; 95% CI 4.80 to 19.32) and jumps (OR 4.29; 95% CI 2.72 to 6.76).\nTerrain parks (TPs) are popular and the majority of snowboarders injured in the TP present to the emergency department.\nWith the identification of potential risk factors for TP injuries among snowboarders, injury prevention programmes can be tailored to those at greatest risk of injury.\nShould these programmes be effective, clinicians can expect to treat fewer snowboarders injured in the TP."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nSnowboarding is a popular albeit risky sport and terrain park (TP) injuries are more severe than regular slope injuries. TPs contain man-made features that facilitate aerial manoeuvres. The objectives of this study were to determine overall and feature-specific injury rates and the potential risk factors for TP injuries.\n\nMethods:\nCase-control study with exposure estimation, conducted in an Alberta TP during two ski seasons. Cases were snowboarders injured in the TP who presented to ski patrol and/or local emergency departments. Controls were uninjured snowboarders in the same TP. κ Statistics were used to measure the reliability of reported risk factor information. Injury rates were calculated and adjusted logistic regression was used to calculate the feature-specific odds of injury.\n\nResults:\nOverall, 333 cases and 1261 controls were enrolled. Reliability of risk factor information was κ>0.60 for 21/24 variables. The overall injury rate was 0.75/1000 runs. Rates were highest for jumps and half-pipe (both 2.56/1000 runs) and lowest for rails (0.43/1000 runs) and quarter-pipes (0.24/1000 runs). Compared with rails, there were increased odds of injury for half-pipe (OR 9.63; 95% CI 4.80 to 19.32), jumps (OR 4.29; 95% CI 2.72 to 6.76), mushroom (OR 2.30; 95% CI 1.20 to 4.41) and kickers (OR 1.99; 95% CI 1.27 to 3.12).\n\nConclusions:\nHigher feature-specific injury rates and increased odds of injury were associated with features that promote aerial manoeuvres or a large drop to the ground. Further research is required to determine ways to increase snowboarder safety in the TP."},"whole_article_text_length":{"kind":"number","value":13827,"string":"13,827"},"whole_article_abstract_length":{"kind":"number","value":317,"string":"317"},"other_sections_lengths":{"kind":"list like","value":[261,193,539,1591,106,246,437,140,1048,379],"string":"[\n 261,\n 193,\n 539,\n 1591,\n 106,\n 246,\n 437,\n 140,\n 1048,\n 379\n]","isConversational":false},"num_sections":{"kind":"number","value":15,"string":"15"},"most_frequent_words":{"kind":"list like","value":["injury","feature","95","snowboarders","tp","age","sex","injuries","severe","use"],"string":"[\n \"injury\",\n \"feature\",\n \"95\",\n \"snowboarders\",\n \"tp\",\n \"age\",\n \"sex\",\n \"injuries\",\n \"severe\",\n \"use\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["skiing snowboarding injuries","tp injuries snowboarders","snowboarders injury prevention","injury snowboarders feature","injury rate 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| sex | injuries | severe | use [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] injury | feature | 95 | snowboarders | tp | age | sex | injuries | severe | use [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] injury | feature | 95 | snowboarders | tp | age | sex | injuries | severe | use [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] injury | feature | 95 | snowboarders | tp | age | sex | injuries | severe | use [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] injury | risk | tps | snowboarding | man | 16 | snowboard | slope | skiing | injury risk [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] feature | injury | 95 cis | cis | calculated | cases | sex | age | 95 | tp [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 95 ci | ci | injury | 95 | odds | versus | significantly | table | odds injury | feature [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] 1000 runs | 1000 | runs | injury | 56 | programmes | 56 1000 runs | 56 1000 | injuries | tp [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] injury | feature | 95 | snowboarders | 95 ci | ci | tp | sex | age | 95 cis [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] injury | feature | 95 | snowboarders | 95 ci | ci | tp | sex | age | 95 cis [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Alberta TP | two ski seasons ||| ||| ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 333 | 1261 ||| κ>0.60 | 21/24 ||| 0.75/1000 ||| half | 2.56/1000 | 0.43/1000 | quarter | 0.24/1000 ||| half | 9.63 | 95% | CI | 4.80 | 19.32 | 4.29 | 95% | 2.72 | 2.30 | 95% | 1.20 | 4.41 | 1.99 | 95% | CI | 1.27 | 3.12 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| Alberta TP | two ski seasons ||| ||| ||| ||| ||| ||| ||| 333 | 1261 ||| κ>0.60 | 21/24 ||| 0.75/1000 ||| half | 2.56/1000 | 0.43/1000 | quarter | 0.24/1000 ||| half | 9.63 | 95% | CI | 4.80 | 19.32 | 4.29 | 95% | 2.72 | 2.30 | 95% | 1.20 | 4.41 | 1.99 | 95% | CI | 1.27 | 3.12 ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| Alberta TP | two ski seasons ||| ||| ||| ||| ||| ||| ||| 333 | 1261 ||| κ>0.60 | 21/24 ||| 0.75/1000 ||| half | 2.56/1000 | 0.43/1000 | quarter | 0.24/1000 ||| half | 9.63 | 95% | CI | 4.80 | 19.32 | 4.29 | 95% | 2.72 | 2.30 | 95% | 1.20 | 4.41 | 1.99 | 95% | CI | 1.27 | 3.12 ||| ||| [SUMMARY]"}}},{"rowIdx":64,"cells":{"title":{"kind":"string","value":"Traditional medicines and their common uses in central region of Syria: Hama and Homs - an ethnomedicinal survey."},"pmid":{"kind":"string","value":"34165371"},"background_abstract":{"kind":"string","value":"Since ancient times, traditional Arabic medicine (TAM) has been used to treat various diseases in Syria. They are cost-effective with fewer side effects and are more suitable for long-term use compared with chemically synthesized medicines. In addition, the scientific importance is manifested, as this survey proceeds, for the purposes of verifying and documenting these traditional medicines and their common uses."},"background_abstract_label":{"kind":"string","value":"CONTEXT"},"methods_abstract":{"kind":"string","value":"Information was collected from 2019 to 2021 from the cities of Homs and Hama and their villages, which are two governorates located in central Syria, after interviews with traditional practitioners called Attarin, and many other people. Plant specimens were collected according to different references concerning medicinal plants of Syria, to document the traditional uses of plants at least two of the traditional healers and three other people were asked."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"In this survey, we listed 76 medicinal plants belonging to 39 families in alphabetical order with the parts used and the method of preparation according to their therapeutic use, which are used to treat 106 ailments."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Many of the uses of medicinal plants mentioned in this survey are still under study. There is no doubt that this study will provide new data that could contribute to further pharmacological discoveries by identifying the active ingredients and their mechanism of effect by doing additional pharmacological work to confirm the alleged biological activities of these plants."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Aged","Ethnobotany","Ethnopharmacology","Female","Humans","Male","Medicine, Traditional","Middle Aged","Plants, Medicinal","Surveys and Questionnaires","Syria"],"string":"[\n \"Adult\",\n \"Aged\",\n \"Ethnobotany\",\n \"Ethnopharmacology\",\n \"Female\",\n \"Humans\",\n \"Male\",\n \"Medicine, Traditional\",\n \"Middle Aged\",\n \"Plants, Medicinal\",\n \"Surveys and Questionnaires\",\n \"Syria\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"8231352"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Traditional medicine (TM), as defined by the World Health Organization (WHO), is the sum total of the knowledge, skills and practices based on the theories, beliefs and experiences indigenous to different cultures, whether explicable or not, used in the maintenance of health as well as in the prevention, diagnosis, improvement or treatment of physical and mental illness (WHO 2002a, 2002b). Some TM systems are supported by huge volumes of literature and records of the theoretical concepts and practical skills; others pass down from generation to generation through verbal teaching. To date, in some parts of the world, the majority of the population continue to rely on their own TM to meet their primary health care needs. When adopted outside of its traditional culture, TM is often referred as complementary and alternative medicine (CAM) (Che et al. 2017).\nAlthough modern medicine is currently available in many developing countries, large proportions of the population in these countries still rely to a large extent on traditional practitioners and medicinal plants for therapeutic purposes. TM is often the first choice for providing primary health care in developing countries, and the WHO estimates that more than 80% of healthcare needs in these countries are met by traditional health care practices, being the cheapest and most accessible (WHO 2002a, 2002b, 2004, 2005).\nThe WHO pays special attention to TM and CAM. Resolution (no. WHA13-6) issued by the World Health Assembly (WHA) in 2009 emphasized the need to update the global TM strategy (WHO 2009), so the WHO issued its strategy for traditional (folk medicine) 2014–2023 (WHO 2013).\nThe United Nations Educational Scientific and Cultural Organization (UNESCO) works to document the medical heritage of peoples within its documentation of the living intangible cultural heritage of peoples and civilizations, through the documentation of TM practices that were included in the UNESCO convention (2003), the convention on biological diversity (1992) and the UNESCO universal declaration on cultural diversity (2001) and the United Nations declaration on the rights of indigenous peoples (2007), and according to the meeting of the UNESCO International Bioethics Committee (IBC) working group on TM and its ethical effects in Paris (2010), it stressed the need to conduct studies that illustrate the use of TM in various regions around the world, and its evaluation in clinical practice and in research and policy (IBC 2013).\nIn 2017, the International Center for Information and Networks for Intangible Cultural Heritage in the Asia-Pacific region (ICHCAP) under the auspices of UNESCO, issued a book entitled Traditional Medicine in which the Syria Trust for Development in Section VII included TM in Syria (Falk 2017).\nEthno-botany is the scientific study of the relationships between people and plants. It was first coined in 1896 by the US botanist John Harshberger; however, the history of ethnobotany began long before that (Campbell et al. 2002; Amjad et al. 2015). It plays an important role in understanding the dynamic relationships between biological diversity and social and cultural systems (Husain et al. 2008; Amjad et al. 2013, 2015). Plants are essential for human beings as they provide food, and medicines (Alam et al. 2011; Ahmad et al. 2012).\nTraditional Arabic medicine (TAM) is one of the famous traditional medical systems, which is occasionally called Unani medicine, Graeco-Arabic medicine, humoral medicine or Islamic medicine. The subject of TM in Syria has received little attention in the literature, and very little is known about the traditional medicinal substances used nowadays by the Syrian population to treat the most common diseases. Throughout ancient times in Syria, as part of the Levantine Nations (Bilad al-Sham), and other lands in the region, humans used various natural materials as sources of medicines (Jaddouh 2004). In the western countryside of Hama, there is a natural reserve for medicinal plants, which is called the Abu Qubais Protected area in Al-Ghab region (which protect the biodiversity rights of indigenous people and affiliated to the general commission for Al-Ghab administration and development), 509 plant species belonging to 72 families have been recorded (Al-Mahmoud and Al-Shater 2010).\nFor these reasons, the present investigation gathered the uses of medicinal plants in the centre region of Syria (Homs and Hama), as a supplement for a national survey, and documents the information concerning the uses of medicinal plants, which may serve as the basis of knowledge for a more intensive scientific research."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"Study area Syria is a country located on the east coast of the Mediterranean Sea in southwestern Asia. Syria is bounded by Turkey to the north, Iraq to the east and southeast, Jordan to the south and Lebanon to the southwest. The study area is the middle region of Syria including Homs Governorate and Hama Governorate.\nThis area has a Mediterranean climate with a long dry season from May to October. In the extreme northwest, there is some light summer rain. On the western region, summers are hot, with mean daily maximum temperatures ranging from low to mid 80 °F, while the mild winters have daily mean minimum reaching temperatures low level of 50 °F. Only above about 1500 m are the summers relatively cool. In inland the climate becomes arid, with colder winters and hotter summers. In the desert, at Tadmur maximum temperatures in the summer, temperature reaches averages in the ranges of upper 90s to low 100 °F, with extremes in the 110 °F. In rural areas, work takes place according to the seasonal rhythm of agriculture. Women generally share in much of the agricultural labour. Agriculture constitutes an important source of income, and fruits and vegetables including onions, olives and grapes. Commercially important forest plants include: pistachio, which is important for its oil-rich fruit, and plants such as olive trees, grapevines, apricot trees and cumin (Hamidé et al. 2021).\nHistorically, the ancient Palmyra, also called Tadmur, is an ancient city in south-central Syria (Homs Governorate). An oasis in the Syrian desert, Palmyra contains the monumental ruins of a great city that was one of the most important cultural centres of the ancient world, from the first to the second century according to World Heritage List (UNESCO 2021).\nSyria is a country located on the east coast of the Mediterranean Sea in southwestern Asia. Syria is bounded by Turkey to the north, Iraq to the east and southeast, Jordan to the south and Lebanon to the southwest. The study area is the middle region of Syria including Homs Governorate and Hama Governorate.\nThis area has a Mediterranean climate with a long dry season from May to October. In the extreme northwest, there is some light summer rain. On the western region, summers are hot, with mean daily maximum temperatures ranging from low to mid 80 °F, while the mild winters have daily mean minimum reaching temperatures low level of 50 °F. Only above about 1500 m are the summers relatively cool. In inland the climate becomes arid, with colder winters and hotter summers. In the desert, at Tadmur maximum temperatures in the summer, temperature reaches averages in the ranges of upper 90s to low 100 °F, with extremes in the 110 °F. In rural areas, work takes place according to the seasonal rhythm of agriculture. Women generally share in much of the agricultural labour. Agriculture constitutes an important source of income, and fruits and vegetables including onions, olives and grapes. Commercially important forest plants include: pistachio, which is important for its oil-rich fruit, and plants such as olive trees, grapevines, apricot trees and cumin (Hamidé et al. 2021).\nHistorically, the ancient Palmyra, also called Tadmur, is an ancient city in south-central Syria (Homs Governorate). An oasis in the Syrian desert, Palmyra contains the monumental ruins of a great city that was one of the most important cultural centres of the ancient world, from the first to the second century according to World Heritage List (UNESCO 2021).\nField work and data collection Field surveys were conducted between December 2019 and January 2021, to document ethnobotanical information through oral interviews and designed semi-structured questionnaire. Thirty-five villages and 59 districts were visited for field research; 235 people were contacted and 151 of those with ethno-botanical experience agreed to become our informants, including 35 local herbalists (Tabib Arabi) and 10 physicians (who hold a general medicine specialty). The queries were repeatedly made to increase the reliability of the data; interviews with the men were usually carried out in the ‘Mukhtar’ house where they come together, and with women in their homes, bazaars and gardens. The Syria Trust for Development (which is a national development organization, and has a program called ‘Mashrouie’ that runs innovative microcredit programs that encourage economic growth in disadvantaged areas) helped us in data collecting. The information gathered during the present study included socio-demographic characteristics of the interviewed informants (age, gender) and ethnopharmacological information, including the local and scientific name of the species, local names, plant parts used, modes of use, conservation method, administration mode and toxicity, all documented data were then translated into English and Latin. Information that had been carried to the region from the outside and that was not used or confirmed was not included and recorded (Weckerle et al. 2018). During the interviews, questions about the following were asked to the participants:Name and surnameAge and sexEducational levelAre plants collected in your region?Do you have any contact with plants?Can you show the plants you use in your region?Can you tell the names of local plants you use in your region?In which season do you collect the plants you use in your region?When collecting plant, which parts of the plant do you collect and how do you collect them?Which parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).How do you prepare and administrate the plants’ parts?How did you diagnose the disease (by physician, by traditional healers, self-treating)?How did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\nName and surname\nAge and sex\nEducational level\nAre plants collected in your region?\nDo you have any contact with plants?\nCan you show the plants you use in your region?\nCan you tell the names of local plants you use in your region?\nIn which season do you collect the plants you use in your region?\nWhen collecting plant, which parts of the plant do you collect and how do you collect them?\nWhich parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).\nHow do you prepare and administrate the plants’ parts?\nHow did you diagnose the disease (by physician, by traditional healers, self-treating)?\nHow did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\nOnly the medication of single herbs was included, while mixtures and multiple recipes were disregarded. The answers that were given with doubt to the questions above were not recorded, and we adopted the information that gave 40% frequency in the collected data, and the data that gave less than that were neglected.\nInformation about diseases diagnosed specifically by physicians and traditional healers was approved, while self-treating diseases were referred to as auxiliary in treatment, nutritional uses were clearly indicated, and some plants were used to prevent disease and improve public health. The information about diseases was divided into three groups when analysing the data (group A: acute diseases, group B: chronic diseases, group C: simple symptoms and signs), and the focus was only on groups B and C.\nField surveys were conducted between December 2019 and January 2021, to document ethnobotanical information through oral interviews and designed semi-structured questionnaire. Thirty-five villages and 59 districts were visited for field research; 235 people were contacted and 151 of those with ethno-botanical experience agreed to become our informants, including 35 local herbalists (Tabib Arabi) and 10 physicians (who hold a general medicine specialty). The queries were repeatedly made to increase the reliability of the data; interviews with the men were usually carried out in the ‘Mukhtar’ house where they come together, and with women in their homes, bazaars and gardens. The Syria Trust for Development (which is a national development organization, and has a program called ‘Mashrouie’ that runs innovative microcredit programs that encourage economic growth in disadvantaged areas) helped us in data collecting. The information gathered during the present study included socio-demographic characteristics of the interviewed informants (age, gender) and ethnopharmacological information, including the local and scientific name of the species, local names, plant parts used, modes of use, conservation method, administration mode and toxicity, all documented data were then translated into English and Latin. Information that had been carried to the region from the outside and that was not used or confirmed was not included and recorded (Weckerle et al. 2018). During the interviews, questions about the following were asked to the participants:Name and surnameAge and sexEducational levelAre plants collected in your region?Do you have any contact with plants?Can you show the plants you use in your region?Can you tell the names of local plants you use in your region?In which season do you collect the plants you use in your region?When collecting plant, which parts of the plant do you collect and how do you collect them?Which parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).How do you prepare and administrate the plants’ parts?How did you diagnose the disease (by physician, by traditional healers, self-treating)?How did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\nName and surname\nAge and sex\nEducational level\nAre plants collected in your region?\nDo you have any contact with plants?\nCan you show the plants you use in your region?\nCan you tell the names of local plants you use in your region?\nIn which season do you collect the plants you use in your region?\nWhen collecting plant, which parts of the plant do you collect and how do you collect them?\nWhich parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).\nHow do you prepare and administrate the plants’ parts?\nHow did you diagnose the disease (by physician, by traditional healers, self-treating)?\nHow did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\nOnly the medication of single herbs was included, while mixtures and multiple recipes were disregarded. The answers that were given with doubt to the questions above were not recorded, and we adopted the information that gave 40% frequency in the collected data, and the data that gave less than that were neglected.\nInformation about diseases diagnosed specifically by physicians and traditional healers was approved, while self-treating diseases were referred to as auxiliary in treatment, nutritional uses were clearly indicated, and some plants were used to prevent disease and improve public health. The information about diseases was divided into three groups when analysing the data (group A: acute diseases, group B: chronic diseases, group C: simple symptoms and signs), and the focus was only on groups B and C.\nTaxonomic identification of the species Medicinal plants being mentioned by the Informants were recorded with local names and photographed. Each reported medicinal plant species was gathered, compressed, dehydrated, mounted on herbarium sheets, and identified; the taxonomic identity of the plants was confirmed by Prof. Abdel Aleem Bello (PhD/Botanical Taxonomy) and Dr. Bayan Tiba (PhD/Botanical Taxonomy) Aleppo University. As far as possible, the name of the plants was updated by consulting the latest literature; generic and species names followed the plant list (http://www.theplantlist.org). All voucher specimens have been preserved during documentation and deposited in the Damascus University, Faculty of Pharmacy, Pharmacognosy Labs Herbarium for future reference, serial numbers were taken from (1ch to 76ch) according to its alphabetical order.\nMedicinal plants being mentioned by the Informants were recorded with local names and photographed. Each reported medicinal plant species was gathered, compressed, dehydrated, mounted on herbarium sheets, and identified; the taxonomic identity of the plants was confirmed by Prof. Abdel Aleem Bello (PhD/Botanical Taxonomy) and Dr. Bayan Tiba (PhD/Botanical Taxonomy) Aleppo University. As far as possible, the name of the plants was updated by consulting the latest literature; generic and species names followed the plant list (http://www.theplantlist.org). All voucher specimens have been preserved during documentation and deposited in the Damascus University, Faculty of Pharmacy, Pharmacognosy Labs Herbarium for future reference, serial numbers were taken from (1ch to 76ch) according to its alphabetical order.\nEthics approval and consent to participate The study was approved by the Ethics Committee of the University of Damascus. Before beginning data collection, we obtained verbal informed permission in each case site-wide and then individually before each interview. We also informed informants that it was an academic project and that the investigation was for research purposes only, and not for any financial or other benefits. Informants provided verbal informed consent to participate in this study; they were free to withdraw their information at any time. These informants freely accepted the interview. All steps of research are consistent with Ethnobiology Code of Ethics (ISE 2006).\nThe study was approved by the Ethics Committee of the University of Damascus. Before beginning data collection, we obtained verbal informed permission in each case site-wide and then individually before each interview. We also informed informants that it was an academic project and that the investigation was for research purposes only, and not for any financial or other benefits. Informants provided verbal informed consent to participate in this study; they were free to withdraw their information at any time. These informants freely accepted the interview. All steps of research are consistent with Ethnobiology Code of Ethics (ISE 2006).\nData analysis The data collected through interviews of the informants were classified and examined with the statistical program IBM® SPSS® Statistics 26 (IBM, Armonk, NY), to determine the proportions of different variables such as ethnopharmacological data. Quantitative value indices were analysed using different statistical quantitative tools, i.e., the use reports (UR) of a species, and use value (UV) (Chaachouaya et al. 2021).\nThe data collected through interviews of the informants were classified and examined with the statistical program IBM® SPSS® Statistics 26 (IBM, Armonk, NY), to determine the proportions of different variables such as ethnopharmacological data. Quantitative value indices were analysed using different statistical quantitative tools, i.e., the use reports (UR) of a species, and use value (UV) (Chaachouaya et al. 2021).\nUse value and use reports The UR of a species or its importance in the culture of a community is denoted by its mentioning rate or its mention frequency by informants. The UR of the species of plants being utilized was evaluated using the formula (Tenté et al 2012):\nUR = Ni/n\nwhere Ni is the number of times a particular species was mentioned; n is the total number of times that all species were mentioned.\nThe UV of recorded medicinal plants was determined by applying the following formula (Tabuti et al. 2003):\nUV=∑ URi/N\nwhere ∑ URi is the total number of UR per plants; N is the total of interviewees questioned for given medicinal species.\nThe UV rate will be more important if there are several useful records for a species, implying that the plant is significant, whereas they will be near 0 if there are few reports compared to its use (Yaseen 2015; Chaachouaya et al. 2021).\nThe UR of a species or its importance in the culture of a community is denoted by its mentioning rate or its mention frequency by informants. The UR of the species of plants being utilized was evaluated using the formula (Tenté et al 2012):\nUR = Ni/n\nwhere Ni is the number of times a particular species was mentioned; n is the total number of times that all species were mentioned.\nThe UV of recorded medicinal plants was determined by applying the following formula (Tabuti et al. 2003):\nUV=∑ URi/N\nwhere ∑ URi is the total number of UR per plants; N is the total of interviewees questioned for given medicinal species.\nThe UV rate will be more important if there are several useful records for a species, implying that the plant is significant, whereas they will be near 0 if there are few reports compared to its use (Yaseen 2015; Chaachouaya et al. 2021)."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Demographic data of informants In total, 151 local inhabitants of 35 villages and 59 districts were selected based on their experience in traditional uses of plants. Table 1 shows the age and gender wise distribution. All of them were interrogated using semi-structured questionnaires. Generally in Syria, both genders were interested in herbal medicines.\nAge and gender distribution.\nIn total, 151 local inhabitants of 35 villages and 59 districts were selected based on their experience in traditional uses of plants. Table 1 shows the age and gender wise distribution. All of them were interrogated using semi-structured questionnaires. Generally in Syria, both genders were interested in herbal medicines.\nAge and gender distribution.\nEthnobotanical and ethnomedicinal uses of plant species The study area is considered one of the Syrian areas rich in medicinal plants. In the east of it there is a desert (badia), which is dominated by desert and thorny plants, and in the west of it, aromatic plants are spread in the mountains near the eastern coast of the Mediterranean Sea. It is a fact that people in some of these rural areas suffer from poverty, so they depend a lot on folk remedies, and folk healers in these areas provide their expertise at small costs, because the medicinal herbs are cheaper than chemical medicines, and most of the medicinal recipes are available around. However, a large portion of the uses of medicinal plants mentioned in the research are still under study.\nA total of 76 medicinal plant species (57.9% are wild and 42.1% are cultivated plants) belonging to 39 families are recorded in the present study; they are being used for a variety of purposes by native people. The detailed inventory is provided in Table 2, which includes botanical names, followed by local name, family and ethnobotanical uses.\nEthnobotanical uses of plant species according to ethnomedicinal survey of central region in Syria (Homs and Hama).\nAp: aerial parts; Bk: bark; Br: branches; Bu: buds; Bb: bulb; Ca: capitulum; Co: cones; Fl: flower; F: fruit; G: gum; Lx: latex; L: leaves; Ps: pedicles; Pe: peel; Ph: phloem; P: pods; Rs: resin; Rm: rhizome; R: root; Sd: seeds; S: stem; St: stylus; T: tubers; W: whole plant.\nC: cultivated plants, W: wild plants.\nHerbarium no.\nThe study area is considered one of the Syrian areas rich in medicinal plants. In the east of it there is a desert (badia), which is dominated by desert and thorny plants, and in the west of it, aromatic plants are spread in the mountains near the eastern coast of the Mediterranean Sea. It is a fact that people in some of these rural areas suffer from poverty, so they depend a lot on folk remedies, and folk healers in these areas provide their expertise at small costs, because the medicinal herbs are cheaper than chemical medicines, and most of the medicinal recipes are available around. However, a large portion of the uses of medicinal plants mentioned in the research are still under study.\nA total of 76 medicinal plant species (57.9% are wild and 42.1% are cultivated plants) belonging to 39 families are recorded in the present study; they are being used for a variety of purposes by native people. The detailed inventory is provided in Table 2, which includes botanical names, followed by local name, family and ethnobotanical uses.\nEthnobotanical uses of plant species according to ethnomedicinal survey of central region in Syria (Homs and Hama).\nAp: aerial parts; Bk: bark; Br: branches; Bu: buds; Bb: bulb; Ca: capitulum; Co: cones; Fl: flower; F: fruit; G: gum; Lx: latex; L: leaves; Ps: pedicles; Pe: peel; Ph: phloem; P: pods; Rs: resin; Rm: rhizome; R: root; Sd: seeds; S: stem; St: stylus; T: tubers; W: whole plant.\nC: cultivated plants, W: wild plants.\nHerbarium no.\nBotanical families of plants used The most commonly mentioned family is Asteraceae (11.84%), followed by Lamiaceae (10.52%), then Rosaceae (7.89%) and Apiaceae (6.57%), Poaceae (5.26%), Anacardiaceae and Fabaceae (3.94%), Fagaceae, Liliaceae, Myrtaceae and Oleaceae (2.63%), then all the other families (1.31%) (Figure 1).\nPlant families commonly used in ethnomedicinal survey of central region in Syria (Homs and Hama).\nThe most commonly mentioned family is Asteraceae (11.84%), followed by Lamiaceae (10.52%), then Rosaceae (7.89%) and Apiaceae (6.57%), Poaceae (5.26%), Anacardiaceae and Fabaceae (3.94%), Fagaceae, Liliaceae, Myrtaceae and Oleaceae (2.63%), then all the other families (1.31%) (Figure 1).\nPlant families commonly used in ethnomedicinal survey of central region in Syria (Homs and Hama).\nUse value of the plants Medicinal use plants (UV) are utilized to find the most frequently used plant species in the study area. Its value ranged from 0.066 to 0.92 (Table 2). The calculated results of UV showed that Cichorium intybus L., Eucalyptus globulus Labill. was ranked first (UV = 0.92) followed by Fraxinus syriaca Boiss. (UV = 0.907), Olea europaea L. (UV = 0.9), then Allium sativum L. (UV = 0.894), Lepidium sativum L. (UV = 0.867), Coriandrum sativum L. (UV = 0.86), Glycyrrhiza glabra L. (UV = 0.854), Dittrichia viscosa (L.) Greuter (UV = 0.841), while the lowest value was found for Plumbago europaea L. (UV = 0.066).\nMedicinal use plants (UV) are utilized to find the most frequently used plant species in the study area. Its value ranged from 0.066 to 0.92 (Table 2). The calculated results of UV showed that Cichorium intybus L., Eucalyptus globulus Labill. was ranked first (UV = 0.92) followed by Fraxinus syriaca Boiss. (UV = 0.907), Olea europaea L. (UV = 0.9), then Allium sativum L. (UV = 0.894), Lepidium sativum L. (UV = 0.867), Coriandrum sativum L. (UV = 0.86), Glycyrrhiza glabra L. (UV = 0.854), Dittrichia viscosa (L.) Greuter (UV = 0.841), while the lowest value was found for Plumbago europaea L. (UV = 0.066).\nMedicinal parts of the plant used The analysis of the ethnobotanical data showed that central region was best suited to the medicinal plant and rangeland. Ethnobotanical use categories showed that leaves were commonly used parts for making indigenous recipes (28.08%), followed by fruits (19.76%) and seeds (15.6%) then roots (10.4%), flowers (9.36%) and aerial parts (4.16%). Then, the others parts of plant are rarely used (Figure 2).\nMedicinal parts of the plants used for ethnomedicinal purposes in the study.\nThe analysis of the ethnobotanical data showed that central region was best suited to the medicinal plant and rangeland. Ethnobotanical use categories showed that leaves were commonly used parts for making indigenous recipes (28.08%), followed by fruits (19.76%) and seeds (15.6%) then roots (10.4%), flowers (9.36%) and aerial parts (4.16%). Then, the others parts of plant are rarely used (Figure 2).\nMedicinal parts of the plants used for ethnomedicinal purposes in the study.\nModes and conditions of medicine preparation The analysis of the ethnobotanical data showed that the recipes in the most cases were obtained from single herb, but some of recipes were prepared together, and there is a famous local mixture called Damask tisane (zhourate Shamieh). A mode of TM preparation reported was a decoction (30%), followed by infusion (23%), and then by other method such as fresh herbs, juice, cooked, powder, vinegar and oils (47%) (Figure 3). Considering according to results, most of the plant preparations are used orally (Table 2).\nModes of ethnomedicines preparation in Homs and Hama.\nThe analysis of the ethnobotanical data showed that the recipes in the most cases were obtained from single herb, but some of recipes were prepared together, and there is a famous local mixture called Damask tisane (zhourate Shamieh). A mode of TM preparation reported was a decoction (30%), followed by infusion (23%), and then by other method such as fresh herbs, juice, cooked, powder, vinegar and oils (47%) (Figure 3). Considering according to results, most of the plant preparations are used orally (Table 2).\nModes of ethnomedicines preparation in Homs and Hama.\nEthnomedicinal information about treatment the different diseases The results of questionnaires showed that 20% of the informants were diagnosed with their diseases by a doctor, and 45% were diagnosed with a conventional therapist, and 35% self-diagnosed their diseases, while the results of the questionnaires showed that the evaluation of the treatment by informants as following (58% relied on the disappearance of symptoms, and 24% through the results of laboratory analysis, 18% adopted other methods such as chest radiography, adopting the attending physician’s opinion and clinical observation of the improvement of skin diseases, and some of them depended on psychological comfort during treatment as evidence of improvement).\nAmong these studied plants, 62 are used to treat digestive disorders, 41 for respiratory diseases, including asthma, bronchitis and coughs, 40 for skin diseases, 16 for diabetes, 36 for kidney and urinary tract disorders, 22 for nervous system disorders, six for enhance the body's immunity, two for haemorrhoids, five for fever, eight for heart disorders, five for infertility and impotence, six for treating several types of cancer, two for increasing breast milk production, five for losing weight, four for lowering cholesterol, and two for increasing weight, and six for anaemia, 15 for blood disorder, two anti-toxicant, 19 for arthritis and pain, one for typhoid disorder, eight for infections, six for gynaecological diseases, one for eye inflammation, two anti-toxicant and four for mouth sores. Many of them are still used today, especially those plants recommended for internal uses such as traditional medicinal teas, which mainly consist of remedies for obesity, weight loss, colds, colds, digestive disorders, abdominal pain, constipation and some skin diseases (Figure 4).\nEthno-medicinal information about treating different diseases related to central region in Syria (Homs and Hama).\nThe results of questionnaires showed that 20% of the informants were diagnosed with their diseases by a doctor, and 45% were diagnosed with a conventional therapist, and 35% self-diagnosed their diseases, while the results of the questionnaires showed that the evaluation of the treatment by informants as following (58% relied on the disappearance of symptoms, and 24% through the results of laboratory analysis, 18% adopted other methods such as chest radiography, adopting the attending physician’s opinion and clinical observation of the improvement of skin diseases, and some of them depended on psychological comfort during treatment as evidence of improvement).\nAmong these studied plants, 62 are used to treat digestive disorders, 41 for respiratory diseases, including asthma, bronchitis and coughs, 40 for skin diseases, 16 for diabetes, 36 for kidney and urinary tract disorders, 22 for nervous system disorders, six for enhance the body's immunity, two for haemorrhoids, five for fever, eight for heart disorders, five for infertility and impotence, six for treating several types of cancer, two for increasing breast milk production, five for losing weight, four for lowering cholesterol, and two for increasing weight, and six for anaemia, 15 for blood disorder, two anti-toxicant, 19 for arthritis and pain, one for typhoid disorder, eight for infections, six for gynaecological diseases, one for eye inflammation, two anti-toxicant and four for mouth sores. Many of them are still used today, especially those plants recommended for internal uses such as traditional medicinal teas, which mainly consist of remedies for obesity, weight loss, colds, colds, digestive disorders, abdominal pain, constipation and some skin diseases (Figure 4).\nEthno-medicinal information about treating different diseases related to central region in Syria (Homs and Hama)."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"Many of the uses of medicinal plants mentioned in Syria are still under study. This study has been conducted with the aim to generate new concepts that could supplement pharmacological work with potentially further pharmacological discoveries; that is, by identifying the active ingredients and their mechanism of effect to confirm the alleged biological activities of these plants."},"other_sections_titles":{"kind":"list like","value":["Study area","Field work and data collection","Taxonomic identification of the species","Ethics approval and consent to participate","Data analysis","Use value and use reports","Demographic data of informants","Ethnobotanical and ethnomedicinal uses of plant species","Botanical families of plants used","Use value of the plants","Medicinal parts of the plant used","Modes and conditions of medicine preparation","Ethnomedicinal information about treatment the different diseases","Limitations"],"string":"[\n \"Study area\",\n \"Field work and data collection\",\n \"Taxonomic identification of the species\",\n \"Ethics approval and consent to participate\",\n \"Data analysis\",\n \"Use value and use reports\",\n \"Demographic data of informants\",\n \"Ethnobotanical and ethnomedicinal uses of plant species\",\n \"Botanical families of plants used\",\n \"Use value of the plants\",\n \"Medicinal parts of the plant used\",\n \"Modes and conditions of medicine preparation\",\n \"Ethnomedicinal information about treatment the different diseases\",\n \"Limitations\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Syria is a country located on the east coast of the Mediterranean Sea in southwestern Asia. Syria is bounded by Turkey to the north, Iraq to the east and southeast, Jordan to the south and Lebanon to the southwest. The study area is the middle region of Syria including Homs Governorate and Hama Governorate.\nThis area has a Mediterranean climate with a long dry season from May to October. In the extreme northwest, there is some light summer rain. On the western region, summers are hot, with mean daily maximum temperatures ranging from low to mid 80 °F, while the mild winters have daily mean minimum reaching temperatures low level of 50 °F. Only above about 1500 m are the summers relatively cool. In inland the climate becomes arid, with colder winters and hotter summers. In the desert, at Tadmur maximum temperatures in the summer, temperature reaches averages in the ranges of upper 90s to low 100 °F, with extremes in the 110 °F. In rural areas, work takes place according to the seasonal rhythm of agriculture. Women generally share in much of the agricultural labour. Agriculture constitutes an important source of income, and fruits and vegetables including onions, olives and grapes. Commercially important forest plants include: pistachio, which is important for its oil-rich fruit, and plants such as olive trees, grapevines, apricot trees and cumin (Hamidé et al. 2021).\nHistorically, the ancient Palmyra, also called Tadmur, is an ancient city in south-central Syria (Homs Governorate). An oasis in the Syrian desert, Palmyra contains the monumental ruins of a great city that was one of the most important cultural centres of the ancient world, from the first to the second century according to World Heritage List (UNESCO 2021).","Field surveys were conducted between December 2019 and January 2021, to document ethnobotanical information through oral interviews and designed semi-structured questionnaire. Thirty-five villages and 59 districts were visited for field research; 235 people were contacted and 151 of those with ethno-botanical experience agreed to become our informants, including 35 local herbalists (Tabib Arabi) and 10 physicians (who hold a general medicine specialty). The queries were repeatedly made to increase the reliability of the data; interviews with the men were usually carried out in the ‘Mukhtar’ house where they come together, and with women in their homes, bazaars and gardens. The Syria Trust for Development (which is a national development organization, and has a program called ‘Mashrouie’ that runs innovative microcredit programs that encourage economic growth in disadvantaged areas) helped us in data collecting. The information gathered during the present study included socio-demographic characteristics of the interviewed informants (age, gender) and ethnopharmacological information, including the local and scientific name of the species, local names, plant parts used, modes of use, conservation method, administration mode and toxicity, all documented data were then translated into English and Latin. Information that had been carried to the region from the outside and that was not used or confirmed was not included and recorded (Weckerle et al. 2018). During the interviews, questions about the following were asked to the participants:Name and surnameAge and sexEducational levelAre plants collected in your region?Do you have any contact with plants?Can you show the plants you use in your region?Can you tell the names of local plants you use in your region?In which season do you collect the plants you use in your region?When collecting plant, which parts of the plant do you collect and how do you collect them?Which parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).How do you prepare and administrate the plants’ parts?How did you diagnose the disease (by physician, by traditional healers, self-treating)?How did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\nName and surname\nAge and sex\nEducational level\nAre plants collected in your region?\nDo you have any contact with plants?\nCan you show the plants you use in your region?\nCan you tell the names of local plants you use in your region?\nIn which season do you collect the plants you use in your region?\nWhen collecting plant, which parts of the plant do you collect and how do you collect them?\nWhich parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).\nHow do you prepare and administrate the plants’ parts?\nHow did you diagnose the disease (by physician, by traditional healers, self-treating)?\nHow did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\nOnly the medication of single herbs was included, while mixtures and multiple recipes were disregarded. The answers that were given with doubt to the questions above were not recorded, and we adopted the information that gave 40% frequency in the collected data, and the data that gave less than that were neglected.\nInformation about diseases diagnosed specifically by physicians and traditional healers was approved, while self-treating diseases were referred to as auxiliary in treatment, nutritional uses were clearly indicated, and some plants were used to prevent disease and improve public health. The information about diseases was divided into three groups when analysing the data (group A: acute diseases, group B: chronic diseases, group C: simple symptoms and signs), and the focus was only on groups B and C.","Medicinal plants being mentioned by the Informants were recorded with local names and photographed. Each reported medicinal plant species was gathered, compressed, dehydrated, mounted on herbarium sheets, and identified; the taxonomic identity of the plants was confirmed by Prof. Abdel Aleem Bello (PhD/Botanical Taxonomy) and Dr. Bayan Tiba (PhD/Botanical Taxonomy) Aleppo University. As far as possible, the name of the plants was updated by consulting the latest literature; generic and species names followed the plant list (http://www.theplantlist.org). All voucher specimens have been preserved during documentation and deposited in the Damascus University, Faculty of Pharmacy, Pharmacognosy Labs Herbarium for future reference, serial numbers were taken from (1ch to 76ch) according to its alphabetical order.","The study was approved by the Ethics Committee of the University of Damascus. Before beginning data collection, we obtained verbal informed permission in each case site-wide and then individually before each interview. We also informed informants that it was an academic project and that the investigation was for research purposes only, and not for any financial or other benefits. Informants provided verbal informed consent to participate in this study; they were free to withdraw their information at any time. These informants freely accepted the interview. All steps of research are consistent with Ethnobiology Code of Ethics (ISE 2006).","The data collected through interviews of the informants were classified and examined with the statistical program IBM® SPSS® Statistics 26 (IBM, Armonk, NY), to determine the proportions of different variables such as ethnopharmacological data. Quantitative value indices were analysed using different statistical quantitative tools, i.e., the use reports (UR) of a species, and use value (UV) (Chaachouaya et al. 2021).","The UR of a species or its importance in the culture of a community is denoted by its mentioning rate or its mention frequency by informants. The UR of the species of plants being utilized was evaluated using the formula (Tenté et al 2012):\nUR = Ni/n\nwhere Ni is the number of times a particular species was mentioned; n is the total number of times that all species were mentioned.\nThe UV of recorded medicinal plants was determined by applying the following formula (Tabuti et al. 2003):\nUV=∑ URi/N\nwhere ∑ URi is the total number of UR per plants; N is the total of interviewees questioned for given medicinal species.\nThe UV rate will be more important if there are several useful records for a species, implying that the plant is significant, whereas they will be near 0 if there are few reports compared to its use (Yaseen 2015; Chaachouaya et al. 2021).","In total, 151 local inhabitants of 35 villages and 59 districts were selected based on their experience in traditional uses of plants. Table 1 shows the age and gender wise distribution. All of them were interrogated using semi-structured questionnaires. Generally in Syria, both genders were interested in herbal medicines.\nAge and gender distribution.","The study area is considered one of the Syrian areas rich in medicinal plants. In the east of it there is a desert (badia), which is dominated by desert and thorny plants, and in the west of it, aromatic plants are spread in the mountains near the eastern coast of the Mediterranean Sea. It is a fact that people in some of these rural areas suffer from poverty, so they depend a lot on folk remedies, and folk healers in these areas provide their expertise at small costs, because the medicinal herbs are cheaper than chemical medicines, and most of the medicinal recipes are available around. However, a large portion of the uses of medicinal plants mentioned in the research are still under study.\nA total of 76 medicinal plant species (57.9% are wild and 42.1% are cultivated plants) belonging to 39 families are recorded in the present study; they are being used for a variety of purposes by native people. The detailed inventory is provided in Table 2, which includes botanical names, followed by local name, family and ethnobotanical uses.\nEthnobotanical uses of plant species according to ethnomedicinal survey of central region in Syria (Homs and Hama).\nAp: aerial parts; Bk: bark; Br: branches; Bu: buds; Bb: bulb; Ca: capitulum; Co: cones; Fl: flower; F: fruit; G: gum; Lx: latex; L: leaves; Ps: pedicles; Pe: peel; Ph: phloem; P: pods; Rs: resin; Rm: rhizome; R: root; Sd: seeds; S: stem; St: stylus; T: tubers; W: whole plant.\nC: cultivated plants, W: wild plants.\nHerbarium no.","The most commonly mentioned family is Asteraceae (11.84%), followed by Lamiaceae (10.52%), then Rosaceae (7.89%) and Apiaceae (6.57%), Poaceae (5.26%), Anacardiaceae and Fabaceae (3.94%), Fagaceae, Liliaceae, Myrtaceae and Oleaceae (2.63%), then all the other families (1.31%) (Figure 1).\nPlant families commonly used in ethnomedicinal survey of central region in Syria (Homs and Hama).","Medicinal use plants (UV) are utilized to find the most frequently used plant species in the study area. Its value ranged from 0.066 to 0.92 (Table 2). The calculated results of UV showed that Cichorium intybus L., Eucalyptus globulus Labill. was ranked first (UV = 0.92) followed by Fraxinus syriaca Boiss. (UV = 0.907), Olea europaea L. (UV = 0.9), then Allium sativum L. (UV = 0.894), Lepidium sativum L. (UV = 0.867), Coriandrum sativum L. (UV = 0.86), Glycyrrhiza glabra L. (UV = 0.854), Dittrichia viscosa (L.) Greuter (UV = 0.841), while the lowest value was found for Plumbago europaea L. (UV = 0.066).","The analysis of the ethnobotanical data showed that central region was best suited to the medicinal plant and rangeland. Ethnobotanical use categories showed that leaves were commonly used parts for making indigenous recipes (28.08%), followed by fruits (19.76%) and seeds (15.6%) then roots (10.4%), flowers (9.36%) and aerial parts (4.16%). Then, the others parts of plant are rarely used (Figure 2).\nMedicinal parts of the plants used for ethnomedicinal purposes in the study.","The analysis of the ethnobotanical data showed that the recipes in the most cases were obtained from single herb, but some of recipes were prepared together, and there is a famous local mixture called Damask tisane (zhourate Shamieh). A mode of TM preparation reported was a decoction (30%), followed by infusion (23%), and then by other method such as fresh herbs, juice, cooked, powder, vinegar and oils (47%) (Figure 3). Considering according to results, most of the plant preparations are used orally (Table 2).\nModes of ethnomedicines preparation in Homs and Hama.","The results of questionnaires showed that 20% of the informants were diagnosed with their diseases by a doctor, and 45% were diagnosed with a conventional therapist, and 35% self-diagnosed their diseases, while the results of the questionnaires showed that the evaluation of the treatment by informants as following (58% relied on the disappearance of symptoms, and 24% through the results of laboratory analysis, 18% adopted other methods such as chest radiography, adopting the attending physician’s opinion and clinical observation of the improvement of skin diseases, and some of them depended on psychological comfort during treatment as evidence of improvement).\nAmong these studied plants, 62 are used to treat digestive disorders, 41 for respiratory diseases, including asthma, bronchitis and coughs, 40 for skin diseases, 16 for diabetes, 36 for kidney and urinary tract disorders, 22 for nervous system disorders, six for enhance the body's immunity, two for haemorrhoids, five for fever, eight for heart disorders, five for infertility and impotence, six for treating several types of cancer, two for increasing breast milk production, five for losing weight, four for lowering cholesterol, and two for increasing weight, and six for anaemia, 15 for blood disorder, two anti-toxicant, 19 for arthritis and pain, one for typhoid disorder, eight for infections, six for gynaecological diseases, one for eye inflammation, two anti-toxicant and four for mouth sores. Many of them are still used today, especially those plants recommended for internal uses such as traditional medicinal teas, which mainly consist of remedies for obesity, weight loss, colds, colds, digestive disorders, abdominal pain, constipation and some skin diseases (Figure 4).\nEthno-medicinal information about treating different diseases related to central region in Syria (Homs and Hama).","There is insufficient information about the pharmacokinetic efficacy of the medicinal plant species in this study. These herbs have reportedly and traditionally been used as adjuvant to relieve and treat some diseases."],"string":"[\n \"Syria is a country located on the east coast of the Mediterranean Sea in southwestern Asia. Syria is bounded by Turkey to the north, Iraq to the east and southeast, Jordan to the south and Lebanon to the southwest. The study area is the middle region of Syria including Homs Governorate and Hama Governorate.\\nThis area has a Mediterranean climate with a long dry season from May to October. In the extreme northwest, there is some light summer rain. On the western region, summers are hot, with mean daily maximum temperatures ranging from low to mid 80 °F, while the mild winters have daily mean minimum reaching temperatures low level of 50 °F. Only above about 1500 m are the summers relatively cool. In inland the climate becomes arid, with colder winters and hotter summers. In the desert, at Tadmur maximum temperatures in the summer, temperature reaches averages in the ranges of upper 90s to low 100 °F, with extremes in the 110 °F. In rural areas, work takes place according to the seasonal rhythm of agriculture. Women generally share in much of the agricultural labour. Agriculture constitutes an important source of income, and fruits and vegetables including onions, olives and grapes. Commercially important forest plants include: pistachio, which is important for its oil-rich fruit, and plants such as olive trees, grapevines, apricot trees and cumin (Hamidé et al. 2021).\\nHistorically, the ancient Palmyra, also called Tadmur, is an ancient city in south-central Syria (Homs Governorate). An oasis in the Syrian desert, Palmyra contains the monumental ruins of a great city that was one of the most important cultural centres of the ancient world, from the first to the second century according to World Heritage List (UNESCO 2021).\",\n \"Field surveys were conducted between December 2019 and January 2021, to document ethnobotanical information through oral interviews and designed semi-structured questionnaire. Thirty-five villages and 59 districts were visited for field research; 235 people were contacted and 151 of those with ethno-botanical experience agreed to become our informants, including 35 local herbalists (Tabib Arabi) and 10 physicians (who hold a general medicine specialty). The queries were repeatedly made to increase the reliability of the data; interviews with the men were usually carried out in the ‘Mukhtar’ house where they come together, and with women in their homes, bazaars and gardens. The Syria Trust for Development (which is a national development organization, and has a program called ‘Mashrouie’ that runs innovative microcredit programs that encourage economic growth in disadvantaged areas) helped us in data collecting. The information gathered during the present study included socio-demographic characteristics of the interviewed informants (age, gender) and ethnopharmacological information, including the local and scientific name of the species, local names, plant parts used, modes of use, conservation method, administration mode and toxicity, all documented data were then translated into English and Latin. Information that had been carried to the region from the outside and that was not used or confirmed was not included and recorded (Weckerle et al. 2018). During the interviews, questions about the following were asked to the participants:Name and surnameAge and sexEducational levelAre plants collected in your region?Do you have any contact with plants?Can you show the plants you use in your region?Can you tell the names of local plants you use in your region?In which season do you collect the plants you use in your region?When collecting plant, which parts of the plant do you collect and how do you collect them?Which parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).How do you prepare and administrate the plants’ parts?How did you diagnose the disease (by physician, by traditional healers, self-treating)?How did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\\nName and surname\\nAge and sex\\nEducational level\\nAre plants collected in your region?\\nDo you have any contact with plants?\\nCan you show the plants you use in your region?\\nCan you tell the names of local plants you use in your region?\\nIn which season do you collect the plants you use in your region?\\nWhen collecting plant, which parts of the plant do you collect and how do you collect them?\\nWhich parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).\\nHow do you prepare and administrate the plants’ parts?\\nHow did you diagnose the disease (by physician, by traditional healers, self-treating)?\\nHow did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\\nOnly the medication of single herbs was included, while mixtures and multiple recipes were disregarded. The answers that were given with doubt to the questions above were not recorded, and we adopted the information that gave 40% frequency in the collected data, and the data that gave less than that were neglected.\\nInformation about diseases diagnosed specifically by physicians and traditional healers was approved, while self-treating diseases were referred to as auxiliary in treatment, nutritional uses were clearly indicated, and some plants were used to prevent disease and improve public health. The information about diseases was divided into three groups when analysing the data (group A: acute diseases, group B: chronic diseases, group C: simple symptoms and signs), and the focus was only on groups B and C.\",\n \"Medicinal plants being mentioned by the Informants were recorded with local names and photographed. Each reported medicinal plant species was gathered, compressed, dehydrated, mounted on herbarium sheets, and identified; the taxonomic identity of the plants was confirmed by Prof. Abdel Aleem Bello (PhD/Botanical Taxonomy) and Dr. Bayan Tiba (PhD/Botanical Taxonomy) Aleppo University. As far as possible, the name of the plants was updated by consulting the latest literature; generic and species names followed the plant list (http://www.theplantlist.org). All voucher specimens have been preserved during documentation and deposited in the Damascus University, Faculty of Pharmacy, Pharmacognosy Labs Herbarium for future reference, serial numbers were taken from (1ch to 76ch) according to its alphabetical order.\",\n \"The study was approved by the Ethics Committee of the University of Damascus. Before beginning data collection, we obtained verbal informed permission in each case site-wide and then individually before each interview. We also informed informants that it was an academic project and that the investigation was for research purposes only, and not for any financial or other benefits. Informants provided verbal informed consent to participate in this study; they were free to withdraw their information at any time. These informants freely accepted the interview. All steps of research are consistent with Ethnobiology Code of Ethics (ISE 2006).\",\n \"The data collected through interviews of the informants were classified and examined with the statistical program IBM® SPSS® Statistics 26 (IBM, Armonk, NY), to determine the proportions of different variables such as ethnopharmacological data. Quantitative value indices were analysed using different statistical quantitative tools, i.e., the use reports (UR) of a species, and use value (UV) (Chaachouaya et al. 2021).\",\n \"The UR of a species or its importance in the culture of a community is denoted by its mentioning rate or its mention frequency by informants. The UR of the species of plants being utilized was evaluated using the formula (Tenté et al 2012):\\nUR = Ni/n\\nwhere Ni is the number of times a particular species was mentioned; n is the total number of times that all species were mentioned.\\nThe UV of recorded medicinal plants was determined by applying the following formula (Tabuti et al. 2003):\\nUV=∑ URi/N\\nwhere ∑ URi is the total number of UR per plants; N is the total of interviewees questioned for given medicinal species.\\nThe UV rate will be more important if there are several useful records for a species, implying that the plant is significant, whereas they will be near 0 if there are few reports compared to its use (Yaseen 2015; Chaachouaya et al. 2021).\",\n \"In total, 151 local inhabitants of 35 villages and 59 districts were selected based on their experience in traditional uses of plants. Table 1 shows the age and gender wise distribution. All of them were interrogated using semi-structured questionnaires. Generally in Syria, both genders were interested in herbal medicines.\\nAge and gender distribution.\",\n \"The study area is considered one of the Syrian areas rich in medicinal plants. In the east of it there is a desert (badia), which is dominated by desert and thorny plants, and in the west of it, aromatic plants are spread in the mountains near the eastern coast of the Mediterranean Sea. It is a fact that people in some of these rural areas suffer from poverty, so they depend a lot on folk remedies, and folk healers in these areas provide their expertise at small costs, because the medicinal herbs are cheaper than chemical medicines, and most of the medicinal recipes are available around. However, a large portion of the uses of medicinal plants mentioned in the research are still under study.\\nA total of 76 medicinal plant species (57.9% are wild and 42.1% are cultivated plants) belonging to 39 families are recorded in the present study; they are being used for a variety of purposes by native people. The detailed inventory is provided in Table 2, which includes botanical names, followed by local name, family and ethnobotanical uses.\\nEthnobotanical uses of plant species according to ethnomedicinal survey of central region in Syria (Homs and Hama).\\nAp: aerial parts; Bk: bark; Br: branches; Bu: buds; Bb: bulb; Ca: capitulum; Co: cones; Fl: flower; F: fruit; G: gum; Lx: latex; L: leaves; Ps: pedicles; Pe: peel; Ph: phloem; P: pods; Rs: resin; Rm: rhizome; R: root; Sd: seeds; S: stem; St: stylus; T: tubers; W: whole plant.\\nC: cultivated plants, W: wild plants.\\nHerbarium no.\",\n \"The most commonly mentioned family is Asteraceae (11.84%), followed by Lamiaceae (10.52%), then Rosaceae (7.89%) and Apiaceae (6.57%), Poaceae (5.26%), Anacardiaceae and Fabaceae (3.94%), Fagaceae, Liliaceae, Myrtaceae and Oleaceae (2.63%), then all the other families (1.31%) (Figure 1).\\nPlant families commonly used in ethnomedicinal survey of central region in Syria (Homs and Hama).\",\n \"Medicinal use plants (UV) are utilized to find the most frequently used plant species in the study area. Its value ranged from 0.066 to 0.92 (Table 2). The calculated results of UV showed that Cichorium intybus L., Eucalyptus globulus Labill. was ranked first (UV = 0.92) followed by Fraxinus syriaca Boiss. (UV = 0.907), Olea europaea L. (UV = 0.9), then Allium sativum L. (UV = 0.894), Lepidium sativum L. (UV = 0.867), Coriandrum sativum L. (UV = 0.86), Glycyrrhiza glabra L. (UV = 0.854), Dittrichia viscosa (L.) Greuter (UV = 0.841), while the lowest value was found for Plumbago europaea L. (UV = 0.066).\",\n \"The analysis of the ethnobotanical data showed that central region was best suited to the medicinal plant and rangeland. Ethnobotanical use categories showed that leaves were commonly used parts for making indigenous recipes (28.08%), followed by fruits (19.76%) and seeds (15.6%) then roots (10.4%), flowers (9.36%) and aerial parts (4.16%). Then, the others parts of plant are rarely used (Figure 2).\\nMedicinal parts of the plants used for ethnomedicinal purposes in the study.\",\n \"The analysis of the ethnobotanical data showed that the recipes in the most cases were obtained from single herb, but some of recipes were prepared together, and there is a famous local mixture called Damask tisane (zhourate Shamieh). A mode of TM preparation reported was a decoction (30%), followed by infusion (23%), and then by other method such as fresh herbs, juice, cooked, powder, vinegar and oils (47%) (Figure 3). Considering according to results, most of the plant preparations are used orally (Table 2).\\nModes of ethnomedicines preparation in Homs and Hama.\",\n \"The results of questionnaires showed that 20% of the informants were diagnosed with their diseases by a doctor, and 45% were diagnosed with a conventional therapist, and 35% self-diagnosed their diseases, while the results of the questionnaires showed that the evaluation of the treatment by informants as following (58% relied on the disappearance of symptoms, and 24% through the results of laboratory analysis, 18% adopted other methods such as chest radiography, adopting the attending physician’s opinion and clinical observation of the improvement of skin diseases, and some of them depended on psychological comfort during treatment as evidence of improvement).\\nAmong these studied plants, 62 are used to treat digestive disorders, 41 for respiratory diseases, including asthma, bronchitis and coughs, 40 for skin diseases, 16 for diabetes, 36 for kidney and urinary tract disorders, 22 for nervous system disorders, six for enhance the body's immunity, two for haemorrhoids, five for fever, eight for heart disorders, five for infertility and impotence, six for treating several types of cancer, two for increasing breast milk production, five for losing weight, four for lowering cholesterol, and two for increasing weight, and six for anaemia, 15 for blood disorder, two anti-toxicant, 19 for arthritis and pain, one for typhoid disorder, eight for infections, six for gynaecological diseases, one for eye inflammation, two anti-toxicant and four for mouth sores. Many of them are still used today, especially those plants recommended for internal uses such as traditional medicinal teas, which mainly consist of remedies for obesity, weight loss, colds, colds, digestive disorders, abdominal pain, constipation and some skin diseases (Figure 4).\\nEthno-medicinal information about treating different diseases related to central region in Syria (Homs and Hama).\",\n \"There is insufficient information about the pharmacokinetic efficacy of the medicinal plant species in this study. These herbs have reportedly and traditionally been used as adjuvant to relieve and treat some diseases.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Introduction","Methods","Study area","Field work and data collection","Taxonomic identification of the species","Ethics approval and consent to participate","Data analysis","Use value and use reports","Results","Demographic data of informants","Ethnobotanical and ethnomedicinal uses of plant species","Botanical families of plants used","Use value of the plants","Medicinal parts of the plant used","Modes and conditions of medicine preparation","Ethnomedicinal information about treatment the different diseases","Discussion","Limitations","Conclusions"],"string":"[\n \"Introduction\",\n \"Methods\",\n \"Study area\",\n \"Field work and data collection\",\n \"Taxonomic identification of the species\",\n \"Ethics approval and consent to participate\",\n \"Data analysis\",\n \"Use value and use reports\",\n \"Results\",\n \"Demographic data of informants\",\n \"Ethnobotanical and ethnomedicinal uses of plant species\",\n \"Botanical families of plants used\",\n \"Use value of the plants\",\n \"Medicinal parts of the plant used\",\n \"Modes and conditions of medicine preparation\",\n \"Ethnomedicinal information about treatment the different diseases\",\n \"Discussion\",\n \"Limitations\",\n \"Conclusions\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Traditional medicine (TM), as defined by the World Health Organization (WHO), is the sum total of the knowledge, skills and practices based on the theories, beliefs and experiences indigenous to different cultures, whether explicable or not, used in the maintenance of health as well as in the prevention, diagnosis, improvement or treatment of physical and mental illness (WHO 2002a, 2002b). Some TM systems are supported by huge volumes of literature and records of the theoretical concepts and practical skills; others pass down from generation to generation through verbal teaching. To date, in some parts of the world, the majority of the population continue to rely on their own TM to meet their primary health care needs. When adopted outside of its traditional culture, TM is often referred as complementary and alternative medicine (CAM) (Che et al. 2017).\nAlthough modern medicine is currently available in many developing countries, large proportions of the population in these countries still rely to a large extent on traditional practitioners and medicinal plants for therapeutic purposes. TM is often the first choice for providing primary health care in developing countries, and the WHO estimates that more than 80% of healthcare needs in these countries are met by traditional health care practices, being the cheapest and most accessible (WHO 2002a, 2002b, 2004, 2005).\nThe WHO pays special attention to TM and CAM. Resolution (no. WHA13-6) issued by the World Health Assembly (WHA) in 2009 emphasized the need to update the global TM strategy (WHO 2009), so the WHO issued its strategy for traditional (folk medicine) 2014–2023 (WHO 2013).\nThe United Nations Educational Scientific and Cultural Organization (UNESCO) works to document the medical heritage of peoples within its documentation of the living intangible cultural heritage of peoples and civilizations, through the documentation of TM practices that were included in the UNESCO convention (2003), the convention on biological diversity (1992) and the UNESCO universal declaration on cultural diversity (2001) and the United Nations declaration on the rights of indigenous peoples (2007), and according to the meeting of the UNESCO International Bioethics Committee (IBC) working group on TM and its ethical effects in Paris (2010), it stressed the need to conduct studies that illustrate the use of TM in various regions around the world, and its evaluation in clinical practice and in research and policy (IBC 2013).\nIn 2017, the International Center for Information and Networks for Intangible Cultural Heritage in the Asia-Pacific region (ICHCAP) under the auspices of UNESCO, issued a book entitled Traditional Medicine in which the Syria Trust for Development in Section VII included TM in Syria (Falk 2017).\nEthno-botany is the scientific study of the relationships between people and plants. It was first coined in 1896 by the US botanist John Harshberger; however, the history of ethnobotany began long before that (Campbell et al. 2002; Amjad et al. 2015). It plays an important role in understanding the dynamic relationships between biological diversity and social and cultural systems (Husain et al. 2008; Amjad et al. 2013, 2015). Plants are essential for human beings as they provide food, and medicines (Alam et al. 2011; Ahmad et al. 2012).\nTraditional Arabic medicine (TAM) is one of the famous traditional medical systems, which is occasionally called Unani medicine, Graeco-Arabic medicine, humoral medicine or Islamic medicine. The subject of TM in Syria has received little attention in the literature, and very little is known about the traditional medicinal substances used nowadays by the Syrian population to treat the most common diseases. Throughout ancient times in Syria, as part of the Levantine Nations (Bilad al-Sham), and other lands in the region, humans used various natural materials as sources of medicines (Jaddouh 2004). In the western countryside of Hama, there is a natural reserve for medicinal plants, which is called the Abu Qubais Protected area in Al-Ghab region (which protect the biodiversity rights of indigenous people and affiliated to the general commission for Al-Ghab administration and development), 509 plant species belonging to 72 families have been recorded (Al-Mahmoud and Al-Shater 2010).\nFor these reasons, the present investigation gathered the uses of medicinal plants in the centre region of Syria (Homs and Hama), as a supplement for a national survey, and documents the information concerning the uses of medicinal plants, which may serve as the basis of knowledge for a more intensive scientific research.","Study area Syria is a country located on the east coast of the Mediterranean Sea in southwestern Asia. Syria is bounded by Turkey to the north, Iraq to the east and southeast, Jordan to the south and Lebanon to the southwest. The study area is the middle region of Syria including Homs Governorate and Hama Governorate.\nThis area has a Mediterranean climate with a long dry season from May to October. In the extreme northwest, there is some light summer rain. On the western region, summers are hot, with mean daily maximum temperatures ranging from low to mid 80 °F, while the mild winters have daily mean minimum reaching temperatures low level of 50 °F. Only above about 1500 m are the summers relatively cool. In inland the climate becomes arid, with colder winters and hotter summers. In the desert, at Tadmur maximum temperatures in the summer, temperature reaches averages in the ranges of upper 90s to low 100 °F, with extremes in the 110 °F. In rural areas, work takes place according to the seasonal rhythm of agriculture. Women generally share in much of the agricultural labour. Agriculture constitutes an important source of income, and fruits and vegetables including onions, olives and grapes. Commercially important forest plants include: pistachio, which is important for its oil-rich fruit, and plants such as olive trees, grapevines, apricot trees and cumin (Hamidé et al. 2021).\nHistorically, the ancient Palmyra, also called Tadmur, is an ancient city in south-central Syria (Homs Governorate). An oasis in the Syrian desert, Palmyra contains the monumental ruins of a great city that was one of the most important cultural centres of the ancient world, from the first to the second century according to World Heritage List (UNESCO 2021).\nSyria is a country located on the east coast of the Mediterranean Sea in southwestern Asia. Syria is bounded by Turkey to the north, Iraq to the east and southeast, Jordan to the south and Lebanon to the southwest. The study area is the middle region of Syria including Homs Governorate and Hama Governorate.\nThis area has a Mediterranean climate with a long dry season from May to October. In the extreme northwest, there is some light summer rain. On the western region, summers are hot, with mean daily maximum temperatures ranging from low to mid 80 °F, while the mild winters have daily mean minimum reaching temperatures low level of 50 °F. Only above about 1500 m are the summers relatively cool. In inland the climate becomes arid, with colder winters and hotter summers. In the desert, at Tadmur maximum temperatures in the summer, temperature reaches averages in the ranges of upper 90s to low 100 °F, with extremes in the 110 °F. In rural areas, work takes place according to the seasonal rhythm of agriculture. Women generally share in much of the agricultural labour. Agriculture constitutes an important source of income, and fruits and vegetables including onions, olives and grapes. Commercially important forest plants include: pistachio, which is important for its oil-rich fruit, and plants such as olive trees, grapevines, apricot trees and cumin (Hamidé et al. 2021).\nHistorically, the ancient Palmyra, also called Tadmur, is an ancient city in south-central Syria (Homs Governorate). An oasis in the Syrian desert, Palmyra contains the monumental ruins of a great city that was one of the most important cultural centres of the ancient world, from the first to the second century according to World Heritage List (UNESCO 2021).\nField work and data collection Field surveys were conducted between December 2019 and January 2021, to document ethnobotanical information through oral interviews and designed semi-structured questionnaire. Thirty-five villages and 59 districts were visited for field research; 235 people were contacted and 151 of those with ethno-botanical experience agreed to become our informants, including 35 local herbalists (Tabib Arabi) and 10 physicians (who hold a general medicine specialty). The queries were repeatedly made to increase the reliability of the data; interviews with the men were usually carried out in the ‘Mukhtar’ house where they come together, and with women in their homes, bazaars and gardens. The Syria Trust for Development (which is a national development organization, and has a program called ‘Mashrouie’ that runs innovative microcredit programs that encourage economic growth in disadvantaged areas) helped us in data collecting. The information gathered during the present study included socio-demographic characteristics of the interviewed informants (age, gender) and ethnopharmacological information, including the local and scientific name of the species, local names, plant parts used, modes of use, conservation method, administration mode and toxicity, all documented data were then translated into English and Latin. Information that had been carried to the region from the outside and that was not used or confirmed was not included and recorded (Weckerle et al. 2018). During the interviews, questions about the following were asked to the participants:Name and surnameAge and sexEducational levelAre plants collected in your region?Do you have any contact with plants?Can you show the plants you use in your region?Can you tell the names of local plants you use in your region?In which season do you collect the plants you use in your region?When collecting plant, which parts of the plant do you collect and how do you collect them?Which parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).How do you prepare and administrate the plants’ parts?How did you diagnose the disease (by physician, by traditional healers, self-treating)?How did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\nName and surname\nAge and sex\nEducational level\nAre plants collected in your region?\nDo you have any contact with plants?\nCan you show the plants you use in your region?\nCan you tell the names of local plants you use in your region?\nIn which season do you collect the plants you use in your region?\nWhen collecting plant, which parts of the plant do you collect and how do you collect them?\nWhich parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).\nHow do you prepare and administrate the plants’ parts?\nHow did you diagnose the disease (by physician, by traditional healers, self-treating)?\nHow did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\nOnly the medication of single herbs was included, while mixtures and multiple recipes were disregarded. The answers that were given with doubt to the questions above were not recorded, and we adopted the information that gave 40% frequency in the collected data, and the data that gave less than that were neglected.\nInformation about diseases diagnosed specifically by physicians and traditional healers was approved, while self-treating diseases were referred to as auxiliary in treatment, nutritional uses were clearly indicated, and some plants were used to prevent disease and improve public health. The information about diseases was divided into three groups when analysing the data (group A: acute diseases, group B: chronic diseases, group C: simple symptoms and signs), and the focus was only on groups B and C.\nField surveys were conducted between December 2019 and January 2021, to document ethnobotanical information through oral interviews and designed semi-structured questionnaire. Thirty-five villages and 59 districts were visited for field research; 235 people were contacted and 151 of those with ethno-botanical experience agreed to become our informants, including 35 local herbalists (Tabib Arabi) and 10 physicians (who hold a general medicine specialty). The queries were repeatedly made to increase the reliability of the data; interviews with the men were usually carried out in the ‘Mukhtar’ house where they come together, and with women in their homes, bazaars and gardens. The Syria Trust for Development (which is a national development organization, and has a program called ‘Mashrouie’ that runs innovative microcredit programs that encourage economic growth in disadvantaged areas) helped us in data collecting. The information gathered during the present study included socio-demographic characteristics of the interviewed informants (age, gender) and ethnopharmacological information, including the local and scientific name of the species, local names, plant parts used, modes of use, conservation method, administration mode and toxicity, all documented data were then translated into English and Latin. Information that had been carried to the region from the outside and that was not used or confirmed was not included and recorded (Weckerle et al. 2018). During the interviews, questions about the following were asked to the participants:Name and surnameAge and sexEducational levelAre plants collected in your region?Do you have any contact with plants?Can you show the plants you use in your region?Can you tell the names of local plants you use in your region?In which season do you collect the plants you use in your region?When collecting plant, which parts of the plant do you collect and how do you collect them?Which parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).How do you prepare and administrate the plants’ parts?How did you diagnose the disease (by physician, by traditional healers, self-treating)?How did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\nName and surname\nAge and sex\nEducational level\nAre plants collected in your region?\nDo you have any contact with plants?\nCan you show the plants you use in your region?\nCan you tell the names of local plants you use in your region?\nIn which season do you collect the plants you use in your region?\nWhen collecting plant, which parts of the plant do you collect and how do you collect them?\nWhich parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).\nHow do you prepare and administrate the plants’ parts?\nHow did you diagnose the disease (by physician, by traditional healers, self-treating)?\nHow did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\nOnly the medication of single herbs was included, while mixtures and multiple recipes were disregarded. The answers that were given with doubt to the questions above were not recorded, and we adopted the information that gave 40% frequency in the collected data, and the data that gave less than that were neglected.\nInformation about diseases diagnosed specifically by physicians and traditional healers was approved, while self-treating diseases were referred to as auxiliary in treatment, nutritional uses were clearly indicated, and some plants were used to prevent disease and improve public health. The information about diseases was divided into three groups when analysing the data (group A: acute diseases, group B: chronic diseases, group C: simple symptoms and signs), and the focus was only on groups B and C.\nTaxonomic identification of the species Medicinal plants being mentioned by the Informants were recorded with local names and photographed. Each reported medicinal plant species was gathered, compressed, dehydrated, mounted on herbarium sheets, and identified; the taxonomic identity of the plants was confirmed by Prof. Abdel Aleem Bello (PhD/Botanical Taxonomy) and Dr. Bayan Tiba (PhD/Botanical Taxonomy) Aleppo University. As far as possible, the name of the plants was updated by consulting the latest literature; generic and species names followed the plant list (http://www.theplantlist.org). All voucher specimens have been preserved during documentation and deposited in the Damascus University, Faculty of Pharmacy, Pharmacognosy Labs Herbarium for future reference, serial numbers were taken from (1ch to 76ch) according to its alphabetical order.\nMedicinal plants being mentioned by the Informants were recorded with local names and photographed. Each reported medicinal plant species was gathered, compressed, dehydrated, mounted on herbarium sheets, and identified; the taxonomic identity of the plants was confirmed by Prof. Abdel Aleem Bello (PhD/Botanical Taxonomy) and Dr. Bayan Tiba (PhD/Botanical Taxonomy) Aleppo University. As far as possible, the name of the plants was updated by consulting the latest literature; generic and species names followed the plant list (http://www.theplantlist.org). All voucher specimens have been preserved during documentation and deposited in the Damascus University, Faculty of Pharmacy, Pharmacognosy Labs Herbarium for future reference, serial numbers were taken from (1ch to 76ch) according to its alphabetical order.\nEthics approval and consent to participate The study was approved by the Ethics Committee of the University of Damascus. Before beginning data collection, we obtained verbal informed permission in each case site-wide and then individually before each interview. We also informed informants that it was an academic project and that the investigation was for research purposes only, and not for any financial or other benefits. Informants provided verbal informed consent to participate in this study; they were free to withdraw their information at any time. These informants freely accepted the interview. All steps of research are consistent with Ethnobiology Code of Ethics (ISE 2006).\nThe study was approved by the Ethics Committee of the University of Damascus. Before beginning data collection, we obtained verbal informed permission in each case site-wide and then individually before each interview. We also informed informants that it was an academic project and that the investigation was for research purposes only, and not for any financial or other benefits. Informants provided verbal informed consent to participate in this study; they were free to withdraw their information at any time. These informants freely accepted the interview. All steps of research are consistent with Ethnobiology Code of Ethics (ISE 2006).\nData analysis The data collected through interviews of the informants were classified and examined with the statistical program IBM® SPSS® Statistics 26 (IBM, Armonk, NY), to determine the proportions of different variables such as ethnopharmacological data. Quantitative value indices were analysed using different statistical quantitative tools, i.e., the use reports (UR) of a species, and use value (UV) (Chaachouaya et al. 2021).\nThe data collected through interviews of the informants were classified and examined with the statistical program IBM® SPSS® Statistics 26 (IBM, Armonk, NY), to determine the proportions of different variables such as ethnopharmacological data. Quantitative value indices were analysed using different statistical quantitative tools, i.e., the use reports (UR) of a species, and use value (UV) (Chaachouaya et al. 2021).\nUse value and use reports The UR of a species or its importance in the culture of a community is denoted by its mentioning rate or its mention frequency by informants. The UR of the species of plants being utilized was evaluated using the formula (Tenté et al 2012):\nUR = Ni/n\nwhere Ni is the number of times a particular species was mentioned; n is the total number of times that all species were mentioned.\nThe UV of recorded medicinal plants was determined by applying the following formula (Tabuti et al. 2003):\nUV=∑ URi/N\nwhere ∑ URi is the total number of UR per plants; N is the total of interviewees questioned for given medicinal species.\nThe UV rate will be more important if there are several useful records for a species, implying that the plant is significant, whereas they will be near 0 if there are few reports compared to its use (Yaseen 2015; Chaachouaya et al. 2021).\nThe UR of a species or its importance in the culture of a community is denoted by its mentioning rate or its mention frequency by informants. The UR of the species of plants being utilized was evaluated using the formula (Tenté et al 2012):\nUR = Ni/n\nwhere Ni is the number of times a particular species was mentioned; n is the total number of times that all species were mentioned.\nThe UV of recorded medicinal plants was determined by applying the following formula (Tabuti et al. 2003):\nUV=∑ URi/N\nwhere ∑ URi is the total number of UR per plants; N is the total of interviewees questioned for given medicinal species.\nThe UV rate will be more important if there are several useful records for a species, implying that the plant is significant, whereas they will be near 0 if there are few reports compared to its use (Yaseen 2015; Chaachouaya et al. 2021).","Syria is a country located on the east coast of the Mediterranean Sea in southwestern Asia. Syria is bounded by Turkey to the north, Iraq to the east and southeast, Jordan to the south and Lebanon to the southwest. The study area is the middle region of Syria including Homs Governorate and Hama Governorate.\nThis area has a Mediterranean climate with a long dry season from May to October. In the extreme northwest, there is some light summer rain. On the western region, summers are hot, with mean daily maximum temperatures ranging from low to mid 80 °F, while the mild winters have daily mean minimum reaching temperatures low level of 50 °F. Only above about 1500 m are the summers relatively cool. In inland the climate becomes arid, with colder winters and hotter summers. In the desert, at Tadmur maximum temperatures in the summer, temperature reaches averages in the ranges of upper 90s to low 100 °F, with extremes in the 110 °F. In rural areas, work takes place according to the seasonal rhythm of agriculture. Women generally share in much of the agricultural labour. Agriculture constitutes an important source of income, and fruits and vegetables including onions, olives and grapes. Commercially important forest plants include: pistachio, which is important for its oil-rich fruit, and plants such as olive trees, grapevines, apricot trees and cumin (Hamidé et al. 2021).\nHistorically, the ancient Palmyra, also called Tadmur, is an ancient city in south-central Syria (Homs Governorate). An oasis in the Syrian desert, Palmyra contains the monumental ruins of a great city that was one of the most important cultural centres of the ancient world, from the first to the second century according to World Heritage List (UNESCO 2021).","Field surveys were conducted between December 2019 and January 2021, to document ethnobotanical information through oral interviews and designed semi-structured questionnaire. Thirty-five villages and 59 districts were visited for field research; 235 people were contacted and 151 of those with ethno-botanical experience agreed to become our informants, including 35 local herbalists (Tabib Arabi) and 10 physicians (who hold a general medicine specialty). The queries were repeatedly made to increase the reliability of the data; interviews with the men were usually carried out in the ‘Mukhtar’ house where they come together, and with women in their homes, bazaars and gardens. The Syria Trust for Development (which is a national development organization, and has a program called ‘Mashrouie’ that runs innovative microcredit programs that encourage economic growth in disadvantaged areas) helped us in data collecting. The information gathered during the present study included socio-demographic characteristics of the interviewed informants (age, gender) and ethnopharmacological information, including the local and scientific name of the species, local names, plant parts used, modes of use, conservation method, administration mode and toxicity, all documented data were then translated into English and Latin. Information that had been carried to the region from the outside and that was not used or confirmed was not included and recorded (Weckerle et al. 2018). During the interviews, questions about the following were asked to the participants:Name and surnameAge and sexEducational levelAre plants collected in your region?Do you have any contact with plants?Can you show the plants you use in your region?Can you tell the names of local plants you use in your region?In which season do you collect the plants you use in your region?When collecting plant, which parts of the plant do you collect and how do you collect them?Which parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).How do you prepare and administrate the plants’ parts?How did you diagnose the disease (by physician, by traditional healers, self-treating)?How did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\nName and surname\nAge and sex\nEducational level\nAre plants collected in your region?\nDo you have any contact with plants?\nCan you show the plants you use in your region?\nCan you tell the names of local plants you use in your region?\nIn which season do you collect the plants you use in your region?\nWhen collecting plant, which parts of the plant do you collect and how do you collect them?\nWhich parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).\nHow do you prepare and administrate the plants’ parts?\nHow did you diagnose the disease (by physician, by traditional healers, self-treating)?\nHow did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\nOnly the medication of single herbs was included, while mixtures and multiple recipes were disregarded. The answers that were given with doubt to the questions above were not recorded, and we adopted the information that gave 40% frequency in the collected data, and the data that gave less than that were neglected.\nInformation about diseases diagnosed specifically by physicians and traditional healers was approved, while self-treating diseases were referred to as auxiliary in treatment, nutritional uses were clearly indicated, and some plants were used to prevent disease and improve public health. The information about diseases was divided into three groups when analysing the data (group A: acute diseases, group B: chronic diseases, group C: simple symptoms and signs), and the focus was only on groups B and C.","Medicinal plants being mentioned by the Informants were recorded with local names and photographed. Each reported medicinal plant species was gathered, compressed, dehydrated, mounted on herbarium sheets, and identified; the taxonomic identity of the plants was confirmed by Prof. Abdel Aleem Bello (PhD/Botanical Taxonomy) and Dr. Bayan Tiba (PhD/Botanical Taxonomy) Aleppo University. As far as possible, the name of the plants was updated by consulting the latest literature; generic and species names followed the plant list (http://www.theplantlist.org). All voucher specimens have been preserved during documentation and deposited in the Damascus University, Faculty of Pharmacy, Pharmacognosy Labs Herbarium for future reference, serial numbers were taken from (1ch to 76ch) according to its alphabetical order.","The study was approved by the Ethics Committee of the University of Damascus. Before beginning data collection, we obtained verbal informed permission in each case site-wide and then individually before each interview. We also informed informants that it was an academic project and that the investigation was for research purposes only, and not for any financial or other benefits. Informants provided verbal informed consent to participate in this study; they were free to withdraw their information at any time. These informants freely accepted the interview. All steps of research are consistent with Ethnobiology Code of Ethics (ISE 2006).","The data collected through interviews of the informants were classified and examined with the statistical program IBM® SPSS® Statistics 26 (IBM, Armonk, NY), to determine the proportions of different variables such as ethnopharmacological data. Quantitative value indices were analysed using different statistical quantitative tools, i.e., the use reports (UR) of a species, and use value (UV) (Chaachouaya et al. 2021).","The UR of a species or its importance in the culture of a community is denoted by its mentioning rate or its mention frequency by informants. The UR of the species of plants being utilized was evaluated using the formula (Tenté et al 2012):\nUR = Ni/n\nwhere Ni is the number of times a particular species was mentioned; n is the total number of times that all species were mentioned.\nThe UV of recorded medicinal plants was determined by applying the following formula (Tabuti et al. 2003):\nUV=∑ URi/N\nwhere ∑ URi is the total number of UR per plants; N is the total of interviewees questioned for given medicinal species.\nThe UV rate will be more important if there are several useful records for a species, implying that the plant is significant, whereas they will be near 0 if there are few reports compared to its use (Yaseen 2015; Chaachouaya et al. 2021).","Demographic data of informants In total, 151 local inhabitants of 35 villages and 59 districts were selected based on their experience in traditional uses of plants. Table 1 shows the age and gender wise distribution. All of them were interrogated using semi-structured questionnaires. Generally in Syria, both genders were interested in herbal medicines.\nAge and gender distribution.\nIn total, 151 local inhabitants of 35 villages and 59 districts were selected based on their experience in traditional uses of plants. Table 1 shows the age and gender wise distribution. All of them were interrogated using semi-structured questionnaires. Generally in Syria, both genders were interested in herbal medicines.\nAge and gender distribution.\nEthnobotanical and ethnomedicinal uses of plant species The study area is considered one of the Syrian areas rich in medicinal plants. In the east of it there is a desert (badia), which is dominated by desert and thorny plants, and in the west of it, aromatic plants are spread in the mountains near the eastern coast of the Mediterranean Sea. It is a fact that people in some of these rural areas suffer from poverty, so they depend a lot on folk remedies, and folk healers in these areas provide their expertise at small costs, because the medicinal herbs are cheaper than chemical medicines, and most of the medicinal recipes are available around. However, a large portion of the uses of medicinal plants mentioned in the research are still under study.\nA total of 76 medicinal plant species (57.9% are wild and 42.1% are cultivated plants) belonging to 39 families are recorded in the present study; they are being used for a variety of purposes by native people. The detailed inventory is provided in Table 2, which includes botanical names, followed by local name, family and ethnobotanical uses.\nEthnobotanical uses of plant species according to ethnomedicinal survey of central region in Syria (Homs and Hama).\nAp: aerial parts; Bk: bark; Br: branches; Bu: buds; Bb: bulb; Ca: capitulum; Co: cones; Fl: flower; F: fruit; G: gum; Lx: latex; L: leaves; Ps: pedicles; Pe: peel; Ph: phloem; P: pods; Rs: resin; Rm: rhizome; R: root; Sd: seeds; S: stem; St: stylus; T: tubers; W: whole plant.\nC: cultivated plants, W: wild plants.\nHerbarium no.\nThe study area is considered one of the Syrian areas rich in medicinal plants. In the east of it there is a desert (badia), which is dominated by desert and thorny plants, and in the west of it, aromatic plants are spread in the mountains near the eastern coast of the Mediterranean Sea. It is a fact that people in some of these rural areas suffer from poverty, so they depend a lot on folk remedies, and folk healers in these areas provide their expertise at small costs, because the medicinal herbs are cheaper than chemical medicines, and most of the medicinal recipes are available around. However, a large portion of the uses of medicinal plants mentioned in the research are still under study.\nA total of 76 medicinal plant species (57.9% are wild and 42.1% are cultivated plants) belonging to 39 families are recorded in the present study; they are being used for a variety of purposes by native people. The detailed inventory is provided in Table 2, which includes botanical names, followed by local name, family and ethnobotanical uses.\nEthnobotanical uses of plant species according to ethnomedicinal survey of central region in Syria (Homs and Hama).\nAp: aerial parts; Bk: bark; Br: branches; Bu: buds; Bb: bulb; Ca: capitulum; Co: cones; Fl: flower; F: fruit; G: gum; Lx: latex; L: leaves; Ps: pedicles; Pe: peel; Ph: phloem; P: pods; Rs: resin; Rm: rhizome; R: root; Sd: seeds; S: stem; St: stylus; T: tubers; W: whole plant.\nC: cultivated plants, W: wild plants.\nHerbarium no.\nBotanical families of plants used The most commonly mentioned family is Asteraceae (11.84%), followed by Lamiaceae (10.52%), then Rosaceae (7.89%) and Apiaceae (6.57%), Poaceae (5.26%), Anacardiaceae and Fabaceae (3.94%), Fagaceae, Liliaceae, Myrtaceae and Oleaceae (2.63%), then all the other families (1.31%) (Figure 1).\nPlant families commonly used in ethnomedicinal survey of central region in Syria (Homs and Hama).\nThe most commonly mentioned family is Asteraceae (11.84%), followed by Lamiaceae (10.52%), then Rosaceae (7.89%) and Apiaceae (6.57%), Poaceae (5.26%), Anacardiaceae and Fabaceae (3.94%), Fagaceae, Liliaceae, Myrtaceae and Oleaceae (2.63%), then all the other families (1.31%) (Figure 1).\nPlant families commonly used in ethnomedicinal survey of central region in Syria (Homs and Hama).\nUse value of the plants Medicinal use plants (UV) are utilized to find the most frequently used plant species in the study area. Its value ranged from 0.066 to 0.92 (Table 2). The calculated results of UV showed that Cichorium intybus L., Eucalyptus globulus Labill. was ranked first (UV = 0.92) followed by Fraxinus syriaca Boiss. (UV = 0.907), Olea europaea L. (UV = 0.9), then Allium sativum L. (UV = 0.894), Lepidium sativum L. (UV = 0.867), Coriandrum sativum L. (UV = 0.86), Glycyrrhiza glabra L. (UV = 0.854), Dittrichia viscosa (L.) Greuter (UV = 0.841), while the lowest value was found for Plumbago europaea L. (UV = 0.066).\nMedicinal use plants (UV) are utilized to find the most frequently used plant species in the study area. Its value ranged from 0.066 to 0.92 (Table 2). The calculated results of UV showed that Cichorium intybus L., Eucalyptus globulus Labill. was ranked first (UV = 0.92) followed by Fraxinus syriaca Boiss. (UV = 0.907), Olea europaea L. (UV = 0.9), then Allium sativum L. (UV = 0.894), Lepidium sativum L. (UV = 0.867), Coriandrum sativum L. (UV = 0.86), Glycyrrhiza glabra L. (UV = 0.854), Dittrichia viscosa (L.) Greuter (UV = 0.841), while the lowest value was found for Plumbago europaea L. (UV = 0.066).\nMedicinal parts of the plant used The analysis of the ethnobotanical data showed that central region was best suited to the medicinal plant and rangeland. Ethnobotanical use categories showed that leaves were commonly used parts for making indigenous recipes (28.08%), followed by fruits (19.76%) and seeds (15.6%) then roots (10.4%), flowers (9.36%) and aerial parts (4.16%). Then, the others parts of plant are rarely used (Figure 2).\nMedicinal parts of the plants used for ethnomedicinal purposes in the study.\nThe analysis of the ethnobotanical data showed that central region was best suited to the medicinal plant and rangeland. Ethnobotanical use categories showed that leaves were commonly used parts for making indigenous recipes (28.08%), followed by fruits (19.76%) and seeds (15.6%) then roots (10.4%), flowers (9.36%) and aerial parts (4.16%). Then, the others parts of plant are rarely used (Figure 2).\nMedicinal parts of the plants used for ethnomedicinal purposes in the study.\nModes and conditions of medicine preparation The analysis of the ethnobotanical data showed that the recipes in the most cases were obtained from single herb, but some of recipes were prepared together, and there is a famous local mixture called Damask tisane (zhourate Shamieh). A mode of TM preparation reported was a decoction (30%), followed by infusion (23%), and then by other method such as fresh herbs, juice, cooked, powder, vinegar and oils (47%) (Figure 3). Considering according to results, most of the plant preparations are used orally (Table 2).\nModes of ethnomedicines preparation in Homs and Hama.\nThe analysis of the ethnobotanical data showed that the recipes in the most cases were obtained from single herb, but some of recipes were prepared together, and there is a famous local mixture called Damask tisane (zhourate Shamieh). A mode of TM preparation reported was a decoction (30%), followed by infusion (23%), and then by other method such as fresh herbs, juice, cooked, powder, vinegar and oils (47%) (Figure 3). Considering according to results, most of the plant preparations are used orally (Table 2).\nModes of ethnomedicines preparation in Homs and Hama.\nEthnomedicinal information about treatment the different diseases The results of questionnaires showed that 20% of the informants were diagnosed with their diseases by a doctor, and 45% were diagnosed with a conventional therapist, and 35% self-diagnosed their diseases, while the results of the questionnaires showed that the evaluation of the treatment by informants as following (58% relied on the disappearance of symptoms, and 24% through the results of laboratory analysis, 18% adopted other methods such as chest radiography, adopting the attending physician’s opinion and clinical observation of the improvement of skin diseases, and some of them depended on psychological comfort during treatment as evidence of improvement).\nAmong these studied plants, 62 are used to treat digestive disorders, 41 for respiratory diseases, including asthma, bronchitis and coughs, 40 for skin diseases, 16 for diabetes, 36 for kidney and urinary tract disorders, 22 for nervous system disorders, six for enhance the body's immunity, two for haemorrhoids, five for fever, eight for heart disorders, five for infertility and impotence, six for treating several types of cancer, two for increasing breast milk production, five for losing weight, four for lowering cholesterol, and two for increasing weight, and six for anaemia, 15 for blood disorder, two anti-toxicant, 19 for arthritis and pain, one for typhoid disorder, eight for infections, six for gynaecological diseases, one for eye inflammation, two anti-toxicant and four for mouth sores. Many of them are still used today, especially those plants recommended for internal uses such as traditional medicinal teas, which mainly consist of remedies for obesity, weight loss, colds, colds, digestive disorders, abdominal pain, constipation and some skin diseases (Figure 4).\nEthno-medicinal information about treating different diseases related to central region in Syria (Homs and Hama).\nThe results of questionnaires showed that 20% of the informants were diagnosed with their diseases by a doctor, and 45% were diagnosed with a conventional therapist, and 35% self-diagnosed their diseases, while the results of the questionnaires showed that the evaluation of the treatment by informants as following (58% relied on the disappearance of symptoms, and 24% through the results of laboratory analysis, 18% adopted other methods such as chest radiography, adopting the attending physician’s opinion and clinical observation of the improvement of skin diseases, and some of them depended on psychological comfort during treatment as evidence of improvement).\nAmong these studied plants, 62 are used to treat digestive disorders, 41 for respiratory diseases, including asthma, bronchitis and coughs, 40 for skin diseases, 16 for diabetes, 36 for kidney and urinary tract disorders, 22 for nervous system disorders, six for enhance the body's immunity, two for haemorrhoids, five for fever, eight for heart disorders, five for infertility and impotence, six for treating several types of cancer, two for increasing breast milk production, five for losing weight, four for lowering cholesterol, and two for increasing weight, and six for anaemia, 15 for blood disorder, two anti-toxicant, 19 for arthritis and pain, one for typhoid disorder, eight for infections, six for gynaecological diseases, one for eye inflammation, two anti-toxicant and four for mouth sores. Many of them are still used today, especially those plants recommended for internal uses such as traditional medicinal teas, which mainly consist of remedies for obesity, weight loss, colds, colds, digestive disorders, abdominal pain, constipation and some skin diseases (Figure 4).\nEthno-medicinal information about treating different diseases related to central region in Syria (Homs and Hama).","In total, 151 local inhabitants of 35 villages and 59 districts were selected based on their experience in traditional uses of plants. Table 1 shows the age and gender wise distribution. All of them were interrogated using semi-structured questionnaires. Generally in Syria, both genders were interested in herbal medicines.\nAge and gender distribution.","The study area is considered one of the Syrian areas rich in medicinal plants. In the east of it there is a desert (badia), which is dominated by desert and thorny plants, and in the west of it, aromatic plants are spread in the mountains near the eastern coast of the Mediterranean Sea. It is a fact that people in some of these rural areas suffer from poverty, so they depend a lot on folk remedies, and folk healers in these areas provide their expertise at small costs, because the medicinal herbs are cheaper than chemical medicines, and most of the medicinal recipes are available around. However, a large portion of the uses of medicinal plants mentioned in the research are still under study.\nA total of 76 medicinal plant species (57.9% are wild and 42.1% are cultivated plants) belonging to 39 families are recorded in the present study; they are being used for a variety of purposes by native people. The detailed inventory is provided in Table 2, which includes botanical names, followed by local name, family and ethnobotanical uses.\nEthnobotanical uses of plant species according to ethnomedicinal survey of central region in Syria (Homs and Hama).\nAp: aerial parts; Bk: bark; Br: branches; Bu: buds; Bb: bulb; Ca: capitulum; Co: cones; Fl: flower; F: fruit; G: gum; Lx: latex; L: leaves; Ps: pedicles; Pe: peel; Ph: phloem; P: pods; Rs: resin; Rm: rhizome; R: root; Sd: seeds; S: stem; St: stylus; T: tubers; W: whole plant.\nC: cultivated plants, W: wild plants.\nHerbarium no.","The most commonly mentioned family is Asteraceae (11.84%), followed by Lamiaceae (10.52%), then Rosaceae (7.89%) and Apiaceae (6.57%), Poaceae (5.26%), Anacardiaceae and Fabaceae (3.94%), Fagaceae, Liliaceae, Myrtaceae and Oleaceae (2.63%), then all the other families (1.31%) (Figure 1).\nPlant families commonly used in ethnomedicinal survey of central region in Syria (Homs and Hama).","Medicinal use plants (UV) are utilized to find the most frequently used plant species in the study area. Its value ranged from 0.066 to 0.92 (Table 2). The calculated results of UV showed that Cichorium intybus L., Eucalyptus globulus Labill. was ranked first (UV = 0.92) followed by Fraxinus syriaca Boiss. (UV = 0.907), Olea europaea L. (UV = 0.9), then Allium sativum L. (UV = 0.894), Lepidium sativum L. (UV = 0.867), Coriandrum sativum L. (UV = 0.86), Glycyrrhiza glabra L. (UV = 0.854), Dittrichia viscosa (L.) Greuter (UV = 0.841), while the lowest value was found for Plumbago europaea L. (UV = 0.066).","The analysis of the ethnobotanical data showed that central region was best suited to the medicinal plant and rangeland. Ethnobotanical use categories showed that leaves were commonly used parts for making indigenous recipes (28.08%), followed by fruits (19.76%) and seeds (15.6%) then roots (10.4%), flowers (9.36%) and aerial parts (4.16%). Then, the others parts of plant are rarely used (Figure 2).\nMedicinal parts of the plants used for ethnomedicinal purposes in the study.","The analysis of the ethnobotanical data showed that the recipes in the most cases were obtained from single herb, but some of recipes were prepared together, and there is a famous local mixture called Damask tisane (zhourate Shamieh). A mode of TM preparation reported was a decoction (30%), followed by infusion (23%), and then by other method such as fresh herbs, juice, cooked, powder, vinegar and oils (47%) (Figure 3). Considering according to results, most of the plant preparations are used orally (Table 2).\nModes of ethnomedicines preparation in Homs and Hama.","The results of questionnaires showed that 20% of the informants were diagnosed with their diseases by a doctor, and 45% were diagnosed with a conventional therapist, and 35% self-diagnosed their diseases, while the results of the questionnaires showed that the evaluation of the treatment by informants as following (58% relied on the disappearance of symptoms, and 24% through the results of laboratory analysis, 18% adopted other methods such as chest radiography, adopting the attending physician’s opinion and clinical observation of the improvement of skin diseases, and some of them depended on psychological comfort during treatment as evidence of improvement).\nAmong these studied plants, 62 are used to treat digestive disorders, 41 for respiratory diseases, including asthma, bronchitis and coughs, 40 for skin diseases, 16 for diabetes, 36 for kidney and urinary tract disorders, 22 for nervous system disorders, six for enhance the body's immunity, two for haemorrhoids, five for fever, eight for heart disorders, five for infertility and impotence, six for treating several types of cancer, two for increasing breast milk production, five for losing weight, four for lowering cholesterol, and two for increasing weight, and six for anaemia, 15 for blood disorder, two anti-toxicant, 19 for arthritis and pain, one for typhoid disorder, eight for infections, six for gynaecological diseases, one for eye inflammation, two anti-toxicant and four for mouth sores. Many of them are still used today, especially those plants recommended for internal uses such as traditional medicinal teas, which mainly consist of remedies for obesity, weight loss, colds, colds, digestive disorders, abdominal pain, constipation and some skin diseases (Figure 4).\nEthno-medicinal information about treating different diseases related to central region in Syria (Homs and Hama).","The use of TAM has spread to treat various diseases in Syria since ancient times. They are cost-effective with fewer side effects and are more suitable for long-term use compared with chemically synthesized medicines.\nThe ethnobotanical categories indicated that there is large use of medicinal herbs in the area of study, most of them are wild. There is an increased exploitation of medicinal plants by the local population, collectors and dealers of herbal medicines, in line with the demand from the pharmaceutical industry. This caused a sharp decrease in the occurrence and products of medicinal plants. Grazing, deforestation by cutting down trees for heating, and fires were mainly responsible for the reduction of medicinal plants. That is why the government is working on developing strategies to conserve wild plant diversity. Some people collect the medicinal plants for an income. They uproot and collect each part of the medicinal plants in non-scientific way. Thus, to date, a few articles devoted to TM of Syria, such as a study of folk medicine in Aleppo Governorate (Alachkar et al. 2011), and a study about the use of ‘Zahraa’ (Syrian traditional tisane) (Carmona et al. 2005), and a third one on the medicinal plants in Golan (Said et al. 2002), which is an occupied Syrian territory.\nThis research aspires to genuinely contribute in providing useful information on the conserving and sustaining the natural resources in the area.\nThe perspectives in the questionnaire were compared with other ethnomedicine studies in the countries surrounding Syria such as Lebanon (Taha et al. 2013), Jordan (Lev and Amar 2002; Al-Qura'n 2009), Palestine (Friedman et al. 1986; Kaileh et al. 2007), Iraq (Al-Douri 2000) and Turkey (Yeşilada et al. 1995; Sezik et al. 2001). Similarities in various traditional uses in Syria, Lebanon, Palestine and Jordan were observed. This is mainly due to the mutual history of these areas that were previously called Levantine Nations (Bilad al-Sham) (Lev 2002), and there is some similarity with a smaller number of folk uses both in Syria and Iraq, but there is a difference in the folk uses described between Syria and Turkey (Korkmaz et al. 2016; Yerebasan et al. 2020). We compared the folk uses of plants mentioned in related articles on ethnomedicine in these regions with the plants studied in our research to find out the extent of congruence or difference the uses of similar plants.\nWe did not record significant differences in phytomedicines consumption customs between interviewees of different religions. In general, phytomedicines consumption was often explained and justified by interviewees as family tradition. We did not detect any gender-related differences in phytomedicines consumption. There were no gender differences concerning the common traditional use of medicinal plants. The ethno-medicine data presented herein imply that medicinal plants are important as food and particularly as medicine (traditional healing) for various local people. While chemical medicinal treatments are becoming commonplace, traditional medications are still of huge importance in many rural, poor and remote places.\nThis study will undoubtedly provide new data that could contribute to further pharmacological discoveries by identifying the active ingredients and their mechanism of effect by doing a lot of pharmacological work to confirm the alleged biological activities of these plants, and the possibility of developing new pharmaceutical formulas cannot be excluded depending on Syrian medicinal plants and their folk uses; as the discovery of artemisinin from Artemisia annua, based on ethnobotanical information (Acton and Klayman 1985), serves as evidence that it is possible to find new and effective medicines using data from TM.","There is insufficient information about the pharmacokinetic efficacy of the medicinal plant species in this study. These herbs have reportedly and traditionally been used as adjuvant to relieve and treat some diseases.","Many of the uses of medicinal plants mentioned in Syria are still under study. This study has been conducted with the aim to generate new concepts that could supplement pharmacological work with potentially further pharmacological discoveries; that is, by identifying the active ingredients and their mechanism of effect to confirm the alleged biological activities of these plants."],"string":"[\n \"Traditional medicine (TM), as defined by the World Health Organization (WHO), is the sum total of the knowledge, skills and practices based on the theories, beliefs and experiences indigenous to different cultures, whether explicable or not, used in the maintenance of health as well as in the prevention, diagnosis, improvement or treatment of physical and mental illness (WHO 2002a, 2002b). Some TM systems are supported by huge volumes of literature and records of the theoretical concepts and practical skills; others pass down from generation to generation through verbal teaching. To date, in some parts of the world, the majority of the population continue to rely on their own TM to meet their primary health care needs. When adopted outside of its traditional culture, TM is often referred as complementary and alternative medicine (CAM) (Che et al. 2017).\\nAlthough modern medicine is currently available in many developing countries, large proportions of the population in these countries still rely to a large extent on traditional practitioners and medicinal plants for therapeutic purposes. TM is often the first choice for providing primary health care in developing countries, and the WHO estimates that more than 80% of healthcare needs in these countries are met by traditional health care practices, being the cheapest and most accessible (WHO 2002a, 2002b, 2004, 2005).\\nThe WHO pays special attention to TM and CAM. Resolution (no. WHA13-6) issued by the World Health Assembly (WHA) in 2009 emphasized the need to update the global TM strategy (WHO 2009), so the WHO issued its strategy for traditional (folk medicine) 2014–2023 (WHO 2013).\\nThe United Nations Educational Scientific and Cultural Organization (UNESCO) works to document the medical heritage of peoples within its documentation of the living intangible cultural heritage of peoples and civilizations, through the documentation of TM practices that were included in the UNESCO convention (2003), the convention on biological diversity (1992) and the UNESCO universal declaration on cultural diversity (2001) and the United Nations declaration on the rights of indigenous peoples (2007), and according to the meeting of the UNESCO International Bioethics Committee (IBC) working group on TM and its ethical effects in Paris (2010), it stressed the need to conduct studies that illustrate the use of TM in various regions around the world, and its evaluation in clinical practice and in research and policy (IBC 2013).\\nIn 2017, the International Center for Information and Networks for Intangible Cultural Heritage in the Asia-Pacific region (ICHCAP) under the auspices of UNESCO, issued a book entitled Traditional Medicine in which the Syria Trust for Development in Section VII included TM in Syria (Falk 2017).\\nEthno-botany is the scientific study of the relationships between people and plants. It was first coined in 1896 by the US botanist John Harshberger; however, the history of ethnobotany began long before that (Campbell et al. 2002; Amjad et al. 2015). It plays an important role in understanding the dynamic relationships between biological diversity and social and cultural systems (Husain et al. 2008; Amjad et al. 2013, 2015). Plants are essential for human beings as they provide food, and medicines (Alam et al. 2011; Ahmad et al. 2012).\\nTraditional Arabic medicine (TAM) is one of the famous traditional medical systems, which is occasionally called Unani medicine, Graeco-Arabic medicine, humoral medicine or Islamic medicine. The subject of TM in Syria has received little attention in the literature, and very little is known about the traditional medicinal substances used nowadays by the Syrian population to treat the most common diseases. Throughout ancient times in Syria, as part of the Levantine Nations (Bilad al-Sham), and other lands in the region, humans used various natural materials as sources of medicines (Jaddouh 2004). In the western countryside of Hama, there is a natural reserve for medicinal plants, which is called the Abu Qubais Protected area in Al-Ghab region (which protect the biodiversity rights of indigenous people and affiliated to the general commission for Al-Ghab administration and development), 509 plant species belonging to 72 families have been recorded (Al-Mahmoud and Al-Shater 2010).\\nFor these reasons, the present investigation gathered the uses of medicinal plants in the centre region of Syria (Homs and Hama), as a supplement for a national survey, and documents the information concerning the uses of medicinal plants, which may serve as the basis of knowledge for a more intensive scientific research.\",\n \"Study area Syria is a country located on the east coast of the Mediterranean Sea in southwestern Asia. Syria is bounded by Turkey to the north, Iraq to the east and southeast, Jordan to the south and Lebanon to the southwest. The study area is the middle region of Syria including Homs Governorate and Hama Governorate.\\nThis area has a Mediterranean climate with a long dry season from May to October. In the extreme northwest, there is some light summer rain. On the western region, summers are hot, with mean daily maximum temperatures ranging from low to mid 80 °F, while the mild winters have daily mean minimum reaching temperatures low level of 50 °F. Only above about 1500 m are the summers relatively cool. In inland the climate becomes arid, with colder winters and hotter summers. In the desert, at Tadmur maximum temperatures in the summer, temperature reaches averages in the ranges of upper 90s to low 100 °F, with extremes in the 110 °F. In rural areas, work takes place according to the seasonal rhythm of agriculture. Women generally share in much of the agricultural labour. Agriculture constitutes an important source of income, and fruits and vegetables including onions, olives and grapes. Commercially important forest plants include: pistachio, which is important for its oil-rich fruit, and plants such as olive trees, grapevines, apricot trees and cumin (Hamidé et al. 2021).\\nHistorically, the ancient Palmyra, also called Tadmur, is an ancient city in south-central Syria (Homs Governorate). An oasis in the Syrian desert, Palmyra contains the monumental ruins of a great city that was one of the most important cultural centres of the ancient world, from the first to the second century according to World Heritage List (UNESCO 2021).\\nSyria is a country located on the east coast of the Mediterranean Sea in southwestern Asia. Syria is bounded by Turkey to the north, Iraq to the east and southeast, Jordan to the south and Lebanon to the southwest. The study area is the middle region of Syria including Homs Governorate and Hama Governorate.\\nThis area has a Mediterranean climate with a long dry season from May to October. In the extreme northwest, there is some light summer rain. On the western region, summers are hot, with mean daily maximum temperatures ranging from low to mid 80 °F, while the mild winters have daily mean minimum reaching temperatures low level of 50 °F. Only above about 1500 m are the summers relatively cool. In inland the climate becomes arid, with colder winters and hotter summers. In the desert, at Tadmur maximum temperatures in the summer, temperature reaches averages in the ranges of upper 90s to low 100 °F, with extremes in the 110 °F. In rural areas, work takes place according to the seasonal rhythm of agriculture. Women generally share in much of the agricultural labour. Agriculture constitutes an important source of income, and fruits and vegetables including onions, olives and grapes. Commercially important forest plants include: pistachio, which is important for its oil-rich fruit, and plants such as olive trees, grapevines, apricot trees and cumin (Hamidé et al. 2021).\\nHistorically, the ancient Palmyra, also called Tadmur, is an ancient city in south-central Syria (Homs Governorate). An oasis in the Syrian desert, Palmyra contains the monumental ruins of a great city that was one of the most important cultural centres of the ancient world, from the first to the second century according to World Heritage List (UNESCO 2021).\\nField work and data collection Field surveys were conducted between December 2019 and January 2021, to document ethnobotanical information through oral interviews and designed semi-structured questionnaire. Thirty-five villages and 59 districts were visited for field research; 235 people were contacted and 151 of those with ethno-botanical experience agreed to become our informants, including 35 local herbalists (Tabib Arabi) and 10 physicians (who hold a general medicine specialty). The queries were repeatedly made to increase the reliability of the data; interviews with the men were usually carried out in the ‘Mukhtar’ house where they come together, and with women in their homes, bazaars and gardens. The Syria Trust for Development (which is a national development organization, and has a program called ‘Mashrouie’ that runs innovative microcredit programs that encourage economic growth in disadvantaged areas) helped us in data collecting. The information gathered during the present study included socio-demographic characteristics of the interviewed informants (age, gender) and ethnopharmacological information, including the local and scientific name of the species, local names, plant parts used, modes of use, conservation method, administration mode and toxicity, all documented data were then translated into English and Latin. Information that had been carried to the region from the outside and that was not used or confirmed was not included and recorded (Weckerle et al. 2018). During the interviews, questions about the following were asked to the participants:Name and surnameAge and sexEducational levelAre plants collected in your region?Do you have any contact with plants?Can you show the plants you use in your region?Can you tell the names of local plants you use in your region?In which season do you collect the plants you use in your region?When collecting plant, which parts of the plant do you collect and how do you collect them?Which parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).How do you prepare and administrate the plants’ parts?How did you diagnose the disease (by physician, by traditional healers, self-treating)?How did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\\nName and surname\\nAge and sex\\nEducational level\\nAre plants collected in your region?\\nDo you have any contact with plants?\\nCan you show the plants you use in your region?\\nCan you tell the names of local plants you use in your region?\\nIn which season do you collect the plants you use in your region?\\nWhen collecting plant, which parts of the plant do you collect and how do you collect them?\\nWhich parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).\\nHow do you prepare and administrate the plants’ parts?\\nHow did you diagnose the disease (by physician, by traditional healers, self-treating)?\\nHow did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\\nOnly the medication of single herbs was included, while mixtures and multiple recipes were disregarded. The answers that were given with doubt to the questions above were not recorded, and we adopted the information that gave 40% frequency in the collected data, and the data that gave less than that were neglected.\\nInformation about diseases diagnosed specifically by physicians and traditional healers was approved, while self-treating diseases were referred to as auxiliary in treatment, nutritional uses were clearly indicated, and some plants were used to prevent disease and improve public health. The information about diseases was divided into three groups when analysing the data (group A: acute diseases, group B: chronic diseases, group C: simple symptoms and signs), and the focus was only on groups B and C.\\nField surveys were conducted between December 2019 and January 2021, to document ethnobotanical information through oral interviews and designed semi-structured questionnaire. Thirty-five villages and 59 districts were visited for field research; 235 people were contacted and 151 of those with ethno-botanical experience agreed to become our informants, including 35 local herbalists (Tabib Arabi) and 10 physicians (who hold a general medicine specialty). The queries were repeatedly made to increase the reliability of the data; interviews with the men were usually carried out in the ‘Mukhtar’ house where they come together, and with women in their homes, bazaars and gardens. The Syria Trust for Development (which is a national development organization, and has a program called ‘Mashrouie’ that runs innovative microcredit programs that encourage economic growth in disadvantaged areas) helped us in data collecting. The information gathered during the present study included socio-demographic characteristics of the interviewed informants (age, gender) and ethnopharmacological information, including the local and scientific name of the species, local names, plant parts used, modes of use, conservation method, administration mode and toxicity, all documented data were then translated into English and Latin. Information that had been carried to the region from the outside and that was not used or confirmed was not included and recorded (Weckerle et al. 2018). During the interviews, questions about the following were asked to the participants:Name and surnameAge and sexEducational levelAre plants collected in your region?Do you have any contact with plants?Can you show the plants you use in your region?Can you tell the names of local plants you use in your region?In which season do you collect the plants you use in your region?When collecting plant, which parts of the plant do you collect and how do you collect them?Which parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).How do you prepare and administrate the plants’ parts?How did you diagnose the disease (by physician, by traditional healers, self-treating)?How did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\\nName and surname\\nAge and sex\\nEducational level\\nAre plants collected in your region?\\nDo you have any contact with plants?\\nCan you show the plants you use in your region?\\nCan you tell the names of local plants you use in your region?\\nIn which season do you collect the plants you use in your region?\\nWhen collecting plant, which parts of the plant do you collect and how do you collect them?\\nWhich parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).\\nHow do you prepare and administrate the plants’ parts?\\nHow did you diagnose the disease (by physician, by traditional healers, self-treating)?\\nHow did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\\nOnly the medication of single herbs was included, while mixtures and multiple recipes were disregarded. The answers that were given with doubt to the questions above were not recorded, and we adopted the information that gave 40% frequency in the collected data, and the data that gave less than that were neglected.\\nInformation about diseases diagnosed specifically by physicians and traditional healers was approved, while self-treating diseases were referred to as auxiliary in treatment, nutritional uses were clearly indicated, and some plants were used to prevent disease and improve public health. The information about diseases was divided into three groups when analysing the data (group A: acute diseases, group B: chronic diseases, group C: simple symptoms and signs), and the focus was only on groups B and C.\\nTaxonomic identification of the species Medicinal plants being mentioned by the Informants were recorded with local names and photographed. Each reported medicinal plant species was gathered, compressed, dehydrated, mounted on herbarium sheets, and identified; the taxonomic identity of the plants was confirmed by Prof. Abdel Aleem Bello (PhD/Botanical Taxonomy) and Dr. Bayan Tiba (PhD/Botanical Taxonomy) Aleppo University. As far as possible, the name of the plants was updated by consulting the latest literature; generic and species names followed the plant list (http://www.theplantlist.org). All voucher specimens have been preserved during documentation and deposited in the Damascus University, Faculty of Pharmacy, Pharmacognosy Labs Herbarium for future reference, serial numbers were taken from (1ch to 76ch) according to its alphabetical order.\\nMedicinal plants being mentioned by the Informants were recorded with local names and photographed. Each reported medicinal plant species was gathered, compressed, dehydrated, mounted on herbarium sheets, and identified; the taxonomic identity of the plants was confirmed by Prof. Abdel Aleem Bello (PhD/Botanical Taxonomy) and Dr. Bayan Tiba (PhD/Botanical Taxonomy) Aleppo University. As far as possible, the name of the plants was updated by consulting the latest literature; generic and species names followed the plant list (http://www.theplantlist.org). All voucher specimens have been preserved during documentation and deposited in the Damascus University, Faculty of Pharmacy, Pharmacognosy Labs Herbarium for future reference, serial numbers were taken from (1ch to 76ch) according to its alphabetical order.\\nEthics approval and consent to participate The study was approved by the Ethics Committee of the University of Damascus. Before beginning data collection, we obtained verbal informed permission in each case site-wide and then individually before each interview. We also informed informants that it was an academic project and that the investigation was for research purposes only, and not for any financial or other benefits. Informants provided verbal informed consent to participate in this study; they were free to withdraw their information at any time. These informants freely accepted the interview. All steps of research are consistent with Ethnobiology Code of Ethics (ISE 2006).\\nThe study was approved by the Ethics Committee of the University of Damascus. Before beginning data collection, we obtained verbal informed permission in each case site-wide and then individually before each interview. We also informed informants that it was an academic project and that the investigation was for research purposes only, and not for any financial or other benefits. Informants provided verbal informed consent to participate in this study; they were free to withdraw their information at any time. These informants freely accepted the interview. All steps of research are consistent with Ethnobiology Code of Ethics (ISE 2006).\\nData analysis The data collected through interviews of the informants were classified and examined with the statistical program IBM® SPSS® Statistics 26 (IBM, Armonk, NY), to determine the proportions of different variables such as ethnopharmacological data. Quantitative value indices were analysed using different statistical quantitative tools, i.e., the use reports (UR) of a species, and use value (UV) (Chaachouaya et al. 2021).\\nThe data collected through interviews of the informants were classified and examined with the statistical program IBM® SPSS® Statistics 26 (IBM, Armonk, NY), to determine the proportions of different variables such as ethnopharmacological data. Quantitative value indices were analysed using different statistical quantitative tools, i.e., the use reports (UR) of a species, and use value (UV) (Chaachouaya et al. 2021).\\nUse value and use reports The UR of a species or its importance in the culture of a community is denoted by its mentioning rate or its mention frequency by informants. The UR of the species of plants being utilized was evaluated using the formula (Tenté et al 2012):\\nUR = Ni/n\\nwhere Ni is the number of times a particular species was mentioned; n is the total number of times that all species were mentioned.\\nThe UV of recorded medicinal plants was determined by applying the following formula (Tabuti et al. 2003):\\nUV=∑ URi/N\\nwhere ∑ URi is the total number of UR per plants; N is the total of interviewees questioned for given medicinal species.\\nThe UV rate will be more important if there are several useful records for a species, implying that the plant is significant, whereas they will be near 0 if there are few reports compared to its use (Yaseen 2015; Chaachouaya et al. 2021).\\nThe UR of a species or its importance in the culture of a community is denoted by its mentioning rate or its mention frequency by informants. The UR of the species of plants being utilized was evaluated using the formula (Tenté et al 2012):\\nUR = Ni/n\\nwhere Ni is the number of times a particular species was mentioned; n is the total number of times that all species were mentioned.\\nThe UV of recorded medicinal plants was determined by applying the following formula (Tabuti et al. 2003):\\nUV=∑ URi/N\\nwhere ∑ URi is the total number of UR per plants; N is the total of interviewees questioned for given medicinal species.\\nThe UV rate will be more important if there are several useful records for a species, implying that the plant is significant, whereas they will be near 0 if there are few reports compared to its use (Yaseen 2015; Chaachouaya et al. 2021).\",\n \"Syria is a country located on the east coast of the Mediterranean Sea in southwestern Asia. Syria is bounded by Turkey to the north, Iraq to the east and southeast, Jordan to the south and Lebanon to the southwest. The study area is the middle region of Syria including Homs Governorate and Hama Governorate.\\nThis area has a Mediterranean climate with a long dry season from May to October. In the extreme northwest, there is some light summer rain. On the western region, summers are hot, with mean daily maximum temperatures ranging from low to mid 80 °F, while the mild winters have daily mean minimum reaching temperatures low level of 50 °F. Only above about 1500 m are the summers relatively cool. In inland the climate becomes arid, with colder winters and hotter summers. In the desert, at Tadmur maximum temperatures in the summer, temperature reaches averages in the ranges of upper 90s to low 100 °F, with extremes in the 110 °F. In rural areas, work takes place according to the seasonal rhythm of agriculture. Women generally share in much of the agricultural labour. Agriculture constitutes an important source of income, and fruits and vegetables including onions, olives and grapes. Commercially important forest plants include: pistachio, which is important for its oil-rich fruit, and plants such as olive trees, grapevines, apricot trees and cumin (Hamidé et al. 2021).\\nHistorically, the ancient Palmyra, also called Tadmur, is an ancient city in south-central Syria (Homs Governorate). An oasis in the Syrian desert, Palmyra contains the monumental ruins of a great city that was one of the most important cultural centres of the ancient world, from the first to the second century according to World Heritage List (UNESCO 2021).\",\n \"Field surveys were conducted between December 2019 and January 2021, to document ethnobotanical information through oral interviews and designed semi-structured questionnaire. Thirty-five villages and 59 districts were visited for field research; 235 people were contacted and 151 of those with ethno-botanical experience agreed to become our informants, including 35 local herbalists (Tabib Arabi) and 10 physicians (who hold a general medicine specialty). The queries were repeatedly made to increase the reliability of the data; interviews with the men were usually carried out in the ‘Mukhtar’ house where they come together, and with women in their homes, bazaars and gardens. The Syria Trust for Development (which is a national development organization, and has a program called ‘Mashrouie’ that runs innovative microcredit programs that encourage economic growth in disadvantaged areas) helped us in data collecting. The information gathered during the present study included socio-demographic characteristics of the interviewed informants (age, gender) and ethnopharmacological information, including the local and scientific name of the species, local names, plant parts used, modes of use, conservation method, administration mode and toxicity, all documented data were then translated into English and Latin. Information that had been carried to the region from the outside and that was not used or confirmed was not included and recorded (Weckerle et al. 2018). During the interviews, questions about the following were asked to the participants:Name and surnameAge and sexEducational levelAre plants collected in your region?Do you have any contact with plants?Can you show the plants you use in your region?Can you tell the names of local plants you use in your region?In which season do you collect the plants you use in your region?When collecting plant, which parts of the plant do you collect and how do you collect them?Which parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).How do you prepare and administrate the plants’ parts?How did you diagnose the disease (by physician, by traditional healers, self-treating)?How did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\\nName and surname\\nAge and sex\\nEducational level\\nAre plants collected in your region?\\nDo you have any contact with plants?\\nCan you show the plants you use in your region?\\nCan you tell the names of local plants you use in your region?\\nIn which season do you collect the plants you use in your region?\\nWhen collecting plant, which parts of the plant do you collect and how do you collect them?\\nWhich parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).\\nHow do you prepare and administrate the plants’ parts?\\nHow did you diagnose the disease (by physician, by traditional healers, self-treating)?\\nHow did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\\nOnly the medication of single herbs was included, while mixtures and multiple recipes were disregarded. The answers that were given with doubt to the questions above were not recorded, and we adopted the information that gave 40% frequency in the collected data, and the data that gave less than that were neglected.\\nInformation about diseases diagnosed specifically by physicians and traditional healers was approved, while self-treating diseases were referred to as auxiliary in treatment, nutritional uses were clearly indicated, and some plants were used to prevent disease and improve public health. The information about diseases was divided into three groups when analysing the data (group A: acute diseases, group B: chronic diseases, group C: simple symptoms and signs), and the focus was only on groups B and C.\",\n \"Medicinal plants being mentioned by the Informants were recorded with local names and photographed. Each reported medicinal plant species was gathered, compressed, dehydrated, mounted on herbarium sheets, and identified; the taxonomic identity of the plants was confirmed by Prof. Abdel Aleem Bello (PhD/Botanical Taxonomy) and Dr. Bayan Tiba (PhD/Botanical Taxonomy) Aleppo University. As far as possible, the name of the plants was updated by consulting the latest literature; generic and species names followed the plant list (http://www.theplantlist.org). All voucher specimens have been preserved during documentation and deposited in the Damascus University, Faculty of Pharmacy, Pharmacognosy Labs Herbarium for future reference, serial numbers were taken from (1ch to 76ch) according to its alphabetical order.\",\n \"The study was approved by the Ethics Committee of the University of Damascus. Before beginning data collection, we obtained verbal informed permission in each case site-wide and then individually before each interview. We also informed informants that it was an academic project and that the investigation was for research purposes only, and not for any financial or other benefits. Informants provided verbal informed consent to participate in this study; they were free to withdraw their information at any time. These informants freely accepted the interview. All steps of research are consistent with Ethnobiology Code of Ethics (ISE 2006).\",\n \"The data collected through interviews of the informants were classified and examined with the statistical program IBM® SPSS® Statistics 26 (IBM, Armonk, NY), to determine the proportions of different variables such as ethnopharmacological data. Quantitative value indices were analysed using different statistical quantitative tools, i.e., the use reports (UR) of a species, and use value (UV) (Chaachouaya et al. 2021).\",\n \"The UR of a species or its importance in the culture of a community is denoted by its mentioning rate or its mention frequency by informants. The UR of the species of plants being utilized was evaluated using the formula (Tenté et al 2012):\\nUR = Ni/n\\nwhere Ni is the number of times a particular species was mentioned; n is the total number of times that all species were mentioned.\\nThe UV of recorded medicinal plants was determined by applying the following formula (Tabuti et al. 2003):\\nUV=∑ URi/N\\nwhere ∑ URi is the total number of UR per plants; N is the total of interviewees questioned for given medicinal species.\\nThe UV rate will be more important if there are several useful records for a species, implying that the plant is significant, whereas they will be near 0 if there are few reports compared to its use (Yaseen 2015; Chaachouaya et al. 2021).\",\n \"Demographic data of informants In total, 151 local inhabitants of 35 villages and 59 districts were selected based on their experience in traditional uses of plants. Table 1 shows the age and gender wise distribution. All of them were interrogated using semi-structured questionnaires. Generally in Syria, both genders were interested in herbal medicines.\\nAge and gender distribution.\\nIn total, 151 local inhabitants of 35 villages and 59 districts were selected based on their experience in traditional uses of plants. Table 1 shows the age and gender wise distribution. All of them were interrogated using semi-structured questionnaires. Generally in Syria, both genders were interested in herbal medicines.\\nAge and gender distribution.\\nEthnobotanical and ethnomedicinal uses of plant species The study area is considered one of the Syrian areas rich in medicinal plants. In the east of it there is a desert (badia), which is dominated by desert and thorny plants, and in the west of it, aromatic plants are spread in the mountains near the eastern coast of the Mediterranean Sea. It is a fact that people in some of these rural areas suffer from poverty, so they depend a lot on folk remedies, and folk healers in these areas provide their expertise at small costs, because the medicinal herbs are cheaper than chemical medicines, and most of the medicinal recipes are available around. However, a large portion of the uses of medicinal plants mentioned in the research are still under study.\\nA total of 76 medicinal plant species (57.9% are wild and 42.1% are cultivated plants) belonging to 39 families are recorded in the present study; they are being used for a variety of purposes by native people. The detailed inventory is provided in Table 2, which includes botanical names, followed by local name, family and ethnobotanical uses.\\nEthnobotanical uses of plant species according to ethnomedicinal survey of central region in Syria (Homs and Hama).\\nAp: aerial parts; Bk: bark; Br: branches; Bu: buds; Bb: bulb; Ca: capitulum; Co: cones; Fl: flower; F: fruit; G: gum; Lx: latex; L: leaves; Ps: pedicles; Pe: peel; Ph: phloem; P: pods; Rs: resin; Rm: rhizome; R: root; Sd: seeds; S: stem; St: stylus; T: tubers; W: whole plant.\\nC: cultivated plants, W: wild plants.\\nHerbarium no.\\nThe study area is considered one of the Syrian areas rich in medicinal plants. In the east of it there is a desert (badia), which is dominated by desert and thorny plants, and in the west of it, aromatic plants are spread in the mountains near the eastern coast of the Mediterranean Sea. It is a fact that people in some of these rural areas suffer from poverty, so they depend a lot on folk remedies, and folk healers in these areas provide their expertise at small costs, because the medicinal herbs are cheaper than chemical medicines, and most of the medicinal recipes are available around. However, a large portion of the uses of medicinal plants mentioned in the research are still under study.\\nA total of 76 medicinal plant species (57.9% are wild and 42.1% are cultivated plants) belonging to 39 families are recorded in the present study; they are being used for a variety of purposes by native people. The detailed inventory is provided in Table 2, which includes botanical names, followed by local name, family and ethnobotanical uses.\\nEthnobotanical uses of plant species according to ethnomedicinal survey of central region in Syria (Homs and Hama).\\nAp: aerial parts; Bk: bark; Br: branches; Bu: buds; Bb: bulb; Ca: capitulum; Co: cones; Fl: flower; F: fruit; G: gum; Lx: latex; L: leaves; Ps: pedicles; Pe: peel; Ph: phloem; P: pods; Rs: resin; Rm: rhizome; R: root; Sd: seeds; S: stem; St: stylus; T: tubers; W: whole plant.\\nC: cultivated plants, W: wild plants.\\nHerbarium no.\\nBotanical families of plants used The most commonly mentioned family is Asteraceae (11.84%), followed by Lamiaceae (10.52%), then Rosaceae (7.89%) and Apiaceae (6.57%), Poaceae (5.26%), Anacardiaceae and Fabaceae (3.94%), Fagaceae, Liliaceae, Myrtaceae and Oleaceae (2.63%), then all the other families (1.31%) (Figure 1).\\nPlant families commonly used in ethnomedicinal survey of central region in Syria (Homs and Hama).\\nThe most commonly mentioned family is Asteraceae (11.84%), followed by Lamiaceae (10.52%), then Rosaceae (7.89%) and Apiaceae (6.57%), Poaceae (5.26%), Anacardiaceae and Fabaceae (3.94%), Fagaceae, Liliaceae, Myrtaceae and Oleaceae (2.63%), then all the other families (1.31%) (Figure 1).\\nPlant families commonly used in ethnomedicinal survey of central region in Syria (Homs and Hama).\\nUse value of the plants Medicinal use plants (UV) are utilized to find the most frequently used plant species in the study area. Its value ranged from 0.066 to 0.92 (Table 2). The calculated results of UV showed that Cichorium intybus L., Eucalyptus globulus Labill. was ranked first (UV = 0.92) followed by Fraxinus syriaca Boiss. (UV = 0.907), Olea europaea L. (UV = 0.9), then Allium sativum L. (UV = 0.894), Lepidium sativum L. (UV = 0.867), Coriandrum sativum L. (UV = 0.86), Glycyrrhiza glabra L. (UV = 0.854), Dittrichia viscosa (L.) Greuter (UV = 0.841), while the lowest value was found for Plumbago europaea L. (UV = 0.066).\\nMedicinal use plants (UV) are utilized to find the most frequently used plant species in the study area. Its value ranged from 0.066 to 0.92 (Table 2). The calculated results of UV showed that Cichorium intybus L., Eucalyptus globulus Labill. was ranked first (UV = 0.92) followed by Fraxinus syriaca Boiss. (UV = 0.907), Olea europaea L. (UV = 0.9), then Allium sativum L. (UV = 0.894), Lepidium sativum L. (UV = 0.867), Coriandrum sativum L. (UV = 0.86), Glycyrrhiza glabra L. (UV = 0.854), Dittrichia viscosa (L.) Greuter (UV = 0.841), while the lowest value was found for Plumbago europaea L. (UV = 0.066).\\nMedicinal parts of the plant used The analysis of the ethnobotanical data showed that central region was best suited to the medicinal plant and rangeland. Ethnobotanical use categories showed that leaves were commonly used parts for making indigenous recipes (28.08%), followed by fruits (19.76%) and seeds (15.6%) then roots (10.4%), flowers (9.36%) and aerial parts (4.16%). Then, the others parts of plant are rarely used (Figure 2).\\nMedicinal parts of the plants used for ethnomedicinal purposes in the study.\\nThe analysis of the ethnobotanical data showed that central region was best suited to the medicinal plant and rangeland. Ethnobotanical use categories showed that leaves were commonly used parts for making indigenous recipes (28.08%), followed by fruits (19.76%) and seeds (15.6%) then roots (10.4%), flowers (9.36%) and aerial parts (4.16%). Then, the others parts of plant are rarely used (Figure 2).\\nMedicinal parts of the plants used for ethnomedicinal purposes in the study.\\nModes and conditions of medicine preparation The analysis of the ethnobotanical data showed that the recipes in the most cases were obtained from single herb, but some of recipes were prepared together, and there is a famous local mixture called Damask tisane (zhourate Shamieh). A mode of TM preparation reported was a decoction (30%), followed by infusion (23%), and then by other method such as fresh herbs, juice, cooked, powder, vinegar and oils (47%) (Figure 3). Considering according to results, most of the plant preparations are used orally (Table 2).\\nModes of ethnomedicines preparation in Homs and Hama.\\nThe analysis of the ethnobotanical data showed that the recipes in the most cases were obtained from single herb, but some of recipes were prepared together, and there is a famous local mixture called Damask tisane (zhourate Shamieh). A mode of TM preparation reported was a decoction (30%), followed by infusion (23%), and then by other method such as fresh herbs, juice, cooked, powder, vinegar and oils (47%) (Figure 3). Considering according to results, most of the plant preparations are used orally (Table 2).\\nModes of ethnomedicines preparation in Homs and Hama.\\nEthnomedicinal information about treatment the different diseases The results of questionnaires showed that 20% of the informants were diagnosed with their diseases by a doctor, and 45% were diagnosed with a conventional therapist, and 35% self-diagnosed their diseases, while the results of the questionnaires showed that the evaluation of the treatment by informants as following (58% relied on the disappearance of symptoms, and 24% through the results of laboratory analysis, 18% adopted other methods such as chest radiography, adopting the attending physician’s opinion and clinical observation of the improvement of skin diseases, and some of them depended on psychological comfort during treatment as evidence of improvement).\\nAmong these studied plants, 62 are used to treat digestive disorders, 41 for respiratory diseases, including asthma, bronchitis and coughs, 40 for skin diseases, 16 for diabetes, 36 for kidney and urinary tract disorders, 22 for nervous system disorders, six for enhance the body's immunity, two for haemorrhoids, five for fever, eight for heart disorders, five for infertility and impotence, six for treating several types of cancer, two for increasing breast milk production, five for losing weight, four for lowering cholesterol, and two for increasing weight, and six for anaemia, 15 for blood disorder, two anti-toxicant, 19 for arthritis and pain, one for typhoid disorder, eight for infections, six for gynaecological diseases, one for eye inflammation, two anti-toxicant and four for mouth sores. Many of them are still used today, especially those plants recommended for internal uses such as traditional medicinal teas, which mainly consist of remedies for obesity, weight loss, colds, colds, digestive disorders, abdominal pain, constipation and some skin diseases (Figure 4).\\nEthno-medicinal information about treating different diseases related to central region in Syria (Homs and Hama).\\nThe results of questionnaires showed that 20% of the informants were diagnosed with their diseases by a doctor, and 45% were diagnosed with a conventional therapist, and 35% self-diagnosed their diseases, while the results of the questionnaires showed that the evaluation of the treatment by informants as following (58% relied on the disappearance of symptoms, and 24% through the results of laboratory analysis, 18% adopted other methods such as chest radiography, adopting the attending physician’s opinion and clinical observation of the improvement of skin diseases, and some of them depended on psychological comfort during treatment as evidence of improvement).\\nAmong these studied plants, 62 are used to treat digestive disorders, 41 for respiratory diseases, including asthma, bronchitis and coughs, 40 for skin diseases, 16 for diabetes, 36 for kidney and urinary tract disorders, 22 for nervous system disorders, six for enhance the body's immunity, two for haemorrhoids, five for fever, eight for heart disorders, five for infertility and impotence, six for treating several types of cancer, two for increasing breast milk production, five for losing weight, four for lowering cholesterol, and two for increasing weight, and six for anaemia, 15 for blood disorder, two anti-toxicant, 19 for arthritis and pain, one for typhoid disorder, eight for infections, six for gynaecological diseases, one for eye inflammation, two anti-toxicant and four for mouth sores. Many of them are still used today, especially those plants recommended for internal uses such as traditional medicinal teas, which mainly consist of remedies for obesity, weight loss, colds, colds, digestive disorders, abdominal pain, constipation and some skin diseases (Figure 4).\\nEthno-medicinal information about treating different diseases related to central region in Syria (Homs and Hama).\",\n \"In total, 151 local inhabitants of 35 villages and 59 districts were selected based on their experience in traditional uses of plants. Table 1 shows the age and gender wise distribution. All of them were interrogated using semi-structured questionnaires. Generally in Syria, both genders were interested in herbal medicines.\\nAge and gender distribution.\",\n \"The study area is considered one of the Syrian areas rich in medicinal plants. In the east of it there is a desert (badia), which is dominated by desert and thorny plants, and in the west of it, aromatic plants are spread in the mountains near the eastern coast of the Mediterranean Sea. It is a fact that people in some of these rural areas suffer from poverty, so they depend a lot on folk remedies, and folk healers in these areas provide their expertise at small costs, because the medicinal herbs are cheaper than chemical medicines, and most of the medicinal recipes are available around. However, a large portion of the uses of medicinal plants mentioned in the research are still under study.\\nA total of 76 medicinal plant species (57.9% are wild and 42.1% are cultivated plants) belonging to 39 families are recorded in the present study; they are being used for a variety of purposes by native people. The detailed inventory is provided in Table 2, which includes botanical names, followed by local name, family and ethnobotanical uses.\\nEthnobotanical uses of plant species according to ethnomedicinal survey of central region in Syria (Homs and Hama).\\nAp: aerial parts; Bk: bark; Br: branches; Bu: buds; Bb: bulb; Ca: capitulum; Co: cones; Fl: flower; F: fruit; G: gum; Lx: latex; L: leaves; Ps: pedicles; Pe: peel; Ph: phloem; P: pods; Rs: resin; Rm: rhizome; R: root; Sd: seeds; S: stem; St: stylus; T: tubers; W: whole plant.\\nC: cultivated plants, W: wild plants.\\nHerbarium no.\",\n \"The most commonly mentioned family is Asteraceae (11.84%), followed by Lamiaceae (10.52%), then Rosaceae (7.89%) and Apiaceae (6.57%), Poaceae (5.26%), Anacardiaceae and Fabaceae (3.94%), Fagaceae, Liliaceae, Myrtaceae and Oleaceae (2.63%), then all the other families (1.31%) (Figure 1).\\nPlant families commonly used in ethnomedicinal survey of central region in Syria (Homs and Hama).\",\n \"Medicinal use plants (UV) are utilized to find the most frequently used plant species in the study area. Its value ranged from 0.066 to 0.92 (Table 2). The calculated results of UV showed that Cichorium intybus L., Eucalyptus globulus Labill. was ranked first (UV = 0.92) followed by Fraxinus syriaca Boiss. (UV = 0.907), Olea europaea L. (UV = 0.9), then Allium sativum L. (UV = 0.894), Lepidium sativum L. (UV = 0.867), Coriandrum sativum L. (UV = 0.86), Glycyrrhiza glabra L. (UV = 0.854), Dittrichia viscosa (L.) Greuter (UV = 0.841), while the lowest value was found for Plumbago europaea L. (UV = 0.066).\",\n \"The analysis of the ethnobotanical data showed that central region was best suited to the medicinal plant and rangeland. Ethnobotanical use categories showed that leaves were commonly used parts for making indigenous recipes (28.08%), followed by fruits (19.76%) and seeds (15.6%) then roots (10.4%), flowers (9.36%) and aerial parts (4.16%). Then, the others parts of plant are rarely used (Figure 2).\\nMedicinal parts of the plants used for ethnomedicinal purposes in the study.\",\n \"The analysis of the ethnobotanical data showed that the recipes in the most cases were obtained from single herb, but some of recipes were prepared together, and there is a famous local mixture called Damask tisane (zhourate Shamieh). A mode of TM preparation reported was a decoction (30%), followed by infusion (23%), and then by other method such as fresh herbs, juice, cooked, powder, vinegar and oils (47%) (Figure 3). Considering according to results, most of the plant preparations are used orally (Table 2).\\nModes of ethnomedicines preparation in Homs and Hama.\",\n \"The results of questionnaires showed that 20% of the informants were diagnosed with their diseases by a doctor, and 45% were diagnosed with a conventional therapist, and 35% self-diagnosed their diseases, while the results of the questionnaires showed that the evaluation of the treatment by informants as following (58% relied on the disappearance of symptoms, and 24% through the results of laboratory analysis, 18% adopted other methods such as chest radiography, adopting the attending physician’s opinion and clinical observation of the improvement of skin diseases, and some of them depended on psychological comfort during treatment as evidence of improvement).\\nAmong these studied plants, 62 are used to treat digestive disorders, 41 for respiratory diseases, including asthma, bronchitis and coughs, 40 for skin diseases, 16 for diabetes, 36 for kidney and urinary tract disorders, 22 for nervous system disorders, six for enhance the body's immunity, two for haemorrhoids, five for fever, eight for heart disorders, five for infertility and impotence, six for treating several types of cancer, two for increasing breast milk production, five for losing weight, four for lowering cholesterol, and two for increasing weight, and six for anaemia, 15 for blood disorder, two anti-toxicant, 19 for arthritis and pain, one for typhoid disorder, eight for infections, six for gynaecological diseases, one for eye inflammation, two anti-toxicant and four for mouth sores. Many of them are still used today, especially those plants recommended for internal uses such as traditional medicinal teas, which mainly consist of remedies for obesity, weight loss, colds, colds, digestive disorders, abdominal pain, constipation and some skin diseases (Figure 4).\\nEthno-medicinal information about treating different diseases related to central region in Syria (Homs and Hama).\",\n \"The use of TAM has spread to treat various diseases in Syria since ancient times. They are cost-effective with fewer side effects and are more suitable for long-term use compared with chemically synthesized medicines.\\nThe ethnobotanical categories indicated that there is large use of medicinal herbs in the area of study, most of them are wild. There is an increased exploitation of medicinal plants by the local population, collectors and dealers of herbal medicines, in line with the demand from the pharmaceutical industry. This caused a sharp decrease in the occurrence and products of medicinal plants. Grazing, deforestation by cutting down trees for heating, and fires were mainly responsible for the reduction of medicinal plants. That is why the government is working on developing strategies to conserve wild plant diversity. Some people collect the medicinal plants for an income. They uproot and collect each part of the medicinal plants in non-scientific way. Thus, to date, a few articles devoted to TM of Syria, such as a study of folk medicine in Aleppo Governorate (Alachkar et al. 2011), and a study about the use of ‘Zahraa’ (Syrian traditional tisane) (Carmona et al. 2005), and a third one on the medicinal plants in Golan (Said et al. 2002), which is an occupied Syrian territory.\\nThis research aspires to genuinely contribute in providing useful information on the conserving and sustaining the natural resources in the area.\\nThe perspectives in the questionnaire were compared with other ethnomedicine studies in the countries surrounding Syria such as Lebanon (Taha et al. 2013), Jordan (Lev and Amar 2002; Al-Qura'n 2009), Palestine (Friedman et al. 1986; Kaileh et al. 2007), Iraq (Al-Douri 2000) and Turkey (Yeşilada et al. 1995; Sezik et al. 2001). Similarities in various traditional uses in Syria, Lebanon, Palestine and Jordan were observed. This is mainly due to the mutual history of these areas that were previously called Levantine Nations (Bilad al-Sham) (Lev 2002), and there is some similarity with a smaller number of folk uses both in Syria and Iraq, but there is a difference in the folk uses described between Syria and Turkey (Korkmaz et al. 2016; Yerebasan et al. 2020). We compared the folk uses of plants mentioned in related articles on ethnomedicine in these regions with the plants studied in our research to find out the extent of congruence or difference the uses of similar plants.\\nWe did not record significant differences in phytomedicines consumption customs between interviewees of different religions. In general, phytomedicines consumption was often explained and justified by interviewees as family tradition. We did not detect any gender-related differences in phytomedicines consumption. There were no gender differences concerning the common traditional use of medicinal plants. The ethno-medicine data presented herein imply that medicinal plants are important as food and particularly as medicine (traditional healing) for various local people. While chemical medicinal treatments are becoming commonplace, traditional medications are still of huge importance in many rural, poor and remote places.\\nThis study will undoubtedly provide new data that could contribute to further pharmacological discoveries by identifying the active ingredients and their mechanism of effect by doing a lot of pharmacological work to confirm the alleged biological activities of these plants, and the possibility of developing new pharmaceutical formulas cannot be excluded depending on Syrian medicinal plants and their folk uses; as the discovery of artemisinin from Artemisia annua, based on ethnobotanical information (Acton and Klayman 1985), serves as evidence that it is possible to find new and effective medicines using data from TM.\",\n \"There is insufficient information about the pharmacokinetic efficacy of the medicinal plant species in this study. These herbs have reportedly and traditionally been used as adjuvant to relieve and treat some diseases.\",\n \"Many of the uses of medicinal plants mentioned in Syria are still under study. This study has been conducted with the aim to generate new concepts that could supplement pharmacological work with potentially further pharmacological discoveries; that is, by identifying the active ingredients and their mechanism of effect to confirm the alleged biological activities of these plants.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,null,null,null,null,null,"results",null,null,null,null,null,null,null,"discussion",null,"conclusions"],"string":"[\n \"intro\",\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n null,\n \"conclusions\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["Traditional Arabic medicine (TAM)","herbal medicine","Mediterranean","phytotherapy","medicinal plants","folk uses","ethnobotanical","ethnopharmacology"],"string":"[\n \"Traditional Arabic medicine (TAM)\",\n \"herbal medicine\",\n \"Mediterranean\",\n \"phytotherapy\",\n \"medicinal plants\",\n \"folk uses\",\n \"ethnobotanical\",\n \"ethnopharmacology\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Introduction:\nTraditional medicine (TM), as defined by the World Health Organization (WHO), is the sum total of the knowledge, skills and practices based on the theories, beliefs and experiences indigenous to different cultures, whether explicable or not, used in the maintenance of health as well as in the prevention, diagnosis, improvement or treatment of physical and mental illness (WHO 2002a, 2002b). Some TM systems are supported by huge volumes of literature and records of the theoretical concepts and practical skills; others pass down from generation to generation through verbal teaching. To date, in some parts of the world, the majority of the population continue to rely on their own TM to meet their primary health care needs. When adopted outside of its traditional culture, TM is often referred as complementary and alternative medicine (CAM) (Che et al. 2017).\nAlthough modern medicine is currently available in many developing countries, large proportions of the population in these countries still rely to a large extent on traditional practitioners and medicinal plants for therapeutic purposes. TM is often the first choice for providing primary health care in developing countries, and the WHO estimates that more than 80% of healthcare needs in these countries are met by traditional health care practices, being the cheapest and most accessible (WHO 2002a, 2002b, 2004, 2005).\nThe WHO pays special attention to TM and CAM. Resolution (no. WHA13-6) issued by the World Health Assembly (WHA) in 2009 emphasized the need to update the global TM strategy (WHO 2009), so the WHO issued its strategy for traditional (folk medicine) 2014–2023 (WHO 2013).\nThe United Nations Educational Scientific and Cultural Organization (UNESCO) works to document the medical heritage of peoples within its documentation of the living intangible cultural heritage of peoples and civilizations, through the documentation of TM practices that were included in the UNESCO convention (2003), the convention on biological diversity (1992) and the UNESCO universal declaration on cultural diversity (2001) and the United Nations declaration on the rights of indigenous peoples (2007), and according to the meeting of the UNESCO International Bioethics Committee (IBC) working group on TM and its ethical effects in Paris (2010), it stressed the need to conduct studies that illustrate the use of TM in various regions around the world, and its evaluation in clinical practice and in research and policy (IBC 2013).\nIn 2017, the International Center for Information and Networks for Intangible Cultural Heritage in the Asia-Pacific region (ICHCAP) under the auspices of UNESCO, issued a book entitled Traditional Medicine in which the Syria Trust for Development in Section VII included TM in Syria (Falk 2017).\nEthno-botany is the scientific study of the relationships between people and plants. It was first coined in 1896 by the US botanist John Harshberger; however, the history of ethnobotany began long before that (Campbell et al. 2002; Amjad et al. 2015). It plays an important role in understanding the dynamic relationships between biological diversity and social and cultural systems (Husain et al. 2008; Amjad et al. 2013, 2015). Plants are essential for human beings as they provide food, and medicines (Alam et al. 2011; Ahmad et al. 2012).\nTraditional Arabic medicine (TAM) is one of the famous traditional medical systems, which is occasionally called Unani medicine, Graeco-Arabic medicine, humoral medicine or Islamic medicine. The subject of TM in Syria has received little attention in the literature, and very little is known about the traditional medicinal substances used nowadays by the Syrian population to treat the most common diseases. Throughout ancient times in Syria, as part of the Levantine Nations (Bilad al-Sham), and other lands in the region, humans used various natural materials as sources of medicines (Jaddouh 2004). In the western countryside of Hama, there is a natural reserve for medicinal plants, which is called the Abu Qubais Protected area in Al-Ghab region (which protect the biodiversity rights of indigenous people and affiliated to the general commission for Al-Ghab administration and development), 509 plant species belonging to 72 families have been recorded (Al-Mahmoud and Al-Shater 2010).\nFor these reasons, the present investigation gathered the uses of medicinal plants in the centre region of Syria (Homs and Hama), as a supplement for a national survey, and documents the information concerning the uses of medicinal plants, which may serve as the basis of knowledge for a more intensive scientific research.\n\nMethods:\nStudy area Syria is a country located on the east coast of the Mediterranean Sea in southwestern Asia. Syria is bounded by Turkey to the north, Iraq to the east and southeast, Jordan to the south and Lebanon to the southwest. The study area is the middle region of Syria including Homs Governorate and Hama Governorate.\nThis area has a Mediterranean climate with a long dry season from May to October. In the extreme northwest, there is some light summer rain. On the western region, summers are hot, with mean daily maximum temperatures ranging from low to mid 80 °F, while the mild winters have daily mean minimum reaching temperatures low level of 50 °F. Only above about 1500 m are the summers relatively cool. In inland the climate becomes arid, with colder winters and hotter summers. In the desert, at Tadmur maximum temperatures in the summer, temperature reaches averages in the ranges of upper 90s to low 100 °F, with extremes in the 110 °F. In rural areas, work takes place according to the seasonal rhythm of agriculture. Women generally share in much of the agricultural labour. Agriculture constitutes an important source of income, and fruits and vegetables including onions, olives and grapes. Commercially important forest plants include: pistachio, which is important for its oil-rich fruit, and plants such as olive trees, grapevines, apricot trees and cumin (Hamidé et al. 2021).\nHistorically, the ancient Palmyra, also called Tadmur, is an ancient city in south-central Syria (Homs Governorate). An oasis in the Syrian desert, Palmyra contains the monumental ruins of a great city that was one of the most important cultural centres of the ancient world, from the first to the second century according to World Heritage List (UNESCO 2021).\nSyria is a country located on the east coast of the Mediterranean Sea in southwestern Asia. Syria is bounded by Turkey to the north, Iraq to the east and southeast, Jordan to the south and Lebanon to the southwest. The study area is the middle region of Syria including Homs Governorate and Hama Governorate.\nThis area has a Mediterranean climate with a long dry season from May to October. In the extreme northwest, there is some light summer rain. On the western region, summers are hot, with mean daily maximum temperatures ranging from low to mid 80 °F, while the mild winters have daily mean minimum reaching temperatures low level of 50 °F. Only above about 1500 m are the summers relatively cool. In inland the climate becomes arid, with colder winters and hotter summers. In the desert, at Tadmur maximum temperatures in the summer, temperature reaches averages in the ranges of upper 90s to low 100 °F, with extremes in the 110 °F. In rural areas, work takes place according to the seasonal rhythm of agriculture. Women generally share in much of the agricultural labour. Agriculture constitutes an important source of income, and fruits and vegetables including onions, olives and grapes. Commercially important forest plants include: pistachio, which is important for its oil-rich fruit, and plants such as olive trees, grapevines, apricot trees and cumin (Hamidé et al. 2021).\nHistorically, the ancient Palmyra, also called Tadmur, is an ancient city in south-central Syria (Homs Governorate). An oasis in the Syrian desert, Palmyra contains the monumental ruins of a great city that was one of the most important cultural centres of the ancient world, from the first to the second century according to World Heritage List (UNESCO 2021).\nField work and data collection Field surveys were conducted between December 2019 and January 2021, to document ethnobotanical information through oral interviews and designed semi-structured questionnaire. Thirty-five villages and 59 districts were visited for field research; 235 people were contacted and 151 of those with ethno-botanical experience agreed to become our informants, including 35 local herbalists (Tabib Arabi) and 10 physicians (who hold a general medicine specialty). The queries were repeatedly made to increase the reliability of the data; interviews with the men were usually carried out in the ‘Mukhtar’ house where they come together, and with women in their homes, bazaars and gardens. The Syria Trust for Development (which is a national development organization, and has a program called ‘Mashrouie’ that runs innovative microcredit programs that encourage economic growth in disadvantaged areas) helped us in data collecting. The information gathered during the present study included socio-demographic characteristics of the interviewed informants (age, gender) and ethnopharmacological information, including the local and scientific name of the species, local names, plant parts used, modes of use, conservation method, administration mode and toxicity, all documented data were then translated into English and Latin. Information that had been carried to the region from the outside and that was not used or confirmed was not included and recorded (Weckerle et al. 2018). During the interviews, questions about the following were asked to the participants:Name and surnameAge and sexEducational levelAre plants collected in your region?Do you have any contact with plants?Can you show the plants you use in your region?Can you tell the names of local plants you use in your region?In which season do you collect the plants you use in your region?When collecting plant, which parts of the plant do you collect and how do you collect them?Which parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).How do you prepare and administrate the plants’ parts?How did you diagnose the disease (by physician, by traditional healers, self-treating)?How did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\nName and surname\nAge and sex\nEducational level\nAre plants collected in your region?\nDo you have any contact with plants?\nCan you show the plants you use in your region?\nCan you tell the names of local plants you use in your region?\nIn which season do you collect the plants you use in your region?\nWhen collecting plant, which parts of the plant do you collect and how do you collect them?\nWhich parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).\nHow do you prepare and administrate the plants’ parts?\nHow did you diagnose the disease (by physician, by traditional healers, self-treating)?\nHow did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\nOnly the medication of single herbs was included, while mixtures and multiple recipes were disregarded. The answers that were given with doubt to the questions above were not recorded, and we adopted the information that gave 40% frequency in the collected data, and the data that gave less than that were neglected.\nInformation about diseases diagnosed specifically by physicians and traditional healers was approved, while self-treating diseases were referred to as auxiliary in treatment, nutritional uses were clearly indicated, and some plants were used to prevent disease and improve public health. The information about diseases was divided into three groups when analysing the data (group A: acute diseases, group B: chronic diseases, group C: simple symptoms and signs), and the focus was only on groups B and C.\nField surveys were conducted between December 2019 and January 2021, to document ethnobotanical information through oral interviews and designed semi-structured questionnaire. Thirty-five villages and 59 districts were visited for field research; 235 people were contacted and 151 of those with ethno-botanical experience agreed to become our informants, including 35 local herbalists (Tabib Arabi) and 10 physicians (who hold a general medicine specialty). The queries were repeatedly made to increase the reliability of the data; interviews with the men were usually carried out in the ‘Mukhtar’ house where they come together, and with women in their homes, bazaars and gardens. The Syria Trust for Development (which is a national development organization, and has a program called ‘Mashrouie’ that runs innovative microcredit programs that encourage economic growth in disadvantaged areas) helped us in data collecting. The information gathered during the present study included socio-demographic characteristics of the interviewed informants (age, gender) and ethnopharmacological information, including the local and scientific name of the species, local names, plant parts used, modes of use, conservation method, administration mode and toxicity, all documented data were then translated into English and Latin. Information that had been carried to the region from the outside and that was not used or confirmed was not included and recorded (Weckerle et al. 2018). During the interviews, questions about the following were asked to the participants:Name and surnameAge and sexEducational levelAre plants collected in your region?Do you have any contact with plants?Can you show the plants you use in your region?Can you tell the names of local plants you use in your region?In which season do you collect the plants you use in your region?When collecting plant, which parts of the plant do you collect and how do you collect them?Which parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).How do you prepare and administrate the plants’ parts?How did you diagnose the disease (by physician, by traditional healers, self-treating)?How did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\nName and surname\nAge and sex\nEducational level\nAre plants collected in your region?\nDo you have any contact with plants?\nCan you show the plants you use in your region?\nCan you tell the names of local plants you use in your region?\nIn which season do you collect the plants you use in your region?\nWhen collecting plant, which parts of the plant do you collect and how do you collect them?\nWhich parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).\nHow do you prepare and administrate the plants’ parts?\nHow did you diagnose the disease (by physician, by traditional healers, self-treating)?\nHow did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\nOnly the medication of single herbs was included, while mixtures and multiple recipes were disregarded. The answers that were given with doubt to the questions above were not recorded, and we adopted the information that gave 40% frequency in the collected data, and the data that gave less than that were neglected.\nInformation about diseases diagnosed specifically by physicians and traditional healers was approved, while self-treating diseases were referred to as auxiliary in treatment, nutritional uses were clearly indicated, and some plants were used to prevent disease and improve public health. The information about diseases was divided into three groups when analysing the data (group A: acute diseases, group B: chronic diseases, group C: simple symptoms and signs), and the focus was only on groups B and C.\nTaxonomic identification of the species Medicinal plants being mentioned by the Informants were recorded with local names and photographed. Each reported medicinal plant species was gathered, compressed, dehydrated, mounted on herbarium sheets, and identified; the taxonomic identity of the plants was confirmed by Prof. Abdel Aleem Bello (PhD/Botanical Taxonomy) and Dr. Bayan Tiba (PhD/Botanical Taxonomy) Aleppo University. As far as possible, the name of the plants was updated by consulting the latest literature; generic and species names followed the plant list (http://www.theplantlist.org). All voucher specimens have been preserved during documentation and deposited in the Damascus University, Faculty of Pharmacy, Pharmacognosy Labs Herbarium for future reference, serial numbers were taken from (1ch to 76ch) according to its alphabetical order.\nMedicinal plants being mentioned by the Informants were recorded with local names and photographed. Each reported medicinal plant species was gathered, compressed, dehydrated, mounted on herbarium sheets, and identified; the taxonomic identity of the plants was confirmed by Prof. Abdel Aleem Bello (PhD/Botanical Taxonomy) and Dr. Bayan Tiba (PhD/Botanical Taxonomy) Aleppo University. As far as possible, the name of the plants was updated by consulting the latest literature; generic and species names followed the plant list (http://www.theplantlist.org). All voucher specimens have been preserved during documentation and deposited in the Damascus University, Faculty of Pharmacy, Pharmacognosy Labs Herbarium for future reference, serial numbers were taken from (1ch to 76ch) according to its alphabetical order.\nEthics approval and consent to participate The study was approved by the Ethics Committee of the University of Damascus. Before beginning data collection, we obtained verbal informed permission in each case site-wide and then individually before each interview. We also informed informants that it was an academic project and that the investigation was for research purposes only, and not for any financial or other benefits. Informants provided verbal informed consent to participate in this study; they were free to withdraw their information at any time. These informants freely accepted the interview. All steps of research are consistent with Ethnobiology Code of Ethics (ISE 2006).\nThe study was approved by the Ethics Committee of the University of Damascus. Before beginning data collection, we obtained verbal informed permission in each case site-wide and then individually before each interview. We also informed informants that it was an academic project and that the investigation was for research purposes only, and not for any financial or other benefits. Informants provided verbal informed consent to participate in this study; they were free to withdraw their information at any time. These informants freely accepted the interview. All steps of research are consistent with Ethnobiology Code of Ethics (ISE 2006).\nData analysis The data collected through interviews of the informants were classified and examined with the statistical program IBM® SPSS® Statistics 26 (IBM, Armonk, NY), to determine the proportions of different variables such as ethnopharmacological data. Quantitative value indices were analysed using different statistical quantitative tools, i.e., the use reports (UR) of a species, and use value (UV) (Chaachouaya et al. 2021).\nThe data collected through interviews of the informants were classified and examined with the statistical program IBM® SPSS® Statistics 26 (IBM, Armonk, NY), to determine the proportions of different variables such as ethnopharmacological data. Quantitative value indices were analysed using different statistical quantitative tools, i.e., the use reports (UR) of a species, and use value (UV) (Chaachouaya et al. 2021).\nUse value and use reports The UR of a species or its importance in the culture of a community is denoted by its mentioning rate or its mention frequency by informants. The UR of the species of plants being utilized was evaluated using the formula (Tenté et al 2012):\nUR = Ni/n\nwhere Ni is the number of times a particular species was mentioned; n is the total number of times that all species were mentioned.\nThe UV of recorded medicinal plants was determined by applying the following formula (Tabuti et al. 2003):\nUV=∑ URi/N\nwhere ∑ URi is the total number of UR per plants; N is the total of interviewees questioned for given medicinal species.\nThe UV rate will be more important if there are several useful records for a species, implying that the plant is significant, whereas they will be near 0 if there are few reports compared to its use (Yaseen 2015; Chaachouaya et al. 2021).\nThe UR of a species or its importance in the culture of a community is denoted by its mentioning rate or its mention frequency by informants. The UR of the species of plants being utilized was evaluated using the formula (Tenté et al 2012):\nUR = Ni/n\nwhere Ni is the number of times a particular species was mentioned; n is the total number of times that all species were mentioned.\nThe UV of recorded medicinal plants was determined by applying the following formula (Tabuti et al. 2003):\nUV=∑ URi/N\nwhere ∑ URi is the total number of UR per plants; N is the total of interviewees questioned for given medicinal species.\nThe UV rate will be more important if there are several useful records for a species, implying that the plant is significant, whereas they will be near 0 if there are few reports compared to its use (Yaseen 2015; Chaachouaya et al. 2021).\n\nStudy area:\nSyria is a country located on the east coast of the Mediterranean Sea in southwestern Asia. Syria is bounded by Turkey to the north, Iraq to the east and southeast, Jordan to the south and Lebanon to the southwest. The study area is the middle region of Syria including Homs Governorate and Hama Governorate.\nThis area has a Mediterranean climate with a long dry season from May to October. In the extreme northwest, there is some light summer rain. On the western region, summers are hot, with mean daily maximum temperatures ranging from low to mid 80 °F, while the mild winters have daily mean minimum reaching temperatures low level of 50 °F. Only above about 1500 m are the summers relatively cool. In inland the climate becomes arid, with colder winters and hotter summers. In the desert, at Tadmur maximum temperatures in the summer, temperature reaches averages in the ranges of upper 90s to low 100 °F, with extremes in the 110 °F. In rural areas, work takes place according to the seasonal rhythm of agriculture. Women generally share in much of the agricultural labour. Agriculture constitutes an important source of income, and fruits and vegetables including onions, olives and grapes. Commercially important forest plants include: pistachio, which is important for its oil-rich fruit, and plants such as olive trees, grapevines, apricot trees and cumin (Hamidé et al. 2021).\nHistorically, the ancient Palmyra, also called Tadmur, is an ancient city in south-central Syria (Homs Governorate). An oasis in the Syrian desert, Palmyra contains the monumental ruins of a great city that was one of the most important cultural centres of the ancient world, from the first to the second century according to World Heritage List (UNESCO 2021).\n\nField work and data collection:\nField surveys were conducted between December 2019 and January 2021, to document ethnobotanical information through oral interviews and designed semi-structured questionnaire. Thirty-five villages and 59 districts were visited for field research; 235 people were contacted and 151 of those with ethno-botanical experience agreed to become our informants, including 35 local herbalists (Tabib Arabi) and 10 physicians (who hold a general medicine specialty). The queries were repeatedly made to increase the reliability of the data; interviews with the men were usually carried out in the ‘Mukhtar’ house where they come together, and with women in their homes, bazaars and gardens. The Syria Trust for Development (which is a national development organization, and has a program called ‘Mashrouie’ that runs innovative microcredit programs that encourage economic growth in disadvantaged areas) helped us in data collecting. The information gathered during the present study included socio-demographic characteristics of the interviewed informants (age, gender) and ethnopharmacological information, including the local and scientific name of the species, local names, plant parts used, modes of use, conservation method, administration mode and toxicity, all documented data were then translated into English and Latin. Information that had been carried to the region from the outside and that was not used or confirmed was not included and recorded (Weckerle et al. 2018). During the interviews, questions about the following were asked to the participants:Name and surnameAge and sexEducational levelAre plants collected in your region?Do you have any contact with plants?Can you show the plants you use in your region?Can you tell the names of local plants you use in your region?In which season do you collect the plants you use in your region?When collecting plant, which parts of the plant do you collect and how do you collect them?Which parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).How do you prepare and administrate the plants’ parts?How did you diagnose the disease (by physician, by traditional healers, self-treating)?How did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\nName and surname\nAge and sex\nEducational level\nAre plants collected in your region?\nDo you have any contact with plants?\nCan you show the plants you use in your region?\nCan you tell the names of local plants you use in your region?\nIn which season do you collect the plants you use in your region?\nWhen collecting plant, which parts of the plant do you collect and how do you collect them?\nWhich parts of the plants do you use? (flowers, fruits, leaves, roots, tubers, young shoots, branches, stems, aerial parts, etc.).\nHow do you prepare and administrate the plants’ parts?\nHow did you diagnose the disease (by physician, by traditional healers, self-treating)?\nHow did you check the effectiveness of the treatment (disappearance of symptoms, by laboratory analysis, other methods)?\nOnly the medication of single herbs was included, while mixtures and multiple recipes were disregarded. The answers that were given with doubt to the questions above were not recorded, and we adopted the information that gave 40% frequency in the collected data, and the data that gave less than that were neglected.\nInformation about diseases diagnosed specifically by physicians and traditional healers was approved, while self-treating diseases were referred to as auxiliary in treatment, nutritional uses were clearly indicated, and some plants were used to prevent disease and improve public health. The information about diseases was divided into three groups when analysing the data (group A: acute diseases, group B: chronic diseases, group C: simple symptoms and signs), and the focus was only on groups B and C.\n\nTaxonomic identification of the species:\nMedicinal plants being mentioned by the Informants were recorded with local names and photographed. Each reported medicinal plant species was gathered, compressed, dehydrated, mounted on herbarium sheets, and identified; the taxonomic identity of the plants was confirmed by Prof. Abdel Aleem Bello (PhD/Botanical Taxonomy) and Dr. Bayan Tiba (PhD/Botanical Taxonomy) Aleppo University. As far as possible, the name of the plants was updated by consulting the latest literature; generic and species names followed the plant list (http://www.theplantlist.org). All voucher specimens have been preserved during documentation and deposited in the Damascus University, Faculty of Pharmacy, Pharmacognosy Labs Herbarium for future reference, serial numbers were taken from (1ch to 76ch) according to its alphabetical order.\n\nEthics approval and consent to participate:\nThe study was approved by the Ethics Committee of the University of Damascus. Before beginning data collection, we obtained verbal informed permission in each case site-wide and then individually before each interview. We also informed informants that it was an academic project and that the investigation was for research purposes only, and not for any financial or other benefits. Informants provided verbal informed consent to participate in this study; they were free to withdraw their information at any time. These informants freely accepted the interview. All steps of research are consistent with Ethnobiology Code of Ethics (ISE 2006).\n\nData analysis:\nThe data collected through interviews of the informants were classified and examined with the statistical program IBM® SPSS® Statistics 26 (IBM, Armonk, NY), to determine the proportions of different variables such as ethnopharmacological data. Quantitative value indices were analysed using different statistical quantitative tools, i.e., the use reports (UR) of a species, and use value (UV) (Chaachouaya et al. 2021).\n\nUse value and use reports:\nThe UR of a species or its importance in the culture of a community is denoted by its mentioning rate or its mention frequency by informants. The UR of the species of plants being utilized was evaluated using the formula (Tenté et al 2012):\nUR = Ni/n\nwhere Ni is the number of times a particular species was mentioned; n is the total number of times that all species were mentioned.\nThe UV of recorded medicinal plants was determined by applying the following formula (Tabuti et al. 2003):\nUV=∑ URi/N\nwhere ∑ URi is the total number of UR per plants; N is the total of interviewees questioned for given medicinal species.\nThe UV rate will be more important if there are several useful records for a species, implying that the plant is significant, whereas they will be near 0 if there are few reports compared to its use (Yaseen 2015; Chaachouaya et al. 2021).\n\nResults:\nDemographic data of informants In total, 151 local inhabitants of 35 villages and 59 districts were selected based on their experience in traditional uses of plants. Table 1 shows the age and gender wise distribution. All of them were interrogated using semi-structured questionnaires. Generally in Syria, both genders were interested in herbal medicines.\nAge and gender distribution.\nIn total, 151 local inhabitants of 35 villages and 59 districts were selected based on their experience in traditional uses of plants. Table 1 shows the age and gender wise distribution. All of them were interrogated using semi-structured questionnaires. Generally in Syria, both genders were interested in herbal medicines.\nAge and gender distribution.\nEthnobotanical and ethnomedicinal uses of plant species The study area is considered one of the Syrian areas rich in medicinal plants. In the east of it there is a desert (badia), which is dominated by desert and thorny plants, and in the west of it, aromatic plants are spread in the mountains near the eastern coast of the Mediterranean Sea. It is a fact that people in some of these rural areas suffer from poverty, so they depend a lot on folk remedies, and folk healers in these areas provide their expertise at small costs, because the medicinal herbs are cheaper than chemical medicines, and most of the medicinal recipes are available around. However, a large portion of the uses of medicinal plants mentioned in the research are still under study.\nA total of 76 medicinal plant species (57.9% are wild and 42.1% are cultivated plants) belonging to 39 families are recorded in the present study; they are being used for a variety of purposes by native people. The detailed inventory is provided in Table 2, which includes botanical names, followed by local name, family and ethnobotanical uses.\nEthnobotanical uses of plant species according to ethnomedicinal survey of central region in Syria (Homs and Hama).\nAp: aerial parts; Bk: bark; Br: branches; Bu: buds; Bb: bulb; Ca: capitulum; Co: cones; Fl: flower; F: fruit; G: gum; Lx: latex; L: leaves; Ps: pedicles; Pe: peel; Ph: phloem; P: pods; Rs: resin; Rm: rhizome; R: root; Sd: seeds; S: stem; St: stylus; T: tubers; W: whole plant.\nC: cultivated plants, W: wild plants.\nHerbarium no.\nThe study area is considered one of the Syrian areas rich in medicinal plants. In the east of it there is a desert (badia), which is dominated by desert and thorny plants, and in the west of it, aromatic plants are spread in the mountains near the eastern coast of the Mediterranean Sea. It is a fact that people in some of these rural areas suffer from poverty, so they depend a lot on folk remedies, and folk healers in these areas provide their expertise at small costs, because the medicinal herbs are cheaper than chemical medicines, and most of the medicinal recipes are available around. However, a large portion of the uses of medicinal plants mentioned in the research are still under study.\nA total of 76 medicinal plant species (57.9% are wild and 42.1% are cultivated plants) belonging to 39 families are recorded in the present study; they are being used for a variety of purposes by native people. The detailed inventory is provided in Table 2, which includes botanical names, followed by local name, family and ethnobotanical uses.\nEthnobotanical uses of plant species according to ethnomedicinal survey of central region in Syria (Homs and Hama).\nAp: aerial parts; Bk: bark; Br: branches; Bu: buds; Bb: bulb; Ca: capitulum; Co: cones; Fl: flower; F: fruit; G: gum; Lx: latex; L: leaves; Ps: pedicles; Pe: peel; Ph: phloem; P: pods; Rs: resin; Rm: rhizome; R: root; Sd: seeds; S: stem; St: stylus; T: tubers; W: whole plant.\nC: cultivated plants, W: wild plants.\nHerbarium no.\nBotanical families of plants used The most commonly mentioned family is Asteraceae (11.84%), followed by Lamiaceae (10.52%), then Rosaceae (7.89%) and Apiaceae (6.57%), Poaceae (5.26%), Anacardiaceae and Fabaceae (3.94%), Fagaceae, Liliaceae, Myrtaceae and Oleaceae (2.63%), then all the other families (1.31%) (Figure 1).\nPlant families commonly used in ethnomedicinal survey of central region in Syria (Homs and Hama).\nThe most commonly mentioned family is Asteraceae (11.84%), followed by Lamiaceae (10.52%), then Rosaceae (7.89%) and Apiaceae (6.57%), Poaceae (5.26%), Anacardiaceae and Fabaceae (3.94%), Fagaceae, Liliaceae, Myrtaceae and Oleaceae (2.63%), then all the other families (1.31%) (Figure 1).\nPlant families commonly used in ethnomedicinal survey of central region in Syria (Homs and Hama).\nUse value of the plants Medicinal use plants (UV) are utilized to find the most frequently used plant species in the study area. Its value ranged from 0.066 to 0.92 (Table 2). The calculated results of UV showed that Cichorium intybus L., Eucalyptus globulus Labill. was ranked first (UV = 0.92) followed by Fraxinus syriaca Boiss. (UV = 0.907), Olea europaea L. (UV = 0.9), then Allium sativum L. (UV = 0.894), Lepidium sativum L. (UV = 0.867), Coriandrum sativum L. (UV = 0.86), Glycyrrhiza glabra L. (UV = 0.854), Dittrichia viscosa (L.) Greuter (UV = 0.841), while the lowest value was found for Plumbago europaea L. (UV = 0.066).\nMedicinal use plants (UV) are utilized to find the most frequently used plant species in the study area. Its value ranged from 0.066 to 0.92 (Table 2). The calculated results of UV showed that Cichorium intybus L., Eucalyptus globulus Labill. was ranked first (UV = 0.92) followed by Fraxinus syriaca Boiss. (UV = 0.907), Olea europaea L. (UV = 0.9), then Allium sativum L. (UV = 0.894), Lepidium sativum L. (UV = 0.867), Coriandrum sativum L. (UV = 0.86), Glycyrrhiza glabra L. (UV = 0.854), Dittrichia viscosa (L.) Greuter (UV = 0.841), while the lowest value was found for Plumbago europaea L. (UV = 0.066).\nMedicinal parts of the plant used The analysis of the ethnobotanical data showed that central region was best suited to the medicinal plant and rangeland. Ethnobotanical use categories showed that leaves were commonly used parts for making indigenous recipes (28.08%), followed by fruits (19.76%) and seeds (15.6%) then roots (10.4%), flowers (9.36%) and aerial parts (4.16%). Then, the others parts of plant are rarely used (Figure 2).\nMedicinal parts of the plants used for ethnomedicinal purposes in the study.\nThe analysis of the ethnobotanical data showed that central region was best suited to the medicinal plant and rangeland. Ethnobotanical use categories showed that leaves were commonly used parts for making indigenous recipes (28.08%), followed by fruits (19.76%) and seeds (15.6%) then roots (10.4%), flowers (9.36%) and aerial parts (4.16%). Then, the others parts of plant are rarely used (Figure 2).\nMedicinal parts of the plants used for ethnomedicinal purposes in the study.\nModes and conditions of medicine preparation The analysis of the ethnobotanical data showed that the recipes in the most cases were obtained from single herb, but some of recipes were prepared together, and there is a famous local mixture called Damask tisane (zhourate Shamieh). A mode of TM preparation reported was a decoction (30%), followed by infusion (23%), and then by other method such as fresh herbs, juice, cooked, powder, vinegar and oils (47%) (Figure 3). Considering according to results, most of the plant preparations are used orally (Table 2).\nModes of ethnomedicines preparation in Homs and Hama.\nThe analysis of the ethnobotanical data showed that the recipes in the most cases were obtained from single herb, but some of recipes were prepared together, and there is a famous local mixture called Damask tisane (zhourate Shamieh). A mode of TM preparation reported was a decoction (30%), followed by infusion (23%), and then by other method such as fresh herbs, juice, cooked, powder, vinegar and oils (47%) (Figure 3). Considering according to results, most of the plant preparations are used orally (Table 2).\nModes of ethnomedicines preparation in Homs and Hama.\nEthnomedicinal information about treatment the different diseases The results of questionnaires showed that 20% of the informants were diagnosed with their diseases by a doctor, and 45% were diagnosed with a conventional therapist, and 35% self-diagnosed their diseases, while the results of the questionnaires showed that the evaluation of the treatment by informants as following (58% relied on the disappearance of symptoms, and 24% through the results of laboratory analysis, 18% adopted other methods such as chest radiography, adopting the attending physician’s opinion and clinical observation of the improvement of skin diseases, and some of them depended on psychological comfort during treatment as evidence of improvement).\nAmong these studied plants, 62 are used to treat digestive disorders, 41 for respiratory diseases, including asthma, bronchitis and coughs, 40 for skin diseases, 16 for diabetes, 36 for kidney and urinary tract disorders, 22 for nervous system disorders, six for enhance the body's immunity, two for haemorrhoids, five for fever, eight for heart disorders, five for infertility and impotence, six for treating several types of cancer, two for increasing breast milk production, five for losing weight, four for lowering cholesterol, and two for increasing weight, and six for anaemia, 15 for blood disorder, two anti-toxicant, 19 for arthritis and pain, one for typhoid disorder, eight for infections, six for gynaecological diseases, one for eye inflammation, two anti-toxicant and four for mouth sores. Many of them are still used today, especially those plants recommended for internal uses such as traditional medicinal teas, which mainly consist of remedies for obesity, weight loss, colds, colds, digestive disorders, abdominal pain, constipation and some skin diseases (Figure 4).\nEthno-medicinal information about treating different diseases related to central region in Syria (Homs and Hama).\nThe results of questionnaires showed that 20% of the informants were diagnosed with their diseases by a doctor, and 45% were diagnosed with a conventional therapist, and 35% self-diagnosed their diseases, while the results of the questionnaires showed that the evaluation of the treatment by informants as following (58% relied on the disappearance of symptoms, and 24% through the results of laboratory analysis, 18% adopted other methods such as chest radiography, adopting the attending physician’s opinion and clinical observation of the improvement of skin diseases, and some of them depended on psychological comfort during treatment as evidence of improvement).\nAmong these studied plants, 62 are used to treat digestive disorders, 41 for respiratory diseases, including asthma, bronchitis and coughs, 40 for skin diseases, 16 for diabetes, 36 for kidney and urinary tract disorders, 22 for nervous system disorders, six for enhance the body's immunity, two for haemorrhoids, five for fever, eight for heart disorders, five for infertility and impotence, six for treating several types of cancer, two for increasing breast milk production, five for losing weight, four for lowering cholesterol, and two for increasing weight, and six for anaemia, 15 for blood disorder, two anti-toxicant, 19 for arthritis and pain, one for typhoid disorder, eight for infections, six for gynaecological diseases, one for eye inflammation, two anti-toxicant and four for mouth sores. Many of them are still used today, especially those plants recommended for internal uses such as traditional medicinal teas, which mainly consist of remedies for obesity, weight loss, colds, colds, digestive disorders, abdominal pain, constipation and some skin diseases (Figure 4).\nEthno-medicinal information about treating different diseases related to central region in Syria (Homs and Hama).\n\nDemographic data of informants:\nIn total, 151 local inhabitants of 35 villages and 59 districts were selected based on their experience in traditional uses of plants. Table 1 shows the age and gender wise distribution. All of them were interrogated using semi-structured questionnaires. Generally in Syria, both genders were interested in herbal medicines.\nAge and gender distribution.\n\nEthnobotanical and ethnomedicinal uses of plant species:\nThe study area is considered one of the Syrian areas rich in medicinal plants. In the east of it there is a desert (badia), which is dominated by desert and thorny plants, and in the west of it, aromatic plants are spread in the mountains near the eastern coast of the Mediterranean Sea. It is a fact that people in some of these rural areas suffer from poverty, so they depend a lot on folk remedies, and folk healers in these areas provide their expertise at small costs, because the medicinal herbs are cheaper than chemical medicines, and most of the medicinal recipes are available around. However, a large portion of the uses of medicinal plants mentioned in the research are still under study.\nA total of 76 medicinal plant species (57.9% are wild and 42.1% are cultivated plants) belonging to 39 families are recorded in the present study; they are being used for a variety of purposes by native people. The detailed inventory is provided in Table 2, which includes botanical names, followed by local name, family and ethnobotanical uses.\nEthnobotanical uses of plant species according to ethnomedicinal survey of central region in Syria (Homs and Hama).\nAp: aerial parts; Bk: bark; Br: branches; Bu: buds; Bb: bulb; Ca: capitulum; Co: cones; Fl: flower; F: fruit; G: gum; Lx: latex; L: leaves; Ps: pedicles; Pe: peel; Ph: phloem; P: pods; Rs: resin; Rm: rhizome; R: root; Sd: seeds; S: stem; St: stylus; T: tubers; W: whole plant.\nC: cultivated plants, W: wild plants.\nHerbarium no.\n\nBotanical families of plants used:\nThe most commonly mentioned family is Asteraceae (11.84%), followed by Lamiaceae (10.52%), then Rosaceae (7.89%) and Apiaceae (6.57%), Poaceae (5.26%), Anacardiaceae and Fabaceae (3.94%), Fagaceae, Liliaceae, Myrtaceae and Oleaceae (2.63%), then all the other families (1.31%) (Figure 1).\nPlant families commonly used in ethnomedicinal survey of central region in Syria (Homs and Hama).\n\nUse value of the plants:\nMedicinal use plants (UV) are utilized to find the most frequently used plant species in the study area. Its value ranged from 0.066 to 0.92 (Table 2). The calculated results of UV showed that Cichorium intybus L., Eucalyptus globulus Labill. was ranked first (UV = 0.92) followed by Fraxinus syriaca Boiss. (UV = 0.907), Olea europaea L. (UV = 0.9), then Allium sativum L. (UV = 0.894), Lepidium sativum L. (UV = 0.867), Coriandrum sativum L. (UV = 0.86), Glycyrrhiza glabra L. (UV = 0.854), Dittrichia viscosa (L.) Greuter (UV = 0.841), while the lowest value was found for Plumbago europaea L. (UV = 0.066).\n\nMedicinal parts of the plant used:\nThe analysis of the ethnobotanical data showed that central region was best suited to the medicinal plant and rangeland. Ethnobotanical use categories showed that leaves were commonly used parts for making indigenous recipes (28.08%), followed by fruits (19.76%) and seeds (15.6%) then roots (10.4%), flowers (9.36%) and aerial parts (4.16%). Then, the others parts of plant are rarely used (Figure 2).\nMedicinal parts of the plants used for ethnomedicinal purposes in the study.\n\nModes and conditions of medicine preparation:\nThe analysis of the ethnobotanical data showed that the recipes in the most cases were obtained from single herb, but some of recipes were prepared together, and there is a famous local mixture called Damask tisane (zhourate Shamieh). A mode of TM preparation reported was a decoction (30%), followed by infusion (23%), and then by other method such as fresh herbs, juice, cooked, powder, vinegar and oils (47%) (Figure 3). Considering according to results, most of the plant preparations are used orally (Table 2).\nModes of ethnomedicines preparation in Homs and Hama.\n\nEthnomedicinal information about treatment the different diseases:\nThe results of questionnaires showed that 20% of the informants were diagnosed with their diseases by a doctor, and 45% were diagnosed with a conventional therapist, and 35% self-diagnosed their diseases, while the results of the questionnaires showed that the evaluation of the treatment by informants as following (58% relied on the disappearance of symptoms, and 24% through the results of laboratory analysis, 18% adopted other methods such as chest radiography, adopting the attending physician’s opinion and clinical observation of the improvement of skin diseases, and some of them depended on psychological comfort during treatment as evidence of improvement).\nAmong these studied plants, 62 are used to treat digestive disorders, 41 for respiratory diseases, including asthma, bronchitis and coughs, 40 for skin diseases, 16 for diabetes, 36 for kidney and urinary tract disorders, 22 for nervous system disorders, six for enhance the body's immunity, two for haemorrhoids, five for fever, eight for heart disorders, five for infertility and impotence, six for treating several types of cancer, two for increasing breast milk production, five for losing weight, four for lowering cholesterol, and two for increasing weight, and six for anaemia, 15 for blood disorder, two anti-toxicant, 19 for arthritis and pain, one for typhoid disorder, eight for infections, six for gynaecological diseases, one for eye inflammation, two anti-toxicant and four for mouth sores. Many of them are still used today, especially those plants recommended for internal uses such as traditional medicinal teas, which mainly consist of remedies for obesity, weight loss, colds, colds, digestive disorders, abdominal pain, constipation and some skin diseases (Figure 4).\nEthno-medicinal information about treating different diseases related to central region in Syria (Homs and Hama).\n\nDiscussion:\nThe use of TAM has spread to treat various diseases in Syria since ancient times. They are cost-effective with fewer side effects and are more suitable for long-term use compared with chemically synthesized medicines.\nThe ethnobotanical categories indicated that there is large use of medicinal herbs in the area of study, most of them are wild. There is an increased exploitation of medicinal plants by the local population, collectors and dealers of herbal medicines, in line with the demand from the pharmaceutical industry. This caused a sharp decrease in the occurrence and products of medicinal plants. Grazing, deforestation by cutting down trees for heating, and fires were mainly responsible for the reduction of medicinal plants. That is why the government is working on developing strategies to conserve wild plant diversity. Some people collect the medicinal plants for an income. They uproot and collect each part of the medicinal plants in non-scientific way. Thus, to date, a few articles devoted to TM of Syria, such as a study of folk medicine in Aleppo Governorate (Alachkar et al. 2011), and a study about the use of ‘Zahraa’ (Syrian traditional tisane) (Carmona et al. 2005), and a third one on the medicinal plants in Golan (Said et al. 2002), which is an occupied Syrian territory.\nThis research aspires to genuinely contribute in providing useful information on the conserving and sustaining the natural resources in the area.\nThe perspectives in the questionnaire were compared with other ethnomedicine studies in the countries surrounding Syria such as Lebanon (Taha et al. 2013), Jordan (Lev and Amar 2002; Al-Qura'n 2009), Palestine (Friedman et al. 1986; Kaileh et al. 2007), Iraq (Al-Douri 2000) and Turkey (Yeşilada et al. 1995; Sezik et al. 2001). Similarities in various traditional uses in Syria, Lebanon, Palestine and Jordan were observed. This is mainly due to the mutual history of these areas that were previously called Levantine Nations (Bilad al-Sham) (Lev 2002), and there is some similarity with a smaller number of folk uses both in Syria and Iraq, but there is a difference in the folk uses described between Syria and Turkey (Korkmaz et al. 2016; Yerebasan et al. 2020). We compared the folk uses of plants mentioned in related articles on ethnomedicine in these regions with the plants studied in our research to find out the extent of congruence or difference the uses of similar plants.\nWe did not record significant differences in phytomedicines consumption customs between interviewees of different religions. In general, phytomedicines consumption was often explained and justified by interviewees as family tradition. We did not detect any gender-related differences in phytomedicines consumption. There were no gender differences concerning the common traditional use of medicinal plants. The ethno-medicine data presented herein imply that medicinal plants are important as food and particularly as medicine (traditional healing) for various local people. While chemical medicinal treatments are becoming commonplace, traditional medications are still of huge importance in many rural, poor and remote places.\nThis study will undoubtedly provide new data that could contribute to further pharmacological discoveries by identifying the active ingredients and their mechanism of effect by doing a lot of pharmacological work to confirm the alleged biological activities of these plants, and the possibility of developing new pharmaceutical formulas cannot be excluded depending on Syrian medicinal plants and their folk uses; as the discovery of artemisinin from Artemisia annua, based on ethnobotanical information (Acton and Klayman 1985), serves as evidence that it is possible to find new and effective medicines using data from TM.\n\nLimitations:\nThere is insufficient information about the pharmacokinetic efficacy of the medicinal plant species in this study. These herbs have reportedly and traditionally been used as adjuvant to relieve and treat some diseases.\n\nConclusions:\nMany of the uses of medicinal plants mentioned in Syria are still under study. This study has been conducted with the aim to generate new concepts that could supplement pharmacological work with potentially further pharmacological discoveries; that is, by identifying the active ingredients and their mechanism of effect to confirm the alleged biological activities of these plants.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nSince ancient times, traditional Arabic medicine (TAM) has been used to treat various diseases in Syria. They are cost-effective with fewer side effects and are more suitable for long-term use compared with chemically synthesized medicines. In addition, the scientific importance is manifested, as this survey proceeds, for the purposes of verifying and documenting these traditional medicines and their common uses.\n\nMethods:\nInformation was collected from 2019 to 2021 from the cities of Homs and Hama and their villages, which are two governorates located in central Syria, after interviews with traditional practitioners called Attarin, and many other people. Plant specimens were collected according to different references concerning medicinal plants of Syria, to document the traditional uses of plants at least two of the traditional healers and three other people were asked.\n\nResults:\nIn this survey, we listed 76 medicinal plants belonging to 39 families in alphabetical order with the parts used and the method of preparation according to their therapeutic use, which are used to treat 106 ailments.\n\nConclusions:\nMany of the uses of medicinal plants mentioned in this survey are still under study. There is no doubt that this study will provide new data that could contribute to further pharmacological discoveries by identifying the active ingredients and their mechanism of effect by doing additional pharmacological work to confirm the alleged biological activities of these plants."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nTraditional medicine (TM), as defined by the World Health Organization (WHO), is the sum total of the knowledge, skills and practices based on the theories, beliefs and experiences indigenous to different cultures, whether explicable or not, used in the maintenance of health as well as in the prevention, diagnosis, improvement or treatment of physical and mental illness (WHO 2002a, 2002b). Some TM systems are supported by huge volumes of literature and records of the theoretical concepts and practical skills; others pass down from generation to generation through verbal teaching. To date, in some parts of the world, the majority of the population continue to rely on their own TM to meet their primary health care needs. When adopted outside of its traditional culture, TM is often referred as complementary and alternative medicine (CAM) (Che et al. 2017).\nAlthough modern medicine is currently available in many developing countries, large proportions of the population in these countries still rely to a large extent on traditional practitioners and medicinal plants for therapeutic purposes. TM is often the first choice for providing primary health care in developing countries, and the WHO estimates that more than 80% of healthcare needs in these countries are met by traditional health care practices, being the cheapest and most accessible (WHO 2002a, 2002b, 2004, 2005).\nThe WHO pays special attention to TM and CAM. Resolution (no. WHA13-6) issued by the World Health Assembly (WHA) in 2009 emphasized the need to update the global TM strategy (WHO 2009), so the WHO issued its strategy for traditional (folk medicine) 2014–2023 (WHO 2013).\nThe United Nations Educational Scientific and Cultural Organization (UNESCO) works to document the medical heritage of peoples within its documentation of the living intangible cultural heritage of peoples and civilizations, through the documentation of TM practices that were included in the UNESCO convention (2003), the convention on biological diversity (1992) and the UNESCO universal declaration on cultural diversity (2001) and the United Nations declaration on the rights of indigenous peoples (2007), and according to the meeting of the UNESCO International Bioethics Committee (IBC) working group on TM and its ethical effects in Paris (2010), it stressed the need to conduct studies that illustrate the use of TM in various regions around the world, and its evaluation in clinical practice and in research and policy (IBC 2013).\nIn 2017, the International Center for Information and Networks for Intangible Cultural Heritage in the Asia-Pacific region (ICHCAP) under the auspices of UNESCO, issued a book entitled Traditional Medicine in which the Syria Trust for Development in Section VII included TM in Syria (Falk 2017).\nEthno-botany is the scientific study of the relationships between people and plants. It was first coined in 1896 by the US botanist John Harshberger; however, the history of ethnobotany began long before that (Campbell et al. 2002; Amjad et al. 2015). It plays an important role in understanding the dynamic relationships between biological diversity and social and cultural systems (Husain et al. 2008; Amjad et al. 2013, 2015). Plants are essential for human beings as they provide food, and medicines (Alam et al. 2011; Ahmad et al. 2012).\nTraditional Arabic medicine (TAM) is one of the famous traditional medical systems, which is occasionally called Unani medicine, Graeco-Arabic medicine, humoral medicine or Islamic medicine. The subject of TM in Syria has received little attention in the literature, and very little is known about the traditional medicinal substances used nowadays by the Syrian population to treat the most common diseases. Throughout ancient times in Syria, as part of the Levantine Nations (Bilad al-Sham), and other lands in the region, humans used various natural materials as sources of medicines (Jaddouh 2004). In the western countryside of Hama, there is a natural reserve for medicinal plants, which is called the Abu Qubais Protected area in Al-Ghab region (which protect the biodiversity rights of indigenous people and affiliated to the general commission for Al-Ghab administration and development), 509 plant species belonging to 72 families have been recorded (Al-Mahmoud and Al-Shater 2010).\nFor these reasons, the present investigation gathered the uses of medicinal plants in the centre region of Syria (Homs and Hama), as a supplement for a national survey, and documents the information concerning the uses of medicinal plants, which may serve as the basis of knowledge for a more intensive scientific research.\n\nConclusions:\nMany of the uses of medicinal plants mentioned in Syria are still under study. This study has been conducted with the aim to generate new concepts that could supplement pharmacological work with potentially further pharmacological discoveries; that is, by identifying the active ingredients and their mechanism of effect to confirm the alleged biological activities of these plants."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nSince ancient times, traditional Arabic medicine (TAM) has been used to treat various diseases in Syria. They are cost-effective with fewer side effects and are more suitable for long-term use compared with chemically synthesized medicines. In addition, the scientific importance is manifested, as this survey proceeds, for the purposes of verifying and documenting these traditional medicines and their common uses.\n\nMethods:\nInformation was collected from 2019 to 2021 from the cities of Homs and Hama and their villages, which are two governorates located in central Syria, after interviews with traditional practitioners called Attarin, and many other people. Plant specimens were collected according to different references concerning medicinal plants of Syria, to document the traditional uses of plants at least two of the traditional healers and three other people were asked.\n\nResults:\nIn this survey, we listed 76 medicinal plants belonging to 39 families in alphabetical order with the parts used and the method of preparation according to their therapeutic use, which are used to treat 106 ailments.\n\nConclusions:\nMany of the uses of medicinal plants mentioned in this survey are still under study. There is no doubt that this study will provide new data that could contribute to further pharmacological discoveries by identifying the active ingredients and their mechanism of effect by doing additional pharmacological work to confirm the alleged biological activities of these plants."},"whole_article_text_length":{"kind":"number","value":10363,"string":"10,363"},"whole_article_abstract_length":{"kind":"number","value":261,"string":"261"},"other_sections_lengths":{"kind":"list like","value":[349,744,139,111,80,188,63,341,92,142,101,121,348,34],"string":"[\n 349,\n 744,\n 139,\n 111,\n 80,\n 188,\n 63,\n 341,\n 92,\n 142,\n 101,\n 121,\n 348,\n 34\n]","isConversational":false},"num_sections":{"kind":"number","value":19,"string":"19"},"most_frequent_words":{"kind":"list like","value":["plants","medicinal","plant","use","region","uv","species","parts","diseases","data"],"string":"[\n \"plants\",\n \"medicinal\",\n \"plant\",\n \"use\",\n \"region\",\n \"uv\",\n \"species\",\n \"parts\",\n \"diseases\",\n \"data\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["traditional practitioners","met traditional health","traditional health care","entitled traditional medicine","traditional medicine tm"],"string":"[\n \"traditional practitioners\",\n \"met traditional health\",\n \"traditional health care\",\n \"entitled traditional medicine\",\n \"traditional medicine tm\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Traditional Arabic medicine (TAM) | herbal medicine | Mediterranean | phytotherapy | medicinal plants | folk uses | ethnobotanical | ethnopharmacology [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Traditional Arabic medicine (TAM) | herbal medicine | Mediterranean | phytotherapy | medicinal plants | folk uses | ethnobotanical | ethnopharmacology [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Traditional Arabic medicine (TAM) | herbal medicine | Mediterranean | phytotherapy | medicinal plants | folk uses | ethnobotanical | ethnopharmacology [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Traditional Arabic medicine (TAM) | herbal medicine | Mediterranean | phytotherapy | medicinal plants | folk uses | ethnobotanical | ethnopharmacology [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Traditional Arabic medicine (TAM) | herbal medicine | Mediterranean | phytotherapy | medicinal plants | folk uses | ethnobotanical | ethnopharmacology [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Traditional Arabic medicine (TAM) | herbal medicine | Mediterranean | phytotherapy | medicinal plants | folk uses | ethnobotanical | ethnopharmacology [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Ethnobotany | Ethnopharmacology | Female | Humans | Male | Medicine, Traditional | Middle Aged | Plants, Medicinal | Surveys and Questionnaires | Syria [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Ethnobotany | Ethnopharmacology | Female | Humans | Male | Medicine, Traditional | Middle Aged | Plants, Medicinal | Surveys and Questionnaires | Syria [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Ethnobotany | Ethnopharmacology | Female | Humans | Male | Medicine, Traditional | Middle Aged | Plants, Medicinal | Surveys and Questionnaires | Syria [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Ethnobotany | Ethnopharmacology | Female | Humans | Male | Medicine, Traditional | Middle Aged | Plants, Medicinal | Surveys and Questionnaires | Syria [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Ethnobotany | Ethnopharmacology | Female | Humans | Male | Medicine, Traditional | Middle Aged | Plants, Medicinal | Surveys and Questionnaires | Syria [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Ethnobotany | Ethnopharmacology | Female | Humans | Male | Medicine, Traditional | Middle Aged | Plants, Medicinal | Surveys and Questionnaires | Syria [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] traditional practitioners | met traditional health | traditional health care | entitled traditional medicine | traditional medicine tm [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] traditional practitioners | met traditional health | traditional health care | entitled traditional medicine | traditional medicine tm [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] traditional practitioners | met traditional health | traditional health care | entitled traditional medicine | traditional medicine tm [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] traditional practitioners | met traditional health | traditional health care | entitled traditional medicine | traditional medicine tm [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] traditional practitioners | met traditional health | traditional health care | entitled traditional medicine | traditional medicine tm [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] traditional practitioners | met traditional health | traditional health care | entitled traditional medicine | traditional medicine tm [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] plants | medicinal | plant | use | region | uv | species | parts | diseases | data [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] plants | medicinal | plant | use | region | uv | species | parts | diseases | data [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] plants | medicinal | plant | use | region | uv | species | parts | diseases | data [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] plants | medicinal | plant | use | region | uv | species | parts | diseases | data [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] plants | medicinal | plant | use | region | uv | species | parts | diseases | data [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] plants | medicinal | plant | use | region | uv | species | parts | diseases | data [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] tm | medicine | traditional | health | unesco | cultural | countries | world | health care | peoples [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] plants | plants use | use | region | parts | data | plants use region | use region | species | collect [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] uv | plants | diseases | medicinal | disorders | showed | plant | results | parts | ethnobotanical [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] pharmacological | pharmacological work potentially | new concepts supplement | plants mentioned syria | study conducted | study conducted aim | study conducted aim generate | study study | study study conducted | study study conducted aim [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] plants | uv | medicinal | species | plant | parts | use | diseases | study | region [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] plants | uv | medicinal | species | plant | parts | use | diseases | study | region [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Arabic | TAM | Syria ||| ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 2019 | 2021 | Homs | Hama | two | Syria | Attarin ||| Syria | at least two | three [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 76 | 39 | 106 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Arabic | TAM | Syria ||| ||| ||| 2019 | 2021 | Homs | Hama | two | Syria | Attarin ||| Syria | at least two | three ||| ||| 76 | 39 | 106 ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Arabic | TAM | Syria ||| ||| ||| 2019 | 2021 | Homs | Hama | two | Syria | Attarin ||| Syria | at least two | three ||| ||| 76 | 39 | 106 ||| ||| [SUMMARY]"}}},{"rowIdx":65,"cells":{"title":{"kind":"string","value":"Mental health and suicidal ideation in US military veterans with histories of COVID-19 infection."},"pmid":{"kind":"string","value":"34035155"},"background_abstract":{"kind":"string","value":"There have been reports of increased prevalence in psychiatric conditions in non-veteran survivors of COVID-19. To date, however, no known study has examined the prevalence, risk and protective factors of psychiatric conditions among US military veterans who survived COVID-19."},"background_abstract_label":{"kind":"string","value":"INTRODUCTION"},"methods_abstract":{"kind":"string","value":"Data were analysed from the 2019 to 2020 National Health and Resilience in Veterans Study, which surveyed a nationally representative, prospective cohort of 3078 US veterans. Prepandemic and 1-year peripandemic risk and protective factors associated with positive screens for peripandemic internalising (major depressive, generalised anxiety and/or posttraumatic stress disorders) and externalising psychiatric disorders (alcohol and/or drug use disorders) and suicidal ideation were examined using bivariate and multivariate logistic regression analyses."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"A total of 233 veterans (8.6%) reported having been infected with COVID-19. Relative to veterans who were not infected, veterans who were infected were more likely to screen positive for internalising disorders (20.5% vs 13.9%, p=0.005), externalising disorders (23.2% vs 14.8%, p=0.001) and current suicidal ideation (12.0% vs 7.6%, p=0.015) at peripandemic. Multivariable analyses revealed that greater prepandemic psychiatric symptom severity and COVID-related stressors were the strongest independent predictors of peripandemic internalising disorders, while prepandemic trauma burden was protective. Prepandemic suicidal ideation, greater loneliness and lower household income were the strongest independent predictors of peripandemic suicidal ideation, whereas prepandemic community integration was protective."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Psychiatric symptoms and suicidal ideation are prevalent in veterans who have survived COVID-19. Veterans with greater prepandemic psychiatric and substance use problems, COVID-related stressors and fewer psychosocial resources may be at increased risk of these outcomes."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["COVID-19","Depressive Disorder, Major","Humans","Mental Health","Prospective Studies","SARS-CoV-2","Suicidal Ideation","Veterans"],"string":"[\n \"COVID-19\",\n \"Depressive Disorder, Major\",\n \"Humans\",\n \"Mental Health\",\n \"Prospective Studies\",\n \"SARS-CoV-2\",\n \"Suicidal Ideation\",\n \"Veterans\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"8154290"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"The COVID-19 pandemic has been linked to increased social isolation, economic recession and psychological distress.1 Numerous studies have reported a substantial increase in the prevalence of depression and anxiety in the general public during the pandemic.2 3 Previous psychiatric diagnoses, COVID-related stressors, such as worries about being infected with COVID-19, financial stressors and actual COVID-19 infection have been proposed as possible risk factors of both the development and worsening of mental illness during the pandemic.1\nVeterans may be a highly vulnerable group to the negative mental health effects of the pandemic due to their older age, and higher prepandemic prevalence of physical and psychiatric risk factors and conditions relative to the general population.4 5i Further, previous studies on veterans have found that stressful events such as combat deployments were associated with increased rates of depression and substance use disorders in veterans.6\nAs of April 2021, in the USA, over 31 million people have been infected with COVID-19 and over half a million people have died of COVID-19-related complications.7 Previous studies on survivors of COVID-19 have reported high prevalence of mental disorders, including depression, anxiety and post-traumatic stress disorder (PTSD8–10). Thus, there has been increasing attention in closely monitoring for psychiatric symptoms and functioning in those who have been infected with COVID-19 in order to mitigate distress and risk of suicide. However, due to the high burden of COVID-19 infections in the USA and the limited capacity of mental health treatment services, it is challenging to monitor everyone who has been infected. These challenges have been further complicated by infection-control measures implemented by almost all mental health service providers during the pandemic.11\nOne potentially effective way of informing the allocation of limited mental health treatment resources may be to identify subgroups that are at high risk of developing or experiencing a worsening of psychiatric conditions after COVID-19 infection. Identifying potential risk and protective factors of psychiatric conditions among COVID-19 survivors in populations with elevated risk, such as US military veterans, may help inform more targeted population-based prevention, screening and intervention strategies.\nToward this end, we analysed data from the National Health and Resilience in Veterans Study (NHRVS12) to examine (1) the prevalence of psychiatric conditions in US veterans who have and have not been infected with COVID-19, and (2) prepandemic and peripandemic risk and protective factors of peripandemic psychiatric conditions in veterans who survived COVID-19 infection."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"Participants Data were analysed from the 2019 to 2020 NHRVS, which surveyed a nationally representative, prospective sample of US military veterans. A total of 4069 veterans completed the prepandemic survey (median completion date: 21 November 2019) prior to the first documented COVID-19 cases in the USA (19 January 2020), and 3078 (75.6%) completed a 1-year peripandemic follow-up assessment (median completion date 14 November 2020). Both surveys were online, 45–60 min, self-report surveys. All participants received 15 000 points (equivalent to $15) for participating in the prepandemic survey, and 20 000 points (equivalent to $20) for participating in the peripandemic survey. Details of the study have been described previously.12 Briefly, the NHRVS sample was drawn from KnowledgePanel, a survey research panel of more than 50 000 US households maintained by Ipsos, a survey research firm. To promote generalisability of the results to the US veteran population, poststratification weights based on the demographic distribution of veterans in the contemporaneous US Census Current Population Survey Veterans Supplement were applied in inferential analyses.\nData were analysed from the 2019 to 2020 NHRVS, which surveyed a nationally representative, prospective sample of US military veterans. A total of 4069 veterans completed the prepandemic survey (median completion date: 21 November 2019) prior to the first documented COVID-19 cases in the USA (19 January 2020), and 3078 (75.6%) completed a 1-year peripandemic follow-up assessment (median completion date 14 November 2020). Both surveys were online, 45–60 min, self-report surveys. All participants received 15 000 points (equivalent to $15) for participating in the prepandemic survey, and 20 000 points (equivalent to $20) for participating in the peripandemic survey. Details of the study have been described previously.12 Briefly, the NHRVS sample was drawn from KnowledgePanel, a survey research panel of more than 50 000 US households maintained by Ipsos, a survey research firm. To promote generalisability of the results to the US veteran population, poststratification weights based on the demographic distribution of veterans in the contemporaneous US Census Current Population Survey Veterans Supplement were applied in inferential analyses.\nAssessments Online supplemental table 1 presents a detailed description of measures used to assess potential risk and protective factors of peripandemic psychiatric conditions in veterans who survived COVID-19 infection.\n\n\n\nOnline supplemental table 1 presents a detailed description of measures used to assess potential risk and protective factors of peripandemic psychiatric conditions in veterans who survived COVID-19 infection.\n\n\n\nData analysis χ2 analyses were conducted to compare the prevalence of psychiatric disorders and suicidal ideation between veterans who were and were not infected with COVID-19. Subsequent analyses focused on veterans who self-reported a history of COVID-19 infection at the peripandemic assessment. Analyses in this subcohort proceeded in two steps. First, we conducted Pearson correlations between participant characteristics and peripandemic internalising psychiatric disorders (ie, major depressive disorder, generalised anxiety disorder and/or pandemic-related stress symptoms); externalising disorders (ie, alcohol and/or drug use disorder); and suicidal ideation; internalising and externalising disorders were grouped together to increase statistical power. Second, we conducted multivariable binary logistic regression analyses to identify factors that independently predicted the aforementioned three categories of peripandemic psychiatric conditions. Variables that differed at the p<0.05 level in bivariate analyses for each set of comparisons were entered into each regression analysis. Third, we conducted relative importance analyses to determine the relative contribution of each significant variable in predicting positive screens for the three outcomes. These analyses partitioned the explained variance in the study outcome variables that is explained by each independent variable while simultaneously accounting for intercorrelations among these independent variables.13\nχ2 analyses were conducted to compare the prevalence of psychiatric disorders and suicidal ideation between veterans who were and were not infected with COVID-19. Subsequent analyses focused on veterans who self-reported a history of COVID-19 infection at the peripandemic assessment. Analyses in this subcohort proceeded in two steps. First, we conducted Pearson correlations between participant characteristics and peripandemic internalising psychiatric disorders (ie, major depressive disorder, generalised anxiety disorder and/or pandemic-related stress symptoms); externalising disorders (ie, alcohol and/or drug use disorder); and suicidal ideation; internalising and externalising disorders were grouped together to increase statistical power. Second, we conducted multivariable binary logistic regression analyses to identify factors that independently predicted the aforementioned three categories of peripandemic psychiatric conditions. Variables that differed at the p<0.05 level in bivariate analyses for each set of comparisons were entered into each regression analysis. Third, we conducted relative importance analyses to determine the relative contribution of each significant variable in predicting positive screens for the three outcomes. These analyses partitioned the explained variance in the study outcome variables that is explained by each independent variable while simultaneously accounting for intercorrelations among these independent variables.13"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"The final sample included 3078 veterans who completed both prepandemic and 1-year peripandemic assessments. The average age of the sample was 62.2 years old (SD=15.7, range 22–99); the majority was male (90.2%) and Caucasian (78.1%); and 35.0% were combat veterans. A total of 233 veterans (8.6%) reported that they had been infected with COVID-19. The majority (70.5%) reported mild-to-moderate symptoms; 6.3% reported requiring a visit to the emergency room; 4.5% reported severe symptoms with serious concerns about not surviving; 1.9% reported being admitted to an intensive care unit; 1.2% reported requiring an overnight stay at a hospital, and 0.8% reported requiring intubation/mechanical ventilation; the remaining 14.8% of the sample did not provide details about the severity of their illness.\nPrevalence of peripandemic psychiatric conditions of veterans who were infected by COVID-19 Relative to veterans who were not infected with COVID-19, veterans who were infected were significantly more likely to screen positive at the peripandemic assessment for any internalising psychiatric disorder (20.5% vs 13.9%, χ2=7.85, p=0.005), alcohol or drug use disorder (23.2% vs 14.8%, χ2=11.51, p=0.001) and current suicidal ideation (12.0% vs 7.6%, χ2=5.92, p=0.015). With regard to specific disorders, veterans with histories of COVID-19 infection were more likely to screen positive for generalised anxiety disorder (14.3% vs 9.1%, χ2=6.99, p=0.008), pandemic-related stress symptoms (7.3% vs 4.0 %, χ2=5.99, p=0.014), alcohol use disorder (14.8% vs 9.2%, χ2=8.03, p=0.005) and drug use disorder (13.9% vs 7.8%, χ2=10.27, p=0.001) relative to veterans who were not infected. The differences in major depressive disorder (11.8% vs 8.6%, χ2=2.93, p=0.087) and past-year suicide plan/attempt (4.9% vs 4.6%, χ2=1.04, p=0.59) were not significant.\nRelative to veterans who were not infected with COVID-19, veterans who were infected were significantly more likely to screen positive at the peripandemic assessment for any internalising psychiatric disorder (20.5% vs 13.9%, χ2=7.85, p=0.005), alcohol or drug use disorder (23.2% vs 14.8%, χ2=11.51, p=0.001) and current suicidal ideation (12.0% vs 7.6%, χ2=5.92, p=0.015). With regard to specific disorders, veterans with histories of COVID-19 infection were more likely to screen positive for generalised anxiety disorder (14.3% vs 9.1%, χ2=6.99, p=0.008), pandemic-related stress symptoms (7.3% vs 4.0 %, χ2=5.99, p=0.014), alcohol use disorder (14.8% vs 9.2%, χ2=8.03, p=0.005) and drug use disorder (13.9% vs 7.8%, χ2=10.27, p=0.001) relative to veterans who were not infected. The differences in major depressive disorder (11.8% vs 8.6%, χ2=2.93, p=0.087) and past-year suicide plan/attempt (4.9% vs 4.6%, χ2=1.04, p=0.59) were not significant.\nCorrelates of peripandemic psychiatric conditions In table 1, columns 2–4 show bivariate correlates of peripandemic positive screens for psychiatric conditions. Prepandemic psychiatric symptom severity, alcohol use problem severity, psychosocial difficulties and loneliness were positively correlated with all three outcomes, while perceived social support and protective psychosocial characteristics were negatively correlated with these outcomes. With regard to COVID-related variables, pandemic-related stress symptoms were positively correlated with all three outcomes.\nCorrelates of positive screens for peripandemic psychiatric outcomes among US veterans with histories of COVID-19 infection\n*P<0.05, **P<0.01, ***P<0.001.\nr, Pearson correlation coefficient.\nIn table 1, columns 2–4 show bivariate correlates of peripandemic positive screens for psychiatric conditions. Prepandemic psychiatric symptom severity, alcohol use problem severity, psychosocial difficulties and loneliness were positively correlated with all three outcomes, while perceived social support and protective psychosocial characteristics were negatively correlated with these outcomes. With regard to COVID-related variables, pandemic-related stress symptoms were positively correlated with all three outcomes.\nCorrelates of positive screens for peripandemic psychiatric outcomes among US veterans with histories of COVID-19 infection\n*P<0.05, **P<0.01, ***P<0.001.\nr, Pearson correlation coefficient.\nMultivariable logistic regression models Multivariable logistic regression analyses (table 1, columns 5–7) revealed that greater prepandemic psychiatric symptom severity, non-prescription drug use, adverse childhood experiences, perceived social support, and COVID-related worries, social restriction stress and financial stress were independent risk factors for peripandemic internalising psychiatric disorders, whereas greater trauma burden was protective. Relative importance analysis indicated that prepandemic psychiatric symptom severity (34.9%), trauma burden (18.6%), COVID-related worries (14.9%), financial stress (14.5%) and social restriction stress (11.6%) explained the majority of variance in this outcome, with drug use severity (5.5%) explaining the remainder.\nPrepandemic severity of alcohol and non-prescription drug use were the only independent risk factors for peripandemic externalising psychiatric disorders, each explaining 48.0% and 52.0% of the variance, respectively.\nPrepandemic alcohol use severity, past-year suicidal ideation, loneliness, impulsivity, perceived social support and having a household member infected with COVID-19 were independent risk factors for peripandemic suicidal ideation, whereas greater protective psychosocial characteristics (specifically greater community integration: OR=0.51, 95% CI 0.34 to 0.79) and higher household income were protective. Relative importance analysis showed that prepandemic suicidal ideation (29.3%), loneliness (25.2%), household income (12.0%), community integration (10.8%) and impulsivity (9.4%) explained the majority of variance in this outcome, with household member COVID-19 infection (5.0%), prepandemic social support (4.9%) and alcohol use severity (3.4%) explaining the remaining variance.\nMultivariable logistic regression analyses (table 1, columns 5–7) revealed that greater prepandemic psychiatric symptom severity, non-prescription drug use, adverse childhood experiences, perceived social support, and COVID-related worries, social restriction stress and financial stress were independent risk factors for peripandemic internalising psychiatric disorders, whereas greater trauma burden was protective. Relative importance analysis indicated that prepandemic psychiatric symptom severity (34.9%), trauma burden (18.6%), COVID-related worries (14.9%), financial stress (14.5%) and social restriction stress (11.6%) explained the majority of variance in this outcome, with drug use severity (5.5%) explaining the remainder.\nPrepandemic severity of alcohol and non-prescription drug use were the only independent risk factors for peripandemic externalising psychiatric disorders, each explaining 48.0% and 52.0% of the variance, respectively.\nPrepandemic alcohol use severity, past-year suicidal ideation, loneliness, impulsivity, perceived social support and having a household member infected with COVID-19 were independent risk factors for peripandemic suicidal ideation, whereas greater protective psychosocial characteristics (specifically greater community integration: OR=0.51, 95% CI 0.34 to 0.79) and higher household income were protective. Relative importance analysis showed that prepandemic suicidal ideation (29.3%), loneliness (25.2%), household income (12.0%), community integration (10.8%) and impulsivity (9.4%) explained the majority of variance in this outcome, with household member COVID-19 infection (5.0%), prepandemic social support (4.9%) and alcohol use severity (3.4%) explaining the remaining variance."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Participants","Assessments","Data analysis","Prevalence of peripandemic psychiatric conditions of veterans who were infected by COVID-19","Correlates of peripandemic psychiatric conditions","Multivariable logistic regression models"],"string":"[\n \"Participants\",\n \"Assessments\",\n \"Data analysis\",\n \"Prevalence of peripandemic psychiatric conditions of veterans who were infected by COVID-19\",\n \"Correlates of peripandemic psychiatric conditions\",\n \"Multivariable logistic regression models\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Data were analysed from the 2019 to 2020 NHRVS, which surveyed a nationally representative, prospective sample of US military veterans. A total of 4069 veterans completed the prepandemic survey (median completion date: 21 November 2019) prior to the first documented COVID-19 cases in the USA (19 January 2020), and 3078 (75.6%) completed a 1-year peripandemic follow-up assessment (median completion date 14 November 2020). Both surveys were online, 45–60 min, self-report surveys. All participants received 15 000 points (equivalent to $15) for participating in the prepandemic survey, and 20 000 points (equivalent to $20) for participating in the peripandemic survey. Details of the study have been described previously.12 Briefly, the NHRVS sample was drawn from KnowledgePanel, a survey research panel of more than 50 000 US households maintained by Ipsos, a survey research firm. To promote generalisability of the results to the US veteran population, poststratification weights based on the demographic distribution of veterans in the contemporaneous US Census Current Population Survey Veterans Supplement were applied in inferential analyses.","Online supplemental table 1 presents a detailed description of measures used to assess potential risk and protective factors of peripandemic psychiatric conditions in veterans who survived COVID-19 infection.\n\n\n","χ2 analyses were conducted to compare the prevalence of psychiatric disorders and suicidal ideation between veterans who were and were not infected with COVID-19. Subsequent analyses focused on veterans who self-reported a history of COVID-19 infection at the peripandemic assessment. Analyses in this subcohort proceeded in two steps. First, we conducted Pearson correlations between participant characteristics and peripandemic internalising psychiatric disorders (ie, major depressive disorder, generalised anxiety disorder and/or pandemic-related stress symptoms); externalising disorders (ie, alcohol and/or drug use disorder); and suicidal ideation; internalising and externalising disorders were grouped together to increase statistical power. Second, we conducted multivariable binary logistic regression analyses to identify factors that independently predicted the aforementioned three categories of peripandemic psychiatric conditions. Variables that differed at the p<0.05 level in bivariate analyses for each set of comparisons were entered into each regression analysis. Third, we conducted relative importance analyses to determine the relative contribution of each significant variable in predicting positive screens for the three outcomes. These analyses partitioned the explained variance in the study outcome variables that is explained by each independent variable while simultaneously accounting for intercorrelations among these independent variables.13","Relative to veterans who were not infected with COVID-19, veterans who were infected were significantly more likely to screen positive at the peripandemic assessment for any internalising psychiatric disorder (20.5% vs 13.9%, χ2=7.85, p=0.005), alcohol or drug use disorder (23.2% vs 14.8%, χ2=11.51, p=0.001) and current suicidal ideation (12.0% vs 7.6%, χ2=5.92, p=0.015). With regard to specific disorders, veterans with histories of COVID-19 infection were more likely to screen positive for generalised anxiety disorder (14.3% vs 9.1%, χ2=6.99, p=0.008), pandemic-related stress symptoms (7.3% vs 4.0 %, χ2=5.99, p=0.014), alcohol use disorder (14.8% vs 9.2%, χ2=8.03, p=0.005) and drug use disorder (13.9% vs 7.8%, χ2=10.27, p=0.001) relative to veterans who were not infected. The differences in major depressive disorder (11.8% vs 8.6%, χ2=2.93, p=0.087) and past-year suicide plan/attempt (4.9% vs 4.6%, χ2=1.04, p=0.59) were not significant.","In table 1, columns 2–4 show bivariate correlates of peripandemic positive screens for psychiatric conditions. Prepandemic psychiatric symptom severity, alcohol use problem severity, psychosocial difficulties and loneliness were positively correlated with all three outcomes, while perceived social support and protective psychosocial characteristics were negatively correlated with these outcomes. With regard to COVID-related variables, pandemic-related stress symptoms were positively correlated with all three outcomes.\nCorrelates of positive screens for peripandemic psychiatric outcomes among US veterans with histories of COVID-19 infection\n*P<0.05, **P<0.01, ***P<0.001.\nr, Pearson correlation coefficient.","Multivariable logistic regression analyses (table 1, columns 5–7) revealed that greater prepandemic psychiatric symptom severity, non-prescription drug use, adverse childhood experiences, perceived social support, and COVID-related worries, social restriction stress and financial stress were independent risk factors for peripandemic internalising psychiatric disorders, whereas greater trauma burden was protective. Relative importance analysis indicated that prepandemic psychiatric symptom severity (34.9%), trauma burden (18.6%), COVID-related worries (14.9%), financial stress (14.5%) and social restriction stress (11.6%) explained the majority of variance in this outcome, with drug use severity (5.5%) explaining the remainder.\nPrepandemic severity of alcohol and non-prescription drug use were the only independent risk factors for peripandemic externalising psychiatric disorders, each explaining 48.0% and 52.0% of the variance, respectively.\nPrepandemic alcohol use severity, past-year suicidal ideation, loneliness, impulsivity, perceived social support and having a household member infected with COVID-19 were independent risk factors for peripandemic suicidal ideation, whereas greater protective psychosocial characteristics (specifically greater community integration: OR=0.51, 95% CI 0.34 to 0.79) and higher household income were protective. Relative importance analysis showed that prepandemic suicidal ideation (29.3%), loneliness (25.2%), household income (12.0%), community integration (10.8%) and impulsivity (9.4%) explained the majority of variance in this outcome, with household member COVID-19 infection (5.0%), prepandemic social support (4.9%) and alcohol use severity (3.4%) explaining the remaining variance."],"string":"[\n \"Data were analysed from the 2019 to 2020 NHRVS, which surveyed a nationally representative, prospective sample of US military veterans. A total of 4069 veterans completed the prepandemic survey (median completion date: 21 November 2019) prior to the first documented COVID-19 cases in the USA (19 January 2020), and 3078 (75.6%) completed a 1-year peripandemic follow-up assessment (median completion date 14 November 2020). Both surveys were online, 45–60 min, self-report surveys. All participants received 15 000 points (equivalent to $15) for participating in the prepandemic survey, and 20 000 points (equivalent to $20) for participating in the peripandemic survey. Details of the study have been described previously.12 Briefly, the NHRVS sample was drawn from KnowledgePanel, a survey research panel of more than 50 000 US households maintained by Ipsos, a survey research firm. To promote generalisability of the results to the US veteran population, poststratification weights based on the demographic distribution of veterans in the contemporaneous US Census Current Population Survey Veterans Supplement were applied in inferential analyses.\",\n \"Online supplemental table 1 presents a detailed description of measures used to assess potential risk and protective factors of peripandemic psychiatric conditions in veterans who survived COVID-19 infection.\\n\\n\\n\",\n \"χ2 analyses were conducted to compare the prevalence of psychiatric disorders and suicidal ideation between veterans who were and were not infected with COVID-19. Subsequent analyses focused on veterans who self-reported a history of COVID-19 infection at the peripandemic assessment. Analyses in this subcohort proceeded in two steps. First, we conducted Pearson correlations between participant characteristics and peripandemic internalising psychiatric disorders (ie, major depressive disorder, generalised anxiety disorder and/or pandemic-related stress symptoms); externalising disorders (ie, alcohol and/or drug use disorder); and suicidal ideation; internalising and externalising disorders were grouped together to increase statistical power. Second, we conducted multivariable binary logistic regression analyses to identify factors that independently predicted the aforementioned three categories of peripandemic psychiatric conditions. Variables that differed at the p<0.05 level in bivariate analyses for each set of comparisons were entered into each regression analysis. Third, we conducted relative importance analyses to determine the relative contribution of each significant variable in predicting positive screens for the three outcomes. These analyses partitioned the explained variance in the study outcome variables that is explained by each independent variable while simultaneously accounting for intercorrelations among these independent variables.13\",\n \"Relative to veterans who were not infected with COVID-19, veterans who were infected were significantly more likely to screen positive at the peripandemic assessment for any internalising psychiatric disorder (20.5% vs 13.9%, χ2=7.85, p=0.005), alcohol or drug use disorder (23.2% vs 14.8%, χ2=11.51, p=0.001) and current suicidal ideation (12.0% vs 7.6%, χ2=5.92, p=0.015). With regard to specific disorders, veterans with histories of COVID-19 infection were more likely to screen positive for generalised anxiety disorder (14.3% vs 9.1%, χ2=6.99, p=0.008), pandemic-related stress symptoms (7.3% vs 4.0 %, χ2=5.99, p=0.014), alcohol use disorder (14.8% vs 9.2%, χ2=8.03, p=0.005) and drug use disorder (13.9% vs 7.8%, χ2=10.27, p=0.001) relative to veterans who were not infected. The differences in major depressive disorder (11.8% vs 8.6%, χ2=2.93, p=0.087) and past-year suicide plan/attempt (4.9% vs 4.6%, χ2=1.04, p=0.59) were not significant.\",\n \"In table 1, columns 2–4 show bivariate correlates of peripandemic positive screens for psychiatric conditions. Prepandemic psychiatric symptom severity, alcohol use problem severity, psychosocial difficulties and loneliness were positively correlated with all three outcomes, while perceived social support and protective psychosocial characteristics were negatively correlated with these outcomes. With regard to COVID-related variables, pandemic-related stress symptoms were positively correlated with all three outcomes.\\nCorrelates of positive screens for peripandemic psychiatric outcomes among US veterans with histories of COVID-19 infection\\n*P<0.05, **P<0.01, ***P<0.001.\\nr, Pearson correlation coefficient.\",\n \"Multivariable logistic regression analyses (table 1, columns 5–7) revealed that greater prepandemic psychiatric symptom severity, non-prescription drug use, adverse childhood experiences, perceived social support, and COVID-related worries, social restriction stress and financial stress were independent risk factors for peripandemic internalising psychiatric disorders, whereas greater trauma burden was protective. Relative importance analysis indicated that prepandemic psychiatric symptom severity (34.9%), trauma burden (18.6%), COVID-related worries (14.9%), financial stress (14.5%) and social restriction stress (11.6%) explained the majority of variance in this outcome, with drug use severity (5.5%) explaining the remainder.\\nPrepandemic severity of alcohol and non-prescription drug use were the only independent risk factors for peripandemic externalising psychiatric disorders, each explaining 48.0% and 52.0% of the variance, respectively.\\nPrepandemic alcohol use severity, past-year suicidal ideation, loneliness, impulsivity, perceived social support and having a household member infected with COVID-19 were independent risk factors for peripandemic suicidal ideation, whereas greater protective psychosocial characteristics (specifically greater community integration: OR=0.51, 95% CI 0.34 to 0.79) and higher household income were protective. Relative importance analysis showed that prepandemic suicidal ideation (29.3%), loneliness (25.2%), household income (12.0%), community integration (10.8%) and impulsivity (9.4%) explained the majority of variance in this outcome, with household member COVID-19 infection (5.0%), prepandemic social support (4.9%) and alcohol use severity (3.4%) explaining the remaining variance.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Introduction","Methods","Participants","Assessments","Data analysis","Results","Prevalence of peripandemic psychiatric conditions of veterans who were infected by COVID-19","Correlates of peripandemic psychiatric conditions","Multivariable logistic regression models","Discussion"],"string":"[\n \"Introduction\",\n \"Methods\",\n \"Participants\",\n \"Assessments\",\n \"Data analysis\",\n \"Results\",\n \"Prevalence of peripandemic psychiatric conditions of veterans who were infected by COVID-19\",\n \"Correlates of peripandemic psychiatric conditions\",\n \"Multivariable logistic regression models\",\n \"Discussion\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["The COVID-19 pandemic has been linked to increased social isolation, economic recession and psychological distress.1 Numerous studies have reported a substantial increase in the prevalence of depression and anxiety in the general public during the pandemic.2 3 Previous psychiatric diagnoses, COVID-related stressors, such as worries about being infected with COVID-19, financial stressors and actual COVID-19 infection have been proposed as possible risk factors of both the development and worsening of mental illness during the pandemic.1\nVeterans may be a highly vulnerable group to the negative mental health effects of the pandemic due to their older age, and higher prepandemic prevalence of physical and psychiatric risk factors and conditions relative to the general population.4 5i Further, previous studies on veterans have found that stressful events such as combat deployments were associated with increased rates of depression and substance use disorders in veterans.6\nAs of April 2021, in the USA, over 31 million people have been infected with COVID-19 and over half a million people have died of COVID-19-related complications.7 Previous studies on survivors of COVID-19 have reported high prevalence of mental disorders, including depression, anxiety and post-traumatic stress disorder (PTSD8–10). Thus, there has been increasing attention in closely monitoring for psychiatric symptoms and functioning in those who have been infected with COVID-19 in order to mitigate distress and risk of suicide. However, due to the high burden of COVID-19 infections in the USA and the limited capacity of mental health treatment services, it is challenging to monitor everyone who has been infected. These challenges have been further complicated by infection-control measures implemented by almost all mental health service providers during the pandemic.11\nOne potentially effective way of informing the allocation of limited mental health treatment resources may be to identify subgroups that are at high risk of developing or experiencing a worsening of psychiatric conditions after COVID-19 infection. Identifying potential risk and protective factors of psychiatric conditions among COVID-19 survivors in populations with elevated risk, such as US military veterans, may help inform more targeted population-based prevention, screening and intervention strategies.\nToward this end, we analysed data from the National Health and Resilience in Veterans Study (NHRVS12) to examine (1) the prevalence of psychiatric conditions in US veterans who have and have not been infected with COVID-19, and (2) prepandemic and peripandemic risk and protective factors of peripandemic psychiatric conditions in veterans who survived COVID-19 infection.","Participants Data were analysed from the 2019 to 2020 NHRVS, which surveyed a nationally representative, prospective sample of US military veterans. A total of 4069 veterans completed the prepandemic survey (median completion date: 21 November 2019) prior to the first documented COVID-19 cases in the USA (19 January 2020), and 3078 (75.6%) completed a 1-year peripandemic follow-up assessment (median completion date 14 November 2020). Both surveys were online, 45–60 min, self-report surveys. All participants received 15 000 points (equivalent to $15) for participating in the prepandemic survey, and 20 000 points (equivalent to $20) for participating in the peripandemic survey. Details of the study have been described previously.12 Briefly, the NHRVS sample was drawn from KnowledgePanel, a survey research panel of more than 50 000 US households maintained by Ipsos, a survey research firm. To promote generalisability of the results to the US veteran population, poststratification weights based on the demographic distribution of veterans in the contemporaneous US Census Current Population Survey Veterans Supplement were applied in inferential analyses.\nData were analysed from the 2019 to 2020 NHRVS, which surveyed a nationally representative, prospective sample of US military veterans. A total of 4069 veterans completed the prepandemic survey (median completion date: 21 November 2019) prior to the first documented COVID-19 cases in the USA (19 January 2020), and 3078 (75.6%) completed a 1-year peripandemic follow-up assessment (median completion date 14 November 2020). Both surveys were online, 45–60 min, self-report surveys. All participants received 15 000 points (equivalent to $15) for participating in the prepandemic survey, and 20 000 points (equivalent to $20) for participating in the peripandemic survey. Details of the study have been described previously.12 Briefly, the NHRVS sample was drawn from KnowledgePanel, a survey research panel of more than 50 000 US households maintained by Ipsos, a survey research firm. To promote generalisability of the results to the US veteran population, poststratification weights based on the demographic distribution of veterans in the contemporaneous US Census Current Population Survey Veterans Supplement were applied in inferential analyses.\nAssessments Online supplemental table 1 presents a detailed description of measures used to assess potential risk and protective factors of peripandemic psychiatric conditions in veterans who survived COVID-19 infection.\n\n\n\nOnline supplemental table 1 presents a detailed description of measures used to assess potential risk and protective factors of peripandemic psychiatric conditions in veterans who survived COVID-19 infection.\n\n\n\nData analysis χ2 analyses were conducted to compare the prevalence of psychiatric disorders and suicidal ideation between veterans who were and were not infected with COVID-19. Subsequent analyses focused on veterans who self-reported a history of COVID-19 infection at the peripandemic assessment. Analyses in this subcohort proceeded in two steps. First, we conducted Pearson correlations between participant characteristics and peripandemic internalising psychiatric disorders (ie, major depressive disorder, generalised anxiety disorder and/or pandemic-related stress symptoms); externalising disorders (ie, alcohol and/or drug use disorder); and suicidal ideation; internalising and externalising disorders were grouped together to increase statistical power. Second, we conducted multivariable binary logistic regression analyses to identify factors that independently predicted the aforementioned three categories of peripandemic psychiatric conditions. Variables that differed at the p<0.05 level in bivariate analyses for each set of comparisons were entered into each regression analysis. Third, we conducted relative importance analyses to determine the relative contribution of each significant variable in predicting positive screens for the three outcomes. These analyses partitioned the explained variance in the study outcome variables that is explained by each independent variable while simultaneously accounting for intercorrelations among these independent variables.13\nχ2 analyses were conducted to compare the prevalence of psychiatric disorders and suicidal ideation between veterans who were and were not infected with COVID-19. Subsequent analyses focused on veterans who self-reported a history of COVID-19 infection at the peripandemic assessment. Analyses in this subcohort proceeded in two steps. First, we conducted Pearson correlations between participant characteristics and peripandemic internalising psychiatric disorders (ie, major depressive disorder, generalised anxiety disorder and/or pandemic-related stress symptoms); externalising disorders (ie, alcohol and/or drug use disorder); and suicidal ideation; internalising and externalising disorders were grouped together to increase statistical power. Second, we conducted multivariable binary logistic regression analyses to identify factors that independently predicted the aforementioned three categories of peripandemic psychiatric conditions. Variables that differed at the p<0.05 level in bivariate analyses for each set of comparisons were entered into each regression analysis. Third, we conducted relative importance analyses to determine the relative contribution of each significant variable in predicting positive screens for the three outcomes. These analyses partitioned the explained variance in the study outcome variables that is explained by each independent variable while simultaneously accounting for intercorrelations among these independent variables.13","Data were analysed from the 2019 to 2020 NHRVS, which surveyed a nationally representative, prospective sample of US military veterans. A total of 4069 veterans completed the prepandemic survey (median completion date: 21 November 2019) prior to the first documented COVID-19 cases in the USA (19 January 2020), and 3078 (75.6%) completed a 1-year peripandemic follow-up assessment (median completion date 14 November 2020). Both surveys were online, 45–60 min, self-report surveys. All participants received 15 000 points (equivalent to $15) for participating in the prepandemic survey, and 20 000 points (equivalent to $20) for participating in the peripandemic survey. Details of the study have been described previously.12 Briefly, the NHRVS sample was drawn from KnowledgePanel, a survey research panel of more than 50 000 US households maintained by Ipsos, a survey research firm. To promote generalisability of the results to the US veteran population, poststratification weights based on the demographic distribution of veterans in the contemporaneous US Census Current Population Survey Veterans Supplement were applied in inferential analyses.","Online supplemental table 1 presents a detailed description of measures used to assess potential risk and protective factors of peripandemic psychiatric conditions in veterans who survived COVID-19 infection.\n\n\n","χ2 analyses were conducted to compare the prevalence of psychiatric disorders and suicidal ideation between veterans who were and were not infected with COVID-19. Subsequent analyses focused on veterans who self-reported a history of COVID-19 infection at the peripandemic assessment. Analyses in this subcohort proceeded in two steps. First, we conducted Pearson correlations between participant characteristics and peripandemic internalising psychiatric disorders (ie, major depressive disorder, generalised anxiety disorder and/or pandemic-related stress symptoms); externalising disorders (ie, alcohol and/or drug use disorder); and suicidal ideation; internalising and externalising disorders were grouped together to increase statistical power. Second, we conducted multivariable binary logistic regression analyses to identify factors that independently predicted the aforementioned three categories of peripandemic psychiatric conditions. Variables that differed at the p<0.05 level in bivariate analyses for each set of comparisons were entered into each regression analysis. Third, we conducted relative importance analyses to determine the relative contribution of each significant variable in predicting positive screens for the three outcomes. These analyses partitioned the explained variance in the study outcome variables that is explained by each independent variable while simultaneously accounting for intercorrelations among these independent variables.13","The final sample included 3078 veterans who completed both prepandemic and 1-year peripandemic assessments. The average age of the sample was 62.2 years old (SD=15.7, range 22–99); the majority was male (90.2%) and Caucasian (78.1%); and 35.0% were combat veterans. A total of 233 veterans (8.6%) reported that they had been infected with COVID-19. The majority (70.5%) reported mild-to-moderate symptoms; 6.3% reported requiring a visit to the emergency room; 4.5% reported severe symptoms with serious concerns about not surviving; 1.9% reported being admitted to an intensive care unit; 1.2% reported requiring an overnight stay at a hospital, and 0.8% reported requiring intubation/mechanical ventilation; the remaining 14.8% of the sample did not provide details about the severity of their illness.\nPrevalence of peripandemic psychiatric conditions of veterans who were infected by COVID-19 Relative to veterans who were not infected with COVID-19, veterans who were infected were significantly more likely to screen positive at the peripandemic assessment for any internalising psychiatric disorder (20.5% vs 13.9%, χ2=7.85, p=0.005), alcohol or drug use disorder (23.2% vs 14.8%, χ2=11.51, p=0.001) and current suicidal ideation (12.0% vs 7.6%, χ2=5.92, p=0.015). With regard to specific disorders, veterans with histories of COVID-19 infection were more likely to screen positive for generalised anxiety disorder (14.3% vs 9.1%, χ2=6.99, p=0.008), pandemic-related stress symptoms (7.3% vs 4.0 %, χ2=5.99, p=0.014), alcohol use disorder (14.8% vs 9.2%, χ2=8.03, p=0.005) and drug use disorder (13.9% vs 7.8%, χ2=10.27, p=0.001) relative to veterans who were not infected. The differences in major depressive disorder (11.8% vs 8.6%, χ2=2.93, p=0.087) and past-year suicide plan/attempt (4.9% vs 4.6%, χ2=1.04, p=0.59) were not significant.\nRelative to veterans who were not infected with COVID-19, veterans who were infected were significantly more likely to screen positive at the peripandemic assessment for any internalising psychiatric disorder (20.5% vs 13.9%, χ2=7.85, p=0.005), alcohol or drug use disorder (23.2% vs 14.8%, χ2=11.51, p=0.001) and current suicidal ideation (12.0% vs 7.6%, χ2=5.92, p=0.015). With regard to specific disorders, veterans with histories of COVID-19 infection were more likely to screen positive for generalised anxiety disorder (14.3% vs 9.1%, χ2=6.99, p=0.008), pandemic-related stress symptoms (7.3% vs 4.0 %, χ2=5.99, p=0.014), alcohol use disorder (14.8% vs 9.2%, χ2=8.03, p=0.005) and drug use disorder (13.9% vs 7.8%, χ2=10.27, p=0.001) relative to veterans who were not infected. The differences in major depressive disorder (11.8% vs 8.6%, χ2=2.93, p=0.087) and past-year suicide plan/attempt (4.9% vs 4.6%, χ2=1.04, p=0.59) were not significant.\nCorrelates of peripandemic psychiatric conditions In table 1, columns 2–4 show bivariate correlates of peripandemic positive screens for psychiatric conditions. Prepandemic psychiatric symptom severity, alcohol use problem severity, psychosocial difficulties and loneliness were positively correlated with all three outcomes, while perceived social support and protective psychosocial characteristics were negatively correlated with these outcomes. With regard to COVID-related variables, pandemic-related stress symptoms were positively correlated with all three outcomes.\nCorrelates of positive screens for peripandemic psychiatric outcomes among US veterans with histories of COVID-19 infection\n*P<0.05, **P<0.01, ***P<0.001.\nr, Pearson correlation coefficient.\nIn table 1, columns 2–4 show bivariate correlates of peripandemic positive screens for psychiatric conditions. Prepandemic psychiatric symptom severity, alcohol use problem severity, psychosocial difficulties and loneliness were positively correlated with all three outcomes, while perceived social support and protective psychosocial characteristics were negatively correlated with these outcomes. With regard to COVID-related variables, pandemic-related stress symptoms were positively correlated with all three outcomes.\nCorrelates of positive screens for peripandemic psychiatric outcomes among US veterans with histories of COVID-19 infection\n*P<0.05, **P<0.01, ***P<0.001.\nr, Pearson correlation coefficient.\nMultivariable logistic regression models Multivariable logistic regression analyses (table 1, columns 5–7) revealed that greater prepandemic psychiatric symptom severity, non-prescription drug use, adverse childhood experiences, perceived social support, and COVID-related worries, social restriction stress and financial stress were independent risk factors for peripandemic internalising psychiatric disorders, whereas greater trauma burden was protective. Relative importance analysis indicated that prepandemic psychiatric symptom severity (34.9%), trauma burden (18.6%), COVID-related worries (14.9%), financial stress (14.5%) and social restriction stress (11.6%) explained the majority of variance in this outcome, with drug use severity (5.5%) explaining the remainder.\nPrepandemic severity of alcohol and non-prescription drug use were the only independent risk factors for peripandemic externalising psychiatric disorders, each explaining 48.0% and 52.0% of the variance, respectively.\nPrepandemic alcohol use severity, past-year suicidal ideation, loneliness, impulsivity, perceived social support and having a household member infected with COVID-19 were independent risk factors for peripandemic suicidal ideation, whereas greater protective psychosocial characteristics (specifically greater community integration: OR=0.51, 95% CI 0.34 to 0.79) and higher household income were protective. Relative importance analysis showed that prepandemic suicidal ideation (29.3%), loneliness (25.2%), household income (12.0%), community integration (10.8%) and impulsivity (9.4%) explained the majority of variance in this outcome, with household member COVID-19 infection (5.0%), prepandemic social support (4.9%) and alcohol use severity (3.4%) explaining the remaining variance.\nMultivariable logistic regression analyses (table 1, columns 5–7) revealed that greater prepandemic psychiatric symptom severity, non-prescription drug use, adverse childhood experiences, perceived social support, and COVID-related worries, social restriction stress and financial stress were independent risk factors for peripandemic internalising psychiatric disorders, whereas greater trauma burden was protective. Relative importance analysis indicated that prepandemic psychiatric symptom severity (34.9%), trauma burden (18.6%), COVID-related worries (14.9%), financial stress (14.5%) and social restriction stress (11.6%) explained the majority of variance in this outcome, with drug use severity (5.5%) explaining the remainder.\nPrepandemic severity of alcohol and non-prescription drug use were the only independent risk factors for peripandemic externalising psychiatric disorders, each explaining 48.0% and 52.0% of the variance, respectively.\nPrepandemic alcohol use severity, past-year suicidal ideation, loneliness, impulsivity, perceived social support and having a household member infected with COVID-19 were independent risk factors for peripandemic suicidal ideation, whereas greater protective psychosocial characteristics (specifically greater community integration: OR=0.51, 95% CI 0.34 to 0.79) and higher household income were protective. Relative importance analysis showed that prepandemic suicidal ideation (29.3%), loneliness (25.2%), household income (12.0%), community integration (10.8%) and impulsivity (9.4%) explained the majority of variance in this outcome, with household member COVID-19 infection (5.0%), prepandemic social support (4.9%) and alcohol use severity (3.4%) explaining the remaining variance.","Relative to veterans who were not infected with COVID-19, veterans who were infected were significantly more likely to screen positive at the peripandemic assessment for any internalising psychiatric disorder (20.5% vs 13.9%, χ2=7.85, p=0.005), alcohol or drug use disorder (23.2% vs 14.8%, χ2=11.51, p=0.001) and current suicidal ideation (12.0% vs 7.6%, χ2=5.92, p=0.015). With regard to specific disorders, veterans with histories of COVID-19 infection were more likely to screen positive for generalised anxiety disorder (14.3% vs 9.1%, χ2=6.99, p=0.008), pandemic-related stress symptoms (7.3% vs 4.0 %, χ2=5.99, p=0.014), alcohol use disorder (14.8% vs 9.2%, χ2=8.03, p=0.005) and drug use disorder (13.9% vs 7.8%, χ2=10.27, p=0.001) relative to veterans who were not infected. The differences in major depressive disorder (11.8% vs 8.6%, χ2=2.93, p=0.087) and past-year suicide plan/attempt (4.9% vs 4.6%, χ2=1.04, p=0.59) were not significant.","In table 1, columns 2–4 show bivariate correlates of peripandemic positive screens for psychiatric conditions. Prepandemic psychiatric symptom severity, alcohol use problem severity, psychosocial difficulties and loneliness were positively correlated with all three outcomes, while perceived social support and protective psychosocial characteristics were negatively correlated with these outcomes. With regard to COVID-related variables, pandemic-related stress symptoms were positively correlated with all three outcomes.\nCorrelates of positive screens for peripandemic psychiatric outcomes among US veterans with histories of COVID-19 infection\n*P<0.05, **P<0.01, ***P<0.001.\nr, Pearson correlation coefficient.","Multivariable logistic regression analyses (table 1, columns 5–7) revealed that greater prepandemic psychiatric symptom severity, non-prescription drug use, adverse childhood experiences, perceived social support, and COVID-related worries, social restriction stress and financial stress were independent risk factors for peripandemic internalising psychiatric disorders, whereas greater trauma burden was protective. Relative importance analysis indicated that prepandemic psychiatric symptom severity (34.9%), trauma burden (18.6%), COVID-related worries (14.9%), financial stress (14.5%) and social restriction stress (11.6%) explained the majority of variance in this outcome, with drug use severity (5.5%) explaining the remainder.\nPrepandemic severity of alcohol and non-prescription drug use were the only independent risk factors for peripandemic externalising psychiatric disorders, each explaining 48.0% and 52.0% of the variance, respectively.\nPrepandemic alcohol use severity, past-year suicidal ideation, loneliness, impulsivity, perceived social support and having a household member infected with COVID-19 were independent risk factors for peripandemic suicidal ideation, whereas greater protective psychosocial characteristics (specifically greater community integration: OR=0.51, 95% CI 0.34 to 0.79) and higher household income were protective. Relative importance analysis showed that prepandemic suicidal ideation (29.3%), loneliness (25.2%), household income (12.0%), community integration (10.8%) and impulsivity (9.4%) explained the majority of variance in this outcome, with household member COVID-19 infection (5.0%), prepandemic social support (4.9%) and alcohol use severity (3.4%) explaining the remaining variance.","This study compared the prevalence of peripandemic psychiatric disorders and suicidal ideation between US veterans with and without histories of COVID-19 infection, and examined prepandemic and peripandemic risk and protective factors for these outcomes among COVID-19 survivors. Results revealed that veterans who were infected with COVID-19 had a significantly higher prevalence of positive screens for psychiatric conditions, as well as suicidal ideation relative to those who were not infected. Prepandemic greater psychiatric symptom severity and COVID-related stressors were the strongest predictors of peripandemic internalising psychiatric disorders in veterans who survived COVID-19, while previous trauma exposure was found to be protective. Prepandemic suicidal ideation, loneliness and impulsivity were the strongest independent risk factors of peripandemic suicidal ideation, while greater prepandemic community integration and higher annual household income were protective.\nResults of this study are largely consistent with previous studies of non-representative patient samples who were hospitalised for COVID-19 infection, which observed a higher prevalence of psychiatric conditions, including depression, anxiety and PTSD.8 It is possible that being infected with COVID-19 may have aggravated psychological distress during the pandemic. Further, it is also plausible that those who had prepandemic psychiatric conditions and more psychosocial difficulties may have been at higher risk of infection given less resources and protective measures to avoid infection.9 Of note, the strongest predictors of peripandemic internalising disorders and externalising disorders in those who survived COVID-19 were prepandemic psychiatric symptom and substance use severity, respectively. This finding suggests that veterans with greater severity of prepandemic psychiatric distress and substance use may sensitise veterans to the deleterious mental health effects of COVID-19 infection.\nA growing body of literature suggests that COVID-related stressors may contribute to risk of pandemic-related psychiatric distress.14 15 Results of the current study extend this work to suggest that COVID-related worries, financial distress and social restriction stress were independently associated with increased risk of screening positive for any internalising psychiatric disorders in veterans who survived COVID-19 infection.\nPrevious research has identified previous suicidal behaviour as a strong predictor of future suicidal behaviour.16 The results of this study are consistent with these findings, as prepandemic suicidal ideation was the strongest predictor of peripandemic suicidal ideation. The negative impact of loneliness on mental health outcomes, including suicidal ideation, has been well established.17 In addition, due to the increased social restrictions imposed during the pandemic, increasing loneliness has been suggested as a possible contributor to worsening mental health.1 In this study, greater prepandemic loneliness was the second strongest predictor of peripandemic suicidal ideation. This finding suggests that loneliness may be a clinically relevant intervention target to help mitigate risk of suicidal behaviour in veterans during the pandemic. Moreover, impulsivity was a strong predictor of peripandemic suicidal ideation, which is consistent with previous research that reported strong positive association between impulsivity and suicidal behaviour.18 Of note, all of these factors were assessed prior to the pandemic, suggesting that they may represent increased vulnerability for suicidal ideation during the pandemic in US military veterans who survived COVID-19 infection.\nGreater prepandemic community integration was protective against peripandemic suicidal ideation, even after controlling for other variables. This finding accords with previous research describing the protective effects of community integration in promoting psychological resilience and mitigation of risk for suicidal behaviour.19 20 Further, higher prepandemic household income was protective against risk of peripandemic suicidal ideation. This finding, which is consistent with the conservation of resources theory,21 suggests that fewer prepandemic financial resources may have led to increased peripandemic distress and suicidal ideation. This finding underscores the importance of assessing, monitoring and ameliorating financial distress brought on by the pandemic in veterans with histories of COVID-19 infection.\nGreater trauma burden was identified as a protective factor to internalising psychiatric disorders during the pandemic. While previous trauma is a well known risk factor for psychiatric distress,22 previous exposure to traumatic events may also have ‘stress inoculating’ effects and help protect against risk of adverse mental health outcomes.23 Specifically, exposure to prior traumas may help promote engagement in adaptive coping strategies and may give rise to positive psychological changes (eg, greater sense of personal strength)24 25 that may in turn help foster resilience to new stressors, such as COVID-19 infection.\nFurther, it is noteworthy that perceived social support was found to be associated with increased risk of both internalising psychiatric disorders and suicidal ideation during the pandemic. One interpretation of this finding is that greater prepandemic severity of psychiatric distress may have increased support-seeking behaviours during the pandemic. Further research is needed to elucidate prospective associations between aspects of social support and risk for pandemic-related psychiatric conditions, particularly given the imposition of social restrictions during the pandemic.\nThis study has several limitations. First, history of COVID-19 infection was based on self-report and may be subject to response bias, as well as access to and use of testing for COVID-19 infection. Thus, it is possible that the prevalence of self-reported COVID-19 infection may not capture the accurate prevalence of infections in US veterans. Second, due to the survey design of this study, we used screening instruments to assess psychiatric conditions. Further research using diagnostic instruments such as structured clinical interviews are needed to replicate the results reported herein. Third, while nationally representative, our sample was composed entirely of US military veterans who are predominantly older, male and white, which makes it difficult to generalise results to non-veteran and more diverse veteran populations. Fourth, although this was a prospective cohort study, given the cross-sectional nature of the surveys and limited follow-up time frame, it is difficult to draw conclusions regarding temporal/causal associations between independent variables, and peripandemic psychiatric disorders and suicidal ideation.\nDespite these limitations, this study provides the first known nationally representative data on the prevalence and prepandemic and pandemic-related risk and protective factors of peripandemic psychiatric outcomes in US military veterans who survived COVID-19 infection. Given the enormous toll of COVID-19 infections in the USA,4 this information may help inform targeted population-based prevention, monitoring and intervention strategies. For example, US veterans who are COVID-19 survivors may be at elevated risk of peripandemic psychiatric conditions, and those with less socioeconomic resources and greater symptom severity at baseline may require higher clinical attention. Further, veterans who endorse greater COVID-related stressors may be at increased risk of peripandemic psychiatric disorders and suicidal ideation. Further research is needed to replicate and extend these results to other populations who are also at higher risk of psychiatric conditions; identify mechanisms leading to increased risk of psychiatric conditions during the pandemic; and assess the efficacy of interventions targeting such evidence-based risk and protective factors in mitigating risk of psychiatric conditions in veterans and other populations."],"string":"[\n \"The COVID-19 pandemic has been linked to increased social isolation, economic recession and psychological distress.1 Numerous studies have reported a substantial increase in the prevalence of depression and anxiety in the general public during the pandemic.2 3 Previous psychiatric diagnoses, COVID-related stressors, such as worries about being infected with COVID-19, financial stressors and actual COVID-19 infection have been proposed as possible risk factors of both the development and worsening of mental illness during the pandemic.1\\nVeterans may be a highly vulnerable group to the negative mental health effects of the pandemic due to their older age, and higher prepandemic prevalence of physical and psychiatric risk factors and conditions relative to the general population.4 5i Further, previous studies on veterans have found that stressful events such as combat deployments were associated with increased rates of depression and substance use disorders in veterans.6\\nAs of April 2021, in the USA, over 31 million people have been infected with COVID-19 and over half a million people have died of COVID-19-related complications.7 Previous studies on survivors of COVID-19 have reported high prevalence of mental disorders, including depression, anxiety and post-traumatic stress disorder (PTSD8–10). Thus, there has been increasing attention in closely monitoring for psychiatric symptoms and functioning in those who have been infected with COVID-19 in order to mitigate distress and risk of suicide. However, due to the high burden of COVID-19 infections in the USA and the limited capacity of mental health treatment services, it is challenging to monitor everyone who has been infected. These challenges have been further complicated by infection-control measures implemented by almost all mental health service providers during the pandemic.11\\nOne potentially effective way of informing the allocation of limited mental health treatment resources may be to identify subgroups that are at high risk of developing or experiencing a worsening of psychiatric conditions after COVID-19 infection. Identifying potential risk and protective factors of psychiatric conditions among COVID-19 survivors in populations with elevated risk, such as US military veterans, may help inform more targeted population-based prevention, screening and intervention strategies.\\nToward this end, we analysed data from the National Health and Resilience in Veterans Study (NHRVS12) to examine (1) the prevalence of psychiatric conditions in US veterans who have and have not been infected with COVID-19, and (2) prepandemic and peripandemic risk and protective factors of peripandemic psychiatric conditions in veterans who survived COVID-19 infection.\",\n \"Participants Data were analysed from the 2019 to 2020 NHRVS, which surveyed a nationally representative, prospective sample of US military veterans. A total of 4069 veterans completed the prepandemic survey (median completion date: 21 November 2019) prior to the first documented COVID-19 cases in the USA (19 January 2020), and 3078 (75.6%) completed a 1-year peripandemic follow-up assessment (median completion date 14 November 2020). Both surveys were online, 45–60 min, self-report surveys. All participants received 15 000 points (equivalent to $15) for participating in the prepandemic survey, and 20 000 points (equivalent to $20) for participating in the peripandemic survey. Details of the study have been described previously.12 Briefly, the NHRVS sample was drawn from KnowledgePanel, a survey research panel of more than 50 000 US households maintained by Ipsos, a survey research firm. To promote generalisability of the results to the US veteran population, poststratification weights based on the demographic distribution of veterans in the contemporaneous US Census Current Population Survey Veterans Supplement were applied in inferential analyses.\\nData were analysed from the 2019 to 2020 NHRVS, which surveyed a nationally representative, prospective sample of US military veterans. A total of 4069 veterans completed the prepandemic survey (median completion date: 21 November 2019) prior to the first documented COVID-19 cases in the USA (19 January 2020), and 3078 (75.6%) completed a 1-year peripandemic follow-up assessment (median completion date 14 November 2020). Both surveys were online, 45–60 min, self-report surveys. All participants received 15 000 points (equivalent to $15) for participating in the prepandemic survey, and 20 000 points (equivalent to $20) for participating in the peripandemic survey. Details of the study have been described previously.12 Briefly, the NHRVS sample was drawn from KnowledgePanel, a survey research panel of more than 50 000 US households maintained by Ipsos, a survey research firm. To promote generalisability of the results to the US veteran population, poststratification weights based on the demographic distribution of veterans in the contemporaneous US Census Current Population Survey Veterans Supplement were applied in inferential analyses.\\nAssessments Online supplemental table 1 presents a detailed description of measures used to assess potential risk and protective factors of peripandemic psychiatric conditions in veterans who survived COVID-19 infection.\\n\\n\\n\\nOnline supplemental table 1 presents a detailed description of measures used to assess potential risk and protective factors of peripandemic psychiatric conditions in veterans who survived COVID-19 infection.\\n\\n\\n\\nData analysis χ2 analyses were conducted to compare the prevalence of psychiatric disorders and suicidal ideation between veterans who were and were not infected with COVID-19. Subsequent analyses focused on veterans who self-reported a history of COVID-19 infection at the peripandemic assessment. Analyses in this subcohort proceeded in two steps. First, we conducted Pearson correlations between participant characteristics and peripandemic internalising psychiatric disorders (ie, major depressive disorder, generalised anxiety disorder and/or pandemic-related stress symptoms); externalising disorders (ie, alcohol and/or drug use disorder); and suicidal ideation; internalising and externalising disorders were grouped together to increase statistical power. Second, we conducted multivariable binary logistic regression analyses to identify factors that independently predicted the aforementioned three categories of peripandemic psychiatric conditions. Variables that differed at the p<0.05 level in bivariate analyses for each set of comparisons were entered into each regression analysis. Third, we conducted relative importance analyses to determine the relative contribution of each significant variable in predicting positive screens for the three outcomes. These analyses partitioned the explained variance in the study outcome variables that is explained by each independent variable while simultaneously accounting for intercorrelations among these independent variables.13\\nχ2 analyses were conducted to compare the prevalence of psychiatric disorders and suicidal ideation between veterans who were and were not infected with COVID-19. Subsequent analyses focused on veterans who self-reported a history of COVID-19 infection at the peripandemic assessment. Analyses in this subcohort proceeded in two steps. First, we conducted Pearson correlations between participant characteristics and peripandemic internalising psychiatric disorders (ie, major depressive disorder, generalised anxiety disorder and/or pandemic-related stress symptoms); externalising disorders (ie, alcohol and/or drug use disorder); and suicidal ideation; internalising and externalising disorders were grouped together to increase statistical power. Second, we conducted multivariable binary logistic regression analyses to identify factors that independently predicted the aforementioned three categories of peripandemic psychiatric conditions. Variables that differed at the p<0.05 level in bivariate analyses for each set of comparisons were entered into each regression analysis. Third, we conducted relative importance analyses to determine the relative contribution of each significant variable in predicting positive screens for the three outcomes. These analyses partitioned the explained variance in the study outcome variables that is explained by each independent variable while simultaneously accounting for intercorrelations among these independent variables.13\",\n \"Data were analysed from the 2019 to 2020 NHRVS, which surveyed a nationally representative, prospective sample of US military veterans. A total of 4069 veterans completed the prepandemic survey (median completion date: 21 November 2019) prior to the first documented COVID-19 cases in the USA (19 January 2020), and 3078 (75.6%) completed a 1-year peripandemic follow-up assessment (median completion date 14 November 2020). Both surveys were online, 45–60 min, self-report surveys. All participants received 15 000 points (equivalent to $15) for participating in the prepandemic survey, and 20 000 points (equivalent to $20) for participating in the peripandemic survey. Details of the study have been described previously.12 Briefly, the NHRVS sample was drawn from KnowledgePanel, a survey research panel of more than 50 000 US households maintained by Ipsos, a survey research firm. To promote generalisability of the results to the US veteran population, poststratification weights based on the demographic distribution of veterans in the contemporaneous US Census Current Population Survey Veterans Supplement were applied in inferential analyses.\",\n \"Online supplemental table 1 presents a detailed description of measures used to assess potential risk and protective factors of peripandemic psychiatric conditions in veterans who survived COVID-19 infection.\\n\\n\\n\",\n \"χ2 analyses were conducted to compare the prevalence of psychiatric disorders and suicidal ideation between veterans who were and were not infected with COVID-19. Subsequent analyses focused on veterans who self-reported a history of COVID-19 infection at the peripandemic assessment. Analyses in this subcohort proceeded in two steps. First, we conducted Pearson correlations between participant characteristics and peripandemic internalising psychiatric disorders (ie, major depressive disorder, generalised anxiety disorder and/or pandemic-related stress symptoms); externalising disorders (ie, alcohol and/or drug use disorder); and suicidal ideation; internalising and externalising disorders were grouped together to increase statistical power. Second, we conducted multivariable binary logistic regression analyses to identify factors that independently predicted the aforementioned three categories of peripandemic psychiatric conditions. Variables that differed at the p<0.05 level in bivariate analyses for each set of comparisons were entered into each regression analysis. Third, we conducted relative importance analyses to determine the relative contribution of each significant variable in predicting positive screens for the three outcomes. These analyses partitioned the explained variance in the study outcome variables that is explained by each independent variable while simultaneously accounting for intercorrelations among these independent variables.13\",\n \"The final sample included 3078 veterans who completed both prepandemic and 1-year peripandemic assessments. The average age of the sample was 62.2 years old (SD=15.7, range 22–99); the majority was male (90.2%) and Caucasian (78.1%); and 35.0% were combat veterans. A total of 233 veterans (8.6%) reported that they had been infected with COVID-19. The majority (70.5%) reported mild-to-moderate symptoms; 6.3% reported requiring a visit to the emergency room; 4.5% reported severe symptoms with serious concerns about not surviving; 1.9% reported being admitted to an intensive care unit; 1.2% reported requiring an overnight stay at a hospital, and 0.8% reported requiring intubation/mechanical ventilation; the remaining 14.8% of the sample did not provide details about the severity of their illness.\\nPrevalence of peripandemic psychiatric conditions of veterans who were infected by COVID-19 Relative to veterans who were not infected with COVID-19, veterans who were infected were significantly more likely to screen positive at the peripandemic assessment for any internalising psychiatric disorder (20.5% vs 13.9%, χ2=7.85, p=0.005), alcohol or drug use disorder (23.2% vs 14.8%, χ2=11.51, p=0.001) and current suicidal ideation (12.0% vs 7.6%, χ2=5.92, p=0.015). With regard to specific disorders, veterans with histories of COVID-19 infection were more likely to screen positive for generalised anxiety disorder (14.3% vs 9.1%, χ2=6.99, p=0.008), pandemic-related stress symptoms (7.3% vs 4.0 %, χ2=5.99, p=0.014), alcohol use disorder (14.8% vs 9.2%, χ2=8.03, p=0.005) and drug use disorder (13.9% vs 7.8%, χ2=10.27, p=0.001) relative to veterans who were not infected. The differences in major depressive disorder (11.8% vs 8.6%, χ2=2.93, p=0.087) and past-year suicide plan/attempt (4.9% vs 4.6%, χ2=1.04, p=0.59) were not significant.\\nRelative to veterans who were not infected with COVID-19, veterans who were infected were significantly more likely to screen positive at the peripandemic assessment for any internalising psychiatric disorder (20.5% vs 13.9%, χ2=7.85, p=0.005), alcohol or drug use disorder (23.2% vs 14.8%, χ2=11.51, p=0.001) and current suicidal ideation (12.0% vs 7.6%, χ2=5.92, p=0.015). With regard to specific disorders, veterans with histories of COVID-19 infection were more likely to screen positive for generalised anxiety disorder (14.3% vs 9.1%, χ2=6.99, p=0.008), pandemic-related stress symptoms (7.3% vs 4.0 %, χ2=5.99, p=0.014), alcohol use disorder (14.8% vs 9.2%, χ2=8.03, p=0.005) and drug use disorder (13.9% vs 7.8%, χ2=10.27, p=0.001) relative to veterans who were not infected. The differences in major depressive disorder (11.8% vs 8.6%, χ2=2.93, p=0.087) and past-year suicide plan/attempt (4.9% vs 4.6%, χ2=1.04, p=0.59) were not significant.\\nCorrelates of peripandemic psychiatric conditions In table 1, columns 2–4 show bivariate correlates of peripandemic positive screens for psychiatric conditions. Prepandemic psychiatric symptom severity, alcohol use problem severity, psychosocial difficulties and loneliness were positively correlated with all three outcomes, while perceived social support and protective psychosocial characteristics were negatively correlated with these outcomes. With regard to COVID-related variables, pandemic-related stress symptoms were positively correlated with all three outcomes.\\nCorrelates of positive screens for peripandemic psychiatric outcomes among US veterans with histories of COVID-19 infection\\n*P<0.05, **P<0.01, ***P<0.001.\\nr, Pearson correlation coefficient.\\nIn table 1, columns 2–4 show bivariate correlates of peripandemic positive screens for psychiatric conditions. Prepandemic psychiatric symptom severity, alcohol use problem severity, psychosocial difficulties and loneliness were positively correlated with all three outcomes, while perceived social support and protective psychosocial characteristics were negatively correlated with these outcomes. With regard to COVID-related variables, pandemic-related stress symptoms were positively correlated with all three outcomes.\\nCorrelates of positive screens for peripandemic psychiatric outcomes among US veterans with histories of COVID-19 infection\\n*P<0.05, **P<0.01, ***P<0.001.\\nr, Pearson correlation coefficient.\\nMultivariable logistic regression models Multivariable logistic regression analyses (table 1, columns 5–7) revealed that greater prepandemic psychiatric symptom severity, non-prescription drug use, adverse childhood experiences, perceived social support, and COVID-related worries, social restriction stress and financial stress were independent risk factors for peripandemic internalising psychiatric disorders, whereas greater trauma burden was protective. Relative importance analysis indicated that prepandemic psychiatric symptom severity (34.9%), trauma burden (18.6%), COVID-related worries (14.9%), financial stress (14.5%) and social restriction stress (11.6%) explained the majority of variance in this outcome, with drug use severity (5.5%) explaining the remainder.\\nPrepandemic severity of alcohol and non-prescription drug use were the only independent risk factors for peripandemic externalising psychiatric disorders, each explaining 48.0% and 52.0% of the variance, respectively.\\nPrepandemic alcohol use severity, past-year suicidal ideation, loneliness, impulsivity, perceived social support and having a household member infected with COVID-19 were independent risk factors for peripandemic suicidal ideation, whereas greater protective psychosocial characteristics (specifically greater community integration: OR=0.51, 95% CI 0.34 to 0.79) and higher household income were protective. Relative importance analysis showed that prepandemic suicidal ideation (29.3%), loneliness (25.2%), household income (12.0%), community integration (10.8%) and impulsivity (9.4%) explained the majority of variance in this outcome, with household member COVID-19 infection (5.0%), prepandemic social support (4.9%) and alcohol use severity (3.4%) explaining the remaining variance.\\nMultivariable logistic regression analyses (table 1, columns 5–7) revealed that greater prepandemic psychiatric symptom severity, non-prescription drug use, adverse childhood experiences, perceived social support, and COVID-related worries, social restriction stress and financial stress were independent risk factors for peripandemic internalising psychiatric disorders, whereas greater trauma burden was protective. Relative importance analysis indicated that prepandemic psychiatric symptom severity (34.9%), trauma burden (18.6%), COVID-related worries (14.9%), financial stress (14.5%) and social restriction stress (11.6%) explained the majority of variance in this outcome, with drug use severity (5.5%) explaining the remainder.\\nPrepandemic severity of alcohol and non-prescription drug use were the only independent risk factors for peripandemic externalising psychiatric disorders, each explaining 48.0% and 52.0% of the variance, respectively.\\nPrepandemic alcohol use severity, past-year suicidal ideation, loneliness, impulsivity, perceived social support and having a household member infected with COVID-19 were independent risk factors for peripandemic suicidal ideation, whereas greater protective psychosocial characteristics (specifically greater community integration: OR=0.51, 95% CI 0.34 to 0.79) and higher household income were protective. Relative importance analysis showed that prepandemic suicidal ideation (29.3%), loneliness (25.2%), household income (12.0%), community integration (10.8%) and impulsivity (9.4%) explained the majority of variance in this outcome, with household member COVID-19 infection (5.0%), prepandemic social support (4.9%) and alcohol use severity (3.4%) explaining the remaining variance.\",\n \"Relative to veterans who were not infected with COVID-19, veterans who were infected were significantly more likely to screen positive at the peripandemic assessment for any internalising psychiatric disorder (20.5% vs 13.9%, χ2=7.85, p=0.005), alcohol or drug use disorder (23.2% vs 14.8%, χ2=11.51, p=0.001) and current suicidal ideation (12.0% vs 7.6%, χ2=5.92, p=0.015). With regard to specific disorders, veterans with histories of COVID-19 infection were more likely to screen positive for generalised anxiety disorder (14.3% vs 9.1%, χ2=6.99, p=0.008), pandemic-related stress symptoms (7.3% vs 4.0 %, χ2=5.99, p=0.014), alcohol use disorder (14.8% vs 9.2%, χ2=8.03, p=0.005) and drug use disorder (13.9% vs 7.8%, χ2=10.27, p=0.001) relative to veterans who were not infected. The differences in major depressive disorder (11.8% vs 8.6%, χ2=2.93, p=0.087) and past-year suicide plan/attempt (4.9% vs 4.6%, χ2=1.04, p=0.59) were not significant.\",\n \"In table 1, columns 2–4 show bivariate correlates of peripandemic positive screens for psychiatric conditions. Prepandemic psychiatric symptom severity, alcohol use problem severity, psychosocial difficulties and loneliness were positively correlated with all three outcomes, while perceived social support and protective psychosocial characteristics were negatively correlated with these outcomes. With regard to COVID-related variables, pandemic-related stress symptoms were positively correlated with all three outcomes.\\nCorrelates of positive screens for peripandemic psychiatric outcomes among US veterans with histories of COVID-19 infection\\n*P<0.05, **P<0.01, ***P<0.001.\\nr, Pearson correlation coefficient.\",\n \"Multivariable logistic regression analyses (table 1, columns 5–7) revealed that greater prepandemic psychiatric symptom severity, non-prescription drug use, adverse childhood experiences, perceived social support, and COVID-related worries, social restriction stress and financial stress were independent risk factors for peripandemic internalising psychiatric disorders, whereas greater trauma burden was protective. Relative importance analysis indicated that prepandemic psychiatric symptom severity (34.9%), trauma burden (18.6%), COVID-related worries (14.9%), financial stress (14.5%) and social restriction stress (11.6%) explained the majority of variance in this outcome, with drug use severity (5.5%) explaining the remainder.\\nPrepandemic severity of alcohol and non-prescription drug use were the only independent risk factors for peripandemic externalising psychiatric disorders, each explaining 48.0% and 52.0% of the variance, respectively.\\nPrepandemic alcohol use severity, past-year suicidal ideation, loneliness, impulsivity, perceived social support and having a household member infected with COVID-19 were independent risk factors for peripandemic suicidal ideation, whereas greater protective psychosocial characteristics (specifically greater community integration: OR=0.51, 95% CI 0.34 to 0.79) and higher household income were protective. Relative importance analysis showed that prepandemic suicidal ideation (29.3%), loneliness (25.2%), household income (12.0%), community integration (10.8%) and impulsivity (9.4%) explained the majority of variance in this outcome, with household member COVID-19 infection (5.0%), prepandemic social support (4.9%) and alcohol use severity (3.4%) explaining the remaining variance.\",\n \"This study compared the prevalence of peripandemic psychiatric disorders and suicidal ideation between US veterans with and without histories of COVID-19 infection, and examined prepandemic and peripandemic risk and protective factors for these outcomes among COVID-19 survivors. Results revealed that veterans who were infected with COVID-19 had a significantly higher prevalence of positive screens for psychiatric conditions, as well as suicidal ideation relative to those who were not infected. Prepandemic greater psychiatric symptom severity and COVID-related stressors were the strongest predictors of peripandemic internalising psychiatric disorders in veterans who survived COVID-19, while previous trauma exposure was found to be protective. Prepandemic suicidal ideation, loneliness and impulsivity were the strongest independent risk factors of peripandemic suicidal ideation, while greater prepandemic community integration and higher annual household income were protective.\\nResults of this study are largely consistent with previous studies of non-representative patient samples who were hospitalised for COVID-19 infection, which observed a higher prevalence of psychiatric conditions, including depression, anxiety and PTSD.8 It is possible that being infected with COVID-19 may have aggravated psychological distress during the pandemic. Further, it is also plausible that those who had prepandemic psychiatric conditions and more psychosocial difficulties may have been at higher risk of infection given less resources and protective measures to avoid infection.9 Of note, the strongest predictors of peripandemic internalising disorders and externalising disorders in those who survived COVID-19 were prepandemic psychiatric symptom and substance use severity, respectively. This finding suggests that veterans with greater severity of prepandemic psychiatric distress and substance use may sensitise veterans to the deleterious mental health effects of COVID-19 infection.\\nA growing body of literature suggests that COVID-related stressors may contribute to risk of pandemic-related psychiatric distress.14 15 Results of the current study extend this work to suggest that COVID-related worries, financial distress and social restriction stress were independently associated with increased risk of screening positive for any internalising psychiatric disorders in veterans who survived COVID-19 infection.\\nPrevious research has identified previous suicidal behaviour as a strong predictor of future suicidal behaviour.16 The results of this study are consistent with these findings, as prepandemic suicidal ideation was the strongest predictor of peripandemic suicidal ideation. The negative impact of loneliness on mental health outcomes, including suicidal ideation, has been well established.17 In addition, due to the increased social restrictions imposed during the pandemic, increasing loneliness has been suggested as a possible contributor to worsening mental health.1 In this study, greater prepandemic loneliness was the second strongest predictor of peripandemic suicidal ideation. This finding suggests that loneliness may be a clinically relevant intervention target to help mitigate risk of suicidal behaviour in veterans during the pandemic. Moreover, impulsivity was a strong predictor of peripandemic suicidal ideation, which is consistent with previous research that reported strong positive association between impulsivity and suicidal behaviour.18 Of note, all of these factors were assessed prior to the pandemic, suggesting that they may represent increased vulnerability for suicidal ideation during the pandemic in US military veterans who survived COVID-19 infection.\\nGreater prepandemic community integration was protective against peripandemic suicidal ideation, even after controlling for other variables. This finding accords with previous research describing the protective effects of community integration in promoting psychological resilience and mitigation of risk for suicidal behaviour.19 20 Further, higher prepandemic household income was protective against risk of peripandemic suicidal ideation. This finding, which is consistent with the conservation of resources theory,21 suggests that fewer prepandemic financial resources may have led to increased peripandemic distress and suicidal ideation. This finding underscores the importance of assessing, monitoring and ameliorating financial distress brought on by the pandemic in veterans with histories of COVID-19 infection.\\nGreater trauma burden was identified as a protective factor to internalising psychiatric disorders during the pandemic. While previous trauma is a well known risk factor for psychiatric distress,22 previous exposure to traumatic events may also have ‘stress inoculating’ effects and help protect against risk of adverse mental health outcomes.23 Specifically, exposure to prior traumas may help promote engagement in adaptive coping strategies and may give rise to positive psychological changes (eg, greater sense of personal strength)24 25 that may in turn help foster resilience to new stressors, such as COVID-19 infection.\\nFurther, it is noteworthy that perceived social support was found to be associated with increased risk of both internalising psychiatric disorders and suicidal ideation during the pandemic. One interpretation of this finding is that greater prepandemic severity of psychiatric distress may have increased support-seeking behaviours during the pandemic. Further research is needed to elucidate prospective associations between aspects of social support and risk for pandemic-related psychiatric conditions, particularly given the imposition of social restrictions during the pandemic.\\nThis study has several limitations. First, history of COVID-19 infection was based on self-report and may be subject to response bias, as well as access to and use of testing for COVID-19 infection. Thus, it is possible that the prevalence of self-reported COVID-19 infection may not capture the accurate prevalence of infections in US veterans. Second, due to the survey design of this study, we used screening instruments to assess psychiatric conditions. Further research using diagnostic instruments such as structured clinical interviews are needed to replicate the results reported herein. Third, while nationally representative, our sample was composed entirely of US military veterans who are predominantly older, male and white, which makes it difficult to generalise results to non-veteran and more diverse veteran populations. Fourth, although this was a prospective cohort study, given the cross-sectional nature of the surveys and limited follow-up time frame, it is difficult to draw conclusions regarding temporal/causal associations between independent variables, and peripandemic psychiatric disorders and suicidal ideation.\\nDespite these limitations, this study provides the first known nationally representative data on the prevalence and prepandemic and pandemic-related risk and protective factors of peripandemic psychiatric outcomes in US military veterans who survived COVID-19 infection. Given the enormous toll of COVID-19 infections in the USA,4 this information may help inform targeted population-based prevention, monitoring and intervention strategies. For example, US veterans who are COVID-19 survivors may be at elevated risk of peripandemic psychiatric conditions, and those with less socioeconomic resources and greater symptom severity at baseline may require higher clinical attention. Further, veterans who endorse greater COVID-related stressors may be at increased risk of peripandemic psychiatric disorders and suicidal ideation. Further research is needed to replicate and extend these results to other populations who are also at higher risk of psychiatric conditions; identify mechanisms leading to increased risk of psychiatric conditions during the pandemic; and assess the efficacy of interventions targeting such evidence-based risk and protective factors in mitigating risk of psychiatric conditions in veterans and other populations.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,null,null,"results",null,null,null,"discussion"],"string":"[\n \"intro\",\n \"methods\",\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n \"discussion\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["epidemiology","mental health","depression & mood disorders","substance misuse","suicide & self-harm","adult psychiatry"],"string":"[\n \"epidemiology\",\n \"mental health\",\n \"depression & mood disorders\",\n \"substance misuse\",\n \"suicide & self-harm\",\n \"adult psychiatry\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Introduction:\nThe COVID-19 pandemic has been linked to increased social isolation, economic recession and psychological distress.1 Numerous studies have reported a substantial increase in the prevalence of depression and anxiety in the general public during the pandemic.2 3 Previous psychiatric diagnoses, COVID-related stressors, such as worries about being infected with COVID-19, financial stressors and actual COVID-19 infection have been proposed as possible risk factors of both the development and worsening of mental illness during the pandemic.1\nVeterans may be a highly vulnerable group to the negative mental health effects of the pandemic due to their older age, and higher prepandemic prevalence of physical and psychiatric risk factors and conditions relative to the general population.4 5i Further, previous studies on veterans have found that stressful events such as combat deployments were associated with increased rates of depression and substance use disorders in veterans.6\nAs of April 2021, in the USA, over 31 million people have been infected with COVID-19 and over half a million people have died of COVID-19-related complications.7 Previous studies on survivors of COVID-19 have reported high prevalence of mental disorders, including depression, anxiety and post-traumatic stress disorder (PTSD8–10). Thus, there has been increasing attention in closely monitoring for psychiatric symptoms and functioning in those who have been infected with COVID-19 in order to mitigate distress and risk of suicide. However, due to the high burden of COVID-19 infections in the USA and the limited capacity of mental health treatment services, it is challenging to monitor everyone who has been infected. These challenges have been further complicated by infection-control measures implemented by almost all mental health service providers during the pandemic.11\nOne potentially effective way of informing the allocation of limited mental health treatment resources may be to identify subgroups that are at high risk of developing or experiencing a worsening of psychiatric conditions after COVID-19 infection. Identifying potential risk and protective factors of psychiatric conditions among COVID-19 survivors in populations with elevated risk, such as US military veterans, may help inform more targeted population-based prevention, screening and intervention strategies.\nToward this end, we analysed data from the National Health and Resilience in Veterans Study (NHRVS12) to examine (1) the prevalence of psychiatric conditions in US veterans who have and have not been infected with COVID-19, and (2) prepandemic and peripandemic risk and protective factors of peripandemic psychiatric conditions in veterans who survived COVID-19 infection.\n\nMethods:\nParticipants Data were analysed from the 2019 to 2020 NHRVS, which surveyed a nationally representative, prospective sample of US military veterans. A total of 4069 veterans completed the prepandemic survey (median completion date: 21 November 2019) prior to the first documented COVID-19 cases in the USA (19 January 2020), and 3078 (75.6%) completed a 1-year peripandemic follow-up assessment (median completion date 14 November 2020). Both surveys were online, 45–60 min, self-report surveys. All participants received 15 000 points (equivalent to $15) for participating in the prepandemic survey, and 20 000 points (equivalent to $20) for participating in the peripandemic survey. Details of the study have been described previously.12 Briefly, the NHRVS sample was drawn from KnowledgePanel, a survey research panel of more than 50 000 US households maintained by Ipsos, a survey research firm. To promote generalisability of the results to the US veteran population, poststratification weights based on the demographic distribution of veterans in the contemporaneous US Census Current Population Survey Veterans Supplement were applied in inferential analyses.\nData were analysed from the 2019 to 2020 NHRVS, which surveyed a nationally representative, prospective sample of US military veterans. A total of 4069 veterans completed the prepandemic survey (median completion date: 21 November 2019) prior to the first documented COVID-19 cases in the USA (19 January 2020), and 3078 (75.6%) completed a 1-year peripandemic follow-up assessment (median completion date 14 November 2020). Both surveys were online, 45–60 min, self-report surveys. All participants received 15 000 points (equivalent to $15) for participating in the prepandemic survey, and 20 000 points (equivalent to $20) for participating in the peripandemic survey. Details of the study have been described previously.12 Briefly, the NHRVS sample was drawn from KnowledgePanel, a survey research panel of more than 50 000 US households maintained by Ipsos, a survey research firm. To promote generalisability of the results to the US veteran population, poststratification weights based on the demographic distribution of veterans in the contemporaneous US Census Current Population Survey Veterans Supplement were applied in inferential analyses.\nAssessments Online supplemental table 1 presents a detailed description of measures used to assess potential risk and protective factors of peripandemic psychiatric conditions in veterans who survived COVID-19 infection.\n\n\n\nOnline supplemental table 1 presents a detailed description of measures used to assess potential risk and protective factors of peripandemic psychiatric conditions in veterans who survived COVID-19 infection.\n\n\n\nData analysis χ2 analyses were conducted to compare the prevalence of psychiatric disorders and suicidal ideation between veterans who were and were not infected with COVID-19. Subsequent analyses focused on veterans who self-reported a history of COVID-19 infection at the peripandemic assessment. Analyses in this subcohort proceeded in two steps. First, we conducted Pearson correlations between participant characteristics and peripandemic internalising psychiatric disorders (ie, major depressive disorder, generalised anxiety disorder and/or pandemic-related stress symptoms); externalising disorders (ie, alcohol and/or drug use disorder); and suicidal ideation; internalising and externalising disorders were grouped together to increase statistical power. Second, we conducted multivariable binary logistic regression analyses to identify factors that independently predicted the aforementioned three categories of peripandemic psychiatric conditions. Variables that differed at the p<0.05 level in bivariate analyses for each set of comparisons were entered into each regression analysis. Third, we conducted relative importance analyses to determine the relative contribution of each significant variable in predicting positive screens for the three outcomes. These analyses partitioned the explained variance in the study outcome variables that is explained by each independent variable while simultaneously accounting for intercorrelations among these independent variables.13\nχ2 analyses were conducted to compare the prevalence of psychiatric disorders and suicidal ideation between veterans who were and were not infected with COVID-19. Subsequent analyses focused on veterans who self-reported a history of COVID-19 infection at the peripandemic assessment. Analyses in this subcohort proceeded in two steps. First, we conducted Pearson correlations between participant characteristics and peripandemic internalising psychiatric disorders (ie, major depressive disorder, generalised anxiety disorder and/or pandemic-related stress symptoms); externalising disorders (ie, alcohol and/or drug use disorder); and suicidal ideation; internalising and externalising disorders were grouped together to increase statistical power. Second, we conducted multivariable binary logistic regression analyses to identify factors that independently predicted the aforementioned three categories of peripandemic psychiatric conditions. Variables that differed at the p<0.05 level in bivariate analyses for each set of comparisons were entered into each regression analysis. Third, we conducted relative importance analyses to determine the relative contribution of each significant variable in predicting positive screens for the three outcomes. These analyses partitioned the explained variance in the study outcome variables that is explained by each independent variable while simultaneously accounting for intercorrelations among these independent variables.13\n\nParticipants:\nData were analysed from the 2019 to 2020 NHRVS, which surveyed a nationally representative, prospective sample of US military veterans. A total of 4069 veterans completed the prepandemic survey (median completion date: 21 November 2019) prior to the first documented COVID-19 cases in the USA (19 January 2020), and 3078 (75.6%) completed a 1-year peripandemic follow-up assessment (median completion date 14 November 2020). Both surveys were online, 45–60 min, self-report surveys. All participants received 15 000 points (equivalent to $15) for participating in the prepandemic survey, and 20 000 points (equivalent to $20) for participating in the peripandemic survey. Details of the study have been described previously.12 Briefly, the NHRVS sample was drawn from KnowledgePanel, a survey research panel of more than 50 000 US households maintained by Ipsos, a survey research firm. To promote generalisability of the results to the US veteran population, poststratification weights based on the demographic distribution of veterans in the contemporaneous US Census Current Population Survey Veterans Supplement were applied in inferential analyses.\n\nAssessments:\nOnline supplemental table 1 presents a detailed description of measures used to assess potential risk and protective factors of peripandemic psychiatric conditions in veterans who survived COVID-19 infection.\n\n\n\n\nData analysis:\nχ2 analyses were conducted to compare the prevalence of psychiatric disorders and suicidal ideation between veterans who were and were not infected with COVID-19. Subsequent analyses focused on veterans who self-reported a history of COVID-19 infection at the peripandemic assessment. Analyses in this subcohort proceeded in two steps. First, we conducted Pearson correlations between participant characteristics and peripandemic internalising psychiatric disorders (ie, major depressive disorder, generalised anxiety disorder and/or pandemic-related stress symptoms); externalising disorders (ie, alcohol and/or drug use disorder); and suicidal ideation; internalising and externalising disorders were grouped together to increase statistical power. Second, we conducted multivariable binary logistic regression analyses to identify factors that independently predicted the aforementioned three categories of peripandemic psychiatric conditions. Variables that differed at the p<0.05 level in bivariate analyses for each set of comparisons were entered into each regression analysis. Third, we conducted relative importance analyses to determine the relative contribution of each significant variable in predicting positive screens for the three outcomes. These analyses partitioned the explained variance in the study outcome variables that is explained by each independent variable while simultaneously accounting for intercorrelations among these independent variables.13\n\nResults:\nThe final sample included 3078 veterans who completed both prepandemic and 1-year peripandemic assessments. The average age of the sample was 62.2 years old (SD=15.7, range 22–99); the majority was male (90.2%) and Caucasian (78.1%); and 35.0% were combat veterans. A total of 233 veterans (8.6%) reported that they had been infected with COVID-19. The majority (70.5%) reported mild-to-moderate symptoms; 6.3% reported requiring a visit to the emergency room; 4.5% reported severe symptoms with serious concerns about not surviving; 1.9% reported being admitted to an intensive care unit; 1.2% reported requiring an overnight stay at a hospital, and 0.8% reported requiring intubation/mechanical ventilation; the remaining 14.8% of the sample did not provide details about the severity of their illness.\nPrevalence of peripandemic psychiatric conditions of veterans who were infected by COVID-19 Relative to veterans who were not infected with COVID-19, veterans who were infected were significantly more likely to screen positive at the peripandemic assessment for any internalising psychiatric disorder (20.5% vs 13.9%, χ2=7.85, p=0.005), alcohol or drug use disorder (23.2% vs 14.8%, χ2=11.51, p=0.001) and current suicidal ideation (12.0% vs 7.6%, χ2=5.92, p=0.015). With regard to specific disorders, veterans with histories of COVID-19 infection were more likely to screen positive for generalised anxiety disorder (14.3% vs 9.1%, χ2=6.99, p=0.008), pandemic-related stress symptoms (7.3% vs 4.0 %, χ2=5.99, p=0.014), alcohol use disorder (14.8% vs 9.2%, χ2=8.03, p=0.005) and drug use disorder (13.9% vs 7.8%, χ2=10.27, p=0.001) relative to veterans who were not infected. The differences in major depressive disorder (11.8% vs 8.6%, χ2=2.93, p=0.087) and past-year suicide plan/attempt (4.9% vs 4.6%, χ2=1.04, p=0.59) were not significant.\nRelative to veterans who were not infected with COVID-19, veterans who were infected were significantly more likely to screen positive at the peripandemic assessment for any internalising psychiatric disorder (20.5% vs 13.9%, χ2=7.85, p=0.005), alcohol or drug use disorder (23.2% vs 14.8%, χ2=11.51, p=0.001) and current suicidal ideation (12.0% vs 7.6%, χ2=5.92, p=0.015). With regard to specific disorders, veterans with histories of COVID-19 infection were more likely to screen positive for generalised anxiety disorder (14.3% vs 9.1%, χ2=6.99, p=0.008), pandemic-related stress symptoms (7.3% vs 4.0 %, χ2=5.99, p=0.014), alcohol use disorder (14.8% vs 9.2%, χ2=8.03, p=0.005) and drug use disorder (13.9% vs 7.8%, χ2=10.27, p=0.001) relative to veterans who were not infected. The differences in major depressive disorder (11.8% vs 8.6%, χ2=2.93, p=0.087) and past-year suicide plan/attempt (4.9% vs 4.6%, χ2=1.04, p=0.59) were not significant.\nCorrelates of peripandemic psychiatric conditions In table 1, columns 2–4 show bivariate correlates of peripandemic positive screens for psychiatric conditions. Prepandemic psychiatric symptom severity, alcohol use problem severity, psychosocial difficulties and loneliness were positively correlated with all three outcomes, while perceived social support and protective psychosocial characteristics were negatively correlated with these outcomes. With regard to COVID-related variables, pandemic-related stress symptoms were positively correlated with all three outcomes.\nCorrelates of positive screens for peripandemic psychiatric outcomes among US veterans with histories of COVID-19 infection\n*P<0.05, **P<0.01, ***P<0.001.\nr, Pearson correlation coefficient.\nIn table 1, columns 2–4 show bivariate correlates of peripandemic positive screens for psychiatric conditions. Prepandemic psychiatric symptom severity, alcohol use problem severity, psychosocial difficulties and loneliness were positively correlated with all three outcomes, while perceived social support and protective psychosocial characteristics were negatively correlated with these outcomes. With regard to COVID-related variables, pandemic-related stress symptoms were positively correlated with all three outcomes.\nCorrelates of positive screens for peripandemic psychiatric outcomes among US veterans with histories of COVID-19 infection\n*P<0.05, **P<0.01, ***P<0.001.\nr, Pearson correlation coefficient.\nMultivariable logistic regression models Multivariable logistic regression analyses (table 1, columns 5–7) revealed that greater prepandemic psychiatric symptom severity, non-prescription drug use, adverse childhood experiences, perceived social support, and COVID-related worries, social restriction stress and financial stress were independent risk factors for peripandemic internalising psychiatric disorders, whereas greater trauma burden was protective. Relative importance analysis indicated that prepandemic psychiatric symptom severity (34.9%), trauma burden (18.6%), COVID-related worries (14.9%), financial stress (14.5%) and social restriction stress (11.6%) explained the majority of variance in this outcome, with drug use severity (5.5%) explaining the remainder.\nPrepandemic severity of alcohol and non-prescription drug use were the only independent risk factors for peripandemic externalising psychiatric disorders, each explaining 48.0% and 52.0% of the variance, respectively.\nPrepandemic alcohol use severity, past-year suicidal ideation, loneliness, impulsivity, perceived social support and having a household member infected with COVID-19 were independent risk factors for peripandemic suicidal ideation, whereas greater protective psychosocial characteristics (specifically greater community integration: OR=0.51, 95% CI 0.34 to 0.79) and higher household income were protective. Relative importance analysis showed that prepandemic suicidal ideation (29.3%), loneliness (25.2%), household income (12.0%), community integration (10.8%) and impulsivity (9.4%) explained the majority of variance in this outcome, with household member COVID-19 infection (5.0%), prepandemic social support (4.9%) and alcohol use severity (3.4%) explaining the remaining variance.\nMultivariable logistic regression analyses (table 1, columns 5–7) revealed that greater prepandemic psychiatric symptom severity, non-prescription drug use, adverse childhood experiences, perceived social support, and COVID-related worries, social restriction stress and financial stress were independent risk factors for peripandemic internalising psychiatric disorders, whereas greater trauma burden was protective. Relative importance analysis indicated that prepandemic psychiatric symptom severity (34.9%), trauma burden (18.6%), COVID-related worries (14.9%), financial stress (14.5%) and social restriction stress (11.6%) explained the majority of variance in this outcome, with drug use severity (5.5%) explaining the remainder.\nPrepandemic severity of alcohol and non-prescription drug use were the only independent risk factors for peripandemic externalising psychiatric disorders, each explaining 48.0% and 52.0% of the variance, respectively.\nPrepandemic alcohol use severity, past-year suicidal ideation, loneliness, impulsivity, perceived social support and having a household member infected with COVID-19 were independent risk factors for peripandemic suicidal ideation, whereas greater protective psychosocial characteristics (specifically greater community integration: OR=0.51, 95% CI 0.34 to 0.79) and higher household income were protective. Relative importance analysis showed that prepandemic suicidal ideation (29.3%), loneliness (25.2%), household income (12.0%), community integration (10.8%) and impulsivity (9.4%) explained the majority of variance in this outcome, with household member COVID-19 infection (5.0%), prepandemic social support (4.9%) and alcohol use severity (3.4%) explaining the remaining variance.\n\nPrevalence of peripandemic psychiatric conditions of veterans who were infected by COVID-19:\nRelative to veterans who were not infected with COVID-19, veterans who were infected were significantly more likely to screen positive at the peripandemic assessment for any internalising psychiatric disorder (20.5% vs 13.9%, χ2=7.85, p=0.005), alcohol or drug use disorder (23.2% vs 14.8%, χ2=11.51, p=0.001) and current suicidal ideation (12.0% vs 7.6%, χ2=5.92, p=0.015). With regard to specific disorders, veterans with histories of COVID-19 infection were more likely to screen positive for generalised anxiety disorder (14.3% vs 9.1%, χ2=6.99, p=0.008), pandemic-related stress symptoms (7.3% vs 4.0 %, χ2=5.99, p=0.014), alcohol use disorder (14.8% vs 9.2%, χ2=8.03, p=0.005) and drug use disorder (13.9% vs 7.8%, χ2=10.27, p=0.001) relative to veterans who were not infected. The differences in major depressive disorder (11.8% vs 8.6%, χ2=2.93, p=0.087) and past-year suicide plan/attempt (4.9% vs 4.6%, χ2=1.04, p=0.59) were not significant.\n\nCorrelates of peripandemic psychiatric conditions:\nIn table 1, columns 2–4 show bivariate correlates of peripandemic positive screens for psychiatric conditions. Prepandemic psychiatric symptom severity, alcohol use problem severity, psychosocial difficulties and loneliness were positively correlated with all three outcomes, while perceived social support and protective psychosocial characteristics were negatively correlated with these outcomes. With regard to COVID-related variables, pandemic-related stress symptoms were positively correlated with all three outcomes.\nCorrelates of positive screens for peripandemic psychiatric outcomes among US veterans with histories of COVID-19 infection\n*P<0.05, **P<0.01, ***P<0.001.\nr, Pearson correlation coefficient.\n\nMultivariable logistic regression models:\nMultivariable logistic regression analyses (table 1, columns 5–7) revealed that greater prepandemic psychiatric symptom severity, non-prescription drug use, adverse childhood experiences, perceived social support, and COVID-related worries, social restriction stress and financial stress were independent risk factors for peripandemic internalising psychiatric disorders, whereas greater trauma burden was protective. Relative importance analysis indicated that prepandemic psychiatric symptom severity (34.9%), trauma burden (18.6%), COVID-related worries (14.9%), financial stress (14.5%) and social restriction stress (11.6%) explained the majority of variance in this outcome, with drug use severity (5.5%) explaining the remainder.\nPrepandemic severity of alcohol and non-prescription drug use were the only independent risk factors for peripandemic externalising psychiatric disorders, each explaining 48.0% and 52.0% of the variance, respectively.\nPrepandemic alcohol use severity, past-year suicidal ideation, loneliness, impulsivity, perceived social support and having a household member infected with COVID-19 were independent risk factors for peripandemic suicidal ideation, whereas greater protective psychosocial characteristics (specifically greater community integration: OR=0.51, 95% CI 0.34 to 0.79) and higher household income were protective. Relative importance analysis showed that prepandemic suicidal ideation (29.3%), loneliness (25.2%), household income (12.0%), community integration (10.8%) and impulsivity (9.4%) explained the majority of variance in this outcome, with household member COVID-19 infection (5.0%), prepandemic social support (4.9%) and alcohol use severity (3.4%) explaining the remaining variance.\n\nDiscussion:\nThis study compared the prevalence of peripandemic psychiatric disorders and suicidal ideation between US veterans with and without histories of COVID-19 infection, and examined prepandemic and peripandemic risk and protective factors for these outcomes among COVID-19 survivors. Results revealed that veterans who were infected with COVID-19 had a significantly higher prevalence of positive screens for psychiatric conditions, as well as suicidal ideation relative to those who were not infected. Prepandemic greater psychiatric symptom severity and COVID-related stressors were the strongest predictors of peripandemic internalising psychiatric disorders in veterans who survived COVID-19, while previous trauma exposure was found to be protective. Prepandemic suicidal ideation, loneliness and impulsivity were the strongest independent risk factors of peripandemic suicidal ideation, while greater prepandemic community integration and higher annual household income were protective.\nResults of this study are largely consistent with previous studies of non-representative patient samples who were hospitalised for COVID-19 infection, which observed a higher prevalence of psychiatric conditions, including depression, anxiety and PTSD.8 It is possible that being infected with COVID-19 may have aggravated psychological distress during the pandemic. Further, it is also plausible that those who had prepandemic psychiatric conditions and more psychosocial difficulties may have been at higher risk of infection given less resources and protective measures to avoid infection.9 Of note, the strongest predictors of peripandemic internalising disorders and externalising disorders in those who survived COVID-19 were prepandemic psychiatric symptom and substance use severity, respectively. This finding suggests that veterans with greater severity of prepandemic psychiatric distress and substance use may sensitise veterans to the deleterious mental health effects of COVID-19 infection.\nA growing body of literature suggests that COVID-related stressors may contribute to risk of pandemic-related psychiatric distress.14 15 Results of the current study extend this work to suggest that COVID-related worries, financial distress and social restriction stress were independently associated with increased risk of screening positive for any internalising psychiatric disorders in veterans who survived COVID-19 infection.\nPrevious research has identified previous suicidal behaviour as a strong predictor of future suicidal behaviour.16 The results of this study are consistent with these findings, as prepandemic suicidal ideation was the strongest predictor of peripandemic suicidal ideation. The negative impact of loneliness on mental health outcomes, including suicidal ideation, has been well established.17 In addition, due to the increased social restrictions imposed during the pandemic, increasing loneliness has been suggested as a possible contributor to worsening mental health.1 In this study, greater prepandemic loneliness was the second strongest predictor of peripandemic suicidal ideation. This finding suggests that loneliness may be a clinically relevant intervention target to help mitigate risk of suicidal behaviour in veterans during the pandemic. Moreover, impulsivity was a strong predictor of peripandemic suicidal ideation, which is consistent with previous research that reported strong positive association between impulsivity and suicidal behaviour.18 Of note, all of these factors were assessed prior to the pandemic, suggesting that they may represent increased vulnerability for suicidal ideation during the pandemic in US military veterans who survived COVID-19 infection.\nGreater prepandemic community integration was protective against peripandemic suicidal ideation, even after controlling for other variables. This finding accords with previous research describing the protective effects of community integration in promoting psychological resilience and mitigation of risk for suicidal behaviour.19 20 Further, higher prepandemic household income was protective against risk of peripandemic suicidal ideation. This finding, which is consistent with the conservation of resources theory,21 suggests that fewer prepandemic financial resources may have led to increased peripandemic distress and suicidal ideation. This finding underscores the importance of assessing, monitoring and ameliorating financial distress brought on by the pandemic in veterans with histories of COVID-19 infection.\nGreater trauma burden was identified as a protective factor to internalising psychiatric disorders during the pandemic. While previous trauma is a well known risk factor for psychiatric distress,22 previous exposure to traumatic events may also have ‘stress inoculating’ effects and help protect against risk of adverse mental health outcomes.23 Specifically, exposure to prior traumas may help promote engagement in adaptive coping strategies and may give rise to positive psychological changes (eg, greater sense of personal strength)24 25 that may in turn help foster resilience to new stressors, such as COVID-19 infection.\nFurther, it is noteworthy that perceived social support was found to be associated with increased risk of both internalising psychiatric disorders and suicidal ideation during the pandemic. One interpretation of this finding is that greater prepandemic severity of psychiatric distress may have increased support-seeking behaviours during the pandemic. Further research is needed to elucidate prospective associations between aspects of social support and risk for pandemic-related psychiatric conditions, particularly given the imposition of social restrictions during the pandemic.\nThis study has several limitations. First, history of COVID-19 infection was based on self-report and may be subject to response bias, as well as access to and use of testing for COVID-19 infection. Thus, it is possible that the prevalence of self-reported COVID-19 infection may not capture the accurate prevalence of infections in US veterans. Second, due to the survey design of this study, we used screening instruments to assess psychiatric conditions. Further research using diagnostic instruments such as structured clinical interviews are needed to replicate the results reported herein. Third, while nationally representative, our sample was composed entirely of US military veterans who are predominantly older, male and white, which makes it difficult to generalise results to non-veteran and more diverse veteran populations. Fourth, although this was a prospective cohort study, given the cross-sectional nature of the surveys and limited follow-up time frame, it is difficult to draw conclusions regarding temporal/causal associations between independent variables, and peripandemic psychiatric disorders and suicidal ideation.\nDespite these limitations, this study provides the first known nationally representative data on the prevalence and prepandemic and pandemic-related risk and protective factors of peripandemic psychiatric outcomes in US military veterans who survived COVID-19 infection. Given the enormous toll of COVID-19 infections in the USA,4 this information may help inform targeted population-based prevention, monitoring and intervention strategies. For example, US veterans who are COVID-19 survivors may be at elevated risk of peripandemic psychiatric conditions, and those with less socioeconomic resources and greater symptom severity at baseline may require higher clinical attention. Further, veterans who endorse greater COVID-related stressors may be at increased risk of peripandemic psychiatric disorders and suicidal ideation. Further research is needed to replicate and extend these results to other populations who are also at higher risk of psychiatric conditions; identify mechanisms leading to increased risk of psychiatric conditions during the pandemic; and assess the efficacy of interventions targeting such evidence-based risk and protective factors in mitigating risk of psychiatric conditions in veterans and other populations.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThere have been reports of increased prevalence in psychiatric conditions in non-veteran survivors of COVID-19. To date, however, no known study has examined the prevalence, risk and protective factors of psychiatric conditions among US military veterans who survived COVID-19.\n\nMethods:\nData were analysed from the 2019 to 2020 National Health and Resilience in Veterans Study, which surveyed a nationally representative, prospective cohort of 3078 US veterans. Prepandemic and 1-year peripandemic risk and protective factors associated with positive screens for peripandemic internalising (major depressive, generalised anxiety and/or posttraumatic stress disorders) and externalising psychiatric disorders (alcohol and/or drug use disorders) and suicidal ideation were examined using bivariate and multivariate logistic regression analyses.\n\nResults:\nA total of 233 veterans (8.6%) reported having been infected with COVID-19. Relative to veterans who were not infected, veterans who were infected were more likely to screen positive for internalising disorders (20.5% vs 13.9%, p=0.005), externalising disorders (23.2% vs 14.8%, p=0.001) and current suicidal ideation (12.0% vs 7.6%, p=0.015) at peripandemic. Multivariable analyses revealed that greater prepandemic psychiatric symptom severity and COVID-related stressors were the strongest independent predictors of peripandemic internalising disorders, while prepandemic trauma burden was protective. Prepandemic suicidal ideation, greater loneliness and lower household income were the strongest independent predictors of peripandemic suicidal ideation, whereas prepandemic community integration was protective.\n\nConclusions:\nPsychiatric symptoms and suicidal ideation are prevalent in veterans who have survived COVID-19. Veterans with greater prepandemic psychiatric and substance use problems, COVID-related stressors and fewer psychosocial resources may be at increased risk of these outcomes."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":5128,"string":"5,128"},"whole_article_abstract_length":{"kind":"number","value":315,"string":"315"},"other_sections_lengths":{"kind":"list like","value":[210,30,211,202,112,303],"string":"[\n 210,\n 30,\n 211,\n 202,\n 112,\n 303\n]","isConversational":false},"num_sections":{"kind":"number","value":10,"string":"10"},"most_frequent_words":{"kind":"list like","value":["covid","psychiatric","19","veterans","covid 19","peripandemic","prepandemic","suicidal","risk","ideation"],"string":"[\n \"covid\",\n \"psychiatric\",\n \"19\",\n \"veterans\",\n \"covid 19\",\n \"peripandemic\",\n \"prepandemic\",\n \"suicidal\",\n \"risk\",\n 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Caffeine."},"pmid":{"kind":"string","value":"32033103"},"background_abstract":{"kind":"string","value":"The main objective of the current investigation was to evaluate the effects of caffeine on power output and bar velocity during an explosive bench press throw in athletes habituated to caffeine."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Twelve resistance trained individuals habituated to caffeine ingestion participated in a randomized double-blind experimental design. Each participant performed three identical experimental sessions 60 min after the intake of a placebo, 3, and 6 mg/kg/b.m. of caffeine. In each experimental session, the participants performed 5 sets of 2 repetitions of the bench press throw (with a load equivalent to 30% repetition maximum (RM), measured in a familiarization trial) on a Smith machine, while bar velocity and power output were registered with a rotatory encoder."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"In comparison to the placebo, the intake of caffeine increased mean bar velocity during 5 sets of the bench press throw (1.37 ± 0.05 vs. 1.41 ± 0.05 and 1.41 ± 0.06 m/s for placebo, 3, and 6 mg/kg/b.m., respectively; p < 0.01), as well as mean power output (545 ± 117 vs. 562 ± 118 and 560 ± 107 W; p < 0.01). However, caffeine was not effective at increasing peak velocity (p = 0.09) nor peak power output (p = 0.07) during the explosive exercise."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The acute doses of caffeine before resistance exercise may increase mean power output and mean bar velocity during the bench press throw training session in a group of habitual caffeine users. Thus, caffeine prior to ballistic exercises enhances performance during a power-specific resistance training session."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Athletes","Athletic Performance","Caffeine","Cross-Over Studies","Double-Blind Method","Healthy Volunteers","Humans","Male","Performance-Enhancing Substances","Resistance Training","Weight Lifting"],"string":"[\n \"Adult\",\n \"Athletes\",\n \"Athletic Performance\",\n \"Caffeine\",\n \"Cross-Over Studies\",\n \"Double-Blind Method\",\n \"Healthy Volunteers\",\n \"Humans\",\n \"Male\",\n \"Performance-Enhancing Substances\",\n \"Resistance Training\",\n \"Weight Lifting\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"7071256"},"background_title":{"kind":"string","value":"1. Introduction"},"background_text":{"kind":"string","value":"Caffeine (CAF) is one of the most common substances used in sport which enhances physical performance [1]. Although CAF may affect various body tissues [2,3], there is a growing body of evidence in animal [4] and human models [5] sustaining the ability of CAF to act as an adenosine antagonist, as the main mechanism behind CAF ergogenic effect. Current research recommends doses of CAF ranging from 3 to 6 mg/kg body mass to elicit ergogenic benefits, while the time of ingestion (from 30 to 90 min before exercise) and the form of ingestion (pills, liquid, chewing gum) are less significant as CAF is rapidly absorbed after ingestion [6]. However, the optimal protocol of CAF supplementation may differ based on the type and duration of exercise, previous habituation to CAF and the type of muscle contraction [6,7,8,9,10].\nAcute CAF intake causes a slightly different response to upper and lower body exercise [10], despite the lack of a mechanisms explaining this phenomenon. A recent meta-analysis about the effects of CAF on muscle strength and power output found that CAF significantly improved upper but not lower body strength [7]. Although the outcomes of the first investigations were contradictory [11,12,13], more recent studies have found a clear effect of CAF on several forms of upper body muscle performance [14,15,16], when the used dose was at least 3 mg/kg/b.m. [17]. Interestingly, the effect of CAF on upper body muscle performance may be partially dampened in athletes habituated to CAF because the regular use of CAF-containing products [18] may develop tolerance to this substance. It must be noticed that this decreased ergogenic effect of CAF is not removed even with the ingestion of doses >9 mg/kg/b.m. [19,20].\nMost studies on the acute effects of CAF on upper body muscle performance used the bench press exercise. The bench press exercise is widely used as a means of developing strength and power of the upper limbs [21,22]. However, other authors, recommend the use of ballistic exercises for the development of upper body power, such as the bench press throw (BPT) [23,24]. Specifically, to increase power output, the loads ranging from 0% to 50% of one-repetition maximum (1 RM) moved at maximal speed are recommended as the most potent loading stimuli for improving power output [23]. However, this type of routine can only be performed on a Smith machine while using the BPT exercise (i.e., maximal bar velocity is obtained at the moment of throw). The traditional free-weight bench press exercise does not allow for the attainment of maximal velocity of execution (i.e., velocity equals 0 m/s at maximal arm extension). Thus, ballistic exercises could be an optimal choice for power training as they allow for greater velocity, and muscle activation in comparison to similar traditional resistance training routines [21]. Perhaps, the main asset of the BPT is the maximum acceleration of a given load, which ultimately produces high movement velocity in a short time [25]. In this respect, the loads applied in ballistic exercises during training will depend on the specific requirements of particular sport disciplines and will determine success in numerous power-based competitions [23]. Furthermore, the BPT performance has been associated with overall performance in different sport-specific tasks [26,27,28,29]. Therefore, it seems reasonable to use the BPT as a means of testing upper-body ballistic performance. Although the BPT is indicated as the most effective exercise for developing power of the upper limbs [30], previous studies have not determined the acute effects of CAF on power output and bar velocity during this type of exercise.\nBurke [31] has suggested that, in the current literature, there is a lack of data about the practical use of supplements in competitive sports because experimental protocols are often different from sports practice. In case of CAF, most studies considering the acute effects of this supplement were assessed on the basis of only a single set of an exercise [7,8,32], while real resistance training sessions in trained individuals rarely contain only one set of a particular exercise [31]. On the other hand, the investigations analyzing the acute effects of CAF on performance during successive sets of resistance exercises are scarce. Bowtell et al. [3] showed that pre-exercise CAF (6 mg/kg/b.m.) intake improved total exercise time during 5 sets of one-legged knee extensions performed to failure in comparison to a placebo (PLAC). It is worth noting that this ergogenic effect was achieved despite significantly lower muscle phosphocreatine concentration (PCr) and pH in the latter sets of an exercise in the CAF trial. Further, CAF ingestion (6 mg/kg/b.m.) attenuated the increase in interstitial potassium during one-legged knee extensions at 20 W (10 min) and 50 W (3 × 3 min) measured using microdialysis [33], which resulted in a 16% improvement in high-intensity exercise capacity.\nMost studies related to the acute impact of CAF intake on power output and bar velocity have used participants unhabituated to CAF or individuals with low-to-moderate daily consumption of this stimulant [11,16,34]. However, the analysis of urinary CAF concentrations after official competitions suggests that CAF is widely employed before or during exercise to enhance performance [35,36]. This would mean that it is highly likely that some athletes are habituated to CAF due to daily consumption of caffeine-containing products. The existence of athletes habituated to CAF may be particularly common in sports such as cycling, rowing, triathlon, athletics, and weightlifting, sport disciplines that benefit from the use of ballistic exercises during training to increase power output. Habitual CAF intake modifies physiological responses to acute ingestion of this stimulant by the up-regulation of adenosine receptors [37,38]. This effect would produce a progressive reduction of CAF ergogenicity in those athletes consuming CAF on a regular basis, because the newly created adenosine receptors may bind to adenosine and induce fatigue. However, the fact of habituation to the ergogenic benefits of CAF is still inconclusive. The studies by Dodd et al. [39] and de Souza [40] showed similar responses to endurance exercise after acute CAF intake between low and habitual CAF consumers, although this is not always the case [41]. Considering the above, the use of cross-sectional investigations including participants with different degrees of habituation to CAF may explain the lack of consistency when concluding about tolerance to the ergogenic effect of CAF. Lara’s et al. [42] crossover design showed that the ergogenicity of CAF was reduced when the substance was consumed daily for 20 days, yet afterwards the ergogenic properties of CAF were maintained. On the contrary, tolerance to some of the side-effects associated to CAF has been observed in habitual consumers of CAF [43]. However, only two previous studies analyzed the impact of CAF intake in habitual CAF consumers using resistance exercise test protocols [18,19,20]. These investigations indicate that CAF ergogenicity to power output is mostly reduced in individuals habituated to CAF, while only high doses (>9 mg/kg/b.m.) may exert some benefit in maximal strength [19,20]. However, to date, there is no available data regarding the influence of acute CAF intake on power output and bar velocity during ballistic exercises in athletes habitually consuming CAF.\nGiven the widespread use of the BPT exercise as a mean of developing power output in the upper limbs [44,45] and the widespread use of CAF in sport, it would be interesting to investigate whether acute CAF intake affects power output and bar velocity in athletes habituated to CAF. For this reason, the aim of the present study was to evaluate the effects of the acute intake of 3 and 6 mg/kg/b.m. of CAF on power output and bar velocity during five sets of the BPT in participants habituated to CAF. It was hypothesized that acute CAF intake would increase power output and bar velocity during the BPT training session when compared to a control situation, even in participants habituated to CAF."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"3. Results"},"results_text":{"kind":"string","value":"The two-way repeated measures ANOVA indicated no significant substance × set main interaction effect for MP (F = 1.19; p = 0.32); MV (F = 1.18; p = 0.32); PP (F = 1.05; p = 0.40); PV (F = 1.09; p = 0.38). However, there was a significant main effect of substance in MP (F = 7.27; p < 0.01) and MV (F = 6.75; p < 0.01). No statistically significant main effect of substance was revealed in PP (F = 2.91; p = 0.07) and PV (F = 2.63; p = 0.09; Table 1).\nPost hoc analyses for main effect of substance indicated significant increases in MP (p < 0.01; ES = 0.14) and MV (p = 0.01; ES = 0.78) in BPT (mean of the 5 sets) after the intake of CAF-3 compared to PLAC as well as significant increases in MP (p = 0.01; ES = 0.13) and MV (p = 0.01; ES = 0.72) in the BPT (mean of the 5 sets) after the intake of CAF-6 compared to PLAC. There were no significant differences in MP and MV between the two doses of CAF (CAF-3 vs. CAF-6). The results of particular sets in MP, MV, PP, and PV as well ES between PLAC and CAF-3, CAF-6 in each set are presented in Table 2.\nFigure 2 and Figure 3 represent the individual responses induced by CAF-3 and CAF-6, in comparison to the placebo, for MP and MV. The 11 out of 12 participants showed an increase in MP and MV after the ingestion of CAF-3, while CAF-6 produced higher values for MP and MV in 10 out of 12 participants.\nThe y-axis represents the difference in mean bar velocity output during the 5 sets of BPT between PLAC–CAF-3; PLAC–CAF-6 for each individual."},"conclusions_title":{"kind":"string","value":"5. Conclusions"},"conclusions_text":{"kind":"string","value":"The results of the present study indicate that acute doses of CAF, between 3 and 6 mg/kg/b.m., ingested before the onset of an explosive resistance exercise produced an overall effect on mean power output and mean bar velocity during a BPT training session in a group of habitual CAF users. The main effect in mean power and bar velocity was found in several sets during the trial which may indicate that the use of CAF was effective in increasing performance in the whole training session. In contrast, no significant changes were observed for peak power output and peak bar velocity. These results suggest that the ingestion of CAF prior to ballistic exercise can enhance the outcomes of resistance training. However, the results of our study refer only to power output and bar velocity of the upper limbs during the BPT with an external load of 30% 1 RM and further investigations should consider the effect of CAF with different loads or the use of lower-body exercises."},"other_sections_titles":{"kind":"list like","value":["2. Materials and Methods","2.2. Habitual Caffeine Intake Assessment","2.3. Familiarization Session and One Repetition Maximum Test","2.4. Experimental Sessions","2.5. Statistical Analysis"],"string":"[\n \"2. Materials and Methods\",\n \"2.2. Habitual Caffeine Intake Assessment\",\n \"2.3. Familiarization Session and One Repetition Maximum Test\",\n \"2.4. Experimental Sessions\",\n \"2.5. Statistical Analysis\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["A randomized, double-blind, PLAC-controlled crossover design was used for this investigation. Initially, the participants performed a familiarization session with the experimental protocols that included a 1 RM measurement for the bench press exercise. Afterwards, they performed 3 different experimental sessions with a one-week interval between sessions to allow for complete recovery and a wash-out of ingested substances (Figure 1). During the 3 experimental sessions, the study participants either ingested a PLAC, 3 mg/kg/b.m. of CAF (CAF-3) or 6 mg/kg/b.m. of CAF (CAF-6). One hour after ingestion of CAF or PLAC, they performed 5 sets of 2 repetitions of the BPT exercise at 30% 1 RM. Both CAF and PLAC were administered orally to allow for peak blood CAF concentration during the training session and at least 2 h after the last meal to avoid any interference of the diet with the absorption of the experimental substances. CAF supplementation was provided to participants in the form of unidentifiable capsules (Caffeine Kick®, Olimp Laboratories, Debica, Poland). The manufacturer of the CAF capsules also provided identical PLAC capsules filled with all-purpose flour. Participants refrained from strenuous physical activity the day before the experimental trials but they maintained their training routines during the duration of the experiment to avoid any performance decrement due to inactivity. Additionally, the participants maintained their dietary habits during the study period, including daily CAF intake. They received a list of products containing CAF which could not be consumed within 12 h of each experimental trial. Compliance was tested verbally and by using dietary records. Additionally, the participants were required to refrain from alcohol and tobacco, medications or dietary supplements for two weeks prior to the experiment. All subjects registered their calorie intake using the “Myfitness pal” software [46] (Under Armour, Baltimore, MD, USA) every 24 h before the testing procedure, to ensure that the caloric intake was similar between experimental sessions.\n 2.1. Participants Twelve healthy strength-trained male athletes (age: 25.3 ± 1.7 years., body mass: 88.4 ± 16.5 kg, body mass index (BMI): 26.5 ± 4.7, bench press 1 RM: 128.6 ± 36.0 kg; mean ± SD) volunteered to participate in the study. All participants completed a written consent form after they had been informed of the risks and benefits of the study protocols. The participants had a minimum of 3 years of strength training experience (4.4 ± 1.6 years). All of them were classified as high habitual CAF consumers according to the classification recently proposed by de Souza Gonçalves et al. [40]. They self-reported their daily ingestion of CAF (5.0 ± 0.95 mg/kg/b.m./day, 443 ± 142 mg/day) based on a Food Frequency Questionnaire (FFQ). The inclusion criteria were as follows: (a) free from neuromuscular and musculoskeletal disorders, (b) 1 RM bench press performance with a load of at least 120% body mass, (c) habitual CAF intake in the range of 4–6 mg/day/kg/b.m. The athletes were excluded from the study when they suffered from any pathology or injury or when they were unable to perform the exercise protocol at the maximum effort. The investigation protocols were approved by the Bioethics Committee for Scientific Research at the Academy of Physical Education in Katowice (March 2019), Poland, according to the ethical standards of the latest version of the Declaration of Helsinki, 2013.\nTwelve healthy strength-trained male athletes (age: 25.3 ± 1.7 years., body mass: 88.4 ± 16.5 kg, body mass index (BMI): 26.5 ± 4.7, bench press 1 RM: 128.6 ± 36.0 kg; mean ± SD) volunteered to participate in the study. All participants completed a written consent form after they had been informed of the risks and benefits of the study protocols. The participants had a minimum of 3 years of strength training experience (4.4 ± 1.6 years). All of them were classified as high habitual CAF consumers according to the classification recently proposed by de Souza Gonçalves et al. [40]. They self-reported their daily ingestion of CAF (5.0 ± 0.95 mg/kg/b.m./day, 443 ± 142 mg/day) based on a Food Frequency Questionnaire (FFQ). The inclusion criteria were as follows: (a) free from neuromuscular and musculoskeletal disorders, (b) 1 RM bench press performance with a load of at least 120% body mass, (c) habitual CAF intake in the range of 4–6 mg/day/kg/b.m. The athletes were excluded from the study when they suffered from any pathology or injury or when they were unable to perform the exercise protocol at the maximum effort. The investigation protocols were approved by the Bioethics Committee for Scientific Research at the Academy of Physical Education in Katowice (March 2019), Poland, according to the ethical standards of the latest version of the Declaration of Helsinki, 2013.\n 2.2. Habitual Caffeine Intake Assessment Daily CAF intake was measured by an adapted version of the Food Frequency Questionnaire (FFQ) proposed by Bühler et al. [47]. Household measures were employed to individually assess the amount of food consumed during a day, week and month. The list was composed of dietary products with moderate-to-high CAF content including different types of coffee, tea, energy drinks, cocoa products, popular beverages, medications, and CAF supplements. Nutritional tables were used for database construction [48,49,50] and an experienced nutritionist calculated the daily CAF intake for each participant.\nDaily CAF intake was measured by an adapted version of the Food Frequency Questionnaire (FFQ) proposed by Bühler et al. [47]. Household measures were employed to individually assess the amount of food consumed during a day, week and month. The list was composed of dietary products with moderate-to-high CAF content including different types of coffee, tea, energy drinks, cocoa products, popular beverages, medications, and CAF supplements. Nutritional tables were used for database construction [48,49,50] and an experienced nutritionist calculated the daily CAF intake for each participant.\n 2.3. Familiarization Session and One Repetition Maximum Test A familiarization session with the experimental procedures preceded 1 RM testing in the bench press exercise. In this session, the athletes arrived at the laboratory between 9:00 and 10:00 am. and cycled on an ergometer for 5 min. Afterwards, they performed 15 repetitions at 20% of their estimated 1 RM in the barbell bench press exercise followed by 10 repetitions at 40% 1 RM, 5 repetitions at 60% 1 RM and 3 repetitions at 80% 1 RM. Then they executed single repetitions of the bench press exercise with a 5 min rest interval between successful attempts. The load for each subsequent attempt was increased by 2.5 to 10 kg, and the process was repeated until failure. Hand placement on the barbell was individually selected grip width (~150% individual bi-acromial distance). After completing the 1 RM test in the bench press exercise, the participants performed a maximal BPT on a Smith machine with a load of 30% 1 RM from 1 RM bench press test result, with a maximal tempo of movement.\nA familiarization session with the experimental procedures preceded 1 RM testing in the bench press exercise. In this session, the athletes arrived at the laboratory between 9:00 and 10:00 am. and cycled on an ergometer for 5 min. Afterwards, they performed 15 repetitions at 20% of their estimated 1 RM in the barbell bench press exercise followed by 10 repetitions at 40% 1 RM, 5 repetitions at 60% 1 RM and 3 repetitions at 80% 1 RM. Then they executed single repetitions of the bench press exercise with a 5 min rest interval between successful attempts. The load for each subsequent attempt was increased by 2.5 to 10 kg, and the process was repeated until failure. Hand placement on the barbell was individually selected grip width (~150% individual bi-acromial distance). After completing the 1 RM test in the bench press exercise, the participants performed a maximal BPT on a Smith machine with a load of 30% 1 RM from 1 RM bench press test result, with a maximal tempo of movement.\n 2.4. Experimental Sessions During experimental sessions, the athletes participated in three identical training trials. All trials took place between 9.00 and 11.00 am. to avoid the effect of circadian variations on the outcomes of the investigation. After replicating the warm-up procedures of the familiarization trial, the athletes performed 5 sets of the 2 BPT repetitions at 30% 1 RM on the Smith machine. The repetitions were performed without rest to produce a ballistic movement while the rest interval between sets was 3 min. The participants were encouraged to produce maximal velocity during both the eccentric and concentric phase of the BPT movement. Two spotters were present on each side of the bar during the exercise protocol to ensure safety. To standardize the exercise protocol for all trials, each BPT was performed without bouncing the barbell off the chest, with the lower back in contact with the bench and without any pause between the eccentric and concentric phases of the movement. A rotatory encoder (Tendo Power Analyzer, Tendo Sport Machines, Trencin, Slovakia) was used for instantaneous recording of bar velocity during the whole range of motion, as in previous investigations [51]. During each BPT, peak power output (PP, in W) mean power output (MP, in W); peak bar velocity (PV, in m/s); and mean bar velocity (MV, in m/s) were registered. MP and MV were obtained as the mean of the two repetitions while PP and PV were obtained from the best repetition.\nDuring experimental sessions, the athletes participated in three identical training trials. All trials took place between 9.00 and 11.00 am. to avoid the effect of circadian variations on the outcomes of the investigation. After replicating the warm-up procedures of the familiarization trial, the athletes performed 5 sets of the 2 BPT repetitions at 30% 1 RM on the Smith machine. The repetitions were performed without rest to produce a ballistic movement while the rest interval between sets was 3 min. The participants were encouraged to produce maximal velocity during both the eccentric and concentric phase of the BPT movement. Two spotters were present on each side of the bar during the exercise protocol to ensure safety. To standardize the exercise protocol for all trials, each BPT was performed without bouncing the barbell off the chest, with the lower back in contact with the bench and without any pause between the eccentric and concentric phases of the movement. A rotatory encoder (Tendo Power Analyzer, Tendo Sport Machines, Trencin, Slovakia) was used for instantaneous recording of bar velocity during the whole range of motion, as in previous investigations [51]. During each BPT, peak power output (PP, in W) mean power output (MP, in W); peak bar velocity (PV, in m/s); and mean bar velocity (MV, in m/s) were registered. MP and MV were obtained as the mean of the two repetitions while PP and PV were obtained from the best repetition.\n 2.5. Statistical Analysis Data are presented as the mean ± SD. All variables presented a normal distribution according to the Shapiro-Wilk test. Verification of differences in peak power output (PP), mean power output (MP), peak bar velocity (PV), and mean bar velocity (MV) was performed using a two-way (substance × set) analysis of variance (ANOVA) with repeated measures. In the event of a significant main effect, post-hoc comparisons were conducted using the Tukey’s test. Percent changes and 95% confidence intervals were also calculated. Effect sizes (Cohen’s d) were reported where appropriate and interpreted as large (d ≥ 0.80); moderate (d between 0.79 and 0.50); small (d between 0.49 and 0.20); and trivial (d < 0.20); [52].\nData are presented as the mean ± SD. All variables presented a normal distribution according to the Shapiro-Wilk test. Verification of differences in peak power output (PP), mean power output (MP), peak bar velocity (PV), and mean bar velocity (MV) was performed using a two-way (substance × set) analysis of variance (ANOVA) with repeated measures. In the event of a significant main effect, post-hoc comparisons were conducted using the Tukey’s test. Percent changes and 95% confidence intervals were also calculated. Effect sizes (Cohen’s d) were reported where appropriate and interpreted as large (d ≥ 0.80); moderate (d between 0.79 and 0.50); small (d between 0.49 and 0.20); and trivial (d < 0.20); [52].","Daily CAF intake was measured by an adapted version of the Food Frequency Questionnaire (FFQ) proposed by Bühler et al. [47]. Household measures were employed to individually assess the amount of food consumed during a day, week and month. The list was composed of dietary products with moderate-to-high CAF content including different types of coffee, tea, energy drinks, cocoa products, popular beverages, medications, and CAF supplements. Nutritional tables were used for database construction [48,49,50] and an experienced nutritionist calculated the daily CAF intake for each participant.","A familiarization session with the experimental procedures preceded 1 RM testing in the bench press exercise. In this session, the athletes arrived at the laboratory between 9:00 and 10:00 am. and cycled on an ergometer for 5 min. Afterwards, they performed 15 repetitions at 20% of their estimated 1 RM in the barbell bench press exercise followed by 10 repetitions at 40% 1 RM, 5 repetitions at 60% 1 RM and 3 repetitions at 80% 1 RM. Then they executed single repetitions of the bench press exercise with a 5 min rest interval between successful attempts. The load for each subsequent attempt was increased by 2.5 to 10 kg, and the process was repeated until failure. Hand placement on the barbell was individually selected grip width (~150% individual bi-acromial distance). After completing the 1 RM test in the bench press exercise, the participants performed a maximal BPT on a Smith machine with a load of 30% 1 RM from 1 RM bench press test result, with a maximal tempo of movement.","During experimental sessions, the athletes participated in three identical training trials. All trials took place between 9.00 and 11.00 am. to avoid the effect of circadian variations on the outcomes of the investigation. After replicating the warm-up procedures of the familiarization trial, the athletes performed 5 sets of the 2 BPT repetitions at 30% 1 RM on the Smith machine. The repetitions were performed without rest to produce a ballistic movement while the rest interval between sets was 3 min. The participants were encouraged to produce maximal velocity during both the eccentric and concentric phase of the BPT movement. Two spotters were present on each side of the bar during the exercise protocol to ensure safety. To standardize the exercise protocol for all trials, each BPT was performed without bouncing the barbell off the chest, with the lower back in contact with the bench and without any pause between the eccentric and concentric phases of the movement. A rotatory encoder (Tendo Power Analyzer, Tendo Sport Machines, Trencin, Slovakia) was used for instantaneous recording of bar velocity during the whole range of motion, as in previous investigations [51]. During each BPT, peak power output (PP, in W) mean power output (MP, in W); peak bar velocity (PV, in m/s); and mean bar velocity (MV, in m/s) were registered. MP and MV were obtained as the mean of the two repetitions while PP and PV were obtained from the best repetition.","Data are presented as the mean ± SD. All variables presented a normal distribution according to the Shapiro-Wilk test. Verification of differences in peak power output (PP), mean power output (MP), peak bar velocity (PV), and mean bar velocity (MV) was performed using a two-way (substance × set) analysis of variance (ANOVA) with repeated measures. In the event of a significant main effect, post-hoc comparisons were conducted using the Tukey’s test. Percent changes and 95% confidence intervals were also calculated. Effect sizes (Cohen’s d) were reported where appropriate and interpreted as large (d ≥ 0.80); moderate (d between 0.79 and 0.50); small (d between 0.49 and 0.20); and trivial (d < 0.20); [52]."],"string":"[\n \"A randomized, double-blind, PLAC-controlled crossover design was used for this investigation. Initially, the participants performed a familiarization session with the experimental protocols that included a 1 RM measurement for the bench press exercise. Afterwards, they performed 3 different experimental sessions with a one-week interval between sessions to allow for complete recovery and a wash-out of ingested substances (Figure 1). During the 3 experimental sessions, the study participants either ingested a PLAC, 3 mg/kg/b.m. of CAF (CAF-3) or 6 mg/kg/b.m. of CAF (CAF-6). One hour after ingestion of CAF or PLAC, they performed 5 sets of 2 repetitions of the BPT exercise at 30% 1 RM. Both CAF and PLAC were administered orally to allow for peak blood CAF concentration during the training session and at least 2 h after the last meal to avoid any interference of the diet with the absorption of the experimental substances. CAF supplementation was provided to participants in the form of unidentifiable capsules (Caffeine Kick®, Olimp Laboratories, Debica, Poland). The manufacturer of the CAF capsules also provided identical PLAC capsules filled with all-purpose flour. Participants refrained from strenuous physical activity the day before the experimental trials but they maintained their training routines during the duration of the experiment to avoid any performance decrement due to inactivity. Additionally, the participants maintained their dietary habits during the study period, including daily CAF intake. They received a list of products containing CAF which could not be consumed within 12 h of each experimental trial. Compliance was tested verbally and by using dietary records. Additionally, the participants were required to refrain from alcohol and tobacco, medications or dietary supplements for two weeks prior to the experiment. All subjects registered their calorie intake using the “Myfitness pal” software [46] (Under Armour, Baltimore, MD, USA) every 24 h before the testing procedure, to ensure that the caloric intake was similar between experimental sessions.\\n 2.1. Participants Twelve healthy strength-trained male athletes (age: 25.3 ± 1.7 years., body mass: 88.4 ± 16.5 kg, body mass index (BMI): 26.5 ± 4.7, bench press 1 RM: 128.6 ± 36.0 kg; mean ± SD) volunteered to participate in the study. All participants completed a written consent form after they had been informed of the risks and benefits of the study protocols. The participants had a minimum of 3 years of strength training experience (4.4 ± 1.6 years). All of them were classified as high habitual CAF consumers according to the classification recently proposed by de Souza Gonçalves et al. [40]. They self-reported their daily ingestion of CAF (5.0 ± 0.95 mg/kg/b.m./day, 443 ± 142 mg/day) based on a Food Frequency Questionnaire (FFQ). The inclusion criteria were as follows: (a) free from neuromuscular and musculoskeletal disorders, (b) 1 RM bench press performance with a load of at least 120% body mass, (c) habitual CAF intake in the range of 4–6 mg/day/kg/b.m. The athletes were excluded from the study when they suffered from any pathology or injury or when they were unable to perform the exercise protocol at the maximum effort. The investigation protocols were approved by the Bioethics Committee for Scientific Research at the Academy of Physical Education in Katowice (March 2019), Poland, according to the ethical standards of the latest version of the Declaration of Helsinki, 2013.\\nTwelve healthy strength-trained male athletes (age: 25.3 ± 1.7 years., body mass: 88.4 ± 16.5 kg, body mass index (BMI): 26.5 ± 4.7, bench press 1 RM: 128.6 ± 36.0 kg; mean ± SD) volunteered to participate in the study. All participants completed a written consent form after they had been informed of the risks and benefits of the study protocols. The participants had a minimum of 3 years of strength training experience (4.4 ± 1.6 years). All of them were classified as high habitual CAF consumers according to the classification recently proposed by de Souza Gonçalves et al. [40]. They self-reported their daily ingestion of CAF (5.0 ± 0.95 mg/kg/b.m./day, 443 ± 142 mg/day) based on a Food Frequency Questionnaire (FFQ). The inclusion criteria were as follows: (a) free from neuromuscular and musculoskeletal disorders, (b) 1 RM bench press performance with a load of at least 120% body mass, (c) habitual CAF intake in the range of 4–6 mg/day/kg/b.m. The athletes were excluded from the study when they suffered from any pathology or injury or when they were unable to perform the exercise protocol at the maximum effort. The investigation protocols were approved by the Bioethics Committee for Scientific Research at the Academy of Physical Education in Katowice (March 2019), Poland, according to the ethical standards of the latest version of the Declaration of Helsinki, 2013.\\n 2.2. Habitual Caffeine Intake Assessment Daily CAF intake was measured by an adapted version of the Food Frequency Questionnaire (FFQ) proposed by Bühler et al. [47]. Household measures were employed to individually assess the amount of food consumed during a day, week and month. The list was composed of dietary products with moderate-to-high CAF content including different types of coffee, tea, energy drinks, cocoa products, popular beverages, medications, and CAF supplements. Nutritional tables were used for database construction [48,49,50] and an experienced nutritionist calculated the daily CAF intake for each participant.\\nDaily CAF intake was measured by an adapted version of the Food Frequency Questionnaire (FFQ) proposed by Bühler et al. [47]. Household measures were employed to individually assess the amount of food consumed during a day, week and month. The list was composed of dietary products with moderate-to-high CAF content including different types of coffee, tea, energy drinks, cocoa products, popular beverages, medications, and CAF supplements. Nutritional tables were used for database construction [48,49,50] and an experienced nutritionist calculated the daily CAF intake for each participant.\\n 2.3. Familiarization Session and One Repetition Maximum Test A familiarization session with the experimental procedures preceded 1 RM testing in the bench press exercise. In this session, the athletes arrived at the laboratory between 9:00 and 10:00 am. and cycled on an ergometer for 5 min. Afterwards, they performed 15 repetitions at 20% of their estimated 1 RM in the barbell bench press exercise followed by 10 repetitions at 40% 1 RM, 5 repetitions at 60% 1 RM and 3 repetitions at 80% 1 RM. Then they executed single repetitions of the bench press exercise with a 5 min rest interval between successful attempts. The load for each subsequent attempt was increased by 2.5 to 10 kg, and the process was repeated until failure. Hand placement on the barbell was individually selected grip width (~150% individual bi-acromial distance). After completing the 1 RM test in the bench press exercise, the participants performed a maximal BPT on a Smith machine with a load of 30% 1 RM from 1 RM bench press test result, with a maximal tempo of movement.\\nA familiarization session with the experimental procedures preceded 1 RM testing in the bench press exercise. In this session, the athletes arrived at the laboratory between 9:00 and 10:00 am. and cycled on an ergometer for 5 min. Afterwards, they performed 15 repetitions at 20% of their estimated 1 RM in the barbell bench press exercise followed by 10 repetitions at 40% 1 RM, 5 repetitions at 60% 1 RM and 3 repetitions at 80% 1 RM. Then they executed single repetitions of the bench press exercise with a 5 min rest interval between successful attempts. The load for each subsequent attempt was increased by 2.5 to 10 kg, and the process was repeated until failure. Hand placement on the barbell was individually selected grip width (~150% individual bi-acromial distance). After completing the 1 RM test in the bench press exercise, the participants performed a maximal BPT on a Smith machine with a load of 30% 1 RM from 1 RM bench press test result, with a maximal tempo of movement.\\n 2.4. Experimental Sessions During experimental sessions, the athletes participated in three identical training trials. All trials took place between 9.00 and 11.00 am. to avoid the effect of circadian variations on the outcomes of the investigation. After replicating the warm-up procedures of the familiarization trial, the athletes performed 5 sets of the 2 BPT repetitions at 30% 1 RM on the Smith machine. The repetitions were performed without rest to produce a ballistic movement while the rest interval between sets was 3 min. The participants were encouraged to produce maximal velocity during both the eccentric and concentric phase of the BPT movement. Two spotters were present on each side of the bar during the exercise protocol to ensure safety. To standardize the exercise protocol for all trials, each BPT was performed without bouncing the barbell off the chest, with the lower back in contact with the bench and without any pause between the eccentric and concentric phases of the movement. A rotatory encoder (Tendo Power Analyzer, Tendo Sport Machines, Trencin, Slovakia) was used for instantaneous recording of bar velocity during the whole range of motion, as in previous investigations [51]. During each BPT, peak power output (PP, in W) mean power output (MP, in W); peak bar velocity (PV, in m/s); and mean bar velocity (MV, in m/s) were registered. MP and MV were obtained as the mean of the two repetitions while PP and PV were obtained from the best repetition.\\nDuring experimental sessions, the athletes participated in three identical training trials. All trials took place between 9.00 and 11.00 am. to avoid the effect of circadian variations on the outcomes of the investigation. After replicating the warm-up procedures of the familiarization trial, the athletes performed 5 sets of the 2 BPT repetitions at 30% 1 RM on the Smith machine. The repetitions were performed without rest to produce a ballistic movement while the rest interval between sets was 3 min. The participants were encouraged to produce maximal velocity during both the eccentric and concentric phase of the BPT movement. Two spotters were present on each side of the bar during the exercise protocol to ensure safety. To standardize the exercise protocol for all trials, each BPT was performed without bouncing the barbell off the chest, with the lower back in contact with the bench and without any pause between the eccentric and concentric phases of the movement. A rotatory encoder (Tendo Power Analyzer, Tendo Sport Machines, Trencin, Slovakia) was used for instantaneous recording of bar velocity during the whole range of motion, as in previous investigations [51]. During each BPT, peak power output (PP, in W) mean power output (MP, in W); peak bar velocity (PV, in m/s); and mean bar velocity (MV, in m/s) were registered. MP and MV were obtained as the mean of the two repetitions while PP and PV were obtained from the best repetition.\\n 2.5. Statistical Analysis Data are presented as the mean ± SD. All variables presented a normal distribution according to the Shapiro-Wilk test. Verification of differences in peak power output (PP), mean power output (MP), peak bar velocity (PV), and mean bar velocity (MV) was performed using a two-way (substance × set) analysis of variance (ANOVA) with repeated measures. In the event of a significant main effect, post-hoc comparisons were conducted using the Tukey’s test. Percent changes and 95% confidence intervals were also calculated. Effect sizes (Cohen’s d) were reported where appropriate and interpreted as large (d ≥ 0.80); moderate (d between 0.79 and 0.50); small (d between 0.49 and 0.20); and trivial (d < 0.20); [52].\\nData are presented as the mean ± SD. All variables presented a normal distribution according to the Shapiro-Wilk test. Verification of differences in peak power output (PP), mean power output (MP), peak bar velocity (PV), and mean bar velocity (MV) was performed using a two-way (substance × set) analysis of variance (ANOVA) with repeated measures. In the event of a significant main effect, post-hoc comparisons were conducted using the Tukey’s test. Percent changes and 95% confidence intervals were also calculated. Effect sizes (Cohen’s d) were reported where appropriate and interpreted as large (d ≥ 0.80); moderate (d between 0.79 and 0.50); small (d between 0.49 and 0.20); and trivial (d < 0.20); [52].\",\n \"Daily CAF intake was measured by an adapted version of the Food Frequency Questionnaire (FFQ) proposed by Bühler et al. [47]. Household measures were employed to individually assess the amount of food consumed during a day, week and month. The list was composed of dietary products with moderate-to-high CAF content including different types of coffee, tea, energy drinks, cocoa products, popular beverages, medications, and CAF supplements. Nutritional tables were used for database construction [48,49,50] and an experienced nutritionist calculated the daily CAF intake for each participant.\",\n \"A familiarization session with the experimental procedures preceded 1 RM testing in the bench press exercise. In this session, the athletes arrived at the laboratory between 9:00 and 10:00 am. and cycled on an ergometer for 5 min. Afterwards, they performed 15 repetitions at 20% of their estimated 1 RM in the barbell bench press exercise followed by 10 repetitions at 40% 1 RM, 5 repetitions at 60% 1 RM and 3 repetitions at 80% 1 RM. Then they executed single repetitions of the bench press exercise with a 5 min rest interval between successful attempts. The load for each subsequent attempt was increased by 2.5 to 10 kg, and the process was repeated until failure. Hand placement on the barbell was individually selected grip width (~150% individual bi-acromial distance). After completing the 1 RM test in the bench press exercise, the participants performed a maximal BPT on a Smith machine with a load of 30% 1 RM from 1 RM bench press test result, with a maximal tempo of movement.\",\n \"During experimental sessions, the athletes participated in three identical training trials. All trials took place between 9.00 and 11.00 am. to avoid the effect of circadian variations on the outcomes of the investigation. After replicating the warm-up procedures of the familiarization trial, the athletes performed 5 sets of the 2 BPT repetitions at 30% 1 RM on the Smith machine. The repetitions were performed without rest to produce a ballistic movement while the rest interval between sets was 3 min. The participants were encouraged to produce maximal velocity during both the eccentric and concentric phase of the BPT movement. Two spotters were present on each side of the bar during the exercise protocol to ensure safety. To standardize the exercise protocol for all trials, each BPT was performed without bouncing the barbell off the chest, with the lower back in contact with the bench and without any pause between the eccentric and concentric phases of the movement. A rotatory encoder (Tendo Power Analyzer, Tendo Sport Machines, Trencin, Slovakia) was used for instantaneous recording of bar velocity during the whole range of motion, as in previous investigations [51]. During each BPT, peak power output (PP, in W) mean power output (MP, in W); peak bar velocity (PV, in m/s); and mean bar velocity (MV, in m/s) were registered. MP and MV were obtained as the mean of the two repetitions while PP and PV were obtained from the best repetition.\",\n \"Data are presented as the mean ± SD. All variables presented a normal distribution according to the Shapiro-Wilk test. Verification of differences in peak power output (PP), mean power output (MP), peak bar velocity (PV), and mean bar velocity (MV) was performed using a two-way (substance × set) analysis of variance (ANOVA) with repeated measures. In the event of a significant main effect, post-hoc comparisons were conducted using the Tukey’s test. Percent changes and 95% confidence intervals were also calculated. Effect sizes (Cohen’s d) were reported where appropriate and interpreted as large (d ≥ 0.80); moderate (d between 0.79 and 0.50); small (d between 0.49 and 0.20); and trivial (d < 0.20); [52].\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["1. Introduction","2. Materials and Methods","2.1. Participants","2.2. Habitual Caffeine Intake Assessment","2.3. Familiarization Session and One Repetition Maximum Test","2.4. Experimental Sessions","2.5. Statistical Analysis","3. Results","4. Discussion","5. Conclusions"],"string":"[\n \"1. Introduction\",\n \"2. Materials and Methods\",\n \"2.1. Participants\",\n \"2.2. Habitual Caffeine Intake Assessment\",\n \"2.3. Familiarization Session and One Repetition Maximum Test\",\n \"2.4. Experimental Sessions\",\n \"2.5. Statistical Analysis\",\n \"3. Results\",\n \"4. Discussion\",\n \"5. Conclusions\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Caffeine (CAF) is one of the most common substances used in sport which enhances physical performance [1]. Although CAF may affect various body tissues [2,3], there is a growing body of evidence in animal [4] and human models [5] sustaining the ability of CAF to act as an adenosine antagonist, as the main mechanism behind CAF ergogenic effect. Current research recommends doses of CAF ranging from 3 to 6 mg/kg body mass to elicit ergogenic benefits, while the time of ingestion (from 30 to 90 min before exercise) and the form of ingestion (pills, liquid, chewing gum) are less significant as CAF is rapidly absorbed after ingestion [6]. However, the optimal protocol of CAF supplementation may differ based on the type and duration of exercise, previous habituation to CAF and the type of muscle contraction [6,7,8,9,10].\nAcute CAF intake causes a slightly different response to upper and lower body exercise [10], despite the lack of a mechanisms explaining this phenomenon. A recent meta-analysis about the effects of CAF on muscle strength and power output found that CAF significantly improved upper but not lower body strength [7]. Although the outcomes of the first investigations were contradictory [11,12,13], more recent studies have found a clear effect of CAF on several forms of upper body muscle performance [14,15,16], when the used dose was at least 3 mg/kg/b.m. [17]. Interestingly, the effect of CAF on upper body muscle performance may be partially dampened in athletes habituated to CAF because the regular use of CAF-containing products [18] may develop tolerance to this substance. It must be noticed that this decreased ergogenic effect of CAF is not removed even with the ingestion of doses >9 mg/kg/b.m. [19,20].\nMost studies on the acute effects of CAF on upper body muscle performance used the bench press exercise. The bench press exercise is widely used as a means of developing strength and power of the upper limbs [21,22]. However, other authors, recommend the use of ballistic exercises for the development of upper body power, such as the bench press throw (BPT) [23,24]. Specifically, to increase power output, the loads ranging from 0% to 50% of one-repetition maximum (1 RM) moved at maximal speed are recommended as the most potent loading stimuli for improving power output [23]. However, this type of routine can only be performed on a Smith machine while using the BPT exercise (i.e., maximal bar velocity is obtained at the moment of throw). The traditional free-weight bench press exercise does not allow for the attainment of maximal velocity of execution (i.e., velocity equals 0 m/s at maximal arm extension). Thus, ballistic exercises could be an optimal choice for power training as they allow for greater velocity, and muscle activation in comparison to similar traditional resistance training routines [21]. Perhaps, the main asset of the BPT is the maximum acceleration of a given load, which ultimately produces high movement velocity in a short time [25]. In this respect, the loads applied in ballistic exercises during training will depend on the specific requirements of particular sport disciplines and will determine success in numerous power-based competitions [23]. Furthermore, the BPT performance has been associated with overall performance in different sport-specific tasks [26,27,28,29]. Therefore, it seems reasonable to use the BPT as a means of testing upper-body ballistic performance. Although the BPT is indicated as the most effective exercise for developing power of the upper limbs [30], previous studies have not determined the acute effects of CAF on power output and bar velocity during this type of exercise.\nBurke [31] has suggested that, in the current literature, there is a lack of data about the practical use of supplements in competitive sports because experimental protocols are often different from sports practice. In case of CAF, most studies considering the acute effects of this supplement were assessed on the basis of only a single set of an exercise [7,8,32], while real resistance training sessions in trained individuals rarely contain only one set of a particular exercise [31]. On the other hand, the investigations analyzing the acute effects of CAF on performance during successive sets of resistance exercises are scarce. Bowtell et al. [3] showed that pre-exercise CAF (6 mg/kg/b.m.) intake improved total exercise time during 5 sets of one-legged knee extensions performed to failure in comparison to a placebo (PLAC). It is worth noting that this ergogenic effect was achieved despite significantly lower muscle phosphocreatine concentration (PCr) and pH in the latter sets of an exercise in the CAF trial. Further, CAF ingestion (6 mg/kg/b.m.) attenuated the increase in interstitial potassium during one-legged knee extensions at 20 W (10 min) and 50 W (3 × 3 min) measured using microdialysis [33], which resulted in a 16% improvement in high-intensity exercise capacity.\nMost studies related to the acute impact of CAF intake on power output and bar velocity have used participants unhabituated to CAF or individuals with low-to-moderate daily consumption of this stimulant [11,16,34]. However, the analysis of urinary CAF concentrations after official competitions suggests that CAF is widely employed before or during exercise to enhance performance [35,36]. This would mean that it is highly likely that some athletes are habituated to CAF due to daily consumption of caffeine-containing products. The existence of athletes habituated to CAF may be particularly common in sports such as cycling, rowing, triathlon, athletics, and weightlifting, sport disciplines that benefit from the use of ballistic exercises during training to increase power output. Habitual CAF intake modifies physiological responses to acute ingestion of this stimulant by the up-regulation of adenosine receptors [37,38]. This effect would produce a progressive reduction of CAF ergogenicity in those athletes consuming CAF on a regular basis, because the newly created adenosine receptors may bind to adenosine and induce fatigue. However, the fact of habituation to the ergogenic benefits of CAF is still inconclusive. The studies by Dodd et al. [39] and de Souza [40] showed similar responses to endurance exercise after acute CAF intake between low and habitual CAF consumers, although this is not always the case [41]. Considering the above, the use of cross-sectional investigations including participants with different degrees of habituation to CAF may explain the lack of consistency when concluding about tolerance to the ergogenic effect of CAF. Lara’s et al. [42] crossover design showed that the ergogenicity of CAF was reduced when the substance was consumed daily for 20 days, yet afterwards the ergogenic properties of CAF were maintained. On the contrary, tolerance to some of the side-effects associated to CAF has been observed in habitual consumers of CAF [43]. However, only two previous studies analyzed the impact of CAF intake in habitual CAF consumers using resistance exercise test protocols [18,19,20]. These investigations indicate that CAF ergogenicity to power output is mostly reduced in individuals habituated to CAF, while only high doses (>9 mg/kg/b.m.) may exert some benefit in maximal strength [19,20]. However, to date, there is no available data regarding the influence of acute CAF intake on power output and bar velocity during ballistic exercises in athletes habitually consuming CAF.\nGiven the widespread use of the BPT exercise as a mean of developing power output in the upper limbs [44,45] and the widespread use of CAF in sport, it would be interesting to investigate whether acute CAF intake affects power output and bar velocity in athletes habituated to CAF. For this reason, the aim of the present study was to evaluate the effects of the acute intake of 3 and 6 mg/kg/b.m. of CAF on power output and bar velocity during five sets of the BPT in participants habituated to CAF. It was hypothesized that acute CAF intake would increase power output and bar velocity during the BPT training session when compared to a control situation, even in participants habituated to CAF.","A randomized, double-blind, PLAC-controlled crossover design was used for this investigation. Initially, the participants performed a familiarization session with the experimental protocols that included a 1 RM measurement for the bench press exercise. Afterwards, they performed 3 different experimental sessions with a one-week interval between sessions to allow for complete recovery and a wash-out of ingested substances (Figure 1). During the 3 experimental sessions, the study participants either ingested a PLAC, 3 mg/kg/b.m. of CAF (CAF-3) or 6 mg/kg/b.m. of CAF (CAF-6). One hour after ingestion of CAF or PLAC, they performed 5 sets of 2 repetitions of the BPT exercise at 30% 1 RM. Both CAF and PLAC were administered orally to allow for peak blood CAF concentration during the training session and at least 2 h after the last meal to avoid any interference of the diet with the absorption of the experimental substances. CAF supplementation was provided to participants in the form of unidentifiable capsules (Caffeine Kick®, Olimp Laboratories, Debica, Poland). The manufacturer of the CAF capsules also provided identical PLAC capsules filled with all-purpose flour. Participants refrained from strenuous physical activity the day before the experimental trials but they maintained their training routines during the duration of the experiment to avoid any performance decrement due to inactivity. Additionally, the participants maintained their dietary habits during the study period, including daily CAF intake. They received a list of products containing CAF which could not be consumed within 12 h of each experimental trial. Compliance was tested verbally and by using dietary records. Additionally, the participants were required to refrain from alcohol and tobacco, medications or dietary supplements for two weeks prior to the experiment. All subjects registered their calorie intake using the “Myfitness pal” software [46] (Under Armour, Baltimore, MD, USA) every 24 h before the testing procedure, to ensure that the caloric intake was similar between experimental sessions.\n 2.1. Participants Twelve healthy strength-trained male athletes (age: 25.3 ± 1.7 years., body mass: 88.4 ± 16.5 kg, body mass index (BMI): 26.5 ± 4.7, bench press 1 RM: 128.6 ± 36.0 kg; mean ± SD) volunteered to participate in the study. All participants completed a written consent form after they had been informed of the risks and benefits of the study protocols. The participants had a minimum of 3 years of strength training experience (4.4 ± 1.6 years). All of them were classified as high habitual CAF consumers according to the classification recently proposed by de Souza Gonçalves et al. [40]. They self-reported their daily ingestion of CAF (5.0 ± 0.95 mg/kg/b.m./day, 443 ± 142 mg/day) based on a Food Frequency Questionnaire (FFQ). The inclusion criteria were as follows: (a) free from neuromuscular and musculoskeletal disorders, (b) 1 RM bench press performance with a load of at least 120% body mass, (c) habitual CAF intake in the range of 4–6 mg/day/kg/b.m. The athletes were excluded from the study when they suffered from any pathology or injury or when they were unable to perform the exercise protocol at the maximum effort. The investigation protocols were approved by the Bioethics Committee for Scientific Research at the Academy of Physical Education in Katowice (March 2019), Poland, according to the ethical standards of the latest version of the Declaration of Helsinki, 2013.\nTwelve healthy strength-trained male athletes (age: 25.3 ± 1.7 years., body mass: 88.4 ± 16.5 kg, body mass index (BMI): 26.5 ± 4.7, bench press 1 RM: 128.6 ± 36.0 kg; mean ± SD) volunteered to participate in the study. All participants completed a written consent form after they had been informed of the risks and benefits of the study protocols. The participants had a minimum of 3 years of strength training experience (4.4 ± 1.6 years). All of them were classified as high habitual CAF consumers according to the classification recently proposed by de Souza Gonçalves et al. [40]. They self-reported their daily ingestion of CAF (5.0 ± 0.95 mg/kg/b.m./day, 443 ± 142 mg/day) based on a Food Frequency Questionnaire (FFQ). The inclusion criteria were as follows: (a) free from neuromuscular and musculoskeletal disorders, (b) 1 RM bench press performance with a load of at least 120% body mass, (c) habitual CAF intake in the range of 4–6 mg/day/kg/b.m. The athletes were excluded from the study when they suffered from any pathology or injury or when they were unable to perform the exercise protocol at the maximum effort. The investigation protocols were approved by the Bioethics Committee for Scientific Research at the Academy of Physical Education in Katowice (March 2019), Poland, according to the ethical standards of the latest version of the Declaration of Helsinki, 2013.\n 2.2. Habitual Caffeine Intake Assessment Daily CAF intake was measured by an adapted version of the Food Frequency Questionnaire (FFQ) proposed by Bühler et al. [47]. Household measures were employed to individually assess the amount of food consumed during a day, week and month. The list was composed of dietary products with moderate-to-high CAF content including different types of coffee, tea, energy drinks, cocoa products, popular beverages, medications, and CAF supplements. Nutritional tables were used for database construction [48,49,50] and an experienced nutritionist calculated the daily CAF intake for each participant.\nDaily CAF intake was measured by an adapted version of the Food Frequency Questionnaire (FFQ) proposed by Bühler et al. [47]. Household measures were employed to individually assess the amount of food consumed during a day, week and month. The list was composed of dietary products with moderate-to-high CAF content including different types of coffee, tea, energy drinks, cocoa products, popular beverages, medications, and CAF supplements. Nutritional tables were used for database construction [48,49,50] and an experienced nutritionist calculated the daily CAF intake for each participant.\n 2.3. Familiarization Session and One Repetition Maximum Test A familiarization session with the experimental procedures preceded 1 RM testing in the bench press exercise. In this session, the athletes arrived at the laboratory between 9:00 and 10:00 am. and cycled on an ergometer for 5 min. Afterwards, they performed 15 repetitions at 20% of their estimated 1 RM in the barbell bench press exercise followed by 10 repetitions at 40% 1 RM, 5 repetitions at 60% 1 RM and 3 repetitions at 80% 1 RM. Then they executed single repetitions of the bench press exercise with a 5 min rest interval between successful attempts. The load for each subsequent attempt was increased by 2.5 to 10 kg, and the process was repeated until failure. Hand placement on the barbell was individually selected grip width (~150% individual bi-acromial distance). After completing the 1 RM test in the bench press exercise, the participants performed a maximal BPT on a Smith machine with a load of 30% 1 RM from 1 RM bench press test result, with a maximal tempo of movement.\nA familiarization session with the experimental procedures preceded 1 RM testing in the bench press exercise. In this session, the athletes arrived at the laboratory between 9:00 and 10:00 am. and cycled on an ergometer for 5 min. Afterwards, they performed 15 repetitions at 20% of their estimated 1 RM in the barbell bench press exercise followed by 10 repetitions at 40% 1 RM, 5 repetitions at 60% 1 RM and 3 repetitions at 80% 1 RM. Then they executed single repetitions of the bench press exercise with a 5 min rest interval between successful attempts. The load for each subsequent attempt was increased by 2.5 to 10 kg, and the process was repeated until failure. Hand placement on the barbell was individually selected grip width (~150% individual bi-acromial distance). After completing the 1 RM test in the bench press exercise, the participants performed a maximal BPT on a Smith machine with a load of 30% 1 RM from 1 RM bench press test result, with a maximal tempo of movement.\n 2.4. Experimental Sessions During experimental sessions, the athletes participated in three identical training trials. All trials took place between 9.00 and 11.00 am. to avoid the effect of circadian variations on the outcomes of the investigation. After replicating the warm-up procedures of the familiarization trial, the athletes performed 5 sets of the 2 BPT repetitions at 30% 1 RM on the Smith machine. The repetitions were performed without rest to produce a ballistic movement while the rest interval between sets was 3 min. The participants were encouraged to produce maximal velocity during both the eccentric and concentric phase of the BPT movement. Two spotters were present on each side of the bar during the exercise protocol to ensure safety. To standardize the exercise protocol for all trials, each BPT was performed without bouncing the barbell off the chest, with the lower back in contact with the bench and without any pause between the eccentric and concentric phases of the movement. A rotatory encoder (Tendo Power Analyzer, Tendo Sport Machines, Trencin, Slovakia) was used for instantaneous recording of bar velocity during the whole range of motion, as in previous investigations [51]. During each BPT, peak power output (PP, in W) mean power output (MP, in W); peak bar velocity (PV, in m/s); and mean bar velocity (MV, in m/s) were registered. MP and MV were obtained as the mean of the two repetitions while PP and PV were obtained from the best repetition.\nDuring experimental sessions, the athletes participated in three identical training trials. All trials took place between 9.00 and 11.00 am. to avoid the effect of circadian variations on the outcomes of the investigation. After replicating the warm-up procedures of the familiarization trial, the athletes performed 5 sets of the 2 BPT repetitions at 30% 1 RM on the Smith machine. The repetitions were performed without rest to produce a ballistic movement while the rest interval between sets was 3 min. The participants were encouraged to produce maximal velocity during both the eccentric and concentric phase of the BPT movement. Two spotters were present on each side of the bar during the exercise protocol to ensure safety. To standardize the exercise protocol for all trials, each BPT was performed without bouncing the barbell off the chest, with the lower back in contact with the bench and without any pause between the eccentric and concentric phases of the movement. A rotatory encoder (Tendo Power Analyzer, Tendo Sport Machines, Trencin, Slovakia) was used for instantaneous recording of bar velocity during the whole range of motion, as in previous investigations [51]. During each BPT, peak power output (PP, in W) mean power output (MP, in W); peak bar velocity (PV, in m/s); and mean bar velocity (MV, in m/s) were registered. MP and MV were obtained as the mean of the two repetitions while PP and PV were obtained from the best repetition.\n 2.5. Statistical Analysis Data are presented as the mean ± SD. All variables presented a normal distribution according to the Shapiro-Wilk test. Verification of differences in peak power output (PP), mean power output (MP), peak bar velocity (PV), and mean bar velocity (MV) was performed using a two-way (substance × set) analysis of variance (ANOVA) with repeated measures. In the event of a significant main effect, post-hoc comparisons were conducted using the Tukey’s test. Percent changes and 95% confidence intervals were also calculated. Effect sizes (Cohen’s d) were reported where appropriate and interpreted as large (d ≥ 0.80); moderate (d between 0.79 and 0.50); small (d between 0.49 and 0.20); and trivial (d < 0.20); [52].\nData are presented as the mean ± SD. All variables presented a normal distribution according to the Shapiro-Wilk test. Verification of differences in peak power output (PP), mean power output (MP), peak bar velocity (PV), and mean bar velocity (MV) was performed using a two-way (substance × set) analysis of variance (ANOVA) with repeated measures. In the event of a significant main effect, post-hoc comparisons were conducted using the Tukey’s test. Percent changes and 95% confidence intervals were also calculated. Effect sizes (Cohen’s d) were reported where appropriate and interpreted as large (d ≥ 0.80); moderate (d between 0.79 and 0.50); small (d between 0.49 and 0.20); and trivial (d < 0.20); [52].","Twelve healthy strength-trained male athletes (age: 25.3 ± 1.7 years., body mass: 88.4 ± 16.5 kg, body mass index (BMI): 26.5 ± 4.7, bench press 1 RM: 128.6 ± 36.0 kg; mean ± SD) volunteered to participate in the study. All participants completed a written consent form after they had been informed of the risks and benefits of the study protocols. The participants had a minimum of 3 years of strength training experience (4.4 ± 1.6 years). All of them were classified as high habitual CAF consumers according to the classification recently proposed by de Souza Gonçalves et al. [40]. They self-reported their daily ingestion of CAF (5.0 ± 0.95 mg/kg/b.m./day, 443 ± 142 mg/day) based on a Food Frequency Questionnaire (FFQ). The inclusion criteria were as follows: (a) free from neuromuscular and musculoskeletal disorders, (b) 1 RM bench press performance with a load of at least 120% body mass, (c) habitual CAF intake in the range of 4–6 mg/day/kg/b.m. The athletes were excluded from the study when they suffered from any pathology or injury or when they were unable to perform the exercise protocol at the maximum effort. The investigation protocols were approved by the Bioethics Committee for Scientific Research at the Academy of Physical Education in Katowice (March 2019), Poland, according to the ethical standards of the latest version of the Declaration of Helsinki, 2013.","Daily CAF intake was measured by an adapted version of the Food Frequency Questionnaire (FFQ) proposed by Bühler et al. [47]. Household measures were employed to individually assess the amount of food consumed during a day, week and month. The list was composed of dietary products with moderate-to-high CAF content including different types of coffee, tea, energy drinks, cocoa products, popular beverages, medications, and CAF supplements. Nutritional tables were used for database construction [48,49,50] and an experienced nutritionist calculated the daily CAF intake for each participant.","A familiarization session with the experimental procedures preceded 1 RM testing in the bench press exercise. In this session, the athletes arrived at the laboratory between 9:00 and 10:00 am. and cycled on an ergometer for 5 min. Afterwards, they performed 15 repetitions at 20% of their estimated 1 RM in the barbell bench press exercise followed by 10 repetitions at 40% 1 RM, 5 repetitions at 60% 1 RM and 3 repetitions at 80% 1 RM. Then they executed single repetitions of the bench press exercise with a 5 min rest interval between successful attempts. The load for each subsequent attempt was increased by 2.5 to 10 kg, and the process was repeated until failure. Hand placement on the barbell was individually selected grip width (~150% individual bi-acromial distance). After completing the 1 RM test in the bench press exercise, the participants performed a maximal BPT on a Smith machine with a load of 30% 1 RM from 1 RM bench press test result, with a maximal tempo of movement.","During experimental sessions, the athletes participated in three identical training trials. All trials took place between 9.00 and 11.00 am. to avoid the effect of circadian variations on the outcomes of the investigation. After replicating the warm-up procedures of the familiarization trial, the athletes performed 5 sets of the 2 BPT repetitions at 30% 1 RM on the Smith machine. The repetitions were performed without rest to produce a ballistic movement while the rest interval between sets was 3 min. The participants were encouraged to produce maximal velocity during both the eccentric and concentric phase of the BPT movement. Two spotters were present on each side of the bar during the exercise protocol to ensure safety. To standardize the exercise protocol for all trials, each BPT was performed without bouncing the barbell off the chest, with the lower back in contact with the bench and without any pause between the eccentric and concentric phases of the movement. A rotatory encoder (Tendo Power Analyzer, Tendo Sport Machines, Trencin, Slovakia) was used for instantaneous recording of bar velocity during the whole range of motion, as in previous investigations [51]. During each BPT, peak power output (PP, in W) mean power output (MP, in W); peak bar velocity (PV, in m/s); and mean bar velocity (MV, in m/s) were registered. MP and MV were obtained as the mean of the two repetitions while PP and PV were obtained from the best repetition.","Data are presented as the mean ± SD. All variables presented a normal distribution according to the Shapiro-Wilk test. Verification of differences in peak power output (PP), mean power output (MP), peak bar velocity (PV), and mean bar velocity (MV) was performed using a two-way (substance × set) analysis of variance (ANOVA) with repeated measures. In the event of a significant main effect, post-hoc comparisons were conducted using the Tukey’s test. Percent changes and 95% confidence intervals were also calculated. Effect sizes (Cohen’s d) were reported where appropriate and interpreted as large (d ≥ 0.80); moderate (d between 0.79 and 0.50); small (d between 0.49 and 0.20); and trivial (d < 0.20); [52].","The two-way repeated measures ANOVA indicated no significant substance × set main interaction effect for MP (F = 1.19; p = 0.32); MV (F = 1.18; p = 0.32); PP (F = 1.05; p = 0.40); PV (F = 1.09; p = 0.38). However, there was a significant main effect of substance in MP (F = 7.27; p < 0.01) and MV (F = 6.75; p < 0.01). No statistically significant main effect of substance was revealed in PP (F = 2.91; p = 0.07) and PV (F = 2.63; p = 0.09; Table 1).\nPost hoc analyses for main effect of substance indicated significant increases in MP (p < 0.01; ES = 0.14) and MV (p = 0.01; ES = 0.78) in BPT (mean of the 5 sets) after the intake of CAF-3 compared to PLAC as well as significant increases in MP (p = 0.01; ES = 0.13) and MV (p = 0.01; ES = 0.72) in the BPT (mean of the 5 sets) after the intake of CAF-6 compared to PLAC. There were no significant differences in MP and MV between the two doses of CAF (CAF-3 vs. CAF-6). The results of particular sets in MP, MV, PP, and PV as well ES between PLAC and CAF-3, CAF-6 in each set are presented in Table 2.\nFigure 2 and Figure 3 represent the individual responses induced by CAF-3 and CAF-6, in comparison to the placebo, for MP and MV. The 11 out of 12 participants showed an increase in MP and MV after the ingestion of CAF-3, while CAF-6 produced higher values for MP and MV in 10 out of 12 participants.\nThe y-axis represents the difference in mean bar velocity output during the 5 sets of BPT between PLAC–CAF-3; PLAC–CAF-6 for each individual.","The main finding of this study was that acute CAF intake has a positive effect on MP and MV during a training session of the BPT performed at 30% 1 RM. Interestingly, both 3 and 6 mg/kg/b.m. doses of CAF had similar effectiveness in enhancing performance when compared to PLAC. Additionally, the ergogenic effect of CAF on MP and MV was evident in most of participants, as all of them responded by improving performance with either CAF-3 or CAF-6, even when they were catalogued as individuals habituated to CAF (Figure 2 and Figure 3). However, the study did not show significant changes in PP and PV after CAF intake with either dose of CAF (3 and 6 mg/kg/b.m.) compared to PLAC. These outcomes suggest that acute CAF intake in a moderate dose (from 3 to 6 mg/kg/b.m.) is effective in increasing mean power and bar velocity during the BPT without a significant influence on peak values of these variables. These results suggest that CAF can be effectively used to acutely improve this power-specific training routine even with individuals habituated to CAF, although the long-term training effects with CAF should be further investigated.\nThe y-axis represents the difference in mean power output during the 5 sets of BPT between PLAC–CAF-3; PLAC–CAF-6 for each individual.\nPrevious research showed that acute CAF intake increases power output during the bench press exercise [8,14,15,16]. However, most of these studies included only one set of the exercise which is not the habitual practice during sports training, where several sets of a particular exercise are performed in order to obtain significant adaptations derived from training. In presented study, the main effect of increase in MP and MV after the intake of CAF-3 and CAF-6 over the placebo has occurred for training session consisting several sets (Table 2). The ergogenic effect of CAF observed during the BPT is partly consistent with the results of previous findings [8,16]. However, it should be emphasized that this is the first study investigating the effects of CAF during a training session that includes several sets of a ballistic exercise. Experimental procedures with the use of CAF in which more than one set of an upper body exercise are used are scarce [18,53,54]. The study of Lane and Byrd [53] showed that the intake of 300 mg of CAF, representing 3.5 mg/kg/b.m. increased peak velocity during 10 sets of the bench press exercise at 80% 1 RM compared to PLAC. Wilk et al. [18] did not show significant changes after the intake of different doses of CAF (3, 6, and 9 mg/kg/b.m.) in both, mean and peak power output and bar velocity during the BP exercise at 50% 1 RM (3 sets of 5 repetitions), although this investigation was carried out in athletes habituated to CAF. No changes in mean and peak bar velocity after CAF intake of 150 mg, representing 1.74 mg/kg/b.m. were observed in a study by Lane et al. [54] where 10 sets of 3 repetitions of the bench press exercise were performed at 80% 1 RM. The current study is quite innovative because it is the first investigation geared to assess the effects of CAF intake on power output by using a ballistic upper body exercise with a low external load (30% 1 RM), geared for power training of athletes [55]. The current state of the literature, indicates that CAF is useful in increasing power during one or multiple sets of the bench press exercise when the dose ingested is >3 mg/kg/b.m., but it seems particularly effective when using low and moderate loads during an explosive exercise, such as the BPT.\nThe overall increase of MP and MV during the training session of the BPT after ingestion of CAF-3 and CAF-6 can be also attributed to increased pre-exercise central excitability. Specifically, the pre-exercise ingestion of CAF would allow the athletes to maintain a certain amount of force even in the presence of biochemical changes within the working muscle that lead to fatigue [3]. Under this theory, CAF intake would allow a higher physical performance because it would help to maintain the neural response even in the presence of metabolic perturbations such as low muscle pH. This effect may be accompanied by reductions in interstitial potassium accumulation found after CAF intake [2], that ultimately leads to the maintenance of excitability during exercise [33,56]. In the central nervous system (CNS), CAF binds to adenosine receptors that influence the release of neurotransmitters, such as noradrenaline and acetylcholine [4,57,58,59] and consequently, increase muscle tension [60]. However, in the current investigation, this purported effect of CAF on CNS was not sufficient to enhance PP and PV during the BPT at 30% 1 RM. Thus, reduced fatigue through CAF-induced modulation of both peripheral and central neural processes may explain the obtained results and higher MP and MV of the bar during the BPT training session. Nevertheless, the association of the ergogenic effect with the mechanisms that allowed this ergogenic effect is speculative at this moment because no measurements were carried out to test the origin of caffeine’s ergogenic effects.\nIt should be taken into consideration that the study participants in the current study were habitual CAF users. In contrast, most of the investigations aimed at determination of the ergogenic effect of CAF on muscle performance have selected individuals unhabituated to this stimulant or with low-to-moderate daily consumption of CAF (e.g., from 58 to 250 mg/day), [11,16,34], to avoid the effects that tolerance to CAF may. However, CAF is an ergogenic aid frequently used in training and competition and it is likely that some athletes seeking for ergogenic benefits of CAF are already habituated to this substance due to the chronic use of caffeine-containing supplements during training and competition. In fact, previous investigations have suggested that between 75% and 90% of athletes use CAF in competitive and training settings [35,36,61], suggesting that studies on the effect of acute CAF intake on physical performance during real training and competition settings are particularly important in athletes habituated to CAF. In this respect, previous research using well-controlled CAF treatments has suggested that the habitual intake of this stimulant may progressively reduce its ergogenic effect on exercise performance [42,62] and then, it has been speculated that the ergogenic effect of CAF could be dampened in habitual CAF users.\nTo the authors’ knowledge, only three previous studies analyzed power output of the upper limbs in a group of participants habituated to CAF [9,18,19,20]. The study of Sabol et al. [9] showed an increase in medicine ball throwing distance after the acute intake of 6 mg/kg/b.m. of CAF but the doses of 2 and 4 mg/kg/b.m. did not show any differences with the PLAC. The study by Wilk et al. [18] did not show increases in power output and bar velocity during the bench press exercise in high habitual CAF users that ingested from 3 to 9 mg/kg/b.m. Although it has been theorized that the reduction in the ergogenic effects of CAF in habitual users can be modified using doses greater than the daily habitual intake [63], previous investigations indicate that athletes habituated to CAF do not benefit from the acute ingestion of CAF in doses above their habitual intake while the prevalence of side effects is greatly increased [19,20]. Interestingly, participants in the presented study self-reported their daily ingestion of CAF, which amounted to 5.0 ± 0.95 mg/kg/b.m., (443 ± 142 mg of CAF per day), and the acute CAF doses (especially CAF-3) and some performance enhancements were obtained even when de dose of CAF did not exceed the value of habitual consumption. In any case, although the current investigation found a positive effect of CAF on mean power output and mean bar velocity during the BPT in athletes habituated to CAF, it is still possible that the effect of this substance is higher in unhabituated individuals.\nIn addition to its strengths, the current study presents limitations that should be addressed. Although the results showed a significant main effect on MP and MV after CAF intake, the direct causes of these changes cannot be determined and explained. The study did not include biochemical analysis which could explain the obtained results. In addition, blood samples were not obtained and thus, we have no data about serum CAF concentrations with each of the dosages of CAF employed in this investigation. Further, we did not analyze the genetic intolerance on CAF in the tested subjects. However, the participants of this study did not report any side effects after consuming CAF in the six months prior to the experiment. Due to the fact that the response to CAF is related to the individual tolerance of this substance [42], the dose [19,20], and gender [64] therefore the results of this study should only be translated to males habituated to CAF who use low to moderate CAF doses to enhance performance. Another limitation of the study was that the 1 RM test was performed using the barbell bench press exercise while the BPT was performed on a Smith machine during the experimental trials to increase the security of participants and investigators. Although there is a high transfer between the results obtained in both types of exercise, the calculation of loading would be more reliable if both evaluations were performed on the same resistance exercise. In any case, this limitation did not affect the outcomes of the investigation because the load was the same for all experimental trials.","The results of the present study indicate that acute doses of CAF, between 3 and 6 mg/kg/b.m., ingested before the onset of an explosive resistance exercise produced an overall effect on mean power output and mean bar velocity during a BPT training session in a group of habitual CAF users. The main effect in mean power and bar velocity was found in several sets during the trial which may indicate that the use of CAF was effective in increasing performance in the whole training session. In contrast, no significant changes were observed for peak power output and peak bar velocity. These results suggest that the ingestion of CAF prior to ballistic exercise can enhance the outcomes of resistance training. However, the results of our study refer only to power output and bar velocity of the upper limbs during the BPT with an external load of 30% 1 RM and further investigations should consider the effect of CAF with different loads or the use of lower-body exercises."],"string":"[\n \"Caffeine (CAF) is one of the most common substances used in sport which enhances physical performance [1]. Although CAF may affect various body tissues [2,3], there is a growing body of evidence in animal [4] and human models [5] sustaining the ability of CAF to act as an adenosine antagonist, as the main mechanism behind CAF ergogenic effect. Current research recommends doses of CAF ranging from 3 to 6 mg/kg body mass to elicit ergogenic benefits, while the time of ingestion (from 30 to 90 min before exercise) and the form of ingestion (pills, liquid, chewing gum) are less significant as CAF is rapidly absorbed after ingestion [6]. However, the optimal protocol of CAF supplementation may differ based on the type and duration of exercise, previous habituation to CAF and the type of muscle contraction [6,7,8,9,10].\\nAcute CAF intake causes a slightly different response to upper and lower body exercise [10], despite the lack of a mechanisms explaining this phenomenon. A recent meta-analysis about the effects of CAF on muscle strength and power output found that CAF significantly improved upper but not lower body strength [7]. Although the outcomes of the first investigations were contradictory [11,12,13], more recent studies have found a clear effect of CAF on several forms of upper body muscle performance [14,15,16], when the used dose was at least 3 mg/kg/b.m. [17]. Interestingly, the effect of CAF on upper body muscle performance may be partially dampened in athletes habituated to CAF because the regular use of CAF-containing products [18] may develop tolerance to this substance. It must be noticed that this decreased ergogenic effect of CAF is not removed even with the ingestion of doses >9 mg/kg/b.m. [19,20].\\nMost studies on the acute effects of CAF on upper body muscle performance used the bench press exercise. The bench press exercise is widely used as a means of developing strength and power of the upper limbs [21,22]. However, other authors, recommend the use of ballistic exercises for the development of upper body power, such as the bench press throw (BPT) [23,24]. Specifically, to increase power output, the loads ranging from 0% to 50% of one-repetition maximum (1 RM) moved at maximal speed are recommended as the most potent loading stimuli for improving power output [23]. However, this type of routine can only be performed on a Smith machine while using the BPT exercise (i.e., maximal bar velocity is obtained at the moment of throw). The traditional free-weight bench press exercise does not allow for the attainment of maximal velocity of execution (i.e., velocity equals 0 m/s at maximal arm extension). Thus, ballistic exercises could be an optimal choice for power training as they allow for greater velocity, and muscle activation in comparison to similar traditional resistance training routines [21]. Perhaps, the main asset of the BPT is the maximum acceleration of a given load, which ultimately produces high movement velocity in a short time [25]. In this respect, the loads applied in ballistic exercises during training will depend on the specific requirements of particular sport disciplines and will determine success in numerous power-based competitions [23]. Furthermore, the BPT performance has been associated with overall performance in different sport-specific tasks [26,27,28,29]. Therefore, it seems reasonable to use the BPT as a means of testing upper-body ballistic performance. Although the BPT is indicated as the most effective exercise for developing power of the upper limbs [30], previous studies have not determined the acute effects of CAF on power output and bar velocity during this type of exercise.\\nBurke [31] has suggested that, in the current literature, there is a lack of data about the practical use of supplements in competitive sports because experimental protocols are often different from sports practice. In case of CAF, most studies considering the acute effects of this supplement were assessed on the basis of only a single set of an exercise [7,8,32], while real resistance training sessions in trained individuals rarely contain only one set of a particular exercise [31]. On the other hand, the investigations analyzing the acute effects of CAF on performance during successive sets of resistance exercises are scarce. Bowtell et al. [3] showed that pre-exercise CAF (6 mg/kg/b.m.) intake improved total exercise time during 5 sets of one-legged knee extensions performed to failure in comparison to a placebo (PLAC). It is worth noting that this ergogenic effect was achieved despite significantly lower muscle phosphocreatine concentration (PCr) and pH in the latter sets of an exercise in the CAF trial. Further, CAF ingestion (6 mg/kg/b.m.) attenuated the increase in interstitial potassium during one-legged knee extensions at 20 W (10 min) and 50 W (3 × 3 min) measured using microdialysis [33], which resulted in a 16% improvement in high-intensity exercise capacity.\\nMost studies related to the acute impact of CAF intake on power output and bar velocity have used participants unhabituated to CAF or individuals with low-to-moderate daily consumption of this stimulant [11,16,34]. However, the analysis of urinary CAF concentrations after official competitions suggests that CAF is widely employed before or during exercise to enhance performance [35,36]. This would mean that it is highly likely that some athletes are habituated to CAF due to daily consumption of caffeine-containing products. The existence of athletes habituated to CAF may be particularly common in sports such as cycling, rowing, triathlon, athletics, and weightlifting, sport disciplines that benefit from the use of ballistic exercises during training to increase power output. Habitual CAF intake modifies physiological responses to acute ingestion of this stimulant by the up-regulation of adenosine receptors [37,38]. This effect would produce a progressive reduction of CAF ergogenicity in those athletes consuming CAF on a regular basis, because the newly created adenosine receptors may bind to adenosine and induce fatigue. However, the fact of habituation to the ergogenic benefits of CAF is still inconclusive. The studies by Dodd et al. [39] and de Souza [40] showed similar responses to endurance exercise after acute CAF intake between low and habitual CAF consumers, although this is not always the case [41]. Considering the above, the use of cross-sectional investigations including participants with different degrees of habituation to CAF may explain the lack of consistency when concluding about tolerance to the ergogenic effect of CAF. Lara’s et al. [42] crossover design showed that the ergogenicity of CAF was reduced when the substance was consumed daily for 20 days, yet afterwards the ergogenic properties of CAF were maintained. On the contrary, tolerance to some of the side-effects associated to CAF has been observed in habitual consumers of CAF [43]. However, only two previous studies analyzed the impact of CAF intake in habitual CAF consumers using resistance exercise test protocols [18,19,20]. These investigations indicate that CAF ergogenicity to power output is mostly reduced in individuals habituated to CAF, while only high doses (>9 mg/kg/b.m.) may exert some benefit in maximal strength [19,20]. However, to date, there is no available data regarding the influence of acute CAF intake on power output and bar velocity during ballistic exercises in athletes habitually consuming CAF.\\nGiven the widespread use of the BPT exercise as a mean of developing power output in the upper limbs [44,45] and the widespread use of CAF in sport, it would be interesting to investigate whether acute CAF intake affects power output and bar velocity in athletes habituated to CAF. For this reason, the aim of the present study was to evaluate the effects of the acute intake of 3 and 6 mg/kg/b.m. of CAF on power output and bar velocity during five sets of the BPT in participants habituated to CAF. It was hypothesized that acute CAF intake would increase power output and bar velocity during the BPT training session when compared to a control situation, even in participants habituated to CAF.\",\n \"A randomized, double-blind, PLAC-controlled crossover design was used for this investigation. Initially, the participants performed a familiarization session with the experimental protocols that included a 1 RM measurement for the bench press exercise. Afterwards, they performed 3 different experimental sessions with a one-week interval between sessions to allow for complete recovery and a wash-out of ingested substances (Figure 1). During the 3 experimental sessions, the study participants either ingested a PLAC, 3 mg/kg/b.m. of CAF (CAF-3) or 6 mg/kg/b.m. of CAF (CAF-6). One hour after ingestion of CAF or PLAC, they performed 5 sets of 2 repetitions of the BPT exercise at 30% 1 RM. Both CAF and PLAC were administered orally to allow for peak blood CAF concentration during the training session and at least 2 h after the last meal to avoid any interference of the diet with the absorption of the experimental substances. CAF supplementation was provided to participants in the form of unidentifiable capsules (Caffeine Kick®, Olimp Laboratories, Debica, Poland). The manufacturer of the CAF capsules also provided identical PLAC capsules filled with all-purpose flour. Participants refrained from strenuous physical activity the day before the experimental trials but they maintained their training routines during the duration of the experiment to avoid any performance decrement due to inactivity. Additionally, the participants maintained their dietary habits during the study period, including daily CAF intake. They received a list of products containing CAF which could not be consumed within 12 h of each experimental trial. Compliance was tested verbally and by using dietary records. Additionally, the participants were required to refrain from alcohol and tobacco, medications or dietary supplements for two weeks prior to the experiment. All subjects registered their calorie intake using the “Myfitness pal” software [46] (Under Armour, Baltimore, MD, USA) every 24 h before the testing procedure, to ensure that the caloric intake was similar between experimental sessions.\\n 2.1. Participants Twelve healthy strength-trained male athletes (age: 25.3 ± 1.7 years., body mass: 88.4 ± 16.5 kg, body mass index (BMI): 26.5 ± 4.7, bench press 1 RM: 128.6 ± 36.0 kg; mean ± SD) volunteered to participate in the study. All participants completed a written consent form after they had been informed of the risks and benefits of the study protocols. The participants had a minimum of 3 years of strength training experience (4.4 ± 1.6 years). All of them were classified as high habitual CAF consumers according to the classification recently proposed by de Souza Gonçalves et al. [40]. They self-reported their daily ingestion of CAF (5.0 ± 0.95 mg/kg/b.m./day, 443 ± 142 mg/day) based on a Food Frequency Questionnaire (FFQ). The inclusion criteria were as follows: (a) free from neuromuscular and musculoskeletal disorders, (b) 1 RM bench press performance with a load of at least 120% body mass, (c) habitual CAF intake in the range of 4–6 mg/day/kg/b.m. The athletes were excluded from the study when they suffered from any pathology or injury or when they were unable to perform the exercise protocol at the maximum effort. The investigation protocols were approved by the Bioethics Committee for Scientific Research at the Academy of Physical Education in Katowice (March 2019), Poland, according to the ethical standards of the latest version of the Declaration of Helsinki, 2013.\\nTwelve healthy strength-trained male athletes (age: 25.3 ± 1.7 years., body mass: 88.4 ± 16.5 kg, body mass index (BMI): 26.5 ± 4.7, bench press 1 RM: 128.6 ± 36.0 kg; mean ± SD) volunteered to participate in the study. All participants completed a written consent form after they had been informed of the risks and benefits of the study protocols. The participants had a minimum of 3 years of strength training experience (4.4 ± 1.6 years). All of them were classified as high habitual CAF consumers according to the classification recently proposed by de Souza Gonçalves et al. [40]. They self-reported their daily ingestion of CAF (5.0 ± 0.95 mg/kg/b.m./day, 443 ± 142 mg/day) based on a Food Frequency Questionnaire (FFQ). The inclusion criteria were as follows: (a) free from neuromuscular and musculoskeletal disorders, (b) 1 RM bench press performance with a load of at least 120% body mass, (c) habitual CAF intake in the range of 4–6 mg/day/kg/b.m. The athletes were excluded from the study when they suffered from any pathology or injury or when they were unable to perform the exercise protocol at the maximum effort. The investigation protocols were approved by the Bioethics Committee for Scientific Research at the Academy of Physical Education in Katowice (March 2019), Poland, according to the ethical standards of the latest version of the Declaration of Helsinki, 2013.\\n 2.2. Habitual Caffeine Intake Assessment Daily CAF intake was measured by an adapted version of the Food Frequency Questionnaire (FFQ) proposed by Bühler et al. [47]. Household measures were employed to individually assess the amount of food consumed during a day, week and month. The list was composed of dietary products with moderate-to-high CAF content including different types of coffee, tea, energy drinks, cocoa products, popular beverages, medications, and CAF supplements. Nutritional tables were used for database construction [48,49,50] and an experienced nutritionist calculated the daily CAF intake for each participant.\\nDaily CAF intake was measured by an adapted version of the Food Frequency Questionnaire (FFQ) proposed by Bühler et al. [47]. Household measures were employed to individually assess the amount of food consumed during a day, week and month. The list was composed of dietary products with moderate-to-high CAF content including different types of coffee, tea, energy drinks, cocoa products, popular beverages, medications, and CAF supplements. Nutritional tables were used for database construction [48,49,50] and an experienced nutritionist calculated the daily CAF intake for each participant.\\n 2.3. Familiarization Session and One Repetition Maximum Test A familiarization session with the experimental procedures preceded 1 RM testing in the bench press exercise. In this session, the athletes arrived at the laboratory between 9:00 and 10:00 am. and cycled on an ergometer for 5 min. Afterwards, they performed 15 repetitions at 20% of their estimated 1 RM in the barbell bench press exercise followed by 10 repetitions at 40% 1 RM, 5 repetitions at 60% 1 RM and 3 repetitions at 80% 1 RM. Then they executed single repetitions of the bench press exercise with a 5 min rest interval between successful attempts. The load for each subsequent attempt was increased by 2.5 to 10 kg, and the process was repeated until failure. Hand placement on the barbell was individually selected grip width (~150% individual bi-acromial distance). After completing the 1 RM test in the bench press exercise, the participants performed a maximal BPT on a Smith machine with a load of 30% 1 RM from 1 RM bench press test result, with a maximal tempo of movement.\\nA familiarization session with the experimental procedures preceded 1 RM testing in the bench press exercise. In this session, the athletes arrived at the laboratory between 9:00 and 10:00 am. and cycled on an ergometer for 5 min. Afterwards, they performed 15 repetitions at 20% of their estimated 1 RM in the barbell bench press exercise followed by 10 repetitions at 40% 1 RM, 5 repetitions at 60% 1 RM and 3 repetitions at 80% 1 RM. Then they executed single repetitions of the bench press exercise with a 5 min rest interval between successful attempts. The load for each subsequent attempt was increased by 2.5 to 10 kg, and the process was repeated until failure. Hand placement on the barbell was individually selected grip width (~150% individual bi-acromial distance). After completing the 1 RM test in the bench press exercise, the participants performed a maximal BPT on a Smith machine with a load of 30% 1 RM from 1 RM bench press test result, with a maximal tempo of movement.\\n 2.4. Experimental Sessions During experimental sessions, the athletes participated in three identical training trials. All trials took place between 9.00 and 11.00 am. to avoid the effect of circadian variations on the outcomes of the investigation. After replicating the warm-up procedures of the familiarization trial, the athletes performed 5 sets of the 2 BPT repetitions at 30% 1 RM on the Smith machine. The repetitions were performed without rest to produce a ballistic movement while the rest interval between sets was 3 min. The participants were encouraged to produce maximal velocity during both the eccentric and concentric phase of the BPT movement. Two spotters were present on each side of the bar during the exercise protocol to ensure safety. To standardize the exercise protocol for all trials, each BPT was performed without bouncing the barbell off the chest, with the lower back in contact with the bench and without any pause between the eccentric and concentric phases of the movement. A rotatory encoder (Tendo Power Analyzer, Tendo Sport Machines, Trencin, Slovakia) was used for instantaneous recording of bar velocity during the whole range of motion, as in previous investigations [51]. During each BPT, peak power output (PP, in W) mean power output (MP, in W); peak bar velocity (PV, in m/s); and mean bar velocity (MV, in m/s) were registered. MP and MV were obtained as the mean of the two repetitions while PP and PV were obtained from the best repetition.\\nDuring experimental sessions, the athletes participated in three identical training trials. All trials took place between 9.00 and 11.00 am. to avoid the effect of circadian variations on the outcomes of the investigation. After replicating the warm-up procedures of the familiarization trial, the athletes performed 5 sets of the 2 BPT repetitions at 30% 1 RM on the Smith machine. The repetitions were performed without rest to produce a ballistic movement while the rest interval between sets was 3 min. The participants were encouraged to produce maximal velocity during both the eccentric and concentric phase of the BPT movement. Two spotters were present on each side of the bar during the exercise protocol to ensure safety. To standardize the exercise protocol for all trials, each BPT was performed without bouncing the barbell off the chest, with the lower back in contact with the bench and without any pause between the eccentric and concentric phases of the movement. A rotatory encoder (Tendo Power Analyzer, Tendo Sport Machines, Trencin, Slovakia) was used for instantaneous recording of bar velocity during the whole range of motion, as in previous investigations [51]. During each BPT, peak power output (PP, in W) mean power output (MP, in W); peak bar velocity (PV, in m/s); and mean bar velocity (MV, in m/s) were registered. MP and MV were obtained as the mean of the two repetitions while PP and PV were obtained from the best repetition.\\n 2.5. Statistical Analysis Data are presented as the mean ± SD. All variables presented a normal distribution according to the Shapiro-Wilk test. Verification of differences in peak power output (PP), mean power output (MP), peak bar velocity (PV), and mean bar velocity (MV) was performed using a two-way (substance × set) analysis of variance (ANOVA) with repeated measures. In the event of a significant main effect, post-hoc comparisons were conducted using the Tukey’s test. Percent changes and 95% confidence intervals were also calculated. Effect sizes (Cohen’s d) were reported where appropriate and interpreted as large (d ≥ 0.80); moderate (d between 0.79 and 0.50); small (d between 0.49 and 0.20); and trivial (d < 0.20); [52].\\nData are presented as the mean ± SD. All variables presented a normal distribution according to the Shapiro-Wilk test. Verification of differences in peak power output (PP), mean power output (MP), peak bar velocity (PV), and mean bar velocity (MV) was performed using a two-way (substance × set) analysis of variance (ANOVA) with repeated measures. In the event of a significant main effect, post-hoc comparisons were conducted using the Tukey’s test. Percent changes and 95% confidence intervals were also calculated. Effect sizes (Cohen’s d) were reported where appropriate and interpreted as large (d ≥ 0.80); moderate (d between 0.79 and 0.50); small (d between 0.49 and 0.20); and trivial (d < 0.20); [52].\",\n \"Twelve healthy strength-trained male athletes (age: 25.3 ± 1.7 years., body mass: 88.4 ± 16.5 kg, body mass index (BMI): 26.5 ± 4.7, bench press 1 RM: 128.6 ± 36.0 kg; mean ± SD) volunteered to participate in the study. All participants completed a written consent form after they had been informed of the risks and benefits of the study protocols. The participants had a minimum of 3 years of strength training experience (4.4 ± 1.6 years). All of them were classified as high habitual CAF consumers according to the classification recently proposed by de Souza Gonçalves et al. [40]. They self-reported their daily ingestion of CAF (5.0 ± 0.95 mg/kg/b.m./day, 443 ± 142 mg/day) based on a Food Frequency Questionnaire (FFQ). The inclusion criteria were as follows: (a) free from neuromuscular and musculoskeletal disorders, (b) 1 RM bench press performance with a load of at least 120% body mass, (c) habitual CAF intake in the range of 4–6 mg/day/kg/b.m. The athletes were excluded from the study when they suffered from any pathology or injury or when they were unable to perform the exercise protocol at the maximum effort. The investigation protocols were approved by the Bioethics Committee for Scientific Research at the Academy of Physical Education in Katowice (March 2019), Poland, according to the ethical standards of the latest version of the Declaration of Helsinki, 2013.\",\n \"Daily CAF intake was measured by an adapted version of the Food Frequency Questionnaire (FFQ) proposed by Bühler et al. [47]. Household measures were employed to individually assess the amount of food consumed during a day, week and month. The list was composed of dietary products with moderate-to-high CAF content including different types of coffee, tea, energy drinks, cocoa products, popular beverages, medications, and CAF supplements. Nutritional tables were used for database construction [48,49,50] and an experienced nutritionist calculated the daily CAF intake for each participant.\",\n \"A familiarization session with the experimental procedures preceded 1 RM testing in the bench press exercise. In this session, the athletes arrived at the laboratory between 9:00 and 10:00 am. and cycled on an ergometer for 5 min. Afterwards, they performed 15 repetitions at 20% of their estimated 1 RM in the barbell bench press exercise followed by 10 repetitions at 40% 1 RM, 5 repetitions at 60% 1 RM and 3 repetitions at 80% 1 RM. Then they executed single repetitions of the bench press exercise with a 5 min rest interval between successful attempts. The load for each subsequent attempt was increased by 2.5 to 10 kg, and the process was repeated until failure. Hand placement on the barbell was individually selected grip width (~150% individual bi-acromial distance). After completing the 1 RM test in the bench press exercise, the participants performed a maximal BPT on a Smith machine with a load of 30% 1 RM from 1 RM bench press test result, with a maximal tempo of movement.\",\n \"During experimental sessions, the athletes participated in three identical training trials. All trials took place between 9.00 and 11.00 am. to avoid the effect of circadian variations on the outcomes of the investigation. After replicating the warm-up procedures of the familiarization trial, the athletes performed 5 sets of the 2 BPT repetitions at 30% 1 RM on the Smith machine. The repetitions were performed without rest to produce a ballistic movement while the rest interval between sets was 3 min. The participants were encouraged to produce maximal velocity during both the eccentric and concentric phase of the BPT movement. Two spotters were present on each side of the bar during the exercise protocol to ensure safety. To standardize the exercise protocol for all trials, each BPT was performed without bouncing the barbell off the chest, with the lower back in contact with the bench and without any pause between the eccentric and concentric phases of the movement. A rotatory encoder (Tendo Power Analyzer, Tendo Sport Machines, Trencin, Slovakia) was used for instantaneous recording of bar velocity during the whole range of motion, as in previous investigations [51]. During each BPT, peak power output (PP, in W) mean power output (MP, in W); peak bar velocity (PV, in m/s); and mean bar velocity (MV, in m/s) were registered. MP and MV were obtained as the mean of the two repetitions while PP and PV were obtained from the best repetition.\",\n \"Data are presented as the mean ± SD. All variables presented a normal distribution according to the Shapiro-Wilk test. Verification of differences in peak power output (PP), mean power output (MP), peak bar velocity (PV), and mean bar velocity (MV) was performed using a two-way (substance × set) analysis of variance (ANOVA) with repeated measures. In the event of a significant main effect, post-hoc comparisons were conducted using the Tukey’s test. Percent changes and 95% confidence intervals were also calculated. Effect sizes (Cohen’s d) were reported where appropriate and interpreted as large (d ≥ 0.80); moderate (d between 0.79 and 0.50); small (d between 0.49 and 0.20); and trivial (d < 0.20); [52].\",\n \"The two-way repeated measures ANOVA indicated no significant substance × set main interaction effect for MP (F = 1.19; p = 0.32); MV (F = 1.18; p = 0.32); PP (F = 1.05; p = 0.40); PV (F = 1.09; p = 0.38). However, there was a significant main effect of substance in MP (F = 7.27; p < 0.01) and MV (F = 6.75; p < 0.01). No statistically significant main effect of substance was revealed in PP (F = 2.91; p = 0.07) and PV (F = 2.63; p = 0.09; Table 1).\\nPost hoc analyses for main effect of substance indicated significant increases in MP (p < 0.01; ES = 0.14) and MV (p = 0.01; ES = 0.78) in BPT (mean of the 5 sets) after the intake of CAF-3 compared to PLAC as well as significant increases in MP (p = 0.01; ES = 0.13) and MV (p = 0.01; ES = 0.72) in the BPT (mean of the 5 sets) after the intake of CAF-6 compared to PLAC. There were no significant differences in MP and MV between the two doses of CAF (CAF-3 vs. CAF-6). The results of particular sets in MP, MV, PP, and PV as well ES between PLAC and CAF-3, CAF-6 in each set are presented in Table 2.\\nFigure 2 and Figure 3 represent the individual responses induced by CAF-3 and CAF-6, in comparison to the placebo, for MP and MV. The 11 out of 12 participants showed an increase in MP and MV after the ingestion of CAF-3, while CAF-6 produced higher values for MP and MV in 10 out of 12 participants.\\nThe y-axis represents the difference in mean bar velocity output during the 5 sets of BPT between PLAC–CAF-3; PLAC–CAF-6 for each individual.\",\n \"The main finding of this study was that acute CAF intake has a positive effect on MP and MV during a training session of the BPT performed at 30% 1 RM. Interestingly, both 3 and 6 mg/kg/b.m. doses of CAF had similar effectiveness in enhancing performance when compared to PLAC. Additionally, the ergogenic effect of CAF on MP and MV was evident in most of participants, as all of them responded by improving performance with either CAF-3 or CAF-6, even when they were catalogued as individuals habituated to CAF (Figure 2 and Figure 3). However, the study did not show significant changes in PP and PV after CAF intake with either dose of CAF (3 and 6 mg/kg/b.m.) compared to PLAC. These outcomes suggest that acute CAF intake in a moderate dose (from 3 to 6 mg/kg/b.m.) is effective in increasing mean power and bar velocity during the BPT without a significant influence on peak values of these variables. These results suggest that CAF can be effectively used to acutely improve this power-specific training routine even with individuals habituated to CAF, although the long-term training effects with CAF should be further investigated.\\nThe y-axis represents the difference in mean power output during the 5 sets of BPT between PLAC–CAF-3; PLAC–CAF-6 for each individual.\\nPrevious research showed that acute CAF intake increases power output during the bench press exercise [8,14,15,16]. However, most of these studies included only one set of the exercise which is not the habitual practice during sports training, where several sets of a particular exercise are performed in order to obtain significant adaptations derived from training. In presented study, the main effect of increase in MP and MV after the intake of CAF-3 and CAF-6 over the placebo has occurred for training session consisting several sets (Table 2). The ergogenic effect of CAF observed during the BPT is partly consistent with the results of previous findings [8,16]. However, it should be emphasized that this is the first study investigating the effects of CAF during a training session that includes several sets of a ballistic exercise. Experimental procedures with the use of CAF in which more than one set of an upper body exercise are used are scarce [18,53,54]. The study of Lane and Byrd [53] showed that the intake of 300 mg of CAF, representing 3.5 mg/kg/b.m. increased peak velocity during 10 sets of the bench press exercise at 80% 1 RM compared to PLAC. Wilk et al. [18] did not show significant changes after the intake of different doses of CAF (3, 6, and 9 mg/kg/b.m.) in both, mean and peak power output and bar velocity during the BP exercise at 50% 1 RM (3 sets of 5 repetitions), although this investigation was carried out in athletes habituated to CAF. No changes in mean and peak bar velocity after CAF intake of 150 mg, representing 1.74 mg/kg/b.m. were observed in a study by Lane et al. [54] where 10 sets of 3 repetitions of the bench press exercise were performed at 80% 1 RM. The current study is quite innovative because it is the first investigation geared to assess the effects of CAF intake on power output by using a ballistic upper body exercise with a low external load (30% 1 RM), geared for power training of athletes [55]. The current state of the literature, indicates that CAF is useful in increasing power during one or multiple sets of the bench press exercise when the dose ingested is >3 mg/kg/b.m., but it seems particularly effective when using low and moderate loads during an explosive exercise, such as the BPT.\\nThe overall increase of MP and MV during the training session of the BPT after ingestion of CAF-3 and CAF-6 can be also attributed to increased pre-exercise central excitability. Specifically, the pre-exercise ingestion of CAF would allow the athletes to maintain a certain amount of force even in the presence of biochemical changes within the working muscle that lead to fatigue [3]. Under this theory, CAF intake would allow a higher physical performance because it would help to maintain the neural response even in the presence of metabolic perturbations such as low muscle pH. This effect may be accompanied by reductions in interstitial potassium accumulation found after CAF intake [2], that ultimately leads to the maintenance of excitability during exercise [33,56]. In the central nervous system (CNS), CAF binds to adenosine receptors that influence the release of neurotransmitters, such as noradrenaline and acetylcholine [4,57,58,59] and consequently, increase muscle tension [60]. However, in the current investigation, this purported effect of CAF on CNS was not sufficient to enhance PP and PV during the BPT at 30% 1 RM. Thus, reduced fatigue through CAF-induced modulation of both peripheral and central neural processes may explain the obtained results and higher MP and MV of the bar during the BPT training session. Nevertheless, the association of the ergogenic effect with the mechanisms that allowed this ergogenic effect is speculative at this moment because no measurements were carried out to test the origin of caffeine’s ergogenic effects.\\nIt should be taken into consideration that the study participants in the current study were habitual CAF users. In contrast, most of the investigations aimed at determination of the ergogenic effect of CAF on muscle performance have selected individuals unhabituated to this stimulant or with low-to-moderate daily consumption of CAF (e.g., from 58 to 250 mg/day), [11,16,34], to avoid the effects that tolerance to CAF may. However, CAF is an ergogenic aid frequently used in training and competition and it is likely that some athletes seeking for ergogenic benefits of CAF are already habituated to this substance due to the chronic use of caffeine-containing supplements during training and competition. In fact, previous investigations have suggested that between 75% and 90% of athletes use CAF in competitive and training settings [35,36,61], suggesting that studies on the effect of acute CAF intake on physical performance during real training and competition settings are particularly important in athletes habituated to CAF. In this respect, previous research using well-controlled CAF treatments has suggested that the habitual intake of this stimulant may progressively reduce its ergogenic effect on exercise performance [42,62] and then, it has been speculated that the ergogenic effect of CAF could be dampened in habitual CAF users.\\nTo the authors’ knowledge, only three previous studies analyzed power output of the upper limbs in a group of participants habituated to CAF [9,18,19,20]. The study of Sabol et al. [9] showed an increase in medicine ball throwing distance after the acute intake of 6 mg/kg/b.m. of CAF but the doses of 2 and 4 mg/kg/b.m. did not show any differences with the PLAC. The study by Wilk et al. [18] did not show increases in power output and bar velocity during the bench press exercise in high habitual CAF users that ingested from 3 to 9 mg/kg/b.m. Although it has been theorized that the reduction in the ergogenic effects of CAF in habitual users can be modified using doses greater than the daily habitual intake [63], previous investigations indicate that athletes habituated to CAF do not benefit from the acute ingestion of CAF in doses above their habitual intake while the prevalence of side effects is greatly increased [19,20]. Interestingly, participants in the presented study self-reported their daily ingestion of CAF, which amounted to 5.0 ± 0.95 mg/kg/b.m., (443 ± 142 mg of CAF per day), and the acute CAF doses (especially CAF-3) and some performance enhancements were obtained even when de dose of CAF did not exceed the value of habitual consumption. In any case, although the current investigation found a positive effect of CAF on mean power output and mean bar velocity during the BPT in athletes habituated to CAF, it is still possible that the effect of this substance is higher in unhabituated individuals.\\nIn addition to its strengths, the current study presents limitations that should be addressed. Although the results showed a significant main effect on MP and MV after CAF intake, the direct causes of these changes cannot be determined and explained. The study did not include biochemical analysis which could explain the obtained results. In addition, blood samples were not obtained and thus, we have no data about serum CAF concentrations with each of the dosages of CAF employed in this investigation. Further, we did not analyze the genetic intolerance on CAF in the tested subjects. However, the participants of this study did not report any side effects after consuming CAF in the six months prior to the experiment. Due to the fact that the response to CAF is related to the individual tolerance of this substance [42], the dose [19,20], and gender [64] therefore the results of this study should only be translated to males habituated to CAF who use low to moderate CAF doses to enhance performance. Another limitation of the study was that the 1 RM test was performed using the barbell bench press exercise while the BPT was performed on a Smith machine during the experimental trials to increase the security of participants and investigators. Although there is a high transfer between the results obtained in both types of exercise, the calculation of loading would be more reliable if both evaluations were performed on the same resistance exercise. In any case, this limitation did not affect the outcomes of the investigation because the load was the same for all experimental trials.\",\n \"The results of the present study indicate that acute doses of CAF, between 3 and 6 mg/kg/b.m., ingested before the onset of an explosive resistance exercise produced an overall effect on mean power output and mean bar velocity during a BPT training session in a group of habitual CAF users. The main effect in mean power and bar velocity was found in several sets during the trial which may indicate that the use of CAF was effective in increasing performance in the whole training session. In contrast, no significant changes were observed for peak power output and peak bar velocity. These results suggest that the ingestion of CAF prior to ballistic exercise can enhance the outcomes of resistance training. However, the results of our study refer only to power output and bar velocity of the upper limbs during the BPT with an external load of 30% 1 RM and further investigations should consider the effect of CAF with different loads or the use of lower-body exercises.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["intro",null,"subjects",null,null,null,null,"results","discussion","conclusions"],"string":"[\n \"intro\",\n null,\n \"subjects\",\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\",\n \"conclusions\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["ballistic exercise","upper limbs","resistance exercise","ergogenic substances","sport performance"],"string":"[\n \"ballistic exercise\",\n \"upper limbs\",\n \"resistance exercise\",\n \"ergogenic substances\",\n \"sport performance\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"1. Introduction:\nCaffeine (CAF) is one of the most common substances used in sport which enhances physical performance [1]. Although CAF may affect various body tissues [2,3], there is a growing body of evidence in animal [4] and human models [5] sustaining the ability of CAF to act as an adenosine antagonist, as the main mechanism behind CAF ergogenic effect. Current research recommends doses of CAF ranging from 3 to 6 mg/kg body mass to elicit ergogenic benefits, while the time of ingestion (from 30 to 90 min before exercise) and the form of ingestion (pills, liquid, chewing gum) are less significant as CAF is rapidly absorbed after ingestion [6]. However, the optimal protocol of CAF supplementation may differ based on the type and duration of exercise, previous habituation to CAF and the type of muscle contraction [6,7,8,9,10].\nAcute CAF intake causes a slightly different response to upper and lower body exercise [10], despite the lack of a mechanisms explaining this phenomenon. A recent meta-analysis about the effects of CAF on muscle strength and power output found that CAF significantly improved upper but not lower body strength [7]. Although the outcomes of the first investigations were contradictory [11,12,13], more recent studies have found a clear effect of CAF on several forms of upper body muscle performance [14,15,16], when the used dose was at least 3 mg/kg/b.m. [17]. Interestingly, the effect of CAF on upper body muscle performance may be partially dampened in athletes habituated to CAF because the regular use of CAF-containing products [18] may develop tolerance to this substance. It must be noticed that this decreased ergogenic effect of CAF is not removed even with the ingestion of doses >9 mg/kg/b.m. [19,20].\nMost studies on the acute effects of CAF on upper body muscle performance used the bench press exercise. The bench press exercise is widely used as a means of developing strength and power of the upper limbs [21,22]. However, other authors, recommend the use of ballistic exercises for the development of upper body power, such as the bench press throw (BPT) [23,24]. Specifically, to increase power output, the loads ranging from 0% to 50% of one-repetition maximum (1 RM) moved at maximal speed are recommended as the most potent loading stimuli for improving power output [23]. However, this type of routine can only be performed on a Smith machine while using the BPT exercise (i.e., maximal bar velocity is obtained at the moment of throw). The traditional free-weight bench press exercise does not allow for the attainment of maximal velocity of execution (i.e., velocity equals 0 m/s at maximal arm extension). Thus, ballistic exercises could be an optimal choice for power training as they allow for greater velocity, and muscle activation in comparison to similar traditional resistance training routines [21]. Perhaps, the main asset of the BPT is the maximum acceleration of a given load, which ultimately produces high movement velocity in a short time [25]. In this respect, the loads applied in ballistic exercises during training will depend on the specific requirements of particular sport disciplines and will determine success in numerous power-based competitions [23]. Furthermore, the BPT performance has been associated with overall performance in different sport-specific tasks [26,27,28,29]. Therefore, it seems reasonable to use the BPT as a means of testing upper-body ballistic performance. Although the BPT is indicated as the most effective exercise for developing power of the upper limbs [30], previous studies have not determined the acute effects of CAF on power output and bar velocity during this type of exercise.\nBurke [31] has suggested that, in the current literature, there is a lack of data about the practical use of supplements in competitive sports because experimental protocols are often different from sports practice. In case of CAF, most studies considering the acute effects of this supplement were assessed on the basis of only a single set of an exercise [7,8,32], while real resistance training sessions in trained individuals rarely contain only one set of a particular exercise [31]. On the other hand, the investigations analyzing the acute effects of CAF on performance during successive sets of resistance exercises are scarce. Bowtell et al. [3] showed that pre-exercise CAF (6 mg/kg/b.m.) intake improved total exercise time during 5 sets of one-legged knee extensions performed to failure in comparison to a placebo (PLAC). It is worth noting that this ergogenic effect was achieved despite significantly lower muscle phosphocreatine concentration (PCr) and pH in the latter sets of an exercise in the CAF trial. Further, CAF ingestion (6 mg/kg/b.m.) attenuated the increase in interstitial potassium during one-legged knee extensions at 20 W (10 min) and 50 W (3 × 3 min) measured using microdialysis [33], which resulted in a 16% improvement in high-intensity exercise capacity.\nMost studies related to the acute impact of CAF intake on power output and bar velocity have used participants unhabituated to CAF or individuals with low-to-moderate daily consumption of this stimulant [11,16,34]. However, the analysis of urinary CAF concentrations after official competitions suggests that CAF is widely employed before or during exercise to enhance performance [35,36]. This would mean that it is highly likely that some athletes are habituated to CAF due to daily consumption of caffeine-containing products. The existence of athletes habituated to CAF may be particularly common in sports such as cycling, rowing, triathlon, athletics, and weightlifting, sport disciplines that benefit from the use of ballistic exercises during training to increase power output. Habitual CAF intake modifies physiological responses to acute ingestion of this stimulant by the up-regulation of adenosine receptors [37,38]. This effect would produce a progressive reduction of CAF ergogenicity in those athletes consuming CAF on a regular basis, because the newly created adenosine receptors may bind to adenosine and induce fatigue. However, the fact of habituation to the ergogenic benefits of CAF is still inconclusive. The studies by Dodd et al. [39] and de Souza [40] showed similar responses to endurance exercise after acute CAF intake between low and habitual CAF consumers, although this is not always the case [41]. Considering the above, the use of cross-sectional investigations including participants with different degrees of habituation to CAF may explain the lack of consistency when concluding about tolerance to the ergogenic effect of CAF. Lara’s et al. [42] crossover design showed that the ergogenicity of CAF was reduced when the substance was consumed daily for 20 days, yet afterwards the ergogenic properties of CAF were maintained. On the contrary, tolerance to some of the side-effects associated to CAF has been observed in habitual consumers of CAF [43]. However, only two previous studies analyzed the impact of CAF intake in habitual CAF consumers using resistance exercise test protocols [18,19,20]. These investigations indicate that CAF ergogenicity to power output is mostly reduced in individuals habituated to CAF, while only high doses (>9 mg/kg/b.m.) may exert some benefit in maximal strength [19,20]. However, to date, there is no available data regarding the influence of acute CAF intake on power output and bar velocity during ballistic exercises in athletes habitually consuming CAF.\nGiven the widespread use of the BPT exercise as a mean of developing power output in the upper limbs [44,45] and the widespread use of CAF in sport, it would be interesting to investigate whether acute CAF intake affects power output and bar velocity in athletes habituated to CAF. For this reason, the aim of the present study was to evaluate the effects of the acute intake of 3 and 6 mg/kg/b.m. of CAF on power output and bar velocity during five sets of the BPT in participants habituated to CAF. It was hypothesized that acute CAF intake would increase power output and bar velocity during the BPT training session when compared to a control situation, even in participants habituated to CAF.\n\n2. Materials and Methods:\nA randomized, double-blind, PLAC-controlled crossover design was used for this investigation. Initially, the participants performed a familiarization session with the experimental protocols that included a 1 RM measurement for the bench press exercise. Afterwards, they performed 3 different experimental sessions with a one-week interval between sessions to allow for complete recovery and a wash-out of ingested substances (Figure 1). During the 3 experimental sessions, the study participants either ingested a PLAC, 3 mg/kg/b.m. of CAF (CAF-3) or 6 mg/kg/b.m. of CAF (CAF-6). One hour after ingestion of CAF or PLAC, they performed 5 sets of 2 repetitions of the BPT exercise at 30% 1 RM. Both CAF and PLAC were administered orally to allow for peak blood CAF concentration during the training session and at least 2 h after the last meal to avoid any interference of the diet with the absorption of the experimental substances. CAF supplementation was provided to participants in the form of unidentifiable capsules (Caffeine Kick®, Olimp Laboratories, Debica, Poland). The manufacturer of the CAF capsules also provided identical PLAC capsules filled with all-purpose flour. Participants refrained from strenuous physical activity the day before the experimental trials but they maintained their training routines during the duration of the experiment to avoid any performance decrement due to inactivity. Additionally, the participants maintained their dietary habits during the study period, including daily CAF intake. They received a list of products containing CAF which could not be consumed within 12 h of each experimental trial. Compliance was tested verbally and by using dietary records. Additionally, the participants were required to refrain from alcohol and tobacco, medications or dietary supplements for two weeks prior to the experiment. All subjects registered their calorie intake using the “Myfitness pal” software [46] (Under Armour, Baltimore, MD, USA) every 24 h before the testing procedure, to ensure that the caloric intake was similar between experimental sessions.\n 2.1. Participants Twelve healthy strength-trained male athletes (age: 25.3 ± 1.7 years., body mass: 88.4 ± 16.5 kg, body mass index (BMI): 26.5 ± 4.7, bench press 1 RM: 128.6 ± 36.0 kg; mean ± SD) volunteered to participate in the study. All participants completed a written consent form after they had been informed of the risks and benefits of the study protocols. The participants had a minimum of 3 years of strength training experience (4.4 ± 1.6 years). All of them were classified as high habitual CAF consumers according to the classification recently proposed by de Souza Gonçalves et al. [40]. They self-reported their daily ingestion of CAF (5.0 ± 0.95 mg/kg/b.m./day, 443 ± 142 mg/day) based on a Food Frequency Questionnaire (FFQ). The inclusion criteria were as follows: (a) free from neuromuscular and musculoskeletal disorders, (b) 1 RM bench press performance with a load of at least 120% body mass, (c) habitual CAF intake in the range of 4–6 mg/day/kg/b.m. The athletes were excluded from the study when they suffered from any pathology or injury or when they were unable to perform the exercise protocol at the maximum effort. The investigation protocols were approved by the Bioethics Committee for Scientific Research at the Academy of Physical Education in Katowice (March 2019), Poland, according to the ethical standards of the latest version of the Declaration of Helsinki, 2013.\nTwelve healthy strength-trained male athletes (age: 25.3 ± 1.7 years., body mass: 88.4 ± 16.5 kg, body mass index (BMI): 26.5 ± 4.7, bench press 1 RM: 128.6 ± 36.0 kg; mean ± SD) volunteered to participate in the study. All participants completed a written consent form after they had been informed of the risks and benefits of the study protocols. The participants had a minimum of 3 years of strength training experience (4.4 ± 1.6 years). All of them were classified as high habitual CAF consumers according to the classification recently proposed by de Souza Gonçalves et al. [40]. They self-reported their daily ingestion of CAF (5.0 ± 0.95 mg/kg/b.m./day, 443 ± 142 mg/day) based on a Food Frequency Questionnaire (FFQ). The inclusion criteria were as follows: (a) free from neuromuscular and musculoskeletal disorders, (b) 1 RM bench press performance with a load of at least 120% body mass, (c) habitual CAF intake in the range of 4–6 mg/day/kg/b.m. The athletes were excluded from the study when they suffered from any pathology or injury or when they were unable to perform the exercise protocol at the maximum effort. The investigation protocols were approved by the Bioethics Committee for Scientific Research at the Academy of Physical Education in Katowice (March 2019), Poland, according to the ethical standards of the latest version of the Declaration of Helsinki, 2013.\n 2.2. Habitual Caffeine Intake Assessment Daily CAF intake was measured by an adapted version of the Food Frequency Questionnaire (FFQ) proposed by Bühler et al. [47]. Household measures were employed to individually assess the amount of food consumed during a day, week and month. The list was composed of dietary products with moderate-to-high CAF content including different types of coffee, tea, energy drinks, cocoa products, popular beverages, medications, and CAF supplements. Nutritional tables were used for database construction [48,49,50] and an experienced nutritionist calculated the daily CAF intake for each participant.\nDaily CAF intake was measured by an adapted version of the Food Frequency Questionnaire (FFQ) proposed by Bühler et al. [47]. Household measures were employed to individually assess the amount of food consumed during a day, week and month. The list was composed of dietary products with moderate-to-high CAF content including different types of coffee, tea, energy drinks, cocoa products, popular beverages, medications, and CAF supplements. Nutritional tables were used for database construction [48,49,50] and an experienced nutritionist calculated the daily CAF intake for each participant.\n 2.3. Familiarization Session and One Repetition Maximum Test A familiarization session with the experimental procedures preceded 1 RM testing in the bench press exercise. In this session, the athletes arrived at the laboratory between 9:00 and 10:00 am. and cycled on an ergometer for 5 min. Afterwards, they performed 15 repetitions at 20% of their estimated 1 RM in the barbell bench press exercise followed by 10 repetitions at 40% 1 RM, 5 repetitions at 60% 1 RM and 3 repetitions at 80% 1 RM. Then they executed single repetitions of the bench press exercise with a 5 min rest interval between successful attempts. The load for each subsequent attempt was increased by 2.5 to 10 kg, and the process was repeated until failure. Hand placement on the barbell was individually selected grip width (~150% individual bi-acromial distance). After completing the 1 RM test in the bench press exercise, the participants performed a maximal BPT on a Smith machine with a load of 30% 1 RM from 1 RM bench press test result, with a maximal tempo of movement.\nA familiarization session with the experimental procedures preceded 1 RM testing in the bench press exercise. In this session, the athletes arrived at the laboratory between 9:00 and 10:00 am. and cycled on an ergometer for 5 min. Afterwards, they performed 15 repetitions at 20% of their estimated 1 RM in the barbell bench press exercise followed by 10 repetitions at 40% 1 RM, 5 repetitions at 60% 1 RM and 3 repetitions at 80% 1 RM. Then they executed single repetitions of the bench press exercise with a 5 min rest interval between successful attempts. The load for each subsequent attempt was increased by 2.5 to 10 kg, and the process was repeated until failure. Hand placement on the barbell was individually selected grip width (~150% individual bi-acromial distance). After completing the 1 RM test in the bench press exercise, the participants performed a maximal BPT on a Smith machine with a load of 30% 1 RM from 1 RM bench press test result, with a maximal tempo of movement.\n 2.4. Experimental Sessions During experimental sessions, the athletes participated in three identical training trials. All trials took place between 9.00 and 11.00 am. to avoid the effect of circadian variations on the outcomes of the investigation. After replicating the warm-up procedures of the familiarization trial, the athletes performed 5 sets of the 2 BPT repetitions at 30% 1 RM on the Smith machine. The repetitions were performed without rest to produce a ballistic movement while the rest interval between sets was 3 min. The participants were encouraged to produce maximal velocity during both the eccentric and concentric phase of the BPT movement. Two spotters were present on each side of the bar during the exercise protocol to ensure safety. To standardize the exercise protocol for all trials, each BPT was performed without bouncing the barbell off the chest, with the lower back in contact with the bench and without any pause between the eccentric and concentric phases of the movement. A rotatory encoder (Tendo Power Analyzer, Tendo Sport Machines, Trencin, Slovakia) was used for instantaneous recording of bar velocity during the whole range of motion, as in previous investigations [51]. During each BPT, peak power output (PP, in W) mean power output (MP, in W); peak bar velocity (PV, in m/s); and mean bar velocity (MV, in m/s) were registered. MP and MV were obtained as the mean of the two repetitions while PP and PV were obtained from the best repetition.\nDuring experimental sessions, the athletes participated in three identical training trials. All trials took place between 9.00 and 11.00 am. to avoid the effect of circadian variations on the outcomes of the investigation. After replicating the warm-up procedures of the familiarization trial, the athletes performed 5 sets of the 2 BPT repetitions at 30% 1 RM on the Smith machine. The repetitions were performed without rest to produce a ballistic movement while the rest interval between sets was 3 min. The participants were encouraged to produce maximal velocity during both the eccentric and concentric phase of the BPT movement. Two spotters were present on each side of the bar during the exercise protocol to ensure safety. To standardize the exercise protocol for all trials, each BPT was performed without bouncing the barbell off the chest, with the lower back in contact with the bench and without any pause between the eccentric and concentric phases of the movement. A rotatory encoder (Tendo Power Analyzer, Tendo Sport Machines, Trencin, Slovakia) was used for instantaneous recording of bar velocity during the whole range of motion, as in previous investigations [51]. During each BPT, peak power output (PP, in W) mean power output (MP, in W); peak bar velocity (PV, in m/s); and mean bar velocity (MV, in m/s) were registered. MP and MV were obtained as the mean of the two repetitions while PP and PV were obtained from the best repetition.\n 2.5. Statistical Analysis Data are presented as the mean ± SD. All variables presented a normal distribution according to the Shapiro-Wilk test. Verification of differences in peak power output (PP), mean power output (MP), peak bar velocity (PV), and mean bar velocity (MV) was performed using a two-way (substance × set) analysis of variance (ANOVA) with repeated measures. In the event of a significant main effect, post-hoc comparisons were conducted using the Tukey’s test. Percent changes and 95% confidence intervals were also calculated. Effect sizes (Cohen’s d) were reported where appropriate and interpreted as large (d ≥ 0.80); moderate (d between 0.79 and 0.50); small (d between 0.49 and 0.20); and trivial (d < 0.20); [52].\nData are presented as the mean ± SD. All variables presented a normal distribution according to the Shapiro-Wilk test. Verification of differences in peak power output (PP), mean power output (MP), peak bar velocity (PV), and mean bar velocity (MV) was performed using a two-way (substance × set) analysis of variance (ANOVA) with repeated measures. In the event of a significant main effect, post-hoc comparisons were conducted using the Tukey’s test. Percent changes and 95% confidence intervals were also calculated. Effect sizes (Cohen’s d) were reported where appropriate and interpreted as large (d ≥ 0.80); moderate (d between 0.79 and 0.50); small (d between 0.49 and 0.20); and trivial (d < 0.20); [52].\n\n2.1. Participants:\nTwelve healthy strength-trained male athletes (age: 25.3 ± 1.7 years., body mass: 88.4 ± 16.5 kg, body mass index (BMI): 26.5 ± 4.7, bench press 1 RM: 128.6 ± 36.0 kg; mean ± SD) volunteered to participate in the study. All participants completed a written consent form after they had been informed of the risks and benefits of the study protocols. The participants had a minimum of 3 years of strength training experience (4.4 ± 1.6 years). All of them were classified as high habitual CAF consumers according to the classification recently proposed by de Souza Gonçalves et al. [40]. They self-reported their daily ingestion of CAF (5.0 ± 0.95 mg/kg/b.m./day, 443 ± 142 mg/day) based on a Food Frequency Questionnaire (FFQ). The inclusion criteria were as follows: (a) free from neuromuscular and musculoskeletal disorders, (b) 1 RM bench press performance with a load of at least 120% body mass, (c) habitual CAF intake in the range of 4–6 mg/day/kg/b.m. The athletes were excluded from the study when they suffered from any pathology or injury or when they were unable to perform the exercise protocol at the maximum effort. The investigation protocols were approved by the Bioethics Committee for Scientific Research at the Academy of Physical Education in Katowice (March 2019), Poland, according to the ethical standards of the latest version of the Declaration of Helsinki, 2013.\n\n2.2. Habitual Caffeine Intake Assessment:\nDaily CAF intake was measured by an adapted version of the Food Frequency Questionnaire (FFQ) proposed by Bühler et al. [47]. Household measures were employed to individually assess the amount of food consumed during a day, week and month. The list was composed of dietary products with moderate-to-high CAF content including different types of coffee, tea, energy drinks, cocoa products, popular beverages, medications, and CAF supplements. Nutritional tables were used for database construction [48,49,50] and an experienced nutritionist calculated the daily CAF intake for each participant.\n\n2.3. Familiarization Session and One Repetition Maximum Test:\nA familiarization session with the experimental procedures preceded 1 RM testing in the bench press exercise. In this session, the athletes arrived at the laboratory between 9:00 and 10:00 am. and cycled on an ergometer for 5 min. Afterwards, they performed 15 repetitions at 20% of their estimated 1 RM in the barbell bench press exercise followed by 10 repetitions at 40% 1 RM, 5 repetitions at 60% 1 RM and 3 repetitions at 80% 1 RM. Then they executed single repetitions of the bench press exercise with a 5 min rest interval between successful attempts. The load for each subsequent attempt was increased by 2.5 to 10 kg, and the process was repeated until failure. Hand placement on the barbell was individually selected grip width (~150% individual bi-acromial distance). After completing the 1 RM test in the bench press exercise, the participants performed a maximal BPT on a Smith machine with a load of 30% 1 RM from 1 RM bench press test result, with a maximal tempo of movement.\n\n2.4. Experimental Sessions:\nDuring experimental sessions, the athletes participated in three identical training trials. All trials took place between 9.00 and 11.00 am. to avoid the effect of circadian variations on the outcomes of the investigation. After replicating the warm-up procedures of the familiarization trial, the athletes performed 5 sets of the 2 BPT repetitions at 30% 1 RM on the Smith machine. The repetitions were performed without rest to produce a ballistic movement while the rest interval between sets was 3 min. The participants were encouraged to produce maximal velocity during both the eccentric and concentric phase of the BPT movement. Two spotters were present on each side of the bar during the exercise protocol to ensure safety. To standardize the exercise protocol for all trials, each BPT was performed without bouncing the barbell off the chest, with the lower back in contact with the bench and without any pause between the eccentric and concentric phases of the movement. A rotatory encoder (Tendo Power Analyzer, Tendo Sport Machines, Trencin, Slovakia) was used for instantaneous recording of bar velocity during the whole range of motion, as in previous investigations [51]. During each BPT, peak power output (PP, in W) mean power output (MP, in W); peak bar velocity (PV, in m/s); and mean bar velocity (MV, in m/s) were registered. MP and MV were obtained as the mean of the two repetitions while PP and PV were obtained from the best repetition.\n\n2.5. Statistical Analysis:\nData are presented as the mean ± SD. All variables presented a normal distribution according to the Shapiro-Wilk test. Verification of differences in peak power output (PP), mean power output (MP), peak bar velocity (PV), and mean bar velocity (MV) was performed using a two-way (substance × set) analysis of variance (ANOVA) with repeated measures. In the event of a significant main effect, post-hoc comparisons were conducted using the Tukey’s test. Percent changes and 95% confidence intervals were also calculated. Effect sizes (Cohen’s d) were reported where appropriate and interpreted as large (d ≥ 0.80); moderate (d between 0.79 and 0.50); small (d between 0.49 and 0.20); and trivial (d < 0.20); [52].\n\n3. Results:\nThe two-way repeated measures ANOVA indicated no significant substance × set main interaction effect for MP (F = 1.19; p = 0.32); MV (F = 1.18; p = 0.32); PP (F = 1.05; p = 0.40); PV (F = 1.09; p = 0.38). However, there was a significant main effect of substance in MP (F = 7.27; p < 0.01) and MV (F = 6.75; p < 0.01). No statistically significant main effect of substance was revealed in PP (F = 2.91; p = 0.07) and PV (F = 2.63; p = 0.09; Table 1).\nPost hoc analyses for main effect of substance indicated significant increases in MP (p < 0.01; ES = 0.14) and MV (p = 0.01; ES = 0.78) in BPT (mean of the 5 sets) after the intake of CAF-3 compared to PLAC as well as significant increases in MP (p = 0.01; ES = 0.13) and MV (p = 0.01; ES = 0.72) in the BPT (mean of the 5 sets) after the intake of CAF-6 compared to PLAC. There were no significant differences in MP and MV between the two doses of CAF (CAF-3 vs. CAF-6). The results of particular sets in MP, MV, PP, and PV as well ES between PLAC and CAF-3, CAF-6 in each set are presented in Table 2.\nFigure 2 and Figure 3 represent the individual responses induced by CAF-3 and CAF-6, in comparison to the placebo, for MP and MV. The 11 out of 12 participants showed an increase in MP and MV after the ingestion of CAF-3, while CAF-6 produced higher values for MP and MV in 10 out of 12 participants.\nThe y-axis represents the difference in mean bar velocity output during the 5 sets of BPT between PLAC–CAF-3; PLAC–CAF-6 for each individual.\n\n4. Discussion:\nThe main finding of this study was that acute CAF intake has a positive effect on MP and MV during a training session of the BPT performed at 30% 1 RM. Interestingly, both 3 and 6 mg/kg/b.m. doses of CAF had similar effectiveness in enhancing performance when compared to PLAC. Additionally, the ergogenic effect of CAF on MP and MV was evident in most of participants, as all of them responded by improving performance with either CAF-3 or CAF-6, even when they were catalogued as individuals habituated to CAF (Figure 2 and Figure 3). However, the study did not show significant changes in PP and PV after CAF intake with either dose of CAF (3 and 6 mg/kg/b.m.) compared to PLAC. These outcomes suggest that acute CAF intake in a moderate dose (from 3 to 6 mg/kg/b.m.) is effective in increasing mean power and bar velocity during the BPT without a significant influence on peak values of these variables. These results suggest that CAF can be effectively used to acutely improve this power-specific training routine even with individuals habituated to CAF, although the long-term training effects with CAF should be further investigated.\nThe y-axis represents the difference in mean power output during the 5 sets of BPT between PLAC–CAF-3; PLAC–CAF-6 for each individual.\nPrevious research showed that acute CAF intake increases power output during the bench press exercise [8,14,15,16]. However, most of these studies included only one set of the exercise which is not the habitual practice during sports training, where several sets of a particular exercise are performed in order to obtain significant adaptations derived from training. In presented study, the main effect of increase in MP and MV after the intake of CAF-3 and CAF-6 over the placebo has occurred for training session consisting several sets (Table 2). The ergogenic effect of CAF observed during the BPT is partly consistent with the results of previous findings [8,16]. However, it should be emphasized that this is the first study investigating the effects of CAF during a training session that includes several sets of a ballistic exercise. Experimental procedures with the use of CAF in which more than one set of an upper body exercise are used are scarce [18,53,54]. The study of Lane and Byrd [53] showed that the intake of 300 mg of CAF, representing 3.5 mg/kg/b.m. increased peak velocity during 10 sets of the bench press exercise at 80% 1 RM compared to PLAC. Wilk et al. [18] did not show significant changes after the intake of different doses of CAF (3, 6, and 9 mg/kg/b.m.) in both, mean and peak power output and bar velocity during the BP exercise at 50% 1 RM (3 sets of 5 repetitions), although this investigation was carried out in athletes habituated to CAF. No changes in mean and peak bar velocity after CAF intake of 150 mg, representing 1.74 mg/kg/b.m. were observed in a study by Lane et al. [54] where 10 sets of 3 repetitions of the bench press exercise were performed at 80% 1 RM. The current study is quite innovative because it is the first investigation geared to assess the effects of CAF intake on power output by using a ballistic upper body exercise with a low external load (30% 1 RM), geared for power training of athletes [55]. The current state of the literature, indicates that CAF is useful in increasing power during one or multiple sets of the bench press exercise when the dose ingested is >3 mg/kg/b.m., but it seems particularly effective when using low and moderate loads during an explosive exercise, such as the BPT.\nThe overall increase of MP and MV during the training session of the BPT after ingestion of CAF-3 and CAF-6 can be also attributed to increased pre-exercise central excitability. Specifically, the pre-exercise ingestion of CAF would allow the athletes to maintain a certain amount of force even in the presence of biochemical changes within the working muscle that lead to fatigue [3]. Under this theory, CAF intake would allow a higher physical performance because it would help to maintain the neural response even in the presence of metabolic perturbations such as low muscle pH. This effect may be accompanied by reductions in interstitial potassium accumulation found after CAF intake [2], that ultimately leads to the maintenance of excitability during exercise [33,56]. In the central nervous system (CNS), CAF binds to adenosine receptors that influence the release of neurotransmitters, such as noradrenaline and acetylcholine [4,57,58,59] and consequently, increase muscle tension [60]. However, in the current investigation, this purported effect of CAF on CNS was not sufficient to enhance PP and PV during the BPT at 30% 1 RM. Thus, reduced fatigue through CAF-induced modulation of both peripheral and central neural processes may explain the obtained results and higher MP and MV of the bar during the BPT training session. Nevertheless, the association of the ergogenic effect with the mechanisms that allowed this ergogenic effect is speculative at this moment because no measurements were carried out to test the origin of caffeine’s ergogenic effects.\nIt should be taken into consideration that the study participants in the current study were habitual CAF users. In contrast, most of the investigations aimed at determination of the ergogenic effect of CAF on muscle performance have selected individuals unhabituated to this stimulant or with low-to-moderate daily consumption of CAF (e.g., from 58 to 250 mg/day), [11,16,34], to avoid the effects that tolerance to CAF may. However, CAF is an ergogenic aid frequently used in training and competition and it is likely that some athletes seeking for ergogenic benefits of CAF are already habituated to this substance due to the chronic use of caffeine-containing supplements during training and competition. In fact, previous investigations have suggested that between 75% and 90% of athletes use CAF in competitive and training settings [35,36,61], suggesting that studies on the effect of acute CAF intake on physical performance during real training and competition settings are particularly important in athletes habituated to CAF. In this respect, previous research using well-controlled CAF treatments has suggested that the habitual intake of this stimulant may progressively reduce its ergogenic effect on exercise performance [42,62] and then, it has been speculated that the ergogenic effect of CAF could be dampened in habitual CAF users.\nTo the authors’ knowledge, only three previous studies analyzed power output of the upper limbs in a group of participants habituated to CAF [9,18,19,20]. The study of Sabol et al. [9] showed an increase in medicine ball throwing distance after the acute intake of 6 mg/kg/b.m. of CAF but the doses of 2 and 4 mg/kg/b.m. did not show any differences with the PLAC. The study by Wilk et al. [18] did not show increases in power output and bar velocity during the bench press exercise in high habitual CAF users that ingested from 3 to 9 mg/kg/b.m. Although it has been theorized that the reduction in the ergogenic effects of CAF in habitual users can be modified using doses greater than the daily habitual intake [63], previous investigations indicate that athletes habituated to CAF do not benefit from the acute ingestion of CAF in doses above their habitual intake while the prevalence of side effects is greatly increased [19,20]. Interestingly, participants in the presented study self-reported their daily ingestion of CAF, which amounted to 5.0 ± 0.95 mg/kg/b.m., (443 ± 142 mg of CAF per day), and the acute CAF doses (especially CAF-3) and some performance enhancements were obtained even when de dose of CAF did not exceed the value of habitual consumption. In any case, although the current investigation found a positive effect of CAF on mean power output and mean bar velocity during the BPT in athletes habituated to CAF, it is still possible that the effect of this substance is higher in unhabituated individuals.\nIn addition to its strengths, the current study presents limitations that should be addressed. Although the results showed a significant main effect on MP and MV after CAF intake, the direct causes of these changes cannot be determined and explained. The study did not include biochemical analysis which could explain the obtained results. In addition, blood samples were not obtained and thus, we have no data about serum CAF concentrations with each of the dosages of CAF employed in this investigation. Further, we did not analyze the genetic intolerance on CAF in the tested subjects. However, the participants of this study did not report any side effects after consuming CAF in the six months prior to the experiment. Due to the fact that the response to CAF is related to the individual tolerance of this substance [42], the dose [19,20], and gender [64] therefore the results of this study should only be translated to males habituated to CAF who use low to moderate CAF doses to enhance performance. Another limitation of the study was that the 1 RM test was performed using the barbell bench press exercise while the BPT was performed on a Smith machine during the experimental trials to increase the security of participants and investigators. Although there is a high transfer between the results obtained in both types of exercise, the calculation of loading would be more reliable if both evaluations were performed on the same resistance exercise. In any case, this limitation did not affect the outcomes of the investigation because the load was the same for all experimental trials.\n\n5. Conclusions:\nThe results of the present study indicate that acute doses of CAF, between 3 and 6 mg/kg/b.m., ingested before the onset of an explosive resistance exercise produced an overall effect on mean power output and mean bar velocity during a BPT training session in a group of habitual CAF users. The main effect in mean power and bar velocity was found in several sets during the trial which may indicate that the use of CAF was effective in increasing performance in the whole training session. In contrast, no significant changes were observed for peak power output and peak bar velocity. These results suggest that the ingestion of CAF prior to ballistic exercise can enhance the outcomes of resistance training. However, the results of our study refer only to power output and bar velocity of the upper limbs during the BPT with an external load of 30% 1 RM and further investigations should consider the effect of CAF with different loads or the use of lower-body exercises.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe main objective of the current investigation was to evaluate the effects of caffeine on power output and bar velocity during an explosive bench press throw in athletes habituated to caffeine.\n\nMethods:\nTwelve resistance trained individuals habituated to caffeine ingestion participated in a randomized double-blind experimental design. Each participant performed three identical experimental sessions 60 min after the intake of a placebo, 3, and 6 mg/kg/b.m. of caffeine. In each experimental session, the participants performed 5 sets of 2 repetitions of the bench press throw (with a load equivalent to 30% repetition maximum (RM), measured in a familiarization trial) on a Smith machine, while bar velocity and power output were registered with a rotatory encoder.\n\nResults:\nIn comparison to the placebo, the intake of caffeine increased mean bar velocity during 5 sets of the bench press throw (1.37 ± 0.05 vs. 1.41 ± 0.05 and 1.41 ± 0.06 m/s for placebo, 3, and 6 mg/kg/b.m., respectively; p < 0.01), as well as mean power output (545 ± 117 vs. 562 ± 118 and 560 ± 107 W; p < 0.01). However, caffeine was not effective at increasing peak velocity (p = 0.09) nor peak power output (p = 0.07) during the explosive exercise.\n\nConclusions:\nThe acute doses of caffeine before resistance exercise may increase mean power output and mean bar velocity during the bench press throw training session in a group of habitual caffeine users. Thus, caffeine prior to ballistic exercises enhances performance during a power-specific resistance training session."},"background_conclusion_text":{"kind":"string","value":"1. Introduction:\nCaffeine (CAF) is one of the most common substances used in sport which enhances physical performance [1]. Although CAF may affect various body tissues [2,3], there is a growing body of evidence in animal [4] and human models [5] sustaining the ability of CAF to act as an adenosine antagonist, as the main mechanism behind CAF ergogenic effect. Current research recommends doses of CAF ranging from 3 to 6 mg/kg body mass to elicit ergogenic benefits, while the time of ingestion (from 30 to 90 min before exercise) and the form of ingestion (pills, liquid, chewing gum) are less significant as CAF is rapidly absorbed after ingestion [6]. However, the optimal protocol of CAF supplementation may differ based on the type and duration of exercise, previous habituation to CAF and the type of muscle contraction [6,7,8,9,10].\nAcute CAF intake causes a slightly different response to upper and lower body exercise [10], despite the lack of a mechanisms explaining this phenomenon. A recent meta-analysis about the effects of CAF on muscle strength and power output found that CAF significantly improved upper but not lower body strength [7]. Although the outcomes of the first investigations were contradictory [11,12,13], more recent studies have found a clear effect of CAF on several forms of upper body muscle performance [14,15,16], when the used dose was at least 3 mg/kg/b.m. [17]. Interestingly, the effect of CAF on upper body muscle performance may be partially dampened in athletes habituated to CAF because the regular use of CAF-containing products [18] may develop tolerance to this substance. It must be noticed that this decreased ergogenic effect of CAF is not removed even with the ingestion of doses >9 mg/kg/b.m. [19,20].\nMost studies on the acute effects of CAF on upper body muscle performance used the bench press exercise. The bench press exercise is widely used as a means of developing strength and power of the upper limbs [21,22]. However, other authors, recommend the use of ballistic exercises for the development of upper body power, such as the bench press throw (BPT) [23,24]. Specifically, to increase power output, the loads ranging from 0% to 50% of one-repetition maximum (1 RM) moved at maximal speed are recommended as the most potent loading stimuli for improving power output [23]. However, this type of routine can only be performed on a Smith machine while using the BPT exercise (i.e., maximal bar velocity is obtained at the moment of throw). The traditional free-weight bench press exercise does not allow for the attainment of maximal velocity of execution (i.e., velocity equals 0 m/s at maximal arm extension). Thus, ballistic exercises could be an optimal choice for power training as they allow for greater velocity, and muscle activation in comparison to similar traditional resistance training routines [21]. Perhaps, the main asset of the BPT is the maximum acceleration of a given load, which ultimately produces high movement velocity in a short time [25]. In this respect, the loads applied in ballistic exercises during training will depend on the specific requirements of particular sport disciplines and will determine success in numerous power-based competitions [23]. Furthermore, the BPT performance has been associated with overall performance in different sport-specific tasks [26,27,28,29]. Therefore, it seems reasonable to use the BPT as a means of testing upper-body ballistic performance. Although the BPT is indicated as the most effective exercise for developing power of the upper limbs [30], previous studies have not determined the acute effects of CAF on power output and bar velocity during this type of exercise.\nBurke [31] has suggested that, in the current literature, there is a lack of data about the practical use of supplements in competitive sports because experimental protocols are often different from sports practice. In case of CAF, most studies considering the acute effects of this supplement were assessed on the basis of only a single set of an exercise [7,8,32], while real resistance training sessions in trained individuals rarely contain only one set of a particular exercise [31]. On the other hand, the investigations analyzing the acute effects of CAF on performance during successive sets of resistance exercises are scarce. Bowtell et al. [3] showed that pre-exercise CAF (6 mg/kg/b.m.) intake improved total exercise time during 5 sets of one-legged knee extensions performed to failure in comparison to a placebo (PLAC). It is worth noting that this ergogenic effect was achieved despite significantly lower muscle phosphocreatine concentration (PCr) and pH in the latter sets of an exercise in the CAF trial. Further, CAF ingestion (6 mg/kg/b.m.) attenuated the increase in interstitial potassium during one-legged knee extensions at 20 W (10 min) and 50 W (3 × 3 min) measured using microdialysis [33], which resulted in a 16% improvement in high-intensity exercise capacity.\nMost studies related to the acute impact of CAF intake on power output and bar velocity have used participants unhabituated to CAF or individuals with low-to-moderate daily consumption of this stimulant [11,16,34]. However, the analysis of urinary CAF concentrations after official competitions suggests that CAF is widely employed before or during exercise to enhance performance [35,36]. This would mean that it is highly likely that some athletes are habituated to CAF due to daily consumption of caffeine-containing products. The existence of athletes habituated to CAF may be particularly common in sports such as cycling, rowing, triathlon, athletics, and weightlifting, sport disciplines that benefit from the use of ballistic exercises during training to increase power output. Habitual CAF intake modifies physiological responses to acute ingestion of this stimulant by the up-regulation of adenosine receptors [37,38]. This effect would produce a progressive reduction of CAF ergogenicity in those athletes consuming CAF on a regular basis, because the newly created adenosine receptors may bind to adenosine and induce fatigue. However, the fact of habituation to the ergogenic benefits of CAF is still inconclusive. The studies by Dodd et al. [39] and de Souza [40] showed similar responses to endurance exercise after acute CAF intake between low and habitual CAF consumers, although this is not always the case [41]. Considering the above, the use of cross-sectional investigations including participants with different degrees of habituation to CAF may explain the lack of consistency when concluding about tolerance to the ergogenic effect of CAF. Lara’s et al. [42] crossover design showed that the ergogenicity of CAF was reduced when the substance was consumed daily for 20 days, yet afterwards the ergogenic properties of CAF were maintained. On the contrary, tolerance to some of the side-effects associated to CAF has been observed in habitual consumers of CAF [43]. However, only two previous studies analyzed the impact of CAF intake in habitual CAF consumers using resistance exercise test protocols [18,19,20]. These investigations indicate that CAF ergogenicity to power output is mostly reduced in individuals habituated to CAF, while only high doses (>9 mg/kg/b.m.) may exert some benefit in maximal strength [19,20]. However, to date, there is no available data regarding the influence of acute CAF intake on power output and bar velocity during ballistic exercises in athletes habitually consuming CAF.\nGiven the widespread use of the BPT exercise as a mean of developing power output in the upper limbs [44,45] and the widespread use of CAF in sport, it would be interesting to investigate whether acute CAF intake affects power output and bar velocity in athletes habituated to CAF. For this reason, the aim of the present study was to evaluate the effects of the acute intake of 3 and 6 mg/kg/b.m. of CAF on power output and bar velocity during five sets of the BPT in participants habituated to CAF. It was hypothesized that acute CAF intake would increase power output and bar velocity during the BPT training session when compared to a control situation, even in participants habituated to CAF.\n\n5. Conclusions:\nThe results of the present study indicate that acute doses of CAF, between 3 and 6 mg/kg/b.m., ingested before the onset of an explosive resistance exercise produced an overall effect on mean power output and mean bar velocity during a BPT training session in a group of habitual CAF users. The main effect in mean power and bar velocity was found in several sets during the trial which may indicate that the use of CAF was effective in increasing performance in the whole training session. In contrast, no significant changes were observed for peak power output and peak bar velocity. These results suggest that the ingestion of CAF prior to ballistic exercise can enhance the outcomes of resistance training. However, the results of our study refer only to power output and bar velocity of the upper limbs during the BPT with an external load of 30% 1 RM and further investigations should consider the effect of CAF with different loads or the use of lower-body exercises."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nThe main objective of the current investigation was to evaluate the effects of caffeine on power output and bar velocity during an explosive bench press throw in athletes habituated to caffeine.\n\nMethods:\nTwelve resistance trained individuals habituated to caffeine ingestion participated in a randomized double-blind experimental design. Each participant performed three identical experimental sessions 60 min after the intake of a placebo, 3, and 6 mg/kg/b.m. of caffeine. In each experimental session, the participants performed 5 sets of 2 repetitions of the bench press throw (with a load equivalent to 30% repetition maximum (RM), measured in a familiarization trial) on a Smith machine, while bar velocity and power output were registered with a rotatory encoder.\n\nResults:\nIn comparison to the placebo, the intake of caffeine increased mean bar velocity during 5 sets of the bench press throw (1.37 ± 0.05 vs. 1.41 ± 0.05 and 1.41 ± 0.06 m/s for placebo, 3, and 6 mg/kg/b.m., respectively; p < 0.01), as well as mean power output (545 ± 117 vs. 562 ± 118 and 560 ± 107 W; p < 0.01). However, caffeine was not effective at increasing peak velocity (p = 0.09) nor peak power output (p = 0.07) during the explosive exercise.\n\nConclusions:\nThe acute doses of caffeine before resistance exercise may increase mean power output and mean bar velocity during the bench press throw training session in a group of habitual caffeine users. Thus, caffeine prior to ballistic exercises enhances performance during a power-specific resistance training session."},"whole_article_text_length":{"kind":"number","value":7580,"string":"7,580"},"whole_article_abstract_length":{"kind":"number","value":312,"string":"312"},"other_sections_lengths":{"kind":"list like","value":[2491,108,195,284,160],"string":"[\n 2491,\n 108,\n 195,\n 284,\n 160\n]","isConversational":false},"num_sections":{"kind":"number","value":10,"string":"10"},"most_frequent_words":{"kind":"list like","value":["caf","exercise","power","rm","intake","bpt","velocity","effect","kg","bar"],"string":"[\n \"caf\",\n \"exercise\",\n \"power\",\n \"rm\",\n \"intake\",\n \"bpt\",\n \"velocity\",\n \"effect\",\n \"kg\",\n \"bar\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["caf ingestion","caf muscle performance","caffeine ergogenic effects","effect caf muscle","exercise ingestion caf"],"string":"[\n \"caf ingestion\",\n \"caf muscle 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[SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] caf ingestion | caf muscle performance | caffeine ergogenic effects | effect caf muscle | exercise ingestion caf [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] caf ingestion | caf muscle performance | caffeine ergogenic effects | effect caf muscle | exercise ingestion caf [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] caf | exercise | power | rm | intake | bpt | velocity | effect | kg | bar [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] caf | exercise | power | rm | intake | bpt | velocity | effect | kg | bar [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] caf | exercise | power | rm | intake | bpt | velocity | 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| power output | training [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] caf | exercise | power | rm | velocity | bar | repetitions | mean | effect | bpt [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] caf | exercise | power | rm | velocity | bar | repetitions | mean | effect | bpt [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 5 | 1.37 | 1.41 | 0.05 | 1.41 | 0.06 | 3 | 6 mg/kg/b.m | 545 | 117 | 562 | 118 | 560 | 107 ||| 0.09 | 0.07 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Twelve ||| three | 60 | 3 | 6 mg/kg/b.m ||| 5 | 2 | 30% | RM | Smith ||| ||| 5 | 1.37 | 1.41 | 0.05 | 1.41 | 0.06 | 3 | 6 mg/kg/b.m | 545 | 117 | 562 | 118 | 560 | 107 ||| 0.09 | 0.07 ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Twelve ||| three | 60 | 3 | 6 mg/kg/b.m ||| 5 | 2 | 30% | RM | Smith ||| ||| 5 | 1.37 | 1.41 | 0.05 | 1.41 | 0.06 | 3 | 6 mg/kg/b.m | 545 | 117 | 562 | 118 | 560 | 107 ||| 0.09 | 0.07 ||| ||| [SUMMARY]"}}},{"rowIdx":67,"cells":{"title":{"kind":"string","value":"The varicella vaccination pattern among children under 5 years old in selected areas in China."},"pmid":{"kind":"string","value":"28487494"},"background_abstract":{"kind":"string","value":"Vaccine is the most effective way to protect susceptible children from varicella. Few published literature or reports on varicella vaccination of Chinese children exist. Thus, in order to obtain specific information on varicella vaccination of this population, we conducted this survey."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We first used purposive sampling methods to select 6 provinces 10 counties from eastern, middle and western parts of China with high quality of Immunization Information Management System (IIMS), and then randomly select children from population in the IIMS, then we checked vaccination certificate on-site."},"methods_abstract_label":{"kind":"string","value":"METHODOLOGY"},"results_abstract":{"kind":"string","value":"Based on the varicella vaccination information collected from 481 children's vaccination certificates from all ten selected counties in China, overall coverage of the first dose of varicella vaccine was 73.6%. There is a positive linear correlation between per capita GDP and vaccine coverage at county level (r=0.929, P < 0.01). The cumulative vaccine coverage among children at 1 year, 2 years and ≥3 years old were 67.6%, 71.9% and 73.6% respectively (X2=4.53, P =0.10). The age of vaccination was mainly concentrated in 12-17 months."},"results_abstract_label":{"kind":"string","value":"PRINCIPAL FINDINGS"},"conclusions_abstract":{"kind":"string","value":"The coverage rate of the first dose of varicella vaccine in selected areas was lower than that recommended by WHO position paper. The coverage rate was relatively low in areas of low social-economic status. The cumulative coverage had no significant statistical difference among different age group. Most children received varicella vaccine before 3 years old. We suggest introducing the varicella vaccine into routine immunization program, to ensure universal high coverage among children in China. We also suggest that varicella vaccination information should be checked before entering school, in order to control and prevent varicella outbreaks in schools."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Chickenpox","Chickenpox Vaccine","Child, Preschool","China","Geography, Medical","Humans","Infant","Infant, Newborn","Population Surveillance","Seasons","Socioeconomic Factors","Vaccination"],"string":"[\n \"Chickenpox\",\n \"Chickenpox Vaccine\",\n \"Child, Preschool\",\n \"China\",\n \"Geography, Medical\",\n \"Humans\",\n \"Infant\",\n \"Infant, Newborn\",\n \"Population Surveillance\",\n \"Seasons\",\n \"Socioeconomic Factors\",\n \"Vaccination\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"5542212"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Varicella is an infectious disease with high transmissibility, common in children [1]. The disease is prone to resulting in large scale outbreak in institutional units, such as nurseries, kindergartens, and schools [2]. Varicella vaccine has been demonstrated good safety and effect [3], and varicella vaccination is the most effective measure to protect susceptible people from the disease. Although varicella case information has been collected nationally since 2005 through National Disease Supervision Information Management System(NDSIMS), its limitation in quality of local clinic reporting poses a challenge in accurate analysis and evaluation of the true situation in China [4]. Based on the estimation of varicella incidence in Shandong, Gansu and Hunan provinces, 4,705,000 cases were reported in 2007 in China, and the cost estimation in 2007 was 2.31 billion RMB for outpatients and 103 million RMB for inpatients [5, 6]. “Varicella and Herpes Zoster Vaccines: WHO Position Paper” recommends that countries where varicella is an important public health burden could consider introducing varicella vaccination in the routine childhood immunization programme. Resources should be sufficient to ensure reaching and sustaining vaccine coverage ≥ 80%. Vaccine coverage that remains <80% over the long term is expected to shift varicella infection to older ages in some settings, which may result in an increase of morbidity and mortality despite reduction in total number of cases [3]. Varicella vaccine was licensed in China in 1996 [7]. Currently, varicella vaccine is sold on the private market, meaning it is voluntary and must be self-paid. There is no national recommended immunization schedule available for varicella vaccine, and the vaccine is administered to eligible children at a vaccination clinic according to instructions provided by the vaccine manufacturers. Based on the immunization regulation, the record of vaccination information is kept by both the parents and the clinic [8–9]. In this study, we investigated the varicella vaccination information among children under 5 years old in selected areas in China, in order to understand the varicella vaccination situation for children in China. This information will provide evidence for authority department of China introducing varicella vaccine into the national routine immunization program."},"methods_title":{"kind":"string","value":"MATERIALS AND METHODS"},"methods_text":{"kind":"string","value":" Sampling methods We used purposive sampling methods to select counties (districts) from Shanghai, Jiangsu, Heilongjiang, Jiangxi, Chongqing and Gansu provinces. We selected these investigation sites based on two factors: (i) representation of east, middle and western parts of China; (ii) having good records of private vaccinations in children's vaccination certificates and with a high quality Immunization Information Management System (IIMS). From Nov 1th 2013 to Nov 15th 2013, we used the simple random sampling method to randomly select children from the population whose birth date was between Jan 1st 2008 and Dec 31th 2012 in the IIMS in selected counties (districts), then from January 1th 2014 to July 15th2014, we checked hand-held vaccination certificates on-site to collect varicella vaccination information.\nWe used the following formula to calculate the sample size:\nn=(ta/2Δp)2P(1−P),ta/2=1.96,Δp=0.0045,p=0.45\nn: the sample size, P: estimate coverage of varicella vaccine, based on past experience, we used 45%. After calculation, a sample size of 470 was needed for this survey.\nConsidering a loss to follow-up of about 10%, a total of 520 children needed to be investigated. The sample size was divided by 10 (10 counties), and 52 children in each county were selected to participate in the investigation. Finally, 39 children were excluded because their parents did not have a hand-held vaccination certificate or we were unable to contact them.\nWe used purposive sampling methods to select counties (districts) from Shanghai, Jiangsu, Heilongjiang, Jiangxi, Chongqing and Gansu provinces. We selected these investigation sites based on two factors: (i) representation of east, middle and western parts of China; (ii) having good records of private vaccinations in children's vaccination certificates and with a high quality Immunization Information Management System (IIMS). From Nov 1th 2013 to Nov 15th 2013, we used the simple random sampling method to randomly select children from the population whose birth date was between Jan 1st 2008 and Dec 31th 2012 in the IIMS in selected counties (districts), then from January 1th 2014 to July 15th2014, we checked hand-held vaccination certificates on-site to collect varicella vaccination information.\nWe used the following formula to calculate the sample size:\nn=(ta/2Δp)2P(1−P),ta/2=1.96,Δp=0.0045,p=0.45\nn: the sample size, P: estimate coverage of varicella vaccine, based on past experience, we used 45%. After calculation, a sample size of 470 was needed for this survey.\nConsidering a loss to follow-up of about 10%, a total of 520 children needed to be investigated. The sample size was divided by 10 (10 counties), and 52 children in each county were selected to participate in the investigation. Finally, 39 children were excluded because their parents did not have a hand-held vaccination certificate or we were unable to contact them.\n Calculation of per capita gross domestic product (GDP) Per capita GDP=GDP/domicile population. GDP and household population data came from the “2012 Per Capita National Economic and Social Development Statistical Bulletin” on the selected County or District's Bureau of Statistics website. The data was not available for Jianye in Jiangsu, Ning'an in Heilongjiang, Chengguan and Qilihe in Gansu, so the per capita GDP in the corresponding city level (higher administrative level) was used for these counties.\nPer capita GDP=GDP/domicile population. GDP and household population data came from the “2012 Per Capita National Economic and Social Development Statistical Bulletin” on the selected County or District's Bureau of Statistics website. The data was not available for Jianye in Jiangsu, Ning'an in Heilongjiang, Chengguan and Qilihe in Gansu, so the per capita GDP in the corresponding city level (higher administrative level) was used for these counties.\n Survey content and data processing We conducted this survey to obtain the basic information of children (such as gender, date of birth) and varicella vaccination (such as inoculation date and doses). We used Excel to enter the data, ArcGIS to draw the map, and Epi Info software to analyze the data.\nWe conducted this survey to obtain the basic information of children (such as gender, date of birth) and varicella vaccination (such as inoculation date and doses). We used Excel to enter the data, ArcGIS to draw the map, and Epi Info software to analyze the data.\n Ethics statement We obtained oral informed consent from all children's guardians before the investigation, and two investigators checked vaccination certificates on-site together. We would continue the investigation only when the children's guardians gave consent. We didn't go through the institutional review board approval in the case of this study for ethical issues, because monitoring vaccine coverage is part of routine program work, in this context, there is no risk for participants and it is not required to get IRB approval. However, we strictly protected the private information of participants in the field, such as the name and contact information. During the analysis, we also took out the personal identification information.\nWe obtained oral informed consent from all children's guardians before the investigation, and two investigators checked vaccination certificates on-site together. We would continue the investigation only when the children's guardians gave consent. We didn't go through the institutional review board approval in the case of this study for ethical issues, because monitoring vaccine coverage is part of routine program work, in this context, there is no risk for participants and it is not required to get IRB approval. However, we strictly protected the private information of participants in the field, such as the name and contact information. During the analysis, we also took out the personal identification information."},"results_title":{"kind":"null"},"results_text":{"kind":"null"},"conclusions_title":{"kind":"string","value":"DISCUSSION"},"conclusions_text":{"kind":"string","value":"We obtained oral informed consent from all children's guardians before the investigation, and two investigators checked vaccination certificates on-site together. We would continue the investigation only when the children's guardians gave consent. We didn't go through the institutional review board approval in the case of this study for ethical issues, because monitoring vaccine coverage is part of routine program work, in this context, there is no risk for participants and it is not required to get IRB approval. However, we strictly protected the private information of participants in the field, such as the name and contact information. During the analysis, we also took out the personal identification information."},"other_sections_titles":{"kind":"list like","value":["INTRODUCTION","RESULTS","Basic information and vaccine coverage in selected counties","Relationship between varicella vaccine coverage and the economic situation","Cumulative coverage of varicella vaccine and distribution of vaccinated age","DISCUSSION"],"string":"[\n \"INTRODUCTION\",\n \"RESULTS\",\n \"Basic information and vaccine coverage in selected counties\",\n \"Relationship between varicella vaccine coverage and the economic situation\",\n \"Cumulative coverage of varicella vaccine and distribution of vaccinated age\",\n \"DISCUSSION\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Varicella is an infectious disease with high transmissibility, common in children [1]. The disease is prone to resulting in large scale outbreak in institutional units, such as nurseries, kindergartens, and schools [2]. Varicella vaccine has been demonstrated good safety and effect [3], and varicella vaccination is the most effective measure to protect susceptible people from the disease. Although varicella case information has been collected nationally since 2005 through National Disease Supervision Information Management System(NDSIMS), its limitation in quality of local clinic reporting poses a challenge in accurate analysis and evaluation of the true situation in China [4]. Based on the estimation of varicella incidence in Shandong, Gansu and Hunan provinces, 4,705,000 cases were reported in 2007 in China, and the cost estimation in 2007 was 2.31 billion RMB for outpatients and 103 million RMB for inpatients [5, 6]. “Varicella and Herpes Zoster Vaccines: WHO Position Paper” recommends that countries where varicella is an important public health burden could consider introducing varicella vaccination in the routine childhood immunization programme. Resources should be sufficient to ensure reaching and sustaining vaccine coverage ≥ 80%. Vaccine coverage that remains <80% over the long term is expected to shift varicella infection to older ages in some settings, which may result in an increase of morbidity and mortality despite reduction in total number of cases [3]. Varicella vaccine was licensed in China in 1996 [7]. Currently, varicella vaccine is sold on the private market, meaning it is voluntary and must be self-paid. There is no national recommended immunization schedule available for varicella vaccine, and the vaccine is administered to eligible children at a vaccination clinic according to instructions provided by the vaccine manufacturers. Based on the immunization regulation, the record of vaccination information is kept by both the parents and the clinic [8–9]. In this study, we investigated the varicella vaccination information among children under 5 years old in selected areas in China, in order to understand the varicella vaccination situation for children in China. This information will provide evidence for authority department of China introducing varicella vaccine into the national routine immunization program."," Basic information and vaccine coverage in selected counties In total, we collected varicella vaccination information from 481 caregivers’ vaccination certificates: 96 in Shanghai, 48 in Jiangsu, 96 in Heilongjiang, 52 in Jiangxi, 93 in Gansu and 96 in Chongqing, the geographical distribution of selected provinces see Figure 1; Male: 263 and female: 218, with a male to female ratio of 1.2:1. The varicella vaccine coverage of the first dose was 73.6% (354/481) for all children surveyed. The coverage in selected counties is described in Table 1. 1.2% (6/481) received the second dose of varicella vaccine.\nIn total, we collected varicella vaccination information from 481 caregivers’ vaccination certificates: 96 in Shanghai, 48 in Jiangsu, 96 in Heilongjiang, 52 in Jiangxi, 93 in Gansu and 96 in Chongqing, the geographical distribution of selected provinces see Figure 1; Male: 263 and female: 218, with a male to female ratio of 1.2:1. The varicella vaccine coverage of the first dose was 73.6% (354/481) for all children surveyed. The coverage in selected counties is described in Table 1. 1.2% (6/481) received the second dose of varicella vaccine.\n Relationship between varicella vaccine coverage and the economic situation The median coverage of varicella vaccine in areas with per capita GDP ≥ 100,000 RMB is 92.7% (85.4% - 97.9%), while median coverage of varicella vaccine in areas with per capita GDP < 100,000RMB is 55.9% (44.2% - 80.9%) (Wilcoxon rank sum test P < 0.05). There is a positive linear correlation between per capita GDP and vaccine coverage at county level (r = 0.929, P < 0.01). The equation of linear regression is y = 0.0004x + 37.08 (F = 50.426, P < 0.001) (Figure 2).\nThe median coverage of varicella vaccine in areas with per capita GDP ≥ 100,000 RMB is 92.7% (85.4% - 97.9%), while median coverage of varicella vaccine in areas with per capita GDP < 100,000RMB is 55.9% (44.2% - 80.9%) (Wilcoxon rank sum test P < 0.05). There is a positive linear correlation between per capita GDP and vaccine coverage at county level (r = 0.929, P < 0.01). The equation of linear regression is y = 0.0004x + 37.08 (F = 50.426, P < 0.001) (Figure 2).\n Cumulative coverage of varicella vaccine and distribution of vaccinated age At age of 1 year old(12-23 months), the vaccine coverage were 70.8%, 70.0%, 65.7%, 70.0%, 60.5% among children born in 2008, 2009, 2010, 2011 and 2012 respectively with no significant difference (X 2 = 3.15, P =0.53). The cumulative vaccine coverage ≥3 years old (≥ 36 months) were 79.2%, 78.0% and 74.7 % among children born in 2008, 2009 and 2010 respectively with no significant difference (X 2=0.59, P =0.75). The children born in 2012 were under 2 years of age during investigation for whom the vaccine coverage was lower (60.5%). In total, the cumulative coverage among children at 1 year, 2 years(24-35 months) and≥3 years old were 67.6%, 71.9% and 73.6% (X 2=4.53, P =0.10), with 4.3% increasing at 2 years old, and 1.7% at 3 years old and later compared to 1 year old (Table 2).\nNote:* 1 year old=”12-23 months”, 2 years old=”24-35 months”, ≥3 years old=”≥36 months”\nAmong all vaccinated children, 91.8% (325/354) received the vaccine at 1 year old, 5.9% (21/354) received at 2 years old, and 2.3% (8/354) at 3 years old and later. Among those receiving the vaccine at 1 year old, the vaccinated age was mainly concentrated in 12-17 months, accounting for 85.3% (302/325) (Figure 3).\nn=(ta/2Δp)2P(1−P),ta/2=1.96,Δp=0.0045,p=0.45\nAt age of 1 year old(12-23 months), the vaccine coverage were 70.8%, 70.0%, 65.7%, 70.0%, 60.5% among children born in 2008, 2009, 2010, 2011 and 2012 respectively with no significant difference (X 2 = 3.15, P =0.53). The cumulative vaccine coverage ≥3 years old (≥ 36 months) were 79.2%, 78.0% and 74.7 % among children born in 2008, 2009 and 2010 respectively with no significant difference (X 2=0.59, P =0.75). The children born in 2012 were under 2 years of age during investigation for whom the vaccine coverage was lower (60.5%). In total, the cumulative coverage among children at 1 year, 2 years(24-35 months) and≥3 years old were 67.6%, 71.9% and 73.6% (X 2=4.53, P =0.10), with 4.3% increasing at 2 years old, and 1.7% at 3 years old and later compared to 1 year old (Table 2).\nNote:* 1 year old=”12-23 months”, 2 years old=”24-35 months”, ≥3 years old=”≥36 months”\nAmong all vaccinated children, 91.8% (325/354) received the vaccine at 1 year old, 5.9% (21/354) received at 2 years old, and 2.3% (8/354) at 3 years old and later. Among those receiving the vaccine at 1 year old, the vaccinated age was mainly concentrated in 12-17 months, accounting for 85.3% (302/325) (Figure 3).\nn=(ta/2Δp)2P(1−P),ta/2=1.96,Δp=0.0045,p=0.45","In total, we collected varicella vaccination information from 481 caregivers’ vaccination certificates: 96 in Shanghai, 48 in Jiangsu, 96 in Heilongjiang, 52 in Jiangxi, 93 in Gansu and 96 in Chongqing, the geographical distribution of selected provinces see Figure 1; Male: 263 and female: 218, with a male to female ratio of 1.2:1. The varicella vaccine coverage of the first dose was 73.6% (354/481) for all children surveyed. The coverage in selected counties is described in Table 1. 1.2% (6/481) received the second dose of varicella vaccine.","The median coverage of varicella vaccine in areas with per capita GDP ≥ 100,000 RMB is 92.7% (85.4% - 97.9%), while median coverage of varicella vaccine in areas with per capita GDP < 100,000RMB is 55.9% (44.2% - 80.9%) (Wilcoxon rank sum test P < 0.05). There is a positive linear correlation between per capita GDP and vaccine coverage at county level (r = 0.929, P < 0.01). The equation of linear regression is y = 0.0004x + 37.08 (F = 50.426, P < 0.001) (Figure 2).","At age of 1 year old(12-23 months), the vaccine coverage were 70.8%, 70.0%, 65.7%, 70.0%, 60.5% among children born in 2008, 2009, 2010, 2011 and 2012 respectively with no significant difference (X 2 = 3.15, P =0.53). The cumulative vaccine coverage ≥3 years old (≥ 36 months) were 79.2%, 78.0% and 74.7 % among children born in 2008, 2009 and 2010 respectively with no significant difference (X 2=0.59, P =0.75). The children born in 2012 were under 2 years of age during investigation for whom the vaccine coverage was lower (60.5%). In total, the cumulative coverage among children at 1 year, 2 years(24-35 months) and≥3 years old were 67.6%, 71.9% and 73.6% (X 2=4.53, P =0.10), with 4.3% increasing at 2 years old, and 1.7% at 3 years old and later compared to 1 year old (Table 2).\nNote:* 1 year old=”12-23 months”, 2 years old=”24-35 months”, ≥3 years old=”≥36 months”\nAmong all vaccinated children, 91.8% (325/354) received the vaccine at 1 year old, 5.9% (21/354) received at 2 years old, and 2.3% (8/354) at 3 years old and later. Among those receiving the vaccine at 1 year old, the vaccinated age was mainly concentrated in 12-17 months, accounting for 85.3% (302/325) (Figure 3).\nn=(ta/2Δp)2P(1−P),ta/2=1.96,Δp=0.0045,p=0.45","The basic reproduction number (R0) is about 8 ∼ 10 for varicella, which is higher than influenza [10], it is easy for varicella to cause an outbreak in a nursery, kindergarten or school. Therefore, it is very difficult to block the spreading of varicella disease in areas with low vaccine coverage, and it is necessary to maintain high coverage to prevent the disease from becoming an epidemic. The World Health Organization position paper states that varicella may transit to older age groups when the rate of vaccination coverage is lower than 80% for a significant amount of time, which may result in increased morbidity and mortality [3]. During this survey we found that the coverage of the varicella vaccine was higher in counties with higher per capita GDP, and was relatively lower in counties with lower per capita GDP. The coverage of selected areas varies from 44% to 98%, and basing on the linear regression modeling of the relationship between GDP and coverage, per capita GDP of China was 38,354 RMB in 2012 [11], we estimated the average coverage rate for children under 5 years old in China might be about 52%. A national survey conducted in 2011 in China showed that the varicella vaccine coverage rate under 2 years old was 47% [12], this is similarly with our estimates.\nVaricella vaccine is a private vaccine in China, the parents of children voluntarily select and pay for the vaccine for their children. In areas with low coverage rate, a child will be individually protected due to the vaccination, however, herd immunity may not be established at the population level. Furthermore, as a private vaccine, the vaccination fee may become a barrier to vaccinate for children who live in financially poor resource environments [13, 14]. The inequalities of immunization coverage due to socioeconomic differences also exist in other countries [15, 16]. Varicella vaccine has been demonstrated good safety and effect [3], and one study in China showed that introducing varicella vaccination into the routine childhood immunization programme is quite cost effective: an one-dose strategy could save 8 billion RMB in one year [17]. We suggest introducing the varicella vaccine into the routine childhood immunization program as soon as possible to improve the equity in access to immunization services in areas of low socioeconomic status, and ensure a coverage rate of more than 80% in all areas according to the recommendations by WHO.\nOur study showed that the coverage rate for different birth cohorts remained stable. The vaccine coverage rate was 68% at 1 year old, and increased slightly with age, cumulative vaccine coverage rate was 72% at 2 years old and 74% ≥ 3 years old, suggesting that after 3 years of age the access to varicella vaccination service is limited. The peak seasons for incidence of varicella in China are winter and spring seasons, and mainly effect preschool and school-age children of 3 ∼ 10 years old [18]. In China, varicella outbreaks account for a relatively high proportion of the school emergency public health events. For example, reported outbreaks due to varicella in Zhejiang province accounted for 35% in the school emergency public health events from 2005 to 2008 [19]. Therefore, it is necessary to provide an opportunity for access to varicella vaccination service for those children over 3 years of age, who missed the opportunity for vaccination at a younger age. School entry check of immunization certificates and providing vaccination services for eligible children during entry of kindergarten and school may increase vaccine coverage rate among older children and reduce the amount of outbreaks in nurseries and schools [20].\nAt present, the one-dose schedule for varicella vaccine is used in most provinces in China, and the one-dose schedule is used in the 6 provinces selected for this investigation. This survey found that most children completed the schedule before 2 years of age, with the majority (85%) receiving the vaccine at 12-17 months old. Research in the United States showed that the vaccine efficacy decreased significantly (only 73%) during the first year after vaccination if the vaccine is provided before 15 months old [10]. Before 2006, the one-dose schedule was recommended in the United States. After 2006, in order to strengthen the control of varicella, a two-dose schedule was introduced, the first dose is administered at 12-15 months old and the second dose administered at preschool age (4-6 years old) [21]. Geometric mean titer (GMT) showed a particularly high boost after the second dose when the interval between doses was more than one year [22]. A two-dose schedule was introduced in Beijing in 2012: the first dose at 18 months of age and the second dose at 4 years of age [7]. To further improve and optimize the varicella vaccine schedule, we suggest further developing the recommendations regarding the varicella immunization schedule by considering disease risk and the effectiveness of vaccines.\nLimitations: We used purposive sampling methods to select provinces and counties (districts) [23], the selected provinces cannot represent all of China. And we obtained the sample based on Immunization Information Management System (IIMS), according to the national requirements, all immunization information for children should be reported to IIMS, however, the quality of the IIMS data varies across areas, it is possible that some migrant children's information was not recorded in IIMS, which may overestimate the coverage rate of varicella vaccine."],"string":"[\n \"Varicella is an infectious disease with high transmissibility, common in children [1]. The disease is prone to resulting in large scale outbreak in institutional units, such as nurseries, kindergartens, and schools [2]. Varicella vaccine has been demonstrated good safety and effect [3], and varicella vaccination is the most effective measure to protect susceptible people from the disease. Although varicella case information has been collected nationally since 2005 through National Disease Supervision Information Management System(NDSIMS), its limitation in quality of local clinic reporting poses a challenge in accurate analysis and evaluation of the true situation in China [4]. Based on the estimation of varicella incidence in Shandong, Gansu and Hunan provinces, 4,705,000 cases were reported in 2007 in China, and the cost estimation in 2007 was 2.31 billion RMB for outpatients and 103 million RMB for inpatients [5, 6]. “Varicella and Herpes Zoster Vaccines: WHO Position Paper” recommends that countries where varicella is an important public health burden could consider introducing varicella vaccination in the routine childhood immunization programme. Resources should be sufficient to ensure reaching and sustaining vaccine coverage ≥ 80%. Vaccine coverage that remains <80% over the long term is expected to shift varicella infection to older ages in some settings, which may result in an increase of morbidity and mortality despite reduction in total number of cases [3]. Varicella vaccine was licensed in China in 1996 [7]. Currently, varicella vaccine is sold on the private market, meaning it is voluntary and must be self-paid. There is no national recommended immunization schedule available for varicella vaccine, and the vaccine is administered to eligible children at a vaccination clinic according to instructions provided by the vaccine manufacturers. Based on the immunization regulation, the record of vaccination information is kept by both the parents and the clinic [8–9]. In this study, we investigated the varicella vaccination information among children under 5 years old in selected areas in China, in order to understand the varicella vaccination situation for children in China. This information will provide evidence for authority department of China introducing varicella vaccine into the national routine immunization program.\",\n \" Basic information and vaccine coverage in selected counties In total, we collected varicella vaccination information from 481 caregivers’ vaccination certificates: 96 in Shanghai, 48 in Jiangsu, 96 in Heilongjiang, 52 in Jiangxi, 93 in Gansu and 96 in Chongqing, the geographical distribution of selected provinces see Figure 1; Male: 263 and female: 218, with a male to female ratio of 1.2:1. The varicella vaccine coverage of the first dose was 73.6% (354/481) for all children surveyed. The coverage in selected counties is described in Table 1. 1.2% (6/481) received the second dose of varicella vaccine.\\nIn total, we collected varicella vaccination information from 481 caregivers’ vaccination certificates: 96 in Shanghai, 48 in Jiangsu, 96 in Heilongjiang, 52 in Jiangxi, 93 in Gansu and 96 in Chongqing, the geographical distribution of selected provinces see Figure 1; Male: 263 and female: 218, with a male to female ratio of 1.2:1. The varicella vaccine coverage of the first dose was 73.6% (354/481) for all children surveyed. The coverage in selected counties is described in Table 1. 1.2% (6/481) received the second dose of varicella vaccine.\\n Relationship between varicella vaccine coverage and the economic situation The median coverage of varicella vaccine in areas with per capita GDP ≥ 100,000 RMB is 92.7% (85.4% - 97.9%), while median coverage of varicella vaccine in areas with per capita GDP < 100,000RMB is 55.9% (44.2% - 80.9%) (Wilcoxon rank sum test P < 0.05). There is a positive linear correlation between per capita GDP and vaccine coverage at county level (r = 0.929, P < 0.01). The equation of linear regression is y = 0.0004x + 37.08 (F = 50.426, P < 0.001) (Figure 2).\\nThe median coverage of varicella vaccine in areas with per capita GDP ≥ 100,000 RMB is 92.7% (85.4% - 97.9%), while median coverage of varicella vaccine in areas with per capita GDP < 100,000RMB is 55.9% (44.2% - 80.9%) (Wilcoxon rank sum test P < 0.05). There is a positive linear correlation between per capita GDP and vaccine coverage at county level (r = 0.929, P < 0.01). The equation of linear regression is y = 0.0004x + 37.08 (F = 50.426, P < 0.001) (Figure 2).\\n Cumulative coverage of varicella vaccine and distribution of vaccinated age At age of 1 year old(12-23 months), the vaccine coverage were 70.8%, 70.0%, 65.7%, 70.0%, 60.5% among children born in 2008, 2009, 2010, 2011 and 2012 respectively with no significant difference (X 2 = 3.15, P =0.53). The cumulative vaccine coverage ≥3 years old (≥ 36 months) were 79.2%, 78.0% and 74.7 % among children born in 2008, 2009 and 2010 respectively with no significant difference (X 2=0.59, P =0.75). The children born in 2012 were under 2 years of age during investigation for whom the vaccine coverage was lower (60.5%). In total, the cumulative coverage among children at 1 year, 2 years(24-35 months) and≥3 years old were 67.6%, 71.9% and 73.6% (X 2=4.53, P =0.10), with 4.3% increasing at 2 years old, and 1.7% at 3 years old and later compared to 1 year old (Table 2).\\nNote:* 1 year old=”12-23 months”, 2 years old=”24-35 months”, ≥3 years old=”≥36 months”\\nAmong all vaccinated children, 91.8% (325/354) received the vaccine at 1 year old, 5.9% (21/354) received at 2 years old, and 2.3% (8/354) at 3 years old and later. Among those receiving the vaccine at 1 year old, the vaccinated age was mainly concentrated in 12-17 months, accounting for 85.3% (302/325) (Figure 3).\\nn=(ta/2Δp)2P(1−P),ta/2=1.96,Δp=0.0045,p=0.45\\nAt age of 1 year old(12-23 months), the vaccine coverage were 70.8%, 70.0%, 65.7%, 70.0%, 60.5% among children born in 2008, 2009, 2010, 2011 and 2012 respectively with no significant difference (X 2 = 3.15, P =0.53). The cumulative vaccine coverage ≥3 years old (≥ 36 months) were 79.2%, 78.0% and 74.7 % among children born in 2008, 2009 and 2010 respectively with no significant difference (X 2=0.59, P =0.75). The children born in 2012 were under 2 years of age during investigation for whom the vaccine coverage was lower (60.5%). In total, the cumulative coverage among children at 1 year, 2 years(24-35 months) and≥3 years old were 67.6%, 71.9% and 73.6% (X 2=4.53, P =0.10), with 4.3% increasing at 2 years old, and 1.7% at 3 years old and later compared to 1 year old (Table 2).\\nNote:* 1 year old=”12-23 months”, 2 years old=”24-35 months”, ≥3 years old=”≥36 months”\\nAmong all vaccinated children, 91.8% (325/354) received the vaccine at 1 year old, 5.9% (21/354) received at 2 years old, and 2.3% (8/354) at 3 years old and later. Among those receiving the vaccine at 1 year old, the vaccinated age was mainly concentrated in 12-17 months, accounting for 85.3% (302/325) (Figure 3).\\nn=(ta/2Δp)2P(1−P),ta/2=1.96,Δp=0.0045,p=0.45\",\n \"In total, we collected varicella vaccination information from 481 caregivers’ vaccination certificates: 96 in Shanghai, 48 in Jiangsu, 96 in Heilongjiang, 52 in Jiangxi, 93 in Gansu and 96 in Chongqing, the geographical distribution of selected provinces see Figure 1; Male: 263 and female: 218, with a male to female ratio of 1.2:1. The varicella vaccine coverage of the first dose was 73.6% (354/481) for all children surveyed. The coverage in selected counties is described in Table 1. 1.2% (6/481) received the second dose of varicella vaccine.\",\n \"The median coverage of varicella vaccine in areas with per capita GDP ≥ 100,000 RMB is 92.7% (85.4% - 97.9%), while median coverage of varicella vaccine in areas with per capita GDP < 100,000RMB is 55.9% (44.2% - 80.9%) (Wilcoxon rank sum test P < 0.05). There is a positive linear correlation between per capita GDP and vaccine coverage at county level (r = 0.929, P < 0.01). The equation of linear regression is y = 0.0004x + 37.08 (F = 50.426, P < 0.001) (Figure 2).\",\n \"At age of 1 year old(12-23 months), the vaccine coverage were 70.8%, 70.0%, 65.7%, 70.0%, 60.5% among children born in 2008, 2009, 2010, 2011 and 2012 respectively with no significant difference (X 2 = 3.15, P =0.53). The cumulative vaccine coverage ≥3 years old (≥ 36 months) were 79.2%, 78.0% and 74.7 % among children born in 2008, 2009 and 2010 respectively with no significant difference (X 2=0.59, P =0.75). The children born in 2012 were under 2 years of age during investigation for whom the vaccine coverage was lower (60.5%). In total, the cumulative coverage among children at 1 year, 2 years(24-35 months) and≥3 years old were 67.6%, 71.9% and 73.6% (X 2=4.53, P =0.10), with 4.3% increasing at 2 years old, and 1.7% at 3 years old and later compared to 1 year old (Table 2).\\nNote:* 1 year old=”12-23 months”, 2 years old=”24-35 months”, ≥3 years old=”≥36 months”\\nAmong all vaccinated children, 91.8% (325/354) received the vaccine at 1 year old, 5.9% (21/354) received at 2 years old, and 2.3% (8/354) at 3 years old and later. Among those receiving the vaccine at 1 year old, the vaccinated age was mainly concentrated in 12-17 months, accounting for 85.3% (302/325) (Figure 3).\\nn=(ta/2Δp)2P(1−P),ta/2=1.96,Δp=0.0045,p=0.45\",\n \"The basic reproduction number (R0) is about 8 ∼ 10 for varicella, which is higher than influenza [10], it is easy for varicella to cause an outbreak in a nursery, kindergarten or school. Therefore, it is very difficult to block the spreading of varicella disease in areas with low vaccine coverage, and it is necessary to maintain high coverage to prevent the disease from becoming an epidemic. The World Health Organization position paper states that varicella may transit to older age groups when the rate of vaccination coverage is lower than 80% for a significant amount of time, which may result in increased morbidity and mortality [3]. During this survey we found that the coverage of the varicella vaccine was higher in counties with higher per capita GDP, and was relatively lower in counties with lower per capita GDP. The coverage of selected areas varies from 44% to 98%, and basing on the linear regression modeling of the relationship between GDP and coverage, per capita GDP of China was 38,354 RMB in 2012 [11], we estimated the average coverage rate for children under 5 years old in China might be about 52%. A national survey conducted in 2011 in China showed that the varicella vaccine coverage rate under 2 years old was 47% [12], this is similarly with our estimates.\\nVaricella vaccine is a private vaccine in China, the parents of children voluntarily select and pay for the vaccine for their children. In areas with low coverage rate, a child will be individually protected due to the vaccination, however, herd immunity may not be established at the population level. Furthermore, as a private vaccine, the vaccination fee may become a barrier to vaccinate for children who live in financially poor resource environments [13, 14]. The inequalities of immunization coverage due to socioeconomic differences also exist in other countries [15, 16]. Varicella vaccine has been demonstrated good safety and effect [3], and one study in China showed that introducing varicella vaccination into the routine childhood immunization programme is quite cost effective: an one-dose strategy could save 8 billion RMB in one year [17]. We suggest introducing the varicella vaccine into the routine childhood immunization program as soon as possible to improve the equity in access to immunization services in areas of low socioeconomic status, and ensure a coverage rate of more than 80% in all areas according to the recommendations by WHO.\\nOur study showed that the coverage rate for different birth cohorts remained stable. The vaccine coverage rate was 68% at 1 year old, and increased slightly with age, cumulative vaccine coverage rate was 72% at 2 years old and 74% ≥ 3 years old, suggesting that after 3 years of age the access to varicella vaccination service is limited. The peak seasons for incidence of varicella in China are winter and spring seasons, and mainly effect preschool and school-age children of 3 ∼ 10 years old [18]. In China, varicella outbreaks account for a relatively high proportion of the school emergency public health events. For example, reported outbreaks due to varicella in Zhejiang province accounted for 35% in the school emergency public health events from 2005 to 2008 [19]. Therefore, it is necessary to provide an opportunity for access to varicella vaccination service for those children over 3 years of age, who missed the opportunity for vaccination at a younger age. School entry check of immunization certificates and providing vaccination services for eligible children during entry of kindergarten and school may increase vaccine coverage rate among older children and reduce the amount of outbreaks in nurseries and schools [20].\\nAt present, the one-dose schedule for varicella vaccine is used in most provinces in China, and the one-dose schedule is used in the 6 provinces selected for this investigation. This survey found that most children completed the schedule before 2 years of age, with the majority (85%) receiving the vaccine at 12-17 months old. Research in the United States showed that the vaccine efficacy decreased significantly (only 73%) during the first year after vaccination if the vaccine is provided before 15 months old [10]. Before 2006, the one-dose schedule was recommended in the United States. After 2006, in order to strengthen the control of varicella, a two-dose schedule was introduced, the first dose is administered at 12-15 months old and the second dose administered at preschool age (4-6 years old) [21]. Geometric mean titer (GMT) showed a particularly high boost after the second dose when the interval between doses was more than one year [22]. A two-dose schedule was introduced in Beijing in 2012: the first dose at 18 months of age and the second dose at 4 years of age [7]. To further improve and optimize the varicella vaccine schedule, we suggest further developing the recommendations regarding the varicella immunization schedule by considering disease risk and the effectiveness of vaccines.\\nLimitations: We used purposive sampling methods to select provinces and counties (districts) [23], the selected provinces cannot represent all of China. And we obtained the sample based on Immunization Information Management System (IIMS), according to the national requirements, all immunization information for children should be reported to IIMS, however, the quality of the IIMS data varies across areas, it is possible that some migrant children's information was not recorded in IIMS, which may overestimate the coverage rate of varicella vaccine.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","RESULTS","Basic information and vaccine coverage in selected counties","Relationship between varicella vaccine coverage and the economic situation","Cumulative coverage of varicella vaccine and distribution of vaccinated age","DISCUSSION","MATERIALS AND METHODS"],"string":"[\n \"INTRODUCTION\",\n \"RESULTS\",\n \"Basic information and vaccine coverage in selected counties\",\n \"Relationship between varicella vaccine coverage and the economic situation\",\n \"Cumulative coverage of varicella vaccine and distribution of vaccinated age\",\n \"DISCUSSION\",\n \"MATERIALS AND METHODS\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Varicella is an infectious disease with high transmissibility, common in children [1]. The disease is prone to resulting in large scale outbreak in institutional units, such as nurseries, kindergartens, and schools [2]. Varicella vaccine has been demonstrated good safety and effect [3], and varicella vaccination is the most effective measure to protect susceptible people from the disease. Although varicella case information has been collected nationally since 2005 through National Disease Supervision Information Management System(NDSIMS), its limitation in quality of local clinic reporting poses a challenge in accurate analysis and evaluation of the true situation in China [4]. Based on the estimation of varicella incidence in Shandong, Gansu and Hunan provinces, 4,705,000 cases were reported in 2007 in China, and the cost estimation in 2007 was 2.31 billion RMB for outpatients and 103 million RMB for inpatients [5, 6]. “Varicella and Herpes Zoster Vaccines: WHO Position Paper” recommends that countries where varicella is an important public health burden could consider introducing varicella vaccination in the routine childhood immunization programme. Resources should be sufficient to ensure reaching and sustaining vaccine coverage ≥ 80%. Vaccine coverage that remains <80% over the long term is expected to shift varicella infection to older ages in some settings, which may result in an increase of morbidity and mortality despite reduction in total number of cases [3]. Varicella vaccine was licensed in China in 1996 [7]. Currently, varicella vaccine is sold on the private market, meaning it is voluntary and must be self-paid. There is no national recommended immunization schedule available for varicella vaccine, and the vaccine is administered to eligible children at a vaccination clinic according to instructions provided by the vaccine manufacturers. Based on the immunization regulation, the record of vaccination information is kept by both the parents and the clinic [8–9]. In this study, we investigated the varicella vaccination information among children under 5 years old in selected areas in China, in order to understand the varicella vaccination situation for children in China. This information will provide evidence for authority department of China introducing varicella vaccine into the national routine immunization program."," Basic information and vaccine coverage in selected counties In total, we collected varicella vaccination information from 481 caregivers’ vaccination certificates: 96 in Shanghai, 48 in Jiangsu, 96 in Heilongjiang, 52 in Jiangxi, 93 in Gansu and 96 in Chongqing, the geographical distribution of selected provinces see Figure 1; Male: 263 and female: 218, with a male to female ratio of 1.2:1. The varicella vaccine coverage of the first dose was 73.6% (354/481) for all children surveyed. The coverage in selected counties is described in Table 1. 1.2% (6/481) received the second dose of varicella vaccine.\nIn total, we collected varicella vaccination information from 481 caregivers’ vaccination certificates: 96 in Shanghai, 48 in Jiangsu, 96 in Heilongjiang, 52 in Jiangxi, 93 in Gansu and 96 in Chongqing, the geographical distribution of selected provinces see Figure 1; Male: 263 and female: 218, with a male to female ratio of 1.2:1. The varicella vaccine coverage of the first dose was 73.6% (354/481) for all children surveyed. The coverage in selected counties is described in Table 1. 1.2% (6/481) received the second dose of varicella vaccine.\n Relationship between varicella vaccine coverage and the economic situation The median coverage of varicella vaccine in areas with per capita GDP ≥ 100,000 RMB is 92.7% (85.4% - 97.9%), while median coverage of varicella vaccine in areas with per capita GDP < 100,000RMB is 55.9% (44.2% - 80.9%) (Wilcoxon rank sum test P < 0.05). There is a positive linear correlation between per capita GDP and vaccine coverage at county level (r = 0.929, P < 0.01). The equation of linear regression is y = 0.0004x + 37.08 (F = 50.426, P < 0.001) (Figure 2).\nThe median coverage of varicella vaccine in areas with per capita GDP ≥ 100,000 RMB is 92.7% (85.4% - 97.9%), while median coverage of varicella vaccine in areas with per capita GDP < 100,000RMB is 55.9% (44.2% - 80.9%) (Wilcoxon rank sum test P < 0.05). There is a positive linear correlation between per capita GDP and vaccine coverage at county level (r = 0.929, P < 0.01). The equation of linear regression is y = 0.0004x + 37.08 (F = 50.426, P < 0.001) (Figure 2).\n Cumulative coverage of varicella vaccine and distribution of vaccinated age At age of 1 year old(12-23 months), the vaccine coverage were 70.8%, 70.0%, 65.7%, 70.0%, 60.5% among children born in 2008, 2009, 2010, 2011 and 2012 respectively with no significant difference (X 2 = 3.15, P =0.53). The cumulative vaccine coverage ≥3 years old (≥ 36 months) were 79.2%, 78.0% and 74.7 % among children born in 2008, 2009 and 2010 respectively with no significant difference (X 2=0.59, P =0.75). The children born in 2012 were under 2 years of age during investigation for whom the vaccine coverage was lower (60.5%). In total, the cumulative coverage among children at 1 year, 2 years(24-35 months) and≥3 years old were 67.6%, 71.9% and 73.6% (X 2=4.53, P =0.10), with 4.3% increasing at 2 years old, and 1.7% at 3 years old and later compared to 1 year old (Table 2).\nNote:* 1 year old=”12-23 months”, 2 years old=”24-35 months”, ≥3 years old=”≥36 months”\nAmong all vaccinated children, 91.8% (325/354) received the vaccine at 1 year old, 5.9% (21/354) received at 2 years old, and 2.3% (8/354) at 3 years old and later. Among those receiving the vaccine at 1 year old, the vaccinated age was mainly concentrated in 12-17 months, accounting for 85.3% (302/325) (Figure 3).\nn=(ta/2Δp)2P(1−P),ta/2=1.96,Δp=0.0045,p=0.45\nAt age of 1 year old(12-23 months), the vaccine coverage were 70.8%, 70.0%, 65.7%, 70.0%, 60.5% among children born in 2008, 2009, 2010, 2011 and 2012 respectively with no significant difference (X 2 = 3.15, P =0.53). The cumulative vaccine coverage ≥3 years old (≥ 36 months) were 79.2%, 78.0% and 74.7 % among children born in 2008, 2009 and 2010 respectively with no significant difference (X 2=0.59, P =0.75). The children born in 2012 were under 2 years of age during investigation for whom the vaccine coverage was lower (60.5%). In total, the cumulative coverage among children at 1 year, 2 years(24-35 months) and≥3 years old were 67.6%, 71.9% and 73.6% (X 2=4.53, P =0.10), with 4.3% increasing at 2 years old, and 1.7% at 3 years old and later compared to 1 year old (Table 2).\nNote:* 1 year old=”12-23 months”, 2 years old=”24-35 months”, ≥3 years old=”≥36 months”\nAmong all vaccinated children, 91.8% (325/354) received the vaccine at 1 year old, 5.9% (21/354) received at 2 years old, and 2.3% (8/354) at 3 years old and later. Among those receiving the vaccine at 1 year old, the vaccinated age was mainly concentrated in 12-17 months, accounting for 85.3% (302/325) (Figure 3).\nn=(ta/2Δp)2P(1−P),ta/2=1.96,Δp=0.0045,p=0.45","In total, we collected varicella vaccination information from 481 caregivers’ vaccination certificates: 96 in Shanghai, 48 in Jiangsu, 96 in Heilongjiang, 52 in Jiangxi, 93 in Gansu and 96 in Chongqing, the geographical distribution of selected provinces see Figure 1; Male: 263 and female: 218, with a male to female ratio of 1.2:1. The varicella vaccine coverage of the first dose was 73.6% (354/481) for all children surveyed. The coverage in selected counties is described in Table 1. 1.2% (6/481) received the second dose of varicella vaccine.","The median coverage of varicella vaccine in areas with per capita GDP ≥ 100,000 RMB is 92.7% (85.4% - 97.9%), while median coverage of varicella vaccine in areas with per capita GDP < 100,000RMB is 55.9% (44.2% - 80.9%) (Wilcoxon rank sum test P < 0.05). There is a positive linear correlation between per capita GDP and vaccine coverage at county level (r = 0.929, P < 0.01). The equation of linear regression is y = 0.0004x + 37.08 (F = 50.426, P < 0.001) (Figure 2).","At age of 1 year old(12-23 months), the vaccine coverage were 70.8%, 70.0%, 65.7%, 70.0%, 60.5% among children born in 2008, 2009, 2010, 2011 and 2012 respectively with no significant difference (X 2 = 3.15, P =0.53). The cumulative vaccine coverage ≥3 years old (≥ 36 months) were 79.2%, 78.0% and 74.7 % among children born in 2008, 2009 and 2010 respectively with no significant difference (X 2=0.59, P =0.75). The children born in 2012 were under 2 years of age during investigation for whom the vaccine coverage was lower (60.5%). In total, the cumulative coverage among children at 1 year, 2 years(24-35 months) and≥3 years old were 67.6%, 71.9% and 73.6% (X 2=4.53, P =0.10), with 4.3% increasing at 2 years old, and 1.7% at 3 years old and later compared to 1 year old (Table 2).\nNote:* 1 year old=”12-23 months”, 2 years old=”24-35 months”, ≥3 years old=”≥36 months”\nAmong all vaccinated children, 91.8% (325/354) received the vaccine at 1 year old, 5.9% (21/354) received at 2 years old, and 2.3% (8/354) at 3 years old and later. Among those receiving the vaccine at 1 year old, the vaccinated age was mainly concentrated in 12-17 months, accounting for 85.3% (302/325) (Figure 3).\nn=(ta/2Δp)2P(1−P),ta/2=1.96,Δp=0.0045,p=0.45","The basic reproduction number (R0) is about 8 ∼ 10 for varicella, which is higher than influenza [10], it is easy for varicella to cause an outbreak in a nursery, kindergarten or school. Therefore, it is very difficult to block the spreading of varicella disease in areas with low vaccine coverage, and it is necessary to maintain high coverage to prevent the disease from becoming an epidemic. The World Health Organization position paper states that varicella may transit to older age groups when the rate of vaccination coverage is lower than 80% for a significant amount of time, which may result in increased morbidity and mortality [3]. During this survey we found that the coverage of the varicella vaccine was higher in counties with higher per capita GDP, and was relatively lower in counties with lower per capita GDP. The coverage of selected areas varies from 44% to 98%, and basing on the linear regression modeling of the relationship between GDP and coverage, per capita GDP of China was 38,354 RMB in 2012 [11], we estimated the average coverage rate for children under 5 years old in China might be about 52%. A national survey conducted in 2011 in China showed that the varicella vaccine coverage rate under 2 years old was 47% [12], this is similarly with our estimates.\nVaricella vaccine is a private vaccine in China, the parents of children voluntarily select and pay for the vaccine for their children. In areas with low coverage rate, a child will be individually protected due to the vaccination, however, herd immunity may not be established at the population level. Furthermore, as a private vaccine, the vaccination fee may become a barrier to vaccinate for children who live in financially poor resource environments [13, 14]. The inequalities of immunization coverage due to socioeconomic differences also exist in other countries [15, 16]. Varicella vaccine has been demonstrated good safety and effect [3], and one study in China showed that introducing varicella vaccination into the routine childhood immunization programme is quite cost effective: an one-dose strategy could save 8 billion RMB in one year [17]. We suggest introducing the varicella vaccine into the routine childhood immunization program as soon as possible to improve the equity in access to immunization services in areas of low socioeconomic status, and ensure a coverage rate of more than 80% in all areas according to the recommendations by WHO.\nOur study showed that the coverage rate for different birth cohorts remained stable. The vaccine coverage rate was 68% at 1 year old, and increased slightly with age, cumulative vaccine coverage rate was 72% at 2 years old and 74% ≥ 3 years old, suggesting that after 3 years of age the access to varicella vaccination service is limited. The peak seasons for incidence of varicella in China are winter and spring seasons, and mainly effect preschool and school-age children of 3 ∼ 10 years old [18]. In China, varicella outbreaks account for a relatively high proportion of the school emergency public health events. For example, reported outbreaks due to varicella in Zhejiang province accounted for 35% in the school emergency public health events from 2005 to 2008 [19]. Therefore, it is necessary to provide an opportunity for access to varicella vaccination service for those children over 3 years of age, who missed the opportunity for vaccination at a younger age. School entry check of immunization certificates and providing vaccination services for eligible children during entry of kindergarten and school may increase vaccine coverage rate among older children and reduce the amount of outbreaks in nurseries and schools [20].\nAt present, the one-dose schedule for varicella vaccine is used in most provinces in China, and the one-dose schedule is used in the 6 provinces selected for this investigation. This survey found that most children completed the schedule before 2 years of age, with the majority (85%) receiving the vaccine at 12-17 months old. Research in the United States showed that the vaccine efficacy decreased significantly (only 73%) during the first year after vaccination if the vaccine is provided before 15 months old [10]. Before 2006, the one-dose schedule was recommended in the United States. After 2006, in order to strengthen the control of varicella, a two-dose schedule was introduced, the first dose is administered at 12-15 months old and the second dose administered at preschool age (4-6 years old) [21]. Geometric mean titer (GMT) showed a particularly high boost after the second dose when the interval between doses was more than one year [22]. A two-dose schedule was introduced in Beijing in 2012: the first dose at 18 months of age and the second dose at 4 years of age [7]. To further improve and optimize the varicella vaccine schedule, we suggest further developing the recommendations regarding the varicella immunization schedule by considering disease risk and the effectiveness of vaccines.\nLimitations: We used purposive sampling methods to select provinces and counties (districts) [23], the selected provinces cannot represent all of China. And we obtained the sample based on Immunization Information Management System (IIMS), according to the national requirements, all immunization information for children should be reported to IIMS, however, the quality of the IIMS data varies across areas, it is possible that some migrant children's information was not recorded in IIMS, which may overestimate the coverage rate of varicella vaccine."," Sampling methods We used purposive sampling methods to select counties (districts) from Shanghai, Jiangsu, Heilongjiang, Jiangxi, Chongqing and Gansu provinces. We selected these investigation sites based on two factors: (i) representation of east, middle and western parts of China; (ii) having good records of private vaccinations in children's vaccination certificates and with a high quality Immunization Information Management System (IIMS). From Nov 1th 2013 to Nov 15th 2013, we used the simple random sampling method to randomly select children from the population whose birth date was between Jan 1st 2008 and Dec 31th 2012 in the IIMS in selected counties (districts), then from January 1th 2014 to July 15th2014, we checked hand-held vaccination certificates on-site to collect varicella vaccination information.\nWe used the following formula to calculate the sample size:\nn=(ta/2Δp)2P(1−P),ta/2=1.96,Δp=0.0045,p=0.45\nn: the sample size, P: estimate coverage of varicella vaccine, based on past experience, we used 45%. After calculation, a sample size of 470 was needed for this survey.\nConsidering a loss to follow-up of about 10%, a total of 520 children needed to be investigated. The sample size was divided by 10 (10 counties), and 52 children in each county were selected to participate in the investigation. Finally, 39 children were excluded because their parents did not have a hand-held vaccination certificate or we were unable to contact them.\nWe used purposive sampling methods to select counties (districts) from Shanghai, Jiangsu, Heilongjiang, Jiangxi, Chongqing and Gansu provinces. We selected these investigation sites based on two factors: (i) representation of east, middle and western parts of China; (ii) having good records of private vaccinations in children's vaccination certificates and with a high quality Immunization Information Management System (IIMS). From Nov 1th 2013 to Nov 15th 2013, we used the simple random sampling method to randomly select children from the population whose birth date was between Jan 1st 2008 and Dec 31th 2012 in the IIMS in selected counties (districts), then from January 1th 2014 to July 15th2014, we checked hand-held vaccination certificates on-site to collect varicella vaccination information.\nWe used the following formula to calculate the sample size:\nn=(ta/2Δp)2P(1−P),ta/2=1.96,Δp=0.0045,p=0.45\nn: the sample size, P: estimate coverage of varicella vaccine, based on past experience, we used 45%. After calculation, a sample size of 470 was needed for this survey.\nConsidering a loss to follow-up of about 10%, a total of 520 children needed to be investigated. The sample size was divided by 10 (10 counties), and 52 children in each county were selected to participate in the investigation. Finally, 39 children were excluded because their parents did not have a hand-held vaccination certificate or we were unable to contact them.\n Calculation of per capita gross domestic product (GDP) Per capita GDP=GDP/domicile population. GDP and household population data came from the “2012 Per Capita National Economic and Social Development Statistical Bulletin” on the selected County or District's Bureau of Statistics website. The data was not available for Jianye in Jiangsu, Ning'an in Heilongjiang, Chengguan and Qilihe in Gansu, so the per capita GDP in the corresponding city level (higher administrative level) was used for these counties.\nPer capita GDP=GDP/domicile population. GDP and household population data came from the “2012 Per Capita National Economic and Social Development Statistical Bulletin” on the selected County or District's Bureau of Statistics website. The data was not available for Jianye in Jiangsu, Ning'an in Heilongjiang, Chengguan and Qilihe in Gansu, so the per capita GDP in the corresponding city level (higher administrative level) was used for these counties.\n Survey content and data processing We conducted this survey to obtain the basic information of children (such as gender, date of birth) and varicella vaccination (such as inoculation date and doses). We used Excel to enter the data, ArcGIS to draw the map, and Epi Info software to analyze the data.\nWe conducted this survey to obtain the basic information of children (such as gender, date of birth) and varicella vaccination (such as inoculation date and doses). We used Excel to enter the data, ArcGIS to draw the map, and Epi Info software to analyze the data.\n Ethics statement We obtained oral informed consent from all children's guardians before the investigation, and two investigators checked vaccination certificates on-site together. We would continue the investigation only when the children's guardians gave consent. We didn't go through the institutional review board approval in the case of this study for ethical issues, because monitoring vaccine coverage is part of routine program work, in this context, there is no risk for participants and it is not required to get IRB approval. However, we strictly protected the private information of participants in the field, such as the name and contact information. During the analysis, we also took out the personal identification information.\nWe obtained oral informed consent from all children's guardians before the investigation, and two investigators checked vaccination certificates on-site together. We would continue the investigation only when the children's guardians gave consent. We didn't go through the institutional review board approval in the case of this study for ethical issues, because monitoring vaccine coverage is part of routine program work, in this context, there is no risk for participants and it is not required to get IRB approval. However, we strictly protected the private information of participants in the field, such as the name and contact information. During the analysis, we also took out the personal identification information."],"string":"[\n \"Varicella is an infectious disease with high transmissibility, common in children [1]. The disease is prone to resulting in large scale outbreak in institutional units, such as nurseries, kindergartens, and schools [2]. Varicella vaccine has been demonstrated good safety and effect [3], and varicella vaccination is the most effective measure to protect susceptible people from the disease. Although varicella case information has been collected nationally since 2005 through National Disease Supervision Information Management System(NDSIMS), its limitation in quality of local clinic reporting poses a challenge in accurate analysis and evaluation of the true situation in China [4]. Based on the estimation of varicella incidence in Shandong, Gansu and Hunan provinces, 4,705,000 cases were reported in 2007 in China, and the cost estimation in 2007 was 2.31 billion RMB for outpatients and 103 million RMB for inpatients [5, 6]. “Varicella and Herpes Zoster Vaccines: WHO Position Paper” recommends that countries where varicella is an important public health burden could consider introducing varicella vaccination in the routine childhood immunization programme. Resources should be sufficient to ensure reaching and sustaining vaccine coverage ≥ 80%. Vaccine coverage that remains <80% over the long term is expected to shift varicella infection to older ages in some settings, which may result in an increase of morbidity and mortality despite reduction in total number of cases [3]. Varicella vaccine was licensed in China in 1996 [7]. Currently, varicella vaccine is sold on the private market, meaning it is voluntary and must be self-paid. There is no national recommended immunization schedule available for varicella vaccine, and the vaccine is administered to eligible children at a vaccination clinic according to instructions provided by the vaccine manufacturers. Based on the immunization regulation, the record of vaccination information is kept by both the parents and the clinic [8–9]. In this study, we investigated the varicella vaccination information among children under 5 years old in selected areas in China, in order to understand the varicella vaccination situation for children in China. This information will provide evidence for authority department of China introducing varicella vaccine into the national routine immunization program.\",\n \" Basic information and vaccine coverage in selected counties In total, we collected varicella vaccination information from 481 caregivers’ vaccination certificates: 96 in Shanghai, 48 in Jiangsu, 96 in Heilongjiang, 52 in Jiangxi, 93 in Gansu and 96 in Chongqing, the geographical distribution of selected provinces see Figure 1; Male: 263 and female: 218, with a male to female ratio of 1.2:1. The varicella vaccine coverage of the first dose was 73.6% (354/481) for all children surveyed. The coverage in selected counties is described in Table 1. 1.2% (6/481) received the second dose of varicella vaccine.\\nIn total, we collected varicella vaccination information from 481 caregivers’ vaccination certificates: 96 in Shanghai, 48 in Jiangsu, 96 in Heilongjiang, 52 in Jiangxi, 93 in Gansu and 96 in Chongqing, the geographical distribution of selected provinces see Figure 1; Male: 263 and female: 218, with a male to female ratio of 1.2:1. The varicella vaccine coverage of the first dose was 73.6% (354/481) for all children surveyed. The coverage in selected counties is described in Table 1. 1.2% (6/481) received the second dose of varicella vaccine.\\n Relationship between varicella vaccine coverage and the economic situation The median coverage of varicella vaccine in areas with per capita GDP ≥ 100,000 RMB is 92.7% (85.4% - 97.9%), while median coverage of varicella vaccine in areas with per capita GDP < 100,000RMB is 55.9% (44.2% - 80.9%) (Wilcoxon rank sum test P < 0.05). There is a positive linear correlation between per capita GDP and vaccine coverage at county level (r = 0.929, P < 0.01). The equation of linear regression is y = 0.0004x + 37.08 (F = 50.426, P < 0.001) (Figure 2).\\nThe median coverage of varicella vaccine in areas with per capita GDP ≥ 100,000 RMB is 92.7% (85.4% - 97.9%), while median coverage of varicella vaccine in areas with per capita GDP < 100,000RMB is 55.9% (44.2% - 80.9%) (Wilcoxon rank sum test P < 0.05). There is a positive linear correlation between per capita GDP and vaccine coverage at county level (r = 0.929, P < 0.01). The equation of linear regression is y = 0.0004x + 37.08 (F = 50.426, P < 0.001) (Figure 2).\\n Cumulative coverage of varicella vaccine and distribution of vaccinated age At age of 1 year old(12-23 months), the vaccine coverage were 70.8%, 70.0%, 65.7%, 70.0%, 60.5% among children born in 2008, 2009, 2010, 2011 and 2012 respectively with no significant difference (X 2 = 3.15, P =0.53). The cumulative vaccine coverage ≥3 years old (≥ 36 months) were 79.2%, 78.0% and 74.7 % among children born in 2008, 2009 and 2010 respectively with no significant difference (X 2=0.59, P =0.75). The children born in 2012 were under 2 years of age during investigation for whom the vaccine coverage was lower (60.5%). In total, the cumulative coverage among children at 1 year, 2 years(24-35 months) and≥3 years old were 67.6%, 71.9% and 73.6% (X 2=4.53, P =0.10), with 4.3% increasing at 2 years old, and 1.7% at 3 years old and later compared to 1 year old (Table 2).\\nNote:* 1 year old=”12-23 months”, 2 years old=”24-35 months”, ≥3 years old=”≥36 months”\\nAmong all vaccinated children, 91.8% (325/354) received the vaccine at 1 year old, 5.9% (21/354) received at 2 years old, and 2.3% (8/354) at 3 years old and later. Among those receiving the vaccine at 1 year old, the vaccinated age was mainly concentrated in 12-17 months, accounting for 85.3% (302/325) (Figure 3).\\nn=(ta/2Δp)2P(1−P),ta/2=1.96,Δp=0.0045,p=0.45\\nAt age of 1 year old(12-23 months), the vaccine coverage were 70.8%, 70.0%, 65.7%, 70.0%, 60.5% among children born in 2008, 2009, 2010, 2011 and 2012 respectively with no significant difference (X 2 = 3.15, P =0.53). The cumulative vaccine coverage ≥3 years old (≥ 36 months) were 79.2%, 78.0% and 74.7 % among children born in 2008, 2009 and 2010 respectively with no significant difference (X 2=0.59, P =0.75). The children born in 2012 were under 2 years of age during investigation for whom the vaccine coverage was lower (60.5%). In total, the cumulative coverage among children at 1 year, 2 years(24-35 months) and≥3 years old were 67.6%, 71.9% and 73.6% (X 2=4.53, P =0.10), with 4.3% increasing at 2 years old, and 1.7% at 3 years old and later compared to 1 year old (Table 2).\\nNote:* 1 year old=”12-23 months”, 2 years old=”24-35 months”, ≥3 years old=”≥36 months”\\nAmong all vaccinated children, 91.8% (325/354) received the vaccine at 1 year old, 5.9% (21/354) received at 2 years old, and 2.3% (8/354) at 3 years old and later. Among those receiving the vaccine at 1 year old, the vaccinated age was mainly concentrated in 12-17 months, accounting for 85.3% (302/325) (Figure 3).\\nn=(ta/2Δp)2P(1−P),ta/2=1.96,Δp=0.0045,p=0.45\",\n \"In total, we collected varicella vaccination information from 481 caregivers’ vaccination certificates: 96 in Shanghai, 48 in Jiangsu, 96 in Heilongjiang, 52 in Jiangxi, 93 in Gansu and 96 in Chongqing, the geographical distribution of selected provinces see Figure 1; Male: 263 and female: 218, with a male to female ratio of 1.2:1. The varicella vaccine coverage of the first dose was 73.6% (354/481) for all children surveyed. The coverage in selected counties is described in Table 1. 1.2% (6/481) received the second dose of varicella vaccine.\",\n \"The median coverage of varicella vaccine in areas with per capita GDP ≥ 100,000 RMB is 92.7% (85.4% - 97.9%), while median coverage of varicella vaccine in areas with per capita GDP < 100,000RMB is 55.9% (44.2% - 80.9%) (Wilcoxon rank sum test P < 0.05). There is a positive linear correlation between per capita GDP and vaccine coverage at county level (r = 0.929, P < 0.01). The equation of linear regression is y = 0.0004x + 37.08 (F = 50.426, P < 0.001) (Figure 2).\",\n \"At age of 1 year old(12-23 months), the vaccine coverage were 70.8%, 70.0%, 65.7%, 70.0%, 60.5% among children born in 2008, 2009, 2010, 2011 and 2012 respectively with no significant difference (X 2 = 3.15, P =0.53). The cumulative vaccine coverage ≥3 years old (≥ 36 months) were 79.2%, 78.0% and 74.7 % among children born in 2008, 2009 and 2010 respectively with no significant difference (X 2=0.59, P =0.75). The children born in 2012 were under 2 years of age during investigation for whom the vaccine coverage was lower (60.5%). In total, the cumulative coverage among children at 1 year, 2 years(24-35 months) and≥3 years old were 67.6%, 71.9% and 73.6% (X 2=4.53, P =0.10), with 4.3% increasing at 2 years old, and 1.7% at 3 years old and later compared to 1 year old (Table 2).\\nNote:* 1 year old=”12-23 months”, 2 years old=”24-35 months”, ≥3 years old=”≥36 months”\\nAmong all vaccinated children, 91.8% (325/354) received the vaccine at 1 year old, 5.9% (21/354) received at 2 years old, and 2.3% (8/354) at 3 years old and later. Among those receiving the vaccine at 1 year old, the vaccinated age was mainly concentrated in 12-17 months, accounting for 85.3% (302/325) (Figure 3).\\nn=(ta/2Δp)2P(1−P),ta/2=1.96,Δp=0.0045,p=0.45\",\n \"The basic reproduction number (R0) is about 8 ∼ 10 for varicella, which is higher than influenza [10], it is easy for varicella to cause an outbreak in a nursery, kindergarten or school. Therefore, it is very difficult to block the spreading of varicella disease in areas with low vaccine coverage, and it is necessary to maintain high coverage to prevent the disease from becoming an epidemic. The World Health Organization position paper states that varicella may transit to older age groups when the rate of vaccination coverage is lower than 80% for a significant amount of time, which may result in increased morbidity and mortality [3]. During this survey we found that the coverage of the varicella vaccine was higher in counties with higher per capita GDP, and was relatively lower in counties with lower per capita GDP. The coverage of selected areas varies from 44% to 98%, and basing on the linear regression modeling of the relationship between GDP and coverage, per capita GDP of China was 38,354 RMB in 2012 [11], we estimated the average coverage rate for children under 5 years old in China might be about 52%. A national survey conducted in 2011 in China showed that the varicella vaccine coverage rate under 2 years old was 47% [12], this is similarly with our estimates.\\nVaricella vaccine is a private vaccine in China, the parents of children voluntarily select and pay for the vaccine for their children. In areas with low coverage rate, a child will be individually protected due to the vaccination, however, herd immunity may not be established at the population level. Furthermore, as a private vaccine, the vaccination fee may become a barrier to vaccinate for children who live in financially poor resource environments [13, 14]. The inequalities of immunization coverage due to socioeconomic differences also exist in other countries [15, 16]. Varicella vaccine has been demonstrated good safety and effect [3], and one study in China showed that introducing varicella vaccination into the routine childhood immunization programme is quite cost effective: an one-dose strategy could save 8 billion RMB in one year [17]. We suggest introducing the varicella vaccine into the routine childhood immunization program as soon as possible to improve the equity in access to immunization services in areas of low socioeconomic status, and ensure a coverage rate of more than 80% in all areas according to the recommendations by WHO.\\nOur study showed that the coverage rate for different birth cohorts remained stable. The vaccine coverage rate was 68% at 1 year old, and increased slightly with age, cumulative vaccine coverage rate was 72% at 2 years old and 74% ≥ 3 years old, suggesting that after 3 years of age the access to varicella vaccination service is limited. The peak seasons for incidence of varicella in China are winter and spring seasons, and mainly effect preschool and school-age children of 3 ∼ 10 years old [18]. In China, varicella outbreaks account for a relatively high proportion of the school emergency public health events. For example, reported outbreaks due to varicella in Zhejiang province accounted for 35% in the school emergency public health events from 2005 to 2008 [19]. Therefore, it is necessary to provide an opportunity for access to varicella vaccination service for those children over 3 years of age, who missed the opportunity for vaccination at a younger age. School entry check of immunization certificates and providing vaccination services for eligible children during entry of kindergarten and school may increase vaccine coverage rate among older children and reduce the amount of outbreaks in nurseries and schools [20].\\nAt present, the one-dose schedule for varicella vaccine is used in most provinces in China, and the one-dose schedule is used in the 6 provinces selected for this investigation. This survey found that most children completed the schedule before 2 years of age, with the majority (85%) receiving the vaccine at 12-17 months old. Research in the United States showed that the vaccine efficacy decreased significantly (only 73%) during the first year after vaccination if the vaccine is provided before 15 months old [10]. Before 2006, the one-dose schedule was recommended in the United States. After 2006, in order to strengthen the control of varicella, a two-dose schedule was introduced, the first dose is administered at 12-15 months old and the second dose administered at preschool age (4-6 years old) [21]. Geometric mean titer (GMT) showed a particularly high boost after the second dose when the interval between doses was more than one year [22]. A two-dose schedule was introduced in Beijing in 2012: the first dose at 18 months of age and the second dose at 4 years of age [7]. To further improve and optimize the varicella vaccine schedule, we suggest further developing the recommendations regarding the varicella immunization schedule by considering disease risk and the effectiveness of vaccines.\\nLimitations: We used purposive sampling methods to select provinces and counties (districts) [23], the selected provinces cannot represent all of China. And we obtained the sample based on Immunization Information Management System (IIMS), according to the national requirements, all immunization information for children should be reported to IIMS, however, the quality of the IIMS data varies across areas, it is possible that some migrant children's information was not recorded in IIMS, which may overestimate the coverage rate of varicella vaccine.\",\n \" Sampling methods We used purposive sampling methods to select counties (districts) from Shanghai, Jiangsu, Heilongjiang, Jiangxi, Chongqing and Gansu provinces. We selected these investigation sites based on two factors: (i) representation of east, middle and western parts of China; (ii) having good records of private vaccinations in children's vaccination certificates and with a high quality Immunization Information Management System (IIMS). From Nov 1th 2013 to Nov 15th 2013, we used the simple random sampling method to randomly select children from the population whose birth date was between Jan 1st 2008 and Dec 31th 2012 in the IIMS in selected counties (districts), then from January 1th 2014 to July 15th2014, we checked hand-held vaccination certificates on-site to collect varicella vaccination information.\\nWe used the following formula to calculate the sample size:\\nn=(ta/2Δp)2P(1−P),ta/2=1.96,Δp=0.0045,p=0.45\\nn: the sample size, P: estimate coverage of varicella vaccine, based on past experience, we used 45%. After calculation, a sample size of 470 was needed for this survey.\\nConsidering a loss to follow-up of about 10%, a total of 520 children needed to be investigated. The sample size was divided by 10 (10 counties), and 52 children in each county were selected to participate in the investigation. Finally, 39 children were excluded because their parents did not have a hand-held vaccination certificate or we were unable to contact them.\\nWe used purposive sampling methods to select counties (districts) from Shanghai, Jiangsu, Heilongjiang, Jiangxi, Chongqing and Gansu provinces. We selected these investigation sites based on two factors: (i) representation of east, middle and western parts of China; (ii) having good records of private vaccinations in children's vaccination certificates and with a high quality Immunization Information Management System (IIMS). From Nov 1th 2013 to Nov 15th 2013, we used the simple random sampling method to randomly select children from the population whose birth date was between Jan 1st 2008 and Dec 31th 2012 in the IIMS in selected counties (districts), then from January 1th 2014 to July 15th2014, we checked hand-held vaccination certificates on-site to collect varicella vaccination information.\\nWe used the following formula to calculate the sample size:\\nn=(ta/2Δp)2P(1−P),ta/2=1.96,Δp=0.0045,p=0.45\\nn: the sample size, P: estimate coverage of varicella vaccine, based on past experience, we used 45%. After calculation, a sample size of 470 was needed for this survey.\\nConsidering a loss to follow-up of about 10%, a total of 520 children needed to be investigated. The sample size was divided by 10 (10 counties), and 52 children in each county were selected to participate in the investigation. Finally, 39 children were excluded because their parents did not have a hand-held vaccination certificate or we were unable to contact them.\\n Calculation of per capita gross domestic product (GDP) Per capita GDP=GDP/domicile population. GDP and household population data came from the “2012 Per Capita National Economic and Social Development Statistical Bulletin” on the selected County or District's Bureau of Statistics website. The data was not available for Jianye in Jiangsu, Ning'an in Heilongjiang, Chengguan and Qilihe in Gansu, so the per capita GDP in the corresponding city level (higher administrative level) was used for these counties.\\nPer capita GDP=GDP/domicile population. GDP and household population data came from the “2012 Per Capita National Economic and Social Development Statistical Bulletin” on the selected County or District's Bureau of Statistics website. The data was not available for Jianye in Jiangsu, Ning'an in Heilongjiang, Chengguan and Qilihe in Gansu, so the per capita GDP in the corresponding city level (higher administrative level) was used for these counties.\\n Survey content and data processing We conducted this survey to obtain the basic information of children (such as gender, date of birth) and varicella vaccination (such as inoculation date and doses). We used Excel to enter the data, ArcGIS to draw the map, and Epi Info software to analyze the data.\\nWe conducted this survey to obtain the basic information of children (such as gender, date of birth) and varicella vaccination (such as inoculation date and doses). We used Excel to enter the data, ArcGIS to draw the map, and Epi Info software to analyze the data.\\n Ethics statement We obtained oral informed consent from all children's guardians before the investigation, and two investigators checked vaccination certificates on-site together. We would continue the investigation only when the children's guardians gave consent. We didn't go through the institutional review board approval in the case of this study for ethical issues, because monitoring vaccine coverage is part of routine program work, in this context, there is no risk for participants and it is not required to get IRB approval. However, we strictly protected the private information of participants in the field, such as the name and contact information. During the analysis, we also took out the personal identification information.\\nWe obtained oral informed consent from all children's guardians before the investigation, and two investigators checked vaccination certificates on-site together. We would continue the investigation only when the children's guardians gave consent. We didn't go through the institutional review board approval in the case of this study for ethical issues, because monitoring vaccine coverage is part of routine program work, in this context, there is no risk for participants and it is not required to get IRB approval. However, we strictly protected the private information of participants in the field, such as the name and contact information. During the analysis, we also took out the personal identification information.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,"methods"],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n \"methods\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["varicella vaccine","vaccination","coverage","children","GDP"],"string":"[\n \"varicella vaccine\",\n \"vaccination\",\n \"coverage\",\n \"children\",\n \"GDP\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nVaricella is an infectious disease with high transmissibility, common in children [1]. The disease is prone to resulting in large scale outbreak in institutional units, such as nurseries, kindergartens, and schools [2]. Varicella vaccine has been demonstrated good safety and effect [3], and varicella vaccination is the most effective measure to protect susceptible people from the disease. Although varicella case information has been collected nationally since 2005 through National Disease Supervision Information Management System(NDSIMS), its limitation in quality of local clinic reporting poses a challenge in accurate analysis and evaluation of the true situation in China [4]. Based on the estimation of varicella incidence in Shandong, Gansu and Hunan provinces, 4,705,000 cases were reported in 2007 in China, and the cost estimation in 2007 was 2.31 billion RMB for outpatients and 103 million RMB for inpatients [5, 6]. “Varicella and Herpes Zoster Vaccines: WHO Position Paper” recommends that countries where varicella is an important public health burden could consider introducing varicella vaccination in the routine childhood immunization programme. Resources should be sufficient to ensure reaching and sustaining vaccine coverage ≥ 80%. Vaccine coverage that remains <80% over the long term is expected to shift varicella infection to older ages in some settings, which may result in an increase of morbidity and mortality despite reduction in total number of cases [3]. Varicella vaccine was licensed in China in 1996 [7]. Currently, varicella vaccine is sold on the private market, meaning it is voluntary and must be self-paid. There is no national recommended immunization schedule available for varicella vaccine, and the vaccine is administered to eligible children at a vaccination clinic according to instructions provided by the vaccine manufacturers. Based on the immunization regulation, the record of vaccination information is kept by both the parents and the clinic [8–9]. In this study, we investigated the varicella vaccination information among children under 5 years old in selected areas in China, in order to understand the varicella vaccination situation for children in China. This information will provide evidence for authority department of China introducing varicella vaccine into the national routine immunization program.\n\nRESULTS:\n Basic information and vaccine coverage in selected counties In total, we collected varicella vaccination information from 481 caregivers’ vaccination certificates: 96 in Shanghai, 48 in Jiangsu, 96 in Heilongjiang, 52 in Jiangxi, 93 in Gansu and 96 in Chongqing, the geographical distribution of selected provinces see Figure 1; Male: 263 and female: 218, with a male to female ratio of 1.2:1. The varicella vaccine coverage of the first dose was 73.6% (354/481) for all children surveyed. The coverage in selected counties is described in Table 1. 1.2% (6/481) received the second dose of varicella vaccine.\nIn total, we collected varicella vaccination information from 481 caregivers’ vaccination certificates: 96 in Shanghai, 48 in Jiangsu, 96 in Heilongjiang, 52 in Jiangxi, 93 in Gansu and 96 in Chongqing, the geographical distribution of selected provinces see Figure 1; Male: 263 and female: 218, with a male to female ratio of 1.2:1. The varicella vaccine coverage of the first dose was 73.6% (354/481) for all children surveyed. The coverage in selected counties is described in Table 1. 1.2% (6/481) received the second dose of varicella vaccine.\n Relationship between varicella vaccine coverage and the economic situation The median coverage of varicella vaccine in areas with per capita GDP ≥ 100,000 RMB is 92.7% (85.4% - 97.9%), while median coverage of varicella vaccine in areas with per capita GDP < 100,000RMB is 55.9% (44.2% - 80.9%) (Wilcoxon rank sum test P < 0.05). There is a positive linear correlation between per capita GDP and vaccine coverage at county level (r = 0.929, P < 0.01). The equation of linear regression is y = 0.0004x + 37.08 (F = 50.426, P < 0.001) (Figure 2).\nThe median coverage of varicella vaccine in areas with per capita GDP ≥ 100,000 RMB is 92.7% (85.4% - 97.9%), while median coverage of varicella vaccine in areas with per capita GDP < 100,000RMB is 55.9% (44.2% - 80.9%) (Wilcoxon rank sum test P < 0.05). There is a positive linear correlation between per capita GDP and vaccine coverage at county level (r = 0.929, P < 0.01). The equation of linear regression is y = 0.0004x + 37.08 (F = 50.426, P < 0.001) (Figure 2).\n Cumulative coverage of varicella vaccine and distribution of vaccinated age At age of 1 year old(12-23 months), the vaccine coverage were 70.8%, 70.0%, 65.7%, 70.0%, 60.5% among children born in 2008, 2009, 2010, 2011 and 2012 respectively with no significant difference (X 2 = 3.15, P =0.53). The cumulative vaccine coverage ≥3 years old (≥ 36 months) were 79.2%, 78.0% and 74.7 % among children born in 2008, 2009 and 2010 respectively with no significant difference (X 2=0.59, P =0.75). The children born in 2012 were under 2 years of age during investigation for whom the vaccine coverage was lower (60.5%). In total, the cumulative coverage among children at 1 year, 2 years(24-35 months) and≥3 years old were 67.6%, 71.9% and 73.6% (X 2=4.53, P =0.10), with 4.3% increasing at 2 years old, and 1.7% at 3 years old and later compared to 1 year old (Table 2).\nNote:* 1 year old=”12-23 months”, 2 years old=”24-35 months”, ≥3 years old=”≥36 months”\nAmong all vaccinated children, 91.8% (325/354) received the vaccine at 1 year old, 5.9% (21/354) received at 2 years old, and 2.3% (8/354) at 3 years old and later. Among those receiving the vaccine at 1 year old, the vaccinated age was mainly concentrated in 12-17 months, accounting for 85.3% (302/325) (Figure 3).\nn=(ta/2Δp)2P(1−P),ta/2=1.96,Δp=0.0045,p=0.45\nAt age of 1 year old(12-23 months), the vaccine coverage were 70.8%, 70.0%, 65.7%, 70.0%, 60.5% among children born in 2008, 2009, 2010, 2011 and 2012 respectively with no significant difference (X 2 = 3.15, P =0.53). The cumulative vaccine coverage ≥3 years old (≥ 36 months) were 79.2%, 78.0% and 74.7 % among children born in 2008, 2009 and 2010 respectively with no significant difference (X 2=0.59, P =0.75). The children born in 2012 were under 2 years of age during investigation for whom the vaccine coverage was lower (60.5%). In total, the cumulative coverage among children at 1 year, 2 years(24-35 months) and≥3 years old were 67.6%, 71.9% and 73.6% (X 2=4.53, P =0.10), with 4.3% increasing at 2 years old, and 1.7% at 3 years old and later compared to 1 year old (Table 2).\nNote:* 1 year old=”12-23 months”, 2 years old=”24-35 months”, ≥3 years old=”≥36 months”\nAmong all vaccinated children, 91.8% (325/354) received the vaccine at 1 year old, 5.9% (21/354) received at 2 years old, and 2.3% (8/354) at 3 years old and later. Among those receiving the vaccine at 1 year old, the vaccinated age was mainly concentrated in 12-17 months, accounting for 85.3% (302/325) (Figure 3).\nn=(ta/2Δp)2P(1−P),ta/2=1.96,Δp=0.0045,p=0.45\n\nBasic information and vaccine coverage in selected counties:\nIn total, we collected varicella vaccination information from 481 caregivers’ vaccination certificates: 96 in Shanghai, 48 in Jiangsu, 96 in Heilongjiang, 52 in Jiangxi, 93 in Gansu and 96 in Chongqing, the geographical distribution of selected provinces see Figure 1; Male: 263 and female: 218, with a male to female ratio of 1.2:1. The varicella vaccine coverage of the first dose was 73.6% (354/481) for all children surveyed. The coverage in selected counties is described in Table 1. 1.2% (6/481) received the second dose of varicella vaccine.\n\nRelationship between varicella vaccine coverage and the economic situation:\nThe median coverage of varicella vaccine in areas with per capita GDP ≥ 100,000 RMB is 92.7% (85.4% - 97.9%), while median coverage of varicella vaccine in areas with per capita GDP < 100,000RMB is 55.9% (44.2% - 80.9%) (Wilcoxon rank sum test P < 0.05). There is a positive linear correlation between per capita GDP and vaccine coverage at county level (r = 0.929, P < 0.01). The equation of linear regression is y = 0.0004x + 37.08 (F = 50.426, P < 0.001) (Figure 2).\n\nCumulative coverage of varicella vaccine and distribution of vaccinated age:\nAt age of 1 year old(12-23 months), the vaccine coverage were 70.8%, 70.0%, 65.7%, 70.0%, 60.5% among children born in 2008, 2009, 2010, 2011 and 2012 respectively with no significant difference (X 2 = 3.15, P =0.53). The cumulative vaccine coverage ≥3 years old (≥ 36 months) were 79.2%, 78.0% and 74.7 % among children born in 2008, 2009 and 2010 respectively with no significant difference (X 2=0.59, P =0.75). The children born in 2012 were under 2 years of age during investigation for whom the vaccine coverage was lower (60.5%). In total, the cumulative coverage among children at 1 year, 2 years(24-35 months) and≥3 years old were 67.6%, 71.9% and 73.6% (X 2=4.53, P =0.10), with 4.3% increasing at 2 years old, and 1.7% at 3 years old and later compared to 1 year old (Table 2).\nNote:* 1 year old=”12-23 months”, 2 years old=”24-35 months”, ≥3 years old=”≥36 months”\nAmong all vaccinated children, 91.8% (325/354) received the vaccine at 1 year old, 5.9% (21/354) received at 2 years old, and 2.3% (8/354) at 3 years old and later. Among those receiving the vaccine at 1 year old, the vaccinated age was mainly concentrated in 12-17 months, accounting for 85.3% (302/325) (Figure 3).\nn=(ta/2Δp)2P(1−P),ta/2=1.96,Δp=0.0045,p=0.45\n\nDISCUSSION:\nThe basic reproduction number (R0) is about 8 ∼ 10 for varicella, which is higher than influenza [10], it is easy for varicella to cause an outbreak in a nursery, kindergarten or school. Therefore, it is very difficult to block the spreading of varicella disease in areas with low vaccine coverage, and it is necessary to maintain high coverage to prevent the disease from becoming an epidemic. The World Health Organization position paper states that varicella may transit to older age groups when the rate of vaccination coverage is lower than 80% for a significant amount of time, which may result in increased morbidity and mortality [3]. During this survey we found that the coverage of the varicella vaccine was higher in counties with higher per capita GDP, and was relatively lower in counties with lower per capita GDP. The coverage of selected areas varies from 44% to 98%, and basing on the linear regression modeling of the relationship between GDP and coverage, per capita GDP of China was 38,354 RMB in 2012 [11], we estimated the average coverage rate for children under 5 years old in China might be about 52%. A national survey conducted in 2011 in China showed that the varicella vaccine coverage rate under 2 years old was 47% [12], this is similarly with our estimates.\nVaricella vaccine is a private vaccine in China, the parents of children voluntarily select and pay for the vaccine for their children. In areas with low coverage rate, a child will be individually protected due to the vaccination, however, herd immunity may not be established at the population level. Furthermore, as a private vaccine, the vaccination fee may become a barrier to vaccinate for children who live in financially poor resource environments [13, 14]. The inequalities of immunization coverage due to socioeconomic differences also exist in other countries [15, 16]. Varicella vaccine has been demonstrated good safety and effect [3], and one study in China showed that introducing varicella vaccination into the routine childhood immunization programme is quite cost effective: an one-dose strategy could save 8 billion RMB in one year [17]. We suggest introducing the varicella vaccine into the routine childhood immunization program as soon as possible to improve the equity in access to immunization services in areas of low socioeconomic status, and ensure a coverage rate of more than 80% in all areas according to the recommendations by WHO.\nOur study showed that the coverage rate for different birth cohorts remained stable. The vaccine coverage rate was 68% at 1 year old, and increased slightly with age, cumulative vaccine coverage rate was 72% at 2 years old and 74% ≥ 3 years old, suggesting that after 3 years of age the access to varicella vaccination service is limited. The peak seasons for incidence of varicella in China are winter and spring seasons, and mainly effect preschool and school-age children of 3 ∼ 10 years old [18]. In China, varicella outbreaks account for a relatively high proportion of the school emergency public health events. For example, reported outbreaks due to varicella in Zhejiang province accounted for 35% in the school emergency public health events from 2005 to 2008 [19]. Therefore, it is necessary to provide an opportunity for access to varicella vaccination service for those children over 3 years of age, who missed the opportunity for vaccination at a younger age. School entry check of immunization certificates and providing vaccination services for eligible children during entry of kindergarten and school may increase vaccine coverage rate among older children and reduce the amount of outbreaks in nurseries and schools [20].\nAt present, the one-dose schedule for varicella vaccine is used in most provinces in China, and the one-dose schedule is used in the 6 provinces selected for this investigation. This survey found that most children completed the schedule before 2 years of age, with the majority (85%) receiving the vaccine at 12-17 months old. Research in the United States showed that the vaccine efficacy decreased significantly (only 73%) during the first year after vaccination if the vaccine is provided before 15 months old [10]. Before 2006, the one-dose schedule was recommended in the United States. After 2006, in order to strengthen the control of varicella, a two-dose schedule was introduced, the first dose is administered at 12-15 months old and the second dose administered at preschool age (4-6 years old) [21]. Geometric mean titer (GMT) showed a particularly high boost after the second dose when the interval between doses was more than one year [22]. A two-dose schedule was introduced in Beijing in 2012: the first dose at 18 months of age and the second dose at 4 years of age [7]. To further improve and optimize the varicella vaccine schedule, we suggest further developing the recommendations regarding the varicella immunization schedule by considering disease risk and the effectiveness of vaccines.\nLimitations: We used purposive sampling methods to select provinces and counties (districts) [23], the selected provinces cannot represent all of China. And we obtained the sample based on Immunization Information Management System (IIMS), according to the national requirements, all immunization information for children should be reported to IIMS, however, the quality of the IIMS data varies across areas, it is possible that some migrant children's information was not recorded in IIMS, which may overestimate the coverage rate of varicella vaccine.\n\nMATERIALS AND METHODS:\n Sampling methods We used purposive sampling methods to select counties (districts) from Shanghai, Jiangsu, Heilongjiang, Jiangxi, Chongqing and Gansu provinces. We selected these investigation sites based on two factors: (i) representation of east, middle and western parts of China; (ii) having good records of private vaccinations in children's vaccination certificates and with a high quality Immunization Information Management System (IIMS). From Nov 1th 2013 to Nov 15th 2013, we used the simple random sampling method to randomly select children from the population whose birth date was between Jan 1st 2008 and Dec 31th 2012 in the IIMS in selected counties (districts), then from January 1th 2014 to July 15th2014, we checked hand-held vaccination certificates on-site to collect varicella vaccination information.\nWe used the following formula to calculate the sample size:\nn=(ta/2Δp)2P(1−P),ta/2=1.96,Δp=0.0045,p=0.45\nn: the sample size, P: estimate coverage of varicella vaccine, based on past experience, we used 45%. After calculation, a sample size of 470 was needed for this survey.\nConsidering a loss to follow-up of about 10%, a total of 520 children needed to be investigated. The sample size was divided by 10 (10 counties), and 52 children in each county were selected to participate in the investigation. Finally, 39 children were excluded because their parents did not have a hand-held vaccination certificate or we were unable to contact them.\nWe used purposive sampling methods to select counties (districts) from Shanghai, Jiangsu, Heilongjiang, Jiangxi, Chongqing and Gansu provinces. We selected these investigation sites based on two factors: (i) representation of east, middle and western parts of China; (ii) having good records of private vaccinations in children's vaccination certificates and with a high quality Immunization Information Management System (IIMS). From Nov 1th 2013 to Nov 15th 2013, we used the simple random sampling method to randomly select children from the population whose birth date was between Jan 1st 2008 and Dec 31th 2012 in the IIMS in selected counties (districts), then from January 1th 2014 to July 15th2014, we checked hand-held vaccination certificates on-site to collect varicella vaccination information.\nWe used the following formula to calculate the sample size:\nn=(ta/2Δp)2P(1−P),ta/2=1.96,Δp=0.0045,p=0.45\nn: the sample size, P: estimate coverage of varicella vaccine, based on past experience, we used 45%. After calculation, a sample size of 470 was needed for this survey.\nConsidering a loss to follow-up of about 10%, a total of 520 children needed to be investigated. The sample size was divided by 10 (10 counties), and 52 children in each county were selected to participate in the investigation. Finally, 39 children were excluded because their parents did not have a hand-held vaccination certificate or we were unable to contact them.\n Calculation of per capita gross domestic product (GDP) Per capita GDP=GDP/domicile population. GDP and household population data came from the “2012 Per Capita National Economic and Social Development Statistical Bulletin” on the selected County or District's Bureau of Statistics website. The data was not available for Jianye in Jiangsu, Ning'an in Heilongjiang, Chengguan and Qilihe in Gansu, so the per capita GDP in the corresponding city level (higher administrative level) was used for these counties.\nPer capita GDP=GDP/domicile population. GDP and household population data came from the “2012 Per Capita National Economic and Social Development Statistical Bulletin” on the selected County or District's Bureau of Statistics website. The data was not available for Jianye in Jiangsu, Ning'an in Heilongjiang, Chengguan and Qilihe in Gansu, so the per capita GDP in the corresponding city level (higher administrative level) was used for these counties.\n Survey content and data processing We conducted this survey to obtain the basic information of children (such as gender, date of birth) and varicella vaccination (such as inoculation date and doses). We used Excel to enter the data, ArcGIS to draw the map, and Epi Info software to analyze the data.\nWe conducted this survey to obtain the basic information of children (such as gender, date of birth) and varicella vaccination (such as inoculation date and doses). We used Excel to enter the data, ArcGIS to draw the map, and Epi Info software to analyze the data.\n Ethics statement We obtained oral informed consent from all children's guardians before the investigation, and two investigators checked vaccination certificates on-site together. We would continue the investigation only when the children's guardians gave consent. We didn't go through the institutional review board approval in the case of this study for ethical issues, because monitoring vaccine coverage is part of routine program work, in this context, there is no risk for participants and it is not required to get IRB approval. However, we strictly protected the private information of participants in the field, such as the name and contact information. During the analysis, we also took out the personal identification information.\nWe obtained oral informed consent from all children's guardians before the investigation, and two investigators checked vaccination certificates on-site together. We would continue the investigation only when the children's guardians gave consent. We didn't go through the institutional review board approval in the case of this study for ethical issues, because monitoring vaccine coverage is part of routine program work, in this context, there is no risk for participants and it is not required to get IRB approval. However, we strictly protected the private information of participants in the field, such as the name and contact information. During the analysis, we also took out the personal identification information.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nVaccine is the most effective way to protect susceptible children from varicella. Few published literature or reports on varicella vaccination of Chinese children exist. Thus, in order to obtain specific information on varicella vaccination of this population, we conducted this survey.\n\nMethods:\nWe first used purposive sampling methods to select 6 provinces 10 counties from eastern, middle and western parts of China with high quality of Immunization Information Management System (IIMS), and then randomly select children from population in the IIMS, then we checked vaccination certificate on-site.\n\nResults:\nBased on the varicella vaccination information collected from 481 children's vaccination certificates from all ten selected counties in China, overall coverage of the first dose of varicella vaccine was 73.6%. There is a positive linear correlation between per capita GDP and vaccine coverage at county level (r=0.929, P < 0.01). The cumulative vaccine coverage among children at 1 year, 2 years and ≥3 years old were 67.6%, 71.9% and 73.6% respectively (X2=4.53, P =0.10). The age of vaccination was mainly concentrated in 12-17 months.\n\nConclusions:\nThe coverage rate of the first dose of varicella vaccine in selected areas was lower than that recommended by WHO position paper. The coverage rate was relatively low in areas of low social-economic status. The cumulative coverage had no significant statistical difference among different age group. Most children received varicella vaccine before 3 years old. We suggest introducing the varicella vaccine into routine immunization program, to ensure universal high coverage among children in China. We also suggest that varicella vaccination information should be checked before entering school, in order to control and prevent varicella outbreaks in schools."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nVaricella is an infectious disease with high transmissibility, common in children [1]. The disease is prone to resulting in large scale outbreak in institutional units, such as nurseries, kindergartens, and schools [2]. Varicella vaccine has been demonstrated good safety and effect [3], and varicella vaccination is the most effective measure to protect susceptible people from the disease. Although varicella case information has been collected nationally since 2005 through National Disease Supervision Information Management System(NDSIMS), its limitation in quality of local clinic reporting poses a challenge in accurate analysis and evaluation of the true situation in China [4]. Based on the estimation of varicella incidence in Shandong, Gansu and Hunan provinces, 4,705,000 cases were reported in 2007 in China, and the cost estimation in 2007 was 2.31 billion RMB for outpatients and 103 million RMB for inpatients [5, 6]. “Varicella and Herpes Zoster Vaccines: WHO Position Paper” recommends that countries where varicella is an important public health burden could consider introducing varicella vaccination in the routine childhood immunization programme. Resources should be sufficient to ensure reaching and sustaining vaccine coverage ≥ 80%. Vaccine coverage that remains <80% over the long term is expected to shift varicella infection to older ages in some settings, which may result in an increase of morbidity and mortality despite reduction in total number of cases [3]. Varicella vaccine was licensed in China in 1996 [7]. Currently, varicella vaccine is sold on the private market, meaning it is voluntary and must be self-paid. There is no national recommended immunization schedule available for varicella vaccine, and the vaccine is administered to eligible children at a vaccination clinic according to instructions provided by the vaccine manufacturers. Based on the immunization regulation, the record of vaccination information is kept by both the parents and the clinic [8–9]. In this study, we investigated the varicella vaccination information among children under 5 years old in selected areas in China, in order to understand the varicella vaccination situation for children in China. This information will provide evidence for authority department of China introducing varicella vaccine into the national routine immunization program.\n\nDISCUSSION:\nWe obtained oral informed consent from all children's guardians before the investigation, and two investigators checked vaccination certificates on-site together. We would continue the investigation only when the children's guardians gave consent. We didn't go through the institutional review board approval in the case of this study for ethical issues, because monitoring vaccine coverage is part of routine program work, in this context, there is no risk for participants and it is not required to get IRB approval. However, we strictly protected the private information of participants in the field, such as the name and contact information. During the analysis, we also took out the personal identification information."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nVaccine is the most effective way to protect susceptible children from varicella. Few published literature or reports on varicella vaccination of Chinese children exist. Thus, in order to obtain specific information on varicella vaccination of this population, we conducted this survey.\n\nMethods:\nWe first used purposive sampling methods to select 6 provinces 10 counties from eastern, middle and western parts of China with high quality of Immunization Information Management System (IIMS), and then randomly select children from population in the IIMS, then we checked vaccination certificate on-site.\n\nResults:\nBased on the varicella vaccination information collected from 481 children's vaccination certificates from all ten selected counties in China, overall coverage of the first dose of varicella vaccine was 73.6%. There is a positive linear correlation between per capita GDP and vaccine coverage at county level (r=0.929, P < 0.01). The cumulative vaccine coverage among children at 1 year, 2 years and ≥3 years old were 67.6%, 71.9% and 73.6% respectively (X2=4.53, P =0.10). The age of vaccination was mainly concentrated in 12-17 months.\n\nConclusions:\nThe coverage rate of the first dose of varicella vaccine in selected areas was lower than that recommended by WHO position paper. The coverage rate was relatively low in areas of low social-economic status. The cumulative coverage had no significant statistical difference among different age group. Most children received varicella vaccine before 3 years old. We suggest introducing the varicella vaccine into routine immunization program, to ensure universal high coverage among children in China. We also suggest that varicella vaccination information should be checked before entering school, in order to control and prevent varicella outbreaks in schools."},"whole_article_text_length":{"kind":"number","value":4206,"string":"4,206"},"whole_article_abstract_length":{"kind":"number","value":326,"string":"326"},"other_sections_lengths":{"kind":"list like","value":[404,1076,109,111,300,1051],"string":"[\n 404,\n 1076,\n 109,\n 111,\n 300,\n 1051\n]","isConversational":false},"num_sections":{"kind":"number","value":7,"string":"7"},"most_frequent_words":{"kind":"list like","value":["vaccine","varicella","coverage","old","children","years","vaccination","varicella vaccine","years old","vaccine coverage"],"string":"[\n \"vaccine\",\n \"varicella\",\n \"coverage\",\n \"old\",\n \"children\",\n \"years\",\n \"vaccination\",\n \"varicella vaccine\",\n \"years old\",\n \"vaccine coverage\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["varicella vaccine 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varicella vaccine | china varicella outbreaks | varicella vaccination situation [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] varicella vaccine national | estimates varicella vaccine | schools varicella vaccine | china varicella outbreaks | varicella vaccination situation [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] varicella vaccine national | estimates varicella vaccine | schools varicella vaccine | china varicella outbreaks | varicella vaccination situation [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] vaccine | varicella | coverage | old | children | years | vaccination | varicella vaccine | years old | vaccine coverage [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] vaccine | varicella | coverage | old | children | years | vaccination | varicella vaccine | years old 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[SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 6 | 10 | eastern | China | Immunization Information Management System [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"null"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] first ||| ||| ||| 3 years old ||| China ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Vaccine ||| Chinese ||| ||| 6 | 10 | eastern | China | Immunization Information Management System ||| 481 | ten | China | first | 73.6% ||| 0.01 ||| 1 year | 2 years | years | 67.6% | 71.9% | 73.6% | X2=4.53 | 0.10 ||| 12-17 months ||| first ||| ||| ||| 3 years old ||| China ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Vaccine ||| Chinese ||| ||| 6 | 10 | eastern | China | Immunization Information Management System ||| 481 | ten | China | first | 73.6% ||| 0.01 ||| 1 year | 2 years | years | 67.6% | 71.9% | 73.6% | X2=4.53 | 0.10 ||| 12-17 months ||| first ||| ||| ||| 3 years old ||| China ||| [SUMMARY]"}}},{"rowIdx":68,"cells":{"title":{"kind":"string","value":"Ischemia-Selective Cardioprotection by Malonate for Ischemia/Reperfusion Injury."},"pmid":{"kind":"string","value":"35959683"},"background_abstract":{"kind":"string","value":"Inhibiting SDH (succinate dehydrogenase), with the competitive inhibitor malonate, has shown promise in ameliorating ischemia/reperfusion injury. However, key for translation to the clinic is understanding the mechanism of malonate entry into cells to enable inhibition of SDH, its mitochondrial target, as malonate itself poorly permeates cellular membranes. The possibility of malonate selectively entering the at-risk heart tissue on reperfusion, however, remains unexplored."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"C57BL/6J mice, C2C12 and H9c2 myoblasts, and HeLa cells were used to elucidate the mechanism of selective malonate uptake into the ischemic heart upon reperfusion. Cells were treated with malonate while varying pH or together with transport inhibitors. Mouse hearts were either perfused ex vivo (Langendorff) or subjected to in vivo left anterior descending coronary artery ligation as models of ischemia/reperfusion injury. Succinate and malonate levels were assessed by liquid chromatography-tandem mass spectrometry LC-MS/MS, in vivo by mass spectrometry imaging, and infarct size by TTC (2,3,5-triphenyl-2H-tetrazolium chloride) staining."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Malonate was robustly protective against cardiac ischemia/reperfusion injury, but only if administered at reperfusion and not when infused before ischemia. The extent of malonate uptake into the heart was proportional to the duration of ischemia. Malonate entry into cardiomyocytes in vivo and in vitro was dramatically increased at the low pH (≈6.5) associated with ischemia. This increased uptake of malonate was blocked by selective inhibition of MCT1 (monocarboxylate transporter 1). Reperfusion of the ischemic heart region with malonate led to selective SDH inhibition in the at-risk region. Acid-formulation greatly enhances the cardioprotective potency of malonate."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Cardioprotection by malonate is dependent on its entry into cardiomyocytes. This is facilitated by the local decrease in pH that occurs during ischemia, leading to its selective uptake upon reperfusion into the at-risk tissue, via MCT1. Thus, malonate's preferential uptake in reperfused tissue means it is an at-risk tissue-selective drug that protects against cardiac ischemia/reperfusion injury."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Animals","Chromatography, Liquid","HeLa Cells","Humans","Ischemia","Malonates","Mice","Mice, Inbred C57BL","Myocardial Reperfusion Injury","Myocytes, Cardiac","Tandem Mass Spectrometry"],"string":"[\n \"Animals\",\n \"Chromatography, Liquid\",\n \"HeLa Cells\",\n \"Humans\",\n \"Ischemia\",\n \"Malonates\",\n \"Mice\",\n \"Mice, Inbred C57BL\",\n \"Myocardial Reperfusion Injury\",\n \"Myocytes, Cardiac\",\n \"Tandem Mass Spectrometry\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"9426742"},"background_title":{"kind":"null"},"background_text":{"kind":"null"},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Data Availability Detailed methods and Major Resources Table can be found in the Supplemental Material. Data will be made available upon reasonable request, by contacting a corresponding author.\nDetailed methods and Major Resources Table can be found in the Supplemental Material. Data will be made available upon reasonable request, by contacting a corresponding author."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Malonate Is Protective in an MI Model and Is Taken Up Selectively into the Ischemic Region of the Heart DSM is protective against cardiac IR injury, but its mechanism of protection remains uncertain. To address this gap in our knowledge we first tested the therapeutic range of DSM in IR injury. We infused DSM at a range of concentrations (1.6–160 mg/kg equivalent to ≈11-1100 µmol/kg) at reperfusion in an in vivo murine LAD (left anterior descending coronary artery) ligation model of MI. DSM infusion during this clinically relevant reperfusion period led to a dose-dependent decrease in infarct size (Figure 1C), with 160 and 16 mg/kg showing robust cardioprotection. However, when DSM was infused before the onset of ischemia it was not protective (Figure 1D), even at 160 mg/kg which gave the smallest infarct when administered at reperfusion. This contrasted with the cell-permeable dimethyl malonate, which can diffuse into the tissue and generate malonate, preventing succinate accumulation and thereby reducing IR injury.18,25 This suggests that either DSM is minimally taken up by tissues during normoxia and is only taken up by tissues after ischemia or that DSM protects against acute IR injury by an extracellular mechanism.\nWe next measured malonate uptake into the ischemic and healthy heart tissue upon reperfusion in the LAD MI model. This showed that malonate was indeed taken up into cells within the infarct region to a level of ≈400 pmol/mg tissue and to a far greater extent than into cells in the healthy tissue (Figure 1E). Furthermore, reperfusing with malonate slowed succinate oxidation within the infarct region with succinate remaining significantly elevated in the infarct region following 1-minute reperfusion, compared with control reperfusion with saline (Figure 1F). Limiting succinate oxidation at reperfusion with malonate also blunted ROS production in the at-risk tissue (Figure 1G). We next assessed how SDH inhibition compared to direct inhibition of the mitochondrial permeability transition pore with CsA (cyclosporin A). Here we found that the cardioprotection from malonate was additive to cyclosporin A alone (Figure 1H), hence targeting the upstream mechanism of permeability transition pore opening is an increasingly attractive option for IR injury treatment.\nTo better understand the metabolic differences between the healthy and infarcted tissue upon malonate treatment, we investigated the in vivo MI model using mass spectrometry imaging. To do this, we subjected hearts to either 30-minute ischemia, 30-minute ischemia before reperfusing the heart for 15-minute, or 30-minute ischemia before reperfusing with DSM for 15 minutes (to mimic the cardioprotective malonate infusion) before snap freezing and processing for mass spectrometry imaging (Figure 2A). Infarct lesions were demarcated using hematoxylin and eosin (H&E), silver infarct staining and metabolite principal component analysis to differentiate the risk and nonrisk regions. mass spectrometry imaging coupled with the demarcated risk areas showed striking changes in the levels of succinate in the infarct region. During ischemia, succinate was significantly elevated in the at-risk tissue, with considerable succinate accumulation in the core of the infarct (Figure 2B and 2C). After 15-minute reperfusion, the succinate levels in the infarct lesion returned to healthy tissue levels, due to succinate oxidation and efflux.21 When malonate was infused at the time of reperfusion, succinate levels remained higher in all regions of the heart than in the nonmalonate treated heart, consistent with the prevention of its oxidation by SDH inhibition by malonate.\nLocalization of succinate accumulation in heart tissue identified by mass spectrometry imaging. A, Outline of experimental groups for mass spectrometry imaging. B, Representative images of succinate abundance detected by mass spectrometry imaging in myocardial sections. Black outer line indicates the edge of the tissue slice, white inner line indicates infarct region. C, Quantification of succinate (C) identified by MSI (mean±SEM, n=3 biological replicates, statistics: Friedman paired test for healthy vs lesion and Kruskal-Wallis with Dunn post hoc test between conditions). au indicates arbitrary units; DSM, disodium malonate; H, healthy tissue; IR, ischemia/reperfusion; L, infarct lesion; and rep, reperfusion.\nWe conclude that malonate is indeed taken up by the heart, but this is greatly enhanced in ischemic region upon reperfusion. Therefore, malonate provides protection against cardiac IR injury in vivo over a range of concentrations following its selective entry into the infarct region, where it slows the oxidation of succinate during reperfusion, preventing the production of RET-derived ROS.\nDSM is protective against cardiac IR injury, but its mechanism of protection remains uncertain. To address this gap in our knowledge we first tested the therapeutic range of DSM in IR injury. We infused DSM at a range of concentrations (1.6–160 mg/kg equivalent to ≈11-1100 µmol/kg) at reperfusion in an in vivo murine LAD (left anterior descending coronary artery) ligation model of MI. DSM infusion during this clinically relevant reperfusion period led to a dose-dependent decrease in infarct size (Figure 1C), with 160 and 16 mg/kg showing robust cardioprotection. However, when DSM was infused before the onset of ischemia it was not protective (Figure 1D), even at 160 mg/kg which gave the smallest infarct when administered at reperfusion. This contrasted with the cell-permeable dimethyl malonate, which can diffuse into the tissue and generate malonate, preventing succinate accumulation and thereby reducing IR injury.18,25 This suggests that either DSM is minimally taken up by tissues during normoxia and is only taken up by tissues after ischemia or that DSM protects against acute IR injury by an extracellular mechanism.\nWe next measured malonate uptake into the ischemic and healthy heart tissue upon reperfusion in the LAD MI model. This showed that malonate was indeed taken up into cells within the infarct region to a level of ≈400 pmol/mg tissue and to a far greater extent than into cells in the healthy tissue (Figure 1E). Furthermore, reperfusing with malonate slowed succinate oxidation within the infarct region with succinate remaining significantly elevated in the infarct region following 1-minute reperfusion, compared with control reperfusion with saline (Figure 1F). Limiting succinate oxidation at reperfusion with malonate also blunted ROS production in the at-risk tissue (Figure 1G). We next assessed how SDH inhibition compared to direct inhibition of the mitochondrial permeability transition pore with CsA (cyclosporin A). Here we found that the cardioprotection from malonate was additive to cyclosporin A alone (Figure 1H), hence targeting the upstream mechanism of permeability transition pore opening is an increasingly attractive option for IR injury treatment.\nTo better understand the metabolic differences between the healthy and infarcted tissue upon malonate treatment, we investigated the in vivo MI model using mass spectrometry imaging. To do this, we subjected hearts to either 30-minute ischemia, 30-minute ischemia before reperfusing the heart for 15-minute, or 30-minute ischemia before reperfusing with DSM for 15 minutes (to mimic the cardioprotective malonate infusion) before snap freezing and processing for mass spectrometry imaging (Figure 2A). Infarct lesions were demarcated using hematoxylin and eosin (H&E), silver infarct staining and metabolite principal component analysis to differentiate the risk and nonrisk regions. mass spectrometry imaging coupled with the demarcated risk areas showed striking changes in the levels of succinate in the infarct region. During ischemia, succinate was significantly elevated in the at-risk tissue, with considerable succinate accumulation in the core of the infarct (Figure 2B and 2C). After 15-minute reperfusion, the succinate levels in the infarct lesion returned to healthy tissue levels, due to succinate oxidation and efflux.21 When malonate was infused at the time of reperfusion, succinate levels remained higher in all regions of the heart than in the nonmalonate treated heart, consistent with the prevention of its oxidation by SDH inhibition by malonate.\nLocalization of succinate accumulation in heart tissue identified by mass spectrometry imaging. A, Outline of experimental groups for mass spectrometry imaging. B, Representative images of succinate abundance detected by mass spectrometry imaging in myocardial sections. Black outer line indicates the edge of the tissue slice, white inner line indicates infarct region. C, Quantification of succinate (C) identified by MSI (mean±SEM, n=3 biological replicates, statistics: Friedman paired test for healthy vs lesion and Kruskal-Wallis with Dunn post hoc test between conditions). au indicates arbitrary units; DSM, disodium malonate; H, healthy tissue; IR, ischemia/reperfusion; L, infarct lesion; and rep, reperfusion.\nWe conclude that malonate is indeed taken up by the heart, but this is greatly enhanced in ischemic region upon reperfusion. Therefore, malonate provides protection against cardiac IR injury in vivo over a range of concentrations following its selective entry into the infarct region, where it slows the oxidation of succinate during reperfusion, preventing the production of RET-derived ROS.\n Malonate Uptake into Cells Is Inefficient at pH 7.4 To explore the mechanism of malonate uptake in vitro, we incubated C2C12 and H9c2 myoblasts with DSM and measured intracellular malonate levels by LC-MS/MS (liquid chromatography-tandem mass spectrometry). Incubation with DSM for 15 minutes at pH 7.4, led to a dose-dependent increase in intracellular malonate (Figure S1A and S1D). Intracellular succinate also accumulated in a malonate-dependent manner (Figure S1B and S1E), confirming that once malonate enters the cell it is rapidly transported into mitochondria and inhibits SDH. However, incubating cells with 1 mmol/L DSM led to variable malonate uptake and little SDH inhibition, indicating that malonate uptake across biological membranes is inefficient. Moreover, the intracellular levels of malonate achieved by 250 µmol/L of the malonate ester prodrug diacetoxymethyl malonate, are 80-fold more than that generated by incubation with 5 mmol/L DSM.25 As DSM was protective in the LAD model when infused at 16 mg/kg, corresponding to a maximum possible blood malonate concentration of ≈1.8 mmol/L (assuming a blood volume of 1.5 mL in a 25 g mouse),29 this compares to the dose range tested in cells (furthermore, far more malonate is available for uptake in vitro than in vivo due to the large reservoir in the incubation medium). Even so, the intracellular malonate levels were far lower with DSM compared to diacetoxymethyl malonate.25\nThe kinetics of cell uptake upon incubation with 5 mmol/L DSM showed time-dependent uptake, although extended incubation times were required for succinate elevation (Figure S1C and S1F). This is likely due to the initial concentrations of malonate entering the cell being insufficient to inhibit SDH. These data are consistent with the lack of protection by DSM delivered before ischemia in vivo, as well as its limited uptake into normoxic tissues. Together these suggest that exposure to ischemia may facilitate malonate entry into the heart.\nTo explore the mechanism of malonate uptake in vitro, we incubated C2C12 and H9c2 myoblasts with DSM and measured intracellular malonate levels by LC-MS/MS (liquid chromatography-tandem mass spectrometry). Incubation with DSM for 15 minutes at pH 7.4, led to a dose-dependent increase in intracellular malonate (Figure S1A and S1D). Intracellular succinate also accumulated in a malonate-dependent manner (Figure S1B and S1E), confirming that once malonate enters the cell it is rapidly transported into mitochondria and inhibits SDH. However, incubating cells with 1 mmol/L DSM led to variable malonate uptake and little SDH inhibition, indicating that malonate uptake across biological membranes is inefficient. Moreover, the intracellular levels of malonate achieved by 250 µmol/L of the malonate ester prodrug diacetoxymethyl malonate, are 80-fold more than that generated by incubation with 5 mmol/L DSM.25 As DSM was protective in the LAD model when infused at 16 mg/kg, corresponding to a maximum possible blood malonate concentration of ≈1.8 mmol/L (assuming a blood volume of 1.5 mL in a 25 g mouse),29 this compares to the dose range tested in cells (furthermore, far more malonate is available for uptake in vitro than in vivo due to the large reservoir in the incubation medium). Even so, the intracellular malonate levels were far lower with DSM compared to diacetoxymethyl malonate.25\nThe kinetics of cell uptake upon incubation with 5 mmol/L DSM showed time-dependent uptake, although extended incubation times were required for succinate elevation (Figure S1C and S1F). This is likely due to the initial concentrations of malonate entering the cell being insufficient to inhibit SDH. These data are consistent with the lack of protection by DSM delivered before ischemia in vivo, as well as its limited uptake into normoxic tissues. Together these suggest that exposure to ischemia may facilitate malonate entry into the heart.\n Malonate Uptake into Cells and the Heart Can Be Modulated by pH Malonate is a dicarboxylate at physiological pH (Figure 1A, pKa=2.83 and 5.69), suggesting that the pH decrease in ischemic tissue9,11,30 may enhance malonate uptake into the heart during early reperfusion, by increasing the concentration of its monocarboxylic form. Incubating cells with DSM at either pH 6, 7.4, or 8 for 15 minutes, led to large differences in malonate uptake (Figure 3A and Figure S2A). At pH 6, the levels of malonate in the cell were significantly higher than at pH 7.4 or 8, thus malonate uptake is favored by acidic pH. Succinate levels mirrored those of malonate, with greater succinate accumulation as a result of increased malonate-dependent SDH inhibition at low pH (Figure 3B and Figure S2B). Low pH alone had no effect on succinate levels (Figure 3B and Figure S2B), suggesting that it was due to malonate entry into the cells followed by SDH inhibition.\nMalonate uptake is enhanced at low pH. A and B, C2C12 cells were incubated with disodium malonate (DSM; 0, 1, or 5 mmol/L) for 15 min at either pH 6, 7.4, or 8 before measuring intracellular malonate (A) and succinate (B) by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). C, Malonate levels in C2C12 cells after incubation with DSM (5 mmol/L) for 15 min at various (patho)physiological pH (mean±SEM, n=3 biological replicates). D, Malonate levels in murine isolated Langendorff-perfused hearts treated with 5 mmol/L DSM infused at either pH 7.4 or 6 for 5 min (mean±SEM, n=4 biological replicates, statistics: unpaired, 2-tailed Mann-Whitney U test). E to H, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at either pH 6 or 7.4 in the presence of FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) (E), gramicidin (F), nigericin (G), or BAM15 (N5,N6-bis(2-Fluorophenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine) (H) before measuring intracellular malonate by LC-MS/MS (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). I, Structures of malonic acid and malonamic acid. J, Quantification of levels of malonate and malonamate in C2C12 cells after incubation (5 mmol/L DSM or disodium malonamate; 15 min) at pH 7.4 (n=6 biological replicates). K, C2C12 cells were incubated with either DSM or disodium malonamate (both 5 mmol/L) at pH 6 or 7.4 for 15 min and the intracellular levels measured by LC-MS/MS. (mean±SEM of the fold-enhancement of uptake at pH 6 vs pH 7.4, n=6 biological replicates). Statistics for J and K: unpaired, 2-tailed Student t test).\nIncubating cells with malonate over a range of pH values between pH 7.4 and 6 clearly showed the pH-dependent uptake of malonate, in line with the increase in monocarboxylate malonate proportion (Figure 3C). This was mirrored by succinate levels (Figure S2C). Therefore, even a small drop in pH can lead to a substantial increase in malonate uptake, and thus is likely to be relevant for its entry into ischemic tissue upon reperfusion.\nAs the cultured cells used are noncontractile and substantially differ in their properties from in situ contracting cardiomyocytes,15 importantly, we next assessed whether decreasing the pH also enhanced malonate uptake into the ex vivo perfused Langendorff heart. Malonate was infused into the heart at either pH 7.4 or 6 for 5 minutes, before briefly flushing at pH 7.4 to remove nonmyocardial malonate. Remarkably, the levels of malonate were significantly elevated when infused at pH 6 compared to 7.4, confirming that the in vitro results translate to the heart (Figure 3D). We conclude that low pH conditions facilitate the entry of malonate into cardiomyocytes.\nMalonate is a dicarboxylate at physiological pH (Figure 1A, pKa=2.83 and 5.69), suggesting that the pH decrease in ischemic tissue9,11,30 may enhance malonate uptake into the heart during early reperfusion, by increasing the concentration of its monocarboxylic form. Incubating cells with DSM at either pH 6, 7.4, or 8 for 15 minutes, led to large differences in malonate uptake (Figure 3A and Figure S2A). At pH 6, the levels of malonate in the cell were significantly higher than at pH 7.4 or 8, thus malonate uptake is favored by acidic pH. Succinate levels mirrored those of malonate, with greater succinate accumulation as a result of increased malonate-dependent SDH inhibition at low pH (Figure 3B and Figure S2B). Low pH alone had no effect on succinate levels (Figure 3B and Figure S2B), suggesting that it was due to malonate entry into the cells followed by SDH inhibition.\nMalonate uptake is enhanced at low pH. A and B, C2C12 cells were incubated with disodium malonate (DSM; 0, 1, or 5 mmol/L) for 15 min at either pH 6, 7.4, or 8 before measuring intracellular malonate (A) and succinate (B) by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). C, Malonate levels in C2C12 cells after incubation with DSM (5 mmol/L) for 15 min at various (patho)physiological pH (mean±SEM, n=3 biological replicates). D, Malonate levels in murine isolated Langendorff-perfused hearts treated with 5 mmol/L DSM infused at either pH 7.4 or 6 for 5 min (mean±SEM, n=4 biological replicates, statistics: unpaired, 2-tailed Mann-Whitney U test). E to H, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at either pH 6 or 7.4 in the presence of FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) (E), gramicidin (F), nigericin (G), or BAM15 (N5,N6-bis(2-Fluorophenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine) (H) before measuring intracellular malonate by LC-MS/MS (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). I, Structures of malonic acid and malonamic acid. J, Quantification of levels of malonate and malonamate in C2C12 cells after incubation (5 mmol/L DSM or disodium malonamate; 15 min) at pH 7.4 (n=6 biological replicates). K, C2C12 cells were incubated with either DSM or disodium malonamate (both 5 mmol/L) at pH 6 or 7.4 for 15 min and the intracellular levels measured by LC-MS/MS. (mean±SEM of the fold-enhancement of uptake at pH 6 vs pH 7.4, n=6 biological replicates). Statistics for J and K: unpaired, 2-tailed Student t test).\nIncubating cells with malonate over a range of pH values between pH 7.4 and 6 clearly showed the pH-dependent uptake of malonate, in line with the increase in monocarboxylate malonate proportion (Figure 3C). This was mirrored by succinate levels (Figure S2C). Therefore, even a small drop in pH can lead to a substantial increase in malonate uptake, and thus is likely to be relevant for its entry into ischemic tissue upon reperfusion.\nAs the cultured cells used are noncontractile and substantially differ in their properties from in situ contracting cardiomyocytes,15 importantly, we next assessed whether decreasing the pH also enhanced malonate uptake into the ex vivo perfused Langendorff heart. Malonate was infused into the heart at either pH 7.4 or 6 for 5 minutes, before briefly flushing at pH 7.4 to remove nonmyocardial malonate. Remarkably, the levels of malonate were significantly elevated when infused at pH 6 compared to 7.4, confirming that the in vitro results translate to the heart (Figure 3D). We conclude that low pH conditions facilitate the entry of malonate into cardiomyocytes.\n Malonate Uptake Can Be Perturbed by Modulating the Plasma Membrane H+ Gradient As pH modulated malonate entry into cells in vitro and in the heart, we next probed the factors affecting malonate uptake into cells. Malonate entry into cells at either pH 7.4 or 6 was blocked at 4 °C and was associated with negligible succinate levels (Figure S2D and S2E). That a decrease in extracellular pH increased malonate entry into cells, suggested uptake driven by the proton gradient. Therefore, we next abolished the plasma membrane proton gradient using ionophores21 which prevented the cellular uptake of malonate (Figure 3E–3G and Figure S3A) and subsequent increase in succinate levels (Figure S3B through S3E). However, the uncoupler BAM15, which is selective for the mitochondrial inner membrane over the plasma membrane,31 had little effect, consistent with the plasma membrane proton gradient driving malonate uptake (Figure 3H and Figure S3F).\nWe next used nonspecific transport inhibitors to assess whether we could block malonate uptake at low pH. DIDS (4,4’-Diisothiocyano-2,2’-stilbenedisulfonic acid), an irreversible inhibitor of chloride/bicarbonate exchange that inhibits malonate uptake into erythrocytes,32,33 led to a dose-dependent inhibition of malonate uptake, although succinate remained high (Figure S3G and S3H). Thus, malonate uptake into cardiomyocytes at low pH is driven by a proton gradient through a transporter-dependent process.\nMalonate’s pKa’s are 2.83 and 5.69, so at pH 6.4, ≈16% of the malonate would be in its monocarboxylate form. To determine whether the uptake of malonate was dependent on its protonation to a monocarboxylate form, we assessed the uptake of a compound that mimics the monocarboxylate malonate. In 3-amino-3-oxopropionate (malonamate; Figure 3I), one carboxylic acid has been replaced with a neutral amido group leaving a single carboxylic acid of pKa ≈4.75. Thus, at pH 7.4 malonamate resembles the monocarboxylate form of malonate. This led to more of malonamate being taken up into cells at pH 7.4 than malonate (Figure 3J). Lowering the pH to 6 led to ≈15-fold increase in malonate uptake while malonamate uptake changed negligibly (Figure 3K) because it will remain as a monocarboxylate across this pH range.\nTogether, these data support transport of the monoanionic form of malonate, under the conditions that occur during early reperfusion. Therefore, a pH gradient can drive the uptake of malonate via protonation and transport in its monocarboxylate form.\nAs pH modulated malonate entry into cells in vitro and in the heart, we next probed the factors affecting malonate uptake into cells. Malonate entry into cells at either pH 7.4 or 6 was blocked at 4 °C and was associated with negligible succinate levels (Figure S2D and S2E). That a decrease in extracellular pH increased malonate entry into cells, suggested uptake driven by the proton gradient. Therefore, we next abolished the plasma membrane proton gradient using ionophores21 which prevented the cellular uptake of malonate (Figure 3E–3G and Figure S3A) and subsequent increase in succinate levels (Figure S3B through S3E). However, the uncoupler BAM15, which is selective for the mitochondrial inner membrane over the plasma membrane,31 had little effect, consistent with the plasma membrane proton gradient driving malonate uptake (Figure 3H and Figure S3F).\nWe next used nonspecific transport inhibitors to assess whether we could block malonate uptake at low pH. DIDS (4,4’-Diisothiocyano-2,2’-stilbenedisulfonic acid), an irreversible inhibitor of chloride/bicarbonate exchange that inhibits malonate uptake into erythrocytes,32,33 led to a dose-dependent inhibition of malonate uptake, although succinate remained high (Figure S3G and S3H). Thus, malonate uptake into cardiomyocytes at low pH is driven by a proton gradient through a transporter-dependent process.\nMalonate’s pKa’s are 2.83 and 5.69, so at pH 6.4, ≈16% of the malonate would be in its monocarboxylate form. To determine whether the uptake of malonate was dependent on its protonation to a monocarboxylate form, we assessed the uptake of a compound that mimics the monocarboxylate malonate. In 3-amino-3-oxopropionate (malonamate; Figure 3I), one carboxylic acid has been replaced with a neutral amido group leaving a single carboxylic acid of pKa ≈4.75. Thus, at pH 7.4 malonamate resembles the monocarboxylate form of malonate. This led to more of malonamate being taken up into cells at pH 7.4 than malonate (Figure 3J). Lowering the pH to 6 led to ≈15-fold increase in malonate uptake while malonamate uptake changed negligibly (Figure 3K) because it will remain as a monocarboxylate across this pH range.\nTogether, these data support transport of the monoanionic form of malonate, under the conditions that occur during early reperfusion. Therefore, a pH gradient can drive the uptake of malonate via protonation and transport in its monocarboxylate form.\n Malonate Uptake Under Reperfusion Conditions Is Dependent on MCT1 The inhibitory effect of DIDS, which also inhibits MCT1,34 raised the possibility of the uptake of the protonated form of malonate being catalyzed by a monocarboxylate transporter (MCT; SLC16 (solute carrier family member 16)). MCT1 (SLC16A1) is the principal transporter of lactate, which is carried in symport with a proton, both of which are elevated during ischemia, making MCT1 an attractive candidate transporter for malonate uptake upon reperfusion. Additionally, it was recently shown that MCT1 mediates the efflux of succinate from the ischemic heart during reperfusion and from exercising muscle.21,35 As MCT1 is a lactate transporter, we first assessed whether high concentrations of lactate in the incubation medium compete with malonate. Excess lactate led to a concentration-dependent decrease in malonate uptake into the cell and a corresponding decrease in succinate accumulation (Figure 4A and Figure S4A). This suggested that at a low pH, malonate uptake into the cells is via MCT1, which is highly expressed in cardiomyocytes.36\nInhibition of MCT1 (monocarboxylate transporter 1) prevents the enhanced uptake of malonate at lowered pH. A, Malonate uptake (5 mmol/L disodium malonate [DSM], 15 min) in C2C12 cells in the presence of lactate (0, 10 or 50 mmol/L). B, Lactate levels in C2C12 cells after 15 min treatment with varying concentrations of MCT1 inhibitors. C and D, Effect of MCT1 inhibition by AR-C141990 or AZD3965 on malonate (5 mmol/L DSM, 15 min) uptake (C) at pH 6 and subsequent succinate levels (D). E, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at various pH±10 µmol/L AR-C141990. F, MCT1 inhibition by AR-C141990 on malonate (5 mmol/L DSM, 15 min) uptake at pH 7.4 (A to F, mean±SEM, n=3 biological replicates, statistics: (A) Kruskal-Wallis with Dunn post hoc test). G and H, Effect of malonate (5 mmol/L DSM) on cellular oxygen consumption at pH 7.4 (G) or 6 (H) ±MCT1 inhibitor (10 µmol/L AR-C141990; data presented as nonmitochondrial respiration normalized mean oxygen consumption rate (OCR)±SEM of 3 biological replicates (n=12–16 technical replicates per biological replicate), statistics: Kruskal-Wallis with Dunn post hoc test). ATP indicates OCR in the presence of 1.5 µmol/L oligomycin; BL, baseline OCR; MAX, OCR in the presence of 1 µmol/L FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone); and NM, OCR in the presence of 4 µg/mL rotenone and 10 µmol/L antimycin A.\nTo confirm that MCT1 was the malonate transporter, we assessed the potent and selective MCT1 inhibitors, AR-C141990 and AZD3965.21,35,37,38 When cells were incubated with these MCT1 inhibitors, they led to a dramatic dose-dependent increase in lactate levels within the cell, consistent with preventing lactate efflux via MCT1 (Figure 4B). MCT1 inhibition by either AR-C141990 or AZD3965 led to a profound dose-dependent decrease in malonate uptake (Figure 4C). Inhibition of malonate uptake by MCT1 inhibition led to a corresponding decrease in succinate accumulation within cells (Figure 4D). Additionally, a time course of malonate uptake at low pH, showed that MCT1 inhibition largely abolished the increase in malonate over time, along with the corresponding increase in succinate (Figure S4B and S4C).\nMCT1 inhibition blocked the pH-dependent increase in malonate uptake (Figure 4E) and in parallel prevented succinate accumulation (Figure 4D). The MCT1 inhibitor led to a dose-dependent inhibition of malonate uptake even at pH 7.4 (Figure 4F). Therefore, malonate can be transported by MCT1 at pH 7.4, but this is greatly enhanced at lower pH due to the increased proportion of malonate in its monocarboxylate form.\nWe next assessed the impact of the malonate taken up into cells on mitochondrial function by measuring respiration. Lowering pH itself had little effect; however, addition of malonate severely reduced cellular respiration (Figure 4G and H and Figure S4E and S4F) and this effect was rescued by MCT1 inhibition (Figure 4G and H and Figure S4E and S4F). MCT1 inhibition led to a small increase in oxygen consumption at baseline, which may be due to lactate accumulation increasing the NADH/NAD+ ratio driving mitochondrial respiration.39\nTo confirm the pharmacological effects of MCT1 inhibition on malonate uptake genetically, we knocked down (KD) MCT1 using siRNA in both HeLa and C2C12 cells (Figure S5A through S5D). Consequently, lactate levels in MCT1 KD cells were significantly elevated compared to the control siRNA (Figure 5A). When MCT1 KD cells were incubated with malonate at low pH, malonate uptake was dramatically blocked (Figure 5B through 5E and Figure S5C), confirming MCT1 is directly responsible for malonate transport at low pH. Residual malonate uptake in the MCT1 KD cells was inhibited by cotreatment with an MCT1 inhibitor, further confirming its MCT1 dependence (Figure 5F and Figure S5F). Additionally, succinate was elevated in the MCT1 KD cells compared with control siRNA cells, a result not seen with acute pharmacological MCT1 inhibition (Figure 5D). This suggests that MCT1 may also play an important role in normoxic succinate metabolism and homeostasis and not just during myocardial reperfusion and intense exercise.21,35 Intriguingly, the succinate levels after malonate treatment over time differed between the 2 cell types, with succinate levels remaining low in C2C12 KD but elevated in HeLa MCT1 KDs compared to controls, suggesting a reliance on MCT1 for succinate efflux (Figure 5G and Figure S5G).\nGenetic knockdown of MCT1 (monocarboxylate transporter 1) prevents the uptake of malonate. A, Relative lactate levels in C2C12 cells treated with control or MCT1 siRNA (mean±SEM of lactate levels relative to control, n=6 biological replicates, statistics: 1-way ANOVA with Bonferroni post hoc test). B to E, Incubation of malonate (5 mmol/L disodium malonate [DSM]) in MCT1 KD cells at pH 7.4 (B and D) or 6 (C and E) for 15 min±MCT1i (10 µmol/L, MCT1 inhibitor AR-C141990). Levels of malonate (B and C) and succinate (D and E) quantified by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). F and G, Time course of malonate uptake (5 mmol/L DSM) at pH 6 in MCT1 KD cells (F) and corresponding succinate levels (G; mean±SEM, n=3 biological replicates). H, Malonate levels in murine Langendorff hearts perfused at pH 6 for 5 min with 5 mmol/L DSM±lactate (50 mmol/L) or MCT1 inhibitor (10 µmol/L AR-C141990; MCT1i; mean±SEM, n=4, statistics: unpaired, 2-tailed Mann-Whitney U test vs control).\nAs HeLa cells constitutively express MCT4, which is under the control of HIF (hypoxia-inducible factor)-1α and may be implicated in the transport of metabolites in IR, we KD MCT4 independently of MCT1 (Figure S5D and S5E). MCT4 KD had little effect on malonate uptake, with malonate and succinate levels mirroring those of control siRNA-treated cells (Figure S5F and S5G). Thus, MCT1 is the main driver of malonate uptake at lowered pH.\nOverall, diminishing MCT1 activity, either by pharmacological inhibition or genetic knockdown, impairs malonate uptake at both acidic and normal physiological pH. In addition, this is the first evidence that MCT1 can transport a 3-carbon chain length dicarboxylate, which may also have implications in normal physiology and other pathologies.\nFinally, we assessed if MCT1 was responsible for malonate uptake at lowered pH in the heart. Malonate uptake into the Langendorff-perfused heart was blocked by either excess lactate, or the MCT1 inhibitor AR-C141990, in the perfusion medium (Figure 5H). Together these findings confirm that MCT1 is responsible for the low pH uptake of malonate, both in cells and in the intact heart.\nThe inhibitory effect of DIDS, which also inhibits MCT1,34 raised the possibility of the uptake of the protonated form of malonate being catalyzed by a monocarboxylate transporter (MCT; SLC16 (solute carrier family member 16)). MCT1 (SLC16A1) is the principal transporter of lactate, which is carried in symport with a proton, both of which are elevated during ischemia, making MCT1 an attractive candidate transporter for malonate uptake upon reperfusion. Additionally, it was recently shown that MCT1 mediates the efflux of succinate from the ischemic heart during reperfusion and from exercising muscle.21,35 As MCT1 is a lactate transporter, we first assessed whether high concentrations of lactate in the incubation medium compete with malonate. Excess lactate led to a concentration-dependent decrease in malonate uptake into the cell and a corresponding decrease in succinate accumulation (Figure 4A and Figure S4A). This suggested that at a low pH, malonate uptake into the cells is via MCT1, which is highly expressed in cardiomyocytes.36\nInhibition of MCT1 (monocarboxylate transporter 1) prevents the enhanced uptake of malonate at lowered pH. A, Malonate uptake (5 mmol/L disodium malonate [DSM], 15 min) in C2C12 cells in the presence of lactate (0, 10 or 50 mmol/L). B, Lactate levels in C2C12 cells after 15 min treatment with varying concentrations of MCT1 inhibitors. C and D, Effect of MCT1 inhibition by AR-C141990 or AZD3965 on malonate (5 mmol/L DSM, 15 min) uptake (C) at pH 6 and subsequent succinate levels (D). E, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at various pH±10 µmol/L AR-C141990. F, MCT1 inhibition by AR-C141990 on malonate (5 mmol/L DSM, 15 min) uptake at pH 7.4 (A to F, mean±SEM, n=3 biological replicates, statistics: (A) Kruskal-Wallis with Dunn post hoc test). G and H, Effect of malonate (5 mmol/L DSM) on cellular oxygen consumption at pH 7.4 (G) or 6 (H) ±MCT1 inhibitor (10 µmol/L AR-C141990; data presented as nonmitochondrial respiration normalized mean oxygen consumption rate (OCR)±SEM of 3 biological replicates (n=12–16 technical replicates per biological replicate), statistics: Kruskal-Wallis with Dunn post hoc test). ATP indicates OCR in the presence of 1.5 µmol/L oligomycin; BL, baseline OCR; MAX, OCR in the presence of 1 µmol/L FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone); and NM, OCR in the presence of 4 µg/mL rotenone and 10 µmol/L antimycin A.\nTo confirm that MCT1 was the malonate transporter, we assessed the potent and selective MCT1 inhibitors, AR-C141990 and AZD3965.21,35,37,38 When cells were incubated with these MCT1 inhibitors, they led to a dramatic dose-dependent increase in lactate levels within the cell, consistent with preventing lactate efflux via MCT1 (Figure 4B). MCT1 inhibition by either AR-C141990 or AZD3965 led to a profound dose-dependent decrease in malonate uptake (Figure 4C). Inhibition of malonate uptake by MCT1 inhibition led to a corresponding decrease in succinate accumulation within cells (Figure 4D). Additionally, a time course of malonate uptake at low pH, showed that MCT1 inhibition largely abolished the increase in malonate over time, along with the corresponding increase in succinate (Figure S4B and S4C).\nMCT1 inhibition blocked the pH-dependent increase in malonate uptake (Figure 4E) and in parallel prevented succinate accumulation (Figure 4D). The MCT1 inhibitor led to a dose-dependent inhibition of malonate uptake even at pH 7.4 (Figure 4F). Therefore, malonate can be transported by MCT1 at pH 7.4, but this is greatly enhanced at lower pH due to the increased proportion of malonate in its monocarboxylate form.\nWe next assessed the impact of the malonate taken up into cells on mitochondrial function by measuring respiration. Lowering pH itself had little effect; however, addition of malonate severely reduced cellular respiration (Figure 4G and H and Figure S4E and S4F) and this effect was rescued by MCT1 inhibition (Figure 4G and H and Figure S4E and S4F). MCT1 inhibition led to a small increase in oxygen consumption at baseline, which may be due to lactate accumulation increasing the NADH/NAD+ ratio driving mitochondrial respiration.39\nTo confirm the pharmacological effects of MCT1 inhibition on malonate uptake genetically, we knocked down (KD) MCT1 using siRNA in both HeLa and C2C12 cells (Figure S5A through S5D). Consequently, lactate levels in MCT1 KD cells were significantly elevated compared to the control siRNA (Figure 5A). When MCT1 KD cells were incubated with malonate at low pH, malonate uptake was dramatically blocked (Figure 5B through 5E and Figure S5C), confirming MCT1 is directly responsible for malonate transport at low pH. Residual malonate uptake in the MCT1 KD cells was inhibited by cotreatment with an MCT1 inhibitor, further confirming its MCT1 dependence (Figure 5F and Figure S5F). Additionally, succinate was elevated in the MCT1 KD cells compared with control siRNA cells, a result not seen with acute pharmacological MCT1 inhibition (Figure 5D). This suggests that MCT1 may also play an important role in normoxic succinate metabolism and homeostasis and not just during myocardial reperfusion and intense exercise.21,35 Intriguingly, the succinate levels after malonate treatment over time differed between the 2 cell types, with succinate levels remaining low in C2C12 KD but elevated in HeLa MCT1 KDs compared to controls, suggesting a reliance on MCT1 for succinate efflux (Figure 5G and Figure S5G).\nGenetic knockdown of MCT1 (monocarboxylate transporter 1) prevents the uptake of malonate. A, Relative lactate levels in C2C12 cells treated with control or MCT1 siRNA (mean±SEM of lactate levels relative to control, n=6 biological replicates, statistics: 1-way ANOVA with Bonferroni post hoc test). B to E, Incubation of malonate (5 mmol/L disodium malonate [DSM]) in MCT1 KD cells at pH 7.4 (B and D) or 6 (C and E) for 15 min±MCT1i (10 µmol/L, MCT1 inhibitor AR-C141990). Levels of malonate (B and C) and succinate (D and E) quantified by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). F and G, Time course of malonate uptake (5 mmol/L DSM) at pH 6 in MCT1 KD cells (F) and corresponding succinate levels (G; mean±SEM, n=3 biological replicates). H, Malonate levels in murine Langendorff hearts perfused at pH 6 for 5 min with 5 mmol/L DSM±lactate (50 mmol/L) or MCT1 inhibitor (10 µmol/L AR-C141990; MCT1i; mean±SEM, n=4, statistics: unpaired, 2-tailed Mann-Whitney U test vs control).\nAs HeLa cells constitutively express MCT4, which is under the control of HIF (hypoxia-inducible factor)-1α and may be implicated in the transport of metabolites in IR, we KD MCT4 independently of MCT1 (Figure S5D and S5E). MCT4 KD had little effect on malonate uptake, with malonate and succinate levels mirroring those of control siRNA-treated cells (Figure S5F and S5G). Thus, MCT1 is the main driver of malonate uptake at lowered pH.\nOverall, diminishing MCT1 activity, either by pharmacological inhibition or genetic knockdown, impairs malonate uptake at both acidic and normal physiological pH. In addition, this is the first evidence that MCT1 can transport a 3-carbon chain length dicarboxylate, which may also have implications in normal physiology and other pathologies.\nFinally, we assessed if MCT1 was responsible for malonate uptake at lowered pH in the heart. Malonate uptake into the Langendorff-perfused heart was blocked by either excess lactate, or the MCT1 inhibitor AR-C141990, in the perfusion medium (Figure 5H). Together these findings confirm that MCT1 is responsible for the low pH uptake of malonate, both in cells and in the intact heart.\n Ischemic Conditions Are Sufficient to Drive Malonate Uptake into the Heart Upon Reperfusion That low pH enhanced malonate entry into heart cells was consistent with the protection afforded by DSM in IR injury being due to the local decrease in pH during ischemia and initial reperfusion. To test this hypothesis, Langendorff-perfused hearts were held ischemic for various times before DSM was infused for 5 minutes, followed by flushing, and measurement of malonate. The malonate levels in heart tissue were dependent on ischemia-time, with the highest occurring after 20 minutes ischemia and being ≈10-fold greater than in the normoxic heart (Figure 6A). This malonate uptake into the ischemic Langendorff-perfused heart was dramatically reduced by the MCT1 inhibitor AR-C141990 (Figure 6B). This confirmed that malonate uptake into the ischemic heart upon reperfusion is a selective process, driven by the low pH and facilitated by the MCT1.\nIschemia drives malonate protonation, uptake, and cardioprotection. A, Langendorff-perfused murine hearts were held ischemic for either 0, 5, 10, or 20 min and reperfused with 5 mmol/L disodium malonate (DSM; pH 7.4) before malonate levels measured in the heart by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 (control, 5 min) or 6 (10 and 20 min) biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). B, Malonate levels in murine Langendorff hearts exposed to 20 min ischemia and reperfused with 5 mmol/L malonate (pH 7.4)±MCT1 (monocarboxylate transporter 1) inhibitor (10 or 50 µmol/L AR-C141990; mean±SEM, n=4–5 biological replicates, statistical significance assessed by unpaired, 2-tailed Mann-Whitney U test vs control). C, Model of potential lactate and malonate exchange during reperfusion. D, Lactate levels in the Langendorff heart after 20 min ischemia and 1 min reperfusion±5 mmol/L DSM (mean, n=4 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test). E, Infarct size in murine LAD (left anterior descending coronary artery) ligation MI model with 100 µl bolus of 8 mg/kg DSM, pH 4 acid control or 8 mg/kg pH 4 formulated malonate at reperfusion after 30 min ischemia (mean, n=5 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test vs acid malonate).\nDuring ischemia, the pH of ischemic tissue lowers but in addition, lactate accumulates and upon reperfusion can be transported out of cardiomyocytes by MCT1. This could enhance malonate uptake, as the MCT1 may then act as a lactate-H+/monocarboxylate malonate-H+ exchanger (Figure 6C). To assess this, we measured lactate levels in malonate-perfused IR Langendorff hearts. We found that compared to reperfusion alone, lactate levels decreased in the malonate-treated hearts (Figure 6D). Furthermore, malonate treatment decreased lactate levels in cells that had been treated with the mitochondrial inhibitor antimycin A to enhance lactate production (Figure S6A). This suggests that extracellular malonate and a proton exchange for intracellular lactate, help drive malonate entry into cardiomyocytes upon reperfusion and contribute to its at-risk tissue-selective effect.\nTherefore, malonate uptake into the heart upon reperfusion is dependent on the tissue having first undergone a period of ischemia, leading to a drop in pH and an accumulation of lactate.\nThat low pH enhanced malonate entry into heart cells was consistent with the protection afforded by DSM in IR injury being due to the local decrease in pH during ischemia and initial reperfusion. To test this hypothesis, Langendorff-perfused hearts were held ischemic for various times before DSM was infused for 5 minutes, followed by flushing, and measurement of malonate. The malonate levels in heart tissue were dependent on ischemia-time, with the highest occurring after 20 minutes ischemia and being ≈10-fold greater than in the normoxic heart (Figure 6A). This malonate uptake into the ischemic Langendorff-perfused heart was dramatically reduced by the MCT1 inhibitor AR-C141990 (Figure 6B). This confirmed that malonate uptake into the ischemic heart upon reperfusion is a selective process, driven by the low pH and facilitated by the MCT1.\nIschemia drives malonate protonation, uptake, and cardioprotection. A, Langendorff-perfused murine hearts were held ischemic for either 0, 5, 10, or 20 min and reperfused with 5 mmol/L disodium malonate (DSM; pH 7.4) before malonate levels measured in the heart by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 (control, 5 min) or 6 (10 and 20 min) biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). B, Malonate levels in murine Langendorff hearts exposed to 20 min ischemia and reperfused with 5 mmol/L malonate (pH 7.4)±MCT1 (monocarboxylate transporter 1) inhibitor (10 or 50 µmol/L AR-C141990; mean±SEM, n=4–5 biological replicates, statistical significance assessed by unpaired, 2-tailed Mann-Whitney U test vs control). C, Model of potential lactate and malonate exchange during reperfusion. D, Lactate levels in the Langendorff heart after 20 min ischemia and 1 min reperfusion±5 mmol/L DSM (mean, n=4 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test). E, Infarct size in murine LAD (left anterior descending coronary artery) ligation MI model with 100 µl bolus of 8 mg/kg DSM, pH 4 acid control or 8 mg/kg pH 4 formulated malonate at reperfusion after 30 min ischemia (mean, n=5 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test vs acid malonate).\nDuring ischemia, the pH of ischemic tissue lowers but in addition, lactate accumulates and upon reperfusion can be transported out of cardiomyocytes by MCT1. This could enhance malonate uptake, as the MCT1 may then act as a lactate-H+/monocarboxylate malonate-H+ exchanger (Figure 6C). To assess this, we measured lactate levels in malonate-perfused IR Langendorff hearts. We found that compared to reperfusion alone, lactate levels decreased in the malonate-treated hearts (Figure 6D). Furthermore, malonate treatment decreased lactate levels in cells that had been treated with the mitochondrial inhibitor antimycin A to enhance lactate production (Figure S6A). This suggests that extracellular malonate and a proton exchange for intracellular lactate, help drive malonate entry into cardiomyocytes upon reperfusion and contribute to its at-risk tissue-selective effect.\nTherefore, malonate uptake into the heart upon reperfusion is dependent on the tissue having first undergone a period of ischemia, leading to a drop in pH and an accumulation of lactate.\n Low pH Formulation Improves Cardioprotection by Malonate The mechanism of malonate uptake into cardiac tissue suggested that lowering the pH of the malonate infusion would increase the proportion in the monocarboxylate form and thereby increase its potency as a cardioprotective agent. To assess this, we used a malonate dose that was not protective (8 mg/kg) when administered at a neutral pH in the in vivo LAD MI model. When this dose of malonate was reformulated at pH 4 (a pH currently used in Food and Drug Administration–approved parenteral formulations)40 and administered as a bolus, this conferred significant protection that was not due to the low pH alone (Figure 6E and Figure S6B). Furthermore, although neutral malonate administered before ischemia was not protective, acidified malonate drove its uptake into cardiomyocytes and significantly reduced infarct size when infused before ischemia (Figure S6C).\nThe mechanism of malonate uptake into cardiac tissue suggested that lowering the pH of the malonate infusion would increase the proportion in the monocarboxylate form and thereby increase its potency as a cardioprotective agent. To assess this, we used a malonate dose that was not protective (8 mg/kg) when administered at a neutral pH in the in vivo LAD MI model. When this dose of malonate was reformulated at pH 4 (a pH currently used in Food and Drug Administration–approved parenteral formulations)40 and administered as a bolus, this conferred significant protection that was not due to the low pH alone (Figure 6E and Figure S6B). Furthermore, although neutral malonate administered before ischemia was not protective, acidified malonate drove its uptake into cardiomyocytes and significantly reduced infarct size when infused before ischemia (Figure S6C)."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"We have shown that malonate is an ischemia-selective drug, due to the lowered pH and lactate accumulation of ischemic tissue driving its uptake via the MCT1 (Figure 7). Furthermore, we show that a low pH formulation of malonate enhances its therapeutic potency. Malonate is the first at-risk tissue-selective cardioprotective drug and represents a significant step toward the treatment of IR injury."},"other_sections_titles":{"kind":"list like","value":["What Is Known?","What New Information Does This Article Contribute?","Malonate Is Protective in an MI Model and Is Taken Up Selectively into the Ischemic Region of the Heart","Malonate Uptake into Cells Is Inefficient at pH 7.4","Malonate Uptake into Cells and the Heart Can Be Modulated by pH","Malonate Uptake Can Be Perturbed by Modulating the Plasma Membrane H+ Gradient","Malonate Uptake Under Reperfusion Conditions Is Dependent on MCT1","Ischemic Conditions Are Sufficient to Drive Malonate Uptake into the Heart Upon Reperfusion","Low pH Formulation Improves Cardioprotection by Malonate","Article Information","Acknowledgments","Sources of Funding","Supplemental Materials"],"string":"[\n \"What Is Known?\",\n \"What New Information Does This Article Contribute?\",\n \"Malonate Is Protective in an MI Model and Is Taken Up Selectively into the Ischemic Region of the Heart\",\n \"Malonate Uptake into Cells Is Inefficient at pH 7.4\",\n \"Malonate Uptake into Cells and the Heart Can Be Modulated by pH\",\n \"Malonate Uptake Can Be Perturbed by Modulating the Plasma Membrane H+ Gradient\",\n \"Malonate Uptake Under Reperfusion Conditions Is Dependent on MCT1\",\n \"Ischemic Conditions Are Sufficient to Drive Malonate Uptake into the Heart Upon Reperfusion\",\n \"Low pH Formulation Improves Cardioprotection by Malonate\",\n \"Article Information\",\n \"Acknowledgments\",\n \"Sources of Funding\",\n \"Supplemental Materials\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Extensive succinate accumulation during ischemia and its subsequent rapid oxidation on reperfusion drives ischemia/reperfusion injury.\nPreventing succinate accumulation during ischemia reduces damage on reperfusion.\nInhibiting succinate dehydrogenase with malonate is protective against ischemia/reperfusion injury, although its mechanism of entry into cardiomyocytes is undefined.","The cardioprotective effect of malonate is dependent on its selective uptake into cardiomyocytes on reperfusion after an ischemic period.\nMalonate entry into cardiomyocytes upon reperfusion is facilitated by the lowered pH and lactate exchange, which selectively drives malonate into cardiomyocytes as a monoanion via the monocarboxylate transporter MCT1 (monocarboxylate transporter 1). This is the first time malonate has been shown to be a substrate for MCT1.\nMalonate selectively enters the at-risk tissue, sparing the nonischemic area on reperfusion. Thus, malonate is the first example of an at-risk tissue-selective, cardioprotective drug.\nDetermining the molecular basis for selective malonate entry via MCT1 into the ischemic heart upon reperfusion is a significant step toward treating cardiac ischemia/reperfusion injury. Malonate is cardioprotective in small and large animal models and MCT1 is highly expressed in the human heart. Furthermore, this mechanism can be exploited to increase malonate potency using an acidic formulation. The next step is to assess malonate as a treatment for cardiac ischemia/reperfusion injury in patients.","DSM is protective against cardiac IR injury, but its mechanism of protection remains uncertain. To address this gap in our knowledge we first tested the therapeutic range of DSM in IR injury. We infused DSM at a range of concentrations (1.6–160 mg/kg equivalent to ≈11-1100 µmol/kg) at reperfusion in an in vivo murine LAD (left anterior descending coronary artery) ligation model of MI. DSM infusion during this clinically relevant reperfusion period led to a dose-dependent decrease in infarct size (Figure 1C), with 160 and 16 mg/kg showing robust cardioprotection. However, when DSM was infused before the onset of ischemia it was not protective (Figure 1D), even at 160 mg/kg which gave the smallest infarct when administered at reperfusion. This contrasted with the cell-permeable dimethyl malonate, which can diffuse into the tissue and generate malonate, preventing succinate accumulation and thereby reducing IR injury.18,25 This suggests that either DSM is minimally taken up by tissues during normoxia and is only taken up by tissues after ischemia or that DSM protects against acute IR injury by an extracellular mechanism.\nWe next measured malonate uptake into the ischemic and healthy heart tissue upon reperfusion in the LAD MI model. This showed that malonate was indeed taken up into cells within the infarct region to a level of ≈400 pmol/mg tissue and to a far greater extent than into cells in the healthy tissue (Figure 1E). Furthermore, reperfusing with malonate slowed succinate oxidation within the infarct region with succinate remaining significantly elevated in the infarct region following 1-minute reperfusion, compared with control reperfusion with saline (Figure 1F). Limiting succinate oxidation at reperfusion with malonate also blunted ROS production in the at-risk tissue (Figure 1G). We next assessed how SDH inhibition compared to direct inhibition of the mitochondrial permeability transition pore with CsA (cyclosporin A). Here we found that the cardioprotection from malonate was additive to cyclosporin A alone (Figure 1H), hence targeting the upstream mechanism of permeability transition pore opening is an increasingly attractive option for IR injury treatment.\nTo better understand the metabolic differences between the healthy and infarcted tissue upon malonate treatment, we investigated the in vivo MI model using mass spectrometry imaging. To do this, we subjected hearts to either 30-minute ischemia, 30-minute ischemia before reperfusing the heart for 15-minute, or 30-minute ischemia before reperfusing with DSM for 15 minutes (to mimic the cardioprotective malonate infusion) before snap freezing and processing for mass spectrometry imaging (Figure 2A). Infarct lesions were demarcated using hematoxylin and eosin (H&E), silver infarct staining and metabolite principal component analysis to differentiate the risk and nonrisk regions. mass spectrometry imaging coupled with the demarcated risk areas showed striking changes in the levels of succinate in the infarct region. During ischemia, succinate was significantly elevated in the at-risk tissue, with considerable succinate accumulation in the core of the infarct (Figure 2B and 2C). After 15-minute reperfusion, the succinate levels in the infarct lesion returned to healthy tissue levels, due to succinate oxidation and efflux.21 When malonate was infused at the time of reperfusion, succinate levels remained higher in all regions of the heart than in the nonmalonate treated heart, consistent with the prevention of its oxidation by SDH inhibition by malonate.\nLocalization of succinate accumulation in heart tissue identified by mass spectrometry imaging. A, Outline of experimental groups for mass spectrometry imaging. B, Representative images of succinate abundance detected by mass spectrometry imaging in myocardial sections. Black outer line indicates the edge of the tissue slice, white inner line indicates infarct region. C, Quantification of succinate (C) identified by MSI (mean±SEM, n=3 biological replicates, statistics: Friedman paired test for healthy vs lesion and Kruskal-Wallis with Dunn post hoc test between conditions). au indicates arbitrary units; DSM, disodium malonate; H, healthy tissue; IR, ischemia/reperfusion; L, infarct lesion; and rep, reperfusion.\nWe conclude that malonate is indeed taken up by the heart, but this is greatly enhanced in ischemic region upon reperfusion. Therefore, malonate provides protection against cardiac IR injury in vivo over a range of concentrations following its selective entry into the infarct region, where it slows the oxidation of succinate during reperfusion, preventing the production of RET-derived ROS.","To explore the mechanism of malonate uptake in vitro, we incubated C2C12 and H9c2 myoblasts with DSM and measured intracellular malonate levels by LC-MS/MS (liquid chromatography-tandem mass spectrometry). Incubation with DSM for 15 minutes at pH 7.4, led to a dose-dependent increase in intracellular malonate (Figure S1A and S1D). Intracellular succinate also accumulated in a malonate-dependent manner (Figure S1B and S1E), confirming that once malonate enters the cell it is rapidly transported into mitochondria and inhibits SDH. However, incubating cells with 1 mmol/L DSM led to variable malonate uptake and little SDH inhibition, indicating that malonate uptake across biological membranes is inefficient. Moreover, the intracellular levels of malonate achieved by 250 µmol/L of the malonate ester prodrug diacetoxymethyl malonate, are 80-fold more than that generated by incubation with 5 mmol/L DSM.25 As DSM was protective in the LAD model when infused at 16 mg/kg, corresponding to a maximum possible blood malonate concentration of ≈1.8 mmol/L (assuming a blood volume of 1.5 mL in a 25 g mouse),29 this compares to the dose range tested in cells (furthermore, far more malonate is available for uptake in vitro than in vivo due to the large reservoir in the incubation medium). Even so, the intracellular malonate levels were far lower with DSM compared to diacetoxymethyl malonate.25\nThe kinetics of cell uptake upon incubation with 5 mmol/L DSM showed time-dependent uptake, although extended incubation times were required for succinate elevation (Figure S1C and S1F). This is likely due to the initial concentrations of malonate entering the cell being insufficient to inhibit SDH. These data are consistent with the lack of protection by DSM delivered before ischemia in vivo, as well as its limited uptake into normoxic tissues. Together these suggest that exposure to ischemia may facilitate malonate entry into the heart.","Malonate is a dicarboxylate at physiological pH (Figure 1A, pKa=2.83 and 5.69), suggesting that the pH decrease in ischemic tissue9,11,30 may enhance malonate uptake into the heart during early reperfusion, by increasing the concentration of its monocarboxylic form. Incubating cells with DSM at either pH 6, 7.4, or 8 for 15 minutes, led to large differences in malonate uptake (Figure 3A and Figure S2A). At pH 6, the levels of malonate in the cell were significantly higher than at pH 7.4 or 8, thus malonate uptake is favored by acidic pH. Succinate levels mirrored those of malonate, with greater succinate accumulation as a result of increased malonate-dependent SDH inhibition at low pH (Figure 3B and Figure S2B). Low pH alone had no effect on succinate levels (Figure 3B and Figure S2B), suggesting that it was due to malonate entry into the cells followed by SDH inhibition.\nMalonate uptake is enhanced at low pH. A and B, C2C12 cells were incubated with disodium malonate (DSM; 0, 1, or 5 mmol/L) for 15 min at either pH 6, 7.4, or 8 before measuring intracellular malonate (A) and succinate (B) by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). C, Malonate levels in C2C12 cells after incubation with DSM (5 mmol/L) for 15 min at various (patho)physiological pH (mean±SEM, n=3 biological replicates). D, Malonate levels in murine isolated Langendorff-perfused hearts treated with 5 mmol/L DSM infused at either pH 7.4 or 6 for 5 min (mean±SEM, n=4 biological replicates, statistics: unpaired, 2-tailed Mann-Whitney U test). E to H, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at either pH 6 or 7.4 in the presence of FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) (E), gramicidin (F), nigericin (G), or BAM15 (N5,N6-bis(2-Fluorophenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine) (H) before measuring intracellular malonate by LC-MS/MS (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). I, Structures of malonic acid and malonamic acid. J, Quantification of levels of malonate and malonamate in C2C12 cells after incubation (5 mmol/L DSM or disodium malonamate; 15 min) at pH 7.4 (n=6 biological replicates). K, C2C12 cells were incubated with either DSM or disodium malonamate (both 5 mmol/L) at pH 6 or 7.4 for 15 min and the intracellular levels measured by LC-MS/MS. (mean±SEM of the fold-enhancement of uptake at pH 6 vs pH 7.4, n=6 biological replicates). Statistics for J and K: unpaired, 2-tailed Student t test).\nIncubating cells with malonate over a range of pH values between pH 7.4 and 6 clearly showed the pH-dependent uptake of malonate, in line with the increase in monocarboxylate malonate proportion (Figure 3C). This was mirrored by succinate levels (Figure S2C). Therefore, even a small drop in pH can lead to a substantial increase in malonate uptake, and thus is likely to be relevant for its entry into ischemic tissue upon reperfusion.\nAs the cultured cells used are noncontractile and substantially differ in their properties from in situ contracting cardiomyocytes,15 importantly, we next assessed whether decreasing the pH also enhanced malonate uptake into the ex vivo perfused Langendorff heart. Malonate was infused into the heart at either pH 7.4 or 6 for 5 minutes, before briefly flushing at pH 7.4 to remove nonmyocardial malonate. Remarkably, the levels of malonate were significantly elevated when infused at pH 6 compared to 7.4, confirming that the in vitro results translate to the heart (Figure 3D). We conclude that low pH conditions facilitate the entry of malonate into cardiomyocytes.","As pH modulated malonate entry into cells in vitro and in the heart, we next probed the factors affecting malonate uptake into cells. Malonate entry into cells at either pH 7.4 or 6 was blocked at 4 °C and was associated with negligible succinate levels (Figure S2D and S2E). That a decrease in extracellular pH increased malonate entry into cells, suggested uptake driven by the proton gradient. Therefore, we next abolished the plasma membrane proton gradient using ionophores21 which prevented the cellular uptake of malonate (Figure 3E–3G and Figure S3A) and subsequent increase in succinate levels (Figure S3B through S3E). However, the uncoupler BAM15, which is selective for the mitochondrial inner membrane over the plasma membrane,31 had little effect, consistent with the plasma membrane proton gradient driving malonate uptake (Figure 3H and Figure S3F).\nWe next used nonspecific transport inhibitors to assess whether we could block malonate uptake at low pH. DIDS (4,4’-Diisothiocyano-2,2’-stilbenedisulfonic acid), an irreversible inhibitor of chloride/bicarbonate exchange that inhibits malonate uptake into erythrocytes,32,33 led to a dose-dependent inhibition of malonate uptake, although succinate remained high (Figure S3G and S3H). Thus, malonate uptake into cardiomyocytes at low pH is driven by a proton gradient through a transporter-dependent process.\nMalonate’s pKa’s are 2.83 and 5.69, so at pH 6.4, ≈16% of the malonate would be in its monocarboxylate form. To determine whether the uptake of malonate was dependent on its protonation to a monocarboxylate form, we assessed the uptake of a compound that mimics the monocarboxylate malonate. In 3-amino-3-oxopropionate (malonamate; Figure 3I), one carboxylic acid has been replaced with a neutral amido group leaving a single carboxylic acid of pKa ≈4.75. Thus, at pH 7.4 malonamate resembles the monocarboxylate form of malonate. This led to more of malonamate being taken up into cells at pH 7.4 than malonate (Figure 3J). Lowering the pH to 6 led to ≈15-fold increase in malonate uptake while malonamate uptake changed negligibly (Figure 3K) because it will remain as a monocarboxylate across this pH range.\nTogether, these data support transport of the monoanionic form of malonate, under the conditions that occur during early reperfusion. Therefore, a pH gradient can drive the uptake of malonate via protonation and transport in its monocarboxylate form.","The inhibitory effect of DIDS, which also inhibits MCT1,34 raised the possibility of the uptake of the protonated form of malonate being catalyzed by a monocarboxylate transporter (MCT; SLC16 (solute carrier family member 16)). MCT1 (SLC16A1) is the principal transporter of lactate, which is carried in symport with a proton, both of which are elevated during ischemia, making MCT1 an attractive candidate transporter for malonate uptake upon reperfusion. Additionally, it was recently shown that MCT1 mediates the efflux of succinate from the ischemic heart during reperfusion and from exercising muscle.21,35 As MCT1 is a lactate transporter, we first assessed whether high concentrations of lactate in the incubation medium compete with malonate. Excess lactate led to a concentration-dependent decrease in malonate uptake into the cell and a corresponding decrease in succinate accumulation (Figure 4A and Figure S4A). This suggested that at a low pH, malonate uptake into the cells is via MCT1, which is highly expressed in cardiomyocytes.36\nInhibition of MCT1 (monocarboxylate transporter 1) prevents the enhanced uptake of malonate at lowered pH. A, Malonate uptake (5 mmol/L disodium malonate [DSM], 15 min) in C2C12 cells in the presence of lactate (0, 10 or 50 mmol/L). B, Lactate levels in C2C12 cells after 15 min treatment with varying concentrations of MCT1 inhibitors. C and D, Effect of MCT1 inhibition by AR-C141990 or AZD3965 on malonate (5 mmol/L DSM, 15 min) uptake (C) at pH 6 and subsequent succinate levels (D). E, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at various pH±10 µmol/L AR-C141990. F, MCT1 inhibition by AR-C141990 on malonate (5 mmol/L DSM, 15 min) uptake at pH 7.4 (A to F, mean±SEM, n=3 biological replicates, statistics: (A) Kruskal-Wallis with Dunn post hoc test). G and H, Effect of malonate (5 mmol/L DSM) on cellular oxygen consumption at pH 7.4 (G) or 6 (H) ±MCT1 inhibitor (10 µmol/L AR-C141990; data presented as nonmitochondrial respiration normalized mean oxygen consumption rate (OCR)±SEM of 3 biological replicates (n=12–16 technical replicates per biological replicate), statistics: Kruskal-Wallis with Dunn post hoc test). ATP indicates OCR in the presence of 1.5 µmol/L oligomycin; BL, baseline OCR; MAX, OCR in the presence of 1 µmol/L FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone); and NM, OCR in the presence of 4 µg/mL rotenone and 10 µmol/L antimycin A.\nTo confirm that MCT1 was the malonate transporter, we assessed the potent and selective MCT1 inhibitors, AR-C141990 and AZD3965.21,35,37,38 When cells were incubated with these MCT1 inhibitors, they led to a dramatic dose-dependent increase in lactate levels within the cell, consistent with preventing lactate efflux via MCT1 (Figure 4B). MCT1 inhibition by either AR-C141990 or AZD3965 led to a profound dose-dependent decrease in malonate uptake (Figure 4C). Inhibition of malonate uptake by MCT1 inhibition led to a corresponding decrease in succinate accumulation within cells (Figure 4D). Additionally, a time course of malonate uptake at low pH, showed that MCT1 inhibition largely abolished the increase in malonate over time, along with the corresponding increase in succinate (Figure S4B and S4C).\nMCT1 inhibition blocked the pH-dependent increase in malonate uptake (Figure 4E) and in parallel prevented succinate accumulation (Figure 4D). The MCT1 inhibitor led to a dose-dependent inhibition of malonate uptake even at pH 7.4 (Figure 4F). Therefore, malonate can be transported by MCT1 at pH 7.4, but this is greatly enhanced at lower pH due to the increased proportion of malonate in its monocarboxylate form.\nWe next assessed the impact of the malonate taken up into cells on mitochondrial function by measuring respiration. Lowering pH itself had little effect; however, addition of malonate severely reduced cellular respiration (Figure 4G and H and Figure S4E and S4F) and this effect was rescued by MCT1 inhibition (Figure 4G and H and Figure S4E and S4F). MCT1 inhibition led to a small increase in oxygen consumption at baseline, which may be due to lactate accumulation increasing the NADH/NAD+ ratio driving mitochondrial respiration.39\nTo confirm the pharmacological effects of MCT1 inhibition on malonate uptake genetically, we knocked down (KD) MCT1 using siRNA in both HeLa and C2C12 cells (Figure S5A through S5D). Consequently, lactate levels in MCT1 KD cells were significantly elevated compared to the control siRNA (Figure 5A). When MCT1 KD cells were incubated with malonate at low pH, malonate uptake was dramatically blocked (Figure 5B through 5E and Figure S5C), confirming MCT1 is directly responsible for malonate transport at low pH. Residual malonate uptake in the MCT1 KD cells was inhibited by cotreatment with an MCT1 inhibitor, further confirming its MCT1 dependence (Figure 5F and Figure S5F). Additionally, succinate was elevated in the MCT1 KD cells compared with control siRNA cells, a result not seen with acute pharmacological MCT1 inhibition (Figure 5D). This suggests that MCT1 may also play an important role in normoxic succinate metabolism and homeostasis and not just during myocardial reperfusion and intense exercise.21,35 Intriguingly, the succinate levels after malonate treatment over time differed between the 2 cell types, with succinate levels remaining low in C2C12 KD but elevated in HeLa MCT1 KDs compared to controls, suggesting a reliance on MCT1 for succinate efflux (Figure 5G and Figure S5G).\nGenetic knockdown of MCT1 (monocarboxylate transporter 1) prevents the uptake of malonate. A, Relative lactate levels in C2C12 cells treated with control or MCT1 siRNA (mean±SEM of lactate levels relative to control, n=6 biological replicates, statistics: 1-way ANOVA with Bonferroni post hoc test). B to E, Incubation of malonate (5 mmol/L disodium malonate [DSM]) in MCT1 KD cells at pH 7.4 (B and D) or 6 (C and E) for 15 min±MCT1i (10 µmol/L, MCT1 inhibitor AR-C141990). Levels of malonate (B and C) and succinate (D and E) quantified by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). F and G, Time course of malonate uptake (5 mmol/L DSM) at pH 6 in MCT1 KD cells (F) and corresponding succinate levels (G; mean±SEM, n=3 biological replicates). H, Malonate levels in murine Langendorff hearts perfused at pH 6 for 5 min with 5 mmol/L DSM±lactate (50 mmol/L) or MCT1 inhibitor (10 µmol/L AR-C141990; MCT1i; mean±SEM, n=4, statistics: unpaired, 2-tailed Mann-Whitney U test vs control).\nAs HeLa cells constitutively express MCT4, which is under the control of HIF (hypoxia-inducible factor)-1α and may be implicated in the transport of metabolites in IR, we KD MCT4 independently of MCT1 (Figure S5D and S5E). MCT4 KD had little effect on malonate uptake, with malonate and succinate levels mirroring those of control siRNA-treated cells (Figure S5F and S5G). Thus, MCT1 is the main driver of malonate uptake at lowered pH.\nOverall, diminishing MCT1 activity, either by pharmacological inhibition or genetic knockdown, impairs malonate uptake at both acidic and normal physiological pH. In addition, this is the first evidence that MCT1 can transport a 3-carbon chain length dicarboxylate, which may also have implications in normal physiology and other pathologies.\nFinally, we assessed if MCT1 was responsible for malonate uptake at lowered pH in the heart. Malonate uptake into the Langendorff-perfused heart was blocked by either excess lactate, or the MCT1 inhibitor AR-C141990, in the perfusion medium (Figure 5H). Together these findings confirm that MCT1 is responsible for the low pH uptake of malonate, both in cells and in the intact heart.","That low pH enhanced malonate entry into heart cells was consistent with the protection afforded by DSM in IR injury being due to the local decrease in pH during ischemia and initial reperfusion. To test this hypothesis, Langendorff-perfused hearts were held ischemic for various times before DSM was infused for 5 minutes, followed by flushing, and measurement of malonate. The malonate levels in heart tissue were dependent on ischemia-time, with the highest occurring after 20 minutes ischemia and being ≈10-fold greater than in the normoxic heart (Figure 6A). This malonate uptake into the ischemic Langendorff-perfused heart was dramatically reduced by the MCT1 inhibitor AR-C141990 (Figure 6B). This confirmed that malonate uptake into the ischemic heart upon reperfusion is a selective process, driven by the low pH and facilitated by the MCT1.\nIschemia drives malonate protonation, uptake, and cardioprotection. A, Langendorff-perfused murine hearts were held ischemic for either 0, 5, 10, or 20 min and reperfused with 5 mmol/L disodium malonate (DSM; pH 7.4) before malonate levels measured in the heart by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 (control, 5 min) or 6 (10 and 20 min) biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). B, Malonate levels in murine Langendorff hearts exposed to 20 min ischemia and reperfused with 5 mmol/L malonate (pH 7.4)±MCT1 (monocarboxylate transporter 1) inhibitor (10 or 50 µmol/L AR-C141990; mean±SEM, n=4–5 biological replicates, statistical significance assessed by unpaired, 2-tailed Mann-Whitney U test vs control). C, Model of potential lactate and malonate exchange during reperfusion. D, Lactate levels in the Langendorff heart after 20 min ischemia and 1 min reperfusion±5 mmol/L DSM (mean, n=4 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test). E, Infarct size in murine LAD (left anterior descending coronary artery) ligation MI model with 100 µl bolus of 8 mg/kg DSM, pH 4 acid control or 8 mg/kg pH 4 formulated malonate at reperfusion after 30 min ischemia (mean, n=5 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test vs acid malonate).\nDuring ischemia, the pH of ischemic tissue lowers but in addition, lactate accumulates and upon reperfusion can be transported out of cardiomyocytes by MCT1. This could enhance malonate uptake, as the MCT1 may then act as a lactate-H+/monocarboxylate malonate-H+ exchanger (Figure 6C). To assess this, we measured lactate levels in malonate-perfused IR Langendorff hearts. We found that compared to reperfusion alone, lactate levels decreased in the malonate-treated hearts (Figure 6D). Furthermore, malonate treatment decreased lactate levels in cells that had been treated with the mitochondrial inhibitor antimycin A to enhance lactate production (Figure S6A). This suggests that extracellular malonate and a proton exchange for intracellular lactate, help drive malonate entry into cardiomyocytes upon reperfusion and contribute to its at-risk tissue-selective effect.\nTherefore, malonate uptake into the heart upon reperfusion is dependent on the tissue having first undergone a period of ischemia, leading to a drop in pH and an accumulation of lactate.","The mechanism of malonate uptake into cardiac tissue suggested that lowering the pH of the malonate infusion would increase the proportion in the monocarboxylate form and thereby increase its potency as a cardioprotective agent. To assess this, we used a malonate dose that was not protective (8 mg/kg) when administered at a neutral pH in the in vivo LAD MI model. When this dose of malonate was reformulated at pH 4 (a pH currently used in Food and Drug Administration–approved parenteral formulations)40 and administered as a bolus, this conferred significant protection that was not due to the low pH alone (Figure 6E and Figure S6B). Furthermore, although neutral malonate administered before ischemia was not protective, acidified malonate drove its uptake into cardiomyocytes and significantly reduced infarct size when infused before ischemia (Figure S6C)."," Acknowledgments The authors thank Stephen Large, Fouad Taghavi, and Margaret M. Huang (Department of Surgery, University of Cambridge) for obtaining human heart tissue and Benjamin Thackray (Department of Medicine, University of Cambridge) for assistance with initial experiments.\nThe authors thank Stephen Large, Fouad Taghavi, and Margaret M. Huang (Department of Surgery, University of Cambridge) for obtaining human heart tissue and Benjamin Thackray (Department of Medicine, University of Cambridge) for assistance with initial experiments.\n Sources of Funding This work was supported by the British Heart Foundation (PG/20/10025 to T. Krieg); the Medical Research Council (MC_UU_00015/3 to M.P. Murphy and MR/P000320/1 to T. Krieg), the Wellcome Trust (220257/Z/20/Z to M.P. Murphy, 221604/Z/20/Z to D. Aksentijevic), Barts Charity (MRC0215 to D. Aksentijevic).\nThis work was supported by the British Heart Foundation (PG/20/10025 to T. Krieg); the Medical Research Council (MC_UU_00015/3 to M.P. Murphy and MR/P000320/1 to T. Krieg), the Wellcome Trust (220257/Z/20/Z to M.P. Murphy, 221604/Z/20/Z to D. Aksentijevic), Barts Charity (MRC0215 to D. Aksentijevic).\n Disclosures Some authors currently hold a patent on the use of malonate esters in cardiac ischemia/reperfusion (IR) injury (M.P. Murphy and T. Krieg) and have submitted patent applications on the use of malonate in IR injury associated with ischemic stroke (M.P. Murphy and T. Krieg) and the pH-enhancement of malonate described in this article (H.A. Prag, M.P. Murphy, and T. Krieg). The other authors report no conflicts.\nSome authors currently hold a patent on the use of malonate esters in cardiac ischemia/reperfusion (IR) injury (M.P. Murphy and T. Krieg) and have submitted patent applications on the use of malonate in IR injury associated with ischemic stroke (M.P. Murphy and T. Krieg) and the pH-enhancement of malonate described in this article (H.A. Prag, M.P. Murphy, and T. Krieg). The other authors report no conflicts.\n Supplemental Materials Supplemental Methods\nMajor Resources Table\nFigures S1–S6\nReferences 49–56\nSupplemental Methods\nMajor Resources Table\nFigures S1–S6\nReferences 49–56","The authors thank Stephen Large, Fouad Taghavi, and Margaret M. Huang (Department of Surgery, University of Cambridge) for obtaining human heart tissue and Benjamin Thackray (Department of Medicine, University of Cambridge) for assistance with initial experiments.","This work was supported by the British Heart Foundation (PG/20/10025 to T. Krieg); the Medical Research Council (MC_UU_00015/3 to M.P. Murphy and MR/P000320/1 to T. Krieg), the Wellcome Trust (220257/Z/20/Z to M.P. Murphy, 221604/Z/20/Z to D. Aksentijevic), Barts Charity (MRC0215 to D. Aksentijevic).","Supplemental Methods\nMajor Resources Table\nFigures S1–S6\nReferences 49–56"],"string":"[\n \"Extensive succinate accumulation during ischemia and its subsequent rapid oxidation on reperfusion drives ischemia/reperfusion injury.\\nPreventing succinate accumulation during ischemia reduces damage on reperfusion.\\nInhibiting succinate dehydrogenase with malonate is protective against ischemia/reperfusion injury, although its mechanism of entry into cardiomyocytes is undefined.\",\n \"The cardioprotective effect of malonate is dependent on its selective uptake into cardiomyocytes on reperfusion after an ischemic period.\\nMalonate entry into cardiomyocytes upon reperfusion is facilitated by the lowered pH and lactate exchange, which selectively drives malonate into cardiomyocytes as a monoanion via the monocarboxylate transporter MCT1 (monocarboxylate transporter 1). This is the first time malonate has been shown to be a substrate for MCT1.\\nMalonate selectively enters the at-risk tissue, sparing the nonischemic area on reperfusion. Thus, malonate is the first example of an at-risk tissue-selective, cardioprotective drug.\\nDetermining the molecular basis for selective malonate entry via MCT1 into the ischemic heart upon reperfusion is a significant step toward treating cardiac ischemia/reperfusion injury. Malonate is cardioprotective in small and large animal models and MCT1 is highly expressed in the human heart. Furthermore, this mechanism can be exploited to increase malonate potency using an acidic formulation. The next step is to assess malonate as a treatment for cardiac ischemia/reperfusion injury in patients.\",\n \"DSM is protective against cardiac IR injury, but its mechanism of protection remains uncertain. To address this gap in our knowledge we first tested the therapeutic range of DSM in IR injury. We infused DSM at a range of concentrations (1.6–160 mg/kg equivalent to ≈11-1100 µmol/kg) at reperfusion in an in vivo murine LAD (left anterior descending coronary artery) ligation model of MI. DSM infusion during this clinically relevant reperfusion period led to a dose-dependent decrease in infarct size (Figure 1C), with 160 and 16 mg/kg showing robust cardioprotection. However, when DSM was infused before the onset of ischemia it was not protective (Figure 1D), even at 160 mg/kg which gave the smallest infarct when administered at reperfusion. This contrasted with the cell-permeable dimethyl malonate, which can diffuse into the tissue and generate malonate, preventing succinate accumulation and thereby reducing IR injury.18,25 This suggests that either DSM is minimally taken up by tissues during normoxia and is only taken up by tissues after ischemia or that DSM protects against acute IR injury by an extracellular mechanism.\\nWe next measured malonate uptake into the ischemic and healthy heart tissue upon reperfusion in the LAD MI model. This showed that malonate was indeed taken up into cells within the infarct region to a level of ≈400 pmol/mg tissue and to a far greater extent than into cells in the healthy tissue (Figure 1E). Furthermore, reperfusing with malonate slowed succinate oxidation within the infarct region with succinate remaining significantly elevated in the infarct region following 1-minute reperfusion, compared with control reperfusion with saline (Figure 1F). Limiting succinate oxidation at reperfusion with malonate also blunted ROS production in the at-risk tissue (Figure 1G). We next assessed how SDH inhibition compared to direct inhibition of the mitochondrial permeability transition pore with CsA (cyclosporin A). Here we found that the cardioprotection from malonate was additive to cyclosporin A alone (Figure 1H), hence targeting the upstream mechanism of permeability transition pore opening is an increasingly attractive option for IR injury treatment.\\nTo better understand the metabolic differences between the healthy and infarcted tissue upon malonate treatment, we investigated the in vivo MI model using mass spectrometry imaging. To do this, we subjected hearts to either 30-minute ischemia, 30-minute ischemia before reperfusing the heart for 15-minute, or 30-minute ischemia before reperfusing with DSM for 15 minutes (to mimic the cardioprotective malonate infusion) before snap freezing and processing for mass spectrometry imaging (Figure 2A). Infarct lesions were demarcated using hematoxylin and eosin (H&E), silver infarct staining and metabolite principal component analysis to differentiate the risk and nonrisk regions. mass spectrometry imaging coupled with the demarcated risk areas showed striking changes in the levels of succinate in the infarct region. During ischemia, succinate was significantly elevated in the at-risk tissue, with considerable succinate accumulation in the core of the infarct (Figure 2B and 2C). After 15-minute reperfusion, the succinate levels in the infarct lesion returned to healthy tissue levels, due to succinate oxidation and efflux.21 When malonate was infused at the time of reperfusion, succinate levels remained higher in all regions of the heart than in the nonmalonate treated heart, consistent with the prevention of its oxidation by SDH inhibition by malonate.\\nLocalization of succinate accumulation in heart tissue identified by mass spectrometry imaging. A, Outline of experimental groups for mass spectrometry imaging. B, Representative images of succinate abundance detected by mass spectrometry imaging in myocardial sections. Black outer line indicates the edge of the tissue slice, white inner line indicates infarct region. C, Quantification of succinate (C) identified by MSI (mean±SEM, n=3 biological replicates, statistics: Friedman paired test for healthy vs lesion and Kruskal-Wallis with Dunn post hoc test between conditions). au indicates arbitrary units; DSM, disodium malonate; H, healthy tissue; IR, ischemia/reperfusion; L, infarct lesion; and rep, reperfusion.\\nWe conclude that malonate is indeed taken up by the heart, but this is greatly enhanced in ischemic region upon reperfusion. Therefore, malonate provides protection against cardiac IR injury in vivo over a range of concentrations following its selective entry into the infarct region, where it slows the oxidation of succinate during reperfusion, preventing the production of RET-derived ROS.\",\n \"To explore the mechanism of malonate uptake in vitro, we incubated C2C12 and H9c2 myoblasts with DSM and measured intracellular malonate levels by LC-MS/MS (liquid chromatography-tandem mass spectrometry). Incubation with DSM for 15 minutes at pH 7.4, led to a dose-dependent increase in intracellular malonate (Figure S1A and S1D). Intracellular succinate also accumulated in a malonate-dependent manner (Figure S1B and S1E), confirming that once malonate enters the cell it is rapidly transported into mitochondria and inhibits SDH. However, incubating cells with 1 mmol/L DSM led to variable malonate uptake and little SDH inhibition, indicating that malonate uptake across biological membranes is inefficient. Moreover, the intracellular levels of malonate achieved by 250 µmol/L of the malonate ester prodrug diacetoxymethyl malonate, are 80-fold more than that generated by incubation with 5 mmol/L DSM.25 As DSM was protective in the LAD model when infused at 16 mg/kg, corresponding to a maximum possible blood malonate concentration of ≈1.8 mmol/L (assuming a blood volume of 1.5 mL in a 25 g mouse),29 this compares to the dose range tested in cells (furthermore, far more malonate is available for uptake in vitro than in vivo due to the large reservoir in the incubation medium). Even so, the intracellular malonate levels were far lower with DSM compared to diacetoxymethyl malonate.25\\nThe kinetics of cell uptake upon incubation with 5 mmol/L DSM showed time-dependent uptake, although extended incubation times were required for succinate elevation (Figure S1C and S1F). This is likely due to the initial concentrations of malonate entering the cell being insufficient to inhibit SDH. These data are consistent with the lack of protection by DSM delivered before ischemia in vivo, as well as its limited uptake into normoxic tissues. Together these suggest that exposure to ischemia may facilitate malonate entry into the heart.\",\n \"Malonate is a dicarboxylate at physiological pH (Figure 1A, pKa=2.83 and 5.69), suggesting that the pH decrease in ischemic tissue9,11,30 may enhance malonate uptake into the heart during early reperfusion, by increasing the concentration of its monocarboxylic form. Incubating cells with DSM at either pH 6, 7.4, or 8 for 15 minutes, led to large differences in malonate uptake (Figure 3A and Figure S2A). At pH 6, the levels of malonate in the cell were significantly higher than at pH 7.4 or 8, thus malonate uptake is favored by acidic pH. Succinate levels mirrored those of malonate, with greater succinate accumulation as a result of increased malonate-dependent SDH inhibition at low pH (Figure 3B and Figure S2B). Low pH alone had no effect on succinate levels (Figure 3B and Figure S2B), suggesting that it was due to malonate entry into the cells followed by SDH inhibition.\\nMalonate uptake is enhanced at low pH. A and B, C2C12 cells were incubated with disodium malonate (DSM; 0, 1, or 5 mmol/L) for 15 min at either pH 6, 7.4, or 8 before measuring intracellular malonate (A) and succinate (B) by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). C, Malonate levels in C2C12 cells after incubation with DSM (5 mmol/L) for 15 min at various (patho)physiological pH (mean±SEM, n=3 biological replicates). D, Malonate levels in murine isolated Langendorff-perfused hearts treated with 5 mmol/L DSM infused at either pH 7.4 or 6 for 5 min (mean±SEM, n=4 biological replicates, statistics: unpaired, 2-tailed Mann-Whitney U test). E to H, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at either pH 6 or 7.4 in the presence of FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) (E), gramicidin (F), nigericin (G), or BAM15 (N5,N6-bis(2-Fluorophenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine) (H) before measuring intracellular malonate by LC-MS/MS (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). I, Structures of malonic acid and malonamic acid. J, Quantification of levels of malonate and malonamate in C2C12 cells after incubation (5 mmol/L DSM or disodium malonamate; 15 min) at pH 7.4 (n=6 biological replicates). K, C2C12 cells were incubated with either DSM or disodium malonamate (both 5 mmol/L) at pH 6 or 7.4 for 15 min and the intracellular levels measured by LC-MS/MS. (mean±SEM of the fold-enhancement of uptake at pH 6 vs pH 7.4, n=6 biological replicates). Statistics for J and K: unpaired, 2-tailed Student t test).\\nIncubating cells with malonate over a range of pH values between pH 7.4 and 6 clearly showed the pH-dependent uptake of malonate, in line with the increase in monocarboxylate malonate proportion (Figure 3C). This was mirrored by succinate levels (Figure S2C). Therefore, even a small drop in pH can lead to a substantial increase in malonate uptake, and thus is likely to be relevant for its entry into ischemic tissue upon reperfusion.\\nAs the cultured cells used are noncontractile and substantially differ in their properties from in situ contracting cardiomyocytes,15 importantly, we next assessed whether decreasing the pH also enhanced malonate uptake into the ex vivo perfused Langendorff heart. Malonate was infused into the heart at either pH 7.4 or 6 for 5 minutes, before briefly flushing at pH 7.4 to remove nonmyocardial malonate. Remarkably, the levels of malonate were significantly elevated when infused at pH 6 compared to 7.4, confirming that the in vitro results translate to the heart (Figure 3D). We conclude that low pH conditions facilitate the entry of malonate into cardiomyocytes.\",\n \"As pH modulated malonate entry into cells in vitro and in the heart, we next probed the factors affecting malonate uptake into cells. Malonate entry into cells at either pH 7.4 or 6 was blocked at 4 °C and was associated with negligible succinate levels (Figure S2D and S2E). That a decrease in extracellular pH increased malonate entry into cells, suggested uptake driven by the proton gradient. Therefore, we next abolished the plasma membrane proton gradient using ionophores21 which prevented the cellular uptake of malonate (Figure 3E–3G and Figure S3A) and subsequent increase in succinate levels (Figure S3B through S3E). However, the uncoupler BAM15, which is selective for the mitochondrial inner membrane over the plasma membrane,31 had little effect, consistent with the plasma membrane proton gradient driving malonate uptake (Figure 3H and Figure S3F).\\nWe next used nonspecific transport inhibitors to assess whether we could block malonate uptake at low pH. DIDS (4,4’-Diisothiocyano-2,2’-stilbenedisulfonic acid), an irreversible inhibitor of chloride/bicarbonate exchange that inhibits malonate uptake into erythrocytes,32,33 led to a dose-dependent inhibition of malonate uptake, although succinate remained high (Figure S3G and S3H). Thus, malonate uptake into cardiomyocytes at low pH is driven by a proton gradient through a transporter-dependent process.\\nMalonate’s pKa’s are 2.83 and 5.69, so at pH 6.4, ≈16% of the malonate would be in its monocarboxylate form. To determine whether the uptake of malonate was dependent on its protonation to a monocarboxylate form, we assessed the uptake of a compound that mimics the monocarboxylate malonate. In 3-amino-3-oxopropionate (malonamate; Figure 3I), one carboxylic acid has been replaced with a neutral amido group leaving a single carboxylic acid of pKa ≈4.75. Thus, at pH 7.4 malonamate resembles the monocarboxylate form of malonate. This led to more of malonamate being taken up into cells at pH 7.4 than malonate (Figure 3J). Lowering the pH to 6 led to ≈15-fold increase in malonate uptake while malonamate uptake changed negligibly (Figure 3K) because it will remain as a monocarboxylate across this pH range.\\nTogether, these data support transport of the monoanionic form of malonate, under the conditions that occur during early reperfusion. Therefore, a pH gradient can drive the uptake of malonate via protonation and transport in its monocarboxylate form.\",\n \"The inhibitory effect of DIDS, which also inhibits MCT1,34 raised the possibility of the uptake of the protonated form of malonate being catalyzed by a monocarboxylate transporter (MCT; SLC16 (solute carrier family member 16)). MCT1 (SLC16A1) is the principal transporter of lactate, which is carried in symport with a proton, both of which are elevated during ischemia, making MCT1 an attractive candidate transporter for malonate uptake upon reperfusion. Additionally, it was recently shown that MCT1 mediates the efflux of succinate from the ischemic heart during reperfusion and from exercising muscle.21,35 As MCT1 is a lactate transporter, we first assessed whether high concentrations of lactate in the incubation medium compete with malonate. Excess lactate led to a concentration-dependent decrease in malonate uptake into the cell and a corresponding decrease in succinate accumulation (Figure 4A and Figure S4A). This suggested that at a low pH, malonate uptake into the cells is via MCT1, which is highly expressed in cardiomyocytes.36\\nInhibition of MCT1 (monocarboxylate transporter 1) prevents the enhanced uptake of malonate at lowered pH. A, Malonate uptake (5 mmol/L disodium malonate [DSM], 15 min) in C2C12 cells in the presence of lactate (0, 10 or 50 mmol/L). B, Lactate levels in C2C12 cells after 15 min treatment with varying concentrations of MCT1 inhibitors. C and D, Effect of MCT1 inhibition by AR-C141990 or AZD3965 on malonate (5 mmol/L DSM, 15 min) uptake (C) at pH 6 and subsequent succinate levels (D). E, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at various pH±10 µmol/L AR-C141990. F, MCT1 inhibition by AR-C141990 on malonate (5 mmol/L DSM, 15 min) uptake at pH 7.4 (A to F, mean±SEM, n=3 biological replicates, statistics: (A) Kruskal-Wallis with Dunn post hoc test). G and H, Effect of malonate (5 mmol/L DSM) on cellular oxygen consumption at pH 7.4 (G) or 6 (H) ±MCT1 inhibitor (10 µmol/L AR-C141990; data presented as nonmitochondrial respiration normalized mean oxygen consumption rate (OCR)±SEM of 3 biological replicates (n=12–16 technical replicates per biological replicate), statistics: Kruskal-Wallis with Dunn post hoc test). ATP indicates OCR in the presence of 1.5 µmol/L oligomycin; BL, baseline OCR; MAX, OCR in the presence of 1 µmol/L FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone); and NM, OCR in the presence of 4 µg/mL rotenone and 10 µmol/L antimycin A.\\nTo confirm that MCT1 was the malonate transporter, we assessed the potent and selective MCT1 inhibitors, AR-C141990 and AZD3965.21,35,37,38 When cells were incubated with these MCT1 inhibitors, they led to a dramatic dose-dependent increase in lactate levels within the cell, consistent with preventing lactate efflux via MCT1 (Figure 4B). MCT1 inhibition by either AR-C141990 or AZD3965 led to a profound dose-dependent decrease in malonate uptake (Figure 4C). Inhibition of malonate uptake by MCT1 inhibition led to a corresponding decrease in succinate accumulation within cells (Figure 4D). Additionally, a time course of malonate uptake at low pH, showed that MCT1 inhibition largely abolished the increase in malonate over time, along with the corresponding increase in succinate (Figure S4B and S4C).\\nMCT1 inhibition blocked the pH-dependent increase in malonate uptake (Figure 4E) and in parallel prevented succinate accumulation (Figure 4D). The MCT1 inhibitor led to a dose-dependent inhibition of malonate uptake even at pH 7.4 (Figure 4F). Therefore, malonate can be transported by MCT1 at pH 7.4, but this is greatly enhanced at lower pH due to the increased proportion of malonate in its monocarboxylate form.\\nWe next assessed the impact of the malonate taken up into cells on mitochondrial function by measuring respiration. Lowering pH itself had little effect; however, addition of malonate severely reduced cellular respiration (Figure 4G and H and Figure S4E and S4F) and this effect was rescued by MCT1 inhibition (Figure 4G and H and Figure S4E and S4F). MCT1 inhibition led to a small increase in oxygen consumption at baseline, which may be due to lactate accumulation increasing the NADH/NAD+ ratio driving mitochondrial respiration.39\\nTo confirm the pharmacological effects of MCT1 inhibition on malonate uptake genetically, we knocked down (KD) MCT1 using siRNA in both HeLa and C2C12 cells (Figure S5A through S5D). Consequently, lactate levels in MCT1 KD cells were significantly elevated compared to the control siRNA (Figure 5A). When MCT1 KD cells were incubated with malonate at low pH, malonate uptake was dramatically blocked (Figure 5B through 5E and Figure S5C), confirming MCT1 is directly responsible for malonate transport at low pH. Residual malonate uptake in the MCT1 KD cells was inhibited by cotreatment with an MCT1 inhibitor, further confirming its MCT1 dependence (Figure 5F and Figure S5F). Additionally, succinate was elevated in the MCT1 KD cells compared with control siRNA cells, a result not seen with acute pharmacological MCT1 inhibition (Figure 5D). This suggests that MCT1 may also play an important role in normoxic succinate metabolism and homeostasis and not just during myocardial reperfusion and intense exercise.21,35 Intriguingly, the succinate levels after malonate treatment over time differed between the 2 cell types, with succinate levels remaining low in C2C12 KD but elevated in HeLa MCT1 KDs compared to controls, suggesting a reliance on MCT1 for succinate efflux (Figure 5G and Figure S5G).\\nGenetic knockdown of MCT1 (monocarboxylate transporter 1) prevents the uptake of malonate. A, Relative lactate levels in C2C12 cells treated with control or MCT1 siRNA (mean±SEM of lactate levels relative to control, n=6 biological replicates, statistics: 1-way ANOVA with Bonferroni post hoc test). B to E, Incubation of malonate (5 mmol/L disodium malonate [DSM]) in MCT1 KD cells at pH 7.4 (B and D) or 6 (C and E) for 15 min±MCT1i (10 µmol/L, MCT1 inhibitor AR-C141990). Levels of malonate (B and C) and succinate (D and E) quantified by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). F and G, Time course of malonate uptake (5 mmol/L DSM) at pH 6 in MCT1 KD cells (F) and corresponding succinate levels (G; mean±SEM, n=3 biological replicates). H, Malonate levels in murine Langendorff hearts perfused at pH 6 for 5 min with 5 mmol/L DSM±lactate (50 mmol/L) or MCT1 inhibitor (10 µmol/L AR-C141990; MCT1i; mean±SEM, n=4, statistics: unpaired, 2-tailed Mann-Whitney U test vs control).\\nAs HeLa cells constitutively express MCT4, which is under the control of HIF (hypoxia-inducible factor)-1α and may be implicated in the transport of metabolites in IR, we KD MCT4 independently of MCT1 (Figure S5D and S5E). MCT4 KD had little effect on malonate uptake, with malonate and succinate levels mirroring those of control siRNA-treated cells (Figure S5F and S5G). Thus, MCT1 is the main driver of malonate uptake at lowered pH.\\nOverall, diminishing MCT1 activity, either by pharmacological inhibition or genetic knockdown, impairs malonate uptake at both acidic and normal physiological pH. In addition, this is the first evidence that MCT1 can transport a 3-carbon chain length dicarboxylate, which may also have implications in normal physiology and other pathologies.\\nFinally, we assessed if MCT1 was responsible for malonate uptake at lowered pH in the heart. Malonate uptake into the Langendorff-perfused heart was blocked by either excess lactate, or the MCT1 inhibitor AR-C141990, in the perfusion medium (Figure 5H). Together these findings confirm that MCT1 is responsible for the low pH uptake of malonate, both in cells and in the intact heart.\",\n \"That low pH enhanced malonate entry into heart cells was consistent with the protection afforded by DSM in IR injury being due to the local decrease in pH during ischemia and initial reperfusion. To test this hypothesis, Langendorff-perfused hearts were held ischemic for various times before DSM was infused for 5 minutes, followed by flushing, and measurement of malonate. The malonate levels in heart tissue were dependent on ischemia-time, with the highest occurring after 20 minutes ischemia and being ≈10-fold greater than in the normoxic heart (Figure 6A). This malonate uptake into the ischemic Langendorff-perfused heart was dramatically reduced by the MCT1 inhibitor AR-C141990 (Figure 6B). This confirmed that malonate uptake into the ischemic heart upon reperfusion is a selective process, driven by the low pH and facilitated by the MCT1.\\nIschemia drives malonate protonation, uptake, and cardioprotection. A, Langendorff-perfused murine hearts were held ischemic for either 0, 5, 10, or 20 min and reperfused with 5 mmol/L disodium malonate (DSM; pH 7.4) before malonate levels measured in the heart by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 (control, 5 min) or 6 (10 and 20 min) biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). B, Malonate levels in murine Langendorff hearts exposed to 20 min ischemia and reperfused with 5 mmol/L malonate (pH 7.4)±MCT1 (monocarboxylate transporter 1) inhibitor (10 or 50 µmol/L AR-C141990; mean±SEM, n=4–5 biological replicates, statistical significance assessed by unpaired, 2-tailed Mann-Whitney U test vs control). C, Model of potential lactate and malonate exchange during reperfusion. D, Lactate levels in the Langendorff heart after 20 min ischemia and 1 min reperfusion±5 mmol/L DSM (mean, n=4 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test). E, Infarct size in murine LAD (left anterior descending coronary artery) ligation MI model with 100 µl bolus of 8 mg/kg DSM, pH 4 acid control or 8 mg/kg pH 4 formulated malonate at reperfusion after 30 min ischemia (mean, n=5 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test vs acid malonate).\\nDuring ischemia, the pH of ischemic tissue lowers but in addition, lactate accumulates and upon reperfusion can be transported out of cardiomyocytes by MCT1. This could enhance malonate uptake, as the MCT1 may then act as a lactate-H+/monocarboxylate malonate-H+ exchanger (Figure 6C). To assess this, we measured lactate levels in malonate-perfused IR Langendorff hearts. We found that compared to reperfusion alone, lactate levels decreased in the malonate-treated hearts (Figure 6D). Furthermore, malonate treatment decreased lactate levels in cells that had been treated with the mitochondrial inhibitor antimycin A to enhance lactate production (Figure S6A). This suggests that extracellular malonate and a proton exchange for intracellular lactate, help drive malonate entry into cardiomyocytes upon reperfusion and contribute to its at-risk tissue-selective effect.\\nTherefore, malonate uptake into the heart upon reperfusion is dependent on the tissue having first undergone a period of ischemia, leading to a drop in pH and an accumulation of lactate.\",\n \"The mechanism of malonate uptake into cardiac tissue suggested that lowering the pH of the malonate infusion would increase the proportion in the monocarboxylate form and thereby increase its potency as a cardioprotective agent. To assess this, we used a malonate dose that was not protective (8 mg/kg) when administered at a neutral pH in the in vivo LAD MI model. When this dose of malonate was reformulated at pH 4 (a pH currently used in Food and Drug Administration–approved parenteral formulations)40 and administered as a bolus, this conferred significant protection that was not due to the low pH alone (Figure 6E and Figure S6B). Furthermore, although neutral malonate administered before ischemia was not protective, acidified malonate drove its uptake into cardiomyocytes and significantly reduced infarct size when infused before ischemia (Figure S6C).\",\n \" Acknowledgments The authors thank Stephen Large, Fouad Taghavi, and Margaret M. Huang (Department of Surgery, University of Cambridge) for obtaining human heart tissue and Benjamin Thackray (Department of Medicine, University of Cambridge) for assistance with initial experiments.\\nThe authors thank Stephen Large, Fouad Taghavi, and Margaret M. Huang (Department of Surgery, University of Cambridge) for obtaining human heart tissue and Benjamin Thackray (Department of Medicine, University of Cambridge) for assistance with initial experiments.\\n Sources of Funding This work was supported by the British Heart Foundation (PG/20/10025 to T. Krieg); the Medical Research Council (MC_UU_00015/3 to M.P. Murphy and MR/P000320/1 to T. Krieg), the Wellcome Trust (220257/Z/20/Z to M.P. Murphy, 221604/Z/20/Z to D. Aksentijevic), Barts Charity (MRC0215 to D. Aksentijevic).\\nThis work was supported by the British Heart Foundation (PG/20/10025 to T. Krieg); the Medical Research Council (MC_UU_00015/3 to M.P. Murphy and MR/P000320/1 to T. Krieg), the Wellcome Trust (220257/Z/20/Z to M.P. Murphy, 221604/Z/20/Z to D. Aksentijevic), Barts Charity (MRC0215 to D. Aksentijevic).\\n Disclosures Some authors currently hold a patent on the use of malonate esters in cardiac ischemia/reperfusion (IR) injury (M.P. Murphy and T. Krieg) and have submitted patent applications on the use of malonate in IR injury associated with ischemic stroke (M.P. Murphy and T. Krieg) and the pH-enhancement of malonate described in this article (H.A. Prag, M.P. Murphy, and T. Krieg). The other authors report no conflicts.\\nSome authors currently hold a patent on the use of malonate esters in cardiac ischemia/reperfusion (IR) injury (M.P. Murphy and T. Krieg) and have submitted patent applications on the use of malonate in IR injury associated with ischemic stroke (M.P. Murphy and T. Krieg) and the pH-enhancement of malonate described in this article (H.A. Prag, M.P. Murphy, and T. Krieg). The other authors report no conflicts.\\n Supplemental Materials Supplemental Methods\\nMajor Resources Table\\nFigures S1–S6\\nReferences 49–56\\nSupplemental Methods\\nMajor Resources Table\\nFigures S1–S6\\nReferences 49–56\",\n \"The authors thank Stephen Large, Fouad Taghavi, and Margaret M. Huang (Department of Surgery, University of Cambridge) for obtaining human heart tissue and Benjamin Thackray (Department of Medicine, University of Cambridge) for assistance with initial experiments.\",\n \"This work was supported by the British Heart Foundation (PG/20/10025 to T. Krieg); the Medical Research Council (MC_UU_00015/3 to M.P. Murphy and MR/P000320/1 to T. Krieg), the Wellcome Trust (220257/Z/20/Z to M.P. Murphy, 221604/Z/20/Z to D. Aksentijevic), Barts Charity (MRC0215 to D. Aksentijevic).\",\n \"Supplemental Methods\\nMajor Resources Table\\nFigures S1–S6\\nReferences 49–56\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["What Is Known?","What New Information Does This Article Contribute?","Methods","Data Availability","Results","Malonate Is Protective in an MI Model and Is Taken Up Selectively into the Ischemic Region of the Heart","Malonate Uptake into Cells Is Inefficient at pH 7.4","Malonate Uptake into Cells and the Heart Can Be Modulated by pH","Malonate Uptake Can Be Perturbed by Modulating the Plasma Membrane H+ Gradient","Malonate Uptake Under Reperfusion Conditions Is Dependent on MCT1","Ischemic Conditions Are Sufficient to Drive Malonate Uptake into the Heart Upon Reperfusion","Low pH Formulation Improves Cardioprotection by Malonate","Discussion","Conclusions","Article Information","Acknowledgments","Sources of Funding","Disclosures","Supplemental Materials","Supplementary Material"],"string":"[\n \"What Is Known?\",\n \"What New Information Does This Article Contribute?\",\n \"Methods\",\n \"Data Availability\",\n \"Results\",\n \"Malonate Is Protective in an MI Model and Is Taken Up Selectively into the Ischemic Region of the Heart\",\n \"Malonate Uptake into Cells Is Inefficient at pH 7.4\",\n \"Malonate Uptake into Cells and the Heart Can Be Modulated by pH\",\n \"Malonate Uptake Can Be Perturbed by Modulating the Plasma Membrane H+ Gradient\",\n \"Malonate Uptake Under Reperfusion Conditions Is Dependent on MCT1\",\n \"Ischemic Conditions Are Sufficient to Drive Malonate Uptake into the Heart Upon Reperfusion\",\n \"Low pH Formulation Improves Cardioprotection by Malonate\",\n \"Discussion\",\n \"Conclusions\",\n \"Article Information\",\n \"Acknowledgments\",\n \"Sources of Funding\",\n \"Disclosures\",\n \"Supplemental Materials\",\n \"Supplementary Material\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Extensive succinate accumulation during ischemia and its subsequent rapid oxidation on reperfusion drives ischemia/reperfusion injury.\nPreventing succinate accumulation during ischemia reduces damage on reperfusion.\nInhibiting succinate dehydrogenase with malonate is protective against ischemia/reperfusion injury, although its mechanism of entry into cardiomyocytes is undefined.","The cardioprotective effect of malonate is dependent on its selective uptake into cardiomyocytes on reperfusion after an ischemic period.\nMalonate entry into cardiomyocytes upon reperfusion is facilitated by the lowered pH and lactate exchange, which selectively drives malonate into cardiomyocytes as a monoanion via the monocarboxylate transporter MCT1 (monocarboxylate transporter 1). This is the first time malonate has been shown to be a substrate for MCT1.\nMalonate selectively enters the at-risk tissue, sparing the nonischemic area on reperfusion. Thus, malonate is the first example of an at-risk tissue-selective, cardioprotective drug.\nDetermining the molecular basis for selective malonate entry via MCT1 into the ischemic heart upon reperfusion is a significant step toward treating cardiac ischemia/reperfusion injury. Malonate is cardioprotective in small and large animal models and MCT1 is highly expressed in the human heart. Furthermore, this mechanism can be exploited to increase malonate potency using an acidic formulation. The next step is to assess malonate as a treatment for cardiac ischemia/reperfusion injury in patients."," Data Availability Detailed methods and Major Resources Table can be found in the Supplemental Material. Data will be made available upon reasonable request, by contacting a corresponding author.\nDetailed methods and Major Resources Table can be found in the Supplemental Material. Data will be made available upon reasonable request, by contacting a corresponding author.","Detailed methods and Major Resources Table can be found in the Supplemental Material. Data will be made available upon reasonable request, by contacting a corresponding author."," Malonate Is Protective in an MI Model and Is Taken Up Selectively into the Ischemic Region of the Heart DSM is protective against cardiac IR injury, but its mechanism of protection remains uncertain. To address this gap in our knowledge we first tested the therapeutic range of DSM in IR injury. We infused DSM at a range of concentrations (1.6–160 mg/kg equivalent to ≈11-1100 µmol/kg) at reperfusion in an in vivo murine LAD (left anterior descending coronary artery) ligation model of MI. DSM infusion during this clinically relevant reperfusion period led to a dose-dependent decrease in infarct size (Figure 1C), with 160 and 16 mg/kg showing robust cardioprotection. However, when DSM was infused before the onset of ischemia it was not protective (Figure 1D), even at 160 mg/kg which gave the smallest infarct when administered at reperfusion. This contrasted with the cell-permeable dimethyl malonate, which can diffuse into the tissue and generate malonate, preventing succinate accumulation and thereby reducing IR injury.18,25 This suggests that either DSM is minimally taken up by tissues during normoxia and is only taken up by tissues after ischemia or that DSM protects against acute IR injury by an extracellular mechanism.\nWe next measured malonate uptake into the ischemic and healthy heart tissue upon reperfusion in the LAD MI model. This showed that malonate was indeed taken up into cells within the infarct region to a level of ≈400 pmol/mg tissue and to a far greater extent than into cells in the healthy tissue (Figure 1E). Furthermore, reperfusing with malonate slowed succinate oxidation within the infarct region with succinate remaining significantly elevated in the infarct region following 1-minute reperfusion, compared with control reperfusion with saline (Figure 1F). Limiting succinate oxidation at reperfusion with malonate also blunted ROS production in the at-risk tissue (Figure 1G). We next assessed how SDH inhibition compared to direct inhibition of the mitochondrial permeability transition pore with CsA (cyclosporin A). Here we found that the cardioprotection from malonate was additive to cyclosporin A alone (Figure 1H), hence targeting the upstream mechanism of permeability transition pore opening is an increasingly attractive option for IR injury treatment.\nTo better understand the metabolic differences between the healthy and infarcted tissue upon malonate treatment, we investigated the in vivo MI model using mass spectrometry imaging. To do this, we subjected hearts to either 30-minute ischemia, 30-minute ischemia before reperfusing the heart for 15-minute, or 30-minute ischemia before reperfusing with DSM for 15 minutes (to mimic the cardioprotective malonate infusion) before snap freezing and processing for mass spectrometry imaging (Figure 2A). Infarct lesions were demarcated using hematoxylin and eosin (H&E), silver infarct staining and metabolite principal component analysis to differentiate the risk and nonrisk regions. mass spectrometry imaging coupled with the demarcated risk areas showed striking changes in the levels of succinate in the infarct region. During ischemia, succinate was significantly elevated in the at-risk tissue, with considerable succinate accumulation in the core of the infarct (Figure 2B and 2C). After 15-minute reperfusion, the succinate levels in the infarct lesion returned to healthy tissue levels, due to succinate oxidation and efflux.21 When malonate was infused at the time of reperfusion, succinate levels remained higher in all regions of the heart than in the nonmalonate treated heart, consistent with the prevention of its oxidation by SDH inhibition by malonate.\nLocalization of succinate accumulation in heart tissue identified by mass spectrometry imaging. A, Outline of experimental groups for mass spectrometry imaging. B, Representative images of succinate abundance detected by mass spectrometry imaging in myocardial sections. Black outer line indicates the edge of the tissue slice, white inner line indicates infarct region. C, Quantification of succinate (C) identified by MSI (mean±SEM, n=3 biological replicates, statistics: Friedman paired test for healthy vs lesion and Kruskal-Wallis with Dunn post hoc test between conditions). au indicates arbitrary units; DSM, disodium malonate; H, healthy tissue; IR, ischemia/reperfusion; L, infarct lesion; and rep, reperfusion.\nWe conclude that malonate is indeed taken up by the heart, but this is greatly enhanced in ischemic region upon reperfusion. Therefore, malonate provides protection against cardiac IR injury in vivo over a range of concentrations following its selective entry into the infarct region, where it slows the oxidation of succinate during reperfusion, preventing the production of RET-derived ROS.\nDSM is protective against cardiac IR injury, but its mechanism of protection remains uncertain. To address this gap in our knowledge we first tested the therapeutic range of DSM in IR injury. We infused DSM at a range of concentrations (1.6–160 mg/kg equivalent to ≈11-1100 µmol/kg) at reperfusion in an in vivo murine LAD (left anterior descending coronary artery) ligation model of MI. DSM infusion during this clinically relevant reperfusion period led to a dose-dependent decrease in infarct size (Figure 1C), with 160 and 16 mg/kg showing robust cardioprotection. However, when DSM was infused before the onset of ischemia it was not protective (Figure 1D), even at 160 mg/kg which gave the smallest infarct when administered at reperfusion. This contrasted with the cell-permeable dimethyl malonate, which can diffuse into the tissue and generate malonate, preventing succinate accumulation and thereby reducing IR injury.18,25 This suggests that either DSM is minimally taken up by tissues during normoxia and is only taken up by tissues after ischemia or that DSM protects against acute IR injury by an extracellular mechanism.\nWe next measured malonate uptake into the ischemic and healthy heart tissue upon reperfusion in the LAD MI model. This showed that malonate was indeed taken up into cells within the infarct region to a level of ≈400 pmol/mg tissue and to a far greater extent than into cells in the healthy tissue (Figure 1E). Furthermore, reperfusing with malonate slowed succinate oxidation within the infarct region with succinate remaining significantly elevated in the infarct region following 1-minute reperfusion, compared with control reperfusion with saline (Figure 1F). Limiting succinate oxidation at reperfusion with malonate also blunted ROS production in the at-risk tissue (Figure 1G). We next assessed how SDH inhibition compared to direct inhibition of the mitochondrial permeability transition pore with CsA (cyclosporin A). Here we found that the cardioprotection from malonate was additive to cyclosporin A alone (Figure 1H), hence targeting the upstream mechanism of permeability transition pore opening is an increasingly attractive option for IR injury treatment.\nTo better understand the metabolic differences between the healthy and infarcted tissue upon malonate treatment, we investigated the in vivo MI model using mass spectrometry imaging. To do this, we subjected hearts to either 30-minute ischemia, 30-minute ischemia before reperfusing the heart for 15-minute, or 30-minute ischemia before reperfusing with DSM for 15 minutes (to mimic the cardioprotective malonate infusion) before snap freezing and processing for mass spectrometry imaging (Figure 2A). Infarct lesions were demarcated using hematoxylin and eosin (H&E), silver infarct staining and metabolite principal component analysis to differentiate the risk and nonrisk regions. mass spectrometry imaging coupled with the demarcated risk areas showed striking changes in the levels of succinate in the infarct region. During ischemia, succinate was significantly elevated in the at-risk tissue, with considerable succinate accumulation in the core of the infarct (Figure 2B and 2C). After 15-minute reperfusion, the succinate levels in the infarct lesion returned to healthy tissue levels, due to succinate oxidation and efflux.21 When malonate was infused at the time of reperfusion, succinate levels remained higher in all regions of the heart than in the nonmalonate treated heart, consistent with the prevention of its oxidation by SDH inhibition by malonate.\nLocalization of succinate accumulation in heart tissue identified by mass spectrometry imaging. A, Outline of experimental groups for mass spectrometry imaging. B, Representative images of succinate abundance detected by mass spectrometry imaging in myocardial sections. Black outer line indicates the edge of the tissue slice, white inner line indicates infarct region. C, Quantification of succinate (C) identified by MSI (mean±SEM, n=3 biological replicates, statistics: Friedman paired test for healthy vs lesion and Kruskal-Wallis with Dunn post hoc test between conditions). au indicates arbitrary units; DSM, disodium malonate; H, healthy tissue; IR, ischemia/reperfusion; L, infarct lesion; and rep, reperfusion.\nWe conclude that malonate is indeed taken up by the heart, but this is greatly enhanced in ischemic region upon reperfusion. Therefore, malonate provides protection against cardiac IR injury in vivo over a range of concentrations following its selective entry into the infarct region, where it slows the oxidation of succinate during reperfusion, preventing the production of RET-derived ROS.\n Malonate Uptake into Cells Is Inefficient at pH 7.4 To explore the mechanism of malonate uptake in vitro, we incubated C2C12 and H9c2 myoblasts with DSM and measured intracellular malonate levels by LC-MS/MS (liquid chromatography-tandem mass spectrometry). Incubation with DSM for 15 minutes at pH 7.4, led to a dose-dependent increase in intracellular malonate (Figure S1A and S1D). Intracellular succinate also accumulated in a malonate-dependent manner (Figure S1B and S1E), confirming that once malonate enters the cell it is rapidly transported into mitochondria and inhibits SDH. However, incubating cells with 1 mmol/L DSM led to variable malonate uptake and little SDH inhibition, indicating that malonate uptake across biological membranes is inefficient. Moreover, the intracellular levels of malonate achieved by 250 µmol/L of the malonate ester prodrug diacetoxymethyl malonate, are 80-fold more than that generated by incubation with 5 mmol/L DSM.25 As DSM was protective in the LAD model when infused at 16 mg/kg, corresponding to a maximum possible blood malonate concentration of ≈1.8 mmol/L (assuming a blood volume of 1.5 mL in a 25 g mouse),29 this compares to the dose range tested in cells (furthermore, far more malonate is available for uptake in vitro than in vivo due to the large reservoir in the incubation medium). Even so, the intracellular malonate levels were far lower with DSM compared to diacetoxymethyl malonate.25\nThe kinetics of cell uptake upon incubation with 5 mmol/L DSM showed time-dependent uptake, although extended incubation times were required for succinate elevation (Figure S1C and S1F). This is likely due to the initial concentrations of malonate entering the cell being insufficient to inhibit SDH. These data are consistent with the lack of protection by DSM delivered before ischemia in vivo, as well as its limited uptake into normoxic tissues. Together these suggest that exposure to ischemia may facilitate malonate entry into the heart.\nTo explore the mechanism of malonate uptake in vitro, we incubated C2C12 and H9c2 myoblasts with DSM and measured intracellular malonate levels by LC-MS/MS (liquid chromatography-tandem mass spectrometry). Incubation with DSM for 15 minutes at pH 7.4, led to a dose-dependent increase in intracellular malonate (Figure S1A and S1D). Intracellular succinate also accumulated in a malonate-dependent manner (Figure S1B and S1E), confirming that once malonate enters the cell it is rapidly transported into mitochondria and inhibits SDH. However, incubating cells with 1 mmol/L DSM led to variable malonate uptake and little SDH inhibition, indicating that malonate uptake across biological membranes is inefficient. Moreover, the intracellular levels of malonate achieved by 250 µmol/L of the malonate ester prodrug diacetoxymethyl malonate, are 80-fold more than that generated by incubation with 5 mmol/L DSM.25 As DSM was protective in the LAD model when infused at 16 mg/kg, corresponding to a maximum possible blood malonate concentration of ≈1.8 mmol/L (assuming a blood volume of 1.5 mL in a 25 g mouse),29 this compares to the dose range tested in cells (furthermore, far more malonate is available for uptake in vitro than in vivo due to the large reservoir in the incubation medium). Even so, the intracellular malonate levels were far lower with DSM compared to diacetoxymethyl malonate.25\nThe kinetics of cell uptake upon incubation with 5 mmol/L DSM showed time-dependent uptake, although extended incubation times were required for succinate elevation (Figure S1C and S1F). This is likely due to the initial concentrations of malonate entering the cell being insufficient to inhibit SDH. These data are consistent with the lack of protection by DSM delivered before ischemia in vivo, as well as its limited uptake into normoxic tissues. Together these suggest that exposure to ischemia may facilitate malonate entry into the heart.\n Malonate Uptake into Cells and the Heart Can Be Modulated by pH Malonate is a dicarboxylate at physiological pH (Figure 1A, pKa=2.83 and 5.69), suggesting that the pH decrease in ischemic tissue9,11,30 may enhance malonate uptake into the heart during early reperfusion, by increasing the concentration of its monocarboxylic form. Incubating cells with DSM at either pH 6, 7.4, or 8 for 15 minutes, led to large differences in malonate uptake (Figure 3A and Figure S2A). At pH 6, the levels of malonate in the cell were significantly higher than at pH 7.4 or 8, thus malonate uptake is favored by acidic pH. Succinate levels mirrored those of malonate, with greater succinate accumulation as a result of increased malonate-dependent SDH inhibition at low pH (Figure 3B and Figure S2B). Low pH alone had no effect on succinate levels (Figure 3B and Figure S2B), suggesting that it was due to malonate entry into the cells followed by SDH inhibition.\nMalonate uptake is enhanced at low pH. A and B, C2C12 cells were incubated with disodium malonate (DSM; 0, 1, or 5 mmol/L) for 15 min at either pH 6, 7.4, or 8 before measuring intracellular malonate (A) and succinate (B) by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). C, Malonate levels in C2C12 cells after incubation with DSM (5 mmol/L) for 15 min at various (patho)physiological pH (mean±SEM, n=3 biological replicates). D, Malonate levels in murine isolated Langendorff-perfused hearts treated with 5 mmol/L DSM infused at either pH 7.4 or 6 for 5 min (mean±SEM, n=4 biological replicates, statistics: unpaired, 2-tailed Mann-Whitney U test). E to H, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at either pH 6 or 7.4 in the presence of FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) (E), gramicidin (F), nigericin (G), or BAM15 (N5,N6-bis(2-Fluorophenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine) (H) before measuring intracellular malonate by LC-MS/MS (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). I, Structures of malonic acid and malonamic acid. J, Quantification of levels of malonate and malonamate in C2C12 cells after incubation (5 mmol/L DSM or disodium malonamate; 15 min) at pH 7.4 (n=6 biological replicates). K, C2C12 cells were incubated with either DSM or disodium malonamate (both 5 mmol/L) at pH 6 or 7.4 for 15 min and the intracellular levels measured by LC-MS/MS. (mean±SEM of the fold-enhancement of uptake at pH 6 vs pH 7.4, n=6 biological replicates). Statistics for J and K: unpaired, 2-tailed Student t test).\nIncubating cells with malonate over a range of pH values between pH 7.4 and 6 clearly showed the pH-dependent uptake of malonate, in line with the increase in monocarboxylate malonate proportion (Figure 3C). This was mirrored by succinate levels (Figure S2C). Therefore, even a small drop in pH can lead to a substantial increase in malonate uptake, and thus is likely to be relevant for its entry into ischemic tissue upon reperfusion.\nAs the cultured cells used are noncontractile and substantially differ in their properties from in situ contracting cardiomyocytes,15 importantly, we next assessed whether decreasing the pH also enhanced malonate uptake into the ex vivo perfused Langendorff heart. Malonate was infused into the heart at either pH 7.4 or 6 for 5 minutes, before briefly flushing at pH 7.4 to remove nonmyocardial malonate. Remarkably, the levels of malonate were significantly elevated when infused at pH 6 compared to 7.4, confirming that the in vitro results translate to the heart (Figure 3D). We conclude that low pH conditions facilitate the entry of malonate into cardiomyocytes.\nMalonate is a dicarboxylate at physiological pH (Figure 1A, pKa=2.83 and 5.69), suggesting that the pH decrease in ischemic tissue9,11,30 may enhance malonate uptake into the heart during early reperfusion, by increasing the concentration of its monocarboxylic form. Incubating cells with DSM at either pH 6, 7.4, or 8 for 15 minutes, led to large differences in malonate uptake (Figure 3A and Figure S2A). At pH 6, the levels of malonate in the cell were significantly higher than at pH 7.4 or 8, thus malonate uptake is favored by acidic pH. Succinate levels mirrored those of malonate, with greater succinate accumulation as a result of increased malonate-dependent SDH inhibition at low pH (Figure 3B and Figure S2B). Low pH alone had no effect on succinate levels (Figure 3B and Figure S2B), suggesting that it was due to malonate entry into the cells followed by SDH inhibition.\nMalonate uptake is enhanced at low pH. A and B, C2C12 cells were incubated with disodium malonate (DSM; 0, 1, or 5 mmol/L) for 15 min at either pH 6, 7.4, or 8 before measuring intracellular malonate (A) and succinate (B) by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). C, Malonate levels in C2C12 cells after incubation with DSM (5 mmol/L) for 15 min at various (patho)physiological pH (mean±SEM, n=3 biological replicates). D, Malonate levels in murine isolated Langendorff-perfused hearts treated with 5 mmol/L DSM infused at either pH 7.4 or 6 for 5 min (mean±SEM, n=4 biological replicates, statistics: unpaired, 2-tailed Mann-Whitney U test). E to H, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at either pH 6 or 7.4 in the presence of FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) (E), gramicidin (F), nigericin (G), or BAM15 (N5,N6-bis(2-Fluorophenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine) (H) before measuring intracellular malonate by LC-MS/MS (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). I, Structures of malonic acid and malonamic acid. J, Quantification of levels of malonate and malonamate in C2C12 cells after incubation (5 mmol/L DSM or disodium malonamate; 15 min) at pH 7.4 (n=6 biological replicates). K, C2C12 cells were incubated with either DSM or disodium malonamate (both 5 mmol/L) at pH 6 or 7.4 for 15 min and the intracellular levels measured by LC-MS/MS. (mean±SEM of the fold-enhancement of uptake at pH 6 vs pH 7.4, n=6 biological replicates). Statistics for J and K: unpaired, 2-tailed Student t test).\nIncubating cells with malonate over a range of pH values between pH 7.4 and 6 clearly showed the pH-dependent uptake of malonate, in line with the increase in monocarboxylate malonate proportion (Figure 3C). This was mirrored by succinate levels (Figure S2C). Therefore, even a small drop in pH can lead to a substantial increase in malonate uptake, and thus is likely to be relevant for its entry into ischemic tissue upon reperfusion.\nAs the cultured cells used are noncontractile and substantially differ in their properties from in situ contracting cardiomyocytes,15 importantly, we next assessed whether decreasing the pH also enhanced malonate uptake into the ex vivo perfused Langendorff heart. Malonate was infused into the heart at either pH 7.4 or 6 for 5 minutes, before briefly flushing at pH 7.4 to remove nonmyocardial malonate. Remarkably, the levels of malonate were significantly elevated when infused at pH 6 compared to 7.4, confirming that the in vitro results translate to the heart (Figure 3D). We conclude that low pH conditions facilitate the entry of malonate into cardiomyocytes.\n Malonate Uptake Can Be Perturbed by Modulating the Plasma Membrane H+ Gradient As pH modulated malonate entry into cells in vitro and in the heart, we next probed the factors affecting malonate uptake into cells. Malonate entry into cells at either pH 7.4 or 6 was blocked at 4 °C and was associated with negligible succinate levels (Figure S2D and S2E). That a decrease in extracellular pH increased malonate entry into cells, suggested uptake driven by the proton gradient. Therefore, we next abolished the plasma membrane proton gradient using ionophores21 which prevented the cellular uptake of malonate (Figure 3E–3G and Figure S3A) and subsequent increase in succinate levels (Figure S3B through S3E). However, the uncoupler BAM15, which is selective for the mitochondrial inner membrane over the plasma membrane,31 had little effect, consistent with the plasma membrane proton gradient driving malonate uptake (Figure 3H and Figure S3F).\nWe next used nonspecific transport inhibitors to assess whether we could block malonate uptake at low pH. DIDS (4,4’-Diisothiocyano-2,2’-stilbenedisulfonic acid), an irreversible inhibitor of chloride/bicarbonate exchange that inhibits malonate uptake into erythrocytes,32,33 led to a dose-dependent inhibition of malonate uptake, although succinate remained high (Figure S3G and S3H). Thus, malonate uptake into cardiomyocytes at low pH is driven by a proton gradient through a transporter-dependent process.\nMalonate’s pKa’s are 2.83 and 5.69, so at pH 6.4, ≈16% of the malonate would be in its monocarboxylate form. To determine whether the uptake of malonate was dependent on its protonation to a monocarboxylate form, we assessed the uptake of a compound that mimics the monocarboxylate malonate. In 3-amino-3-oxopropionate (malonamate; Figure 3I), one carboxylic acid has been replaced with a neutral amido group leaving a single carboxylic acid of pKa ≈4.75. Thus, at pH 7.4 malonamate resembles the monocarboxylate form of malonate. This led to more of malonamate being taken up into cells at pH 7.4 than malonate (Figure 3J). Lowering the pH to 6 led to ≈15-fold increase in malonate uptake while malonamate uptake changed negligibly (Figure 3K) because it will remain as a monocarboxylate across this pH range.\nTogether, these data support transport of the monoanionic form of malonate, under the conditions that occur during early reperfusion. Therefore, a pH gradient can drive the uptake of malonate via protonation and transport in its monocarboxylate form.\nAs pH modulated malonate entry into cells in vitro and in the heart, we next probed the factors affecting malonate uptake into cells. Malonate entry into cells at either pH 7.4 or 6 was blocked at 4 °C and was associated with negligible succinate levels (Figure S2D and S2E). That a decrease in extracellular pH increased malonate entry into cells, suggested uptake driven by the proton gradient. Therefore, we next abolished the plasma membrane proton gradient using ionophores21 which prevented the cellular uptake of malonate (Figure 3E–3G and Figure S3A) and subsequent increase in succinate levels (Figure S3B through S3E). However, the uncoupler BAM15, which is selective for the mitochondrial inner membrane over the plasma membrane,31 had little effect, consistent with the plasma membrane proton gradient driving malonate uptake (Figure 3H and Figure S3F).\nWe next used nonspecific transport inhibitors to assess whether we could block malonate uptake at low pH. DIDS (4,4’-Diisothiocyano-2,2’-stilbenedisulfonic acid), an irreversible inhibitor of chloride/bicarbonate exchange that inhibits malonate uptake into erythrocytes,32,33 led to a dose-dependent inhibition of malonate uptake, although succinate remained high (Figure S3G and S3H). Thus, malonate uptake into cardiomyocytes at low pH is driven by a proton gradient through a transporter-dependent process.\nMalonate’s pKa’s are 2.83 and 5.69, so at pH 6.4, ≈16% of the malonate would be in its monocarboxylate form. To determine whether the uptake of malonate was dependent on its protonation to a monocarboxylate form, we assessed the uptake of a compound that mimics the monocarboxylate malonate. In 3-amino-3-oxopropionate (malonamate; Figure 3I), one carboxylic acid has been replaced with a neutral amido group leaving a single carboxylic acid of pKa ≈4.75. Thus, at pH 7.4 malonamate resembles the monocarboxylate form of malonate. This led to more of malonamate being taken up into cells at pH 7.4 than malonate (Figure 3J). Lowering the pH to 6 led to ≈15-fold increase in malonate uptake while malonamate uptake changed negligibly (Figure 3K) because it will remain as a monocarboxylate across this pH range.\nTogether, these data support transport of the monoanionic form of malonate, under the conditions that occur during early reperfusion. Therefore, a pH gradient can drive the uptake of malonate via protonation and transport in its monocarboxylate form.\n Malonate Uptake Under Reperfusion Conditions Is Dependent on MCT1 The inhibitory effect of DIDS, which also inhibits MCT1,34 raised the possibility of the uptake of the protonated form of malonate being catalyzed by a monocarboxylate transporter (MCT; SLC16 (solute carrier family member 16)). MCT1 (SLC16A1) is the principal transporter of lactate, which is carried in symport with a proton, both of which are elevated during ischemia, making MCT1 an attractive candidate transporter for malonate uptake upon reperfusion. Additionally, it was recently shown that MCT1 mediates the efflux of succinate from the ischemic heart during reperfusion and from exercising muscle.21,35 As MCT1 is a lactate transporter, we first assessed whether high concentrations of lactate in the incubation medium compete with malonate. Excess lactate led to a concentration-dependent decrease in malonate uptake into the cell and a corresponding decrease in succinate accumulation (Figure 4A and Figure S4A). This suggested that at a low pH, malonate uptake into the cells is via MCT1, which is highly expressed in cardiomyocytes.36\nInhibition of MCT1 (monocarboxylate transporter 1) prevents the enhanced uptake of malonate at lowered pH. A, Malonate uptake (5 mmol/L disodium malonate [DSM], 15 min) in C2C12 cells in the presence of lactate (0, 10 or 50 mmol/L). B, Lactate levels in C2C12 cells after 15 min treatment with varying concentrations of MCT1 inhibitors. C and D, Effect of MCT1 inhibition by AR-C141990 or AZD3965 on malonate (5 mmol/L DSM, 15 min) uptake (C) at pH 6 and subsequent succinate levels (D). E, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at various pH±10 µmol/L AR-C141990. F, MCT1 inhibition by AR-C141990 on malonate (5 mmol/L DSM, 15 min) uptake at pH 7.4 (A to F, mean±SEM, n=3 biological replicates, statistics: (A) Kruskal-Wallis with Dunn post hoc test). G and H, Effect of malonate (5 mmol/L DSM) on cellular oxygen consumption at pH 7.4 (G) or 6 (H) ±MCT1 inhibitor (10 µmol/L AR-C141990; data presented as nonmitochondrial respiration normalized mean oxygen consumption rate (OCR)±SEM of 3 biological replicates (n=12–16 technical replicates per biological replicate), statistics: Kruskal-Wallis with Dunn post hoc test). ATP indicates OCR in the presence of 1.5 µmol/L oligomycin; BL, baseline OCR; MAX, OCR in the presence of 1 µmol/L FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone); and NM, OCR in the presence of 4 µg/mL rotenone and 10 µmol/L antimycin A.\nTo confirm that MCT1 was the malonate transporter, we assessed the potent and selective MCT1 inhibitors, AR-C141990 and AZD3965.21,35,37,38 When cells were incubated with these MCT1 inhibitors, they led to a dramatic dose-dependent increase in lactate levels within the cell, consistent with preventing lactate efflux via MCT1 (Figure 4B). MCT1 inhibition by either AR-C141990 or AZD3965 led to a profound dose-dependent decrease in malonate uptake (Figure 4C). Inhibition of malonate uptake by MCT1 inhibition led to a corresponding decrease in succinate accumulation within cells (Figure 4D). Additionally, a time course of malonate uptake at low pH, showed that MCT1 inhibition largely abolished the increase in malonate over time, along with the corresponding increase in succinate (Figure S4B and S4C).\nMCT1 inhibition blocked the pH-dependent increase in malonate uptake (Figure 4E) and in parallel prevented succinate accumulation (Figure 4D). The MCT1 inhibitor led to a dose-dependent inhibition of malonate uptake even at pH 7.4 (Figure 4F). Therefore, malonate can be transported by MCT1 at pH 7.4, but this is greatly enhanced at lower pH due to the increased proportion of malonate in its monocarboxylate form.\nWe next assessed the impact of the malonate taken up into cells on mitochondrial function by measuring respiration. Lowering pH itself had little effect; however, addition of malonate severely reduced cellular respiration (Figure 4G and H and Figure S4E and S4F) and this effect was rescued by MCT1 inhibition (Figure 4G and H and Figure S4E and S4F). MCT1 inhibition led to a small increase in oxygen consumption at baseline, which may be due to lactate accumulation increasing the NADH/NAD+ ratio driving mitochondrial respiration.39\nTo confirm the pharmacological effects of MCT1 inhibition on malonate uptake genetically, we knocked down (KD) MCT1 using siRNA in both HeLa and C2C12 cells (Figure S5A through S5D). Consequently, lactate levels in MCT1 KD cells were significantly elevated compared to the control siRNA (Figure 5A). When MCT1 KD cells were incubated with malonate at low pH, malonate uptake was dramatically blocked (Figure 5B through 5E and Figure S5C), confirming MCT1 is directly responsible for malonate transport at low pH. Residual malonate uptake in the MCT1 KD cells was inhibited by cotreatment with an MCT1 inhibitor, further confirming its MCT1 dependence (Figure 5F and Figure S5F). Additionally, succinate was elevated in the MCT1 KD cells compared with control siRNA cells, a result not seen with acute pharmacological MCT1 inhibition (Figure 5D). This suggests that MCT1 may also play an important role in normoxic succinate metabolism and homeostasis and not just during myocardial reperfusion and intense exercise.21,35 Intriguingly, the succinate levels after malonate treatment over time differed between the 2 cell types, with succinate levels remaining low in C2C12 KD but elevated in HeLa MCT1 KDs compared to controls, suggesting a reliance on MCT1 for succinate efflux (Figure 5G and Figure S5G).\nGenetic knockdown of MCT1 (monocarboxylate transporter 1) prevents the uptake of malonate. A, Relative lactate levels in C2C12 cells treated with control or MCT1 siRNA (mean±SEM of lactate levels relative to control, n=6 biological replicates, statistics: 1-way ANOVA with Bonferroni post hoc test). B to E, Incubation of malonate (5 mmol/L disodium malonate [DSM]) in MCT1 KD cells at pH 7.4 (B and D) or 6 (C and E) for 15 min±MCT1i (10 µmol/L, MCT1 inhibitor AR-C141990). Levels of malonate (B and C) and succinate (D and E) quantified by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). F and G, Time course of malonate uptake (5 mmol/L DSM) at pH 6 in MCT1 KD cells (F) and corresponding succinate levels (G; mean±SEM, n=3 biological replicates). H, Malonate levels in murine Langendorff hearts perfused at pH 6 for 5 min with 5 mmol/L DSM±lactate (50 mmol/L) or MCT1 inhibitor (10 µmol/L AR-C141990; MCT1i; mean±SEM, n=4, statistics: unpaired, 2-tailed Mann-Whitney U test vs control).\nAs HeLa cells constitutively express MCT4, which is under the control of HIF (hypoxia-inducible factor)-1α and may be implicated in the transport of metabolites in IR, we KD MCT4 independently of MCT1 (Figure S5D and S5E). MCT4 KD had little effect on malonate uptake, with malonate and succinate levels mirroring those of control siRNA-treated cells (Figure S5F and S5G). Thus, MCT1 is the main driver of malonate uptake at lowered pH.\nOverall, diminishing MCT1 activity, either by pharmacological inhibition or genetic knockdown, impairs malonate uptake at both acidic and normal physiological pH. In addition, this is the first evidence that MCT1 can transport a 3-carbon chain length dicarboxylate, which may also have implications in normal physiology and other pathologies.\nFinally, we assessed if MCT1 was responsible for malonate uptake at lowered pH in the heart. Malonate uptake into the Langendorff-perfused heart was blocked by either excess lactate, or the MCT1 inhibitor AR-C141990, in the perfusion medium (Figure 5H). Together these findings confirm that MCT1 is responsible for the low pH uptake of malonate, both in cells and in the intact heart.\nThe inhibitory effect of DIDS, which also inhibits MCT1,34 raised the possibility of the uptake of the protonated form of malonate being catalyzed by a monocarboxylate transporter (MCT; SLC16 (solute carrier family member 16)). MCT1 (SLC16A1) is the principal transporter of lactate, which is carried in symport with a proton, both of which are elevated during ischemia, making MCT1 an attractive candidate transporter for malonate uptake upon reperfusion. Additionally, it was recently shown that MCT1 mediates the efflux of succinate from the ischemic heart during reperfusion and from exercising muscle.21,35 As MCT1 is a lactate transporter, we first assessed whether high concentrations of lactate in the incubation medium compete with malonate. Excess lactate led to a concentration-dependent decrease in malonate uptake into the cell and a corresponding decrease in succinate accumulation (Figure 4A and Figure S4A). This suggested that at a low pH, malonate uptake into the cells is via MCT1, which is highly expressed in cardiomyocytes.36\nInhibition of MCT1 (monocarboxylate transporter 1) prevents the enhanced uptake of malonate at lowered pH. A, Malonate uptake (5 mmol/L disodium malonate [DSM], 15 min) in C2C12 cells in the presence of lactate (0, 10 or 50 mmol/L). B, Lactate levels in C2C12 cells after 15 min treatment with varying concentrations of MCT1 inhibitors. C and D, Effect of MCT1 inhibition by AR-C141990 or AZD3965 on malonate (5 mmol/L DSM, 15 min) uptake (C) at pH 6 and subsequent succinate levels (D). E, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at various pH±10 µmol/L AR-C141990. F, MCT1 inhibition by AR-C141990 on malonate (5 mmol/L DSM, 15 min) uptake at pH 7.4 (A to F, mean±SEM, n=3 biological replicates, statistics: (A) Kruskal-Wallis with Dunn post hoc test). G and H, Effect of malonate (5 mmol/L DSM) on cellular oxygen consumption at pH 7.4 (G) or 6 (H) ±MCT1 inhibitor (10 µmol/L AR-C141990; data presented as nonmitochondrial respiration normalized mean oxygen consumption rate (OCR)±SEM of 3 biological replicates (n=12–16 technical replicates per biological replicate), statistics: Kruskal-Wallis with Dunn post hoc test). ATP indicates OCR in the presence of 1.5 µmol/L oligomycin; BL, baseline OCR; MAX, OCR in the presence of 1 µmol/L FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone); and NM, OCR in the presence of 4 µg/mL rotenone and 10 µmol/L antimycin A.\nTo confirm that MCT1 was the malonate transporter, we assessed the potent and selective MCT1 inhibitors, AR-C141990 and AZD3965.21,35,37,38 When cells were incubated with these MCT1 inhibitors, they led to a dramatic dose-dependent increase in lactate levels within the cell, consistent with preventing lactate efflux via MCT1 (Figure 4B). MCT1 inhibition by either AR-C141990 or AZD3965 led to a profound dose-dependent decrease in malonate uptake (Figure 4C). Inhibition of malonate uptake by MCT1 inhibition led to a corresponding decrease in succinate accumulation within cells (Figure 4D). Additionally, a time course of malonate uptake at low pH, showed that MCT1 inhibition largely abolished the increase in malonate over time, along with the corresponding increase in succinate (Figure S4B and S4C).\nMCT1 inhibition blocked the pH-dependent increase in malonate uptake (Figure 4E) and in parallel prevented succinate accumulation (Figure 4D). The MCT1 inhibitor led to a dose-dependent inhibition of malonate uptake even at pH 7.4 (Figure 4F). Therefore, malonate can be transported by MCT1 at pH 7.4, but this is greatly enhanced at lower pH due to the increased proportion of malonate in its monocarboxylate form.\nWe next assessed the impact of the malonate taken up into cells on mitochondrial function by measuring respiration. Lowering pH itself had little effect; however, addition of malonate severely reduced cellular respiration (Figure 4G and H and Figure S4E and S4F) and this effect was rescued by MCT1 inhibition (Figure 4G and H and Figure S4E and S4F). MCT1 inhibition led to a small increase in oxygen consumption at baseline, which may be due to lactate accumulation increasing the NADH/NAD+ ratio driving mitochondrial respiration.39\nTo confirm the pharmacological effects of MCT1 inhibition on malonate uptake genetically, we knocked down (KD) MCT1 using siRNA in both HeLa and C2C12 cells (Figure S5A through S5D). Consequently, lactate levels in MCT1 KD cells were significantly elevated compared to the control siRNA (Figure 5A). When MCT1 KD cells were incubated with malonate at low pH, malonate uptake was dramatically blocked (Figure 5B through 5E and Figure S5C), confirming MCT1 is directly responsible for malonate transport at low pH. Residual malonate uptake in the MCT1 KD cells was inhibited by cotreatment with an MCT1 inhibitor, further confirming its MCT1 dependence (Figure 5F and Figure S5F). Additionally, succinate was elevated in the MCT1 KD cells compared with control siRNA cells, a result not seen with acute pharmacological MCT1 inhibition (Figure 5D). This suggests that MCT1 may also play an important role in normoxic succinate metabolism and homeostasis and not just during myocardial reperfusion and intense exercise.21,35 Intriguingly, the succinate levels after malonate treatment over time differed between the 2 cell types, with succinate levels remaining low in C2C12 KD but elevated in HeLa MCT1 KDs compared to controls, suggesting a reliance on MCT1 for succinate efflux (Figure 5G and Figure S5G).\nGenetic knockdown of MCT1 (monocarboxylate transporter 1) prevents the uptake of malonate. A, Relative lactate levels in C2C12 cells treated with control or MCT1 siRNA (mean±SEM of lactate levels relative to control, n=6 biological replicates, statistics: 1-way ANOVA with Bonferroni post hoc test). B to E, Incubation of malonate (5 mmol/L disodium malonate [DSM]) in MCT1 KD cells at pH 7.4 (B and D) or 6 (C and E) for 15 min±MCT1i (10 µmol/L, MCT1 inhibitor AR-C141990). Levels of malonate (B and C) and succinate (D and E) quantified by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). F and G, Time course of malonate uptake (5 mmol/L DSM) at pH 6 in MCT1 KD cells (F) and corresponding succinate levels (G; mean±SEM, n=3 biological replicates). H, Malonate levels in murine Langendorff hearts perfused at pH 6 for 5 min with 5 mmol/L DSM±lactate (50 mmol/L) or MCT1 inhibitor (10 µmol/L AR-C141990; MCT1i; mean±SEM, n=4, statistics: unpaired, 2-tailed Mann-Whitney U test vs control).\nAs HeLa cells constitutively express MCT4, which is under the control of HIF (hypoxia-inducible factor)-1α and may be implicated in the transport of metabolites in IR, we KD MCT4 independently of MCT1 (Figure S5D and S5E). MCT4 KD had little effect on malonate uptake, with malonate and succinate levels mirroring those of control siRNA-treated cells (Figure S5F and S5G). Thus, MCT1 is the main driver of malonate uptake at lowered pH.\nOverall, diminishing MCT1 activity, either by pharmacological inhibition or genetic knockdown, impairs malonate uptake at both acidic and normal physiological pH. In addition, this is the first evidence that MCT1 can transport a 3-carbon chain length dicarboxylate, which may also have implications in normal physiology and other pathologies.\nFinally, we assessed if MCT1 was responsible for malonate uptake at lowered pH in the heart. Malonate uptake into the Langendorff-perfused heart was blocked by either excess lactate, or the MCT1 inhibitor AR-C141990, in the perfusion medium (Figure 5H). Together these findings confirm that MCT1 is responsible for the low pH uptake of malonate, both in cells and in the intact heart.\n Ischemic Conditions Are Sufficient to Drive Malonate Uptake into the Heart Upon Reperfusion That low pH enhanced malonate entry into heart cells was consistent with the protection afforded by DSM in IR injury being due to the local decrease in pH during ischemia and initial reperfusion. To test this hypothesis, Langendorff-perfused hearts were held ischemic for various times before DSM was infused for 5 minutes, followed by flushing, and measurement of malonate. The malonate levels in heart tissue were dependent on ischemia-time, with the highest occurring after 20 minutes ischemia and being ≈10-fold greater than in the normoxic heart (Figure 6A). This malonate uptake into the ischemic Langendorff-perfused heart was dramatically reduced by the MCT1 inhibitor AR-C141990 (Figure 6B). This confirmed that malonate uptake into the ischemic heart upon reperfusion is a selective process, driven by the low pH and facilitated by the MCT1.\nIschemia drives malonate protonation, uptake, and cardioprotection. A, Langendorff-perfused murine hearts were held ischemic for either 0, 5, 10, or 20 min and reperfused with 5 mmol/L disodium malonate (DSM; pH 7.4) before malonate levels measured in the heart by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 (control, 5 min) or 6 (10 and 20 min) biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). B, Malonate levels in murine Langendorff hearts exposed to 20 min ischemia and reperfused with 5 mmol/L malonate (pH 7.4)±MCT1 (monocarboxylate transporter 1) inhibitor (10 or 50 µmol/L AR-C141990; mean±SEM, n=4–5 biological replicates, statistical significance assessed by unpaired, 2-tailed Mann-Whitney U test vs control). C, Model of potential lactate and malonate exchange during reperfusion. D, Lactate levels in the Langendorff heart after 20 min ischemia and 1 min reperfusion±5 mmol/L DSM (mean, n=4 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test). E, Infarct size in murine LAD (left anterior descending coronary artery) ligation MI model with 100 µl bolus of 8 mg/kg DSM, pH 4 acid control or 8 mg/kg pH 4 formulated malonate at reperfusion after 30 min ischemia (mean, n=5 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test vs acid malonate).\nDuring ischemia, the pH of ischemic tissue lowers but in addition, lactate accumulates and upon reperfusion can be transported out of cardiomyocytes by MCT1. This could enhance malonate uptake, as the MCT1 may then act as a lactate-H+/monocarboxylate malonate-H+ exchanger (Figure 6C). To assess this, we measured lactate levels in malonate-perfused IR Langendorff hearts. We found that compared to reperfusion alone, lactate levels decreased in the malonate-treated hearts (Figure 6D). Furthermore, malonate treatment decreased lactate levels in cells that had been treated with the mitochondrial inhibitor antimycin A to enhance lactate production (Figure S6A). This suggests that extracellular malonate and a proton exchange for intracellular lactate, help drive malonate entry into cardiomyocytes upon reperfusion and contribute to its at-risk tissue-selective effect.\nTherefore, malonate uptake into the heart upon reperfusion is dependent on the tissue having first undergone a period of ischemia, leading to a drop in pH and an accumulation of lactate.\nThat low pH enhanced malonate entry into heart cells was consistent with the protection afforded by DSM in IR injury being due to the local decrease in pH during ischemia and initial reperfusion. To test this hypothesis, Langendorff-perfused hearts were held ischemic for various times before DSM was infused for 5 minutes, followed by flushing, and measurement of malonate. The malonate levels in heart tissue were dependent on ischemia-time, with the highest occurring after 20 minutes ischemia and being ≈10-fold greater than in the normoxic heart (Figure 6A). This malonate uptake into the ischemic Langendorff-perfused heart was dramatically reduced by the MCT1 inhibitor AR-C141990 (Figure 6B). This confirmed that malonate uptake into the ischemic heart upon reperfusion is a selective process, driven by the low pH and facilitated by the MCT1.\nIschemia drives malonate protonation, uptake, and cardioprotection. A, Langendorff-perfused murine hearts were held ischemic for either 0, 5, 10, or 20 min and reperfused with 5 mmol/L disodium malonate (DSM; pH 7.4) before malonate levels measured in the heart by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 (control, 5 min) or 6 (10 and 20 min) biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). B, Malonate levels in murine Langendorff hearts exposed to 20 min ischemia and reperfused with 5 mmol/L malonate (pH 7.4)±MCT1 (monocarboxylate transporter 1) inhibitor (10 or 50 µmol/L AR-C141990; mean±SEM, n=4–5 biological replicates, statistical significance assessed by unpaired, 2-tailed Mann-Whitney U test vs control). C, Model of potential lactate and malonate exchange during reperfusion. D, Lactate levels in the Langendorff heart after 20 min ischemia and 1 min reperfusion±5 mmol/L DSM (mean, n=4 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test). E, Infarct size in murine LAD (left anterior descending coronary artery) ligation MI model with 100 µl bolus of 8 mg/kg DSM, pH 4 acid control or 8 mg/kg pH 4 formulated malonate at reperfusion after 30 min ischemia (mean, n=5 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test vs acid malonate).\nDuring ischemia, the pH of ischemic tissue lowers but in addition, lactate accumulates and upon reperfusion can be transported out of cardiomyocytes by MCT1. This could enhance malonate uptake, as the MCT1 may then act as a lactate-H+/monocarboxylate malonate-H+ exchanger (Figure 6C). To assess this, we measured lactate levels in malonate-perfused IR Langendorff hearts. We found that compared to reperfusion alone, lactate levels decreased in the malonate-treated hearts (Figure 6D). Furthermore, malonate treatment decreased lactate levels in cells that had been treated with the mitochondrial inhibitor antimycin A to enhance lactate production (Figure S6A). This suggests that extracellular malonate and a proton exchange for intracellular lactate, help drive malonate entry into cardiomyocytes upon reperfusion and contribute to its at-risk tissue-selective effect.\nTherefore, malonate uptake into the heart upon reperfusion is dependent on the tissue having first undergone a period of ischemia, leading to a drop in pH and an accumulation of lactate.\n Low pH Formulation Improves Cardioprotection by Malonate The mechanism of malonate uptake into cardiac tissue suggested that lowering the pH of the malonate infusion would increase the proportion in the monocarboxylate form and thereby increase its potency as a cardioprotective agent. To assess this, we used a malonate dose that was not protective (8 mg/kg) when administered at a neutral pH in the in vivo LAD MI model. When this dose of malonate was reformulated at pH 4 (a pH currently used in Food and Drug Administration–approved parenteral formulations)40 and administered as a bolus, this conferred significant protection that was not due to the low pH alone (Figure 6E and Figure S6B). Furthermore, although neutral malonate administered before ischemia was not protective, acidified malonate drove its uptake into cardiomyocytes and significantly reduced infarct size when infused before ischemia (Figure S6C).\nThe mechanism of malonate uptake into cardiac tissue suggested that lowering the pH of the malonate infusion would increase the proportion in the monocarboxylate form and thereby increase its potency as a cardioprotective agent. To assess this, we used a malonate dose that was not protective (8 mg/kg) when administered at a neutral pH in the in vivo LAD MI model. When this dose of malonate was reformulated at pH 4 (a pH currently used in Food and Drug Administration–approved parenteral formulations)40 and administered as a bolus, this conferred significant protection that was not due to the low pH alone (Figure 6E and Figure S6B). Furthermore, although neutral malonate administered before ischemia was not protective, acidified malonate drove its uptake into cardiomyocytes and significantly reduced infarct size when infused before ischemia (Figure S6C).","DSM is protective against cardiac IR injury, but its mechanism of protection remains uncertain. To address this gap in our knowledge we first tested the therapeutic range of DSM in IR injury. We infused DSM at a range of concentrations (1.6–160 mg/kg equivalent to ≈11-1100 µmol/kg) at reperfusion in an in vivo murine LAD (left anterior descending coronary artery) ligation model of MI. DSM infusion during this clinically relevant reperfusion period led to a dose-dependent decrease in infarct size (Figure 1C), with 160 and 16 mg/kg showing robust cardioprotection. However, when DSM was infused before the onset of ischemia it was not protective (Figure 1D), even at 160 mg/kg which gave the smallest infarct when administered at reperfusion. This contrasted with the cell-permeable dimethyl malonate, which can diffuse into the tissue and generate malonate, preventing succinate accumulation and thereby reducing IR injury.18,25 This suggests that either DSM is minimally taken up by tissues during normoxia and is only taken up by tissues after ischemia or that DSM protects against acute IR injury by an extracellular mechanism.\nWe next measured malonate uptake into the ischemic and healthy heart tissue upon reperfusion in the LAD MI model. This showed that malonate was indeed taken up into cells within the infarct region to a level of ≈400 pmol/mg tissue and to a far greater extent than into cells in the healthy tissue (Figure 1E). Furthermore, reperfusing with malonate slowed succinate oxidation within the infarct region with succinate remaining significantly elevated in the infarct region following 1-minute reperfusion, compared with control reperfusion with saline (Figure 1F). Limiting succinate oxidation at reperfusion with malonate also blunted ROS production in the at-risk tissue (Figure 1G). We next assessed how SDH inhibition compared to direct inhibition of the mitochondrial permeability transition pore with CsA (cyclosporin A). Here we found that the cardioprotection from malonate was additive to cyclosporin A alone (Figure 1H), hence targeting the upstream mechanism of permeability transition pore opening is an increasingly attractive option for IR injury treatment.\nTo better understand the metabolic differences between the healthy and infarcted tissue upon malonate treatment, we investigated the in vivo MI model using mass spectrometry imaging. To do this, we subjected hearts to either 30-minute ischemia, 30-minute ischemia before reperfusing the heart for 15-minute, or 30-minute ischemia before reperfusing with DSM for 15 minutes (to mimic the cardioprotective malonate infusion) before snap freezing and processing for mass spectrometry imaging (Figure 2A). Infarct lesions were demarcated using hematoxylin and eosin (H&E), silver infarct staining and metabolite principal component analysis to differentiate the risk and nonrisk regions. mass spectrometry imaging coupled with the demarcated risk areas showed striking changes in the levels of succinate in the infarct region. During ischemia, succinate was significantly elevated in the at-risk tissue, with considerable succinate accumulation in the core of the infarct (Figure 2B and 2C). After 15-minute reperfusion, the succinate levels in the infarct lesion returned to healthy tissue levels, due to succinate oxidation and efflux.21 When malonate was infused at the time of reperfusion, succinate levels remained higher in all regions of the heart than in the nonmalonate treated heart, consistent with the prevention of its oxidation by SDH inhibition by malonate.\nLocalization of succinate accumulation in heart tissue identified by mass spectrometry imaging. A, Outline of experimental groups for mass spectrometry imaging. B, Representative images of succinate abundance detected by mass spectrometry imaging in myocardial sections. Black outer line indicates the edge of the tissue slice, white inner line indicates infarct region. C, Quantification of succinate (C) identified by MSI (mean±SEM, n=3 biological replicates, statistics: Friedman paired test for healthy vs lesion and Kruskal-Wallis with Dunn post hoc test between conditions). au indicates arbitrary units; DSM, disodium malonate; H, healthy tissue; IR, ischemia/reperfusion; L, infarct lesion; and rep, reperfusion.\nWe conclude that malonate is indeed taken up by the heart, but this is greatly enhanced in ischemic region upon reperfusion. Therefore, malonate provides protection against cardiac IR injury in vivo over a range of concentrations following its selective entry into the infarct region, where it slows the oxidation of succinate during reperfusion, preventing the production of RET-derived ROS.","To explore the mechanism of malonate uptake in vitro, we incubated C2C12 and H9c2 myoblasts with DSM and measured intracellular malonate levels by LC-MS/MS (liquid chromatography-tandem mass spectrometry). Incubation with DSM for 15 minutes at pH 7.4, led to a dose-dependent increase in intracellular malonate (Figure S1A and S1D). Intracellular succinate also accumulated in a malonate-dependent manner (Figure S1B and S1E), confirming that once malonate enters the cell it is rapidly transported into mitochondria and inhibits SDH. However, incubating cells with 1 mmol/L DSM led to variable malonate uptake and little SDH inhibition, indicating that malonate uptake across biological membranes is inefficient. Moreover, the intracellular levels of malonate achieved by 250 µmol/L of the malonate ester prodrug diacetoxymethyl malonate, are 80-fold more than that generated by incubation with 5 mmol/L DSM.25 As DSM was protective in the LAD model when infused at 16 mg/kg, corresponding to a maximum possible blood malonate concentration of ≈1.8 mmol/L (assuming a blood volume of 1.5 mL in a 25 g mouse),29 this compares to the dose range tested in cells (furthermore, far more malonate is available for uptake in vitro than in vivo due to the large reservoir in the incubation medium). Even so, the intracellular malonate levels were far lower with DSM compared to diacetoxymethyl malonate.25\nThe kinetics of cell uptake upon incubation with 5 mmol/L DSM showed time-dependent uptake, although extended incubation times were required for succinate elevation (Figure S1C and S1F). This is likely due to the initial concentrations of malonate entering the cell being insufficient to inhibit SDH. These data are consistent with the lack of protection by DSM delivered before ischemia in vivo, as well as its limited uptake into normoxic tissues. Together these suggest that exposure to ischemia may facilitate malonate entry into the heart.","Malonate is a dicarboxylate at physiological pH (Figure 1A, pKa=2.83 and 5.69), suggesting that the pH decrease in ischemic tissue9,11,30 may enhance malonate uptake into the heart during early reperfusion, by increasing the concentration of its monocarboxylic form. Incubating cells with DSM at either pH 6, 7.4, or 8 for 15 minutes, led to large differences in malonate uptake (Figure 3A and Figure S2A). At pH 6, the levels of malonate in the cell were significantly higher than at pH 7.4 or 8, thus malonate uptake is favored by acidic pH. Succinate levels mirrored those of malonate, with greater succinate accumulation as a result of increased malonate-dependent SDH inhibition at low pH (Figure 3B and Figure S2B). Low pH alone had no effect on succinate levels (Figure 3B and Figure S2B), suggesting that it was due to malonate entry into the cells followed by SDH inhibition.\nMalonate uptake is enhanced at low pH. A and B, C2C12 cells were incubated with disodium malonate (DSM; 0, 1, or 5 mmol/L) for 15 min at either pH 6, 7.4, or 8 before measuring intracellular malonate (A) and succinate (B) by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). C, Malonate levels in C2C12 cells after incubation with DSM (5 mmol/L) for 15 min at various (patho)physiological pH (mean±SEM, n=3 biological replicates). D, Malonate levels in murine isolated Langendorff-perfused hearts treated with 5 mmol/L DSM infused at either pH 7.4 or 6 for 5 min (mean±SEM, n=4 biological replicates, statistics: unpaired, 2-tailed Mann-Whitney U test). E to H, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at either pH 6 or 7.4 in the presence of FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) (E), gramicidin (F), nigericin (G), or BAM15 (N5,N6-bis(2-Fluorophenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine) (H) before measuring intracellular malonate by LC-MS/MS (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). I, Structures of malonic acid and malonamic acid. J, Quantification of levels of malonate and malonamate in C2C12 cells after incubation (5 mmol/L DSM or disodium malonamate; 15 min) at pH 7.4 (n=6 biological replicates). K, C2C12 cells were incubated with either DSM or disodium malonamate (both 5 mmol/L) at pH 6 or 7.4 for 15 min and the intracellular levels measured by LC-MS/MS. (mean±SEM of the fold-enhancement of uptake at pH 6 vs pH 7.4, n=6 biological replicates). Statistics for J and K: unpaired, 2-tailed Student t test).\nIncubating cells with malonate over a range of pH values between pH 7.4 and 6 clearly showed the pH-dependent uptake of malonate, in line with the increase in monocarboxylate malonate proportion (Figure 3C). This was mirrored by succinate levels (Figure S2C). Therefore, even a small drop in pH can lead to a substantial increase in malonate uptake, and thus is likely to be relevant for its entry into ischemic tissue upon reperfusion.\nAs the cultured cells used are noncontractile and substantially differ in their properties from in situ contracting cardiomyocytes,15 importantly, we next assessed whether decreasing the pH also enhanced malonate uptake into the ex vivo perfused Langendorff heart. Malonate was infused into the heart at either pH 7.4 or 6 for 5 minutes, before briefly flushing at pH 7.4 to remove nonmyocardial malonate. Remarkably, the levels of malonate were significantly elevated when infused at pH 6 compared to 7.4, confirming that the in vitro results translate to the heart (Figure 3D). We conclude that low pH conditions facilitate the entry of malonate into cardiomyocytes.","As pH modulated malonate entry into cells in vitro and in the heart, we next probed the factors affecting malonate uptake into cells. Malonate entry into cells at either pH 7.4 or 6 was blocked at 4 °C and was associated with negligible succinate levels (Figure S2D and S2E). That a decrease in extracellular pH increased malonate entry into cells, suggested uptake driven by the proton gradient. Therefore, we next abolished the plasma membrane proton gradient using ionophores21 which prevented the cellular uptake of malonate (Figure 3E–3G and Figure S3A) and subsequent increase in succinate levels (Figure S3B through S3E). However, the uncoupler BAM15, which is selective for the mitochondrial inner membrane over the plasma membrane,31 had little effect, consistent with the plasma membrane proton gradient driving malonate uptake (Figure 3H and Figure S3F).\nWe next used nonspecific transport inhibitors to assess whether we could block malonate uptake at low pH. DIDS (4,4’-Diisothiocyano-2,2’-stilbenedisulfonic acid), an irreversible inhibitor of chloride/bicarbonate exchange that inhibits malonate uptake into erythrocytes,32,33 led to a dose-dependent inhibition of malonate uptake, although succinate remained high (Figure S3G and S3H). Thus, malonate uptake into cardiomyocytes at low pH is driven by a proton gradient through a transporter-dependent process.\nMalonate’s pKa’s are 2.83 and 5.69, so at pH 6.4, ≈16% of the malonate would be in its monocarboxylate form. To determine whether the uptake of malonate was dependent on its protonation to a monocarboxylate form, we assessed the uptake of a compound that mimics the monocarboxylate malonate. In 3-amino-3-oxopropionate (malonamate; Figure 3I), one carboxylic acid has been replaced with a neutral amido group leaving a single carboxylic acid of pKa ≈4.75. Thus, at pH 7.4 malonamate resembles the monocarboxylate form of malonate. This led to more of malonamate being taken up into cells at pH 7.4 than malonate (Figure 3J). Lowering the pH to 6 led to ≈15-fold increase in malonate uptake while malonamate uptake changed negligibly (Figure 3K) because it will remain as a monocarboxylate across this pH range.\nTogether, these data support transport of the monoanionic form of malonate, under the conditions that occur during early reperfusion. Therefore, a pH gradient can drive the uptake of malonate via protonation and transport in its monocarboxylate form.","The inhibitory effect of DIDS, which also inhibits MCT1,34 raised the possibility of the uptake of the protonated form of malonate being catalyzed by a monocarboxylate transporter (MCT; SLC16 (solute carrier family member 16)). MCT1 (SLC16A1) is the principal transporter of lactate, which is carried in symport with a proton, both of which are elevated during ischemia, making MCT1 an attractive candidate transporter for malonate uptake upon reperfusion. Additionally, it was recently shown that MCT1 mediates the efflux of succinate from the ischemic heart during reperfusion and from exercising muscle.21,35 As MCT1 is a lactate transporter, we first assessed whether high concentrations of lactate in the incubation medium compete with malonate. Excess lactate led to a concentration-dependent decrease in malonate uptake into the cell and a corresponding decrease in succinate accumulation (Figure 4A and Figure S4A). This suggested that at a low pH, malonate uptake into the cells is via MCT1, which is highly expressed in cardiomyocytes.36\nInhibition of MCT1 (monocarboxylate transporter 1) prevents the enhanced uptake of malonate at lowered pH. A, Malonate uptake (5 mmol/L disodium malonate [DSM], 15 min) in C2C12 cells in the presence of lactate (0, 10 or 50 mmol/L). B, Lactate levels in C2C12 cells after 15 min treatment with varying concentrations of MCT1 inhibitors. C and D, Effect of MCT1 inhibition by AR-C141990 or AZD3965 on malonate (5 mmol/L DSM, 15 min) uptake (C) at pH 6 and subsequent succinate levels (D). E, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at various pH±10 µmol/L AR-C141990. F, MCT1 inhibition by AR-C141990 on malonate (5 mmol/L DSM, 15 min) uptake at pH 7.4 (A to F, mean±SEM, n=3 biological replicates, statistics: (A) Kruskal-Wallis with Dunn post hoc test). G and H, Effect of malonate (5 mmol/L DSM) on cellular oxygen consumption at pH 7.4 (G) or 6 (H) ±MCT1 inhibitor (10 µmol/L AR-C141990; data presented as nonmitochondrial respiration normalized mean oxygen consumption rate (OCR)±SEM of 3 biological replicates (n=12–16 technical replicates per biological replicate), statistics: Kruskal-Wallis with Dunn post hoc test). ATP indicates OCR in the presence of 1.5 µmol/L oligomycin; BL, baseline OCR; MAX, OCR in the presence of 1 µmol/L FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone); and NM, OCR in the presence of 4 µg/mL rotenone and 10 µmol/L antimycin A.\nTo confirm that MCT1 was the malonate transporter, we assessed the potent and selective MCT1 inhibitors, AR-C141990 and AZD3965.21,35,37,38 When cells were incubated with these MCT1 inhibitors, they led to a dramatic dose-dependent increase in lactate levels within the cell, consistent with preventing lactate efflux via MCT1 (Figure 4B). MCT1 inhibition by either AR-C141990 or AZD3965 led to a profound dose-dependent decrease in malonate uptake (Figure 4C). Inhibition of malonate uptake by MCT1 inhibition led to a corresponding decrease in succinate accumulation within cells (Figure 4D). Additionally, a time course of malonate uptake at low pH, showed that MCT1 inhibition largely abolished the increase in malonate over time, along with the corresponding increase in succinate (Figure S4B and S4C).\nMCT1 inhibition blocked the pH-dependent increase in malonate uptake (Figure 4E) and in parallel prevented succinate accumulation (Figure 4D). The MCT1 inhibitor led to a dose-dependent inhibition of malonate uptake even at pH 7.4 (Figure 4F). Therefore, malonate can be transported by MCT1 at pH 7.4, but this is greatly enhanced at lower pH due to the increased proportion of malonate in its monocarboxylate form.\nWe next assessed the impact of the malonate taken up into cells on mitochondrial function by measuring respiration. Lowering pH itself had little effect; however, addition of malonate severely reduced cellular respiration (Figure 4G and H and Figure S4E and S4F) and this effect was rescued by MCT1 inhibition (Figure 4G and H and Figure S4E and S4F). MCT1 inhibition led to a small increase in oxygen consumption at baseline, which may be due to lactate accumulation increasing the NADH/NAD+ ratio driving mitochondrial respiration.39\nTo confirm the pharmacological effects of MCT1 inhibition on malonate uptake genetically, we knocked down (KD) MCT1 using siRNA in both HeLa and C2C12 cells (Figure S5A through S5D). Consequently, lactate levels in MCT1 KD cells were significantly elevated compared to the control siRNA (Figure 5A). When MCT1 KD cells were incubated with malonate at low pH, malonate uptake was dramatically blocked (Figure 5B through 5E and Figure S5C), confirming MCT1 is directly responsible for malonate transport at low pH. Residual malonate uptake in the MCT1 KD cells was inhibited by cotreatment with an MCT1 inhibitor, further confirming its MCT1 dependence (Figure 5F and Figure S5F). Additionally, succinate was elevated in the MCT1 KD cells compared with control siRNA cells, a result not seen with acute pharmacological MCT1 inhibition (Figure 5D). This suggests that MCT1 may also play an important role in normoxic succinate metabolism and homeostasis and not just during myocardial reperfusion and intense exercise.21,35 Intriguingly, the succinate levels after malonate treatment over time differed between the 2 cell types, with succinate levels remaining low in C2C12 KD but elevated in HeLa MCT1 KDs compared to controls, suggesting a reliance on MCT1 for succinate efflux (Figure 5G and Figure S5G).\nGenetic knockdown of MCT1 (monocarboxylate transporter 1) prevents the uptake of malonate. A, Relative lactate levels in C2C12 cells treated with control or MCT1 siRNA (mean±SEM of lactate levels relative to control, n=6 biological replicates, statistics: 1-way ANOVA with Bonferroni post hoc test). B to E, Incubation of malonate (5 mmol/L disodium malonate [DSM]) in MCT1 KD cells at pH 7.4 (B and D) or 6 (C and E) for 15 min±MCT1i (10 µmol/L, MCT1 inhibitor AR-C141990). Levels of malonate (B and C) and succinate (D and E) quantified by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). F and G, Time course of malonate uptake (5 mmol/L DSM) at pH 6 in MCT1 KD cells (F) and corresponding succinate levels (G; mean±SEM, n=3 biological replicates). H, Malonate levels in murine Langendorff hearts perfused at pH 6 for 5 min with 5 mmol/L DSM±lactate (50 mmol/L) or MCT1 inhibitor (10 µmol/L AR-C141990; MCT1i; mean±SEM, n=4, statistics: unpaired, 2-tailed Mann-Whitney U test vs control).\nAs HeLa cells constitutively express MCT4, which is under the control of HIF (hypoxia-inducible factor)-1α and may be implicated in the transport of metabolites in IR, we KD MCT4 independently of MCT1 (Figure S5D and S5E). MCT4 KD had little effect on malonate uptake, with malonate and succinate levels mirroring those of control siRNA-treated cells (Figure S5F and S5G). Thus, MCT1 is the main driver of malonate uptake at lowered pH.\nOverall, diminishing MCT1 activity, either by pharmacological inhibition or genetic knockdown, impairs malonate uptake at both acidic and normal physiological pH. In addition, this is the first evidence that MCT1 can transport a 3-carbon chain length dicarboxylate, which may also have implications in normal physiology and other pathologies.\nFinally, we assessed if MCT1 was responsible for malonate uptake at lowered pH in the heart. Malonate uptake into the Langendorff-perfused heart was blocked by either excess lactate, or the MCT1 inhibitor AR-C141990, in the perfusion medium (Figure 5H). Together these findings confirm that MCT1 is responsible for the low pH uptake of malonate, both in cells and in the intact heart.","That low pH enhanced malonate entry into heart cells was consistent with the protection afforded by DSM in IR injury being due to the local decrease in pH during ischemia and initial reperfusion. To test this hypothesis, Langendorff-perfused hearts were held ischemic for various times before DSM was infused for 5 minutes, followed by flushing, and measurement of malonate. The malonate levels in heart tissue were dependent on ischemia-time, with the highest occurring after 20 minutes ischemia and being ≈10-fold greater than in the normoxic heart (Figure 6A). This malonate uptake into the ischemic Langendorff-perfused heart was dramatically reduced by the MCT1 inhibitor AR-C141990 (Figure 6B). This confirmed that malonate uptake into the ischemic heart upon reperfusion is a selective process, driven by the low pH and facilitated by the MCT1.\nIschemia drives malonate protonation, uptake, and cardioprotection. A, Langendorff-perfused murine hearts were held ischemic for either 0, 5, 10, or 20 min and reperfused with 5 mmol/L disodium malonate (DSM; pH 7.4) before malonate levels measured in the heart by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 (control, 5 min) or 6 (10 and 20 min) biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). B, Malonate levels in murine Langendorff hearts exposed to 20 min ischemia and reperfused with 5 mmol/L malonate (pH 7.4)±MCT1 (monocarboxylate transporter 1) inhibitor (10 or 50 µmol/L AR-C141990; mean±SEM, n=4–5 biological replicates, statistical significance assessed by unpaired, 2-tailed Mann-Whitney U test vs control). C, Model of potential lactate and malonate exchange during reperfusion. D, Lactate levels in the Langendorff heart after 20 min ischemia and 1 min reperfusion±5 mmol/L DSM (mean, n=4 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test). E, Infarct size in murine LAD (left anterior descending coronary artery) ligation MI model with 100 µl bolus of 8 mg/kg DSM, pH 4 acid control or 8 mg/kg pH 4 formulated malonate at reperfusion after 30 min ischemia (mean, n=5 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test vs acid malonate).\nDuring ischemia, the pH of ischemic tissue lowers but in addition, lactate accumulates and upon reperfusion can be transported out of cardiomyocytes by MCT1. This could enhance malonate uptake, as the MCT1 may then act as a lactate-H+/monocarboxylate malonate-H+ exchanger (Figure 6C). To assess this, we measured lactate levels in malonate-perfused IR Langendorff hearts. We found that compared to reperfusion alone, lactate levels decreased in the malonate-treated hearts (Figure 6D). Furthermore, malonate treatment decreased lactate levels in cells that had been treated with the mitochondrial inhibitor antimycin A to enhance lactate production (Figure S6A). This suggests that extracellular malonate and a proton exchange for intracellular lactate, help drive malonate entry into cardiomyocytes upon reperfusion and contribute to its at-risk tissue-selective effect.\nTherefore, malonate uptake into the heart upon reperfusion is dependent on the tissue having first undergone a period of ischemia, leading to a drop in pH and an accumulation of lactate.","The mechanism of malonate uptake into cardiac tissue suggested that lowering the pH of the malonate infusion would increase the proportion in the monocarboxylate form and thereby increase its potency as a cardioprotective agent. To assess this, we used a malonate dose that was not protective (8 mg/kg) when administered at a neutral pH in the in vivo LAD MI model. When this dose of malonate was reformulated at pH 4 (a pH currently used in Food and Drug Administration–approved parenteral formulations)40 and administered as a bolus, this conferred significant protection that was not due to the low pH alone (Figure 6E and Figure S6B). Furthermore, although neutral malonate administered before ischemia was not protective, acidified malonate drove its uptake into cardiomyocytes and significantly reduced infarct size when infused before ischemia (Figure S6C).","No medicine is currently available that can be given at reperfusion to prevent cardiac IR injury.1,6 Drugs that prevent cardiac IR injury should both reduce MI damage and the subsequent development of heart failure.1,12 Targeting succinate metabolism has been shown to be a promising therapeutic approach. Inhibiting SDH using the reversible, competitive inhibitor malonate reduces infarct size in small and large animal models of cardiac IR injury, despite its mechanism of entry into heart tissue being unknown.26,27\nHere, we found that malonate uptake into cells and the heart at pH 7.4 was inefficient. However, the uptake of malonate into cells was dramatically enhanced at the lower levels of pH that occur during ischemia and by lactate accumulation within cells. Thus, ischemia provides an environment that will protonate malonate and thereby enable its uptake by the MCT1 transporter. Interestingly, this transporter undergoes a trans-acceleration phenomenon, whereby its transition from the outward-facing to the inward-facing conformation occurs more rapidly in the presence of a trans-substrate.41,42 In this case, the uptake of malonate on the extracellular side of the plasma membrane may be accelerated by lactate efflux. In the LAD MI model of IR injury, malonate is readily available at the point of reperfusion, thus a proportion of the malonate would be protonated and accessible for transport by MCT1. This enables malonate entry into the heart in an at-risk tissue-selective manner.\nAdditionally, as well as the inhibition of SDH, accelerating lactate efflux from the heart may also play a role in the reduction of IR injury. By shifting the equilibrium to facilitate anaerobic ATP production, this may promote the early restoration of ionic gradients through ATP-dependent transporters.43,44 Furthermore, facilitating lactate efflux may promote the extrusion of protons from the intracellular environment, thus reducing the activity of H+ transporters such as the Na+-H+ exchanger, though further investigation into these mechanisms is warranted.43,45\nThis is the first account of an ischemia-selective cardioprotective agent, utilizing the pathological differences between risk and nonrisk tissue to drive uptake. As MCT1 is highly expressed in the hearts of mice, rats, pigs, and humans (Figure S6D) and the characteristics of ischemia are conserved between these species,19,24 these drivers for malonate uptake and the plasma membrane transporter may facilitate malonate cardioprotection in humans. In addition to its place in the treatment of IR injury in MI, the mechanism of ischemia-enhanced delivery of malonate may provide a novel treatment option for IR injury under many other circumstances.\nThere is much interest in targeted drug delivery and the ability to engage the intended site while reducing off-target effects. Here, we have shown that malonate is not only a cardioprotective agent, but it does this while its entry into the rest of the heart is limited. Thus, malonate is a potent and ischemic tissue-targeted drug, explaining how large doses can be delivered acutely with minimal toxic effects, which is likely to be important bearing in mind the many comorbidities associated with MI.46 Furthermore, as malonate can be efficiently metabolised28,47 and has the potential to promote cardiomyocyte regeneration.48\nMalonate is robustly protective in acute IR injury, though further work is now required to understand the tractability of malonate treatment in chronic IR injury models; in particular, conducting a double-blind chronic large animal IR injury study.49 This would provide the greatest insight into the cardioprotection capabilities of malonate post-MI and its effect on the development of heart failure and define its potential for translation to the clinic.\nSchematic of ischemia-dependent malonate uptake via MCT1 (monocarboxylate transporter 1). The accumulation of lactate and protons in ischemic tissue and equilibration with the extracellular space facilitates protonation of malonate to its monocarboxylate form. This enables it to be an MCT1 substrate and enter cardiomyocytes upon reperfusion. Here, the malonate is transported into mitochondria by the mitochondrial dicarboxylate carrier where it can subsequently go on to inhibit SDH (succinate dehydrogenase). SDH inhibition reduces succinate oxidation, reactive oxygen species (ROS) production by reverse electron transport (RET) through complex I and opening of the mitochondrial permeability transition pore, thereby reducing cell death from ischemia/reperfusion (IR) injury. CxI indicates complex I; DIC, mitochondrial dicarboxylate carrier (SLC25A10); mPTP, mitochondrial permeability transition pore; and TCA, tricarboxylic acid.","We have shown that malonate is an ischemia-selective drug, due to the lowered pH and lactate accumulation of ischemic tissue driving its uptake via the MCT1 (Figure 7). Furthermore, we show that a low pH formulation of malonate enhances its therapeutic potency. Malonate is the first at-risk tissue-selective cardioprotective drug and represents a significant step toward the treatment of IR injury."," Acknowledgments The authors thank Stephen Large, Fouad Taghavi, and Margaret M. Huang (Department of Surgery, University of Cambridge) for obtaining human heart tissue and Benjamin Thackray (Department of Medicine, University of Cambridge) for assistance with initial experiments.\nThe authors thank Stephen Large, Fouad Taghavi, and Margaret M. Huang (Department of Surgery, University of Cambridge) for obtaining human heart tissue and Benjamin Thackray (Department of Medicine, University of Cambridge) for assistance with initial experiments.\n Sources of Funding This work was supported by the British Heart Foundation (PG/20/10025 to T. Krieg); the Medical Research Council (MC_UU_00015/3 to M.P. Murphy and MR/P000320/1 to T. Krieg), the Wellcome Trust (220257/Z/20/Z to M.P. Murphy, 221604/Z/20/Z to D. Aksentijevic), Barts Charity (MRC0215 to D. Aksentijevic).\nThis work was supported by the British Heart Foundation (PG/20/10025 to T. Krieg); the Medical Research Council (MC_UU_00015/3 to M.P. Murphy and MR/P000320/1 to T. Krieg), the Wellcome Trust (220257/Z/20/Z to M.P. Murphy, 221604/Z/20/Z to D. Aksentijevic), Barts Charity (MRC0215 to D. Aksentijevic).\n Disclosures Some authors currently hold a patent on the use of malonate esters in cardiac ischemia/reperfusion (IR) injury (M.P. Murphy and T. Krieg) and have submitted patent applications on the use of malonate in IR injury associated with ischemic stroke (M.P. Murphy and T. Krieg) and the pH-enhancement of malonate described in this article (H.A. Prag, M.P. Murphy, and T. Krieg). The other authors report no conflicts.\nSome authors currently hold a patent on the use of malonate esters in cardiac ischemia/reperfusion (IR) injury (M.P. Murphy and T. Krieg) and have submitted patent applications on the use of malonate in IR injury associated with ischemic stroke (M.P. Murphy and T. Krieg) and the pH-enhancement of malonate described in this article (H.A. Prag, M.P. Murphy, and T. Krieg). The other authors report no conflicts.\n Supplemental Materials Supplemental Methods\nMajor Resources Table\nFigures S1–S6\nReferences 49–56\nSupplemental Methods\nMajor Resources Table\nFigures S1–S6\nReferences 49–56","The authors thank Stephen Large, Fouad Taghavi, and Margaret M. Huang (Department of Surgery, University of Cambridge) for obtaining human heart tissue and Benjamin Thackray (Department of Medicine, University of Cambridge) for assistance with initial experiments.","This work was supported by the British Heart Foundation (PG/20/10025 to T. Krieg); the Medical Research Council (MC_UU_00015/3 to M.P. Murphy and MR/P000320/1 to T. Krieg), the Wellcome Trust (220257/Z/20/Z to M.P. Murphy, 221604/Z/20/Z to D. Aksentijevic), Barts Charity (MRC0215 to D. Aksentijevic).","Some authors currently hold a patent on the use of malonate esters in cardiac ischemia/reperfusion (IR) injury (M.P. Murphy and T. Krieg) and have submitted patent applications on the use of malonate in IR injury associated with ischemic stroke (M.P. Murphy and T. Krieg) and the pH-enhancement of malonate described in this article (H.A. Prag, M.P. Murphy, and T. Krieg). The other authors report no conflicts.","Supplemental Methods\nMajor Resources Table\nFigures S1–S6\nReferences 49–56",""],"string":"[\n \"Extensive succinate accumulation during ischemia and its subsequent rapid oxidation on reperfusion drives ischemia/reperfusion injury.\\nPreventing succinate accumulation during ischemia reduces damage on reperfusion.\\nInhibiting succinate dehydrogenase with malonate is protective against ischemia/reperfusion injury, although its mechanism of entry into cardiomyocytes is undefined.\",\n \"The cardioprotective effect of malonate is dependent on its selective uptake into cardiomyocytes on reperfusion after an ischemic period.\\nMalonate entry into cardiomyocytes upon reperfusion is facilitated by the lowered pH and lactate exchange, which selectively drives malonate into cardiomyocytes as a monoanion via the monocarboxylate transporter MCT1 (monocarboxylate transporter 1). This is the first time malonate has been shown to be a substrate for MCT1.\\nMalonate selectively enters the at-risk tissue, sparing the nonischemic area on reperfusion. Thus, malonate is the first example of an at-risk tissue-selective, cardioprotective drug.\\nDetermining the molecular basis for selective malonate entry via MCT1 into the ischemic heart upon reperfusion is a significant step toward treating cardiac ischemia/reperfusion injury. Malonate is cardioprotective in small and large animal models and MCT1 is highly expressed in the human heart. Furthermore, this mechanism can be exploited to increase malonate potency using an acidic formulation. The next step is to assess malonate as a treatment for cardiac ischemia/reperfusion injury in patients.\",\n \" Data Availability Detailed methods and Major Resources Table can be found in the Supplemental Material. Data will be made available upon reasonable request, by contacting a corresponding author.\\nDetailed methods and Major Resources Table can be found in the Supplemental Material. Data will be made available upon reasonable request, by contacting a corresponding author.\",\n \"Detailed methods and Major Resources Table can be found in the Supplemental Material. Data will be made available upon reasonable request, by contacting a corresponding author.\",\n \" Malonate Is Protective in an MI Model and Is Taken Up Selectively into the Ischemic Region of the Heart DSM is protective against cardiac IR injury, but its mechanism of protection remains uncertain. To address this gap in our knowledge we first tested the therapeutic range of DSM in IR injury. We infused DSM at a range of concentrations (1.6–160 mg/kg equivalent to ≈11-1100 µmol/kg) at reperfusion in an in vivo murine LAD (left anterior descending coronary artery) ligation model of MI. DSM infusion during this clinically relevant reperfusion period led to a dose-dependent decrease in infarct size (Figure 1C), with 160 and 16 mg/kg showing robust cardioprotection. However, when DSM was infused before the onset of ischemia it was not protective (Figure 1D), even at 160 mg/kg which gave the smallest infarct when administered at reperfusion. This contrasted with the cell-permeable dimethyl malonate, which can diffuse into the tissue and generate malonate, preventing succinate accumulation and thereby reducing IR injury.18,25 This suggests that either DSM is minimally taken up by tissues during normoxia and is only taken up by tissues after ischemia or that DSM protects against acute IR injury by an extracellular mechanism.\\nWe next measured malonate uptake into the ischemic and healthy heart tissue upon reperfusion in the LAD MI model. This showed that malonate was indeed taken up into cells within the infarct region to a level of ≈400 pmol/mg tissue and to a far greater extent than into cells in the healthy tissue (Figure 1E). Furthermore, reperfusing with malonate slowed succinate oxidation within the infarct region with succinate remaining significantly elevated in the infarct region following 1-minute reperfusion, compared with control reperfusion with saline (Figure 1F). Limiting succinate oxidation at reperfusion with malonate also blunted ROS production in the at-risk tissue (Figure 1G). We next assessed how SDH inhibition compared to direct inhibition of the mitochondrial permeability transition pore with CsA (cyclosporin A). Here we found that the cardioprotection from malonate was additive to cyclosporin A alone (Figure 1H), hence targeting the upstream mechanism of permeability transition pore opening is an increasingly attractive option for IR injury treatment.\\nTo better understand the metabolic differences between the healthy and infarcted tissue upon malonate treatment, we investigated the in vivo MI model using mass spectrometry imaging. To do this, we subjected hearts to either 30-minute ischemia, 30-minute ischemia before reperfusing the heart for 15-minute, or 30-minute ischemia before reperfusing with DSM for 15 minutes (to mimic the cardioprotective malonate infusion) before snap freezing and processing for mass spectrometry imaging (Figure 2A). Infarct lesions were demarcated using hematoxylin and eosin (H&E), silver infarct staining and metabolite principal component analysis to differentiate the risk and nonrisk regions. mass spectrometry imaging coupled with the demarcated risk areas showed striking changes in the levels of succinate in the infarct region. During ischemia, succinate was significantly elevated in the at-risk tissue, with considerable succinate accumulation in the core of the infarct (Figure 2B and 2C). After 15-minute reperfusion, the succinate levels in the infarct lesion returned to healthy tissue levels, due to succinate oxidation and efflux.21 When malonate was infused at the time of reperfusion, succinate levels remained higher in all regions of the heart than in the nonmalonate treated heart, consistent with the prevention of its oxidation by SDH inhibition by malonate.\\nLocalization of succinate accumulation in heart tissue identified by mass spectrometry imaging. A, Outline of experimental groups for mass spectrometry imaging. B, Representative images of succinate abundance detected by mass spectrometry imaging in myocardial sections. Black outer line indicates the edge of the tissue slice, white inner line indicates infarct region. C, Quantification of succinate (C) identified by MSI (mean±SEM, n=3 biological replicates, statistics: Friedman paired test for healthy vs lesion and Kruskal-Wallis with Dunn post hoc test between conditions). au indicates arbitrary units; DSM, disodium malonate; H, healthy tissue; IR, ischemia/reperfusion; L, infarct lesion; and rep, reperfusion.\\nWe conclude that malonate is indeed taken up by the heart, but this is greatly enhanced in ischemic region upon reperfusion. Therefore, malonate provides protection against cardiac IR injury in vivo over a range of concentrations following its selective entry into the infarct region, where it slows the oxidation of succinate during reperfusion, preventing the production of RET-derived ROS.\\nDSM is protective against cardiac IR injury, but its mechanism of protection remains uncertain. To address this gap in our knowledge we first tested the therapeutic range of DSM in IR injury. We infused DSM at a range of concentrations (1.6–160 mg/kg equivalent to ≈11-1100 µmol/kg) at reperfusion in an in vivo murine LAD (left anterior descending coronary artery) ligation model of MI. DSM infusion during this clinically relevant reperfusion period led to a dose-dependent decrease in infarct size (Figure 1C), with 160 and 16 mg/kg showing robust cardioprotection. However, when DSM was infused before the onset of ischemia it was not protective (Figure 1D), even at 160 mg/kg which gave the smallest infarct when administered at reperfusion. This contrasted with the cell-permeable dimethyl malonate, which can diffuse into the tissue and generate malonate, preventing succinate accumulation and thereby reducing IR injury.18,25 This suggests that either DSM is minimally taken up by tissues during normoxia and is only taken up by tissues after ischemia or that DSM protects against acute IR injury by an extracellular mechanism.\\nWe next measured malonate uptake into the ischemic and healthy heart tissue upon reperfusion in the LAD MI model. This showed that malonate was indeed taken up into cells within the infarct region to a level of ≈400 pmol/mg tissue and to a far greater extent than into cells in the healthy tissue (Figure 1E). Furthermore, reperfusing with malonate slowed succinate oxidation within the infarct region with succinate remaining significantly elevated in the infarct region following 1-minute reperfusion, compared with control reperfusion with saline (Figure 1F). Limiting succinate oxidation at reperfusion with malonate also blunted ROS production in the at-risk tissue (Figure 1G). We next assessed how SDH inhibition compared to direct inhibition of the mitochondrial permeability transition pore with CsA (cyclosporin A). Here we found that the cardioprotection from malonate was additive to cyclosporin A alone (Figure 1H), hence targeting the upstream mechanism of permeability transition pore opening is an increasingly attractive option for IR injury treatment.\\nTo better understand the metabolic differences between the healthy and infarcted tissue upon malonate treatment, we investigated the in vivo MI model using mass spectrometry imaging. To do this, we subjected hearts to either 30-minute ischemia, 30-minute ischemia before reperfusing the heart for 15-minute, or 30-minute ischemia before reperfusing with DSM for 15 minutes (to mimic the cardioprotective malonate infusion) before snap freezing and processing for mass spectrometry imaging (Figure 2A). Infarct lesions were demarcated using hematoxylin and eosin (H&E), silver infarct staining and metabolite principal component analysis to differentiate the risk and nonrisk regions. mass spectrometry imaging coupled with the demarcated risk areas showed striking changes in the levels of succinate in the infarct region. During ischemia, succinate was significantly elevated in the at-risk tissue, with considerable succinate accumulation in the core of the infarct (Figure 2B and 2C). After 15-minute reperfusion, the succinate levels in the infarct lesion returned to healthy tissue levels, due to succinate oxidation and efflux.21 When malonate was infused at the time of reperfusion, succinate levels remained higher in all regions of the heart than in the nonmalonate treated heart, consistent with the prevention of its oxidation by SDH inhibition by malonate.\\nLocalization of succinate accumulation in heart tissue identified by mass spectrometry imaging. A, Outline of experimental groups for mass spectrometry imaging. B, Representative images of succinate abundance detected by mass spectrometry imaging in myocardial sections. Black outer line indicates the edge of the tissue slice, white inner line indicates infarct region. C, Quantification of succinate (C) identified by MSI (mean±SEM, n=3 biological replicates, statistics: Friedman paired test for healthy vs lesion and Kruskal-Wallis with Dunn post hoc test between conditions). au indicates arbitrary units; DSM, disodium malonate; H, healthy tissue; IR, ischemia/reperfusion; L, infarct lesion; and rep, reperfusion.\\nWe conclude that malonate is indeed taken up by the heart, but this is greatly enhanced in ischemic region upon reperfusion. Therefore, malonate provides protection against cardiac IR injury in vivo over a range of concentrations following its selective entry into the infarct region, where it slows the oxidation of succinate during reperfusion, preventing the production of RET-derived ROS.\\n Malonate Uptake into Cells Is Inefficient at pH 7.4 To explore the mechanism of malonate uptake in vitro, we incubated C2C12 and H9c2 myoblasts with DSM and measured intracellular malonate levels by LC-MS/MS (liquid chromatography-tandem mass spectrometry). Incubation with DSM for 15 minutes at pH 7.4, led to a dose-dependent increase in intracellular malonate (Figure S1A and S1D). Intracellular succinate also accumulated in a malonate-dependent manner (Figure S1B and S1E), confirming that once malonate enters the cell it is rapidly transported into mitochondria and inhibits SDH. However, incubating cells with 1 mmol/L DSM led to variable malonate uptake and little SDH inhibition, indicating that malonate uptake across biological membranes is inefficient. Moreover, the intracellular levels of malonate achieved by 250 µmol/L of the malonate ester prodrug diacetoxymethyl malonate, are 80-fold more than that generated by incubation with 5 mmol/L DSM.25 As DSM was protective in the LAD model when infused at 16 mg/kg, corresponding to a maximum possible blood malonate concentration of ≈1.8 mmol/L (assuming a blood volume of 1.5 mL in a 25 g mouse),29 this compares to the dose range tested in cells (furthermore, far more malonate is available for uptake in vitro than in vivo due to the large reservoir in the incubation medium). Even so, the intracellular malonate levels were far lower with DSM compared to diacetoxymethyl malonate.25\\nThe kinetics of cell uptake upon incubation with 5 mmol/L DSM showed time-dependent uptake, although extended incubation times were required for succinate elevation (Figure S1C and S1F). This is likely due to the initial concentrations of malonate entering the cell being insufficient to inhibit SDH. These data are consistent with the lack of protection by DSM delivered before ischemia in vivo, as well as its limited uptake into normoxic tissues. Together these suggest that exposure to ischemia may facilitate malonate entry into the heart.\\nTo explore the mechanism of malonate uptake in vitro, we incubated C2C12 and H9c2 myoblasts with DSM and measured intracellular malonate levels by LC-MS/MS (liquid chromatography-tandem mass spectrometry). Incubation with DSM for 15 minutes at pH 7.4, led to a dose-dependent increase in intracellular malonate (Figure S1A and S1D). Intracellular succinate also accumulated in a malonate-dependent manner (Figure S1B and S1E), confirming that once malonate enters the cell it is rapidly transported into mitochondria and inhibits SDH. However, incubating cells with 1 mmol/L DSM led to variable malonate uptake and little SDH inhibition, indicating that malonate uptake across biological membranes is inefficient. Moreover, the intracellular levels of malonate achieved by 250 µmol/L of the malonate ester prodrug diacetoxymethyl malonate, are 80-fold more than that generated by incubation with 5 mmol/L DSM.25 As DSM was protective in the LAD model when infused at 16 mg/kg, corresponding to a maximum possible blood malonate concentration of ≈1.8 mmol/L (assuming a blood volume of 1.5 mL in a 25 g mouse),29 this compares to the dose range tested in cells (furthermore, far more malonate is available for uptake in vitro than in vivo due to the large reservoir in the incubation medium). Even so, the intracellular malonate levels were far lower with DSM compared to diacetoxymethyl malonate.25\\nThe kinetics of cell uptake upon incubation with 5 mmol/L DSM showed time-dependent uptake, although extended incubation times were required for succinate elevation (Figure S1C and S1F). This is likely due to the initial concentrations of malonate entering the cell being insufficient to inhibit SDH. These data are consistent with the lack of protection by DSM delivered before ischemia in vivo, as well as its limited uptake into normoxic tissues. Together these suggest that exposure to ischemia may facilitate malonate entry into the heart.\\n Malonate Uptake into Cells and the Heart Can Be Modulated by pH Malonate is a dicarboxylate at physiological pH (Figure 1A, pKa=2.83 and 5.69), suggesting that the pH decrease in ischemic tissue9,11,30 may enhance malonate uptake into the heart during early reperfusion, by increasing the concentration of its monocarboxylic form. Incubating cells with DSM at either pH 6, 7.4, or 8 for 15 minutes, led to large differences in malonate uptake (Figure 3A and Figure S2A). At pH 6, the levels of malonate in the cell were significantly higher than at pH 7.4 or 8, thus malonate uptake is favored by acidic pH. Succinate levels mirrored those of malonate, with greater succinate accumulation as a result of increased malonate-dependent SDH inhibition at low pH (Figure 3B and Figure S2B). Low pH alone had no effect on succinate levels (Figure 3B and Figure S2B), suggesting that it was due to malonate entry into the cells followed by SDH inhibition.\\nMalonate uptake is enhanced at low pH. A and B, C2C12 cells were incubated with disodium malonate (DSM; 0, 1, or 5 mmol/L) for 15 min at either pH 6, 7.4, or 8 before measuring intracellular malonate (A) and succinate (B) by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). C, Malonate levels in C2C12 cells after incubation with DSM (5 mmol/L) for 15 min at various (patho)physiological pH (mean±SEM, n=3 biological replicates). D, Malonate levels in murine isolated Langendorff-perfused hearts treated with 5 mmol/L DSM infused at either pH 7.4 or 6 for 5 min (mean±SEM, n=4 biological replicates, statistics: unpaired, 2-tailed Mann-Whitney U test). E to H, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at either pH 6 or 7.4 in the presence of FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) (E), gramicidin (F), nigericin (G), or BAM15 (N5,N6-bis(2-Fluorophenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine) (H) before measuring intracellular malonate by LC-MS/MS (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). I, Structures of malonic acid and malonamic acid. J, Quantification of levels of malonate and malonamate in C2C12 cells after incubation (5 mmol/L DSM or disodium malonamate; 15 min) at pH 7.4 (n=6 biological replicates). K, C2C12 cells were incubated with either DSM or disodium malonamate (both 5 mmol/L) at pH 6 or 7.4 for 15 min and the intracellular levels measured by LC-MS/MS. (mean±SEM of the fold-enhancement of uptake at pH 6 vs pH 7.4, n=6 biological replicates). Statistics for J and K: unpaired, 2-tailed Student t test).\\nIncubating cells with malonate over a range of pH values between pH 7.4 and 6 clearly showed the pH-dependent uptake of malonate, in line with the increase in monocarboxylate malonate proportion (Figure 3C). This was mirrored by succinate levels (Figure S2C). Therefore, even a small drop in pH can lead to a substantial increase in malonate uptake, and thus is likely to be relevant for its entry into ischemic tissue upon reperfusion.\\nAs the cultured cells used are noncontractile and substantially differ in their properties from in situ contracting cardiomyocytes,15 importantly, we next assessed whether decreasing the pH also enhanced malonate uptake into the ex vivo perfused Langendorff heart. Malonate was infused into the heart at either pH 7.4 or 6 for 5 minutes, before briefly flushing at pH 7.4 to remove nonmyocardial malonate. Remarkably, the levels of malonate were significantly elevated when infused at pH 6 compared to 7.4, confirming that the in vitro results translate to the heart (Figure 3D). We conclude that low pH conditions facilitate the entry of malonate into cardiomyocytes.\\nMalonate is a dicarboxylate at physiological pH (Figure 1A, pKa=2.83 and 5.69), suggesting that the pH decrease in ischemic tissue9,11,30 may enhance malonate uptake into the heart during early reperfusion, by increasing the concentration of its monocarboxylic form. Incubating cells with DSM at either pH 6, 7.4, or 8 for 15 minutes, led to large differences in malonate uptake (Figure 3A and Figure S2A). At pH 6, the levels of malonate in the cell were significantly higher than at pH 7.4 or 8, thus malonate uptake is favored by acidic pH. Succinate levels mirrored those of malonate, with greater succinate accumulation as a result of increased malonate-dependent SDH inhibition at low pH (Figure 3B and Figure S2B). Low pH alone had no effect on succinate levels (Figure 3B and Figure S2B), suggesting that it was due to malonate entry into the cells followed by SDH inhibition.\\nMalonate uptake is enhanced at low pH. A and B, C2C12 cells were incubated with disodium malonate (DSM; 0, 1, or 5 mmol/L) for 15 min at either pH 6, 7.4, or 8 before measuring intracellular malonate (A) and succinate (B) by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). C, Malonate levels in C2C12 cells after incubation with DSM (5 mmol/L) for 15 min at various (patho)physiological pH (mean±SEM, n=3 biological replicates). D, Malonate levels in murine isolated Langendorff-perfused hearts treated with 5 mmol/L DSM infused at either pH 7.4 or 6 for 5 min (mean±SEM, n=4 biological replicates, statistics: unpaired, 2-tailed Mann-Whitney U test). E to H, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at either pH 6 or 7.4 in the presence of FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) (E), gramicidin (F), nigericin (G), or BAM15 (N5,N6-bis(2-Fluorophenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine) (H) before measuring intracellular malonate by LC-MS/MS (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). I, Structures of malonic acid and malonamic acid. J, Quantification of levels of malonate and malonamate in C2C12 cells after incubation (5 mmol/L DSM or disodium malonamate; 15 min) at pH 7.4 (n=6 biological replicates). K, C2C12 cells were incubated with either DSM or disodium malonamate (both 5 mmol/L) at pH 6 or 7.4 for 15 min and the intracellular levels measured by LC-MS/MS. (mean±SEM of the fold-enhancement of uptake at pH 6 vs pH 7.4, n=6 biological replicates). Statistics for J and K: unpaired, 2-tailed Student t test).\\nIncubating cells with malonate over a range of pH values between pH 7.4 and 6 clearly showed the pH-dependent uptake of malonate, in line with the increase in monocarboxylate malonate proportion (Figure 3C). This was mirrored by succinate levels (Figure S2C). Therefore, even a small drop in pH can lead to a substantial increase in malonate uptake, and thus is likely to be relevant for its entry into ischemic tissue upon reperfusion.\\nAs the cultured cells used are noncontractile and substantially differ in their properties from in situ contracting cardiomyocytes,15 importantly, we next assessed whether decreasing the pH also enhanced malonate uptake into the ex vivo perfused Langendorff heart. Malonate was infused into the heart at either pH 7.4 or 6 for 5 minutes, before briefly flushing at pH 7.4 to remove nonmyocardial malonate. Remarkably, the levels of malonate were significantly elevated when infused at pH 6 compared to 7.4, confirming that the in vitro results translate to the heart (Figure 3D). We conclude that low pH conditions facilitate the entry of malonate into cardiomyocytes.\\n Malonate Uptake Can Be Perturbed by Modulating the Plasma Membrane H+ Gradient As pH modulated malonate entry into cells in vitro and in the heart, we next probed the factors affecting malonate uptake into cells. Malonate entry into cells at either pH 7.4 or 6 was blocked at 4 °C and was associated with negligible succinate levels (Figure S2D and S2E). That a decrease in extracellular pH increased malonate entry into cells, suggested uptake driven by the proton gradient. Therefore, we next abolished the plasma membrane proton gradient using ionophores21 which prevented the cellular uptake of malonate (Figure 3E–3G and Figure S3A) and subsequent increase in succinate levels (Figure S3B through S3E). However, the uncoupler BAM15, which is selective for the mitochondrial inner membrane over the plasma membrane,31 had little effect, consistent with the plasma membrane proton gradient driving malonate uptake (Figure 3H and Figure S3F).\\nWe next used nonspecific transport inhibitors to assess whether we could block malonate uptake at low pH. DIDS (4,4’-Diisothiocyano-2,2’-stilbenedisulfonic acid), an irreversible inhibitor of chloride/bicarbonate exchange that inhibits malonate uptake into erythrocytes,32,33 led to a dose-dependent inhibition of malonate uptake, although succinate remained high (Figure S3G and S3H). Thus, malonate uptake into cardiomyocytes at low pH is driven by a proton gradient through a transporter-dependent process.\\nMalonate’s pKa’s are 2.83 and 5.69, so at pH 6.4, ≈16% of the malonate would be in its monocarboxylate form. To determine whether the uptake of malonate was dependent on its protonation to a monocarboxylate form, we assessed the uptake of a compound that mimics the monocarboxylate malonate. In 3-amino-3-oxopropionate (malonamate; Figure 3I), one carboxylic acid has been replaced with a neutral amido group leaving a single carboxylic acid of pKa ≈4.75. Thus, at pH 7.4 malonamate resembles the monocarboxylate form of malonate. This led to more of malonamate being taken up into cells at pH 7.4 than malonate (Figure 3J). Lowering the pH to 6 led to ≈15-fold increase in malonate uptake while malonamate uptake changed negligibly (Figure 3K) because it will remain as a monocarboxylate across this pH range.\\nTogether, these data support transport of the monoanionic form of malonate, under the conditions that occur during early reperfusion. Therefore, a pH gradient can drive the uptake of malonate via protonation and transport in its monocarboxylate form.\\nAs pH modulated malonate entry into cells in vitro and in the heart, we next probed the factors affecting malonate uptake into cells. Malonate entry into cells at either pH 7.4 or 6 was blocked at 4 °C and was associated with negligible succinate levels (Figure S2D and S2E). That a decrease in extracellular pH increased malonate entry into cells, suggested uptake driven by the proton gradient. Therefore, we next abolished the plasma membrane proton gradient using ionophores21 which prevented the cellular uptake of malonate (Figure 3E–3G and Figure S3A) and subsequent increase in succinate levels (Figure S3B through S3E). However, the uncoupler BAM15, which is selective for the mitochondrial inner membrane over the plasma membrane,31 had little effect, consistent with the plasma membrane proton gradient driving malonate uptake (Figure 3H and Figure S3F).\\nWe next used nonspecific transport inhibitors to assess whether we could block malonate uptake at low pH. DIDS (4,4’-Diisothiocyano-2,2’-stilbenedisulfonic acid), an irreversible inhibitor of chloride/bicarbonate exchange that inhibits malonate uptake into erythrocytes,32,33 led to a dose-dependent inhibition of malonate uptake, although succinate remained high (Figure S3G and S3H). Thus, malonate uptake into cardiomyocytes at low pH is driven by a proton gradient through a transporter-dependent process.\\nMalonate’s pKa’s are 2.83 and 5.69, so at pH 6.4, ≈16% of the malonate would be in its monocarboxylate form. To determine whether the uptake of malonate was dependent on its protonation to a monocarboxylate form, we assessed the uptake of a compound that mimics the monocarboxylate malonate. In 3-amino-3-oxopropionate (malonamate; Figure 3I), one carboxylic acid has been replaced with a neutral amido group leaving a single carboxylic acid of pKa ≈4.75. Thus, at pH 7.4 malonamate resembles the monocarboxylate form of malonate. This led to more of malonamate being taken up into cells at pH 7.4 than malonate (Figure 3J). Lowering the pH to 6 led to ≈15-fold increase in malonate uptake while malonamate uptake changed negligibly (Figure 3K) because it will remain as a monocarboxylate across this pH range.\\nTogether, these data support transport of the monoanionic form of malonate, under the conditions that occur during early reperfusion. Therefore, a pH gradient can drive the uptake of malonate via protonation and transport in its monocarboxylate form.\\n Malonate Uptake Under Reperfusion Conditions Is Dependent on MCT1 The inhibitory effect of DIDS, which also inhibits MCT1,34 raised the possibility of the uptake of the protonated form of malonate being catalyzed by a monocarboxylate transporter (MCT; SLC16 (solute carrier family member 16)). MCT1 (SLC16A1) is the principal transporter of lactate, which is carried in symport with a proton, both of which are elevated during ischemia, making MCT1 an attractive candidate transporter for malonate uptake upon reperfusion. Additionally, it was recently shown that MCT1 mediates the efflux of succinate from the ischemic heart during reperfusion and from exercising muscle.21,35 As MCT1 is a lactate transporter, we first assessed whether high concentrations of lactate in the incubation medium compete with malonate. Excess lactate led to a concentration-dependent decrease in malonate uptake into the cell and a corresponding decrease in succinate accumulation (Figure 4A and Figure S4A). This suggested that at a low pH, malonate uptake into the cells is via MCT1, which is highly expressed in cardiomyocytes.36\\nInhibition of MCT1 (monocarboxylate transporter 1) prevents the enhanced uptake of malonate at lowered pH. A, Malonate uptake (5 mmol/L disodium malonate [DSM], 15 min) in C2C12 cells in the presence of lactate (0, 10 or 50 mmol/L). B, Lactate levels in C2C12 cells after 15 min treatment with varying concentrations of MCT1 inhibitors. C and D, Effect of MCT1 inhibition by AR-C141990 or AZD3965 on malonate (5 mmol/L DSM, 15 min) uptake (C) at pH 6 and subsequent succinate levels (D). E, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at various pH±10 µmol/L AR-C141990. F, MCT1 inhibition by AR-C141990 on malonate (5 mmol/L DSM, 15 min) uptake at pH 7.4 (A to F, mean±SEM, n=3 biological replicates, statistics: (A) Kruskal-Wallis with Dunn post hoc test). G and H, Effect of malonate (5 mmol/L DSM) on cellular oxygen consumption at pH 7.4 (G) or 6 (H) ±MCT1 inhibitor (10 µmol/L AR-C141990; data presented as nonmitochondrial respiration normalized mean oxygen consumption rate (OCR)±SEM of 3 biological replicates (n=12–16 technical replicates per biological replicate), statistics: Kruskal-Wallis with Dunn post hoc test). ATP indicates OCR in the presence of 1.5 µmol/L oligomycin; BL, baseline OCR; MAX, OCR in the presence of 1 µmol/L FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone); and NM, OCR in the presence of 4 µg/mL rotenone and 10 µmol/L antimycin A.\\nTo confirm that MCT1 was the malonate transporter, we assessed the potent and selective MCT1 inhibitors, AR-C141990 and AZD3965.21,35,37,38 When cells were incubated with these MCT1 inhibitors, they led to a dramatic dose-dependent increase in lactate levels within the cell, consistent with preventing lactate efflux via MCT1 (Figure 4B). MCT1 inhibition by either AR-C141990 or AZD3965 led to a profound dose-dependent decrease in malonate uptake (Figure 4C). Inhibition of malonate uptake by MCT1 inhibition led to a corresponding decrease in succinate accumulation within cells (Figure 4D). Additionally, a time course of malonate uptake at low pH, showed that MCT1 inhibition largely abolished the increase in malonate over time, along with the corresponding increase in succinate (Figure S4B and S4C).\\nMCT1 inhibition blocked the pH-dependent increase in malonate uptake (Figure 4E) and in parallel prevented succinate accumulation (Figure 4D). The MCT1 inhibitor led to a dose-dependent inhibition of malonate uptake even at pH 7.4 (Figure 4F). Therefore, malonate can be transported by MCT1 at pH 7.4, but this is greatly enhanced at lower pH due to the increased proportion of malonate in its monocarboxylate form.\\nWe next assessed the impact of the malonate taken up into cells on mitochondrial function by measuring respiration. Lowering pH itself had little effect; however, addition of malonate severely reduced cellular respiration (Figure 4G and H and Figure S4E and S4F) and this effect was rescued by MCT1 inhibition (Figure 4G and H and Figure S4E and S4F). MCT1 inhibition led to a small increase in oxygen consumption at baseline, which may be due to lactate accumulation increasing the NADH/NAD+ ratio driving mitochondrial respiration.39\\nTo confirm the pharmacological effects of MCT1 inhibition on malonate uptake genetically, we knocked down (KD) MCT1 using siRNA in both HeLa and C2C12 cells (Figure S5A through S5D). Consequently, lactate levels in MCT1 KD cells were significantly elevated compared to the control siRNA (Figure 5A). When MCT1 KD cells were incubated with malonate at low pH, malonate uptake was dramatically blocked (Figure 5B through 5E and Figure S5C), confirming MCT1 is directly responsible for malonate transport at low pH. Residual malonate uptake in the MCT1 KD cells was inhibited by cotreatment with an MCT1 inhibitor, further confirming its MCT1 dependence (Figure 5F and Figure S5F). Additionally, succinate was elevated in the MCT1 KD cells compared with control siRNA cells, a result not seen with acute pharmacological MCT1 inhibition (Figure 5D). This suggests that MCT1 may also play an important role in normoxic succinate metabolism and homeostasis and not just during myocardial reperfusion and intense exercise.21,35 Intriguingly, the succinate levels after malonate treatment over time differed between the 2 cell types, with succinate levels remaining low in C2C12 KD but elevated in HeLa MCT1 KDs compared to controls, suggesting a reliance on MCT1 for succinate efflux (Figure 5G and Figure S5G).\\nGenetic knockdown of MCT1 (monocarboxylate transporter 1) prevents the uptake of malonate. A, Relative lactate levels in C2C12 cells treated with control or MCT1 siRNA (mean±SEM of lactate levels relative to control, n=6 biological replicates, statistics: 1-way ANOVA with Bonferroni post hoc test). B to E, Incubation of malonate (5 mmol/L disodium malonate [DSM]) in MCT1 KD cells at pH 7.4 (B and D) or 6 (C and E) for 15 min±MCT1i (10 µmol/L, MCT1 inhibitor AR-C141990). Levels of malonate (B and C) and succinate (D and E) quantified by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). F and G, Time course of malonate uptake (5 mmol/L DSM) at pH 6 in MCT1 KD cells (F) and corresponding succinate levels (G; mean±SEM, n=3 biological replicates). H, Malonate levels in murine Langendorff hearts perfused at pH 6 for 5 min with 5 mmol/L DSM±lactate (50 mmol/L) or MCT1 inhibitor (10 µmol/L AR-C141990; MCT1i; mean±SEM, n=4, statistics: unpaired, 2-tailed Mann-Whitney U test vs control).\\nAs HeLa cells constitutively express MCT4, which is under the control of HIF (hypoxia-inducible factor)-1α and may be implicated in the transport of metabolites in IR, we KD MCT4 independently of MCT1 (Figure S5D and S5E). MCT4 KD had little effect on malonate uptake, with malonate and succinate levels mirroring those of control siRNA-treated cells (Figure S5F and S5G). Thus, MCT1 is the main driver of malonate uptake at lowered pH.\\nOverall, diminishing MCT1 activity, either by pharmacological inhibition or genetic knockdown, impairs malonate uptake at both acidic and normal physiological pH. In addition, this is the first evidence that MCT1 can transport a 3-carbon chain length dicarboxylate, which may also have implications in normal physiology and other pathologies.\\nFinally, we assessed if MCT1 was responsible for malonate uptake at lowered pH in the heart. Malonate uptake into the Langendorff-perfused heart was blocked by either excess lactate, or the MCT1 inhibitor AR-C141990, in the perfusion medium (Figure 5H). Together these findings confirm that MCT1 is responsible for the low pH uptake of malonate, both in cells and in the intact heart.\\nThe inhibitory effect of DIDS, which also inhibits MCT1,34 raised the possibility of the uptake of the protonated form of malonate being catalyzed by a monocarboxylate transporter (MCT; SLC16 (solute carrier family member 16)). MCT1 (SLC16A1) is the principal transporter of lactate, which is carried in symport with a proton, both of which are elevated during ischemia, making MCT1 an attractive candidate transporter for malonate uptake upon reperfusion. Additionally, it was recently shown that MCT1 mediates the efflux of succinate from the ischemic heart during reperfusion and from exercising muscle.21,35 As MCT1 is a lactate transporter, we first assessed whether high concentrations of lactate in the incubation medium compete with malonate. Excess lactate led to a concentration-dependent decrease in malonate uptake into the cell and a corresponding decrease in succinate accumulation (Figure 4A and Figure S4A). This suggested that at a low pH, malonate uptake into the cells is via MCT1, which is highly expressed in cardiomyocytes.36\\nInhibition of MCT1 (monocarboxylate transporter 1) prevents the enhanced uptake of malonate at lowered pH. A, Malonate uptake (5 mmol/L disodium malonate [DSM], 15 min) in C2C12 cells in the presence of lactate (0, 10 or 50 mmol/L). B, Lactate levels in C2C12 cells after 15 min treatment with varying concentrations of MCT1 inhibitors. C and D, Effect of MCT1 inhibition by AR-C141990 or AZD3965 on malonate (5 mmol/L DSM, 15 min) uptake (C) at pH 6 and subsequent succinate levels (D). E, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at various pH±10 µmol/L AR-C141990. F, MCT1 inhibition by AR-C141990 on malonate (5 mmol/L DSM, 15 min) uptake at pH 7.4 (A to F, mean±SEM, n=3 biological replicates, statistics: (A) Kruskal-Wallis with Dunn post hoc test). G and H, Effect of malonate (5 mmol/L DSM) on cellular oxygen consumption at pH 7.4 (G) or 6 (H) ±MCT1 inhibitor (10 µmol/L AR-C141990; data presented as nonmitochondrial respiration normalized mean oxygen consumption rate (OCR)±SEM of 3 biological replicates (n=12–16 technical replicates per biological replicate), statistics: Kruskal-Wallis with Dunn post hoc test). ATP indicates OCR in the presence of 1.5 µmol/L oligomycin; BL, baseline OCR; MAX, OCR in the presence of 1 µmol/L FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone); and NM, OCR in the presence of 4 µg/mL rotenone and 10 µmol/L antimycin A.\\nTo confirm that MCT1 was the malonate transporter, we assessed the potent and selective MCT1 inhibitors, AR-C141990 and AZD3965.21,35,37,38 When cells were incubated with these MCT1 inhibitors, they led to a dramatic dose-dependent increase in lactate levels within the cell, consistent with preventing lactate efflux via MCT1 (Figure 4B). MCT1 inhibition by either AR-C141990 or AZD3965 led to a profound dose-dependent decrease in malonate uptake (Figure 4C). Inhibition of malonate uptake by MCT1 inhibition led to a corresponding decrease in succinate accumulation within cells (Figure 4D). Additionally, a time course of malonate uptake at low pH, showed that MCT1 inhibition largely abolished the increase in malonate over time, along with the corresponding increase in succinate (Figure S4B and S4C).\\nMCT1 inhibition blocked the pH-dependent increase in malonate uptake (Figure 4E) and in parallel prevented succinate accumulation (Figure 4D). The MCT1 inhibitor led to a dose-dependent inhibition of malonate uptake even at pH 7.4 (Figure 4F). Therefore, malonate can be transported by MCT1 at pH 7.4, but this is greatly enhanced at lower pH due to the increased proportion of malonate in its monocarboxylate form.\\nWe next assessed the impact of the malonate taken up into cells on mitochondrial function by measuring respiration. Lowering pH itself had little effect; however, addition of malonate severely reduced cellular respiration (Figure 4G and H and Figure S4E and S4F) and this effect was rescued by MCT1 inhibition (Figure 4G and H and Figure S4E and S4F). MCT1 inhibition led to a small increase in oxygen consumption at baseline, which may be due to lactate accumulation increasing the NADH/NAD+ ratio driving mitochondrial respiration.39\\nTo confirm the pharmacological effects of MCT1 inhibition on malonate uptake genetically, we knocked down (KD) MCT1 using siRNA in both HeLa and C2C12 cells (Figure S5A through S5D). Consequently, lactate levels in MCT1 KD cells were significantly elevated compared to the control siRNA (Figure 5A). When MCT1 KD cells were incubated with malonate at low pH, malonate uptake was dramatically blocked (Figure 5B through 5E and Figure S5C), confirming MCT1 is directly responsible for malonate transport at low pH. Residual malonate uptake in the MCT1 KD cells was inhibited by cotreatment with an MCT1 inhibitor, further confirming its MCT1 dependence (Figure 5F and Figure S5F). Additionally, succinate was elevated in the MCT1 KD cells compared with control siRNA cells, a result not seen with acute pharmacological MCT1 inhibition (Figure 5D). This suggests that MCT1 may also play an important role in normoxic succinate metabolism and homeostasis and not just during myocardial reperfusion and intense exercise.21,35 Intriguingly, the succinate levels after malonate treatment over time differed between the 2 cell types, with succinate levels remaining low in C2C12 KD but elevated in HeLa MCT1 KDs compared to controls, suggesting a reliance on MCT1 for succinate efflux (Figure 5G and Figure S5G).\\nGenetic knockdown of MCT1 (monocarboxylate transporter 1) prevents the uptake of malonate. A, Relative lactate levels in C2C12 cells treated with control or MCT1 siRNA (mean±SEM of lactate levels relative to control, n=6 biological replicates, statistics: 1-way ANOVA with Bonferroni post hoc test). B to E, Incubation of malonate (5 mmol/L disodium malonate [DSM]) in MCT1 KD cells at pH 7.4 (B and D) or 6 (C and E) for 15 min±MCT1i (10 µmol/L, MCT1 inhibitor AR-C141990). Levels of malonate (B and C) and succinate (D and E) quantified by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). F and G, Time course of malonate uptake (5 mmol/L DSM) at pH 6 in MCT1 KD cells (F) and corresponding succinate levels (G; mean±SEM, n=3 biological replicates). H, Malonate levels in murine Langendorff hearts perfused at pH 6 for 5 min with 5 mmol/L DSM±lactate (50 mmol/L) or MCT1 inhibitor (10 µmol/L AR-C141990; MCT1i; mean±SEM, n=4, statistics: unpaired, 2-tailed Mann-Whitney U test vs control).\\nAs HeLa cells constitutively express MCT4, which is under the control of HIF (hypoxia-inducible factor)-1α and may be implicated in the transport of metabolites in IR, we KD MCT4 independently of MCT1 (Figure S5D and S5E). MCT4 KD had little effect on malonate uptake, with malonate and succinate levels mirroring those of control siRNA-treated cells (Figure S5F and S5G). Thus, MCT1 is the main driver of malonate uptake at lowered pH.\\nOverall, diminishing MCT1 activity, either by pharmacological inhibition or genetic knockdown, impairs malonate uptake at both acidic and normal physiological pH. In addition, this is the first evidence that MCT1 can transport a 3-carbon chain length dicarboxylate, which may also have implications in normal physiology and other pathologies.\\nFinally, we assessed if MCT1 was responsible for malonate uptake at lowered pH in the heart. Malonate uptake into the Langendorff-perfused heart was blocked by either excess lactate, or the MCT1 inhibitor AR-C141990, in the perfusion medium (Figure 5H). Together these findings confirm that MCT1 is responsible for the low pH uptake of malonate, both in cells and in the intact heart.\\n Ischemic Conditions Are Sufficient to Drive Malonate Uptake into the Heart Upon Reperfusion That low pH enhanced malonate entry into heart cells was consistent with the protection afforded by DSM in IR injury being due to the local decrease in pH during ischemia and initial reperfusion. To test this hypothesis, Langendorff-perfused hearts were held ischemic for various times before DSM was infused for 5 minutes, followed by flushing, and measurement of malonate. The malonate levels in heart tissue were dependent on ischemia-time, with the highest occurring after 20 minutes ischemia and being ≈10-fold greater than in the normoxic heart (Figure 6A). This malonate uptake into the ischemic Langendorff-perfused heart was dramatically reduced by the MCT1 inhibitor AR-C141990 (Figure 6B). This confirmed that malonate uptake into the ischemic heart upon reperfusion is a selective process, driven by the low pH and facilitated by the MCT1.\\nIschemia drives malonate protonation, uptake, and cardioprotection. A, Langendorff-perfused murine hearts were held ischemic for either 0, 5, 10, or 20 min and reperfused with 5 mmol/L disodium malonate (DSM; pH 7.4) before malonate levels measured in the heart by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 (control, 5 min) or 6 (10 and 20 min) biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). B, Malonate levels in murine Langendorff hearts exposed to 20 min ischemia and reperfused with 5 mmol/L malonate (pH 7.4)±MCT1 (monocarboxylate transporter 1) inhibitor (10 or 50 µmol/L AR-C141990; mean±SEM, n=4–5 biological replicates, statistical significance assessed by unpaired, 2-tailed Mann-Whitney U test vs control). C, Model of potential lactate and malonate exchange during reperfusion. D, Lactate levels in the Langendorff heart after 20 min ischemia and 1 min reperfusion±5 mmol/L DSM (mean, n=4 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test). E, Infarct size in murine LAD (left anterior descending coronary artery) ligation MI model with 100 µl bolus of 8 mg/kg DSM, pH 4 acid control or 8 mg/kg pH 4 formulated malonate at reperfusion after 30 min ischemia (mean, n=5 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test vs acid malonate).\\nDuring ischemia, the pH of ischemic tissue lowers but in addition, lactate accumulates and upon reperfusion can be transported out of cardiomyocytes by MCT1. This could enhance malonate uptake, as the MCT1 may then act as a lactate-H+/monocarboxylate malonate-H+ exchanger (Figure 6C). To assess this, we measured lactate levels in malonate-perfused IR Langendorff hearts. We found that compared to reperfusion alone, lactate levels decreased in the malonate-treated hearts (Figure 6D). Furthermore, malonate treatment decreased lactate levels in cells that had been treated with the mitochondrial inhibitor antimycin A to enhance lactate production (Figure S6A). This suggests that extracellular malonate and a proton exchange for intracellular lactate, help drive malonate entry into cardiomyocytes upon reperfusion and contribute to its at-risk tissue-selective effect.\\nTherefore, malonate uptake into the heart upon reperfusion is dependent on the tissue having first undergone a period of ischemia, leading to a drop in pH and an accumulation of lactate.\\nThat low pH enhanced malonate entry into heart cells was consistent with the protection afforded by DSM in IR injury being due to the local decrease in pH during ischemia and initial reperfusion. To test this hypothesis, Langendorff-perfused hearts were held ischemic for various times before DSM was infused for 5 minutes, followed by flushing, and measurement of malonate. The malonate levels in heart tissue were dependent on ischemia-time, with the highest occurring after 20 minutes ischemia and being ≈10-fold greater than in the normoxic heart (Figure 6A). This malonate uptake into the ischemic Langendorff-perfused heart was dramatically reduced by the MCT1 inhibitor AR-C141990 (Figure 6B). This confirmed that malonate uptake into the ischemic heart upon reperfusion is a selective process, driven by the low pH and facilitated by the MCT1.\\nIschemia drives malonate protonation, uptake, and cardioprotection. A, Langendorff-perfused murine hearts were held ischemic for either 0, 5, 10, or 20 min and reperfused with 5 mmol/L disodium malonate (DSM; pH 7.4) before malonate levels measured in the heart by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 (control, 5 min) or 6 (10 and 20 min) biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). B, Malonate levels in murine Langendorff hearts exposed to 20 min ischemia and reperfused with 5 mmol/L malonate (pH 7.4)±MCT1 (monocarboxylate transporter 1) inhibitor (10 or 50 µmol/L AR-C141990; mean±SEM, n=4–5 biological replicates, statistical significance assessed by unpaired, 2-tailed Mann-Whitney U test vs control). C, Model of potential lactate and malonate exchange during reperfusion. D, Lactate levels in the Langendorff heart after 20 min ischemia and 1 min reperfusion±5 mmol/L DSM (mean, n=4 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test). E, Infarct size in murine LAD (left anterior descending coronary artery) ligation MI model with 100 µl bolus of 8 mg/kg DSM, pH 4 acid control or 8 mg/kg pH 4 formulated malonate at reperfusion after 30 min ischemia (mean, n=5 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test vs acid malonate).\\nDuring ischemia, the pH of ischemic tissue lowers but in addition, lactate accumulates and upon reperfusion can be transported out of cardiomyocytes by MCT1. This could enhance malonate uptake, as the MCT1 may then act as a lactate-H+/monocarboxylate malonate-H+ exchanger (Figure 6C). To assess this, we measured lactate levels in malonate-perfused IR Langendorff hearts. We found that compared to reperfusion alone, lactate levels decreased in the malonate-treated hearts (Figure 6D). Furthermore, malonate treatment decreased lactate levels in cells that had been treated with the mitochondrial inhibitor antimycin A to enhance lactate production (Figure S6A). This suggests that extracellular malonate and a proton exchange for intracellular lactate, help drive malonate entry into cardiomyocytes upon reperfusion and contribute to its at-risk tissue-selective effect.\\nTherefore, malonate uptake into the heart upon reperfusion is dependent on the tissue having first undergone a period of ischemia, leading to a drop in pH and an accumulation of lactate.\\n Low pH Formulation Improves Cardioprotection by Malonate The mechanism of malonate uptake into cardiac tissue suggested that lowering the pH of the malonate infusion would increase the proportion in the monocarboxylate form and thereby increase its potency as a cardioprotective agent. To assess this, we used a malonate dose that was not protective (8 mg/kg) when administered at a neutral pH in the in vivo LAD MI model. When this dose of malonate was reformulated at pH 4 (a pH currently used in Food and Drug Administration–approved parenteral formulations)40 and administered as a bolus, this conferred significant protection that was not due to the low pH alone (Figure 6E and Figure S6B). Furthermore, although neutral malonate administered before ischemia was not protective, acidified malonate drove its uptake into cardiomyocytes and significantly reduced infarct size when infused before ischemia (Figure S6C).\\nThe mechanism of malonate uptake into cardiac tissue suggested that lowering the pH of the malonate infusion would increase the proportion in the monocarboxylate form and thereby increase its potency as a cardioprotective agent. To assess this, we used a malonate dose that was not protective (8 mg/kg) when administered at a neutral pH in the in vivo LAD MI model. When this dose of malonate was reformulated at pH 4 (a pH currently used in Food and Drug Administration–approved parenteral formulations)40 and administered as a bolus, this conferred significant protection that was not due to the low pH alone (Figure 6E and Figure S6B). Furthermore, although neutral malonate administered before ischemia was not protective, acidified malonate drove its uptake into cardiomyocytes and significantly reduced infarct size when infused before ischemia (Figure S6C).\",\n \"DSM is protective against cardiac IR injury, but its mechanism of protection remains uncertain. To address this gap in our knowledge we first tested the therapeutic range of DSM in IR injury. We infused DSM at a range of concentrations (1.6–160 mg/kg equivalent to ≈11-1100 µmol/kg) at reperfusion in an in vivo murine LAD (left anterior descending coronary artery) ligation model of MI. DSM infusion during this clinically relevant reperfusion period led to a dose-dependent decrease in infarct size (Figure 1C), with 160 and 16 mg/kg showing robust cardioprotection. However, when DSM was infused before the onset of ischemia it was not protective (Figure 1D), even at 160 mg/kg which gave the smallest infarct when administered at reperfusion. This contrasted with the cell-permeable dimethyl malonate, which can diffuse into the tissue and generate malonate, preventing succinate accumulation and thereby reducing IR injury.18,25 This suggests that either DSM is minimally taken up by tissues during normoxia and is only taken up by tissues after ischemia or that DSM protects against acute IR injury by an extracellular mechanism.\\nWe next measured malonate uptake into the ischemic and healthy heart tissue upon reperfusion in the LAD MI model. This showed that malonate was indeed taken up into cells within the infarct region to a level of ≈400 pmol/mg tissue and to a far greater extent than into cells in the healthy tissue (Figure 1E). Furthermore, reperfusing with malonate slowed succinate oxidation within the infarct region with succinate remaining significantly elevated in the infarct region following 1-minute reperfusion, compared with control reperfusion with saline (Figure 1F). Limiting succinate oxidation at reperfusion with malonate also blunted ROS production in the at-risk tissue (Figure 1G). We next assessed how SDH inhibition compared to direct inhibition of the mitochondrial permeability transition pore with CsA (cyclosporin A). Here we found that the cardioprotection from malonate was additive to cyclosporin A alone (Figure 1H), hence targeting the upstream mechanism of permeability transition pore opening is an increasingly attractive option for IR injury treatment.\\nTo better understand the metabolic differences between the healthy and infarcted tissue upon malonate treatment, we investigated the in vivo MI model using mass spectrometry imaging. To do this, we subjected hearts to either 30-minute ischemia, 30-minute ischemia before reperfusing the heart for 15-minute, or 30-minute ischemia before reperfusing with DSM for 15 minutes (to mimic the cardioprotective malonate infusion) before snap freezing and processing for mass spectrometry imaging (Figure 2A). Infarct lesions were demarcated using hematoxylin and eosin (H&E), silver infarct staining and metabolite principal component analysis to differentiate the risk and nonrisk regions. mass spectrometry imaging coupled with the demarcated risk areas showed striking changes in the levels of succinate in the infarct region. During ischemia, succinate was significantly elevated in the at-risk tissue, with considerable succinate accumulation in the core of the infarct (Figure 2B and 2C). After 15-minute reperfusion, the succinate levels in the infarct lesion returned to healthy tissue levels, due to succinate oxidation and efflux.21 When malonate was infused at the time of reperfusion, succinate levels remained higher in all regions of the heart than in the nonmalonate treated heart, consistent with the prevention of its oxidation by SDH inhibition by malonate.\\nLocalization of succinate accumulation in heart tissue identified by mass spectrometry imaging. A, Outline of experimental groups for mass spectrometry imaging. B, Representative images of succinate abundance detected by mass spectrometry imaging in myocardial sections. Black outer line indicates the edge of the tissue slice, white inner line indicates infarct region. C, Quantification of succinate (C) identified by MSI (mean±SEM, n=3 biological replicates, statistics: Friedman paired test for healthy vs lesion and Kruskal-Wallis with Dunn post hoc test between conditions). au indicates arbitrary units; DSM, disodium malonate; H, healthy tissue; IR, ischemia/reperfusion; L, infarct lesion; and rep, reperfusion.\\nWe conclude that malonate is indeed taken up by the heart, but this is greatly enhanced in ischemic region upon reperfusion. Therefore, malonate provides protection against cardiac IR injury in vivo over a range of concentrations following its selective entry into the infarct region, where it slows the oxidation of succinate during reperfusion, preventing the production of RET-derived ROS.\",\n \"To explore the mechanism of malonate uptake in vitro, we incubated C2C12 and H9c2 myoblasts with DSM and measured intracellular malonate levels by LC-MS/MS (liquid chromatography-tandem mass spectrometry). Incubation with DSM for 15 minutes at pH 7.4, led to a dose-dependent increase in intracellular malonate (Figure S1A and S1D). Intracellular succinate also accumulated in a malonate-dependent manner (Figure S1B and S1E), confirming that once malonate enters the cell it is rapidly transported into mitochondria and inhibits SDH. However, incubating cells with 1 mmol/L DSM led to variable malonate uptake and little SDH inhibition, indicating that malonate uptake across biological membranes is inefficient. Moreover, the intracellular levels of malonate achieved by 250 µmol/L of the malonate ester prodrug diacetoxymethyl malonate, are 80-fold more than that generated by incubation with 5 mmol/L DSM.25 As DSM was protective in the LAD model when infused at 16 mg/kg, corresponding to a maximum possible blood malonate concentration of ≈1.8 mmol/L (assuming a blood volume of 1.5 mL in a 25 g mouse),29 this compares to the dose range tested in cells (furthermore, far more malonate is available for uptake in vitro than in vivo due to the large reservoir in the incubation medium). Even so, the intracellular malonate levels were far lower with DSM compared to diacetoxymethyl malonate.25\\nThe kinetics of cell uptake upon incubation with 5 mmol/L DSM showed time-dependent uptake, although extended incubation times were required for succinate elevation (Figure S1C and S1F). This is likely due to the initial concentrations of malonate entering the cell being insufficient to inhibit SDH. These data are consistent with the lack of protection by DSM delivered before ischemia in vivo, as well as its limited uptake into normoxic tissues. Together these suggest that exposure to ischemia may facilitate malonate entry into the heart.\",\n \"Malonate is a dicarboxylate at physiological pH (Figure 1A, pKa=2.83 and 5.69), suggesting that the pH decrease in ischemic tissue9,11,30 may enhance malonate uptake into the heart during early reperfusion, by increasing the concentration of its monocarboxylic form. Incubating cells with DSM at either pH 6, 7.4, or 8 for 15 minutes, led to large differences in malonate uptake (Figure 3A and Figure S2A). At pH 6, the levels of malonate in the cell were significantly higher than at pH 7.4 or 8, thus malonate uptake is favored by acidic pH. Succinate levels mirrored those of malonate, with greater succinate accumulation as a result of increased malonate-dependent SDH inhibition at low pH (Figure 3B and Figure S2B). Low pH alone had no effect on succinate levels (Figure 3B and Figure S2B), suggesting that it was due to malonate entry into the cells followed by SDH inhibition.\\nMalonate uptake is enhanced at low pH. A and B, C2C12 cells were incubated with disodium malonate (DSM; 0, 1, or 5 mmol/L) for 15 min at either pH 6, 7.4, or 8 before measuring intracellular malonate (A) and succinate (B) by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). C, Malonate levels in C2C12 cells after incubation with DSM (5 mmol/L) for 15 min at various (patho)physiological pH (mean±SEM, n=3 biological replicates). D, Malonate levels in murine isolated Langendorff-perfused hearts treated with 5 mmol/L DSM infused at either pH 7.4 or 6 for 5 min (mean±SEM, n=4 biological replicates, statistics: unpaired, 2-tailed Mann-Whitney U test). E to H, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at either pH 6 or 7.4 in the presence of FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) (E), gramicidin (F), nigericin (G), or BAM15 (N5,N6-bis(2-Fluorophenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine) (H) before measuring intracellular malonate by LC-MS/MS (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). I, Structures of malonic acid and malonamic acid. J, Quantification of levels of malonate and malonamate in C2C12 cells after incubation (5 mmol/L DSM or disodium malonamate; 15 min) at pH 7.4 (n=6 biological replicates). K, C2C12 cells were incubated with either DSM or disodium malonamate (both 5 mmol/L) at pH 6 or 7.4 for 15 min and the intracellular levels measured by LC-MS/MS. (mean±SEM of the fold-enhancement of uptake at pH 6 vs pH 7.4, n=6 biological replicates). Statistics for J and K: unpaired, 2-tailed Student t test).\\nIncubating cells with malonate over a range of pH values between pH 7.4 and 6 clearly showed the pH-dependent uptake of malonate, in line with the increase in monocarboxylate malonate proportion (Figure 3C). This was mirrored by succinate levels (Figure S2C). Therefore, even a small drop in pH can lead to a substantial increase in malonate uptake, and thus is likely to be relevant for its entry into ischemic tissue upon reperfusion.\\nAs the cultured cells used are noncontractile and substantially differ in their properties from in situ contracting cardiomyocytes,15 importantly, we next assessed whether decreasing the pH also enhanced malonate uptake into the ex vivo perfused Langendorff heart. Malonate was infused into the heart at either pH 7.4 or 6 for 5 minutes, before briefly flushing at pH 7.4 to remove nonmyocardial malonate. Remarkably, the levels of malonate were significantly elevated when infused at pH 6 compared to 7.4, confirming that the in vitro results translate to the heart (Figure 3D). We conclude that low pH conditions facilitate the entry of malonate into cardiomyocytes.\",\n \"As pH modulated malonate entry into cells in vitro and in the heart, we next probed the factors affecting malonate uptake into cells. Malonate entry into cells at either pH 7.4 or 6 was blocked at 4 °C and was associated with negligible succinate levels (Figure S2D and S2E). That a decrease in extracellular pH increased malonate entry into cells, suggested uptake driven by the proton gradient. Therefore, we next abolished the plasma membrane proton gradient using ionophores21 which prevented the cellular uptake of malonate (Figure 3E–3G and Figure S3A) and subsequent increase in succinate levels (Figure S3B through S3E). However, the uncoupler BAM15, which is selective for the mitochondrial inner membrane over the plasma membrane,31 had little effect, consistent with the plasma membrane proton gradient driving malonate uptake (Figure 3H and Figure S3F).\\nWe next used nonspecific transport inhibitors to assess whether we could block malonate uptake at low pH. DIDS (4,4’-Diisothiocyano-2,2’-stilbenedisulfonic acid), an irreversible inhibitor of chloride/bicarbonate exchange that inhibits malonate uptake into erythrocytes,32,33 led to a dose-dependent inhibition of malonate uptake, although succinate remained high (Figure S3G and S3H). Thus, malonate uptake into cardiomyocytes at low pH is driven by a proton gradient through a transporter-dependent process.\\nMalonate’s pKa’s are 2.83 and 5.69, so at pH 6.4, ≈16% of the malonate would be in its monocarboxylate form. To determine whether the uptake of malonate was dependent on its protonation to a monocarboxylate form, we assessed the uptake of a compound that mimics the monocarboxylate malonate. In 3-amino-3-oxopropionate (malonamate; Figure 3I), one carboxylic acid has been replaced with a neutral amido group leaving a single carboxylic acid of pKa ≈4.75. Thus, at pH 7.4 malonamate resembles the monocarboxylate form of malonate. This led to more of malonamate being taken up into cells at pH 7.4 than malonate (Figure 3J). Lowering the pH to 6 led to ≈15-fold increase in malonate uptake while malonamate uptake changed negligibly (Figure 3K) because it will remain as a monocarboxylate across this pH range.\\nTogether, these data support transport of the monoanionic form of malonate, under the conditions that occur during early reperfusion. Therefore, a pH gradient can drive the uptake of malonate via protonation and transport in its monocarboxylate form.\",\n \"The inhibitory effect of DIDS, which also inhibits MCT1,34 raised the possibility of the uptake of the protonated form of malonate being catalyzed by a monocarboxylate transporter (MCT; SLC16 (solute carrier family member 16)). MCT1 (SLC16A1) is the principal transporter of lactate, which is carried in symport with a proton, both of which are elevated during ischemia, making MCT1 an attractive candidate transporter for malonate uptake upon reperfusion. Additionally, it was recently shown that MCT1 mediates the efflux of succinate from the ischemic heart during reperfusion and from exercising muscle.21,35 As MCT1 is a lactate transporter, we first assessed whether high concentrations of lactate in the incubation medium compete with malonate. Excess lactate led to a concentration-dependent decrease in malonate uptake into the cell and a corresponding decrease in succinate accumulation (Figure 4A and Figure S4A). This suggested that at a low pH, malonate uptake into the cells is via MCT1, which is highly expressed in cardiomyocytes.36\\nInhibition of MCT1 (monocarboxylate transporter 1) prevents the enhanced uptake of malonate at lowered pH. A, Malonate uptake (5 mmol/L disodium malonate [DSM], 15 min) in C2C12 cells in the presence of lactate (0, 10 or 50 mmol/L). B, Lactate levels in C2C12 cells after 15 min treatment with varying concentrations of MCT1 inhibitors. C and D, Effect of MCT1 inhibition by AR-C141990 or AZD3965 on malonate (5 mmol/L DSM, 15 min) uptake (C) at pH 6 and subsequent succinate levels (D). E, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at various pH±10 µmol/L AR-C141990. F, MCT1 inhibition by AR-C141990 on malonate (5 mmol/L DSM, 15 min) uptake at pH 7.4 (A to F, mean±SEM, n=3 biological replicates, statistics: (A) Kruskal-Wallis with Dunn post hoc test). G and H, Effect of malonate (5 mmol/L DSM) on cellular oxygen consumption at pH 7.4 (G) or 6 (H) ±MCT1 inhibitor (10 µmol/L AR-C141990; data presented as nonmitochondrial respiration normalized mean oxygen consumption rate (OCR)±SEM of 3 biological replicates (n=12–16 technical replicates per biological replicate), statistics: Kruskal-Wallis with Dunn post hoc test). ATP indicates OCR in the presence of 1.5 µmol/L oligomycin; BL, baseline OCR; MAX, OCR in the presence of 1 µmol/L FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone); and NM, OCR in the presence of 4 µg/mL rotenone and 10 µmol/L antimycin A.\\nTo confirm that MCT1 was the malonate transporter, we assessed the potent and selective MCT1 inhibitors, AR-C141990 and AZD3965.21,35,37,38 When cells were incubated with these MCT1 inhibitors, they led to a dramatic dose-dependent increase in lactate levels within the cell, consistent with preventing lactate efflux via MCT1 (Figure 4B). MCT1 inhibition by either AR-C141990 or AZD3965 led to a profound dose-dependent decrease in malonate uptake (Figure 4C). Inhibition of malonate uptake by MCT1 inhibition led to a corresponding decrease in succinate accumulation within cells (Figure 4D). Additionally, a time course of malonate uptake at low pH, showed that MCT1 inhibition largely abolished the increase in malonate over time, along with the corresponding increase in succinate (Figure S4B and S4C).\\nMCT1 inhibition blocked the pH-dependent increase in malonate uptake (Figure 4E) and in parallel prevented succinate accumulation (Figure 4D). The MCT1 inhibitor led to a dose-dependent inhibition of malonate uptake even at pH 7.4 (Figure 4F). Therefore, malonate can be transported by MCT1 at pH 7.4, but this is greatly enhanced at lower pH due to the increased proportion of malonate in its monocarboxylate form.\\nWe next assessed the impact of the malonate taken up into cells on mitochondrial function by measuring respiration. Lowering pH itself had little effect; however, addition of malonate severely reduced cellular respiration (Figure 4G and H and Figure S4E and S4F) and this effect was rescued by MCT1 inhibition (Figure 4G and H and Figure S4E and S4F). MCT1 inhibition led to a small increase in oxygen consumption at baseline, which may be due to lactate accumulation increasing the NADH/NAD+ ratio driving mitochondrial respiration.39\\nTo confirm the pharmacological effects of MCT1 inhibition on malonate uptake genetically, we knocked down (KD) MCT1 using siRNA in both HeLa and C2C12 cells (Figure S5A through S5D). Consequently, lactate levels in MCT1 KD cells were significantly elevated compared to the control siRNA (Figure 5A). When MCT1 KD cells were incubated with malonate at low pH, malonate uptake was dramatically blocked (Figure 5B through 5E and Figure S5C), confirming MCT1 is directly responsible for malonate transport at low pH. Residual malonate uptake in the MCT1 KD cells was inhibited by cotreatment with an MCT1 inhibitor, further confirming its MCT1 dependence (Figure 5F and Figure S5F). Additionally, succinate was elevated in the MCT1 KD cells compared with control siRNA cells, a result not seen with acute pharmacological MCT1 inhibition (Figure 5D). This suggests that MCT1 may also play an important role in normoxic succinate metabolism and homeostasis and not just during myocardial reperfusion and intense exercise.21,35 Intriguingly, the succinate levels after malonate treatment over time differed between the 2 cell types, with succinate levels remaining low in C2C12 KD but elevated in HeLa MCT1 KDs compared to controls, suggesting a reliance on MCT1 for succinate efflux (Figure 5G and Figure S5G).\\nGenetic knockdown of MCT1 (monocarboxylate transporter 1) prevents the uptake of malonate. A, Relative lactate levels in C2C12 cells treated with control or MCT1 siRNA (mean±SEM of lactate levels relative to control, n=6 biological replicates, statistics: 1-way ANOVA with Bonferroni post hoc test). B to E, Incubation of malonate (5 mmol/L disodium malonate [DSM]) in MCT1 KD cells at pH 7.4 (B and D) or 6 (C and E) for 15 min±MCT1i (10 µmol/L, MCT1 inhibitor AR-C141990). Levels of malonate (B and C) and succinate (D and E) quantified by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). F and G, Time course of malonate uptake (5 mmol/L DSM) at pH 6 in MCT1 KD cells (F) and corresponding succinate levels (G; mean±SEM, n=3 biological replicates). H, Malonate levels in murine Langendorff hearts perfused at pH 6 for 5 min with 5 mmol/L DSM±lactate (50 mmol/L) or MCT1 inhibitor (10 µmol/L AR-C141990; MCT1i; mean±SEM, n=4, statistics: unpaired, 2-tailed Mann-Whitney U test vs control).\\nAs HeLa cells constitutively express MCT4, which is under the control of HIF (hypoxia-inducible factor)-1α and may be implicated in the transport of metabolites in IR, we KD MCT4 independently of MCT1 (Figure S5D and S5E). MCT4 KD had little effect on malonate uptake, with malonate and succinate levels mirroring those of control siRNA-treated cells (Figure S5F and S5G). Thus, MCT1 is the main driver of malonate uptake at lowered pH.\\nOverall, diminishing MCT1 activity, either by pharmacological inhibition or genetic knockdown, impairs malonate uptake at both acidic and normal physiological pH. In addition, this is the first evidence that MCT1 can transport a 3-carbon chain length dicarboxylate, which may also have implications in normal physiology and other pathologies.\\nFinally, we assessed if MCT1 was responsible for malonate uptake at lowered pH in the heart. Malonate uptake into the Langendorff-perfused heart was blocked by either excess lactate, or the MCT1 inhibitor AR-C141990, in the perfusion medium (Figure 5H). Together these findings confirm that MCT1 is responsible for the low pH uptake of malonate, both in cells and in the intact heart.\",\n \"That low pH enhanced malonate entry into heart cells was consistent with the protection afforded by DSM in IR injury being due to the local decrease in pH during ischemia and initial reperfusion. To test this hypothesis, Langendorff-perfused hearts were held ischemic for various times before DSM was infused for 5 minutes, followed by flushing, and measurement of malonate. The malonate levels in heart tissue were dependent on ischemia-time, with the highest occurring after 20 minutes ischemia and being ≈10-fold greater than in the normoxic heart (Figure 6A). This malonate uptake into the ischemic Langendorff-perfused heart was dramatically reduced by the MCT1 inhibitor AR-C141990 (Figure 6B). This confirmed that malonate uptake into the ischemic heart upon reperfusion is a selective process, driven by the low pH and facilitated by the MCT1.\\nIschemia drives malonate protonation, uptake, and cardioprotection. A, Langendorff-perfused murine hearts were held ischemic for either 0, 5, 10, or 20 min and reperfused with 5 mmol/L disodium malonate (DSM; pH 7.4) before malonate levels measured in the heart by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 (control, 5 min) or 6 (10 and 20 min) biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). B, Malonate levels in murine Langendorff hearts exposed to 20 min ischemia and reperfused with 5 mmol/L malonate (pH 7.4)±MCT1 (monocarboxylate transporter 1) inhibitor (10 or 50 µmol/L AR-C141990; mean±SEM, n=4–5 biological replicates, statistical significance assessed by unpaired, 2-tailed Mann-Whitney U test vs control). C, Model of potential lactate and malonate exchange during reperfusion. D, Lactate levels in the Langendorff heart after 20 min ischemia and 1 min reperfusion±5 mmol/L DSM (mean, n=4 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test). E, Infarct size in murine LAD (left anterior descending coronary artery) ligation MI model with 100 µl bolus of 8 mg/kg DSM, pH 4 acid control or 8 mg/kg pH 4 formulated malonate at reperfusion after 30 min ischemia (mean, n=5 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test vs acid malonate).\\nDuring ischemia, the pH of ischemic tissue lowers but in addition, lactate accumulates and upon reperfusion can be transported out of cardiomyocytes by MCT1. This could enhance malonate uptake, as the MCT1 may then act as a lactate-H+/monocarboxylate malonate-H+ exchanger (Figure 6C). To assess this, we measured lactate levels in malonate-perfused IR Langendorff hearts. We found that compared to reperfusion alone, lactate levels decreased in the malonate-treated hearts (Figure 6D). Furthermore, malonate treatment decreased lactate levels in cells that had been treated with the mitochondrial inhibitor antimycin A to enhance lactate production (Figure S6A). This suggests that extracellular malonate and a proton exchange for intracellular lactate, help drive malonate entry into cardiomyocytes upon reperfusion and contribute to its at-risk tissue-selective effect.\\nTherefore, malonate uptake into the heart upon reperfusion is dependent on the tissue having first undergone a period of ischemia, leading to a drop in pH and an accumulation of lactate.\",\n \"The mechanism of malonate uptake into cardiac tissue suggested that lowering the pH of the malonate infusion would increase the proportion in the monocarboxylate form and thereby increase its potency as a cardioprotective agent. To assess this, we used a malonate dose that was not protective (8 mg/kg) when administered at a neutral pH in the in vivo LAD MI model. When this dose of malonate was reformulated at pH 4 (a pH currently used in Food and Drug Administration–approved parenteral formulations)40 and administered as a bolus, this conferred significant protection that was not due to the low pH alone (Figure 6E and Figure S6B). Furthermore, although neutral malonate administered before ischemia was not protective, acidified malonate drove its uptake into cardiomyocytes and significantly reduced infarct size when infused before ischemia (Figure S6C).\",\n \"No medicine is currently available that can be given at reperfusion to prevent cardiac IR injury.1,6 Drugs that prevent cardiac IR injury should both reduce MI damage and the subsequent development of heart failure.1,12 Targeting succinate metabolism has been shown to be a promising therapeutic approach. Inhibiting SDH using the reversible, competitive inhibitor malonate reduces infarct size in small and large animal models of cardiac IR injury, despite its mechanism of entry into heart tissue being unknown.26,27\\nHere, we found that malonate uptake into cells and the heart at pH 7.4 was inefficient. However, the uptake of malonate into cells was dramatically enhanced at the lower levels of pH that occur during ischemia and by lactate accumulation within cells. Thus, ischemia provides an environment that will protonate malonate and thereby enable its uptake by the MCT1 transporter. Interestingly, this transporter undergoes a trans-acceleration phenomenon, whereby its transition from the outward-facing to the inward-facing conformation occurs more rapidly in the presence of a trans-substrate.41,42 In this case, the uptake of malonate on the extracellular side of the plasma membrane may be accelerated by lactate efflux. In the LAD MI model of IR injury, malonate is readily available at the point of reperfusion, thus a proportion of the malonate would be protonated and accessible for transport by MCT1. This enables malonate entry into the heart in an at-risk tissue-selective manner.\\nAdditionally, as well as the inhibition of SDH, accelerating lactate efflux from the heart may also play a role in the reduction of IR injury. By shifting the equilibrium to facilitate anaerobic ATP production, this may promote the early restoration of ionic gradients through ATP-dependent transporters.43,44 Furthermore, facilitating lactate efflux may promote the extrusion of protons from the intracellular environment, thus reducing the activity of H+ transporters such as the Na+-H+ exchanger, though further investigation into these mechanisms is warranted.43,45\\nThis is the first account of an ischemia-selective cardioprotective agent, utilizing the pathological differences between risk and nonrisk tissue to drive uptake. As MCT1 is highly expressed in the hearts of mice, rats, pigs, and humans (Figure S6D) and the characteristics of ischemia are conserved between these species,19,24 these drivers for malonate uptake and the plasma membrane transporter may facilitate malonate cardioprotection in humans. In addition to its place in the treatment of IR injury in MI, the mechanism of ischemia-enhanced delivery of malonate may provide a novel treatment option for IR injury under many other circumstances.\\nThere is much interest in targeted drug delivery and the ability to engage the intended site while reducing off-target effects. Here, we have shown that malonate is not only a cardioprotective agent, but it does this while its entry into the rest of the heart is limited. Thus, malonate is a potent and ischemic tissue-targeted drug, explaining how large doses can be delivered acutely with minimal toxic effects, which is likely to be important bearing in mind the many comorbidities associated with MI.46 Furthermore, as malonate can be efficiently metabolised28,47 and has the potential to promote cardiomyocyte regeneration.48\\nMalonate is robustly protective in acute IR injury, though further work is now required to understand the tractability of malonate treatment in chronic IR injury models; in particular, conducting a double-blind chronic large animal IR injury study.49 This would provide the greatest insight into the cardioprotection capabilities of malonate post-MI and its effect on the development of heart failure and define its potential for translation to the clinic.\\nSchematic of ischemia-dependent malonate uptake via MCT1 (monocarboxylate transporter 1). The accumulation of lactate and protons in ischemic tissue and equilibration with the extracellular space facilitates protonation of malonate to its monocarboxylate form. This enables it to be an MCT1 substrate and enter cardiomyocytes upon reperfusion. Here, the malonate is transported into mitochondria by the mitochondrial dicarboxylate carrier where it can subsequently go on to inhibit SDH (succinate dehydrogenase). SDH inhibition reduces succinate oxidation, reactive oxygen species (ROS) production by reverse electron transport (RET) through complex I and opening of the mitochondrial permeability transition pore, thereby reducing cell death from ischemia/reperfusion (IR) injury. CxI indicates complex I; DIC, mitochondrial dicarboxylate carrier (SLC25A10); mPTP, mitochondrial permeability transition pore; and TCA, tricarboxylic acid.\",\n \"We have shown that malonate is an ischemia-selective drug, due to the lowered pH and lactate accumulation of ischemic tissue driving its uptake via the MCT1 (Figure 7). Furthermore, we show that a low pH formulation of malonate enhances its therapeutic potency. Malonate is the first at-risk tissue-selective cardioprotective drug and represents a significant step toward the treatment of IR injury.\",\n \" Acknowledgments The authors thank Stephen Large, Fouad Taghavi, and Margaret M. Huang (Department of Surgery, University of Cambridge) for obtaining human heart tissue and Benjamin Thackray (Department of Medicine, University of Cambridge) for assistance with initial experiments.\\nThe authors thank Stephen Large, Fouad Taghavi, and Margaret M. Huang (Department of Surgery, University of Cambridge) for obtaining human heart tissue and Benjamin Thackray (Department of Medicine, University of Cambridge) for assistance with initial experiments.\\n Sources of Funding This work was supported by the British Heart Foundation (PG/20/10025 to T. Krieg); the Medical Research Council (MC_UU_00015/3 to M.P. Murphy and MR/P000320/1 to T. Krieg), the Wellcome Trust (220257/Z/20/Z to M.P. Murphy, 221604/Z/20/Z to D. Aksentijevic), Barts Charity (MRC0215 to D. Aksentijevic).\\nThis work was supported by the British Heart Foundation (PG/20/10025 to T. Krieg); the Medical Research Council (MC_UU_00015/3 to M.P. Murphy and MR/P000320/1 to T. Krieg), the Wellcome Trust (220257/Z/20/Z to M.P. Murphy, 221604/Z/20/Z to D. Aksentijevic), Barts Charity (MRC0215 to D. Aksentijevic).\\n Disclosures Some authors currently hold a patent on the use of malonate esters in cardiac ischemia/reperfusion (IR) injury (M.P. Murphy and T. Krieg) and have submitted patent applications on the use of malonate in IR injury associated with ischemic stroke (M.P. Murphy and T. Krieg) and the pH-enhancement of malonate described in this article (H.A. Prag, M.P. Murphy, and T. Krieg). The other authors report no conflicts.\\nSome authors currently hold a patent on the use of malonate esters in cardiac ischemia/reperfusion (IR) injury (M.P. Murphy and T. Krieg) and have submitted patent applications on the use of malonate in IR injury associated with ischemic stroke (M.P. Murphy and T. Krieg) and the pH-enhancement of malonate described in this article (H.A. Prag, M.P. Murphy, and T. Krieg). The other authors report no conflicts.\\n Supplemental Materials Supplemental Methods\\nMajor Resources Table\\nFigures S1–S6\\nReferences 49–56\\nSupplemental Methods\\nMajor Resources Table\\nFigures S1–S6\\nReferences 49–56\",\n \"The authors thank Stephen Large, Fouad Taghavi, and Margaret M. Huang (Department of Surgery, University of Cambridge) for obtaining human heart tissue and Benjamin Thackray (Department of Medicine, University of Cambridge) for assistance with initial experiments.\",\n \"This work was supported by the British Heart Foundation (PG/20/10025 to T. Krieg); the Medical Research Council (MC_UU_00015/3 to M.P. Murphy and MR/P000320/1 to T. Krieg), the Wellcome Trust (220257/Z/20/Z to M.P. Murphy, 221604/Z/20/Z to D. Aksentijevic), Barts Charity (MRC0215 to D. Aksentijevic).\",\n \"Some authors currently hold a patent on the use of malonate esters in cardiac ischemia/reperfusion (IR) injury (M.P. Murphy and T. Krieg) and have submitted patent applications on the use of malonate in IR injury associated with ischemic stroke (M.P. Murphy and T. Krieg) and the pH-enhancement of malonate described in this article (H.A. Prag, M.P. Murphy, and T. Krieg). The other authors report no conflicts.\",\n \"Supplemental Methods\\nMajor Resources Table\\nFigures S1–S6\\nReferences 49–56\",\n \"\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":[null,null,"methods","data-availability","results",null,null,null,null,null,null,null,"discussion","conclusions",null,null,null,"COI-Statement",null,"supplementary-material"],"string":"[\n null,\n null,\n \"methods\",\n \"data-availability\",\n \"results\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null,\n \"COI-Statement\",\n null,\n \"supplementary-material\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["ischemia","mitochondria","myocardial infarction","reactive oxygen species","reperfusion"],"string":"[\n \"ischemia\",\n \"mitochondria\",\n \"myocardial infarction\",\n \"reactive oxygen species\",\n \"reperfusion\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"What Is Known?:\nExtensive succinate accumulation during ischemia and its subsequent rapid oxidation on reperfusion drives ischemia/reperfusion injury.\nPreventing succinate accumulation during ischemia reduces damage on reperfusion.\nInhibiting succinate dehydrogenase with malonate is protective against ischemia/reperfusion injury, although its mechanism of entry into cardiomyocytes is undefined.\n\nWhat New Information Does This Article Contribute?:\nThe cardioprotective effect of malonate is dependent on its selective uptake into cardiomyocytes on reperfusion after an ischemic period.\nMalonate entry into cardiomyocytes upon reperfusion is facilitated by the lowered pH and lactate exchange, which selectively drives malonate into cardiomyocytes as a monoanion via the monocarboxylate transporter MCT1 (monocarboxylate transporter 1). This is the first time malonate has been shown to be a substrate for MCT1.\nMalonate selectively enters the at-risk tissue, sparing the nonischemic area on reperfusion. Thus, malonate is the first example of an at-risk tissue-selective, cardioprotective drug.\nDetermining the molecular basis for selective malonate entry via MCT1 into the ischemic heart upon reperfusion is a significant step toward treating cardiac ischemia/reperfusion injury. Malonate is cardioprotective in small and large animal models and MCT1 is highly expressed in the human heart. Furthermore, this mechanism can be exploited to increase malonate potency using an acidic formulation. The next step is to assess malonate as a treatment for cardiac ischemia/reperfusion injury in patients.\n\nMethods:\n Data Availability Detailed methods and Major Resources Table can be found in the Supplemental Material. Data will be made available upon reasonable request, by contacting a corresponding author.\nDetailed methods and Major Resources Table can be found in the Supplemental Material. Data will be made available upon reasonable request, by contacting a corresponding author.\n\nData Availability:\nDetailed methods and Major Resources Table can be found in the Supplemental Material. Data will be made available upon reasonable request, by contacting a corresponding author.\n\nResults:\n Malonate Is Protective in an MI Model and Is Taken Up Selectively into the Ischemic Region of the Heart DSM is protective against cardiac IR injury, but its mechanism of protection remains uncertain. To address this gap in our knowledge we first tested the therapeutic range of DSM in IR injury. We infused DSM at a range of concentrations (1.6–160 mg/kg equivalent to ≈11-1100 µmol/kg) at reperfusion in an in vivo murine LAD (left anterior descending coronary artery) ligation model of MI. DSM infusion during this clinically relevant reperfusion period led to a dose-dependent decrease in infarct size (Figure 1C), with 160 and 16 mg/kg showing robust cardioprotection. However, when DSM was infused before the onset of ischemia it was not protective (Figure 1D), even at 160 mg/kg which gave the smallest infarct when administered at reperfusion. This contrasted with the cell-permeable dimethyl malonate, which can diffuse into the tissue and generate malonate, preventing succinate accumulation and thereby reducing IR injury.18,25 This suggests that either DSM is minimally taken up by tissues during normoxia and is only taken up by tissues after ischemia or that DSM protects against acute IR injury by an extracellular mechanism.\nWe next measured malonate uptake into the ischemic and healthy heart tissue upon reperfusion in the LAD MI model. This showed that malonate was indeed taken up into cells within the infarct region to a level of ≈400 pmol/mg tissue and to a far greater extent than into cells in the healthy tissue (Figure 1E). Furthermore, reperfusing with malonate slowed succinate oxidation within the infarct region with succinate remaining significantly elevated in the infarct region following 1-minute reperfusion, compared with control reperfusion with saline (Figure 1F). Limiting succinate oxidation at reperfusion with malonate also blunted ROS production in the at-risk tissue (Figure 1G). We next assessed how SDH inhibition compared to direct inhibition of the mitochondrial permeability transition pore with CsA (cyclosporin A). Here we found that the cardioprotection from malonate was additive to cyclosporin A alone (Figure 1H), hence targeting the upstream mechanism of permeability transition pore opening is an increasingly attractive option for IR injury treatment.\nTo better understand the metabolic differences between the healthy and infarcted tissue upon malonate treatment, we investigated the in vivo MI model using mass spectrometry imaging. To do this, we subjected hearts to either 30-minute ischemia, 30-minute ischemia before reperfusing the heart for 15-minute, or 30-minute ischemia before reperfusing with DSM for 15 minutes (to mimic the cardioprotective malonate infusion) before snap freezing and processing for mass spectrometry imaging (Figure 2A). Infarct lesions were demarcated using hematoxylin and eosin (H&E), silver infarct staining and metabolite principal component analysis to differentiate the risk and nonrisk regions. mass spectrometry imaging coupled with the demarcated risk areas showed striking changes in the levels of succinate in the infarct region. During ischemia, succinate was significantly elevated in the at-risk tissue, with considerable succinate accumulation in the core of the infarct (Figure 2B and 2C). After 15-minute reperfusion, the succinate levels in the infarct lesion returned to healthy tissue levels, due to succinate oxidation and efflux.21 When malonate was infused at the time of reperfusion, succinate levels remained higher in all regions of the heart than in the nonmalonate treated heart, consistent with the prevention of its oxidation by SDH inhibition by malonate.\nLocalization of succinate accumulation in heart tissue identified by mass spectrometry imaging. A, Outline of experimental groups for mass spectrometry imaging. B, Representative images of succinate abundance detected by mass spectrometry imaging in myocardial sections. Black outer line indicates the edge of the tissue slice, white inner line indicates infarct region. C, Quantification of succinate (C) identified by MSI (mean±SEM, n=3 biological replicates, statistics: Friedman paired test for healthy vs lesion and Kruskal-Wallis with Dunn post hoc test between conditions). au indicates arbitrary units; DSM, disodium malonate; H, healthy tissue; IR, ischemia/reperfusion; L, infarct lesion; and rep, reperfusion.\nWe conclude that malonate is indeed taken up by the heart, but this is greatly enhanced in ischemic region upon reperfusion. Therefore, malonate provides protection against cardiac IR injury in vivo over a range of concentrations following its selective entry into the infarct region, where it slows the oxidation of succinate during reperfusion, preventing the production of RET-derived ROS.\nDSM is protective against cardiac IR injury, but its mechanism of protection remains uncertain. To address this gap in our knowledge we first tested the therapeutic range of DSM in IR injury. We infused DSM at a range of concentrations (1.6–160 mg/kg equivalent to ≈11-1100 µmol/kg) at reperfusion in an in vivo murine LAD (left anterior descending coronary artery) ligation model of MI. DSM infusion during this clinically relevant reperfusion period led to a dose-dependent decrease in infarct size (Figure 1C), with 160 and 16 mg/kg showing robust cardioprotection. However, when DSM was infused before the onset of ischemia it was not protective (Figure 1D), even at 160 mg/kg which gave the smallest infarct when administered at reperfusion. This contrasted with the cell-permeable dimethyl malonate, which can diffuse into the tissue and generate malonate, preventing succinate accumulation and thereby reducing IR injury.18,25 This suggests that either DSM is minimally taken up by tissues during normoxia and is only taken up by tissues after ischemia or that DSM protects against acute IR injury by an extracellular mechanism.\nWe next measured malonate uptake into the ischemic and healthy heart tissue upon reperfusion in the LAD MI model. This showed that malonate was indeed taken up into cells within the infarct region to a level of ≈400 pmol/mg tissue and to a far greater extent than into cells in the healthy tissue (Figure 1E). Furthermore, reperfusing with malonate slowed succinate oxidation within the infarct region with succinate remaining significantly elevated in the infarct region following 1-minute reperfusion, compared with control reperfusion with saline (Figure 1F). Limiting succinate oxidation at reperfusion with malonate also blunted ROS production in the at-risk tissue (Figure 1G). We next assessed how SDH inhibition compared to direct inhibition of the mitochondrial permeability transition pore with CsA (cyclosporin A). Here we found that the cardioprotection from malonate was additive to cyclosporin A alone (Figure 1H), hence targeting the upstream mechanism of permeability transition pore opening is an increasingly attractive option for IR injury treatment.\nTo better understand the metabolic differences between the healthy and infarcted tissue upon malonate treatment, we investigated the in vivo MI model using mass spectrometry imaging. To do this, we subjected hearts to either 30-minute ischemia, 30-minute ischemia before reperfusing the heart for 15-minute, or 30-minute ischemia before reperfusing with DSM for 15 minutes (to mimic the cardioprotective malonate infusion) before snap freezing and processing for mass spectrometry imaging (Figure 2A). Infarct lesions were demarcated using hematoxylin and eosin (H&E), silver infarct staining and metabolite principal component analysis to differentiate the risk and nonrisk regions. mass spectrometry imaging coupled with the demarcated risk areas showed striking changes in the levels of succinate in the infarct region. During ischemia, succinate was significantly elevated in the at-risk tissue, with considerable succinate accumulation in the core of the infarct (Figure 2B and 2C). After 15-minute reperfusion, the succinate levels in the infarct lesion returned to healthy tissue levels, due to succinate oxidation and efflux.21 When malonate was infused at the time of reperfusion, succinate levels remained higher in all regions of the heart than in the nonmalonate treated heart, consistent with the prevention of its oxidation by SDH inhibition by malonate.\nLocalization of succinate accumulation in heart tissue identified by mass spectrometry imaging. A, Outline of experimental groups for mass spectrometry imaging. B, Representative images of succinate abundance detected by mass spectrometry imaging in myocardial sections. Black outer line indicates the edge of the tissue slice, white inner line indicates infarct region. C, Quantification of succinate (C) identified by MSI (mean±SEM, n=3 biological replicates, statistics: Friedman paired test for healthy vs lesion and Kruskal-Wallis with Dunn post hoc test between conditions). au indicates arbitrary units; DSM, disodium malonate; H, healthy tissue; IR, ischemia/reperfusion; L, infarct lesion; and rep, reperfusion.\nWe conclude that malonate is indeed taken up by the heart, but this is greatly enhanced in ischemic region upon reperfusion. Therefore, malonate provides protection against cardiac IR injury in vivo over a range of concentrations following its selective entry into the infarct region, where it slows the oxidation of succinate during reperfusion, preventing the production of RET-derived ROS.\n Malonate Uptake into Cells Is Inefficient at pH 7.4 To explore the mechanism of malonate uptake in vitro, we incubated C2C12 and H9c2 myoblasts with DSM and measured intracellular malonate levels by LC-MS/MS (liquid chromatography-tandem mass spectrometry). Incubation with DSM for 15 minutes at pH 7.4, led to a dose-dependent increase in intracellular malonate (Figure S1A and S1D). Intracellular succinate also accumulated in a malonate-dependent manner (Figure S1B and S1E), confirming that once malonate enters the cell it is rapidly transported into mitochondria and inhibits SDH. However, incubating cells with 1 mmol/L DSM led to variable malonate uptake and little SDH inhibition, indicating that malonate uptake across biological membranes is inefficient. Moreover, the intracellular levels of malonate achieved by 250 µmol/L of the malonate ester prodrug diacetoxymethyl malonate, are 80-fold more than that generated by incubation with 5 mmol/L DSM.25 As DSM was protective in the LAD model when infused at 16 mg/kg, corresponding to a maximum possible blood malonate concentration of ≈1.8 mmol/L (assuming a blood volume of 1.5 mL in a 25 g mouse),29 this compares to the dose range tested in cells (furthermore, far more malonate is available for uptake in vitro than in vivo due to the large reservoir in the incubation medium). Even so, the intracellular malonate levels were far lower with DSM compared to diacetoxymethyl malonate.25\nThe kinetics of cell uptake upon incubation with 5 mmol/L DSM showed time-dependent uptake, although extended incubation times were required for succinate elevation (Figure S1C and S1F). This is likely due to the initial concentrations of malonate entering the cell being insufficient to inhibit SDH. These data are consistent with the lack of protection by DSM delivered before ischemia in vivo, as well as its limited uptake into normoxic tissues. Together these suggest that exposure to ischemia may facilitate malonate entry into the heart.\nTo explore the mechanism of malonate uptake in vitro, we incubated C2C12 and H9c2 myoblasts with DSM and measured intracellular malonate levels by LC-MS/MS (liquid chromatography-tandem mass spectrometry). Incubation with DSM for 15 minutes at pH 7.4, led to a dose-dependent increase in intracellular malonate (Figure S1A and S1D). Intracellular succinate also accumulated in a malonate-dependent manner (Figure S1B and S1E), confirming that once malonate enters the cell it is rapidly transported into mitochondria and inhibits SDH. However, incubating cells with 1 mmol/L DSM led to variable malonate uptake and little SDH inhibition, indicating that malonate uptake across biological membranes is inefficient. Moreover, the intracellular levels of malonate achieved by 250 µmol/L of the malonate ester prodrug diacetoxymethyl malonate, are 80-fold more than that generated by incubation with 5 mmol/L DSM.25 As DSM was protective in the LAD model when infused at 16 mg/kg, corresponding to a maximum possible blood malonate concentration of ≈1.8 mmol/L (assuming a blood volume of 1.5 mL in a 25 g mouse),29 this compares to the dose range tested in cells (furthermore, far more malonate is available for uptake in vitro than in vivo due to the large reservoir in the incubation medium). Even so, the intracellular malonate levels were far lower with DSM compared to diacetoxymethyl malonate.25\nThe kinetics of cell uptake upon incubation with 5 mmol/L DSM showed time-dependent uptake, although extended incubation times were required for succinate elevation (Figure S1C and S1F). This is likely due to the initial concentrations of malonate entering the cell being insufficient to inhibit SDH. These data are consistent with the lack of protection by DSM delivered before ischemia in vivo, as well as its limited uptake into normoxic tissues. Together these suggest that exposure to ischemia may facilitate malonate entry into the heart.\n Malonate Uptake into Cells and the Heart Can Be Modulated by pH Malonate is a dicarboxylate at physiological pH (Figure 1A, pKa=2.83 and 5.69), suggesting that the pH decrease in ischemic tissue9,11,30 may enhance malonate uptake into the heart during early reperfusion, by increasing the concentration of its monocarboxylic form. Incubating cells with DSM at either pH 6, 7.4, or 8 for 15 minutes, led to large differences in malonate uptake (Figure 3A and Figure S2A). At pH 6, the levels of malonate in the cell were significantly higher than at pH 7.4 or 8, thus malonate uptake is favored by acidic pH. Succinate levels mirrored those of malonate, with greater succinate accumulation as a result of increased malonate-dependent SDH inhibition at low pH (Figure 3B and Figure S2B). Low pH alone had no effect on succinate levels (Figure 3B and Figure S2B), suggesting that it was due to malonate entry into the cells followed by SDH inhibition.\nMalonate uptake is enhanced at low pH. A and B, C2C12 cells were incubated with disodium malonate (DSM; 0, 1, or 5 mmol/L) for 15 min at either pH 6, 7.4, or 8 before measuring intracellular malonate (A) and succinate (B) by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). C, Malonate levels in C2C12 cells after incubation with DSM (5 mmol/L) for 15 min at various (patho)physiological pH (mean±SEM, n=3 biological replicates). D, Malonate levels in murine isolated Langendorff-perfused hearts treated with 5 mmol/L DSM infused at either pH 7.4 or 6 for 5 min (mean±SEM, n=4 biological replicates, statistics: unpaired, 2-tailed Mann-Whitney U test). E to H, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at either pH 6 or 7.4 in the presence of FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) (E), gramicidin (F), nigericin (G), or BAM15 (N5,N6-bis(2-Fluorophenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine) (H) before measuring intracellular malonate by LC-MS/MS (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). I, Structures of malonic acid and malonamic acid. J, Quantification of levels of malonate and malonamate in C2C12 cells after incubation (5 mmol/L DSM or disodium malonamate; 15 min) at pH 7.4 (n=6 biological replicates). K, C2C12 cells were incubated with either DSM or disodium malonamate (both 5 mmol/L) at pH 6 or 7.4 for 15 min and the intracellular levels measured by LC-MS/MS. (mean±SEM of the fold-enhancement of uptake at pH 6 vs pH 7.4, n=6 biological replicates). Statistics for J and K: unpaired, 2-tailed Student t test).\nIncubating cells with malonate over a range of pH values between pH 7.4 and 6 clearly showed the pH-dependent uptake of malonate, in line with the increase in monocarboxylate malonate proportion (Figure 3C). This was mirrored by succinate levels (Figure S2C). Therefore, even a small drop in pH can lead to a substantial increase in malonate uptake, and thus is likely to be relevant for its entry into ischemic tissue upon reperfusion.\nAs the cultured cells used are noncontractile and substantially differ in their properties from in situ contracting cardiomyocytes,15 importantly, we next assessed whether decreasing the pH also enhanced malonate uptake into the ex vivo perfused Langendorff heart. Malonate was infused into the heart at either pH 7.4 or 6 for 5 minutes, before briefly flushing at pH 7.4 to remove nonmyocardial malonate. Remarkably, the levels of malonate were significantly elevated when infused at pH 6 compared to 7.4, confirming that the in vitro results translate to the heart (Figure 3D). We conclude that low pH conditions facilitate the entry of malonate into cardiomyocytes.\nMalonate is a dicarboxylate at physiological pH (Figure 1A, pKa=2.83 and 5.69), suggesting that the pH decrease in ischemic tissue9,11,30 may enhance malonate uptake into the heart during early reperfusion, by increasing the concentration of its monocarboxylic form. Incubating cells with DSM at either pH 6, 7.4, or 8 for 15 minutes, led to large differences in malonate uptake (Figure 3A and Figure S2A). At pH 6, the levels of malonate in the cell were significantly higher than at pH 7.4 or 8, thus malonate uptake is favored by acidic pH. Succinate levels mirrored those of malonate, with greater succinate accumulation as a result of increased malonate-dependent SDH inhibition at low pH (Figure 3B and Figure S2B). Low pH alone had no effect on succinate levels (Figure 3B and Figure S2B), suggesting that it was due to malonate entry into the cells followed by SDH inhibition.\nMalonate uptake is enhanced at low pH. A and B, C2C12 cells were incubated with disodium malonate (DSM; 0, 1, or 5 mmol/L) for 15 min at either pH 6, 7.4, or 8 before measuring intracellular malonate (A) and succinate (B) by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). C, Malonate levels in C2C12 cells after incubation with DSM (5 mmol/L) for 15 min at various (patho)physiological pH (mean±SEM, n=3 biological replicates). D, Malonate levels in murine isolated Langendorff-perfused hearts treated with 5 mmol/L DSM infused at either pH 7.4 or 6 for 5 min (mean±SEM, n=4 biological replicates, statistics: unpaired, 2-tailed Mann-Whitney U test). E to H, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at either pH 6 or 7.4 in the presence of FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) (E), gramicidin (F), nigericin (G), or BAM15 (N5,N6-bis(2-Fluorophenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine) (H) before measuring intracellular malonate by LC-MS/MS (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). I, Structures of malonic acid and malonamic acid. J, Quantification of levels of malonate and malonamate in C2C12 cells after incubation (5 mmol/L DSM or disodium malonamate; 15 min) at pH 7.4 (n=6 biological replicates). K, C2C12 cells were incubated with either DSM or disodium malonamate (both 5 mmol/L) at pH 6 or 7.4 for 15 min and the intracellular levels measured by LC-MS/MS. (mean±SEM of the fold-enhancement of uptake at pH 6 vs pH 7.4, n=6 biological replicates). Statistics for J and K: unpaired, 2-tailed Student t test).\nIncubating cells with malonate over a range of pH values between pH 7.4 and 6 clearly showed the pH-dependent uptake of malonate, in line with the increase in monocarboxylate malonate proportion (Figure 3C). This was mirrored by succinate levels (Figure S2C). Therefore, even a small drop in pH can lead to a substantial increase in malonate uptake, and thus is likely to be relevant for its entry into ischemic tissue upon reperfusion.\nAs the cultured cells used are noncontractile and substantially differ in their properties from in situ contracting cardiomyocytes,15 importantly, we next assessed whether decreasing the pH also enhanced malonate uptake into the ex vivo perfused Langendorff heart. Malonate was infused into the heart at either pH 7.4 or 6 for 5 minutes, before briefly flushing at pH 7.4 to remove nonmyocardial malonate. Remarkably, the levels of malonate were significantly elevated when infused at pH 6 compared to 7.4, confirming that the in vitro results translate to the heart (Figure 3D). We conclude that low pH conditions facilitate the entry of malonate into cardiomyocytes.\n Malonate Uptake Can Be Perturbed by Modulating the Plasma Membrane H+ Gradient As pH modulated malonate entry into cells in vitro and in the heart, we next probed the factors affecting malonate uptake into cells. Malonate entry into cells at either pH 7.4 or 6 was blocked at 4 °C and was associated with negligible succinate levels (Figure S2D and S2E). That a decrease in extracellular pH increased malonate entry into cells, suggested uptake driven by the proton gradient. Therefore, we next abolished the plasma membrane proton gradient using ionophores21 which prevented the cellular uptake of malonate (Figure 3E–3G and Figure S3A) and subsequent increase in succinate levels (Figure S3B through S3E). However, the uncoupler BAM15, which is selective for the mitochondrial inner membrane over the plasma membrane,31 had little effect, consistent with the plasma membrane proton gradient driving malonate uptake (Figure 3H and Figure S3F).\nWe next used nonspecific transport inhibitors to assess whether we could block malonate uptake at low pH. DIDS (4,4’-Diisothiocyano-2,2’-stilbenedisulfonic acid), an irreversible inhibitor of chloride/bicarbonate exchange that inhibits malonate uptake into erythrocytes,32,33 led to a dose-dependent inhibition of malonate uptake, although succinate remained high (Figure S3G and S3H). Thus, malonate uptake into cardiomyocytes at low pH is driven by a proton gradient through a transporter-dependent process.\nMalonate’s pKa’s are 2.83 and 5.69, so at pH 6.4, ≈16% of the malonate would be in its monocarboxylate form. To determine whether the uptake of malonate was dependent on its protonation to a monocarboxylate form, we assessed the uptake of a compound that mimics the monocarboxylate malonate. In 3-amino-3-oxopropionate (malonamate; Figure 3I), one carboxylic acid has been replaced with a neutral amido group leaving a single carboxylic acid of pKa ≈4.75. Thus, at pH 7.4 malonamate resembles the monocarboxylate form of malonate. This led to more of malonamate being taken up into cells at pH 7.4 than malonate (Figure 3J). Lowering the pH to 6 led to ≈15-fold increase in malonate uptake while malonamate uptake changed negligibly (Figure 3K) because it will remain as a monocarboxylate across this pH range.\nTogether, these data support transport of the monoanionic form of malonate, under the conditions that occur during early reperfusion. Therefore, a pH gradient can drive the uptake of malonate via protonation and transport in its monocarboxylate form.\nAs pH modulated malonate entry into cells in vitro and in the heart, we next probed the factors affecting malonate uptake into cells. Malonate entry into cells at either pH 7.4 or 6 was blocked at 4 °C and was associated with negligible succinate levels (Figure S2D and S2E). That a decrease in extracellular pH increased malonate entry into cells, suggested uptake driven by the proton gradient. Therefore, we next abolished the plasma membrane proton gradient using ionophores21 which prevented the cellular uptake of malonate (Figure 3E–3G and Figure S3A) and subsequent increase in succinate levels (Figure S3B through S3E). However, the uncoupler BAM15, which is selective for the mitochondrial inner membrane over the plasma membrane,31 had little effect, consistent with the plasma membrane proton gradient driving malonate uptake (Figure 3H and Figure S3F).\nWe next used nonspecific transport inhibitors to assess whether we could block malonate uptake at low pH. DIDS (4,4’-Diisothiocyano-2,2’-stilbenedisulfonic acid), an irreversible inhibitor of chloride/bicarbonate exchange that inhibits malonate uptake into erythrocytes,32,33 led to a dose-dependent inhibition of malonate uptake, although succinate remained high (Figure S3G and S3H). Thus, malonate uptake into cardiomyocytes at low pH is driven by a proton gradient through a transporter-dependent process.\nMalonate’s pKa’s are 2.83 and 5.69, so at pH 6.4, ≈16% of the malonate would be in its monocarboxylate form. To determine whether the uptake of malonate was dependent on its protonation to a monocarboxylate form, we assessed the uptake of a compound that mimics the monocarboxylate malonate. In 3-amino-3-oxopropionate (malonamate; Figure 3I), one carboxylic acid has been replaced with a neutral amido group leaving a single carboxylic acid of pKa ≈4.75. Thus, at pH 7.4 malonamate resembles the monocarboxylate form of malonate. This led to more of malonamate being taken up into cells at pH 7.4 than malonate (Figure 3J). Lowering the pH to 6 led to ≈15-fold increase in malonate uptake while malonamate uptake changed negligibly (Figure 3K) because it will remain as a monocarboxylate across this pH range.\nTogether, these data support transport of the monoanionic form of malonate, under the conditions that occur during early reperfusion. Therefore, a pH gradient can drive the uptake of malonate via protonation and transport in its monocarboxylate form.\n Malonate Uptake Under Reperfusion Conditions Is Dependent on MCT1 The inhibitory effect of DIDS, which also inhibits MCT1,34 raised the possibility of the uptake of the protonated form of malonate being catalyzed by a monocarboxylate transporter (MCT; SLC16 (solute carrier family member 16)). MCT1 (SLC16A1) is the principal transporter of lactate, which is carried in symport with a proton, both of which are elevated during ischemia, making MCT1 an attractive candidate transporter for malonate uptake upon reperfusion. Additionally, it was recently shown that MCT1 mediates the efflux of succinate from the ischemic heart during reperfusion and from exercising muscle.21,35 As MCT1 is a lactate transporter, we first assessed whether high concentrations of lactate in the incubation medium compete with malonate. Excess lactate led to a concentration-dependent decrease in malonate uptake into the cell and a corresponding decrease in succinate accumulation (Figure 4A and Figure S4A). This suggested that at a low pH, malonate uptake into the cells is via MCT1, which is highly expressed in cardiomyocytes.36\nInhibition of MCT1 (monocarboxylate transporter 1) prevents the enhanced uptake of malonate at lowered pH. A, Malonate uptake (5 mmol/L disodium malonate [DSM], 15 min) in C2C12 cells in the presence of lactate (0, 10 or 50 mmol/L). B, Lactate levels in C2C12 cells after 15 min treatment with varying concentrations of MCT1 inhibitors. C and D, Effect of MCT1 inhibition by AR-C141990 or AZD3965 on malonate (5 mmol/L DSM, 15 min) uptake (C) at pH 6 and subsequent succinate levels (D). E, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at various pH±10 µmol/L AR-C141990. F, MCT1 inhibition by AR-C141990 on malonate (5 mmol/L DSM, 15 min) uptake at pH 7.4 (A to F, mean±SEM, n=3 biological replicates, statistics: (A) Kruskal-Wallis with Dunn post hoc test). G and H, Effect of malonate (5 mmol/L DSM) on cellular oxygen consumption at pH 7.4 (G) or 6 (H) ±MCT1 inhibitor (10 µmol/L AR-C141990; data presented as nonmitochondrial respiration normalized mean oxygen consumption rate (OCR)±SEM of 3 biological replicates (n=12–16 technical replicates per biological replicate), statistics: Kruskal-Wallis with Dunn post hoc test). ATP indicates OCR in the presence of 1.5 µmol/L oligomycin; BL, baseline OCR; MAX, OCR in the presence of 1 µmol/L FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone); and NM, OCR in the presence of 4 µg/mL rotenone and 10 µmol/L antimycin A.\nTo confirm that MCT1 was the malonate transporter, we assessed the potent and selective MCT1 inhibitors, AR-C141990 and AZD3965.21,35,37,38 When cells were incubated with these MCT1 inhibitors, they led to a dramatic dose-dependent increase in lactate levels within the cell, consistent with preventing lactate efflux via MCT1 (Figure 4B). MCT1 inhibition by either AR-C141990 or AZD3965 led to a profound dose-dependent decrease in malonate uptake (Figure 4C). Inhibition of malonate uptake by MCT1 inhibition led to a corresponding decrease in succinate accumulation within cells (Figure 4D). Additionally, a time course of malonate uptake at low pH, showed that MCT1 inhibition largely abolished the increase in malonate over time, along with the corresponding increase in succinate (Figure S4B and S4C).\nMCT1 inhibition blocked the pH-dependent increase in malonate uptake (Figure 4E) and in parallel prevented succinate accumulation (Figure 4D). The MCT1 inhibitor led to a dose-dependent inhibition of malonate uptake even at pH 7.4 (Figure 4F). Therefore, malonate can be transported by MCT1 at pH 7.4, but this is greatly enhanced at lower pH due to the increased proportion of malonate in its monocarboxylate form.\nWe next assessed the impact of the malonate taken up into cells on mitochondrial function by measuring respiration. Lowering pH itself had little effect; however, addition of malonate severely reduced cellular respiration (Figure 4G and H and Figure S4E and S4F) and this effect was rescued by MCT1 inhibition (Figure 4G and H and Figure S4E and S4F). MCT1 inhibition led to a small increase in oxygen consumption at baseline, which may be due to lactate accumulation increasing the NADH/NAD+ ratio driving mitochondrial respiration.39\nTo confirm the pharmacological effects of MCT1 inhibition on malonate uptake genetically, we knocked down (KD) MCT1 using siRNA in both HeLa and C2C12 cells (Figure S5A through S5D). Consequently, lactate levels in MCT1 KD cells were significantly elevated compared to the control siRNA (Figure 5A). When MCT1 KD cells were incubated with malonate at low pH, malonate uptake was dramatically blocked (Figure 5B through 5E and Figure S5C), confirming MCT1 is directly responsible for malonate transport at low pH. Residual malonate uptake in the MCT1 KD cells was inhibited by cotreatment with an MCT1 inhibitor, further confirming its MCT1 dependence (Figure 5F and Figure S5F). Additionally, succinate was elevated in the MCT1 KD cells compared with control siRNA cells, a result not seen with acute pharmacological MCT1 inhibition (Figure 5D). This suggests that MCT1 may also play an important role in normoxic succinate metabolism and homeostasis and not just during myocardial reperfusion and intense exercise.21,35 Intriguingly, the succinate levels after malonate treatment over time differed between the 2 cell types, with succinate levels remaining low in C2C12 KD but elevated in HeLa MCT1 KDs compared to controls, suggesting a reliance on MCT1 for succinate efflux (Figure 5G and Figure S5G).\nGenetic knockdown of MCT1 (monocarboxylate transporter 1) prevents the uptake of malonate. A, Relative lactate levels in C2C12 cells treated with control or MCT1 siRNA (mean±SEM of lactate levels relative to control, n=6 biological replicates, statistics: 1-way ANOVA with Bonferroni post hoc test). B to E, Incubation of malonate (5 mmol/L disodium malonate [DSM]) in MCT1 KD cells at pH 7.4 (B and D) or 6 (C and E) for 15 min±MCT1i (10 µmol/L, MCT1 inhibitor AR-C141990). Levels of malonate (B and C) and succinate (D and E) quantified by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). F and G, Time course of malonate uptake (5 mmol/L DSM) at pH 6 in MCT1 KD cells (F) and corresponding succinate levels (G; mean±SEM, n=3 biological replicates). H, Malonate levels in murine Langendorff hearts perfused at pH 6 for 5 min with 5 mmol/L DSM±lactate (50 mmol/L) or MCT1 inhibitor (10 µmol/L AR-C141990; MCT1i; mean±SEM, n=4, statistics: unpaired, 2-tailed Mann-Whitney U test vs control).\nAs HeLa cells constitutively express MCT4, which is under the control of HIF (hypoxia-inducible factor)-1α and may be implicated in the transport of metabolites in IR, we KD MCT4 independently of MCT1 (Figure S5D and S5E). MCT4 KD had little effect on malonate uptake, with malonate and succinate levels mirroring those of control siRNA-treated cells (Figure S5F and S5G). Thus, MCT1 is the main driver of malonate uptake at lowered pH.\nOverall, diminishing MCT1 activity, either by pharmacological inhibition or genetic knockdown, impairs malonate uptake at both acidic and normal physiological pH. In addition, this is the first evidence that MCT1 can transport a 3-carbon chain length dicarboxylate, which may also have implications in normal physiology and other pathologies.\nFinally, we assessed if MCT1 was responsible for malonate uptake at lowered pH in the heart. Malonate uptake into the Langendorff-perfused heart was blocked by either excess lactate, or the MCT1 inhibitor AR-C141990, in the perfusion medium (Figure 5H). Together these findings confirm that MCT1 is responsible for the low pH uptake of malonate, both in cells and in the intact heart.\nThe inhibitory effect of DIDS, which also inhibits MCT1,34 raised the possibility of the uptake of the protonated form of malonate being catalyzed by a monocarboxylate transporter (MCT; SLC16 (solute carrier family member 16)). MCT1 (SLC16A1) is the principal transporter of lactate, which is carried in symport with a proton, both of which are elevated during ischemia, making MCT1 an attractive candidate transporter for malonate uptake upon reperfusion. Additionally, it was recently shown that MCT1 mediates the efflux of succinate from the ischemic heart during reperfusion and from exercising muscle.21,35 As MCT1 is a lactate transporter, we first assessed whether high concentrations of lactate in the incubation medium compete with malonate. Excess lactate led to a concentration-dependent decrease in malonate uptake into the cell and a corresponding decrease in succinate accumulation (Figure 4A and Figure S4A). This suggested that at a low pH, malonate uptake into the cells is via MCT1, which is highly expressed in cardiomyocytes.36\nInhibition of MCT1 (monocarboxylate transporter 1) prevents the enhanced uptake of malonate at lowered pH. A, Malonate uptake (5 mmol/L disodium malonate [DSM], 15 min) in C2C12 cells in the presence of lactate (0, 10 or 50 mmol/L). B, Lactate levels in C2C12 cells after 15 min treatment with varying concentrations of MCT1 inhibitors. C and D, Effect of MCT1 inhibition by AR-C141990 or AZD3965 on malonate (5 mmol/L DSM, 15 min) uptake (C) at pH 6 and subsequent succinate levels (D). E, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at various pH±10 µmol/L AR-C141990. F, MCT1 inhibition by AR-C141990 on malonate (5 mmol/L DSM, 15 min) uptake at pH 7.4 (A to F, mean±SEM, n=3 biological replicates, statistics: (A) Kruskal-Wallis with Dunn post hoc test). G and H, Effect of malonate (5 mmol/L DSM) on cellular oxygen consumption at pH 7.4 (G) or 6 (H) ±MCT1 inhibitor (10 µmol/L AR-C141990; data presented as nonmitochondrial respiration normalized mean oxygen consumption rate (OCR)±SEM of 3 biological replicates (n=12–16 technical replicates per biological replicate), statistics: Kruskal-Wallis with Dunn post hoc test). ATP indicates OCR in the presence of 1.5 µmol/L oligomycin; BL, baseline OCR; MAX, OCR in the presence of 1 µmol/L FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone); and NM, OCR in the presence of 4 µg/mL rotenone and 10 µmol/L antimycin A.\nTo confirm that MCT1 was the malonate transporter, we assessed the potent and selective MCT1 inhibitors, AR-C141990 and AZD3965.21,35,37,38 When cells were incubated with these MCT1 inhibitors, they led to a dramatic dose-dependent increase in lactate levels within the cell, consistent with preventing lactate efflux via MCT1 (Figure 4B). MCT1 inhibition by either AR-C141990 or AZD3965 led to a profound dose-dependent decrease in malonate uptake (Figure 4C). Inhibition of malonate uptake by MCT1 inhibition led to a corresponding decrease in succinate accumulation within cells (Figure 4D). Additionally, a time course of malonate uptake at low pH, showed that MCT1 inhibition largely abolished the increase in malonate over time, along with the corresponding increase in succinate (Figure S4B and S4C).\nMCT1 inhibition blocked the pH-dependent increase in malonate uptake (Figure 4E) and in parallel prevented succinate accumulation (Figure 4D). The MCT1 inhibitor led to a dose-dependent inhibition of malonate uptake even at pH 7.4 (Figure 4F). Therefore, malonate can be transported by MCT1 at pH 7.4, but this is greatly enhanced at lower pH due to the increased proportion of malonate in its monocarboxylate form.\nWe next assessed the impact of the malonate taken up into cells on mitochondrial function by measuring respiration. Lowering pH itself had little effect; however, addition of malonate severely reduced cellular respiration (Figure 4G and H and Figure S4E and S4F) and this effect was rescued by MCT1 inhibition (Figure 4G and H and Figure S4E and S4F). MCT1 inhibition led to a small increase in oxygen consumption at baseline, which may be due to lactate accumulation increasing the NADH/NAD+ ratio driving mitochondrial respiration.39\nTo confirm the pharmacological effects of MCT1 inhibition on malonate uptake genetically, we knocked down (KD) MCT1 using siRNA in both HeLa and C2C12 cells (Figure S5A through S5D). Consequently, lactate levels in MCT1 KD cells were significantly elevated compared to the control siRNA (Figure 5A). When MCT1 KD cells were incubated with malonate at low pH, malonate uptake was dramatically blocked (Figure 5B through 5E and Figure S5C), confirming MCT1 is directly responsible for malonate transport at low pH. Residual malonate uptake in the MCT1 KD cells was inhibited by cotreatment with an MCT1 inhibitor, further confirming its MCT1 dependence (Figure 5F and Figure S5F). Additionally, succinate was elevated in the MCT1 KD cells compared with control siRNA cells, a result not seen with acute pharmacological MCT1 inhibition (Figure 5D). This suggests that MCT1 may also play an important role in normoxic succinate metabolism and homeostasis and not just during myocardial reperfusion and intense exercise.21,35 Intriguingly, the succinate levels after malonate treatment over time differed between the 2 cell types, with succinate levels remaining low in C2C12 KD but elevated in HeLa MCT1 KDs compared to controls, suggesting a reliance on MCT1 for succinate efflux (Figure 5G and Figure S5G).\nGenetic knockdown of MCT1 (monocarboxylate transporter 1) prevents the uptake of malonate. A, Relative lactate levels in C2C12 cells treated with control or MCT1 siRNA (mean±SEM of lactate levels relative to control, n=6 biological replicates, statistics: 1-way ANOVA with Bonferroni post hoc test). B to E, Incubation of malonate (5 mmol/L disodium malonate [DSM]) in MCT1 KD cells at pH 7.4 (B and D) or 6 (C and E) for 15 min±MCT1i (10 µmol/L, MCT1 inhibitor AR-C141990). Levels of malonate (B and C) and succinate (D and E) quantified by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). F and G, Time course of malonate uptake (5 mmol/L DSM) at pH 6 in MCT1 KD cells (F) and corresponding succinate levels (G; mean±SEM, n=3 biological replicates). H, Malonate levels in murine Langendorff hearts perfused at pH 6 for 5 min with 5 mmol/L DSM±lactate (50 mmol/L) or MCT1 inhibitor (10 µmol/L AR-C141990; MCT1i; mean±SEM, n=4, statistics: unpaired, 2-tailed Mann-Whitney U test vs control).\nAs HeLa cells constitutively express MCT4, which is under the control of HIF (hypoxia-inducible factor)-1α and may be implicated in the transport of metabolites in IR, we KD MCT4 independently of MCT1 (Figure S5D and S5E). MCT4 KD had little effect on malonate uptake, with malonate and succinate levels mirroring those of control siRNA-treated cells (Figure S5F and S5G). Thus, MCT1 is the main driver of malonate uptake at lowered pH.\nOverall, diminishing MCT1 activity, either by pharmacological inhibition or genetic knockdown, impairs malonate uptake at both acidic and normal physiological pH. In addition, this is the first evidence that MCT1 can transport a 3-carbon chain length dicarboxylate, which may also have implications in normal physiology and other pathologies.\nFinally, we assessed if MCT1 was responsible for malonate uptake at lowered pH in the heart. Malonate uptake into the Langendorff-perfused heart was blocked by either excess lactate, or the MCT1 inhibitor AR-C141990, in the perfusion medium (Figure 5H). Together these findings confirm that MCT1 is responsible for the low pH uptake of malonate, both in cells and in the intact heart.\n Ischemic Conditions Are Sufficient to Drive Malonate Uptake into the Heart Upon Reperfusion That low pH enhanced malonate entry into heart cells was consistent with the protection afforded by DSM in IR injury being due to the local decrease in pH during ischemia and initial reperfusion. To test this hypothesis, Langendorff-perfused hearts were held ischemic for various times before DSM was infused for 5 minutes, followed by flushing, and measurement of malonate. The malonate levels in heart tissue were dependent on ischemia-time, with the highest occurring after 20 minutes ischemia and being ≈10-fold greater than in the normoxic heart (Figure 6A). This malonate uptake into the ischemic Langendorff-perfused heart was dramatically reduced by the MCT1 inhibitor AR-C141990 (Figure 6B). This confirmed that malonate uptake into the ischemic heart upon reperfusion is a selective process, driven by the low pH and facilitated by the MCT1.\nIschemia drives malonate protonation, uptake, and cardioprotection. A, Langendorff-perfused murine hearts were held ischemic for either 0, 5, 10, or 20 min and reperfused with 5 mmol/L disodium malonate (DSM; pH 7.4) before malonate levels measured in the heart by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 (control, 5 min) or 6 (10 and 20 min) biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). B, Malonate levels in murine Langendorff hearts exposed to 20 min ischemia and reperfused with 5 mmol/L malonate (pH 7.4)±MCT1 (monocarboxylate transporter 1) inhibitor (10 or 50 µmol/L AR-C141990; mean±SEM, n=4–5 biological replicates, statistical significance assessed by unpaired, 2-tailed Mann-Whitney U test vs control). C, Model of potential lactate and malonate exchange during reperfusion. D, Lactate levels in the Langendorff heart after 20 min ischemia and 1 min reperfusion±5 mmol/L DSM (mean, n=4 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test). E, Infarct size in murine LAD (left anterior descending coronary artery) ligation MI model with 100 µl bolus of 8 mg/kg DSM, pH 4 acid control or 8 mg/kg pH 4 formulated malonate at reperfusion after 30 min ischemia (mean, n=5 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test vs acid malonate).\nDuring ischemia, the pH of ischemic tissue lowers but in addition, lactate accumulates and upon reperfusion can be transported out of cardiomyocytes by MCT1. This could enhance malonate uptake, as the MCT1 may then act as a lactate-H+/monocarboxylate malonate-H+ exchanger (Figure 6C). To assess this, we measured lactate levels in malonate-perfused IR Langendorff hearts. We found that compared to reperfusion alone, lactate levels decreased in the malonate-treated hearts (Figure 6D). Furthermore, malonate treatment decreased lactate levels in cells that had been treated with the mitochondrial inhibitor antimycin A to enhance lactate production (Figure S6A). This suggests that extracellular malonate and a proton exchange for intracellular lactate, help drive malonate entry into cardiomyocytes upon reperfusion and contribute to its at-risk tissue-selective effect.\nTherefore, malonate uptake into the heart upon reperfusion is dependent on the tissue having first undergone a period of ischemia, leading to a drop in pH and an accumulation of lactate.\nThat low pH enhanced malonate entry into heart cells was consistent with the protection afforded by DSM in IR injury being due to the local decrease in pH during ischemia and initial reperfusion. To test this hypothesis, Langendorff-perfused hearts were held ischemic for various times before DSM was infused for 5 minutes, followed by flushing, and measurement of malonate. The malonate levels in heart tissue were dependent on ischemia-time, with the highest occurring after 20 minutes ischemia and being ≈10-fold greater than in the normoxic heart (Figure 6A). This malonate uptake into the ischemic Langendorff-perfused heart was dramatically reduced by the MCT1 inhibitor AR-C141990 (Figure 6B). This confirmed that malonate uptake into the ischemic heart upon reperfusion is a selective process, driven by the low pH and facilitated by the MCT1.\nIschemia drives malonate protonation, uptake, and cardioprotection. A, Langendorff-perfused murine hearts were held ischemic for either 0, 5, 10, or 20 min and reperfused with 5 mmol/L disodium malonate (DSM; pH 7.4) before malonate levels measured in the heart by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 (control, 5 min) or 6 (10 and 20 min) biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). B, Malonate levels in murine Langendorff hearts exposed to 20 min ischemia and reperfused with 5 mmol/L malonate (pH 7.4)±MCT1 (monocarboxylate transporter 1) inhibitor (10 or 50 µmol/L AR-C141990; mean±SEM, n=4–5 biological replicates, statistical significance assessed by unpaired, 2-tailed Mann-Whitney U test vs control). C, Model of potential lactate and malonate exchange during reperfusion. D, Lactate levels in the Langendorff heart after 20 min ischemia and 1 min reperfusion±5 mmol/L DSM (mean, n=4 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test). E, Infarct size in murine LAD (left anterior descending coronary artery) ligation MI model with 100 µl bolus of 8 mg/kg DSM, pH 4 acid control or 8 mg/kg pH 4 formulated malonate at reperfusion after 30 min ischemia (mean, n=5 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test vs acid malonate).\nDuring ischemia, the pH of ischemic tissue lowers but in addition, lactate accumulates and upon reperfusion can be transported out of cardiomyocytes by MCT1. This could enhance malonate uptake, as the MCT1 may then act as a lactate-H+/monocarboxylate malonate-H+ exchanger (Figure 6C). To assess this, we measured lactate levels in malonate-perfused IR Langendorff hearts. We found that compared to reperfusion alone, lactate levels decreased in the malonate-treated hearts (Figure 6D). Furthermore, malonate treatment decreased lactate levels in cells that had been treated with the mitochondrial inhibitor antimycin A to enhance lactate production (Figure S6A). This suggests that extracellular malonate and a proton exchange for intracellular lactate, help drive malonate entry into cardiomyocytes upon reperfusion and contribute to its at-risk tissue-selective effect.\nTherefore, malonate uptake into the heart upon reperfusion is dependent on the tissue having first undergone a period of ischemia, leading to a drop in pH and an accumulation of lactate.\n Low pH Formulation Improves Cardioprotection by Malonate The mechanism of malonate uptake into cardiac tissue suggested that lowering the pH of the malonate infusion would increase the proportion in the monocarboxylate form and thereby increase its potency as a cardioprotective agent. To assess this, we used a malonate dose that was not protective (8 mg/kg) when administered at a neutral pH in the in vivo LAD MI model. When this dose of malonate was reformulated at pH 4 (a pH currently used in Food and Drug Administration–approved parenteral formulations)40 and administered as a bolus, this conferred significant protection that was not due to the low pH alone (Figure 6E and Figure S6B). Furthermore, although neutral malonate administered before ischemia was not protective, acidified malonate drove its uptake into cardiomyocytes and significantly reduced infarct size when infused before ischemia (Figure S6C).\nThe mechanism of malonate uptake into cardiac tissue suggested that lowering the pH of the malonate infusion would increase the proportion in the monocarboxylate form and thereby increase its potency as a cardioprotective agent. To assess this, we used a malonate dose that was not protective (8 mg/kg) when administered at a neutral pH in the in vivo LAD MI model. When this dose of malonate was reformulated at pH 4 (a pH currently used in Food and Drug Administration–approved parenteral formulations)40 and administered as a bolus, this conferred significant protection that was not due to the low pH alone (Figure 6E and Figure S6B). Furthermore, although neutral malonate administered before ischemia was not protective, acidified malonate drove its uptake into cardiomyocytes and significantly reduced infarct size when infused before ischemia (Figure S6C).\n\nMalonate Is Protective in an MI Model and Is Taken Up Selectively into the Ischemic Region of the Heart:\nDSM is protective against cardiac IR injury, but its mechanism of protection remains uncertain. To address this gap in our knowledge we first tested the therapeutic range of DSM in IR injury. We infused DSM at a range of concentrations (1.6–160 mg/kg equivalent to ≈11-1100 µmol/kg) at reperfusion in an in vivo murine LAD (left anterior descending coronary artery) ligation model of MI. DSM infusion during this clinically relevant reperfusion period led to a dose-dependent decrease in infarct size (Figure 1C), with 160 and 16 mg/kg showing robust cardioprotection. However, when DSM was infused before the onset of ischemia it was not protective (Figure 1D), even at 160 mg/kg which gave the smallest infarct when administered at reperfusion. This contrasted with the cell-permeable dimethyl malonate, which can diffuse into the tissue and generate malonate, preventing succinate accumulation and thereby reducing IR injury.18,25 This suggests that either DSM is minimally taken up by tissues during normoxia and is only taken up by tissues after ischemia or that DSM protects against acute IR injury by an extracellular mechanism.\nWe next measured malonate uptake into the ischemic and healthy heart tissue upon reperfusion in the LAD MI model. This showed that malonate was indeed taken up into cells within the infarct region to a level of ≈400 pmol/mg tissue and to a far greater extent than into cells in the healthy tissue (Figure 1E). Furthermore, reperfusing with malonate slowed succinate oxidation within the infarct region with succinate remaining significantly elevated in the infarct region following 1-minute reperfusion, compared with control reperfusion with saline (Figure 1F). Limiting succinate oxidation at reperfusion with malonate also blunted ROS production in the at-risk tissue (Figure 1G). We next assessed how SDH inhibition compared to direct inhibition of the mitochondrial permeability transition pore with CsA (cyclosporin A). Here we found that the cardioprotection from malonate was additive to cyclosporin A alone (Figure 1H), hence targeting the upstream mechanism of permeability transition pore opening is an increasingly attractive option for IR injury treatment.\nTo better understand the metabolic differences between the healthy and infarcted tissue upon malonate treatment, we investigated the in vivo MI model using mass spectrometry imaging. To do this, we subjected hearts to either 30-minute ischemia, 30-minute ischemia before reperfusing the heart for 15-minute, or 30-minute ischemia before reperfusing with DSM for 15 minutes (to mimic the cardioprotective malonate infusion) before snap freezing and processing for mass spectrometry imaging (Figure 2A). Infarct lesions were demarcated using hematoxylin and eosin (H&E), silver infarct staining and metabolite principal component analysis to differentiate the risk and nonrisk regions. mass spectrometry imaging coupled with the demarcated risk areas showed striking changes in the levels of succinate in the infarct region. During ischemia, succinate was significantly elevated in the at-risk tissue, with considerable succinate accumulation in the core of the infarct (Figure 2B and 2C). After 15-minute reperfusion, the succinate levels in the infarct lesion returned to healthy tissue levels, due to succinate oxidation and efflux.21 When malonate was infused at the time of reperfusion, succinate levels remained higher in all regions of the heart than in the nonmalonate treated heart, consistent with the prevention of its oxidation by SDH inhibition by malonate.\nLocalization of succinate accumulation in heart tissue identified by mass spectrometry imaging. A, Outline of experimental groups for mass spectrometry imaging. B, Representative images of succinate abundance detected by mass spectrometry imaging in myocardial sections. Black outer line indicates the edge of the tissue slice, white inner line indicates infarct region. C, Quantification of succinate (C) identified by MSI (mean±SEM, n=3 biological replicates, statistics: Friedman paired test for healthy vs lesion and Kruskal-Wallis with Dunn post hoc test between conditions). au indicates arbitrary units; DSM, disodium malonate; H, healthy tissue; IR, ischemia/reperfusion; L, infarct lesion; and rep, reperfusion.\nWe conclude that malonate is indeed taken up by the heart, but this is greatly enhanced in ischemic region upon reperfusion. Therefore, malonate provides protection against cardiac IR injury in vivo over a range of concentrations following its selective entry into the infarct region, where it slows the oxidation of succinate during reperfusion, preventing the production of RET-derived ROS.\n\nMalonate Uptake into Cells Is Inefficient at pH 7.4:\nTo explore the mechanism of malonate uptake in vitro, we incubated C2C12 and H9c2 myoblasts with DSM and measured intracellular malonate levels by LC-MS/MS (liquid chromatography-tandem mass spectrometry). Incubation with DSM for 15 minutes at pH 7.4, led to a dose-dependent increase in intracellular malonate (Figure S1A and S1D). Intracellular succinate also accumulated in a malonate-dependent manner (Figure S1B and S1E), confirming that once malonate enters the cell it is rapidly transported into mitochondria and inhibits SDH. However, incubating cells with 1 mmol/L DSM led to variable malonate uptake and little SDH inhibition, indicating that malonate uptake across biological membranes is inefficient. Moreover, the intracellular levels of malonate achieved by 250 µmol/L of the malonate ester prodrug diacetoxymethyl malonate, are 80-fold more than that generated by incubation with 5 mmol/L DSM.25 As DSM was protective in the LAD model when infused at 16 mg/kg, corresponding to a maximum possible blood malonate concentration of ≈1.8 mmol/L (assuming a blood volume of 1.5 mL in a 25 g mouse),29 this compares to the dose range tested in cells (furthermore, far more malonate is available for uptake in vitro than in vivo due to the large reservoir in the incubation medium). Even so, the intracellular malonate levels were far lower with DSM compared to diacetoxymethyl malonate.25\nThe kinetics of cell uptake upon incubation with 5 mmol/L DSM showed time-dependent uptake, although extended incubation times were required for succinate elevation (Figure S1C and S1F). This is likely due to the initial concentrations of malonate entering the cell being insufficient to inhibit SDH. These data are consistent with the lack of protection by DSM delivered before ischemia in vivo, as well as its limited uptake into normoxic tissues. Together these suggest that exposure to ischemia may facilitate malonate entry into the heart.\n\nMalonate Uptake into Cells and the Heart Can Be Modulated by pH:\nMalonate is a dicarboxylate at physiological pH (Figure 1A, pKa=2.83 and 5.69), suggesting that the pH decrease in ischemic tissue9,11,30 may enhance malonate uptake into the heart during early reperfusion, by increasing the concentration of its monocarboxylic form. Incubating cells with DSM at either pH 6, 7.4, or 8 for 15 minutes, led to large differences in malonate uptake (Figure 3A and Figure S2A). At pH 6, the levels of malonate in the cell were significantly higher than at pH 7.4 or 8, thus malonate uptake is favored by acidic pH. Succinate levels mirrored those of malonate, with greater succinate accumulation as a result of increased malonate-dependent SDH inhibition at low pH (Figure 3B and Figure S2B). Low pH alone had no effect on succinate levels (Figure 3B and Figure S2B), suggesting that it was due to malonate entry into the cells followed by SDH inhibition.\nMalonate uptake is enhanced at low pH. A and B, C2C12 cells were incubated with disodium malonate (DSM; 0, 1, or 5 mmol/L) for 15 min at either pH 6, 7.4, or 8 before measuring intracellular malonate (A) and succinate (B) by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). C, Malonate levels in C2C12 cells after incubation with DSM (5 mmol/L) for 15 min at various (patho)physiological pH (mean±SEM, n=3 biological replicates). D, Malonate levels in murine isolated Langendorff-perfused hearts treated with 5 mmol/L DSM infused at either pH 7.4 or 6 for 5 min (mean±SEM, n=4 biological replicates, statistics: unpaired, 2-tailed Mann-Whitney U test). E to H, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at either pH 6 or 7.4 in the presence of FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) (E), gramicidin (F), nigericin (G), or BAM15 (N5,N6-bis(2-Fluorophenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine) (H) before measuring intracellular malonate by LC-MS/MS (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). I, Structures of malonic acid and malonamic acid. J, Quantification of levels of malonate and malonamate in C2C12 cells after incubation (5 mmol/L DSM or disodium malonamate; 15 min) at pH 7.4 (n=6 biological replicates). K, C2C12 cells were incubated with either DSM or disodium malonamate (both 5 mmol/L) at pH 6 or 7.4 for 15 min and the intracellular levels measured by LC-MS/MS. (mean±SEM of the fold-enhancement of uptake at pH 6 vs pH 7.4, n=6 biological replicates). Statistics for J and K: unpaired, 2-tailed Student t test).\nIncubating cells with malonate over a range of pH values between pH 7.4 and 6 clearly showed the pH-dependent uptake of malonate, in line with the increase in monocarboxylate malonate proportion (Figure 3C). This was mirrored by succinate levels (Figure S2C). Therefore, even a small drop in pH can lead to a substantial increase in malonate uptake, and thus is likely to be relevant for its entry into ischemic tissue upon reperfusion.\nAs the cultured cells used are noncontractile and substantially differ in their properties from in situ contracting cardiomyocytes,15 importantly, we next assessed whether decreasing the pH also enhanced malonate uptake into the ex vivo perfused Langendorff heart. Malonate was infused into the heart at either pH 7.4 or 6 for 5 minutes, before briefly flushing at pH 7.4 to remove nonmyocardial malonate. Remarkably, the levels of malonate were significantly elevated when infused at pH 6 compared to 7.4, confirming that the in vitro results translate to the heart (Figure 3D). We conclude that low pH conditions facilitate the entry of malonate into cardiomyocytes.\n\nMalonate Uptake Can Be Perturbed by Modulating the Plasma Membrane H+ Gradient:\nAs pH modulated malonate entry into cells in vitro and in the heart, we next probed the factors affecting malonate uptake into cells. Malonate entry into cells at either pH 7.4 or 6 was blocked at 4 °C and was associated with negligible succinate levels (Figure S2D and S2E). That a decrease in extracellular pH increased malonate entry into cells, suggested uptake driven by the proton gradient. Therefore, we next abolished the plasma membrane proton gradient using ionophores21 which prevented the cellular uptake of malonate (Figure 3E–3G and Figure S3A) and subsequent increase in succinate levels (Figure S3B through S3E). However, the uncoupler BAM15, which is selective for the mitochondrial inner membrane over the plasma membrane,31 had little effect, consistent with the plasma membrane proton gradient driving malonate uptake (Figure 3H and Figure S3F).\nWe next used nonspecific transport inhibitors to assess whether we could block malonate uptake at low pH. DIDS (4,4’-Diisothiocyano-2,2’-stilbenedisulfonic acid), an irreversible inhibitor of chloride/bicarbonate exchange that inhibits malonate uptake into erythrocytes,32,33 led to a dose-dependent inhibition of malonate uptake, although succinate remained high (Figure S3G and S3H). Thus, malonate uptake into cardiomyocytes at low pH is driven by a proton gradient through a transporter-dependent process.\nMalonate’s pKa’s are 2.83 and 5.69, so at pH 6.4, ≈16% of the malonate would be in its monocarboxylate form. To determine whether the uptake of malonate was dependent on its protonation to a monocarboxylate form, we assessed the uptake of a compound that mimics the monocarboxylate malonate. In 3-amino-3-oxopropionate (malonamate; Figure 3I), one carboxylic acid has been replaced with a neutral amido group leaving a single carboxylic acid of pKa ≈4.75. Thus, at pH 7.4 malonamate resembles the monocarboxylate form of malonate. This led to more of malonamate being taken up into cells at pH 7.4 than malonate (Figure 3J). Lowering the pH to 6 led to ≈15-fold increase in malonate uptake while malonamate uptake changed negligibly (Figure 3K) because it will remain as a monocarboxylate across this pH range.\nTogether, these data support transport of the monoanionic form of malonate, under the conditions that occur during early reperfusion. Therefore, a pH gradient can drive the uptake of malonate via protonation and transport in its monocarboxylate form.\n\nMalonate Uptake Under Reperfusion Conditions Is Dependent on MCT1:\nThe inhibitory effect of DIDS, which also inhibits MCT1,34 raised the possibility of the uptake of the protonated form of malonate being catalyzed by a monocarboxylate transporter (MCT; SLC16 (solute carrier family member 16)). MCT1 (SLC16A1) is the principal transporter of lactate, which is carried in symport with a proton, both of which are elevated during ischemia, making MCT1 an attractive candidate transporter for malonate uptake upon reperfusion. Additionally, it was recently shown that MCT1 mediates the efflux of succinate from the ischemic heart during reperfusion and from exercising muscle.21,35 As MCT1 is a lactate transporter, we first assessed whether high concentrations of lactate in the incubation medium compete with malonate. Excess lactate led to a concentration-dependent decrease in malonate uptake into the cell and a corresponding decrease in succinate accumulation (Figure 4A and Figure S4A). This suggested that at a low pH, malonate uptake into the cells is via MCT1, which is highly expressed in cardiomyocytes.36\nInhibition of MCT1 (monocarboxylate transporter 1) prevents the enhanced uptake of malonate at lowered pH. A, Malonate uptake (5 mmol/L disodium malonate [DSM], 15 min) in C2C12 cells in the presence of lactate (0, 10 or 50 mmol/L). B, Lactate levels in C2C12 cells after 15 min treatment with varying concentrations of MCT1 inhibitors. C and D, Effect of MCT1 inhibition by AR-C141990 or AZD3965 on malonate (5 mmol/L DSM, 15 min) uptake (C) at pH 6 and subsequent succinate levels (D). E, C2C12 cells were incubated with DSM (5 mmol/L) for 15 min at various pH±10 µmol/L AR-C141990. F, MCT1 inhibition by AR-C141990 on malonate (5 mmol/L DSM, 15 min) uptake at pH 7.4 (A to F, mean±SEM, n=3 biological replicates, statistics: (A) Kruskal-Wallis with Dunn post hoc test). G and H, Effect of malonate (5 mmol/L DSM) on cellular oxygen consumption at pH 7.4 (G) or 6 (H) ±MCT1 inhibitor (10 µmol/L AR-C141990; data presented as nonmitochondrial respiration normalized mean oxygen consumption rate (OCR)±SEM of 3 biological replicates (n=12–16 technical replicates per biological replicate), statistics: Kruskal-Wallis with Dunn post hoc test). ATP indicates OCR in the presence of 1.5 µmol/L oligomycin; BL, baseline OCR; MAX, OCR in the presence of 1 µmol/L FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone); and NM, OCR in the presence of 4 µg/mL rotenone and 10 µmol/L antimycin A.\nTo confirm that MCT1 was the malonate transporter, we assessed the potent and selective MCT1 inhibitors, AR-C141990 and AZD3965.21,35,37,38 When cells were incubated with these MCT1 inhibitors, they led to a dramatic dose-dependent increase in lactate levels within the cell, consistent with preventing lactate efflux via MCT1 (Figure 4B). MCT1 inhibition by either AR-C141990 or AZD3965 led to a profound dose-dependent decrease in malonate uptake (Figure 4C). Inhibition of malonate uptake by MCT1 inhibition led to a corresponding decrease in succinate accumulation within cells (Figure 4D). Additionally, a time course of malonate uptake at low pH, showed that MCT1 inhibition largely abolished the increase in malonate over time, along with the corresponding increase in succinate (Figure S4B and S4C).\nMCT1 inhibition blocked the pH-dependent increase in malonate uptake (Figure 4E) and in parallel prevented succinate accumulation (Figure 4D). The MCT1 inhibitor led to a dose-dependent inhibition of malonate uptake even at pH 7.4 (Figure 4F). Therefore, malonate can be transported by MCT1 at pH 7.4, but this is greatly enhanced at lower pH due to the increased proportion of malonate in its monocarboxylate form.\nWe next assessed the impact of the malonate taken up into cells on mitochondrial function by measuring respiration. Lowering pH itself had little effect; however, addition of malonate severely reduced cellular respiration (Figure 4G and H and Figure S4E and S4F) and this effect was rescued by MCT1 inhibition (Figure 4G and H and Figure S4E and S4F). MCT1 inhibition led to a small increase in oxygen consumption at baseline, which may be due to lactate accumulation increasing the NADH/NAD+ ratio driving mitochondrial respiration.39\nTo confirm the pharmacological effects of MCT1 inhibition on malonate uptake genetically, we knocked down (KD) MCT1 using siRNA in both HeLa and C2C12 cells (Figure S5A through S5D). Consequently, lactate levels in MCT1 KD cells were significantly elevated compared to the control siRNA (Figure 5A). When MCT1 KD cells were incubated with malonate at low pH, malonate uptake was dramatically blocked (Figure 5B through 5E and Figure S5C), confirming MCT1 is directly responsible for malonate transport at low pH. Residual malonate uptake in the MCT1 KD cells was inhibited by cotreatment with an MCT1 inhibitor, further confirming its MCT1 dependence (Figure 5F and Figure S5F). Additionally, succinate was elevated in the MCT1 KD cells compared with control siRNA cells, a result not seen with acute pharmacological MCT1 inhibition (Figure 5D). This suggests that MCT1 may also play an important role in normoxic succinate metabolism and homeostasis and not just during myocardial reperfusion and intense exercise.21,35 Intriguingly, the succinate levels after malonate treatment over time differed between the 2 cell types, with succinate levels remaining low in C2C12 KD but elevated in HeLa MCT1 KDs compared to controls, suggesting a reliance on MCT1 for succinate efflux (Figure 5G and Figure S5G).\nGenetic knockdown of MCT1 (monocarboxylate transporter 1) prevents the uptake of malonate. A, Relative lactate levels in C2C12 cells treated with control or MCT1 siRNA (mean±SEM of lactate levels relative to control, n=6 biological replicates, statistics: 1-way ANOVA with Bonferroni post hoc test). B to E, Incubation of malonate (5 mmol/L disodium malonate [DSM]) in MCT1 KD cells at pH 7.4 (B and D) or 6 (C and E) for 15 min±MCT1i (10 µmol/L, MCT1 inhibitor AR-C141990). Levels of malonate (B and C) and succinate (D and E) quantified by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=3 biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). F and G, Time course of malonate uptake (5 mmol/L DSM) at pH 6 in MCT1 KD cells (F) and corresponding succinate levels (G; mean±SEM, n=3 biological replicates). H, Malonate levels in murine Langendorff hearts perfused at pH 6 for 5 min with 5 mmol/L DSM±lactate (50 mmol/L) or MCT1 inhibitor (10 µmol/L AR-C141990; MCT1i; mean±SEM, n=4, statistics: unpaired, 2-tailed Mann-Whitney U test vs control).\nAs HeLa cells constitutively express MCT4, which is under the control of HIF (hypoxia-inducible factor)-1α and may be implicated in the transport of metabolites in IR, we KD MCT4 independently of MCT1 (Figure S5D and S5E). MCT4 KD had little effect on malonate uptake, with malonate and succinate levels mirroring those of control siRNA-treated cells (Figure S5F and S5G). Thus, MCT1 is the main driver of malonate uptake at lowered pH.\nOverall, diminishing MCT1 activity, either by pharmacological inhibition or genetic knockdown, impairs malonate uptake at both acidic and normal physiological pH. In addition, this is the first evidence that MCT1 can transport a 3-carbon chain length dicarboxylate, which may also have implications in normal physiology and other pathologies.\nFinally, we assessed if MCT1 was responsible for malonate uptake at lowered pH in the heart. Malonate uptake into the Langendorff-perfused heart was blocked by either excess lactate, or the MCT1 inhibitor AR-C141990, in the perfusion medium (Figure 5H). Together these findings confirm that MCT1 is responsible for the low pH uptake of malonate, both in cells and in the intact heart.\n\nIschemic Conditions Are Sufficient to Drive Malonate Uptake into the Heart Upon Reperfusion:\nThat low pH enhanced malonate entry into heart cells was consistent with the protection afforded by DSM in IR injury being due to the local decrease in pH during ischemia and initial reperfusion. To test this hypothesis, Langendorff-perfused hearts were held ischemic for various times before DSM was infused for 5 minutes, followed by flushing, and measurement of malonate. The malonate levels in heart tissue were dependent on ischemia-time, with the highest occurring after 20 minutes ischemia and being ≈10-fold greater than in the normoxic heart (Figure 6A). This malonate uptake into the ischemic Langendorff-perfused heart was dramatically reduced by the MCT1 inhibitor AR-C141990 (Figure 6B). This confirmed that malonate uptake into the ischemic heart upon reperfusion is a selective process, driven by the low pH and facilitated by the MCT1.\nIschemia drives malonate protonation, uptake, and cardioprotection. A, Langendorff-perfused murine hearts were held ischemic for either 0, 5, 10, or 20 min and reperfused with 5 mmol/L disodium malonate (DSM; pH 7.4) before malonate levels measured in the heart by LC-MS/MS (liquid chromatography-tandem mass spectrometry) (mean±SEM, n=4 (control, 5 min) or 6 (10 and 20 min) biological replicates, statistics: Kruskal-Wallis with Dunn post hoc test). B, Malonate levels in murine Langendorff hearts exposed to 20 min ischemia and reperfused with 5 mmol/L malonate (pH 7.4)±MCT1 (monocarboxylate transporter 1) inhibitor (10 or 50 µmol/L AR-C141990; mean±SEM, n=4–5 biological replicates, statistical significance assessed by unpaired, 2-tailed Mann-Whitney U test vs control). C, Model of potential lactate and malonate exchange during reperfusion. D, Lactate levels in the Langendorff heart after 20 min ischemia and 1 min reperfusion±5 mmol/L DSM (mean, n=4 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test). E, Infarct size in murine LAD (left anterior descending coronary artery) ligation MI model with 100 µl bolus of 8 mg/kg DSM, pH 4 acid control or 8 mg/kg pH 4 formulated malonate at reperfusion after 30 min ischemia (mean, n=5 biological replicates, statistics: 2-tailed, unpaired Mann-Whitney U test vs acid malonate).\nDuring ischemia, the pH of ischemic tissue lowers but in addition, lactate accumulates and upon reperfusion can be transported out of cardiomyocytes by MCT1. This could enhance malonate uptake, as the MCT1 may then act as a lactate-H+/monocarboxylate malonate-H+ exchanger (Figure 6C). To assess this, we measured lactate levels in malonate-perfused IR Langendorff hearts. We found that compared to reperfusion alone, lactate levels decreased in the malonate-treated hearts (Figure 6D). Furthermore, malonate treatment decreased lactate levels in cells that had been treated with the mitochondrial inhibitor antimycin A to enhance lactate production (Figure S6A). This suggests that extracellular malonate and a proton exchange for intracellular lactate, help drive malonate entry into cardiomyocytes upon reperfusion and contribute to its at-risk tissue-selective effect.\nTherefore, malonate uptake into the heart upon reperfusion is dependent on the tissue having first undergone a period of ischemia, leading to a drop in pH and an accumulation of lactate.\n\nLow pH Formulation Improves Cardioprotection by Malonate:\nThe mechanism of malonate uptake into cardiac tissue suggested that lowering the pH of the malonate infusion would increase the proportion in the monocarboxylate form and thereby increase its potency as a cardioprotective agent. To assess this, we used a malonate dose that was not protective (8 mg/kg) when administered at a neutral pH in the in vivo LAD MI model. When this dose of malonate was reformulated at pH 4 (a pH currently used in Food and Drug Administration–approved parenteral formulations)40 and administered as a bolus, this conferred significant protection that was not due to the low pH alone (Figure 6E and Figure S6B). Furthermore, although neutral malonate administered before ischemia was not protective, acidified malonate drove its uptake into cardiomyocytes and significantly reduced infarct size when infused before ischemia (Figure S6C).\n\nDiscussion:\nNo medicine is currently available that can be given at reperfusion to prevent cardiac IR injury.1,6 Drugs that prevent cardiac IR injury should both reduce MI damage and the subsequent development of heart failure.1,12 Targeting succinate metabolism has been shown to be a promising therapeutic approach. Inhibiting SDH using the reversible, competitive inhibitor malonate reduces infarct size in small and large animal models of cardiac IR injury, despite its mechanism of entry into heart tissue being unknown.26,27\nHere, we found that malonate uptake into cells and the heart at pH 7.4 was inefficient. However, the uptake of malonate into cells was dramatically enhanced at the lower levels of pH that occur during ischemia and by lactate accumulation within cells. Thus, ischemia provides an environment that will protonate malonate and thereby enable its uptake by the MCT1 transporter. Interestingly, this transporter undergoes a trans-acceleration phenomenon, whereby its transition from the outward-facing to the inward-facing conformation occurs more rapidly in the presence of a trans-substrate.41,42 In this case, the uptake of malonate on the extracellular side of the plasma membrane may be accelerated by lactate efflux. In the LAD MI model of IR injury, malonate is readily available at the point of reperfusion, thus a proportion of the malonate would be protonated and accessible for transport by MCT1. This enables malonate entry into the heart in an at-risk tissue-selective manner.\nAdditionally, as well as the inhibition of SDH, accelerating lactate efflux from the heart may also play a role in the reduction of IR injury. By shifting the equilibrium to facilitate anaerobic ATP production, this may promote the early restoration of ionic gradients through ATP-dependent transporters.43,44 Furthermore, facilitating lactate efflux may promote the extrusion of protons from the intracellular environment, thus reducing the activity of H+ transporters such as the Na+-H+ exchanger, though further investigation into these mechanisms is warranted.43,45\nThis is the first account of an ischemia-selective cardioprotective agent, utilizing the pathological differences between risk and nonrisk tissue to drive uptake. As MCT1 is highly expressed in the hearts of mice, rats, pigs, and humans (Figure S6D) and the characteristics of ischemia are conserved between these species,19,24 these drivers for malonate uptake and the plasma membrane transporter may facilitate malonate cardioprotection in humans. In addition to its place in the treatment of IR injury in MI, the mechanism of ischemia-enhanced delivery of malonate may provide a novel treatment option for IR injury under many other circumstances.\nThere is much interest in targeted drug delivery and the ability to engage the intended site while reducing off-target effects. Here, we have shown that malonate is not only a cardioprotective agent, but it does this while its entry into the rest of the heart is limited. Thus, malonate is a potent and ischemic tissue-targeted drug, explaining how large doses can be delivered acutely with minimal toxic effects, which is likely to be important bearing in mind the many comorbidities associated with MI.46 Furthermore, as malonate can be efficiently metabolised28,47 and has the potential to promote cardiomyocyte regeneration.48\nMalonate is robustly protective in acute IR injury, though further work is now required to understand the tractability of malonate treatment in chronic IR injury models; in particular, conducting a double-blind chronic large animal IR injury study.49 This would provide the greatest insight into the cardioprotection capabilities of malonate post-MI and its effect on the development of heart failure and define its potential for translation to the clinic.\nSchematic of ischemia-dependent malonate uptake via MCT1 (monocarboxylate transporter 1). The accumulation of lactate and protons in ischemic tissue and equilibration with the extracellular space facilitates protonation of malonate to its monocarboxylate form. This enables it to be an MCT1 substrate and enter cardiomyocytes upon reperfusion. Here, the malonate is transported into mitochondria by the mitochondrial dicarboxylate carrier where it can subsequently go on to inhibit SDH (succinate dehydrogenase). SDH inhibition reduces succinate oxidation, reactive oxygen species (ROS) production by reverse electron transport (RET) through complex I and opening of the mitochondrial permeability transition pore, thereby reducing cell death from ischemia/reperfusion (IR) injury. CxI indicates complex I; DIC, mitochondrial dicarboxylate carrier (SLC25A10); mPTP, mitochondrial permeability transition pore; and TCA, tricarboxylic acid.\n\nConclusions:\nWe have shown that malonate is an ischemia-selective drug, due to the lowered pH and lactate accumulation of ischemic tissue driving its uptake via the MCT1 (Figure 7). Furthermore, we show that a low pH formulation of malonate enhances its therapeutic potency. Malonate is the first at-risk tissue-selective cardioprotective drug and represents a significant step toward the treatment of IR injury.\n\nArticle Information:\n Acknowledgments The authors thank Stephen Large, Fouad Taghavi, and Margaret M. Huang (Department of Surgery, University of Cambridge) for obtaining human heart tissue and Benjamin Thackray (Department of Medicine, University of Cambridge) for assistance with initial experiments.\nThe authors thank Stephen Large, Fouad Taghavi, and Margaret M. Huang (Department of Surgery, University of Cambridge) for obtaining human heart tissue and Benjamin Thackray (Department of Medicine, University of Cambridge) for assistance with initial experiments.\n Sources of Funding This work was supported by the British Heart Foundation (PG/20/10025 to T. Krieg); the Medical Research Council (MC_UU_00015/3 to M.P. Murphy and MR/P000320/1 to T. Krieg), the Wellcome Trust (220257/Z/20/Z to M.P. Murphy, 221604/Z/20/Z to D. Aksentijevic), Barts Charity (MRC0215 to D. Aksentijevic).\nThis work was supported by the British Heart Foundation (PG/20/10025 to T. Krieg); the Medical Research Council (MC_UU_00015/3 to M.P. Murphy and MR/P000320/1 to T. Krieg), the Wellcome Trust (220257/Z/20/Z to M.P. Murphy, 221604/Z/20/Z to D. Aksentijevic), Barts Charity (MRC0215 to D. Aksentijevic).\n Disclosures Some authors currently hold a patent on the use of malonate esters in cardiac ischemia/reperfusion (IR) injury (M.P. Murphy and T. Krieg) and have submitted patent applications on the use of malonate in IR injury associated with ischemic stroke (M.P. Murphy and T. Krieg) and the pH-enhancement of malonate described in this article (H.A. Prag, M.P. Murphy, and T. Krieg). The other authors report no conflicts.\nSome authors currently hold a patent on the use of malonate esters in cardiac ischemia/reperfusion (IR) injury (M.P. Murphy and T. Krieg) and have submitted patent applications on the use of malonate in IR injury associated with ischemic stroke (M.P. Murphy and T. Krieg) and the pH-enhancement of malonate described in this article (H.A. Prag, M.P. Murphy, and T. Krieg). The other authors report no conflicts.\n Supplemental Materials Supplemental Methods\nMajor Resources Table\nFigures S1–S6\nReferences 49–56\nSupplemental Methods\nMajor Resources Table\nFigures S1–S6\nReferences 49–56\n\nAcknowledgments:\nThe authors thank Stephen Large, Fouad Taghavi, and Margaret M. Huang (Department of Surgery, University of Cambridge) for obtaining human heart tissue and Benjamin Thackray (Department of Medicine, University of Cambridge) for assistance with initial experiments.\n\nSources of Funding:\nThis work was supported by the British Heart Foundation (PG/20/10025 to T. Krieg); the Medical Research Council (MC_UU_00015/3 to M.P. Murphy and MR/P000320/1 to T. Krieg), the Wellcome Trust (220257/Z/20/Z to M.P. Murphy, 221604/Z/20/Z to D. Aksentijevic), Barts Charity (MRC0215 to D. Aksentijevic).\n\nDisclosures:\nSome authors currently hold a patent on the use of malonate esters in cardiac ischemia/reperfusion (IR) injury (M.P. Murphy and T. Krieg) and have submitted patent applications on the use of malonate in IR injury associated with ischemic stroke (M.P. Murphy and T. Krieg) and the pH-enhancement of malonate described in this article (H.A. Prag, M.P. Murphy, and T. Krieg). The other authors report no conflicts.\n\nSupplemental Materials:\nSupplemental Methods\nMajor Resources Table\nFigures S1–S6\nReferences 49–56\n\nSupplementary Material:\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nInhibiting SDH (succinate dehydrogenase), with the competitive inhibitor malonate, has shown promise in ameliorating ischemia/reperfusion injury. However, key for translation to the clinic is understanding the mechanism of malonate entry into cells to enable inhibition of SDH, its mitochondrial target, as malonate itself poorly permeates cellular membranes. The possibility of malonate selectively entering the at-risk heart tissue on reperfusion, however, remains unexplored.\n\nMethods:\nC57BL/6J mice, C2C12 and H9c2 myoblasts, and HeLa cells were used to elucidate the mechanism of selective malonate uptake into the ischemic heart upon reperfusion. Cells were treated with malonate while varying pH or together with transport inhibitors. Mouse hearts were either perfused ex vivo (Langendorff) or subjected to in vivo left anterior descending coronary artery ligation as models of ischemia/reperfusion injury. Succinate and malonate levels were assessed by liquid chromatography-tandem mass spectrometry LC-MS/MS, in vivo by mass spectrometry imaging, and infarct size by TTC (2,3,5-triphenyl-2H-tetrazolium chloride) staining.\n\nResults:\nMalonate was robustly protective against cardiac ischemia/reperfusion injury, but only if administered at reperfusion and not when infused before ischemia. The extent of malonate uptake into the heart was proportional to the duration of ischemia. Malonate entry into cardiomyocytes in vivo and in vitro was dramatically increased at the low pH (≈6.5) associated with ischemia. This increased uptake of malonate was blocked by selective inhibition of MCT1 (monocarboxylate transporter 1). Reperfusion of the ischemic heart region with malonate led to selective SDH inhibition in the at-risk region. Acid-formulation greatly enhances the cardioprotective potency of malonate.\n\nConclusions:\nCardioprotection by malonate is dependent on its entry into cardiomyocytes. This is facilitated by the local decrease in pH that occurs during ischemia, leading to its selective uptake upon reperfusion into the at-risk tissue, via MCT1. Thus, malonate's preferential uptake in reperfused tissue means it is an at-risk tissue-selective drug that protects against cardiac ischemia/reperfusion injury."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":16271,"string":"16,271"},"whole_article_abstract_length":{"kind":"number","value":388,"string":"388"},"other_sections_lengths":{"kind":"list like","value":[53,192,829,356,759,442,1546,633,152,433,45,66,14],"string":"[\n 53,\n 192,\n 829,\n 356,\n 759,\n 442,\n 1546,\n 633,\n 152,\n 433,\n 45,\n 66,\n 14\n]","isConversational":false},"num_sections":{"kind":"number","value":20,"string":"20"},"most_frequent_words":{"kind":"list like","value":["malonate","ph","uptake","figure","mct1","malonate uptake","cells","succinate","levels","dsm"],"string":"[\n \"malonate\",\n \"ph\",\n \"uptake\",\n \"figure\",\n \"mct1\",\n \"malonate uptake\",\n \"cells\",\n \"succinate\",\n \"levels\",\n \"dsm\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["ischemia facilitate malonate","facilitate malonate cardioprotection","cardioprotection malonate mechanism","malonate ischemia selective","cardiomyocytes reperfusion malonate"],"string":"[\n \"ischemia facilitate malonate\",\n \"facilitate malonate cardioprotection\",\n \"cardioprotection malonate mechanism\",\n \"malonate ischemia selective\",\n \"cardiomyocytes reperfusion malonate\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"null"},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"null"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] ischemia | mitochondria | myocardial infarction | reactive oxygen species | reperfusion [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ischemia | mitochondria | myocardial infarction | reactive oxygen species | reperfusion [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ischemia | mitochondria | myocardial infarction | reactive oxygen species | reperfusion [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ischemia | mitochondria | myocardial infarction | reactive oxygen species | reperfusion [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"null"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Chromatography, Liquid | HeLa Cells | Humans | Ischemia | Malonates | Mice | Mice, Inbred C57BL | Myocardial Reperfusion Injury | Myocytes, Cardiac | Tandem Mass Spectrometry [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | Chromatography, Liquid | HeLa Cells | Humans | Ischemia | Malonates | Mice | Mice, Inbred C57BL | Myocardial Reperfusion Injury | Myocytes, Cardiac | Tandem Mass Spectrometry 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[SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ischemia facilitate malonate | facilitate malonate cardioprotection | cardioprotection malonate mechanism | malonate ischemia selective | cardiomyocytes reperfusion malonate [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ischemia facilitate malonate | facilitate malonate cardioprotection | cardioprotection malonate mechanism | malonate ischemia selective | cardiomyocytes reperfusion malonate [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ischemia facilitate malonate | facilitate malonate cardioprotection | cardioprotection malonate mechanism | malonate ischemia selective | cardiomyocytes reperfusion malonate 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[SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] malonate | ph | uptake | figure | mct1 | reperfusion | succinate | dsm | ischemia | malonate uptake [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"null"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] C2C12 | HeLa ||| ||| Mouse | Langendorff ||| LC-MS/MS | TTC [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Malonate ||| ||| Malonate ||| MCT1 | 1 ||| ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| SDH ||| ||| C2C12 | HeLa ||| ||| Mouse | Langendorff ||| LC-MS/MS | TTC ||| ||| ||| Malonate ||| MCT1 | 1 ||| ||| ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":69,"cells":{"title":{"kind":"string","value":"The Evolution of HIV Patient Retention and Care in French Guiana: A Broader View From the Système National des Données de Santé."},"pmid":{"kind":"string","value":"35252098"},"background_abstract":{"kind":"string","value":"Although the simplification of antiretroviral (AVR) treatment regimens and follow-up has led to fewer constraints for patients with HIV, their follow-up remains of paramount importance to optimize AVR therapy, to detect and prevent HIV-related morbidity, and prevent secondary infections. The problem of follow-up interruption in French Guiana has been persistent and seemingly impervious to efforts to alleviate it."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Using the complementary lenses of the hospital HIV cohort and the health insurance information system, we looked at the incidence of follow-up interruption and the proportion of patients followed by private practitioners."},"methods_abstract_label":{"kind":"string","value":"METHOD"},"results_abstract":{"kind":"string","value":"We tallied 803 persons that were not known to have died and who were lost to follow-up. Over time, hospital outpatients were lost to follow-up significantly sooner. By contrast, there was a significant trend with more and more patients exclusively followed by private practitioners."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"While hospital outpatient care remains by far the most common mode of patient care, there seems to be a gradual erosion of this model in favor of private practice."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Anti-Retroviral Agents","Cohort Studies","French Guiana","HIV Infections","Humans","Incidence"],"string":"[\n \"Anti-Retroviral Agents\",\n \"Cohort Studies\",\n \"French Guiana\",\n \"HIV Infections\",\n \"Humans\",\n \"Incidence\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"8891454"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"French Guiana is the French overseas territory where the HIV epidemic is most prevalent (1). Recent modeling using the European Center for Disease Control (ECDC) modeling tool estimated the number of persons living with HIV to be over 3,200, 3,000 of whom knew their diagnosis (2). HIV transmission in French Guiana is mostly heterosexual and three of four patients are foreign citizens (3). French Guiana has the highest gross domestic product (GDP) per capita in South America and, as such, it is attractive for the poor Caribbean and South American populations in search of a better life. In a context of precariousness and sexual vulnerability, a large proportion of infected immigrants actually acquire the virus after their arrival in French Guiana (4, 5). The standards of health care are those of mainland France. Since 2013, antiretroviral (AVR) treatment is recommended for all patients irrespective of their CD4 count. All persons living with HIV receive free or paid AVR treatments regardless of their origin or socio-economic level. Undocumented immigrants with HIV are eligible for residence permits for medical reasons and health insurance coverage. Although the simplification of treatment regimens and follow-up has led to fewer constraints for the patients, their follow-up remains of paramount importance to optimize anti-retroviral therapy to detect and prevent HIV-related morbidity and prevent secondary infections. In 2006, we had studied follow-up interruption and its risk factors in French Guiana (6). Younger patients, foreigners, untreated patients, and non-immunosuppressed patients at the time of diagnosis were more likely to interrupt follow-up. In addition, the greatest risk of interruption was usually within the first 6 months after diagnosis. Since then, nearly all patients are given AVRs with regimens that are more potent but better tolerated, which usually simplifies medical follow-up. In the post 90-90-90 context, French Guiana aims to optimize the cascade of care by improving early testing and treatment and retaining patients in the healthcare system so they can continue to benefit from virological suppression. Despite progress, this remains a problem in France (7, 8). For the specific case of French Guiana, our hypothesis was that different and novel forces were at play and may have modified the adherence to outpatient follow-up. First, in the widespread early treatment with powerful and well-tolerated drugs, the simplification of follow-up would predict that patient retention should increase over time. Second, because patient follow-up is much simpler than before, general practitioners are more inclined to accept such a task, and patients –always weary of being spotted in a specialized HIV outpatient care center—may prefer to be followed closely to their home, in the “stigma-free” practice of their family physician. We therefore looked at the proportion of patients lost to follow-up –from the point of view of hospital outpatient care in different time periods. We also looked, for the first time, at the data from the health insurance system (9), which notably reimburses all antiretroviral drugs (ARVs) to determine the proportion of patients for whom ambulatory care was given from a specialized hospital department and those who only received ambulatory care from a private practitioner."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Patients From the Hospital Outpatient Clinics The information systems for HIV in French Guiana, mandatory reporting of new HIV infections and AIDS cases, and the French Hospital Database of HIV, a cohort for which specifically trained research technicians have collected data on HIV patients since 1989 {first in the DMI2 government program until 2008, then in eNADIS/DATAIDS [Dat'AIDS cohort (clinicaltrials.gov ref. NCT02898987)]}, were used for the study.\nThe information systems for HIV in French Guiana, mandatory reporting of new HIV infections and AIDS cases, and the French Hospital Database of HIV, a cohort for which specifically trained research technicians have collected data on HIV patients since 1989 {first in the DMI2 government program until 2008, then in eNADIS/DATAIDS [Dat'AIDS cohort (clinicaltrials.gov ref. NCT02898987)]}, were used for the study.\n Lost to Follow-Up This cohort allowed us to obtain the number and proportion of patients lost to follow-up (not having consulted in more than 12 months) from the point of view of the hospital system.\nThis cohort allowed us to obtain the number and proportion of patients lost to follow-up (not having consulted in more than 12 months) from the point of view of the hospital system.\n Determination of the Proportion of Patients Receiving Treatment in a Public Hospital or in a Private Practice Since 2017, health administrations and researchers may, with permission, access data from the Système National des Données de Santé which now combines health care reimbursement data (SNIIRAM), hospital data (PMSI), and death certificate data (CepiDC). For French Guiana, this allowed us to obtain, for the first time, the proportion of persons treated for HIV in a public hospital structure or in private practice, or both.\nSince 2017, health administrations and researchers may, with permission, access data from the Système National des Données de Santé which now combines health care reimbursement data (SNIIRAM), hospital data (PMSI), and death certificate data (CepiDC). For French Guiana, this allowed us to obtain, for the first time, the proportion of persons treated for HIV in a public hospital structure or in private practice, or both.\n Selection of ARV Treatments The ARV treatments were selected from the code identifiant de présentation (CIP) code of the Drug Database [base de données publique des médicaments (BDM)], the reference database of drugs reimbursed by the French Health Insurance.\nThe ARV treatments were selected from the code identifiant de présentation (CIP) code of the Drug Database [base de données publique des médicaments (BDM)], the reference database of drugs reimbursed by the French Health Insurance.\n Patient Selection The data used to select the people on ARVs were taken from the inter-scheme consumption datamart (DCIR). All the health insurance beneficiaries were included in the study if they received at least one reimbursement for the delivery of ARVs during the year, even when the treatments were prescribed and/or delivered outside French Guiana. The specific insurance regimens included the General Health Insurance Scheme, including beneficiaries of the free complementary health insurance coverage for the poor French residents or legal migrants (CMUC/CSS) and the state medical aid for undocumented foreigners (AME); the social security system for the self-employed (RSI); and the agricultural social mutual insurance (MSA) of French Guiana.\nA subsample was also made up of patients who received at least one reimbursement for ARVs under the AME or CMUC/CSS during the year. Since AME or CMUC/CSS reflect poverty, these selection criteria allowed to test the hypothesis that trends for private or public follow-up differed between socially precarious and non-precarious populations.\nThe data used to select the people on ARVs were taken from the inter-scheme consumption datamart (DCIR). All the health insurance beneficiaries were included in the study if they received at least one reimbursement for the delivery of ARVs during the year, even when the treatments were prescribed and/or delivered outside French Guiana. The specific insurance regimens included the General Health Insurance Scheme, including beneficiaries of the free complementary health insurance coverage for the poor French residents or legal migrants (CMUC/CSS) and the state medical aid for undocumented foreigners (AME); the social security system for the self-employed (RSI); and the agricultural social mutual insurance (MSA) of French Guiana.\nA subsample was also made up of patients who received at least one reimbursement for ARVs under the AME or CMUC/CSS during the year. Since AME or CMUC/CSS reflect poverty, these selection criteria allowed to test the hypothesis that trends for private or public follow-up differed between socially precarious and non-precarious populations.\n Description of the Evolution of the Type of Care The type of care was defined as follows: exclusively hospital-based when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the hospital sector, exclusively private when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the private sector and mixed when the ARV treatments for which the patient was reimbursed during the year are prescribed by the hospital sector and the private sector.\nThe annual evolution of the distribution of the type of care was studied over the 2016–2020 period in the population of all patients on ARVs at the regional level and in the population, including only CMUC/CSS or AME patients (precarious persons) at the regional level.\nThe type of care was defined as follows: exclusively hospital-based when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the hospital sector, exclusively private when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the private sector and mixed when the ARV treatments for which the patient was reimbursed during the year are prescribed by the hospital sector and the private sector.\nThe annual evolution of the distribution of the type of care was studied over the 2016–2020 period in the population of all patients on ARVs at the regional level and in the population, including only CMUC/CSS or AME patients (precarious persons) at the regional level.\n Data Analysis Using the DAT'AIDS cohort data, single failure survival analyses were performed with follow-up interruption of more than 12 months (not due to death) as a failure event for a patient without knowledge of his or her present follow-up status. Kaplan Meier curves were plotted. Log Rank tests were performed. These analyses allowed us to quantify and visualize the time until follow-up interruption for different periods. For the health insurance data, a linear trend Chi-square test was used to test the statistical significance of the annual change in the different types of care, observed graphically over the period studied. This trend analysis aimed to determine whether the process was gradual.\nUsing the DAT'AIDS cohort data, single failure survival analyses were performed with follow-up interruption of more than 12 months (not due to death) as a failure event for a patient without knowledge of his or her present follow-up status. Kaplan Meier curves were plotted. Log Rank tests were performed. These analyses allowed us to quantify and visualize the time until follow-up interruption for different periods. For the health insurance data, a linear trend Chi-square test was used to test the statistical significance of the annual change in the different types of care, observed graphically over the period studied. This trend analysis aimed to determine whether the process was gradual.\n Regulatory and Ethical Issues Anonymized individual data from the DAT'AIDS database was used (clinicaltrials.gov ref. NCT02898987). For the proportion followed up in private or public structure, the anonymized data from the système national des données de santé (SNDS) is accessible for certain organizations with a public service mission. Particularly, these organizations, listed by decree by the Conseil d'Etat, issued after the opinion of the Commission Nationale Informatique et Libertés (CNIL), may access certain data on a permanent basis in order to carry out their missions. This is the case for researchers of Academic hospitals and the Institut National de la Santé et de la Recherche Médicale (Inserm).\nAnonymized individual data from the DAT'AIDS database was used (clinicaltrials.gov ref. NCT02898987). For the proportion followed up in private or public structure, the anonymized data from the système national des données de santé (SNDS) is accessible for certain organizations with a public service mission. Particularly, these organizations, listed by decree by the Conseil d'Etat, issued after the opinion of the Commission Nationale Informatique et Libertés (CNIL), may access certain data on a permanent basis in order to carry out their missions. This is the case for researchers of Academic hospitals and the Institut National de la Santé et de la Recherche Médicale (Inserm)."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Hospital Follow-Up in Time We tallied 803 persons that were not known to have died and who were lost to follow-up. Figure 1 shows that over time, patients seemed to be lost to follow-up increasingly sooner (Log-rank test, p < 0.001, Table 1).\nIncidence of follow-up interruption in French Guiana from the point of view of the hospital HIV outpatient cohort.\nLog-rank test comparing the incidence of follow-up interruption for different consecutive years.\nCHI2 (4) = 307.45; p = 0.0000.\nWe tallied 803 persons that were not known to have died and who were lost to follow-up. Figure 1 shows that over time, patients seemed to be lost to follow-up increasingly sooner (Log-rank test, p < 0.001, Table 1).\nIncidence of follow-up interruption in French Guiana from the point of view of the hospital HIV outpatient cohort.\nLog-rank test comparing the incidence of follow-up interruption for different consecutive years.\nCHI2 (4) = 307.45; p = 0.0000.\n The Total Number of Persons on ARV in French Guiana When looking at the health insurance data, in 2020, there were 3,047 patients who benefitted from health insurance payments for ARV treatment. This total number has been steadily increasing since 2016 (see Table 2). The evolution rate over the period was 33.35% with an average annual evolution rate of 7.46%. However, the evolution of the number of patients was more moderate in 2020, a year when activity was completely disorganized by coronavirus disease 2019 (COVID-19) and lockdowns.\nDistribution of the type of antiretroviral (ARV) follow-up (private, hospital, or mixed) in Guiana between 2016 and 2020.\nSource: SNDS/DCIR—Beneficiaries of the RG (CMU/CSS and AME).\nRSI and MSA affiliated in French Guiana, excluding complementary insurances not managed by the computer.\nWhen looking at the health insurance data, in 2020, there were 3,047 patients who benefitted from health insurance payments for ARV treatment. This total number has been steadily increasing since 2016 (see Table 2). The evolution rate over the period was 33.35% with an average annual evolution rate of 7.46%. However, the evolution of the number of patients was more moderate in 2020, a year when activity was completely disorganized by coronavirus disease 2019 (COVID-19) and lockdowns.\nDistribution of the type of antiretroviral (ARV) follow-up (private, hospital, or mixed) in Guiana between 2016 and 2020.\nSource: SNDS/DCIR—Beneficiaries of the RG (CMU/CSS and AME).\nRSI and MSA affiliated in French Guiana, excluding complementary insurances not managed by the computer.\n Hospital and/or Private Follow-Up In 2020, 57.43% (n = 1,750) of patients were followed exclusively by the hospital sector. The proportion of patients treated exclusively in the private sector was 16.31% (n = 497), and that of patients with mixed treatment prescription (private and hospital) was 26.26% (n = 800) (Table 1).\nUntil 2019, the number of patients followed up increased whatever the type of care. In 2020, only the number of patients followed up exclusively in the private sector continued to increase (+138 patients), whereas the number of patients followed up in the hospital sector or with mixed follow-up decreased slightly (see Table 2). Furthermore, the proportion of patients followed up exclusively by the hospital sector has decreased over the last 3 years, particularly in 2020. The change over the entire period was statistically significant (p < 0.0001). Conversely, the proportion of patients followed exclusively by the private sector has been steadily increasing since 2018 (the trend was statistically significant; p < 0.0001). Finally, the proportion of patients with follow-up in both the hospital and private sectors has varied little since 2016. However, there was a slight decrease in 2020 compared to 2018 and 2019, years in which the proportion had stabilized at around 28% of patients (the trend over the whole period being statistically significant; p = 0.006). Because COVID-19 in 2020 led to dramatic changes (lock-downs, cancelation of hospital consultations, etc.), we also computed the trends for 2016–2019, and the observation of a linear increase of the trend toward outpatients was still significant: p = 0.004 and p = 0.002, respectively.\nFocus on patients with indicators of precariousness (Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire or Aide Médicale Etat).\nIn 2020, 1,873 patients who had been reimbursed for ARVs were beneficiaries of the CMUC/CSS or the AME (i.e., 61.47% of all patients on ARVs) insurance plans for socially precarious persons. The number of patients in this situation increased until 2019 but decreased in 2020. Overall, the increase over the whole period was 24.53%, with an average annual growth rate of 5.64%. In 2020, 1,082 of the 1,873 patients receiving CMUC/CSS or AME on ARVs had exclusive hospital follow-up (i.e., 57.77%). This proportion steadily decreased over the last 3 years after an initial increase in 2017 (the change over the whole period being statistically significant; p < 0.0001). Conversely, the proportions of patients managed exclusively by the private sector or benefiting from mixed public and private follow-up, after having decreased over the year 2017, increased over the years 2018, 2019, and 2020. The proportion of precarious patients on ARVs exclusively followed up in the private sector thus rose to reach 14.15% in 2020 (a statistically significant trend for the period 2016–2020; p < 0.0001). The proportion of patients with mixed management (combination of public and private follow-up) increased by + 2 points between 2016 and 2020 (the trend was statistically significant over the period; p = 0.0036) to reach more than 28% in 2020 (Table 3).\nDistribution of the type of antiretroviral follow-up (private practice, hospital, or mixed) in French Guiana between 2016 and 2020 among precarious patients.\nSource: SNDS/DCIR—Beneficiaries of Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire and Aide Médicale Etat in French Guiana.\nLinear trend chi2 (private vs. hospital only, 2016–2020): p < 0.001;\nLinear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.003.\nBecause of trend distortions resulting from COVID-19, the trend was recalculated for 2016–2019, excluding 2020.\n*Linear trend chi2 (private vs. hospital only, 2016–2009): p = 0.004; **Linear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.002.\nIn 2020, 57.43% (n = 1,750) of patients were followed exclusively by the hospital sector. The proportion of patients treated exclusively in the private sector was 16.31% (n = 497), and that of patients with mixed treatment prescription (private and hospital) was 26.26% (n = 800) (Table 1).\nUntil 2019, the number of patients followed up increased whatever the type of care. In 2020, only the number of patients followed up exclusively in the private sector continued to increase (+138 patients), whereas the number of patients followed up in the hospital sector or with mixed follow-up decreased slightly (see Table 2). Furthermore, the proportion of patients followed up exclusively by the hospital sector has decreased over the last 3 years, particularly in 2020. The change over the entire period was statistically significant (p < 0.0001). Conversely, the proportion of patients followed exclusively by the private sector has been steadily increasing since 2018 (the trend was statistically significant; p < 0.0001). Finally, the proportion of patients with follow-up in both the hospital and private sectors has varied little since 2016. However, there was a slight decrease in 2020 compared to 2018 and 2019, years in which the proportion had stabilized at around 28% of patients (the trend over the whole period being statistically significant; p = 0.006). Because COVID-19 in 2020 led to dramatic changes (lock-downs, cancelation of hospital consultations, etc.), we also computed the trends for 2016–2019, and the observation of a linear increase of the trend toward outpatients was still significant: p = 0.004 and p = 0.002, respectively.\nFocus on patients with indicators of precariousness (Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire or Aide Médicale Etat).\nIn 2020, 1,873 patients who had been reimbursed for ARVs were beneficiaries of the CMUC/CSS or the AME (i.e., 61.47% of all patients on ARVs) insurance plans for socially precarious persons. The number of patients in this situation increased until 2019 but decreased in 2020. Overall, the increase over the whole period was 24.53%, with an average annual growth rate of 5.64%. In 2020, 1,082 of the 1,873 patients receiving CMUC/CSS or AME on ARVs had exclusive hospital follow-up (i.e., 57.77%). This proportion steadily decreased over the last 3 years after an initial increase in 2017 (the change over the whole period being statistically significant; p < 0.0001). Conversely, the proportions of patients managed exclusively by the private sector or benefiting from mixed public and private follow-up, after having decreased over the year 2017, increased over the years 2018, 2019, and 2020. The proportion of precarious patients on ARVs exclusively followed up in the private sector thus rose to reach 14.15% in 2020 (a statistically significant trend for the period 2016–2020; p < 0.0001). The proportion of patients with mixed management (combination of public and private follow-up) increased by + 2 points between 2016 and 2020 (the trend was statistically significant over the period; p = 0.0036) to reach more than 28% in 2020 (Table 3).\nDistribution of the type of antiretroviral follow-up (private practice, hospital, or mixed) in French Guiana between 2016 and 2020 among precarious patients.\nSource: SNDS/DCIR—Beneficiaries of Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire and Aide Médicale Etat in French Guiana.\nLinear trend chi2 (private vs. hospital only, 2016–2020): p < 0.001;\nLinear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.003.\nBecause of trend distortions resulting from COVID-19, the trend was recalculated for 2016–2019, excluding 2020.\n*Linear trend chi2 (private vs. hospital only, 2016–2009): p = 0.004; **Linear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.002."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Patients From the Hospital Outpatient Clinics","Lost to Follow-Up","Determination of the Proportion of Patients Receiving Treatment in a Public Hospital or in a Private Practice","Selection of ARV Treatments","Patient Selection","Description of the Evolution of the Type of Care","Data Analysis","Regulatory and Ethical Issues","Hospital Follow-Up in Time","The Total Number of Persons on ARV in French Guiana","Hospital and/or Private Follow-Up","Ethics Statement","Author Contributions"],"string":"[\n \"Patients From the Hospital Outpatient Clinics\",\n \"Lost to Follow-Up\",\n \"Determination of the Proportion of Patients Receiving Treatment in a Public Hospital or in a Private Practice\",\n \"Selection of ARV Treatments\",\n \"Patient Selection\",\n \"Description of the Evolution of the Type of Care\",\n \"Data Analysis\",\n \"Regulatory and Ethical Issues\",\n \"Hospital Follow-Up in Time\",\n \"The Total Number of Persons on ARV in French Guiana\",\n \"Hospital and/or Private Follow-Up\",\n \"Ethics Statement\",\n \"Author Contributions\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["The information systems for HIV in French Guiana, mandatory reporting of new HIV infections and AIDS cases, and the French Hospital Database of HIV, a cohort for which specifically trained research technicians have collected data on HIV patients since 1989 {first in the DMI2 government program until 2008, then in eNADIS/DATAIDS [Dat'AIDS cohort (clinicaltrials.gov ref. NCT02898987)]}, were used for the study.","This cohort allowed us to obtain the number and proportion of patients lost to follow-up (not having consulted in more than 12 months) from the point of view of the hospital system.","Since 2017, health administrations and researchers may, with permission, access data from the Système National des Données de Santé which now combines health care reimbursement data (SNIIRAM), hospital data (PMSI), and death certificate data (CepiDC). For French Guiana, this allowed us to obtain, for the first time, the proportion of persons treated for HIV in a public hospital structure or in private practice, or both.","The ARV treatments were selected from the code identifiant de présentation (CIP) code of the Drug Database [base de données publique des médicaments (BDM)], the reference database of drugs reimbursed by the French Health Insurance.","The data used to select the people on ARVs were taken from the inter-scheme consumption datamart (DCIR). All the health insurance beneficiaries were included in the study if they received at least one reimbursement for the delivery of ARVs during the year, even when the treatments were prescribed and/or delivered outside French Guiana. The specific insurance regimens included the General Health Insurance Scheme, including beneficiaries of the free complementary health insurance coverage for the poor French residents or legal migrants (CMUC/CSS) and the state medical aid for undocumented foreigners (AME); the social security system for the self-employed (RSI); and the agricultural social mutual insurance (MSA) of French Guiana.\nA subsample was also made up of patients who received at least one reimbursement for ARVs under the AME or CMUC/CSS during the year. Since AME or CMUC/CSS reflect poverty, these selection criteria allowed to test the hypothesis that trends for private or public follow-up differed between socially precarious and non-precarious populations.","The type of care was defined as follows: exclusively hospital-based when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the hospital sector, exclusively private when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the private sector and mixed when the ARV treatments for which the patient was reimbursed during the year are prescribed by the hospital sector and the private sector.\nThe annual evolution of the distribution of the type of care was studied over the 2016–2020 period in the population of all patients on ARVs at the regional level and in the population, including only CMUC/CSS or AME patients (precarious persons) at the regional level.","Using the DAT'AIDS cohort data, single failure survival analyses were performed with follow-up interruption of more than 12 months (not due to death) as a failure event for a patient without knowledge of his or her present follow-up status. Kaplan Meier curves were plotted. Log Rank tests were performed. These analyses allowed us to quantify and visualize the time until follow-up interruption for different periods. For the health insurance data, a linear trend Chi-square test was used to test the statistical significance of the annual change in the different types of care, observed graphically over the period studied. This trend analysis aimed to determine whether the process was gradual.","Anonymized individual data from the DAT'AIDS database was used (clinicaltrials.gov ref. NCT02898987). For the proportion followed up in private or public structure, the anonymized data from the système national des données de santé (SNDS) is accessible for certain organizations with a public service mission. Particularly, these organizations, listed by decree by the Conseil d'Etat, issued after the opinion of the Commission Nationale Informatique et Libertés (CNIL), may access certain data on a permanent basis in order to carry out their missions. This is the case for researchers of Academic hospitals and the Institut National de la Santé et de la Recherche Médicale (Inserm).","We tallied 803 persons that were not known to have died and who were lost to follow-up. Figure 1 shows that over time, patients seemed to be lost to follow-up increasingly sooner (Log-rank test, p < 0.001, Table 1).\nIncidence of follow-up interruption in French Guiana from the point of view of the hospital HIV outpatient cohort.\nLog-rank test comparing the incidence of follow-up interruption for different consecutive years.\nCHI2 (4) = 307.45; p = 0.0000.","When looking at the health insurance data, in 2020, there were 3,047 patients who benefitted from health insurance payments for ARV treatment. This total number has been steadily increasing since 2016 (see Table 2). The evolution rate over the period was 33.35% with an average annual evolution rate of 7.46%. However, the evolution of the number of patients was more moderate in 2020, a year when activity was completely disorganized by coronavirus disease 2019 (COVID-19) and lockdowns.\nDistribution of the type of antiretroviral (ARV) follow-up (private, hospital, or mixed) in Guiana between 2016 and 2020.\nSource: SNDS/DCIR—Beneficiaries of the RG (CMU/CSS and AME).\nRSI and MSA affiliated in French Guiana, excluding complementary insurances not managed by the computer.","In 2020, 57.43% (n = 1,750) of patients were followed exclusively by the hospital sector. The proportion of patients treated exclusively in the private sector was 16.31% (n = 497), and that of patients with mixed treatment prescription (private and hospital) was 26.26% (n = 800) (Table 1).\nUntil 2019, the number of patients followed up increased whatever the type of care. In 2020, only the number of patients followed up exclusively in the private sector continued to increase (+138 patients), whereas the number of patients followed up in the hospital sector or with mixed follow-up decreased slightly (see Table 2). Furthermore, the proportion of patients followed up exclusively by the hospital sector has decreased over the last 3 years, particularly in 2020. The change over the entire period was statistically significant (p < 0.0001). Conversely, the proportion of patients followed exclusively by the private sector has been steadily increasing since 2018 (the trend was statistically significant; p < 0.0001). Finally, the proportion of patients with follow-up in both the hospital and private sectors has varied little since 2016. However, there was a slight decrease in 2020 compared to 2018 and 2019, years in which the proportion had stabilized at around 28% of patients (the trend over the whole period being statistically significant; p = 0.006). Because COVID-19 in 2020 led to dramatic changes (lock-downs, cancelation of hospital consultations, etc.), we also computed the trends for 2016–2019, and the observation of a linear increase of the trend toward outpatients was still significant: p = 0.004 and p = 0.002, respectively.\nFocus on patients with indicators of precariousness (Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire or Aide Médicale Etat).\nIn 2020, 1,873 patients who had been reimbursed for ARVs were beneficiaries of the CMUC/CSS or the AME (i.e., 61.47% of all patients on ARVs) insurance plans for socially precarious persons. The number of patients in this situation increased until 2019 but decreased in 2020. Overall, the increase over the whole period was 24.53%, with an average annual growth rate of 5.64%. In 2020, 1,082 of the 1,873 patients receiving CMUC/CSS or AME on ARVs had exclusive hospital follow-up (i.e., 57.77%). This proportion steadily decreased over the last 3 years after an initial increase in 2017 (the change over the whole period being statistically significant; p < 0.0001). Conversely, the proportions of patients managed exclusively by the private sector or benefiting from mixed public and private follow-up, after having decreased over the year 2017, increased over the years 2018, 2019, and 2020. The proportion of precarious patients on ARVs exclusively followed up in the private sector thus rose to reach 14.15% in 2020 (a statistically significant trend for the period 2016–2020; p < 0.0001). The proportion of patients with mixed management (combination of public and private follow-up) increased by + 2 points between 2016 and 2020 (the trend was statistically significant over the period; p = 0.0036) to reach more than 28% in 2020 (Table 3).\nDistribution of the type of antiretroviral follow-up (private practice, hospital, or mixed) in French Guiana between 2016 and 2020 among precarious patients.\nSource: SNDS/DCIR—Beneficiaries of Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire and Aide Médicale Etat in French Guiana.\nLinear trend chi2 (private vs. hospital only, 2016–2020): p < 0.001;\nLinear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.003.\nBecause of trend distortions resulting from COVID-19, the trend was recalculated for 2016–2019, excluding 2020.\n*Linear trend chi2 (private vs. hospital only, 2016–2009): p = 0.004; **Linear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.002.","The studies involving human participants were reviewed and approved by Commission Nationale Informatique et Libertés. The patients/participants provided their written informed consent to participate in this study.","MN: study design. HD and NV: data analysis. HD and MN: first draft writing. CS, LA, AL, FH, LM, JW, NV, EP, and AA: review and editing. All authors contributed to the article and approved the submitted version."],"string":"[\n \"The information systems for HIV in French Guiana, mandatory reporting of new HIV infections and AIDS cases, and the French Hospital Database of HIV, a cohort for which specifically trained research technicians have collected data on HIV patients since 1989 {first in the DMI2 government program until 2008, then in eNADIS/DATAIDS [Dat'AIDS cohort (clinicaltrials.gov ref. NCT02898987)]}, were used for the study.\",\n \"This cohort allowed us to obtain the number and proportion of patients lost to follow-up (not having consulted in more than 12 months) from the point of view of the hospital system.\",\n \"Since 2017, health administrations and researchers may, with permission, access data from the Système National des Données de Santé which now combines health care reimbursement data (SNIIRAM), hospital data (PMSI), and death certificate data (CepiDC). For French Guiana, this allowed us to obtain, for the first time, the proportion of persons treated for HIV in a public hospital structure or in private practice, or both.\",\n \"The ARV treatments were selected from the code identifiant de présentation (CIP) code of the Drug Database [base de données publique des médicaments (BDM)], the reference database of drugs reimbursed by the French Health Insurance.\",\n \"The data used to select the people on ARVs were taken from the inter-scheme consumption datamart (DCIR). All the health insurance beneficiaries were included in the study if they received at least one reimbursement for the delivery of ARVs during the year, even when the treatments were prescribed and/or delivered outside French Guiana. The specific insurance regimens included the General Health Insurance Scheme, including beneficiaries of the free complementary health insurance coverage for the poor French residents or legal migrants (CMUC/CSS) and the state medical aid for undocumented foreigners (AME); the social security system for the self-employed (RSI); and the agricultural social mutual insurance (MSA) of French Guiana.\\nA subsample was also made up of patients who received at least one reimbursement for ARVs under the AME or CMUC/CSS during the year. Since AME or CMUC/CSS reflect poverty, these selection criteria allowed to test the hypothesis that trends for private or public follow-up differed between socially precarious and non-precarious populations.\",\n \"The type of care was defined as follows: exclusively hospital-based when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the hospital sector, exclusively private when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the private sector and mixed when the ARV treatments for which the patient was reimbursed during the year are prescribed by the hospital sector and the private sector.\\nThe annual evolution of the distribution of the type of care was studied over the 2016–2020 period in the population of all patients on ARVs at the regional level and in the population, including only CMUC/CSS or AME patients (precarious persons) at the regional level.\",\n \"Using the DAT'AIDS cohort data, single failure survival analyses were performed with follow-up interruption of more than 12 months (not due to death) as a failure event for a patient without knowledge of his or her present follow-up status. Kaplan Meier curves were plotted. Log Rank tests were performed. These analyses allowed us to quantify and visualize the time until follow-up interruption for different periods. For the health insurance data, a linear trend Chi-square test was used to test the statistical significance of the annual change in the different types of care, observed graphically over the period studied. This trend analysis aimed to determine whether the process was gradual.\",\n \"Anonymized individual data from the DAT'AIDS database was used (clinicaltrials.gov ref. NCT02898987). For the proportion followed up in private or public structure, the anonymized data from the système national des données de santé (SNDS) is accessible for certain organizations with a public service mission. Particularly, these organizations, listed by decree by the Conseil d'Etat, issued after the opinion of the Commission Nationale Informatique et Libertés (CNIL), may access certain data on a permanent basis in order to carry out their missions. This is the case for researchers of Academic hospitals and the Institut National de la Santé et de la Recherche Médicale (Inserm).\",\n \"We tallied 803 persons that were not known to have died and who were lost to follow-up. Figure 1 shows that over time, patients seemed to be lost to follow-up increasingly sooner (Log-rank test, p < 0.001, Table 1).\\nIncidence of follow-up interruption in French Guiana from the point of view of the hospital HIV outpatient cohort.\\nLog-rank test comparing the incidence of follow-up interruption for different consecutive years.\\nCHI2 (4) = 307.45; p = 0.0000.\",\n \"When looking at the health insurance data, in 2020, there were 3,047 patients who benefitted from health insurance payments for ARV treatment. This total number has been steadily increasing since 2016 (see Table 2). The evolution rate over the period was 33.35% with an average annual evolution rate of 7.46%. However, the evolution of the number of patients was more moderate in 2020, a year when activity was completely disorganized by coronavirus disease 2019 (COVID-19) and lockdowns.\\nDistribution of the type of antiretroviral (ARV) follow-up (private, hospital, or mixed) in Guiana between 2016 and 2020.\\nSource: SNDS/DCIR—Beneficiaries of the RG (CMU/CSS and AME).\\nRSI and MSA affiliated in French Guiana, excluding complementary insurances not managed by the computer.\",\n \"In 2020, 57.43% (n = 1,750) of patients were followed exclusively by the hospital sector. The proportion of patients treated exclusively in the private sector was 16.31% (n = 497), and that of patients with mixed treatment prescription (private and hospital) was 26.26% (n = 800) (Table 1).\\nUntil 2019, the number of patients followed up increased whatever the type of care. In 2020, only the number of patients followed up exclusively in the private sector continued to increase (+138 patients), whereas the number of patients followed up in the hospital sector or with mixed follow-up decreased slightly (see Table 2). Furthermore, the proportion of patients followed up exclusively by the hospital sector has decreased over the last 3 years, particularly in 2020. The change over the entire period was statistically significant (p < 0.0001). Conversely, the proportion of patients followed exclusively by the private sector has been steadily increasing since 2018 (the trend was statistically significant; p < 0.0001). Finally, the proportion of patients with follow-up in both the hospital and private sectors has varied little since 2016. However, there was a slight decrease in 2020 compared to 2018 and 2019, years in which the proportion had stabilized at around 28% of patients (the trend over the whole period being statistically significant; p = 0.006). Because COVID-19 in 2020 led to dramatic changes (lock-downs, cancelation of hospital consultations, etc.), we also computed the trends for 2016–2019, and the observation of a linear increase of the trend toward outpatients was still significant: p = 0.004 and p = 0.002, respectively.\\nFocus on patients with indicators of precariousness (Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire or Aide Médicale Etat).\\nIn 2020, 1,873 patients who had been reimbursed for ARVs were beneficiaries of the CMUC/CSS or the AME (i.e., 61.47% of all patients on ARVs) insurance plans for socially precarious persons. The number of patients in this situation increased until 2019 but decreased in 2020. Overall, the increase over the whole period was 24.53%, with an average annual growth rate of 5.64%. In 2020, 1,082 of the 1,873 patients receiving CMUC/CSS or AME on ARVs had exclusive hospital follow-up (i.e., 57.77%). This proportion steadily decreased over the last 3 years after an initial increase in 2017 (the change over the whole period being statistically significant; p < 0.0001). Conversely, the proportions of patients managed exclusively by the private sector or benefiting from mixed public and private follow-up, after having decreased over the year 2017, increased over the years 2018, 2019, and 2020. The proportion of precarious patients on ARVs exclusively followed up in the private sector thus rose to reach 14.15% in 2020 (a statistically significant trend for the period 2016–2020; p < 0.0001). The proportion of patients with mixed management (combination of public and private follow-up) increased by + 2 points between 2016 and 2020 (the trend was statistically significant over the period; p = 0.0036) to reach more than 28% in 2020 (Table 3).\\nDistribution of the type of antiretroviral follow-up (private practice, hospital, or mixed) in French Guiana between 2016 and 2020 among precarious patients.\\nSource: SNDS/DCIR—Beneficiaries of Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire and Aide Médicale Etat in French Guiana.\\nLinear trend chi2 (private vs. hospital only, 2016–2020): p < 0.001;\\nLinear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.003.\\nBecause of trend distortions resulting from COVID-19, the trend was recalculated for 2016–2019, excluding 2020.\\n*Linear trend chi2 (private vs. hospital only, 2016–2009): p = 0.004; **Linear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.002.\",\n \"The studies involving human participants were reviewed and approved by Commission Nationale Informatique et Libertés. The patients/participants provided their written informed consent to participate in this study.\",\n \"MN: study design. HD and NV: data analysis. HD and MN: first draft writing. CS, LA, AL, FH, LM, JW, NV, EP, and AA: review and editing. All authors contributed to the article and approved the submitted version.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Introduction","Methods","Patients From the Hospital Outpatient Clinics","Lost to Follow-Up","Determination of the Proportion of Patients Receiving Treatment in a Public Hospital or in a Private Practice","Selection of ARV Treatments","Patient Selection","Description of the Evolution of the Type of Care","Data Analysis","Regulatory and Ethical Issues","Results","Hospital Follow-Up in Time","The Total Number of Persons on ARV in French Guiana","Hospital and/or Private Follow-Up","Discussion","Data Availability Statement","Ethics Statement","Author Contributions","Conflict of Interest","Publisher's Note"],"string":"[\n \"Introduction\",\n \"Methods\",\n \"Patients From the Hospital Outpatient Clinics\",\n \"Lost to Follow-Up\",\n \"Determination of the Proportion of Patients Receiving Treatment in a Public Hospital or in a Private Practice\",\n \"Selection of ARV Treatments\",\n \"Patient Selection\",\n \"Description of the Evolution of the Type of Care\",\n \"Data Analysis\",\n \"Regulatory and Ethical Issues\",\n \"Results\",\n \"Hospital Follow-Up in Time\",\n \"The Total Number of Persons on ARV in French Guiana\",\n \"Hospital and/or Private Follow-Up\",\n \"Discussion\",\n \"Data Availability Statement\",\n \"Ethics Statement\",\n \"Author Contributions\",\n \"Conflict of Interest\",\n \"Publisher's Note\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["French Guiana is the French overseas territory where the HIV epidemic is most prevalent (1). Recent modeling using the European Center for Disease Control (ECDC) modeling tool estimated the number of persons living with HIV to be over 3,200, 3,000 of whom knew their diagnosis (2). HIV transmission in French Guiana is mostly heterosexual and three of four patients are foreign citizens (3). French Guiana has the highest gross domestic product (GDP) per capita in South America and, as such, it is attractive for the poor Caribbean and South American populations in search of a better life. In a context of precariousness and sexual vulnerability, a large proportion of infected immigrants actually acquire the virus after their arrival in French Guiana (4, 5). The standards of health care are those of mainland France. Since 2013, antiretroviral (AVR) treatment is recommended for all patients irrespective of their CD4 count. All persons living with HIV receive free or paid AVR treatments regardless of their origin or socio-economic level. Undocumented immigrants with HIV are eligible for residence permits for medical reasons and health insurance coverage. Although the simplification of treatment regimens and follow-up has led to fewer constraints for the patients, their follow-up remains of paramount importance to optimize anti-retroviral therapy to detect and prevent HIV-related morbidity and prevent secondary infections. In 2006, we had studied follow-up interruption and its risk factors in French Guiana (6). Younger patients, foreigners, untreated patients, and non-immunosuppressed patients at the time of diagnosis were more likely to interrupt follow-up. In addition, the greatest risk of interruption was usually within the first 6 months after diagnosis. Since then, nearly all patients are given AVRs with regimens that are more potent but better tolerated, which usually simplifies medical follow-up. In the post 90-90-90 context, French Guiana aims to optimize the cascade of care by improving early testing and treatment and retaining patients in the healthcare system so they can continue to benefit from virological suppression. Despite progress, this remains a problem in France (7, 8). For the specific case of French Guiana, our hypothesis was that different and novel forces were at play and may have modified the adherence to outpatient follow-up. First, in the widespread early treatment with powerful and well-tolerated drugs, the simplification of follow-up would predict that patient retention should increase over time. Second, because patient follow-up is much simpler than before, general practitioners are more inclined to accept such a task, and patients –always weary of being spotted in a specialized HIV outpatient care center—may prefer to be followed closely to their home, in the “stigma-free” practice of their family physician. We therefore looked at the proportion of patients lost to follow-up –from the point of view of hospital outpatient care in different time periods. We also looked, for the first time, at the data from the health insurance system (9), which notably reimburses all antiretroviral drugs (ARVs) to determine the proportion of patients for whom ambulatory care was given from a specialized hospital department and those who only received ambulatory care from a private practitioner."," Patients From the Hospital Outpatient Clinics The information systems for HIV in French Guiana, mandatory reporting of new HIV infections and AIDS cases, and the French Hospital Database of HIV, a cohort for which specifically trained research technicians have collected data on HIV patients since 1989 {first in the DMI2 government program until 2008, then in eNADIS/DATAIDS [Dat'AIDS cohort (clinicaltrials.gov ref. NCT02898987)]}, were used for the study.\nThe information systems for HIV in French Guiana, mandatory reporting of new HIV infections and AIDS cases, and the French Hospital Database of HIV, a cohort for which specifically trained research technicians have collected data on HIV patients since 1989 {first in the DMI2 government program until 2008, then in eNADIS/DATAIDS [Dat'AIDS cohort (clinicaltrials.gov ref. NCT02898987)]}, were used for the study.\n Lost to Follow-Up This cohort allowed us to obtain the number and proportion of patients lost to follow-up (not having consulted in more than 12 months) from the point of view of the hospital system.\nThis cohort allowed us to obtain the number and proportion of patients lost to follow-up (not having consulted in more than 12 months) from the point of view of the hospital system.\n Determination of the Proportion of Patients Receiving Treatment in a Public Hospital or in a Private Practice Since 2017, health administrations and researchers may, with permission, access data from the Système National des Données de Santé which now combines health care reimbursement data (SNIIRAM), hospital data (PMSI), and death certificate data (CepiDC). For French Guiana, this allowed us to obtain, for the first time, the proportion of persons treated for HIV in a public hospital structure or in private practice, or both.\nSince 2017, health administrations and researchers may, with permission, access data from the Système National des Données de Santé which now combines health care reimbursement data (SNIIRAM), hospital data (PMSI), and death certificate data (CepiDC). For French Guiana, this allowed us to obtain, for the first time, the proportion of persons treated for HIV in a public hospital structure or in private practice, or both.\n Selection of ARV Treatments The ARV treatments were selected from the code identifiant de présentation (CIP) code of the Drug Database [base de données publique des médicaments (BDM)], the reference database of drugs reimbursed by the French Health Insurance.\nThe ARV treatments were selected from the code identifiant de présentation (CIP) code of the Drug Database [base de données publique des médicaments (BDM)], the reference database of drugs reimbursed by the French Health Insurance.\n Patient Selection The data used to select the people on ARVs were taken from the inter-scheme consumption datamart (DCIR). All the health insurance beneficiaries were included in the study if they received at least one reimbursement for the delivery of ARVs during the year, even when the treatments were prescribed and/or delivered outside French Guiana. The specific insurance regimens included the General Health Insurance Scheme, including beneficiaries of the free complementary health insurance coverage for the poor French residents or legal migrants (CMUC/CSS) and the state medical aid for undocumented foreigners (AME); the social security system for the self-employed (RSI); and the agricultural social mutual insurance (MSA) of French Guiana.\nA subsample was also made up of patients who received at least one reimbursement for ARVs under the AME or CMUC/CSS during the year. Since AME or CMUC/CSS reflect poverty, these selection criteria allowed to test the hypothesis that trends for private or public follow-up differed between socially precarious and non-precarious populations.\nThe data used to select the people on ARVs were taken from the inter-scheme consumption datamart (DCIR). All the health insurance beneficiaries were included in the study if they received at least one reimbursement for the delivery of ARVs during the year, even when the treatments were prescribed and/or delivered outside French Guiana. The specific insurance regimens included the General Health Insurance Scheme, including beneficiaries of the free complementary health insurance coverage for the poor French residents or legal migrants (CMUC/CSS) and the state medical aid for undocumented foreigners (AME); the social security system for the self-employed (RSI); and the agricultural social mutual insurance (MSA) of French Guiana.\nA subsample was also made up of patients who received at least one reimbursement for ARVs under the AME or CMUC/CSS during the year. Since AME or CMUC/CSS reflect poverty, these selection criteria allowed to test the hypothesis that trends for private or public follow-up differed between socially precarious and non-precarious populations.\n Description of the Evolution of the Type of Care The type of care was defined as follows: exclusively hospital-based when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the hospital sector, exclusively private when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the private sector and mixed when the ARV treatments for which the patient was reimbursed during the year are prescribed by the hospital sector and the private sector.\nThe annual evolution of the distribution of the type of care was studied over the 2016–2020 period in the population of all patients on ARVs at the regional level and in the population, including only CMUC/CSS or AME patients (precarious persons) at the regional level.\nThe type of care was defined as follows: exclusively hospital-based when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the hospital sector, exclusively private when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the private sector and mixed when the ARV treatments for which the patient was reimbursed during the year are prescribed by the hospital sector and the private sector.\nThe annual evolution of the distribution of the type of care was studied over the 2016–2020 period in the population of all patients on ARVs at the regional level and in the population, including only CMUC/CSS or AME patients (precarious persons) at the regional level.\n Data Analysis Using the DAT'AIDS cohort data, single failure survival analyses were performed with follow-up interruption of more than 12 months (not due to death) as a failure event for a patient without knowledge of his or her present follow-up status. Kaplan Meier curves were plotted. Log Rank tests were performed. These analyses allowed us to quantify and visualize the time until follow-up interruption for different periods. For the health insurance data, a linear trend Chi-square test was used to test the statistical significance of the annual change in the different types of care, observed graphically over the period studied. This trend analysis aimed to determine whether the process was gradual.\nUsing the DAT'AIDS cohort data, single failure survival analyses were performed with follow-up interruption of more than 12 months (not due to death) as a failure event for a patient without knowledge of his or her present follow-up status. Kaplan Meier curves were plotted. Log Rank tests were performed. These analyses allowed us to quantify and visualize the time until follow-up interruption for different periods. For the health insurance data, a linear trend Chi-square test was used to test the statistical significance of the annual change in the different types of care, observed graphically over the period studied. This trend analysis aimed to determine whether the process was gradual.\n Regulatory and Ethical Issues Anonymized individual data from the DAT'AIDS database was used (clinicaltrials.gov ref. NCT02898987). For the proportion followed up in private or public structure, the anonymized data from the système national des données de santé (SNDS) is accessible for certain organizations with a public service mission. Particularly, these organizations, listed by decree by the Conseil d'Etat, issued after the opinion of the Commission Nationale Informatique et Libertés (CNIL), may access certain data on a permanent basis in order to carry out their missions. This is the case for researchers of Academic hospitals and the Institut National de la Santé et de la Recherche Médicale (Inserm).\nAnonymized individual data from the DAT'AIDS database was used (clinicaltrials.gov ref. NCT02898987). For the proportion followed up in private or public structure, the anonymized data from the système national des données de santé (SNDS) is accessible for certain organizations with a public service mission. Particularly, these organizations, listed by decree by the Conseil d'Etat, issued after the opinion of the Commission Nationale Informatique et Libertés (CNIL), may access certain data on a permanent basis in order to carry out their missions. This is the case for researchers of Academic hospitals and the Institut National de la Santé et de la Recherche Médicale (Inserm).","The information systems for HIV in French Guiana, mandatory reporting of new HIV infections and AIDS cases, and the French Hospital Database of HIV, a cohort for which specifically trained research technicians have collected data on HIV patients since 1989 {first in the DMI2 government program until 2008, then in eNADIS/DATAIDS [Dat'AIDS cohort (clinicaltrials.gov ref. NCT02898987)]}, were used for the study.","This cohort allowed us to obtain the number and proportion of patients lost to follow-up (not having consulted in more than 12 months) from the point of view of the hospital system.","Since 2017, health administrations and researchers may, with permission, access data from the Système National des Données de Santé which now combines health care reimbursement data (SNIIRAM), hospital data (PMSI), and death certificate data (CepiDC). For French Guiana, this allowed us to obtain, for the first time, the proportion of persons treated for HIV in a public hospital structure or in private practice, or both.","The ARV treatments were selected from the code identifiant de présentation (CIP) code of the Drug Database [base de données publique des médicaments (BDM)], the reference database of drugs reimbursed by the French Health Insurance.","The data used to select the people on ARVs were taken from the inter-scheme consumption datamart (DCIR). All the health insurance beneficiaries were included in the study if they received at least one reimbursement for the delivery of ARVs during the year, even when the treatments were prescribed and/or delivered outside French Guiana. The specific insurance regimens included the General Health Insurance Scheme, including beneficiaries of the free complementary health insurance coverage for the poor French residents or legal migrants (CMUC/CSS) and the state medical aid for undocumented foreigners (AME); the social security system for the self-employed (RSI); and the agricultural social mutual insurance (MSA) of French Guiana.\nA subsample was also made up of patients who received at least one reimbursement for ARVs under the AME or CMUC/CSS during the year. Since AME or CMUC/CSS reflect poverty, these selection criteria allowed to test the hypothesis that trends for private or public follow-up differed between socially precarious and non-precarious populations.","The type of care was defined as follows: exclusively hospital-based when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the hospital sector, exclusively private when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the private sector and mixed when the ARV treatments for which the patient was reimbursed during the year are prescribed by the hospital sector and the private sector.\nThe annual evolution of the distribution of the type of care was studied over the 2016–2020 period in the population of all patients on ARVs at the regional level and in the population, including only CMUC/CSS or AME patients (precarious persons) at the regional level.","Using the DAT'AIDS cohort data, single failure survival analyses were performed with follow-up interruption of more than 12 months (not due to death) as a failure event for a patient without knowledge of his or her present follow-up status. Kaplan Meier curves were plotted. Log Rank tests were performed. These analyses allowed us to quantify and visualize the time until follow-up interruption for different periods. For the health insurance data, a linear trend Chi-square test was used to test the statistical significance of the annual change in the different types of care, observed graphically over the period studied. This trend analysis aimed to determine whether the process was gradual.","Anonymized individual data from the DAT'AIDS database was used (clinicaltrials.gov ref. NCT02898987). For the proportion followed up in private or public structure, the anonymized data from the système national des données de santé (SNDS) is accessible for certain organizations with a public service mission. Particularly, these organizations, listed by decree by the Conseil d'Etat, issued after the opinion of the Commission Nationale Informatique et Libertés (CNIL), may access certain data on a permanent basis in order to carry out their missions. This is the case for researchers of Academic hospitals and the Institut National de la Santé et de la Recherche Médicale (Inserm)."," Hospital Follow-Up in Time We tallied 803 persons that were not known to have died and who were lost to follow-up. Figure 1 shows that over time, patients seemed to be lost to follow-up increasingly sooner (Log-rank test, p < 0.001, Table 1).\nIncidence of follow-up interruption in French Guiana from the point of view of the hospital HIV outpatient cohort.\nLog-rank test comparing the incidence of follow-up interruption for different consecutive years.\nCHI2 (4) = 307.45; p = 0.0000.\nWe tallied 803 persons that were not known to have died and who were lost to follow-up. Figure 1 shows that over time, patients seemed to be lost to follow-up increasingly sooner (Log-rank test, p < 0.001, Table 1).\nIncidence of follow-up interruption in French Guiana from the point of view of the hospital HIV outpatient cohort.\nLog-rank test comparing the incidence of follow-up interruption for different consecutive years.\nCHI2 (4) = 307.45; p = 0.0000.\n The Total Number of Persons on ARV in French Guiana When looking at the health insurance data, in 2020, there were 3,047 patients who benefitted from health insurance payments for ARV treatment. This total number has been steadily increasing since 2016 (see Table 2). The evolution rate over the period was 33.35% with an average annual evolution rate of 7.46%. However, the evolution of the number of patients was more moderate in 2020, a year when activity was completely disorganized by coronavirus disease 2019 (COVID-19) and lockdowns.\nDistribution of the type of antiretroviral (ARV) follow-up (private, hospital, or mixed) in Guiana between 2016 and 2020.\nSource: SNDS/DCIR—Beneficiaries of the RG (CMU/CSS and AME).\nRSI and MSA affiliated in French Guiana, excluding complementary insurances not managed by the computer.\nWhen looking at the health insurance data, in 2020, there were 3,047 patients who benefitted from health insurance payments for ARV treatment. This total number has been steadily increasing since 2016 (see Table 2). The evolution rate over the period was 33.35% with an average annual evolution rate of 7.46%. However, the evolution of the number of patients was more moderate in 2020, a year when activity was completely disorganized by coronavirus disease 2019 (COVID-19) and lockdowns.\nDistribution of the type of antiretroviral (ARV) follow-up (private, hospital, or mixed) in Guiana between 2016 and 2020.\nSource: SNDS/DCIR—Beneficiaries of the RG (CMU/CSS and AME).\nRSI and MSA affiliated in French Guiana, excluding complementary insurances not managed by the computer.\n Hospital and/or Private Follow-Up In 2020, 57.43% (n = 1,750) of patients were followed exclusively by the hospital sector. The proportion of patients treated exclusively in the private sector was 16.31% (n = 497), and that of patients with mixed treatment prescription (private and hospital) was 26.26% (n = 800) (Table 1).\nUntil 2019, the number of patients followed up increased whatever the type of care. In 2020, only the number of patients followed up exclusively in the private sector continued to increase (+138 patients), whereas the number of patients followed up in the hospital sector or with mixed follow-up decreased slightly (see Table 2). Furthermore, the proportion of patients followed up exclusively by the hospital sector has decreased over the last 3 years, particularly in 2020. The change over the entire period was statistically significant (p < 0.0001). Conversely, the proportion of patients followed exclusively by the private sector has been steadily increasing since 2018 (the trend was statistically significant; p < 0.0001). Finally, the proportion of patients with follow-up in both the hospital and private sectors has varied little since 2016. However, there was a slight decrease in 2020 compared to 2018 and 2019, years in which the proportion had stabilized at around 28% of patients (the trend over the whole period being statistically significant; p = 0.006). Because COVID-19 in 2020 led to dramatic changes (lock-downs, cancelation of hospital consultations, etc.), we also computed the trends for 2016–2019, and the observation of a linear increase of the trend toward outpatients was still significant: p = 0.004 and p = 0.002, respectively.\nFocus on patients with indicators of precariousness (Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire or Aide Médicale Etat).\nIn 2020, 1,873 patients who had been reimbursed for ARVs were beneficiaries of the CMUC/CSS or the AME (i.e., 61.47% of all patients on ARVs) insurance plans for socially precarious persons. The number of patients in this situation increased until 2019 but decreased in 2020. Overall, the increase over the whole period was 24.53%, with an average annual growth rate of 5.64%. In 2020, 1,082 of the 1,873 patients receiving CMUC/CSS or AME on ARVs had exclusive hospital follow-up (i.e., 57.77%). This proportion steadily decreased over the last 3 years after an initial increase in 2017 (the change over the whole period being statistically significant; p < 0.0001). Conversely, the proportions of patients managed exclusively by the private sector or benefiting from mixed public and private follow-up, after having decreased over the year 2017, increased over the years 2018, 2019, and 2020. The proportion of precarious patients on ARVs exclusively followed up in the private sector thus rose to reach 14.15% in 2020 (a statistically significant trend for the period 2016–2020; p < 0.0001). The proportion of patients with mixed management (combination of public and private follow-up) increased by + 2 points between 2016 and 2020 (the trend was statistically significant over the period; p = 0.0036) to reach more than 28% in 2020 (Table 3).\nDistribution of the type of antiretroviral follow-up (private practice, hospital, or mixed) in French Guiana between 2016 and 2020 among precarious patients.\nSource: SNDS/DCIR—Beneficiaries of Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire and Aide Médicale Etat in French Guiana.\nLinear trend chi2 (private vs. hospital only, 2016–2020): p < 0.001;\nLinear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.003.\nBecause of trend distortions resulting from COVID-19, the trend was recalculated for 2016–2019, excluding 2020.\n*Linear trend chi2 (private vs. hospital only, 2016–2009): p = 0.004; **Linear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.002.\nIn 2020, 57.43% (n = 1,750) of patients were followed exclusively by the hospital sector. The proportion of patients treated exclusively in the private sector was 16.31% (n = 497), and that of patients with mixed treatment prescription (private and hospital) was 26.26% (n = 800) (Table 1).\nUntil 2019, the number of patients followed up increased whatever the type of care. In 2020, only the number of patients followed up exclusively in the private sector continued to increase (+138 patients), whereas the number of patients followed up in the hospital sector or with mixed follow-up decreased slightly (see Table 2). Furthermore, the proportion of patients followed up exclusively by the hospital sector has decreased over the last 3 years, particularly in 2020. The change over the entire period was statistically significant (p < 0.0001). Conversely, the proportion of patients followed exclusively by the private sector has been steadily increasing since 2018 (the trend was statistically significant; p < 0.0001). Finally, the proportion of patients with follow-up in both the hospital and private sectors has varied little since 2016. However, there was a slight decrease in 2020 compared to 2018 and 2019, years in which the proportion had stabilized at around 28% of patients (the trend over the whole period being statistically significant; p = 0.006). Because COVID-19 in 2020 led to dramatic changes (lock-downs, cancelation of hospital consultations, etc.), we also computed the trends for 2016–2019, and the observation of a linear increase of the trend toward outpatients was still significant: p = 0.004 and p = 0.002, respectively.\nFocus on patients with indicators of precariousness (Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire or Aide Médicale Etat).\nIn 2020, 1,873 patients who had been reimbursed for ARVs were beneficiaries of the CMUC/CSS or the AME (i.e., 61.47% of all patients on ARVs) insurance plans for socially precarious persons. The number of patients in this situation increased until 2019 but decreased in 2020. Overall, the increase over the whole period was 24.53%, with an average annual growth rate of 5.64%. In 2020, 1,082 of the 1,873 patients receiving CMUC/CSS or AME on ARVs had exclusive hospital follow-up (i.e., 57.77%). This proportion steadily decreased over the last 3 years after an initial increase in 2017 (the change over the whole period being statistically significant; p < 0.0001). Conversely, the proportions of patients managed exclusively by the private sector or benefiting from mixed public and private follow-up, after having decreased over the year 2017, increased over the years 2018, 2019, and 2020. The proportion of precarious patients on ARVs exclusively followed up in the private sector thus rose to reach 14.15% in 2020 (a statistically significant trend for the period 2016–2020; p < 0.0001). The proportion of patients with mixed management (combination of public and private follow-up) increased by + 2 points between 2016 and 2020 (the trend was statistically significant over the period; p = 0.0036) to reach more than 28% in 2020 (Table 3).\nDistribution of the type of antiretroviral follow-up (private practice, hospital, or mixed) in French Guiana between 2016 and 2020 among precarious patients.\nSource: SNDS/DCIR—Beneficiaries of Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire and Aide Médicale Etat in French Guiana.\nLinear trend chi2 (private vs. hospital only, 2016–2020): p < 0.001;\nLinear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.003.\nBecause of trend distortions resulting from COVID-19, the trend was recalculated for 2016–2019, excluding 2020.\n*Linear trend chi2 (private vs. hospital only, 2016–2009): p = 0.004; **Linear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.002.","We tallied 803 persons that were not known to have died and who were lost to follow-up. Figure 1 shows that over time, patients seemed to be lost to follow-up increasingly sooner (Log-rank test, p < 0.001, Table 1).\nIncidence of follow-up interruption in French Guiana from the point of view of the hospital HIV outpatient cohort.\nLog-rank test comparing the incidence of follow-up interruption for different consecutive years.\nCHI2 (4) = 307.45; p = 0.0000.","When looking at the health insurance data, in 2020, there were 3,047 patients who benefitted from health insurance payments for ARV treatment. This total number has been steadily increasing since 2016 (see Table 2). The evolution rate over the period was 33.35% with an average annual evolution rate of 7.46%. However, the evolution of the number of patients was more moderate in 2020, a year when activity was completely disorganized by coronavirus disease 2019 (COVID-19) and lockdowns.\nDistribution of the type of antiretroviral (ARV) follow-up (private, hospital, or mixed) in Guiana between 2016 and 2020.\nSource: SNDS/DCIR—Beneficiaries of the RG (CMU/CSS and AME).\nRSI and MSA affiliated in French Guiana, excluding complementary insurances not managed by the computer.","In 2020, 57.43% (n = 1,750) of patients were followed exclusively by the hospital sector. The proportion of patients treated exclusively in the private sector was 16.31% (n = 497), and that of patients with mixed treatment prescription (private and hospital) was 26.26% (n = 800) (Table 1).\nUntil 2019, the number of patients followed up increased whatever the type of care. In 2020, only the number of patients followed up exclusively in the private sector continued to increase (+138 patients), whereas the number of patients followed up in the hospital sector or with mixed follow-up decreased slightly (see Table 2). Furthermore, the proportion of patients followed up exclusively by the hospital sector has decreased over the last 3 years, particularly in 2020. The change over the entire period was statistically significant (p < 0.0001). Conversely, the proportion of patients followed exclusively by the private sector has been steadily increasing since 2018 (the trend was statistically significant; p < 0.0001). Finally, the proportion of patients with follow-up in both the hospital and private sectors has varied little since 2016. However, there was a slight decrease in 2020 compared to 2018 and 2019, years in which the proportion had stabilized at around 28% of patients (the trend over the whole period being statistically significant; p = 0.006). Because COVID-19 in 2020 led to dramatic changes (lock-downs, cancelation of hospital consultations, etc.), we also computed the trends for 2016–2019, and the observation of a linear increase of the trend toward outpatients was still significant: p = 0.004 and p = 0.002, respectively.\nFocus on patients with indicators of precariousness (Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire or Aide Médicale Etat).\nIn 2020, 1,873 patients who had been reimbursed for ARVs were beneficiaries of the CMUC/CSS or the AME (i.e., 61.47% of all patients on ARVs) insurance plans for socially precarious persons. The number of patients in this situation increased until 2019 but decreased in 2020. Overall, the increase over the whole period was 24.53%, with an average annual growth rate of 5.64%. In 2020, 1,082 of the 1,873 patients receiving CMUC/CSS or AME on ARVs had exclusive hospital follow-up (i.e., 57.77%). This proportion steadily decreased over the last 3 years after an initial increase in 2017 (the change over the whole period being statistically significant; p < 0.0001). Conversely, the proportions of patients managed exclusively by the private sector or benefiting from mixed public and private follow-up, after having decreased over the year 2017, increased over the years 2018, 2019, and 2020. The proportion of precarious patients on ARVs exclusively followed up in the private sector thus rose to reach 14.15% in 2020 (a statistically significant trend for the period 2016–2020; p < 0.0001). The proportion of patients with mixed management (combination of public and private follow-up) increased by + 2 points between 2016 and 2020 (the trend was statistically significant over the period; p = 0.0036) to reach more than 28% in 2020 (Table 3).\nDistribution of the type of antiretroviral follow-up (private practice, hospital, or mixed) in French Guiana between 2016 and 2020 among precarious patients.\nSource: SNDS/DCIR—Beneficiaries of Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire and Aide Médicale Etat in French Guiana.\nLinear trend chi2 (private vs. hospital only, 2016–2020): p < 0.001;\nLinear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.003.\nBecause of trend distortions resulting from COVID-19, the trend was recalculated for 2016–2019, excluding 2020.\n*Linear trend chi2 (private vs. hospital only, 2016–2009): p = 0.004; **Linear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.002.","For the first time, the use of health insurance data allowed us to see the whole picture of HIV care in French Guiana. The actual number of persons benefiting from antiretroviral treatment in French Guiana was actually very close to the recent estimations using HIV-cohort data for the ECDC modeling tool (1). The number of persons on AVR treatment has substantially increased over the study time but slowed down in 2020. Given the simultaneous disruption of information systems and HIV testing and care, it is difficult to tease out whether this reflects the slowing down of an epidemic where increasing numbers of persons are virologically controlled, or the COVID-19-related gap in new diagnoses and patient referrals. Data from 2021 is also likely to be impacted by COVID-19, so only future post-COVID-19 years may allow us to find out a better explanation for our observation.\nWhile the organization of care and virological results had gradually improved over the years, we were initially surprised to see the rate of persons lost to follow-up from the hospital outpatient cohort actually increased over time. However, clinicians had perceived the feedback from patients increasingly tempted by a follow-up by their private practitioner for proximity reasons and because going to specialized HIV outpatient departments was more stigmatizing than a waiting room of a general practitioner. At the same time, treatment regimens and follow-up are far simpler than they used to be, which expands the number of physicians willing to take-on the task of being the referent physician of patients with HIV. Furthermore, the aging HIV-cohort (39.5% aged over 50 years) increasingly requires general practitioners–not infectious disease specialists—to manage comorbidities, which may be another incentive to have an integral follow-up by one's family physician instead of a specialist. This perception seems to be vindicated by the health insurance data, which shows a growing proportion of persons choosing to be exclusively treated by their private practitioner. Furthermore, about a quarter used both the private and public sectors, suggesting pragmatic convenience was behind this behavior. The COVID-19 disruption of HIV follow-up may have further tilted follow-up toward private practice in 2020, and it remains to be seen whether this will be corrected in the future or if it has accelerated an ongoing trend toward decentralization of HIV care.\nAt first view, one could have hypothesized that this trend toward private practice did not include the most precarious cases which in French Guiana's HIV cohort, represents 2/3rds of patients if we only look at the type of Health Insurance. However, this was refuted by the data which shows the exact same trend among the more precarious and shows that, contrary to widespread beliefs, poor populations do have access to private practice and use it readily as soon as they have health coverage. Since 2000, the Public Health Law has ensured that the most precarious cases do not have to advance any fees for medical consultations, and thus there are no financial obstacles to consult a private practitioner instead of a hospital physician.\nThe study has a number of limitations. First, the use of health insurance regimens as a proxy for poverty is legitimate, but it may actually underestimate the degree of precariousness. Particularly, the SNDS did not provide other indicators, such as country of birth or nationality, which may have served as another proxy for precariousness. Another limitation is that the simultaneous accelerating rate of persons being lost to follow-up from the hospital system and the increasing proportion of patients exclusively followed by private practitioners do not formally prove that those that do not come back in the outpatient ward are actually consulting private practitioners. Instead, they could have left the territory, or stopped treatment and follow-up, or died. However, for deaths or treatment interruptions, this seems largely unlikely because opportunistic infections and deaths –which eventually are counted—have substantially decreased over time suggesting that indeed many of the patients who do not come back to the hospital outpatient clinic are receiving care in private practices.\nIn conclusion, while hospital outpatient care remains by far the most common mode of patient care, there seems to be a gradual erosion of this model in favor of private practice. For the first time, the Health Insurance information system allows obtaining a broader view of patient care than hospital cohort data alone. In the rapidly evolving therapeutic and conceptual landscape of HIV care, in the context of aging cohorts and shifting patient preferences, coordinating continuous medical education with private practitioners seems to be crucially important so that every caregiver is up to date with the current guidelines. This slow gradual process may even have accelerated in the transformational context of the COVID-19 pandemic.","The data analyzed in this study is subject to the following licenses/restrictions: The DATAIDS data may be shared upon reasonable request. The health Insurance data requires additional permissions. Requests to access these datasets should be directed to corevih@ch-cayenne.","The studies involving human participants were reviewed and approved by Commission Nationale Informatique et Libertés. The patients/participants provided their written informed consent to participate in this study.","MN: study design. HD and NV: data analysis. HD and MN: first draft writing. CS, LA, AL, FH, LM, JW, NV, EP, and AA: review and editing. All authors contributed to the article and approved the submitted version.","The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.","All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."],"string":"[\n \"French Guiana is the French overseas territory where the HIV epidemic is most prevalent (1). Recent modeling using the European Center for Disease Control (ECDC) modeling tool estimated the number of persons living with HIV to be over 3,200, 3,000 of whom knew their diagnosis (2). HIV transmission in French Guiana is mostly heterosexual and three of four patients are foreign citizens (3). French Guiana has the highest gross domestic product (GDP) per capita in South America and, as such, it is attractive for the poor Caribbean and South American populations in search of a better life. In a context of precariousness and sexual vulnerability, a large proportion of infected immigrants actually acquire the virus after their arrival in French Guiana (4, 5). The standards of health care are those of mainland France. Since 2013, antiretroviral (AVR) treatment is recommended for all patients irrespective of their CD4 count. All persons living with HIV receive free or paid AVR treatments regardless of their origin or socio-economic level. Undocumented immigrants with HIV are eligible for residence permits for medical reasons and health insurance coverage. Although the simplification of treatment regimens and follow-up has led to fewer constraints for the patients, their follow-up remains of paramount importance to optimize anti-retroviral therapy to detect and prevent HIV-related morbidity and prevent secondary infections. In 2006, we had studied follow-up interruption and its risk factors in French Guiana (6). Younger patients, foreigners, untreated patients, and non-immunosuppressed patients at the time of diagnosis were more likely to interrupt follow-up. In addition, the greatest risk of interruption was usually within the first 6 months after diagnosis. Since then, nearly all patients are given AVRs with regimens that are more potent but better tolerated, which usually simplifies medical follow-up. In the post 90-90-90 context, French Guiana aims to optimize the cascade of care by improving early testing and treatment and retaining patients in the healthcare system so they can continue to benefit from virological suppression. Despite progress, this remains a problem in France (7, 8). For the specific case of French Guiana, our hypothesis was that different and novel forces were at play and may have modified the adherence to outpatient follow-up. First, in the widespread early treatment with powerful and well-tolerated drugs, the simplification of follow-up would predict that patient retention should increase over time. Second, because patient follow-up is much simpler than before, general practitioners are more inclined to accept such a task, and patients –always weary of being spotted in a specialized HIV outpatient care center—may prefer to be followed closely to their home, in the “stigma-free” practice of their family physician. We therefore looked at the proportion of patients lost to follow-up –from the point of view of hospital outpatient care in different time periods. We also looked, for the first time, at the data from the health insurance system (9), which notably reimburses all antiretroviral drugs (ARVs) to determine the proportion of patients for whom ambulatory care was given from a specialized hospital department and those who only received ambulatory care from a private practitioner.\",\n \" Patients From the Hospital Outpatient Clinics The information systems for HIV in French Guiana, mandatory reporting of new HIV infections and AIDS cases, and the French Hospital Database of HIV, a cohort for which specifically trained research technicians have collected data on HIV patients since 1989 {first in the DMI2 government program until 2008, then in eNADIS/DATAIDS [Dat'AIDS cohort (clinicaltrials.gov ref. NCT02898987)]}, were used for the study.\\nThe information systems for HIV in French Guiana, mandatory reporting of new HIV infections and AIDS cases, and the French Hospital Database of HIV, a cohort for which specifically trained research technicians have collected data on HIV patients since 1989 {first in the DMI2 government program until 2008, then in eNADIS/DATAIDS [Dat'AIDS cohort (clinicaltrials.gov ref. NCT02898987)]}, were used for the study.\\n Lost to Follow-Up This cohort allowed us to obtain the number and proportion of patients lost to follow-up (not having consulted in more than 12 months) from the point of view of the hospital system.\\nThis cohort allowed us to obtain the number and proportion of patients lost to follow-up (not having consulted in more than 12 months) from the point of view of the hospital system.\\n Determination of the Proportion of Patients Receiving Treatment in a Public Hospital or in a Private Practice Since 2017, health administrations and researchers may, with permission, access data from the Système National des Données de Santé which now combines health care reimbursement data (SNIIRAM), hospital data (PMSI), and death certificate data (CepiDC). For French Guiana, this allowed us to obtain, for the first time, the proportion of persons treated for HIV in a public hospital structure or in private practice, or both.\\nSince 2017, health administrations and researchers may, with permission, access data from the Système National des Données de Santé which now combines health care reimbursement data (SNIIRAM), hospital data (PMSI), and death certificate data (CepiDC). For French Guiana, this allowed us to obtain, for the first time, the proportion of persons treated for HIV in a public hospital structure or in private practice, or both.\\n Selection of ARV Treatments The ARV treatments were selected from the code identifiant de présentation (CIP) code of the Drug Database [base de données publique des médicaments (BDM)], the reference database of drugs reimbursed by the French Health Insurance.\\nThe ARV treatments were selected from the code identifiant de présentation (CIP) code of the Drug Database [base de données publique des médicaments (BDM)], the reference database of drugs reimbursed by the French Health Insurance.\\n Patient Selection The data used to select the people on ARVs were taken from the inter-scheme consumption datamart (DCIR). All the health insurance beneficiaries were included in the study if they received at least one reimbursement for the delivery of ARVs during the year, even when the treatments were prescribed and/or delivered outside French Guiana. The specific insurance regimens included the General Health Insurance Scheme, including beneficiaries of the free complementary health insurance coverage for the poor French residents or legal migrants (CMUC/CSS) and the state medical aid for undocumented foreigners (AME); the social security system for the self-employed (RSI); and the agricultural social mutual insurance (MSA) of French Guiana.\\nA subsample was also made up of patients who received at least one reimbursement for ARVs under the AME or CMUC/CSS during the year. Since AME or CMUC/CSS reflect poverty, these selection criteria allowed to test the hypothesis that trends for private or public follow-up differed between socially precarious and non-precarious populations.\\nThe data used to select the people on ARVs were taken from the inter-scheme consumption datamart (DCIR). All the health insurance beneficiaries were included in the study if they received at least one reimbursement for the delivery of ARVs during the year, even when the treatments were prescribed and/or delivered outside French Guiana. The specific insurance regimens included the General Health Insurance Scheme, including beneficiaries of the free complementary health insurance coverage for the poor French residents or legal migrants (CMUC/CSS) and the state medical aid for undocumented foreigners (AME); the social security system for the self-employed (RSI); and the agricultural social mutual insurance (MSA) of French Guiana.\\nA subsample was also made up of patients who received at least one reimbursement for ARVs under the AME or CMUC/CSS during the year. Since AME or CMUC/CSS reflect poverty, these selection criteria allowed to test the hypothesis that trends for private or public follow-up differed between socially precarious and non-precarious populations.\\n Description of the Evolution of the Type of Care The type of care was defined as follows: exclusively hospital-based when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the hospital sector, exclusively private when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the private sector and mixed when the ARV treatments for which the patient was reimbursed during the year are prescribed by the hospital sector and the private sector.\\nThe annual evolution of the distribution of the type of care was studied over the 2016–2020 period in the population of all patients on ARVs at the regional level and in the population, including only CMUC/CSS or AME patients (precarious persons) at the regional level.\\nThe type of care was defined as follows: exclusively hospital-based when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the hospital sector, exclusively private when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the private sector and mixed when the ARV treatments for which the patient was reimbursed during the year are prescribed by the hospital sector and the private sector.\\nThe annual evolution of the distribution of the type of care was studied over the 2016–2020 period in the population of all patients on ARVs at the regional level and in the population, including only CMUC/CSS or AME patients (precarious persons) at the regional level.\\n Data Analysis Using the DAT'AIDS cohort data, single failure survival analyses were performed with follow-up interruption of more than 12 months (not due to death) as a failure event for a patient without knowledge of his or her present follow-up status. Kaplan Meier curves were plotted. Log Rank tests were performed. These analyses allowed us to quantify and visualize the time until follow-up interruption for different periods. For the health insurance data, a linear trend Chi-square test was used to test the statistical significance of the annual change in the different types of care, observed graphically over the period studied. This trend analysis aimed to determine whether the process was gradual.\\nUsing the DAT'AIDS cohort data, single failure survival analyses were performed with follow-up interruption of more than 12 months (not due to death) as a failure event for a patient without knowledge of his or her present follow-up status. Kaplan Meier curves were plotted. Log Rank tests were performed. These analyses allowed us to quantify and visualize the time until follow-up interruption for different periods. For the health insurance data, a linear trend Chi-square test was used to test the statistical significance of the annual change in the different types of care, observed graphically over the period studied. This trend analysis aimed to determine whether the process was gradual.\\n Regulatory and Ethical Issues Anonymized individual data from the DAT'AIDS database was used (clinicaltrials.gov ref. NCT02898987). For the proportion followed up in private or public structure, the anonymized data from the système national des données de santé (SNDS) is accessible for certain organizations with a public service mission. Particularly, these organizations, listed by decree by the Conseil d'Etat, issued after the opinion of the Commission Nationale Informatique et Libertés (CNIL), may access certain data on a permanent basis in order to carry out their missions. This is the case for researchers of Academic hospitals and the Institut National de la Santé et de la Recherche Médicale (Inserm).\\nAnonymized individual data from the DAT'AIDS database was used (clinicaltrials.gov ref. NCT02898987). For the proportion followed up in private or public structure, the anonymized data from the système national des données de santé (SNDS) is accessible for certain organizations with a public service mission. Particularly, these organizations, listed by decree by the Conseil d'Etat, issued after the opinion of the Commission Nationale Informatique et Libertés (CNIL), may access certain data on a permanent basis in order to carry out their missions. This is the case for researchers of Academic hospitals and the Institut National de la Santé et de la Recherche Médicale (Inserm).\",\n \"The information systems for HIV in French Guiana, mandatory reporting of new HIV infections and AIDS cases, and the French Hospital Database of HIV, a cohort for which specifically trained research technicians have collected data on HIV patients since 1989 {first in the DMI2 government program until 2008, then in eNADIS/DATAIDS [Dat'AIDS cohort (clinicaltrials.gov ref. NCT02898987)]}, were used for the study.\",\n \"This cohort allowed us to obtain the number and proportion of patients lost to follow-up (not having consulted in more than 12 months) from the point of view of the hospital system.\",\n \"Since 2017, health administrations and researchers may, with permission, access data from the Système National des Données de Santé which now combines health care reimbursement data (SNIIRAM), hospital data (PMSI), and death certificate data (CepiDC). For French Guiana, this allowed us to obtain, for the first time, the proportion of persons treated for HIV in a public hospital structure or in private practice, or both.\",\n \"The ARV treatments were selected from the code identifiant de présentation (CIP) code of the Drug Database [base de données publique des médicaments (BDM)], the reference database of drugs reimbursed by the French Health Insurance.\",\n \"The data used to select the people on ARVs were taken from the inter-scheme consumption datamart (DCIR). All the health insurance beneficiaries were included in the study if they received at least one reimbursement for the delivery of ARVs during the year, even when the treatments were prescribed and/or delivered outside French Guiana. The specific insurance regimens included the General Health Insurance Scheme, including beneficiaries of the free complementary health insurance coverage for the poor French residents or legal migrants (CMUC/CSS) and the state medical aid for undocumented foreigners (AME); the social security system for the self-employed (RSI); and the agricultural social mutual insurance (MSA) of French Guiana.\\nA subsample was also made up of patients who received at least one reimbursement for ARVs under the AME or CMUC/CSS during the year. Since AME or CMUC/CSS reflect poverty, these selection criteria allowed to test the hypothesis that trends for private or public follow-up differed between socially precarious and non-precarious populations.\",\n \"The type of care was defined as follows: exclusively hospital-based when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the hospital sector, exclusively private when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the private sector and mixed when the ARV treatments for which the patient was reimbursed during the year are prescribed by the hospital sector and the private sector.\\nThe annual evolution of the distribution of the type of care was studied over the 2016–2020 period in the population of all patients on ARVs at the regional level and in the population, including only CMUC/CSS or AME patients (precarious persons) at the regional level.\",\n \"Using the DAT'AIDS cohort data, single failure survival analyses were performed with follow-up interruption of more than 12 months (not due to death) as a failure event for a patient without knowledge of his or her present follow-up status. Kaplan Meier curves were plotted. Log Rank tests were performed. These analyses allowed us to quantify and visualize the time until follow-up interruption for different periods. For the health insurance data, a linear trend Chi-square test was used to test the statistical significance of the annual change in the different types of care, observed graphically over the period studied. This trend analysis aimed to determine whether the process was gradual.\",\n \"Anonymized individual data from the DAT'AIDS database was used (clinicaltrials.gov ref. NCT02898987). For the proportion followed up in private or public structure, the anonymized data from the système national des données de santé (SNDS) is accessible for certain organizations with a public service mission. Particularly, these organizations, listed by decree by the Conseil d'Etat, issued after the opinion of the Commission Nationale Informatique et Libertés (CNIL), may access certain data on a permanent basis in order to carry out their missions. This is the case for researchers of Academic hospitals and the Institut National de la Santé et de la Recherche Médicale (Inserm).\",\n \" Hospital Follow-Up in Time We tallied 803 persons that were not known to have died and who were lost to follow-up. Figure 1 shows that over time, patients seemed to be lost to follow-up increasingly sooner (Log-rank test, p < 0.001, Table 1).\\nIncidence of follow-up interruption in French Guiana from the point of view of the hospital HIV outpatient cohort.\\nLog-rank test comparing the incidence of follow-up interruption for different consecutive years.\\nCHI2 (4) = 307.45; p = 0.0000.\\nWe tallied 803 persons that were not known to have died and who were lost to follow-up. Figure 1 shows that over time, patients seemed to be lost to follow-up increasingly sooner (Log-rank test, p < 0.001, Table 1).\\nIncidence of follow-up interruption in French Guiana from the point of view of the hospital HIV outpatient cohort.\\nLog-rank test comparing the incidence of follow-up interruption for different consecutive years.\\nCHI2 (4) = 307.45; p = 0.0000.\\n The Total Number of Persons on ARV in French Guiana When looking at the health insurance data, in 2020, there were 3,047 patients who benefitted from health insurance payments for ARV treatment. This total number has been steadily increasing since 2016 (see Table 2). The evolution rate over the period was 33.35% with an average annual evolution rate of 7.46%. However, the evolution of the number of patients was more moderate in 2020, a year when activity was completely disorganized by coronavirus disease 2019 (COVID-19) and lockdowns.\\nDistribution of the type of antiretroviral (ARV) follow-up (private, hospital, or mixed) in Guiana between 2016 and 2020.\\nSource: SNDS/DCIR—Beneficiaries of the RG (CMU/CSS and AME).\\nRSI and MSA affiliated in French Guiana, excluding complementary insurances not managed by the computer.\\nWhen looking at the health insurance data, in 2020, there were 3,047 patients who benefitted from health insurance payments for ARV treatment. This total number has been steadily increasing since 2016 (see Table 2). The evolution rate over the period was 33.35% with an average annual evolution rate of 7.46%. However, the evolution of the number of patients was more moderate in 2020, a year when activity was completely disorganized by coronavirus disease 2019 (COVID-19) and lockdowns.\\nDistribution of the type of antiretroviral (ARV) follow-up (private, hospital, or mixed) in Guiana between 2016 and 2020.\\nSource: SNDS/DCIR—Beneficiaries of the RG (CMU/CSS and AME).\\nRSI and MSA affiliated in French Guiana, excluding complementary insurances not managed by the computer.\\n Hospital and/or Private Follow-Up In 2020, 57.43% (n = 1,750) of patients were followed exclusively by the hospital sector. The proportion of patients treated exclusively in the private sector was 16.31% (n = 497), and that of patients with mixed treatment prescription (private and hospital) was 26.26% (n = 800) (Table 1).\\nUntil 2019, the number of patients followed up increased whatever the type of care. In 2020, only the number of patients followed up exclusively in the private sector continued to increase (+138 patients), whereas the number of patients followed up in the hospital sector or with mixed follow-up decreased slightly (see Table 2). Furthermore, the proportion of patients followed up exclusively by the hospital sector has decreased over the last 3 years, particularly in 2020. The change over the entire period was statistically significant (p < 0.0001). Conversely, the proportion of patients followed exclusively by the private sector has been steadily increasing since 2018 (the trend was statistically significant; p < 0.0001). Finally, the proportion of patients with follow-up in both the hospital and private sectors has varied little since 2016. However, there was a slight decrease in 2020 compared to 2018 and 2019, years in which the proportion had stabilized at around 28% of patients (the trend over the whole period being statistically significant; p = 0.006). Because COVID-19 in 2020 led to dramatic changes (lock-downs, cancelation of hospital consultations, etc.), we also computed the trends for 2016–2019, and the observation of a linear increase of the trend toward outpatients was still significant: p = 0.004 and p = 0.002, respectively.\\nFocus on patients with indicators of precariousness (Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire or Aide Médicale Etat).\\nIn 2020, 1,873 patients who had been reimbursed for ARVs were beneficiaries of the CMUC/CSS or the AME (i.e., 61.47% of all patients on ARVs) insurance plans for socially precarious persons. The number of patients in this situation increased until 2019 but decreased in 2020. Overall, the increase over the whole period was 24.53%, with an average annual growth rate of 5.64%. In 2020, 1,082 of the 1,873 patients receiving CMUC/CSS or AME on ARVs had exclusive hospital follow-up (i.e., 57.77%). This proportion steadily decreased over the last 3 years after an initial increase in 2017 (the change over the whole period being statistically significant; p < 0.0001). Conversely, the proportions of patients managed exclusively by the private sector or benefiting from mixed public and private follow-up, after having decreased over the year 2017, increased over the years 2018, 2019, and 2020. The proportion of precarious patients on ARVs exclusively followed up in the private sector thus rose to reach 14.15% in 2020 (a statistically significant trend for the period 2016–2020; p < 0.0001). The proportion of patients with mixed management (combination of public and private follow-up) increased by + 2 points between 2016 and 2020 (the trend was statistically significant over the period; p = 0.0036) to reach more than 28% in 2020 (Table 3).\\nDistribution of the type of antiretroviral follow-up (private practice, hospital, or mixed) in French Guiana between 2016 and 2020 among precarious patients.\\nSource: SNDS/DCIR—Beneficiaries of Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire and Aide Médicale Etat in French Guiana.\\nLinear trend chi2 (private vs. hospital only, 2016–2020): p < 0.001;\\nLinear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.003.\\nBecause of trend distortions resulting from COVID-19, the trend was recalculated for 2016–2019, excluding 2020.\\n*Linear trend chi2 (private vs. hospital only, 2016–2009): p = 0.004; **Linear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.002.\\nIn 2020, 57.43% (n = 1,750) of patients were followed exclusively by the hospital sector. The proportion of patients treated exclusively in the private sector was 16.31% (n = 497), and that of patients with mixed treatment prescription (private and hospital) was 26.26% (n = 800) (Table 1).\\nUntil 2019, the number of patients followed up increased whatever the type of care. In 2020, only the number of patients followed up exclusively in the private sector continued to increase (+138 patients), whereas the number of patients followed up in the hospital sector or with mixed follow-up decreased slightly (see Table 2). Furthermore, the proportion of patients followed up exclusively by the hospital sector has decreased over the last 3 years, particularly in 2020. The change over the entire period was statistically significant (p < 0.0001). Conversely, the proportion of patients followed exclusively by the private sector has been steadily increasing since 2018 (the trend was statistically significant; p < 0.0001). Finally, the proportion of patients with follow-up in both the hospital and private sectors has varied little since 2016. However, there was a slight decrease in 2020 compared to 2018 and 2019, years in which the proportion had stabilized at around 28% of patients (the trend over the whole period being statistically significant; p = 0.006). Because COVID-19 in 2020 led to dramatic changes (lock-downs, cancelation of hospital consultations, etc.), we also computed the trends for 2016–2019, and the observation of a linear increase of the trend toward outpatients was still significant: p = 0.004 and p = 0.002, respectively.\\nFocus on patients with indicators of precariousness (Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire or Aide Médicale Etat).\\nIn 2020, 1,873 patients who had been reimbursed for ARVs were beneficiaries of the CMUC/CSS or the AME (i.e., 61.47% of all patients on ARVs) insurance plans for socially precarious persons. The number of patients in this situation increased until 2019 but decreased in 2020. Overall, the increase over the whole period was 24.53%, with an average annual growth rate of 5.64%. In 2020, 1,082 of the 1,873 patients receiving CMUC/CSS or AME on ARVs had exclusive hospital follow-up (i.e., 57.77%). This proportion steadily decreased over the last 3 years after an initial increase in 2017 (the change over the whole period being statistically significant; p < 0.0001). Conversely, the proportions of patients managed exclusively by the private sector or benefiting from mixed public and private follow-up, after having decreased over the year 2017, increased over the years 2018, 2019, and 2020. The proportion of precarious patients on ARVs exclusively followed up in the private sector thus rose to reach 14.15% in 2020 (a statistically significant trend for the period 2016–2020; p < 0.0001). The proportion of patients with mixed management (combination of public and private follow-up) increased by + 2 points between 2016 and 2020 (the trend was statistically significant over the period; p = 0.0036) to reach more than 28% in 2020 (Table 3).\\nDistribution of the type of antiretroviral follow-up (private practice, hospital, or mixed) in French Guiana between 2016 and 2020 among precarious patients.\\nSource: SNDS/DCIR—Beneficiaries of Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire and Aide Médicale Etat in French Guiana.\\nLinear trend chi2 (private vs. hospital only, 2016–2020): p < 0.001;\\nLinear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.003.\\nBecause of trend distortions resulting from COVID-19, the trend was recalculated for 2016–2019, excluding 2020.\\n*Linear trend chi2 (private vs. hospital only, 2016–2009): p = 0.004; **Linear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.002.\",\n \"We tallied 803 persons that were not known to have died and who were lost to follow-up. Figure 1 shows that over time, patients seemed to be lost to follow-up increasingly sooner (Log-rank test, p < 0.001, Table 1).\\nIncidence of follow-up interruption in French Guiana from the point of view of the hospital HIV outpatient cohort.\\nLog-rank test comparing the incidence of follow-up interruption for different consecutive years.\\nCHI2 (4) = 307.45; p = 0.0000.\",\n \"When looking at the health insurance data, in 2020, there were 3,047 patients who benefitted from health insurance payments for ARV treatment. This total number has been steadily increasing since 2016 (see Table 2). The evolution rate over the period was 33.35% with an average annual evolution rate of 7.46%. However, the evolution of the number of patients was more moderate in 2020, a year when activity was completely disorganized by coronavirus disease 2019 (COVID-19) and lockdowns.\\nDistribution of the type of antiretroviral (ARV) follow-up (private, hospital, or mixed) in Guiana between 2016 and 2020.\\nSource: SNDS/DCIR—Beneficiaries of the RG (CMU/CSS and AME).\\nRSI and MSA affiliated in French Guiana, excluding complementary insurances not managed by the computer.\",\n \"In 2020, 57.43% (n = 1,750) of patients were followed exclusively by the hospital sector. The proportion of patients treated exclusively in the private sector was 16.31% (n = 497), and that of patients with mixed treatment prescription (private and hospital) was 26.26% (n = 800) (Table 1).\\nUntil 2019, the number of patients followed up increased whatever the type of care. In 2020, only the number of patients followed up exclusively in the private sector continued to increase (+138 patients), whereas the number of patients followed up in the hospital sector or with mixed follow-up decreased slightly (see Table 2). Furthermore, the proportion of patients followed up exclusively by the hospital sector has decreased over the last 3 years, particularly in 2020. The change over the entire period was statistically significant (p < 0.0001). Conversely, the proportion of patients followed exclusively by the private sector has been steadily increasing since 2018 (the trend was statistically significant; p < 0.0001). Finally, the proportion of patients with follow-up in both the hospital and private sectors has varied little since 2016. However, there was a slight decrease in 2020 compared to 2018 and 2019, years in which the proportion had stabilized at around 28% of patients (the trend over the whole period being statistically significant; p = 0.006). Because COVID-19 in 2020 led to dramatic changes (lock-downs, cancelation of hospital consultations, etc.), we also computed the trends for 2016–2019, and the observation of a linear increase of the trend toward outpatients was still significant: p = 0.004 and p = 0.002, respectively.\\nFocus on patients with indicators of precariousness (Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire or Aide Médicale Etat).\\nIn 2020, 1,873 patients who had been reimbursed for ARVs were beneficiaries of the CMUC/CSS or the AME (i.e., 61.47% of all patients on ARVs) insurance plans for socially precarious persons. The number of patients in this situation increased until 2019 but decreased in 2020. Overall, the increase over the whole period was 24.53%, with an average annual growth rate of 5.64%. In 2020, 1,082 of the 1,873 patients receiving CMUC/CSS or AME on ARVs had exclusive hospital follow-up (i.e., 57.77%). This proportion steadily decreased over the last 3 years after an initial increase in 2017 (the change over the whole period being statistically significant; p < 0.0001). Conversely, the proportions of patients managed exclusively by the private sector or benefiting from mixed public and private follow-up, after having decreased over the year 2017, increased over the years 2018, 2019, and 2020. The proportion of precarious patients on ARVs exclusively followed up in the private sector thus rose to reach 14.15% in 2020 (a statistically significant trend for the period 2016–2020; p < 0.0001). The proportion of patients with mixed management (combination of public and private follow-up) increased by + 2 points between 2016 and 2020 (the trend was statistically significant over the period; p = 0.0036) to reach more than 28% in 2020 (Table 3).\\nDistribution of the type of antiretroviral follow-up (private practice, hospital, or mixed) in French Guiana between 2016 and 2020 among precarious patients.\\nSource: SNDS/DCIR—Beneficiaries of Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire and Aide Médicale Etat in French Guiana.\\nLinear trend chi2 (private vs. hospital only, 2016–2020): p < 0.001;\\nLinear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.003.\\nBecause of trend distortions resulting from COVID-19, the trend was recalculated for 2016–2019, excluding 2020.\\n*Linear trend chi2 (private vs. hospital only, 2016–2009): p = 0.004; **Linear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.002.\",\n \"For the first time, the use of health insurance data allowed us to see the whole picture of HIV care in French Guiana. The actual number of persons benefiting from antiretroviral treatment in French Guiana was actually very close to the recent estimations using HIV-cohort data for the ECDC modeling tool (1). The number of persons on AVR treatment has substantially increased over the study time but slowed down in 2020. Given the simultaneous disruption of information systems and HIV testing and care, it is difficult to tease out whether this reflects the slowing down of an epidemic where increasing numbers of persons are virologically controlled, or the COVID-19-related gap in new diagnoses and patient referrals. Data from 2021 is also likely to be impacted by COVID-19, so only future post-COVID-19 years may allow us to find out a better explanation for our observation.\\nWhile the organization of care and virological results had gradually improved over the years, we were initially surprised to see the rate of persons lost to follow-up from the hospital outpatient cohort actually increased over time. However, clinicians had perceived the feedback from patients increasingly tempted by a follow-up by their private practitioner for proximity reasons and because going to specialized HIV outpatient departments was more stigmatizing than a waiting room of a general practitioner. At the same time, treatment regimens and follow-up are far simpler than they used to be, which expands the number of physicians willing to take-on the task of being the referent physician of patients with HIV. Furthermore, the aging HIV-cohort (39.5% aged over 50 years) increasingly requires general practitioners–not infectious disease specialists—to manage comorbidities, which may be another incentive to have an integral follow-up by one's family physician instead of a specialist. This perception seems to be vindicated by the health insurance data, which shows a growing proportion of persons choosing to be exclusively treated by their private practitioner. Furthermore, about a quarter used both the private and public sectors, suggesting pragmatic convenience was behind this behavior. The COVID-19 disruption of HIV follow-up may have further tilted follow-up toward private practice in 2020, and it remains to be seen whether this will be corrected in the future or if it has accelerated an ongoing trend toward decentralization of HIV care.\\nAt first view, one could have hypothesized that this trend toward private practice did not include the most precarious cases which in French Guiana's HIV cohort, represents 2/3rds of patients if we only look at the type of Health Insurance. However, this was refuted by the data which shows the exact same trend among the more precarious and shows that, contrary to widespread beliefs, poor populations do have access to private practice and use it readily as soon as they have health coverage. Since 2000, the Public Health Law has ensured that the most precarious cases do not have to advance any fees for medical consultations, and thus there are no financial obstacles to consult a private practitioner instead of a hospital physician.\\nThe study has a number of limitations. First, the use of health insurance regimens as a proxy for poverty is legitimate, but it may actually underestimate the degree of precariousness. Particularly, the SNDS did not provide other indicators, such as country of birth or nationality, which may have served as another proxy for precariousness. Another limitation is that the simultaneous accelerating rate of persons being lost to follow-up from the hospital system and the increasing proportion of patients exclusively followed by private practitioners do not formally prove that those that do not come back in the outpatient ward are actually consulting private practitioners. Instead, they could have left the territory, or stopped treatment and follow-up, or died. However, for deaths or treatment interruptions, this seems largely unlikely because opportunistic infections and deaths –which eventually are counted—have substantially decreased over time suggesting that indeed many of the patients who do not come back to the hospital outpatient clinic are receiving care in private practices.\\nIn conclusion, while hospital outpatient care remains by far the most common mode of patient care, there seems to be a gradual erosion of this model in favor of private practice. For the first time, the Health Insurance information system allows obtaining a broader view of patient care than hospital cohort data alone. In the rapidly evolving therapeutic and conceptual landscape of HIV care, in the context of aging cohorts and shifting patient preferences, coordinating continuous medical education with private practitioners seems to be crucially important so that every caregiver is up to date with the current guidelines. This slow gradual process may even have accelerated in the transformational context of the COVID-19 pandemic.\",\n \"The data analyzed in this study is subject to the following licenses/restrictions: The DATAIDS data may be shared upon reasonable request. The health Insurance data requires additional permissions. Requests to access these datasets should be directed to corevih@ch-cayenne.\",\n \"The studies involving human participants were reviewed and approved by Commission Nationale Informatique et Libertés. The patients/participants provided their written informed consent to participate in this study.\",\n \"MN: study design. HD and NV: data analysis. HD and MN: first draft writing. CS, LA, AL, FH, LM, JW, NV, EP, and AA: review and editing. All authors contributed to the article and approved the submitted version.\",\n \"The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\",\n \"All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,null,null,null,null,null,null,null,"results",null,null,null,"discussion","data-availability",null,null,"COI-statement","disclaimer"],"string":"[\n \"intro\",\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n \"discussion\",\n \"data-availability\",\n null,\n null,\n \"COI-statement\",\n \"disclaimer\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["HIV","cascade of care","follow-up interruption","private practice","French Guiana"],"string":"[\n \"HIV\",\n \"cascade of care\",\n \"follow-up interruption\",\n \"private practice\",\n \"French Guiana\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Introduction:\nFrench Guiana is the French overseas territory where the HIV epidemic is most prevalent (1). Recent modeling using the European Center for Disease Control (ECDC) modeling tool estimated the number of persons living with HIV to be over 3,200, 3,000 of whom knew their diagnosis (2). HIV transmission in French Guiana is mostly heterosexual and three of four patients are foreign citizens (3). French Guiana has the highest gross domestic product (GDP) per capita in South America and, as such, it is attractive for the poor Caribbean and South American populations in search of a better life. In a context of precariousness and sexual vulnerability, a large proportion of infected immigrants actually acquire the virus after their arrival in French Guiana (4, 5). The standards of health care are those of mainland France. Since 2013, antiretroviral (AVR) treatment is recommended for all patients irrespective of their CD4 count. All persons living with HIV receive free or paid AVR treatments regardless of their origin or socio-economic level. Undocumented immigrants with HIV are eligible for residence permits for medical reasons and health insurance coverage. Although the simplification of treatment regimens and follow-up has led to fewer constraints for the patients, their follow-up remains of paramount importance to optimize anti-retroviral therapy to detect and prevent HIV-related morbidity and prevent secondary infections. In 2006, we had studied follow-up interruption and its risk factors in French Guiana (6). Younger patients, foreigners, untreated patients, and non-immunosuppressed patients at the time of diagnosis were more likely to interrupt follow-up. In addition, the greatest risk of interruption was usually within the first 6 months after diagnosis. Since then, nearly all patients are given AVRs with regimens that are more potent but better tolerated, which usually simplifies medical follow-up. In the post 90-90-90 context, French Guiana aims to optimize the cascade of care by improving early testing and treatment and retaining patients in the healthcare system so they can continue to benefit from virological suppression. Despite progress, this remains a problem in France (7, 8). For the specific case of French Guiana, our hypothesis was that different and novel forces were at play and may have modified the adherence to outpatient follow-up. First, in the widespread early treatment with powerful and well-tolerated drugs, the simplification of follow-up would predict that patient retention should increase over time. Second, because patient follow-up is much simpler than before, general practitioners are more inclined to accept such a task, and patients –always weary of being spotted in a specialized HIV outpatient care center—may prefer to be followed closely to their home, in the “stigma-free” practice of their family physician. We therefore looked at the proportion of patients lost to follow-up –from the point of view of hospital outpatient care in different time periods. We also looked, for the first time, at the data from the health insurance system (9), which notably reimburses all antiretroviral drugs (ARVs) to determine the proportion of patients for whom ambulatory care was given from a specialized hospital department and those who only received ambulatory care from a private practitioner.\n\nMethods:\n Patients From the Hospital Outpatient Clinics The information systems for HIV in French Guiana, mandatory reporting of new HIV infections and AIDS cases, and the French Hospital Database of HIV, a cohort for which specifically trained research technicians have collected data on HIV patients since 1989 {first in the DMI2 government program until 2008, then in eNADIS/DATAIDS [Dat'AIDS cohort (clinicaltrials.gov ref. NCT02898987)]}, were used for the study.\nThe information systems for HIV in French Guiana, mandatory reporting of new HIV infections and AIDS cases, and the French Hospital Database of HIV, a cohort for which specifically trained research technicians have collected data on HIV patients since 1989 {first in the DMI2 government program until 2008, then in eNADIS/DATAIDS [Dat'AIDS cohort (clinicaltrials.gov ref. NCT02898987)]}, were used for the study.\n Lost to Follow-Up This cohort allowed us to obtain the number and proportion of patients lost to follow-up (not having consulted in more than 12 months) from the point of view of the hospital system.\nThis cohort allowed us to obtain the number and proportion of patients lost to follow-up (not having consulted in more than 12 months) from the point of view of the hospital system.\n Determination of the Proportion of Patients Receiving Treatment in a Public Hospital or in a Private Practice Since 2017, health administrations and researchers may, with permission, access data from the Système National des Données de Santé which now combines health care reimbursement data (SNIIRAM), hospital data (PMSI), and death certificate data (CepiDC). For French Guiana, this allowed us to obtain, for the first time, the proportion of persons treated for HIV in a public hospital structure or in private practice, or both.\nSince 2017, health administrations and researchers may, with permission, access data from the Système National des Données de Santé which now combines health care reimbursement data (SNIIRAM), hospital data (PMSI), and death certificate data (CepiDC). For French Guiana, this allowed us to obtain, for the first time, the proportion of persons treated for HIV in a public hospital structure or in private practice, or both.\n Selection of ARV Treatments The ARV treatments were selected from the code identifiant de présentation (CIP) code of the Drug Database [base de données publique des médicaments (BDM)], the reference database of drugs reimbursed by the French Health Insurance.\nThe ARV treatments were selected from the code identifiant de présentation (CIP) code of the Drug Database [base de données publique des médicaments (BDM)], the reference database of drugs reimbursed by the French Health Insurance.\n Patient Selection The data used to select the people on ARVs were taken from the inter-scheme consumption datamart (DCIR). All the health insurance beneficiaries were included in the study if they received at least one reimbursement for the delivery of ARVs during the year, even when the treatments were prescribed and/or delivered outside French Guiana. The specific insurance regimens included the General Health Insurance Scheme, including beneficiaries of the free complementary health insurance coverage for the poor French residents or legal migrants (CMUC/CSS) and the state medical aid for undocumented foreigners (AME); the social security system for the self-employed (RSI); and the agricultural social mutual insurance (MSA) of French Guiana.\nA subsample was also made up of patients who received at least one reimbursement for ARVs under the AME or CMUC/CSS during the year. Since AME or CMUC/CSS reflect poverty, these selection criteria allowed to test the hypothesis that trends for private or public follow-up differed between socially precarious and non-precarious populations.\nThe data used to select the people on ARVs were taken from the inter-scheme consumption datamart (DCIR). All the health insurance beneficiaries were included in the study if they received at least one reimbursement for the delivery of ARVs during the year, even when the treatments were prescribed and/or delivered outside French Guiana. The specific insurance regimens included the General Health Insurance Scheme, including beneficiaries of the free complementary health insurance coverage for the poor French residents or legal migrants (CMUC/CSS) and the state medical aid for undocumented foreigners (AME); the social security system for the self-employed (RSI); and the agricultural social mutual insurance (MSA) of French Guiana.\nA subsample was also made up of patients who received at least one reimbursement for ARVs under the AME or CMUC/CSS during the year. Since AME or CMUC/CSS reflect poverty, these selection criteria allowed to test the hypothesis that trends for private or public follow-up differed between socially precarious and non-precarious populations.\n Description of the Evolution of the Type of Care The type of care was defined as follows: exclusively hospital-based when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the hospital sector, exclusively private when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the private sector and mixed when the ARV treatments for which the patient was reimbursed during the year are prescribed by the hospital sector and the private sector.\nThe annual evolution of the distribution of the type of care was studied over the 2016–2020 period in the population of all patients on ARVs at the regional level and in the population, including only CMUC/CSS or AME patients (precarious persons) at the regional level.\nThe type of care was defined as follows: exclusively hospital-based when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the hospital sector, exclusively private when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the private sector and mixed when the ARV treatments for which the patient was reimbursed during the year are prescribed by the hospital sector and the private sector.\nThe annual evolution of the distribution of the type of care was studied over the 2016–2020 period in the population of all patients on ARVs at the regional level and in the population, including only CMUC/CSS or AME patients (precarious persons) at the regional level.\n Data Analysis Using the DAT'AIDS cohort data, single failure survival analyses were performed with follow-up interruption of more than 12 months (not due to death) as a failure event for a patient without knowledge of his or her present follow-up status. Kaplan Meier curves were plotted. Log Rank tests were performed. These analyses allowed us to quantify and visualize the time until follow-up interruption for different periods. For the health insurance data, a linear trend Chi-square test was used to test the statistical significance of the annual change in the different types of care, observed graphically over the period studied. This trend analysis aimed to determine whether the process was gradual.\nUsing the DAT'AIDS cohort data, single failure survival analyses were performed with follow-up interruption of more than 12 months (not due to death) as a failure event for a patient without knowledge of his or her present follow-up status. Kaplan Meier curves were plotted. Log Rank tests were performed. These analyses allowed us to quantify and visualize the time until follow-up interruption for different periods. For the health insurance data, a linear trend Chi-square test was used to test the statistical significance of the annual change in the different types of care, observed graphically over the period studied. This trend analysis aimed to determine whether the process was gradual.\n Regulatory and Ethical Issues Anonymized individual data from the DAT'AIDS database was used (clinicaltrials.gov ref. NCT02898987). For the proportion followed up in private or public structure, the anonymized data from the système national des données de santé (SNDS) is accessible for certain organizations with a public service mission. Particularly, these organizations, listed by decree by the Conseil d'Etat, issued after the opinion of the Commission Nationale Informatique et Libertés (CNIL), may access certain data on a permanent basis in order to carry out their missions. This is the case for researchers of Academic hospitals and the Institut National de la Santé et de la Recherche Médicale (Inserm).\nAnonymized individual data from the DAT'AIDS database was used (clinicaltrials.gov ref. NCT02898987). For the proportion followed up in private or public structure, the anonymized data from the système national des données de santé (SNDS) is accessible for certain organizations with a public service mission. Particularly, these organizations, listed by decree by the Conseil d'Etat, issued after the opinion of the Commission Nationale Informatique et Libertés (CNIL), may access certain data on a permanent basis in order to carry out their missions. This is the case for researchers of Academic hospitals and the Institut National de la Santé et de la Recherche Médicale (Inserm).\n\nPatients From the Hospital Outpatient Clinics:\nThe information systems for HIV in French Guiana, mandatory reporting of new HIV infections and AIDS cases, and the French Hospital Database of HIV, a cohort for which specifically trained research technicians have collected data on HIV patients since 1989 {first in the DMI2 government program until 2008, then in eNADIS/DATAIDS [Dat'AIDS cohort (clinicaltrials.gov ref. NCT02898987)]}, were used for the study.\n\nLost to Follow-Up:\nThis cohort allowed us to obtain the number and proportion of patients lost to follow-up (not having consulted in more than 12 months) from the point of view of the hospital system.\n\nDetermination of the Proportion of Patients Receiving Treatment in a Public Hospital or in a Private Practice:\nSince 2017, health administrations and researchers may, with permission, access data from the Système National des Données de Santé which now combines health care reimbursement data (SNIIRAM), hospital data (PMSI), and death certificate data (CepiDC). For French Guiana, this allowed us to obtain, for the first time, the proportion of persons treated for HIV in a public hospital structure or in private practice, or both.\n\nSelection of ARV Treatments:\nThe ARV treatments were selected from the code identifiant de présentation (CIP) code of the Drug Database [base de données publique des médicaments (BDM)], the reference database of drugs reimbursed by the French Health Insurance.\n\nPatient Selection:\nThe data used to select the people on ARVs were taken from the inter-scheme consumption datamart (DCIR). All the health insurance beneficiaries were included in the study if they received at least one reimbursement for the delivery of ARVs during the year, even when the treatments were prescribed and/or delivered outside French Guiana. The specific insurance regimens included the General Health Insurance Scheme, including beneficiaries of the free complementary health insurance coverage for the poor French residents or legal migrants (CMUC/CSS) and the state medical aid for undocumented foreigners (AME); the social security system for the self-employed (RSI); and the agricultural social mutual insurance (MSA) of French Guiana.\nA subsample was also made up of patients who received at least one reimbursement for ARVs under the AME or CMUC/CSS during the year. Since AME or CMUC/CSS reflect poverty, these selection criteria allowed to test the hypothesis that trends for private or public follow-up differed between socially precarious and non-precarious populations.\n\nDescription of the Evolution of the Type of Care:\nThe type of care was defined as follows: exclusively hospital-based when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the hospital sector, exclusively private when the ARV treatments for which the patient was reimbursed during the year were exclusively prescribed by the private sector and mixed when the ARV treatments for which the patient was reimbursed during the year are prescribed by the hospital sector and the private sector.\nThe annual evolution of the distribution of the type of care was studied over the 2016–2020 period in the population of all patients on ARVs at the regional level and in the population, including only CMUC/CSS or AME patients (precarious persons) at the regional level.\n\nData Analysis:\nUsing the DAT'AIDS cohort data, single failure survival analyses were performed with follow-up interruption of more than 12 months (not due to death) as a failure event for a patient without knowledge of his or her present follow-up status. Kaplan Meier curves were plotted. Log Rank tests were performed. These analyses allowed us to quantify and visualize the time until follow-up interruption for different periods. For the health insurance data, a linear trend Chi-square test was used to test the statistical significance of the annual change in the different types of care, observed graphically over the period studied. This trend analysis aimed to determine whether the process was gradual.\n\nRegulatory and Ethical Issues:\nAnonymized individual data from the DAT'AIDS database was used (clinicaltrials.gov ref. NCT02898987). For the proportion followed up in private or public structure, the anonymized data from the système national des données de santé (SNDS) is accessible for certain organizations with a public service mission. Particularly, these organizations, listed by decree by the Conseil d'Etat, issued after the opinion of the Commission Nationale Informatique et Libertés (CNIL), may access certain data on a permanent basis in order to carry out their missions. This is the case for researchers of Academic hospitals and the Institut National de la Santé et de la Recherche Médicale (Inserm).\n\nResults:\n Hospital Follow-Up in Time We tallied 803 persons that were not known to have died and who were lost to follow-up. Figure 1 shows that over time, patients seemed to be lost to follow-up increasingly sooner (Log-rank test, p < 0.001, Table 1).\nIncidence of follow-up interruption in French Guiana from the point of view of the hospital HIV outpatient cohort.\nLog-rank test comparing the incidence of follow-up interruption for different consecutive years.\nCHI2 (4) = 307.45; p = 0.0000.\nWe tallied 803 persons that were not known to have died and who were lost to follow-up. Figure 1 shows that over time, patients seemed to be lost to follow-up increasingly sooner (Log-rank test, p < 0.001, Table 1).\nIncidence of follow-up interruption in French Guiana from the point of view of the hospital HIV outpatient cohort.\nLog-rank test comparing the incidence of follow-up interruption for different consecutive years.\nCHI2 (4) = 307.45; p = 0.0000.\n The Total Number of Persons on ARV in French Guiana When looking at the health insurance data, in 2020, there were 3,047 patients who benefitted from health insurance payments for ARV treatment. This total number has been steadily increasing since 2016 (see Table 2). The evolution rate over the period was 33.35% with an average annual evolution rate of 7.46%. However, the evolution of the number of patients was more moderate in 2020, a year when activity was completely disorganized by coronavirus disease 2019 (COVID-19) and lockdowns.\nDistribution of the type of antiretroviral (ARV) follow-up (private, hospital, or mixed) in Guiana between 2016 and 2020.\nSource: SNDS/DCIR—Beneficiaries of the RG (CMU/CSS and AME).\nRSI and MSA affiliated in French Guiana, excluding complementary insurances not managed by the computer.\nWhen looking at the health insurance data, in 2020, there were 3,047 patients who benefitted from health insurance payments for ARV treatment. This total number has been steadily increasing since 2016 (see Table 2). The evolution rate over the period was 33.35% with an average annual evolution rate of 7.46%. However, the evolution of the number of patients was more moderate in 2020, a year when activity was completely disorganized by coronavirus disease 2019 (COVID-19) and lockdowns.\nDistribution of the type of antiretroviral (ARV) follow-up (private, hospital, or mixed) in Guiana between 2016 and 2020.\nSource: SNDS/DCIR—Beneficiaries of the RG (CMU/CSS and AME).\nRSI and MSA affiliated in French Guiana, excluding complementary insurances not managed by the computer.\n Hospital and/or Private Follow-Up In 2020, 57.43% (n = 1,750) of patients were followed exclusively by the hospital sector. The proportion of patients treated exclusively in the private sector was 16.31% (n = 497), and that of patients with mixed treatment prescription (private and hospital) was 26.26% (n = 800) (Table 1).\nUntil 2019, the number of patients followed up increased whatever the type of care. In 2020, only the number of patients followed up exclusively in the private sector continued to increase (+138 patients), whereas the number of patients followed up in the hospital sector or with mixed follow-up decreased slightly (see Table 2). Furthermore, the proportion of patients followed up exclusively by the hospital sector has decreased over the last 3 years, particularly in 2020. The change over the entire period was statistically significant (p < 0.0001). Conversely, the proportion of patients followed exclusively by the private sector has been steadily increasing since 2018 (the trend was statistically significant; p < 0.0001). Finally, the proportion of patients with follow-up in both the hospital and private sectors has varied little since 2016. However, there was a slight decrease in 2020 compared to 2018 and 2019, years in which the proportion had stabilized at around 28% of patients (the trend over the whole period being statistically significant; p = 0.006). Because COVID-19 in 2020 led to dramatic changes (lock-downs, cancelation of hospital consultations, etc.), we also computed the trends for 2016–2019, and the observation of a linear increase of the trend toward outpatients was still significant: p = 0.004 and p = 0.002, respectively.\nFocus on patients with indicators of precariousness (Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire or Aide Médicale Etat).\nIn 2020, 1,873 patients who had been reimbursed for ARVs were beneficiaries of the CMUC/CSS or the AME (i.e., 61.47% of all patients on ARVs) insurance plans for socially precarious persons. The number of patients in this situation increased until 2019 but decreased in 2020. Overall, the increase over the whole period was 24.53%, with an average annual growth rate of 5.64%. In 2020, 1,082 of the 1,873 patients receiving CMUC/CSS or AME on ARVs had exclusive hospital follow-up (i.e., 57.77%). This proportion steadily decreased over the last 3 years after an initial increase in 2017 (the change over the whole period being statistically significant; p < 0.0001). Conversely, the proportions of patients managed exclusively by the private sector or benefiting from mixed public and private follow-up, after having decreased over the year 2017, increased over the years 2018, 2019, and 2020. The proportion of precarious patients on ARVs exclusively followed up in the private sector thus rose to reach 14.15% in 2020 (a statistically significant trend for the period 2016–2020; p < 0.0001). The proportion of patients with mixed management (combination of public and private follow-up) increased by + 2 points between 2016 and 2020 (the trend was statistically significant over the period; p = 0.0036) to reach more than 28% in 2020 (Table 3).\nDistribution of the type of antiretroviral follow-up (private practice, hospital, or mixed) in French Guiana between 2016 and 2020 among precarious patients.\nSource: SNDS/DCIR—Beneficiaries of Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire and Aide Médicale Etat in French Guiana.\nLinear trend chi2 (private vs. hospital only, 2016–2020): p < 0.001;\nLinear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.003.\nBecause of trend distortions resulting from COVID-19, the trend was recalculated for 2016–2019, excluding 2020.\n*Linear trend chi2 (private vs. hospital only, 2016–2009): p = 0.004; **Linear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.002.\nIn 2020, 57.43% (n = 1,750) of patients were followed exclusively by the hospital sector. The proportion of patients treated exclusively in the private sector was 16.31% (n = 497), and that of patients with mixed treatment prescription (private and hospital) was 26.26% (n = 800) (Table 1).\nUntil 2019, the number of patients followed up increased whatever the type of care. In 2020, only the number of patients followed up exclusively in the private sector continued to increase (+138 patients), whereas the number of patients followed up in the hospital sector or with mixed follow-up decreased slightly (see Table 2). Furthermore, the proportion of patients followed up exclusively by the hospital sector has decreased over the last 3 years, particularly in 2020. The change over the entire period was statistically significant (p < 0.0001). Conversely, the proportion of patients followed exclusively by the private sector has been steadily increasing since 2018 (the trend was statistically significant; p < 0.0001). Finally, the proportion of patients with follow-up in both the hospital and private sectors has varied little since 2016. However, there was a slight decrease in 2020 compared to 2018 and 2019, years in which the proportion had stabilized at around 28% of patients (the trend over the whole period being statistically significant; p = 0.006). Because COVID-19 in 2020 led to dramatic changes (lock-downs, cancelation of hospital consultations, etc.), we also computed the trends for 2016–2019, and the observation of a linear increase of the trend toward outpatients was still significant: p = 0.004 and p = 0.002, respectively.\nFocus on patients with indicators of precariousness (Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire or Aide Médicale Etat).\nIn 2020, 1,873 patients who had been reimbursed for ARVs were beneficiaries of the CMUC/CSS or the AME (i.e., 61.47% of all patients on ARVs) insurance plans for socially precarious persons. The number of patients in this situation increased until 2019 but decreased in 2020. Overall, the increase over the whole period was 24.53%, with an average annual growth rate of 5.64%. In 2020, 1,082 of the 1,873 patients receiving CMUC/CSS or AME on ARVs had exclusive hospital follow-up (i.e., 57.77%). This proportion steadily decreased over the last 3 years after an initial increase in 2017 (the change over the whole period being statistically significant; p < 0.0001). Conversely, the proportions of patients managed exclusively by the private sector or benefiting from mixed public and private follow-up, after having decreased over the year 2017, increased over the years 2018, 2019, and 2020. The proportion of precarious patients on ARVs exclusively followed up in the private sector thus rose to reach 14.15% in 2020 (a statistically significant trend for the period 2016–2020; p < 0.0001). The proportion of patients with mixed management (combination of public and private follow-up) increased by + 2 points between 2016 and 2020 (the trend was statistically significant over the period; p = 0.0036) to reach more than 28% in 2020 (Table 3).\nDistribution of the type of antiretroviral follow-up (private practice, hospital, or mixed) in French Guiana between 2016 and 2020 among precarious patients.\nSource: SNDS/DCIR—Beneficiaries of Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire and Aide Médicale Etat in French Guiana.\nLinear trend chi2 (private vs. hospital only, 2016–2020): p < 0.001;\nLinear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.003.\nBecause of trend distortions resulting from COVID-19, the trend was recalculated for 2016–2019, excluding 2020.\n*Linear trend chi2 (private vs. hospital only, 2016–2009): p = 0.004; **Linear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.002.\n\nHospital Follow-Up in Time:\nWe tallied 803 persons that were not known to have died and who were lost to follow-up. Figure 1 shows that over time, patients seemed to be lost to follow-up increasingly sooner (Log-rank test, p < 0.001, Table 1).\nIncidence of follow-up interruption in French Guiana from the point of view of the hospital HIV outpatient cohort.\nLog-rank test comparing the incidence of follow-up interruption for different consecutive years.\nCHI2 (4) = 307.45; p = 0.0000.\n\nThe Total Number of Persons on ARV in French Guiana:\nWhen looking at the health insurance data, in 2020, there were 3,047 patients who benefitted from health insurance payments for ARV treatment. This total number has been steadily increasing since 2016 (see Table 2). The evolution rate over the period was 33.35% with an average annual evolution rate of 7.46%. However, the evolution of the number of patients was more moderate in 2020, a year when activity was completely disorganized by coronavirus disease 2019 (COVID-19) and lockdowns.\nDistribution of the type of antiretroviral (ARV) follow-up (private, hospital, or mixed) in Guiana between 2016 and 2020.\nSource: SNDS/DCIR—Beneficiaries of the RG (CMU/CSS and AME).\nRSI and MSA affiliated in French Guiana, excluding complementary insurances not managed by the computer.\n\nHospital and/or Private Follow-Up:\nIn 2020, 57.43% (n = 1,750) of patients were followed exclusively by the hospital sector. The proportion of patients treated exclusively in the private sector was 16.31% (n = 497), and that of patients with mixed treatment prescription (private and hospital) was 26.26% (n = 800) (Table 1).\nUntil 2019, the number of patients followed up increased whatever the type of care. In 2020, only the number of patients followed up exclusively in the private sector continued to increase (+138 patients), whereas the number of patients followed up in the hospital sector or with mixed follow-up decreased slightly (see Table 2). Furthermore, the proportion of patients followed up exclusively by the hospital sector has decreased over the last 3 years, particularly in 2020. The change over the entire period was statistically significant (p < 0.0001). Conversely, the proportion of patients followed exclusively by the private sector has been steadily increasing since 2018 (the trend was statistically significant; p < 0.0001). Finally, the proportion of patients with follow-up in both the hospital and private sectors has varied little since 2016. However, there was a slight decrease in 2020 compared to 2018 and 2019, years in which the proportion had stabilized at around 28% of patients (the trend over the whole period being statistically significant; p = 0.006). Because COVID-19 in 2020 led to dramatic changes (lock-downs, cancelation of hospital consultations, etc.), we also computed the trends for 2016–2019, and the observation of a linear increase of the trend toward outpatients was still significant: p = 0.004 and p = 0.002, respectively.\nFocus on patients with indicators of precariousness (Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire or Aide Médicale Etat).\nIn 2020, 1,873 patients who had been reimbursed for ARVs were beneficiaries of the CMUC/CSS or the AME (i.e., 61.47% of all patients on ARVs) insurance plans for socially precarious persons. The number of patients in this situation increased until 2019 but decreased in 2020. Overall, the increase over the whole period was 24.53%, with an average annual growth rate of 5.64%. In 2020, 1,082 of the 1,873 patients receiving CMUC/CSS or AME on ARVs had exclusive hospital follow-up (i.e., 57.77%). This proportion steadily decreased over the last 3 years after an initial increase in 2017 (the change over the whole period being statistically significant; p < 0.0001). Conversely, the proportions of patients managed exclusively by the private sector or benefiting from mixed public and private follow-up, after having decreased over the year 2017, increased over the years 2018, 2019, and 2020. The proportion of precarious patients on ARVs exclusively followed up in the private sector thus rose to reach 14.15% in 2020 (a statistically significant trend for the period 2016–2020; p < 0.0001). The proportion of patients with mixed management (combination of public and private follow-up) increased by + 2 points between 2016 and 2020 (the trend was statistically significant over the period; p = 0.0036) to reach more than 28% in 2020 (Table 3).\nDistribution of the type of antiretroviral follow-up (private practice, hospital, or mixed) in French Guiana between 2016 and 2020 among precarious patients.\nSource: SNDS/DCIR—Beneficiaries of Couverture Maladie Universelle Complémentaire/Complémentaire Santé Solidaire and Aide Médicale Etat in French Guiana.\nLinear trend chi2 (private vs. hospital only, 2016–2020): p < 0.001;\nLinear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.003.\nBecause of trend distortions resulting from COVID-19, the trend was recalculated for 2016–2019, excluding 2020.\n*Linear trend chi2 (private vs. hospital only, 2016–2009): p = 0.004; **Linear trend chi2 (mixed private+hospital vs. hospital only, 2016–2020): p = 0.002.\n\nDiscussion:\nFor the first time, the use of health insurance data allowed us to see the whole picture of HIV care in French Guiana. The actual number of persons benefiting from antiretroviral treatment in French Guiana was actually very close to the recent estimations using HIV-cohort data for the ECDC modeling tool (1). The number of persons on AVR treatment has substantially increased over the study time but slowed down in 2020. Given the simultaneous disruption of information systems and HIV testing and care, it is difficult to tease out whether this reflects the slowing down of an epidemic where increasing numbers of persons are virologically controlled, or the COVID-19-related gap in new diagnoses and patient referrals. Data from 2021 is also likely to be impacted by COVID-19, so only future post-COVID-19 years may allow us to find out a better explanation for our observation.\nWhile the organization of care and virological results had gradually improved over the years, we were initially surprised to see the rate of persons lost to follow-up from the hospital outpatient cohort actually increased over time. However, clinicians had perceived the feedback from patients increasingly tempted by a follow-up by their private practitioner for proximity reasons and because going to specialized HIV outpatient departments was more stigmatizing than a waiting room of a general practitioner. At the same time, treatment regimens and follow-up are far simpler than they used to be, which expands the number of physicians willing to take-on the task of being the referent physician of patients with HIV. Furthermore, the aging HIV-cohort (39.5% aged over 50 years) increasingly requires general practitioners–not infectious disease specialists—to manage comorbidities, which may be another incentive to have an integral follow-up by one's family physician instead of a specialist. This perception seems to be vindicated by the health insurance data, which shows a growing proportion of persons choosing to be exclusively treated by their private practitioner. Furthermore, about a quarter used both the private and public sectors, suggesting pragmatic convenience was behind this behavior. The COVID-19 disruption of HIV follow-up may have further tilted follow-up toward private practice in 2020, and it remains to be seen whether this will be corrected in the future or if it has accelerated an ongoing trend toward decentralization of HIV care.\nAt first view, one could have hypothesized that this trend toward private practice did not include the most precarious cases which in French Guiana's HIV cohort, represents 2/3rds of patients if we only look at the type of Health Insurance. However, this was refuted by the data which shows the exact same trend among the more precarious and shows that, contrary to widespread beliefs, poor populations do have access to private practice and use it readily as soon as they have health coverage. Since 2000, the Public Health Law has ensured that the most precarious cases do not have to advance any fees for medical consultations, and thus there are no financial obstacles to consult a private practitioner instead of a hospital physician.\nThe study has a number of limitations. First, the use of health insurance regimens as a proxy for poverty is legitimate, but it may actually underestimate the degree of precariousness. Particularly, the SNDS did not provide other indicators, such as country of birth or nationality, which may have served as another proxy for precariousness. Another limitation is that the simultaneous accelerating rate of persons being lost to follow-up from the hospital system and the increasing proportion of patients exclusively followed by private practitioners do not formally prove that those that do not come back in the outpatient ward are actually consulting private practitioners. Instead, they could have left the territory, or stopped treatment and follow-up, or died. However, for deaths or treatment interruptions, this seems largely unlikely because opportunistic infections and deaths –which eventually are counted—have substantially decreased over time suggesting that indeed many of the patients who do not come back to the hospital outpatient clinic are receiving care in private practices.\nIn conclusion, while hospital outpatient care remains by far the most common mode of patient care, there seems to be a gradual erosion of this model in favor of private practice. For the first time, the Health Insurance information system allows obtaining a broader view of patient care than hospital cohort data alone. In the rapidly evolving therapeutic and conceptual landscape of HIV care, in the context of aging cohorts and shifting patient preferences, coordinating continuous medical education with private practitioners seems to be crucially important so that every caregiver is up to date with the current guidelines. This slow gradual process may even have accelerated in the transformational context of the COVID-19 pandemic.\n\nData Availability Statement:\nThe data analyzed in this study is subject to the following licenses/restrictions: The DATAIDS data may be shared upon reasonable request. The health Insurance data requires additional permissions. Requests to access these datasets should be directed to corevih@ch-cayenne.\n\nEthics Statement:\nThe studies involving human participants were reviewed and approved by Commission Nationale Informatique et Libertés. The patients/participants provided their written informed consent to participate in this study.\n\nAuthor Contributions:\nMN: study design. HD and NV: data analysis. HD and MN: first draft writing. CS, LA, AL, FH, LM, JW, NV, EP, and AA: review and editing. All authors contributed to the article and approved the submitted version.\n\nConflict of Interest:\nThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\n\nPublisher's Note:\nAll claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nAlthough the simplification of antiretroviral (AVR) treatment regimens and follow-up has led to fewer constraints for patients with HIV, their follow-up remains of paramount importance to optimize AVR therapy, to detect and prevent HIV-related morbidity, and prevent secondary infections. The problem of follow-up interruption in French Guiana has been persistent and seemingly impervious to efforts to alleviate it.\n\nMethods:\nUsing the complementary lenses of the hospital HIV cohort and the health insurance information system, we looked at the incidence of follow-up interruption and the proportion of patients followed by private practitioners.\n\nResults:\nWe tallied 803 persons that were not known to have died and who were lost to follow-up. Over time, hospital outpatients were lost to follow-up significantly sooner. By contrast, there was a significant trend with more and more patients exclusively followed by private practitioners.\n\nConclusions:\nWhile hospital outpatient care remains by far the most common mode of patient care, there seems to be a gradual erosion of this model in favor of private practice."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":7477,"string":"7,477"},"whole_article_abstract_length":{"kind":"number","value":208,"string":"208"},"other_sections_lengths":{"kind":"list like","value":[76,37,82,43,195,134,128,121,104,156,757,31,55],"string":"[\n 76,\n 37,\n 82,\n 43,\n 195,\n 134,\n 128,\n 121,\n 104,\n 156,\n 757,\n 31,\n 55\n]","isConversational":false},"num_sections":{"kind":"number","value":20,"string":"20"},"most_frequent_words":{"kind":"list like","value":["patients","hospital","private","follow","2020","data","french","trend","proportion","2016"],"string":"[\n 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[SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":70,"cells":{"title":{"kind":"string","value":"Spectral content (colour) of noise exposure affects work efficiency."},"pmid":{"kind":"string","value":"33243964"},"background_abstract":{"kind":"string","value":"As part of an effort to enhance the efficiency of workers, experiments relating to three types of noise exposure were conducted. Previous studies have proved that pink noise can cause a brain wave to reach a lower potential. In this study, we utilized physical methods, in cognitive experiments, to understand the impacts that three colour noises have on working efficiency."},"background_abstract_label":{"kind":"string","value":"INTRODUCTION"},"methods_abstract":{"kind":"string","value":"All 22 participants were exposed to a sound environment of quiet, red, pink and white noises respectively. After a laboratory experiment, details of psychomotor speed, continuous performance, executive function and working memory were recorded."},"methods_abstract_label":{"kind":"string","value":"SUBJECTS AND METHODS"},"results_abstract":{"kind":"string","value":"Red, pink and white noises were significantly positive in comparison with the quiet environment of the psychomotor speed test. As for the continuous performance test, pink noise gave the only significantly positive result. Red, pink and white noise resulted in a better executive function test. Red and pink noise showed significantly positive improvement, while white noise was significantly positive in comparison with the quiet environment of the working memory test. In addition, the results from the comfort questionnaires showed that red and pink noise increase the possibility of better judgment, implementation, and overall environment."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"At present time, it is considered that noise has negative effects on hearing and health. However, experimental results show that certain noise can enhance environmental comfort. It is feasible, in the future, to use knowledge of colour noises to improve productivity in a workplace with a healthy environment."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Audiometry, Pure-Tone","Efficiency","Executive Function","Female","Healthy Volunteers","Hearing","Humans","Male","Memory, Short-Term","Noise","Psychomotor Performance","Reaction Time","Sound","Work","Young Adult"],"string":"[\n \"Audiometry, Pure-Tone\",\n \"Efficiency\",\n \"Executive Function\",\n \"Female\",\n \"Healthy Volunteers\",\n \"Hearing\",\n \"Humans\",\n \"Male\",\n \"Memory, Short-Term\",\n \"Noise\",\n \"Psychomotor Performance\",\n \"Reaction Time\",\n \"Sound\",\n \"Work\",\n \"Young Adult\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"7986458"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Exposure to high levels of noise in industry factory is associated with a number of effects on health manifested in various psychosocial responses such as annoyance, disturbance of daily activities, sleep and performance and in physical responses, such as hearing loss, hypertension and ischemic heart disease.[12] While the open-plan office is now widespread in the workplace, it appears that noise is a major nuisance factor in open-plan offices in spite of a relatively low noise level(less than 65 dB(A)).[3] In general, the higher the decibel numbers of sound, the greater the health hazard. However, when the number of sound decibels is not high, the degree of influence on people may vary depending on the sound characteristics.\nIn audio engineering, physics, and many other fields, the colour of noise refers to the power spectrum of a noise signal. The practice of naming kinds of noise after colours started with white noise, a signal whose spectrum has equal power within any equal interval of frequencies. That name was given by analogy with white light, which was assumed to have such a flat power spectrum over the visible range. Other colour names, like pink, red, and blue were then given to noise with other spectral profiles, often (but not always) in reference to the colour of light with similar spectra.\nNoise often has detrimental effects on performance. Under most circumstances, information processing is disturbed by environmental noise and other non-task compatible distracters.[45] However, researchers have recently reported that under certain circumstances individuals with attention problems appear to benefit from the addition of specific forms of environmental noise. Typically, this facilitative effect has been limited to non-vocal background music on simple arithmetic task performance,[6] but Stansfeld et al.[7] found just that under certain conditions even road traffic noise can improve performance on episodic memory tasks, particularly in children at risk of attention problems and academic underachievement. Furthermore, Söderlund et al.[8] have demonstrated that adding auditory white noise to the environment enhanced the memory performance of children with Attention Deficit Hyperactivity Disorder (ADHD)-type problems but disrupted that of non-ADHD control children. Stochastic resonance, a phenomenon whereby signal processing is enhanced by the addition of random noise, has been widely demonstrated across various modalities, including visual, auditory, tactile and cross-modal forms of processing.[910] Of particular interest are findings that auditory noise has the capacity to enhance some aspects of human cognitive performance, such as the speed of arithmetical computations.[11]\n\nMeanwhile, some recent studies showed related evidence. For example, in 2010, light music, an example belonging to pink noise, is proved beneficial for elder people to improve their sleep quality as a long term effect.[12] Moreover, in a previous study,[13] the steady pink noise with intensity of 35 dB, 40 dB, 50 dB and 60 dB was used to stimulate four participants during sleep and they believed that pink noise could improve the sleep quality of participants. However, it is clear that this conclusion was based on significant increase of light sleep period (especially stage 2) accompanied with declined duration of rapid eye movement. In the brain synchronization study, it has demonstrated that the pink noise could synchronize brain wave and induce brain activity into a specialized state, that is, in a lower complexity level.[14]\n\nSezici’s study showed that playing of white noise significantly decreased the daily crying durations and increased the sleeping durations of the colicky babies compared to swinging in both groups.[15] In the literature, parents emphasized that playing of white noise was quite beneficial in relieving pain among crying colicky babies.[16] In their studies, Kucukoglu et al.[17] and Karakoc and Turker[18] revealed that the pain scores of the newborns who were made to listen to white noise were observed to decrease compared with those of the newborns in the control group. White noise encompasses all characteristics of sounds within the range of human hearing. It has been used in the treatment of tinnitus, insomnia, masking unwanted sounds, and provision of relaxation.[1920]\n\nZhou et al.[14] demonstrated that steady pink noise has significant effect on reducing brain wave complexity and inducing more stable sleep time to improve sleep quality of individuals. In total, 42 elderly people (21 using music and 21 controls) completed the study, and there was some indication that soft slow music yielder higher improvement on some of the parameters, which are worthy of further investigation.[12] A tabulated list of the effects observed in previous studies with colour noise exposures is shown in Table 1.\nA tabulated list of the effects observed in previous studies with colour noise exposures\nSeveral experiments have demonstrated that the detrimental effect of office noise on worker attitude and performance can be significantly reduced through the use of masking noise.[2122232425] For instance the experimental study performed by Haka[24] demonstrated that the performance of operation span tasks, serial recall and long term memory tasks were all improved when masking noise was used; and the field study performed by Hongisto[23] indicated that masking noise significantly reduced disturbance of worker attitudes towards concentration caused by office noise. Masking noise achieves this improvement through reducing the intelligibility of nearby speech.[25] In addition, Vassie et al.[26] indicated that participants rejected the brown masking noise delivered through earphones as it caused irritation and discomfort. Future studies should also consider other types of masking noise and should measure the level and duration of the masking noise.[26]\n\nIt is well-known that any human action results from brain activity, which is affected by light and sound. Too much acoustic energy causes hearing loss, impaired human nerves, and emotional disorders.[2728] However, sound play also very important role for people. According to previous researchers, not only white noise can create shading effect, but pink noise can improve sleep quality. Most people in the workplace are in the office or in the service industry, not in the noisy working environment for the factory. Creating a quiet environment is sometimes not easy. However, adding some sounds to enhance the work efficiency and reduce the work pressure of low-noise environment should be an issue worth exploring. Therefore, this study wanted to understand the effects of three different colours noise exposure. In the past, there were not many studies in this regard. Therefore, the goal of the current study is to test the hypothesis that adding another sound in a low background noise environment may increases productivity and comfort."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"The results of the four tests conducted in this current study are summarized in Table 2. For each simple reaction time test, 10 sets of tests were completed. In the simple reaction time test, the average response time was better in red noise exposure scenario (0.565 seconds) than in the pink noise exposure scenario (0.602 seconds) and in the white noise exposure scenario (0.609 seconds). The variances of red noise exposure scenario (0.005 seconds) and pink noise exposure scenario (0.008 seconds) were lower than the white noise exposure scenario (0.013 seconds) and the quiet scenario (0.023 seconds). From the average of 10 test results, there was a very significant difference between the three noise exposure scenarios, red noise exposure (P < 0.001), pink noise exposure (P < 0.001), white noise exposure (P < 0.001) and the quiet scenario, as shown in Figure 2.\nMeans and variances of SRT, CPT-IP, TMT, TOENST in four different sound field environments\nPaired t-test analysis of Simple Reaction Time (SRT) in four different sound field exposures.\nCompared with the average correct rate in the continuous performance test, 93.6% of the pink noise exposure situation is better than 91.7% of the red noise exposure situation and 91.1% of the white noise exposure situation, and the three kinds of noise exposure are better than 88.8% on the quiet situation. Among the 22 participants, half of the respondents achieved 93.3% correct rate on red noise exposure, 93.3% on pink noise exposure, 93.3% on white noise exposure, and they all scored better than 90.0% in the quiet situation. The average correct rate of the test showed that there was a slight improvement over the quiet scenario for all of the red noise scenario, the pink noise scenario, and the white noise scenario. The paired t-test showed significant differences (P < 0.05) between the pink noise exposure scenario and the quiet scenario in the continuous performance test, Figure 3.\nPaired t-test analysis of continuous performance test-identical pairs (CPT-IP) in four different sound field exposures.\nIn the executive function tests, comparing the average time it takes to complete the test, it shows the red noise exposure scenario (21.37 seconds) is superior to the pink noise exposure scenario (22.31 seconds) and the white noise exposure scenario (23.11 seconds), all of which are better than the quiet scenario (24.54 seconds). The best test results (minimum) showed that the red noise exposure scenario (12.00 seconds), pink noise exposure scenario (10.73 seconds), white noise exposure scenario (13.13 seconds) were also better than the quiet scenario (15.33 seconds). The average finishing time of the tests showed that the red noise exposure situation, the pink noise exposure situation and the white noise exposure situation are better than the quiet situation. As shown on Figure 4, the paired t-test of executive function tests showed significant differences between the red noise exposure scenario (P < 0.001), the pink noise exposure scenario (P < 0.001), white noise exposure scenario (P < 0.001) and the quiet scenario.\nPaired t-test analysis of Trail Making Test (TMT) in four different sound field exposures.\nThe average score of the working memory test showed that the red noise exposure situation (18.8 points) was superior to the pink noise exposure situation (17.3 points) and the white noise exposure situation (15.9 points), all of which were better than the quiet situation (14.4 points). In a comparison of variance, the red noise exposure (5.1 points) and the pink noise exposure (6.9 points) were significantly lower than the white noise exposure (11.2 points) and noiseless exposure (9.3 points). The paired t-test analysis showed that in the working memory test, the red noise exposure (P < 0.001), the pink noise exposure (P < 0.001), the white noise exposure (P < 0.01), the three noise exposure all had a significant difference as compared with the quiet situation, as shown on Figure 5.\nPaired t-test analysis of Taiwan’s Odd-Even Number Sequencing Test (TOENST) in four different sound field exposures.\nAs shown in Table 3, compared with the quiet situation, both the red noise and the pink noise exposure in the simple reaction time test, the executive function test and the working memory test all showed significant differences (P < 0.001). The white noise exposure was very significantly different (P < 0.001) in the simple reaction time test, and there was a significant difference (P < 0.01) in the working memory test. However, only pink noise exposure was significantly different (P < 0.05) in the continuous performance test.\nPaired t-test analysis of three colour noise exposure situations compared with quiet situations\nA statistical analysis of the sound field comfort questionnaire showed that, the questionnaire had a Cronbach’s alpha score of 0.772. Generally, a questionnaire with a Cronbach’s alpha score between 0.7 and 0.9 is considered to be of good reliability. As shown in Table 4, the subjective comfort evaluation statistics of the sound field environment indicated a highest and lowest score of 0 and −2, a little worse in the quiet scenario, while the scores between −1 and 3 were slightly worse to good in the red noise scenario; the scores between −1 and 2 were slightly worse to better in pink noise scenario; however, the scores between −3 and 1 represented a worse to slightly better result for the white noise scenario. The sum of the scores of the subjective evaluation in the sound field were −40 (average rating −0.26), +81 (average rating 0.53), +38 (average rating 0.25), −94 (average rating −0.61) for quiet, red noise, pink noise, and white noise scenarios, respectively.\nSubjective comfort evaluations of four sound environments"},"conclusions_title":{"kind":"string","value":"CONCLUSION"},"conclusions_text":{"kind":"string","value":"The results of this study show that participants perform significantly better in Psychomotor Speed Test, Continuous Performance Test, Executive Function Test and Working Memory Test. Therefore, colour noise may be used in the future to improve a workplace sound field environment, so that white-collar workers have a better working environment. Using physical stimuli of red, pink and white noises to improve worker productivity may prove to be a new approach towards productivity improvement that may provide fewer side effects than traditional methods such as drinking refreshing beverages. The introduction of the sounds represents a simple, low-cost, non-invasive improvement.\n Financial support and sponsorship Nil.\nNil.\n Conflicts of interest There are no conflicts of interest.\nThere are no conflicts of interest."},"other_sections_titles":{"kind":"list like","value":["Participants","Presentation of noise","Outcome measures","Experimental protocol","Statistical analysis of data","Financial support and sponsorship"],"string":"[\n \"Participants\",\n \"Presentation of noise\",\n \"Outcome measures\",\n \"Experimental protocol\",\n \"Statistical analysis of data\",\n \"Financial support and sponsorship\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Twenty-two healthy college students were enrolled in this study. The mean age of the participants was 22 years. All participants reported no medical history of neurological disease, hypertension or heart disease. A machine called an audiometer was used to produce sounds at various volumes and frequencies. The participants being tested listened to sounds through headphones and responded when they hear them by pressing a button. A pure tone audiogram was performed by a trained health care professional at the beginning of the study for each participant, and the hearing acuity of the participants was determined to be normal, with the thresholds of 500 Hz, 1000 Hz, 2000 Hz and 4000 Hz being less than 15 dB. The participants received financial compensation for their participation in the experiment. Informed consent was obtained from all participants.","During this experiment, four sound conditions were used, relating to the three types of masking sound (red noise, pink noise, white noise) and a condition, which consisted of a wide band background noise. White noise is a signal with a flat frequency spectrum when plotted as a linear function of frequency. Red noise will refer to a power density which decreases 6 dB per octave with increasing frequency (density proportional to 1/f2). The frequency spectrum of pink noise is linear in logarithmic scale, and the spectral power density, compared with white noise, decreases by 3 dB per octave (density proportional to 1/f). The noise level within the room used in the study was measured using a Cirrus Type 832C sound level meter. Thirty second SPL samples, LAeq,30s, were measured over a period of an hour. Noise levels ranged from an Lmin of 41 dBA to an Lmax of 50 dBA. Measurements performed on successive days identified similar ranges of Lmin and Lmax.\nThe signal source of masking sound was sent through a desktop computer to a Bluetooth speaker (BOSE SoundLink Mini). The sound level of each colour noise exposure was about 47 dBA when turning on the speaker and the background noise (without adding colour noise) was 44 dBA. Background noise had a decibel (dBA) value of three decibels (dBA) difference compared to the noise exposure scenario of three different colours, so that means that the sound energy of the colour noise exposure scenario was double that of the original silence.","Over the years, different scholars have given their definitions of efficiency, and different scholars also have different views and explanations for it. Therefore, it is not easy to offer a precise definition of work efficiency. Among many offerings, the definition of work efficiency includes “work response time”, the time when the work instruction is received plus the response time, and “work execution processing time” the time it takes for each task from the reaction to the completion. “Working memory” refers to the processing of tasks for the content of the current short-term memories. Cognitive function is concept that covers several functions of the brain such as attention, executive function, processing speed, learning and memory. Information processing and response speed are basic cognitive function, which is needed for more complex functions such as working memory. We measured cognitive performance via a battery of standardized cognitive tests. If improvements were observed in cognitive performance, this would not only have important benefits for employees, but benefits for the organization in terms of the potential for increased productivity. The main outcome measurers for this study were those of four laboratory tests: Psychomotor Speed Test, Continuous Performance Test, Executive Function Test, and Working Memory Test. These tests were presented in a random order. Participants completed them on individual laptops in a laboratory with one of the testers present. The Psychomotor Speed Test measures Simple Reaction Time (SRT), general alertness and motor speed through delivery of a known stimulus to a known location to elicit a known response. The Continuous Performance Test − Identical Pairs (CPT-IP) requires the identification of identical stimulus pairs within a continuously presented series of stimuli. The Trail Making Test (TMT) is a widely used test to assess executive function, and it is a neuropsychological test of visual attention and task switching. It consists of two parts in which the participant is instructed to connect a set of 25 dots as quickly as possible while still maintaining accuracy. The test can provide information about visual search speed, scanning, speed of processing, mental flexibility, as well as executive functioning. Currently, the Wechsler Adult Intelligence Scale (WAIS) is the most commonly used instrument in the armamentarium of clinical neuropsychologists.[2930] This test involves letter sequences and tests one’s ability to think logically and analytically. However, the Wechsler Adult Intelligence Scale-III and IV Letter Number Sequencing are not appropriate for non-alphabetic cultures. The Taiwan’s Odd-Even Number Sequencing Test (TOENST), as proposed by the Department of Psychology, National Taiwan University,[31] was adopted to replace the Letter Number Test. Participants need to rearrange and read the Arabic numerals in a series. The way to read out the series is: “read out the odd numbers in ascending order and then the even numbers in descending order.” For example, the random numbers 7, 2, 8, 6, 3 is incremented by an odd number (3, 7) and decrements by an even number (8, 6, 2), so the answer is 3 7 8 6 2.\nThe comfort of the sound field environment was evaluated by a closed questionnaire, and the subjective comfort of the participant’s sound field environment was measured by the semantic differential method. There were mainly seven closed questionnaires in the questionnaire design, with “no feeling” in the middle (0 point). From middle to the left indicates that the ability deteriorates and worsens. Each left one grid is −1, the left two grids are −2, and so on. The middle to the right means that the ability to become better, every right one grid is +1, the right two grids is +2, and so on.","Participants were tested in the same laboratory. Subject to the participants’ available time, the experiment was conducted under conditions of quiet (without noise), red noise, pink noise and white noise exposure. Before each experiment, the participants adopted a random method to determine the sound field exposure situation and the participants were not informed of the kind of sound scenario. The order of the four sound conditions (quiet, red, pink, white) was randomized. Ten minutes before the experiment was started, the participants were physiologically and psychologically adapted to the laboratory environment before they started the experiment. Each participant had to perform four tests and complete questionnaires in each of the four sessions in the experiment, and the order of the four tests was randomized. There was a five-minute break between each test. During the break, the sound exposure was on and the sound exposure was not terminated until all sound field exposure conditions had ended. After finishing four tests, participants filled in the sound field comfort evaluation questionnaire. During the filling in process, the sound continued. The flowchart in Figure 1 illustrates the experimental protocol.\nFlow chart of the experimental protocol.\nA within-subject design was used in the experiment with one independent variable, i.e. sound condition. Five dependent variables, including four objective parameters (psychomotor speed test, continuous performance test, executive function test, and working memory test) and one subjective perception, were collected to explore work efficiency in this study.","The study was interested in evaluating the effectiveness of colour noise exposure. The approach considered was to measure the performance of a sample of participants before and after colour noise exposure, and analyse the differences using a paired sample t-test. IBM SPSS Statistics Version 20 was used to determine whether there were any statistically significant differences between the means of independent groups. Data from the completed questionnaires were stored in a database and transported in and subsequently analysed using the statistical program SPSS.","Nil."],"string":"[\n \"Twenty-two healthy college students were enrolled in this study. The mean age of the participants was 22 years. All participants reported no medical history of neurological disease, hypertension or heart disease. A machine called an audiometer was used to produce sounds at various volumes and frequencies. The participants being tested listened to sounds through headphones and responded when they hear them by pressing a button. A pure tone audiogram was performed by a trained health care professional at the beginning of the study for each participant, and the hearing acuity of the participants was determined to be normal, with the thresholds of 500 Hz, 1000 Hz, 2000 Hz and 4000 Hz being less than 15 dB. The participants received financial compensation for their participation in the experiment. Informed consent was obtained from all participants.\",\n \"During this experiment, four sound conditions were used, relating to the three types of masking sound (red noise, pink noise, white noise) and a condition, which consisted of a wide band background noise. White noise is a signal with a flat frequency spectrum when plotted as a linear function of frequency. Red noise will refer to a power density which decreases 6 dB per octave with increasing frequency (density proportional to 1/f2). The frequency spectrum of pink noise is linear in logarithmic scale, and the spectral power density, compared with white noise, decreases by 3 dB per octave (density proportional to 1/f). The noise level within the room used in the study was measured using a Cirrus Type 832C sound level meter. Thirty second SPL samples, LAeq,30s, were measured over a period of an hour. Noise levels ranged from an Lmin of 41 dBA to an Lmax of 50 dBA. Measurements performed on successive days identified similar ranges of Lmin and Lmax.\\nThe signal source of masking sound was sent through a desktop computer to a Bluetooth speaker (BOSE SoundLink Mini). The sound level of each colour noise exposure was about 47 dBA when turning on the speaker and the background noise (without adding colour noise) was 44 dBA. Background noise had a decibel (dBA) value of three decibels (dBA) difference compared to the noise exposure scenario of three different colours, so that means that the sound energy of the colour noise exposure scenario was double that of the original silence.\",\n \"Over the years, different scholars have given their definitions of efficiency, and different scholars also have different views and explanations for it. Therefore, it is not easy to offer a precise definition of work efficiency. Among many offerings, the definition of work efficiency includes “work response time”, the time when the work instruction is received plus the response time, and “work execution processing time” the time it takes for each task from the reaction to the completion. “Working memory” refers to the processing of tasks for the content of the current short-term memories. Cognitive function is concept that covers several functions of the brain such as attention, executive function, processing speed, learning and memory. Information processing and response speed are basic cognitive function, which is needed for more complex functions such as working memory. We measured cognitive performance via a battery of standardized cognitive tests. If improvements were observed in cognitive performance, this would not only have important benefits for employees, but benefits for the organization in terms of the potential for increased productivity. The main outcome measurers for this study were those of four laboratory tests: Psychomotor Speed Test, Continuous Performance Test, Executive Function Test, and Working Memory Test. These tests were presented in a random order. Participants completed them on individual laptops in a laboratory with one of the testers present. The Psychomotor Speed Test measures Simple Reaction Time (SRT), general alertness and motor speed through delivery of a known stimulus to a known location to elicit a known response. The Continuous Performance Test − Identical Pairs (CPT-IP) requires the identification of identical stimulus pairs within a continuously presented series of stimuli. The Trail Making Test (TMT) is a widely used test to assess executive function, and it is a neuropsychological test of visual attention and task switching. It consists of two parts in which the participant is instructed to connect a set of 25 dots as quickly as possible while still maintaining accuracy. The test can provide information about visual search speed, scanning, speed of processing, mental flexibility, as well as executive functioning. Currently, the Wechsler Adult Intelligence Scale (WAIS) is the most commonly used instrument in the armamentarium of clinical neuropsychologists.[2930] This test involves letter sequences and tests one’s ability to think logically and analytically. However, the Wechsler Adult Intelligence Scale-III and IV Letter Number Sequencing are not appropriate for non-alphabetic cultures. The Taiwan’s Odd-Even Number Sequencing Test (TOENST), as proposed by the Department of Psychology, National Taiwan University,[31] was adopted to replace the Letter Number Test. Participants need to rearrange and read the Arabic numerals in a series. The way to read out the series is: “read out the odd numbers in ascending order and then the even numbers in descending order.” For example, the random numbers 7, 2, 8, 6, 3 is incremented by an odd number (3, 7) and decrements by an even number (8, 6, 2), so the answer is 3 7 8 6 2.\\nThe comfort of the sound field environment was evaluated by a closed questionnaire, and the subjective comfort of the participant’s sound field environment was measured by the semantic differential method. There were mainly seven closed questionnaires in the questionnaire design, with “no feeling” in the middle (0 point). From middle to the left indicates that the ability deteriorates and worsens. Each left one grid is −1, the left two grids are −2, and so on. The middle to the right means that the ability to become better, every right one grid is +1, the right two grids is +2, and so on.\",\n \"Participants were tested in the same laboratory. Subject to the participants’ available time, the experiment was conducted under conditions of quiet (without noise), red noise, pink noise and white noise exposure. Before each experiment, the participants adopted a random method to determine the sound field exposure situation and the participants were not informed of the kind of sound scenario. The order of the four sound conditions (quiet, red, pink, white) was randomized. Ten minutes before the experiment was started, the participants were physiologically and psychologically adapted to the laboratory environment before they started the experiment. Each participant had to perform four tests and complete questionnaires in each of the four sessions in the experiment, and the order of the four tests was randomized. There was a five-minute break between each test. During the break, the sound exposure was on and the sound exposure was not terminated until all sound field exposure conditions had ended. After finishing four tests, participants filled in the sound field comfort evaluation questionnaire. During the filling in process, the sound continued. The flowchart in Figure 1 illustrates the experimental protocol.\\nFlow chart of the experimental protocol.\\nA within-subject design was used in the experiment with one independent variable, i.e. sound condition. Five dependent variables, including four objective parameters (psychomotor speed test, continuous performance test, executive function test, and working memory test) and one subjective perception, were collected to explore work efficiency in this study.\",\n \"The study was interested in evaluating the effectiveness of colour noise exposure. The approach considered was to measure the performance of a sample of participants before and after colour noise exposure, and analyse the differences using a paired sample t-test. IBM SPSS Statistics Version 20 was used to determine whether there were any statistically significant differences between the means of independent groups. Data from the completed questionnaires were stored in a database and transported in and subsequently analysed using the statistical program SPSS.\",\n \"Nil.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","SUBJECTS AND METHODS","Participants","Presentation of noise","Outcome measures","Experimental protocol","Statistical analysis of data","RESULTS","DISCUSSION","CONCLUSION","Financial support and sponsorship","Conflicts of interest"],"string":"[\n \"INTRODUCTION\",\n \"SUBJECTS AND METHODS\",\n \"Participants\",\n \"Presentation of noise\",\n \"Outcome measures\",\n \"Experimental protocol\",\n \"Statistical analysis of data\",\n \"RESULTS\",\n \"DISCUSSION\",\n \"CONCLUSION\",\n \"Financial support and sponsorship\",\n \"Conflicts of interest\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Exposure to high levels of noise in industry factory is associated with a number of effects on health manifested in various psychosocial responses such as annoyance, disturbance of daily activities, sleep and performance and in physical responses, such as hearing loss, hypertension and ischemic heart disease.[12] While the open-plan office is now widespread in the workplace, it appears that noise is a major nuisance factor in open-plan offices in spite of a relatively low noise level(less than 65 dB(A)).[3] In general, the higher the decibel numbers of sound, the greater the health hazard. However, when the number of sound decibels is not high, the degree of influence on people may vary depending on the sound characteristics.\nIn audio engineering, physics, and many other fields, the colour of noise refers to the power spectrum of a noise signal. The practice of naming kinds of noise after colours started with white noise, a signal whose spectrum has equal power within any equal interval of frequencies. That name was given by analogy with white light, which was assumed to have such a flat power spectrum over the visible range. Other colour names, like pink, red, and blue were then given to noise with other spectral profiles, often (but not always) in reference to the colour of light with similar spectra.\nNoise often has detrimental effects on performance. Under most circumstances, information processing is disturbed by environmental noise and other non-task compatible distracters.[45] However, researchers have recently reported that under certain circumstances individuals with attention problems appear to benefit from the addition of specific forms of environmental noise. Typically, this facilitative effect has been limited to non-vocal background music on simple arithmetic task performance,[6] but Stansfeld et al.[7] found just that under certain conditions even road traffic noise can improve performance on episodic memory tasks, particularly in children at risk of attention problems and academic underachievement. Furthermore, Söderlund et al.[8] have demonstrated that adding auditory white noise to the environment enhanced the memory performance of children with Attention Deficit Hyperactivity Disorder (ADHD)-type problems but disrupted that of non-ADHD control children. Stochastic resonance, a phenomenon whereby signal processing is enhanced by the addition of random noise, has been widely demonstrated across various modalities, including visual, auditory, tactile and cross-modal forms of processing.[910] Of particular interest are findings that auditory noise has the capacity to enhance some aspects of human cognitive performance, such as the speed of arithmetical computations.[11]\n\nMeanwhile, some recent studies showed related evidence. For example, in 2010, light music, an example belonging to pink noise, is proved beneficial for elder people to improve their sleep quality as a long term effect.[12] Moreover, in a previous study,[13] the steady pink noise with intensity of 35 dB, 40 dB, 50 dB and 60 dB was used to stimulate four participants during sleep and they believed that pink noise could improve the sleep quality of participants. However, it is clear that this conclusion was based on significant increase of light sleep period (especially stage 2) accompanied with declined duration of rapid eye movement. In the brain synchronization study, it has demonstrated that the pink noise could synchronize brain wave and induce brain activity into a specialized state, that is, in a lower complexity level.[14]\n\nSezici’s study showed that playing of white noise significantly decreased the daily crying durations and increased the sleeping durations of the colicky babies compared to swinging in both groups.[15] In the literature, parents emphasized that playing of white noise was quite beneficial in relieving pain among crying colicky babies.[16] In their studies, Kucukoglu et al.[17] and Karakoc and Turker[18] revealed that the pain scores of the newborns who were made to listen to white noise were observed to decrease compared with those of the newborns in the control group. White noise encompasses all characteristics of sounds within the range of human hearing. It has been used in the treatment of tinnitus, insomnia, masking unwanted sounds, and provision of relaxation.[1920]\n\nZhou et al.[14] demonstrated that steady pink noise has significant effect on reducing brain wave complexity and inducing more stable sleep time to improve sleep quality of individuals. In total, 42 elderly people (21 using music and 21 controls) completed the study, and there was some indication that soft slow music yielder higher improvement on some of the parameters, which are worthy of further investigation.[12] A tabulated list of the effects observed in previous studies with colour noise exposures is shown in Table 1.\nA tabulated list of the effects observed in previous studies with colour noise exposures\nSeveral experiments have demonstrated that the detrimental effect of office noise on worker attitude and performance can be significantly reduced through the use of masking noise.[2122232425] For instance the experimental study performed by Haka[24] demonstrated that the performance of operation span tasks, serial recall and long term memory tasks were all improved when masking noise was used; and the field study performed by Hongisto[23] indicated that masking noise significantly reduced disturbance of worker attitudes towards concentration caused by office noise. Masking noise achieves this improvement through reducing the intelligibility of nearby speech.[25] In addition, Vassie et al.[26] indicated that participants rejected the brown masking noise delivered through earphones as it caused irritation and discomfort. Future studies should also consider other types of masking noise and should measure the level and duration of the masking noise.[26]\n\nIt is well-known that any human action results from brain activity, which is affected by light and sound. Too much acoustic energy causes hearing loss, impaired human nerves, and emotional disorders.[2728] However, sound play also very important role for people. According to previous researchers, not only white noise can create shading effect, but pink noise can improve sleep quality. Most people in the workplace are in the office or in the service industry, not in the noisy working environment for the factory. Creating a quiet environment is sometimes not easy. However, adding some sounds to enhance the work efficiency and reduce the work pressure of low-noise environment should be an issue worth exploring. Therefore, this study wanted to understand the effects of three different colours noise exposure. In the past, there were not many studies in this regard. Therefore, the goal of the current study is to test the hypothesis that adding another sound in a low background noise environment may increases productivity and comfort."," Participants Twenty-two healthy college students were enrolled in this study. The mean age of the participants was 22 years. All participants reported no medical history of neurological disease, hypertension or heart disease. A machine called an audiometer was used to produce sounds at various volumes and frequencies. The participants being tested listened to sounds through headphones and responded when they hear them by pressing a button. A pure tone audiogram was performed by a trained health care professional at the beginning of the study for each participant, and the hearing acuity of the participants was determined to be normal, with the thresholds of 500 Hz, 1000 Hz, 2000 Hz and 4000 Hz being less than 15 dB. The participants received financial compensation for their participation in the experiment. Informed consent was obtained from all participants.\nTwenty-two healthy college students were enrolled in this study. The mean age of the participants was 22 years. All participants reported no medical history of neurological disease, hypertension or heart disease. A machine called an audiometer was used to produce sounds at various volumes and frequencies. The participants being tested listened to sounds through headphones and responded when they hear them by pressing a button. A pure tone audiogram was performed by a trained health care professional at the beginning of the study for each participant, and the hearing acuity of the participants was determined to be normal, with the thresholds of 500 Hz, 1000 Hz, 2000 Hz and 4000 Hz being less than 15 dB. The participants received financial compensation for their participation in the experiment. Informed consent was obtained from all participants.\n Presentation of noise During this experiment, four sound conditions were used, relating to the three types of masking sound (red noise, pink noise, white noise) and a condition, which consisted of a wide band background noise. White noise is a signal with a flat frequency spectrum when plotted as a linear function of frequency. Red noise will refer to a power density which decreases 6 dB per octave with increasing frequency (density proportional to 1/f2). The frequency spectrum of pink noise is linear in logarithmic scale, and the spectral power density, compared with white noise, decreases by 3 dB per octave (density proportional to 1/f). The noise level within the room used in the study was measured using a Cirrus Type 832C sound level meter. Thirty second SPL samples, LAeq,30s, were measured over a period of an hour. Noise levels ranged from an Lmin of 41 dBA to an Lmax of 50 dBA. Measurements performed on successive days identified similar ranges of Lmin and Lmax.\nThe signal source of masking sound was sent through a desktop computer to a Bluetooth speaker (BOSE SoundLink Mini). The sound level of each colour noise exposure was about 47 dBA when turning on the speaker and the background noise (without adding colour noise) was 44 dBA. Background noise had a decibel (dBA) value of three decibels (dBA) difference compared to the noise exposure scenario of three different colours, so that means that the sound energy of the colour noise exposure scenario was double that of the original silence.\nDuring this experiment, four sound conditions were used, relating to the three types of masking sound (red noise, pink noise, white noise) and a condition, which consisted of a wide band background noise. White noise is a signal with a flat frequency spectrum when plotted as a linear function of frequency. Red noise will refer to a power density which decreases 6 dB per octave with increasing frequency (density proportional to 1/f2). The frequency spectrum of pink noise is linear in logarithmic scale, and the spectral power density, compared with white noise, decreases by 3 dB per octave (density proportional to 1/f). The noise level within the room used in the study was measured using a Cirrus Type 832C sound level meter. Thirty second SPL samples, LAeq,30s, were measured over a period of an hour. Noise levels ranged from an Lmin of 41 dBA to an Lmax of 50 dBA. Measurements performed on successive days identified similar ranges of Lmin and Lmax.\nThe signal source of masking sound was sent through a desktop computer to a Bluetooth speaker (BOSE SoundLink Mini). The sound level of each colour noise exposure was about 47 dBA when turning on the speaker and the background noise (without adding colour noise) was 44 dBA. Background noise had a decibel (dBA) value of three decibels (dBA) difference compared to the noise exposure scenario of three different colours, so that means that the sound energy of the colour noise exposure scenario was double that of the original silence.\n Outcome measures Over the years, different scholars have given their definitions of efficiency, and different scholars also have different views and explanations for it. Therefore, it is not easy to offer a precise definition of work efficiency. Among many offerings, the definition of work efficiency includes “work response time”, the time when the work instruction is received plus the response time, and “work execution processing time” the time it takes for each task from the reaction to the completion. “Working memory” refers to the processing of tasks for the content of the current short-term memories. Cognitive function is concept that covers several functions of the brain such as attention, executive function, processing speed, learning and memory. Information processing and response speed are basic cognitive function, which is needed for more complex functions such as working memory. We measured cognitive performance via a battery of standardized cognitive tests. If improvements were observed in cognitive performance, this would not only have important benefits for employees, but benefits for the organization in terms of the potential for increased productivity. The main outcome measurers for this study were those of four laboratory tests: Psychomotor Speed Test, Continuous Performance Test, Executive Function Test, and Working Memory Test. These tests were presented in a random order. Participants completed them on individual laptops in a laboratory with one of the testers present. The Psychomotor Speed Test measures Simple Reaction Time (SRT), general alertness and motor speed through delivery of a known stimulus to a known location to elicit a known response. The Continuous Performance Test − Identical Pairs (CPT-IP) requires the identification of identical stimulus pairs within a continuously presented series of stimuli. The Trail Making Test (TMT) is a widely used test to assess executive function, and it is a neuropsychological test of visual attention and task switching. It consists of two parts in which the participant is instructed to connect a set of 25 dots as quickly as possible while still maintaining accuracy. The test can provide information about visual search speed, scanning, speed of processing, mental flexibility, as well as executive functioning. Currently, the Wechsler Adult Intelligence Scale (WAIS) is the most commonly used instrument in the armamentarium of clinical neuropsychologists.[2930] This test involves letter sequences and tests one’s ability to think logically and analytically. However, the Wechsler Adult Intelligence Scale-III and IV Letter Number Sequencing are not appropriate for non-alphabetic cultures. The Taiwan’s Odd-Even Number Sequencing Test (TOENST), as proposed by the Department of Psychology, National Taiwan University,[31] was adopted to replace the Letter Number Test. Participants need to rearrange and read the Arabic numerals in a series. The way to read out the series is: “read out the odd numbers in ascending order and then the even numbers in descending order.” For example, the random numbers 7, 2, 8, 6, 3 is incremented by an odd number (3, 7) and decrements by an even number (8, 6, 2), so the answer is 3 7 8 6 2.\nThe comfort of the sound field environment was evaluated by a closed questionnaire, and the subjective comfort of the participant’s sound field environment was measured by the semantic differential method. There were mainly seven closed questionnaires in the questionnaire design, with “no feeling” in the middle (0 point). From middle to the left indicates that the ability deteriorates and worsens. Each left one grid is −1, the left two grids are −2, and so on. The middle to the right means that the ability to become better, every right one grid is +1, the right two grids is +2, and so on.\nOver the years, different scholars have given their definitions of efficiency, and different scholars also have different views and explanations for it. Therefore, it is not easy to offer a precise definition of work efficiency. Among many offerings, the definition of work efficiency includes “work response time”, the time when the work instruction is received plus the response time, and “work execution processing time” the time it takes for each task from the reaction to the completion. “Working memory” refers to the processing of tasks for the content of the current short-term memories. Cognitive function is concept that covers several functions of the brain such as attention, executive function, processing speed, learning and memory. Information processing and response speed are basic cognitive function, which is needed for more complex functions such as working memory. We measured cognitive performance via a battery of standardized cognitive tests. If improvements were observed in cognitive performance, this would not only have important benefits for employees, but benefits for the organization in terms of the potential for increased productivity. The main outcome measurers for this study were those of four laboratory tests: Psychomotor Speed Test, Continuous Performance Test, Executive Function Test, and Working Memory Test. These tests were presented in a random order. Participants completed them on individual laptops in a laboratory with one of the testers present. The Psychomotor Speed Test measures Simple Reaction Time (SRT), general alertness and motor speed through delivery of a known stimulus to a known location to elicit a known response. The Continuous Performance Test − Identical Pairs (CPT-IP) requires the identification of identical stimulus pairs within a continuously presented series of stimuli. The Trail Making Test (TMT) is a widely used test to assess executive function, and it is a neuropsychological test of visual attention and task switching. It consists of two parts in which the participant is instructed to connect a set of 25 dots as quickly as possible while still maintaining accuracy. The test can provide information about visual search speed, scanning, speed of processing, mental flexibility, as well as executive functioning. Currently, the Wechsler Adult Intelligence Scale (WAIS) is the most commonly used instrument in the armamentarium of clinical neuropsychologists.[2930] This test involves letter sequences and tests one’s ability to think logically and analytically. However, the Wechsler Adult Intelligence Scale-III and IV Letter Number Sequencing are not appropriate for non-alphabetic cultures. The Taiwan’s Odd-Even Number Sequencing Test (TOENST), as proposed by the Department of Psychology, National Taiwan University,[31] was adopted to replace the Letter Number Test. Participants need to rearrange and read the Arabic numerals in a series. The way to read out the series is: “read out the odd numbers in ascending order and then the even numbers in descending order.” For example, the random numbers 7, 2, 8, 6, 3 is incremented by an odd number (3, 7) and decrements by an even number (8, 6, 2), so the answer is 3 7 8 6 2.\nThe comfort of the sound field environment was evaluated by a closed questionnaire, and the subjective comfort of the participant’s sound field environment was measured by the semantic differential method. There were mainly seven closed questionnaires in the questionnaire design, with “no feeling” in the middle (0 point). From middle to the left indicates that the ability deteriorates and worsens. Each left one grid is −1, the left two grids are −2, and so on. The middle to the right means that the ability to become better, every right one grid is +1, the right two grids is +2, and so on.\n Experimental protocol Participants were tested in the same laboratory. Subject to the participants’ available time, the experiment was conducted under conditions of quiet (without noise), red noise, pink noise and white noise exposure. Before each experiment, the participants adopted a random method to determine the sound field exposure situation and the participants were not informed of the kind of sound scenario. The order of the four sound conditions (quiet, red, pink, white) was randomized. Ten minutes before the experiment was started, the participants were physiologically and psychologically adapted to the laboratory environment before they started the experiment. Each participant had to perform four tests and complete questionnaires in each of the four sessions in the experiment, and the order of the four tests was randomized. There was a five-minute break between each test. During the break, the sound exposure was on and the sound exposure was not terminated until all sound field exposure conditions had ended. After finishing four tests, participants filled in the sound field comfort evaluation questionnaire. During the filling in process, the sound continued. The flowchart in Figure 1 illustrates the experimental protocol.\nFlow chart of the experimental protocol.\nA within-subject design was used in the experiment with one independent variable, i.e. sound condition. Five dependent variables, including four objective parameters (psychomotor speed test, continuous performance test, executive function test, and working memory test) and one subjective perception, were collected to explore work efficiency in this study.\nParticipants were tested in the same laboratory. Subject to the participants’ available time, the experiment was conducted under conditions of quiet (without noise), red noise, pink noise and white noise exposure. Before each experiment, the participants adopted a random method to determine the sound field exposure situation and the participants were not informed of the kind of sound scenario. The order of the four sound conditions (quiet, red, pink, white) was randomized. Ten minutes before the experiment was started, the participants were physiologically and psychologically adapted to the laboratory environment before they started the experiment. Each participant had to perform four tests and complete questionnaires in each of the four sessions in the experiment, and the order of the four tests was randomized. There was a five-minute break between each test. During the break, the sound exposure was on and the sound exposure was not terminated until all sound field exposure conditions had ended. After finishing four tests, participants filled in the sound field comfort evaluation questionnaire. During the filling in process, the sound continued. The flowchart in Figure 1 illustrates the experimental protocol.\nFlow chart of the experimental protocol.\nA within-subject design was used in the experiment with one independent variable, i.e. sound condition. Five dependent variables, including four objective parameters (psychomotor speed test, continuous performance test, executive function test, and working memory test) and one subjective perception, were collected to explore work efficiency in this study.\n Statistical analysis of data The study was interested in evaluating the effectiveness of colour noise exposure. The approach considered was to measure the performance of a sample of participants before and after colour noise exposure, and analyse the differences using a paired sample t-test. IBM SPSS Statistics Version 20 was used to determine whether there were any statistically significant differences between the means of independent groups. Data from the completed questionnaires were stored in a database and transported in and subsequently analysed using the statistical program SPSS.\nThe study was interested in evaluating the effectiveness of colour noise exposure. The approach considered was to measure the performance of a sample of participants before and after colour noise exposure, and analyse the differences using a paired sample t-test. IBM SPSS Statistics Version 20 was used to determine whether there were any statistically significant differences between the means of independent groups. Data from the completed questionnaires were stored in a database and transported in and subsequently analysed using the statistical program SPSS.","Twenty-two healthy college students were enrolled in this study. The mean age of the participants was 22 years. All participants reported no medical history of neurological disease, hypertension or heart disease. A machine called an audiometer was used to produce sounds at various volumes and frequencies. The participants being tested listened to sounds through headphones and responded when they hear them by pressing a button. A pure tone audiogram was performed by a trained health care professional at the beginning of the study for each participant, and the hearing acuity of the participants was determined to be normal, with the thresholds of 500 Hz, 1000 Hz, 2000 Hz and 4000 Hz being less than 15 dB. The participants received financial compensation for their participation in the experiment. Informed consent was obtained from all participants.","During this experiment, four sound conditions were used, relating to the three types of masking sound (red noise, pink noise, white noise) and a condition, which consisted of a wide band background noise. White noise is a signal with a flat frequency spectrum when plotted as a linear function of frequency. Red noise will refer to a power density which decreases 6 dB per octave with increasing frequency (density proportional to 1/f2). The frequency spectrum of pink noise is linear in logarithmic scale, and the spectral power density, compared with white noise, decreases by 3 dB per octave (density proportional to 1/f). The noise level within the room used in the study was measured using a Cirrus Type 832C sound level meter. Thirty second SPL samples, LAeq,30s, were measured over a period of an hour. Noise levels ranged from an Lmin of 41 dBA to an Lmax of 50 dBA. Measurements performed on successive days identified similar ranges of Lmin and Lmax.\nThe signal source of masking sound was sent through a desktop computer to a Bluetooth speaker (BOSE SoundLink Mini). The sound level of each colour noise exposure was about 47 dBA when turning on the speaker and the background noise (without adding colour noise) was 44 dBA. Background noise had a decibel (dBA) value of three decibels (dBA) difference compared to the noise exposure scenario of three different colours, so that means that the sound energy of the colour noise exposure scenario was double that of the original silence.","Over the years, different scholars have given their definitions of efficiency, and different scholars also have different views and explanations for it. Therefore, it is not easy to offer a precise definition of work efficiency. Among many offerings, the definition of work efficiency includes “work response time”, the time when the work instruction is received plus the response time, and “work execution processing time” the time it takes for each task from the reaction to the completion. “Working memory” refers to the processing of tasks for the content of the current short-term memories. Cognitive function is concept that covers several functions of the brain such as attention, executive function, processing speed, learning and memory. Information processing and response speed are basic cognitive function, which is needed for more complex functions such as working memory. We measured cognitive performance via a battery of standardized cognitive tests. If improvements were observed in cognitive performance, this would not only have important benefits for employees, but benefits for the organization in terms of the potential for increased productivity. The main outcome measurers for this study were those of four laboratory tests: Psychomotor Speed Test, Continuous Performance Test, Executive Function Test, and Working Memory Test. These tests were presented in a random order. Participants completed them on individual laptops in a laboratory with one of the testers present. The Psychomotor Speed Test measures Simple Reaction Time (SRT), general alertness and motor speed through delivery of a known stimulus to a known location to elicit a known response. The Continuous Performance Test − Identical Pairs (CPT-IP) requires the identification of identical stimulus pairs within a continuously presented series of stimuli. The Trail Making Test (TMT) is a widely used test to assess executive function, and it is a neuropsychological test of visual attention and task switching. It consists of two parts in which the participant is instructed to connect a set of 25 dots as quickly as possible while still maintaining accuracy. The test can provide information about visual search speed, scanning, speed of processing, mental flexibility, as well as executive functioning. Currently, the Wechsler Adult Intelligence Scale (WAIS) is the most commonly used instrument in the armamentarium of clinical neuropsychologists.[2930] This test involves letter sequences and tests one’s ability to think logically and analytically. However, the Wechsler Adult Intelligence Scale-III and IV Letter Number Sequencing are not appropriate for non-alphabetic cultures. The Taiwan’s Odd-Even Number Sequencing Test (TOENST), as proposed by the Department of Psychology, National Taiwan University,[31] was adopted to replace the Letter Number Test. Participants need to rearrange and read the Arabic numerals in a series. The way to read out the series is: “read out the odd numbers in ascending order and then the even numbers in descending order.” For example, the random numbers 7, 2, 8, 6, 3 is incremented by an odd number (3, 7) and decrements by an even number (8, 6, 2), so the answer is 3 7 8 6 2.\nThe comfort of the sound field environment was evaluated by a closed questionnaire, and the subjective comfort of the participant’s sound field environment was measured by the semantic differential method. There were mainly seven closed questionnaires in the questionnaire design, with “no feeling” in the middle (0 point). From middle to the left indicates that the ability deteriorates and worsens. Each left one grid is −1, the left two grids are −2, and so on. The middle to the right means that the ability to become better, every right one grid is +1, the right two grids is +2, and so on.","Participants were tested in the same laboratory. Subject to the participants’ available time, the experiment was conducted under conditions of quiet (without noise), red noise, pink noise and white noise exposure. Before each experiment, the participants adopted a random method to determine the sound field exposure situation and the participants were not informed of the kind of sound scenario. The order of the four sound conditions (quiet, red, pink, white) was randomized. Ten minutes before the experiment was started, the participants were physiologically and psychologically adapted to the laboratory environment before they started the experiment. Each participant had to perform four tests and complete questionnaires in each of the four sessions in the experiment, and the order of the four tests was randomized. There was a five-minute break between each test. During the break, the sound exposure was on and the sound exposure was not terminated until all sound field exposure conditions had ended. After finishing four tests, participants filled in the sound field comfort evaluation questionnaire. During the filling in process, the sound continued. The flowchart in Figure 1 illustrates the experimental protocol.\nFlow chart of the experimental protocol.\nA within-subject design was used in the experiment with one independent variable, i.e. sound condition. Five dependent variables, including four objective parameters (psychomotor speed test, continuous performance test, executive function test, and working memory test) and one subjective perception, were collected to explore work efficiency in this study.","The study was interested in evaluating the effectiveness of colour noise exposure. The approach considered was to measure the performance of a sample of participants before and after colour noise exposure, and analyse the differences using a paired sample t-test. IBM SPSS Statistics Version 20 was used to determine whether there were any statistically significant differences between the means of independent groups. Data from the completed questionnaires were stored in a database and transported in and subsequently analysed using the statistical program SPSS.","The results of the four tests conducted in this current study are summarized in Table 2. For each simple reaction time test, 10 sets of tests were completed. In the simple reaction time test, the average response time was better in red noise exposure scenario (0.565 seconds) than in the pink noise exposure scenario (0.602 seconds) and in the white noise exposure scenario (0.609 seconds). The variances of red noise exposure scenario (0.005 seconds) and pink noise exposure scenario (0.008 seconds) were lower than the white noise exposure scenario (0.013 seconds) and the quiet scenario (0.023 seconds). From the average of 10 test results, there was a very significant difference between the three noise exposure scenarios, red noise exposure (P < 0.001), pink noise exposure (P < 0.001), white noise exposure (P < 0.001) and the quiet scenario, as shown in Figure 2.\nMeans and variances of SRT, CPT-IP, TMT, TOENST in four different sound field environments\nPaired t-test analysis of Simple Reaction Time (SRT) in four different sound field exposures.\nCompared with the average correct rate in the continuous performance test, 93.6% of the pink noise exposure situation is better than 91.7% of the red noise exposure situation and 91.1% of the white noise exposure situation, and the three kinds of noise exposure are better than 88.8% on the quiet situation. Among the 22 participants, half of the respondents achieved 93.3% correct rate on red noise exposure, 93.3% on pink noise exposure, 93.3% on white noise exposure, and they all scored better than 90.0% in the quiet situation. The average correct rate of the test showed that there was a slight improvement over the quiet scenario for all of the red noise scenario, the pink noise scenario, and the white noise scenario. The paired t-test showed significant differences (P < 0.05) between the pink noise exposure scenario and the quiet scenario in the continuous performance test, Figure 3.\nPaired t-test analysis of continuous performance test-identical pairs (CPT-IP) in four different sound field exposures.\nIn the executive function tests, comparing the average time it takes to complete the test, it shows the red noise exposure scenario (21.37 seconds) is superior to the pink noise exposure scenario (22.31 seconds) and the white noise exposure scenario (23.11 seconds), all of which are better than the quiet scenario (24.54 seconds). The best test results (minimum) showed that the red noise exposure scenario (12.00 seconds), pink noise exposure scenario (10.73 seconds), white noise exposure scenario (13.13 seconds) were also better than the quiet scenario (15.33 seconds). The average finishing time of the tests showed that the red noise exposure situation, the pink noise exposure situation and the white noise exposure situation are better than the quiet situation. As shown on Figure 4, the paired t-test of executive function tests showed significant differences between the red noise exposure scenario (P < 0.001), the pink noise exposure scenario (P < 0.001), white noise exposure scenario (P < 0.001) and the quiet scenario.\nPaired t-test analysis of Trail Making Test (TMT) in four different sound field exposures.\nThe average score of the working memory test showed that the red noise exposure situation (18.8 points) was superior to the pink noise exposure situation (17.3 points) and the white noise exposure situation (15.9 points), all of which were better than the quiet situation (14.4 points). In a comparison of variance, the red noise exposure (5.1 points) and the pink noise exposure (6.9 points) were significantly lower than the white noise exposure (11.2 points) and noiseless exposure (9.3 points). The paired t-test analysis showed that in the working memory test, the red noise exposure (P < 0.001), the pink noise exposure (P < 0.001), the white noise exposure (P < 0.01), the three noise exposure all had a significant difference as compared with the quiet situation, as shown on Figure 5.\nPaired t-test analysis of Taiwan’s Odd-Even Number Sequencing Test (TOENST) in four different sound field exposures.\nAs shown in Table 3, compared with the quiet situation, both the red noise and the pink noise exposure in the simple reaction time test, the executive function test and the working memory test all showed significant differences (P < 0.001). The white noise exposure was very significantly different (P < 0.001) in the simple reaction time test, and there was a significant difference (P < 0.01) in the working memory test. However, only pink noise exposure was significantly different (P < 0.05) in the continuous performance test.\nPaired t-test analysis of three colour noise exposure situations compared with quiet situations\nA statistical analysis of the sound field comfort questionnaire showed that, the questionnaire had a Cronbach’s alpha score of 0.772. Generally, a questionnaire with a Cronbach’s alpha score between 0.7 and 0.9 is considered to be of good reliability. As shown in Table 4, the subjective comfort evaluation statistics of the sound field environment indicated a highest and lowest score of 0 and −2, a little worse in the quiet scenario, while the scores between −1 and 3 were slightly worse to good in the red noise scenario; the scores between −1 and 2 were slightly worse to better in pink noise scenario; however, the scores between −3 and 1 represented a worse to slightly better result for the white noise scenario. The sum of the scores of the subjective evaluation in the sound field were −40 (average rating −0.26), +81 (average rating 0.53), +38 (average rating 0.25), −94 (average rating −0.61) for quiet, red noise, pink noise, and white noise scenarios, respectively.\nSubjective comfort evaluations of four sound environments","Of the four trials, there were three significant (P < 0.001) increases in scores for red noise exposure and pink noise exposure, possibly reflecting improvements in work efficiency. Literature explains that white noise exposure can serve as a masking sound, and this may improve work efficiency. Two tests showed a significant (P < 0.001) increase in performance, with a significant (P < 0.01) increase in one trial also corroborating previous studies. In brain synchronization studies, pink noise exposure has been shown to synchronize the brain and induce brain activity to lower its potential,[13] and to exclude the effects of the outside world for purposes of concentrated processing. Except for the slight significant difference (P < 0.05) in white noise exposure, the continuous performance test was the only one that did not show very significant results. Participants may all have been preoccupied with the test, so the average correct rates are above 90%. Even the correct rates of two participants reached 100% when exposed to all four sound situations, and there were three participants with a 100% correct rate when exposed to three scenarios.\nIn the continuous performance test, the correct rate of non-exposure was about 88.8%, which of red noise, pink noise, and white noise were 91.7%, 93.6%, and 91.1%, respectively. Half of the 22 participants achieved a correct rate of 93.3% under the conditions of red noise exposure, pink noise exposure and white noise exposure, all of which were higher than the 88.8% correct rate of no noise exposure. The average score of the test results showed that a red noise exposure scenario, pink noise exposure scenario, and white noise exposure scenario are slightly better than the quiet scenario, which means that with the exposure to colour noise, the correct rate will rise and attention will be slightly improved.\nBecause the exposure of the participant’s living environment to the noise itself, might affect the questionnaire data, the type of accommodation should be indicated on the comfort questionnaire, and whether or not there is any noise exposure should be indicated in the closed questionnaire. In this study, there were three home participants exposed to noise, there were three home participants who sometimes had noise exposure. Of these six participants, five of the participants had different answers from the others, indicating a larger numerical increase in white noise.\nThe scores of the questionnaire results in the comfort of colour noises environment did not fully match the test data. Participants were randomly interviewed after the experiment. They said that with the additional sound increases, they felt that the sound interference, and the selection value should be less favourable. The laboratory background sound level was 44 dBA in the quiet scenario. The participants experienced significant changes to sound of 47 dBA when exposed to colour noise. Moreover, because the experimental exposure scenario was determined in a random manner, it was difficult to remember which previous test scores were selected. Scenario comfort evaluation is felt on a subjective basis, but participants may have had preconceptions when completing the questionnaire, thus affecting the final score. Although the results of the environmental comfort questionnaire were not as expected, the overall percentage of participants who felt slightly better or above were 48.8%, 32%, 9% for participants exposed to red, pink, white noise, respectively.\nThe results of this study show that exposure to the three kinds of colour noises, red, pink and white noise yields significantly better results in the Psychomotor Speed Test, Continuous Performance Test, Executive Function Test and Working Memory Test, which proves that these three kinds of sound field environments on the office work-type job site might improve work efficiency. The sum of the questionnaire scores on subjective comfort of the sound field environment showed that red, pink, and white noise exposure, were better than the original no noise exposure of the sound field, and this result implies that the three colour noise sound fields seem to make the white-collar work patterns a better workplaces not only by improving productivity, but also by making workers feel comfortable. Because white noise is equal energy at each frequency, it can cover the sounds that do not want to be heard around the environment. Pink noise might be due to neurons in the hypothalamus synchronously resonating with pink noises in the low oscillation frequency band, making slow rhythms and dominating brain waves. In addition, the power density of high frequency in red noise is even lower than these in pink noise and may therefore affect neurons in the hypothalamus even more, leading to information more speedily reaching the cerebral cortex through the hypothalamus and to faster transmission of motor messages through the hypothalamus.\nThe concept of the open-plan office is now widespread in the workplace. While offering many advantages in terms of layout and facilitating communication between colleagues, this way of organising the workspace had two major disadvantages. The feeling of privacy is lessened and noise level is increased causing discomfort for individuals working in this type of environment. It appears that noise is a major nuisance factor in open-plan offices in spite of a relatively low noise level. The noise nuisance experienced in open-plan offices affects work satisfaction, and this exposure to noise can reduce employee performance depending on the types of task to be carried out and the characteristics of the noise present in the workplace. The current research suggests that creating a colour noise environment through a loudspeaker or headphone might improve some aspects of cognitive performance in different workplaces. Many studies in the past have proved that noise has a negative influence on human hearing, cardiovascular systems, and emotions and so on. However, the three colour noises are applied to the original sound field environment. Therefore, if the background noise of the workplace is already too high, it would not be in order, for health reasons, to further add colour noise.","The results of this study show that participants perform significantly better in Psychomotor Speed Test, Continuous Performance Test, Executive Function Test and Working Memory Test. Therefore, colour noise may be used in the future to improve a workplace sound field environment, so that white-collar workers have a better working environment. Using physical stimuli of red, pink and white noises to improve worker productivity may prove to be a new approach towards productivity improvement that may provide fewer side effects than traditional methods such as drinking refreshing beverages. The introduction of the sounds represents a simple, low-cost, non-invasive improvement.\n Financial support and sponsorship Nil.\nNil.\n Conflicts of interest There are no conflicts of interest.\nThere are no conflicts of interest.","Nil.","There are no conflicts of interest."],"string":"[\n \"Exposure to high levels of noise in industry factory is associated with a number of effects on health manifested in various psychosocial responses such as annoyance, disturbance of daily activities, sleep and performance and in physical responses, such as hearing loss, hypertension and ischemic heart disease.[12] While the open-plan office is now widespread in the workplace, it appears that noise is a major nuisance factor in open-plan offices in spite of a relatively low noise level(less than 65 dB(A)).[3] In general, the higher the decibel numbers of sound, the greater the health hazard. However, when the number of sound decibels is not high, the degree of influence on people may vary depending on the sound characteristics.\\nIn audio engineering, physics, and many other fields, the colour of noise refers to the power spectrum of a noise signal. The practice of naming kinds of noise after colours started with white noise, a signal whose spectrum has equal power within any equal interval of frequencies. That name was given by analogy with white light, which was assumed to have such a flat power spectrum over the visible range. Other colour names, like pink, red, and blue were then given to noise with other spectral profiles, often (but not always) in reference to the colour of light with similar spectra.\\nNoise often has detrimental effects on performance. Under most circumstances, information processing is disturbed by environmental noise and other non-task compatible distracters.[45] However, researchers have recently reported that under certain circumstances individuals with attention problems appear to benefit from the addition of specific forms of environmental noise. Typically, this facilitative effect has been limited to non-vocal background music on simple arithmetic task performance,[6] but Stansfeld et al.[7] found just that under certain conditions even road traffic noise can improve performance on episodic memory tasks, particularly in children at risk of attention problems and academic underachievement. Furthermore, Söderlund et al.[8] have demonstrated that adding auditory white noise to the environment enhanced the memory performance of children with Attention Deficit Hyperactivity Disorder (ADHD)-type problems but disrupted that of non-ADHD control children. Stochastic resonance, a phenomenon whereby signal processing is enhanced by the addition of random noise, has been widely demonstrated across various modalities, including visual, auditory, tactile and cross-modal forms of processing.[910] Of particular interest are findings that auditory noise has the capacity to enhance some aspects of human cognitive performance, such as the speed of arithmetical computations.[11]\\n\\nMeanwhile, some recent studies showed related evidence. For example, in 2010, light music, an example belonging to pink noise, is proved beneficial for elder people to improve their sleep quality as a long term effect.[12] Moreover, in a previous study,[13] the steady pink noise with intensity of 35 dB, 40 dB, 50 dB and 60 dB was used to stimulate four participants during sleep and they believed that pink noise could improve the sleep quality of participants. However, it is clear that this conclusion was based on significant increase of light sleep period (especially stage 2) accompanied with declined duration of rapid eye movement. In the brain synchronization study, it has demonstrated that the pink noise could synchronize brain wave and induce brain activity into a specialized state, that is, in a lower complexity level.[14]\\n\\nSezici’s study showed that playing of white noise significantly decreased the daily crying durations and increased the sleeping durations of the colicky babies compared to swinging in both groups.[15] In the literature, parents emphasized that playing of white noise was quite beneficial in relieving pain among crying colicky babies.[16] In their studies, Kucukoglu et al.[17] and Karakoc and Turker[18] revealed that the pain scores of the newborns who were made to listen to white noise were observed to decrease compared with those of the newborns in the control group. White noise encompasses all characteristics of sounds within the range of human hearing. It has been used in the treatment of tinnitus, insomnia, masking unwanted sounds, and provision of relaxation.[1920]\\n\\nZhou et al.[14] demonstrated that steady pink noise has significant effect on reducing brain wave complexity and inducing more stable sleep time to improve sleep quality of individuals. In total, 42 elderly people (21 using music and 21 controls) completed the study, and there was some indication that soft slow music yielder higher improvement on some of the parameters, which are worthy of further investigation.[12] A tabulated list of the effects observed in previous studies with colour noise exposures is shown in Table 1.\\nA tabulated list of the effects observed in previous studies with colour noise exposures\\nSeveral experiments have demonstrated that the detrimental effect of office noise on worker attitude and performance can be significantly reduced through the use of masking noise.[2122232425] For instance the experimental study performed by Haka[24] demonstrated that the performance of operation span tasks, serial recall and long term memory tasks were all improved when masking noise was used; and the field study performed by Hongisto[23] indicated that masking noise significantly reduced disturbance of worker attitudes towards concentration caused by office noise. Masking noise achieves this improvement through reducing the intelligibility of nearby speech.[25] In addition, Vassie et al.[26] indicated that participants rejected the brown masking noise delivered through earphones as it caused irritation and discomfort. Future studies should also consider other types of masking noise and should measure the level and duration of the masking noise.[26]\\n\\nIt is well-known that any human action results from brain activity, which is affected by light and sound. Too much acoustic energy causes hearing loss, impaired human nerves, and emotional disorders.[2728] However, sound play also very important role for people. According to previous researchers, not only white noise can create shading effect, but pink noise can improve sleep quality. Most people in the workplace are in the office or in the service industry, not in the noisy working environment for the factory. Creating a quiet environment is sometimes not easy. However, adding some sounds to enhance the work efficiency and reduce the work pressure of low-noise environment should be an issue worth exploring. Therefore, this study wanted to understand the effects of three different colours noise exposure. In the past, there were not many studies in this regard. Therefore, the goal of the current study is to test the hypothesis that adding another sound in a low background noise environment may increases productivity and comfort.\",\n \" Participants Twenty-two healthy college students were enrolled in this study. The mean age of the participants was 22 years. All participants reported no medical history of neurological disease, hypertension or heart disease. A machine called an audiometer was used to produce sounds at various volumes and frequencies. The participants being tested listened to sounds through headphones and responded when they hear them by pressing a button. A pure tone audiogram was performed by a trained health care professional at the beginning of the study for each participant, and the hearing acuity of the participants was determined to be normal, with the thresholds of 500 Hz, 1000 Hz, 2000 Hz and 4000 Hz being less than 15 dB. The participants received financial compensation for their participation in the experiment. Informed consent was obtained from all participants.\\nTwenty-two healthy college students were enrolled in this study. The mean age of the participants was 22 years. All participants reported no medical history of neurological disease, hypertension or heart disease. A machine called an audiometer was used to produce sounds at various volumes and frequencies. The participants being tested listened to sounds through headphones and responded when they hear them by pressing a button. A pure tone audiogram was performed by a trained health care professional at the beginning of the study for each participant, and the hearing acuity of the participants was determined to be normal, with the thresholds of 500 Hz, 1000 Hz, 2000 Hz and 4000 Hz being less than 15 dB. The participants received financial compensation for their participation in the experiment. Informed consent was obtained from all participants.\\n Presentation of noise During this experiment, four sound conditions were used, relating to the three types of masking sound (red noise, pink noise, white noise) and a condition, which consisted of a wide band background noise. White noise is a signal with a flat frequency spectrum when plotted as a linear function of frequency. Red noise will refer to a power density which decreases 6 dB per octave with increasing frequency (density proportional to 1/f2). The frequency spectrum of pink noise is linear in logarithmic scale, and the spectral power density, compared with white noise, decreases by 3 dB per octave (density proportional to 1/f). The noise level within the room used in the study was measured using a Cirrus Type 832C sound level meter. Thirty second SPL samples, LAeq,30s, were measured over a period of an hour. Noise levels ranged from an Lmin of 41 dBA to an Lmax of 50 dBA. Measurements performed on successive days identified similar ranges of Lmin and Lmax.\\nThe signal source of masking sound was sent through a desktop computer to a Bluetooth speaker (BOSE SoundLink Mini). The sound level of each colour noise exposure was about 47 dBA when turning on the speaker and the background noise (without adding colour noise) was 44 dBA. Background noise had a decibel (dBA) value of three decibels (dBA) difference compared to the noise exposure scenario of three different colours, so that means that the sound energy of the colour noise exposure scenario was double that of the original silence.\\nDuring this experiment, four sound conditions were used, relating to the three types of masking sound (red noise, pink noise, white noise) and a condition, which consisted of a wide band background noise. White noise is a signal with a flat frequency spectrum when plotted as a linear function of frequency. Red noise will refer to a power density which decreases 6 dB per octave with increasing frequency (density proportional to 1/f2). The frequency spectrum of pink noise is linear in logarithmic scale, and the spectral power density, compared with white noise, decreases by 3 dB per octave (density proportional to 1/f). The noise level within the room used in the study was measured using a Cirrus Type 832C sound level meter. Thirty second SPL samples, LAeq,30s, were measured over a period of an hour. Noise levels ranged from an Lmin of 41 dBA to an Lmax of 50 dBA. Measurements performed on successive days identified similar ranges of Lmin and Lmax.\\nThe signal source of masking sound was sent through a desktop computer to a Bluetooth speaker (BOSE SoundLink Mini). The sound level of each colour noise exposure was about 47 dBA when turning on the speaker and the background noise (without adding colour noise) was 44 dBA. Background noise had a decibel (dBA) value of three decibels (dBA) difference compared to the noise exposure scenario of three different colours, so that means that the sound energy of the colour noise exposure scenario was double that of the original silence.\\n Outcome measures Over the years, different scholars have given their definitions of efficiency, and different scholars also have different views and explanations for it. Therefore, it is not easy to offer a precise definition of work efficiency. Among many offerings, the definition of work efficiency includes “work response time”, the time when the work instruction is received plus the response time, and “work execution processing time” the time it takes for each task from the reaction to the completion. “Working memory” refers to the processing of tasks for the content of the current short-term memories. Cognitive function is concept that covers several functions of the brain such as attention, executive function, processing speed, learning and memory. Information processing and response speed are basic cognitive function, which is needed for more complex functions such as working memory. We measured cognitive performance via a battery of standardized cognitive tests. If improvements were observed in cognitive performance, this would not only have important benefits for employees, but benefits for the organization in terms of the potential for increased productivity. The main outcome measurers for this study were those of four laboratory tests: Psychomotor Speed Test, Continuous Performance Test, Executive Function Test, and Working Memory Test. These tests were presented in a random order. Participants completed them on individual laptops in a laboratory with one of the testers present. The Psychomotor Speed Test measures Simple Reaction Time (SRT), general alertness and motor speed through delivery of a known stimulus to a known location to elicit a known response. The Continuous Performance Test − Identical Pairs (CPT-IP) requires the identification of identical stimulus pairs within a continuously presented series of stimuli. The Trail Making Test (TMT) is a widely used test to assess executive function, and it is a neuropsychological test of visual attention and task switching. It consists of two parts in which the participant is instructed to connect a set of 25 dots as quickly as possible while still maintaining accuracy. The test can provide information about visual search speed, scanning, speed of processing, mental flexibility, as well as executive functioning. Currently, the Wechsler Adult Intelligence Scale (WAIS) is the most commonly used instrument in the armamentarium of clinical neuropsychologists.[2930] This test involves letter sequences and tests one’s ability to think logically and analytically. However, the Wechsler Adult Intelligence Scale-III and IV Letter Number Sequencing are not appropriate for non-alphabetic cultures. The Taiwan’s Odd-Even Number Sequencing Test (TOENST), as proposed by the Department of Psychology, National Taiwan University,[31] was adopted to replace the Letter Number Test. Participants need to rearrange and read the Arabic numerals in a series. The way to read out the series is: “read out the odd numbers in ascending order and then the even numbers in descending order.” For example, the random numbers 7, 2, 8, 6, 3 is incremented by an odd number (3, 7) and decrements by an even number (8, 6, 2), so the answer is 3 7 8 6 2.\\nThe comfort of the sound field environment was evaluated by a closed questionnaire, and the subjective comfort of the participant’s sound field environment was measured by the semantic differential method. There were mainly seven closed questionnaires in the questionnaire design, with “no feeling” in the middle (0 point). From middle to the left indicates that the ability deteriorates and worsens. Each left one grid is −1, the left two grids are −2, and so on. The middle to the right means that the ability to become better, every right one grid is +1, the right two grids is +2, and so on.\\nOver the years, different scholars have given their definitions of efficiency, and different scholars also have different views and explanations for it. Therefore, it is not easy to offer a precise definition of work efficiency. Among many offerings, the definition of work efficiency includes “work response time”, the time when the work instruction is received plus the response time, and “work execution processing time” the time it takes for each task from the reaction to the completion. “Working memory” refers to the processing of tasks for the content of the current short-term memories. Cognitive function is concept that covers several functions of the brain such as attention, executive function, processing speed, learning and memory. Information processing and response speed are basic cognitive function, which is needed for more complex functions such as working memory. We measured cognitive performance via a battery of standardized cognitive tests. If improvements were observed in cognitive performance, this would not only have important benefits for employees, but benefits for the organization in terms of the potential for increased productivity. The main outcome measurers for this study were those of four laboratory tests: Psychomotor Speed Test, Continuous Performance Test, Executive Function Test, and Working Memory Test. These tests were presented in a random order. Participants completed them on individual laptops in a laboratory with one of the testers present. The Psychomotor Speed Test measures Simple Reaction Time (SRT), general alertness and motor speed through delivery of a known stimulus to a known location to elicit a known response. The Continuous Performance Test − Identical Pairs (CPT-IP) requires the identification of identical stimulus pairs within a continuously presented series of stimuli. The Trail Making Test (TMT) is a widely used test to assess executive function, and it is a neuropsychological test of visual attention and task switching. It consists of two parts in which the participant is instructed to connect a set of 25 dots as quickly as possible while still maintaining accuracy. The test can provide information about visual search speed, scanning, speed of processing, mental flexibility, as well as executive functioning. Currently, the Wechsler Adult Intelligence Scale (WAIS) is the most commonly used instrument in the armamentarium of clinical neuropsychologists.[2930] This test involves letter sequences and tests one’s ability to think logically and analytically. However, the Wechsler Adult Intelligence Scale-III and IV Letter Number Sequencing are not appropriate for non-alphabetic cultures. The Taiwan’s Odd-Even Number Sequencing Test (TOENST), as proposed by the Department of Psychology, National Taiwan University,[31] was adopted to replace the Letter Number Test. Participants need to rearrange and read the Arabic numerals in a series. The way to read out the series is: “read out the odd numbers in ascending order and then the even numbers in descending order.” For example, the random numbers 7, 2, 8, 6, 3 is incremented by an odd number (3, 7) and decrements by an even number (8, 6, 2), so the answer is 3 7 8 6 2.\\nThe comfort of the sound field environment was evaluated by a closed questionnaire, and the subjective comfort of the participant’s sound field environment was measured by the semantic differential method. There were mainly seven closed questionnaires in the questionnaire design, with “no feeling” in the middle (0 point). From middle to the left indicates that the ability deteriorates and worsens. Each left one grid is −1, the left two grids are −2, and so on. The middle to the right means that the ability to become better, every right one grid is +1, the right two grids is +2, and so on.\\n Experimental protocol Participants were tested in the same laboratory. Subject to the participants’ available time, the experiment was conducted under conditions of quiet (without noise), red noise, pink noise and white noise exposure. Before each experiment, the participants adopted a random method to determine the sound field exposure situation and the participants were not informed of the kind of sound scenario. The order of the four sound conditions (quiet, red, pink, white) was randomized. Ten minutes before the experiment was started, the participants were physiologically and psychologically adapted to the laboratory environment before they started the experiment. Each participant had to perform four tests and complete questionnaires in each of the four sessions in the experiment, and the order of the four tests was randomized. There was a five-minute break between each test. During the break, the sound exposure was on and the sound exposure was not terminated until all sound field exposure conditions had ended. After finishing four tests, participants filled in the sound field comfort evaluation questionnaire. During the filling in process, the sound continued. The flowchart in Figure 1 illustrates the experimental protocol.\\nFlow chart of the experimental protocol.\\nA within-subject design was used in the experiment with one independent variable, i.e. sound condition. Five dependent variables, including four objective parameters (psychomotor speed test, continuous performance test, executive function test, and working memory test) and one subjective perception, were collected to explore work efficiency in this study.\\nParticipants were tested in the same laboratory. Subject to the participants’ available time, the experiment was conducted under conditions of quiet (without noise), red noise, pink noise and white noise exposure. Before each experiment, the participants adopted a random method to determine the sound field exposure situation and the participants were not informed of the kind of sound scenario. The order of the four sound conditions (quiet, red, pink, white) was randomized. Ten minutes before the experiment was started, the participants were physiologically and psychologically adapted to the laboratory environment before they started the experiment. Each participant had to perform four tests and complete questionnaires in each of the four sessions in the experiment, and the order of the four tests was randomized. There was a five-minute break between each test. During the break, the sound exposure was on and the sound exposure was not terminated until all sound field exposure conditions had ended. After finishing four tests, participants filled in the sound field comfort evaluation questionnaire. During the filling in process, the sound continued. The flowchart in Figure 1 illustrates the experimental protocol.\\nFlow chart of the experimental protocol.\\nA within-subject design was used in the experiment with one independent variable, i.e. sound condition. Five dependent variables, including four objective parameters (psychomotor speed test, continuous performance test, executive function test, and working memory test) and one subjective perception, were collected to explore work efficiency in this study.\\n Statistical analysis of data The study was interested in evaluating the effectiveness of colour noise exposure. The approach considered was to measure the performance of a sample of participants before and after colour noise exposure, and analyse the differences using a paired sample t-test. IBM SPSS Statistics Version 20 was used to determine whether there were any statistically significant differences between the means of independent groups. Data from the completed questionnaires were stored in a database and transported in and subsequently analysed using the statistical program SPSS.\\nThe study was interested in evaluating the effectiveness of colour noise exposure. The approach considered was to measure the performance of a sample of participants before and after colour noise exposure, and analyse the differences using a paired sample t-test. IBM SPSS Statistics Version 20 was used to determine whether there were any statistically significant differences between the means of independent groups. Data from the completed questionnaires were stored in a database and transported in and subsequently analysed using the statistical program SPSS.\",\n \"Twenty-two healthy college students were enrolled in this study. The mean age of the participants was 22 years. All participants reported no medical history of neurological disease, hypertension or heart disease. A machine called an audiometer was used to produce sounds at various volumes and frequencies. The participants being tested listened to sounds through headphones and responded when they hear them by pressing a button. A pure tone audiogram was performed by a trained health care professional at the beginning of the study for each participant, and the hearing acuity of the participants was determined to be normal, with the thresholds of 500 Hz, 1000 Hz, 2000 Hz and 4000 Hz being less than 15 dB. The participants received financial compensation for their participation in the experiment. Informed consent was obtained from all participants.\",\n \"During this experiment, four sound conditions were used, relating to the three types of masking sound (red noise, pink noise, white noise) and a condition, which consisted of a wide band background noise. White noise is a signal with a flat frequency spectrum when plotted as a linear function of frequency. Red noise will refer to a power density which decreases 6 dB per octave with increasing frequency (density proportional to 1/f2). The frequency spectrum of pink noise is linear in logarithmic scale, and the spectral power density, compared with white noise, decreases by 3 dB per octave (density proportional to 1/f). The noise level within the room used in the study was measured using a Cirrus Type 832C sound level meter. Thirty second SPL samples, LAeq,30s, were measured over a period of an hour. Noise levels ranged from an Lmin of 41 dBA to an Lmax of 50 dBA. Measurements performed on successive days identified similar ranges of Lmin and Lmax.\\nThe signal source of masking sound was sent through a desktop computer to a Bluetooth speaker (BOSE SoundLink Mini). The sound level of each colour noise exposure was about 47 dBA when turning on the speaker and the background noise (without adding colour noise) was 44 dBA. Background noise had a decibel (dBA) value of three decibels (dBA) difference compared to the noise exposure scenario of three different colours, so that means that the sound energy of the colour noise exposure scenario was double that of the original silence.\",\n \"Over the years, different scholars have given their definitions of efficiency, and different scholars also have different views and explanations for it. Therefore, it is not easy to offer a precise definition of work efficiency. Among many offerings, the definition of work efficiency includes “work response time”, the time when the work instruction is received plus the response time, and “work execution processing time” the time it takes for each task from the reaction to the completion. “Working memory” refers to the processing of tasks for the content of the current short-term memories. Cognitive function is concept that covers several functions of the brain such as attention, executive function, processing speed, learning and memory. Information processing and response speed are basic cognitive function, which is needed for more complex functions such as working memory. We measured cognitive performance via a battery of standardized cognitive tests. If improvements were observed in cognitive performance, this would not only have important benefits for employees, but benefits for the organization in terms of the potential for increased productivity. The main outcome measurers for this study were those of four laboratory tests: Psychomotor Speed Test, Continuous Performance Test, Executive Function Test, and Working Memory Test. These tests were presented in a random order. Participants completed them on individual laptops in a laboratory with one of the testers present. The Psychomotor Speed Test measures Simple Reaction Time (SRT), general alertness and motor speed through delivery of a known stimulus to a known location to elicit a known response. The Continuous Performance Test − Identical Pairs (CPT-IP) requires the identification of identical stimulus pairs within a continuously presented series of stimuli. The Trail Making Test (TMT) is a widely used test to assess executive function, and it is a neuropsychological test of visual attention and task switching. It consists of two parts in which the participant is instructed to connect a set of 25 dots as quickly as possible while still maintaining accuracy. The test can provide information about visual search speed, scanning, speed of processing, mental flexibility, as well as executive functioning. Currently, the Wechsler Adult Intelligence Scale (WAIS) is the most commonly used instrument in the armamentarium of clinical neuropsychologists.[2930] This test involves letter sequences and tests one’s ability to think logically and analytically. However, the Wechsler Adult Intelligence Scale-III and IV Letter Number Sequencing are not appropriate for non-alphabetic cultures. The Taiwan’s Odd-Even Number Sequencing Test (TOENST), as proposed by the Department of Psychology, National Taiwan University,[31] was adopted to replace the Letter Number Test. Participants need to rearrange and read the Arabic numerals in a series. The way to read out the series is: “read out the odd numbers in ascending order and then the even numbers in descending order.” For example, the random numbers 7, 2, 8, 6, 3 is incremented by an odd number (3, 7) and decrements by an even number (8, 6, 2), so the answer is 3 7 8 6 2.\\nThe comfort of the sound field environment was evaluated by a closed questionnaire, and the subjective comfort of the participant’s sound field environment was measured by the semantic differential method. There were mainly seven closed questionnaires in the questionnaire design, with “no feeling” in the middle (0 point). From middle to the left indicates that the ability deteriorates and worsens. Each left one grid is −1, the left two grids are −2, and so on. The middle to the right means that the ability to become better, every right one grid is +1, the right two grids is +2, and so on.\",\n \"Participants were tested in the same laboratory. Subject to the participants’ available time, the experiment was conducted under conditions of quiet (without noise), red noise, pink noise and white noise exposure. Before each experiment, the participants adopted a random method to determine the sound field exposure situation and the participants were not informed of the kind of sound scenario. The order of the four sound conditions (quiet, red, pink, white) was randomized. Ten minutes before the experiment was started, the participants were physiologically and psychologically adapted to the laboratory environment before they started the experiment. Each participant had to perform four tests and complete questionnaires in each of the four sessions in the experiment, and the order of the four tests was randomized. There was a five-minute break between each test. During the break, the sound exposure was on and the sound exposure was not terminated until all sound field exposure conditions had ended. After finishing four tests, participants filled in the sound field comfort evaluation questionnaire. During the filling in process, the sound continued. The flowchart in Figure 1 illustrates the experimental protocol.\\nFlow chart of the experimental protocol.\\nA within-subject design was used in the experiment with one independent variable, i.e. sound condition. Five dependent variables, including four objective parameters (psychomotor speed test, continuous performance test, executive function test, and working memory test) and one subjective perception, were collected to explore work efficiency in this study.\",\n \"The study was interested in evaluating the effectiveness of colour noise exposure. The approach considered was to measure the performance of a sample of participants before and after colour noise exposure, and analyse the differences using a paired sample t-test. IBM SPSS Statistics Version 20 was used to determine whether there were any statistically significant differences between the means of independent groups. Data from the completed questionnaires were stored in a database and transported in and subsequently analysed using the statistical program SPSS.\",\n \"The results of the four tests conducted in this current study are summarized in Table 2. For each simple reaction time test, 10 sets of tests were completed. In the simple reaction time test, the average response time was better in red noise exposure scenario (0.565 seconds) than in the pink noise exposure scenario (0.602 seconds) and in the white noise exposure scenario (0.609 seconds). The variances of red noise exposure scenario (0.005 seconds) and pink noise exposure scenario (0.008 seconds) were lower than the white noise exposure scenario (0.013 seconds) and the quiet scenario (0.023 seconds). From the average of 10 test results, there was a very significant difference between the three noise exposure scenarios, red noise exposure (P < 0.001), pink noise exposure (P < 0.001), white noise exposure (P < 0.001) and the quiet scenario, as shown in Figure 2.\\nMeans and variances of SRT, CPT-IP, TMT, TOENST in four different sound field environments\\nPaired t-test analysis of Simple Reaction Time (SRT) in four different sound field exposures.\\nCompared with the average correct rate in the continuous performance test, 93.6% of the pink noise exposure situation is better than 91.7% of the red noise exposure situation and 91.1% of the white noise exposure situation, and the three kinds of noise exposure are better than 88.8% on the quiet situation. Among the 22 participants, half of the respondents achieved 93.3% correct rate on red noise exposure, 93.3% on pink noise exposure, 93.3% on white noise exposure, and they all scored better than 90.0% in the quiet situation. The average correct rate of the test showed that there was a slight improvement over the quiet scenario for all of the red noise scenario, the pink noise scenario, and the white noise scenario. The paired t-test showed significant differences (P < 0.05) between the pink noise exposure scenario and the quiet scenario in the continuous performance test, Figure 3.\\nPaired t-test analysis of continuous performance test-identical pairs (CPT-IP) in four different sound field exposures.\\nIn the executive function tests, comparing the average time it takes to complete the test, it shows the red noise exposure scenario (21.37 seconds) is superior to the pink noise exposure scenario (22.31 seconds) and the white noise exposure scenario (23.11 seconds), all of which are better than the quiet scenario (24.54 seconds). The best test results (minimum) showed that the red noise exposure scenario (12.00 seconds), pink noise exposure scenario (10.73 seconds), white noise exposure scenario (13.13 seconds) were also better than the quiet scenario (15.33 seconds). The average finishing time of the tests showed that the red noise exposure situation, the pink noise exposure situation and the white noise exposure situation are better than the quiet situation. As shown on Figure 4, the paired t-test of executive function tests showed significant differences between the red noise exposure scenario (P < 0.001), the pink noise exposure scenario (P < 0.001), white noise exposure scenario (P < 0.001) and the quiet scenario.\\nPaired t-test analysis of Trail Making Test (TMT) in four different sound field exposures.\\nThe average score of the working memory test showed that the red noise exposure situation (18.8 points) was superior to the pink noise exposure situation (17.3 points) and the white noise exposure situation (15.9 points), all of which were better than the quiet situation (14.4 points). In a comparison of variance, the red noise exposure (5.1 points) and the pink noise exposure (6.9 points) were significantly lower than the white noise exposure (11.2 points) and noiseless exposure (9.3 points). The paired t-test analysis showed that in the working memory test, the red noise exposure (P < 0.001), the pink noise exposure (P < 0.001), the white noise exposure (P < 0.01), the three noise exposure all had a significant difference as compared with the quiet situation, as shown on Figure 5.\\nPaired t-test analysis of Taiwan’s Odd-Even Number Sequencing Test (TOENST) in four different sound field exposures.\\nAs shown in Table 3, compared with the quiet situation, both the red noise and the pink noise exposure in the simple reaction time test, the executive function test and the working memory test all showed significant differences (P < 0.001). The white noise exposure was very significantly different (P < 0.001) in the simple reaction time test, and there was a significant difference (P < 0.01) in the working memory test. However, only pink noise exposure was significantly different (P < 0.05) in the continuous performance test.\\nPaired t-test analysis of three colour noise exposure situations compared with quiet situations\\nA statistical analysis of the sound field comfort questionnaire showed that, the questionnaire had a Cronbach’s alpha score of 0.772. Generally, a questionnaire with a Cronbach’s alpha score between 0.7 and 0.9 is considered to be of good reliability. As shown in Table 4, the subjective comfort evaluation statistics of the sound field environment indicated a highest and lowest score of 0 and −2, a little worse in the quiet scenario, while the scores between −1 and 3 were slightly worse to good in the red noise scenario; the scores between −1 and 2 were slightly worse to better in pink noise scenario; however, the scores between −3 and 1 represented a worse to slightly better result for the white noise scenario. The sum of the scores of the subjective evaluation in the sound field were −40 (average rating −0.26), +81 (average rating 0.53), +38 (average rating 0.25), −94 (average rating −0.61) for quiet, red noise, pink noise, and white noise scenarios, respectively.\\nSubjective comfort evaluations of four sound environments\",\n \"Of the four trials, there were three significant (P < 0.001) increases in scores for red noise exposure and pink noise exposure, possibly reflecting improvements in work efficiency. Literature explains that white noise exposure can serve as a masking sound, and this may improve work efficiency. Two tests showed a significant (P < 0.001) increase in performance, with a significant (P < 0.01) increase in one trial also corroborating previous studies. In brain synchronization studies, pink noise exposure has been shown to synchronize the brain and induce brain activity to lower its potential,[13] and to exclude the effects of the outside world for purposes of concentrated processing. Except for the slight significant difference (P < 0.05) in white noise exposure, the continuous performance test was the only one that did not show very significant results. Participants may all have been preoccupied with the test, so the average correct rates are above 90%. Even the correct rates of two participants reached 100% when exposed to all four sound situations, and there were three participants with a 100% correct rate when exposed to three scenarios.\\nIn the continuous performance test, the correct rate of non-exposure was about 88.8%, which of red noise, pink noise, and white noise were 91.7%, 93.6%, and 91.1%, respectively. Half of the 22 participants achieved a correct rate of 93.3% under the conditions of red noise exposure, pink noise exposure and white noise exposure, all of which were higher than the 88.8% correct rate of no noise exposure. The average score of the test results showed that a red noise exposure scenario, pink noise exposure scenario, and white noise exposure scenario are slightly better than the quiet scenario, which means that with the exposure to colour noise, the correct rate will rise and attention will be slightly improved.\\nBecause the exposure of the participant’s living environment to the noise itself, might affect the questionnaire data, the type of accommodation should be indicated on the comfort questionnaire, and whether or not there is any noise exposure should be indicated in the closed questionnaire. In this study, there were three home participants exposed to noise, there were three home participants who sometimes had noise exposure. Of these six participants, five of the participants had different answers from the others, indicating a larger numerical increase in white noise.\\nThe scores of the questionnaire results in the comfort of colour noises environment did not fully match the test data. Participants were randomly interviewed after the experiment. They said that with the additional sound increases, they felt that the sound interference, and the selection value should be less favourable. The laboratory background sound level was 44 dBA in the quiet scenario. The participants experienced significant changes to sound of 47 dBA when exposed to colour noise. Moreover, because the experimental exposure scenario was determined in a random manner, it was difficult to remember which previous test scores were selected. Scenario comfort evaluation is felt on a subjective basis, but participants may have had preconceptions when completing the questionnaire, thus affecting the final score. Although the results of the environmental comfort questionnaire were not as expected, the overall percentage of participants who felt slightly better or above were 48.8%, 32%, 9% for participants exposed to red, pink, white noise, respectively.\\nThe results of this study show that exposure to the three kinds of colour noises, red, pink and white noise yields significantly better results in the Psychomotor Speed Test, Continuous Performance Test, Executive Function Test and Working Memory Test, which proves that these three kinds of sound field environments on the office work-type job site might improve work efficiency. The sum of the questionnaire scores on subjective comfort of the sound field environment showed that red, pink, and white noise exposure, were better than the original no noise exposure of the sound field, and this result implies that the three colour noise sound fields seem to make the white-collar work patterns a better workplaces not only by improving productivity, but also by making workers feel comfortable. Because white noise is equal energy at each frequency, it can cover the sounds that do not want to be heard around the environment. Pink noise might be due to neurons in the hypothalamus synchronously resonating with pink noises in the low oscillation frequency band, making slow rhythms and dominating brain waves. In addition, the power density of high frequency in red noise is even lower than these in pink noise and may therefore affect neurons in the hypothalamus even more, leading to information more speedily reaching the cerebral cortex through the hypothalamus and to faster transmission of motor messages through the hypothalamus.\\nThe concept of the open-plan office is now widespread in the workplace. While offering many advantages in terms of layout and facilitating communication between colleagues, this way of organising the workspace had two major disadvantages. The feeling of privacy is lessened and noise level is increased causing discomfort for individuals working in this type of environment. It appears that noise is a major nuisance factor in open-plan offices in spite of a relatively low noise level. The noise nuisance experienced in open-plan offices affects work satisfaction, and this exposure to noise can reduce employee performance depending on the types of task to be carried out and the characteristics of the noise present in the workplace. The current research suggests that creating a colour noise environment through a loudspeaker or headphone might improve some aspects of cognitive performance in different workplaces. Many studies in the past have proved that noise has a negative influence on human hearing, cardiovascular systems, and emotions and so on. However, the three colour noises are applied to the original sound field environment. Therefore, if the background noise of the workplace is already too high, it would not be in order, for health reasons, to further add colour noise.\",\n \"The results of this study show that participants perform significantly better in Psychomotor Speed Test, Continuous Performance Test, Executive Function Test and Working Memory Test. Therefore, colour noise may be used in the future to improve a workplace sound field environment, so that white-collar workers have a better working environment. Using physical stimuli of red, pink and white noises to improve worker productivity may prove to be a new approach towards productivity improvement that may provide fewer side effects than traditional methods such as drinking refreshing beverages. The introduction of the sounds represents a simple, low-cost, non-invasive improvement.\\n Financial support and sponsorship Nil.\\nNil.\\n Conflicts of interest There are no conflicts of interest.\\nThere are no conflicts of interest.\",\n \"Nil.\",\n \"There are no conflicts of interest.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["intro","subjects|methods",null,null,null,null,null,"results","discussion","conclusion",null,"COI-statement"],"string":"[\n \"intro\",\n \"subjects|methods\",\n null,\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\",\n \"conclusion\",\n null,\n \"COI-statement\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["Human performance","pink noise","red noise","subjective comfort","white noise"],"string":"[\n \"Human performance\",\n \"pink noise\",\n \"red noise\",\n \"subjective comfort\",\n \"white noise\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nExposure to high levels of noise in industry factory is associated with a number of effects on health manifested in various psychosocial responses such as annoyance, disturbance of daily activities, sleep and performance and in physical responses, such as hearing loss, hypertension and ischemic heart disease.[12] While the open-plan office is now widespread in the workplace, it appears that noise is a major nuisance factor in open-plan offices in spite of a relatively low noise level(less than 65 dB(A)).[3] In general, the higher the decibel numbers of sound, the greater the health hazard. However, when the number of sound decibels is not high, the degree of influence on people may vary depending on the sound characteristics.\nIn audio engineering, physics, and many other fields, the colour of noise refers to the power spectrum of a noise signal. The practice of naming kinds of noise after colours started with white noise, a signal whose spectrum has equal power within any equal interval of frequencies. That name was given by analogy with white light, which was assumed to have such a flat power spectrum over the visible range. Other colour names, like pink, red, and blue were then given to noise with other spectral profiles, often (but not always) in reference to the colour of light with similar spectra.\nNoise often has detrimental effects on performance. Under most circumstances, information processing is disturbed by environmental noise and other non-task compatible distracters.[45] However, researchers have recently reported that under certain circumstances individuals with attention problems appear to benefit from the addition of specific forms of environmental noise. Typically, this facilitative effect has been limited to non-vocal background music on simple arithmetic task performance,[6] but Stansfeld et al.[7] found just that under certain conditions even road traffic noise can improve performance on episodic memory tasks, particularly in children at risk of attention problems and academic underachievement. Furthermore, Söderlund et al.[8] have demonstrated that adding auditory white noise to the environment enhanced the memory performance of children with Attention Deficit Hyperactivity Disorder (ADHD)-type problems but disrupted that of non-ADHD control children. Stochastic resonance, a phenomenon whereby signal processing is enhanced by the addition of random noise, has been widely demonstrated across various modalities, including visual, auditory, tactile and cross-modal forms of processing.[910] Of particular interest are findings that auditory noise has the capacity to enhance some aspects of human cognitive performance, such as the speed of arithmetical computations.[11]\n\nMeanwhile, some recent studies showed related evidence. For example, in 2010, light music, an example belonging to pink noise, is proved beneficial for elder people to improve their sleep quality as a long term effect.[12] Moreover, in a previous study,[13] the steady pink noise with intensity of 35 dB, 40 dB, 50 dB and 60 dB was used to stimulate four participants during sleep and they believed that pink noise could improve the sleep quality of participants. However, it is clear that this conclusion was based on significant increase of light sleep period (especially stage 2) accompanied with declined duration of rapid eye movement. In the brain synchronization study, it has demonstrated that the pink noise could synchronize brain wave and induce brain activity into a specialized state, that is, in a lower complexity level.[14]\n\nSezici’s study showed that playing of white noise significantly decreased the daily crying durations and increased the sleeping durations of the colicky babies compared to swinging in both groups.[15] In the literature, parents emphasized that playing of white noise was quite beneficial in relieving pain among crying colicky babies.[16] In their studies, Kucukoglu et al.[17] and Karakoc and Turker[18] revealed that the pain scores of the newborns who were made to listen to white noise were observed to decrease compared with those of the newborns in the control group. White noise encompasses all characteristics of sounds within the range of human hearing. It has been used in the treatment of tinnitus, insomnia, masking unwanted sounds, and provision of relaxation.[1920]\n\nZhou et al.[14] demonstrated that steady pink noise has significant effect on reducing brain wave complexity and inducing more stable sleep time to improve sleep quality of individuals. In total, 42 elderly people (21 using music and 21 controls) completed the study, and there was some indication that soft slow music yielder higher improvement on some of the parameters, which are worthy of further investigation.[12] A tabulated list of the effects observed in previous studies with colour noise exposures is shown in Table 1.\nA tabulated list of the effects observed in previous studies with colour noise exposures\nSeveral experiments have demonstrated that the detrimental effect of office noise on worker attitude and performance can be significantly reduced through the use of masking noise.[2122232425] For instance the experimental study performed by Haka[24] demonstrated that the performance of operation span tasks, serial recall and long term memory tasks were all improved when masking noise was used; and the field study performed by Hongisto[23] indicated that masking noise significantly reduced disturbance of worker attitudes towards concentration caused by office noise. Masking noise achieves this improvement through reducing the intelligibility of nearby speech.[25] In addition, Vassie et al.[26] indicated that participants rejected the brown masking noise delivered through earphones as it caused irritation and discomfort. Future studies should also consider other types of masking noise and should measure the level and duration of the masking noise.[26]\n\nIt is well-known that any human action results from brain activity, which is affected by light and sound. Too much acoustic energy causes hearing loss, impaired human nerves, and emotional disorders.[2728] However, sound play also very important role for people. According to previous researchers, not only white noise can create shading effect, but pink noise can improve sleep quality. Most people in the workplace are in the office or in the service industry, not in the noisy working environment for the factory. Creating a quiet environment is sometimes not easy. However, adding some sounds to enhance the work efficiency and reduce the work pressure of low-noise environment should be an issue worth exploring. Therefore, this study wanted to understand the effects of three different colours noise exposure. In the past, there were not many studies in this regard. Therefore, the goal of the current study is to test the hypothesis that adding another sound in a low background noise environment may increases productivity and comfort.\n\nSUBJECTS AND METHODS:\n Participants Twenty-two healthy college students were enrolled in this study. The mean age of the participants was 22 years. All participants reported no medical history of neurological disease, hypertension or heart disease. A machine called an audiometer was used to produce sounds at various volumes and frequencies. The participants being tested listened to sounds through headphones and responded when they hear them by pressing a button. A pure tone audiogram was performed by a trained health care professional at the beginning of the study for each participant, and the hearing acuity of the participants was determined to be normal, with the thresholds of 500 Hz, 1000 Hz, 2000 Hz and 4000 Hz being less than 15 dB. The participants received financial compensation for their participation in the experiment. Informed consent was obtained from all participants.\nTwenty-two healthy college students were enrolled in this study. The mean age of the participants was 22 years. All participants reported no medical history of neurological disease, hypertension or heart disease. A machine called an audiometer was used to produce sounds at various volumes and frequencies. The participants being tested listened to sounds through headphones and responded when they hear them by pressing a button. A pure tone audiogram was performed by a trained health care professional at the beginning of the study for each participant, and the hearing acuity of the participants was determined to be normal, with the thresholds of 500 Hz, 1000 Hz, 2000 Hz and 4000 Hz being less than 15 dB. The participants received financial compensation for their participation in the experiment. Informed consent was obtained from all participants.\n Presentation of noise During this experiment, four sound conditions were used, relating to the three types of masking sound (red noise, pink noise, white noise) and a condition, which consisted of a wide band background noise. White noise is a signal with a flat frequency spectrum when plotted as a linear function of frequency. Red noise will refer to a power density which decreases 6 dB per octave with increasing frequency (density proportional to 1/f2). The frequency spectrum of pink noise is linear in logarithmic scale, and the spectral power density, compared with white noise, decreases by 3 dB per octave (density proportional to 1/f). The noise level within the room used in the study was measured using a Cirrus Type 832C sound level meter. Thirty second SPL samples, LAeq,30s, were measured over a period of an hour. Noise levels ranged from an Lmin of 41 dBA to an Lmax of 50 dBA. Measurements performed on successive days identified similar ranges of Lmin and Lmax.\nThe signal source of masking sound was sent through a desktop computer to a Bluetooth speaker (BOSE SoundLink Mini). The sound level of each colour noise exposure was about 47 dBA when turning on the speaker and the background noise (without adding colour noise) was 44 dBA. Background noise had a decibel (dBA) value of three decibels (dBA) difference compared to the noise exposure scenario of three different colours, so that means that the sound energy of the colour noise exposure scenario was double that of the original silence.\nDuring this experiment, four sound conditions were used, relating to the three types of masking sound (red noise, pink noise, white noise) and a condition, which consisted of a wide band background noise. White noise is a signal with a flat frequency spectrum when plotted as a linear function of frequency. Red noise will refer to a power density which decreases 6 dB per octave with increasing frequency (density proportional to 1/f2). The frequency spectrum of pink noise is linear in logarithmic scale, and the spectral power density, compared with white noise, decreases by 3 dB per octave (density proportional to 1/f). The noise level within the room used in the study was measured using a Cirrus Type 832C sound level meter. Thirty second SPL samples, LAeq,30s, were measured over a period of an hour. Noise levels ranged from an Lmin of 41 dBA to an Lmax of 50 dBA. Measurements performed on successive days identified similar ranges of Lmin and Lmax.\nThe signal source of masking sound was sent through a desktop computer to a Bluetooth speaker (BOSE SoundLink Mini). The sound level of each colour noise exposure was about 47 dBA when turning on the speaker and the background noise (without adding colour noise) was 44 dBA. Background noise had a decibel (dBA) value of three decibels (dBA) difference compared to the noise exposure scenario of three different colours, so that means that the sound energy of the colour noise exposure scenario was double that of the original silence.\n Outcome measures Over the years, different scholars have given their definitions of efficiency, and different scholars also have different views and explanations for it. Therefore, it is not easy to offer a precise definition of work efficiency. Among many offerings, the definition of work efficiency includes “work response time”, the time when the work instruction is received plus the response time, and “work execution processing time” the time it takes for each task from the reaction to the completion. “Working memory” refers to the processing of tasks for the content of the current short-term memories. Cognitive function is concept that covers several functions of the brain such as attention, executive function, processing speed, learning and memory. Information processing and response speed are basic cognitive function, which is needed for more complex functions such as working memory. We measured cognitive performance via a battery of standardized cognitive tests. If improvements were observed in cognitive performance, this would not only have important benefits for employees, but benefits for the organization in terms of the potential for increased productivity. The main outcome measurers for this study were those of four laboratory tests: Psychomotor Speed Test, Continuous Performance Test, Executive Function Test, and Working Memory Test. These tests were presented in a random order. Participants completed them on individual laptops in a laboratory with one of the testers present. The Psychomotor Speed Test measures Simple Reaction Time (SRT), general alertness and motor speed through delivery of a known stimulus to a known location to elicit a known response. The Continuous Performance Test − Identical Pairs (CPT-IP) requires the identification of identical stimulus pairs within a continuously presented series of stimuli. The Trail Making Test (TMT) is a widely used test to assess executive function, and it is a neuropsychological test of visual attention and task switching. It consists of two parts in which the participant is instructed to connect a set of 25 dots as quickly as possible while still maintaining accuracy. The test can provide information about visual search speed, scanning, speed of processing, mental flexibility, as well as executive functioning. Currently, the Wechsler Adult Intelligence Scale (WAIS) is the most commonly used instrument in the armamentarium of clinical neuropsychologists.[2930] This test involves letter sequences and tests one’s ability to think logically and analytically. However, the Wechsler Adult Intelligence Scale-III and IV Letter Number Sequencing are not appropriate for non-alphabetic cultures. The Taiwan’s Odd-Even Number Sequencing Test (TOENST), as proposed by the Department of Psychology, National Taiwan University,[31] was adopted to replace the Letter Number Test. Participants need to rearrange and read the Arabic numerals in a series. The way to read out the series is: “read out the odd numbers in ascending order and then the even numbers in descending order.” For example, the random numbers 7, 2, 8, 6, 3 is incremented by an odd number (3, 7) and decrements by an even number (8, 6, 2), so the answer is 3 7 8 6 2.\nThe comfort of the sound field environment was evaluated by a closed questionnaire, and the subjective comfort of the participant’s sound field environment was measured by the semantic differential method. There were mainly seven closed questionnaires in the questionnaire design, with “no feeling” in the middle (0 point). From middle to the left indicates that the ability deteriorates and worsens. Each left one grid is −1, the left two grids are −2, and so on. The middle to the right means that the ability to become better, every right one grid is +1, the right two grids is +2, and so on.\nOver the years, different scholars have given their definitions of efficiency, and different scholars also have different views and explanations for it. Therefore, it is not easy to offer a precise definition of work efficiency. Among many offerings, the definition of work efficiency includes “work response time”, the time when the work instruction is received plus the response time, and “work execution processing time” the time it takes for each task from the reaction to the completion. “Working memory” refers to the processing of tasks for the content of the current short-term memories. Cognitive function is concept that covers several functions of the brain such as attention, executive function, processing speed, learning and memory. Information processing and response speed are basic cognitive function, which is needed for more complex functions such as working memory. We measured cognitive performance via a battery of standardized cognitive tests. If improvements were observed in cognitive performance, this would not only have important benefits for employees, but benefits for the organization in terms of the potential for increased productivity. The main outcome measurers for this study were those of four laboratory tests: Psychomotor Speed Test, Continuous Performance Test, Executive Function Test, and Working Memory Test. These tests were presented in a random order. Participants completed them on individual laptops in a laboratory with one of the testers present. The Psychomotor Speed Test measures Simple Reaction Time (SRT), general alertness and motor speed through delivery of a known stimulus to a known location to elicit a known response. The Continuous Performance Test − Identical Pairs (CPT-IP) requires the identification of identical stimulus pairs within a continuously presented series of stimuli. The Trail Making Test (TMT) is a widely used test to assess executive function, and it is a neuropsychological test of visual attention and task switching. It consists of two parts in which the participant is instructed to connect a set of 25 dots as quickly as possible while still maintaining accuracy. The test can provide information about visual search speed, scanning, speed of processing, mental flexibility, as well as executive functioning. Currently, the Wechsler Adult Intelligence Scale (WAIS) is the most commonly used instrument in the armamentarium of clinical neuropsychologists.[2930] This test involves letter sequences and tests one’s ability to think logically and analytically. However, the Wechsler Adult Intelligence Scale-III and IV Letter Number Sequencing are not appropriate for non-alphabetic cultures. The Taiwan’s Odd-Even Number Sequencing Test (TOENST), as proposed by the Department of Psychology, National Taiwan University,[31] was adopted to replace the Letter Number Test. Participants need to rearrange and read the Arabic numerals in a series. The way to read out the series is: “read out the odd numbers in ascending order and then the even numbers in descending order.” For example, the random numbers 7, 2, 8, 6, 3 is incremented by an odd number (3, 7) and decrements by an even number (8, 6, 2), so the answer is 3 7 8 6 2.\nThe comfort of the sound field environment was evaluated by a closed questionnaire, and the subjective comfort of the participant’s sound field environment was measured by the semantic differential method. There were mainly seven closed questionnaires in the questionnaire design, with “no feeling” in the middle (0 point). From middle to the left indicates that the ability deteriorates and worsens. Each left one grid is −1, the left two grids are −2, and so on. The middle to the right means that the ability to become better, every right one grid is +1, the right two grids is +2, and so on.\n Experimental protocol Participants were tested in the same laboratory. Subject to the participants’ available time, the experiment was conducted under conditions of quiet (without noise), red noise, pink noise and white noise exposure. Before each experiment, the participants adopted a random method to determine the sound field exposure situation and the participants were not informed of the kind of sound scenario. The order of the four sound conditions (quiet, red, pink, white) was randomized. Ten minutes before the experiment was started, the participants were physiologically and psychologically adapted to the laboratory environment before they started the experiment. Each participant had to perform four tests and complete questionnaires in each of the four sessions in the experiment, and the order of the four tests was randomized. There was a five-minute break between each test. During the break, the sound exposure was on and the sound exposure was not terminated until all sound field exposure conditions had ended. After finishing four tests, participants filled in the sound field comfort evaluation questionnaire. During the filling in process, the sound continued. The flowchart in Figure 1 illustrates the experimental protocol.\nFlow chart of the experimental protocol.\nA within-subject design was used in the experiment with one independent variable, i.e. sound condition. Five dependent variables, including four objective parameters (psychomotor speed test, continuous performance test, executive function test, and working memory test) and one subjective perception, were collected to explore work efficiency in this study.\nParticipants were tested in the same laboratory. Subject to the participants’ available time, the experiment was conducted under conditions of quiet (without noise), red noise, pink noise and white noise exposure. Before each experiment, the participants adopted a random method to determine the sound field exposure situation and the participants were not informed of the kind of sound scenario. The order of the four sound conditions (quiet, red, pink, white) was randomized. Ten minutes before the experiment was started, the participants were physiologically and psychologically adapted to the laboratory environment before they started the experiment. Each participant had to perform four tests and complete questionnaires in each of the four sessions in the experiment, and the order of the four tests was randomized. There was a five-minute break between each test. During the break, the sound exposure was on and the sound exposure was not terminated until all sound field exposure conditions had ended. After finishing four tests, participants filled in the sound field comfort evaluation questionnaire. During the filling in process, the sound continued. The flowchart in Figure 1 illustrates the experimental protocol.\nFlow chart of the experimental protocol.\nA within-subject design was used in the experiment with one independent variable, i.e. sound condition. Five dependent variables, including four objective parameters (psychomotor speed test, continuous performance test, executive function test, and working memory test) and one subjective perception, were collected to explore work efficiency in this study.\n Statistical analysis of data The study was interested in evaluating the effectiveness of colour noise exposure. The approach considered was to measure the performance of a sample of participants before and after colour noise exposure, and analyse the differences using a paired sample t-test. IBM SPSS Statistics Version 20 was used to determine whether there were any statistically significant differences between the means of independent groups. Data from the completed questionnaires were stored in a database and transported in and subsequently analysed using the statistical program SPSS.\nThe study was interested in evaluating the effectiveness of colour noise exposure. The approach considered was to measure the performance of a sample of participants before and after colour noise exposure, and analyse the differences using a paired sample t-test. IBM SPSS Statistics Version 20 was used to determine whether there were any statistically significant differences between the means of independent groups. Data from the completed questionnaires were stored in a database and transported in and subsequently analysed using the statistical program SPSS.\n\nParticipants:\nTwenty-two healthy college students were enrolled in this study. The mean age of the participants was 22 years. All participants reported no medical history of neurological disease, hypertension or heart disease. A machine called an audiometer was used to produce sounds at various volumes and frequencies. The participants being tested listened to sounds through headphones and responded when they hear them by pressing a button. A pure tone audiogram was performed by a trained health care professional at the beginning of the study for each participant, and the hearing acuity of the participants was determined to be normal, with the thresholds of 500 Hz, 1000 Hz, 2000 Hz and 4000 Hz being less than 15 dB. The participants received financial compensation for their participation in the experiment. Informed consent was obtained from all participants.\n\nPresentation of noise:\nDuring this experiment, four sound conditions were used, relating to the three types of masking sound (red noise, pink noise, white noise) and a condition, which consisted of a wide band background noise. White noise is a signal with a flat frequency spectrum when plotted as a linear function of frequency. Red noise will refer to a power density which decreases 6 dB per octave with increasing frequency (density proportional to 1/f2). The frequency spectrum of pink noise is linear in logarithmic scale, and the spectral power density, compared with white noise, decreases by 3 dB per octave (density proportional to 1/f). The noise level within the room used in the study was measured using a Cirrus Type 832C sound level meter. Thirty second SPL samples, LAeq,30s, were measured over a period of an hour. Noise levels ranged from an Lmin of 41 dBA to an Lmax of 50 dBA. Measurements performed on successive days identified similar ranges of Lmin and Lmax.\nThe signal source of masking sound was sent through a desktop computer to a Bluetooth speaker (BOSE SoundLink Mini). The sound level of each colour noise exposure was about 47 dBA when turning on the speaker and the background noise (without adding colour noise) was 44 dBA. Background noise had a decibel (dBA) value of three decibels (dBA) difference compared to the noise exposure scenario of three different colours, so that means that the sound energy of the colour noise exposure scenario was double that of the original silence.\n\nOutcome measures:\nOver the years, different scholars have given their definitions of efficiency, and different scholars also have different views and explanations for it. Therefore, it is not easy to offer a precise definition of work efficiency. Among many offerings, the definition of work efficiency includes “work response time”, the time when the work instruction is received plus the response time, and “work execution processing time” the time it takes for each task from the reaction to the completion. “Working memory” refers to the processing of tasks for the content of the current short-term memories. Cognitive function is concept that covers several functions of the brain such as attention, executive function, processing speed, learning and memory. Information processing and response speed are basic cognitive function, which is needed for more complex functions such as working memory. We measured cognitive performance via a battery of standardized cognitive tests. If improvements were observed in cognitive performance, this would not only have important benefits for employees, but benefits for the organization in terms of the potential for increased productivity. The main outcome measurers for this study were those of four laboratory tests: Psychomotor Speed Test, Continuous Performance Test, Executive Function Test, and Working Memory Test. These tests were presented in a random order. Participants completed them on individual laptops in a laboratory with one of the testers present. The Psychomotor Speed Test measures Simple Reaction Time (SRT), general alertness and motor speed through delivery of a known stimulus to a known location to elicit a known response. The Continuous Performance Test − Identical Pairs (CPT-IP) requires the identification of identical stimulus pairs within a continuously presented series of stimuli. The Trail Making Test (TMT) is a widely used test to assess executive function, and it is a neuropsychological test of visual attention and task switching. It consists of two parts in which the participant is instructed to connect a set of 25 dots as quickly as possible while still maintaining accuracy. The test can provide information about visual search speed, scanning, speed of processing, mental flexibility, as well as executive functioning. Currently, the Wechsler Adult Intelligence Scale (WAIS) is the most commonly used instrument in the armamentarium of clinical neuropsychologists.[2930] This test involves letter sequences and tests one’s ability to think logically and analytically. However, the Wechsler Adult Intelligence Scale-III and IV Letter Number Sequencing are not appropriate for non-alphabetic cultures. The Taiwan’s Odd-Even Number Sequencing Test (TOENST), as proposed by the Department of Psychology, National Taiwan University,[31] was adopted to replace the Letter Number Test. Participants need to rearrange and read the Arabic numerals in a series. The way to read out the series is: “read out the odd numbers in ascending order and then the even numbers in descending order.” For example, the random numbers 7, 2, 8, 6, 3 is incremented by an odd number (3, 7) and decrements by an even number (8, 6, 2), so the answer is 3 7 8 6 2.\nThe comfort of the sound field environment was evaluated by a closed questionnaire, and the subjective comfort of the participant’s sound field environment was measured by the semantic differential method. There were mainly seven closed questionnaires in the questionnaire design, with “no feeling” in the middle (0 point). From middle to the left indicates that the ability deteriorates and worsens. Each left one grid is −1, the left two grids are −2, and so on. The middle to the right means that the ability to become better, every right one grid is +1, the right two grids is +2, and so on.\n\nExperimental protocol:\nParticipants were tested in the same laboratory. Subject to the participants’ available time, the experiment was conducted under conditions of quiet (without noise), red noise, pink noise and white noise exposure. Before each experiment, the participants adopted a random method to determine the sound field exposure situation and the participants were not informed of the kind of sound scenario. The order of the four sound conditions (quiet, red, pink, white) was randomized. Ten minutes before the experiment was started, the participants were physiologically and psychologically adapted to the laboratory environment before they started the experiment. Each participant had to perform four tests and complete questionnaires in each of the four sessions in the experiment, and the order of the four tests was randomized. There was a five-minute break between each test. During the break, the sound exposure was on and the sound exposure was not terminated until all sound field exposure conditions had ended. After finishing four tests, participants filled in the sound field comfort evaluation questionnaire. During the filling in process, the sound continued. The flowchart in Figure 1 illustrates the experimental protocol.\nFlow chart of the experimental protocol.\nA within-subject design was used in the experiment with one independent variable, i.e. sound condition. Five dependent variables, including four objective parameters (psychomotor speed test, continuous performance test, executive function test, and working memory test) and one subjective perception, were collected to explore work efficiency in this study.\n\nStatistical analysis of data:\nThe study was interested in evaluating the effectiveness of colour noise exposure. The approach considered was to measure the performance of a sample of participants before and after colour noise exposure, and analyse the differences using a paired sample t-test. IBM SPSS Statistics Version 20 was used to determine whether there were any statistically significant differences between the means of independent groups. Data from the completed questionnaires were stored in a database and transported in and subsequently analysed using the statistical program SPSS.\n\nRESULTS:\nThe results of the four tests conducted in this current study are summarized in Table 2. For each simple reaction time test, 10 sets of tests were completed. In the simple reaction time test, the average response time was better in red noise exposure scenario (0.565 seconds) than in the pink noise exposure scenario (0.602 seconds) and in the white noise exposure scenario (0.609 seconds). The variances of red noise exposure scenario (0.005 seconds) and pink noise exposure scenario (0.008 seconds) were lower than the white noise exposure scenario (0.013 seconds) and the quiet scenario (0.023 seconds). From the average of 10 test results, there was a very significant difference between the three noise exposure scenarios, red noise exposure (P < 0.001), pink noise exposure (P < 0.001), white noise exposure (P < 0.001) and the quiet scenario, as shown in Figure 2.\nMeans and variances of SRT, CPT-IP, TMT, TOENST in four different sound field environments\nPaired t-test analysis of Simple Reaction Time (SRT) in four different sound field exposures.\nCompared with the average correct rate in the continuous performance test, 93.6% of the pink noise exposure situation is better than 91.7% of the red noise exposure situation and 91.1% of the white noise exposure situation, and the three kinds of noise exposure are better than 88.8% on the quiet situation. Among the 22 participants, half of the respondents achieved 93.3% correct rate on red noise exposure, 93.3% on pink noise exposure, 93.3% on white noise exposure, and they all scored better than 90.0% in the quiet situation. The average correct rate of the test showed that there was a slight improvement over the quiet scenario for all of the red noise scenario, the pink noise scenario, and the white noise scenario. The paired t-test showed significant differences (P < 0.05) between the pink noise exposure scenario and the quiet scenario in the continuous performance test, Figure 3.\nPaired t-test analysis of continuous performance test-identical pairs (CPT-IP) in four different sound field exposures.\nIn the executive function tests, comparing the average time it takes to complete the test, it shows the red noise exposure scenario (21.37 seconds) is superior to the pink noise exposure scenario (22.31 seconds) and the white noise exposure scenario (23.11 seconds), all of which are better than the quiet scenario (24.54 seconds). The best test results (minimum) showed that the red noise exposure scenario (12.00 seconds), pink noise exposure scenario (10.73 seconds), white noise exposure scenario (13.13 seconds) were also better than the quiet scenario (15.33 seconds). The average finishing time of the tests showed that the red noise exposure situation, the pink noise exposure situation and the white noise exposure situation are better than the quiet situation. As shown on Figure 4, the paired t-test of executive function tests showed significant differences between the red noise exposure scenario (P < 0.001), the pink noise exposure scenario (P < 0.001), white noise exposure scenario (P < 0.001) and the quiet scenario.\nPaired t-test analysis of Trail Making Test (TMT) in four different sound field exposures.\nThe average score of the working memory test showed that the red noise exposure situation (18.8 points) was superior to the pink noise exposure situation (17.3 points) and the white noise exposure situation (15.9 points), all of which were better than the quiet situation (14.4 points). In a comparison of variance, the red noise exposure (5.1 points) and the pink noise exposure (6.9 points) were significantly lower than the white noise exposure (11.2 points) and noiseless exposure (9.3 points). The paired t-test analysis showed that in the working memory test, the red noise exposure (P < 0.001), the pink noise exposure (P < 0.001), the white noise exposure (P < 0.01), the three noise exposure all had a significant difference as compared with the quiet situation, as shown on Figure 5.\nPaired t-test analysis of Taiwan’s Odd-Even Number Sequencing Test (TOENST) in four different sound field exposures.\nAs shown in Table 3, compared with the quiet situation, both the red noise and the pink noise exposure in the simple reaction time test, the executive function test and the working memory test all showed significant differences (P < 0.001). The white noise exposure was very significantly different (P < 0.001) in the simple reaction time test, and there was a significant difference (P < 0.01) in the working memory test. However, only pink noise exposure was significantly different (P < 0.05) in the continuous performance test.\nPaired t-test analysis of three colour noise exposure situations compared with quiet situations\nA statistical analysis of the sound field comfort questionnaire showed that, the questionnaire had a Cronbach’s alpha score of 0.772. Generally, a questionnaire with a Cronbach’s alpha score between 0.7 and 0.9 is considered to be of good reliability. As shown in Table 4, the subjective comfort evaluation statistics of the sound field environment indicated a highest and lowest score of 0 and −2, a little worse in the quiet scenario, while the scores between −1 and 3 were slightly worse to good in the red noise scenario; the scores between −1 and 2 were slightly worse to better in pink noise scenario; however, the scores between −3 and 1 represented a worse to slightly better result for the white noise scenario. The sum of the scores of the subjective evaluation in the sound field were −40 (average rating −0.26), +81 (average rating 0.53), +38 (average rating 0.25), −94 (average rating −0.61) for quiet, red noise, pink noise, and white noise scenarios, respectively.\nSubjective comfort evaluations of four sound environments\n\nDISCUSSION:\nOf the four trials, there were three significant (P < 0.001) increases in scores for red noise exposure and pink noise exposure, possibly reflecting improvements in work efficiency. Literature explains that white noise exposure can serve as a masking sound, and this may improve work efficiency. Two tests showed a significant (P < 0.001) increase in performance, with a significant (P < 0.01) increase in one trial also corroborating previous studies. In brain synchronization studies, pink noise exposure has been shown to synchronize the brain and induce brain activity to lower its potential,[13] and to exclude the effects of the outside world for purposes of concentrated processing. Except for the slight significant difference (P < 0.05) in white noise exposure, the continuous performance test was the only one that did not show very significant results. Participants may all have been preoccupied with the test, so the average correct rates are above 90%. Even the correct rates of two participants reached 100% when exposed to all four sound situations, and there were three participants with a 100% correct rate when exposed to three scenarios.\nIn the continuous performance test, the correct rate of non-exposure was about 88.8%, which of red noise, pink noise, and white noise were 91.7%, 93.6%, and 91.1%, respectively. Half of the 22 participants achieved a correct rate of 93.3% under the conditions of red noise exposure, pink noise exposure and white noise exposure, all of which were higher than the 88.8% correct rate of no noise exposure. The average score of the test results showed that a red noise exposure scenario, pink noise exposure scenario, and white noise exposure scenario are slightly better than the quiet scenario, which means that with the exposure to colour noise, the correct rate will rise and attention will be slightly improved.\nBecause the exposure of the participant’s living environment to the noise itself, might affect the questionnaire data, the type of accommodation should be indicated on the comfort questionnaire, and whether or not there is any noise exposure should be indicated in the closed questionnaire. In this study, there were three home participants exposed to noise, there were three home participants who sometimes had noise exposure. Of these six participants, five of the participants had different answers from the others, indicating a larger numerical increase in white noise.\nThe scores of the questionnaire results in the comfort of colour noises environment did not fully match the test data. Participants were randomly interviewed after the experiment. They said that with the additional sound increases, they felt that the sound interference, and the selection value should be less favourable. The laboratory background sound level was 44 dBA in the quiet scenario. The participants experienced significant changes to sound of 47 dBA when exposed to colour noise. Moreover, because the experimental exposure scenario was determined in a random manner, it was difficult to remember which previous test scores were selected. Scenario comfort evaluation is felt on a subjective basis, but participants may have had preconceptions when completing the questionnaire, thus affecting the final score. Although the results of the environmental comfort questionnaire were not as expected, the overall percentage of participants who felt slightly better or above were 48.8%, 32%, 9% for participants exposed to red, pink, white noise, respectively.\nThe results of this study show that exposure to the three kinds of colour noises, red, pink and white noise yields significantly better results in the Psychomotor Speed Test, Continuous Performance Test, Executive Function Test and Working Memory Test, which proves that these three kinds of sound field environments on the office work-type job site might improve work efficiency. The sum of the questionnaire scores on subjective comfort of the sound field environment showed that red, pink, and white noise exposure, were better than the original no noise exposure of the sound field, and this result implies that the three colour noise sound fields seem to make the white-collar work patterns a better workplaces not only by improving productivity, but also by making workers feel comfortable. Because white noise is equal energy at each frequency, it can cover the sounds that do not want to be heard around the environment. Pink noise might be due to neurons in the hypothalamus synchronously resonating with pink noises in the low oscillation frequency band, making slow rhythms and dominating brain waves. In addition, the power density of high frequency in red noise is even lower than these in pink noise and may therefore affect neurons in the hypothalamus even more, leading to information more speedily reaching the cerebral cortex through the hypothalamus and to faster transmission of motor messages through the hypothalamus.\nThe concept of the open-plan office is now widespread in the workplace. While offering many advantages in terms of layout and facilitating communication between colleagues, this way of organising the workspace had two major disadvantages. The feeling of privacy is lessened and noise level is increased causing discomfort for individuals working in this type of environment. It appears that noise is a major nuisance factor in open-plan offices in spite of a relatively low noise level. The noise nuisance experienced in open-plan offices affects work satisfaction, and this exposure to noise can reduce employee performance depending on the types of task to be carried out and the characteristics of the noise present in the workplace. The current research suggests that creating a colour noise environment through a loudspeaker or headphone might improve some aspects of cognitive performance in different workplaces. Many studies in the past have proved that noise has a negative influence on human hearing, cardiovascular systems, and emotions and so on. However, the three colour noises are applied to the original sound field environment. Therefore, if the background noise of the workplace is already too high, it would not be in order, for health reasons, to further add colour noise.\n\nCONCLUSION:\nThe results of this study show that participants perform significantly better in Psychomotor Speed Test, Continuous Performance Test, Executive Function Test and Working Memory Test. Therefore, colour noise may be used in the future to improve a workplace sound field environment, so that white-collar workers have a better working environment. Using physical stimuli of red, pink and white noises to improve worker productivity may prove to be a new approach towards productivity improvement that may provide fewer side effects than traditional methods such as drinking refreshing beverages. The introduction of the sounds represents a simple, low-cost, non-invasive improvement.\n Financial support and sponsorship Nil.\nNil.\n Conflicts of interest There are no conflicts of interest.\nThere are no conflicts of interest.\n\nFinancial support and sponsorship:\nNil.\n\nConflicts of interest:\nThere are no conflicts of interest.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nAs part of an effort to enhance the efficiency of workers, experiments relating to three types of noise exposure were conducted. Previous studies have proved that pink noise can cause a brain wave to reach a lower potential. In this study, we utilized physical methods, in cognitive experiments, to understand the impacts that three colour noises have on working efficiency.\n\nMethods:\nAll 22 participants were exposed to a sound environment of quiet, red, pink and white noises respectively. After a laboratory experiment, details of psychomotor speed, continuous performance, executive function and working memory were recorded.\n\nResults:\nRed, pink and white noises were significantly positive in comparison with the quiet environment of the psychomotor speed test. As for the continuous performance test, pink noise gave the only significantly positive result. Red, pink and white noise resulted in a better executive function test. Red and pink noise showed significantly positive improvement, while white noise was significantly positive in comparison with the quiet environment of the working memory test. In addition, the results from the comfort questionnaires showed that red and pink noise increase the possibility of better judgment, implementation, and overall environment.\n\nConclusions:\nAt present time, it is considered that noise has negative effects on hearing and health. However, experimental results show that certain noise can enhance environmental comfort. It is feasible, in the future, to use knowledge of colour noises to improve productivity in a workplace with a healthy environment."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nExposure to high levels of noise in industry factory is associated with a number of effects on health manifested in various psychosocial responses such as annoyance, disturbance of daily activities, sleep and performance and in physical responses, such as hearing loss, hypertension and ischemic heart disease.[12] While the open-plan office is now widespread in the workplace, it appears that noise is a major nuisance factor in open-plan offices in spite of a relatively low noise level(less than 65 dB(A)).[3] In general, the higher the decibel numbers of sound, the greater the health hazard. However, when the number of sound decibels is not high, the degree of influence on people may vary depending on the sound characteristics.\nIn audio engineering, physics, and many other fields, the colour of noise refers to the power spectrum of a noise signal. The practice of naming kinds of noise after colours started with white noise, a signal whose spectrum has equal power within any equal interval of frequencies. That name was given by analogy with white light, which was assumed to have such a flat power spectrum over the visible range. Other colour names, like pink, red, and blue were then given to noise with other spectral profiles, often (but not always) in reference to the colour of light with similar spectra.\nNoise often has detrimental effects on performance. Under most circumstances, information processing is disturbed by environmental noise and other non-task compatible distracters.[45] However, researchers have recently reported that under certain circumstances individuals with attention problems appear to benefit from the addition of specific forms of environmental noise. Typically, this facilitative effect has been limited to non-vocal background music on simple arithmetic task performance,[6] but Stansfeld et al.[7] found just that under certain conditions even road traffic noise can improve performance on episodic memory tasks, particularly in children at risk of attention problems and academic underachievement. Furthermore, Söderlund et al.[8] have demonstrated that adding auditory white noise to the environment enhanced the memory performance of children with Attention Deficit Hyperactivity Disorder (ADHD)-type problems but disrupted that of non-ADHD control children. Stochastic resonance, a phenomenon whereby signal processing is enhanced by the addition of random noise, has been widely demonstrated across various modalities, including visual, auditory, tactile and cross-modal forms of processing.[910] Of particular interest are findings that auditory noise has the capacity to enhance some aspects of human cognitive performance, such as the speed of arithmetical computations.[11]\n\nMeanwhile, some recent studies showed related evidence. For example, in 2010, light music, an example belonging to pink noise, is proved beneficial for elder people to improve their sleep quality as a long term effect.[12] Moreover, in a previous study,[13] the steady pink noise with intensity of 35 dB, 40 dB, 50 dB and 60 dB was used to stimulate four participants during sleep and they believed that pink noise could improve the sleep quality of participants. However, it is clear that this conclusion was based on significant increase of light sleep period (especially stage 2) accompanied with declined duration of rapid eye movement. In the brain synchronization study, it has demonstrated that the pink noise could synchronize brain wave and induce brain activity into a specialized state, that is, in a lower complexity level.[14]\n\nSezici’s study showed that playing of white noise significantly decreased the daily crying durations and increased the sleeping durations of the colicky babies compared to swinging in both groups.[15] In the literature, parents emphasized that playing of white noise was quite beneficial in relieving pain among crying colicky babies.[16] In their studies, Kucukoglu et al.[17] and Karakoc and Turker[18] revealed that the pain scores of the newborns who were made to listen to white noise were observed to decrease compared with those of the newborns in the control group. White noise encompasses all characteristics of sounds within the range of human hearing. It has been used in the treatment of tinnitus, insomnia, masking unwanted sounds, and provision of relaxation.[1920]\n\nZhou et al.[14] demonstrated that steady pink noise has significant effect on reducing brain wave complexity and inducing more stable sleep time to improve sleep quality of individuals. In total, 42 elderly people (21 using music and 21 controls) completed the study, and there was some indication that soft slow music yielder higher improvement on some of the parameters, which are worthy of further investigation.[12] A tabulated list of the effects observed in previous studies with colour noise exposures is shown in Table 1.\nA tabulated list of the effects observed in previous studies with colour noise exposures\nSeveral experiments have demonstrated that the detrimental effect of office noise on worker attitude and performance can be significantly reduced through the use of masking noise.[2122232425] For instance the experimental study performed by Haka[24] demonstrated that the performance of operation span tasks, serial recall and long term memory tasks were all improved when masking noise was used; and the field study performed by Hongisto[23] indicated that masking noise significantly reduced disturbance of worker attitudes towards concentration caused by office noise. Masking noise achieves this improvement through reducing the intelligibility of nearby speech.[25] In addition, Vassie et al.[26] indicated that participants rejected the brown masking noise delivered through earphones as it caused irritation and discomfort. Future studies should also consider other types of masking noise and should measure the level and duration of the masking noise.[26]\n\nIt is well-known that any human action results from brain activity, which is affected by light and sound. Too much acoustic energy causes hearing loss, impaired human nerves, and emotional disorders.[2728] However, sound play also very important role for people. According to previous researchers, not only white noise can create shading effect, but pink noise can improve sleep quality. Most people in the workplace are in the office or in the service industry, not in the noisy working environment for the factory. Creating a quiet environment is sometimes not easy. However, adding some sounds to enhance the work efficiency and reduce the work pressure of low-noise environment should be an issue worth exploring. Therefore, this study wanted to understand the effects of three different colours noise exposure. In the past, there were not many studies in this regard. Therefore, the goal of the current study is to test the hypothesis that adding another sound in a low background noise environment may increases productivity and comfort.\n\nCONCLUSION:\nThe results of this study show that participants perform significantly better in Psychomotor Speed Test, Continuous Performance Test, Executive Function Test and Working Memory Test. Therefore, colour noise may be used in the future to improve a workplace sound field environment, so that white-collar workers have a better working environment. Using physical stimuli of red, pink and white noises to improve worker productivity may prove to be a new approach towards productivity improvement that may provide fewer side effects than traditional methods such as drinking refreshing beverages. The introduction of the sounds represents a simple, low-cost, non-invasive improvement.\n Financial support and sponsorship Nil.\nNil.\n Conflicts of interest There are no conflicts of interest.\nThere are no conflicts of interest."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nAs part of an effort to enhance the efficiency of workers, experiments relating to three types of noise exposure were conducted. Previous studies have proved that pink noise can cause a brain wave to reach a lower potential. In this study, we utilized physical methods, in cognitive experiments, to understand the impacts that three colour noises have on working efficiency.\n\nMethods:\nAll 22 participants were exposed to a sound environment of quiet, red, pink and white noises respectively. After a laboratory experiment, details of psychomotor speed, continuous performance, executive function and working memory were recorded.\n\nResults:\nRed, pink and white noises were significantly positive in comparison with the quiet environment of the psychomotor speed test. As for the continuous performance test, pink noise gave the only significantly positive result. Red, pink and white noise resulted in a better executive function test. Red and pink noise showed significantly positive improvement, while white noise was significantly positive in comparison with the quiet environment of the working memory test. In addition, the results from the comfort questionnaires showed that red and pink noise increase the possibility of better judgment, implementation, and overall environment.\n\nConclusions:\nAt present time, it is considered that noise has negative effects on hearing and health. However, experimental results show that certain noise can enhance environmental comfort. It is feasible, in the future, to use knowledge of colour noises to improve productivity in a workplace with a healthy environment."},"whole_article_text_length":{"kind":"number","value":8294,"string":"8,294"},"whole_article_abstract_length":{"kind":"number","value":284,"string":"284"},"other_sections_lengths":{"kind":"list like","value":[153,294,707,283,90,2],"string":"[\n 153,\n 294,\n 707,\n 283,\n 90,\n 2\n]","isConversational":false},"num_sections":{"kind":"number","value":12,"string":"12"},"most_frequent_words":{"kind":"list like","value":["noise","test","exposure","noise exposure","sound","participants","white","pink","scenario","white noise"],"string":"[\n \"noise\",\n \"test\",\n \"exposure\",\n \"noise exposure\",\n \"sound\",\n \"participants\",\n \"white\",\n \"pink\",\n \"scenario\",\n \"white noise\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["noise workplace","white noise beneficial","kinds noise colours","noise workplace high","colour 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||| ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] three ||| ||| three ||| 22 ||| ||| ||| ||| ||| ||| ||| ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] three ||| ||| three ||| 22 ||| ||| ||| ||| ||| ||| ||| ||| ||| [SUMMARY]"}}},{"rowIdx":71,"cells":{"title":{"kind":"string","value":"Diverting colostomy is an effective and reversible option for severe hemorrhagic radiation proctopathy."},"pmid":{"kind":"string","value":"32148382"},"background_abstract":{"kind":"string","value":"Severe chronic radiation proctopathy (CRP) is difficult to treat."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"To assess the efficacy of colostomy in CRP, patients with severe hemorrhagic CRP who underwent colostomy or conservative treatment were enrolled. Patients with tumor recurrence, rectal-vaginal fistula or other types of rectal fistulas, or who were lost to follow-up were excluded. Rectal bleeding, hemoglobin (Hb), endoscopic features, endo-ultrasound, rectal manometry, and magnetic resonance imaging findings were recorded. Quality of life before stoma and after closure reversal was scored with questionnaires. Anorectal functions were assessed using the CRP symptom scale, which contains the following items: Watery stool, urgency, perianal pain, tenesmus, rectal bleeding, and fecal/gas incontinence."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"A total of 738 continual CRP patients were screened. After exclusion, 14 patients in the colostomy group and 25 in the conservative group were included in the final analysis. Preoperative Hb was only 63 g/L ± 17.8 g/L in the colostomy group compared to 88.2 g/L ± 19.3 g/L (P < 0.001) in the conservative group. All 14 patients in the former group achieved complete remission of bleeding, and the colostomy was successfully reversed in 13 of 14 (93%), excepting one very old patient. The median duration of stoma was 16 (range: 9-53) mo. The Hb level increased gradually from 75 g/L at 3 mo, 99 g/L at 6 mo, and 107 g/L at 9 mo to 111 g/L at 1 year and 117 g/L at 2 years after the stoma, but no bleeding cessation or significant increase in Hb levels was observed in the conservative group. Endoscopic telangiectasia and bleeding were greatly improved. Endo-ultrasound showed decreased vascularity, and magnetic resonance imaging revealed an increasing presarcal space and thickened rectal wall. Anorectal functions and quality of life were significantly improved after stoma reversal, when compared to those before stoma creation."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Diverting colostomy is a very effective method in the remission of refractory hemorrhagic CRP. Stoma can be reversed, and anorectal functions can be recovered after reversal."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Aged","Colostomy","Female","Gastrointestinal Hemorrhage","Humans","Male","Middle Aged","Quality of Life","Radiation Injuries","Rectal Diseases","Rectum","Retrospective Studies","Surgical Stomas","Treatment Outcome"],"string":"[\n \"Aged\",\n \"Colostomy\",\n \"Female\",\n \"Gastrointestinal Hemorrhage\",\n \"Humans\",\n \"Male\",\n \"Middle Aged\",\n \"Quality of Life\",\n \"Radiation Injuries\",\n \"Rectal Diseases\",\n \"Rectum\",\n \"Retrospective Studies\",\n \"Surgical Stomas\",\n \"Treatment Outcome\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"7052535"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Chronic radiation proctopathy (CRP) is a common and sometimes difficult issue after radiotherapy for pelvic malignancies. A permanent change in bowel habits may occur in 90% of patients. After pelvic radiotherapy, 20%-50% of patients will develop difficult bowel function, affecting their quality of life (QOL)[1,2]. CRP can cause more than 20 different symptoms. Rectal bleeding is the most common symptom and occurs in > 50% of CRP patients[1,3]. Transfusion-dependent severe bleeding occurs in 1%-5% of patients[1]. Pathologically, ischemia in the submucosa due to obliterative endarteritis and progressive fibrosis in macroscopic changes are the main causes[4].\nMedical treatment consists of topical sucralfate enemas[5], oral or topical sulfasalazine[6], and rebamipide[7]. However, these reagents are only effective in acute or mild CRP, whereas their efficacy is usually disappointing in CRP patients with moderate to severe hemorrhage. More invasive modalities include endoscopic argon plasma coagulation (APC)[8], topical 4%-10% formalin[9-12], radiofrequency ablation[13], and hyperbaric oxygen therapy[14,15], and are currently popular optional treatments for CRP. Most of these modalities lack randomized trial evidence. These treatment options are reported to be effective in controlling mild to moderate bleeding in most of the literature. Nonetheless, severe and refractory bleeding is difficult to manage[1,16]. Furthermore, APC or topical formalin requires multiple sessions for severe bleeding and can cause severe side effects, including perforation, strictures, and perianal pain[17].\nFecal diversion is reported to be effective in the management of severe CRP bleeding[16,18,19], as it can reduce irritation injury to the lesions to decrease hemorrhage[16]. However, the usage of fecal diversion is not adopted as widely as is APC or formalin. Most of the fecal diversion data are from the 1980s, and this option has not been well studied to date. We previously reported one retrospective cohort of CRP patients with severe hemorrhage who received colostomy[16]. The results showed that colostomy can bring much higher remission of severe bleeding (94%) than can conservative treatment (11%) with APC or topical formalin. Pathologically, chronic inflammation and progressive fibrosis are observed after stoma. Diverting stoma is usually thought to be permanent according to Ayerdi et al[20].\nAnorectal function and QOL after stoma reversal remain unclear. During the past 3 years, we have successfully performed colostomy reversals in a large cohort of CRP patients with severe bleeding and have follow-up data after colostomy reversal. Here, we report the efficacy of colostomy, the rate of stoma closure of diverting colostomy, and anorectal function after reversal for this series of CRP patients with severe bleeding."},"methods_title":{"kind":"string","value":"MATERIALS AND METHODS"},"methods_text":{"kind":"string","value":" Patients and ethical statements Patients with hemorrhagic CRP who were treated after admission at The Sixth Affiliated Hospital of Sun Yat-Sen University from March 2008 to December 2018 were enrolled. Medical records and imaging and clinicopathological data were extracted from our electronic database. This study was approved by the Ethical Committee of The Sixth Affiliated Hospital of Sun Yat-Sen University and was performed according to the provisions of the World Medical Association’s Declaration of Helsinki in 1995 (updated in Tokyo, 2004). Due to the nature of a retrospective study, informed consent was waived.\nPatients with hemorrhagic CRP who were treated after admission at The Sixth Affiliated Hospital of Sun Yat-Sen University from March 2008 to December 2018 were enrolled. Medical records and imaging and clinicopathological data were extracted from our electronic database. This study was approved by the Ethical Committee of The Sixth Affiliated Hospital of Sun Yat-Sen University and was performed according to the provisions of the World Medical Association’s Declaration of Helsinki in 1995 (updated in Tokyo, 2004). Due to the nature of a retrospective study, informed consent was waived.\n Inclusion and exclusion criteria CRP patients with refractory rectal bleeding who received diverting colostomy or conservative treatments for severe anemia and who needed transfusions were enrolled. Patients who were lost to follow-up, had tumor recurrence, rectal-vaginal fistula or other types of rectal fistulas, or underwent rectal resection with preventive colostomy were excluded because these conditions would affect the evaluation of remission of rectal bleeding.\nCRP patients with refractory rectal bleeding who received diverting colostomy or conservative treatments for severe anemia and who needed transfusions were enrolled. Patients who were lost to follow-up, had tumor recurrence, rectal-vaginal fistula or other types of rectal fistulas, or underwent rectal resection with preventive colostomy were excluded because these conditions would affect the evaluation of remission of rectal bleeding.\n Diagnosis, definitions, and scores CRP was diagnosed by the combination of pelvic radiation history for malignancies, symptoms such as rectal bleeding, and endoscopic findings of CRP-specific changes such as telangiectasia and active bleeding in the rectum, as well as exclusion of other bleeding diseases. Hemorrhagic problems, such as tumor recurrence and hemorrhoids, were excluded. Refractory severe bleeding was defined as frequent bleeding and severe anemia with the need for transfusions. At admission, CRP patients in our center were first referred to noninvasive enemas, and if there was no response or recurrence, invasive treatment, such as APC (1-2 rounds) or formalin topical irrigation, was utilized. Conservative treatment failure was defined as no response or recurrence of frequent or severe rectal bleeding after invasive treatments.\nIn this study, we used a modified subjective-objective management analysis (known as mSOMA) system that we designed previously to assess the severity of rectal bleeding[16]. The advantages of the mSOMA system include both subjective complaints of patients and objective hemoglobin (Hb) levels according to laboratory tests. Remission of bleeding was defined as complete cessation or occasional bleeding that needed no further treatment. The CTCAE score of bleeding was used to assess its severity.\nCRP was diagnosed by the combination of pelvic radiation history for malignancies, symptoms such as rectal bleeding, and endoscopic findings of CRP-specific changes such as telangiectasia and active bleeding in the rectum, as well as exclusion of other bleeding diseases. Hemorrhagic problems, such as tumor recurrence and hemorrhoids, were excluded. Refractory severe bleeding was defined as frequent bleeding and severe anemia with the need for transfusions. At admission, CRP patients in our center were first referred to noninvasive enemas, and if there was no response or recurrence, invasive treatment, such as APC (1-2 rounds) or formalin topical irrigation, was utilized. Conservative treatment failure was defined as no response or recurrence of frequent or severe rectal bleeding after invasive treatments.\nIn this study, we used a modified subjective-objective management analysis (known as mSOMA) system that we designed previously to assess the severity of rectal bleeding[16]. The advantages of the mSOMA system include both subjective complaints of patients and objective hemoglobin (Hb) levels according to laboratory tests. Remission of bleeding was defined as complete cessation or occasional bleeding that needed no further treatment. The CTCAE score of bleeding was used to assess its severity.\n Indications for fecal diversion and stoma closure The indications for diverting diversion in hemorrhagic CRP contained the following conditions: Recurrent bleeding and unresponsive to conservative treatments such as endoscopic APC or topical formalin, accompanying severe anemia, and transfusions needed. Transverse loop colostomy and “gunsight” or “cross‘’ type of stoma closure were created according to the standard protocols used in our previous studies[16]. The criteria of stoma closure were as follows: Remission or occasional rectal bleeding, regressive edema in rectal mucosa, good performance of anorectal function, exclusion of tumor recurrence, and no severe CRP complications such as fistula, stricture, and deep ulcer[16].\nThe indications for diverting diversion in hemorrhagic CRP contained the following conditions: Recurrent bleeding and unresponsive to conservative treatments such as endoscopic APC or topical formalin, accompanying severe anemia, and transfusions needed. Transverse loop colostomy and “gunsight” or “cross‘’ type of stoma closure were created according to the standard protocols used in our previous studies[16]. The criteria of stoma closure were as follows: Remission or occasional rectal bleeding, regressive edema in rectal mucosa, good performance of anorectal function, exclusion of tumor recurrence, and no severe CRP complications such as fistula, stricture, and deep ulcer[16].\n Follow-up Patients were scheduled for follow-up through outpatient visits or telephone questionnaires at 6 mo, 9 mo, 1 year, 1.5 years, and 2 years after colostomy. QOL after stoma closure was evaluated according to EORTC QLQ C30 questionnaires[21]. We focused on the following items in this study: The remission rate of bleeding, the rate of stoma reversal, QOL after stoma reversal, dynamic changes of Hb levels, stoma-related complications, and severe CRP complications.\nPatients were scheduled for follow-up through outpatient visits or telephone questionnaires at 6 mo, 9 mo, 1 year, 1.5 years, and 2 years after colostomy. QOL after stoma closure was evaluated according to EORTC QLQ C30 questionnaires[21]. We focused on the following items in this study: The remission rate of bleeding, the rate of stoma reversal, QOL after stoma reversal, dynamic changes of Hb levels, stoma-related complications, and severe CRP complications.\n Anorectal functions In our previous study, tenesmus, stool frequency, and anorectal pain were common symptoms in active CRP[22]. Because there are no standard scales or scores to assess anorectal function precisely in CRP patients, we developed a new scale system that is a patient self-reported scale of outcomes, namely, the chronic radiation proctopathy symptom scale (CRPSS). The CRPSS considers the following items: Watery stool, urgency, perianal pain, tenesmus, rectal bleeding, and fecal/gas incontinence. The details are provided in Supplementary File 1. For CRP patients treated by colostomy, CRPSS scores were evaluated before stoma and at reversal.\nIn our previous study, tenesmus, stool frequency, and anorectal pain were common symptoms in active CRP[22]. Because there are no standard scales or scores to assess anorectal function precisely in CRP patients, we developed a new scale system that is a patient self-reported scale of outcomes, namely, the chronic radiation proctopathy symptom scale (CRPSS). The CRPSS considers the following items: Watery stool, urgency, perianal pain, tenesmus, rectal bleeding, and fecal/gas incontinence. The details are provided in Supplementary File 1. For CRP patients treated by colostomy, CRPSS scores were evaluated before stoma and at reversal.\n Statistical analysis Analyses for continuous variables were performed by Student’s t-test. The χ2 test was used for categorical variables. Fisher’s exact test was conducted when appropriate. For non-parameter variables, the Wilcoxon rank sum tests were used. All statistical analyses were performed using SPSS version 23.0 (Chicago, IL, United States). P < 0.05 (two-tailed) was considered statistically significant.\nAnalyses for continuous variables were performed by Student’s t-test. The χ2 test was used for categorical variables. Fisher’s exact test was conducted when appropriate. For non-parameter variables, the Wilcoxon rank sum tests were used. All statistical analyses were performed using SPSS version 23.0 (Chicago, IL, United States). P < 0.05 (two-tailed) was considered statistically significant."},"results_title":{"kind":"null"},"results_text":{"kind":"null"},"conclusions_title":{"kind":"string","value":"Research conclusions"},"conclusions_text":{"kind":"string","value":"We thank Dr. Jervoise Andreyev (a famous international leader in radiation proctitis, Consultant Gastroenterologist in Pelvic Radiation Disease, Royal Marsden NHS Foundation Trust, London SW3 6JJ, United Kingdom) for critical revisions of our paper."},"other_sections_titles":{"kind":"list like","value":["INTRODUCTION","Patients and ethical statements","Inclusion and exclusion criteria","Diagnosis, definitions, and scores","Indications for fecal diversion and stoma closure","Follow-up","Anorectal functions","Statistical analysis","RESULTS","Demographics and clinical outcomes","Dynamically increasing hemoglobin levels after colostomy","Stoma closure and endoscopic features","Ultrasound findings before stoma and at reversal","Improved magnetic resonance imaging features at stoma reversal","Good restored anorectal functions after stoma reversal","Improved quality of life at colostomy reversal","DISCUSSION","ARTICLE HIGHLIGHTS","Research background","Research motivation","Research objectives","Research methods","Research results","Research conclusions"],"string":"[\n \"INTRODUCTION\",\n \"Patients and ethical statements\",\n \"Inclusion and exclusion criteria\",\n \"Diagnosis, definitions, and scores\",\n \"Indications for fecal diversion and stoma closure\",\n \"Follow-up\",\n \"Anorectal functions\",\n \"Statistical analysis\",\n \"RESULTS\",\n \"Demographics and clinical outcomes\",\n \"Dynamically increasing hemoglobin levels after colostomy\",\n \"Stoma closure and endoscopic features\",\n \"Ultrasound findings before stoma and at reversal\",\n \"Improved magnetic resonance imaging features at stoma reversal\",\n \"Good restored anorectal functions after stoma reversal\",\n \"Improved quality of life at colostomy reversal\",\n \"DISCUSSION\",\n \"ARTICLE HIGHLIGHTS\",\n \"Research background\",\n \"Research motivation\",\n \"Research objectives\",\n \"Research methods\",\n \"Research results\",\n \"Research conclusions\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Chronic radiation proctopathy (CRP) is a common and sometimes difficult issue after radiotherapy for pelvic malignancies. A permanent change in bowel habits may occur in 90% of patients. After pelvic radiotherapy, 20%-50% of patients will develop difficult bowel function, affecting their quality of life (QOL)[1,2]. CRP can cause more than 20 different symptoms. Rectal bleeding is the most common symptom and occurs in > 50% of CRP patients[1,3]. Transfusion-dependent severe bleeding occurs in 1%-5% of patients[1]. Pathologically, ischemia in the submucosa due to obliterative endarteritis and progressive fibrosis in macroscopic changes are the main causes[4].\nMedical treatment consists of topical sucralfate enemas[5], oral or topical sulfasalazine[6], and rebamipide[7]. However, these reagents are only effective in acute or mild CRP, whereas their efficacy is usually disappointing in CRP patients with moderate to severe hemorrhage. More invasive modalities include endoscopic argon plasma coagulation (APC)[8], topical 4%-10% formalin[9-12], radiofrequency ablation[13], and hyperbaric oxygen therapy[14,15], and are currently popular optional treatments for CRP. Most of these modalities lack randomized trial evidence. These treatment options are reported to be effective in controlling mild to moderate bleeding in most of the literature. Nonetheless, severe and refractory bleeding is difficult to manage[1,16]. Furthermore, APC or topical formalin requires multiple sessions for severe bleeding and can cause severe side effects, including perforation, strictures, and perianal pain[17].\nFecal diversion is reported to be effective in the management of severe CRP bleeding[16,18,19], as it can reduce irritation injury to the lesions to decrease hemorrhage[16]. However, the usage of fecal diversion is not adopted as widely as is APC or formalin. Most of the fecal diversion data are from the 1980s, and this option has not been well studied to date. We previously reported one retrospective cohort of CRP patients with severe hemorrhage who received colostomy[16]. The results showed that colostomy can bring much higher remission of severe bleeding (94%) than can conservative treatment (11%) with APC or topical formalin. Pathologically, chronic inflammation and progressive fibrosis are observed after stoma. Diverting stoma is usually thought to be permanent according to Ayerdi et al[20].\nAnorectal function and QOL after stoma reversal remain unclear. During the past 3 years, we have successfully performed colostomy reversals in a large cohort of CRP patients with severe bleeding and have follow-up data after colostomy reversal. Here, we report the efficacy of colostomy, the rate of stoma closure of diverting colostomy, and anorectal function after reversal for this series of CRP patients with severe bleeding.","Patients with hemorrhagic CRP who were treated after admission at The Sixth Affiliated Hospital of Sun Yat-Sen University from March 2008 to December 2018 were enrolled. Medical records and imaging and clinicopathological data were extracted from our electronic database. This study was approved by the Ethical Committee of The Sixth Affiliated Hospital of Sun Yat-Sen University and was performed according to the provisions of the World Medical Association’s Declaration of Helsinki in 1995 (updated in Tokyo, 2004). Due to the nature of a retrospective study, informed consent was waived.","CRP patients with refractory rectal bleeding who received diverting colostomy or conservative treatments for severe anemia and who needed transfusions were enrolled. Patients who were lost to follow-up, had tumor recurrence, rectal-vaginal fistula or other types of rectal fistulas, or underwent rectal resection with preventive colostomy were excluded because these conditions would affect the evaluation of remission of rectal bleeding.","CRP was diagnosed by the combination of pelvic radiation history for malignancies, symptoms such as rectal bleeding, and endoscopic findings of CRP-specific changes such as telangiectasia and active bleeding in the rectum, as well as exclusion of other bleeding diseases. Hemorrhagic problems, such as tumor recurrence and hemorrhoids, were excluded. Refractory severe bleeding was defined as frequent bleeding and severe anemia with the need for transfusions. At admission, CRP patients in our center were first referred to noninvasive enemas, and if there was no response or recurrence, invasive treatment, such as APC (1-2 rounds) or formalin topical irrigation, was utilized. Conservative treatment failure was defined as no response or recurrence of frequent or severe rectal bleeding after invasive treatments.\nIn this study, we used a modified subjective-objective management analysis (known as mSOMA) system that we designed previously to assess the severity of rectal bleeding[16]. The advantages of the mSOMA system include both subjective complaints of patients and objective hemoglobin (Hb) levels according to laboratory tests. Remission of bleeding was defined as complete cessation or occasional bleeding that needed no further treatment. The CTCAE score of bleeding was used to assess its severity.","The indications for diverting diversion in hemorrhagic CRP contained the following conditions: Recurrent bleeding and unresponsive to conservative treatments such as endoscopic APC or topical formalin, accompanying severe anemia, and transfusions needed. Transverse loop colostomy and “gunsight” or “cross‘’ type of stoma closure were created according to the standard protocols used in our previous studies[16]. The criteria of stoma closure were as follows: Remission or occasional rectal bleeding, regressive edema in rectal mucosa, good performance of anorectal function, exclusion of tumor recurrence, and no severe CRP complications such as fistula, stricture, and deep ulcer[16].","Patients were scheduled for follow-up through outpatient visits or telephone questionnaires at 6 mo, 9 mo, 1 year, 1.5 years, and 2 years after colostomy. QOL after stoma closure was evaluated according to EORTC QLQ C30 questionnaires[21]. We focused on the following items in this study: The remission rate of bleeding, the rate of stoma reversal, QOL after stoma reversal, dynamic changes of Hb levels, stoma-related complications, and severe CRP complications.","In our previous study, tenesmus, stool frequency, and anorectal pain were common symptoms in active CRP[22]. Because there are no standard scales or scores to assess anorectal function precisely in CRP patients, we developed a new scale system that is a patient self-reported scale of outcomes, namely, the chronic radiation proctopathy symptom scale (CRPSS). The CRPSS considers the following items: Watery stool, urgency, perianal pain, tenesmus, rectal bleeding, and fecal/gas incontinence. The details are provided in Supplementary File 1. For CRP patients treated by colostomy, CRPSS scores were evaluated before stoma and at reversal.","Analyses for continuous variables were performed by Student’s t-test. The χ2 test was used for categorical variables. Fisher’s exact test was conducted when appropriate. For non-parameter variables, the Wilcoxon rank sum tests were used. All statistical analyses were performed using SPSS version 23.0 (Chicago, IL, United States). P < 0.05 (two-tailed) was considered statistically significant."," Demographics and clinical outcomes From March 2008 to Feb 2019, 738 continual CRP patients treated in our center were screened. After exclusion of 644 patients with mild or moderate CRP, 94 with refractory bleeding and moderate to severe anemia were further screened. Among them, 50 patients were treated by diverted colostomy and 44 patients were treated with conservative therapies. Among the 50 patients treated by colostomy, 32 were excluded due to recto-vaginal fistulas or deep rectal ulcer with refractory perianal pain before stoma. The remaining 18 patients with refractory bleeding were enrolled. Among them, two died from cancer recurrence and two were lost to follow-up. The remaining 14 patients were enrolled in the final analysis as the colostomy group. For the conservative group, 19 patients were excluded, including 11 with tumor relapse and 8 who were lost to follow-up. Thus, 25 were enrolled in the final analysis (Figure 1). Demographic and baseline characteristics were collected, including the intention-to-treat (ITT) group of 50 colostomies. In the enrolled patients, 14 patients comprised the diverting colostomy group, and 25 cases comprised the conservative treatment group. The primary tumors in most of the patients (33/39, 85%) were cervical cancers. No significant differences in age, sex, type of primary tumor, or radiation dosage were found between the diverting colostomy group and the conservative treatment group or between the ITT colostomy group and the colostomy group (Table 1).\nDemographics and characteristics\nFisher's exact test. ITT: Intention-to-treat; Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin.\nFlow chart of patient selection.\nIn the ITT group, no postoperative follow-ups were conducted other than for the 14 investigated colostomy patients. Higher bleeding scores (P = 0.033) and relatively decreased preoperative Hb levels (P = 0.051, no significant difference) were found in the diverting colostomy group compared to the ITT group because 36 patients in the ITT group underwent colostomy for recto-vaginal fistulas or deep rectal ulcers instead of severe bleeding.\nFrom March 2008 to Feb 2019, 738 continual CRP patients treated in our center were screened. After exclusion of 644 patients with mild or moderate CRP, 94 with refractory bleeding and moderate to severe anemia were further screened. Among them, 50 patients were treated by diverted colostomy and 44 patients were treated with conservative therapies. Among the 50 patients treated by colostomy, 32 were excluded due to recto-vaginal fistulas or deep rectal ulcer with refractory perianal pain before stoma. The remaining 18 patients with refractory bleeding were enrolled. Among them, two died from cancer recurrence and two were lost to follow-up. The remaining 14 patients were enrolled in the final analysis as the colostomy group. For the conservative group, 19 patients were excluded, including 11 with tumor relapse and 8 who were lost to follow-up. Thus, 25 were enrolled in the final analysis (Figure 1). Demographic and baseline characteristics were collected, including the intention-to-treat (ITT) group of 50 colostomies. In the enrolled patients, 14 patients comprised the diverting colostomy group, and 25 cases comprised the conservative treatment group. The primary tumors in most of the patients (33/39, 85%) were cervical cancers. No significant differences in age, sex, type of primary tumor, or radiation dosage were found between the diverting colostomy group and the conservative treatment group or between the ITT colostomy group and the colostomy group (Table 1).\nDemographics and characteristics\nFisher's exact test. ITT: Intention-to-treat; Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin.\nFlow chart of patient selection.\nIn the ITT group, no postoperative follow-ups were conducted other than for the 14 investigated colostomy patients. Higher bleeding scores (P = 0.033) and relatively decreased preoperative Hb levels (P = 0.051, no significant difference) were found in the diverting colostomy group compared to the ITT group because 36 patients in the ITT group underwent colostomy for recto-vaginal fistulas or deep rectal ulcers instead of severe bleeding.\n Dynamically increasing hemoglobin levels after colostomy In the colostomy group, the median duration of bleeding was 13.0 mo ± 4.3 mo, and 12 (86%) patients received transfusion before stoma compared to 8.0 mo ± 1.8 mo (P = 0.042) of bleeding duration and 6 (24%) (P < 0.001) transfusions in the conservative group. In the conservative group, 8 patients received formalin irrigation and 4 patients received APC treatments. The preoperative (Pre-Op) Hb was only 63 g/L ± 17.8 g/L in the colostomy group compared to 88.2 g/L ± 19.3 g/L (P < 0.001) in the conservative group. One patient with moderate anemia (Hb of 87 g/L) received colostomy for perianal pain. Another patient with severe anemia (Hb of 78 g/L) did not receive a transfusion due to unavailability of a blood supply before colostomy. The details of the 14 patients are listed in Table 2. Increasing Hb levels were observed from 3 mo (median Hb of 75 g/L), 6 mo (Hb of 99 g/L), and 9 mo (Hb of 107 g/L) to 1 year (Hb 111 of g/L) and 2 years (Hb 117 g/L) after colostomy (Figure 2). Postoperative transfusions were conducted in 3 of 14 patients (transfusion requirements for 2 patients in other hospitals was unknown) in the colostomy group. The dynamic remission rates between the colostomy and conservative groups after surgery were as follows: 86% vs 4% at 3 mo (P < 0.001), 86% vs 12% at 6 mo (P < 0.001), 93% vs 20% at 9 mo (P < 0.001), 100% vs 24% at 1 year (P < 0.001), and 100% vs 20% at 2 years (P < 0.001) (Table 1).\nClinical details and outcomes of 14 colostomy patients\nMedian value. Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin; RTD: Rectal telangiectasia density; APC: Argon plasma coagulation.\nDynamic changes in hemoglobin levels. Pre-Op: Pre-operative.\nIn the colostomy group, the median duration of bleeding was 13.0 mo ± 4.3 mo, and 12 (86%) patients received transfusion before stoma compared to 8.0 mo ± 1.8 mo (P = 0.042) of bleeding duration and 6 (24%) (P < 0.001) transfusions in the conservative group. In the conservative group, 8 patients received formalin irrigation and 4 patients received APC treatments. The preoperative (Pre-Op) Hb was only 63 g/L ± 17.8 g/L in the colostomy group compared to 88.2 g/L ± 19.3 g/L (P < 0.001) in the conservative group. One patient with moderate anemia (Hb of 87 g/L) received colostomy for perianal pain. Another patient with severe anemia (Hb of 78 g/L) did not receive a transfusion due to unavailability of a blood supply before colostomy. The details of the 14 patients are listed in Table 2. Increasing Hb levels were observed from 3 mo (median Hb of 75 g/L), 6 mo (Hb of 99 g/L), and 9 mo (Hb of 107 g/L) to 1 year (Hb 111 of g/L) and 2 years (Hb 117 g/L) after colostomy (Figure 2). Postoperative transfusions were conducted in 3 of 14 patients (transfusion requirements for 2 patients in other hospitals was unknown) in the colostomy group. The dynamic remission rates between the colostomy and conservative groups after surgery were as follows: 86% vs 4% at 3 mo (P < 0.001), 86% vs 12% at 6 mo (P < 0.001), 93% vs 20% at 9 mo (P < 0.001), 100% vs 24% at 1 year (P < 0.001), and 100% vs 20% at 2 years (P < 0.001) (Table 1).\nClinical details and outcomes of 14 colostomy patients\nMedian value. Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin; RTD: Rectal telangiectasia density; APC: Argon plasma coagulation.\nDynamic changes in hemoglobin levels. Pre-Op: Pre-operative.\n Stoma closure and endoscopic features Among the 14 patients with severe bleeding treated by colostomy, all obtained complete remission of bleeding during follow-up. Only 3 (23%) of 13 patients received one round of APC after stoma to control bleeding, and 1 patient received 4% formalin irrigation after colostomy (Table 2). Among the 14 patients, 13 (93%) underwent stoma closure, and the remaining patient did not undergo stoma reversal due to concerns of surgical risks because of old age (83 years old). The median duration of stoma was 16 mo (range: 9-53 mo). Stoma complications occurred in 3 (21%) cases, including 1 para-stoma hernia, 1 stoma prolapse, and 1 stoma obstruction (Grade II by Clavien-Dindo classification)[23]. All 3 patients recovered well after stoma closure.\nEndoscopic findings of bleeding, confluent telangiectasia, and congested mucosa improved dramatically at stoma reversal compared to before stoma creation (Figure 3). The bleeding score by CTCAE decreased from Pre-Op 2.7 points ± 0.5 points to 0.8 points ± 0.5 points (P < 0.001) at 1 year after stoma in the colostomy group; the bleeding score was 2.0 ± 0.5 at initial diagnosis and 1.7 ± 0.9 (P = 0.1282) at 1 year after treatments in the conservative group (Table 1 and Figure 4).\nEndoscopic changes before stoma and at reversal.\nCTC bleeding scores before stoma and at stoma reversal.\nAmong the 14 patients with severe bleeding treated by colostomy, all obtained complete remission of bleeding during follow-up. Only 3 (23%) of 13 patients received one round of APC after stoma to control bleeding, and 1 patient received 4% formalin irrigation after colostomy (Table 2). Among the 14 patients, 13 (93%) underwent stoma closure, and the remaining patient did not undergo stoma reversal due to concerns of surgical risks because of old age (83 years old). The median duration of stoma was 16 mo (range: 9-53 mo). Stoma complications occurred in 3 (21%) cases, including 1 para-stoma hernia, 1 stoma prolapse, and 1 stoma obstruction (Grade II by Clavien-Dindo classification)[23]. All 3 patients recovered well after stoma closure.\nEndoscopic findings of bleeding, confluent telangiectasia, and congested mucosa improved dramatically at stoma reversal compared to before stoma creation (Figure 3). The bleeding score by CTCAE decreased from Pre-Op 2.7 points ± 0.5 points to 0.8 points ± 0.5 points (P < 0.001) at 1 year after stoma in the colostomy group; the bleeding score was 2.0 ± 0.5 at initial diagnosis and 1.7 ± 0.9 (P = 0.1282) at 1 year after treatments in the conservative group (Table 1 and Figure 4).\nEndoscopic changes before stoma and at reversal.\nCTC bleeding scores before stoma and at stoma reversal.\n Ultrasound findings before stoma and at reversal Endorectal ultrasound (EUS) was analyzed retrospectively before stoma and at reversal in 5 patients; the remaining patients did not receive EUS at either of these two time points. In our previous study, thickening of the rectal wall, blurred wall stratification, and increased vascularity were EUS features in CRP[24]. In this study, we found similar features before stoma creation and a tremendous decrease in superficial vascularity, which can explain the cessation of bleeding (Figure 5).\nEndoscopic ultrasound features before stoma and at stoma reversal.\nEndorectal ultrasound (EUS) was analyzed retrospectively before stoma and at reversal in 5 patients; the remaining patients did not receive EUS at either of these two time points. In our previous study, thickening of the rectal wall, blurred wall stratification, and increased vascularity were EUS features in CRP[24]. In this study, we found similar features before stoma creation and a tremendous decrease in superficial vascularity, which can explain the cessation of bleeding (Figure 5).\nEndoscopic ultrasound features before stoma and at stoma reversal.\n Improved magnetic resonance imaging features at stoma reversal To evaluate the severity of the effect on the rectal wall and pelvic floor in the colostomy group, we also report magnetic resonance imaging (MRI) parameters related to CRP, including the thickness of the rectal wall, width of the presacral space, and thicknesses of the levator ani, the gluteus maximus muscle, the obturator intemus, and the distal part of the sigmoid colon (Table 3), which was referred to in our previous study of radiation injury to the pelvis[25]. MRI scans both at pre-colostomy and at reversal were analyzed in 5 patients, as the remaining 9 did not receive MRI scans both pre-colostomy and at reversal. The thickness of the rectal wall was significantly decreased at reversal (8.91 ± 1.61) compared with pre-colostomy (10.7 ± 4.29) (P = 0.047) (Table 3).\nMagnetic resonance imaging findings at post-colostomy and at reversal compared in 5 patients with chronic radiation proctopathy\nTo evaluate the severity of the effect on the rectal wall and pelvic floor in the colostomy group, we also report magnetic resonance imaging (MRI) parameters related to CRP, including the thickness of the rectal wall, width of the presacral space, and thicknesses of the levator ani, the gluteus maximus muscle, the obturator intemus, and the distal part of the sigmoid colon (Table 3), which was referred to in our previous study of radiation injury to the pelvis[25]. MRI scans both at pre-colostomy and at reversal were analyzed in 5 patients, as the remaining 9 did not receive MRI scans both pre-colostomy and at reversal. The thickness of the rectal wall was significantly decreased at reversal (8.91 ± 1.61) compared with pre-colostomy (10.7 ± 4.29) (P = 0.047) (Table 3).\nMagnetic resonance imaging findings at post-colostomy and at reversal compared in 5 patients with chronic radiation proctopathy\n Good restored anorectal functions after stoma reversal Anorectal functions before stoma and after reversal were evaluated by CRPSS scores in all 12 patients in the colostomy group. The results showed that rectal bleeding, peritoneal pain, tenesmus, urgency and fecal/gas incontinency were significantly improved after stoma reversal compared to those before stoma creation. Additionally, all of these scores were < 2 points, which indicated that anorectal function recovered very well after stoma reversal (Figure 6). Complete remission of bleeding and good performance of anorectal function compared to the normal population (scores ≤ 1 point) were observed. We also analyzed anorectal manometry to objectively assess anorectal function before reversal in 6 of the 14 patients who received it. Increased sensitivity of the rectal mucosa and decreased rectal volume were observed. Decreased sphincter functions were found in 2 of 6 cases (Table 4). These patients fulfilled the indications for reversal, and we found that these patients obtained good performance of anorectal function after reversal. Rectal defecography was also conducted before reversal, and no stricture was observed.\nRectal manometry assessment of anorectal functions before stoma reversals\nAnorectal functions before stoma and after stoma reversal.\nAnorectal functions before stoma and after reversal were evaluated by CRPSS scores in all 12 patients in the colostomy group. The results showed that rectal bleeding, peritoneal pain, tenesmus, urgency and fecal/gas incontinency were significantly improved after stoma reversal compared to those before stoma creation. Additionally, all of these scores were < 2 points, which indicated that anorectal function recovered very well after stoma reversal (Figure 6). Complete remission of bleeding and good performance of anorectal function compared to the normal population (scores ≤ 1 point) were observed. We also analyzed anorectal manometry to objectively assess anorectal function before reversal in 6 of the 14 patients who received it. Increased sensitivity of the rectal mucosa and decreased rectal volume were observed. Decreased sphincter functions were found in 2 of 6 cases (Table 4). These patients fulfilled the indications for reversal, and we found that these patients obtained good performance of anorectal function after reversal. Rectal defecography was also conducted before reversal, and no stricture was observed.\nRectal manometry assessment of anorectal functions before stoma reversals\nAnorectal functions before stoma and after stoma reversal.\n Improved quality of life at colostomy reversal QOL after stoma was assessed in the colostomy and conservative groups using EORTC QLQ-C30 questionnaires. As there are no similar reports in the Chinese population, we referred to the normal German population[26]. Osoba et al[26] suggested that a difference of ≥ 20 points in global health was considered to be clinically relevant. In the panel of patients who underwent stoma closure, QOL was dramatically improved compared to Pre-Op baseline QOL before stoma creation, including improved global health (difference of 40, P < 0.001), physical function (difference of 36.4, P < 0.001), role function (difference of 55, P < 0.001), emotional function (difference of 39.4, P < 0.001), social function (difference of 36.3, P < 0.001), and improved symptoms, such as fatigue (difference of -62.5, P < 0.001), pain (difference of -34.9, P < 0.001), dyspnea (difference of -37.8, P < 0.001), insomnia (difference of -36.2, P < 0.001), diarrhea (difference of -23.8, P < 0.001), and financial problems (difference of -35.5, P < 0.001). However, in the conservative group, no improved QOL variables with a difference of ≥ 20 were observed (Table 5). QOL after stoma closure was also compared to the normal population reference, with similar scores for functions and symptoms (Table 5).\nQuality of life in the colostomy group and conservative group of chronic radiation proctopathy patients based on the EORTC QLQ-C30 scale\nWilcoxon rank-sum test. \nPoint (follow-up) – point (pretreatment). Δ(FU): Change in follow-up; SD: Standard deviation.\nQOL after stoma was assessed in the colostomy and conservative groups using EORTC QLQ-C30 questionnaires. As there are no similar reports in the Chinese population, we referred to the normal German population[26]. Osoba et al[26] suggested that a difference of ≥ 20 points in global health was considered to be clinically relevant. In the panel of patients who underwent stoma closure, QOL was dramatically improved compared to Pre-Op baseline QOL before stoma creation, including improved global health (difference of 40, P < 0.001), physical function (difference of 36.4, P < 0.001), role function (difference of 55, P < 0.001), emotional function (difference of 39.4, P < 0.001), social function (difference of 36.3, P < 0.001), and improved symptoms, such as fatigue (difference of -62.5, P < 0.001), pain (difference of -34.9, P < 0.001), dyspnea (difference of -37.8, P < 0.001), insomnia (difference of -36.2, P < 0.001), diarrhea (difference of -23.8, P < 0.001), and financial problems (difference of -35.5, P < 0.001). However, in the conservative group, no improved QOL variables with a difference of ≥ 20 were observed (Table 5). QOL after stoma closure was also compared to the normal population reference, with similar scores for functions and symptoms (Table 5).\nQuality of life in the colostomy group and conservative group of chronic radiation proctopathy patients based on the EORTC QLQ-C30 scale\nWilcoxon rank-sum test. \nPoint (follow-up) – point (pretreatment). Δ(FU): Change in follow-up; SD: Standard deviation.","From March 2008 to Feb 2019, 738 continual CRP patients treated in our center were screened. After exclusion of 644 patients with mild or moderate CRP, 94 with refractory bleeding and moderate to severe anemia were further screened. Among them, 50 patients were treated by diverted colostomy and 44 patients were treated with conservative therapies. Among the 50 patients treated by colostomy, 32 were excluded due to recto-vaginal fistulas or deep rectal ulcer with refractory perianal pain before stoma. The remaining 18 patients with refractory bleeding were enrolled. Among them, two died from cancer recurrence and two were lost to follow-up. The remaining 14 patients were enrolled in the final analysis as the colostomy group. For the conservative group, 19 patients were excluded, including 11 with tumor relapse and 8 who were lost to follow-up. Thus, 25 were enrolled in the final analysis (Figure 1). Demographic and baseline characteristics were collected, including the intention-to-treat (ITT) group of 50 colostomies. In the enrolled patients, 14 patients comprised the diverting colostomy group, and 25 cases comprised the conservative treatment group. The primary tumors in most of the patients (33/39, 85%) were cervical cancers. No significant differences in age, sex, type of primary tumor, or radiation dosage were found between the diverting colostomy group and the conservative treatment group or between the ITT colostomy group and the colostomy group (Table 1).\nDemographics and characteristics\nFisher's exact test. ITT: Intention-to-treat; Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin.\nFlow chart of patient selection.\nIn the ITT group, no postoperative follow-ups were conducted other than for the 14 investigated colostomy patients. Higher bleeding scores (P = 0.033) and relatively decreased preoperative Hb levels (P = 0.051, no significant difference) were found in the diverting colostomy group compared to the ITT group because 36 patients in the ITT group underwent colostomy for recto-vaginal fistulas or deep rectal ulcers instead of severe bleeding.","In the colostomy group, the median duration of bleeding was 13.0 mo ± 4.3 mo, and 12 (86%) patients received transfusion before stoma compared to 8.0 mo ± 1.8 mo (P = 0.042) of bleeding duration and 6 (24%) (P < 0.001) transfusions in the conservative group. In the conservative group, 8 patients received formalin irrigation and 4 patients received APC treatments. The preoperative (Pre-Op) Hb was only 63 g/L ± 17.8 g/L in the colostomy group compared to 88.2 g/L ± 19.3 g/L (P < 0.001) in the conservative group. One patient with moderate anemia (Hb of 87 g/L) received colostomy for perianal pain. Another patient with severe anemia (Hb of 78 g/L) did not receive a transfusion due to unavailability of a blood supply before colostomy. The details of the 14 patients are listed in Table 2. Increasing Hb levels were observed from 3 mo (median Hb of 75 g/L), 6 mo (Hb of 99 g/L), and 9 mo (Hb of 107 g/L) to 1 year (Hb 111 of g/L) and 2 years (Hb 117 g/L) after colostomy (Figure 2). Postoperative transfusions were conducted in 3 of 14 patients (transfusion requirements for 2 patients in other hospitals was unknown) in the colostomy group. The dynamic remission rates between the colostomy and conservative groups after surgery were as follows: 86% vs 4% at 3 mo (P < 0.001), 86% vs 12% at 6 mo (P < 0.001), 93% vs 20% at 9 mo (P < 0.001), 100% vs 24% at 1 year (P < 0.001), and 100% vs 20% at 2 years (P < 0.001) (Table 1).\nClinical details and outcomes of 14 colostomy patients\nMedian value. Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin; RTD: Rectal telangiectasia density; APC: Argon plasma coagulation.\nDynamic changes in hemoglobin levels. Pre-Op: Pre-operative.","Among the 14 patients with severe bleeding treated by colostomy, all obtained complete remission of bleeding during follow-up. Only 3 (23%) of 13 patients received one round of APC after stoma to control bleeding, and 1 patient received 4% formalin irrigation after colostomy (Table 2). Among the 14 patients, 13 (93%) underwent stoma closure, and the remaining patient did not undergo stoma reversal due to concerns of surgical risks because of old age (83 years old). The median duration of stoma was 16 mo (range: 9-53 mo). Stoma complications occurred in 3 (21%) cases, including 1 para-stoma hernia, 1 stoma prolapse, and 1 stoma obstruction (Grade II by Clavien-Dindo classification)[23]. All 3 patients recovered well after stoma closure.\nEndoscopic findings of bleeding, confluent telangiectasia, and congested mucosa improved dramatically at stoma reversal compared to before stoma creation (Figure 3). The bleeding score by CTCAE decreased from Pre-Op 2.7 points ± 0.5 points to 0.8 points ± 0.5 points (P < 0.001) at 1 year after stoma in the colostomy group; the bleeding score was 2.0 ± 0.5 at initial diagnosis and 1.7 ± 0.9 (P = 0.1282) at 1 year after treatments in the conservative group (Table 1 and Figure 4).\nEndoscopic changes before stoma and at reversal.\nCTC bleeding scores before stoma and at stoma reversal.","Endorectal ultrasound (EUS) was analyzed retrospectively before stoma and at reversal in 5 patients; the remaining patients did not receive EUS at either of these two time points. In our previous study, thickening of the rectal wall, blurred wall stratification, and increased vascularity were EUS features in CRP[24]. In this study, we found similar features before stoma creation and a tremendous decrease in superficial vascularity, which can explain the cessation of bleeding (Figure 5).\nEndoscopic ultrasound features before stoma and at stoma reversal.","To evaluate the severity of the effect on the rectal wall and pelvic floor in the colostomy group, we also report magnetic resonance imaging (MRI) parameters related to CRP, including the thickness of the rectal wall, width of the presacral space, and thicknesses of the levator ani, the gluteus maximus muscle, the obturator intemus, and the distal part of the sigmoid colon (Table 3), which was referred to in our previous study of radiation injury to the pelvis[25]. MRI scans both at pre-colostomy and at reversal were analyzed in 5 patients, as the remaining 9 did not receive MRI scans both pre-colostomy and at reversal. The thickness of the rectal wall was significantly decreased at reversal (8.91 ± 1.61) compared with pre-colostomy (10.7 ± 4.29) (P = 0.047) (Table 3).\nMagnetic resonance imaging findings at post-colostomy and at reversal compared in 5 patients with chronic radiation proctopathy","Anorectal functions before stoma and after reversal were evaluated by CRPSS scores in all 12 patients in the colostomy group. The results showed that rectal bleeding, peritoneal pain, tenesmus, urgency and fecal/gas incontinency were significantly improved after stoma reversal compared to those before stoma creation. Additionally, all of these scores were < 2 points, which indicated that anorectal function recovered very well after stoma reversal (Figure 6). Complete remission of bleeding and good performance of anorectal function compared to the normal population (scores ≤ 1 point) were observed. We also analyzed anorectal manometry to objectively assess anorectal function before reversal in 6 of the 14 patients who received it. Increased sensitivity of the rectal mucosa and decreased rectal volume were observed. Decreased sphincter functions were found in 2 of 6 cases (Table 4). These patients fulfilled the indications for reversal, and we found that these patients obtained good performance of anorectal function after reversal. Rectal defecography was also conducted before reversal, and no stricture was observed.\nRectal manometry assessment of anorectal functions before stoma reversals\nAnorectal functions before stoma and after stoma reversal.","QOL after stoma was assessed in the colostomy and conservative groups using EORTC QLQ-C30 questionnaires. As there are no similar reports in the Chinese population, we referred to the normal German population[26]. Osoba et al[26] suggested that a difference of ≥ 20 points in global health was considered to be clinically relevant. In the panel of patients who underwent stoma closure, QOL was dramatically improved compared to Pre-Op baseline QOL before stoma creation, including improved global health (difference of 40, P < 0.001), physical function (difference of 36.4, P < 0.001), role function (difference of 55, P < 0.001), emotional function (difference of 39.4, P < 0.001), social function (difference of 36.3, P < 0.001), and improved symptoms, such as fatigue (difference of -62.5, P < 0.001), pain (difference of -34.9, P < 0.001), dyspnea (difference of -37.8, P < 0.001), insomnia (difference of -36.2, P < 0.001), diarrhea (difference of -23.8, P < 0.001), and financial problems (difference of -35.5, P < 0.001). However, in the conservative group, no improved QOL variables with a difference of ≥ 20 were observed (Table 5). QOL after stoma closure was also compared to the normal population reference, with similar scores for functions and symptoms (Table 5).\nQuality of life in the colostomy group and conservative group of chronic radiation proctopathy patients based on the EORTC QLQ-C30 scale\nWilcoxon rank-sum test. \nPoint (follow-up) – point (pretreatment). Δ(FU): Change in follow-up; SD: Standard deviation.","The literature reports that 1%-6% of CRP patients have transfusion-dependent bleeding; many of these cases can be treated with topical formalin, APC, or hyperbaric oxygen treatment[1]. Surgical resection of rectal lesions is usually reserved as the last resort for hemorrhagic CRP because it is associated with high morbidity and mortality[3,13,19]. In our previous study, fecal diversion was found to be a simple, effective, and safe procedure for severe hemorrhagic CRP[16]. Theoretically, fecal diversion can reduce the irritation of stool and accelerate the course of fibrosis and thus relieve CRP bleeding rapidly[16].\nIn this study, we report results for diverting colostomy and conservative treatments of enemas, topical formalin, or APC in a large cohort of patients with severe hemorrhagic CRP and who were followed for at least 1 year. The results showed that stomas can be reversed in 93% of CRP cases with severe hemorrhage. Increased Hb and rapid cessation of bleeding were found in the colostomy group, whereas bleeding and Hb levels were not changed in the conservative group. Moreover, anorectal function was greatly improved after stoma reversal than before stoma creation, reaching the levels of the normal population. In addition, it is important to note that colostomy was performed in a very select population of CRP patients and those patients with CRP stricture and ulcerations were excluded. Stoma complications occurred in 21% of cases, including 1 case of parastomal hernia due to a weakened abdominal wall of the parastomal zone, 1 case of stoma prolapse due to an intestine overly pulled at stoma creation, and 1 case of stoma obstruction due to stricture. All of these complications were recovered after stoma reversal. According to the literature, the complication rate of stoma is approximately 20%-50%, and the complication rate of stoma in this study is thus acceptable. Thus, colostomy is a better option and can bring more benefits to CRP patients with severe bleeding than can conservative treatments.\nIn our previous study, we found complete remission of telangiectasia and edema, recovery of mucosal integrity, and massive fibrosis in the submucosa at 3-4 years after diversion[16]. In this study, anorectal manometry was conducted in some stoma patients and resulted in good compliance, resting pressure, and contraction. Rectal defecography was performed at reversal for all colostomy patients, and no rectal stricture was found. Thus, temporary diverting colostomy and reversal at an appropriate time can be a useful option for these patients.\nIn this study, most patients in the colostomy group presented with severe anemia and needed multiple transfusions. One patient did not receive transfusion, with an Hb level of 78, before colostomy. In China, blood supply is limited, and transfusion is only conducted in large central hospitals when Hb is < 60. At 3 to 6 mo after diverting colostomy, bleeding gradually remitted and increased Hb levels were observed. By 9 mo to 1 year after colostomy, most patients achieved complete remission of bleeding and the Hb level had almost returned to normal. Stoma reversal was conducted at 1 year after colostomy in 4 (31%) of 13 patients. In 5 cases, reversal was performed at more than 2 years after colostomy because the patients did not know colostomy could be reversed until we contacted them via telephone for follow-up. Thus, the median duration of stoma was prolonged to 16 mo. In fact, most stomas in this cohort could be reversed at 1 year after colostomy.\nBased on the results of our retrospective study[16], we started a prospective clinical trial to enroll severe hemorrhagic CRP patients who are suitable for diverting colostomy (Clinical Trial No. NCT03397901). All patients were followed and evaluated for stoma closure for 1 year after colostomy. The indication for diverting colostomy was extended to hemorrhagic CRP with moderate anemia in our series because preventative colostomy can relieve refractory bleeding before it progresses to severe life-threatening anemia and patients can obtain dramatic benefit from preventative fecal diversion.\nTo date, it is still unclear whether anorectal function can be preserved after stoma closure. In this study, we found that anorectal functions, including anal control and release of gas/stool, could be restored, which also indicated that CRP was inactive after a period of diverting colostomy. Although increased rectal sensitivity and decreased rectal volume were observed at reversal by anorectal manometry, good anorectal function was achieved after reversal compared to the normal population and improved compared to that before stoma creation. No additional pelvic biofeedback treatments were required in these patients.\nFor some patients, an additional one to two rounds of APC were required to help control bleeding during the first 6 mo after colostomy. The temporary stoma not only enabled the remission of anal symptoms but also restored the biological functions of the anus, providing evidence to support fecal diversion as a helpful option for severe hemorrhagic CRP patients.\nIn some studies, bile acid malabsorption and small bowel bacterial overgrowth have been thought to be the cause of diarrhea during acute and chronic radiation enteritis[1]. Diverting colostomy cannot reverse these changes. We focused on hemorrhage in CRP and diarrhea, common in small-bowel radiation injury, which is different from tenesmus and is not the major symptom in most CRP cases.\nAccording to our previous study, severe hemorrhagic CRP patients usually experience poor global health and fatigue due to moderate to severe anemia[16]. Their social and emotional functions are also impacted by frequent stool and anxiety of bleeding. They cannot attend social activities due to concern of looking for a lavatory[1]. In this study, after the remission of anal symptoms, we found that patients had good QOL, especially after stoma reversal, and were able to return to almost the same life as the normal population.\nAnorectal functions scored by the CRPSS were assessed before stoma and after reversal. We also analyzed EUS and pelvic MRI before stoma and at reversal to evaluate the severity of CRP. To objectively evaluate anorectal function before reversal, anorectal manometry was performed and retrospectively analyzed in some patients, though only in 6 patients because manometry is not a routine test before stoma reversal.\nThis study presents our serial results of diverting colostomy for severe hemorrhagic CRP. The results showed significant superiority over conservative treatments. However, there are several limitations to this study. First, this study was retrospective, with potential recall bias. Second, the sample size of this cohort was relatively small, which will affect the grade of evidence, and a larger cohort is essential. Finally, EUS, MRI, and anorectal manometry were only performed on patients undergoing colostomy reversal as a limitation. Thus, we started a prospective cohort of diverting colostomy for severe hemorrhagic CRP and will provide more reliable evidence in the future.\nIn conclusion, diverting colostomy is a very effective and rapid method for the remission of severe bleeding in CRP patients. The stoma can be reversed and good anorectal function can be restored after reversal in most patients."," Research background Chronic radiation proctopathy (CRP) is a common and sometimes difficult issue after radiotherapy for pelvic malignancies. Severe and refractory bleeding is hard to manage. Diverting fecal diversion is very effective and fast in remission of rectal bleeding in the management of severe CRP bleeding in our previous study, but diverting stoma is usually thought to be permanent according to the literature. The anorectal function and quality of life after stoma reversal remain unclear.\nChronic radiation proctopathy (CRP) is a common and sometimes difficult issue after radiotherapy for pelvic malignancies. Severe and refractory bleeding is hard to manage. Diverting fecal diversion is very effective and fast in remission of rectal bleeding in the management of severe CRP bleeding in our previous study, but diverting stoma is usually thought to be permanent according to the literature. The anorectal function and quality of life after stoma reversal remain unclear.\n Research motivation During the past 3 years, we have successfully performed colostomy reversals in a larger cohort of CRP patients with severe bleeding and followed patients after colostomy reversals. In this series study, we will report the efficacy of colostomy, the rate of stoma closure of diverting colostomy, and anorectal function after reversals.\nDuring the past 3 years, we have successfully performed colostomy reversals in a larger cohort of CRP patients with severe bleeding and followed patients after colostomy reversals. In this series study, we will report the efficacy of colostomy, the rate of stoma closure of diverting colostomy, and anorectal function after reversals.\n Research objectives The aim of this study was to evaluate the efficacy of colostomy and the rate of stoma reversals in severe hemorrhagic CRP.\nThe aim of this study was to evaluate the efficacy of colostomy and the rate of stoma reversals in severe hemorrhagic CRP.\n Research methods Patients with severe hemorrhagic CRP who underwent colostomy or conservative treatment were enrolled retrospectively. Rectal bleeding, hemoglobin (Hb), endoscopic features, endo-ultrasound (EUS), rectal manometry, and MRI scans were recorded. Anorectal functions and quality of life before stoma and after stoma reversal were scored with EORTC-QOL-C30 questionnaires.\nPatients with severe hemorrhagic CRP who underwent colostomy or conservative treatment were enrolled retrospectively. Rectal bleeding, hemoglobin (Hb), endoscopic features, endo-ultrasound (EUS), rectal manometry, and MRI scans were recorded. Anorectal functions and quality of life before stoma and after stoma reversal were scored with EORTC-QOL-C30 questionnaires.\n Research results After screening 738 continual CRP patients, 14 patients in the colostomy group and 25 patients in the conservative group as controls were enrolled. The Hb was gradually increased to normal levels in two years after colostomy, while no significant increase was observed in the conservative group. All of 14 patients obtained complete remission of bleeding and colostomy was successfully reversed in 13 of 14 (93%), except one with very old age. Improved endoscopic telangiectasia and bleeding, decreased vascularity by EUS, increased presarcal space, and thickened rectal wall by MRI were observed. Anorectal functions and quality of life were significantly improved after stoma reverse.\nAfter screening 738 continual CRP patients, 14 patients in the colostomy group and 25 patients in the conservative group as controls were enrolled. The Hb was gradually increased to normal levels in two years after colostomy, while no significant increase was observed in the conservative group. All of 14 patients obtained complete remission of bleeding and colostomy was successfully reversed in 13 of 14 (93%), except one with very old age. Improved endoscopic telangiectasia and bleeding, decreased vascularity by EUS, increased presarcal space, and thickened rectal wall by MRI were observed. Anorectal functions and quality of life were significantly improved after stoma reverse.\n Research conclusions Diverting colostomy is a very effective method in the remission of refractory hemorrhagic CRP. Meanwhile, stoma can be reversed and anorectal functions can be recovered after reversal.\nDiverting colostomy is a very effective method in the remission of refractory hemorrhagic CRP. Meanwhile, stoma can be reversed and anorectal functions can be recovered after reversal.\n Research perspectives Preventative diverting colostomy can relieve refractory bleeding before it progresses to severe life-threatening anemia and patients can obtain dramatic benefit. Thus, we have started a prospective clinical trial to enroll severe hemorrhagic CRP patients who are suitable for diverting colostomy (Clinical Trial No. NCT03397901), which will provide more evidence to the usage of colostomy in severe CRP patients.\nPreventative diverting colostomy can relieve refractory bleeding before it progresses to severe life-threatening anemia and patients can obtain dramatic benefit. Thus, we have started a prospective clinical trial to enroll severe hemorrhagic CRP patients who are suitable for diverting colostomy (Clinical Trial No. NCT03397901), which will provide more evidence to the usage of colostomy in severe CRP patients.","Chronic radiation proctopathy (CRP) is a common and sometimes difficult issue after radiotherapy for pelvic malignancies. Severe and refractory bleeding is hard to manage. Diverting fecal diversion is very effective and fast in remission of rectal bleeding in the management of severe CRP bleeding in our previous study, but diverting stoma is usually thought to be permanent according to the literature. The anorectal function and quality of life after stoma reversal remain unclear.","During the past 3 years, we have successfully performed colostomy reversals in a larger cohort of CRP patients with severe bleeding and followed patients after colostomy reversals. In this series study, we will report the efficacy of colostomy, the rate of stoma closure of diverting colostomy, and anorectal function after reversals.","The aim of this study was to evaluate the efficacy of colostomy and the rate of stoma reversals in severe hemorrhagic CRP.","Patients with severe hemorrhagic CRP who underwent colostomy or conservative treatment were enrolled retrospectively. Rectal bleeding, hemoglobin (Hb), endoscopic features, endo-ultrasound (EUS), rectal manometry, and MRI scans were recorded. Anorectal functions and quality of life before stoma and after stoma reversal were scored with EORTC-QOL-C30 questionnaires.","After screening 738 continual CRP patients, 14 patients in the colostomy group and 25 patients in the conservative group as controls were enrolled. The Hb was gradually increased to normal levels in two years after colostomy, while no significant increase was observed in the conservative group. All of 14 patients obtained complete remission of bleeding and colostomy was successfully reversed in 13 of 14 (93%), except one with very old age. Improved endoscopic telangiectasia and bleeding, decreased vascularity by EUS, increased presarcal space, and thickened rectal wall by MRI were observed. Anorectal functions and quality of life were significantly improved after stoma reverse.","Diverting colostomy is a very effective method in the remission of refractory hemorrhagic CRP. Meanwhile, stoma can be reversed and anorectal functions can be recovered after reversal."],"string":"[\n \"Chronic radiation proctopathy (CRP) is a common and sometimes difficult issue after radiotherapy for pelvic malignancies. A permanent change in bowel habits may occur in 90% of patients. After pelvic radiotherapy, 20%-50% of patients will develop difficult bowel function, affecting their quality of life (QOL)[1,2]. CRP can cause more than 20 different symptoms. Rectal bleeding is the most common symptom and occurs in > 50% of CRP patients[1,3]. Transfusion-dependent severe bleeding occurs in 1%-5% of patients[1]. Pathologically, ischemia in the submucosa due to obliterative endarteritis and progressive fibrosis in macroscopic changes are the main causes[4].\\nMedical treatment consists of topical sucralfate enemas[5], oral or topical sulfasalazine[6], and rebamipide[7]. However, these reagents are only effective in acute or mild CRP, whereas their efficacy is usually disappointing in CRP patients with moderate to severe hemorrhage. More invasive modalities include endoscopic argon plasma coagulation (APC)[8], topical 4%-10% formalin[9-12], radiofrequency ablation[13], and hyperbaric oxygen therapy[14,15], and are currently popular optional treatments for CRP. Most of these modalities lack randomized trial evidence. These treatment options are reported to be effective in controlling mild to moderate bleeding in most of the literature. Nonetheless, severe and refractory bleeding is difficult to manage[1,16]. Furthermore, APC or topical formalin requires multiple sessions for severe bleeding and can cause severe side effects, including perforation, strictures, and perianal pain[17].\\nFecal diversion is reported to be effective in the management of severe CRP bleeding[16,18,19], as it can reduce irritation injury to the lesions to decrease hemorrhage[16]. However, the usage of fecal diversion is not adopted as widely as is APC or formalin. Most of the fecal diversion data are from the 1980s, and this option has not been well studied to date. We previously reported one retrospective cohort of CRP patients with severe hemorrhage who received colostomy[16]. The results showed that colostomy can bring much higher remission of severe bleeding (94%) than can conservative treatment (11%) with APC or topical formalin. Pathologically, chronic inflammation and progressive fibrosis are observed after stoma. Diverting stoma is usually thought to be permanent according to Ayerdi et al[20].\\nAnorectal function and QOL after stoma reversal remain unclear. During the past 3 years, we have successfully performed colostomy reversals in a large cohort of CRP patients with severe bleeding and have follow-up data after colostomy reversal. Here, we report the efficacy of colostomy, the rate of stoma closure of diverting colostomy, and anorectal function after reversal for this series of CRP patients with severe bleeding.\",\n \"Patients with hemorrhagic CRP who were treated after admission at The Sixth Affiliated Hospital of Sun Yat-Sen University from March 2008 to December 2018 were enrolled. Medical records and imaging and clinicopathological data were extracted from our electronic database. This study was approved by the Ethical Committee of The Sixth Affiliated Hospital of Sun Yat-Sen University and was performed according to the provisions of the World Medical Association’s Declaration of Helsinki in 1995 (updated in Tokyo, 2004). Due to the nature of a retrospective study, informed consent was waived.\",\n \"CRP patients with refractory rectal bleeding who received diverting colostomy or conservative treatments for severe anemia and who needed transfusions were enrolled. Patients who were lost to follow-up, had tumor recurrence, rectal-vaginal fistula or other types of rectal fistulas, or underwent rectal resection with preventive colostomy were excluded because these conditions would affect the evaluation of remission of rectal bleeding.\",\n \"CRP was diagnosed by the combination of pelvic radiation history for malignancies, symptoms such as rectal bleeding, and endoscopic findings of CRP-specific changes such as telangiectasia and active bleeding in the rectum, as well as exclusion of other bleeding diseases. Hemorrhagic problems, such as tumor recurrence and hemorrhoids, were excluded. Refractory severe bleeding was defined as frequent bleeding and severe anemia with the need for transfusions. At admission, CRP patients in our center were first referred to noninvasive enemas, and if there was no response or recurrence, invasive treatment, such as APC (1-2 rounds) or formalin topical irrigation, was utilized. Conservative treatment failure was defined as no response or recurrence of frequent or severe rectal bleeding after invasive treatments.\\nIn this study, we used a modified subjective-objective management analysis (known as mSOMA) system that we designed previously to assess the severity of rectal bleeding[16]. The advantages of the mSOMA system include both subjective complaints of patients and objective hemoglobin (Hb) levels according to laboratory tests. Remission of bleeding was defined as complete cessation or occasional bleeding that needed no further treatment. The CTCAE score of bleeding was used to assess its severity.\",\n \"The indications for diverting diversion in hemorrhagic CRP contained the following conditions: Recurrent bleeding and unresponsive to conservative treatments such as endoscopic APC or topical formalin, accompanying severe anemia, and transfusions needed. Transverse loop colostomy and “gunsight” or “cross‘’ type of stoma closure were created according to the standard protocols used in our previous studies[16]. The criteria of stoma closure were as follows: Remission or occasional rectal bleeding, regressive edema in rectal mucosa, good performance of anorectal function, exclusion of tumor recurrence, and no severe CRP complications such as fistula, stricture, and deep ulcer[16].\",\n \"Patients were scheduled for follow-up through outpatient visits or telephone questionnaires at 6 mo, 9 mo, 1 year, 1.5 years, and 2 years after colostomy. QOL after stoma closure was evaluated according to EORTC QLQ C30 questionnaires[21]. We focused on the following items in this study: The remission rate of bleeding, the rate of stoma reversal, QOL after stoma reversal, dynamic changes of Hb levels, stoma-related complications, and severe CRP complications.\",\n \"In our previous study, tenesmus, stool frequency, and anorectal pain were common symptoms in active CRP[22]. Because there are no standard scales or scores to assess anorectal function precisely in CRP patients, we developed a new scale system that is a patient self-reported scale of outcomes, namely, the chronic radiation proctopathy symptom scale (CRPSS). The CRPSS considers the following items: Watery stool, urgency, perianal pain, tenesmus, rectal bleeding, and fecal/gas incontinence. The details are provided in Supplementary File 1. For CRP patients treated by colostomy, CRPSS scores were evaluated before stoma and at reversal.\",\n \"Analyses for continuous variables were performed by Student’s t-test. The χ2 test was used for categorical variables. Fisher’s exact test was conducted when appropriate. For non-parameter variables, the Wilcoxon rank sum tests were used. All statistical analyses were performed using SPSS version 23.0 (Chicago, IL, United States). P < 0.05 (two-tailed) was considered statistically significant.\",\n \" Demographics and clinical outcomes From March 2008 to Feb 2019, 738 continual CRP patients treated in our center were screened. After exclusion of 644 patients with mild or moderate CRP, 94 with refractory bleeding and moderate to severe anemia were further screened. Among them, 50 patients were treated by diverted colostomy and 44 patients were treated with conservative therapies. Among the 50 patients treated by colostomy, 32 were excluded due to recto-vaginal fistulas or deep rectal ulcer with refractory perianal pain before stoma. The remaining 18 patients with refractory bleeding were enrolled. Among them, two died from cancer recurrence and two were lost to follow-up. The remaining 14 patients were enrolled in the final analysis as the colostomy group. For the conservative group, 19 patients were excluded, including 11 with tumor relapse and 8 who were lost to follow-up. Thus, 25 were enrolled in the final analysis (Figure 1). Demographic and baseline characteristics were collected, including the intention-to-treat (ITT) group of 50 colostomies. In the enrolled patients, 14 patients comprised the diverting colostomy group, and 25 cases comprised the conservative treatment group. The primary tumors in most of the patients (33/39, 85%) were cervical cancers. No significant differences in age, sex, type of primary tumor, or radiation dosage were found between the diverting colostomy group and the conservative treatment group or between the ITT colostomy group and the colostomy group (Table 1).\\nDemographics and characteristics\\nFisher's exact test. ITT: Intention-to-treat; Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin.\\nFlow chart of patient selection.\\nIn the ITT group, no postoperative follow-ups were conducted other than for the 14 investigated colostomy patients. Higher bleeding scores (P = 0.033) and relatively decreased preoperative Hb levels (P = 0.051, no significant difference) were found in the diverting colostomy group compared to the ITT group because 36 patients in the ITT group underwent colostomy for recto-vaginal fistulas or deep rectal ulcers instead of severe bleeding.\\nFrom March 2008 to Feb 2019, 738 continual CRP patients treated in our center were screened. After exclusion of 644 patients with mild or moderate CRP, 94 with refractory bleeding and moderate to severe anemia were further screened. Among them, 50 patients were treated by diverted colostomy and 44 patients were treated with conservative therapies. Among the 50 patients treated by colostomy, 32 were excluded due to recto-vaginal fistulas or deep rectal ulcer with refractory perianal pain before stoma. The remaining 18 patients with refractory bleeding were enrolled. Among them, two died from cancer recurrence and two were lost to follow-up. The remaining 14 patients were enrolled in the final analysis as the colostomy group. For the conservative group, 19 patients were excluded, including 11 with tumor relapse and 8 who were lost to follow-up. Thus, 25 were enrolled in the final analysis (Figure 1). Demographic and baseline characteristics were collected, including the intention-to-treat (ITT) group of 50 colostomies. In the enrolled patients, 14 patients comprised the diverting colostomy group, and 25 cases comprised the conservative treatment group. The primary tumors in most of the patients (33/39, 85%) were cervical cancers. No significant differences in age, sex, type of primary tumor, or radiation dosage were found between the diverting colostomy group and the conservative treatment group or between the ITT colostomy group and the colostomy group (Table 1).\\nDemographics and characteristics\\nFisher's exact test. ITT: Intention-to-treat; Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin.\\nFlow chart of patient selection.\\nIn the ITT group, no postoperative follow-ups were conducted other than for the 14 investigated colostomy patients. Higher bleeding scores (P = 0.033) and relatively decreased preoperative Hb levels (P = 0.051, no significant difference) were found in the diverting colostomy group compared to the ITT group because 36 patients in the ITT group underwent colostomy for recto-vaginal fistulas or deep rectal ulcers instead of severe bleeding.\\n Dynamically increasing hemoglobin levels after colostomy In the colostomy group, the median duration of bleeding was 13.0 mo ± 4.3 mo, and 12 (86%) patients received transfusion before stoma compared to 8.0 mo ± 1.8 mo (P = 0.042) of bleeding duration and 6 (24%) (P < 0.001) transfusions in the conservative group. In the conservative group, 8 patients received formalin irrigation and 4 patients received APC treatments. The preoperative (Pre-Op) Hb was only 63 g/L ± 17.8 g/L in the colostomy group compared to 88.2 g/L ± 19.3 g/L (P < 0.001) in the conservative group. One patient with moderate anemia (Hb of 87 g/L) received colostomy for perianal pain. Another patient with severe anemia (Hb of 78 g/L) did not receive a transfusion due to unavailability of a blood supply before colostomy. The details of the 14 patients are listed in Table 2. Increasing Hb levels were observed from 3 mo (median Hb of 75 g/L), 6 mo (Hb of 99 g/L), and 9 mo (Hb of 107 g/L) to 1 year (Hb 111 of g/L) and 2 years (Hb 117 g/L) after colostomy (Figure 2). Postoperative transfusions were conducted in 3 of 14 patients (transfusion requirements for 2 patients in other hospitals was unknown) in the colostomy group. The dynamic remission rates between the colostomy and conservative groups after surgery were as follows: 86% vs 4% at 3 mo (P < 0.001), 86% vs 12% at 6 mo (P < 0.001), 93% vs 20% at 9 mo (P < 0.001), 100% vs 24% at 1 year (P < 0.001), and 100% vs 20% at 2 years (P < 0.001) (Table 1).\\nClinical details and outcomes of 14 colostomy patients\\nMedian value. Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin; RTD: Rectal telangiectasia density; APC: Argon plasma coagulation.\\nDynamic changes in hemoglobin levels. Pre-Op: Pre-operative.\\nIn the colostomy group, the median duration of bleeding was 13.0 mo ± 4.3 mo, and 12 (86%) patients received transfusion before stoma compared to 8.0 mo ± 1.8 mo (P = 0.042) of bleeding duration and 6 (24%) (P < 0.001) transfusions in the conservative group. In the conservative group, 8 patients received formalin irrigation and 4 patients received APC treatments. The preoperative (Pre-Op) Hb was only 63 g/L ± 17.8 g/L in the colostomy group compared to 88.2 g/L ± 19.3 g/L (P < 0.001) in the conservative group. One patient with moderate anemia (Hb of 87 g/L) received colostomy for perianal pain. Another patient with severe anemia (Hb of 78 g/L) did not receive a transfusion due to unavailability of a blood supply before colostomy. The details of the 14 patients are listed in Table 2. Increasing Hb levels were observed from 3 mo (median Hb of 75 g/L), 6 mo (Hb of 99 g/L), and 9 mo (Hb of 107 g/L) to 1 year (Hb 111 of g/L) and 2 years (Hb 117 g/L) after colostomy (Figure 2). Postoperative transfusions were conducted in 3 of 14 patients (transfusion requirements for 2 patients in other hospitals was unknown) in the colostomy group. The dynamic remission rates between the colostomy and conservative groups after surgery were as follows: 86% vs 4% at 3 mo (P < 0.001), 86% vs 12% at 6 mo (P < 0.001), 93% vs 20% at 9 mo (P < 0.001), 100% vs 24% at 1 year (P < 0.001), and 100% vs 20% at 2 years (P < 0.001) (Table 1).\\nClinical details and outcomes of 14 colostomy patients\\nMedian value. Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin; RTD: Rectal telangiectasia density; APC: Argon plasma coagulation.\\nDynamic changes in hemoglobin levels. Pre-Op: Pre-operative.\\n Stoma closure and endoscopic features Among the 14 patients with severe bleeding treated by colostomy, all obtained complete remission of bleeding during follow-up. Only 3 (23%) of 13 patients received one round of APC after stoma to control bleeding, and 1 patient received 4% formalin irrigation after colostomy (Table 2). Among the 14 patients, 13 (93%) underwent stoma closure, and the remaining patient did not undergo stoma reversal due to concerns of surgical risks because of old age (83 years old). The median duration of stoma was 16 mo (range: 9-53 mo). Stoma complications occurred in 3 (21%) cases, including 1 para-stoma hernia, 1 stoma prolapse, and 1 stoma obstruction (Grade II by Clavien-Dindo classification)[23]. All 3 patients recovered well after stoma closure.\\nEndoscopic findings of bleeding, confluent telangiectasia, and congested mucosa improved dramatically at stoma reversal compared to before stoma creation (Figure 3). The bleeding score by CTCAE decreased from Pre-Op 2.7 points ± 0.5 points to 0.8 points ± 0.5 points (P < 0.001) at 1 year after stoma in the colostomy group; the bleeding score was 2.0 ± 0.5 at initial diagnosis and 1.7 ± 0.9 (P = 0.1282) at 1 year after treatments in the conservative group (Table 1 and Figure 4).\\nEndoscopic changes before stoma and at reversal.\\nCTC bleeding scores before stoma and at stoma reversal.\\nAmong the 14 patients with severe bleeding treated by colostomy, all obtained complete remission of bleeding during follow-up. Only 3 (23%) of 13 patients received one round of APC after stoma to control bleeding, and 1 patient received 4% formalin irrigation after colostomy (Table 2). Among the 14 patients, 13 (93%) underwent stoma closure, and the remaining patient did not undergo stoma reversal due to concerns of surgical risks because of old age (83 years old). The median duration of stoma was 16 mo (range: 9-53 mo). Stoma complications occurred in 3 (21%) cases, including 1 para-stoma hernia, 1 stoma prolapse, and 1 stoma obstruction (Grade II by Clavien-Dindo classification)[23]. All 3 patients recovered well after stoma closure.\\nEndoscopic findings of bleeding, confluent telangiectasia, and congested mucosa improved dramatically at stoma reversal compared to before stoma creation (Figure 3). The bleeding score by CTCAE decreased from Pre-Op 2.7 points ± 0.5 points to 0.8 points ± 0.5 points (P < 0.001) at 1 year after stoma in the colostomy group; the bleeding score was 2.0 ± 0.5 at initial diagnosis and 1.7 ± 0.9 (P = 0.1282) at 1 year after treatments in the conservative group (Table 1 and Figure 4).\\nEndoscopic changes before stoma and at reversal.\\nCTC bleeding scores before stoma and at stoma reversal.\\n Ultrasound findings before stoma and at reversal Endorectal ultrasound (EUS) was analyzed retrospectively before stoma and at reversal in 5 patients; the remaining patients did not receive EUS at either of these two time points. In our previous study, thickening of the rectal wall, blurred wall stratification, and increased vascularity were EUS features in CRP[24]. In this study, we found similar features before stoma creation and a tremendous decrease in superficial vascularity, which can explain the cessation of bleeding (Figure 5).\\nEndoscopic ultrasound features before stoma and at stoma reversal.\\nEndorectal ultrasound (EUS) was analyzed retrospectively before stoma and at reversal in 5 patients; the remaining patients did not receive EUS at either of these two time points. In our previous study, thickening of the rectal wall, blurred wall stratification, and increased vascularity were EUS features in CRP[24]. In this study, we found similar features before stoma creation and a tremendous decrease in superficial vascularity, which can explain the cessation of bleeding (Figure 5).\\nEndoscopic ultrasound features before stoma and at stoma reversal.\\n Improved magnetic resonance imaging features at stoma reversal To evaluate the severity of the effect on the rectal wall and pelvic floor in the colostomy group, we also report magnetic resonance imaging (MRI) parameters related to CRP, including the thickness of the rectal wall, width of the presacral space, and thicknesses of the levator ani, the gluteus maximus muscle, the obturator intemus, and the distal part of the sigmoid colon (Table 3), which was referred to in our previous study of radiation injury to the pelvis[25]. MRI scans both at pre-colostomy and at reversal were analyzed in 5 patients, as the remaining 9 did not receive MRI scans both pre-colostomy and at reversal. The thickness of the rectal wall was significantly decreased at reversal (8.91 ± 1.61) compared with pre-colostomy (10.7 ± 4.29) (P = 0.047) (Table 3).\\nMagnetic resonance imaging findings at post-colostomy and at reversal compared in 5 patients with chronic radiation proctopathy\\nTo evaluate the severity of the effect on the rectal wall and pelvic floor in the colostomy group, we also report magnetic resonance imaging (MRI) parameters related to CRP, including the thickness of the rectal wall, width of the presacral space, and thicknesses of the levator ani, the gluteus maximus muscle, the obturator intemus, and the distal part of the sigmoid colon (Table 3), which was referred to in our previous study of radiation injury to the pelvis[25]. MRI scans both at pre-colostomy and at reversal were analyzed in 5 patients, as the remaining 9 did not receive MRI scans both pre-colostomy and at reversal. The thickness of the rectal wall was significantly decreased at reversal (8.91 ± 1.61) compared with pre-colostomy (10.7 ± 4.29) (P = 0.047) (Table 3).\\nMagnetic resonance imaging findings at post-colostomy and at reversal compared in 5 patients with chronic radiation proctopathy\\n Good restored anorectal functions after stoma reversal Anorectal functions before stoma and after reversal were evaluated by CRPSS scores in all 12 patients in the colostomy group. The results showed that rectal bleeding, peritoneal pain, tenesmus, urgency and fecal/gas incontinency were significantly improved after stoma reversal compared to those before stoma creation. Additionally, all of these scores were < 2 points, which indicated that anorectal function recovered very well after stoma reversal (Figure 6). Complete remission of bleeding and good performance of anorectal function compared to the normal population (scores ≤ 1 point) were observed. We also analyzed anorectal manometry to objectively assess anorectal function before reversal in 6 of the 14 patients who received it. Increased sensitivity of the rectal mucosa and decreased rectal volume were observed. Decreased sphincter functions were found in 2 of 6 cases (Table 4). These patients fulfilled the indications for reversal, and we found that these patients obtained good performance of anorectal function after reversal. Rectal defecography was also conducted before reversal, and no stricture was observed.\\nRectal manometry assessment of anorectal functions before stoma reversals\\nAnorectal functions before stoma and after stoma reversal.\\nAnorectal functions before stoma and after reversal were evaluated by CRPSS scores in all 12 patients in the colostomy group. The results showed that rectal bleeding, peritoneal pain, tenesmus, urgency and fecal/gas incontinency were significantly improved after stoma reversal compared to those before stoma creation. Additionally, all of these scores were < 2 points, which indicated that anorectal function recovered very well after stoma reversal (Figure 6). Complete remission of bleeding and good performance of anorectal function compared to the normal population (scores ≤ 1 point) were observed. We also analyzed anorectal manometry to objectively assess anorectal function before reversal in 6 of the 14 patients who received it. Increased sensitivity of the rectal mucosa and decreased rectal volume were observed. Decreased sphincter functions were found in 2 of 6 cases (Table 4). These patients fulfilled the indications for reversal, and we found that these patients obtained good performance of anorectal function after reversal. Rectal defecography was also conducted before reversal, and no stricture was observed.\\nRectal manometry assessment of anorectal functions before stoma reversals\\nAnorectal functions before stoma and after stoma reversal.\\n Improved quality of life at colostomy reversal QOL after stoma was assessed in the colostomy and conservative groups using EORTC QLQ-C30 questionnaires. As there are no similar reports in the Chinese population, we referred to the normal German population[26]. Osoba et al[26] suggested that a difference of ≥ 20 points in global health was considered to be clinically relevant. In the panel of patients who underwent stoma closure, QOL was dramatically improved compared to Pre-Op baseline QOL before stoma creation, including improved global health (difference of 40, P < 0.001), physical function (difference of 36.4, P < 0.001), role function (difference of 55, P < 0.001), emotional function (difference of 39.4, P < 0.001), social function (difference of 36.3, P < 0.001), and improved symptoms, such as fatigue (difference of -62.5, P < 0.001), pain (difference of -34.9, P < 0.001), dyspnea (difference of -37.8, P < 0.001), insomnia (difference of -36.2, P < 0.001), diarrhea (difference of -23.8, P < 0.001), and financial problems (difference of -35.5, P < 0.001). However, in the conservative group, no improved QOL variables with a difference of ≥ 20 were observed (Table 5). QOL after stoma closure was also compared to the normal population reference, with similar scores for functions and symptoms (Table 5).\\nQuality of life in the colostomy group and conservative group of chronic radiation proctopathy patients based on the EORTC QLQ-C30 scale\\nWilcoxon rank-sum test. \\nPoint (follow-up) – point (pretreatment). Δ(FU): Change in follow-up; SD: Standard deviation.\\nQOL after stoma was assessed in the colostomy and conservative groups using EORTC QLQ-C30 questionnaires. As there are no similar reports in the Chinese population, we referred to the normal German population[26]. Osoba et al[26] suggested that a difference of ≥ 20 points in global health was considered to be clinically relevant. In the panel of patients who underwent stoma closure, QOL was dramatically improved compared to Pre-Op baseline QOL before stoma creation, including improved global health (difference of 40, P < 0.001), physical function (difference of 36.4, P < 0.001), role function (difference of 55, P < 0.001), emotional function (difference of 39.4, P < 0.001), social function (difference of 36.3, P < 0.001), and improved symptoms, such as fatigue (difference of -62.5, P < 0.001), pain (difference of -34.9, P < 0.001), dyspnea (difference of -37.8, P < 0.001), insomnia (difference of -36.2, P < 0.001), diarrhea (difference of -23.8, P < 0.001), and financial problems (difference of -35.5, P < 0.001). However, in the conservative group, no improved QOL variables with a difference of ≥ 20 were observed (Table 5). QOL after stoma closure was also compared to the normal population reference, with similar scores for functions and symptoms (Table 5).\\nQuality of life in the colostomy group and conservative group of chronic radiation proctopathy patients based on the EORTC QLQ-C30 scale\\nWilcoxon rank-sum test. \\nPoint (follow-up) – point (pretreatment). Δ(FU): Change in follow-up; SD: Standard deviation.\",\n \"From March 2008 to Feb 2019, 738 continual CRP patients treated in our center were screened. After exclusion of 644 patients with mild or moderate CRP, 94 with refractory bleeding and moderate to severe anemia were further screened. Among them, 50 patients were treated by diverted colostomy and 44 patients were treated with conservative therapies. Among the 50 patients treated by colostomy, 32 were excluded due to recto-vaginal fistulas or deep rectal ulcer with refractory perianal pain before stoma. The remaining 18 patients with refractory bleeding were enrolled. Among them, two died from cancer recurrence and two were lost to follow-up. The remaining 14 patients were enrolled in the final analysis as the colostomy group. For the conservative group, 19 patients were excluded, including 11 with tumor relapse and 8 who were lost to follow-up. Thus, 25 were enrolled in the final analysis (Figure 1). Demographic and baseline characteristics were collected, including the intention-to-treat (ITT) group of 50 colostomies. In the enrolled patients, 14 patients comprised the diverting colostomy group, and 25 cases comprised the conservative treatment group. The primary tumors in most of the patients (33/39, 85%) were cervical cancers. No significant differences in age, sex, type of primary tumor, or radiation dosage were found between the diverting colostomy group and the conservative treatment group or between the ITT colostomy group and the colostomy group (Table 1).\\nDemographics and characteristics\\nFisher's exact test. ITT: Intention-to-treat; Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin.\\nFlow chart of patient selection.\\nIn the ITT group, no postoperative follow-ups were conducted other than for the 14 investigated colostomy patients. Higher bleeding scores (P = 0.033) and relatively decreased preoperative Hb levels (P = 0.051, no significant difference) were found in the diverting colostomy group compared to the ITT group because 36 patients in the ITT group underwent colostomy for recto-vaginal fistulas or deep rectal ulcers instead of severe bleeding.\",\n \"In the colostomy group, the median duration of bleeding was 13.0 mo ± 4.3 mo, and 12 (86%) patients received transfusion before stoma compared to 8.0 mo ± 1.8 mo (P = 0.042) of bleeding duration and 6 (24%) (P < 0.001) transfusions in the conservative group. In the conservative group, 8 patients received formalin irrigation and 4 patients received APC treatments. The preoperative (Pre-Op) Hb was only 63 g/L ± 17.8 g/L in the colostomy group compared to 88.2 g/L ± 19.3 g/L (P < 0.001) in the conservative group. One patient with moderate anemia (Hb of 87 g/L) received colostomy for perianal pain. Another patient with severe anemia (Hb of 78 g/L) did not receive a transfusion due to unavailability of a blood supply before colostomy. The details of the 14 patients are listed in Table 2. Increasing Hb levels were observed from 3 mo (median Hb of 75 g/L), 6 mo (Hb of 99 g/L), and 9 mo (Hb of 107 g/L) to 1 year (Hb 111 of g/L) and 2 years (Hb 117 g/L) after colostomy (Figure 2). Postoperative transfusions were conducted in 3 of 14 patients (transfusion requirements for 2 patients in other hospitals was unknown) in the colostomy group. The dynamic remission rates between the colostomy and conservative groups after surgery were as follows: 86% vs 4% at 3 mo (P < 0.001), 86% vs 12% at 6 mo (P < 0.001), 93% vs 20% at 9 mo (P < 0.001), 100% vs 24% at 1 year (P < 0.001), and 100% vs 20% at 2 years (P < 0.001) (Table 1).\\nClinical details and outcomes of 14 colostomy patients\\nMedian value. Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin; RTD: Rectal telangiectasia density; APC: Argon plasma coagulation.\\nDynamic changes in hemoglobin levels. Pre-Op: Pre-operative.\",\n \"Among the 14 patients with severe bleeding treated by colostomy, all obtained complete remission of bleeding during follow-up. Only 3 (23%) of 13 patients received one round of APC after stoma to control bleeding, and 1 patient received 4% formalin irrigation after colostomy (Table 2). Among the 14 patients, 13 (93%) underwent stoma closure, and the remaining patient did not undergo stoma reversal due to concerns of surgical risks because of old age (83 years old). The median duration of stoma was 16 mo (range: 9-53 mo). Stoma complications occurred in 3 (21%) cases, including 1 para-stoma hernia, 1 stoma prolapse, and 1 stoma obstruction (Grade II by Clavien-Dindo classification)[23]. All 3 patients recovered well after stoma closure.\\nEndoscopic findings of bleeding, confluent telangiectasia, and congested mucosa improved dramatically at stoma reversal compared to before stoma creation (Figure 3). The bleeding score by CTCAE decreased from Pre-Op 2.7 points ± 0.5 points to 0.8 points ± 0.5 points (P < 0.001) at 1 year after stoma in the colostomy group; the bleeding score was 2.0 ± 0.5 at initial diagnosis and 1.7 ± 0.9 (P = 0.1282) at 1 year after treatments in the conservative group (Table 1 and Figure 4).\\nEndoscopic changes before stoma and at reversal.\\nCTC bleeding scores before stoma and at stoma reversal.\",\n \"Endorectal ultrasound (EUS) was analyzed retrospectively before stoma and at reversal in 5 patients; the remaining patients did not receive EUS at either of these two time points. In our previous study, thickening of the rectal wall, blurred wall stratification, and increased vascularity were EUS features in CRP[24]. In this study, we found similar features before stoma creation and a tremendous decrease in superficial vascularity, which can explain the cessation of bleeding (Figure 5).\\nEndoscopic ultrasound features before stoma and at stoma reversal.\",\n \"To evaluate the severity of the effect on the rectal wall and pelvic floor in the colostomy group, we also report magnetic resonance imaging (MRI) parameters related to CRP, including the thickness of the rectal wall, width of the presacral space, and thicknesses of the levator ani, the gluteus maximus muscle, the obturator intemus, and the distal part of the sigmoid colon (Table 3), which was referred to in our previous study of radiation injury to the pelvis[25]. MRI scans both at pre-colostomy and at reversal were analyzed in 5 patients, as the remaining 9 did not receive MRI scans both pre-colostomy and at reversal. The thickness of the rectal wall was significantly decreased at reversal (8.91 ± 1.61) compared with pre-colostomy (10.7 ± 4.29) (P = 0.047) (Table 3).\\nMagnetic resonance imaging findings at post-colostomy and at reversal compared in 5 patients with chronic radiation proctopathy\",\n \"Anorectal functions before stoma and after reversal were evaluated by CRPSS scores in all 12 patients in the colostomy group. The results showed that rectal bleeding, peritoneal pain, tenesmus, urgency and fecal/gas incontinency were significantly improved after stoma reversal compared to those before stoma creation. Additionally, all of these scores were < 2 points, which indicated that anorectal function recovered very well after stoma reversal (Figure 6). Complete remission of bleeding and good performance of anorectal function compared to the normal population (scores ≤ 1 point) were observed. We also analyzed anorectal manometry to objectively assess anorectal function before reversal in 6 of the 14 patients who received it. Increased sensitivity of the rectal mucosa and decreased rectal volume were observed. Decreased sphincter functions were found in 2 of 6 cases (Table 4). These patients fulfilled the indications for reversal, and we found that these patients obtained good performance of anorectal function after reversal. Rectal defecography was also conducted before reversal, and no stricture was observed.\\nRectal manometry assessment of anorectal functions before stoma reversals\\nAnorectal functions before stoma and after stoma reversal.\",\n \"QOL after stoma was assessed in the colostomy and conservative groups using EORTC QLQ-C30 questionnaires. As there are no similar reports in the Chinese population, we referred to the normal German population[26]. Osoba et al[26] suggested that a difference of ≥ 20 points in global health was considered to be clinically relevant. In the panel of patients who underwent stoma closure, QOL was dramatically improved compared to Pre-Op baseline QOL before stoma creation, including improved global health (difference of 40, P < 0.001), physical function (difference of 36.4, P < 0.001), role function (difference of 55, P < 0.001), emotional function (difference of 39.4, P < 0.001), social function (difference of 36.3, P < 0.001), and improved symptoms, such as fatigue (difference of -62.5, P < 0.001), pain (difference of -34.9, P < 0.001), dyspnea (difference of -37.8, P < 0.001), insomnia (difference of -36.2, P < 0.001), diarrhea (difference of -23.8, P < 0.001), and financial problems (difference of -35.5, P < 0.001). However, in the conservative group, no improved QOL variables with a difference of ≥ 20 were observed (Table 5). QOL after stoma closure was also compared to the normal population reference, with similar scores for functions and symptoms (Table 5).\\nQuality of life in the colostomy group and conservative group of chronic radiation proctopathy patients based on the EORTC QLQ-C30 scale\\nWilcoxon rank-sum test. \\nPoint (follow-up) – point (pretreatment). Δ(FU): Change in follow-up; SD: Standard deviation.\",\n \"The literature reports that 1%-6% of CRP patients have transfusion-dependent bleeding; many of these cases can be treated with topical formalin, APC, or hyperbaric oxygen treatment[1]. Surgical resection of rectal lesions is usually reserved as the last resort for hemorrhagic CRP because it is associated with high morbidity and mortality[3,13,19]. In our previous study, fecal diversion was found to be a simple, effective, and safe procedure for severe hemorrhagic CRP[16]. Theoretically, fecal diversion can reduce the irritation of stool and accelerate the course of fibrosis and thus relieve CRP bleeding rapidly[16].\\nIn this study, we report results for diverting colostomy and conservative treatments of enemas, topical formalin, or APC in a large cohort of patients with severe hemorrhagic CRP and who were followed for at least 1 year. The results showed that stomas can be reversed in 93% of CRP cases with severe hemorrhage. Increased Hb and rapid cessation of bleeding were found in the colostomy group, whereas bleeding and Hb levels were not changed in the conservative group. Moreover, anorectal function was greatly improved after stoma reversal than before stoma creation, reaching the levels of the normal population. In addition, it is important to note that colostomy was performed in a very select population of CRP patients and those patients with CRP stricture and ulcerations were excluded. Stoma complications occurred in 21% of cases, including 1 case of parastomal hernia due to a weakened abdominal wall of the parastomal zone, 1 case of stoma prolapse due to an intestine overly pulled at stoma creation, and 1 case of stoma obstruction due to stricture. All of these complications were recovered after stoma reversal. According to the literature, the complication rate of stoma is approximately 20%-50%, and the complication rate of stoma in this study is thus acceptable. Thus, colostomy is a better option and can bring more benefits to CRP patients with severe bleeding than can conservative treatments.\\nIn our previous study, we found complete remission of telangiectasia and edema, recovery of mucosal integrity, and massive fibrosis in the submucosa at 3-4 years after diversion[16]. In this study, anorectal manometry was conducted in some stoma patients and resulted in good compliance, resting pressure, and contraction. Rectal defecography was performed at reversal for all colostomy patients, and no rectal stricture was found. Thus, temporary diverting colostomy and reversal at an appropriate time can be a useful option for these patients.\\nIn this study, most patients in the colostomy group presented with severe anemia and needed multiple transfusions. One patient did not receive transfusion, with an Hb level of 78, before colostomy. In China, blood supply is limited, and transfusion is only conducted in large central hospitals when Hb is < 60. At 3 to 6 mo after diverting colostomy, bleeding gradually remitted and increased Hb levels were observed. By 9 mo to 1 year after colostomy, most patients achieved complete remission of bleeding and the Hb level had almost returned to normal. Stoma reversal was conducted at 1 year after colostomy in 4 (31%) of 13 patients. In 5 cases, reversal was performed at more than 2 years after colostomy because the patients did not know colostomy could be reversed until we contacted them via telephone for follow-up. Thus, the median duration of stoma was prolonged to 16 mo. In fact, most stomas in this cohort could be reversed at 1 year after colostomy.\\nBased on the results of our retrospective study[16], we started a prospective clinical trial to enroll severe hemorrhagic CRP patients who are suitable for diverting colostomy (Clinical Trial No. NCT03397901). All patients were followed and evaluated for stoma closure for 1 year after colostomy. The indication for diverting colostomy was extended to hemorrhagic CRP with moderate anemia in our series because preventative colostomy can relieve refractory bleeding before it progresses to severe life-threatening anemia and patients can obtain dramatic benefit from preventative fecal diversion.\\nTo date, it is still unclear whether anorectal function can be preserved after stoma closure. In this study, we found that anorectal functions, including anal control and release of gas/stool, could be restored, which also indicated that CRP was inactive after a period of diverting colostomy. Although increased rectal sensitivity and decreased rectal volume were observed at reversal by anorectal manometry, good anorectal function was achieved after reversal compared to the normal population and improved compared to that before stoma creation. No additional pelvic biofeedback treatments were required in these patients.\\nFor some patients, an additional one to two rounds of APC were required to help control bleeding during the first 6 mo after colostomy. The temporary stoma not only enabled the remission of anal symptoms but also restored the biological functions of the anus, providing evidence to support fecal diversion as a helpful option for severe hemorrhagic CRP patients.\\nIn some studies, bile acid malabsorption and small bowel bacterial overgrowth have been thought to be the cause of diarrhea during acute and chronic radiation enteritis[1]. Diverting colostomy cannot reverse these changes. We focused on hemorrhage in CRP and diarrhea, common in small-bowel radiation injury, which is different from tenesmus and is not the major symptom in most CRP cases.\\nAccording to our previous study, severe hemorrhagic CRP patients usually experience poor global health and fatigue due to moderate to severe anemia[16]. Their social and emotional functions are also impacted by frequent stool and anxiety of bleeding. They cannot attend social activities due to concern of looking for a lavatory[1]. In this study, after the remission of anal symptoms, we found that patients had good QOL, especially after stoma reversal, and were able to return to almost the same life as the normal population.\\nAnorectal functions scored by the CRPSS were assessed before stoma and after reversal. We also analyzed EUS and pelvic MRI before stoma and at reversal to evaluate the severity of CRP. To objectively evaluate anorectal function before reversal, anorectal manometry was performed and retrospectively analyzed in some patients, though only in 6 patients because manometry is not a routine test before stoma reversal.\\nThis study presents our serial results of diverting colostomy for severe hemorrhagic CRP. The results showed significant superiority over conservative treatments. However, there are several limitations to this study. First, this study was retrospective, with potential recall bias. Second, the sample size of this cohort was relatively small, which will affect the grade of evidence, and a larger cohort is essential. Finally, EUS, MRI, and anorectal manometry were only performed on patients undergoing colostomy reversal as a limitation. Thus, we started a prospective cohort of diverting colostomy for severe hemorrhagic CRP and will provide more reliable evidence in the future.\\nIn conclusion, diverting colostomy is a very effective and rapid method for the remission of severe bleeding in CRP patients. The stoma can be reversed and good anorectal function can be restored after reversal in most patients.\",\n \" Research background Chronic radiation proctopathy (CRP) is a common and sometimes difficult issue after radiotherapy for pelvic malignancies. Severe and refractory bleeding is hard to manage. Diverting fecal diversion is very effective and fast in remission of rectal bleeding in the management of severe CRP bleeding in our previous study, but diverting stoma is usually thought to be permanent according to the literature. The anorectal function and quality of life after stoma reversal remain unclear.\\nChronic radiation proctopathy (CRP) is a common and sometimes difficult issue after radiotherapy for pelvic malignancies. Severe and refractory bleeding is hard to manage. Diverting fecal diversion is very effective and fast in remission of rectal bleeding in the management of severe CRP bleeding in our previous study, but diverting stoma is usually thought to be permanent according to the literature. The anorectal function and quality of life after stoma reversal remain unclear.\\n Research motivation During the past 3 years, we have successfully performed colostomy reversals in a larger cohort of CRP patients with severe bleeding and followed patients after colostomy reversals. In this series study, we will report the efficacy of colostomy, the rate of stoma closure of diverting colostomy, and anorectal function after reversals.\\nDuring the past 3 years, we have successfully performed colostomy reversals in a larger cohort of CRP patients with severe bleeding and followed patients after colostomy reversals. In this series study, we will report the efficacy of colostomy, the rate of stoma closure of diverting colostomy, and anorectal function after reversals.\\n Research objectives The aim of this study was to evaluate the efficacy of colostomy and the rate of stoma reversals in severe hemorrhagic CRP.\\nThe aim of this study was to evaluate the efficacy of colostomy and the rate of stoma reversals in severe hemorrhagic CRP.\\n Research methods Patients with severe hemorrhagic CRP who underwent colostomy or conservative treatment were enrolled retrospectively. Rectal bleeding, hemoglobin (Hb), endoscopic features, endo-ultrasound (EUS), rectal manometry, and MRI scans were recorded. Anorectal functions and quality of life before stoma and after stoma reversal were scored with EORTC-QOL-C30 questionnaires.\\nPatients with severe hemorrhagic CRP who underwent colostomy or conservative treatment were enrolled retrospectively. Rectal bleeding, hemoglobin (Hb), endoscopic features, endo-ultrasound (EUS), rectal manometry, and MRI scans were recorded. Anorectal functions and quality of life before stoma and after stoma reversal were scored with EORTC-QOL-C30 questionnaires.\\n Research results After screening 738 continual CRP patients, 14 patients in the colostomy group and 25 patients in the conservative group as controls were enrolled. The Hb was gradually increased to normal levels in two years after colostomy, while no significant increase was observed in the conservative group. All of 14 patients obtained complete remission of bleeding and colostomy was successfully reversed in 13 of 14 (93%), except one with very old age. Improved endoscopic telangiectasia and bleeding, decreased vascularity by EUS, increased presarcal space, and thickened rectal wall by MRI were observed. Anorectal functions and quality of life were significantly improved after stoma reverse.\\nAfter screening 738 continual CRP patients, 14 patients in the colostomy group and 25 patients in the conservative group as controls were enrolled. The Hb was gradually increased to normal levels in two years after colostomy, while no significant increase was observed in the conservative group. All of 14 patients obtained complete remission of bleeding and colostomy was successfully reversed in 13 of 14 (93%), except one with very old age. Improved endoscopic telangiectasia and bleeding, decreased vascularity by EUS, increased presarcal space, and thickened rectal wall by MRI were observed. Anorectal functions and quality of life were significantly improved after stoma reverse.\\n Research conclusions Diverting colostomy is a very effective method in the remission of refractory hemorrhagic CRP. Meanwhile, stoma can be reversed and anorectal functions can be recovered after reversal.\\nDiverting colostomy is a very effective method in the remission of refractory hemorrhagic CRP. Meanwhile, stoma can be reversed and anorectal functions can be recovered after reversal.\\n Research perspectives Preventative diverting colostomy can relieve refractory bleeding before it progresses to severe life-threatening anemia and patients can obtain dramatic benefit. Thus, we have started a prospective clinical trial to enroll severe hemorrhagic CRP patients who are suitable for diverting colostomy (Clinical Trial No. NCT03397901), which will provide more evidence to the usage of colostomy in severe CRP patients.\\nPreventative diverting colostomy can relieve refractory bleeding before it progresses to severe life-threatening anemia and patients can obtain dramatic benefit. Thus, we have started a prospective clinical trial to enroll severe hemorrhagic CRP patients who are suitable for diverting colostomy (Clinical Trial No. NCT03397901), which will provide more evidence to the usage of colostomy in severe CRP patients.\",\n \"Chronic radiation proctopathy (CRP) is a common and sometimes difficult issue after radiotherapy for pelvic malignancies. Severe and refractory bleeding is hard to manage. Diverting fecal diversion is very effective and fast in remission of rectal bleeding in the management of severe CRP bleeding in our previous study, but diverting stoma is usually thought to be permanent according to the literature. The anorectal function and quality of life after stoma reversal remain unclear.\",\n \"During the past 3 years, we have successfully performed colostomy reversals in a larger cohort of CRP patients with severe bleeding and followed patients after colostomy reversals. In this series study, we will report the efficacy of colostomy, the rate of stoma closure of diverting colostomy, and anorectal function after reversals.\",\n \"The aim of this study was to evaluate the efficacy of colostomy and the rate of stoma reversals in severe hemorrhagic CRP.\",\n \"Patients with severe hemorrhagic CRP who underwent colostomy or conservative treatment were enrolled retrospectively. Rectal bleeding, hemoglobin (Hb), endoscopic features, endo-ultrasound (EUS), rectal manometry, and MRI scans were recorded. Anorectal functions and quality of life before stoma and after stoma reversal were scored with EORTC-QOL-C30 questionnaires.\",\n \"After screening 738 continual CRP patients, 14 patients in the colostomy group and 25 patients in the conservative group as controls were enrolled. The Hb was gradually increased to normal levels in two years after colostomy, while no significant increase was observed in the conservative group. All of 14 patients obtained complete remission of bleeding and colostomy was successfully reversed in 13 of 14 (93%), except one with very old age. Improved endoscopic telangiectasia and bleeding, decreased vascularity by EUS, increased presarcal space, and thickened rectal wall by MRI were observed. Anorectal functions and quality of life were significantly improved after stoma reverse.\",\n \"Diverting colostomy is a very effective method in the remission of refractory hemorrhagic CRP. Meanwhile, stoma can be reversed and anorectal functions can be recovered after reversal.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","MATERIALS AND METHODS","Patients and ethical statements","Inclusion and exclusion criteria","Diagnosis, definitions, and scores","Indications for fecal diversion and stoma closure","Follow-up","Anorectal functions","Statistical analysis","RESULTS","Demographics and clinical outcomes","Dynamically increasing hemoglobin levels after colostomy","Stoma closure and endoscopic features","Ultrasound findings before stoma and at reversal","Improved magnetic resonance imaging features at stoma reversal","Good restored anorectal functions after stoma reversal","Improved quality of life at colostomy reversal","DISCUSSION","ARTICLE HIGHLIGHTS","Research background","Research motivation","Research objectives","Research methods","Research results","Research conclusions"],"string":"[\n \"INTRODUCTION\",\n \"MATERIALS AND METHODS\",\n \"Patients and ethical statements\",\n \"Inclusion and exclusion criteria\",\n \"Diagnosis, definitions, and scores\",\n \"Indications for fecal diversion and stoma closure\",\n \"Follow-up\",\n \"Anorectal functions\",\n \"Statistical analysis\",\n \"RESULTS\",\n \"Demographics and clinical outcomes\",\n \"Dynamically increasing hemoglobin levels after colostomy\",\n \"Stoma closure and endoscopic features\",\n \"Ultrasound findings before stoma and at reversal\",\n \"Improved magnetic resonance imaging features at stoma reversal\",\n \"Good restored anorectal functions after stoma reversal\",\n \"Improved quality of life at colostomy reversal\",\n \"DISCUSSION\",\n \"ARTICLE HIGHLIGHTS\",\n \"Research background\",\n \"Research motivation\",\n \"Research objectives\",\n \"Research methods\",\n \"Research results\",\n \"Research conclusions\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Chronic radiation proctopathy (CRP) is a common and sometimes difficult issue after radiotherapy for pelvic malignancies. A permanent change in bowel habits may occur in 90% of patients. After pelvic radiotherapy, 20%-50% of patients will develop difficult bowel function, affecting their quality of life (QOL)[1,2]. CRP can cause more than 20 different symptoms. Rectal bleeding is the most common symptom and occurs in > 50% of CRP patients[1,3]. Transfusion-dependent severe bleeding occurs in 1%-5% of patients[1]. Pathologically, ischemia in the submucosa due to obliterative endarteritis and progressive fibrosis in macroscopic changes are the main causes[4].\nMedical treatment consists of topical sucralfate enemas[5], oral or topical sulfasalazine[6], and rebamipide[7]. However, these reagents are only effective in acute or mild CRP, whereas their efficacy is usually disappointing in CRP patients with moderate to severe hemorrhage. More invasive modalities include endoscopic argon plasma coagulation (APC)[8], topical 4%-10% formalin[9-12], radiofrequency ablation[13], and hyperbaric oxygen therapy[14,15], and are currently popular optional treatments for CRP. Most of these modalities lack randomized trial evidence. These treatment options are reported to be effective in controlling mild to moderate bleeding in most of the literature. Nonetheless, severe and refractory bleeding is difficult to manage[1,16]. Furthermore, APC or topical formalin requires multiple sessions for severe bleeding and can cause severe side effects, including perforation, strictures, and perianal pain[17].\nFecal diversion is reported to be effective in the management of severe CRP bleeding[16,18,19], as it can reduce irritation injury to the lesions to decrease hemorrhage[16]. However, the usage of fecal diversion is not adopted as widely as is APC or formalin. Most of the fecal diversion data are from the 1980s, and this option has not been well studied to date. We previously reported one retrospective cohort of CRP patients with severe hemorrhage who received colostomy[16]. The results showed that colostomy can bring much higher remission of severe bleeding (94%) than can conservative treatment (11%) with APC or topical formalin. Pathologically, chronic inflammation and progressive fibrosis are observed after stoma. Diverting stoma is usually thought to be permanent according to Ayerdi et al[20].\nAnorectal function and QOL after stoma reversal remain unclear. During the past 3 years, we have successfully performed colostomy reversals in a large cohort of CRP patients with severe bleeding and have follow-up data after colostomy reversal. Here, we report the efficacy of colostomy, the rate of stoma closure of diverting colostomy, and anorectal function after reversal for this series of CRP patients with severe bleeding."," Patients and ethical statements Patients with hemorrhagic CRP who were treated after admission at The Sixth Affiliated Hospital of Sun Yat-Sen University from March 2008 to December 2018 were enrolled. Medical records and imaging and clinicopathological data were extracted from our electronic database. This study was approved by the Ethical Committee of The Sixth Affiliated Hospital of Sun Yat-Sen University and was performed according to the provisions of the World Medical Association’s Declaration of Helsinki in 1995 (updated in Tokyo, 2004). Due to the nature of a retrospective study, informed consent was waived.\nPatients with hemorrhagic CRP who were treated after admission at The Sixth Affiliated Hospital of Sun Yat-Sen University from March 2008 to December 2018 were enrolled. Medical records and imaging and clinicopathological data were extracted from our electronic database. This study was approved by the Ethical Committee of The Sixth Affiliated Hospital of Sun Yat-Sen University and was performed according to the provisions of the World Medical Association’s Declaration of Helsinki in 1995 (updated in Tokyo, 2004). Due to the nature of a retrospective study, informed consent was waived.\n Inclusion and exclusion criteria CRP patients with refractory rectal bleeding who received diverting colostomy or conservative treatments for severe anemia and who needed transfusions were enrolled. Patients who were lost to follow-up, had tumor recurrence, rectal-vaginal fistula or other types of rectal fistulas, or underwent rectal resection with preventive colostomy were excluded because these conditions would affect the evaluation of remission of rectal bleeding.\nCRP patients with refractory rectal bleeding who received diverting colostomy or conservative treatments for severe anemia and who needed transfusions were enrolled. Patients who were lost to follow-up, had tumor recurrence, rectal-vaginal fistula or other types of rectal fistulas, or underwent rectal resection with preventive colostomy were excluded because these conditions would affect the evaluation of remission of rectal bleeding.\n Diagnosis, definitions, and scores CRP was diagnosed by the combination of pelvic radiation history for malignancies, symptoms such as rectal bleeding, and endoscopic findings of CRP-specific changes such as telangiectasia and active bleeding in the rectum, as well as exclusion of other bleeding diseases. Hemorrhagic problems, such as tumor recurrence and hemorrhoids, were excluded. Refractory severe bleeding was defined as frequent bleeding and severe anemia with the need for transfusions. At admission, CRP patients in our center were first referred to noninvasive enemas, and if there was no response or recurrence, invasive treatment, such as APC (1-2 rounds) or formalin topical irrigation, was utilized. Conservative treatment failure was defined as no response or recurrence of frequent or severe rectal bleeding after invasive treatments.\nIn this study, we used a modified subjective-objective management analysis (known as mSOMA) system that we designed previously to assess the severity of rectal bleeding[16]. The advantages of the mSOMA system include both subjective complaints of patients and objective hemoglobin (Hb) levels according to laboratory tests. Remission of bleeding was defined as complete cessation or occasional bleeding that needed no further treatment. The CTCAE score of bleeding was used to assess its severity.\nCRP was diagnosed by the combination of pelvic radiation history for malignancies, symptoms such as rectal bleeding, and endoscopic findings of CRP-specific changes such as telangiectasia and active bleeding in the rectum, as well as exclusion of other bleeding diseases. Hemorrhagic problems, such as tumor recurrence and hemorrhoids, were excluded. Refractory severe bleeding was defined as frequent bleeding and severe anemia with the need for transfusions. At admission, CRP patients in our center were first referred to noninvasive enemas, and if there was no response or recurrence, invasive treatment, such as APC (1-2 rounds) or formalin topical irrigation, was utilized. Conservative treatment failure was defined as no response or recurrence of frequent or severe rectal bleeding after invasive treatments.\nIn this study, we used a modified subjective-objective management analysis (known as mSOMA) system that we designed previously to assess the severity of rectal bleeding[16]. The advantages of the mSOMA system include both subjective complaints of patients and objective hemoglobin (Hb) levels according to laboratory tests. Remission of bleeding was defined as complete cessation or occasional bleeding that needed no further treatment. The CTCAE score of bleeding was used to assess its severity.\n Indications for fecal diversion and stoma closure The indications for diverting diversion in hemorrhagic CRP contained the following conditions: Recurrent bleeding and unresponsive to conservative treatments such as endoscopic APC or topical formalin, accompanying severe anemia, and transfusions needed. Transverse loop colostomy and “gunsight” or “cross‘’ type of stoma closure were created according to the standard protocols used in our previous studies[16]. The criteria of stoma closure were as follows: Remission or occasional rectal bleeding, regressive edema in rectal mucosa, good performance of anorectal function, exclusion of tumor recurrence, and no severe CRP complications such as fistula, stricture, and deep ulcer[16].\nThe indications for diverting diversion in hemorrhagic CRP contained the following conditions: Recurrent bleeding and unresponsive to conservative treatments such as endoscopic APC or topical formalin, accompanying severe anemia, and transfusions needed. Transverse loop colostomy and “gunsight” or “cross‘’ type of stoma closure were created according to the standard protocols used in our previous studies[16]. The criteria of stoma closure were as follows: Remission or occasional rectal bleeding, regressive edema in rectal mucosa, good performance of anorectal function, exclusion of tumor recurrence, and no severe CRP complications such as fistula, stricture, and deep ulcer[16].\n Follow-up Patients were scheduled for follow-up through outpatient visits or telephone questionnaires at 6 mo, 9 mo, 1 year, 1.5 years, and 2 years after colostomy. QOL after stoma closure was evaluated according to EORTC QLQ C30 questionnaires[21]. We focused on the following items in this study: The remission rate of bleeding, the rate of stoma reversal, QOL after stoma reversal, dynamic changes of Hb levels, stoma-related complications, and severe CRP complications.\nPatients were scheduled for follow-up through outpatient visits or telephone questionnaires at 6 mo, 9 mo, 1 year, 1.5 years, and 2 years after colostomy. QOL after stoma closure was evaluated according to EORTC QLQ C30 questionnaires[21]. We focused on the following items in this study: The remission rate of bleeding, the rate of stoma reversal, QOL after stoma reversal, dynamic changes of Hb levels, stoma-related complications, and severe CRP complications.\n Anorectal functions In our previous study, tenesmus, stool frequency, and anorectal pain were common symptoms in active CRP[22]. Because there are no standard scales or scores to assess anorectal function precisely in CRP patients, we developed a new scale system that is a patient self-reported scale of outcomes, namely, the chronic radiation proctopathy symptom scale (CRPSS). The CRPSS considers the following items: Watery stool, urgency, perianal pain, tenesmus, rectal bleeding, and fecal/gas incontinence. The details are provided in Supplementary File 1. For CRP patients treated by colostomy, CRPSS scores were evaluated before stoma and at reversal.\nIn our previous study, tenesmus, stool frequency, and anorectal pain were common symptoms in active CRP[22]. Because there are no standard scales or scores to assess anorectal function precisely in CRP patients, we developed a new scale system that is a patient self-reported scale of outcomes, namely, the chronic radiation proctopathy symptom scale (CRPSS). The CRPSS considers the following items: Watery stool, urgency, perianal pain, tenesmus, rectal bleeding, and fecal/gas incontinence. The details are provided in Supplementary File 1. For CRP patients treated by colostomy, CRPSS scores were evaluated before stoma and at reversal.\n Statistical analysis Analyses for continuous variables were performed by Student’s t-test. The χ2 test was used for categorical variables. Fisher’s exact test was conducted when appropriate. For non-parameter variables, the Wilcoxon rank sum tests were used. All statistical analyses were performed using SPSS version 23.0 (Chicago, IL, United States). P < 0.05 (two-tailed) was considered statistically significant.\nAnalyses for continuous variables were performed by Student’s t-test. The χ2 test was used for categorical variables. Fisher’s exact test was conducted when appropriate. For non-parameter variables, the Wilcoxon rank sum tests were used. All statistical analyses were performed using SPSS version 23.0 (Chicago, IL, United States). P < 0.05 (two-tailed) was considered statistically significant.","Patients with hemorrhagic CRP who were treated after admission at The Sixth Affiliated Hospital of Sun Yat-Sen University from March 2008 to December 2018 were enrolled. Medical records and imaging and clinicopathological data were extracted from our electronic database. This study was approved by the Ethical Committee of The Sixth Affiliated Hospital of Sun Yat-Sen University and was performed according to the provisions of the World Medical Association’s Declaration of Helsinki in 1995 (updated in Tokyo, 2004). Due to the nature of a retrospective study, informed consent was waived.","CRP patients with refractory rectal bleeding who received diverting colostomy or conservative treatments for severe anemia and who needed transfusions were enrolled. Patients who were lost to follow-up, had tumor recurrence, rectal-vaginal fistula or other types of rectal fistulas, or underwent rectal resection with preventive colostomy were excluded because these conditions would affect the evaluation of remission of rectal bleeding.","CRP was diagnosed by the combination of pelvic radiation history for malignancies, symptoms such as rectal bleeding, and endoscopic findings of CRP-specific changes such as telangiectasia and active bleeding in the rectum, as well as exclusion of other bleeding diseases. Hemorrhagic problems, such as tumor recurrence and hemorrhoids, were excluded. Refractory severe bleeding was defined as frequent bleeding and severe anemia with the need for transfusions. At admission, CRP patients in our center were first referred to noninvasive enemas, and if there was no response or recurrence, invasive treatment, such as APC (1-2 rounds) or formalin topical irrigation, was utilized. Conservative treatment failure was defined as no response or recurrence of frequent or severe rectal bleeding after invasive treatments.\nIn this study, we used a modified subjective-objective management analysis (known as mSOMA) system that we designed previously to assess the severity of rectal bleeding[16]. The advantages of the mSOMA system include both subjective complaints of patients and objective hemoglobin (Hb) levels according to laboratory tests. Remission of bleeding was defined as complete cessation or occasional bleeding that needed no further treatment. The CTCAE score of bleeding was used to assess its severity.","The indications for diverting diversion in hemorrhagic CRP contained the following conditions: Recurrent bleeding and unresponsive to conservative treatments such as endoscopic APC or topical formalin, accompanying severe anemia, and transfusions needed. Transverse loop colostomy and “gunsight” or “cross‘’ type of stoma closure were created according to the standard protocols used in our previous studies[16]. The criteria of stoma closure were as follows: Remission or occasional rectal bleeding, regressive edema in rectal mucosa, good performance of anorectal function, exclusion of tumor recurrence, and no severe CRP complications such as fistula, stricture, and deep ulcer[16].","Patients were scheduled for follow-up through outpatient visits or telephone questionnaires at 6 mo, 9 mo, 1 year, 1.5 years, and 2 years after colostomy. QOL after stoma closure was evaluated according to EORTC QLQ C30 questionnaires[21]. We focused on the following items in this study: The remission rate of bleeding, the rate of stoma reversal, QOL after stoma reversal, dynamic changes of Hb levels, stoma-related complications, and severe CRP complications.","In our previous study, tenesmus, stool frequency, and anorectal pain were common symptoms in active CRP[22]. Because there are no standard scales or scores to assess anorectal function precisely in CRP patients, we developed a new scale system that is a patient self-reported scale of outcomes, namely, the chronic radiation proctopathy symptom scale (CRPSS). The CRPSS considers the following items: Watery stool, urgency, perianal pain, tenesmus, rectal bleeding, and fecal/gas incontinence. The details are provided in Supplementary File 1. For CRP patients treated by colostomy, CRPSS scores were evaluated before stoma and at reversal.","Analyses for continuous variables were performed by Student’s t-test. The χ2 test was used for categorical variables. Fisher’s exact test was conducted when appropriate. For non-parameter variables, the Wilcoxon rank sum tests were used. All statistical analyses were performed using SPSS version 23.0 (Chicago, IL, United States). P < 0.05 (two-tailed) was considered statistically significant."," Demographics and clinical outcomes From March 2008 to Feb 2019, 738 continual CRP patients treated in our center were screened. After exclusion of 644 patients with mild or moderate CRP, 94 with refractory bleeding and moderate to severe anemia were further screened. Among them, 50 patients were treated by diverted colostomy and 44 patients were treated with conservative therapies. Among the 50 patients treated by colostomy, 32 were excluded due to recto-vaginal fistulas or deep rectal ulcer with refractory perianal pain before stoma. The remaining 18 patients with refractory bleeding were enrolled. Among them, two died from cancer recurrence and two were lost to follow-up. The remaining 14 patients were enrolled in the final analysis as the colostomy group. For the conservative group, 19 patients were excluded, including 11 with tumor relapse and 8 who were lost to follow-up. Thus, 25 were enrolled in the final analysis (Figure 1). Demographic and baseline characteristics were collected, including the intention-to-treat (ITT) group of 50 colostomies. In the enrolled patients, 14 patients comprised the diverting colostomy group, and 25 cases comprised the conservative treatment group. The primary tumors in most of the patients (33/39, 85%) were cervical cancers. No significant differences in age, sex, type of primary tumor, or radiation dosage were found between the diverting colostomy group and the conservative treatment group or between the ITT colostomy group and the colostomy group (Table 1).\nDemographics and characteristics\nFisher's exact test. ITT: Intention-to-treat; Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin.\nFlow chart of patient selection.\nIn the ITT group, no postoperative follow-ups were conducted other than for the 14 investigated colostomy patients. Higher bleeding scores (P = 0.033) and relatively decreased preoperative Hb levels (P = 0.051, no significant difference) were found in the diverting colostomy group compared to the ITT group because 36 patients in the ITT group underwent colostomy for recto-vaginal fistulas or deep rectal ulcers instead of severe bleeding.\nFrom March 2008 to Feb 2019, 738 continual CRP patients treated in our center were screened. After exclusion of 644 patients with mild or moderate CRP, 94 with refractory bleeding and moderate to severe anemia were further screened. Among them, 50 patients were treated by diverted colostomy and 44 patients were treated with conservative therapies. Among the 50 patients treated by colostomy, 32 were excluded due to recto-vaginal fistulas or deep rectal ulcer with refractory perianal pain before stoma. The remaining 18 patients with refractory bleeding were enrolled. Among them, two died from cancer recurrence and two were lost to follow-up. The remaining 14 patients were enrolled in the final analysis as the colostomy group. For the conservative group, 19 patients were excluded, including 11 with tumor relapse and 8 who were lost to follow-up. Thus, 25 were enrolled in the final analysis (Figure 1). Demographic and baseline characteristics were collected, including the intention-to-treat (ITT) group of 50 colostomies. In the enrolled patients, 14 patients comprised the diverting colostomy group, and 25 cases comprised the conservative treatment group. The primary tumors in most of the patients (33/39, 85%) were cervical cancers. No significant differences in age, sex, type of primary tumor, or radiation dosage were found between the diverting colostomy group and the conservative treatment group or between the ITT colostomy group and the colostomy group (Table 1).\nDemographics and characteristics\nFisher's exact test. ITT: Intention-to-treat; Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin.\nFlow chart of patient selection.\nIn the ITT group, no postoperative follow-ups were conducted other than for the 14 investigated colostomy patients. Higher bleeding scores (P = 0.033) and relatively decreased preoperative Hb levels (P = 0.051, no significant difference) were found in the diverting colostomy group compared to the ITT group because 36 patients in the ITT group underwent colostomy for recto-vaginal fistulas or deep rectal ulcers instead of severe bleeding.\n Dynamically increasing hemoglobin levels after colostomy In the colostomy group, the median duration of bleeding was 13.0 mo ± 4.3 mo, and 12 (86%) patients received transfusion before stoma compared to 8.0 mo ± 1.8 mo (P = 0.042) of bleeding duration and 6 (24%) (P < 0.001) transfusions in the conservative group. In the conservative group, 8 patients received formalin irrigation and 4 patients received APC treatments. The preoperative (Pre-Op) Hb was only 63 g/L ± 17.8 g/L in the colostomy group compared to 88.2 g/L ± 19.3 g/L (P < 0.001) in the conservative group. One patient with moderate anemia (Hb of 87 g/L) received colostomy for perianal pain. Another patient with severe anemia (Hb of 78 g/L) did not receive a transfusion due to unavailability of a blood supply before colostomy. The details of the 14 patients are listed in Table 2. Increasing Hb levels were observed from 3 mo (median Hb of 75 g/L), 6 mo (Hb of 99 g/L), and 9 mo (Hb of 107 g/L) to 1 year (Hb 111 of g/L) and 2 years (Hb 117 g/L) after colostomy (Figure 2). Postoperative transfusions were conducted in 3 of 14 patients (transfusion requirements for 2 patients in other hospitals was unknown) in the colostomy group. The dynamic remission rates between the colostomy and conservative groups after surgery were as follows: 86% vs 4% at 3 mo (P < 0.001), 86% vs 12% at 6 mo (P < 0.001), 93% vs 20% at 9 mo (P < 0.001), 100% vs 24% at 1 year (P < 0.001), and 100% vs 20% at 2 years (P < 0.001) (Table 1).\nClinical details and outcomes of 14 colostomy patients\nMedian value. Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin; RTD: Rectal telangiectasia density; APC: Argon plasma coagulation.\nDynamic changes in hemoglobin levels. Pre-Op: Pre-operative.\nIn the colostomy group, the median duration of bleeding was 13.0 mo ± 4.3 mo, and 12 (86%) patients received transfusion before stoma compared to 8.0 mo ± 1.8 mo (P = 0.042) of bleeding duration and 6 (24%) (P < 0.001) transfusions in the conservative group. In the conservative group, 8 patients received formalin irrigation and 4 patients received APC treatments. The preoperative (Pre-Op) Hb was only 63 g/L ± 17.8 g/L in the colostomy group compared to 88.2 g/L ± 19.3 g/L (P < 0.001) in the conservative group. One patient with moderate anemia (Hb of 87 g/L) received colostomy for perianal pain. Another patient with severe anemia (Hb of 78 g/L) did not receive a transfusion due to unavailability of a blood supply before colostomy. The details of the 14 patients are listed in Table 2. Increasing Hb levels were observed from 3 mo (median Hb of 75 g/L), 6 mo (Hb of 99 g/L), and 9 mo (Hb of 107 g/L) to 1 year (Hb 111 of g/L) and 2 years (Hb 117 g/L) after colostomy (Figure 2). Postoperative transfusions were conducted in 3 of 14 patients (transfusion requirements for 2 patients in other hospitals was unknown) in the colostomy group. The dynamic remission rates between the colostomy and conservative groups after surgery were as follows: 86% vs 4% at 3 mo (P < 0.001), 86% vs 12% at 6 mo (P < 0.001), 93% vs 20% at 9 mo (P < 0.001), 100% vs 24% at 1 year (P < 0.001), and 100% vs 20% at 2 years (P < 0.001) (Table 1).\nClinical details and outcomes of 14 colostomy patients\nMedian value. Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin; RTD: Rectal telangiectasia density; APC: Argon plasma coagulation.\nDynamic changes in hemoglobin levels. Pre-Op: Pre-operative.\n Stoma closure and endoscopic features Among the 14 patients with severe bleeding treated by colostomy, all obtained complete remission of bleeding during follow-up. Only 3 (23%) of 13 patients received one round of APC after stoma to control bleeding, and 1 patient received 4% formalin irrigation after colostomy (Table 2). Among the 14 patients, 13 (93%) underwent stoma closure, and the remaining patient did not undergo stoma reversal due to concerns of surgical risks because of old age (83 years old). The median duration of stoma was 16 mo (range: 9-53 mo). Stoma complications occurred in 3 (21%) cases, including 1 para-stoma hernia, 1 stoma prolapse, and 1 stoma obstruction (Grade II by Clavien-Dindo classification)[23]. All 3 patients recovered well after stoma closure.\nEndoscopic findings of bleeding, confluent telangiectasia, and congested mucosa improved dramatically at stoma reversal compared to before stoma creation (Figure 3). The bleeding score by CTCAE decreased from Pre-Op 2.7 points ± 0.5 points to 0.8 points ± 0.5 points (P < 0.001) at 1 year after stoma in the colostomy group; the bleeding score was 2.0 ± 0.5 at initial diagnosis and 1.7 ± 0.9 (P = 0.1282) at 1 year after treatments in the conservative group (Table 1 and Figure 4).\nEndoscopic changes before stoma and at reversal.\nCTC bleeding scores before stoma and at stoma reversal.\nAmong the 14 patients with severe bleeding treated by colostomy, all obtained complete remission of bleeding during follow-up. Only 3 (23%) of 13 patients received one round of APC after stoma to control bleeding, and 1 patient received 4% formalin irrigation after colostomy (Table 2). Among the 14 patients, 13 (93%) underwent stoma closure, and the remaining patient did not undergo stoma reversal due to concerns of surgical risks because of old age (83 years old). The median duration of stoma was 16 mo (range: 9-53 mo). Stoma complications occurred in 3 (21%) cases, including 1 para-stoma hernia, 1 stoma prolapse, and 1 stoma obstruction (Grade II by Clavien-Dindo classification)[23]. All 3 patients recovered well after stoma closure.\nEndoscopic findings of bleeding, confluent telangiectasia, and congested mucosa improved dramatically at stoma reversal compared to before stoma creation (Figure 3). The bleeding score by CTCAE decreased from Pre-Op 2.7 points ± 0.5 points to 0.8 points ± 0.5 points (P < 0.001) at 1 year after stoma in the colostomy group; the bleeding score was 2.0 ± 0.5 at initial diagnosis and 1.7 ± 0.9 (P = 0.1282) at 1 year after treatments in the conservative group (Table 1 and Figure 4).\nEndoscopic changes before stoma and at reversal.\nCTC bleeding scores before stoma and at stoma reversal.\n Ultrasound findings before stoma and at reversal Endorectal ultrasound (EUS) was analyzed retrospectively before stoma and at reversal in 5 patients; the remaining patients did not receive EUS at either of these two time points. In our previous study, thickening of the rectal wall, blurred wall stratification, and increased vascularity were EUS features in CRP[24]. In this study, we found similar features before stoma creation and a tremendous decrease in superficial vascularity, which can explain the cessation of bleeding (Figure 5).\nEndoscopic ultrasound features before stoma and at stoma reversal.\nEndorectal ultrasound (EUS) was analyzed retrospectively before stoma and at reversal in 5 patients; the remaining patients did not receive EUS at either of these two time points. In our previous study, thickening of the rectal wall, blurred wall stratification, and increased vascularity were EUS features in CRP[24]. In this study, we found similar features before stoma creation and a tremendous decrease in superficial vascularity, which can explain the cessation of bleeding (Figure 5).\nEndoscopic ultrasound features before stoma and at stoma reversal.\n Improved magnetic resonance imaging features at stoma reversal To evaluate the severity of the effect on the rectal wall and pelvic floor in the colostomy group, we also report magnetic resonance imaging (MRI) parameters related to CRP, including the thickness of the rectal wall, width of the presacral space, and thicknesses of the levator ani, the gluteus maximus muscle, the obturator intemus, and the distal part of the sigmoid colon (Table 3), which was referred to in our previous study of radiation injury to the pelvis[25]. MRI scans both at pre-colostomy and at reversal were analyzed in 5 patients, as the remaining 9 did not receive MRI scans both pre-colostomy and at reversal. The thickness of the rectal wall was significantly decreased at reversal (8.91 ± 1.61) compared with pre-colostomy (10.7 ± 4.29) (P = 0.047) (Table 3).\nMagnetic resonance imaging findings at post-colostomy and at reversal compared in 5 patients with chronic radiation proctopathy\nTo evaluate the severity of the effect on the rectal wall and pelvic floor in the colostomy group, we also report magnetic resonance imaging (MRI) parameters related to CRP, including the thickness of the rectal wall, width of the presacral space, and thicknesses of the levator ani, the gluteus maximus muscle, the obturator intemus, and the distal part of the sigmoid colon (Table 3), which was referred to in our previous study of radiation injury to the pelvis[25]. MRI scans both at pre-colostomy and at reversal were analyzed in 5 patients, as the remaining 9 did not receive MRI scans both pre-colostomy and at reversal. The thickness of the rectal wall was significantly decreased at reversal (8.91 ± 1.61) compared with pre-colostomy (10.7 ± 4.29) (P = 0.047) (Table 3).\nMagnetic resonance imaging findings at post-colostomy and at reversal compared in 5 patients with chronic radiation proctopathy\n Good restored anorectal functions after stoma reversal Anorectal functions before stoma and after reversal were evaluated by CRPSS scores in all 12 patients in the colostomy group. The results showed that rectal bleeding, peritoneal pain, tenesmus, urgency and fecal/gas incontinency were significantly improved after stoma reversal compared to those before stoma creation. Additionally, all of these scores were < 2 points, which indicated that anorectal function recovered very well after stoma reversal (Figure 6). Complete remission of bleeding and good performance of anorectal function compared to the normal population (scores ≤ 1 point) were observed. We also analyzed anorectal manometry to objectively assess anorectal function before reversal in 6 of the 14 patients who received it. Increased sensitivity of the rectal mucosa and decreased rectal volume were observed. Decreased sphincter functions were found in 2 of 6 cases (Table 4). These patients fulfilled the indications for reversal, and we found that these patients obtained good performance of anorectal function after reversal. Rectal defecography was also conducted before reversal, and no stricture was observed.\nRectal manometry assessment of anorectal functions before stoma reversals\nAnorectal functions before stoma and after stoma reversal.\nAnorectal functions before stoma and after reversal were evaluated by CRPSS scores in all 12 patients in the colostomy group. The results showed that rectal bleeding, peritoneal pain, tenesmus, urgency and fecal/gas incontinency were significantly improved after stoma reversal compared to those before stoma creation. Additionally, all of these scores were < 2 points, which indicated that anorectal function recovered very well after stoma reversal (Figure 6). Complete remission of bleeding and good performance of anorectal function compared to the normal population (scores ≤ 1 point) were observed. We also analyzed anorectal manometry to objectively assess anorectal function before reversal in 6 of the 14 patients who received it. Increased sensitivity of the rectal mucosa and decreased rectal volume were observed. Decreased sphincter functions were found in 2 of 6 cases (Table 4). These patients fulfilled the indications for reversal, and we found that these patients obtained good performance of anorectal function after reversal. Rectal defecography was also conducted before reversal, and no stricture was observed.\nRectal manometry assessment of anorectal functions before stoma reversals\nAnorectal functions before stoma and after stoma reversal.\n Improved quality of life at colostomy reversal QOL after stoma was assessed in the colostomy and conservative groups using EORTC QLQ-C30 questionnaires. As there are no similar reports in the Chinese population, we referred to the normal German population[26]. Osoba et al[26] suggested that a difference of ≥ 20 points in global health was considered to be clinically relevant. In the panel of patients who underwent stoma closure, QOL was dramatically improved compared to Pre-Op baseline QOL before stoma creation, including improved global health (difference of 40, P < 0.001), physical function (difference of 36.4, P < 0.001), role function (difference of 55, P < 0.001), emotional function (difference of 39.4, P < 0.001), social function (difference of 36.3, P < 0.001), and improved symptoms, such as fatigue (difference of -62.5, P < 0.001), pain (difference of -34.9, P < 0.001), dyspnea (difference of -37.8, P < 0.001), insomnia (difference of -36.2, P < 0.001), diarrhea (difference of -23.8, P < 0.001), and financial problems (difference of -35.5, P < 0.001). However, in the conservative group, no improved QOL variables with a difference of ≥ 20 were observed (Table 5). QOL after stoma closure was also compared to the normal population reference, with similar scores for functions and symptoms (Table 5).\nQuality of life in the colostomy group and conservative group of chronic radiation proctopathy patients based on the EORTC QLQ-C30 scale\nWilcoxon rank-sum test. \nPoint (follow-up) – point (pretreatment). Δ(FU): Change in follow-up; SD: Standard deviation.\nQOL after stoma was assessed in the colostomy and conservative groups using EORTC QLQ-C30 questionnaires. As there are no similar reports in the Chinese population, we referred to the normal German population[26]. Osoba et al[26] suggested that a difference of ≥ 20 points in global health was considered to be clinically relevant. In the panel of patients who underwent stoma closure, QOL was dramatically improved compared to Pre-Op baseline QOL before stoma creation, including improved global health (difference of 40, P < 0.001), physical function (difference of 36.4, P < 0.001), role function (difference of 55, P < 0.001), emotional function (difference of 39.4, P < 0.001), social function (difference of 36.3, P < 0.001), and improved symptoms, such as fatigue (difference of -62.5, P < 0.001), pain (difference of -34.9, P < 0.001), dyspnea (difference of -37.8, P < 0.001), insomnia (difference of -36.2, P < 0.001), diarrhea (difference of -23.8, P < 0.001), and financial problems (difference of -35.5, P < 0.001). However, in the conservative group, no improved QOL variables with a difference of ≥ 20 were observed (Table 5). QOL after stoma closure was also compared to the normal population reference, with similar scores for functions and symptoms (Table 5).\nQuality of life in the colostomy group and conservative group of chronic radiation proctopathy patients based on the EORTC QLQ-C30 scale\nWilcoxon rank-sum test. \nPoint (follow-up) – point (pretreatment). Δ(FU): Change in follow-up; SD: Standard deviation.","From March 2008 to Feb 2019, 738 continual CRP patients treated in our center were screened. After exclusion of 644 patients with mild or moderate CRP, 94 with refractory bleeding and moderate to severe anemia were further screened. Among them, 50 patients were treated by diverted colostomy and 44 patients were treated with conservative therapies. Among the 50 patients treated by colostomy, 32 were excluded due to recto-vaginal fistulas or deep rectal ulcer with refractory perianal pain before stoma. The remaining 18 patients with refractory bleeding were enrolled. Among them, two died from cancer recurrence and two were lost to follow-up. The remaining 14 patients were enrolled in the final analysis as the colostomy group. For the conservative group, 19 patients were excluded, including 11 with tumor relapse and 8 who were lost to follow-up. Thus, 25 were enrolled in the final analysis (Figure 1). Demographic and baseline characteristics were collected, including the intention-to-treat (ITT) group of 50 colostomies. In the enrolled patients, 14 patients comprised the diverting colostomy group, and 25 cases comprised the conservative treatment group. The primary tumors in most of the patients (33/39, 85%) were cervical cancers. No significant differences in age, sex, type of primary tumor, or radiation dosage were found between the diverting colostomy group and the conservative treatment group or between the ITT colostomy group and the colostomy group (Table 1).\nDemographics and characteristics\nFisher's exact test. ITT: Intention-to-treat; Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin.\nFlow chart of patient selection.\nIn the ITT group, no postoperative follow-ups were conducted other than for the 14 investigated colostomy patients. Higher bleeding scores (P = 0.033) and relatively decreased preoperative Hb levels (P = 0.051, no significant difference) were found in the diverting colostomy group compared to the ITT group because 36 patients in the ITT group underwent colostomy for recto-vaginal fistulas or deep rectal ulcers instead of severe bleeding.","In the colostomy group, the median duration of bleeding was 13.0 mo ± 4.3 mo, and 12 (86%) patients received transfusion before stoma compared to 8.0 mo ± 1.8 mo (P = 0.042) of bleeding duration and 6 (24%) (P < 0.001) transfusions in the conservative group. In the conservative group, 8 patients received formalin irrigation and 4 patients received APC treatments. The preoperative (Pre-Op) Hb was only 63 g/L ± 17.8 g/L in the colostomy group compared to 88.2 g/L ± 19.3 g/L (P < 0.001) in the conservative group. One patient with moderate anemia (Hb of 87 g/L) received colostomy for perianal pain. Another patient with severe anemia (Hb of 78 g/L) did not receive a transfusion due to unavailability of a blood supply before colostomy. The details of the 14 patients are listed in Table 2. Increasing Hb levels were observed from 3 mo (median Hb of 75 g/L), 6 mo (Hb of 99 g/L), and 9 mo (Hb of 107 g/L) to 1 year (Hb 111 of g/L) and 2 years (Hb 117 g/L) after colostomy (Figure 2). Postoperative transfusions were conducted in 3 of 14 patients (transfusion requirements for 2 patients in other hospitals was unknown) in the colostomy group. The dynamic remission rates between the colostomy and conservative groups after surgery were as follows: 86% vs 4% at 3 mo (P < 0.001), 86% vs 12% at 6 mo (P < 0.001), 93% vs 20% at 9 mo (P < 0.001), 100% vs 24% at 1 year (P < 0.001), and 100% vs 20% at 2 years (P < 0.001) (Table 1).\nClinical details and outcomes of 14 colostomy patients\nMedian value. Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin; RTD: Rectal telangiectasia density; APC: Argon plasma coagulation.\nDynamic changes in hemoglobin levels. Pre-Op: Pre-operative.","Among the 14 patients with severe bleeding treated by colostomy, all obtained complete remission of bleeding during follow-up. Only 3 (23%) of 13 patients received one round of APC after stoma to control bleeding, and 1 patient received 4% formalin irrigation after colostomy (Table 2). Among the 14 patients, 13 (93%) underwent stoma closure, and the remaining patient did not undergo stoma reversal due to concerns of surgical risks because of old age (83 years old). The median duration of stoma was 16 mo (range: 9-53 mo). Stoma complications occurred in 3 (21%) cases, including 1 para-stoma hernia, 1 stoma prolapse, and 1 stoma obstruction (Grade II by Clavien-Dindo classification)[23]. All 3 patients recovered well after stoma closure.\nEndoscopic findings of bleeding, confluent telangiectasia, and congested mucosa improved dramatically at stoma reversal compared to before stoma creation (Figure 3). The bleeding score by CTCAE decreased from Pre-Op 2.7 points ± 0.5 points to 0.8 points ± 0.5 points (P < 0.001) at 1 year after stoma in the colostomy group; the bleeding score was 2.0 ± 0.5 at initial diagnosis and 1.7 ± 0.9 (P = 0.1282) at 1 year after treatments in the conservative group (Table 1 and Figure 4).\nEndoscopic changes before stoma and at reversal.\nCTC bleeding scores before stoma and at stoma reversal.","Endorectal ultrasound (EUS) was analyzed retrospectively before stoma and at reversal in 5 patients; the remaining patients did not receive EUS at either of these two time points. In our previous study, thickening of the rectal wall, blurred wall stratification, and increased vascularity were EUS features in CRP[24]. In this study, we found similar features before stoma creation and a tremendous decrease in superficial vascularity, which can explain the cessation of bleeding (Figure 5).\nEndoscopic ultrasound features before stoma and at stoma reversal.","To evaluate the severity of the effect on the rectal wall and pelvic floor in the colostomy group, we also report magnetic resonance imaging (MRI) parameters related to CRP, including the thickness of the rectal wall, width of the presacral space, and thicknesses of the levator ani, the gluteus maximus muscle, the obturator intemus, and the distal part of the sigmoid colon (Table 3), which was referred to in our previous study of radiation injury to the pelvis[25]. MRI scans both at pre-colostomy and at reversal were analyzed in 5 patients, as the remaining 9 did not receive MRI scans both pre-colostomy and at reversal. The thickness of the rectal wall was significantly decreased at reversal (8.91 ± 1.61) compared with pre-colostomy (10.7 ± 4.29) (P = 0.047) (Table 3).\nMagnetic resonance imaging findings at post-colostomy and at reversal compared in 5 patients with chronic radiation proctopathy","Anorectal functions before stoma and after reversal were evaluated by CRPSS scores in all 12 patients in the colostomy group. The results showed that rectal bleeding, peritoneal pain, tenesmus, urgency and fecal/gas incontinency were significantly improved after stoma reversal compared to those before stoma creation. Additionally, all of these scores were < 2 points, which indicated that anorectal function recovered very well after stoma reversal (Figure 6). Complete remission of bleeding and good performance of anorectal function compared to the normal population (scores ≤ 1 point) were observed. We also analyzed anorectal manometry to objectively assess anorectal function before reversal in 6 of the 14 patients who received it. Increased sensitivity of the rectal mucosa and decreased rectal volume were observed. Decreased sphincter functions were found in 2 of 6 cases (Table 4). These patients fulfilled the indications for reversal, and we found that these patients obtained good performance of anorectal function after reversal. Rectal defecography was also conducted before reversal, and no stricture was observed.\nRectal manometry assessment of anorectal functions before stoma reversals\nAnorectal functions before stoma and after stoma reversal.","QOL after stoma was assessed in the colostomy and conservative groups using EORTC QLQ-C30 questionnaires. As there are no similar reports in the Chinese population, we referred to the normal German population[26]. Osoba et al[26] suggested that a difference of ≥ 20 points in global health was considered to be clinically relevant. In the panel of patients who underwent stoma closure, QOL was dramatically improved compared to Pre-Op baseline QOL before stoma creation, including improved global health (difference of 40, P < 0.001), physical function (difference of 36.4, P < 0.001), role function (difference of 55, P < 0.001), emotional function (difference of 39.4, P < 0.001), social function (difference of 36.3, P < 0.001), and improved symptoms, such as fatigue (difference of -62.5, P < 0.001), pain (difference of -34.9, P < 0.001), dyspnea (difference of -37.8, P < 0.001), insomnia (difference of -36.2, P < 0.001), diarrhea (difference of -23.8, P < 0.001), and financial problems (difference of -35.5, P < 0.001). However, in the conservative group, no improved QOL variables with a difference of ≥ 20 were observed (Table 5). QOL after stoma closure was also compared to the normal population reference, with similar scores for functions and symptoms (Table 5).\nQuality of life in the colostomy group and conservative group of chronic radiation proctopathy patients based on the EORTC QLQ-C30 scale\nWilcoxon rank-sum test. \nPoint (follow-up) – point (pretreatment). Δ(FU): Change in follow-up; SD: Standard deviation.","The literature reports that 1%-6% of CRP patients have transfusion-dependent bleeding; many of these cases can be treated with topical formalin, APC, or hyperbaric oxygen treatment[1]. Surgical resection of rectal lesions is usually reserved as the last resort for hemorrhagic CRP because it is associated with high morbidity and mortality[3,13,19]. In our previous study, fecal diversion was found to be a simple, effective, and safe procedure for severe hemorrhagic CRP[16]. Theoretically, fecal diversion can reduce the irritation of stool and accelerate the course of fibrosis and thus relieve CRP bleeding rapidly[16].\nIn this study, we report results for diverting colostomy and conservative treatments of enemas, topical formalin, or APC in a large cohort of patients with severe hemorrhagic CRP and who were followed for at least 1 year. The results showed that stomas can be reversed in 93% of CRP cases with severe hemorrhage. Increased Hb and rapid cessation of bleeding were found in the colostomy group, whereas bleeding and Hb levels were not changed in the conservative group. Moreover, anorectal function was greatly improved after stoma reversal than before stoma creation, reaching the levels of the normal population. In addition, it is important to note that colostomy was performed in a very select population of CRP patients and those patients with CRP stricture and ulcerations were excluded. Stoma complications occurred in 21% of cases, including 1 case of parastomal hernia due to a weakened abdominal wall of the parastomal zone, 1 case of stoma prolapse due to an intestine overly pulled at stoma creation, and 1 case of stoma obstruction due to stricture. All of these complications were recovered after stoma reversal. According to the literature, the complication rate of stoma is approximately 20%-50%, and the complication rate of stoma in this study is thus acceptable. Thus, colostomy is a better option and can bring more benefits to CRP patients with severe bleeding than can conservative treatments.\nIn our previous study, we found complete remission of telangiectasia and edema, recovery of mucosal integrity, and massive fibrosis in the submucosa at 3-4 years after diversion[16]. In this study, anorectal manometry was conducted in some stoma patients and resulted in good compliance, resting pressure, and contraction. Rectal defecography was performed at reversal for all colostomy patients, and no rectal stricture was found. Thus, temporary diverting colostomy and reversal at an appropriate time can be a useful option for these patients.\nIn this study, most patients in the colostomy group presented with severe anemia and needed multiple transfusions. One patient did not receive transfusion, with an Hb level of 78, before colostomy. In China, blood supply is limited, and transfusion is only conducted in large central hospitals when Hb is < 60. At 3 to 6 mo after diverting colostomy, bleeding gradually remitted and increased Hb levels were observed. By 9 mo to 1 year after colostomy, most patients achieved complete remission of bleeding and the Hb level had almost returned to normal. Stoma reversal was conducted at 1 year after colostomy in 4 (31%) of 13 patients. In 5 cases, reversal was performed at more than 2 years after colostomy because the patients did not know colostomy could be reversed until we contacted them via telephone for follow-up. Thus, the median duration of stoma was prolonged to 16 mo. In fact, most stomas in this cohort could be reversed at 1 year after colostomy.\nBased on the results of our retrospective study[16], we started a prospective clinical trial to enroll severe hemorrhagic CRP patients who are suitable for diverting colostomy (Clinical Trial No. NCT03397901). All patients were followed and evaluated for stoma closure for 1 year after colostomy. The indication for diverting colostomy was extended to hemorrhagic CRP with moderate anemia in our series because preventative colostomy can relieve refractory bleeding before it progresses to severe life-threatening anemia and patients can obtain dramatic benefit from preventative fecal diversion.\nTo date, it is still unclear whether anorectal function can be preserved after stoma closure. In this study, we found that anorectal functions, including anal control and release of gas/stool, could be restored, which also indicated that CRP was inactive after a period of diverting colostomy. Although increased rectal sensitivity and decreased rectal volume were observed at reversal by anorectal manometry, good anorectal function was achieved after reversal compared to the normal population and improved compared to that before stoma creation. No additional pelvic biofeedback treatments were required in these patients.\nFor some patients, an additional one to two rounds of APC were required to help control bleeding during the first 6 mo after colostomy. The temporary stoma not only enabled the remission of anal symptoms but also restored the biological functions of the anus, providing evidence to support fecal diversion as a helpful option for severe hemorrhagic CRP patients.\nIn some studies, bile acid malabsorption and small bowel bacterial overgrowth have been thought to be the cause of diarrhea during acute and chronic radiation enteritis[1]. Diverting colostomy cannot reverse these changes. We focused on hemorrhage in CRP and diarrhea, common in small-bowel radiation injury, which is different from tenesmus and is not the major symptom in most CRP cases.\nAccording to our previous study, severe hemorrhagic CRP patients usually experience poor global health and fatigue due to moderate to severe anemia[16]. Their social and emotional functions are also impacted by frequent stool and anxiety of bleeding. They cannot attend social activities due to concern of looking for a lavatory[1]. In this study, after the remission of anal symptoms, we found that patients had good QOL, especially after stoma reversal, and were able to return to almost the same life as the normal population.\nAnorectal functions scored by the CRPSS were assessed before stoma and after reversal. We also analyzed EUS and pelvic MRI before stoma and at reversal to evaluate the severity of CRP. To objectively evaluate anorectal function before reversal, anorectal manometry was performed and retrospectively analyzed in some patients, though only in 6 patients because manometry is not a routine test before stoma reversal.\nThis study presents our serial results of diverting colostomy for severe hemorrhagic CRP. The results showed significant superiority over conservative treatments. However, there are several limitations to this study. First, this study was retrospective, with potential recall bias. Second, the sample size of this cohort was relatively small, which will affect the grade of evidence, and a larger cohort is essential. Finally, EUS, MRI, and anorectal manometry were only performed on patients undergoing colostomy reversal as a limitation. Thus, we started a prospective cohort of diverting colostomy for severe hemorrhagic CRP and will provide more reliable evidence in the future.\nIn conclusion, diverting colostomy is a very effective and rapid method for the remission of severe bleeding in CRP patients. The stoma can be reversed and good anorectal function can be restored after reversal in most patients."," Research background Chronic radiation proctopathy (CRP) is a common and sometimes difficult issue after radiotherapy for pelvic malignancies. Severe and refractory bleeding is hard to manage. Diverting fecal diversion is very effective and fast in remission of rectal bleeding in the management of severe CRP bleeding in our previous study, but diverting stoma is usually thought to be permanent according to the literature. The anorectal function and quality of life after stoma reversal remain unclear.\nChronic radiation proctopathy (CRP) is a common and sometimes difficult issue after radiotherapy for pelvic malignancies. Severe and refractory bleeding is hard to manage. Diverting fecal diversion is very effective and fast in remission of rectal bleeding in the management of severe CRP bleeding in our previous study, but diverting stoma is usually thought to be permanent according to the literature. The anorectal function and quality of life after stoma reversal remain unclear.\n Research motivation During the past 3 years, we have successfully performed colostomy reversals in a larger cohort of CRP patients with severe bleeding and followed patients after colostomy reversals. In this series study, we will report the efficacy of colostomy, the rate of stoma closure of diverting colostomy, and anorectal function after reversals.\nDuring the past 3 years, we have successfully performed colostomy reversals in a larger cohort of CRP patients with severe bleeding and followed patients after colostomy reversals. In this series study, we will report the efficacy of colostomy, the rate of stoma closure of diverting colostomy, and anorectal function after reversals.\n Research objectives The aim of this study was to evaluate the efficacy of colostomy and the rate of stoma reversals in severe hemorrhagic CRP.\nThe aim of this study was to evaluate the efficacy of colostomy and the rate of stoma reversals in severe hemorrhagic CRP.\n Research methods Patients with severe hemorrhagic CRP who underwent colostomy or conservative treatment were enrolled retrospectively. Rectal bleeding, hemoglobin (Hb), endoscopic features, endo-ultrasound (EUS), rectal manometry, and MRI scans were recorded. Anorectal functions and quality of life before stoma and after stoma reversal were scored with EORTC-QOL-C30 questionnaires.\nPatients with severe hemorrhagic CRP who underwent colostomy or conservative treatment were enrolled retrospectively. Rectal bleeding, hemoglobin (Hb), endoscopic features, endo-ultrasound (EUS), rectal manometry, and MRI scans were recorded. Anorectal functions and quality of life before stoma and after stoma reversal were scored with EORTC-QOL-C30 questionnaires.\n Research results After screening 738 continual CRP patients, 14 patients in the colostomy group and 25 patients in the conservative group as controls were enrolled. The Hb was gradually increased to normal levels in two years after colostomy, while no significant increase was observed in the conservative group. All of 14 patients obtained complete remission of bleeding and colostomy was successfully reversed in 13 of 14 (93%), except one with very old age. Improved endoscopic telangiectasia and bleeding, decreased vascularity by EUS, increased presarcal space, and thickened rectal wall by MRI were observed. Anorectal functions and quality of life were significantly improved after stoma reverse.\nAfter screening 738 continual CRP patients, 14 patients in the colostomy group and 25 patients in the conservative group as controls were enrolled. The Hb was gradually increased to normal levels in two years after colostomy, while no significant increase was observed in the conservative group. All of 14 patients obtained complete remission of bleeding and colostomy was successfully reversed in 13 of 14 (93%), except one with very old age. Improved endoscopic telangiectasia and bleeding, decreased vascularity by EUS, increased presarcal space, and thickened rectal wall by MRI were observed. Anorectal functions and quality of life were significantly improved after stoma reverse.\n Research conclusions Diverting colostomy is a very effective method in the remission of refractory hemorrhagic CRP. Meanwhile, stoma can be reversed and anorectal functions can be recovered after reversal.\nDiverting colostomy is a very effective method in the remission of refractory hemorrhagic CRP. Meanwhile, stoma can be reversed and anorectal functions can be recovered after reversal.\n Research perspectives Preventative diverting colostomy can relieve refractory bleeding before it progresses to severe life-threatening anemia and patients can obtain dramatic benefit. Thus, we have started a prospective clinical trial to enroll severe hemorrhagic CRP patients who are suitable for diverting colostomy (Clinical Trial No. NCT03397901), which will provide more evidence to the usage of colostomy in severe CRP patients.\nPreventative diverting colostomy can relieve refractory bleeding before it progresses to severe life-threatening anemia and patients can obtain dramatic benefit. Thus, we have started a prospective clinical trial to enroll severe hemorrhagic CRP patients who are suitable for diverting colostomy (Clinical Trial No. NCT03397901), which will provide more evidence to the usage of colostomy in severe CRP patients.","Chronic radiation proctopathy (CRP) is a common and sometimes difficult issue after radiotherapy for pelvic malignancies. Severe and refractory bleeding is hard to manage. Diverting fecal diversion is very effective and fast in remission of rectal bleeding in the management of severe CRP bleeding in our previous study, but diverting stoma is usually thought to be permanent according to the literature. The anorectal function and quality of life after stoma reversal remain unclear.","During the past 3 years, we have successfully performed colostomy reversals in a larger cohort of CRP patients with severe bleeding and followed patients after colostomy reversals. In this series study, we will report the efficacy of colostomy, the rate of stoma closure of diverting colostomy, and anorectal function after reversals.","The aim of this study was to evaluate the efficacy of colostomy and the rate of stoma reversals in severe hemorrhagic CRP.","Patients with severe hemorrhagic CRP who underwent colostomy or conservative treatment were enrolled retrospectively. Rectal bleeding, hemoglobin (Hb), endoscopic features, endo-ultrasound (EUS), rectal manometry, and MRI scans were recorded. Anorectal functions and quality of life before stoma and after stoma reversal were scored with EORTC-QOL-C30 questionnaires.","After screening 738 continual CRP patients, 14 patients in the colostomy group and 25 patients in the conservative group as controls were enrolled. The Hb was gradually increased to normal levels in two years after colostomy, while no significant increase was observed in the conservative group. All of 14 patients obtained complete remission of bleeding and colostomy was successfully reversed in 13 of 14 (93%), except one with very old age. Improved endoscopic telangiectasia and bleeding, decreased vascularity by EUS, increased presarcal space, and thickened rectal wall by MRI were observed. Anorectal functions and quality of life were significantly improved after stoma reverse.","Diverting colostomy is a very effective method in the remission of refractory hemorrhagic CRP. Meanwhile, stoma can be reversed and anorectal functions can be recovered after reversal."],"string":"[\n \"Chronic radiation proctopathy (CRP) is a common and sometimes difficult issue after radiotherapy for pelvic malignancies. A permanent change in bowel habits may occur in 90% of patients. After pelvic radiotherapy, 20%-50% of patients will develop difficult bowel function, affecting their quality of life (QOL)[1,2]. CRP can cause more than 20 different symptoms. Rectal bleeding is the most common symptom and occurs in > 50% of CRP patients[1,3]. Transfusion-dependent severe bleeding occurs in 1%-5% of patients[1]. Pathologically, ischemia in the submucosa due to obliterative endarteritis and progressive fibrosis in macroscopic changes are the main causes[4].\\nMedical treatment consists of topical sucralfate enemas[5], oral or topical sulfasalazine[6], and rebamipide[7]. However, these reagents are only effective in acute or mild CRP, whereas their efficacy is usually disappointing in CRP patients with moderate to severe hemorrhage. More invasive modalities include endoscopic argon plasma coagulation (APC)[8], topical 4%-10% formalin[9-12], radiofrequency ablation[13], and hyperbaric oxygen therapy[14,15], and are currently popular optional treatments for CRP. Most of these modalities lack randomized trial evidence. These treatment options are reported to be effective in controlling mild to moderate bleeding in most of the literature. Nonetheless, severe and refractory bleeding is difficult to manage[1,16]. Furthermore, APC or topical formalin requires multiple sessions for severe bleeding and can cause severe side effects, including perforation, strictures, and perianal pain[17].\\nFecal diversion is reported to be effective in the management of severe CRP bleeding[16,18,19], as it can reduce irritation injury to the lesions to decrease hemorrhage[16]. However, the usage of fecal diversion is not adopted as widely as is APC or formalin. Most of the fecal diversion data are from the 1980s, and this option has not been well studied to date. We previously reported one retrospective cohort of CRP patients with severe hemorrhage who received colostomy[16]. The results showed that colostomy can bring much higher remission of severe bleeding (94%) than can conservative treatment (11%) with APC or topical formalin. Pathologically, chronic inflammation and progressive fibrosis are observed after stoma. Diverting stoma is usually thought to be permanent according to Ayerdi et al[20].\\nAnorectal function and QOL after stoma reversal remain unclear. During the past 3 years, we have successfully performed colostomy reversals in a large cohort of CRP patients with severe bleeding and have follow-up data after colostomy reversal. Here, we report the efficacy of colostomy, the rate of stoma closure of diverting colostomy, and anorectal function after reversal for this series of CRP patients with severe bleeding.\",\n \" Patients and ethical statements Patients with hemorrhagic CRP who were treated after admission at The Sixth Affiliated Hospital of Sun Yat-Sen University from March 2008 to December 2018 were enrolled. Medical records and imaging and clinicopathological data were extracted from our electronic database. This study was approved by the Ethical Committee of The Sixth Affiliated Hospital of Sun Yat-Sen University and was performed according to the provisions of the World Medical Association’s Declaration of Helsinki in 1995 (updated in Tokyo, 2004). Due to the nature of a retrospective study, informed consent was waived.\\nPatients with hemorrhagic CRP who were treated after admission at The Sixth Affiliated Hospital of Sun Yat-Sen University from March 2008 to December 2018 were enrolled. Medical records and imaging and clinicopathological data were extracted from our electronic database. This study was approved by the Ethical Committee of The Sixth Affiliated Hospital of Sun Yat-Sen University and was performed according to the provisions of the World Medical Association’s Declaration of Helsinki in 1995 (updated in Tokyo, 2004). Due to the nature of a retrospective study, informed consent was waived.\\n Inclusion and exclusion criteria CRP patients with refractory rectal bleeding who received diverting colostomy or conservative treatments for severe anemia and who needed transfusions were enrolled. Patients who were lost to follow-up, had tumor recurrence, rectal-vaginal fistula or other types of rectal fistulas, or underwent rectal resection with preventive colostomy were excluded because these conditions would affect the evaluation of remission of rectal bleeding.\\nCRP patients with refractory rectal bleeding who received diverting colostomy or conservative treatments for severe anemia and who needed transfusions were enrolled. Patients who were lost to follow-up, had tumor recurrence, rectal-vaginal fistula or other types of rectal fistulas, or underwent rectal resection with preventive colostomy were excluded because these conditions would affect the evaluation of remission of rectal bleeding.\\n Diagnosis, definitions, and scores CRP was diagnosed by the combination of pelvic radiation history for malignancies, symptoms such as rectal bleeding, and endoscopic findings of CRP-specific changes such as telangiectasia and active bleeding in the rectum, as well as exclusion of other bleeding diseases. Hemorrhagic problems, such as tumor recurrence and hemorrhoids, were excluded. Refractory severe bleeding was defined as frequent bleeding and severe anemia with the need for transfusions. At admission, CRP patients in our center were first referred to noninvasive enemas, and if there was no response or recurrence, invasive treatment, such as APC (1-2 rounds) or formalin topical irrigation, was utilized. Conservative treatment failure was defined as no response or recurrence of frequent or severe rectal bleeding after invasive treatments.\\nIn this study, we used a modified subjective-objective management analysis (known as mSOMA) system that we designed previously to assess the severity of rectal bleeding[16]. The advantages of the mSOMA system include both subjective complaints of patients and objective hemoglobin (Hb) levels according to laboratory tests. Remission of bleeding was defined as complete cessation or occasional bleeding that needed no further treatment. The CTCAE score of bleeding was used to assess its severity.\\nCRP was diagnosed by the combination of pelvic radiation history for malignancies, symptoms such as rectal bleeding, and endoscopic findings of CRP-specific changes such as telangiectasia and active bleeding in the rectum, as well as exclusion of other bleeding diseases. Hemorrhagic problems, such as tumor recurrence and hemorrhoids, were excluded. Refractory severe bleeding was defined as frequent bleeding and severe anemia with the need for transfusions. At admission, CRP patients in our center were first referred to noninvasive enemas, and if there was no response or recurrence, invasive treatment, such as APC (1-2 rounds) or formalin topical irrigation, was utilized. Conservative treatment failure was defined as no response or recurrence of frequent or severe rectal bleeding after invasive treatments.\\nIn this study, we used a modified subjective-objective management analysis (known as mSOMA) system that we designed previously to assess the severity of rectal bleeding[16]. The advantages of the mSOMA system include both subjective complaints of patients and objective hemoglobin (Hb) levels according to laboratory tests. Remission of bleeding was defined as complete cessation or occasional bleeding that needed no further treatment. The CTCAE score of bleeding was used to assess its severity.\\n Indications for fecal diversion and stoma closure The indications for diverting diversion in hemorrhagic CRP contained the following conditions: Recurrent bleeding and unresponsive to conservative treatments such as endoscopic APC or topical formalin, accompanying severe anemia, and transfusions needed. Transverse loop colostomy and “gunsight” or “cross‘’ type of stoma closure were created according to the standard protocols used in our previous studies[16]. The criteria of stoma closure were as follows: Remission or occasional rectal bleeding, regressive edema in rectal mucosa, good performance of anorectal function, exclusion of tumor recurrence, and no severe CRP complications such as fistula, stricture, and deep ulcer[16].\\nThe indications for diverting diversion in hemorrhagic CRP contained the following conditions: Recurrent bleeding and unresponsive to conservative treatments such as endoscopic APC or topical formalin, accompanying severe anemia, and transfusions needed. Transverse loop colostomy and “gunsight” or “cross‘’ type of stoma closure were created according to the standard protocols used in our previous studies[16]. The criteria of stoma closure were as follows: Remission or occasional rectal bleeding, regressive edema in rectal mucosa, good performance of anorectal function, exclusion of tumor recurrence, and no severe CRP complications such as fistula, stricture, and deep ulcer[16].\\n Follow-up Patients were scheduled for follow-up through outpatient visits or telephone questionnaires at 6 mo, 9 mo, 1 year, 1.5 years, and 2 years after colostomy. QOL after stoma closure was evaluated according to EORTC QLQ C30 questionnaires[21]. We focused on the following items in this study: The remission rate of bleeding, the rate of stoma reversal, QOL after stoma reversal, dynamic changes of Hb levels, stoma-related complications, and severe CRP complications.\\nPatients were scheduled for follow-up through outpatient visits or telephone questionnaires at 6 mo, 9 mo, 1 year, 1.5 years, and 2 years after colostomy. QOL after stoma closure was evaluated according to EORTC QLQ C30 questionnaires[21]. We focused on the following items in this study: The remission rate of bleeding, the rate of stoma reversal, QOL after stoma reversal, dynamic changes of Hb levels, stoma-related complications, and severe CRP complications.\\n Anorectal functions In our previous study, tenesmus, stool frequency, and anorectal pain were common symptoms in active CRP[22]. Because there are no standard scales or scores to assess anorectal function precisely in CRP patients, we developed a new scale system that is a patient self-reported scale of outcomes, namely, the chronic radiation proctopathy symptom scale (CRPSS). The CRPSS considers the following items: Watery stool, urgency, perianal pain, tenesmus, rectal bleeding, and fecal/gas incontinence. The details are provided in Supplementary File 1. For CRP patients treated by colostomy, CRPSS scores were evaluated before stoma and at reversal.\\nIn our previous study, tenesmus, stool frequency, and anorectal pain were common symptoms in active CRP[22]. Because there are no standard scales or scores to assess anorectal function precisely in CRP patients, we developed a new scale system that is a patient self-reported scale of outcomes, namely, the chronic radiation proctopathy symptom scale (CRPSS). The CRPSS considers the following items: Watery stool, urgency, perianal pain, tenesmus, rectal bleeding, and fecal/gas incontinence. The details are provided in Supplementary File 1. For CRP patients treated by colostomy, CRPSS scores were evaluated before stoma and at reversal.\\n Statistical analysis Analyses for continuous variables were performed by Student’s t-test. The χ2 test was used for categorical variables. Fisher’s exact test was conducted when appropriate. For non-parameter variables, the Wilcoxon rank sum tests were used. All statistical analyses were performed using SPSS version 23.0 (Chicago, IL, United States). P < 0.05 (two-tailed) was considered statistically significant.\\nAnalyses for continuous variables were performed by Student’s t-test. The χ2 test was used for categorical variables. Fisher’s exact test was conducted when appropriate. For non-parameter variables, the Wilcoxon rank sum tests were used. All statistical analyses were performed using SPSS version 23.0 (Chicago, IL, United States). P < 0.05 (two-tailed) was considered statistically significant.\",\n \"Patients with hemorrhagic CRP who were treated after admission at The Sixth Affiliated Hospital of Sun Yat-Sen University from March 2008 to December 2018 were enrolled. Medical records and imaging and clinicopathological data were extracted from our electronic database. This study was approved by the Ethical Committee of The Sixth Affiliated Hospital of Sun Yat-Sen University and was performed according to the provisions of the World Medical Association’s Declaration of Helsinki in 1995 (updated in Tokyo, 2004). Due to the nature of a retrospective study, informed consent was waived.\",\n \"CRP patients with refractory rectal bleeding who received diverting colostomy or conservative treatments for severe anemia and who needed transfusions were enrolled. Patients who were lost to follow-up, had tumor recurrence, rectal-vaginal fistula or other types of rectal fistulas, or underwent rectal resection with preventive colostomy were excluded because these conditions would affect the evaluation of remission of rectal bleeding.\",\n \"CRP was diagnosed by the combination of pelvic radiation history for malignancies, symptoms such as rectal bleeding, and endoscopic findings of CRP-specific changes such as telangiectasia and active bleeding in the rectum, as well as exclusion of other bleeding diseases. Hemorrhagic problems, such as tumor recurrence and hemorrhoids, were excluded. Refractory severe bleeding was defined as frequent bleeding and severe anemia with the need for transfusions. At admission, CRP patients in our center were first referred to noninvasive enemas, and if there was no response or recurrence, invasive treatment, such as APC (1-2 rounds) or formalin topical irrigation, was utilized. Conservative treatment failure was defined as no response or recurrence of frequent or severe rectal bleeding after invasive treatments.\\nIn this study, we used a modified subjective-objective management analysis (known as mSOMA) system that we designed previously to assess the severity of rectal bleeding[16]. The advantages of the mSOMA system include both subjective complaints of patients and objective hemoglobin (Hb) levels according to laboratory tests. Remission of bleeding was defined as complete cessation or occasional bleeding that needed no further treatment. The CTCAE score of bleeding was used to assess its severity.\",\n \"The indications for diverting diversion in hemorrhagic CRP contained the following conditions: Recurrent bleeding and unresponsive to conservative treatments such as endoscopic APC or topical formalin, accompanying severe anemia, and transfusions needed. Transverse loop colostomy and “gunsight” or “cross‘’ type of stoma closure were created according to the standard protocols used in our previous studies[16]. The criteria of stoma closure were as follows: Remission or occasional rectal bleeding, regressive edema in rectal mucosa, good performance of anorectal function, exclusion of tumor recurrence, and no severe CRP complications such as fistula, stricture, and deep ulcer[16].\",\n \"Patients were scheduled for follow-up through outpatient visits or telephone questionnaires at 6 mo, 9 mo, 1 year, 1.5 years, and 2 years after colostomy. QOL after stoma closure was evaluated according to EORTC QLQ C30 questionnaires[21]. We focused on the following items in this study: The remission rate of bleeding, the rate of stoma reversal, QOL after stoma reversal, dynamic changes of Hb levels, stoma-related complications, and severe CRP complications.\",\n \"In our previous study, tenesmus, stool frequency, and anorectal pain were common symptoms in active CRP[22]. Because there are no standard scales or scores to assess anorectal function precisely in CRP patients, we developed a new scale system that is a patient self-reported scale of outcomes, namely, the chronic radiation proctopathy symptom scale (CRPSS). The CRPSS considers the following items: Watery stool, urgency, perianal pain, tenesmus, rectal bleeding, and fecal/gas incontinence. The details are provided in Supplementary File 1. For CRP patients treated by colostomy, CRPSS scores were evaluated before stoma and at reversal.\",\n \"Analyses for continuous variables were performed by Student’s t-test. The χ2 test was used for categorical variables. Fisher’s exact test was conducted when appropriate. For non-parameter variables, the Wilcoxon rank sum tests were used. All statistical analyses were performed using SPSS version 23.0 (Chicago, IL, United States). P < 0.05 (two-tailed) was considered statistically significant.\",\n \" Demographics and clinical outcomes From March 2008 to Feb 2019, 738 continual CRP patients treated in our center were screened. After exclusion of 644 patients with mild or moderate CRP, 94 with refractory bleeding and moderate to severe anemia were further screened. Among them, 50 patients were treated by diverted colostomy and 44 patients were treated with conservative therapies. Among the 50 patients treated by colostomy, 32 were excluded due to recto-vaginal fistulas or deep rectal ulcer with refractory perianal pain before stoma. The remaining 18 patients with refractory bleeding were enrolled. Among them, two died from cancer recurrence and two were lost to follow-up. The remaining 14 patients were enrolled in the final analysis as the colostomy group. For the conservative group, 19 patients were excluded, including 11 with tumor relapse and 8 who were lost to follow-up. Thus, 25 were enrolled in the final analysis (Figure 1). Demographic and baseline characteristics were collected, including the intention-to-treat (ITT) group of 50 colostomies. In the enrolled patients, 14 patients comprised the diverting colostomy group, and 25 cases comprised the conservative treatment group. The primary tumors in most of the patients (33/39, 85%) were cervical cancers. No significant differences in age, sex, type of primary tumor, or radiation dosage were found between the diverting colostomy group and the conservative treatment group or between the ITT colostomy group and the colostomy group (Table 1).\\nDemographics and characteristics\\nFisher's exact test. ITT: Intention-to-treat; Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin.\\nFlow chart of patient selection.\\nIn the ITT group, no postoperative follow-ups were conducted other than for the 14 investigated colostomy patients. Higher bleeding scores (P = 0.033) and relatively decreased preoperative Hb levels (P = 0.051, no significant difference) were found in the diverting colostomy group compared to the ITT group because 36 patients in the ITT group underwent colostomy for recto-vaginal fistulas or deep rectal ulcers instead of severe bleeding.\\nFrom March 2008 to Feb 2019, 738 continual CRP patients treated in our center were screened. After exclusion of 644 patients with mild or moderate CRP, 94 with refractory bleeding and moderate to severe anemia were further screened. Among them, 50 patients were treated by diverted colostomy and 44 patients were treated with conservative therapies. Among the 50 patients treated by colostomy, 32 were excluded due to recto-vaginal fistulas or deep rectal ulcer with refractory perianal pain before stoma. The remaining 18 patients with refractory bleeding were enrolled. Among them, two died from cancer recurrence and two were lost to follow-up. The remaining 14 patients were enrolled in the final analysis as the colostomy group. For the conservative group, 19 patients were excluded, including 11 with tumor relapse and 8 who were lost to follow-up. Thus, 25 were enrolled in the final analysis (Figure 1). Demographic and baseline characteristics were collected, including the intention-to-treat (ITT) group of 50 colostomies. In the enrolled patients, 14 patients comprised the diverting colostomy group, and 25 cases comprised the conservative treatment group. The primary tumors in most of the patients (33/39, 85%) were cervical cancers. No significant differences in age, sex, type of primary tumor, or radiation dosage were found between the diverting colostomy group and the conservative treatment group or between the ITT colostomy group and the colostomy group (Table 1).\\nDemographics and characteristics\\nFisher's exact test. ITT: Intention-to-treat; Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin.\\nFlow chart of patient selection.\\nIn the ITT group, no postoperative follow-ups were conducted other than for the 14 investigated colostomy patients. Higher bleeding scores (P = 0.033) and relatively decreased preoperative Hb levels (P = 0.051, no significant difference) were found in the diverting colostomy group compared to the ITT group because 36 patients in the ITT group underwent colostomy for recto-vaginal fistulas or deep rectal ulcers instead of severe bleeding.\\n Dynamically increasing hemoglobin levels after colostomy In the colostomy group, the median duration of bleeding was 13.0 mo ± 4.3 mo, and 12 (86%) patients received transfusion before stoma compared to 8.0 mo ± 1.8 mo (P = 0.042) of bleeding duration and 6 (24%) (P < 0.001) transfusions in the conservative group. In the conservative group, 8 patients received formalin irrigation and 4 patients received APC treatments. The preoperative (Pre-Op) Hb was only 63 g/L ± 17.8 g/L in the colostomy group compared to 88.2 g/L ± 19.3 g/L (P < 0.001) in the conservative group. One patient with moderate anemia (Hb of 87 g/L) received colostomy for perianal pain. Another patient with severe anemia (Hb of 78 g/L) did not receive a transfusion due to unavailability of a blood supply before colostomy. The details of the 14 patients are listed in Table 2. Increasing Hb levels were observed from 3 mo (median Hb of 75 g/L), 6 mo (Hb of 99 g/L), and 9 mo (Hb of 107 g/L) to 1 year (Hb 111 of g/L) and 2 years (Hb 117 g/L) after colostomy (Figure 2). Postoperative transfusions were conducted in 3 of 14 patients (transfusion requirements for 2 patients in other hospitals was unknown) in the colostomy group. The dynamic remission rates between the colostomy and conservative groups after surgery were as follows: 86% vs 4% at 3 mo (P < 0.001), 86% vs 12% at 6 mo (P < 0.001), 93% vs 20% at 9 mo (P < 0.001), 100% vs 24% at 1 year (P < 0.001), and 100% vs 20% at 2 years (P < 0.001) (Table 1).\\nClinical details and outcomes of 14 colostomy patients\\nMedian value. Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin; RTD: Rectal telangiectasia density; APC: Argon plasma coagulation.\\nDynamic changes in hemoglobin levels. Pre-Op: Pre-operative.\\nIn the colostomy group, the median duration of bleeding was 13.0 mo ± 4.3 mo, and 12 (86%) patients received transfusion before stoma compared to 8.0 mo ± 1.8 mo (P = 0.042) of bleeding duration and 6 (24%) (P < 0.001) transfusions in the conservative group. In the conservative group, 8 patients received formalin irrigation and 4 patients received APC treatments. The preoperative (Pre-Op) Hb was only 63 g/L ± 17.8 g/L in the colostomy group compared to 88.2 g/L ± 19.3 g/L (P < 0.001) in the conservative group. One patient with moderate anemia (Hb of 87 g/L) received colostomy for perianal pain. Another patient with severe anemia (Hb of 78 g/L) did not receive a transfusion due to unavailability of a blood supply before colostomy. The details of the 14 patients are listed in Table 2. Increasing Hb levels were observed from 3 mo (median Hb of 75 g/L), 6 mo (Hb of 99 g/L), and 9 mo (Hb of 107 g/L) to 1 year (Hb 111 of g/L) and 2 years (Hb 117 g/L) after colostomy (Figure 2). Postoperative transfusions were conducted in 3 of 14 patients (transfusion requirements for 2 patients in other hospitals was unknown) in the colostomy group. The dynamic remission rates between the colostomy and conservative groups after surgery were as follows: 86% vs 4% at 3 mo (P < 0.001), 86% vs 12% at 6 mo (P < 0.001), 93% vs 20% at 9 mo (P < 0.001), 100% vs 24% at 1 year (P < 0.001), and 100% vs 20% at 2 years (P < 0.001) (Table 1).\\nClinical details and outcomes of 14 colostomy patients\\nMedian value. Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin; RTD: Rectal telangiectasia density; APC: Argon plasma coagulation.\\nDynamic changes in hemoglobin levels. Pre-Op: Pre-operative.\\n Stoma closure and endoscopic features Among the 14 patients with severe bleeding treated by colostomy, all obtained complete remission of bleeding during follow-up. Only 3 (23%) of 13 patients received one round of APC after stoma to control bleeding, and 1 patient received 4% formalin irrigation after colostomy (Table 2). Among the 14 patients, 13 (93%) underwent stoma closure, and the remaining patient did not undergo stoma reversal due to concerns of surgical risks because of old age (83 years old). The median duration of stoma was 16 mo (range: 9-53 mo). Stoma complications occurred in 3 (21%) cases, including 1 para-stoma hernia, 1 stoma prolapse, and 1 stoma obstruction (Grade II by Clavien-Dindo classification)[23]. All 3 patients recovered well after stoma closure.\\nEndoscopic findings of bleeding, confluent telangiectasia, and congested mucosa improved dramatically at stoma reversal compared to before stoma creation (Figure 3). The bleeding score by CTCAE decreased from Pre-Op 2.7 points ± 0.5 points to 0.8 points ± 0.5 points (P < 0.001) at 1 year after stoma in the colostomy group; the bleeding score was 2.0 ± 0.5 at initial diagnosis and 1.7 ± 0.9 (P = 0.1282) at 1 year after treatments in the conservative group (Table 1 and Figure 4).\\nEndoscopic changes before stoma and at reversal.\\nCTC bleeding scores before stoma and at stoma reversal.\\nAmong the 14 patients with severe bleeding treated by colostomy, all obtained complete remission of bleeding during follow-up. Only 3 (23%) of 13 patients received one round of APC after stoma to control bleeding, and 1 patient received 4% formalin irrigation after colostomy (Table 2). Among the 14 patients, 13 (93%) underwent stoma closure, and the remaining patient did not undergo stoma reversal due to concerns of surgical risks because of old age (83 years old). The median duration of stoma was 16 mo (range: 9-53 mo). Stoma complications occurred in 3 (21%) cases, including 1 para-stoma hernia, 1 stoma prolapse, and 1 stoma obstruction (Grade II by Clavien-Dindo classification)[23]. All 3 patients recovered well after stoma closure.\\nEndoscopic findings of bleeding, confluent telangiectasia, and congested mucosa improved dramatically at stoma reversal compared to before stoma creation (Figure 3). The bleeding score by CTCAE decreased from Pre-Op 2.7 points ± 0.5 points to 0.8 points ± 0.5 points (P < 0.001) at 1 year after stoma in the colostomy group; the bleeding score was 2.0 ± 0.5 at initial diagnosis and 1.7 ± 0.9 (P = 0.1282) at 1 year after treatments in the conservative group (Table 1 and Figure 4).\\nEndoscopic changes before stoma and at reversal.\\nCTC bleeding scores before stoma and at stoma reversal.\\n Ultrasound findings before stoma and at reversal Endorectal ultrasound (EUS) was analyzed retrospectively before stoma and at reversal in 5 patients; the remaining patients did not receive EUS at either of these two time points. In our previous study, thickening of the rectal wall, blurred wall stratification, and increased vascularity were EUS features in CRP[24]. In this study, we found similar features before stoma creation and a tremendous decrease in superficial vascularity, which can explain the cessation of bleeding (Figure 5).\\nEndoscopic ultrasound features before stoma and at stoma reversal.\\nEndorectal ultrasound (EUS) was analyzed retrospectively before stoma and at reversal in 5 patients; the remaining patients did not receive EUS at either of these two time points. In our previous study, thickening of the rectal wall, blurred wall stratification, and increased vascularity were EUS features in CRP[24]. In this study, we found similar features before stoma creation and a tremendous decrease in superficial vascularity, which can explain the cessation of bleeding (Figure 5).\\nEndoscopic ultrasound features before stoma and at stoma reversal.\\n Improved magnetic resonance imaging features at stoma reversal To evaluate the severity of the effect on the rectal wall and pelvic floor in the colostomy group, we also report magnetic resonance imaging (MRI) parameters related to CRP, including the thickness of the rectal wall, width of the presacral space, and thicknesses of the levator ani, the gluteus maximus muscle, the obturator intemus, and the distal part of the sigmoid colon (Table 3), which was referred to in our previous study of radiation injury to the pelvis[25]. MRI scans both at pre-colostomy and at reversal were analyzed in 5 patients, as the remaining 9 did not receive MRI scans both pre-colostomy and at reversal. The thickness of the rectal wall was significantly decreased at reversal (8.91 ± 1.61) compared with pre-colostomy (10.7 ± 4.29) (P = 0.047) (Table 3).\\nMagnetic resonance imaging findings at post-colostomy and at reversal compared in 5 patients with chronic radiation proctopathy\\nTo evaluate the severity of the effect on the rectal wall and pelvic floor in the colostomy group, we also report magnetic resonance imaging (MRI) parameters related to CRP, including the thickness of the rectal wall, width of the presacral space, and thicknesses of the levator ani, the gluteus maximus muscle, the obturator intemus, and the distal part of the sigmoid colon (Table 3), which was referred to in our previous study of radiation injury to the pelvis[25]. MRI scans both at pre-colostomy and at reversal were analyzed in 5 patients, as the remaining 9 did not receive MRI scans both pre-colostomy and at reversal. The thickness of the rectal wall was significantly decreased at reversal (8.91 ± 1.61) compared with pre-colostomy (10.7 ± 4.29) (P = 0.047) (Table 3).\\nMagnetic resonance imaging findings at post-colostomy and at reversal compared in 5 patients with chronic radiation proctopathy\\n Good restored anorectal functions after stoma reversal Anorectal functions before stoma and after reversal were evaluated by CRPSS scores in all 12 patients in the colostomy group. The results showed that rectal bleeding, peritoneal pain, tenesmus, urgency and fecal/gas incontinency were significantly improved after stoma reversal compared to those before stoma creation. Additionally, all of these scores were < 2 points, which indicated that anorectal function recovered very well after stoma reversal (Figure 6). Complete remission of bleeding and good performance of anorectal function compared to the normal population (scores ≤ 1 point) were observed. We also analyzed anorectal manometry to objectively assess anorectal function before reversal in 6 of the 14 patients who received it. Increased sensitivity of the rectal mucosa and decreased rectal volume were observed. Decreased sphincter functions were found in 2 of 6 cases (Table 4). These patients fulfilled the indications for reversal, and we found that these patients obtained good performance of anorectal function after reversal. Rectal defecography was also conducted before reversal, and no stricture was observed.\\nRectal manometry assessment of anorectal functions before stoma reversals\\nAnorectal functions before stoma and after stoma reversal.\\nAnorectal functions before stoma and after reversal were evaluated by CRPSS scores in all 12 patients in the colostomy group. The results showed that rectal bleeding, peritoneal pain, tenesmus, urgency and fecal/gas incontinency were significantly improved after stoma reversal compared to those before stoma creation. Additionally, all of these scores were < 2 points, which indicated that anorectal function recovered very well after stoma reversal (Figure 6). Complete remission of bleeding and good performance of anorectal function compared to the normal population (scores ≤ 1 point) were observed. We also analyzed anorectal manometry to objectively assess anorectal function before reversal in 6 of the 14 patients who received it. Increased sensitivity of the rectal mucosa and decreased rectal volume were observed. Decreased sphincter functions were found in 2 of 6 cases (Table 4). These patients fulfilled the indications for reversal, and we found that these patients obtained good performance of anorectal function after reversal. Rectal defecography was also conducted before reversal, and no stricture was observed.\\nRectal manometry assessment of anorectal functions before stoma reversals\\nAnorectal functions before stoma and after stoma reversal.\\n Improved quality of life at colostomy reversal QOL after stoma was assessed in the colostomy and conservative groups using EORTC QLQ-C30 questionnaires. As there are no similar reports in the Chinese population, we referred to the normal German population[26]. Osoba et al[26] suggested that a difference of ≥ 20 points in global health was considered to be clinically relevant. In the panel of patients who underwent stoma closure, QOL was dramatically improved compared to Pre-Op baseline QOL before stoma creation, including improved global health (difference of 40, P < 0.001), physical function (difference of 36.4, P < 0.001), role function (difference of 55, P < 0.001), emotional function (difference of 39.4, P < 0.001), social function (difference of 36.3, P < 0.001), and improved symptoms, such as fatigue (difference of -62.5, P < 0.001), pain (difference of -34.9, P < 0.001), dyspnea (difference of -37.8, P < 0.001), insomnia (difference of -36.2, P < 0.001), diarrhea (difference of -23.8, P < 0.001), and financial problems (difference of -35.5, P < 0.001). However, in the conservative group, no improved QOL variables with a difference of ≥ 20 were observed (Table 5). QOL after stoma closure was also compared to the normal population reference, with similar scores for functions and symptoms (Table 5).\\nQuality of life in the colostomy group and conservative group of chronic radiation proctopathy patients based on the EORTC QLQ-C30 scale\\nWilcoxon rank-sum test. \\nPoint (follow-up) – point (pretreatment). Δ(FU): Change in follow-up; SD: Standard deviation.\\nQOL after stoma was assessed in the colostomy and conservative groups using EORTC QLQ-C30 questionnaires. As there are no similar reports in the Chinese population, we referred to the normal German population[26]. Osoba et al[26] suggested that a difference of ≥ 20 points in global health was considered to be clinically relevant. In the panel of patients who underwent stoma closure, QOL was dramatically improved compared to Pre-Op baseline QOL before stoma creation, including improved global health (difference of 40, P < 0.001), physical function (difference of 36.4, P < 0.001), role function (difference of 55, P < 0.001), emotional function (difference of 39.4, P < 0.001), social function (difference of 36.3, P < 0.001), and improved symptoms, such as fatigue (difference of -62.5, P < 0.001), pain (difference of -34.9, P < 0.001), dyspnea (difference of -37.8, P < 0.001), insomnia (difference of -36.2, P < 0.001), diarrhea (difference of -23.8, P < 0.001), and financial problems (difference of -35.5, P < 0.001). However, in the conservative group, no improved QOL variables with a difference of ≥ 20 were observed (Table 5). QOL after stoma closure was also compared to the normal population reference, with similar scores for functions and symptoms (Table 5).\\nQuality of life in the colostomy group and conservative group of chronic radiation proctopathy patients based on the EORTC QLQ-C30 scale\\nWilcoxon rank-sum test. \\nPoint (follow-up) – point (pretreatment). Δ(FU): Change in follow-up; SD: Standard deviation.\",\n \"From March 2008 to Feb 2019, 738 continual CRP patients treated in our center were screened. After exclusion of 644 patients with mild or moderate CRP, 94 with refractory bleeding and moderate to severe anemia were further screened. Among them, 50 patients were treated by diverted colostomy and 44 patients were treated with conservative therapies. Among the 50 patients treated by colostomy, 32 were excluded due to recto-vaginal fistulas or deep rectal ulcer with refractory perianal pain before stoma. The remaining 18 patients with refractory bleeding were enrolled. Among them, two died from cancer recurrence and two were lost to follow-up. The remaining 14 patients were enrolled in the final analysis as the colostomy group. For the conservative group, 19 patients were excluded, including 11 with tumor relapse and 8 who were lost to follow-up. Thus, 25 were enrolled in the final analysis (Figure 1). Demographic and baseline characteristics were collected, including the intention-to-treat (ITT) group of 50 colostomies. In the enrolled patients, 14 patients comprised the diverting colostomy group, and 25 cases comprised the conservative treatment group. The primary tumors in most of the patients (33/39, 85%) were cervical cancers. No significant differences in age, sex, type of primary tumor, or radiation dosage were found between the diverting colostomy group and the conservative treatment group or between the ITT colostomy group and the colostomy group (Table 1).\\nDemographics and characteristics\\nFisher's exact test. ITT: Intention-to-treat; Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin.\\nFlow chart of patient selection.\\nIn the ITT group, no postoperative follow-ups were conducted other than for the 14 investigated colostomy patients. Higher bleeding scores (P = 0.033) and relatively decreased preoperative Hb levels (P = 0.051, no significant difference) were found in the diverting colostomy group compared to the ITT group because 36 patients in the ITT group underwent colostomy for recto-vaginal fistulas or deep rectal ulcers instead of severe bleeding.\",\n \"In the colostomy group, the median duration of bleeding was 13.0 mo ± 4.3 mo, and 12 (86%) patients received transfusion before stoma compared to 8.0 mo ± 1.8 mo (P = 0.042) of bleeding duration and 6 (24%) (P < 0.001) transfusions in the conservative group. In the conservative group, 8 patients received formalin irrigation and 4 patients received APC treatments. The preoperative (Pre-Op) Hb was only 63 g/L ± 17.8 g/L in the colostomy group compared to 88.2 g/L ± 19.3 g/L (P < 0.001) in the conservative group. One patient with moderate anemia (Hb of 87 g/L) received colostomy for perianal pain. Another patient with severe anemia (Hb of 78 g/L) did not receive a transfusion due to unavailability of a blood supply before colostomy. The details of the 14 patients are listed in Table 2. Increasing Hb levels were observed from 3 mo (median Hb of 75 g/L), 6 mo (Hb of 99 g/L), and 9 mo (Hb of 107 g/L) to 1 year (Hb 111 of g/L) and 2 years (Hb 117 g/L) after colostomy (Figure 2). Postoperative transfusions were conducted in 3 of 14 patients (transfusion requirements for 2 patients in other hospitals was unknown) in the colostomy group. The dynamic remission rates between the colostomy and conservative groups after surgery were as follows: 86% vs 4% at 3 mo (P < 0.001), 86% vs 12% at 6 mo (P < 0.001), 93% vs 20% at 9 mo (P < 0.001), 100% vs 24% at 1 year (P < 0.001), and 100% vs 20% at 2 years (P < 0.001) (Table 1).\\nClinical details and outcomes of 14 colostomy patients\\nMedian value. Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin; RTD: Rectal telangiectasia density; APC: Argon plasma coagulation.\\nDynamic changes in hemoglobin levels. Pre-Op: Pre-operative.\",\n \"Among the 14 patients with severe bleeding treated by colostomy, all obtained complete remission of bleeding during follow-up. Only 3 (23%) of 13 patients received one round of APC after stoma to control bleeding, and 1 patient received 4% formalin irrigation after colostomy (Table 2). Among the 14 patients, 13 (93%) underwent stoma closure, and the remaining patient did not undergo stoma reversal due to concerns of surgical risks because of old age (83 years old). The median duration of stoma was 16 mo (range: 9-53 mo). Stoma complications occurred in 3 (21%) cases, including 1 para-stoma hernia, 1 stoma prolapse, and 1 stoma obstruction (Grade II by Clavien-Dindo classification)[23]. All 3 patients recovered well after stoma closure.\\nEndoscopic findings of bleeding, confluent telangiectasia, and congested mucosa improved dramatically at stoma reversal compared to before stoma creation (Figure 3). The bleeding score by CTCAE decreased from Pre-Op 2.7 points ± 0.5 points to 0.8 points ± 0.5 points (P < 0.001) at 1 year after stoma in the colostomy group; the bleeding score was 2.0 ± 0.5 at initial diagnosis and 1.7 ± 0.9 (P = 0.1282) at 1 year after treatments in the conservative group (Table 1 and Figure 4).\\nEndoscopic changes before stoma and at reversal.\\nCTC bleeding scores before stoma and at stoma reversal.\",\n \"Endorectal ultrasound (EUS) was analyzed retrospectively before stoma and at reversal in 5 patients; the remaining patients did not receive EUS at either of these two time points. In our previous study, thickening of the rectal wall, blurred wall stratification, and increased vascularity were EUS features in CRP[24]. In this study, we found similar features before stoma creation and a tremendous decrease in superficial vascularity, which can explain the cessation of bleeding (Figure 5).\\nEndoscopic ultrasound features before stoma and at stoma reversal.\",\n \"To evaluate the severity of the effect on the rectal wall and pelvic floor in the colostomy group, we also report magnetic resonance imaging (MRI) parameters related to CRP, including the thickness of the rectal wall, width of the presacral space, and thicknesses of the levator ani, the gluteus maximus muscle, the obturator intemus, and the distal part of the sigmoid colon (Table 3), which was referred to in our previous study of radiation injury to the pelvis[25]. MRI scans both at pre-colostomy and at reversal were analyzed in 5 patients, as the remaining 9 did not receive MRI scans both pre-colostomy and at reversal. The thickness of the rectal wall was significantly decreased at reversal (8.91 ± 1.61) compared with pre-colostomy (10.7 ± 4.29) (P = 0.047) (Table 3).\\nMagnetic resonance imaging findings at post-colostomy and at reversal compared in 5 patients with chronic radiation proctopathy\",\n \"Anorectal functions before stoma and after reversal were evaluated by CRPSS scores in all 12 patients in the colostomy group. The results showed that rectal bleeding, peritoneal pain, tenesmus, urgency and fecal/gas incontinency were significantly improved after stoma reversal compared to those before stoma creation. Additionally, all of these scores were < 2 points, which indicated that anorectal function recovered very well after stoma reversal (Figure 6). Complete remission of bleeding and good performance of anorectal function compared to the normal population (scores ≤ 1 point) were observed. We also analyzed anorectal manometry to objectively assess anorectal function before reversal in 6 of the 14 patients who received it. Increased sensitivity of the rectal mucosa and decreased rectal volume were observed. Decreased sphincter functions were found in 2 of 6 cases (Table 4). These patients fulfilled the indications for reversal, and we found that these patients obtained good performance of anorectal function after reversal. Rectal defecography was also conducted before reversal, and no stricture was observed.\\nRectal manometry assessment of anorectal functions before stoma reversals\\nAnorectal functions before stoma and after stoma reversal.\",\n \"QOL after stoma was assessed in the colostomy and conservative groups using EORTC QLQ-C30 questionnaires. As there are no similar reports in the Chinese population, we referred to the normal German population[26]. Osoba et al[26] suggested that a difference of ≥ 20 points in global health was considered to be clinically relevant. In the panel of patients who underwent stoma closure, QOL was dramatically improved compared to Pre-Op baseline QOL before stoma creation, including improved global health (difference of 40, P < 0.001), physical function (difference of 36.4, P < 0.001), role function (difference of 55, P < 0.001), emotional function (difference of 39.4, P < 0.001), social function (difference of 36.3, P < 0.001), and improved symptoms, such as fatigue (difference of -62.5, P < 0.001), pain (difference of -34.9, P < 0.001), dyspnea (difference of -37.8, P < 0.001), insomnia (difference of -36.2, P < 0.001), diarrhea (difference of -23.8, P < 0.001), and financial problems (difference of -35.5, P < 0.001). However, in the conservative group, no improved QOL variables with a difference of ≥ 20 were observed (Table 5). QOL after stoma closure was also compared to the normal population reference, with similar scores for functions and symptoms (Table 5).\\nQuality of life in the colostomy group and conservative group of chronic radiation proctopathy patients based on the EORTC QLQ-C30 scale\\nWilcoxon rank-sum test. \\nPoint (follow-up) – point (pretreatment). Δ(FU): Change in follow-up; SD: Standard deviation.\",\n \"The literature reports that 1%-6% of CRP patients have transfusion-dependent bleeding; many of these cases can be treated with topical formalin, APC, or hyperbaric oxygen treatment[1]. Surgical resection of rectal lesions is usually reserved as the last resort for hemorrhagic CRP because it is associated with high morbidity and mortality[3,13,19]. In our previous study, fecal diversion was found to be a simple, effective, and safe procedure for severe hemorrhagic CRP[16]. Theoretically, fecal diversion can reduce the irritation of stool and accelerate the course of fibrosis and thus relieve CRP bleeding rapidly[16].\\nIn this study, we report results for diverting colostomy and conservative treatments of enemas, topical formalin, or APC in a large cohort of patients with severe hemorrhagic CRP and who were followed for at least 1 year. The results showed that stomas can be reversed in 93% of CRP cases with severe hemorrhage. Increased Hb and rapid cessation of bleeding were found in the colostomy group, whereas bleeding and Hb levels were not changed in the conservative group. Moreover, anorectal function was greatly improved after stoma reversal than before stoma creation, reaching the levels of the normal population. In addition, it is important to note that colostomy was performed in a very select population of CRP patients and those patients with CRP stricture and ulcerations were excluded. Stoma complications occurred in 21% of cases, including 1 case of parastomal hernia due to a weakened abdominal wall of the parastomal zone, 1 case of stoma prolapse due to an intestine overly pulled at stoma creation, and 1 case of stoma obstruction due to stricture. All of these complications were recovered after stoma reversal. According to the literature, the complication rate of stoma is approximately 20%-50%, and the complication rate of stoma in this study is thus acceptable. Thus, colostomy is a better option and can bring more benefits to CRP patients with severe bleeding than can conservative treatments.\\nIn our previous study, we found complete remission of telangiectasia and edema, recovery of mucosal integrity, and massive fibrosis in the submucosa at 3-4 years after diversion[16]. In this study, anorectal manometry was conducted in some stoma patients and resulted in good compliance, resting pressure, and contraction. Rectal defecography was performed at reversal for all colostomy patients, and no rectal stricture was found. Thus, temporary diverting colostomy and reversal at an appropriate time can be a useful option for these patients.\\nIn this study, most patients in the colostomy group presented with severe anemia and needed multiple transfusions. One patient did not receive transfusion, with an Hb level of 78, before colostomy. In China, blood supply is limited, and transfusion is only conducted in large central hospitals when Hb is < 60. At 3 to 6 mo after diverting colostomy, bleeding gradually remitted and increased Hb levels were observed. By 9 mo to 1 year after colostomy, most patients achieved complete remission of bleeding and the Hb level had almost returned to normal. Stoma reversal was conducted at 1 year after colostomy in 4 (31%) of 13 patients. In 5 cases, reversal was performed at more than 2 years after colostomy because the patients did not know colostomy could be reversed until we contacted them via telephone for follow-up. Thus, the median duration of stoma was prolonged to 16 mo. In fact, most stomas in this cohort could be reversed at 1 year after colostomy.\\nBased on the results of our retrospective study[16], we started a prospective clinical trial to enroll severe hemorrhagic CRP patients who are suitable for diverting colostomy (Clinical Trial No. NCT03397901). All patients were followed and evaluated for stoma closure for 1 year after colostomy. The indication for diverting colostomy was extended to hemorrhagic CRP with moderate anemia in our series because preventative colostomy can relieve refractory bleeding before it progresses to severe life-threatening anemia and patients can obtain dramatic benefit from preventative fecal diversion.\\nTo date, it is still unclear whether anorectal function can be preserved after stoma closure. In this study, we found that anorectal functions, including anal control and release of gas/stool, could be restored, which also indicated that CRP was inactive after a period of diverting colostomy. Although increased rectal sensitivity and decreased rectal volume were observed at reversal by anorectal manometry, good anorectal function was achieved after reversal compared to the normal population and improved compared to that before stoma creation. No additional pelvic biofeedback treatments were required in these patients.\\nFor some patients, an additional one to two rounds of APC were required to help control bleeding during the first 6 mo after colostomy. The temporary stoma not only enabled the remission of anal symptoms but also restored the biological functions of the anus, providing evidence to support fecal diversion as a helpful option for severe hemorrhagic CRP patients.\\nIn some studies, bile acid malabsorption and small bowel bacterial overgrowth have been thought to be the cause of diarrhea during acute and chronic radiation enteritis[1]. Diverting colostomy cannot reverse these changes. We focused on hemorrhage in CRP and diarrhea, common in small-bowel radiation injury, which is different from tenesmus and is not the major symptom in most CRP cases.\\nAccording to our previous study, severe hemorrhagic CRP patients usually experience poor global health and fatigue due to moderate to severe anemia[16]. Their social and emotional functions are also impacted by frequent stool and anxiety of bleeding. They cannot attend social activities due to concern of looking for a lavatory[1]. In this study, after the remission of anal symptoms, we found that patients had good QOL, especially after stoma reversal, and were able to return to almost the same life as the normal population.\\nAnorectal functions scored by the CRPSS were assessed before stoma and after reversal. We also analyzed EUS and pelvic MRI before stoma and at reversal to evaluate the severity of CRP. To objectively evaluate anorectal function before reversal, anorectal manometry was performed and retrospectively analyzed in some patients, though only in 6 patients because manometry is not a routine test before stoma reversal.\\nThis study presents our serial results of diverting colostomy for severe hemorrhagic CRP. The results showed significant superiority over conservative treatments. However, there are several limitations to this study. First, this study was retrospective, with potential recall bias. Second, the sample size of this cohort was relatively small, which will affect the grade of evidence, and a larger cohort is essential. Finally, EUS, MRI, and anorectal manometry were only performed on patients undergoing colostomy reversal as a limitation. Thus, we started a prospective cohort of diverting colostomy for severe hemorrhagic CRP and will provide more reliable evidence in the future.\\nIn conclusion, diverting colostomy is a very effective and rapid method for the remission of severe bleeding in CRP patients. The stoma can be reversed and good anorectal function can be restored after reversal in most patients.\",\n \" Research background Chronic radiation proctopathy (CRP) is a common and sometimes difficult issue after radiotherapy for pelvic malignancies. Severe and refractory bleeding is hard to manage. Diverting fecal diversion is very effective and fast in remission of rectal bleeding in the management of severe CRP bleeding in our previous study, but diverting stoma is usually thought to be permanent according to the literature. The anorectal function and quality of life after stoma reversal remain unclear.\\nChronic radiation proctopathy (CRP) is a common and sometimes difficult issue after radiotherapy for pelvic malignancies. Severe and refractory bleeding is hard to manage. Diverting fecal diversion is very effective and fast in remission of rectal bleeding in the management of severe CRP bleeding in our previous study, but diverting stoma is usually thought to be permanent according to the literature. The anorectal function and quality of life after stoma reversal remain unclear.\\n Research motivation During the past 3 years, we have successfully performed colostomy reversals in a larger cohort of CRP patients with severe bleeding and followed patients after colostomy reversals. In this series study, we will report the efficacy of colostomy, the rate of stoma closure of diverting colostomy, and anorectal function after reversals.\\nDuring the past 3 years, we have successfully performed colostomy reversals in a larger cohort of CRP patients with severe bleeding and followed patients after colostomy reversals. In this series study, we will report the efficacy of colostomy, the rate of stoma closure of diverting colostomy, and anorectal function after reversals.\\n Research objectives The aim of this study was to evaluate the efficacy of colostomy and the rate of stoma reversals in severe hemorrhagic CRP.\\nThe aim of this study was to evaluate the efficacy of colostomy and the rate of stoma reversals in severe hemorrhagic CRP.\\n Research methods Patients with severe hemorrhagic CRP who underwent colostomy or conservative treatment were enrolled retrospectively. Rectal bleeding, hemoglobin (Hb), endoscopic features, endo-ultrasound (EUS), rectal manometry, and MRI scans were recorded. Anorectal functions and quality of life before stoma and after stoma reversal were scored with EORTC-QOL-C30 questionnaires.\\nPatients with severe hemorrhagic CRP who underwent colostomy or conservative treatment were enrolled retrospectively. Rectal bleeding, hemoglobin (Hb), endoscopic features, endo-ultrasound (EUS), rectal manometry, and MRI scans were recorded. Anorectal functions and quality of life before stoma and after stoma reversal were scored with EORTC-QOL-C30 questionnaires.\\n Research results After screening 738 continual CRP patients, 14 patients in the colostomy group and 25 patients in the conservative group as controls were enrolled. The Hb was gradually increased to normal levels in two years after colostomy, while no significant increase was observed in the conservative group. All of 14 patients obtained complete remission of bleeding and colostomy was successfully reversed in 13 of 14 (93%), except one with very old age. Improved endoscopic telangiectasia and bleeding, decreased vascularity by EUS, increased presarcal space, and thickened rectal wall by MRI were observed. Anorectal functions and quality of life were significantly improved after stoma reverse.\\nAfter screening 738 continual CRP patients, 14 patients in the colostomy group and 25 patients in the conservative group as controls were enrolled. The Hb was gradually increased to normal levels in two years after colostomy, while no significant increase was observed in the conservative group. All of 14 patients obtained complete remission of bleeding and colostomy was successfully reversed in 13 of 14 (93%), except one with very old age. Improved endoscopic telangiectasia and bleeding, decreased vascularity by EUS, increased presarcal space, and thickened rectal wall by MRI were observed. Anorectal functions and quality of life were significantly improved after stoma reverse.\\n Research conclusions Diverting colostomy is a very effective method in the remission of refractory hemorrhagic CRP. Meanwhile, stoma can be reversed and anorectal functions can be recovered after reversal.\\nDiverting colostomy is a very effective method in the remission of refractory hemorrhagic CRP. Meanwhile, stoma can be reversed and anorectal functions can be recovered after reversal.\\n Research perspectives Preventative diverting colostomy can relieve refractory bleeding before it progresses to severe life-threatening anemia and patients can obtain dramatic benefit. Thus, we have started a prospective clinical trial to enroll severe hemorrhagic CRP patients who are suitable for diverting colostomy (Clinical Trial No. NCT03397901), which will provide more evidence to the usage of colostomy in severe CRP patients.\\nPreventative diverting colostomy can relieve refractory bleeding before it progresses to severe life-threatening anemia and patients can obtain dramatic benefit. Thus, we have started a prospective clinical trial to enroll severe hemorrhagic CRP patients who are suitable for diverting colostomy (Clinical Trial No. NCT03397901), which will provide more evidence to the usage of colostomy in severe CRP patients.\",\n \"Chronic radiation proctopathy (CRP) is a common and sometimes difficult issue after radiotherapy for pelvic malignancies. Severe and refractory bleeding is hard to manage. Diverting fecal diversion is very effective and fast in remission of rectal bleeding in the management of severe CRP bleeding in our previous study, but diverting stoma is usually thought to be permanent according to the literature. The anorectal function and quality of life after stoma reversal remain unclear.\",\n \"During the past 3 years, we have successfully performed colostomy reversals in a larger cohort of CRP patients with severe bleeding and followed patients after colostomy reversals. In this series study, we will report the efficacy of colostomy, the rate of stoma closure of diverting colostomy, and anorectal function after reversals.\",\n \"The aim of this study was to evaluate the efficacy of colostomy and the rate of stoma reversals in severe hemorrhagic CRP.\",\n \"Patients with severe hemorrhagic CRP who underwent colostomy or conservative treatment were enrolled retrospectively. Rectal bleeding, hemoglobin (Hb), endoscopic features, endo-ultrasound (EUS), rectal manometry, and MRI scans were recorded. Anorectal functions and quality of life before stoma and after stoma reversal were scored with EORTC-QOL-C30 questionnaires.\",\n \"After screening 738 continual CRP patients, 14 patients in the colostomy group and 25 patients in the conservative group as controls were enrolled. The Hb was gradually increased to normal levels in two years after colostomy, while no significant increase was observed in the conservative group. All of 14 patients obtained complete remission of bleeding and colostomy was successfully reversed in 13 of 14 (93%), except one with very old age. Improved endoscopic telangiectasia and bleeding, decreased vascularity by EUS, increased presarcal space, and thickened rectal wall by MRI were observed. Anorectal functions and quality of life were significantly improved after stoma reverse.\",\n \"Diverting colostomy is a very effective method in the remission of refractory hemorrhagic CRP. Meanwhile, stoma can be reversed and anorectal functions can be recovered after reversal.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"keywords":{"kind":"list like","value":["Chronic radiation proctitis","Hemorrhage","Colostomy","Anorectal function","Quality of life"],"string":"[\n \"Chronic radiation proctitis\",\n \"Hemorrhage\",\n \"Colostomy\",\n \"Anorectal function\",\n \"Quality of life\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nChronic radiation proctopathy (CRP) is a common and sometimes difficult issue after radiotherapy for pelvic malignancies. A permanent change in bowel habits may occur in 90% of patients. After pelvic radiotherapy, 20%-50% of patients will develop difficult bowel function, affecting their quality of life (QOL)[1,2]. CRP can cause more than 20 different symptoms. Rectal bleeding is the most common symptom and occurs in > 50% of CRP patients[1,3]. Transfusion-dependent severe bleeding occurs in 1%-5% of patients[1]. Pathologically, ischemia in the submucosa due to obliterative endarteritis and progressive fibrosis in macroscopic changes are the main causes[4].\nMedical treatment consists of topical sucralfate enemas[5], oral or topical sulfasalazine[6], and rebamipide[7]. However, these reagents are only effective in acute or mild CRP, whereas their efficacy is usually disappointing in CRP patients with moderate to severe hemorrhage. More invasive modalities include endoscopic argon plasma coagulation (APC)[8], topical 4%-10% formalin[9-12], radiofrequency ablation[13], and hyperbaric oxygen therapy[14,15], and are currently popular optional treatments for CRP. Most of these modalities lack randomized trial evidence. These treatment options are reported to be effective in controlling mild to moderate bleeding in most of the literature. Nonetheless, severe and refractory bleeding is difficult to manage[1,16]. Furthermore, APC or topical formalin requires multiple sessions for severe bleeding and can cause severe side effects, including perforation, strictures, and perianal pain[17].\nFecal diversion is reported to be effective in the management of severe CRP bleeding[16,18,19], as it can reduce irritation injury to the lesions to decrease hemorrhage[16]. However, the usage of fecal diversion is not adopted as widely as is APC or formalin. Most of the fecal diversion data are from the 1980s, and this option has not been well studied to date. We previously reported one retrospective cohort of CRP patients with severe hemorrhage who received colostomy[16]. The results showed that colostomy can bring much higher remission of severe bleeding (94%) than can conservative treatment (11%) with APC or topical formalin. Pathologically, chronic inflammation and progressive fibrosis are observed after stoma. Diverting stoma is usually thought to be permanent according to Ayerdi et al[20].\nAnorectal function and QOL after stoma reversal remain unclear. During the past 3 years, we have successfully performed colostomy reversals in a large cohort of CRP patients with severe bleeding and have follow-up data after colostomy reversal. Here, we report the efficacy of colostomy, the rate of stoma closure of diverting colostomy, and anorectal function after reversal for this series of CRP patients with severe bleeding.\n\nMATERIALS AND METHODS:\n Patients and ethical statements Patients with hemorrhagic CRP who were treated after admission at The Sixth Affiliated Hospital of Sun Yat-Sen University from March 2008 to December 2018 were enrolled. Medical records and imaging and clinicopathological data were extracted from our electronic database. This study was approved by the Ethical Committee of The Sixth Affiliated Hospital of Sun Yat-Sen University and was performed according to the provisions of the World Medical Association’s Declaration of Helsinki in 1995 (updated in Tokyo, 2004). Due to the nature of a retrospective study, informed consent was waived.\nPatients with hemorrhagic CRP who were treated after admission at The Sixth Affiliated Hospital of Sun Yat-Sen University from March 2008 to December 2018 were enrolled. Medical records and imaging and clinicopathological data were extracted from our electronic database. This study was approved by the Ethical Committee of The Sixth Affiliated Hospital of Sun Yat-Sen University and was performed according to the provisions of the World Medical Association’s Declaration of Helsinki in 1995 (updated in Tokyo, 2004). Due to the nature of a retrospective study, informed consent was waived.\n Inclusion and exclusion criteria CRP patients with refractory rectal bleeding who received diverting colostomy or conservative treatments for severe anemia and who needed transfusions were enrolled. Patients who were lost to follow-up, had tumor recurrence, rectal-vaginal fistula or other types of rectal fistulas, or underwent rectal resection with preventive colostomy were excluded because these conditions would affect the evaluation of remission of rectal bleeding.\nCRP patients with refractory rectal bleeding who received diverting colostomy or conservative treatments for severe anemia and who needed transfusions were enrolled. Patients who were lost to follow-up, had tumor recurrence, rectal-vaginal fistula or other types of rectal fistulas, or underwent rectal resection with preventive colostomy were excluded because these conditions would affect the evaluation of remission of rectal bleeding.\n Diagnosis, definitions, and scores CRP was diagnosed by the combination of pelvic radiation history for malignancies, symptoms such as rectal bleeding, and endoscopic findings of CRP-specific changes such as telangiectasia and active bleeding in the rectum, as well as exclusion of other bleeding diseases. Hemorrhagic problems, such as tumor recurrence and hemorrhoids, were excluded. Refractory severe bleeding was defined as frequent bleeding and severe anemia with the need for transfusions. At admission, CRP patients in our center were first referred to noninvasive enemas, and if there was no response or recurrence, invasive treatment, such as APC (1-2 rounds) or formalin topical irrigation, was utilized. Conservative treatment failure was defined as no response or recurrence of frequent or severe rectal bleeding after invasive treatments.\nIn this study, we used a modified subjective-objective management analysis (known as mSOMA) system that we designed previously to assess the severity of rectal bleeding[16]. The advantages of the mSOMA system include both subjective complaints of patients and objective hemoglobin (Hb) levels according to laboratory tests. Remission of bleeding was defined as complete cessation or occasional bleeding that needed no further treatment. The CTCAE score of bleeding was used to assess its severity.\nCRP was diagnosed by the combination of pelvic radiation history for malignancies, symptoms such as rectal bleeding, and endoscopic findings of CRP-specific changes such as telangiectasia and active bleeding in the rectum, as well as exclusion of other bleeding diseases. Hemorrhagic problems, such as tumor recurrence and hemorrhoids, were excluded. Refractory severe bleeding was defined as frequent bleeding and severe anemia with the need for transfusions. At admission, CRP patients in our center were first referred to noninvasive enemas, and if there was no response or recurrence, invasive treatment, such as APC (1-2 rounds) or formalin topical irrigation, was utilized. Conservative treatment failure was defined as no response or recurrence of frequent or severe rectal bleeding after invasive treatments.\nIn this study, we used a modified subjective-objective management analysis (known as mSOMA) system that we designed previously to assess the severity of rectal bleeding[16]. The advantages of the mSOMA system include both subjective complaints of patients and objective hemoglobin (Hb) levels according to laboratory tests. Remission of bleeding was defined as complete cessation or occasional bleeding that needed no further treatment. The CTCAE score of bleeding was used to assess its severity.\n Indications for fecal diversion and stoma closure The indications for diverting diversion in hemorrhagic CRP contained the following conditions: Recurrent bleeding and unresponsive to conservative treatments such as endoscopic APC or topical formalin, accompanying severe anemia, and transfusions needed. Transverse loop colostomy and “gunsight” or “cross‘’ type of stoma closure were created according to the standard protocols used in our previous studies[16]. The criteria of stoma closure were as follows: Remission or occasional rectal bleeding, regressive edema in rectal mucosa, good performance of anorectal function, exclusion of tumor recurrence, and no severe CRP complications such as fistula, stricture, and deep ulcer[16].\nThe indications for diverting diversion in hemorrhagic CRP contained the following conditions: Recurrent bleeding and unresponsive to conservative treatments such as endoscopic APC or topical formalin, accompanying severe anemia, and transfusions needed. Transverse loop colostomy and “gunsight” or “cross‘’ type of stoma closure were created according to the standard protocols used in our previous studies[16]. The criteria of stoma closure were as follows: Remission or occasional rectal bleeding, regressive edema in rectal mucosa, good performance of anorectal function, exclusion of tumor recurrence, and no severe CRP complications such as fistula, stricture, and deep ulcer[16].\n Follow-up Patients were scheduled for follow-up through outpatient visits or telephone questionnaires at 6 mo, 9 mo, 1 year, 1.5 years, and 2 years after colostomy. QOL after stoma closure was evaluated according to EORTC QLQ C30 questionnaires[21]. We focused on the following items in this study: The remission rate of bleeding, the rate of stoma reversal, QOL after stoma reversal, dynamic changes of Hb levels, stoma-related complications, and severe CRP complications.\nPatients were scheduled for follow-up through outpatient visits or telephone questionnaires at 6 mo, 9 mo, 1 year, 1.5 years, and 2 years after colostomy. QOL after stoma closure was evaluated according to EORTC QLQ C30 questionnaires[21]. We focused on the following items in this study: The remission rate of bleeding, the rate of stoma reversal, QOL after stoma reversal, dynamic changes of Hb levels, stoma-related complications, and severe CRP complications.\n Anorectal functions In our previous study, tenesmus, stool frequency, and anorectal pain were common symptoms in active CRP[22]. Because there are no standard scales or scores to assess anorectal function precisely in CRP patients, we developed a new scale system that is a patient self-reported scale of outcomes, namely, the chronic radiation proctopathy symptom scale (CRPSS). The CRPSS considers the following items: Watery stool, urgency, perianal pain, tenesmus, rectal bleeding, and fecal/gas incontinence. The details are provided in Supplementary File 1. For CRP patients treated by colostomy, CRPSS scores were evaluated before stoma and at reversal.\nIn our previous study, tenesmus, stool frequency, and anorectal pain were common symptoms in active CRP[22]. Because there are no standard scales or scores to assess anorectal function precisely in CRP patients, we developed a new scale system that is a patient self-reported scale of outcomes, namely, the chronic radiation proctopathy symptom scale (CRPSS). The CRPSS considers the following items: Watery stool, urgency, perianal pain, tenesmus, rectal bleeding, and fecal/gas incontinence. The details are provided in Supplementary File 1. For CRP patients treated by colostomy, CRPSS scores were evaluated before stoma and at reversal.\n Statistical analysis Analyses for continuous variables were performed by Student’s t-test. The χ2 test was used for categorical variables. Fisher’s exact test was conducted when appropriate. For non-parameter variables, the Wilcoxon rank sum tests were used. All statistical analyses were performed using SPSS version 23.0 (Chicago, IL, United States). P < 0.05 (two-tailed) was considered statistically significant.\nAnalyses for continuous variables were performed by Student’s t-test. The χ2 test was used for categorical variables. Fisher’s exact test was conducted when appropriate. For non-parameter variables, the Wilcoxon rank sum tests were used. All statistical analyses were performed using SPSS version 23.0 (Chicago, IL, United States). P < 0.05 (two-tailed) was considered statistically significant.\n\nPatients and ethical statements:\nPatients with hemorrhagic CRP who were treated after admission at The Sixth Affiliated Hospital of Sun Yat-Sen University from March 2008 to December 2018 were enrolled. Medical records and imaging and clinicopathological data were extracted from our electronic database. This study was approved by the Ethical Committee of The Sixth Affiliated Hospital of Sun Yat-Sen University and was performed according to the provisions of the World Medical Association’s Declaration of Helsinki in 1995 (updated in Tokyo, 2004). Due to the nature of a retrospective study, informed consent was waived.\n\nInclusion and exclusion criteria:\nCRP patients with refractory rectal bleeding who received diverting colostomy or conservative treatments for severe anemia and who needed transfusions were enrolled. Patients who were lost to follow-up, had tumor recurrence, rectal-vaginal fistula or other types of rectal fistulas, or underwent rectal resection with preventive colostomy were excluded because these conditions would affect the evaluation of remission of rectal bleeding.\n\nDiagnosis, definitions, and scores:\nCRP was diagnosed by the combination of pelvic radiation history for malignancies, symptoms such as rectal bleeding, and endoscopic findings of CRP-specific changes such as telangiectasia and active bleeding in the rectum, as well as exclusion of other bleeding diseases. Hemorrhagic problems, such as tumor recurrence and hemorrhoids, were excluded. Refractory severe bleeding was defined as frequent bleeding and severe anemia with the need for transfusions. At admission, CRP patients in our center were first referred to noninvasive enemas, and if there was no response or recurrence, invasive treatment, such as APC (1-2 rounds) or formalin topical irrigation, was utilized. Conservative treatment failure was defined as no response or recurrence of frequent or severe rectal bleeding after invasive treatments.\nIn this study, we used a modified subjective-objective management analysis (known as mSOMA) system that we designed previously to assess the severity of rectal bleeding[16]. The advantages of the mSOMA system include both subjective complaints of patients and objective hemoglobin (Hb) levels according to laboratory tests. Remission of bleeding was defined as complete cessation or occasional bleeding that needed no further treatment. The CTCAE score of bleeding was used to assess its severity.\n\nIndications for fecal diversion and stoma closure:\nThe indications for diverting diversion in hemorrhagic CRP contained the following conditions: Recurrent bleeding and unresponsive to conservative treatments such as endoscopic APC or topical formalin, accompanying severe anemia, and transfusions needed. Transverse loop colostomy and “gunsight” or “cross‘’ type of stoma closure were created according to the standard protocols used in our previous studies[16]. The criteria of stoma closure were as follows: Remission or occasional rectal bleeding, regressive edema in rectal mucosa, good performance of anorectal function, exclusion of tumor recurrence, and no severe CRP complications such as fistula, stricture, and deep ulcer[16].\n\nFollow-up:\nPatients were scheduled for follow-up through outpatient visits or telephone questionnaires at 6 mo, 9 mo, 1 year, 1.5 years, and 2 years after colostomy. QOL after stoma closure was evaluated according to EORTC QLQ C30 questionnaires[21]. We focused on the following items in this study: The remission rate of bleeding, the rate of stoma reversal, QOL after stoma reversal, dynamic changes of Hb levels, stoma-related complications, and severe CRP complications.\n\nAnorectal functions:\nIn our previous study, tenesmus, stool frequency, and anorectal pain were common symptoms in active CRP[22]. Because there are no standard scales or scores to assess anorectal function precisely in CRP patients, we developed a new scale system that is a patient self-reported scale of outcomes, namely, the chronic radiation proctopathy symptom scale (CRPSS). The CRPSS considers the following items: Watery stool, urgency, perianal pain, tenesmus, rectal bleeding, and fecal/gas incontinence. The details are provided in Supplementary File 1. For CRP patients treated by colostomy, CRPSS scores were evaluated before stoma and at reversal.\n\nStatistical analysis:\nAnalyses for continuous variables were performed by Student’s t-test. The χ2 test was used for categorical variables. Fisher’s exact test was conducted when appropriate. For non-parameter variables, the Wilcoxon rank sum tests were used. All statistical analyses were performed using SPSS version 23.0 (Chicago, IL, United States). P < 0.05 (two-tailed) was considered statistically significant.\n\nRESULTS:\n Demographics and clinical outcomes From March 2008 to Feb 2019, 738 continual CRP patients treated in our center were screened. After exclusion of 644 patients with mild or moderate CRP, 94 with refractory bleeding and moderate to severe anemia were further screened. Among them, 50 patients were treated by diverted colostomy and 44 patients were treated with conservative therapies. Among the 50 patients treated by colostomy, 32 were excluded due to recto-vaginal fistulas or deep rectal ulcer with refractory perianal pain before stoma. The remaining 18 patients with refractory bleeding were enrolled. Among them, two died from cancer recurrence and two were lost to follow-up. The remaining 14 patients were enrolled in the final analysis as the colostomy group. For the conservative group, 19 patients were excluded, including 11 with tumor relapse and 8 who were lost to follow-up. Thus, 25 were enrolled in the final analysis (Figure 1). Demographic and baseline characteristics were collected, including the intention-to-treat (ITT) group of 50 colostomies. In the enrolled patients, 14 patients comprised the diverting colostomy group, and 25 cases comprised the conservative treatment group. The primary tumors in most of the patients (33/39, 85%) were cervical cancers. No significant differences in age, sex, type of primary tumor, or radiation dosage were found between the diverting colostomy group and the conservative treatment group or between the ITT colostomy group and the colostomy group (Table 1).\nDemographics and characteristics\nFisher's exact test. ITT: Intention-to-treat; Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin.\nFlow chart of patient selection.\nIn the ITT group, no postoperative follow-ups were conducted other than for the 14 investigated colostomy patients. Higher bleeding scores (P = 0.033) and relatively decreased preoperative Hb levels (P = 0.051, no significant difference) were found in the diverting colostomy group compared to the ITT group because 36 patients in the ITT group underwent colostomy for recto-vaginal fistulas or deep rectal ulcers instead of severe bleeding.\nFrom March 2008 to Feb 2019, 738 continual CRP patients treated in our center were screened. After exclusion of 644 patients with mild or moderate CRP, 94 with refractory bleeding and moderate to severe anemia were further screened. Among them, 50 patients were treated by diverted colostomy and 44 patients were treated with conservative therapies. Among the 50 patients treated by colostomy, 32 were excluded due to recto-vaginal fistulas or deep rectal ulcer with refractory perianal pain before stoma. The remaining 18 patients with refractory bleeding were enrolled. Among them, two died from cancer recurrence and two were lost to follow-up. The remaining 14 patients were enrolled in the final analysis as the colostomy group. For the conservative group, 19 patients were excluded, including 11 with tumor relapse and 8 who were lost to follow-up. Thus, 25 were enrolled in the final analysis (Figure 1). Demographic and baseline characteristics were collected, including the intention-to-treat (ITT) group of 50 colostomies. In the enrolled patients, 14 patients comprised the diverting colostomy group, and 25 cases comprised the conservative treatment group. The primary tumors in most of the patients (33/39, 85%) were cervical cancers. No significant differences in age, sex, type of primary tumor, or radiation dosage were found between the diverting colostomy group and the conservative treatment group or between the ITT colostomy group and the colostomy group (Table 1).\nDemographics and characteristics\nFisher's exact test. ITT: Intention-to-treat; Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin.\nFlow chart of patient selection.\nIn the ITT group, no postoperative follow-ups were conducted other than for the 14 investigated colostomy patients. Higher bleeding scores (P = 0.033) and relatively decreased preoperative Hb levels (P = 0.051, no significant difference) were found in the diverting colostomy group compared to the ITT group because 36 patients in the ITT group underwent colostomy for recto-vaginal fistulas or deep rectal ulcers instead of severe bleeding.\n Dynamically increasing hemoglobin levels after colostomy In the colostomy group, the median duration of bleeding was 13.0 mo ± 4.3 mo, and 12 (86%) patients received transfusion before stoma compared to 8.0 mo ± 1.8 mo (P = 0.042) of bleeding duration and 6 (24%) (P < 0.001) transfusions in the conservative group. In the conservative group, 8 patients received formalin irrigation and 4 patients received APC treatments. The preoperative (Pre-Op) Hb was only 63 g/L ± 17.8 g/L in the colostomy group compared to 88.2 g/L ± 19.3 g/L (P < 0.001) in the conservative group. One patient with moderate anemia (Hb of 87 g/L) received colostomy for perianal pain. Another patient with severe anemia (Hb of 78 g/L) did not receive a transfusion due to unavailability of a blood supply before colostomy. The details of the 14 patients are listed in Table 2. Increasing Hb levels were observed from 3 mo (median Hb of 75 g/L), 6 mo (Hb of 99 g/L), and 9 mo (Hb of 107 g/L) to 1 year (Hb 111 of g/L) and 2 years (Hb 117 g/L) after colostomy (Figure 2). Postoperative transfusions were conducted in 3 of 14 patients (transfusion requirements for 2 patients in other hospitals was unknown) in the colostomy group. The dynamic remission rates between the colostomy and conservative groups after surgery were as follows: 86% vs 4% at 3 mo (P < 0.001), 86% vs 12% at 6 mo (P < 0.001), 93% vs 20% at 9 mo (P < 0.001), 100% vs 24% at 1 year (P < 0.001), and 100% vs 20% at 2 years (P < 0.001) (Table 1).\nClinical details and outcomes of 14 colostomy patients\nMedian value. Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin; RTD: Rectal telangiectasia density; APC: Argon plasma coagulation.\nDynamic changes in hemoglobin levels. Pre-Op: Pre-operative.\nIn the colostomy group, the median duration of bleeding was 13.0 mo ± 4.3 mo, and 12 (86%) patients received transfusion before stoma compared to 8.0 mo ± 1.8 mo (P = 0.042) of bleeding duration and 6 (24%) (P < 0.001) transfusions in the conservative group. In the conservative group, 8 patients received formalin irrigation and 4 patients received APC treatments. The preoperative (Pre-Op) Hb was only 63 g/L ± 17.8 g/L in the colostomy group compared to 88.2 g/L ± 19.3 g/L (P < 0.001) in the conservative group. One patient with moderate anemia (Hb of 87 g/L) received colostomy for perianal pain. Another patient with severe anemia (Hb of 78 g/L) did not receive a transfusion due to unavailability of a blood supply before colostomy. The details of the 14 patients are listed in Table 2. Increasing Hb levels were observed from 3 mo (median Hb of 75 g/L), 6 mo (Hb of 99 g/L), and 9 mo (Hb of 107 g/L) to 1 year (Hb 111 of g/L) and 2 years (Hb 117 g/L) after colostomy (Figure 2). Postoperative transfusions were conducted in 3 of 14 patients (transfusion requirements for 2 patients in other hospitals was unknown) in the colostomy group. The dynamic remission rates between the colostomy and conservative groups after surgery were as follows: 86% vs 4% at 3 mo (P < 0.001), 86% vs 12% at 6 mo (P < 0.001), 93% vs 20% at 9 mo (P < 0.001), 100% vs 24% at 1 year (P < 0.001), and 100% vs 20% at 2 years (P < 0.001) (Table 1).\nClinical details and outcomes of 14 colostomy patients\nMedian value. Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin; RTD: Rectal telangiectasia density; APC: Argon plasma coagulation.\nDynamic changes in hemoglobin levels. Pre-Op: Pre-operative.\n Stoma closure and endoscopic features Among the 14 patients with severe bleeding treated by colostomy, all obtained complete remission of bleeding during follow-up. Only 3 (23%) of 13 patients received one round of APC after stoma to control bleeding, and 1 patient received 4% formalin irrigation after colostomy (Table 2). Among the 14 patients, 13 (93%) underwent stoma closure, and the remaining patient did not undergo stoma reversal due to concerns of surgical risks because of old age (83 years old). The median duration of stoma was 16 mo (range: 9-53 mo). Stoma complications occurred in 3 (21%) cases, including 1 para-stoma hernia, 1 stoma prolapse, and 1 stoma obstruction (Grade II by Clavien-Dindo classification)[23]. All 3 patients recovered well after stoma closure.\nEndoscopic findings of bleeding, confluent telangiectasia, and congested mucosa improved dramatically at stoma reversal compared to before stoma creation (Figure 3). The bleeding score by CTCAE decreased from Pre-Op 2.7 points ± 0.5 points to 0.8 points ± 0.5 points (P < 0.001) at 1 year after stoma in the colostomy group; the bleeding score was 2.0 ± 0.5 at initial diagnosis and 1.7 ± 0.9 (P = 0.1282) at 1 year after treatments in the conservative group (Table 1 and Figure 4).\nEndoscopic changes before stoma and at reversal.\nCTC bleeding scores before stoma and at stoma reversal.\nAmong the 14 patients with severe bleeding treated by colostomy, all obtained complete remission of bleeding during follow-up. Only 3 (23%) of 13 patients received one round of APC after stoma to control bleeding, and 1 patient received 4% formalin irrigation after colostomy (Table 2). Among the 14 patients, 13 (93%) underwent stoma closure, and the remaining patient did not undergo stoma reversal due to concerns of surgical risks because of old age (83 years old). The median duration of stoma was 16 mo (range: 9-53 mo). Stoma complications occurred in 3 (21%) cases, including 1 para-stoma hernia, 1 stoma prolapse, and 1 stoma obstruction (Grade II by Clavien-Dindo classification)[23]. All 3 patients recovered well after stoma closure.\nEndoscopic findings of bleeding, confluent telangiectasia, and congested mucosa improved dramatically at stoma reversal compared to before stoma creation (Figure 3). The bleeding score by CTCAE decreased from Pre-Op 2.7 points ± 0.5 points to 0.8 points ± 0.5 points (P < 0.001) at 1 year after stoma in the colostomy group; the bleeding score was 2.0 ± 0.5 at initial diagnosis and 1.7 ± 0.9 (P = 0.1282) at 1 year after treatments in the conservative group (Table 1 and Figure 4).\nEndoscopic changes before stoma and at reversal.\nCTC bleeding scores before stoma and at stoma reversal.\n Ultrasound findings before stoma and at reversal Endorectal ultrasound (EUS) was analyzed retrospectively before stoma and at reversal in 5 patients; the remaining patients did not receive EUS at either of these two time points. In our previous study, thickening of the rectal wall, blurred wall stratification, and increased vascularity were EUS features in CRP[24]. In this study, we found similar features before stoma creation and a tremendous decrease in superficial vascularity, which can explain the cessation of bleeding (Figure 5).\nEndoscopic ultrasound features before stoma and at stoma reversal.\nEndorectal ultrasound (EUS) was analyzed retrospectively before stoma and at reversal in 5 patients; the remaining patients did not receive EUS at either of these two time points. In our previous study, thickening of the rectal wall, blurred wall stratification, and increased vascularity were EUS features in CRP[24]. In this study, we found similar features before stoma creation and a tremendous decrease in superficial vascularity, which can explain the cessation of bleeding (Figure 5).\nEndoscopic ultrasound features before stoma and at stoma reversal.\n Improved magnetic resonance imaging features at stoma reversal To evaluate the severity of the effect on the rectal wall and pelvic floor in the colostomy group, we also report magnetic resonance imaging (MRI) parameters related to CRP, including the thickness of the rectal wall, width of the presacral space, and thicknesses of the levator ani, the gluteus maximus muscle, the obturator intemus, and the distal part of the sigmoid colon (Table 3), which was referred to in our previous study of radiation injury to the pelvis[25]. MRI scans both at pre-colostomy and at reversal were analyzed in 5 patients, as the remaining 9 did not receive MRI scans both pre-colostomy and at reversal. The thickness of the rectal wall was significantly decreased at reversal (8.91 ± 1.61) compared with pre-colostomy (10.7 ± 4.29) (P = 0.047) (Table 3).\nMagnetic resonance imaging findings at post-colostomy and at reversal compared in 5 patients with chronic radiation proctopathy\nTo evaluate the severity of the effect on the rectal wall and pelvic floor in the colostomy group, we also report magnetic resonance imaging (MRI) parameters related to CRP, including the thickness of the rectal wall, width of the presacral space, and thicknesses of the levator ani, the gluteus maximus muscle, the obturator intemus, and the distal part of the sigmoid colon (Table 3), which was referred to in our previous study of radiation injury to the pelvis[25]. MRI scans both at pre-colostomy and at reversal were analyzed in 5 patients, as the remaining 9 did not receive MRI scans both pre-colostomy and at reversal. The thickness of the rectal wall was significantly decreased at reversal (8.91 ± 1.61) compared with pre-colostomy (10.7 ± 4.29) (P = 0.047) (Table 3).\nMagnetic resonance imaging findings at post-colostomy and at reversal compared in 5 patients with chronic radiation proctopathy\n Good restored anorectal functions after stoma reversal Anorectal functions before stoma and after reversal were evaluated by CRPSS scores in all 12 patients in the colostomy group. The results showed that rectal bleeding, peritoneal pain, tenesmus, urgency and fecal/gas incontinency were significantly improved after stoma reversal compared to those before stoma creation. Additionally, all of these scores were < 2 points, which indicated that anorectal function recovered very well after stoma reversal (Figure 6). Complete remission of bleeding and good performance of anorectal function compared to the normal population (scores ≤ 1 point) were observed. We also analyzed anorectal manometry to objectively assess anorectal function before reversal in 6 of the 14 patients who received it. Increased sensitivity of the rectal mucosa and decreased rectal volume were observed. Decreased sphincter functions were found in 2 of 6 cases (Table 4). These patients fulfilled the indications for reversal, and we found that these patients obtained good performance of anorectal function after reversal. Rectal defecography was also conducted before reversal, and no stricture was observed.\nRectal manometry assessment of anorectal functions before stoma reversals\nAnorectal functions before stoma and after stoma reversal.\nAnorectal functions before stoma and after reversal were evaluated by CRPSS scores in all 12 patients in the colostomy group. The results showed that rectal bleeding, peritoneal pain, tenesmus, urgency and fecal/gas incontinency were significantly improved after stoma reversal compared to those before stoma creation. Additionally, all of these scores were < 2 points, which indicated that anorectal function recovered very well after stoma reversal (Figure 6). Complete remission of bleeding and good performance of anorectal function compared to the normal population (scores ≤ 1 point) were observed. We also analyzed anorectal manometry to objectively assess anorectal function before reversal in 6 of the 14 patients who received it. Increased sensitivity of the rectal mucosa and decreased rectal volume were observed. Decreased sphincter functions were found in 2 of 6 cases (Table 4). These patients fulfilled the indications for reversal, and we found that these patients obtained good performance of anorectal function after reversal. Rectal defecography was also conducted before reversal, and no stricture was observed.\nRectal manometry assessment of anorectal functions before stoma reversals\nAnorectal functions before stoma and after stoma reversal.\n Improved quality of life at colostomy reversal QOL after stoma was assessed in the colostomy and conservative groups using EORTC QLQ-C30 questionnaires. As there are no similar reports in the Chinese population, we referred to the normal German population[26]. Osoba et al[26] suggested that a difference of ≥ 20 points in global health was considered to be clinically relevant. In the panel of patients who underwent stoma closure, QOL was dramatically improved compared to Pre-Op baseline QOL before stoma creation, including improved global health (difference of 40, P < 0.001), physical function (difference of 36.4, P < 0.001), role function (difference of 55, P < 0.001), emotional function (difference of 39.4, P < 0.001), social function (difference of 36.3, P < 0.001), and improved symptoms, such as fatigue (difference of -62.5, P < 0.001), pain (difference of -34.9, P < 0.001), dyspnea (difference of -37.8, P < 0.001), insomnia (difference of -36.2, P < 0.001), diarrhea (difference of -23.8, P < 0.001), and financial problems (difference of -35.5, P < 0.001). However, in the conservative group, no improved QOL variables with a difference of ≥ 20 were observed (Table 5). QOL after stoma closure was also compared to the normal population reference, with similar scores for functions and symptoms (Table 5).\nQuality of life in the colostomy group and conservative group of chronic radiation proctopathy patients based on the EORTC QLQ-C30 scale\nWilcoxon rank-sum test. \nPoint (follow-up) – point (pretreatment). Δ(FU): Change in follow-up; SD: Standard deviation.\nQOL after stoma was assessed in the colostomy and conservative groups using EORTC QLQ-C30 questionnaires. As there are no similar reports in the Chinese population, we referred to the normal German population[26]. Osoba et al[26] suggested that a difference of ≥ 20 points in global health was considered to be clinically relevant. In the panel of patients who underwent stoma closure, QOL was dramatically improved compared to Pre-Op baseline QOL before stoma creation, including improved global health (difference of 40, P < 0.001), physical function (difference of 36.4, P < 0.001), role function (difference of 55, P < 0.001), emotional function (difference of 39.4, P < 0.001), social function (difference of 36.3, P < 0.001), and improved symptoms, such as fatigue (difference of -62.5, P < 0.001), pain (difference of -34.9, P < 0.001), dyspnea (difference of -37.8, P < 0.001), insomnia (difference of -36.2, P < 0.001), diarrhea (difference of -23.8, P < 0.001), and financial problems (difference of -35.5, P < 0.001). However, in the conservative group, no improved QOL variables with a difference of ≥ 20 were observed (Table 5). QOL after stoma closure was also compared to the normal population reference, with similar scores for functions and symptoms (Table 5).\nQuality of life in the colostomy group and conservative group of chronic radiation proctopathy patients based on the EORTC QLQ-C30 scale\nWilcoxon rank-sum test. \nPoint (follow-up) – point (pretreatment). Δ(FU): Change in follow-up; SD: Standard deviation.\n\nDemographics and clinical outcomes:\nFrom March 2008 to Feb 2019, 738 continual CRP patients treated in our center were screened. After exclusion of 644 patients with mild or moderate CRP, 94 with refractory bleeding and moderate to severe anemia were further screened. Among them, 50 patients were treated by diverted colostomy and 44 patients were treated with conservative therapies. Among the 50 patients treated by colostomy, 32 were excluded due to recto-vaginal fistulas or deep rectal ulcer with refractory perianal pain before stoma. The remaining 18 patients with refractory bleeding were enrolled. Among them, two died from cancer recurrence and two were lost to follow-up. The remaining 14 patients were enrolled in the final analysis as the colostomy group. For the conservative group, 19 patients were excluded, including 11 with tumor relapse and 8 who were lost to follow-up. Thus, 25 were enrolled in the final analysis (Figure 1). Demographic and baseline characteristics were collected, including the intention-to-treat (ITT) group of 50 colostomies. In the enrolled patients, 14 patients comprised the diverting colostomy group, and 25 cases comprised the conservative treatment group. The primary tumors in most of the patients (33/39, 85%) were cervical cancers. No significant differences in age, sex, type of primary tumor, or radiation dosage were found between the diverting colostomy group and the conservative treatment group or between the ITT colostomy group and the colostomy group (Table 1).\nDemographics and characteristics\nFisher's exact test. ITT: Intention-to-treat; Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin.\nFlow chart of patient selection.\nIn the ITT group, no postoperative follow-ups were conducted other than for the 14 investigated colostomy patients. Higher bleeding scores (P = 0.033) and relatively decreased preoperative Hb levels (P = 0.051, no significant difference) were found in the diverting colostomy group compared to the ITT group because 36 patients in the ITT group underwent colostomy for recto-vaginal fistulas or deep rectal ulcers instead of severe bleeding.\n\nDynamically increasing hemoglobin levels after colostomy:\nIn the colostomy group, the median duration of bleeding was 13.0 mo ± 4.3 mo, and 12 (86%) patients received transfusion before stoma compared to 8.0 mo ± 1.8 mo (P = 0.042) of bleeding duration and 6 (24%) (P < 0.001) transfusions in the conservative group. In the conservative group, 8 patients received formalin irrigation and 4 patients received APC treatments. The preoperative (Pre-Op) Hb was only 63 g/L ± 17.8 g/L in the colostomy group compared to 88.2 g/L ± 19.3 g/L (P < 0.001) in the conservative group. One patient with moderate anemia (Hb of 87 g/L) received colostomy for perianal pain. Another patient with severe anemia (Hb of 78 g/L) did not receive a transfusion due to unavailability of a blood supply before colostomy. The details of the 14 patients are listed in Table 2. Increasing Hb levels were observed from 3 mo (median Hb of 75 g/L), 6 mo (Hb of 99 g/L), and 9 mo (Hb of 107 g/L) to 1 year (Hb 111 of g/L) and 2 years (Hb 117 g/L) after colostomy (Figure 2). Postoperative transfusions were conducted in 3 of 14 patients (transfusion requirements for 2 patients in other hospitals was unknown) in the colostomy group. The dynamic remission rates between the colostomy and conservative groups after surgery were as follows: 86% vs 4% at 3 mo (P < 0.001), 86% vs 12% at 6 mo (P < 0.001), 93% vs 20% at 9 mo (P < 0.001), 100% vs 24% at 1 year (P < 0.001), and 100% vs 20% at 2 years (P < 0.001) (Table 1).\nClinical details and outcomes of 14 colostomy patients\nMedian value. Pre-Op: Pre-operative; Post-Op: Post-operative; Hb: Hemoglobin; RTD: Rectal telangiectasia density; APC: Argon plasma coagulation.\nDynamic changes in hemoglobin levels. Pre-Op: Pre-operative.\n\nStoma closure and endoscopic features:\nAmong the 14 patients with severe bleeding treated by colostomy, all obtained complete remission of bleeding during follow-up. Only 3 (23%) of 13 patients received one round of APC after stoma to control bleeding, and 1 patient received 4% formalin irrigation after colostomy (Table 2). Among the 14 patients, 13 (93%) underwent stoma closure, and the remaining patient did not undergo stoma reversal due to concerns of surgical risks because of old age (83 years old). The median duration of stoma was 16 mo (range: 9-53 mo). Stoma complications occurred in 3 (21%) cases, including 1 para-stoma hernia, 1 stoma prolapse, and 1 stoma obstruction (Grade II by Clavien-Dindo classification)[23]. All 3 patients recovered well after stoma closure.\nEndoscopic findings of bleeding, confluent telangiectasia, and congested mucosa improved dramatically at stoma reversal compared to before stoma creation (Figure 3). The bleeding score by CTCAE decreased from Pre-Op 2.7 points ± 0.5 points to 0.8 points ± 0.5 points (P < 0.001) at 1 year after stoma in the colostomy group; the bleeding score was 2.0 ± 0.5 at initial diagnosis and 1.7 ± 0.9 (P = 0.1282) at 1 year after treatments in the conservative group (Table 1 and Figure 4).\nEndoscopic changes before stoma and at reversal.\nCTC bleeding scores before stoma and at stoma reversal.\n\nUltrasound findings before stoma and at reversal:\nEndorectal ultrasound (EUS) was analyzed retrospectively before stoma and at reversal in 5 patients; the remaining patients did not receive EUS at either of these two time points. In our previous study, thickening of the rectal wall, blurred wall stratification, and increased vascularity were EUS features in CRP[24]. In this study, we found similar features before stoma creation and a tremendous decrease in superficial vascularity, which can explain the cessation of bleeding (Figure 5).\nEndoscopic ultrasound features before stoma and at stoma reversal.\n\nImproved magnetic resonance imaging features at stoma reversal:\nTo evaluate the severity of the effect on the rectal wall and pelvic floor in the colostomy group, we also report magnetic resonance imaging (MRI) parameters related to CRP, including the thickness of the rectal wall, width of the presacral space, and thicknesses of the levator ani, the gluteus maximus muscle, the obturator intemus, and the distal part of the sigmoid colon (Table 3), which was referred to in our previous study of radiation injury to the pelvis[25]. MRI scans both at pre-colostomy and at reversal were analyzed in 5 patients, as the remaining 9 did not receive MRI scans both pre-colostomy and at reversal. The thickness of the rectal wall was significantly decreased at reversal (8.91 ± 1.61) compared with pre-colostomy (10.7 ± 4.29) (P = 0.047) (Table 3).\nMagnetic resonance imaging findings at post-colostomy and at reversal compared in 5 patients with chronic radiation proctopathy\n\nGood restored anorectal functions after stoma reversal:\nAnorectal functions before stoma and after reversal were evaluated by CRPSS scores in all 12 patients in the colostomy group. The results showed that rectal bleeding, peritoneal pain, tenesmus, urgency and fecal/gas incontinency were significantly improved after stoma reversal compared to those before stoma creation. Additionally, all of these scores were < 2 points, which indicated that anorectal function recovered very well after stoma reversal (Figure 6). Complete remission of bleeding and good performance of anorectal function compared to the normal population (scores ≤ 1 point) were observed. We also analyzed anorectal manometry to objectively assess anorectal function before reversal in 6 of the 14 patients who received it. Increased sensitivity of the rectal mucosa and decreased rectal volume were observed. Decreased sphincter functions were found in 2 of 6 cases (Table 4). These patients fulfilled the indications for reversal, and we found that these patients obtained good performance of anorectal function after reversal. Rectal defecography was also conducted before reversal, and no stricture was observed.\nRectal manometry assessment of anorectal functions before stoma reversals\nAnorectal functions before stoma and after stoma reversal.\n\nImproved quality of life at colostomy reversal:\nQOL after stoma was assessed in the colostomy and conservative groups using EORTC QLQ-C30 questionnaires. As there are no similar reports in the Chinese population, we referred to the normal German population[26]. Osoba et al[26] suggested that a difference of ≥ 20 points in global health was considered to be clinically relevant. In the panel of patients who underwent stoma closure, QOL was dramatically improved compared to Pre-Op baseline QOL before stoma creation, including improved global health (difference of 40, P < 0.001), physical function (difference of 36.4, P < 0.001), role function (difference of 55, P < 0.001), emotional function (difference of 39.4, P < 0.001), social function (difference of 36.3, P < 0.001), and improved symptoms, such as fatigue (difference of -62.5, P < 0.001), pain (difference of -34.9, P < 0.001), dyspnea (difference of -37.8, P < 0.001), insomnia (difference of -36.2, P < 0.001), diarrhea (difference of -23.8, P < 0.001), and financial problems (difference of -35.5, P < 0.001). However, in the conservative group, no improved QOL variables with a difference of ≥ 20 were observed (Table 5). QOL after stoma closure was also compared to the normal population reference, with similar scores for functions and symptoms (Table 5).\nQuality of life in the colostomy group and conservative group of chronic radiation proctopathy patients based on the EORTC QLQ-C30 scale\nWilcoxon rank-sum test. \nPoint (follow-up) – point (pretreatment). Δ(FU): Change in follow-up; SD: Standard deviation.\n\nDISCUSSION:\nThe literature reports that 1%-6% of CRP patients have transfusion-dependent bleeding; many of these cases can be treated with topical formalin, APC, or hyperbaric oxygen treatment[1]. Surgical resection of rectal lesions is usually reserved as the last resort for hemorrhagic CRP because it is associated with high morbidity and mortality[3,13,19]. In our previous study, fecal diversion was found to be a simple, effective, and safe procedure for severe hemorrhagic CRP[16]. Theoretically, fecal diversion can reduce the irritation of stool and accelerate the course of fibrosis and thus relieve CRP bleeding rapidly[16].\nIn this study, we report results for diverting colostomy and conservative treatments of enemas, topical formalin, or APC in a large cohort of patients with severe hemorrhagic CRP and who were followed for at least 1 year. The results showed that stomas can be reversed in 93% of CRP cases with severe hemorrhage. Increased Hb and rapid cessation of bleeding were found in the colostomy group, whereas bleeding and Hb levels were not changed in the conservative group. Moreover, anorectal function was greatly improved after stoma reversal than before stoma creation, reaching the levels of the normal population. In addition, it is important to note that colostomy was performed in a very select population of CRP patients and those patients with CRP stricture and ulcerations were excluded. Stoma complications occurred in 21% of cases, including 1 case of parastomal hernia due to a weakened abdominal wall of the parastomal zone, 1 case of stoma prolapse due to an intestine overly pulled at stoma creation, and 1 case of stoma obstruction due to stricture. All of these complications were recovered after stoma reversal. According to the literature, the complication rate of stoma is approximately 20%-50%, and the complication rate of stoma in this study is thus acceptable. Thus, colostomy is a better option and can bring more benefits to CRP patients with severe bleeding than can conservative treatments.\nIn our previous study, we found complete remission of telangiectasia and edema, recovery of mucosal integrity, and massive fibrosis in the submucosa at 3-4 years after diversion[16]. In this study, anorectal manometry was conducted in some stoma patients and resulted in good compliance, resting pressure, and contraction. Rectal defecography was performed at reversal for all colostomy patients, and no rectal stricture was found. Thus, temporary diverting colostomy and reversal at an appropriate time can be a useful option for these patients.\nIn this study, most patients in the colostomy group presented with severe anemia and needed multiple transfusions. One patient did not receive transfusion, with an Hb level of 78, before colostomy. In China, blood supply is limited, and transfusion is only conducted in large central hospitals when Hb is < 60. At 3 to 6 mo after diverting colostomy, bleeding gradually remitted and increased Hb levels were observed. By 9 mo to 1 year after colostomy, most patients achieved complete remission of bleeding and the Hb level had almost returned to normal. Stoma reversal was conducted at 1 year after colostomy in 4 (31%) of 13 patients. In 5 cases, reversal was performed at more than 2 years after colostomy because the patients did not know colostomy could be reversed until we contacted them via telephone for follow-up. Thus, the median duration of stoma was prolonged to 16 mo. In fact, most stomas in this cohort could be reversed at 1 year after colostomy.\nBased on the results of our retrospective study[16], we started a prospective clinical trial to enroll severe hemorrhagic CRP patients who are suitable for diverting colostomy (Clinical Trial No. NCT03397901). All patients were followed and evaluated for stoma closure for 1 year after colostomy. The indication for diverting colostomy was extended to hemorrhagic CRP with moderate anemia in our series because preventative colostomy can relieve refractory bleeding before it progresses to severe life-threatening anemia and patients can obtain dramatic benefit from preventative fecal diversion.\nTo date, it is still unclear whether anorectal function can be preserved after stoma closure. In this study, we found that anorectal functions, including anal control and release of gas/stool, could be restored, which also indicated that CRP was inactive after a period of diverting colostomy. Although increased rectal sensitivity and decreased rectal volume were observed at reversal by anorectal manometry, good anorectal function was achieved after reversal compared to the normal population and improved compared to that before stoma creation. No additional pelvic biofeedback treatments were required in these patients.\nFor some patients, an additional one to two rounds of APC were required to help control bleeding during the first 6 mo after colostomy. The temporary stoma not only enabled the remission of anal symptoms but also restored the biological functions of the anus, providing evidence to support fecal diversion as a helpful option for severe hemorrhagic CRP patients.\nIn some studies, bile acid malabsorption and small bowel bacterial overgrowth have been thought to be the cause of diarrhea during acute and chronic radiation enteritis[1]. Diverting colostomy cannot reverse these changes. We focused on hemorrhage in CRP and diarrhea, common in small-bowel radiation injury, which is different from tenesmus and is not the major symptom in most CRP cases.\nAccording to our previous study, severe hemorrhagic CRP patients usually experience poor global health and fatigue due to moderate to severe anemia[16]. Their social and emotional functions are also impacted by frequent stool and anxiety of bleeding. They cannot attend social activities due to concern of looking for a lavatory[1]. In this study, after the remission of anal symptoms, we found that patients had good QOL, especially after stoma reversal, and were able to return to almost the same life as the normal population.\nAnorectal functions scored by the CRPSS were assessed before stoma and after reversal. We also analyzed EUS and pelvic MRI before stoma and at reversal to evaluate the severity of CRP. To objectively evaluate anorectal function before reversal, anorectal manometry was performed and retrospectively analyzed in some patients, though only in 6 patients because manometry is not a routine test before stoma reversal.\nThis study presents our serial results of diverting colostomy for severe hemorrhagic CRP. The results showed significant superiority over conservative treatments. However, there are several limitations to this study. First, this study was retrospective, with potential recall bias. Second, the sample size of this cohort was relatively small, which will affect the grade of evidence, and a larger cohort is essential. Finally, EUS, MRI, and anorectal manometry were only performed on patients undergoing colostomy reversal as a limitation. Thus, we started a prospective cohort of diverting colostomy for severe hemorrhagic CRP and will provide more reliable evidence in the future.\nIn conclusion, diverting colostomy is a very effective and rapid method for the remission of severe bleeding in CRP patients. The stoma can be reversed and good anorectal function can be restored after reversal in most patients.\n\nARTICLE HIGHLIGHTS:\n Research background Chronic radiation proctopathy (CRP) is a common and sometimes difficult issue after radiotherapy for pelvic malignancies. Severe and refractory bleeding is hard to manage. Diverting fecal diversion is very effective and fast in remission of rectal bleeding in the management of severe CRP bleeding in our previous study, but diverting stoma is usually thought to be permanent according to the literature. The anorectal function and quality of life after stoma reversal remain unclear.\nChronic radiation proctopathy (CRP) is a common and sometimes difficult issue after radiotherapy for pelvic malignancies. Severe and refractory bleeding is hard to manage. Diverting fecal diversion is very effective and fast in remission of rectal bleeding in the management of severe CRP bleeding in our previous study, but diverting stoma is usually thought to be permanent according to the literature. The anorectal function and quality of life after stoma reversal remain unclear.\n Research motivation During the past 3 years, we have successfully performed colostomy reversals in a larger cohort of CRP patients with severe bleeding and followed patients after colostomy reversals. In this series study, we will report the efficacy of colostomy, the rate of stoma closure of diverting colostomy, and anorectal function after reversals.\nDuring the past 3 years, we have successfully performed colostomy reversals in a larger cohort of CRP patients with severe bleeding and followed patients after colostomy reversals. In this series study, we will report the efficacy of colostomy, the rate of stoma closure of diverting colostomy, and anorectal function after reversals.\n Research objectives The aim of this study was to evaluate the efficacy of colostomy and the rate of stoma reversals in severe hemorrhagic CRP.\nThe aim of this study was to evaluate the efficacy of colostomy and the rate of stoma reversals in severe hemorrhagic CRP.\n Research methods Patients with severe hemorrhagic CRP who underwent colostomy or conservative treatment were enrolled retrospectively. Rectal bleeding, hemoglobin (Hb), endoscopic features, endo-ultrasound (EUS), rectal manometry, and MRI scans were recorded. Anorectal functions and quality of life before stoma and after stoma reversal were scored with EORTC-QOL-C30 questionnaires.\nPatients with severe hemorrhagic CRP who underwent colostomy or conservative treatment were enrolled retrospectively. Rectal bleeding, hemoglobin (Hb), endoscopic features, endo-ultrasound (EUS), rectal manometry, and MRI scans were recorded. Anorectal functions and quality of life before stoma and after stoma reversal were scored with EORTC-QOL-C30 questionnaires.\n Research results After screening 738 continual CRP patients, 14 patients in the colostomy group and 25 patients in the conservative group as controls were enrolled. The Hb was gradually increased to normal levels in two years after colostomy, while no significant increase was observed in the conservative group. All of 14 patients obtained complete remission of bleeding and colostomy was successfully reversed in 13 of 14 (93%), except one with very old age. Improved endoscopic telangiectasia and bleeding, decreased vascularity by EUS, increased presarcal space, and thickened rectal wall by MRI were observed. Anorectal functions and quality of life were significantly improved after stoma reverse.\nAfter screening 738 continual CRP patients, 14 patients in the colostomy group and 25 patients in the conservative group as controls were enrolled. The Hb was gradually increased to normal levels in two years after colostomy, while no significant increase was observed in the conservative group. All of 14 patients obtained complete remission of bleeding and colostomy was successfully reversed in 13 of 14 (93%), except one with very old age. Improved endoscopic telangiectasia and bleeding, decreased vascularity by EUS, increased presarcal space, and thickened rectal wall by MRI were observed. Anorectal functions and quality of life were significantly improved after stoma reverse.\n Research conclusions Diverting colostomy is a very effective method in the remission of refractory hemorrhagic CRP. Meanwhile, stoma can be reversed and anorectal functions can be recovered after reversal.\nDiverting colostomy is a very effective method in the remission of refractory hemorrhagic CRP. Meanwhile, stoma can be reversed and anorectal functions can be recovered after reversal.\n Research perspectives Preventative diverting colostomy can relieve refractory bleeding before it progresses to severe life-threatening anemia and patients can obtain dramatic benefit. Thus, we have started a prospective clinical trial to enroll severe hemorrhagic CRP patients who are suitable for diverting colostomy (Clinical Trial No. NCT03397901), which will provide more evidence to the usage of colostomy in severe CRP patients.\nPreventative diverting colostomy can relieve refractory bleeding before it progresses to severe life-threatening anemia and patients can obtain dramatic benefit. Thus, we have started a prospective clinical trial to enroll severe hemorrhagic CRP patients who are suitable for diverting colostomy (Clinical Trial No. NCT03397901), which will provide more evidence to the usage of colostomy in severe CRP patients.\n\nResearch background:\nChronic radiation proctopathy (CRP) is a common and sometimes difficult issue after radiotherapy for pelvic malignancies. Severe and refractory bleeding is hard to manage. Diverting fecal diversion is very effective and fast in remission of rectal bleeding in the management of severe CRP bleeding in our previous study, but diverting stoma is usually thought to be permanent according to the literature. The anorectal function and quality of life after stoma reversal remain unclear.\n\nResearch motivation:\nDuring the past 3 years, we have successfully performed colostomy reversals in a larger cohort of CRP patients with severe bleeding and followed patients after colostomy reversals. In this series study, we will report the efficacy of colostomy, the rate of stoma closure of diverting colostomy, and anorectal function after reversals.\n\nResearch objectives:\nThe aim of this study was to evaluate the efficacy of colostomy and the rate of stoma reversals in severe hemorrhagic CRP.\n\nResearch methods:\nPatients with severe hemorrhagic CRP who underwent colostomy or conservative treatment were enrolled retrospectively. Rectal bleeding, hemoglobin (Hb), endoscopic features, endo-ultrasound (EUS), rectal manometry, and MRI scans were recorded. Anorectal functions and quality of life before stoma and after stoma reversal were scored with EORTC-QOL-C30 questionnaires.\n\nResearch results:\nAfter screening 738 continual CRP patients, 14 patients in the colostomy group and 25 patients in the conservative group as controls were enrolled. The Hb was gradually increased to normal levels in two years after colostomy, while no significant increase was observed in the conservative group. All of 14 patients obtained complete remission of bleeding and colostomy was successfully reversed in 13 of 14 (93%), except one with very old age. Improved endoscopic telangiectasia and bleeding, decreased vascularity by EUS, increased presarcal space, and thickened rectal wall by MRI were observed. Anorectal functions and quality of life were significantly improved after stoma reverse.\n\nResearch conclusions:\nDiverting colostomy is a very effective method in the remission of refractory hemorrhagic CRP. Meanwhile, stoma can be reversed and anorectal functions can be recovered after reversal.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nSevere chronic radiation proctopathy (CRP) is difficult to treat.\n\nMethods:\nTo assess the efficacy of colostomy in CRP, patients with severe hemorrhagic CRP who underwent colostomy or conservative treatment were enrolled. Patients with tumor recurrence, rectal-vaginal fistula or other types of rectal fistulas, or who were lost to follow-up were excluded. Rectal bleeding, hemoglobin (Hb), endoscopic features, endo-ultrasound, rectal manometry, and magnetic resonance imaging findings were recorded. Quality of life before stoma and after closure reversal was scored with questionnaires. Anorectal functions were assessed using the CRP symptom scale, which contains the following items: Watery stool, urgency, perianal pain, tenesmus, rectal bleeding, and fecal/gas incontinence.\n\nResults:\nA total of 738 continual CRP patients were screened. After exclusion, 14 patients in the colostomy group and 25 in the conservative group were included in the final analysis. Preoperative Hb was only 63 g/L ± 17.8 g/L in the colostomy group compared to 88.2 g/L ± 19.3 g/L (P < 0.001) in the conservative group. All 14 patients in the former group achieved complete remission of bleeding, and the colostomy was successfully reversed in 13 of 14 (93%), excepting one very old patient. The median duration of stoma was 16 (range: 9-53) mo. The Hb level increased gradually from 75 g/L at 3 mo, 99 g/L at 6 mo, and 107 g/L at 9 mo to 111 g/L at 1 year and 117 g/L at 2 years after the stoma, but no bleeding cessation or significant increase in Hb levels was observed in the conservative group. Endoscopic telangiectasia and bleeding were greatly improved. Endo-ultrasound showed decreased vascularity, and magnetic resonance imaging revealed an increasing presarcal space and thickened rectal wall. Anorectal functions and quality of life were significantly improved after stoma reversal, when compared to those before stoma creation.\n\nConclusions:\nDiverting colostomy is a very effective method in the remission of refractory hemorrhagic CRP. Stoma can be reversed, and anorectal functions can be recovered after reversal."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nChronic radiation proctopathy (CRP) is a common and sometimes difficult issue after radiotherapy for pelvic malignancies. A permanent change in bowel habits may occur in 90% of patients. After pelvic radiotherapy, 20%-50% of patients will develop difficult bowel function, affecting their quality of life (QOL)[1,2]. CRP can cause more than 20 different symptoms. Rectal bleeding is the most common symptom and occurs in > 50% of CRP patients[1,3]. Transfusion-dependent severe bleeding occurs in 1%-5% of patients[1]. Pathologically, ischemia in the submucosa due to obliterative endarteritis and progressive fibrosis in macroscopic changes are the main causes[4].\nMedical treatment consists of topical sucralfate enemas[5], oral or topical sulfasalazine[6], and rebamipide[7]. However, these reagents are only effective in acute or mild CRP, whereas their efficacy is usually disappointing in CRP patients with moderate to severe hemorrhage. More invasive modalities include endoscopic argon plasma coagulation (APC)[8], topical 4%-10% formalin[9-12], radiofrequency ablation[13], and hyperbaric oxygen therapy[14,15], and are currently popular optional treatments for CRP. Most of these modalities lack randomized trial evidence. These treatment options are reported to be effective in controlling mild to moderate bleeding in most of the literature. Nonetheless, severe and refractory bleeding is difficult to manage[1,16]. Furthermore, APC or topical formalin requires multiple sessions for severe bleeding and can cause severe side effects, including perforation, strictures, and perianal pain[17].\nFecal diversion is reported to be effective in the management of severe CRP bleeding[16,18,19], as it can reduce irritation injury to the lesions to decrease hemorrhage[16]. However, the usage of fecal diversion is not adopted as widely as is APC or formalin. Most of the fecal diversion data are from the 1980s, and this option has not been well studied to date. We previously reported one retrospective cohort of CRP patients with severe hemorrhage who received colostomy[16]. The results showed that colostomy can bring much higher remission of severe bleeding (94%) than can conservative treatment (11%) with APC or topical formalin. Pathologically, chronic inflammation and progressive fibrosis are observed after stoma. Diverting stoma is usually thought to be permanent according to Ayerdi et al[20].\nAnorectal function and QOL after stoma reversal remain unclear. During the past 3 years, we have successfully performed colostomy reversals in a large cohort of CRP patients with severe bleeding and have follow-up data after colostomy reversal. Here, we report the efficacy of colostomy, the rate of stoma closure of diverting colostomy, and anorectal function after reversal for this series of CRP patients with severe bleeding.\n\nResearch conclusions:\nWe thank Dr. Jervoise Andreyev (a famous international leader in radiation proctitis, Consultant Gastroenterologist in Pelvic Radiation Disease, Royal Marsden NHS Foundation Trust, London SW3 6JJ, United Kingdom) for critical revisions of our paper."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nSevere chronic radiation proctopathy (CRP) is difficult to treat.\n\nMethods:\nTo assess the efficacy of colostomy in CRP, patients with severe hemorrhagic CRP who underwent colostomy or conservative treatment were enrolled. Patients with tumor recurrence, rectal-vaginal fistula or other types of rectal fistulas, or who were lost to follow-up were excluded. Rectal bleeding, hemoglobin (Hb), endoscopic features, endo-ultrasound, rectal manometry, and magnetic resonance imaging findings were recorded. Quality of life before stoma and after closure reversal was scored with questionnaires. Anorectal functions were assessed using the CRP symptom scale, which contains the following items: Watery stool, urgency, perianal pain, tenesmus, rectal bleeding, and fecal/gas incontinence.\n\nResults:\nA total of 738 continual CRP patients were screened. After exclusion, 14 patients in the colostomy group and 25 in the conservative group were included in the final analysis. Preoperative Hb was only 63 g/L ± 17.8 g/L in the colostomy group compared to 88.2 g/L ± 19.3 g/L (P < 0.001) in the conservative group. All 14 patients in the former group achieved complete remission of bleeding, and the colostomy was successfully reversed in 13 of 14 (93%), excepting one very old patient. The median duration of stoma was 16 (range: 9-53) mo. The Hb level increased gradually from 75 g/L at 3 mo, 99 g/L at 6 mo, and 107 g/L at 9 mo to 111 g/L at 1 year and 117 g/L at 2 years after the stoma, but no bleeding cessation or significant increase in Hb levels was observed in the conservative group. Endoscopic telangiectasia and bleeding were greatly improved. Endo-ultrasound showed decreased vascularity, and magnetic resonance imaging revealed an increasing presarcal space and thickened rectal wall. Anorectal functions and quality of life were significantly improved after stoma reversal, when compared to those before stoma creation.\n\nConclusions:\nDiverting colostomy is a very effective method in the remission of refractory hemorrhagic CRP. Stoma can be reversed, and anorectal functions can be recovered after reversal."},"whole_article_text_length":{"kind":"number","value":11506,"string":"11,506"},"whole_article_abstract_length":{"kind":"number","value":418,"string":"418"},"other_sections_lengths":{"kind":"list like","value":[497,102,69,226,114,89,119,76,3903,400,425,277,99,181,210,327,1307,911,80,57,23,64,117,30],"string":"[\n 497,\n 102,\n 69,\n 226,\n 114,\n 89,\n 119,\n 76,\n 3903,\n 400,\n 425,\n 277,\n 99,\n 181,\n 210,\n 327,\n 1307,\n 911,\n 80,\n 57,\n 23,\n 64,\n 117,\n 30\n]","isConversational":false},"num_sections":{"kind":"number","value":25,"string":"25"},"most_frequent_words":{"kind":"list like","value":["patients","stoma","colostomy","bleeding","crp","reversal","group","rectal","severe","anorectal"],"string":"[\n \"patients\",\n \"stoma\",\n \"colostomy\",\n \"bleeding\",\n \"crp\",\n \"reversal\",\n \"group\",\n \"rectal\",\n \"severe\",\n \"anorectal\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["patients pelvic radiotherapy","pelvic radiotherapy","radiation proctopathy good","bowel radiation injury","radiation proctopathy crp"],"string":"[\n \"patients pelvic radiotherapy\",\n \"pelvic radiotherapy\",\n \"radiation proctopathy good\",\n \"bowel radiation injury\",\n \"radiation proctopathy crp\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"null"},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Chronic radiation proctitis | Hemorrhage | Colostomy | Anorectal function | Quality of life [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Chronic radiation proctitis | Hemorrhage | Colostomy | Anorectal function | Quality of life [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"null"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Chronic radiation proctitis | Hemorrhage | Colostomy | Anorectal function | Quality of life [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Chronic radiation proctitis | Hemorrhage | Colostomy | Anorectal function | Quality of life [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Chronic radiation proctitis | Hemorrhage | Colostomy | Anorectal function | Quality of life [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Colostomy | Female | Gastrointestinal Hemorrhage | Humans | Male | Middle Aged | Quality of Life | Radiation Injuries | Rectal Diseases | Rectum | Retrospective Studies | Surgical Stomas | Treatment Outcome [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Colostomy | Female | Gastrointestinal Hemorrhage | Humans | Male | Middle Aged | Quality of Life | Radiation Injuries | Rectal Diseases | Rectum | Retrospective Studies | Surgical Stomas | Treatment Outcome [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"null"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Colostomy | Female | Gastrointestinal Hemorrhage | Humans | Male | Middle Aged | Quality of Life | Radiation Injuries | Rectal Diseases | Rectum | Retrospective Studies | Surgical Stomas | Treatment Outcome [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Colostomy | Female | Gastrointestinal Hemorrhage | Humans | Male | Middle Aged | Quality of Life | Radiation Injuries | Rectal Diseases | Rectum | Retrospective Studies | Surgical Stomas | Treatment Outcome [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Colostomy | Female | Gastrointestinal Hemorrhage | Humans | Male | Middle Aged | Quality of Life | Radiation Injuries | Rectal Diseases | Rectum | Retrospective Studies | Surgical Stomas | Treatment Outcome [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] patients pelvic radiotherapy | pelvic radiotherapy | radiation proctopathy good | bowel radiation injury | radiation proctopathy crp [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] patients pelvic radiotherapy | pelvic radiotherapy | radiation proctopathy good | bowel radiation injury | radiation proctopathy crp [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"null"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] patients pelvic radiotherapy | pelvic radiotherapy | radiation proctopathy good | bowel radiation injury | radiation proctopathy crp [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients pelvic radiotherapy | pelvic radiotherapy | radiation proctopathy good | bowel radiation injury | radiation proctopathy crp [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] patients pelvic radiotherapy | pelvic radiotherapy | radiation proctopathy good | bowel radiation injury | radiation proctopathy crp [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | stoma | colostomy | bleeding | crp | reversal | group | rectal | severe | anorectal [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | stoma | colostomy | bleeding | crp | reversal | group | rectal | severe | anorectal [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"null"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | stoma | colostomy | bleeding | crp | reversal | group | rectal | severe | anorectal [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | stoma | colostomy | bleeding | crp | reversal | group | rectal | severe | anorectal [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | stoma | colostomy | bleeding | crp | reversal | group | rectal | severe | anorectal [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] severe | topical | crp | bleeding | severe bleeding | patients | hemorrhage | crp patients | 16 | difficult [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] bleeding | rectal | crp | rectal bleeding | recurrence | patients | severe | defined | stoma | system [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"null"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] colostomy effective method | remission refractory hemorrhagic | hemorrhagic crp stoma reversed | hemorrhagic crp stoma | method remission refractory | method remission refractory hemorrhagic | anorectal functions recovered | crp stoma reversed anorectal | crp stoma reversed | crp stoma [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] stoma | colostomy | patients | bleeding | crp | reversal | rectal | severe | group | anorectal [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] stoma | colostomy | patients | bleeding | crp | reversal | rectal | severe | group | anorectal [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] CRP [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] CRP | CRP ||| ||| ||| ||| CRP [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"null"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] CRP ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] CRP ||| CRP | CRP ||| ||| ||| ||| CRP ||| 738 | CRP ||| 14 | 25 ||| Preoperative Hb | 17.8 ||| g/L | 88.2 | 19.3 | g/L ||| 14 | 13 | 14 | 93% | one ||| 16 | 9-53 ||| Hb | 75 | 3 mo, | 99 | 6 | 107 | 111 | 1 year | 117 | 2 years | Hb ||| ||| ||| ||| CRP ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] CRP ||| CRP | CRP ||| ||| ||| ||| CRP ||| 738 | CRP ||| 14 | 25 ||| Preoperative Hb | 17.8 ||| g/L | 88.2 | 19.3 | g/L ||| 14 | 13 | 14 | 93% | one ||| 16 | 9-53 ||| Hb | 75 | 3 mo, | 99 | 6 | 107 | 111 | 1 year | 117 | 2 years | Hb ||| ||| ||| ||| CRP ||| [SUMMARY]"}}},{"rowIdx":72,"cells":{"title":{"kind":"null"},"pmid":{"kind":"string","value":"28480430"},"background_abstract":{"kind":"string","value":"The present study evaluates the effect of Spatholobus suberectus stem extract (SS) in the management of pancreatitis alone and in combination with heparin."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Pancreatitis was induced pancreatitis by cerulean (50μg/kg, i.p.) five times at an interval of 1 h without any pretreatment of drug. Rats were treated with SS (100 and 200 mg/kg, p. o.) and heparin (150 U/kg, i.p.) alone and in combination for the duration of a week. Later pancreatic weight and blood flow was estimated and different biochemical parameters like concentration of D-dimer and Interleukin 1β (IL-Ιβ) and activity of amylase and lipase were determined in blood of pancreatitis rats. Moreover effect of drug treatment on DNA synthesis and histopathology was also estimated on cerulean induced pancreatitis rats."},"methods_abstract_label":{"kind":"string","value":"MATERIAL AND METHODS"},"results_abstract":{"kind":"string","value":"Results of this study suggest that treatment with SS alone and in combination with heparin significantly increase in prothrombin time and pancreatic blood flow than negative control group. There was significant decrease in concentration of IL-Ιβ and D-dimer and activity of amylase and lipase in SS and heparin treated group than negative control group. Pancreatic DNA synthesis was also found to be reduced in SS and heparin alone and in combination treated group. Histopathology study also reveals that treatment with SS and heparin alone and in combination reduces edema, hemorrhages, leukocyte infiltration in the TS of pancreatic tissues."},"results_abstract_label":{"kind":"string","value":"RESULT"},"conclusions_abstract":{"kind":"string","value":"Present study concludes that treatment with SS alone effectively manages the pancreatitis by ceasing the inflammatory pathway and potentiates the effect of heparin in the management of pancreatitis."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Amylases","Animals","Anticoagulants","Ceruletide","Drug Therapy, Combination","Fabaceae","Fibrin Fibrinogen Degradation Products","Heparin","Interleukin-1beta","Lipase","Male","Nucleic Acid Synthesis Inhibitors","Pancreas","Pancreatitis","Phytotherapy","Plant Extracts","Plant Stems","Protective Agents","Prothrombin Time","Rats","Rats, Wistar"],"string":"[\n \"Amylases\",\n \"Animals\",\n \"Anticoagulants\",\n \"Ceruletide\",\n \"Drug Therapy, Combination\",\n \"Fabaceae\",\n \"Fibrin Fibrinogen Degradation Products\",\n \"Heparin\",\n \"Interleukin-1beta\",\n \"Lipase\",\n \"Male\",\n \"Nucleic Acid Synthesis Inhibitors\",\n \"Pancreas\",\n \"Pancreatitis\",\n \"Phytotherapy\",\n \"Plant Extracts\",\n \"Plant Stems\",\n \"Protective Agents\",\n \"Prothrombin Time\",\n \"Rats\",\n \"Rats, Wistar\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"5412224"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Pancreatitis is a coagulation-like disorder. However, inflammation and coagulation is closely related to each other. Coagulation is induced by inflammation and activation of process of inflammation equally responsible for the coagulation as thrombosis (Levi et al, 2012). Reported study suggested that disturbance in microcirculation results in formation of pro-inflammatory cytokines; oxygen derived free radicals, release of proteolytic enzyme and activation of leukocytes (Salomone et al, 2003). Thrombin forms as inflammatory cytokines enhances the expression of tissue factor on endothelium and monocytes (Maeda et al, 2006).\nReported study suggested that heparin/anticoagulant shows protective effect in pancreatitis in animal as well as clinical study. Pretreatment with heparin ceases the development of pancreatitis induced by cerulean, taurocholate and bile on various animal studies (Gabryelewicz et al, 1969). Moreover heparin restores the pancreatic function in cerulean induced pancreatitis if it is administered after the induction of pancreatitis (Dobosz et al, 1998; Qiu et al, 2007). Moreover a study suggested that heparin manages the hyperlipidemia induced pancreatitis when co administered with insulin and in sever pancreatitis it protects the encephalopathy (Alagözlü et al, 2006; Kyriakidis et al, 2006). Heparin manages the pancreatitis by inhibiting the formation of thrombin through heparin-antithrombin III complex (Warzecha et al, 2010). However, effect of heparin over inflammatory parameters or pathway is not given in the literatures.\nSpatholobus suberectus belongs to family: Leguminosae used for the management of several disorders such as rheumatism, anemia and abnormal menstruation traditionally in China (Lam et al, 2000; Li et al, 2003; Yen, 1992). Reported study suggested that several compounds like butin, liquiritigenin, dihydroquercetin, plathymenin, eriodictyol and neoisoliquiritigenin were been isolated from stem of SS (Lee et al, 2006). A study on stem extract of SS effectively manages cerebral ischemia by attenuating NF-κΒ p65 and cytokines and thereby prevents the DNA damage (Zhang et al, 2016). Moreover studies like anti rheumatic, anti inflammatory, antioxidant and anti platelet activity of SS are reported in the literature (Zhang et al, 2016). Thus this study evaluates the synergistic effect of Spatholobus suberectus extract when treated with heparin in pancreatitis."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"null"},"results_text":{"kind":"null"},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"Present study concludes that treatment with SS alone effectively manages the pancreatitis by ceasing the inflammatory pathway. Moreover SS in combination with heparin gives synergistic effect as in combination it act on inflammatory and coagulation pathway simultaneously for the pancreatitis."},"other_sections_titles":{"kind":"list like","value":["Animals","Plant Extract","Induction of Pancreatitis","Estimation of pancreatic blood flow","Estimation of Biochemical parameters","Estimation of pancreatic DNA synthesis","Histopathology study","Statistical analysis","Result","Effect of Spatholobus suberectus and heparin on prothrombin time, weight of pancreas and pancreatic blood flow","Effect of Spatholobus suberectus and heparin on biochemical parameters","Effect of Spatholobus suberectus and heparin on pancreatic DNA synthesis","Effect of Spatholobus suberectus and heparin on histopathology of pancreas"],"string":"[\n \"Animals\",\n \"Plant Extract\",\n \"Induction of Pancreatitis\",\n \"Estimation of pancreatic blood flow\",\n \"Estimation of Biochemical parameters\",\n \"Estimation of pancreatic DNA synthesis\",\n \"Histopathology study\",\n \"Statistical analysis\",\n \"Result\",\n \"Effect of Spatholobus suberectus and heparin on prothrombin time, weight of pancreas and pancreatic blood flow\",\n \"Effect of Spatholobus suberectus and heparin on biochemical parameters\",\n \"Effect of Spatholobus suberectus and heparin on pancreatic DNA synthesis\",\n \"Effect of Spatholobus suberectus and heparin on histopathology of pancreas\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Healthy male Wistar rats of 180-200 g body weight were used for the pharmacological screening in the present study. The animals were housed at 25 ±2°C temperature, 12 h light/dark cycle and 60 ± 5 % of relative humidity. Rats were feed with standard diet and water ad libitum. Protocols of the present investigation for all the animal studies were approved by Capital University of Medical Science, Beijing.","Spatholobus suberectus was procured from the local botanist. S. suberectus stem cut into small pieces and dried it under the shade. Later it was boiled for specific duration of time in distilled water and then filters the extract by using filter paper (150 μm). Thereafter the extract was lyophilized. During the experiment at the time of dosing extract of SS was dissolved in distilled H2O and for 5 min centrifuge it at 10000 RPM.","All the rats were separated in to seven different group (n=8) such as Control group which recives only saline; Negative control group which receives cerulean (50 μg/kg, i.p.) five times at an interval of 1 h whithout any pretreatment of drug; Heparin treated group receives 150 U/kg of heparin 1 day after the cerulean by subcutaneous injection twice a day; SS treated group receives 100 and 200 mg/kg of SS extract p. o. 1 day after the cerulean injection; Heparin +SS treated group receives heparin (150 U/kg, s. c.) and SS (100 and 200 mg/kg) 1 day after the cerulean injection for the duration of one week (Baczy’ nska et al, 2004).","All the animals were anesthetized by ketamine (50mg/kg, i.p.) at the end of protocol and pancreatic blood flow was estimated as per previously reported methods in exposed pancreas by using a laser Doppler flowmeter. Interpretation of data was represented as percent change from value obtained in control group without induction of pancreatitis.","Later abdominal aorta was used for the collection of fresh blood sample and prothrombin time was estimated in it as international normalized ratio using test strip (Alere San Diego, Inc., USA).\nImmunoturbidimetric assay method was used for the estimation of concentration of plasma D-Dimer by using automatic coagulation analyzer (BCS XP System, Simens Healthcare Diagnostics, Garmany). Kodak Ectachem DT II System analyzer was used for the estimation of activity of amylase and lipase in serum using using Amyl and Lipa DT Slides. Interlukin 1β (IL-Ιβ) concentration was estimated in the serum by using IL-Ιβ Platinum Elisa (Konturek et al, 1994).","Pancreas isolated from all the rats was weighed. Later for histological examination and pancreatic DNA synthesis was done for the pancreatic tissue. Assessment of labeled thymidine in to DNA was done for the estimation of rate of pancreatic DNA synthesis as per previously reported study. Rate of DNA synthesis was expressed as disintegrations of labeled thymidine per minute per microgram DNA (dpm^g DNA) (Dembi’ nski et al, 2006).","Hematoxylin and eosin (H&E) staining was done for the examination of damage of pancreatic tissue as given in previous report. A scale of range from 0 to 3 was used for histological grading of necrosis, hemorrhages, vacuolization of acinar cells, leukocyte inflammatory infiltration and edema (Tomaszewska et al, 2000).","Data of present study given as mean ± SD (n=8). Statistically analysis was done by one way ANOVA (Dunnett.) In this study values p<0.05 was considered as significant."," Effect of Spatholobus suberectus and heparin on prothrombin time, weight of pancreas and pancreatic blood flow Effect of Spatholobus suberectus in combination with heparin was observed on prothrombin time in cerulean induced pancreatitis as shown in Figure 1. There was significant (p<0.05, p<0.01) increase in the prothrombin time in heparin, SS 100 & 200 mg, heparin+SS 100 mg/kg and heparin+SS 200 mg/kg treated group than negative control group.\nWeight of pancreas was significantly increases in cerulean induced pancreatitis group i.e. negative control group than control group. It was also observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases (p<0.05, p<0.01) the weight of pancreas compared to negative control group (Figure 2). Moreover treatment with Spatholobus suberectus and heparin alone and in combination increases the pancreatic blood flow which is calculated as % of control group than negative control group (Figure 3).\nEffect of Spatholobus suberectus in combination with heparin was observed on prothrombin time as international normalized ratio (INR) in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic weight in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic blood flow (% of control) in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus in combination with heparin was observed on prothrombin time in cerulean induced pancreatitis as shown in Figure 1. There was significant (p<0.05, p<0.01) increase in the prothrombin time in heparin, SS 100 & 200 mg, heparin+SS 100 mg/kg and heparin+SS 200 mg/kg treated group than negative control group.\nWeight of pancreas was significantly increases in cerulean induced pancreatitis group i.e. negative control group than control group. It was also observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases (p<0.05, p<0.01) the weight of pancreas compared to negative control group (Figure 2). Moreover treatment with Spatholobus suberectus and heparin alone and in combination increases the pancreatic blood flow which is calculated as % of control group than negative control group (Figure 3).\nEffect of Spatholobus suberectus in combination with heparin was observed on prothrombin time as international normalized ratio (INR) in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic weight in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic blood flow (% of control) in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\n Effect of Spatholobus suberectus and heparin on biochemical parameters Effect of Spatholobus suberectus and heparin alone and in combination on the concentration of D-dimer in cerulean induced pancreatitis as shown Figure 4. It was observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases the concentration of D-dimer in cerulean induced pancreatitis than negative control group.\nEffect of Spatholobus suberectus in combination with heparin was observed on the concentration of D-dimer in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nIn cerulean induced pancreatitis rats the activity of lipase and amylase enzyme and concentration of Π^-1β significantly increases than control group. There were significant decrease in the activity of lipase and amylase enzyme and concentration of Π^-1β in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 5 a, b & c.\nEffect of Spatholobus suberectus and heparin on biochemical parameter in cerulean induced pancreatitis. (a) Amylase activity; (b) Lipase activity; (c) Interlukin 1 β\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus and heparin alone and in combination on the concentration of D-dimer in cerulean induced pancreatitis as shown Figure 4. It was observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases the concentration of D-dimer in cerulean induced pancreatitis than negative control group.\nEffect of Spatholobus suberectus in combination with heparin was observed on the concentration of D-dimer in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nIn cerulean induced pancreatitis rats the activity of lipase and amylase enzyme and concentration of Π^-1β significantly increases than control group. There were significant decrease in the activity of lipase and amylase enzyme and concentration of Π^-1β in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 5 a, b & c.\nEffect of Spatholobus suberectus and heparin on biochemical parameter in cerulean induced pancreatitis. (a) Amylase activity; (b) Lipase activity; (c) Interlukin 1 β\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\n Effect of Spatholobus suberectus and heparin on pancreatic DNA synthesis Effect of Spatholobus suberectus and heparin alone and in combination on pancreatic DNA synthesis was shown in Figure 6. There were significant (p<0.05, p<0.01) increase in the pancreatic DNA synthesis in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 6.\nEffect of Spatholobus suberectus and heparin on pancreatic DNA synthesis in cerulean induced pancreatitis. Means ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus and heparin alone and in combination on pancreatic DNA synthesis was shown in Figure 6. There were significant (p<0.05, p<0.01) increase in the pancreatic DNA synthesis in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 6.\nEffect of Spatholobus suberectus and heparin on pancreatic DNA synthesis in cerulean induced pancreatitis. Means ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\n Effect of Spatholobus suberectus and heparin on histopathology of pancreas Histopathology study was performed on Spatholobus suberectus and heparin treated cerulean induced pancreatitis rats as shown in Table 1 and Figure 7. It was observed that pancreas of cerulean induced pancreatitis rats shows severe edema, moderate diffuse infiltration of inflammatory leukocyte and hemorrhages. Acinar cell shows more than 50% of vacuolization and signs of necrosis was absent in the TS of pancreas. Treatment with Spatholobus suberectus and heparin alone and in combination attenuates the development of pancreatitis. Histopathological representation shows that heparin in combination with SS significant decline in vacuolization of acinar cells, inflammatory infiltration and pancreatic edema.\nEffect of Spatholobus suberectus and heparin on histopathology signs of pancreas\nEffect of drug treatment on histopathology of pancreatic tissue in cerulean induced pancreatitis rat model. A. Control; B. Negative control; C. SS 200 mg/kg; D. Heparin+SS 200 mg/kg.\nHistopathology study was performed on Spatholobus suberectus and heparin treated cerulean induced pancreatitis rats as shown in Table 1 and Figure 7. It was observed that pancreas of cerulean induced pancreatitis rats shows severe edema, moderate diffuse infiltration of inflammatory leukocyte and hemorrhages. Acinar cell shows more than 50% of vacuolization and signs of necrosis was absent in the TS of pancreas. Treatment with Spatholobus suberectus and heparin alone and in combination attenuates the development of pancreatitis. Histopathological representation shows that heparin in combination with SS significant decline in vacuolization of acinar cells, inflammatory infiltration and pancreatic edema.\nEffect of Spatholobus suberectus and heparin on histopathology signs of pancreas\nEffect of drug treatment on histopathology of pancreatic tissue in cerulean induced pancreatitis rat model. A. Control; B. Negative control; C. SS 200 mg/kg; D. Heparin+SS 200 mg/kg.","Effect of Spatholobus suberectus in combination with heparin was observed on prothrombin time in cerulean induced pancreatitis as shown in Figure 1. There was significant (p<0.05, p<0.01) increase in the prothrombin time in heparin, SS 100 & 200 mg, heparin+SS 100 mg/kg and heparin+SS 200 mg/kg treated group than negative control group.\nWeight of pancreas was significantly increases in cerulean induced pancreatitis group i.e. negative control group than control group. It was also observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases (p<0.05, p<0.01) the weight of pancreas compared to negative control group (Figure 2). Moreover treatment with Spatholobus suberectus and heparin alone and in combination increases the pancreatic blood flow which is calculated as % of control group than negative control group (Figure 3).\nEffect of Spatholobus suberectus in combination with heparin was observed on prothrombin time as international normalized ratio (INR) in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic weight in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic blood flow (% of control) in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control","Effect of Spatholobus suberectus and heparin alone and in combination on the concentration of D-dimer in cerulean induced pancreatitis as shown Figure 4. It was observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases the concentration of D-dimer in cerulean induced pancreatitis than negative control group.\nEffect of Spatholobus suberectus in combination with heparin was observed on the concentration of D-dimer in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nIn cerulean induced pancreatitis rats the activity of lipase and amylase enzyme and concentration of Π^-1β significantly increases than control group. There were significant decrease in the activity of lipase and amylase enzyme and concentration of Π^-1β in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 5 a, b & c.\nEffect of Spatholobus suberectus and heparin on biochemical parameter in cerulean induced pancreatitis. (a) Amylase activity; (b) Lipase activity; (c) Interlukin 1 β\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control","Effect of Spatholobus suberectus and heparin alone and in combination on pancreatic DNA synthesis was shown in Figure 6. There were significant (p<0.05, p<0.01) increase in the pancreatic DNA synthesis in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 6.\nEffect of Spatholobus suberectus and heparin on pancreatic DNA synthesis in cerulean induced pancreatitis. Means ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control","Histopathology study was performed on Spatholobus suberectus and heparin treated cerulean induced pancreatitis rats as shown in Table 1 and Figure 7. It was observed that pancreas of cerulean induced pancreatitis rats shows severe edema, moderate diffuse infiltration of inflammatory leukocyte and hemorrhages. Acinar cell shows more than 50% of vacuolization and signs of necrosis was absent in the TS of pancreas. Treatment with Spatholobus suberectus and heparin alone and in combination attenuates the development of pancreatitis. Histopathological representation shows that heparin in combination with SS significant decline in vacuolization of acinar cells, inflammatory infiltration and pancreatic edema.\nEffect of Spatholobus suberectus and heparin on histopathology signs of pancreas\nEffect of drug treatment on histopathology of pancreatic tissue in cerulean induced pancreatitis rat model. A. Control; B. Negative control; C. SS 200 mg/kg; D. Heparin+SS 200 mg/kg."],"string":"[\n \"Healthy male Wistar rats of 180-200 g body weight were used for the pharmacological screening in the present study. The animals were housed at 25 ±2°C temperature, 12 h light/dark cycle and 60 ± 5 % of relative humidity. Rats were feed with standard diet and water ad libitum. Protocols of the present investigation for all the animal studies were approved by Capital University of Medical Science, Beijing.\",\n \"Spatholobus suberectus was procured from the local botanist. S. suberectus stem cut into small pieces and dried it under the shade. Later it was boiled for specific duration of time in distilled water and then filters the extract by using filter paper (150 μm). Thereafter the extract was lyophilized. During the experiment at the time of dosing extract of SS was dissolved in distilled H2O and for 5 min centrifuge it at 10000 RPM.\",\n \"All the rats were separated in to seven different group (n=8) such as Control group which recives only saline; Negative control group which receives cerulean (50 μg/kg, i.p.) five times at an interval of 1 h whithout any pretreatment of drug; Heparin treated group receives 150 U/kg of heparin 1 day after the cerulean by subcutaneous injection twice a day; SS treated group receives 100 and 200 mg/kg of SS extract p. o. 1 day after the cerulean injection; Heparin +SS treated group receives heparin (150 U/kg, s. c.) and SS (100 and 200 mg/kg) 1 day after the cerulean injection for the duration of one week (Baczy’ nska et al, 2004).\",\n \"All the animals were anesthetized by ketamine (50mg/kg, i.p.) at the end of protocol and pancreatic blood flow was estimated as per previously reported methods in exposed pancreas by using a laser Doppler flowmeter. Interpretation of data was represented as percent change from value obtained in control group without induction of pancreatitis.\",\n \"Later abdominal aorta was used for the collection of fresh blood sample and prothrombin time was estimated in it as international normalized ratio using test strip (Alere San Diego, Inc., USA).\\nImmunoturbidimetric assay method was used for the estimation of concentration of plasma D-Dimer by using automatic coagulation analyzer (BCS XP System, Simens Healthcare Diagnostics, Garmany). Kodak Ectachem DT II System analyzer was used for the estimation of activity of amylase and lipase in serum using using Amyl and Lipa DT Slides. Interlukin 1β (IL-Ιβ) concentration was estimated in the serum by using IL-Ιβ Platinum Elisa (Konturek et al, 1994).\",\n \"Pancreas isolated from all the rats was weighed. Later for histological examination and pancreatic DNA synthesis was done for the pancreatic tissue. Assessment of labeled thymidine in to DNA was done for the estimation of rate of pancreatic DNA synthesis as per previously reported study. Rate of DNA synthesis was expressed as disintegrations of labeled thymidine per minute per microgram DNA (dpm^g DNA) (Dembi’ nski et al, 2006).\",\n \"Hematoxylin and eosin (H&E) staining was done for the examination of damage of pancreatic tissue as given in previous report. A scale of range from 0 to 3 was used for histological grading of necrosis, hemorrhages, vacuolization of acinar cells, leukocyte inflammatory infiltration and edema (Tomaszewska et al, 2000).\",\n \"Data of present study given as mean ± SD (n=8). Statistically analysis was done by one way ANOVA (Dunnett.) In this study values p<0.05 was considered as significant.\",\n \" Effect of Spatholobus suberectus and heparin on prothrombin time, weight of pancreas and pancreatic blood flow Effect of Spatholobus suberectus in combination with heparin was observed on prothrombin time in cerulean induced pancreatitis as shown in Figure 1. There was significant (p<0.05, p<0.01) increase in the prothrombin time in heparin, SS 100 & 200 mg, heparin+SS 100 mg/kg and heparin+SS 200 mg/kg treated group than negative control group.\\nWeight of pancreas was significantly increases in cerulean induced pancreatitis group i.e. negative control group than control group. It was also observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases (p<0.05, p<0.01) the weight of pancreas compared to negative control group (Figure 2). Moreover treatment with Spatholobus suberectus and heparin alone and in combination increases the pancreatic blood flow which is calculated as % of control group than negative control group (Figure 3).\\nEffect of Spatholobus suberectus in combination with heparin was observed on prothrombin time as international normalized ratio (INR) in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic weight in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic blood flow (% of control) in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nEffect of Spatholobus suberectus in combination with heparin was observed on prothrombin time in cerulean induced pancreatitis as shown in Figure 1. There was significant (p<0.05, p<0.01) increase in the prothrombin time in heparin, SS 100 & 200 mg, heparin+SS 100 mg/kg and heparin+SS 200 mg/kg treated group than negative control group.\\nWeight of pancreas was significantly increases in cerulean induced pancreatitis group i.e. negative control group than control group. It was also observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases (p<0.05, p<0.01) the weight of pancreas compared to negative control group (Figure 2). Moreover treatment with Spatholobus suberectus and heparin alone and in combination increases the pancreatic blood flow which is calculated as % of control group than negative control group (Figure 3).\\nEffect of Spatholobus suberectus in combination with heparin was observed on prothrombin time as international normalized ratio (INR) in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic weight in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic blood flow (% of control) in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\n Effect of Spatholobus suberectus and heparin on biochemical parameters Effect of Spatholobus suberectus and heparin alone and in combination on the concentration of D-dimer in cerulean induced pancreatitis as shown Figure 4. It was observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases the concentration of D-dimer in cerulean induced pancreatitis than negative control group.\\nEffect of Spatholobus suberectus in combination with heparin was observed on the concentration of D-dimer in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nIn cerulean induced pancreatitis rats the activity of lipase and amylase enzyme and concentration of Π^-1β significantly increases than control group. There were significant decrease in the activity of lipase and amylase enzyme and concentration of Π^-1β in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 5 a, b & c.\\nEffect of Spatholobus suberectus and heparin on biochemical parameter in cerulean induced pancreatitis. (a) Amylase activity; (b) Lipase activity; (c) Interlukin 1 β\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nEffect of Spatholobus suberectus and heparin alone and in combination on the concentration of D-dimer in cerulean induced pancreatitis as shown Figure 4. It was observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases the concentration of D-dimer in cerulean induced pancreatitis than negative control group.\\nEffect of Spatholobus suberectus in combination with heparin was observed on the concentration of D-dimer in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nIn cerulean induced pancreatitis rats the activity of lipase and amylase enzyme and concentration of Π^-1β significantly increases than control group. There were significant decrease in the activity of lipase and amylase enzyme and concentration of Π^-1β in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 5 a, b & c.\\nEffect of Spatholobus suberectus and heparin on biochemical parameter in cerulean induced pancreatitis. (a) Amylase activity; (b) Lipase activity; (c) Interlukin 1 β\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\n Effect of Spatholobus suberectus and heparin on pancreatic DNA synthesis Effect of Spatholobus suberectus and heparin alone and in combination on pancreatic DNA synthesis was shown in Figure 6. There were significant (p<0.05, p<0.01) increase in the pancreatic DNA synthesis in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 6.\\nEffect of Spatholobus suberectus and heparin on pancreatic DNA synthesis in cerulean induced pancreatitis. Means ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nEffect of Spatholobus suberectus and heparin alone and in combination on pancreatic DNA synthesis was shown in Figure 6. There were significant (p<0.05, p<0.01) increase in the pancreatic DNA synthesis in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 6.\\nEffect of Spatholobus suberectus and heparin on pancreatic DNA synthesis in cerulean induced pancreatitis. Means ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\n Effect of Spatholobus suberectus and heparin on histopathology of pancreas Histopathology study was performed on Spatholobus suberectus and heparin treated cerulean induced pancreatitis rats as shown in Table 1 and Figure 7. It was observed that pancreas of cerulean induced pancreatitis rats shows severe edema, moderate diffuse infiltration of inflammatory leukocyte and hemorrhages. Acinar cell shows more than 50% of vacuolization and signs of necrosis was absent in the TS of pancreas. Treatment with Spatholobus suberectus and heparin alone and in combination attenuates the development of pancreatitis. Histopathological representation shows that heparin in combination with SS significant decline in vacuolization of acinar cells, inflammatory infiltration and pancreatic edema.\\nEffect of Spatholobus suberectus and heparin on histopathology signs of pancreas\\nEffect of drug treatment on histopathology of pancreatic tissue in cerulean induced pancreatitis rat model. A. Control; B. Negative control; C. SS 200 mg/kg; D. Heparin+SS 200 mg/kg.\\nHistopathology study was performed on Spatholobus suberectus and heparin treated cerulean induced pancreatitis rats as shown in Table 1 and Figure 7. It was observed that pancreas of cerulean induced pancreatitis rats shows severe edema, moderate diffuse infiltration of inflammatory leukocyte and hemorrhages. Acinar cell shows more than 50% of vacuolization and signs of necrosis was absent in the TS of pancreas. Treatment with Spatholobus suberectus and heparin alone and in combination attenuates the development of pancreatitis. Histopathological representation shows that heparin in combination with SS significant decline in vacuolization of acinar cells, inflammatory infiltration and pancreatic edema.\\nEffect of Spatholobus suberectus and heparin on histopathology signs of pancreas\\nEffect of drug treatment on histopathology of pancreatic tissue in cerulean induced pancreatitis rat model. A. Control; B. Negative control; C. SS 200 mg/kg; D. Heparin+SS 200 mg/kg.\",\n \"Effect of Spatholobus suberectus in combination with heparin was observed on prothrombin time in cerulean induced pancreatitis as shown in Figure 1. There was significant (p<0.05, p<0.01) increase in the prothrombin time in heparin, SS 100 & 200 mg, heparin+SS 100 mg/kg and heparin+SS 200 mg/kg treated group than negative control group.\\nWeight of pancreas was significantly increases in cerulean induced pancreatitis group i.e. negative control group than control group. It was also observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases (p<0.05, p<0.01) the weight of pancreas compared to negative control group (Figure 2). Moreover treatment with Spatholobus suberectus and heparin alone and in combination increases the pancreatic blood flow which is calculated as % of control group than negative control group (Figure 3).\\nEffect of Spatholobus suberectus in combination with heparin was observed on prothrombin time as international normalized ratio (INR) in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic weight in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic blood flow (% of control) in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\",\n \"Effect of Spatholobus suberectus and heparin alone and in combination on the concentration of D-dimer in cerulean induced pancreatitis as shown Figure 4. It was observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases the concentration of D-dimer in cerulean induced pancreatitis than negative control group.\\nEffect of Spatholobus suberectus in combination with heparin was observed on the concentration of D-dimer in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nIn cerulean induced pancreatitis rats the activity of lipase and amylase enzyme and concentration of Π^-1β significantly increases than control group. There were significant decrease in the activity of lipase and amylase enzyme and concentration of Π^-1β in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 5 a, b & c.\\nEffect of Spatholobus suberectus and heparin on biochemical parameter in cerulean induced pancreatitis. (a) Amylase activity; (b) Lipase activity; (c) Interlukin 1 β\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\",\n \"Effect of Spatholobus suberectus and heparin alone and in combination on pancreatic DNA synthesis was shown in Figure 6. There were significant (p<0.05, p<0.01) increase in the pancreatic DNA synthesis in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 6.\\nEffect of Spatholobus suberectus and heparin on pancreatic DNA synthesis in cerulean induced pancreatitis. Means ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\",\n \"Histopathology study was performed on Spatholobus suberectus and heparin treated cerulean induced pancreatitis rats as shown in Table 1 and Figure 7. It was observed that pancreas of cerulean induced pancreatitis rats shows severe edema, moderate diffuse infiltration of inflammatory leukocyte and hemorrhages. Acinar cell shows more than 50% of vacuolization and signs of necrosis was absent in the TS of pancreas. Treatment with Spatholobus suberectus and heparin alone and in combination attenuates the development of pancreatitis. Histopathological representation shows that heparin in combination with SS significant decline in vacuolization of acinar cells, inflammatory infiltration and pancreatic edema.\\nEffect of Spatholobus suberectus and heparin on histopathology signs of pancreas\\nEffect of drug treatment on histopathology of pancreatic tissue in cerulean induced pancreatitis rat model. A. Control; B. Negative control; C. SS 200 mg/kg; D. Heparin+SS 200 mg/kg.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Introduction","Material and Methods","Animals","Plant Extract","Induction of Pancreatitis","Estimation of pancreatic blood flow","Estimation of Biochemical parameters","Estimation of pancreatic DNA synthesis","Histopathology study","Statistical analysis","Result","Effect of Spatholobus suberectus and heparin on prothrombin time, weight of pancreas and pancreatic blood flow","Effect of Spatholobus suberectus and heparin on biochemical parameters","Effect of Spatholobus suberectus and heparin on pancreatic DNA synthesis","Effect of Spatholobus suberectus and heparin on histopathology of pancreas","Discussion","Conclusion"],"string":"[\n \"Introduction\",\n \"Material and Methods\",\n \"Animals\",\n \"Plant Extract\",\n \"Induction of Pancreatitis\",\n \"Estimation of pancreatic blood flow\",\n \"Estimation of Biochemical parameters\",\n \"Estimation of pancreatic DNA synthesis\",\n \"Histopathology study\",\n \"Statistical analysis\",\n \"Result\",\n \"Effect of Spatholobus suberectus and heparin on prothrombin time, weight of pancreas and pancreatic blood flow\",\n \"Effect of Spatholobus suberectus and heparin on biochemical parameters\",\n \"Effect of Spatholobus suberectus and heparin on pancreatic DNA synthesis\",\n \"Effect of Spatholobus suberectus and heparin on histopathology of pancreas\",\n \"Discussion\",\n \"Conclusion\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Pancreatitis is a coagulation-like disorder. However, inflammation and coagulation is closely related to each other. Coagulation is induced by inflammation and activation of process of inflammation equally responsible for the coagulation as thrombosis (Levi et al, 2012). Reported study suggested that disturbance in microcirculation results in formation of pro-inflammatory cytokines; oxygen derived free radicals, release of proteolytic enzyme and activation of leukocytes (Salomone et al, 2003). Thrombin forms as inflammatory cytokines enhances the expression of tissue factor on endothelium and monocytes (Maeda et al, 2006).\nReported study suggested that heparin/anticoagulant shows protective effect in pancreatitis in animal as well as clinical study. Pretreatment with heparin ceases the development of pancreatitis induced by cerulean, taurocholate and bile on various animal studies (Gabryelewicz et al, 1969). Moreover heparin restores the pancreatic function in cerulean induced pancreatitis if it is administered after the induction of pancreatitis (Dobosz et al, 1998; Qiu et al, 2007). Moreover a study suggested that heparin manages the hyperlipidemia induced pancreatitis when co administered with insulin and in sever pancreatitis it protects the encephalopathy (Alagözlü et al, 2006; Kyriakidis et al, 2006). Heparin manages the pancreatitis by inhibiting the formation of thrombin through heparin-antithrombin III complex (Warzecha et al, 2010). However, effect of heparin over inflammatory parameters or pathway is not given in the literatures.\nSpatholobus suberectus belongs to family: Leguminosae used for the management of several disorders such as rheumatism, anemia and abnormal menstruation traditionally in China (Lam et al, 2000; Li et al, 2003; Yen, 1992). Reported study suggested that several compounds like butin, liquiritigenin, dihydroquercetin, plathymenin, eriodictyol and neoisoliquiritigenin were been isolated from stem of SS (Lee et al, 2006). A study on stem extract of SS effectively manages cerebral ischemia by attenuating NF-κΒ p65 and cytokines and thereby prevents the DNA damage (Zhang et al, 2016). Moreover studies like anti rheumatic, anti inflammatory, antioxidant and anti platelet activity of SS are reported in the literature (Zhang et al, 2016). Thus this study evaluates the synergistic effect of Spatholobus suberectus extract when treated with heparin in pancreatitis."," Animals Healthy male Wistar rats of 180-200 g body weight were used for the pharmacological screening in the present study. The animals were housed at 25 ±2°C temperature, 12 h light/dark cycle and 60 ± 5 % of relative humidity. Rats were feed with standard diet and water ad libitum. Protocols of the present investigation for all the animal studies were approved by Capital University of Medical Science, Beijing.\nHealthy male Wistar rats of 180-200 g body weight were used for the pharmacological screening in the present study. The animals were housed at 25 ±2°C temperature, 12 h light/dark cycle and 60 ± 5 % of relative humidity. Rats were feed with standard diet and water ad libitum. Protocols of the present investigation for all the animal studies were approved by Capital University of Medical Science, Beijing.\n Plant Extract Spatholobus suberectus was procured from the local botanist. S. suberectus stem cut into small pieces and dried it under the shade. Later it was boiled for specific duration of time in distilled water and then filters the extract by using filter paper (150 μm). Thereafter the extract was lyophilized. During the experiment at the time of dosing extract of SS was dissolved in distilled H2O and for 5 min centrifuge it at 10000 RPM.\nSpatholobus suberectus was procured from the local botanist. S. suberectus stem cut into small pieces and dried it under the shade. Later it was boiled for specific duration of time in distilled water and then filters the extract by using filter paper (150 μm). Thereafter the extract was lyophilized. During the experiment at the time of dosing extract of SS was dissolved in distilled H2O and for 5 min centrifuge it at 10000 RPM.\n Induction of Pancreatitis All the rats were separated in to seven different group (n=8) such as Control group which recives only saline; Negative control group which receives cerulean (50 μg/kg, i.p.) five times at an interval of 1 h whithout any pretreatment of drug; Heparin treated group receives 150 U/kg of heparin 1 day after the cerulean by subcutaneous injection twice a day; SS treated group receives 100 and 200 mg/kg of SS extract p. o. 1 day after the cerulean injection; Heparin +SS treated group receives heparin (150 U/kg, s. c.) and SS (100 and 200 mg/kg) 1 day after the cerulean injection for the duration of one week (Baczy’ nska et al, 2004).\nAll the rats were separated in to seven different group (n=8) such as Control group which recives only saline; Negative control group which receives cerulean (50 μg/kg, i.p.) five times at an interval of 1 h whithout any pretreatment of drug; Heparin treated group receives 150 U/kg of heparin 1 day after the cerulean by subcutaneous injection twice a day; SS treated group receives 100 and 200 mg/kg of SS extract p. o. 1 day after the cerulean injection; Heparin +SS treated group receives heparin (150 U/kg, s. c.) and SS (100 and 200 mg/kg) 1 day after the cerulean injection for the duration of one week (Baczy’ nska et al, 2004).\n Estimation of pancreatic blood flow All the animals were anesthetized by ketamine (50mg/kg, i.p.) at the end of protocol and pancreatic blood flow was estimated as per previously reported methods in exposed pancreas by using a laser Doppler flowmeter. Interpretation of data was represented as percent change from value obtained in control group without induction of pancreatitis.\nAll the animals were anesthetized by ketamine (50mg/kg, i.p.) at the end of protocol and pancreatic blood flow was estimated as per previously reported methods in exposed pancreas by using a laser Doppler flowmeter. Interpretation of data was represented as percent change from value obtained in control group without induction of pancreatitis.\n Estimation of Biochemical parameters Later abdominal aorta was used for the collection of fresh blood sample and prothrombin time was estimated in it as international normalized ratio using test strip (Alere San Diego, Inc., USA).\nImmunoturbidimetric assay method was used for the estimation of concentration of plasma D-Dimer by using automatic coagulation analyzer (BCS XP System, Simens Healthcare Diagnostics, Garmany). Kodak Ectachem DT II System analyzer was used for the estimation of activity of amylase and lipase in serum using using Amyl and Lipa DT Slides. Interlukin 1β (IL-Ιβ) concentration was estimated in the serum by using IL-Ιβ Platinum Elisa (Konturek et al, 1994).\nLater abdominal aorta was used for the collection of fresh blood sample and prothrombin time was estimated in it as international normalized ratio using test strip (Alere San Diego, Inc., USA).\nImmunoturbidimetric assay method was used for the estimation of concentration of plasma D-Dimer by using automatic coagulation analyzer (BCS XP System, Simens Healthcare Diagnostics, Garmany). Kodak Ectachem DT II System analyzer was used for the estimation of activity of amylase and lipase in serum using using Amyl and Lipa DT Slides. Interlukin 1β (IL-Ιβ) concentration was estimated in the serum by using IL-Ιβ Platinum Elisa (Konturek et al, 1994).\n Estimation of pancreatic DNA synthesis Pancreas isolated from all the rats was weighed. Later for histological examination and pancreatic DNA synthesis was done for the pancreatic tissue. Assessment of labeled thymidine in to DNA was done for the estimation of rate of pancreatic DNA synthesis as per previously reported study. Rate of DNA synthesis was expressed as disintegrations of labeled thymidine per minute per microgram DNA (dpm^g DNA) (Dembi’ nski et al, 2006).\nPancreas isolated from all the rats was weighed. Later for histological examination and pancreatic DNA synthesis was done for the pancreatic tissue. Assessment of labeled thymidine in to DNA was done for the estimation of rate of pancreatic DNA synthesis as per previously reported study. Rate of DNA synthesis was expressed as disintegrations of labeled thymidine per minute per microgram DNA (dpm^g DNA) (Dembi’ nski et al, 2006).\n Histopathology study Hematoxylin and eosin (H&E) staining was done for the examination of damage of pancreatic tissue as given in previous report. A scale of range from 0 to 3 was used for histological grading of necrosis, hemorrhages, vacuolization of acinar cells, leukocyte inflammatory infiltration and edema (Tomaszewska et al, 2000).\nHematoxylin and eosin (H&E) staining was done for the examination of damage of pancreatic tissue as given in previous report. A scale of range from 0 to 3 was used for histological grading of necrosis, hemorrhages, vacuolization of acinar cells, leukocyte inflammatory infiltration and edema (Tomaszewska et al, 2000).\n Statistical analysis Data of present study given as mean ± SD (n=8). Statistically analysis was done by one way ANOVA (Dunnett.) In this study values p<0.05 was considered as significant.\nData of present study given as mean ± SD (n=8). Statistically analysis was done by one way ANOVA (Dunnett.) In this study values p<0.05 was considered as significant.","Healthy male Wistar rats of 180-200 g body weight were used for the pharmacological screening in the present study. The animals were housed at 25 ±2°C temperature, 12 h light/dark cycle and 60 ± 5 % of relative humidity. Rats were feed with standard diet and water ad libitum. Protocols of the present investigation for all the animal studies were approved by Capital University of Medical Science, Beijing.","Spatholobus suberectus was procured from the local botanist. S. suberectus stem cut into small pieces and dried it under the shade. Later it was boiled for specific duration of time in distilled water and then filters the extract by using filter paper (150 μm). Thereafter the extract was lyophilized. During the experiment at the time of dosing extract of SS was dissolved in distilled H2O and for 5 min centrifuge it at 10000 RPM.","All the rats were separated in to seven different group (n=8) such as Control group which recives only saline; Negative control group which receives cerulean (50 μg/kg, i.p.) five times at an interval of 1 h whithout any pretreatment of drug; Heparin treated group receives 150 U/kg of heparin 1 day after the cerulean by subcutaneous injection twice a day; SS treated group receives 100 and 200 mg/kg of SS extract p. o. 1 day after the cerulean injection; Heparin +SS treated group receives heparin (150 U/kg, s. c.) and SS (100 and 200 mg/kg) 1 day after the cerulean injection for the duration of one week (Baczy’ nska et al, 2004).","All the animals were anesthetized by ketamine (50mg/kg, i.p.) at the end of protocol and pancreatic blood flow was estimated as per previously reported methods in exposed pancreas by using a laser Doppler flowmeter. Interpretation of data was represented as percent change from value obtained in control group without induction of pancreatitis.","Later abdominal aorta was used for the collection of fresh blood sample and prothrombin time was estimated in it as international normalized ratio using test strip (Alere San Diego, Inc., USA).\nImmunoturbidimetric assay method was used for the estimation of concentration of plasma D-Dimer by using automatic coagulation analyzer (BCS XP System, Simens Healthcare Diagnostics, Garmany). Kodak Ectachem DT II System analyzer was used for the estimation of activity of amylase and lipase in serum using using Amyl and Lipa DT Slides. Interlukin 1β (IL-Ιβ) concentration was estimated in the serum by using IL-Ιβ Platinum Elisa (Konturek et al, 1994).","Pancreas isolated from all the rats was weighed. Later for histological examination and pancreatic DNA synthesis was done for the pancreatic tissue. Assessment of labeled thymidine in to DNA was done for the estimation of rate of pancreatic DNA synthesis as per previously reported study. Rate of DNA synthesis was expressed as disintegrations of labeled thymidine per minute per microgram DNA (dpm^g DNA) (Dembi’ nski et al, 2006).","Hematoxylin and eosin (H&E) staining was done for the examination of damage of pancreatic tissue as given in previous report. A scale of range from 0 to 3 was used for histological grading of necrosis, hemorrhages, vacuolization of acinar cells, leukocyte inflammatory infiltration and edema (Tomaszewska et al, 2000).","Data of present study given as mean ± SD (n=8). Statistically analysis was done by one way ANOVA (Dunnett.) In this study values p<0.05 was considered as significant."," Effect of Spatholobus suberectus and heparin on prothrombin time, weight of pancreas and pancreatic blood flow Effect of Spatholobus suberectus in combination with heparin was observed on prothrombin time in cerulean induced pancreatitis as shown in Figure 1. There was significant (p<0.05, p<0.01) increase in the prothrombin time in heparin, SS 100 & 200 mg, heparin+SS 100 mg/kg and heparin+SS 200 mg/kg treated group than negative control group.\nWeight of pancreas was significantly increases in cerulean induced pancreatitis group i.e. negative control group than control group. It was also observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases (p<0.05, p<0.01) the weight of pancreas compared to negative control group (Figure 2). Moreover treatment with Spatholobus suberectus and heparin alone and in combination increases the pancreatic blood flow which is calculated as % of control group than negative control group (Figure 3).\nEffect of Spatholobus suberectus in combination with heparin was observed on prothrombin time as international normalized ratio (INR) in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic weight in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic blood flow (% of control) in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus in combination with heparin was observed on prothrombin time in cerulean induced pancreatitis as shown in Figure 1. There was significant (p<0.05, p<0.01) increase in the prothrombin time in heparin, SS 100 & 200 mg, heparin+SS 100 mg/kg and heparin+SS 200 mg/kg treated group than negative control group.\nWeight of pancreas was significantly increases in cerulean induced pancreatitis group i.e. negative control group than control group. It was also observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases (p<0.05, p<0.01) the weight of pancreas compared to negative control group (Figure 2). Moreover treatment with Spatholobus suberectus and heparin alone and in combination increases the pancreatic blood flow which is calculated as % of control group than negative control group (Figure 3).\nEffect of Spatholobus suberectus in combination with heparin was observed on prothrombin time as international normalized ratio (INR) in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic weight in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic blood flow (% of control) in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\n Effect of Spatholobus suberectus and heparin on biochemical parameters Effect of Spatholobus suberectus and heparin alone and in combination on the concentration of D-dimer in cerulean induced pancreatitis as shown Figure 4. It was observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases the concentration of D-dimer in cerulean induced pancreatitis than negative control group.\nEffect of Spatholobus suberectus in combination with heparin was observed on the concentration of D-dimer in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nIn cerulean induced pancreatitis rats the activity of lipase and amylase enzyme and concentration of Π^-1β significantly increases than control group. There were significant decrease in the activity of lipase and amylase enzyme and concentration of Π^-1β in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 5 a, b & c.\nEffect of Spatholobus suberectus and heparin on biochemical parameter in cerulean induced pancreatitis. (a) Amylase activity; (b) Lipase activity; (c) Interlukin 1 β\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus and heparin alone and in combination on the concentration of D-dimer in cerulean induced pancreatitis as shown Figure 4. It was observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases the concentration of D-dimer in cerulean induced pancreatitis than negative control group.\nEffect of Spatholobus suberectus in combination with heparin was observed on the concentration of D-dimer in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nIn cerulean induced pancreatitis rats the activity of lipase and amylase enzyme and concentration of Π^-1β significantly increases than control group. There were significant decrease in the activity of lipase and amylase enzyme and concentration of Π^-1β in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 5 a, b & c.\nEffect of Spatholobus suberectus and heparin on biochemical parameter in cerulean induced pancreatitis. (a) Amylase activity; (b) Lipase activity; (c) Interlukin 1 β\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\n Effect of Spatholobus suberectus and heparin on pancreatic DNA synthesis Effect of Spatholobus suberectus and heparin alone and in combination on pancreatic DNA synthesis was shown in Figure 6. There were significant (p<0.05, p<0.01) increase in the pancreatic DNA synthesis in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 6.\nEffect of Spatholobus suberectus and heparin on pancreatic DNA synthesis in cerulean induced pancreatitis. Means ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus and heparin alone and in combination on pancreatic DNA synthesis was shown in Figure 6. There were significant (p<0.05, p<0.01) increase in the pancreatic DNA synthesis in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 6.\nEffect of Spatholobus suberectus and heparin on pancreatic DNA synthesis in cerulean induced pancreatitis. Means ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\n Effect of Spatholobus suberectus and heparin on histopathology of pancreas Histopathology study was performed on Spatholobus suberectus and heparin treated cerulean induced pancreatitis rats as shown in Table 1 and Figure 7. It was observed that pancreas of cerulean induced pancreatitis rats shows severe edema, moderate diffuse infiltration of inflammatory leukocyte and hemorrhages. Acinar cell shows more than 50% of vacuolization and signs of necrosis was absent in the TS of pancreas. Treatment with Spatholobus suberectus and heparin alone and in combination attenuates the development of pancreatitis. Histopathological representation shows that heparin in combination with SS significant decline in vacuolization of acinar cells, inflammatory infiltration and pancreatic edema.\nEffect of Spatholobus suberectus and heparin on histopathology signs of pancreas\nEffect of drug treatment on histopathology of pancreatic tissue in cerulean induced pancreatitis rat model. A. Control; B. Negative control; C. SS 200 mg/kg; D. Heparin+SS 200 mg/kg.\nHistopathology study was performed on Spatholobus suberectus and heparin treated cerulean induced pancreatitis rats as shown in Table 1 and Figure 7. It was observed that pancreas of cerulean induced pancreatitis rats shows severe edema, moderate diffuse infiltration of inflammatory leukocyte and hemorrhages. Acinar cell shows more than 50% of vacuolization and signs of necrosis was absent in the TS of pancreas. Treatment with Spatholobus suberectus and heparin alone and in combination attenuates the development of pancreatitis. Histopathological representation shows that heparin in combination with SS significant decline in vacuolization of acinar cells, inflammatory infiltration and pancreatic edema.\nEffect of Spatholobus suberectus and heparin on histopathology signs of pancreas\nEffect of drug treatment on histopathology of pancreatic tissue in cerulean induced pancreatitis rat model. A. Control; B. Negative control; C. SS 200 mg/kg; D. Heparin+SS 200 mg/kg.","Effect of Spatholobus suberectus in combination with heparin was observed on prothrombin time in cerulean induced pancreatitis as shown in Figure 1. There was significant (p<0.05, p<0.01) increase in the prothrombin time in heparin, SS 100 & 200 mg, heparin+SS 100 mg/kg and heparin+SS 200 mg/kg treated group than negative control group.\nWeight of pancreas was significantly increases in cerulean induced pancreatitis group i.e. negative control group than control group. It was also observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases (p<0.05, p<0.01) the weight of pancreas compared to negative control group (Figure 2). Moreover treatment with Spatholobus suberectus and heparin alone and in combination increases the pancreatic blood flow which is calculated as % of control group than negative control group (Figure 3).\nEffect of Spatholobus suberectus in combination with heparin was observed on prothrombin time as international normalized ratio (INR) in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic weight in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic blood flow (% of control) in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control","Effect of Spatholobus suberectus and heparin alone and in combination on the concentration of D-dimer in cerulean induced pancreatitis as shown Figure 4. It was observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases the concentration of D-dimer in cerulean induced pancreatitis than negative control group.\nEffect of Spatholobus suberectus in combination with heparin was observed on the concentration of D-dimer in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nIn cerulean induced pancreatitis rats the activity of lipase and amylase enzyme and concentration of Π^-1β significantly increases than control group. There were significant decrease in the activity of lipase and amylase enzyme and concentration of Π^-1β in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 5 a, b & c.\nEffect of Spatholobus suberectus and heparin on biochemical parameter in cerulean induced pancreatitis. (a) Amylase activity; (b) Lipase activity; (c) Interlukin 1 β\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control","Effect of Spatholobus suberectus and heparin alone and in combination on pancreatic DNA synthesis was shown in Figure 6. There were significant (p<0.05, p<0.01) increase in the pancreatic DNA synthesis in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 6.\nEffect of Spatholobus suberectus and heparin on pancreatic DNA synthesis in cerulean induced pancreatitis. Means ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control","Histopathology study was performed on Spatholobus suberectus and heparin treated cerulean induced pancreatitis rats as shown in Table 1 and Figure 7. It was observed that pancreas of cerulean induced pancreatitis rats shows severe edema, moderate diffuse infiltration of inflammatory leukocyte and hemorrhages. Acinar cell shows more than 50% of vacuolization and signs of necrosis was absent in the TS of pancreas. Treatment with Spatholobus suberectus and heparin alone and in combination attenuates the development of pancreatitis. Histopathological representation shows that heparin in combination with SS significant decline in vacuolization of acinar cells, inflammatory infiltration and pancreatic edema.\nEffect of Spatholobus suberectus and heparin on histopathology signs of pancreas\nEffect of drug treatment on histopathology of pancreatic tissue in cerulean induced pancreatitis rat model. A. Control; B. Negative control; C. SS 200 mg/kg; D. Heparin+SS 200 mg/kg.","Heparin reported to have beneficial effect on pancreatitis. Heparin manages the pancreatitis by inhibiting the formation of thrombin through heparin-antithrombin III complex but its effect over inflammatory pathway is not evaluated yet (Warzecha et al, 2010). Moreover Spatholobus suberectus reported to posses anti inflammatory, antioxidant and anti platelet activity (Zhang et al, 2016). Thus an attempt was made to evaluate the beneficial effect of Spatholobus suberectus in cerulean induced pancreatitis and synergistic effect of SS with heparin in cerulean induced pancreatitis rat model. It was evaluated by estimating the prothrobin time, pancreatic weight and blood flow in cerulean induced pancreatitis. Biochemical parameter such as concentration of IL-1β D-dimer, activity of amylase and lipase and DAN synthesis was estimated in cerulean induced pancreatitis rat model. Moreover histopathology study was also performed.\nThe result of the given study suggested that SS alone and in combination with heparin significantly increase in the prothrombin time and pancreatic blood flow in cerulean induced pancreatitis rat model. Literature suggested that in pancreatitis, pancreatic blood flow and prothrombin time significantly decreases and the anticoagulants reported to enhance the blood flow and thereby effective manages the pancreatitis (Ceranowicz et al, 2015).\nThere is activation of leukocyte which results in inflammatory cytokines release that develops the inflammation in pancreatitis. In pancreatitis cytokines like Π^-1β is responsible for inflammatory response and activates the prostaglandin, platelet activating factor and TNFa. These mediators cease the synthesis of DNA and thereby do damage to the pancreatic cells (Fink and Norman, 1996; Vollmar and Menger, 2003). Histopathology of pancreatic tissue reveals the presence of edema, leukocyte infiltration, hemorrhages and vacuolization in pancreatitis.\nThe investigation reveals that SS alone and in presence of heparin effectively decreases the concentration of Π^1β, D-dimer in blood and activity of amylase and lipase than negative control group. Moreover SS alone and in combination with heparin reduces the DNA synthesis in pancreatitis rats. Effect of SS and heparin alone and in combination on histopathology of pancreas tissue attenuates the pancreatitis by reducing the edema, hemorrhages and leukocyte infiltration.","Present study concludes that treatment with SS alone effectively manages the pancreatitis by ceasing the inflammatory pathway. Moreover SS in combination with heparin gives synergistic effect as in combination it act on inflammatory and coagulation pathway simultaneously for the pancreatitis."],"string":"[\n \"Pancreatitis is a coagulation-like disorder. However, inflammation and coagulation is closely related to each other. Coagulation is induced by inflammation and activation of process of inflammation equally responsible for the coagulation as thrombosis (Levi et al, 2012). Reported study suggested that disturbance in microcirculation results in formation of pro-inflammatory cytokines; oxygen derived free radicals, release of proteolytic enzyme and activation of leukocytes (Salomone et al, 2003). Thrombin forms as inflammatory cytokines enhances the expression of tissue factor on endothelium and monocytes (Maeda et al, 2006).\\nReported study suggested that heparin/anticoagulant shows protective effect in pancreatitis in animal as well as clinical study. Pretreatment with heparin ceases the development of pancreatitis induced by cerulean, taurocholate and bile on various animal studies (Gabryelewicz et al, 1969). Moreover heparin restores the pancreatic function in cerulean induced pancreatitis if it is administered after the induction of pancreatitis (Dobosz et al, 1998; Qiu et al, 2007). Moreover a study suggested that heparin manages the hyperlipidemia induced pancreatitis when co administered with insulin and in sever pancreatitis it protects the encephalopathy (Alagözlü et al, 2006; Kyriakidis et al, 2006). Heparin manages the pancreatitis by inhibiting the formation of thrombin through heparin-antithrombin III complex (Warzecha et al, 2010). However, effect of heparin over inflammatory parameters or pathway is not given in the literatures.\\nSpatholobus suberectus belongs to family: Leguminosae used for the management of several disorders such as rheumatism, anemia and abnormal menstruation traditionally in China (Lam et al, 2000; Li et al, 2003; Yen, 1992). Reported study suggested that several compounds like butin, liquiritigenin, dihydroquercetin, plathymenin, eriodictyol and neoisoliquiritigenin were been isolated from stem of SS (Lee et al, 2006). A study on stem extract of SS effectively manages cerebral ischemia by attenuating NF-κΒ p65 and cytokines and thereby prevents the DNA damage (Zhang et al, 2016). Moreover studies like anti rheumatic, anti inflammatory, antioxidant and anti platelet activity of SS are reported in the literature (Zhang et al, 2016). Thus this study evaluates the synergistic effect of Spatholobus suberectus extract when treated with heparin in pancreatitis.\",\n \" Animals Healthy male Wistar rats of 180-200 g body weight were used for the pharmacological screening in the present study. The animals were housed at 25 ±2°C temperature, 12 h light/dark cycle and 60 ± 5 % of relative humidity. Rats were feed with standard diet and water ad libitum. Protocols of the present investigation for all the animal studies were approved by Capital University of Medical Science, Beijing.\\nHealthy male Wistar rats of 180-200 g body weight were used for the pharmacological screening in the present study. The animals were housed at 25 ±2°C temperature, 12 h light/dark cycle and 60 ± 5 % of relative humidity. Rats were feed with standard diet and water ad libitum. Protocols of the present investigation for all the animal studies were approved by Capital University of Medical Science, Beijing.\\n Plant Extract Spatholobus suberectus was procured from the local botanist. S. suberectus stem cut into small pieces and dried it under the shade. Later it was boiled for specific duration of time in distilled water and then filters the extract by using filter paper (150 μm). Thereafter the extract was lyophilized. During the experiment at the time of dosing extract of SS was dissolved in distilled H2O and for 5 min centrifuge it at 10000 RPM.\\nSpatholobus suberectus was procured from the local botanist. S. suberectus stem cut into small pieces and dried it under the shade. Later it was boiled for specific duration of time in distilled water and then filters the extract by using filter paper (150 μm). Thereafter the extract was lyophilized. During the experiment at the time of dosing extract of SS was dissolved in distilled H2O and for 5 min centrifuge it at 10000 RPM.\\n Induction of Pancreatitis All the rats were separated in to seven different group (n=8) such as Control group which recives only saline; Negative control group which receives cerulean (50 μg/kg, i.p.) five times at an interval of 1 h whithout any pretreatment of drug; Heparin treated group receives 150 U/kg of heparin 1 day after the cerulean by subcutaneous injection twice a day; SS treated group receives 100 and 200 mg/kg of SS extract p. o. 1 day after the cerulean injection; Heparin +SS treated group receives heparin (150 U/kg, s. c.) and SS (100 and 200 mg/kg) 1 day after the cerulean injection for the duration of one week (Baczy’ nska et al, 2004).\\nAll the rats were separated in to seven different group (n=8) such as Control group which recives only saline; Negative control group which receives cerulean (50 μg/kg, i.p.) five times at an interval of 1 h whithout any pretreatment of drug; Heparin treated group receives 150 U/kg of heparin 1 day after the cerulean by subcutaneous injection twice a day; SS treated group receives 100 and 200 mg/kg of SS extract p. o. 1 day after the cerulean injection; Heparin +SS treated group receives heparin (150 U/kg, s. c.) and SS (100 and 200 mg/kg) 1 day after the cerulean injection for the duration of one week (Baczy’ nska et al, 2004).\\n Estimation of pancreatic blood flow All the animals were anesthetized by ketamine (50mg/kg, i.p.) at the end of protocol and pancreatic blood flow was estimated as per previously reported methods in exposed pancreas by using a laser Doppler flowmeter. Interpretation of data was represented as percent change from value obtained in control group without induction of pancreatitis.\\nAll the animals were anesthetized by ketamine (50mg/kg, i.p.) at the end of protocol and pancreatic blood flow was estimated as per previously reported methods in exposed pancreas by using a laser Doppler flowmeter. Interpretation of data was represented as percent change from value obtained in control group without induction of pancreatitis.\\n Estimation of Biochemical parameters Later abdominal aorta was used for the collection of fresh blood sample and prothrombin time was estimated in it as international normalized ratio using test strip (Alere San Diego, Inc., USA).\\nImmunoturbidimetric assay method was used for the estimation of concentration of plasma D-Dimer by using automatic coagulation analyzer (BCS XP System, Simens Healthcare Diagnostics, Garmany). Kodak Ectachem DT II System analyzer was used for the estimation of activity of amylase and lipase in serum using using Amyl and Lipa DT Slides. Interlukin 1β (IL-Ιβ) concentration was estimated in the serum by using IL-Ιβ Platinum Elisa (Konturek et al, 1994).\\nLater abdominal aorta was used for the collection of fresh blood sample and prothrombin time was estimated in it as international normalized ratio using test strip (Alere San Diego, Inc., USA).\\nImmunoturbidimetric assay method was used for the estimation of concentration of plasma D-Dimer by using automatic coagulation analyzer (BCS XP System, Simens Healthcare Diagnostics, Garmany). Kodak Ectachem DT II System analyzer was used for the estimation of activity of amylase and lipase in serum using using Amyl and Lipa DT Slides. Interlukin 1β (IL-Ιβ) concentration was estimated in the serum by using IL-Ιβ Platinum Elisa (Konturek et al, 1994).\\n Estimation of pancreatic DNA synthesis Pancreas isolated from all the rats was weighed. Later for histological examination and pancreatic DNA synthesis was done for the pancreatic tissue. Assessment of labeled thymidine in to DNA was done for the estimation of rate of pancreatic DNA synthesis as per previously reported study. Rate of DNA synthesis was expressed as disintegrations of labeled thymidine per minute per microgram DNA (dpm^g DNA) (Dembi’ nski et al, 2006).\\nPancreas isolated from all the rats was weighed. Later for histological examination and pancreatic DNA synthesis was done for the pancreatic tissue. Assessment of labeled thymidine in to DNA was done for the estimation of rate of pancreatic DNA synthesis as per previously reported study. Rate of DNA synthesis was expressed as disintegrations of labeled thymidine per minute per microgram DNA (dpm^g DNA) (Dembi’ nski et al, 2006).\\n Histopathology study Hematoxylin and eosin (H&E) staining was done for the examination of damage of pancreatic tissue as given in previous report. A scale of range from 0 to 3 was used for histological grading of necrosis, hemorrhages, vacuolization of acinar cells, leukocyte inflammatory infiltration and edema (Tomaszewska et al, 2000).\\nHematoxylin and eosin (H&E) staining was done for the examination of damage of pancreatic tissue as given in previous report. A scale of range from 0 to 3 was used for histological grading of necrosis, hemorrhages, vacuolization of acinar cells, leukocyte inflammatory infiltration and edema (Tomaszewska et al, 2000).\\n Statistical analysis Data of present study given as mean ± SD (n=8). Statistically analysis was done by one way ANOVA (Dunnett.) In this study values p<0.05 was considered as significant.\\nData of present study given as mean ± SD (n=8). Statistically analysis was done by one way ANOVA (Dunnett.) In this study values p<0.05 was considered as significant.\",\n \"Healthy male Wistar rats of 180-200 g body weight were used for the pharmacological screening in the present study. The animals were housed at 25 ±2°C temperature, 12 h light/dark cycle and 60 ± 5 % of relative humidity. Rats were feed with standard diet and water ad libitum. Protocols of the present investigation for all the animal studies were approved by Capital University of Medical Science, Beijing.\",\n \"Spatholobus suberectus was procured from the local botanist. S. suberectus stem cut into small pieces and dried it under the shade. Later it was boiled for specific duration of time in distilled water and then filters the extract by using filter paper (150 μm). Thereafter the extract was lyophilized. During the experiment at the time of dosing extract of SS was dissolved in distilled H2O and for 5 min centrifuge it at 10000 RPM.\",\n \"All the rats were separated in to seven different group (n=8) such as Control group which recives only saline; Negative control group which receives cerulean (50 μg/kg, i.p.) five times at an interval of 1 h whithout any pretreatment of drug; Heparin treated group receives 150 U/kg of heparin 1 day after the cerulean by subcutaneous injection twice a day; SS treated group receives 100 and 200 mg/kg of SS extract p. o. 1 day after the cerulean injection; Heparin +SS treated group receives heparin (150 U/kg, s. c.) and SS (100 and 200 mg/kg) 1 day after the cerulean injection for the duration of one week (Baczy’ nska et al, 2004).\",\n \"All the animals were anesthetized by ketamine (50mg/kg, i.p.) at the end of protocol and pancreatic blood flow was estimated as per previously reported methods in exposed pancreas by using a laser Doppler flowmeter. Interpretation of data was represented as percent change from value obtained in control group without induction of pancreatitis.\",\n \"Later abdominal aorta was used for the collection of fresh blood sample and prothrombin time was estimated in it as international normalized ratio using test strip (Alere San Diego, Inc., USA).\\nImmunoturbidimetric assay method was used for the estimation of concentration of plasma D-Dimer by using automatic coagulation analyzer (BCS XP System, Simens Healthcare Diagnostics, Garmany). Kodak Ectachem DT II System analyzer was used for the estimation of activity of amylase and lipase in serum using using Amyl and Lipa DT Slides. Interlukin 1β (IL-Ιβ) concentration was estimated in the serum by using IL-Ιβ Platinum Elisa (Konturek et al, 1994).\",\n \"Pancreas isolated from all the rats was weighed. Later for histological examination and pancreatic DNA synthesis was done for the pancreatic tissue. Assessment of labeled thymidine in to DNA was done for the estimation of rate of pancreatic DNA synthesis as per previously reported study. Rate of DNA synthesis was expressed as disintegrations of labeled thymidine per minute per microgram DNA (dpm^g DNA) (Dembi’ nski et al, 2006).\",\n \"Hematoxylin and eosin (H&E) staining was done for the examination of damage of pancreatic tissue as given in previous report. A scale of range from 0 to 3 was used for histological grading of necrosis, hemorrhages, vacuolization of acinar cells, leukocyte inflammatory infiltration and edema (Tomaszewska et al, 2000).\",\n \"Data of present study given as mean ± SD (n=8). Statistically analysis was done by one way ANOVA (Dunnett.) In this study values p<0.05 was considered as significant.\",\n \" Effect of Spatholobus suberectus and heparin on prothrombin time, weight of pancreas and pancreatic blood flow Effect of Spatholobus suberectus in combination with heparin was observed on prothrombin time in cerulean induced pancreatitis as shown in Figure 1. There was significant (p<0.05, p<0.01) increase in the prothrombin time in heparin, SS 100 & 200 mg, heparin+SS 100 mg/kg and heparin+SS 200 mg/kg treated group than negative control group.\\nWeight of pancreas was significantly increases in cerulean induced pancreatitis group i.e. negative control group than control group. It was also observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases (p<0.05, p<0.01) the weight of pancreas compared to negative control group (Figure 2). Moreover treatment with Spatholobus suberectus and heparin alone and in combination increases the pancreatic blood flow which is calculated as % of control group than negative control group (Figure 3).\\nEffect of Spatholobus suberectus in combination with heparin was observed on prothrombin time as international normalized ratio (INR) in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic weight in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic blood flow (% of control) in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nEffect of Spatholobus suberectus in combination with heparin was observed on prothrombin time in cerulean induced pancreatitis as shown in Figure 1. There was significant (p<0.05, p<0.01) increase in the prothrombin time in heparin, SS 100 & 200 mg, heparin+SS 100 mg/kg and heparin+SS 200 mg/kg treated group than negative control group.\\nWeight of pancreas was significantly increases in cerulean induced pancreatitis group i.e. negative control group than control group. It was also observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases (p<0.05, p<0.01) the weight of pancreas compared to negative control group (Figure 2). Moreover treatment with Spatholobus suberectus and heparin alone and in combination increases the pancreatic blood flow which is calculated as % of control group than negative control group (Figure 3).\\nEffect of Spatholobus suberectus in combination with heparin was observed on prothrombin time as international normalized ratio (INR) in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic weight in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic blood flow (% of control) in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\n Effect of Spatholobus suberectus and heparin on biochemical parameters Effect of Spatholobus suberectus and heparin alone and in combination on the concentration of D-dimer in cerulean induced pancreatitis as shown Figure 4. It was observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases the concentration of D-dimer in cerulean induced pancreatitis than negative control group.\\nEffect of Spatholobus suberectus in combination with heparin was observed on the concentration of D-dimer in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nIn cerulean induced pancreatitis rats the activity of lipase and amylase enzyme and concentration of Π^-1β significantly increases than control group. There were significant decrease in the activity of lipase and amylase enzyme and concentration of Π^-1β in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 5 a, b & c.\\nEffect of Spatholobus suberectus and heparin on biochemical parameter in cerulean induced pancreatitis. (a) Amylase activity; (b) Lipase activity; (c) Interlukin 1 β\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nEffect of Spatholobus suberectus and heparin alone and in combination on the concentration of D-dimer in cerulean induced pancreatitis as shown Figure 4. It was observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases the concentration of D-dimer in cerulean induced pancreatitis than negative control group.\\nEffect of Spatholobus suberectus in combination with heparin was observed on the concentration of D-dimer in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nIn cerulean induced pancreatitis rats the activity of lipase and amylase enzyme and concentration of Π^-1β significantly increases than control group. There were significant decrease in the activity of lipase and amylase enzyme and concentration of Π^-1β in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 5 a, b & c.\\nEffect of Spatholobus suberectus and heparin on biochemical parameter in cerulean induced pancreatitis. (a) Amylase activity; (b) Lipase activity; (c) Interlukin 1 β\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\n Effect of Spatholobus suberectus and heparin on pancreatic DNA synthesis Effect of Spatholobus suberectus and heparin alone and in combination on pancreatic DNA synthesis was shown in Figure 6. There were significant (p<0.05, p<0.01) increase in the pancreatic DNA synthesis in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 6.\\nEffect of Spatholobus suberectus and heparin on pancreatic DNA synthesis in cerulean induced pancreatitis. Means ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nEffect of Spatholobus suberectus and heparin alone and in combination on pancreatic DNA synthesis was shown in Figure 6. There were significant (p<0.05, p<0.01) increase in the pancreatic DNA synthesis in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 6.\\nEffect of Spatholobus suberectus and heparin on pancreatic DNA synthesis in cerulean induced pancreatitis. Means ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\n Effect of Spatholobus suberectus and heparin on histopathology of pancreas Histopathology study was performed on Spatholobus suberectus and heparin treated cerulean induced pancreatitis rats as shown in Table 1 and Figure 7. It was observed that pancreas of cerulean induced pancreatitis rats shows severe edema, moderate diffuse infiltration of inflammatory leukocyte and hemorrhages. Acinar cell shows more than 50% of vacuolization and signs of necrosis was absent in the TS of pancreas. Treatment with Spatholobus suberectus and heparin alone and in combination attenuates the development of pancreatitis. Histopathological representation shows that heparin in combination with SS significant decline in vacuolization of acinar cells, inflammatory infiltration and pancreatic edema.\\nEffect of Spatholobus suberectus and heparin on histopathology signs of pancreas\\nEffect of drug treatment on histopathology of pancreatic tissue in cerulean induced pancreatitis rat model. A. Control; B. Negative control; C. SS 200 mg/kg; D. Heparin+SS 200 mg/kg.\\nHistopathology study was performed on Spatholobus suberectus and heparin treated cerulean induced pancreatitis rats as shown in Table 1 and Figure 7. It was observed that pancreas of cerulean induced pancreatitis rats shows severe edema, moderate diffuse infiltration of inflammatory leukocyte and hemorrhages. Acinar cell shows more than 50% of vacuolization and signs of necrosis was absent in the TS of pancreas. Treatment with Spatholobus suberectus and heparin alone and in combination attenuates the development of pancreatitis. Histopathological representation shows that heparin in combination with SS significant decline in vacuolization of acinar cells, inflammatory infiltration and pancreatic edema.\\nEffect of Spatholobus suberectus and heparin on histopathology signs of pancreas\\nEffect of drug treatment on histopathology of pancreatic tissue in cerulean induced pancreatitis rat model. A. Control; B. Negative control; C. SS 200 mg/kg; D. Heparin+SS 200 mg/kg.\",\n \"Effect of Spatholobus suberectus in combination with heparin was observed on prothrombin time in cerulean induced pancreatitis as shown in Figure 1. There was significant (p<0.05, p<0.01) increase in the prothrombin time in heparin, SS 100 & 200 mg, heparin+SS 100 mg/kg and heparin+SS 200 mg/kg treated group than negative control group.\\nWeight of pancreas was significantly increases in cerulean induced pancreatitis group i.e. negative control group than control group. It was also observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases (p<0.05, p<0.01) the weight of pancreas compared to negative control group (Figure 2). Moreover treatment with Spatholobus suberectus and heparin alone and in combination increases the pancreatic blood flow which is calculated as % of control group than negative control group (Figure 3).\\nEffect of Spatholobus suberectus in combination with heparin was observed on prothrombin time as international normalized ratio (INR) in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic weight in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic blood flow (% of control) in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\",\n \"Effect of Spatholobus suberectus and heparin alone and in combination on the concentration of D-dimer in cerulean induced pancreatitis as shown Figure 4. It was observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases the concentration of D-dimer in cerulean induced pancreatitis than negative control group.\\nEffect of Spatholobus suberectus in combination with heparin was observed on the concentration of D-dimer in cerulean induced pancreatitis\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\\nIn cerulean induced pancreatitis rats the activity of lipase and amylase enzyme and concentration of Π^-1β significantly increases than control group. There were significant decrease in the activity of lipase and amylase enzyme and concentration of Π^-1β in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 5 a, b & c.\\nEffect of Spatholobus suberectus and heparin on biochemical parameter in cerulean induced pancreatitis. (a) Amylase activity; (b) Lipase activity; (c) Interlukin 1 β\\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\",\n \"Effect of Spatholobus suberectus and heparin alone and in combination on pancreatic DNA synthesis was shown in Figure 6. There were significant (p<0.05, p<0.01) increase in the pancreatic DNA synthesis in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 6.\\nEffect of Spatholobus suberectus and heparin on pancreatic DNA synthesis in cerulean induced pancreatitis. Means ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\",\n \"Histopathology study was performed on Spatholobus suberectus and heparin treated cerulean induced pancreatitis rats as shown in Table 1 and Figure 7. It was observed that pancreas of cerulean induced pancreatitis rats shows severe edema, moderate diffuse infiltration of inflammatory leukocyte and hemorrhages. Acinar cell shows more than 50% of vacuolization and signs of necrosis was absent in the TS of pancreas. Treatment with Spatholobus suberectus and heparin alone and in combination attenuates the development of pancreatitis. Histopathological representation shows that heparin in combination with SS significant decline in vacuolization of acinar cells, inflammatory infiltration and pancreatic edema.\\nEffect of Spatholobus suberectus and heparin on histopathology signs of pancreas\\nEffect of drug treatment on histopathology of pancreatic tissue in cerulean induced pancreatitis rat model. A. Control; B. Negative control; C. SS 200 mg/kg; D. Heparin+SS 200 mg/kg.\",\n \"Heparin reported to have beneficial effect on pancreatitis. Heparin manages the pancreatitis by inhibiting the formation of thrombin through heparin-antithrombin III complex but its effect over inflammatory pathway is not evaluated yet (Warzecha et al, 2010). Moreover Spatholobus suberectus reported to posses anti inflammatory, antioxidant and anti platelet activity (Zhang et al, 2016). Thus an attempt was made to evaluate the beneficial effect of Spatholobus suberectus in cerulean induced pancreatitis and synergistic effect of SS with heparin in cerulean induced pancreatitis rat model. It was evaluated by estimating the prothrobin time, pancreatic weight and blood flow in cerulean induced pancreatitis. Biochemical parameter such as concentration of IL-1β D-dimer, activity of amylase and lipase and DAN synthesis was estimated in cerulean induced pancreatitis rat model. Moreover histopathology study was also performed.\\nThe result of the given study suggested that SS alone and in combination with heparin significantly increase in the prothrombin time and pancreatic blood flow in cerulean induced pancreatitis rat model. Literature suggested that in pancreatitis, pancreatic blood flow and prothrombin time significantly decreases and the anticoagulants reported to enhance the blood flow and thereby effective manages the pancreatitis (Ceranowicz et al, 2015).\\nThere is activation of leukocyte which results in inflammatory cytokines release that develops the inflammation in pancreatitis. In pancreatitis cytokines like Π^-1β is responsible for inflammatory response and activates the prostaglandin, platelet activating factor and TNFa. These mediators cease the synthesis of DNA and thereby do damage to the pancreatic cells (Fink and Norman, 1996; Vollmar and Menger, 2003). Histopathology of pancreatic tissue reveals the presence of edema, leukocyte infiltration, hemorrhages and vacuolization in pancreatitis.\\nThe investigation reveals that SS alone and in presence of heparin effectively decreases the concentration of Π^1β, D-dimer in blood and activity of amylase and lipase than negative control group. Moreover SS alone and in combination with heparin reduces the DNA synthesis in pancreatitis rats. Effect of SS and heparin alone and in combination on histopathology of pancreas tissue attenuates the pancreatitis by reducing the edema, hemorrhages and leukocyte infiltration.\",\n \"Present study concludes that treatment with SS alone effectively manages the pancreatitis by ceasing the inflammatory pathway. Moreover SS in combination with heparin gives synergistic effect as in combination it act on inflammatory and coagulation pathway simultaneously for the pancreatitis.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["intro","material|methods",null,null,null,null,null,null,null,null,null,null,null,null,null,"discussion","conclusion"],"string":"[\n \"intro\",\n \"material|methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusion\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["\nSpatholobus suberectus\n","Pancreatitis","Heparin","Cerulean"],"string":"[\n \"\\nSpatholobus suberectus\\n\",\n \"Pancreatitis\",\n \"Heparin\",\n \"Cerulean\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Introduction:\nPancreatitis is a coagulation-like disorder. However, inflammation and coagulation is closely related to each other. Coagulation is induced by inflammation and activation of process of inflammation equally responsible for the coagulation as thrombosis (Levi et al, 2012). Reported study suggested that disturbance in microcirculation results in formation of pro-inflammatory cytokines; oxygen derived free radicals, release of proteolytic enzyme and activation of leukocytes (Salomone et al, 2003). Thrombin forms as inflammatory cytokines enhances the expression of tissue factor on endothelium and monocytes (Maeda et al, 2006).\nReported study suggested that heparin/anticoagulant shows protective effect in pancreatitis in animal as well as clinical study. Pretreatment with heparin ceases the development of pancreatitis induced by cerulean, taurocholate and bile on various animal studies (Gabryelewicz et al, 1969). Moreover heparin restores the pancreatic function in cerulean induced pancreatitis if it is administered after the induction of pancreatitis (Dobosz et al, 1998; Qiu et al, 2007). Moreover a study suggested that heparin manages the hyperlipidemia induced pancreatitis when co administered with insulin and in sever pancreatitis it protects the encephalopathy (Alagözlü et al, 2006; Kyriakidis et al, 2006). Heparin manages the pancreatitis by inhibiting the formation of thrombin through heparin-antithrombin III complex (Warzecha et al, 2010). However, effect of heparin over inflammatory parameters or pathway is not given in the literatures.\nSpatholobus suberectus belongs to family: Leguminosae used for the management of several disorders such as rheumatism, anemia and abnormal menstruation traditionally in China (Lam et al, 2000; Li et al, 2003; Yen, 1992). Reported study suggested that several compounds like butin, liquiritigenin, dihydroquercetin, plathymenin, eriodictyol and neoisoliquiritigenin were been isolated from stem of SS (Lee et al, 2006). A study on stem extract of SS effectively manages cerebral ischemia by attenuating NF-κΒ p65 and cytokines and thereby prevents the DNA damage (Zhang et al, 2016). Moreover studies like anti rheumatic, anti inflammatory, antioxidant and anti platelet activity of SS are reported in the literature (Zhang et al, 2016). Thus this study evaluates the synergistic effect of Spatholobus suberectus extract when treated with heparin in pancreatitis.\n\nMaterial and Methods:\n Animals Healthy male Wistar rats of 180-200 g body weight were used for the pharmacological screening in the present study. The animals were housed at 25 ±2°C temperature, 12 h light/dark cycle and 60 ± 5 % of relative humidity. Rats were feed with standard diet and water ad libitum. Protocols of the present investigation for all the animal studies were approved by Capital University of Medical Science, Beijing.\nHealthy male Wistar rats of 180-200 g body weight were used for the pharmacological screening in the present study. The animals were housed at 25 ±2°C temperature, 12 h light/dark cycle and 60 ± 5 % of relative humidity. Rats were feed with standard diet and water ad libitum. Protocols of the present investigation for all the animal studies were approved by Capital University of Medical Science, Beijing.\n Plant Extract Spatholobus suberectus was procured from the local botanist. S. suberectus stem cut into small pieces and dried it under the shade. Later it was boiled for specific duration of time in distilled water and then filters the extract by using filter paper (150 μm). Thereafter the extract was lyophilized. During the experiment at the time of dosing extract of SS was dissolved in distilled H2O and for 5 min centrifuge it at 10000 RPM.\nSpatholobus suberectus was procured from the local botanist. S. suberectus stem cut into small pieces and dried it under the shade. Later it was boiled for specific duration of time in distilled water and then filters the extract by using filter paper (150 μm). Thereafter the extract was lyophilized. During the experiment at the time of dosing extract of SS was dissolved in distilled H2O and for 5 min centrifuge it at 10000 RPM.\n Induction of Pancreatitis All the rats were separated in to seven different group (n=8) such as Control group which recives only saline; Negative control group which receives cerulean (50 μg/kg, i.p.) five times at an interval of 1 h whithout any pretreatment of drug; Heparin treated group receives 150 U/kg of heparin 1 day after the cerulean by subcutaneous injection twice a day; SS treated group receives 100 and 200 mg/kg of SS extract p. o. 1 day after the cerulean injection; Heparin +SS treated group receives heparin (150 U/kg, s. c.) and SS (100 and 200 mg/kg) 1 day after the cerulean injection for the duration of one week (Baczy’ nska et al, 2004).\nAll the rats were separated in to seven different group (n=8) such as Control group which recives only saline; Negative control group which receives cerulean (50 μg/kg, i.p.) five times at an interval of 1 h whithout any pretreatment of drug; Heparin treated group receives 150 U/kg of heparin 1 day after the cerulean by subcutaneous injection twice a day; SS treated group receives 100 and 200 mg/kg of SS extract p. o. 1 day after the cerulean injection; Heparin +SS treated group receives heparin (150 U/kg, s. c.) and SS (100 and 200 mg/kg) 1 day after the cerulean injection for the duration of one week (Baczy’ nska et al, 2004).\n Estimation of pancreatic blood flow All the animals were anesthetized by ketamine (50mg/kg, i.p.) at the end of protocol and pancreatic blood flow was estimated as per previously reported methods in exposed pancreas by using a laser Doppler flowmeter. Interpretation of data was represented as percent change from value obtained in control group without induction of pancreatitis.\nAll the animals were anesthetized by ketamine (50mg/kg, i.p.) at the end of protocol and pancreatic blood flow was estimated as per previously reported methods in exposed pancreas by using a laser Doppler flowmeter. Interpretation of data was represented as percent change from value obtained in control group without induction of pancreatitis.\n Estimation of Biochemical parameters Later abdominal aorta was used for the collection of fresh blood sample and prothrombin time was estimated in it as international normalized ratio using test strip (Alere San Diego, Inc., USA).\nImmunoturbidimetric assay method was used for the estimation of concentration of plasma D-Dimer by using automatic coagulation analyzer (BCS XP System, Simens Healthcare Diagnostics, Garmany). Kodak Ectachem DT II System analyzer was used for the estimation of activity of amylase and lipase in serum using using Amyl and Lipa DT Slides. Interlukin 1β (IL-Ιβ) concentration was estimated in the serum by using IL-Ιβ Platinum Elisa (Konturek et al, 1994).\nLater abdominal aorta was used for the collection of fresh blood sample and prothrombin time was estimated in it as international normalized ratio using test strip (Alere San Diego, Inc., USA).\nImmunoturbidimetric assay method was used for the estimation of concentration of plasma D-Dimer by using automatic coagulation analyzer (BCS XP System, Simens Healthcare Diagnostics, Garmany). Kodak Ectachem DT II System analyzer was used for the estimation of activity of amylase and lipase in serum using using Amyl and Lipa DT Slides. Interlukin 1β (IL-Ιβ) concentration was estimated in the serum by using IL-Ιβ Platinum Elisa (Konturek et al, 1994).\n Estimation of pancreatic DNA synthesis Pancreas isolated from all the rats was weighed. Later for histological examination and pancreatic DNA synthesis was done for the pancreatic tissue. Assessment of labeled thymidine in to DNA was done for the estimation of rate of pancreatic DNA synthesis as per previously reported study. Rate of DNA synthesis was expressed as disintegrations of labeled thymidine per minute per microgram DNA (dpm^g DNA) (Dembi’ nski et al, 2006).\nPancreas isolated from all the rats was weighed. Later for histological examination and pancreatic DNA synthesis was done for the pancreatic tissue. Assessment of labeled thymidine in to DNA was done for the estimation of rate of pancreatic DNA synthesis as per previously reported study. Rate of DNA synthesis was expressed as disintegrations of labeled thymidine per minute per microgram DNA (dpm^g DNA) (Dembi’ nski et al, 2006).\n Histopathology study Hematoxylin and eosin (H&E) staining was done for the examination of damage of pancreatic tissue as given in previous report. A scale of range from 0 to 3 was used for histological grading of necrosis, hemorrhages, vacuolization of acinar cells, leukocyte inflammatory infiltration and edema (Tomaszewska et al, 2000).\nHematoxylin and eosin (H&E) staining was done for the examination of damage of pancreatic tissue as given in previous report. A scale of range from 0 to 3 was used for histological grading of necrosis, hemorrhages, vacuolization of acinar cells, leukocyte inflammatory infiltration and edema (Tomaszewska et al, 2000).\n Statistical analysis Data of present study given as mean ± SD (n=8). Statistically analysis was done by one way ANOVA (Dunnett.) In this study values p<0.05 was considered as significant.\nData of present study given as mean ± SD (n=8). Statistically analysis was done by one way ANOVA (Dunnett.) In this study values p<0.05 was considered as significant.\n\nAnimals:\nHealthy male Wistar rats of 180-200 g body weight were used for the pharmacological screening in the present study. The animals were housed at 25 ±2°C temperature, 12 h light/dark cycle and 60 ± 5 % of relative humidity. Rats were feed with standard diet and water ad libitum. Protocols of the present investigation for all the animal studies were approved by Capital University of Medical Science, Beijing.\n\nPlant Extract:\nSpatholobus suberectus was procured from the local botanist. S. suberectus stem cut into small pieces and dried it under the shade. Later it was boiled for specific duration of time in distilled water and then filters the extract by using filter paper (150 μm). Thereafter the extract was lyophilized. During the experiment at the time of dosing extract of SS was dissolved in distilled H2O and for 5 min centrifuge it at 10000 RPM.\n\nInduction of Pancreatitis:\nAll the rats were separated in to seven different group (n=8) such as Control group which recives only saline; Negative control group which receives cerulean (50 μg/kg, i.p.) five times at an interval of 1 h whithout any pretreatment of drug; Heparin treated group receives 150 U/kg of heparin 1 day after the cerulean by subcutaneous injection twice a day; SS treated group receives 100 and 200 mg/kg of SS extract p. o. 1 day after the cerulean injection; Heparin +SS treated group receives heparin (150 U/kg, s. c.) and SS (100 and 200 mg/kg) 1 day after the cerulean injection for the duration of one week (Baczy’ nska et al, 2004).\n\nEstimation of pancreatic blood flow:\nAll the animals were anesthetized by ketamine (50mg/kg, i.p.) at the end of protocol and pancreatic blood flow was estimated as per previously reported methods in exposed pancreas by using a laser Doppler flowmeter. Interpretation of data was represented as percent change from value obtained in control group without induction of pancreatitis.\n\nEstimation of Biochemical parameters:\nLater abdominal aorta was used for the collection of fresh blood sample and prothrombin time was estimated in it as international normalized ratio using test strip (Alere San Diego, Inc., USA).\nImmunoturbidimetric assay method was used for the estimation of concentration of plasma D-Dimer by using automatic coagulation analyzer (BCS XP System, Simens Healthcare Diagnostics, Garmany). Kodak Ectachem DT II System analyzer was used for the estimation of activity of amylase and lipase in serum using using Amyl and Lipa DT Slides. Interlukin 1β (IL-Ιβ) concentration was estimated in the serum by using IL-Ιβ Platinum Elisa (Konturek et al, 1994).\n\nEstimation of pancreatic DNA synthesis:\nPancreas isolated from all the rats was weighed. Later for histological examination and pancreatic DNA synthesis was done for the pancreatic tissue. Assessment of labeled thymidine in to DNA was done for the estimation of rate of pancreatic DNA synthesis as per previously reported study. Rate of DNA synthesis was expressed as disintegrations of labeled thymidine per minute per microgram DNA (dpm^g DNA) (Dembi’ nski et al, 2006).\n\nHistopathology study:\nHematoxylin and eosin (H&E) staining was done for the examination of damage of pancreatic tissue as given in previous report. A scale of range from 0 to 3 was used for histological grading of necrosis, hemorrhages, vacuolization of acinar cells, leukocyte inflammatory infiltration and edema (Tomaszewska et al, 2000).\n\nStatistical analysis:\nData of present study given as mean ± SD (n=8). Statistically analysis was done by one way ANOVA (Dunnett.) In this study values p<0.05 was considered as significant.\n\nResult:\n Effect of Spatholobus suberectus and heparin on prothrombin time, weight of pancreas and pancreatic blood flow Effect of Spatholobus suberectus in combination with heparin was observed on prothrombin time in cerulean induced pancreatitis as shown in Figure 1. There was significant (p<0.05, p<0.01) increase in the prothrombin time in heparin, SS 100 & 200 mg, heparin+SS 100 mg/kg and heparin+SS 200 mg/kg treated group than negative control group.\nWeight of pancreas was significantly increases in cerulean induced pancreatitis group i.e. negative control group than control group. It was also observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases (p<0.05, p<0.01) the weight of pancreas compared to negative control group (Figure 2). Moreover treatment with Spatholobus suberectus and heparin alone and in combination increases the pancreatic blood flow which is calculated as % of control group than negative control group (Figure 3).\nEffect of Spatholobus suberectus in combination with heparin was observed on prothrombin time as international normalized ratio (INR) in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic weight in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic blood flow (% of control) in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus in combination with heparin was observed on prothrombin time in cerulean induced pancreatitis as shown in Figure 1. There was significant (p<0.05, p<0.01) increase in the prothrombin time in heparin, SS 100 & 200 mg, heparin+SS 100 mg/kg and heparin+SS 200 mg/kg treated group than negative control group.\nWeight of pancreas was significantly increases in cerulean induced pancreatitis group i.e. negative control group than control group. It was also observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases (p<0.05, p<0.01) the weight of pancreas compared to negative control group (Figure 2). Moreover treatment with Spatholobus suberectus and heparin alone and in combination increases the pancreatic blood flow which is calculated as % of control group than negative control group (Figure 3).\nEffect of Spatholobus suberectus in combination with heparin was observed on prothrombin time as international normalized ratio (INR) in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic weight in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic blood flow (% of control) in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\n Effect of Spatholobus suberectus and heparin on biochemical parameters Effect of Spatholobus suberectus and heparin alone and in combination on the concentration of D-dimer in cerulean induced pancreatitis as shown Figure 4. It was observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases the concentration of D-dimer in cerulean induced pancreatitis than negative control group.\nEffect of Spatholobus suberectus in combination with heparin was observed on the concentration of D-dimer in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nIn cerulean induced pancreatitis rats the activity of lipase and amylase enzyme and concentration of Π^-1β significantly increases than control group. There were significant decrease in the activity of lipase and amylase enzyme and concentration of Π^-1β in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 5 a, b & c.\nEffect of Spatholobus suberectus and heparin on biochemical parameter in cerulean induced pancreatitis. (a) Amylase activity; (b) Lipase activity; (c) Interlukin 1 β\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus and heparin alone and in combination on the concentration of D-dimer in cerulean induced pancreatitis as shown Figure 4. It was observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases the concentration of D-dimer in cerulean induced pancreatitis than negative control group.\nEffect of Spatholobus suberectus in combination with heparin was observed on the concentration of D-dimer in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nIn cerulean induced pancreatitis rats the activity of lipase and amylase enzyme and concentration of Π^-1β significantly increases than control group. There were significant decrease in the activity of lipase and amylase enzyme and concentration of Π^-1β in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 5 a, b & c.\nEffect of Spatholobus suberectus and heparin on biochemical parameter in cerulean induced pancreatitis. (a) Amylase activity; (b) Lipase activity; (c) Interlukin 1 β\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\n Effect of Spatholobus suberectus and heparin on pancreatic DNA synthesis Effect of Spatholobus suberectus and heparin alone and in combination on pancreatic DNA synthesis was shown in Figure 6. There were significant (p<0.05, p<0.01) increase in the pancreatic DNA synthesis in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 6.\nEffect of Spatholobus suberectus and heparin on pancreatic DNA synthesis in cerulean induced pancreatitis. Means ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus and heparin alone and in combination on pancreatic DNA synthesis was shown in Figure 6. There were significant (p<0.05, p<0.01) increase in the pancreatic DNA synthesis in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 6.\nEffect of Spatholobus suberectus and heparin on pancreatic DNA synthesis in cerulean induced pancreatitis. Means ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\n Effect of Spatholobus suberectus and heparin on histopathology of pancreas Histopathology study was performed on Spatholobus suberectus and heparin treated cerulean induced pancreatitis rats as shown in Table 1 and Figure 7. It was observed that pancreas of cerulean induced pancreatitis rats shows severe edema, moderate diffuse infiltration of inflammatory leukocyte and hemorrhages. Acinar cell shows more than 50% of vacuolization and signs of necrosis was absent in the TS of pancreas. Treatment with Spatholobus suberectus and heparin alone and in combination attenuates the development of pancreatitis. Histopathological representation shows that heparin in combination with SS significant decline in vacuolization of acinar cells, inflammatory infiltration and pancreatic edema.\nEffect of Spatholobus suberectus and heparin on histopathology signs of pancreas\nEffect of drug treatment on histopathology of pancreatic tissue in cerulean induced pancreatitis rat model. A. Control; B. Negative control; C. SS 200 mg/kg; D. Heparin+SS 200 mg/kg.\nHistopathology study was performed on Spatholobus suberectus and heparin treated cerulean induced pancreatitis rats as shown in Table 1 and Figure 7. It was observed that pancreas of cerulean induced pancreatitis rats shows severe edema, moderate diffuse infiltration of inflammatory leukocyte and hemorrhages. Acinar cell shows more than 50% of vacuolization and signs of necrosis was absent in the TS of pancreas. Treatment with Spatholobus suberectus and heparin alone and in combination attenuates the development of pancreatitis. Histopathological representation shows that heparin in combination with SS significant decline in vacuolization of acinar cells, inflammatory infiltration and pancreatic edema.\nEffect of Spatholobus suberectus and heparin on histopathology signs of pancreas\nEffect of drug treatment on histopathology of pancreatic tissue in cerulean induced pancreatitis rat model. A. Control; B. Negative control; C. SS 200 mg/kg; D. Heparin+SS 200 mg/kg.\n\nEffect of Spatholobus suberectus and heparin on prothrombin time, weight of pancreas and pancreatic blood flow:\nEffect of Spatholobus suberectus in combination with heparin was observed on prothrombin time in cerulean induced pancreatitis as shown in Figure 1. There was significant (p<0.05, p<0.01) increase in the prothrombin time in heparin, SS 100 & 200 mg, heparin+SS 100 mg/kg and heparin+SS 200 mg/kg treated group than negative control group.\nWeight of pancreas was significantly increases in cerulean induced pancreatitis group i.e. negative control group than control group. It was also observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases (p<0.05, p<0.01) the weight of pancreas compared to negative control group (Figure 2). Moreover treatment with Spatholobus suberectus and heparin alone and in combination increases the pancreatic blood flow which is calculated as % of control group than negative control group (Figure 3).\nEffect of Spatholobus suberectus in combination with heparin was observed on prothrombin time as international normalized ratio (INR) in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic weight in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nEffect of Spatholobus suberectus in combination with heparin was observed on pancreatic blood flow (% of control) in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\n\nEffect of Spatholobus suberectus and heparin on biochemical parameters:\nEffect of Spatholobus suberectus and heparin alone and in combination on the concentration of D-dimer in cerulean induced pancreatitis as shown Figure 4. It was observed that treatment with Spatholobus suberectus and heparin alone and in combination significantly decreases the concentration of D-dimer in cerulean induced pancreatitis than negative control group.\nEffect of Spatholobus suberectus in combination with heparin was observed on the concentration of D-dimer in cerulean induced pancreatitis\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\nIn cerulean induced pancreatitis rats the activity of lipase and amylase enzyme and concentration of Π^-1β significantly increases than control group. There were significant decrease in the activity of lipase and amylase enzyme and concentration of Π^-1β in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 5 a, b & c.\nEffect of Spatholobus suberectus and heparin on biochemical parameter in cerulean induced pancreatitis. (a) Amylase activity; (b) Lipase activity; (c) Interlukin 1 β\nMeans ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\n\nEffect of Spatholobus suberectus and heparin on pancreatic DNA synthesis:\nEffect of Spatholobus suberectus and heparin alone and in combination on pancreatic DNA synthesis was shown in Figure 6. There were significant (p<0.05, p<0.01) increase in the pancreatic DNA synthesis in Spatholobus suberectus and heparin alone and in combination treated group of rats compared to negative control group as shown in Figure 6.\nEffect of Spatholobus suberectus and heparin on pancreatic DNA synthesis in cerulean induced pancreatitis. Means ± SD (n=8), ##p < 0.01 Vs control, *p<0.05, **p<0.01 Vs Negative control\n\nEffect of Spatholobus suberectus and heparin on histopathology of pancreas:\nHistopathology study was performed on Spatholobus suberectus and heparin treated cerulean induced pancreatitis rats as shown in Table 1 and Figure 7. It was observed that pancreas of cerulean induced pancreatitis rats shows severe edema, moderate diffuse infiltration of inflammatory leukocyte and hemorrhages. Acinar cell shows more than 50% of vacuolization and signs of necrosis was absent in the TS of pancreas. Treatment with Spatholobus suberectus and heparin alone and in combination attenuates the development of pancreatitis. Histopathological representation shows that heparin in combination with SS significant decline in vacuolization of acinar cells, inflammatory infiltration and pancreatic edema.\nEffect of Spatholobus suberectus and heparin on histopathology signs of pancreas\nEffect of drug treatment on histopathology of pancreatic tissue in cerulean induced pancreatitis rat model. A. Control; B. Negative control; C. SS 200 mg/kg; D. Heparin+SS 200 mg/kg.\n\nDiscussion:\nHeparin reported to have beneficial effect on pancreatitis. Heparin manages the pancreatitis by inhibiting the formation of thrombin through heparin-antithrombin III complex but its effect over inflammatory pathway is not evaluated yet (Warzecha et al, 2010). Moreover Spatholobus suberectus reported to posses anti inflammatory, antioxidant and anti platelet activity (Zhang et al, 2016). Thus an attempt was made to evaluate the beneficial effect of Spatholobus suberectus in cerulean induced pancreatitis and synergistic effect of SS with heparin in cerulean induced pancreatitis rat model. It was evaluated by estimating the prothrobin time, pancreatic weight and blood flow in cerulean induced pancreatitis. Biochemical parameter such as concentration of IL-1β D-dimer, activity of amylase and lipase and DAN synthesis was estimated in cerulean induced pancreatitis rat model. Moreover histopathology study was also performed.\nThe result of the given study suggested that SS alone and in combination with heparin significantly increase in the prothrombin time and pancreatic blood flow in cerulean induced pancreatitis rat model. Literature suggested that in pancreatitis, pancreatic blood flow and prothrombin time significantly decreases and the anticoagulants reported to enhance the blood flow and thereby effective manages the pancreatitis (Ceranowicz et al, 2015).\nThere is activation of leukocyte which results in inflammatory cytokines release that develops the inflammation in pancreatitis. In pancreatitis cytokines like Π^-1β is responsible for inflammatory response and activates the prostaglandin, platelet activating factor and TNFa. These mediators cease the synthesis of DNA and thereby do damage to the pancreatic cells (Fink and Norman, 1996; Vollmar and Menger, 2003). Histopathology of pancreatic tissue reveals the presence of edema, leukocyte infiltration, hemorrhages and vacuolization in pancreatitis.\nThe investigation reveals that SS alone and in presence of heparin effectively decreases the concentration of Π^1β, D-dimer in blood and activity of amylase and lipase than negative control group. Moreover SS alone and in combination with heparin reduces the DNA synthesis in pancreatitis rats. Effect of SS and heparin alone and in combination on histopathology of pancreas tissue attenuates the pancreatitis by reducing the edema, hemorrhages and leukocyte infiltration.\n\nConclusion:\nPresent study concludes that treatment with SS alone effectively manages the pancreatitis by ceasing the inflammatory pathway. Moreover SS in combination with heparin gives synergistic effect as in combination it act on inflammatory and coagulation pathway simultaneously for the pancreatitis.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe present study evaluates the effect of Spatholobus suberectus stem extract (SS) in the management of pancreatitis alone and in combination with heparin.\n\nMethods:\nPancreatitis was induced pancreatitis by cerulean (50μg/kg, i.p.) five times at an interval of 1 h without any pretreatment of drug. Rats were treated with SS (100 and 200 mg/kg, p. o.) and heparin (150 U/kg, i.p.) alone and in combination for the duration of a week. Later pancreatic weight and blood flow was estimated and different biochemical parameters like concentration of D-dimer and Interleukin 1β (IL-Ιβ) and activity of amylase and lipase were determined in blood of pancreatitis rats. Moreover effect of drug treatment on DNA synthesis and histopathology was also estimated on cerulean induced pancreatitis rats.\n\nResults:\nResults of this study suggest that treatment with SS alone and in combination with heparin significantly increase in prothrombin time and pancreatic blood flow than negative control group. There was significant decrease in concentration of IL-Ιβ and D-dimer and activity of amylase and lipase in SS and heparin treated group than negative control group. Pancreatic DNA synthesis was also found to be reduced in SS and heparin alone and in combination treated group. Histopathology study also reveals that treatment with SS and heparin alone and in combination reduces edema, hemorrhages, leukocyte infiltration in the TS of pancreatic tissues.\n\nConclusions:\nPresent study concludes that treatment with SS alone effectively manages the pancreatitis by ceasing the inflammatory pathway and potentiates the effect of heparin in the management of pancreatitis."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nPancreatitis is a coagulation-like disorder. However, inflammation and coagulation is closely related to each other. Coagulation is induced by inflammation and activation of process of inflammation equally responsible for the coagulation as thrombosis (Levi et al, 2012). Reported study suggested that disturbance in microcirculation results in formation of pro-inflammatory cytokines; oxygen derived free radicals, release of proteolytic enzyme and activation of leukocytes (Salomone et al, 2003). Thrombin forms as inflammatory cytokines enhances the expression of tissue factor on endothelium and monocytes (Maeda et al, 2006).\nReported study suggested that heparin/anticoagulant shows protective effect in pancreatitis in animal as well as clinical study. Pretreatment with heparin ceases the development of pancreatitis induced by cerulean, taurocholate and bile on various animal studies (Gabryelewicz et al, 1969). Moreover heparin restores the pancreatic function in cerulean induced pancreatitis if it is administered after the induction of pancreatitis (Dobosz et al, 1998; Qiu et al, 2007). Moreover a study suggested that heparin manages the hyperlipidemia induced pancreatitis when co administered with insulin and in sever pancreatitis it protects the encephalopathy (Alagözlü et al, 2006; Kyriakidis et al, 2006). Heparin manages the pancreatitis by inhibiting the formation of thrombin through heparin-antithrombin III complex (Warzecha et al, 2010). However, effect of heparin over inflammatory parameters or pathway is not given in the literatures.\nSpatholobus suberectus belongs to family: Leguminosae used for the management of several disorders such as rheumatism, anemia and abnormal menstruation traditionally in China (Lam et al, 2000; Li et al, 2003; Yen, 1992). Reported study suggested that several compounds like butin, liquiritigenin, dihydroquercetin, plathymenin, eriodictyol and neoisoliquiritigenin were been isolated from stem of SS (Lee et al, 2006). A study on stem extract of SS effectively manages cerebral ischemia by attenuating NF-κΒ p65 and cytokines and thereby prevents the DNA damage (Zhang et al, 2016). Moreover studies like anti rheumatic, anti inflammatory, antioxidant and anti platelet activity of SS are reported in the literature (Zhang et al, 2016). Thus this study evaluates the synergistic effect of Spatholobus suberectus extract when treated with heparin in pancreatitis.\n\nConclusion:\nPresent study concludes that treatment with SS alone effectively manages the pancreatitis by ceasing the inflammatory pathway. Moreover SS in combination with heparin gives synergistic effect as in combination it act on inflammatory and coagulation pathway simultaneously for the pancreatitis."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nThe present study evaluates the effect of Spatholobus suberectus stem extract (SS) in the management of pancreatitis alone and in combination with heparin.\n\nMethods:\nPancreatitis was induced pancreatitis by cerulean (50μg/kg, i.p.) five times at an interval of 1 h without any pretreatment of drug. Rats were treated with SS (100 and 200 mg/kg, p. o.) and heparin (150 U/kg, i.p.) alone and in combination for the duration of a week. Later pancreatic weight and blood flow was estimated and different biochemical parameters like concentration of D-dimer and Interleukin 1β (IL-Ιβ) and activity of amylase and lipase were determined in blood of pancreatitis rats. Moreover effect of drug treatment on DNA synthesis and histopathology was also estimated on cerulean induced pancreatitis rats.\n\nResults:\nResults of this study suggest that treatment with SS alone and in combination with heparin significantly increase in prothrombin time and pancreatic blood flow than negative control group. There was significant decrease in concentration of IL-Ιβ and D-dimer and activity of amylase and lipase in SS and heparin treated group than negative control group. Pancreatic DNA synthesis was also found to be reduced in SS and heparin alone and in combination treated group. Histopathology study also reveals that treatment with SS and heparin alone and in combination reduces edema, hemorrhages, leukocyte infiltration in the TS of pancreatic tissues.\n\nConclusions:\nPresent study concludes that treatment with SS alone effectively manages the pancreatitis by ceasing the inflammatory pathway and potentiates the effect of heparin in the management of pancreatitis."},"whole_article_text_length":{"kind":"number","value":5394,"string":"5,394"},"whole_article_abstract_length":{"kind":"number","value":303,"string":"303"},"other_sections_lengths":{"kind":"list like","value":[80,81,143,60,124,78,59,35,1612,294,229,98,156],"string":"[\n 80,\n 81,\n 143,\n 60,\n 124,\n 78,\n 59,\n 35,\n 1612,\n 294,\n 229,\n 98,\n 156\n]","isConversational":false},"num_sections":{"kind":"number","value":17,"string":"17"},"most_frequent_words":{"kind":"list like","value":["heparin","control","pancreatitis","suberectus","spatholobus suberectus","spatholobus","group","cerulean","pancreatic","induced"],"string":"[\n \"heparin\",\n \"control\",\n \"pancreatitis\",\n \"suberectus\",\n \"spatholobus suberectus\",\n \"spatholobus\",\n \"group\",\n \"cerulean\",\n \"pancreatic\",\n \"induced\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["inflammatory coagulation pathway","pancreatitis heparin manages","effect heparin inflammatory","introduction pancreatitis coagulation","coagulation induced inflammation"],"string":"[\n \"inflammatory coagulation pathway\",\n \"pancreatitis heparin manages\",\n \"effect heparin inflammatory\",\n \"introduction pancreatitis coagulation\",\n \"coagulation induced inflammation\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"null"},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] \nSpatholobus suberectus\n | Pancreatitis | Heparin | Cerulean [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"null"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] \nSpatholobus suberectus\n | Pancreatitis | Heparin | Cerulean [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] \nSpatholobus suberectus\n | Pancreatitis | Heparin | Cerulean [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] \nSpatholobus suberectus\n | Pancreatitis | Heparin | Cerulean [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylases | Animals | Anticoagulants | Ceruletide | Drug Therapy, Combination | Fabaceae | Fibrin Fibrinogen Degradation Products | Heparin | Interleukin-1beta | Lipase | Male | Nucleic Acid Synthesis Inhibitors | Pancreas | Pancreatitis | Phytotherapy | Plant Extracts | Plant Stems | Protective Agents | Prothrombin Time | Rats | Rats, Wistar [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"null"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylases | Animals | Anticoagulants | Ceruletide | Drug Therapy, Combination | Fabaceae | Fibrin Fibrinogen Degradation Products | Heparin | Interleukin-1beta | Lipase | Male | Nucleic Acid Synthesis Inhibitors | Pancreas | Pancreatitis | Phytotherapy | Plant Extracts | Plant Stems | Protective Agents | Prothrombin Time | Rats | Rats, Wistar [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylases | Animals | Anticoagulants | Ceruletide | Drug Therapy, Combination | Fabaceae | Fibrin Fibrinogen Degradation Products | Heparin | Interleukin-1beta | Lipase | Male | Nucleic Acid Synthesis Inhibitors | Pancreas | Pancreatitis | Phytotherapy | Plant Extracts | Plant Stems | Protective Agents | Prothrombin Time | Rats | Rats, Wistar [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Amylases | Animals | Anticoagulants | Ceruletide | Drug Therapy, Combination | Fabaceae | Fibrin Fibrinogen Degradation Products | Heparin | Interleukin-1beta | Lipase | Male | Nucleic Acid Synthesis Inhibitors | Pancreas | Pancreatitis | Phytotherapy | Plant Extracts | Plant Stems | Protective Agents | Prothrombin Time | Rats | Rats, Wistar [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] inflammatory coagulation pathway | pancreatitis heparin manages | effect heparin inflammatory | introduction pancreatitis coagulation | coagulation induced inflammation [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"null"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] inflammatory coagulation pathway | pancreatitis heparin manages | effect heparin inflammatory | introduction pancreatitis coagulation | coagulation induced inflammation [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] inflammatory coagulation pathway | pancreatitis heparin manages | effect heparin inflammatory | introduction pancreatitis coagulation | coagulation induced inflammation [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] inflammatory coagulation pathway | pancreatitis heparin manages | effect heparin inflammatory | introduction pancreatitis coagulation | coagulation induced inflammation [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] heparin | control | pancreatitis | suberectus | spatholobus suberectus | spatholobus | group | cerulean | pancreatic | induced [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"null"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] heparin | control | pancreatitis | suberectus | spatholobus suberectus | spatholobus | group | cerulean | pancreatic | induced [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] heparin | control | pancreatitis | suberectus | spatholobus suberectus | spatholobus | group | cerulean | pancreatic | induced [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] heparin | control | pancreatitis | suberectus | spatholobus suberectus | spatholobus | group | cerulean | pancreatic | induced [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] pancreatitis | heparin | suggested | study suggested | study | 2006 | reported study suggested | coagulation | reported | inflammation [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"null"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] pathway | combination | inflammatory | treatment ss effectively | pathway simultaneously | concludes | concludes treatment | concludes treatment ss | concludes treatment ss effectively | pathway simultaneously pancreatitis [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] heparin | pancreatitis | control | group | suberectus | cerulean | spatholobus suberectus | spatholobus | pancreatic | combination [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] heparin | pancreatitis | control | group | suberectus | cerulean | spatholobus suberectus | spatholobus | pancreatic | combination [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Spatholobus | SS [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"null"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] SS [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Spatholobus | SS ||| Pancreatitis | cerulean | 50μg/kg | i.p. ||| five | 1 ||| SS | 100 | 150 | i.p. ||| a week ||| Interleukin 1β ||| ||| SS ||| SS ||| SS ||| SS ||| SS [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Spatholobus | SS ||| Pancreatitis | cerulean | 50μg/kg | i.p. ||| five | 1 ||| SS | 100 | 150 | i.p. ||| a week ||| Interleukin 1β ||| ||| SS ||| SS ||| SS ||| SS ||| SS [SUMMARY]"}}},{"rowIdx":73,"cells":{"title":{"kind":"string","value":"Serum Osteoprotegerin Is an Independent Marker of Metabolic Complications in Non-DialysisDependent Chronic Kidney Disease Patients."},"pmid":{"kind":"string","value":"34684610"},"background_abstract":{"kind":"string","value":"Osteoprotegerin (OPG) belongs to the tumour necrosis factor superfamily and is known to accelerate endothelial dysfunction and vascular calcification. OPG concentrations are elevated in patients with chronic kidney disease. The aim of this study was to investigate the association between OPG levels and frequent complications of chronic kidney disease (CKD) such as anaemia, protein energy wasting (PEW), inflammation, overhydration, hyperglycaemia and hypertension."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"One hundred non-dialysis-dependent men with CKD stage 3-5 were included in the study. Bioimpedance spectroscopy (BIS) was used to measure overhydration, fat amount and lean body mass. We also measured the serum concentrations of haemoglobin, albumin, total cholesterol, C-reactive protein (CRP), fibrinogen and glycated haemoglobin (HgbA1c), as well as blood pressure."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"We observed a significant, positive correlation between OPG and age, serum creatinine, CRP, fibrinogen, HgbA1c concentrations, systolic blood pressure and overhydration. Negative correlations were observed between OPG and glomerular filtration rate (eGFR), serum albumin concentrations and serum haemoglobin level. Logistic regression models revealed that OPG is an independent marker of metabolic complications such as anaemia, PEW, inflammation and poor renal prognosis (including overhydration, uncontrolled diabetes and hypertension) in the studied population."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Our results suggest that OPG can be an independent marker of PEW, inflammation and vascular metabolic disturbances in patients with chronic kidney disease."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Aged","Anemia","Biomarkers","Humans","Inflammation","Logistic Models","Male","Middle Aged","Osteoprotegerin","Prognosis","Renal Dialysis","Renal Insufficiency, Chronic"],"string":"[\n \"Aged\",\n \"Anemia\",\n \"Biomarkers\",\n \"Humans\",\n \"Inflammation\",\n \"Logistic Models\",\n \"Male\",\n \"Middle Aged\",\n \"Osteoprotegerin\",\n \"Prognosis\",\n \"Renal Dialysis\",\n \"Renal Insufficiency, Chronic\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"8538217"},"background_title":{"kind":"string","value":"1. Introduction"},"background_text":{"kind":"string","value":"Osteoprotegerin (OPG) is a molecule belonging to the tumour necrosis factor (TNF) superfamily. It is a decoy receptor for TNF-related apoptosis-inducing ligand (TRAIL). The TNF superfamily regulates immune responses, homeostasis as well as haematopoiesis [1]. Initially, OPG was identified as a bone resorption inhibitor that blocks the binding of RANK (receptor activator of nuclear factor kappa-β) to its ligand RANKL [2]. Further studies revealed that it has an additional impact on the cardiovascular and immune systems [3]. The OPG/RANKL/RANK system is involved in pathological angiogenesis, inflammatory states and cell survival. OPG is a glycoprotein released by vascular smooth muscle cells, endothelial cells, osteoblasts and immune cells. There is a signalling pathway between endothelial cells and osteoblasts during osteogenesis creating a connection between angiogenesis and osteogenesis [4]. The OPG/RANKL/RANK/TRAIL system, via receptors located on the cell surface, sends intracellular signals and modifies gene expression. As a result, monocytes, neutrophils and endothelial cells are recruited through cytokine production and receptor activation [5]. Serum OPG levels are associated with endothelial dysfunction and mediate vascular calcification [6]. Elevated OPG levels were observed in patients with atherosclerosis, heart failure, metabolic syndrome and diabetes [7,8,9].\nThe role of OPG in kidney pathologies is not well understood. OPG is expressed in kidney samples, cultured tubular cells and urinary exosome-like vesicles. It has been hypothesized that it can play a role in matrix deposition, inflammation and apoptosis [10]. Furthermore, the role of OPG in cardiovascular pathology and vascular calcification, which are common complications of chronic kidney disease, makes OPG an interesting marker also in CKD. When compared to healthy individuals, non-dialysis-dependent CKD patients, haemodialysis and peritoneal dialysis patients and renal transplant recipients present elevated levels of OPG [11,12,13]. As OPG is involved in vascular calcification, it is also associated with cardiovascular mortality in CKD patients [14]. Decreased levels of OPG are observed in nephrotic syndrome, which is probably caused by the loss with urine. Glucocorticosteroid treatment increases RANKL and reduces OPG expression, which also decreases the OPG levels in patients with nephrotic syndrome [15].\nThe purpose of our study was to investigate the association between OPG levels and the main complications of chronic kidney disease, including anaemia, protein energy wasting, inflammation and poor prognosis factors of CKD progression such as overhydration, hyperglycaemia and hypertension."},"methods_title":{"kind":"string","value":"2. Methods"},"methods_text":{"kind":"string","value":" 2.1. Design A prospective, observational study in male patients with chronic kidney disease non-treated with dialysis was performed. The inclusion criterion was eGFR lower than 60 mL/min/1.73 m2.\nA prospective, observational study in male patients with chronic kidney disease non-treated with dialysis was performed. The inclusion criterion was eGFR lower than 60 mL/min/1.73 m2.\n 2.2. Patients Participants were recruited among patients visiting the Nephrological Outpatient Clinic for a routine control during the period between November 2018 and February 2020. If they fulfilled the inclusion criteria, a new visit was arranged for participation in the study. The participants were asked to avoid saunas, physical exertion and alcohol consumption the day before the examination. The visit took place after overnight fasting.\nOne hundred men with chronic kidney disease and eGFR lower than 60 mL/min/1.73 m2 were included in the study. The exclusion criteria were: renal replacement therapy or its requirement within the following 3 months, clinical signs of infection and presence of metal parts in the body. The study protocol was accepted by the local ethics committee (Bioethics Committee in Military Institute of Medicine, IRB acceptance number 120/WIM/2018 obtained 22 August 2018). All participants signed an informed consent.\nBody composition including overhydration (OH), fat amount and lean body mass were measured by bioimpedance spectroscopy with the use of a Body Composition Monitor (Fresenius Medical Care). While being measured, patients stayed in a supine position after a 5 min rest. Electrodes were placed in a tetrapolar configuration (on one hand and one foot).\nBlood samples for standard measurements were collected after an overnight fast and were transported immediately to the local Department of Laboratory Diagnostics. Concentrations of high-sensitivity C-reactive protein were determined by a nephelometry assay (BNII Siemens) with a cut-off point of 0.8 mg/dL. Serum creatinine concentrations were measured using the Jaffe method (Gen.2; Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland), and serum albumin levels using a BCP Albumin Assay Kit (Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland). Samples for measuring osteoprotegerin (OPG) levels were kept frozen at −80 °C. OPG levels were assessed using the Luminex MAGPIX platform.\nGFR was calculated according to the short Modification of Diet in Renal Disease (MDRD) formula.\nGFR in mL/min per 1.73 m2 = 175 × SerumCr−1.154 × age−0.203 × 1.212 (if patient is black) × 0.742 (if female)\nParticipants were recruited among patients visiting the Nephrological Outpatient Clinic for a routine control during the period between November 2018 and February 2020. If they fulfilled the inclusion criteria, a new visit was arranged for participation in the study. The participants were asked to avoid saunas, physical exertion and alcohol consumption the day before the examination. The visit took place after overnight fasting.\nOne hundred men with chronic kidney disease and eGFR lower than 60 mL/min/1.73 m2 were included in the study. The exclusion criteria were: renal replacement therapy or its requirement within the following 3 months, clinical signs of infection and presence of metal parts in the body. The study protocol was accepted by the local ethics committee (Bioethics Committee in Military Institute of Medicine, IRB acceptance number 120/WIM/2018 obtained 22 August 2018). All participants signed an informed consent.\nBody composition including overhydration (OH), fat amount and lean body mass were measured by bioimpedance spectroscopy with the use of a Body Composition Monitor (Fresenius Medical Care). While being measured, patients stayed in a supine position after a 5 min rest. Electrodes were placed in a tetrapolar configuration (on one hand and one foot).\nBlood samples for standard measurements were collected after an overnight fast and were transported immediately to the local Department of Laboratory Diagnostics. Concentrations of high-sensitivity C-reactive protein were determined by a nephelometry assay (BNII Siemens) with a cut-off point of 0.8 mg/dL. Serum creatinine concentrations were measured using the Jaffe method (Gen.2; Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland), and serum albumin levels using a BCP Albumin Assay Kit (Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland). Samples for measuring osteoprotegerin (OPG) levels were kept frozen at −80 °C. OPG levels were assessed using the Luminex MAGPIX platform.\nGFR was calculated according to the short Modification of Diet in Renal Disease (MDRD) formula.\nGFR in mL/min per 1.73 m2 = 175 × SerumCr−1.154 × age−0.203 × 1.212 (if patient is black) × 0.742 (if female)\n 2.3. Defining the Complications of Chronic Kidney Disease Complications of chronic kidney disease were defined as follows:-Anaemia, when serum haemoglobin level was lower than 12 g/dL [16]-Protein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]-Inflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]-Poor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\nAnaemia, when serum haemoglobin level was lower than 12 g/dL [16]\nProtein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]\nInflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]\nPoor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\nComplications of chronic kidney disease were defined as follows:-Anaemia, when serum haemoglobin level was lower than 12 g/dL [16]-Protein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]-Inflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]-Poor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\nAnaemia, when serum haemoglobin level was lower than 12 g/dL [16]\nProtein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]\nInflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]\nPoor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\n 2.4. Statistical Analysis The characteristics of the study population are presented using medians with interquartile ranges (for continuous data with distribution other than normal, tested with the Shapiro–Wilk test). The correlation between OPG and clinical and laboratory parameters was assessed using the Spearman rank correlation coefficient. Logistic regression was used to investigate the significance of OPG levels as a marker of metabolic complications of CKD. Given the known relationship between OPG, age and GFR, we compared the value of logistic models, with and without OPG, for each metabolic complication (anaemia, PEW, inflammation, uncontrolled factors of CKD progression). Suitability of fit of each model was assessed using the area under the curve (AUC) with standard error (SE) and presented using the Receiver Operating Characteristic (ROC) curve. The models were compared using Hanley’s algorithm [19]. The analysis was performed with Statistica 13.1 with p-values < 0.05 considered statistically significant.\nThe characteristics of the study population are presented using medians with interquartile ranges (for continuous data with distribution other than normal, tested with the Shapiro–Wilk test). The correlation between OPG and clinical and laboratory parameters was assessed using the Spearman rank correlation coefficient. Logistic regression was used to investigate the significance of OPG levels as a marker of metabolic complications of CKD. Given the known relationship between OPG, age and GFR, we compared the value of logistic models, with and without OPG, for each metabolic complication (anaemia, PEW, inflammation, uncontrolled factors of CKD progression). Suitability of fit of each model was assessed using the area under the curve (AUC) with standard error (SE) and presented using the Receiver Operating Characteristic (ROC) curve. The models were compared using Hanley’s algorithm [19]. The analysis was performed with Statistica 13.1 with p-values < 0.05 considered statistically significant."},"results_title":{"kind":"string","value":"3. Results"},"results_text":{"kind":"string","value":"The study population consisted of 100 male patients with chronic kidney disease and eGFR lower than 60 mL/min/1.73 m2, non-treated with dialysis. The median age of the studied population was 66 years (59–72). Clinical data are presented in Table 1.\nWe observed significant, positive correlations between OPG and age, serum creatinine concentration, CRP, fibrinogen, HgBA1C, systolic blood pressure and overhydration. Significant, negative correlations were observed between OPG and eGFR, serum albumin concentration as well as serum haemoglobin level. Table 2.\nLogistic regression models, adjusted for age and GFR, were created to evaluate the influence of the OPG level as an independent marker of complications in chronic kidney disease patients, such as anaemia, PEW, an inflammatory state, poor prognostic factors of CKD progression.\n 3.1. Anaemia The OPG level was a significant, independent marker of anaemia in the studied population (p < 0.001).\nThe model including OPG, age and GFR (AUC=0.84 ± 0.05) identified CKD patients with anaemia better than the model including only age and GFR (AUC=0.76 ± 0.06), as shown in Table 3. However, the difference was not of statistical significance (p = 0.096), as shown in Figure 1.\nThe OPG level was a significant, independent marker of anaemia in the studied population (p < 0.001).\nThe model including OPG, age and GFR (AUC=0.84 ± 0.05) identified CKD patients with anaemia better than the model including only age and GFR (AUC=0.76 ± 0.06), as shown in Table 3. However, the difference was not of statistical significance (p = 0.096), as shown in Figure 1.\n 3.2. Protein Energy Wasting (PEW) OPG was a significant, independent risk factor for PEW occurrence in the study population (p < 0.001). However, the model including OPG, age and GFR was not significantly better (AUC = 0.79 ± 0.05) than those considering only age and GFR (AUC = 0.71 ± 0.06, p = 0.142), as shown in Table 4 and Figure 2.\nOPG was a significant, independent risk factor for PEW occurrence in the study population (p < 0.001). However, the model including OPG, age and GFR was not significantly better (AUC = 0.79 ± 0.05) than those considering only age and GFR (AUC = 0.71 ± 0.06, p = 0.142), as shown in Table 4 and Figure 2.\n 3.3. Inflammatory State OPG was a significant, independent risk factor for subclinical inflammation in the study population (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model including only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 5 and Figure 3.\nOPG was a significant, independent risk factor for subclinical inflammation in the study population (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model including only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 5 and Figure 3.\n 3.4. Poor Prognostic Factors (Overhydration, Hyperglycaemia and Hypertension) Overhydration, hyperglycaemia and hypertension were chosen as poor prognostic factors of chronic kidney disease progression and were analysed together in logistic regression analysis. OPG was an independent marker that identified patients with poor prognostic factors (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model which included only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 6 and Figure 4.\nOverhydration, hyperglycaemia and hypertension were chosen as poor prognostic factors of chronic kidney disease progression and were analysed together in logistic regression analysis. OPG was an independent marker that identified patients with poor prognostic factors (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model which included only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 6 and Figure 4."},"conclusions_title":{"kind":"string","value":"5. Conclusions"},"conclusions_text":{"kind":"string","value":"CKD is in itself a cardiovascular risk factor, and cardiovascular mortality in CKD patients is high. CVD mortality in CKD patients is even greater in the presence of diabetes and hypertension [30]. Early detection of such complications and the identification of patients at risk of developing them is vital. Therefore, new markers that can predict metabolic disorders and have an unfavourable effect on vascular damage are necessary. OPG seems to be a possible marker associated with PEW, inflammation and vascular metabolic disturbances which is worth further study."},"other_sections_titles":{"kind":"list like","value":["2.1. Design","2.3. Defining the Complications of Chronic Kidney Disease","2.4. Statistical Analysis","3.1. Anaemia","3.2. Protein Energy Wasting (PEW)","3.3. Inflammatory State","3.4. Poor Prognostic Factors (Overhydration, Hyperglycaemia and Hypertension)"],"string":"[\n \"2.1. Design\",\n \"2.3. Defining the Complications of Chronic Kidney Disease\",\n \"2.4. Statistical Analysis\",\n \"3.1. Anaemia\",\n \"3.2. Protein Energy Wasting (PEW)\",\n \"3.3. Inflammatory State\",\n \"3.4. Poor Prognostic Factors (Overhydration, Hyperglycaemia and Hypertension)\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["A prospective, observational study in male patients with chronic kidney disease non-treated with dialysis was performed. The inclusion criterion was eGFR lower than 60 mL/min/1.73 m2.","Complications of chronic kidney disease were defined as follows:-Anaemia, when serum haemoglobin level was lower than 12 g/dL [16]-Protein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]-Inflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]-Poor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\nAnaemia, when serum haemoglobin level was lower than 12 g/dL [16]\nProtein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]\nInflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]\nPoor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]","The characteristics of the study population are presented using medians with interquartile ranges (for continuous data with distribution other than normal, tested with the Shapiro–Wilk test). The correlation between OPG and clinical and laboratory parameters was assessed using the Spearman rank correlation coefficient. Logistic regression was used to investigate the significance of OPG levels as a marker of metabolic complications of CKD. Given the known relationship between OPG, age and GFR, we compared the value of logistic models, with and without OPG, for each metabolic complication (anaemia, PEW, inflammation, uncontrolled factors of CKD progression). Suitability of fit of each model was assessed using the area under the curve (AUC) with standard error (SE) and presented using the Receiver Operating Characteristic (ROC) curve. The models were compared using Hanley’s algorithm [19]. The analysis was performed with Statistica 13.1 with p-values < 0.05 considered statistically significant.","The OPG level was a significant, independent marker of anaemia in the studied population (p < 0.001).\nThe model including OPG, age and GFR (AUC=0.84 ± 0.05) identified CKD patients with anaemia better than the model including only age and GFR (AUC=0.76 ± 0.06), as shown in Table 3. However, the difference was not of statistical significance (p = 0.096), as shown in Figure 1.","OPG was a significant, independent risk factor for PEW occurrence in the study population (p < 0.001). However, the model including OPG, age and GFR was not significantly better (AUC = 0.79 ± 0.05) than those considering only age and GFR (AUC = 0.71 ± 0.06, p = 0.142), as shown in Table 4 and Figure 2.","OPG was a significant, independent risk factor for subclinical inflammation in the study population (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model including only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 5 and Figure 3.","Overhydration, hyperglycaemia and hypertension were chosen as poor prognostic factors of chronic kidney disease progression and were analysed together in logistic regression analysis. OPG was an independent marker that identified patients with poor prognostic factors (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model which included only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 6 and Figure 4."],"string":"[\n \"A prospective, observational study in male patients with chronic kidney disease non-treated with dialysis was performed. The inclusion criterion was eGFR lower than 60 mL/min/1.73 m2.\",\n \"Complications of chronic kidney disease were defined as follows:-Anaemia, when serum haemoglobin level was lower than 12 g/dL [16]-Protein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]-Inflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]-Poor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\\nAnaemia, when serum haemoglobin level was lower than 12 g/dL [16]\\nProtein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]\\nInflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]\\nPoor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\",\n \"The characteristics of the study population are presented using medians with interquartile ranges (for continuous data with distribution other than normal, tested with the Shapiro–Wilk test). The correlation between OPG and clinical and laboratory parameters was assessed using the Spearman rank correlation coefficient. Logistic regression was used to investigate the significance of OPG levels as a marker of metabolic complications of CKD. Given the known relationship between OPG, age and GFR, we compared the value of logistic models, with and without OPG, for each metabolic complication (anaemia, PEW, inflammation, uncontrolled factors of CKD progression). Suitability of fit of each model was assessed using the area under the curve (AUC) with standard error (SE) and presented using the Receiver Operating Characteristic (ROC) curve. The models were compared using Hanley’s algorithm [19]. The analysis was performed with Statistica 13.1 with p-values < 0.05 considered statistically significant.\",\n \"The OPG level was a significant, independent marker of anaemia in the studied population (p < 0.001).\\nThe model including OPG, age and GFR (AUC=0.84 ± 0.05) identified CKD patients with anaemia better than the model including only age and GFR (AUC=0.76 ± 0.06), as shown in Table 3. However, the difference was not of statistical significance (p = 0.096), as shown in Figure 1.\",\n \"OPG was a significant, independent risk factor for PEW occurrence in the study population (p < 0.001). However, the model including OPG, age and GFR was not significantly better (AUC = 0.79 ± 0.05) than those considering only age and GFR (AUC = 0.71 ± 0.06, p = 0.142), as shown in Table 4 and Figure 2.\",\n \"OPG was a significant, independent risk factor for subclinical inflammation in the study population (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model including only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 5 and Figure 3.\",\n \"Overhydration, hyperglycaemia and hypertension were chosen as poor prognostic factors of chronic kidney disease progression and were analysed together in logistic regression analysis. OPG was an independent marker that identified patients with poor prognostic factors (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model which included only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 6 and Figure 4.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["1. Introduction","2. Methods","2.1. Design","2.2. Patients","2.3. Defining the Complications of Chronic Kidney Disease","2.4. Statistical Analysis","3. Results","3.1. Anaemia","3.2. Protein Energy Wasting (PEW)","3.3. Inflammatory State","3.4. Poor Prognostic Factors (Overhydration, Hyperglycaemia and Hypertension)","4. Discussion","5. Conclusions"],"string":"[\n \"1. Introduction\",\n \"2. Methods\",\n \"2.1. Design\",\n \"2.2. Patients\",\n \"2.3. Defining the Complications of Chronic Kidney Disease\",\n \"2.4. Statistical Analysis\",\n \"3. Results\",\n \"3.1. Anaemia\",\n \"3.2. Protein Energy Wasting (PEW)\",\n \"3.3. Inflammatory State\",\n \"3.4. Poor Prognostic Factors (Overhydration, Hyperglycaemia and Hypertension)\",\n \"4. Discussion\",\n \"5. Conclusions\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Osteoprotegerin (OPG) is a molecule belonging to the tumour necrosis factor (TNF) superfamily. It is a decoy receptor for TNF-related apoptosis-inducing ligand (TRAIL). The TNF superfamily regulates immune responses, homeostasis as well as haematopoiesis [1]. Initially, OPG was identified as a bone resorption inhibitor that blocks the binding of RANK (receptor activator of nuclear factor kappa-β) to its ligand RANKL [2]. Further studies revealed that it has an additional impact on the cardiovascular and immune systems [3]. The OPG/RANKL/RANK system is involved in pathological angiogenesis, inflammatory states and cell survival. OPG is a glycoprotein released by vascular smooth muscle cells, endothelial cells, osteoblasts and immune cells. There is a signalling pathway between endothelial cells and osteoblasts during osteogenesis creating a connection between angiogenesis and osteogenesis [4]. The OPG/RANKL/RANK/TRAIL system, via receptors located on the cell surface, sends intracellular signals and modifies gene expression. As a result, monocytes, neutrophils and endothelial cells are recruited through cytokine production and receptor activation [5]. Serum OPG levels are associated with endothelial dysfunction and mediate vascular calcification [6]. Elevated OPG levels were observed in patients with atherosclerosis, heart failure, metabolic syndrome and diabetes [7,8,9].\nThe role of OPG in kidney pathologies is not well understood. OPG is expressed in kidney samples, cultured tubular cells and urinary exosome-like vesicles. It has been hypothesized that it can play a role in matrix deposition, inflammation and apoptosis [10]. Furthermore, the role of OPG in cardiovascular pathology and vascular calcification, which are common complications of chronic kidney disease, makes OPG an interesting marker also in CKD. When compared to healthy individuals, non-dialysis-dependent CKD patients, haemodialysis and peritoneal dialysis patients and renal transplant recipients present elevated levels of OPG [11,12,13]. As OPG is involved in vascular calcification, it is also associated with cardiovascular mortality in CKD patients [14]. Decreased levels of OPG are observed in nephrotic syndrome, which is probably caused by the loss with urine. Glucocorticosteroid treatment increases RANKL and reduces OPG expression, which also decreases the OPG levels in patients with nephrotic syndrome [15].\nThe purpose of our study was to investigate the association between OPG levels and the main complications of chronic kidney disease, including anaemia, protein energy wasting, inflammation and poor prognosis factors of CKD progression such as overhydration, hyperglycaemia and hypertension."," 2.1. Design A prospective, observational study in male patients with chronic kidney disease non-treated with dialysis was performed. The inclusion criterion was eGFR lower than 60 mL/min/1.73 m2.\nA prospective, observational study in male patients with chronic kidney disease non-treated with dialysis was performed. The inclusion criterion was eGFR lower than 60 mL/min/1.73 m2.\n 2.2. Patients Participants were recruited among patients visiting the Nephrological Outpatient Clinic for a routine control during the period between November 2018 and February 2020. If they fulfilled the inclusion criteria, a new visit was arranged for participation in the study. The participants were asked to avoid saunas, physical exertion and alcohol consumption the day before the examination. The visit took place after overnight fasting.\nOne hundred men with chronic kidney disease and eGFR lower than 60 mL/min/1.73 m2 were included in the study. The exclusion criteria were: renal replacement therapy or its requirement within the following 3 months, clinical signs of infection and presence of metal parts in the body. The study protocol was accepted by the local ethics committee (Bioethics Committee in Military Institute of Medicine, IRB acceptance number 120/WIM/2018 obtained 22 August 2018). All participants signed an informed consent.\nBody composition including overhydration (OH), fat amount and lean body mass were measured by bioimpedance spectroscopy with the use of a Body Composition Monitor (Fresenius Medical Care). While being measured, patients stayed in a supine position after a 5 min rest. Electrodes were placed in a tetrapolar configuration (on one hand and one foot).\nBlood samples for standard measurements were collected after an overnight fast and were transported immediately to the local Department of Laboratory Diagnostics. Concentrations of high-sensitivity C-reactive protein were determined by a nephelometry assay (BNII Siemens) with a cut-off point of 0.8 mg/dL. Serum creatinine concentrations were measured using the Jaffe method (Gen.2; Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland), and serum albumin levels using a BCP Albumin Assay Kit (Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland). Samples for measuring osteoprotegerin (OPG) levels were kept frozen at −80 °C. OPG levels were assessed using the Luminex MAGPIX platform.\nGFR was calculated according to the short Modification of Diet in Renal Disease (MDRD) formula.\nGFR in mL/min per 1.73 m2 = 175 × SerumCr−1.154 × age−0.203 × 1.212 (if patient is black) × 0.742 (if female)\nParticipants were recruited among patients visiting the Nephrological Outpatient Clinic for a routine control during the period between November 2018 and February 2020. If they fulfilled the inclusion criteria, a new visit was arranged for participation in the study. The participants were asked to avoid saunas, physical exertion and alcohol consumption the day before the examination. The visit took place after overnight fasting.\nOne hundred men with chronic kidney disease and eGFR lower than 60 mL/min/1.73 m2 were included in the study. The exclusion criteria were: renal replacement therapy or its requirement within the following 3 months, clinical signs of infection and presence of metal parts in the body. The study protocol was accepted by the local ethics committee (Bioethics Committee in Military Institute of Medicine, IRB acceptance number 120/WIM/2018 obtained 22 August 2018). All participants signed an informed consent.\nBody composition including overhydration (OH), fat amount and lean body mass were measured by bioimpedance spectroscopy with the use of a Body Composition Monitor (Fresenius Medical Care). While being measured, patients stayed in a supine position after a 5 min rest. Electrodes were placed in a tetrapolar configuration (on one hand and one foot).\nBlood samples for standard measurements were collected after an overnight fast and were transported immediately to the local Department of Laboratory Diagnostics. Concentrations of high-sensitivity C-reactive protein were determined by a nephelometry assay (BNII Siemens) with a cut-off point of 0.8 mg/dL. Serum creatinine concentrations were measured using the Jaffe method (Gen.2; Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland), and serum albumin levels using a BCP Albumin Assay Kit (Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland). Samples for measuring osteoprotegerin (OPG) levels were kept frozen at −80 °C. OPG levels were assessed using the Luminex MAGPIX platform.\nGFR was calculated according to the short Modification of Diet in Renal Disease (MDRD) formula.\nGFR in mL/min per 1.73 m2 = 175 × SerumCr−1.154 × age−0.203 × 1.212 (if patient is black) × 0.742 (if female)\n 2.3. Defining the Complications of Chronic Kidney Disease Complications of chronic kidney disease were defined as follows:-Anaemia, when serum haemoglobin level was lower than 12 g/dL [16]-Protein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]-Inflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]-Poor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\nAnaemia, when serum haemoglobin level was lower than 12 g/dL [16]\nProtein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]\nInflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]\nPoor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\nComplications of chronic kidney disease were defined as follows:-Anaemia, when serum haemoglobin level was lower than 12 g/dL [16]-Protein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]-Inflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]-Poor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\nAnaemia, when serum haemoglobin level was lower than 12 g/dL [16]\nProtein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]\nInflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]\nPoor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\n 2.4. Statistical Analysis The characteristics of the study population are presented using medians with interquartile ranges (for continuous data with distribution other than normal, tested with the Shapiro–Wilk test). The correlation between OPG and clinical and laboratory parameters was assessed using the Spearman rank correlation coefficient. Logistic regression was used to investigate the significance of OPG levels as a marker of metabolic complications of CKD. Given the known relationship between OPG, age and GFR, we compared the value of logistic models, with and without OPG, for each metabolic complication (anaemia, PEW, inflammation, uncontrolled factors of CKD progression). Suitability of fit of each model was assessed using the area under the curve (AUC) with standard error (SE) and presented using the Receiver Operating Characteristic (ROC) curve. The models were compared using Hanley’s algorithm [19]. The analysis was performed with Statistica 13.1 with p-values < 0.05 considered statistically significant.\nThe characteristics of the study population are presented using medians with interquartile ranges (for continuous data with distribution other than normal, tested with the Shapiro–Wilk test). The correlation between OPG and clinical and laboratory parameters was assessed using the Spearman rank correlation coefficient. Logistic regression was used to investigate the significance of OPG levels as a marker of metabolic complications of CKD. Given the known relationship between OPG, age and GFR, we compared the value of logistic models, with and without OPG, for each metabolic complication (anaemia, PEW, inflammation, uncontrolled factors of CKD progression). Suitability of fit of each model was assessed using the area under the curve (AUC) with standard error (SE) and presented using the Receiver Operating Characteristic (ROC) curve. The models were compared using Hanley’s algorithm [19]. The analysis was performed with Statistica 13.1 with p-values < 0.05 considered statistically significant.","A prospective, observational study in male patients with chronic kidney disease non-treated with dialysis was performed. The inclusion criterion was eGFR lower than 60 mL/min/1.73 m2.","Participants were recruited among patients visiting the Nephrological Outpatient Clinic for a routine control during the period between November 2018 and February 2020. If they fulfilled the inclusion criteria, a new visit was arranged for participation in the study. The participants were asked to avoid saunas, physical exertion and alcohol consumption the day before the examination. The visit took place after overnight fasting.\nOne hundred men with chronic kidney disease and eGFR lower than 60 mL/min/1.73 m2 were included in the study. The exclusion criteria were: renal replacement therapy or its requirement within the following 3 months, clinical signs of infection and presence of metal parts in the body. The study protocol was accepted by the local ethics committee (Bioethics Committee in Military Institute of Medicine, IRB acceptance number 120/WIM/2018 obtained 22 August 2018). All participants signed an informed consent.\nBody composition including overhydration (OH), fat amount and lean body mass were measured by bioimpedance spectroscopy with the use of a Body Composition Monitor (Fresenius Medical Care). While being measured, patients stayed in a supine position after a 5 min rest. Electrodes were placed in a tetrapolar configuration (on one hand and one foot).\nBlood samples for standard measurements were collected after an overnight fast and were transported immediately to the local Department of Laboratory Diagnostics. Concentrations of high-sensitivity C-reactive protein were determined by a nephelometry assay (BNII Siemens) with a cut-off point of 0.8 mg/dL. Serum creatinine concentrations were measured using the Jaffe method (Gen.2; Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland), and serum albumin levels using a BCP Albumin Assay Kit (Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland). Samples for measuring osteoprotegerin (OPG) levels were kept frozen at −80 °C. OPG levels were assessed using the Luminex MAGPIX platform.\nGFR was calculated according to the short Modification of Diet in Renal Disease (MDRD) formula.\nGFR in mL/min per 1.73 m2 = 175 × SerumCr−1.154 × age−0.203 × 1.212 (if patient is black) × 0.742 (if female)","Complications of chronic kidney disease were defined as follows:-Anaemia, when serum haemoglobin level was lower than 12 g/dL [16]-Protein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]-Inflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]-Poor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\nAnaemia, when serum haemoglobin level was lower than 12 g/dL [16]\nProtein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]\nInflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]\nPoor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]","The characteristics of the study population are presented using medians with interquartile ranges (for continuous data with distribution other than normal, tested with the Shapiro–Wilk test). The correlation between OPG and clinical and laboratory parameters was assessed using the Spearman rank correlation coefficient. Logistic regression was used to investigate the significance of OPG levels as a marker of metabolic complications of CKD. Given the known relationship between OPG, age and GFR, we compared the value of logistic models, with and without OPG, for each metabolic complication (anaemia, PEW, inflammation, uncontrolled factors of CKD progression). Suitability of fit of each model was assessed using the area under the curve (AUC) with standard error (SE) and presented using the Receiver Operating Characteristic (ROC) curve. The models were compared using Hanley’s algorithm [19]. The analysis was performed with Statistica 13.1 with p-values < 0.05 considered statistically significant.","The study population consisted of 100 male patients with chronic kidney disease and eGFR lower than 60 mL/min/1.73 m2, non-treated with dialysis. The median age of the studied population was 66 years (59–72). Clinical data are presented in Table 1.\nWe observed significant, positive correlations between OPG and age, serum creatinine concentration, CRP, fibrinogen, HgBA1C, systolic blood pressure and overhydration. Significant, negative correlations were observed between OPG and eGFR, serum albumin concentration as well as serum haemoglobin level. Table 2.\nLogistic regression models, adjusted for age and GFR, were created to evaluate the influence of the OPG level as an independent marker of complications in chronic kidney disease patients, such as anaemia, PEW, an inflammatory state, poor prognostic factors of CKD progression.\n 3.1. Anaemia The OPG level was a significant, independent marker of anaemia in the studied population (p < 0.001).\nThe model including OPG, age and GFR (AUC=0.84 ± 0.05) identified CKD patients with anaemia better than the model including only age and GFR (AUC=0.76 ± 0.06), as shown in Table 3. However, the difference was not of statistical significance (p = 0.096), as shown in Figure 1.\nThe OPG level was a significant, independent marker of anaemia in the studied population (p < 0.001).\nThe model including OPG, age and GFR (AUC=0.84 ± 0.05) identified CKD patients with anaemia better than the model including only age and GFR (AUC=0.76 ± 0.06), as shown in Table 3. However, the difference was not of statistical significance (p = 0.096), as shown in Figure 1.\n 3.2. Protein Energy Wasting (PEW) OPG was a significant, independent risk factor for PEW occurrence in the study population (p < 0.001). However, the model including OPG, age and GFR was not significantly better (AUC = 0.79 ± 0.05) than those considering only age and GFR (AUC = 0.71 ± 0.06, p = 0.142), as shown in Table 4 and Figure 2.\nOPG was a significant, independent risk factor for PEW occurrence in the study population (p < 0.001). However, the model including OPG, age and GFR was not significantly better (AUC = 0.79 ± 0.05) than those considering only age and GFR (AUC = 0.71 ± 0.06, p = 0.142), as shown in Table 4 and Figure 2.\n 3.3. Inflammatory State OPG was a significant, independent risk factor for subclinical inflammation in the study population (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model including only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 5 and Figure 3.\nOPG was a significant, independent risk factor for subclinical inflammation in the study population (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model including only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 5 and Figure 3.\n 3.4. Poor Prognostic Factors (Overhydration, Hyperglycaemia and Hypertension) Overhydration, hyperglycaemia and hypertension were chosen as poor prognostic factors of chronic kidney disease progression and were analysed together in logistic regression analysis. OPG was an independent marker that identified patients with poor prognostic factors (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model which included only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 6 and Figure 4.\nOverhydration, hyperglycaemia and hypertension were chosen as poor prognostic factors of chronic kidney disease progression and were analysed together in logistic regression analysis. OPG was an independent marker that identified patients with poor prognostic factors (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model which included only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 6 and Figure 4.","The OPG level was a significant, independent marker of anaemia in the studied population (p < 0.001).\nThe model including OPG, age and GFR (AUC=0.84 ± 0.05) identified CKD patients with anaemia better than the model including only age and GFR (AUC=0.76 ± 0.06), as shown in Table 3. However, the difference was not of statistical significance (p = 0.096), as shown in Figure 1.","OPG was a significant, independent risk factor for PEW occurrence in the study population (p < 0.001). However, the model including OPG, age and GFR was not significantly better (AUC = 0.79 ± 0.05) than those considering only age and GFR (AUC = 0.71 ± 0.06, p = 0.142), as shown in Table 4 and Figure 2.","OPG was a significant, independent risk factor for subclinical inflammation in the study population (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model including only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 5 and Figure 3.","Overhydration, hyperglycaemia and hypertension were chosen as poor prognostic factors of chronic kidney disease progression and were analysed together in logistic regression analysis. OPG was an independent marker that identified patients with poor prognostic factors (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model which included only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 6 and Figure 4.","The role of OPG in chronic kidney disease patients is insufficiently comprehended. We know that the OPG level increases together with a decrease in the glomerular filtration rate, as proved in many studies [20,21]. The results of our study also confirm these observations. In a population of non-dialysis-dependent men with stage 3–5 chronic kidney disease, a significant, inverse correlation between OPG levels and eGFR was noted (R = −0.36; p < 0.001).\nIn our study, we also observed significant correlations between OPG and parameters which indicate CKD complications. The OPG level was correlated with the level of haemoglobins and acted as a marker of anaemia occurrence in the studied population. The logistic regression model including OPG was superior to that including only age and GFR in identifying patients with anaemia. However, the difference was not of statistical significance. OPG, as a member of the TNF superfamily, can influence haematopoiesis. Some researchers have observed that TNF superfamily members can regulate the growth of hematopoietic stem cells and progenitor cells [1,22].\nAside from anaemia, protein energy wasting (PEW) is also a complication of chronic kidney disease. The components of PEW are low serum albumin, cholesterol levels as well as low BMI and fat amount [17]. In our study, the OPG level was a marker of PEW in non-dialysis-dependent CKD men. PEW aetiology in CKD patients is associated with many factors such as hormonal disorders, insulin resistance and a subclinical inflammatory state. In our study, the OPG levels also correlated with the CRP and fibrinogen levels. OPG was also a significant marker of instances of inflammation. The logistic regression model that included OPG proved significantly better in in the identification of subclinical inflammation than the model which only included age and GFR. This finding is unsurprising, because OPG is known for its pro-inflammatory action. OPG has been described as a marker of endothelial damage associated with the inflammatory process, and in vitro studies have shown that OPG is involved in inflammatory cell chemotaxis [23].\nMost studies concerning OPG in CKD patients concentrate on the association between OPG and vascular calcification. OPG levels correlate with vascular calcification in non-dialysis-dependent CKD patients as well as in patients treated with renal replacement therapy [24,25]. In animal studies, mice without OPG developed severe vascular calcification [26]. Therefore, it is hypothesized that OPG has a protective role in vascular injury, and the elevation of OPG levels inhibits vascular calcification. However, vascular calcification can also occur in the medial as well as in the intimal layer. In atherosclerosis, intima is thickened, inflamed and calcified, forming plaques with diffuse localisation along the vessel walls, whereas medial calcification occurs along the elastic lamina, leading to stiffness of the artery wall. Studies in animal models, including Apolipoprotein E-deficient mice, suggest that OPG plays a protective, anti-calcification role in both the medial and the intimal (atherosclerotic) calcification process [27].\nThe OPG levels increase rapidly in the early stages of vascular calcification and subsequently remain almost invariant [21]. Therefore, OPG appears to be a marker of the onset of atherosclerosis. However, the development of cardiovascular disease (CVD) begins with endothelial dysfunction. The earliest manifestation of CVD is observed in the area of microcirculation, and in CKD patients, this area is affected by the loss of homeostasis, even in cases of non-severe renal impairment [28]. OPG, as a factor released by endothelial cells, can be a marker of endothelial dysfunction and may probably be involved in a complicated chain of pathophysiological connections between chronic kidney disease and CVD.\nIn our study, we analysed factors of poor prognosis of CKD progression which are associated with vascular damage, such as overhydration, hypertension and glycaemic disturbances expressed by HgbA1c. Positive, significant correlations were observed between OPG levels and all three parameters. In other studies, OPG levels were associated with glycaemic status, and higher OPG levels were observed in patients with poor glycaemic control [29]. In our study, OPG was identified independently, associated with the presence of poor prognostic factors (overhydration, hypertension and glycaemic disturbances) irrespective of age and eGFR.\nThe limitation of our study is its relatively small sample size. An increase in the number of participants could enable a division of the study population into various subgroups according to the different stages of chronic kidney disease. Complications of CKD are more severe in more advanced stages of CKD, and OPG influence could also be more significant.","CKD is in itself a cardiovascular risk factor, and cardiovascular mortality in CKD patients is high. CVD mortality in CKD patients is even greater in the presence of diabetes and hypertension [30]. Early detection of such complications and the identification of patients at risk of developing them is vital. Therefore, new markers that can predict metabolic disorders and have an unfavourable effect on vascular damage are necessary. OPG seems to be a possible marker associated with PEW, inflammation and vascular metabolic disturbances which is worth further study."],"string":"[\n \"Osteoprotegerin (OPG) is a molecule belonging to the tumour necrosis factor (TNF) superfamily. It is a decoy receptor for TNF-related apoptosis-inducing ligand (TRAIL). The TNF superfamily regulates immune responses, homeostasis as well as haematopoiesis [1]. Initially, OPG was identified as a bone resorption inhibitor that blocks the binding of RANK (receptor activator of nuclear factor kappa-β) to its ligand RANKL [2]. Further studies revealed that it has an additional impact on the cardiovascular and immune systems [3]. The OPG/RANKL/RANK system is involved in pathological angiogenesis, inflammatory states and cell survival. OPG is a glycoprotein released by vascular smooth muscle cells, endothelial cells, osteoblasts and immune cells. There is a signalling pathway between endothelial cells and osteoblasts during osteogenesis creating a connection between angiogenesis and osteogenesis [4]. The OPG/RANKL/RANK/TRAIL system, via receptors located on the cell surface, sends intracellular signals and modifies gene expression. As a result, monocytes, neutrophils and endothelial cells are recruited through cytokine production and receptor activation [5]. Serum OPG levels are associated with endothelial dysfunction and mediate vascular calcification [6]. Elevated OPG levels were observed in patients with atherosclerosis, heart failure, metabolic syndrome and diabetes [7,8,9].\\nThe role of OPG in kidney pathologies is not well understood. OPG is expressed in kidney samples, cultured tubular cells and urinary exosome-like vesicles. It has been hypothesized that it can play a role in matrix deposition, inflammation and apoptosis [10]. Furthermore, the role of OPG in cardiovascular pathology and vascular calcification, which are common complications of chronic kidney disease, makes OPG an interesting marker also in CKD. When compared to healthy individuals, non-dialysis-dependent CKD patients, haemodialysis and peritoneal dialysis patients and renal transplant recipients present elevated levels of OPG [11,12,13]. As OPG is involved in vascular calcification, it is also associated with cardiovascular mortality in CKD patients [14]. Decreased levels of OPG are observed in nephrotic syndrome, which is probably caused by the loss with urine. Glucocorticosteroid treatment increases RANKL and reduces OPG expression, which also decreases the OPG levels in patients with nephrotic syndrome [15].\\nThe purpose of our study was to investigate the association between OPG levels and the main complications of chronic kidney disease, including anaemia, protein energy wasting, inflammation and poor prognosis factors of CKD progression such as overhydration, hyperglycaemia and hypertension.\",\n \" 2.1. Design A prospective, observational study in male patients with chronic kidney disease non-treated with dialysis was performed. The inclusion criterion was eGFR lower than 60 mL/min/1.73 m2.\\nA prospective, observational study in male patients with chronic kidney disease non-treated with dialysis was performed. The inclusion criterion was eGFR lower than 60 mL/min/1.73 m2.\\n 2.2. Patients Participants were recruited among patients visiting the Nephrological Outpatient Clinic for a routine control during the period between November 2018 and February 2020. If they fulfilled the inclusion criteria, a new visit was arranged for participation in the study. The participants were asked to avoid saunas, physical exertion and alcohol consumption the day before the examination. The visit took place after overnight fasting.\\nOne hundred men with chronic kidney disease and eGFR lower than 60 mL/min/1.73 m2 were included in the study. The exclusion criteria were: renal replacement therapy or its requirement within the following 3 months, clinical signs of infection and presence of metal parts in the body. The study protocol was accepted by the local ethics committee (Bioethics Committee in Military Institute of Medicine, IRB acceptance number 120/WIM/2018 obtained 22 August 2018). All participants signed an informed consent.\\nBody composition including overhydration (OH), fat amount and lean body mass were measured by bioimpedance spectroscopy with the use of a Body Composition Monitor (Fresenius Medical Care). While being measured, patients stayed in a supine position after a 5 min rest. Electrodes were placed in a tetrapolar configuration (on one hand and one foot).\\nBlood samples for standard measurements were collected after an overnight fast and were transported immediately to the local Department of Laboratory Diagnostics. Concentrations of high-sensitivity C-reactive protein were determined by a nephelometry assay (BNII Siemens) with a cut-off point of 0.8 mg/dL. Serum creatinine concentrations were measured using the Jaffe method (Gen.2; Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland), and serum albumin levels using a BCP Albumin Assay Kit (Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland). Samples for measuring osteoprotegerin (OPG) levels were kept frozen at −80 °C. OPG levels were assessed using the Luminex MAGPIX platform.\\nGFR was calculated according to the short Modification of Diet in Renal Disease (MDRD) formula.\\nGFR in mL/min per 1.73 m2 = 175 × SerumCr−1.154 × age−0.203 × 1.212 (if patient is black) × 0.742 (if female)\\nParticipants were recruited among patients visiting the Nephrological Outpatient Clinic for a routine control during the period between November 2018 and February 2020. If they fulfilled the inclusion criteria, a new visit was arranged for participation in the study. The participants were asked to avoid saunas, physical exertion and alcohol consumption the day before the examination. The visit took place after overnight fasting.\\nOne hundred men with chronic kidney disease and eGFR lower than 60 mL/min/1.73 m2 were included in the study. The exclusion criteria were: renal replacement therapy or its requirement within the following 3 months, clinical signs of infection and presence of metal parts in the body. The study protocol was accepted by the local ethics committee (Bioethics Committee in Military Institute of Medicine, IRB acceptance number 120/WIM/2018 obtained 22 August 2018). All participants signed an informed consent.\\nBody composition including overhydration (OH), fat amount and lean body mass were measured by bioimpedance spectroscopy with the use of a Body Composition Monitor (Fresenius Medical Care). While being measured, patients stayed in a supine position after a 5 min rest. Electrodes were placed in a tetrapolar configuration (on one hand and one foot).\\nBlood samples for standard measurements were collected after an overnight fast and were transported immediately to the local Department of Laboratory Diagnostics. Concentrations of high-sensitivity C-reactive protein were determined by a nephelometry assay (BNII Siemens) with a cut-off point of 0.8 mg/dL. Serum creatinine concentrations were measured using the Jaffe method (Gen.2; Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland), and serum albumin levels using a BCP Albumin Assay Kit (Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland). Samples for measuring osteoprotegerin (OPG) levels were kept frozen at −80 °C. OPG levels were assessed using the Luminex MAGPIX platform.\\nGFR was calculated according to the short Modification of Diet in Renal Disease (MDRD) formula.\\nGFR in mL/min per 1.73 m2 = 175 × SerumCr−1.154 × age−0.203 × 1.212 (if patient is black) × 0.742 (if female)\\n 2.3. Defining the Complications of Chronic Kidney Disease Complications of chronic kidney disease were defined as follows:-Anaemia, when serum haemoglobin level was lower than 12 g/dL [16]-Protein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]-Inflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]-Poor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\\nAnaemia, when serum haemoglobin level was lower than 12 g/dL [16]\\nProtein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]\\nInflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]\\nPoor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\\nComplications of chronic kidney disease were defined as follows:-Anaemia, when serum haemoglobin level was lower than 12 g/dL [16]-Protein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]-Inflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]-Poor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\\nAnaemia, when serum haemoglobin level was lower than 12 g/dL [16]\\nProtein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]\\nInflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]\\nPoor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\\n 2.4. Statistical Analysis The characteristics of the study population are presented using medians with interquartile ranges (for continuous data with distribution other than normal, tested with the Shapiro–Wilk test). The correlation between OPG and clinical and laboratory parameters was assessed using the Spearman rank correlation coefficient. Logistic regression was used to investigate the significance of OPG levels as a marker of metabolic complications of CKD. Given the known relationship between OPG, age and GFR, we compared the value of logistic models, with and without OPG, for each metabolic complication (anaemia, PEW, inflammation, uncontrolled factors of CKD progression). Suitability of fit of each model was assessed using the area under the curve (AUC) with standard error (SE) and presented using the Receiver Operating Characteristic (ROC) curve. The models were compared using Hanley’s algorithm [19]. The analysis was performed with Statistica 13.1 with p-values < 0.05 considered statistically significant.\\nThe characteristics of the study population are presented using medians with interquartile ranges (for continuous data with distribution other than normal, tested with the Shapiro–Wilk test). The correlation between OPG and clinical and laboratory parameters was assessed using the Spearman rank correlation coefficient. Logistic regression was used to investigate the significance of OPG levels as a marker of metabolic complications of CKD. Given the known relationship between OPG, age and GFR, we compared the value of logistic models, with and without OPG, for each metabolic complication (anaemia, PEW, inflammation, uncontrolled factors of CKD progression). Suitability of fit of each model was assessed using the area under the curve (AUC) with standard error (SE) and presented using the Receiver Operating Characteristic (ROC) curve. The models were compared using Hanley’s algorithm [19]. The analysis was performed with Statistica 13.1 with p-values < 0.05 considered statistically significant.\",\n \"A prospective, observational study in male patients with chronic kidney disease non-treated with dialysis was performed. The inclusion criterion was eGFR lower than 60 mL/min/1.73 m2.\",\n \"Participants were recruited among patients visiting the Nephrological Outpatient Clinic for a routine control during the period between November 2018 and February 2020. If they fulfilled the inclusion criteria, a new visit was arranged for participation in the study. The participants were asked to avoid saunas, physical exertion and alcohol consumption the day before the examination. The visit took place after overnight fasting.\\nOne hundred men with chronic kidney disease and eGFR lower than 60 mL/min/1.73 m2 were included in the study. The exclusion criteria were: renal replacement therapy or its requirement within the following 3 months, clinical signs of infection and presence of metal parts in the body. The study protocol was accepted by the local ethics committee (Bioethics Committee in Military Institute of Medicine, IRB acceptance number 120/WIM/2018 obtained 22 August 2018). All participants signed an informed consent.\\nBody composition including overhydration (OH), fat amount and lean body mass were measured by bioimpedance spectroscopy with the use of a Body Composition Monitor (Fresenius Medical Care). While being measured, patients stayed in a supine position after a 5 min rest. Electrodes were placed in a tetrapolar configuration (on one hand and one foot).\\nBlood samples for standard measurements were collected after an overnight fast and were transported immediately to the local Department of Laboratory Diagnostics. Concentrations of high-sensitivity C-reactive protein were determined by a nephelometry assay (BNII Siemens) with a cut-off point of 0.8 mg/dL. Serum creatinine concentrations were measured using the Jaffe method (Gen.2; Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland), and serum albumin levels using a BCP Albumin Assay Kit (Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland). Samples for measuring osteoprotegerin (OPG) levels were kept frozen at −80 °C. OPG levels were assessed using the Luminex MAGPIX platform.\\nGFR was calculated according to the short Modification of Diet in Renal Disease (MDRD) formula.\\nGFR in mL/min per 1.73 m2 = 175 × SerumCr−1.154 × age−0.203 × 1.212 (if patient is black) × 0.742 (if female)\",\n \"Complications of chronic kidney disease were defined as follows:-Anaemia, when serum haemoglobin level was lower than 12 g/dL [16]-Protein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]-Inflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]-Poor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\\nAnaemia, when serum haemoglobin level was lower than 12 g/dL [16]\\nProtein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]\\nInflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]\\nPoor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\",\n \"The characteristics of the study population are presented using medians with interquartile ranges (for continuous data with distribution other than normal, tested with the Shapiro–Wilk test). The correlation between OPG and clinical and laboratory parameters was assessed using the Spearman rank correlation coefficient. Logistic regression was used to investigate the significance of OPG levels as a marker of metabolic complications of CKD. Given the known relationship between OPG, age and GFR, we compared the value of logistic models, with and without OPG, for each metabolic complication (anaemia, PEW, inflammation, uncontrolled factors of CKD progression). Suitability of fit of each model was assessed using the area under the curve (AUC) with standard error (SE) and presented using the Receiver Operating Characteristic (ROC) curve. The models were compared using Hanley’s algorithm [19]. The analysis was performed with Statistica 13.1 with p-values < 0.05 considered statistically significant.\",\n \"The study population consisted of 100 male patients with chronic kidney disease and eGFR lower than 60 mL/min/1.73 m2, non-treated with dialysis. The median age of the studied population was 66 years (59–72). Clinical data are presented in Table 1.\\nWe observed significant, positive correlations between OPG and age, serum creatinine concentration, CRP, fibrinogen, HgBA1C, systolic blood pressure and overhydration. Significant, negative correlations were observed between OPG and eGFR, serum albumin concentration as well as serum haemoglobin level. Table 2.\\nLogistic regression models, adjusted for age and GFR, were created to evaluate the influence of the OPG level as an independent marker of complications in chronic kidney disease patients, such as anaemia, PEW, an inflammatory state, poor prognostic factors of CKD progression.\\n 3.1. Anaemia The OPG level was a significant, independent marker of anaemia in the studied population (p < 0.001).\\nThe model including OPG, age and GFR (AUC=0.84 ± 0.05) identified CKD patients with anaemia better than the model including only age and GFR (AUC=0.76 ± 0.06), as shown in Table 3. However, the difference was not of statistical significance (p = 0.096), as shown in Figure 1.\\nThe OPG level was a significant, independent marker of anaemia in the studied population (p < 0.001).\\nThe model including OPG, age and GFR (AUC=0.84 ± 0.05) identified CKD patients with anaemia better than the model including only age and GFR (AUC=0.76 ± 0.06), as shown in Table 3. However, the difference was not of statistical significance (p = 0.096), as shown in Figure 1.\\n 3.2. Protein Energy Wasting (PEW) OPG was a significant, independent risk factor for PEW occurrence in the study population (p < 0.001). However, the model including OPG, age and GFR was not significantly better (AUC = 0.79 ± 0.05) than those considering only age and GFR (AUC = 0.71 ± 0.06, p = 0.142), as shown in Table 4 and Figure 2.\\nOPG was a significant, independent risk factor for PEW occurrence in the study population (p < 0.001). However, the model including OPG, age and GFR was not significantly better (AUC = 0.79 ± 0.05) than those considering only age and GFR (AUC = 0.71 ± 0.06, p = 0.142), as shown in Table 4 and Figure 2.\\n 3.3. Inflammatory State OPG was a significant, independent risk factor for subclinical inflammation in the study population (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model including only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 5 and Figure 3.\\nOPG was a significant, independent risk factor for subclinical inflammation in the study population (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model including only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 5 and Figure 3.\\n 3.4. Poor Prognostic Factors (Overhydration, Hyperglycaemia and Hypertension) Overhydration, hyperglycaemia and hypertension were chosen as poor prognostic factors of chronic kidney disease progression and were analysed together in logistic regression analysis. OPG was an independent marker that identified patients with poor prognostic factors (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model which included only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 6 and Figure 4.\\nOverhydration, hyperglycaemia and hypertension were chosen as poor prognostic factors of chronic kidney disease progression and were analysed together in logistic regression analysis. OPG was an independent marker that identified patients with poor prognostic factors (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model which included only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 6 and Figure 4.\",\n \"The OPG level was a significant, independent marker of anaemia in the studied population (p < 0.001).\\nThe model including OPG, age and GFR (AUC=0.84 ± 0.05) identified CKD patients with anaemia better than the model including only age and GFR (AUC=0.76 ± 0.06), as shown in Table 3. However, the difference was not of statistical significance (p = 0.096), as shown in Figure 1.\",\n \"OPG was a significant, independent risk factor for PEW occurrence in the study population (p < 0.001). However, the model including OPG, age and GFR was not significantly better (AUC = 0.79 ± 0.05) than those considering only age and GFR (AUC = 0.71 ± 0.06, p = 0.142), as shown in Table 4 and Figure 2.\",\n \"OPG was a significant, independent risk factor for subclinical inflammation in the study population (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model including only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 5 and Figure 3.\",\n \"Overhydration, hyperglycaemia and hypertension were chosen as poor prognostic factors of chronic kidney disease progression and were analysed together in logistic regression analysis. OPG was an independent marker that identified patients with poor prognostic factors (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model which included only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 6 and Figure 4.\",\n \"The role of OPG in chronic kidney disease patients is insufficiently comprehended. We know that the OPG level increases together with a decrease in the glomerular filtration rate, as proved in many studies [20,21]. The results of our study also confirm these observations. In a population of non-dialysis-dependent men with stage 3–5 chronic kidney disease, a significant, inverse correlation between OPG levels and eGFR was noted (R = −0.36; p < 0.001).\\nIn our study, we also observed significant correlations between OPG and parameters which indicate CKD complications. The OPG level was correlated with the level of haemoglobins and acted as a marker of anaemia occurrence in the studied population. The logistic regression model including OPG was superior to that including only age and GFR in identifying patients with anaemia. However, the difference was not of statistical significance. OPG, as a member of the TNF superfamily, can influence haematopoiesis. Some researchers have observed that TNF superfamily members can regulate the growth of hematopoietic stem cells and progenitor cells [1,22].\\nAside from anaemia, protein energy wasting (PEW) is also a complication of chronic kidney disease. The components of PEW are low serum albumin, cholesterol levels as well as low BMI and fat amount [17]. In our study, the OPG level was a marker of PEW in non-dialysis-dependent CKD men. PEW aetiology in CKD patients is associated with many factors such as hormonal disorders, insulin resistance and a subclinical inflammatory state. In our study, the OPG levels also correlated with the CRP and fibrinogen levels. OPG was also a significant marker of instances of inflammation. The logistic regression model that included OPG proved significantly better in in the identification of subclinical inflammation than the model which only included age and GFR. This finding is unsurprising, because OPG is known for its pro-inflammatory action. OPG has been described as a marker of endothelial damage associated with the inflammatory process, and in vitro studies have shown that OPG is involved in inflammatory cell chemotaxis [23].\\nMost studies concerning OPG in CKD patients concentrate on the association between OPG and vascular calcification. OPG levels correlate with vascular calcification in non-dialysis-dependent CKD patients as well as in patients treated with renal replacement therapy [24,25]. In animal studies, mice without OPG developed severe vascular calcification [26]. Therefore, it is hypothesized that OPG has a protective role in vascular injury, and the elevation of OPG levels inhibits vascular calcification. However, vascular calcification can also occur in the medial as well as in the intimal layer. In atherosclerosis, intima is thickened, inflamed and calcified, forming plaques with diffuse localisation along the vessel walls, whereas medial calcification occurs along the elastic lamina, leading to stiffness of the artery wall. Studies in animal models, including Apolipoprotein E-deficient mice, suggest that OPG plays a protective, anti-calcification role in both the medial and the intimal (atherosclerotic) calcification process [27].\\nThe OPG levels increase rapidly in the early stages of vascular calcification and subsequently remain almost invariant [21]. Therefore, OPG appears to be a marker of the onset of atherosclerosis. However, the development of cardiovascular disease (CVD) begins with endothelial dysfunction. The earliest manifestation of CVD is observed in the area of microcirculation, and in CKD patients, this area is affected by the loss of homeostasis, even in cases of non-severe renal impairment [28]. OPG, as a factor released by endothelial cells, can be a marker of endothelial dysfunction and may probably be involved in a complicated chain of pathophysiological connections between chronic kidney disease and CVD.\\nIn our study, we analysed factors of poor prognosis of CKD progression which are associated with vascular damage, such as overhydration, hypertension and glycaemic disturbances expressed by HgbA1c. Positive, significant correlations were observed between OPG levels and all three parameters. In other studies, OPG levels were associated with glycaemic status, and higher OPG levels were observed in patients with poor glycaemic control [29]. In our study, OPG was identified independently, associated with the presence of poor prognostic factors (overhydration, hypertension and glycaemic disturbances) irrespective of age and eGFR.\\nThe limitation of our study is its relatively small sample size. An increase in the number of participants could enable a division of the study population into various subgroups according to the different stages of chronic kidney disease. Complications of CKD are more severe in more advanced stages of CKD, and OPG influence could also be more significant.\",\n \"CKD is in itself a cardiovascular risk factor, and cardiovascular mortality in CKD patients is high. CVD mortality in CKD patients is even greater in the presence of diabetes and hypertension [30]. Early detection of such complications and the identification of patients at risk of developing them is vital. Therefore, new markers that can predict metabolic disorders and have an unfavourable effect on vascular damage are necessary. OPG seems to be a possible marker associated with PEW, inflammation and vascular metabolic disturbances which is worth further study.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,"subjects",null,null,"results",null,null,null,null,"discussion","conclusions"],"string":"[\n \"intro\",\n \"methods\",\n null,\n \"subjects\",\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["chronic kidney disease","kidney","inflammation","protein energy-wasting","metabolic complications","bioimpedance spectroscopy"],"string":"[\n \"chronic kidney disease\",\n \"kidney\",\n \"inflammation\",\n \"protein energy-wasting\",\n \"metabolic complications\",\n \"bioimpedance spectroscopy\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"1. Introduction:\nOsteoprotegerin (OPG) is a molecule belonging to the tumour necrosis factor (TNF) superfamily. It is a decoy receptor for TNF-related apoptosis-inducing ligand (TRAIL). The TNF superfamily regulates immune responses, homeostasis as well as haematopoiesis [1]. Initially, OPG was identified as a bone resorption inhibitor that blocks the binding of RANK (receptor activator of nuclear factor kappa-β) to its ligand RANKL [2]. Further studies revealed that it has an additional impact on the cardiovascular and immune systems [3]. The OPG/RANKL/RANK system is involved in pathological angiogenesis, inflammatory states and cell survival. OPG is a glycoprotein released by vascular smooth muscle cells, endothelial cells, osteoblasts and immune cells. There is a signalling pathway between endothelial cells and osteoblasts during osteogenesis creating a connection between angiogenesis and osteogenesis [4]. The OPG/RANKL/RANK/TRAIL system, via receptors located on the cell surface, sends intracellular signals and modifies gene expression. As a result, monocytes, neutrophils and endothelial cells are recruited through cytokine production and receptor activation [5]. Serum OPG levels are associated with endothelial dysfunction and mediate vascular calcification [6]. Elevated OPG levels were observed in patients with atherosclerosis, heart failure, metabolic syndrome and diabetes [7,8,9].\nThe role of OPG in kidney pathologies is not well understood. OPG is expressed in kidney samples, cultured tubular cells and urinary exosome-like vesicles. It has been hypothesized that it can play a role in matrix deposition, inflammation and apoptosis [10]. Furthermore, the role of OPG in cardiovascular pathology and vascular calcification, which are common complications of chronic kidney disease, makes OPG an interesting marker also in CKD. When compared to healthy individuals, non-dialysis-dependent CKD patients, haemodialysis and peritoneal dialysis patients and renal transplant recipients present elevated levels of OPG [11,12,13]. As OPG is involved in vascular calcification, it is also associated with cardiovascular mortality in CKD patients [14]. Decreased levels of OPG are observed in nephrotic syndrome, which is probably caused by the loss with urine. Glucocorticosteroid treatment increases RANKL and reduces OPG expression, which also decreases the OPG levels in patients with nephrotic syndrome [15].\nThe purpose of our study was to investigate the association between OPG levels and the main complications of chronic kidney disease, including anaemia, protein energy wasting, inflammation and poor prognosis factors of CKD progression such as overhydration, hyperglycaemia and hypertension.\n\n2. Methods:\n 2.1. Design A prospective, observational study in male patients with chronic kidney disease non-treated with dialysis was performed. The inclusion criterion was eGFR lower than 60 mL/min/1.73 m2.\nA prospective, observational study in male patients with chronic kidney disease non-treated with dialysis was performed. The inclusion criterion was eGFR lower than 60 mL/min/1.73 m2.\n 2.2. Patients Participants were recruited among patients visiting the Nephrological Outpatient Clinic for a routine control during the period between November 2018 and February 2020. If they fulfilled the inclusion criteria, a new visit was arranged for participation in the study. The participants were asked to avoid saunas, physical exertion and alcohol consumption the day before the examination. The visit took place after overnight fasting.\nOne hundred men with chronic kidney disease and eGFR lower than 60 mL/min/1.73 m2 were included in the study. The exclusion criteria were: renal replacement therapy or its requirement within the following 3 months, clinical signs of infection and presence of metal parts in the body. The study protocol was accepted by the local ethics committee (Bioethics Committee in Military Institute of Medicine, IRB acceptance number 120/WIM/2018 obtained 22 August 2018). All participants signed an informed consent.\nBody composition including overhydration (OH), fat amount and lean body mass were measured by bioimpedance spectroscopy with the use of a Body Composition Monitor (Fresenius Medical Care). While being measured, patients stayed in a supine position after a 5 min rest. Electrodes were placed in a tetrapolar configuration (on one hand and one foot).\nBlood samples for standard measurements were collected after an overnight fast and were transported immediately to the local Department of Laboratory Diagnostics. Concentrations of high-sensitivity C-reactive protein were determined by a nephelometry assay (BNII Siemens) with a cut-off point of 0.8 mg/dL. Serum creatinine concentrations were measured using the Jaffe method (Gen.2; Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland), and serum albumin levels using a BCP Albumin Assay Kit (Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland). Samples for measuring osteoprotegerin (OPG) levels were kept frozen at −80 °C. OPG levels were assessed using the Luminex MAGPIX platform.\nGFR was calculated according to the short Modification of Diet in Renal Disease (MDRD) formula.\nGFR in mL/min per 1.73 m2 = 175 × SerumCr−1.154 × age−0.203 × 1.212 (if patient is black) × 0.742 (if female)\nParticipants were recruited among patients visiting the Nephrological Outpatient Clinic for a routine control during the period between November 2018 and February 2020. If they fulfilled the inclusion criteria, a new visit was arranged for participation in the study. The participants were asked to avoid saunas, physical exertion and alcohol consumption the day before the examination. The visit took place after overnight fasting.\nOne hundred men with chronic kidney disease and eGFR lower than 60 mL/min/1.73 m2 were included in the study. The exclusion criteria were: renal replacement therapy or its requirement within the following 3 months, clinical signs of infection and presence of metal parts in the body. The study protocol was accepted by the local ethics committee (Bioethics Committee in Military Institute of Medicine, IRB acceptance number 120/WIM/2018 obtained 22 August 2018). All participants signed an informed consent.\nBody composition including overhydration (OH), fat amount and lean body mass were measured by bioimpedance spectroscopy with the use of a Body Composition Monitor (Fresenius Medical Care). While being measured, patients stayed in a supine position after a 5 min rest. Electrodes were placed in a tetrapolar configuration (on one hand and one foot).\nBlood samples for standard measurements were collected after an overnight fast and were transported immediately to the local Department of Laboratory Diagnostics. Concentrations of high-sensitivity C-reactive protein were determined by a nephelometry assay (BNII Siemens) with a cut-off point of 0.8 mg/dL. Serum creatinine concentrations were measured using the Jaffe method (Gen.2; Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland), and serum albumin levels using a BCP Albumin Assay Kit (Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland). Samples for measuring osteoprotegerin (OPG) levels were kept frozen at −80 °C. OPG levels were assessed using the Luminex MAGPIX platform.\nGFR was calculated according to the short Modification of Diet in Renal Disease (MDRD) formula.\nGFR in mL/min per 1.73 m2 = 175 × SerumCr−1.154 × age−0.203 × 1.212 (if patient is black) × 0.742 (if female)\n 2.3. Defining the Complications of Chronic Kidney Disease Complications of chronic kidney disease were defined as follows:-Anaemia, when serum haemoglobin level was lower than 12 g/dL [16]-Protein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]-Inflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]-Poor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\nAnaemia, when serum haemoglobin level was lower than 12 g/dL [16]\nProtein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]\nInflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]\nPoor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\nComplications of chronic kidney disease were defined as follows:-Anaemia, when serum haemoglobin level was lower than 12 g/dL [16]-Protein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]-Inflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]-Poor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\nAnaemia, when serum haemoglobin level was lower than 12 g/dL [16]\nProtein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]\nInflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]\nPoor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\n 2.4. Statistical Analysis The characteristics of the study population are presented using medians with interquartile ranges (for continuous data with distribution other than normal, tested with the Shapiro–Wilk test). The correlation between OPG and clinical and laboratory parameters was assessed using the Spearman rank correlation coefficient. Logistic regression was used to investigate the significance of OPG levels as a marker of metabolic complications of CKD. Given the known relationship between OPG, age and GFR, we compared the value of logistic models, with and without OPG, for each metabolic complication (anaemia, PEW, inflammation, uncontrolled factors of CKD progression). Suitability of fit of each model was assessed using the area under the curve (AUC) with standard error (SE) and presented using the Receiver Operating Characteristic (ROC) curve. The models were compared using Hanley’s algorithm [19]. The analysis was performed with Statistica 13.1 with p-values < 0.05 considered statistically significant.\nThe characteristics of the study population are presented using medians with interquartile ranges (for continuous data with distribution other than normal, tested with the Shapiro–Wilk test). The correlation between OPG and clinical and laboratory parameters was assessed using the Spearman rank correlation coefficient. Logistic regression was used to investigate the significance of OPG levels as a marker of metabolic complications of CKD. Given the known relationship between OPG, age and GFR, we compared the value of logistic models, with and without OPG, for each metabolic complication (anaemia, PEW, inflammation, uncontrolled factors of CKD progression). Suitability of fit of each model was assessed using the area under the curve (AUC) with standard error (SE) and presented using the Receiver Operating Characteristic (ROC) curve. The models were compared using Hanley’s algorithm [19]. The analysis was performed with Statistica 13.1 with p-values < 0.05 considered statistically significant.\n\n2.1. Design:\nA prospective, observational study in male patients with chronic kidney disease non-treated with dialysis was performed. The inclusion criterion was eGFR lower than 60 mL/min/1.73 m2.\n\n2.2. Patients:\nParticipants were recruited among patients visiting the Nephrological Outpatient Clinic for a routine control during the period between November 2018 and February 2020. If they fulfilled the inclusion criteria, a new visit was arranged for participation in the study. The participants were asked to avoid saunas, physical exertion and alcohol consumption the day before the examination. The visit took place after overnight fasting.\nOne hundred men with chronic kidney disease and eGFR lower than 60 mL/min/1.73 m2 were included in the study. The exclusion criteria were: renal replacement therapy or its requirement within the following 3 months, clinical signs of infection and presence of metal parts in the body. The study protocol was accepted by the local ethics committee (Bioethics Committee in Military Institute of Medicine, IRB acceptance number 120/WIM/2018 obtained 22 August 2018). All participants signed an informed consent.\nBody composition including overhydration (OH), fat amount and lean body mass were measured by bioimpedance spectroscopy with the use of a Body Composition Monitor (Fresenius Medical Care). While being measured, patients stayed in a supine position after a 5 min rest. Electrodes were placed in a tetrapolar configuration (on one hand and one foot).\nBlood samples for standard measurements were collected after an overnight fast and were transported immediately to the local Department of Laboratory Diagnostics. Concentrations of high-sensitivity C-reactive protein were determined by a nephelometry assay (BNII Siemens) with a cut-off point of 0.8 mg/dL. Serum creatinine concentrations were measured using the Jaffe method (Gen.2; Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland), and serum albumin levels using a BCP Albumin Assay Kit (Roche Diagnostics GmbH, Risch-Rotkreuz, Switzerland). Samples for measuring osteoprotegerin (OPG) levels were kept frozen at −80 °C. OPG levels were assessed using the Luminex MAGPIX platform.\nGFR was calculated according to the short Modification of Diet in Renal Disease (MDRD) formula.\nGFR in mL/min per 1.73 m2 = 175 × SerumCr−1.154 × age−0.203 × 1.212 (if patient is black) × 0.742 (if female)\n\n2.3. Defining the Complications of Chronic Kidney Disease:\nComplications of chronic kidney disease were defined as follows:-Anaemia, when serum haemoglobin level was lower than 12 g/dL [16]-Protein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]-Inflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]-Poor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\nAnaemia, when serum haemoglobin level was lower than 12 g/dL [16]\nProtein energy wasting (PEW), when one of the following occurred: serum albumin level was lower than 3.8 g/dL, total cholesterol level was lower than 100 mg/dL, BMI was lower than 23 kg/m2 or Fat was lower than 10% [17]\nInflammatory state, when CRP >0.8 mg/dL or Fbg >400 mg/dL [17]\nPoor prognostic factors of CKD progression, when HbA1c >8% or SBP >140 mmHg or presence of overhydration, i.e., (OH) >4 L [18]\n\n2.4. Statistical Analysis:\nThe characteristics of the study population are presented using medians with interquartile ranges (for continuous data with distribution other than normal, tested with the Shapiro–Wilk test). The correlation between OPG and clinical and laboratory parameters was assessed using the Spearman rank correlation coefficient. Logistic regression was used to investigate the significance of OPG levels as a marker of metabolic complications of CKD. Given the known relationship between OPG, age and GFR, we compared the value of logistic models, with and without OPG, for each metabolic complication (anaemia, PEW, inflammation, uncontrolled factors of CKD progression). Suitability of fit of each model was assessed using the area under the curve (AUC) with standard error (SE) and presented using the Receiver Operating Characteristic (ROC) curve. The models were compared using Hanley’s algorithm [19]. The analysis was performed with Statistica 13.1 with p-values < 0.05 considered statistically significant.\n\n3. Results:\nThe study population consisted of 100 male patients with chronic kidney disease and eGFR lower than 60 mL/min/1.73 m2, non-treated with dialysis. The median age of the studied population was 66 years (59–72). Clinical data are presented in Table 1.\nWe observed significant, positive correlations between OPG and age, serum creatinine concentration, CRP, fibrinogen, HgBA1C, systolic blood pressure and overhydration. Significant, negative correlations were observed between OPG and eGFR, serum albumin concentration as well as serum haemoglobin level. Table 2.\nLogistic regression models, adjusted for age and GFR, were created to evaluate the influence of the OPG level as an independent marker of complications in chronic kidney disease patients, such as anaemia, PEW, an inflammatory state, poor prognostic factors of CKD progression.\n 3.1. Anaemia The OPG level was a significant, independent marker of anaemia in the studied population (p < 0.001).\nThe model including OPG, age and GFR (AUC=0.84 ± 0.05) identified CKD patients with anaemia better than the model including only age and GFR (AUC=0.76 ± 0.06), as shown in Table 3. However, the difference was not of statistical significance (p = 0.096), as shown in Figure 1.\nThe OPG level was a significant, independent marker of anaemia in the studied population (p < 0.001).\nThe model including OPG, age and GFR (AUC=0.84 ± 0.05) identified CKD patients with anaemia better than the model including only age and GFR (AUC=0.76 ± 0.06), as shown in Table 3. However, the difference was not of statistical significance (p = 0.096), as shown in Figure 1.\n 3.2. Protein Energy Wasting (PEW) OPG was a significant, independent risk factor for PEW occurrence in the study population (p < 0.001). However, the model including OPG, age and GFR was not significantly better (AUC = 0.79 ± 0.05) than those considering only age and GFR (AUC = 0.71 ± 0.06, p = 0.142), as shown in Table 4 and Figure 2.\nOPG was a significant, independent risk factor for PEW occurrence in the study population (p < 0.001). However, the model including OPG, age and GFR was not significantly better (AUC = 0.79 ± 0.05) than those considering only age and GFR (AUC = 0.71 ± 0.06, p = 0.142), as shown in Table 4 and Figure 2.\n 3.3. Inflammatory State OPG was a significant, independent risk factor for subclinical inflammation in the study population (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model including only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 5 and Figure 3.\nOPG was a significant, independent risk factor for subclinical inflammation in the study population (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model including only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 5 and Figure 3.\n 3.4. Poor Prognostic Factors (Overhydration, Hyperglycaemia and Hypertension) Overhydration, hyperglycaemia and hypertension were chosen as poor prognostic factors of chronic kidney disease progression and were analysed together in logistic regression analysis. OPG was an independent marker that identified patients with poor prognostic factors (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model which included only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 6 and Figure 4.\nOverhydration, hyperglycaemia and hypertension were chosen as poor prognostic factors of chronic kidney disease progression and were analysed together in logistic regression analysis. OPG was an independent marker that identified patients with poor prognostic factors (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model which included only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 6 and Figure 4.\n\n3.1. Anaemia:\nThe OPG level was a significant, independent marker of anaemia in the studied population (p < 0.001).\nThe model including OPG, age and GFR (AUC=0.84 ± 0.05) identified CKD patients with anaemia better than the model including only age and GFR (AUC=0.76 ± 0.06), as shown in Table 3. However, the difference was not of statistical significance (p = 0.096), as shown in Figure 1.\n\n3.2. Protein Energy Wasting (PEW):\nOPG was a significant, independent risk factor for PEW occurrence in the study population (p < 0.001). However, the model including OPG, age and GFR was not significantly better (AUC = 0.79 ± 0.05) than those considering only age and GFR (AUC = 0.71 ± 0.06, p = 0.142), as shown in Table 4 and Figure 2.\n\n3.3. Inflammatory State:\nOPG was a significant, independent risk factor for subclinical inflammation in the study population (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model including only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 5 and Figure 3.\n\n3.4. Poor Prognostic Factors (Overhydration, Hyperglycaemia and Hypertension):\nOverhydration, hyperglycaemia and hypertension were chosen as poor prognostic factors of chronic kidney disease progression and were analysed together in logistic regression analysis. OPG was an independent marker that identified patients with poor prognostic factors (p < 0.001). The model including OPG, age and GFR was significantly better (AUC = 0.77 ± 0.05) than the model which included only age and GFR (AUC = 0.67 ± 0.06; p = 0.041), as shown in Table 6 and Figure 4.\n\n4. Discussion:\nThe role of OPG in chronic kidney disease patients is insufficiently comprehended. We know that the OPG level increases together with a decrease in the glomerular filtration rate, as proved in many studies [20,21]. The results of our study also confirm these observations. In a population of non-dialysis-dependent men with stage 3–5 chronic kidney disease, a significant, inverse correlation between OPG levels and eGFR was noted (R = −0.36; p < 0.001).\nIn our study, we also observed significant correlations between OPG and parameters which indicate CKD complications. The OPG level was correlated with the level of haemoglobins and acted as a marker of anaemia occurrence in the studied population. The logistic regression model including OPG was superior to that including only age and GFR in identifying patients with anaemia. However, the difference was not of statistical significance. OPG, as a member of the TNF superfamily, can influence haematopoiesis. Some researchers have observed that TNF superfamily members can regulate the growth of hematopoietic stem cells and progenitor cells [1,22].\nAside from anaemia, protein energy wasting (PEW) is also a complication of chronic kidney disease. The components of PEW are low serum albumin, cholesterol levels as well as low BMI and fat amount [17]. In our study, the OPG level was a marker of PEW in non-dialysis-dependent CKD men. PEW aetiology in CKD patients is associated with many factors such as hormonal disorders, insulin resistance and a subclinical inflammatory state. In our study, the OPG levels also correlated with the CRP and fibrinogen levels. OPG was also a significant marker of instances of inflammation. The logistic regression model that included OPG proved significantly better in in the identification of subclinical inflammation than the model which only included age and GFR. This finding is unsurprising, because OPG is known for its pro-inflammatory action. OPG has been described as a marker of endothelial damage associated with the inflammatory process, and in vitro studies have shown that OPG is involved in inflammatory cell chemotaxis [23].\nMost studies concerning OPG in CKD patients concentrate on the association between OPG and vascular calcification. OPG levels correlate with vascular calcification in non-dialysis-dependent CKD patients as well as in patients treated with renal replacement therapy [24,25]. In animal studies, mice without OPG developed severe vascular calcification [26]. Therefore, it is hypothesized that OPG has a protective role in vascular injury, and the elevation of OPG levels inhibits vascular calcification. However, vascular calcification can also occur in the medial as well as in the intimal layer. In atherosclerosis, intima is thickened, inflamed and calcified, forming plaques with diffuse localisation along the vessel walls, whereas medial calcification occurs along the elastic lamina, leading to stiffness of the artery wall. Studies in animal models, including Apolipoprotein E-deficient mice, suggest that OPG plays a protective, anti-calcification role in both the medial and the intimal (atherosclerotic) calcification process [27].\nThe OPG levels increase rapidly in the early stages of vascular calcification and subsequently remain almost invariant [21]. Therefore, OPG appears to be a marker of the onset of atherosclerosis. However, the development of cardiovascular disease (CVD) begins with endothelial dysfunction. The earliest manifestation of CVD is observed in the area of microcirculation, and in CKD patients, this area is affected by the loss of homeostasis, even in cases of non-severe renal impairment [28]. OPG, as a factor released by endothelial cells, can be a marker of endothelial dysfunction and may probably be involved in a complicated chain of pathophysiological connections between chronic kidney disease and CVD.\nIn our study, we analysed factors of poor prognosis of CKD progression which are associated with vascular damage, such as overhydration, hypertension and glycaemic disturbances expressed by HgbA1c. Positive, significant correlations were observed between OPG levels and all three parameters. In other studies, OPG levels were associated with glycaemic status, and higher OPG levels were observed in patients with poor glycaemic control [29]. In our study, OPG was identified independently, associated with the presence of poor prognostic factors (overhydration, hypertension and glycaemic disturbances) irrespective of age and eGFR.\nThe limitation of our study is its relatively small sample size. An increase in the number of participants could enable a division of the study population into various subgroups according to the different stages of chronic kidney disease. Complications of CKD are more severe in more advanced stages of CKD, and OPG influence could also be more significant.\n\n5. Conclusions:\nCKD is in itself a cardiovascular risk factor, and cardiovascular mortality in CKD patients is high. CVD mortality in CKD patients is even greater in the presence of diabetes and hypertension [30]. Early detection of such complications and the identification of patients at risk of developing them is vital. Therefore, new markers that can predict metabolic disorders and have an unfavourable effect on vascular damage are necessary. OPG seems to be a possible marker associated with PEW, inflammation and vascular metabolic disturbances which is worth further study.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nOsteoprotegerin (OPG) belongs to the tumour necrosis factor superfamily and is known to accelerate endothelial dysfunction and vascular calcification. OPG concentrations are elevated in patients with chronic kidney disease. The aim of this study was to investigate the association between OPG levels and frequent complications of chronic kidney disease (CKD) such as anaemia, protein energy wasting (PEW), inflammation, overhydration, hyperglycaemia and hypertension.\n\nMethods:\nOne hundred non-dialysis-dependent men with CKD stage 3-5 were included in the study. Bioimpedance spectroscopy (BIS) was used to measure overhydration, fat amount and lean body mass. We also measured the serum concentrations of haemoglobin, albumin, total cholesterol, C-reactive protein (CRP), fibrinogen and glycated haemoglobin (HgbA1c), as well as blood pressure.\n\nResults:\nWe observed a significant, positive correlation between OPG and age, serum creatinine, CRP, fibrinogen, HgbA1c concentrations, systolic blood pressure and overhydration. Negative correlations were observed between OPG and glomerular filtration rate (eGFR), serum albumin concentrations and serum haemoglobin level. Logistic regression models revealed that OPG is an independent marker of metabolic complications such as anaemia, PEW, inflammation and poor renal prognosis (including overhydration, uncontrolled diabetes and hypertension) in the studied population.\n\nConclusions:\nOur results suggest that OPG can be an independent marker of PEW, inflammation and vascular metabolic disturbances in patients with chronic kidney disease."},"background_conclusion_text":{"kind":"string","value":"1. Introduction:\nOsteoprotegerin (OPG) is a molecule belonging to the tumour necrosis factor (TNF) superfamily. It is a decoy receptor for TNF-related apoptosis-inducing ligand (TRAIL). The TNF superfamily regulates immune responses, homeostasis as well as haematopoiesis [1]. Initially, OPG was identified as a bone resorption inhibitor that blocks the binding of RANK (receptor activator of nuclear factor kappa-β) to its ligand RANKL [2]. Further studies revealed that it has an additional impact on the cardiovascular and immune systems [3]. The OPG/RANKL/RANK system is involved in pathological angiogenesis, inflammatory states and cell survival. OPG is a glycoprotein released by vascular smooth muscle cells, endothelial cells, osteoblasts and immune cells. There is a signalling pathway between endothelial cells and osteoblasts during osteogenesis creating a connection between angiogenesis and osteogenesis [4]. The OPG/RANKL/RANK/TRAIL system, via receptors located on the cell surface, sends intracellular signals and modifies gene expression. As a result, monocytes, neutrophils and endothelial cells are recruited through cytokine production and receptor activation [5]. Serum OPG levels are associated with endothelial dysfunction and mediate vascular calcification [6]. Elevated OPG levels were observed in patients with atherosclerosis, heart failure, metabolic syndrome and diabetes [7,8,9].\nThe role of OPG in kidney pathologies is not well understood. OPG is expressed in kidney samples, cultured tubular cells and urinary exosome-like vesicles. It has been hypothesized that it can play a role in matrix deposition, inflammation and apoptosis [10]. Furthermore, the role of OPG in cardiovascular pathology and vascular calcification, which are common complications of chronic kidney disease, makes OPG an interesting marker also in CKD. When compared to healthy individuals, non-dialysis-dependent CKD patients, haemodialysis and peritoneal dialysis patients and renal transplant recipients present elevated levels of OPG [11,12,13]. As OPG is involved in vascular calcification, it is also associated with cardiovascular mortality in CKD patients [14]. Decreased levels of OPG are observed in nephrotic syndrome, which is probably caused by the loss with urine. Glucocorticosteroid treatment increases RANKL and reduces OPG expression, which also decreases the OPG levels in patients with nephrotic syndrome [15].\nThe purpose of our study was to investigate the association between OPG levels and the main complications of chronic kidney disease, including anaemia, protein energy wasting, inflammation and poor prognosis factors of CKD progression such as overhydration, hyperglycaemia and hypertension.\n\n5. Conclusions:\nCKD is in itself a cardiovascular risk factor, and cardiovascular mortality in CKD patients is high. CVD mortality in CKD patients is even greater in the presence of diabetes and hypertension [30]. Early detection of such complications and the identification of patients at risk of developing them is vital. Therefore, new markers that can predict metabolic disorders and have an unfavourable effect on vascular damage are necessary. OPG seems to be a possible marker associated with PEW, inflammation and vascular metabolic disturbances which is worth further study."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nOsteoprotegerin (OPG) belongs to the tumour necrosis factor superfamily and is known to accelerate endothelial dysfunction and vascular calcification. OPG concentrations are elevated in patients with chronic kidney disease. The aim of this study was to investigate the association between OPG levels and frequent complications of chronic kidney disease (CKD) such as anaemia, protein energy wasting (PEW), inflammation, overhydration, hyperglycaemia and hypertension.\n\nMethods:\nOne hundred non-dialysis-dependent men with CKD stage 3-5 were included in the study. Bioimpedance spectroscopy (BIS) was used to measure overhydration, fat amount and lean body mass. We also measured the serum concentrations of haemoglobin, albumin, total cholesterol, C-reactive protein (CRP), fibrinogen and glycated haemoglobin (HgbA1c), as well as blood pressure.\n\nResults:\nWe observed a significant, positive correlation between OPG and age, serum creatinine, CRP, fibrinogen, HgbA1c concentrations, systolic blood pressure and overhydration. Negative correlations were observed between OPG and glomerular filtration rate (eGFR), serum albumin concentrations and serum haemoglobin level. Logistic regression models revealed that OPG is an independent marker of metabolic complications such as anaemia, PEW, inflammation and poor renal prognosis (including overhydration, uncontrolled diabetes and hypertension) in the studied population.\n\nConclusions:\nOur results suggest that OPG can be an independent marker of PEW, inflammation and vascular metabolic disturbances in patients with chronic kidney disease."},"whole_article_text_length":{"kind":"number","value":5272,"string":"5,272"},"whole_article_abstract_length":{"kind":"number","value":277,"string":"277"},"other_sections_lengths":{"kind":"list like","value":[33,252,176,82,70,68,91],"string":"[\n 33,\n 252,\n 176,\n 82,\n 70,\n 68,\n 91\n]","isConversational":false},"num_sections":{"kind":"number","value":13,"string":"13"},"most_frequent_words":{"kind":"list like","value":["opg","lower","age","gfr","patients","dl","ckd","study","age gfr","levels"],"string":"[\n \"opg\",\n \"lower\",\n \"age\",\n \"gfr\",\n 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osteoprotegerin opg | osteoprotegerin opg levels | angiogenesis osteogenesis opg [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] opg | lower | age | gfr | patients | dl | ckd | study | age gfr | levels [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] opg | lower | age | gfr | patients | dl | ckd | study | age gfr | levels [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] opg | lower | age | gfr | patients | dl | ckd | study | age gfr | levels [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] opg | lower | age | gfr | patients | dl | ckd | study | age gfr | levels [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] opg | lower | age | gfr | patients | dl | ckd | study | age gfr | levels 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[SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] opg | auc | gfr | age | age gfr | model | model including | patients | lower | dl [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] opg | auc | gfr | age | age gfr | model | model including | patients | lower | dl [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Osteoprotegerin | OPG ||| OPG ||| OPG | PEW [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] One hundred | CKD | 3-5 ||| Bioimpedance | BIS ||| CRP [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] OPG | CRP | HgbA1c ||| OPG ||| OPG | PEW [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] OPG | PEW [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Osteoprotegerin | OPG ||| OPG ||| OPG | PEW ||| One hundred | CKD | 3-5 ||| Bioimpedance | BIS ||| CRP ||| ||| OPG | CRP | HgbA1c ||| OPG ||| OPG | PEW ||| OPG | PEW [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Osteoprotegerin | OPG ||| OPG ||| OPG | PEW ||| One hundred | CKD | 3-5 ||| Bioimpedance | BIS ||| CRP ||| ||| OPG | CRP | HgbA1c ||| OPG ||| OPG | PEW ||| OPG | PEW [SUMMARY]"}}},{"rowIdx":74,"cells":{"title":{"kind":"string","value":"A pan-Theileria FRET-qPCR survey for Theileria spp. in ruminants from nine provinces of China."},"pmid":{"kind":"string","value":"25175751"},"background_abstract":{"kind":"string","value":"Theileria spp. are tick transmitted protozoa that can infect large and small ruminants causing disease and economic losses. Diagnosis of infections is often challenging, as parasites can be difficult to detect and identify microscopically and serology is unreliable. While there are PCR assays which can identify certain Theileria spp., there is no one PCR that has been designed to identify all recognized species that occur in ruminants and which will greatly simplify the laboratory diagnoses of infections."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Primers and probes for a genus-specific pan-Theileria FRET-qPCR were selected by comparing sequences of recognized Theileria spp. in GenBank and the test validated using reference organisms. The assay was also tested on whole blood samples from large and small ruminants from nine provinces in China."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The pan-Theileria FRET-qPCR detected all recognized species but none of the closely related protozoa. In whole blood samples from animals in China, Theileria spp. DNA was detected in 53.2% of the sheep tested (59/111), 44.4% of the goats (120/270) and 30.8% of the cattle (380/1,235). Water buffaloes (n = 29) were negative. Sequencing of some of the PCR products showed cattle in China were infected with T. orientalis/T. sergenti/T. buffeli group while T. ovis and T. luwenshuni were found in sheep and T. luwenshuni in goats. The prevalence of Theileria DNA was significantly higher in Bos p. indicus than in Bos p. taurus (77.7% vs. 18.3%) and copy numbers were also significantly higher (10(4.88) vs. 10(3.00) Theileria 18S rRNA gene copies/per ml whole blood)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The pan-Theileria FRET-qPCR can detect all recognized Theileria spp. of ruminants in a single reaction. Large and small ruminants in China are commonly infected with a variety of Theileria spp."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Animals","DNA, Protozoan","Molecular Sequence Data","Polymerase Chain Reaction","Prevalence","Ruminants","Sensitivity and Specificity","Theileria","Theileriasis"],"string":"[\n \"Animals\",\n \"DNA, Protozoan\",\n \"Molecular Sequence Data\",\n \"Polymerase Chain Reaction\",\n \"Prevalence\",\n \"Ruminants\",\n \"Sensitivity and Specificity\",\n \"Theileria\",\n \"Theileriasis\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"4262081"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Theileria spp. are tick-transmitted, intracellular protozoan parasites infecting leukocytes and erythrocytes of wild and domestic large and small ruminants. Several Theileria spp., transmitted by ixodid ticks of the genera Rhipicephalus, Hyalomma, Amblyomma and Haemaphysalis, have been described in cattle, water buffaloes, sheep and goats in different geographical zones of the world [1–5]. Theileriosis is primarily limited to tropical and sub-tropical areas of the world, with infections mainly reported in Africa and the Middle East but also in southern Europe and northern Asia [6–11]. Infections by Theileria spp. can cause fever, anemia and hemoglobinuria and, in severe cases, death although many species are benign. Animals recovered from acute or primary infections usually remain persistently infected and may act as reservoirs of infecting ticks [12, 13].\nWhile there have been numerous reports of theileriosis in various animal species in China since 1958 [14–27], many have been reported in Chinese and some were based on microscopic detection of parasites which can be difficult with low parasitemia and does not allow ready differentiation of species. Serological studies, although sensitive and easy to perform, are not specific as there is cross reactivity between Theileria spp. Although molecular studies have been performed, these have been to detect Theileria of specific domestic animal species, for example sheep and goats [27]. There have been no highly sensitive and specific molecular methods described which enable studies on various animals from widely divergent areas of China where different Theileria spp. might occur. To address this problem, we developed and validated a highly sensitive genus-specific Theileria FRET-qPCR that detects the recognized Theileria spp. of domestic animals and investigated the molecular prevalence of Theileria in cattle, water buffaloes, goats and sheep from nine provinces in China."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Animals and blood collection Between 2007 and 2013, whole blood samples (around 6 ml) were collected in EDTA from apparently healthy cattle (n = 1,235), water buffaloes (29), goats (270) and sheep (111) from 9 provinces/municipality of China (Table 1). The Bos primigenius (p.) taurus studied (n = 975) were Holsteins, Simmentals, Bohai blacks, Luxis and Wannans while the Bos. p. indicus (n = 260) were the Yunlings, Minnans, and Leiqiongs (Table 1). The water buffaloes, goats and sheep in the study were bred in China and were indigenous breeds. Gender information was available for cattle from Yunnan province. After collection, the blood samples were frozen at -20°C and shipped on ice (over 2 days) to Yangzhou University where they were frozen at -80°C until thawed at room temperature for DNA extraction as described below. This study was reviewed and approved by the Institutional Animal Care and Use Committee of Yangzhou University and animal owners gave written permissions for blood collection.Table 1\nMolecular prevalence of\nTheileria\nspp. in cattle, water buffalo, goat and sheep\nAnimal speciesSubspecies /breedProvinceCityCoordinate of city\nTheileriapositivitypositive /total\n%\nCattle (n = 1235)\nBos p. taurus\nSimmentalInner MongoliaChifeng42.17°N, 118.58°E19/13214.4%Bohai blackShandongBinzhou37.22°N, 118.02°E4/666.1%LuxiShandongJining35.23°N, 116.33°E40/40100%HolsteinJiangsuYancheng33.22°N, 120.08°E72/32122.4%HolsteinJiangsuYangzhou32.23°N, 119.26°E17/14411.8%HolsteinShanghaiShanghai31.14°N, 121.29°E9/2553.5%WannanAnhuiWuhu31.19°N, 118.22°E17/17100%\nBos p. indicus\nYunlingYunnanKunming25.04°N, 102.42°E124/16177.0%MinnanFujianPutian24.26°N, 119.01°E4/2516.0%LeiqiongHainanHaikou20.02°N, 110.20°E74/74100%Water buffalo (n = 29)HaiziJiangsuYancheng33.22°N, 120.08°E0/290%Goat (n = 270)XinjiangXinjiangUrumqi43.45°N, 87.36°E4/984.1%Yangtse River Delta WhiteJiangsuYangzhou32.23°N, 119.26°E116/17267.4%Sheep (n = 111)WurankeInner MongoliaXilingol43.57°N, 116.03°E36/7250.0%Sishui FurShandongJining35.23°N, 116.33°E23/3959.0%\n\nMolecular prevalence of\nTheileria\nspp. in cattle, water buffalo, goat and sheep\n\nBetween 2007 and 2013, whole blood samples (around 6 ml) were collected in EDTA from apparently healthy cattle (n = 1,235), water buffaloes (29), goats (270) and sheep (111) from 9 provinces/municipality of China (Table 1). The Bos primigenius (p.) taurus studied (n = 975) were Holsteins, Simmentals, Bohai blacks, Luxis and Wannans while the Bos. p. indicus (n = 260) were the Yunlings, Minnans, and Leiqiongs (Table 1). The water buffaloes, goats and sheep in the study were bred in China and were indigenous breeds. Gender information was available for cattle from Yunnan province. After collection, the blood samples were frozen at -20°C and shipped on ice (over 2 days) to Yangzhou University where they were frozen at -80°C until thawed at room temperature for DNA extraction as described below. This study was reviewed and approved by the Institutional Animal Care and Use Committee of Yangzhou University and animal owners gave written permissions for blood collection.Table 1\nMolecular prevalence of\nTheileria\nspp. in cattle, water buffalo, goat and sheep\nAnimal speciesSubspecies /breedProvinceCityCoordinate of city\nTheileriapositivitypositive /total\n%\nCattle (n = 1235)\nBos p. taurus\nSimmentalInner MongoliaChifeng42.17°N, 118.58°E19/13214.4%Bohai blackShandongBinzhou37.22°N, 118.02°E4/666.1%LuxiShandongJining35.23°N, 116.33°E40/40100%HolsteinJiangsuYancheng33.22°N, 120.08°E72/32122.4%HolsteinJiangsuYangzhou32.23°N, 119.26°E17/14411.8%HolsteinShanghaiShanghai31.14°N, 121.29°E9/2553.5%WannanAnhuiWuhu31.19°N, 118.22°E17/17100%\nBos p. indicus\nYunlingYunnanKunming25.04°N, 102.42°E124/16177.0%MinnanFujianPutian24.26°N, 119.01°E4/2516.0%LeiqiongHainanHaikou20.02°N, 110.20°E74/74100%Water buffalo (n = 29)HaiziJiangsuYancheng33.22°N, 120.08°E0/290%Goat (n = 270)XinjiangXinjiangUrumqi43.45°N, 87.36°E4/984.1%Yangtse River Delta WhiteJiangsuYangzhou32.23°N, 119.26°E116/17267.4%Sheep (n = 111)WurankeInner MongoliaXilingol43.57°N, 116.03°E36/7250.0%Sishui FurShandongJining35.23°N, 116.33°E23/3959.0%\n\nMolecular prevalence of\nTheileria\nspp. in cattle, water buffalo, goat and sheep\n\n DNA extraction DNA was extracted from whole blood samples using a standard phenol-chloroform method previously described [28]. Two ml whole blood was used to extract DNA which was resuspended into 200 μl 1 × T10E0.1 buffer. The concentration of the extracted DNA was established with a Microscale Ultraviolet Spectrophotometer. Negative controls consisting of sterile molecular grade water were used to detect cross- contamination during DNA extraction and processing. The HMBS-based FRET-PCR was performed to verify if the extracted DNAs from blood samples were appropriate for molecular detection of tick-borne pathogens [29, 30].\nDNA was extracted from whole blood samples using a standard phenol-chloroform method previously described [28]. Two ml whole blood was used to extract DNA which was resuspended into 200 μl 1 × T10E0.1 buffer. The concentration of the extracted DNA was established with a Microscale Ultraviolet Spectrophotometer. Negative controls consisting of sterile molecular grade water were used to detect cross- contamination during DNA extraction and processing. The HMBS-based FRET-PCR was performed to verify if the extracted DNAs from blood samples were appropriate for molecular detection of tick-borne pathogens [29, 30].\n Theileriaspp.FRET-qPCR Primers and probes The 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\nThe 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n Thermal cycling The Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\nThe Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\n Specificity PCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\nPCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\n Sensitivity For use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\nFor use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\n Primers and probes The 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\nThe 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n Thermal cycling The Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\nThe Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\n Specificity PCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\nPCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\n Sensitivity For use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\nFor use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\n Identification of Theileriaspp. by PCR and sequencing The amplicon of the pan-Theileria FRET-qPCR we established has a sequence which is highly conserved among the different Theileria species. To differentiate Theileria spp. in a positive reaction, we used a standard PCR to amplify a highly polymorphic region of the 18S rRNA gene (591–594 nucleotides for different Theileria spp.) and sequenced the products (GenScript, Nanjing, Jiangsu, China). For the PCR we designed a forward primer (5′-CCTGAGAAACGGCTACCACATCT-3′) that amplified all Theileria species and used a previously described reverse primer (5′-GGACTACGACGGTATCTGATCG-3′) that also amplified all species [31].\nThe amplicon of the pan-Theileria FRET-qPCR we established has a sequence which is highly conserved among the different Theileria species. To differentiate Theileria spp. in a positive reaction, we used a standard PCR to amplify a highly polymorphic region of the 18S rRNA gene (591–594 nucleotides for different Theileria spp.) and sequenced the products (GenScript, Nanjing, Jiangsu, China). For the PCR we designed a forward primer (5′-CCTGAGAAACGGCTACCACATCT-3′) that amplified all Theileria species and used a previously described reverse primer (5′-GGACTACGACGGTATCTGATCG-3′) that also amplified all species [31].\n Statistical analysis Differences in positivity of Theileria spp. were analyzed by Chi-squared Test while numbers of copies of the Theileria 18S rRNA gene determined in the Theileria FRET-qPCR were log10-transformed and analyzed using the Student’s T-test. Differences of P < 0.05 were considered statistically significant.\nDifferences in positivity of Theileria spp. were analyzed by Chi-squared Test while numbers of copies of the Theileria 18S rRNA gene determined in the Theileria FRET-qPCR were log10-transformed and analyzed using the Student’s T-test. Differences of P < 0.05 were considered statistically significant."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Development of the pan-TheileriaFRET-PCR Comparison of the sequences in the highly conserved region of the Theileria spp. we used showed the region is highly conserved, but is substantially different from those in closely related protozoan species (Figure 1). The two primers and two probes we chose for the pan-Theileria FRET-qPCR had 0–4 nucleotide mismatches with the Theileria spp. in GenBank, but had 25, 23, 22, 8, 16 and 15 mismatches with B. bovis, B. divergens, B. bigemina, C. felis, H. americanum and T. gondii, respectively (Figure 1). The specificity of the pan-Theileria FRET-PCR was further confirmed when it gave positive reactions with the T. orientalis control, but gave negative reactions with DNAs of B. canis, C. felis, H. americanum and T. gondii. The pan-Theileria FRET-qPCR had a specific melting curve (Tm 57.5°C) with Theileria spp. DNA. Using the gel-purified PCR products as quantitative standards, we determined the detection limit of the pan-Theileria FRET-qPCR was 2 copies of the Theileria 18S rRNA gene per reaction for T. orientalis, T. sergenti and T. luwenshuni, T. buffeli and T. ovis.\nComparison of the sequences in the highly conserved region of the Theileria spp. we used showed the region is highly conserved, but is substantially different from those in closely related protozoan species (Figure 1). The two primers and two probes we chose for the pan-Theileria FRET-qPCR had 0–4 nucleotide mismatches with the Theileria spp. in GenBank, but had 25, 23, 22, 8, 16 and 15 mismatches with B. bovis, B. divergens, B. bigemina, C. felis, H. americanum and T. gondii, respectively (Figure 1). The specificity of the pan-Theileria FRET-PCR was further confirmed when it gave positive reactions with the T. orientalis control, but gave negative reactions with DNAs of B. canis, C. felis, H. americanum and T. gondii. The pan-Theileria FRET-qPCR had a specific melting curve (Tm 57.5°C) with Theileria spp. DNA. Using the gel-purified PCR products as quantitative standards, we determined the detection limit of the pan-Theileria FRET-qPCR was 2 copies of the Theileria 18S rRNA gene per reaction for T. orientalis, T. sergenti and T. luwenshuni, T. buffeli and T. ovis.\n Prevalence of Theileriaspp. DNA in ruminants Animals positive for Theileria were found in each of the nine provinces sampled with several animals of each species being positive at each location, except in the case of water buffaloes which were all negative in the one site they were studied. The overall prevalences of Theileria spp. DNA in sheep (53.2%; 59/111) and goats (44.4%; 120/270) were significantly higher than in cattle (30.8%; 380/1,235) (two-tailed Chi-squared Test, P < 10-4). The pan-Theileria FRET-PCR showed that sheep had an average of 102.4 copies of Theileria 18S rRNA/ml whole blood which was significantly lower than the 104.3 copies in cattle and 105.8 copies in goats (Student’s t Test, P < 10-4). While the prevalence of Theileria spp. DNA varied greatly from 3.5% (9/255) in Holsteins from Shanghai to 100% in Luxi cattle from Shandong (40/40) and Leiqiong cattle from Hainan (74/74), the prevalence did not differ significantly in sheep from Inner Mongolia and Shandong (Table 1, Figure 2).Figure 2\nSites in China where samples ruminants were tested for\nTheileria\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\n\nSites in China where samples ruminants were tested for\nTheileria\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\nSequencing of 87 randomly selected amplicons (52 from cattle, 14 from goats and 21 from sheep) from Theileria DNA positive samples showed that T. orientalis/ T. sergenti/T. buffeli group [29] were present in cattle while T. luwenshuni was found in goats in Jiangsu province and T. ovis and T. luwenshuni in sheep from Inner Mongolia and Jiangsu province, respectively.\nAnimals positive for Theileria were found in each of the nine provinces sampled with several animals of each species being positive at each location, except in the case of water buffaloes which were all negative in the one site they were studied. The overall prevalences of Theileria spp. DNA in sheep (53.2%; 59/111) and goats (44.4%; 120/270) were significantly higher than in cattle (30.8%; 380/1,235) (two-tailed Chi-squared Test, P < 10-4). The pan-Theileria FRET-PCR showed that sheep had an average of 102.4 copies of Theileria 18S rRNA/ml whole blood which was significantly lower than the 104.3 copies in cattle and 105.8 copies in goats (Student’s t Test, P < 10-4). While the prevalence of Theileria spp. DNA varied greatly from 3.5% (9/255) in Holsteins from Shanghai to 100% in Luxi cattle from Shandong (40/40) and Leiqiong cattle from Hainan (74/74), the prevalence did not differ significantly in sheep from Inner Mongolia and Shandong (Table 1, Figure 2).Figure 2\nSites in China where samples ruminants were tested for\nTheileria\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\n\nSites in China where samples ruminants were tested for\nTheileria\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\nSequencing of 87 randomly selected amplicons (52 from cattle, 14 from goats and 21 from sheep) from Theileria DNA positive samples showed that T. orientalis/ T. sergenti/T. buffeli group [29] were present in cattle while T. luwenshuni was found in goats in Jiangsu province and T. ovis and T. luwenshuni in sheep from Inner Mongolia and Jiangsu province, respectively.\n Factors associated with the occurrence of theileriosis in cattle When we analyzed factors that might be associated with the prevalence of Theileria spp. DNA in cattle we found that Bos p. indicus animals had significantly higher positivity (77.7% vs. 18.3%; P < 10-4) and copy number of the Theileria 18S rRNA gene (104.81 vs. 103.73 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. Similarly, Bos p. indicus cattle were more likely to be positive (65.2% vs. 13.7%; P < 10-4) and have higher copy numbers of the Theileria 18S rRNA gene (104.88 vs. 103.00 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. The cattle from southern China had significantly higher Theileria 18S rRNA gene copy numbers (104.39 vs. 103.87 copies/per ml whole blood; P = 0.02) than those from northern China but this difference in the prevalence was not significant (31.8% vs. 26.5%). In cattle from Yunnan province where gender information was available, female animals were more commonly positive (76.7% vs. 41.3%; P < 10-4) and copy numbers (105.02 vs. 102.93Theileria/per ml whole blood; P < 10-4) than males.\nWhen we analyzed factors that might be associated with the prevalence of Theileria spp. DNA in cattle we found that Bos p. indicus animals had significantly higher positivity (77.7% vs. 18.3%; P < 10-4) and copy number of the Theileria 18S rRNA gene (104.81 vs. 103.73 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. Similarly, Bos p. indicus cattle were more likely to be positive (65.2% vs. 13.7%; P < 10-4) and have higher copy numbers of the Theileria 18S rRNA gene (104.88 vs. 103.00 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. The cattle from southern China had significantly higher Theileria 18S rRNA gene copy numbers (104.39 vs. 103.87 copies/per ml whole blood; P = 0.02) than those from northern China but this difference in the prevalence was not significant (31.8% vs. 26.5%). In cattle from Yunnan province where gender information was available, female animals were more commonly positive (76.7% vs. 41.3%; P < 10-4) and copy numbers (105.02 vs. 102.93Theileria/per ml whole blood; P < 10-4) than males.\n Gene accession numbers The Theileria rRNA nucleotide sequences obtained in this study that were not identical to existing entries in GenBank were deposited with the following gene accession numbers: KJ850933 and KJ850938 (T. sergenti); KJ850936 and KJ850940 (T. buffeli); KJ850934, KJ850937, KJ850943, KJ850939 and KJ850941 (T. orientalis); KJ850942 (T. ovis); KJ850935 and KM016463 (T. luwenshuni). The sequences obtained were very similar (0–4 nucleotide mismatches) to Theileria spp. sequences deposited by other laboratories in China, USA, France, Australia and Iran (Table 2).Table 2\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\nIsolates identified in this studyHighly similar sequences in GenBank\nTheileriaspp.Gene accession #Source/originGene accession #Source/originMismatches\nT. orientalis\nKJ850934Simmental cattle, Inner MongoliaAP011948Cattle, Shintoku of Japan0/547KJ850939Luxi cattle, ShandongHM538220Cattle, Suizhou of China0/547KJ850937Yunling cattle, YunnanAB520956Cattle, New South Wales of Australia2/491KJ850941Yunling cattle, YunnanAB520955Cattle, Raymond of Australia0/509KJ850943Holstein cattle, JiangsuAB520956Cattle, New South Wales of Australia0/549\nT. sergenti\nKJ850933Australian Holstein cattle, JiangsuJQ723015Cattle, Hunan of China0/541KJ850938Yunling cattle, YunnanJQ723015Cattle, Hunan of China0/492\nT. buffeli\nKJ850936Leiqiong cattle, HainanHM538196Cattle, Hubei of China;1/508KJ850940Wannan cattle, AnhuiAY661513Cattle, USA0/545\nT. ovis\nKJ850942Wuranke sheep, Inner MongoliaFJ603460Sheep, Xinjiang of China;0/529\nT. luwenshuni\nKJ850935from Sishui-Fur sheep, ShandongKC769996, JX469518, JF719831Sheep, China0/549KM016463Yangtse River Delta White goat, JiangsuKC769997Goat, Beijing of China0/571\n\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\n\nThe Theileria rRNA nucleotide sequences obtained in this study that were not identical to existing entries in GenBank were deposited with the following gene accession numbers: KJ850933 and KJ850938 (T. sergenti); KJ850936 and KJ850940 (T. buffeli); KJ850934, KJ850937, KJ850943, KJ850939 and KJ850941 (T. orientalis); KJ850942 (T. ovis); KJ850935 and KM016463 (T. luwenshuni). The sequences obtained were very similar (0–4 nucleotide mismatches) to Theileria spp. sequences deposited by other laboratories in China, USA, France, Australia and Iran (Table 2).Table 2\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\nIsolates identified in this studyHighly similar sequences in GenBank\nTheileriaspp.Gene accession #Source/originGene accession #Source/originMismatches\nT. orientalis\nKJ850934Simmental cattle, Inner MongoliaAP011948Cattle, Shintoku of Japan0/547KJ850939Luxi cattle, ShandongHM538220Cattle, Suizhou of China0/547KJ850937Yunling cattle, YunnanAB520956Cattle, New South Wales of Australia2/491KJ850941Yunling cattle, YunnanAB520955Cattle, Raymond of Australia0/509KJ850943Holstein cattle, JiangsuAB520956Cattle, New South Wales of Australia0/549\nT. sergenti\nKJ850933Australian Holstein cattle, JiangsuJQ723015Cattle, Hunan of China0/541KJ850938Yunling cattle, YunnanJQ723015Cattle, Hunan of China0/492\nT. buffeli\nKJ850936Leiqiong cattle, HainanHM538196Cattle, Hubei of China;1/508KJ850940Wannan cattle, AnhuiAY661513Cattle, USA0/545\nT. ovis\nKJ850942Wuranke sheep, Inner MongoliaFJ603460Sheep, Xinjiang of China;0/529\nT. luwenshuni\nKJ850935from Sishui-Fur sheep, ShandongKC769996, JX469518, JF719831Sheep, China0/549KM016463Yangtse River Delta White goat, JiangsuKC769997Goat, Beijing of China0/571\n\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\n"},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"In summary, our study has described the development and testing of a FRET-PCR which can detect recognized Theileria spp. The pan-Theileria FRET-PCR should be a useful diagnostic tool as it will enable diagnostic laboratories to detect infections in all domestic species with a single test."},"other_sections_titles":{"kind":"list like","value":["Background","Animals and blood collection","DNA extraction","Theileriaspp.FRET-qPCR","Primers and probes","Thermal cycling","Specificity","Sensitivity","Identification of Theileriaspp. by PCR and sequencing","Statistical analysis","Development of the pan-TheileriaFRET-PCR","Prevalence of Theileriaspp. DNA in ruminants","Factors associated with the occurrence of theileriosis in cattle","Gene accession numbers"],"string":"[\n \"Background\",\n \"Animals and blood collection\",\n \"DNA extraction\",\n \"Theileriaspp.FRET-qPCR\",\n \"Primers and probes\",\n \"Thermal cycling\",\n \"Specificity\",\n \"Sensitivity\",\n \"Identification of Theileriaspp. by PCR and sequencing\",\n \"Statistical analysis\",\n \"Development of the pan-TheileriaFRET-PCR\",\n \"Prevalence of Theileriaspp. DNA in ruminants\",\n \"Factors associated with the occurrence of theileriosis in cattle\",\n \"Gene accession numbers\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Theileria spp. are tick-transmitted, intracellular protozoan parasites infecting leukocytes and erythrocytes of wild and domestic large and small ruminants. Several Theileria spp., transmitted by ixodid ticks of the genera Rhipicephalus, Hyalomma, Amblyomma and Haemaphysalis, have been described in cattle, water buffaloes, sheep and goats in different geographical zones of the world [1–5]. Theileriosis is primarily limited to tropical and sub-tropical areas of the world, with infections mainly reported in Africa and the Middle East but also in southern Europe and northern Asia [6–11]. Infections by Theileria spp. can cause fever, anemia and hemoglobinuria and, in severe cases, death although many species are benign. Animals recovered from acute or primary infections usually remain persistently infected and may act as reservoirs of infecting ticks [12, 13].\nWhile there have been numerous reports of theileriosis in various animal species in China since 1958 [14–27], many have been reported in Chinese and some were based on microscopic detection of parasites which can be difficult with low parasitemia and does not allow ready differentiation of species. Serological studies, although sensitive and easy to perform, are not specific as there is cross reactivity between Theileria spp. Although molecular studies have been performed, these have been to detect Theileria of specific domestic animal species, for example sheep and goats [27]. There have been no highly sensitive and specific molecular methods described which enable studies on various animals from widely divergent areas of China where different Theileria spp. might occur. To address this problem, we developed and validated a highly sensitive genus-specific Theileria FRET-qPCR that detects the recognized Theileria spp. of domestic animals and investigated the molecular prevalence of Theileria in cattle, water buffaloes, goats and sheep from nine provinces in China.","Between 2007 and 2013, whole blood samples (around 6 ml) were collected in EDTA from apparently healthy cattle (n = 1,235), water buffaloes (29), goats (270) and sheep (111) from 9 provinces/municipality of China (Table 1). The Bos primigenius (p.) taurus studied (n = 975) were Holsteins, Simmentals, Bohai blacks, Luxis and Wannans while the Bos. p. indicus (n = 260) were the Yunlings, Minnans, and Leiqiongs (Table 1). The water buffaloes, goats and sheep in the study were bred in China and were indigenous breeds. Gender information was available for cattle from Yunnan province. After collection, the blood samples were frozen at -20°C and shipped on ice (over 2 days) to Yangzhou University where they were frozen at -80°C until thawed at room temperature for DNA extraction as described below. This study was reviewed and approved by the Institutional Animal Care and Use Committee of Yangzhou University and animal owners gave written permissions for blood collection.Table 1\nMolecular prevalence of\nTheileria\nspp. in cattle, water buffalo, goat and sheep\nAnimal speciesSubspecies /breedProvinceCityCoordinate of city\nTheileriapositivitypositive /total\n%\nCattle (n = 1235)\nBos p. taurus\nSimmentalInner MongoliaChifeng42.17°N, 118.58°E19/13214.4%Bohai blackShandongBinzhou37.22°N, 118.02°E4/666.1%LuxiShandongJining35.23°N, 116.33°E40/40100%HolsteinJiangsuYancheng33.22°N, 120.08°E72/32122.4%HolsteinJiangsuYangzhou32.23°N, 119.26°E17/14411.8%HolsteinShanghaiShanghai31.14°N, 121.29°E9/2553.5%WannanAnhuiWuhu31.19°N, 118.22°E17/17100%\nBos p. indicus\nYunlingYunnanKunming25.04°N, 102.42°E124/16177.0%MinnanFujianPutian24.26°N, 119.01°E4/2516.0%LeiqiongHainanHaikou20.02°N, 110.20°E74/74100%Water buffalo (n = 29)HaiziJiangsuYancheng33.22°N, 120.08°E0/290%Goat (n = 270)XinjiangXinjiangUrumqi43.45°N, 87.36°E4/984.1%Yangtse River Delta WhiteJiangsuYangzhou32.23°N, 119.26°E116/17267.4%Sheep (n = 111)WurankeInner MongoliaXilingol43.57°N, 116.03°E36/7250.0%Sishui FurShandongJining35.23°N, 116.33°E23/3959.0%\n\nMolecular prevalence of\nTheileria\nspp. in cattle, water buffalo, goat and sheep\n","DNA was extracted from whole blood samples using a standard phenol-chloroform method previously described [28]. Two ml whole blood was used to extract DNA which was resuspended into 200 μl 1 × T10E0.1 buffer. The concentration of the extracted DNA was established with a Microscale Ultraviolet Spectrophotometer. Negative controls consisting of sterile molecular grade water were used to detect cross- contamination during DNA extraction and processing. The HMBS-based FRET-PCR was performed to verify if the extracted DNAs from blood samples were appropriate for molecular detection of tick-borne pathogens [29, 30]."," Primers and probes The 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\nThe 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n Thermal cycling The Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\nThe Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\n Specificity PCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\nPCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\n Sensitivity For use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\nFor use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.","The 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.","The Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.","PCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).","For use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.","The amplicon of the pan-Theileria FRET-qPCR we established has a sequence which is highly conserved among the different Theileria species. To differentiate Theileria spp. in a positive reaction, we used a standard PCR to amplify a highly polymorphic region of the 18S rRNA gene (591–594 nucleotides for different Theileria spp.) and sequenced the products (GenScript, Nanjing, Jiangsu, China). For the PCR we designed a forward primer (5′-CCTGAGAAACGGCTACCACATCT-3′) that amplified all Theileria species and used a previously described reverse primer (5′-GGACTACGACGGTATCTGATCG-3′) that also amplified all species [31].","Differences in positivity of Theileria spp. were analyzed by Chi-squared Test while numbers of copies of the Theileria 18S rRNA gene determined in the Theileria FRET-qPCR were log10-transformed and analyzed using the Student’s T-test. Differences of P < 0.05 were considered statistically significant.","Comparison of the sequences in the highly conserved region of the Theileria spp. we used showed the region is highly conserved, but is substantially different from those in closely related protozoan species (Figure 1). The two primers and two probes we chose for the pan-Theileria FRET-qPCR had 0–4 nucleotide mismatches with the Theileria spp. in GenBank, but had 25, 23, 22, 8, 16 and 15 mismatches with B. bovis, B. divergens, B. bigemina, C. felis, H. americanum and T. gondii, respectively (Figure 1). The specificity of the pan-Theileria FRET-PCR was further confirmed when it gave positive reactions with the T. orientalis control, but gave negative reactions with DNAs of B. canis, C. felis, H. americanum and T. gondii. The pan-Theileria FRET-qPCR had a specific melting curve (Tm 57.5°C) with Theileria spp. DNA. Using the gel-purified PCR products as quantitative standards, we determined the detection limit of the pan-Theileria FRET-qPCR was 2 copies of the Theileria 18S rRNA gene per reaction for T. orientalis, T. sergenti and T. luwenshuni, T. buffeli and T. ovis.","Animals positive for Theileria were found in each of the nine provinces sampled with several animals of each species being positive at each location, except in the case of water buffaloes which were all negative in the one site they were studied. The overall prevalences of Theileria spp. DNA in sheep (53.2%; 59/111) and goats (44.4%; 120/270) were significantly higher than in cattle (30.8%; 380/1,235) (two-tailed Chi-squared Test, P < 10-4). The pan-Theileria FRET-PCR showed that sheep had an average of 102.4 copies of Theileria 18S rRNA/ml whole blood which was significantly lower than the 104.3 copies in cattle and 105.8 copies in goats (Student’s t Test, P < 10-4). While the prevalence of Theileria spp. DNA varied greatly from 3.5% (9/255) in Holsteins from Shanghai to 100% in Luxi cattle from Shandong (40/40) and Leiqiong cattle from Hainan (74/74), the prevalence did not differ significantly in sheep from Inner Mongolia and Shandong (Table 1, Figure 2).Figure 2\nSites in China where samples ruminants were tested for\nTheileria\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\n\nSites in China where samples ruminants were tested for\nTheileria\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\nSequencing of 87 randomly selected amplicons (52 from cattle, 14 from goats and 21 from sheep) from Theileria DNA positive samples showed that T. orientalis/ T. sergenti/T. buffeli group [29] were present in cattle while T. luwenshuni was found in goats in Jiangsu province and T. ovis and T. luwenshuni in sheep from Inner Mongolia and Jiangsu province, respectively.","When we analyzed factors that might be associated with the prevalence of Theileria spp. DNA in cattle we found that Bos p. indicus animals had significantly higher positivity (77.7% vs. 18.3%; P < 10-4) and copy number of the Theileria 18S rRNA gene (104.81 vs. 103.73 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. Similarly, Bos p. indicus cattle were more likely to be positive (65.2% vs. 13.7%; P < 10-4) and have higher copy numbers of the Theileria 18S rRNA gene (104.88 vs. 103.00 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. The cattle from southern China had significantly higher Theileria 18S rRNA gene copy numbers (104.39 vs. 103.87 copies/per ml whole blood; P = 0.02) than those from northern China but this difference in the prevalence was not significant (31.8% vs. 26.5%). In cattle from Yunnan province where gender information was available, female animals were more commonly positive (76.7% vs. 41.3%; P < 10-4) and copy numbers (105.02 vs. 102.93Theileria/per ml whole blood; P < 10-4) than males.","The Theileria rRNA nucleotide sequences obtained in this study that were not identical to existing entries in GenBank were deposited with the following gene accession numbers: KJ850933 and KJ850938 (T. sergenti); KJ850936 and KJ850940 (T. buffeli); KJ850934, KJ850937, KJ850943, KJ850939 and KJ850941 (T. orientalis); KJ850942 (T. ovis); KJ850935 and KM016463 (T. luwenshuni). The sequences obtained were very similar (0–4 nucleotide mismatches) to Theileria spp. sequences deposited by other laboratories in China, USA, France, Australia and Iran (Table 2).Table 2\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\nIsolates identified in this studyHighly similar sequences in GenBank\nTheileriaspp.Gene accession #Source/originGene accession #Source/originMismatches\nT. orientalis\nKJ850934Simmental cattle, Inner MongoliaAP011948Cattle, Shintoku of Japan0/547KJ850939Luxi cattle, ShandongHM538220Cattle, Suizhou of China0/547KJ850937Yunling cattle, YunnanAB520956Cattle, New South Wales of Australia2/491KJ850941Yunling cattle, YunnanAB520955Cattle, Raymond of Australia0/509KJ850943Holstein cattle, JiangsuAB520956Cattle, New South Wales of Australia0/549\nT. sergenti\nKJ850933Australian Holstein cattle, JiangsuJQ723015Cattle, Hunan of China0/541KJ850938Yunling cattle, YunnanJQ723015Cattle, Hunan of China0/492\nT. buffeli\nKJ850936Leiqiong cattle, HainanHM538196Cattle, Hubei of China;1/508KJ850940Wannan cattle, AnhuiAY661513Cattle, USA0/545\nT. ovis\nKJ850942Wuranke sheep, Inner MongoliaFJ603460Sheep, Xinjiang of China;0/529\nT. luwenshuni\nKJ850935from Sishui-Fur sheep, ShandongKC769996, JX469518, JF719831Sheep, China0/549KM016463Yangtse River Delta White goat, JiangsuKC769997Goat, Beijing of China0/571\n\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\n"],"string":"[\n \"Theileria spp. are tick-transmitted, intracellular protozoan parasites infecting leukocytes and erythrocytes of wild and domestic large and small ruminants. Several Theileria spp., transmitted by ixodid ticks of the genera Rhipicephalus, Hyalomma, Amblyomma and Haemaphysalis, have been described in cattle, water buffaloes, sheep and goats in different geographical zones of the world [1–5]. Theileriosis is primarily limited to tropical and sub-tropical areas of the world, with infections mainly reported in Africa and the Middle East but also in southern Europe and northern Asia [6–11]. Infections by Theileria spp. can cause fever, anemia and hemoglobinuria and, in severe cases, death although many species are benign. Animals recovered from acute or primary infections usually remain persistently infected and may act as reservoirs of infecting ticks [12, 13].\\nWhile there have been numerous reports of theileriosis in various animal species in China since 1958 [14–27], many have been reported in Chinese and some were based on microscopic detection of parasites which can be difficult with low parasitemia and does not allow ready differentiation of species. Serological studies, although sensitive and easy to perform, are not specific as there is cross reactivity between Theileria spp. Although molecular studies have been performed, these have been to detect Theileria of specific domestic animal species, for example sheep and goats [27]. There have been no highly sensitive and specific molecular methods described which enable studies on various animals from widely divergent areas of China where different Theileria spp. might occur. To address this problem, we developed and validated a highly sensitive genus-specific Theileria FRET-qPCR that detects the recognized Theileria spp. of domestic animals and investigated the molecular prevalence of Theileria in cattle, water buffaloes, goats and sheep from nine provinces in China.\",\n \"Between 2007 and 2013, whole blood samples (around 6 ml) were collected in EDTA from apparently healthy cattle (n = 1,235), water buffaloes (29), goats (270) and sheep (111) from 9 provinces/municipality of China (Table 1). The Bos primigenius (p.) taurus studied (n = 975) were Holsteins, Simmentals, Bohai blacks, Luxis and Wannans while the Bos. p. indicus (n = 260) were the Yunlings, Minnans, and Leiqiongs (Table 1). The water buffaloes, goats and sheep in the study were bred in China and were indigenous breeds. Gender information was available for cattle from Yunnan province. After collection, the blood samples were frozen at -20°C and shipped on ice (over 2 days) to Yangzhou University where they were frozen at -80°C until thawed at room temperature for DNA extraction as described below. This study was reviewed and approved by the Institutional Animal Care and Use Committee of Yangzhou University and animal owners gave written permissions for blood collection.Table 1\\nMolecular prevalence of\\nTheileria\\nspp. in cattle, water buffalo, goat and sheep\\nAnimal speciesSubspecies /breedProvinceCityCoordinate of city\\nTheileriapositivitypositive /total\\n%\\nCattle (n = 1235)\\nBos p. taurus\\nSimmentalInner MongoliaChifeng42.17°N, 118.58°E19/13214.4%Bohai blackShandongBinzhou37.22°N, 118.02°E4/666.1%LuxiShandongJining35.23°N, 116.33°E40/40100%HolsteinJiangsuYancheng33.22°N, 120.08°E72/32122.4%HolsteinJiangsuYangzhou32.23°N, 119.26°E17/14411.8%HolsteinShanghaiShanghai31.14°N, 121.29°E9/2553.5%WannanAnhuiWuhu31.19°N, 118.22°E17/17100%\\nBos p. indicus\\nYunlingYunnanKunming25.04°N, 102.42°E124/16177.0%MinnanFujianPutian24.26°N, 119.01°E4/2516.0%LeiqiongHainanHaikou20.02°N, 110.20°E74/74100%Water buffalo (n = 29)HaiziJiangsuYancheng33.22°N, 120.08°E0/290%Goat (n = 270)XinjiangXinjiangUrumqi43.45°N, 87.36°E4/984.1%Yangtse River Delta WhiteJiangsuYangzhou32.23°N, 119.26°E116/17267.4%Sheep (n = 111)WurankeInner MongoliaXilingol43.57°N, 116.03°E36/7250.0%Sishui FurShandongJining35.23°N, 116.33°E23/3959.0%\\n\\nMolecular prevalence of\\nTheileria\\nspp. in cattle, water buffalo, goat and sheep\\n\",\n \"DNA was extracted from whole blood samples using a standard phenol-chloroform method previously described [28]. Two ml whole blood was used to extract DNA which was resuspended into 200 μl 1 × T10E0.1 buffer. The concentration of the extracted DNA was established with a Microscale Ultraviolet Spectrophotometer. Negative controls consisting of sterile molecular grade water were used to detect cross- contamination during DNA extraction and processing. The HMBS-based FRET-PCR was performed to verify if the extracted DNAs from blood samples were appropriate for molecular detection of tick-borne pathogens [29, 30].\",\n \" Primers and probes The 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\\nAlignment of oligonucleotides for\\nTheileria\\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\\n\\nAlignment of oligonucleotides for\\nTheileria\\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\\nThe 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\\nAlignment of oligonucleotides for\\nTheileria\\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\\n\\nAlignment of oligonucleotides for\\nTheileria\\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\\n Thermal cycling The Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\\nThe Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\\n Specificity PCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\\nPCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\\n Sensitivity For use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\\nFor use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\",\n \"The 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\\nAlignment of oligonucleotides for\\nTheileria\\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\\n\\nAlignment of oligonucleotides for\\nTheileria\\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\",\n \"The Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\",\n \"PCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\",\n \"For use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\",\n \"The amplicon of the pan-Theileria FRET-qPCR we established has a sequence which is highly conserved among the different Theileria species. To differentiate Theileria spp. in a positive reaction, we used a standard PCR to amplify a highly polymorphic region of the 18S rRNA gene (591–594 nucleotides for different Theileria spp.) and sequenced the products (GenScript, Nanjing, Jiangsu, China). For the PCR we designed a forward primer (5′-CCTGAGAAACGGCTACCACATCT-3′) that amplified all Theileria species and used a previously described reverse primer (5′-GGACTACGACGGTATCTGATCG-3′) that also amplified all species [31].\",\n \"Differences in positivity of Theileria spp. were analyzed by Chi-squared Test while numbers of copies of the Theileria 18S rRNA gene determined in the Theileria FRET-qPCR were log10-transformed and analyzed using the Student’s T-test. Differences of P < 0.05 were considered statistically significant.\",\n \"Comparison of the sequences in the highly conserved region of the Theileria spp. we used showed the region is highly conserved, but is substantially different from those in closely related protozoan species (Figure 1). The two primers and two probes we chose for the pan-Theileria FRET-qPCR had 0–4 nucleotide mismatches with the Theileria spp. in GenBank, but had 25, 23, 22, 8, 16 and 15 mismatches with B. bovis, B. divergens, B. bigemina, C. felis, H. americanum and T. gondii, respectively (Figure 1). The specificity of the pan-Theileria FRET-PCR was further confirmed when it gave positive reactions with the T. orientalis control, but gave negative reactions with DNAs of B. canis, C. felis, H. americanum and T. gondii. The pan-Theileria FRET-qPCR had a specific melting curve (Tm 57.5°C) with Theileria spp. DNA. Using the gel-purified PCR products as quantitative standards, we determined the detection limit of the pan-Theileria FRET-qPCR was 2 copies of the Theileria 18S rRNA gene per reaction for T. orientalis, T. sergenti and T. luwenshuni, T. buffeli and T. ovis.\",\n \"Animals positive for Theileria were found in each of the nine provinces sampled with several animals of each species being positive at each location, except in the case of water buffaloes which were all negative in the one site they were studied. The overall prevalences of Theileria spp. DNA in sheep (53.2%; 59/111) and goats (44.4%; 120/270) were significantly higher than in cattle (30.8%; 380/1,235) (two-tailed Chi-squared Test, P < 10-4). The pan-Theileria FRET-PCR showed that sheep had an average of 102.4 copies of Theileria 18S rRNA/ml whole blood which was significantly lower than the 104.3 copies in cattle and 105.8 copies in goats (Student’s t Test, P < 10-4). While the prevalence of Theileria spp. DNA varied greatly from 3.5% (9/255) in Holsteins from Shanghai to 100% in Luxi cattle from Shandong (40/40) and Leiqiong cattle from Hainan (74/74), the prevalence did not differ significantly in sheep from Inner Mongolia and Shandong (Table 1, Figure 2).Figure 2\\nSites in China where samples ruminants were tested for\\nTheileria\\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\\n\\nSites in China where samples ruminants were tested for\\nTheileria\\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\\nSequencing of 87 randomly selected amplicons (52 from cattle, 14 from goats and 21 from sheep) from Theileria DNA positive samples showed that T. orientalis/ T. sergenti/T. buffeli group [29] were present in cattle while T. luwenshuni was found in goats in Jiangsu province and T. ovis and T. luwenshuni in sheep from Inner Mongolia and Jiangsu province, respectively.\",\n \"When we analyzed factors that might be associated with the prevalence of Theileria spp. DNA in cattle we found that Bos p. indicus animals had significantly higher positivity (77.7% vs. 18.3%; P < 10-4) and copy number of the Theileria 18S rRNA gene (104.81 vs. 103.73 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. Similarly, Bos p. indicus cattle were more likely to be positive (65.2% vs. 13.7%; P < 10-4) and have higher copy numbers of the Theileria 18S rRNA gene (104.88 vs. 103.00 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. The cattle from southern China had significantly higher Theileria 18S rRNA gene copy numbers (104.39 vs. 103.87 copies/per ml whole blood; P = 0.02) than those from northern China but this difference in the prevalence was not significant (31.8% vs. 26.5%). In cattle from Yunnan province where gender information was available, female animals were more commonly positive (76.7% vs. 41.3%; P < 10-4) and copy numbers (105.02 vs. 102.93Theileria/per ml whole blood; P < 10-4) than males.\",\n \"The Theileria rRNA nucleotide sequences obtained in this study that were not identical to existing entries in GenBank were deposited with the following gene accession numbers: KJ850933 and KJ850938 (T. sergenti); KJ850936 and KJ850940 (T. buffeli); KJ850934, KJ850937, KJ850943, KJ850939 and KJ850941 (T. orientalis); KJ850942 (T. ovis); KJ850935 and KM016463 (T. luwenshuni). The sequences obtained were very similar (0–4 nucleotide mismatches) to Theileria spp. sequences deposited by other laboratories in China, USA, France, Australia and Iran (Table 2).Table 2\\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\\nIsolates identified in this studyHighly similar sequences in GenBank\\nTheileriaspp.Gene accession #Source/originGene accession #Source/originMismatches\\nT. orientalis\\nKJ850934Simmental cattle, Inner MongoliaAP011948Cattle, Shintoku of Japan0/547KJ850939Luxi cattle, ShandongHM538220Cattle, Suizhou of China0/547KJ850937Yunling cattle, YunnanAB520956Cattle, New South Wales of Australia2/491KJ850941Yunling cattle, YunnanAB520955Cattle, Raymond of Australia0/509KJ850943Holstein cattle, JiangsuAB520956Cattle, New South Wales of Australia0/549\\nT. sergenti\\nKJ850933Australian Holstein cattle, JiangsuJQ723015Cattle, Hunan of China0/541KJ850938Yunling cattle, YunnanJQ723015Cattle, Hunan of China0/492\\nT. buffeli\\nKJ850936Leiqiong cattle, HainanHM538196Cattle, Hubei of China;1/508KJ850940Wannan cattle, AnhuiAY661513Cattle, USA0/545\\nT. ovis\\nKJ850942Wuranke sheep, Inner MongoliaFJ603460Sheep, Xinjiang of China;0/529\\nT. luwenshuni\\nKJ850935from Sishui-Fur sheep, ShandongKC769996, JX469518, JF719831Sheep, China0/549KM016463Yangtse River Delta White goat, JiangsuKC769997Goat, Beijing of China0/571\\n\\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\\n\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Animals and blood collection","DNA extraction","Theileriaspp.FRET-qPCR","Primers and probes","Thermal cycling","Specificity","Sensitivity","Identification of Theileriaspp. by PCR and sequencing","Statistical analysis","Results","Development of the pan-TheileriaFRET-PCR","Prevalence of Theileriaspp. DNA in ruminants","Factors associated with the occurrence of theileriosis in cattle","Gene accession numbers","Discussion","Conclusion"],"string":"[\n \"Background\",\n \"Methods\",\n \"Animals and blood collection\",\n \"DNA extraction\",\n \"Theileriaspp.FRET-qPCR\",\n \"Primers and probes\",\n \"Thermal cycling\",\n \"Specificity\",\n \"Sensitivity\",\n \"Identification of Theileriaspp. by PCR and sequencing\",\n \"Statistical analysis\",\n \"Results\",\n \"Development of the pan-TheileriaFRET-PCR\",\n \"Prevalence of Theileriaspp. DNA in ruminants\",\n \"Factors associated with the occurrence of theileriosis in cattle\",\n \"Gene accession numbers\",\n \"Discussion\",\n \"Conclusion\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Theileria spp. are tick-transmitted, intracellular protozoan parasites infecting leukocytes and erythrocytes of wild and domestic large and small ruminants. Several Theileria spp., transmitted by ixodid ticks of the genera Rhipicephalus, Hyalomma, Amblyomma and Haemaphysalis, have been described in cattle, water buffaloes, sheep and goats in different geographical zones of the world [1–5]. Theileriosis is primarily limited to tropical and sub-tropical areas of the world, with infections mainly reported in Africa and the Middle East but also in southern Europe and northern Asia [6–11]. Infections by Theileria spp. can cause fever, anemia and hemoglobinuria and, in severe cases, death although many species are benign. Animals recovered from acute or primary infections usually remain persistently infected and may act as reservoirs of infecting ticks [12, 13].\nWhile there have been numerous reports of theileriosis in various animal species in China since 1958 [14–27], many have been reported in Chinese and some were based on microscopic detection of parasites which can be difficult with low parasitemia and does not allow ready differentiation of species. Serological studies, although sensitive and easy to perform, are not specific as there is cross reactivity between Theileria spp. Although molecular studies have been performed, these have been to detect Theileria of specific domestic animal species, for example sheep and goats [27]. There have been no highly sensitive and specific molecular methods described which enable studies on various animals from widely divergent areas of China where different Theileria spp. might occur. To address this problem, we developed and validated a highly sensitive genus-specific Theileria FRET-qPCR that detects the recognized Theileria spp. of domestic animals and investigated the molecular prevalence of Theileria in cattle, water buffaloes, goats and sheep from nine provinces in China."," Animals and blood collection Between 2007 and 2013, whole blood samples (around 6 ml) were collected in EDTA from apparently healthy cattle (n = 1,235), water buffaloes (29), goats (270) and sheep (111) from 9 provinces/municipality of China (Table 1). The Bos primigenius (p.) taurus studied (n = 975) were Holsteins, Simmentals, Bohai blacks, Luxis and Wannans while the Bos. p. indicus (n = 260) were the Yunlings, Minnans, and Leiqiongs (Table 1). The water buffaloes, goats and sheep in the study were bred in China and were indigenous breeds. Gender information was available for cattle from Yunnan province. After collection, the blood samples were frozen at -20°C and shipped on ice (over 2 days) to Yangzhou University where they were frozen at -80°C until thawed at room temperature for DNA extraction as described below. This study was reviewed and approved by the Institutional Animal Care and Use Committee of Yangzhou University and animal owners gave written permissions for blood collection.Table 1\nMolecular prevalence of\nTheileria\nspp. in cattle, water buffalo, goat and sheep\nAnimal speciesSubspecies /breedProvinceCityCoordinate of city\nTheileriapositivitypositive /total\n%\nCattle (n = 1235)\nBos p. taurus\nSimmentalInner MongoliaChifeng42.17°N, 118.58°E19/13214.4%Bohai blackShandongBinzhou37.22°N, 118.02°E4/666.1%LuxiShandongJining35.23°N, 116.33°E40/40100%HolsteinJiangsuYancheng33.22°N, 120.08°E72/32122.4%HolsteinJiangsuYangzhou32.23°N, 119.26°E17/14411.8%HolsteinShanghaiShanghai31.14°N, 121.29°E9/2553.5%WannanAnhuiWuhu31.19°N, 118.22°E17/17100%\nBos p. indicus\nYunlingYunnanKunming25.04°N, 102.42°E124/16177.0%MinnanFujianPutian24.26°N, 119.01°E4/2516.0%LeiqiongHainanHaikou20.02°N, 110.20°E74/74100%Water buffalo (n = 29)HaiziJiangsuYancheng33.22°N, 120.08°E0/290%Goat (n = 270)XinjiangXinjiangUrumqi43.45°N, 87.36°E4/984.1%Yangtse River Delta WhiteJiangsuYangzhou32.23°N, 119.26°E116/17267.4%Sheep (n = 111)WurankeInner MongoliaXilingol43.57°N, 116.03°E36/7250.0%Sishui FurShandongJining35.23°N, 116.33°E23/3959.0%\n\nMolecular prevalence of\nTheileria\nspp. in cattle, water buffalo, goat and sheep\n\nBetween 2007 and 2013, whole blood samples (around 6 ml) were collected in EDTA from apparently healthy cattle (n = 1,235), water buffaloes (29), goats (270) and sheep (111) from 9 provinces/municipality of China (Table 1). The Bos primigenius (p.) taurus studied (n = 975) were Holsteins, Simmentals, Bohai blacks, Luxis and Wannans while the Bos. p. indicus (n = 260) were the Yunlings, Minnans, and Leiqiongs (Table 1). The water buffaloes, goats and sheep in the study were bred in China and were indigenous breeds. Gender information was available for cattle from Yunnan province. After collection, the blood samples were frozen at -20°C and shipped on ice (over 2 days) to Yangzhou University where they were frozen at -80°C until thawed at room temperature for DNA extraction as described below. This study was reviewed and approved by the Institutional Animal Care and Use Committee of Yangzhou University and animal owners gave written permissions for blood collection.Table 1\nMolecular prevalence of\nTheileria\nspp. in cattle, water buffalo, goat and sheep\nAnimal speciesSubspecies /breedProvinceCityCoordinate of city\nTheileriapositivitypositive /total\n%\nCattle (n = 1235)\nBos p. taurus\nSimmentalInner MongoliaChifeng42.17°N, 118.58°E19/13214.4%Bohai blackShandongBinzhou37.22°N, 118.02°E4/666.1%LuxiShandongJining35.23°N, 116.33°E40/40100%HolsteinJiangsuYancheng33.22°N, 120.08°E72/32122.4%HolsteinJiangsuYangzhou32.23°N, 119.26°E17/14411.8%HolsteinShanghaiShanghai31.14°N, 121.29°E9/2553.5%WannanAnhuiWuhu31.19°N, 118.22°E17/17100%\nBos p. indicus\nYunlingYunnanKunming25.04°N, 102.42°E124/16177.0%MinnanFujianPutian24.26°N, 119.01°E4/2516.0%LeiqiongHainanHaikou20.02°N, 110.20°E74/74100%Water buffalo (n = 29)HaiziJiangsuYancheng33.22°N, 120.08°E0/290%Goat (n = 270)XinjiangXinjiangUrumqi43.45°N, 87.36°E4/984.1%Yangtse River Delta WhiteJiangsuYangzhou32.23°N, 119.26°E116/17267.4%Sheep (n = 111)WurankeInner MongoliaXilingol43.57°N, 116.03°E36/7250.0%Sishui FurShandongJining35.23°N, 116.33°E23/3959.0%\n\nMolecular prevalence of\nTheileria\nspp. in cattle, water buffalo, goat and sheep\n\n DNA extraction DNA was extracted from whole blood samples using a standard phenol-chloroform method previously described [28]. Two ml whole blood was used to extract DNA which was resuspended into 200 μl 1 × T10E0.1 buffer. The concentration of the extracted DNA was established with a Microscale Ultraviolet Spectrophotometer. Negative controls consisting of sterile molecular grade water were used to detect cross- contamination during DNA extraction and processing. The HMBS-based FRET-PCR was performed to verify if the extracted DNAs from blood samples were appropriate for molecular detection of tick-borne pathogens [29, 30].\nDNA was extracted from whole blood samples using a standard phenol-chloroform method previously described [28]. Two ml whole blood was used to extract DNA which was resuspended into 200 μl 1 × T10E0.1 buffer. The concentration of the extracted DNA was established with a Microscale Ultraviolet Spectrophotometer. Negative controls consisting of sterile molecular grade water were used to detect cross- contamination during DNA extraction and processing. The HMBS-based FRET-PCR was performed to verify if the extracted DNAs from blood samples were appropriate for molecular detection of tick-borne pathogens [29, 30].\n Theileriaspp.FRET-qPCR Primers and probes The 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\nThe 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n Thermal cycling The Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\nThe Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\n Specificity PCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\nPCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\n Sensitivity For use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\nFor use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\n Primers and probes The 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\nThe 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n Thermal cycling The Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\nThe Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\n Specificity PCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\nPCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\n Sensitivity For use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\nFor use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\n Identification of Theileriaspp. by PCR and sequencing The amplicon of the pan-Theileria FRET-qPCR we established has a sequence which is highly conserved among the different Theileria species. To differentiate Theileria spp. in a positive reaction, we used a standard PCR to amplify a highly polymorphic region of the 18S rRNA gene (591–594 nucleotides for different Theileria spp.) and sequenced the products (GenScript, Nanjing, Jiangsu, China). For the PCR we designed a forward primer (5′-CCTGAGAAACGGCTACCACATCT-3′) that amplified all Theileria species and used a previously described reverse primer (5′-GGACTACGACGGTATCTGATCG-3′) that also amplified all species [31].\nThe amplicon of the pan-Theileria FRET-qPCR we established has a sequence which is highly conserved among the different Theileria species. To differentiate Theileria spp. in a positive reaction, we used a standard PCR to amplify a highly polymorphic region of the 18S rRNA gene (591–594 nucleotides for different Theileria spp.) and sequenced the products (GenScript, Nanjing, Jiangsu, China). For the PCR we designed a forward primer (5′-CCTGAGAAACGGCTACCACATCT-3′) that amplified all Theileria species and used a previously described reverse primer (5′-GGACTACGACGGTATCTGATCG-3′) that also amplified all species [31].\n Statistical analysis Differences in positivity of Theileria spp. were analyzed by Chi-squared Test while numbers of copies of the Theileria 18S rRNA gene determined in the Theileria FRET-qPCR were log10-transformed and analyzed using the Student’s T-test. Differences of P < 0.05 were considered statistically significant.\nDifferences in positivity of Theileria spp. were analyzed by Chi-squared Test while numbers of copies of the Theileria 18S rRNA gene determined in the Theileria FRET-qPCR were log10-transformed and analyzed using the Student’s T-test. Differences of P < 0.05 were considered statistically significant.","Between 2007 and 2013, whole blood samples (around 6 ml) were collected in EDTA from apparently healthy cattle (n = 1,235), water buffaloes (29), goats (270) and sheep (111) from 9 provinces/municipality of China (Table 1). The Bos primigenius (p.) taurus studied (n = 975) were Holsteins, Simmentals, Bohai blacks, Luxis and Wannans while the Bos. p. indicus (n = 260) were the Yunlings, Minnans, and Leiqiongs (Table 1). The water buffaloes, goats and sheep in the study were bred in China and were indigenous breeds. Gender information was available for cattle from Yunnan province. After collection, the blood samples were frozen at -20°C and shipped on ice (over 2 days) to Yangzhou University where they were frozen at -80°C until thawed at room temperature for DNA extraction as described below. This study was reviewed and approved by the Institutional Animal Care and Use Committee of Yangzhou University and animal owners gave written permissions for blood collection.Table 1\nMolecular prevalence of\nTheileria\nspp. in cattle, water buffalo, goat and sheep\nAnimal speciesSubspecies /breedProvinceCityCoordinate of city\nTheileriapositivitypositive /total\n%\nCattle (n = 1235)\nBos p. taurus\nSimmentalInner MongoliaChifeng42.17°N, 118.58°E19/13214.4%Bohai blackShandongBinzhou37.22°N, 118.02°E4/666.1%LuxiShandongJining35.23°N, 116.33°E40/40100%HolsteinJiangsuYancheng33.22°N, 120.08°E72/32122.4%HolsteinJiangsuYangzhou32.23°N, 119.26°E17/14411.8%HolsteinShanghaiShanghai31.14°N, 121.29°E9/2553.5%WannanAnhuiWuhu31.19°N, 118.22°E17/17100%\nBos p. indicus\nYunlingYunnanKunming25.04°N, 102.42°E124/16177.0%MinnanFujianPutian24.26°N, 119.01°E4/2516.0%LeiqiongHainanHaikou20.02°N, 110.20°E74/74100%Water buffalo (n = 29)HaiziJiangsuYancheng33.22°N, 120.08°E0/290%Goat (n = 270)XinjiangXinjiangUrumqi43.45°N, 87.36°E4/984.1%Yangtse River Delta WhiteJiangsuYangzhou32.23°N, 119.26°E116/17267.4%Sheep (n = 111)WurankeInner MongoliaXilingol43.57°N, 116.03°E36/7250.0%Sishui FurShandongJining35.23°N, 116.33°E23/3959.0%\n\nMolecular prevalence of\nTheileria\nspp. in cattle, water buffalo, goat and sheep\n","DNA was extracted from whole blood samples using a standard phenol-chloroform method previously described [28]. Two ml whole blood was used to extract DNA which was resuspended into 200 μl 1 × T10E0.1 buffer. The concentration of the extracted DNA was established with a Microscale Ultraviolet Spectrophotometer. Negative controls consisting of sterile molecular grade water were used to detect cross- contamination during DNA extraction and processing. The HMBS-based FRET-PCR was performed to verify if the extracted DNAs from blood samples were appropriate for molecular detection of tick-borne pathogens [29, 30]."," Primers and probes The 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\nThe 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n Thermal cycling The Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\nThe Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\n Specificity PCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\nPCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\n Sensitivity For use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\nFor use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.","The 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.","The Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.","PCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).","For use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.","The amplicon of the pan-Theileria FRET-qPCR we established has a sequence which is highly conserved among the different Theileria species. To differentiate Theileria spp. in a positive reaction, we used a standard PCR to amplify a highly polymorphic region of the 18S rRNA gene (591–594 nucleotides for different Theileria spp.) and sequenced the products (GenScript, Nanjing, Jiangsu, China). For the PCR we designed a forward primer (5′-CCTGAGAAACGGCTACCACATCT-3′) that amplified all Theileria species and used a previously described reverse primer (5′-GGACTACGACGGTATCTGATCG-3′) that also amplified all species [31].","Differences in positivity of Theileria spp. were analyzed by Chi-squared Test while numbers of copies of the Theileria 18S rRNA gene determined in the Theileria FRET-qPCR were log10-transformed and analyzed using the Student’s T-test. Differences of P < 0.05 were considered statistically significant."," Development of the pan-TheileriaFRET-PCR Comparison of the sequences in the highly conserved region of the Theileria spp. we used showed the region is highly conserved, but is substantially different from those in closely related protozoan species (Figure 1). The two primers and two probes we chose for the pan-Theileria FRET-qPCR had 0–4 nucleotide mismatches with the Theileria spp. in GenBank, but had 25, 23, 22, 8, 16 and 15 mismatches with B. bovis, B. divergens, B. bigemina, C. felis, H. americanum and T. gondii, respectively (Figure 1). The specificity of the pan-Theileria FRET-PCR was further confirmed when it gave positive reactions with the T. orientalis control, but gave negative reactions with DNAs of B. canis, C. felis, H. americanum and T. gondii. The pan-Theileria FRET-qPCR had a specific melting curve (Tm 57.5°C) with Theileria spp. DNA. Using the gel-purified PCR products as quantitative standards, we determined the detection limit of the pan-Theileria FRET-qPCR was 2 copies of the Theileria 18S rRNA gene per reaction for T. orientalis, T. sergenti and T. luwenshuni, T. buffeli and T. ovis.\nComparison of the sequences in the highly conserved region of the Theileria spp. we used showed the region is highly conserved, but is substantially different from those in closely related protozoan species (Figure 1). The two primers and two probes we chose for the pan-Theileria FRET-qPCR had 0–4 nucleotide mismatches with the Theileria spp. in GenBank, but had 25, 23, 22, 8, 16 and 15 mismatches with B. bovis, B. divergens, B. bigemina, C. felis, H. americanum and T. gondii, respectively (Figure 1). The specificity of the pan-Theileria FRET-PCR was further confirmed when it gave positive reactions with the T. orientalis control, but gave negative reactions with DNAs of B. canis, C. felis, H. americanum and T. gondii. The pan-Theileria FRET-qPCR had a specific melting curve (Tm 57.5°C) with Theileria spp. DNA. Using the gel-purified PCR products as quantitative standards, we determined the detection limit of the pan-Theileria FRET-qPCR was 2 copies of the Theileria 18S rRNA gene per reaction for T. orientalis, T. sergenti and T. luwenshuni, T. buffeli and T. ovis.\n Prevalence of Theileriaspp. DNA in ruminants Animals positive for Theileria were found in each of the nine provinces sampled with several animals of each species being positive at each location, except in the case of water buffaloes which were all negative in the one site they were studied. The overall prevalences of Theileria spp. DNA in sheep (53.2%; 59/111) and goats (44.4%; 120/270) were significantly higher than in cattle (30.8%; 380/1,235) (two-tailed Chi-squared Test, P < 10-4). The pan-Theileria FRET-PCR showed that sheep had an average of 102.4 copies of Theileria 18S rRNA/ml whole blood which was significantly lower than the 104.3 copies in cattle and 105.8 copies in goats (Student’s t Test, P < 10-4). While the prevalence of Theileria spp. DNA varied greatly from 3.5% (9/255) in Holsteins from Shanghai to 100% in Luxi cattle from Shandong (40/40) and Leiqiong cattle from Hainan (74/74), the prevalence did not differ significantly in sheep from Inner Mongolia and Shandong (Table 1, Figure 2).Figure 2\nSites in China where samples ruminants were tested for\nTheileria\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\n\nSites in China where samples ruminants were tested for\nTheileria\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\nSequencing of 87 randomly selected amplicons (52 from cattle, 14 from goats and 21 from sheep) from Theileria DNA positive samples showed that T. orientalis/ T. sergenti/T. buffeli group [29] were present in cattle while T. luwenshuni was found in goats in Jiangsu province and T. ovis and T. luwenshuni in sheep from Inner Mongolia and Jiangsu province, respectively.\nAnimals positive for Theileria were found in each of the nine provinces sampled with several animals of each species being positive at each location, except in the case of water buffaloes which were all negative in the one site they were studied. The overall prevalences of Theileria spp. DNA in sheep (53.2%; 59/111) and goats (44.4%; 120/270) were significantly higher than in cattle (30.8%; 380/1,235) (two-tailed Chi-squared Test, P < 10-4). The pan-Theileria FRET-PCR showed that sheep had an average of 102.4 copies of Theileria 18S rRNA/ml whole blood which was significantly lower than the 104.3 copies in cattle and 105.8 copies in goats (Student’s t Test, P < 10-4). While the prevalence of Theileria spp. DNA varied greatly from 3.5% (9/255) in Holsteins from Shanghai to 100% in Luxi cattle from Shandong (40/40) and Leiqiong cattle from Hainan (74/74), the prevalence did not differ significantly in sheep from Inner Mongolia and Shandong (Table 1, Figure 2).Figure 2\nSites in China where samples ruminants were tested for\nTheileria\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\n\nSites in China where samples ruminants were tested for\nTheileria\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\nSequencing of 87 randomly selected amplicons (52 from cattle, 14 from goats and 21 from sheep) from Theileria DNA positive samples showed that T. orientalis/ T. sergenti/T. buffeli group [29] were present in cattle while T. luwenshuni was found in goats in Jiangsu province and T. ovis and T. luwenshuni in sheep from Inner Mongolia and Jiangsu province, respectively.\n Factors associated with the occurrence of theileriosis in cattle When we analyzed factors that might be associated with the prevalence of Theileria spp. DNA in cattle we found that Bos p. indicus animals had significantly higher positivity (77.7% vs. 18.3%; P < 10-4) and copy number of the Theileria 18S rRNA gene (104.81 vs. 103.73 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. Similarly, Bos p. indicus cattle were more likely to be positive (65.2% vs. 13.7%; P < 10-4) and have higher copy numbers of the Theileria 18S rRNA gene (104.88 vs. 103.00 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. The cattle from southern China had significantly higher Theileria 18S rRNA gene copy numbers (104.39 vs. 103.87 copies/per ml whole blood; P = 0.02) than those from northern China but this difference in the prevalence was not significant (31.8% vs. 26.5%). In cattle from Yunnan province where gender information was available, female animals were more commonly positive (76.7% vs. 41.3%; P < 10-4) and copy numbers (105.02 vs. 102.93Theileria/per ml whole blood; P < 10-4) than males.\nWhen we analyzed factors that might be associated with the prevalence of Theileria spp. DNA in cattle we found that Bos p. indicus animals had significantly higher positivity (77.7% vs. 18.3%; P < 10-4) and copy number of the Theileria 18S rRNA gene (104.81 vs. 103.73 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. Similarly, Bos p. indicus cattle were more likely to be positive (65.2% vs. 13.7%; P < 10-4) and have higher copy numbers of the Theileria 18S rRNA gene (104.88 vs. 103.00 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. The cattle from southern China had significantly higher Theileria 18S rRNA gene copy numbers (104.39 vs. 103.87 copies/per ml whole blood; P = 0.02) than those from northern China but this difference in the prevalence was not significant (31.8% vs. 26.5%). In cattle from Yunnan province where gender information was available, female animals were more commonly positive (76.7% vs. 41.3%; P < 10-4) and copy numbers (105.02 vs. 102.93Theileria/per ml whole blood; P < 10-4) than males.\n Gene accession numbers The Theileria rRNA nucleotide sequences obtained in this study that were not identical to existing entries in GenBank were deposited with the following gene accession numbers: KJ850933 and KJ850938 (T. sergenti); KJ850936 and KJ850940 (T. buffeli); KJ850934, KJ850937, KJ850943, KJ850939 and KJ850941 (T. orientalis); KJ850942 (T. ovis); KJ850935 and KM016463 (T. luwenshuni). The sequences obtained were very similar (0–4 nucleotide mismatches) to Theileria spp. sequences deposited by other laboratories in China, USA, France, Australia and Iran (Table 2).Table 2\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\nIsolates identified in this studyHighly similar sequences in GenBank\nTheileriaspp.Gene accession #Source/originGene accession #Source/originMismatches\nT. orientalis\nKJ850934Simmental cattle, Inner MongoliaAP011948Cattle, Shintoku of Japan0/547KJ850939Luxi cattle, ShandongHM538220Cattle, Suizhou of China0/547KJ850937Yunling cattle, YunnanAB520956Cattle, New South Wales of Australia2/491KJ850941Yunling cattle, YunnanAB520955Cattle, Raymond of Australia0/509KJ850943Holstein cattle, JiangsuAB520956Cattle, New South Wales of Australia0/549\nT. sergenti\nKJ850933Australian Holstein cattle, JiangsuJQ723015Cattle, Hunan of China0/541KJ850938Yunling cattle, YunnanJQ723015Cattle, Hunan of China0/492\nT. buffeli\nKJ850936Leiqiong cattle, HainanHM538196Cattle, Hubei of China;1/508KJ850940Wannan cattle, AnhuiAY661513Cattle, USA0/545\nT. ovis\nKJ850942Wuranke sheep, Inner MongoliaFJ603460Sheep, Xinjiang of China;0/529\nT. luwenshuni\nKJ850935from Sishui-Fur sheep, ShandongKC769996, JX469518, JF719831Sheep, China0/549KM016463Yangtse River Delta White goat, JiangsuKC769997Goat, Beijing of China0/571\n\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\n\nThe Theileria rRNA nucleotide sequences obtained in this study that were not identical to existing entries in GenBank were deposited with the following gene accession numbers: KJ850933 and KJ850938 (T. sergenti); KJ850936 and KJ850940 (T. buffeli); KJ850934, KJ850937, KJ850943, KJ850939 and KJ850941 (T. orientalis); KJ850942 (T. ovis); KJ850935 and KM016463 (T. luwenshuni). The sequences obtained were very similar (0–4 nucleotide mismatches) to Theileria spp. sequences deposited by other laboratories in China, USA, France, Australia and Iran (Table 2).Table 2\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\nIsolates identified in this studyHighly similar sequences in GenBank\nTheileriaspp.Gene accession #Source/originGene accession #Source/originMismatches\nT. orientalis\nKJ850934Simmental cattle, Inner MongoliaAP011948Cattle, Shintoku of Japan0/547KJ850939Luxi cattle, ShandongHM538220Cattle, Suizhou of China0/547KJ850937Yunling cattle, YunnanAB520956Cattle, New South Wales of Australia2/491KJ850941Yunling cattle, YunnanAB520955Cattle, Raymond of Australia0/509KJ850943Holstein cattle, JiangsuAB520956Cattle, New South Wales of Australia0/549\nT. sergenti\nKJ850933Australian Holstein cattle, JiangsuJQ723015Cattle, Hunan of China0/541KJ850938Yunling cattle, YunnanJQ723015Cattle, Hunan of China0/492\nT. buffeli\nKJ850936Leiqiong cattle, HainanHM538196Cattle, Hubei of China;1/508KJ850940Wannan cattle, AnhuiAY661513Cattle, USA0/545\nT. ovis\nKJ850942Wuranke sheep, Inner MongoliaFJ603460Sheep, Xinjiang of China;0/529\nT. luwenshuni\nKJ850935from Sishui-Fur sheep, ShandongKC769996, JX469518, JF719831Sheep, China0/549KM016463Yangtse River Delta White goat, JiangsuKC769997Goat, Beijing of China0/571\n\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\n","Comparison of the sequences in the highly conserved region of the Theileria spp. we used showed the region is highly conserved, but is substantially different from those in closely related protozoan species (Figure 1). The two primers and two probes we chose for the pan-Theileria FRET-qPCR had 0–4 nucleotide mismatches with the Theileria spp. in GenBank, but had 25, 23, 22, 8, 16 and 15 mismatches with B. bovis, B. divergens, B. bigemina, C. felis, H. americanum and T. gondii, respectively (Figure 1). The specificity of the pan-Theileria FRET-PCR was further confirmed when it gave positive reactions with the T. orientalis control, but gave negative reactions with DNAs of B. canis, C. felis, H. americanum and T. gondii. The pan-Theileria FRET-qPCR had a specific melting curve (Tm 57.5°C) with Theileria spp. DNA. Using the gel-purified PCR products as quantitative standards, we determined the detection limit of the pan-Theileria FRET-qPCR was 2 copies of the Theileria 18S rRNA gene per reaction for T. orientalis, T. sergenti and T. luwenshuni, T. buffeli and T. ovis.","Animals positive for Theileria were found in each of the nine provinces sampled with several animals of each species being positive at each location, except in the case of water buffaloes which were all negative in the one site they were studied. The overall prevalences of Theileria spp. DNA in sheep (53.2%; 59/111) and goats (44.4%; 120/270) were significantly higher than in cattle (30.8%; 380/1,235) (two-tailed Chi-squared Test, P < 10-4). The pan-Theileria FRET-PCR showed that sheep had an average of 102.4 copies of Theileria 18S rRNA/ml whole blood which was significantly lower than the 104.3 copies in cattle and 105.8 copies in goats (Student’s t Test, P < 10-4). While the prevalence of Theileria spp. DNA varied greatly from 3.5% (9/255) in Holsteins from Shanghai to 100% in Luxi cattle from Shandong (40/40) and Leiqiong cattle from Hainan (74/74), the prevalence did not differ significantly in sheep from Inner Mongolia and Shandong (Table 1, Figure 2).Figure 2\nSites in China where samples ruminants were tested for\nTheileria\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\n\nSites in China where samples ruminants were tested for\nTheileria\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\nSequencing of 87 randomly selected amplicons (52 from cattle, 14 from goats and 21 from sheep) from Theileria DNA positive samples showed that T. orientalis/ T. sergenti/T. buffeli group [29] were present in cattle while T. luwenshuni was found in goats in Jiangsu province and T. ovis and T. luwenshuni in sheep from Inner Mongolia and Jiangsu province, respectively.","When we analyzed factors that might be associated with the prevalence of Theileria spp. DNA in cattle we found that Bos p. indicus animals had significantly higher positivity (77.7% vs. 18.3%; P < 10-4) and copy number of the Theileria 18S rRNA gene (104.81 vs. 103.73 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. Similarly, Bos p. indicus cattle were more likely to be positive (65.2% vs. 13.7%; P < 10-4) and have higher copy numbers of the Theileria 18S rRNA gene (104.88 vs. 103.00 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. The cattle from southern China had significantly higher Theileria 18S rRNA gene copy numbers (104.39 vs. 103.87 copies/per ml whole blood; P = 0.02) than those from northern China but this difference in the prevalence was not significant (31.8% vs. 26.5%). In cattle from Yunnan province where gender information was available, female animals were more commonly positive (76.7% vs. 41.3%; P < 10-4) and copy numbers (105.02 vs. 102.93Theileria/per ml whole blood; P < 10-4) than males.","The Theileria rRNA nucleotide sequences obtained in this study that were not identical to existing entries in GenBank were deposited with the following gene accession numbers: KJ850933 and KJ850938 (T. sergenti); KJ850936 and KJ850940 (T. buffeli); KJ850934, KJ850937, KJ850943, KJ850939 and KJ850941 (T. orientalis); KJ850942 (T. ovis); KJ850935 and KM016463 (T. luwenshuni). The sequences obtained were very similar (0–4 nucleotide mismatches) to Theileria spp. sequences deposited by other laboratories in China, USA, France, Australia and Iran (Table 2).Table 2\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\nIsolates identified in this studyHighly similar sequences in GenBank\nTheileriaspp.Gene accession #Source/originGene accession #Source/originMismatches\nT. orientalis\nKJ850934Simmental cattle, Inner MongoliaAP011948Cattle, Shintoku of Japan0/547KJ850939Luxi cattle, ShandongHM538220Cattle, Suizhou of China0/547KJ850937Yunling cattle, YunnanAB520956Cattle, New South Wales of Australia2/491KJ850941Yunling cattle, YunnanAB520955Cattle, Raymond of Australia0/509KJ850943Holstein cattle, JiangsuAB520956Cattle, New South Wales of Australia0/549\nT. sergenti\nKJ850933Australian Holstein cattle, JiangsuJQ723015Cattle, Hunan of China0/541KJ850938Yunling cattle, YunnanJQ723015Cattle, Hunan of China0/492\nT. buffeli\nKJ850936Leiqiong cattle, HainanHM538196Cattle, Hubei of China;1/508KJ850940Wannan cattle, AnhuiAY661513Cattle, USA0/545\nT. ovis\nKJ850942Wuranke sheep, Inner MongoliaFJ603460Sheep, Xinjiang of China;0/529\nT. luwenshuni\nKJ850935from Sishui-Fur sheep, ShandongKC769996, JX469518, JF719831Sheep, China0/549KM016463Yangtse River Delta White goat, JiangsuKC769997Goat, Beijing of China0/571\n\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\n","By systematically aligning the 18S rRNA sequences of representative Theileria spp. and other related protozoa, we identified a highly conserved region to place primers and probes for a pan-Theileria FRET-qPCR. The primers and probes we designed enabled us to amplify all our reference Theileria species with a detection limit of at least 2 Theileria 18S rRNA gene copies per PCR system. None of the other protozoan species tested gave reaction products. To the best of our knowledge, this is the first FRET-qPCR which specifically detects all Theileria species.\nOur data indicate infections with Theileria spp. are very widespread and common in cattle in China. We found positive animals in each province where we tested and an overall average of 30.77% of animals being positive. This relatively high level of positivity is similar to that obtained in the only other molecular survey for Theileria in China which showed 13.46% being positive in the northeast [32]. Sequencing data showed cattle are infected with the T. orientalis/T. sergenti/T. buffeli group which are generally recognized to be benign species [33] although some strains might cause economic losses [34].\nAlthough we sequenced relatively few positive amplicons, we found no evidence of the more pathogenic strains, T. annulata and T. parva, which is not unexpected in the case of T. parva which has only been reported from Africa [8]. T. annulata, however, has been reported in H. asiaticum in northwestern China [35]. Our failure to demonstrate the organism in our study might be because T. annulata has an uneven distribution in China.\nAlthough we found no evidence of Theileria spp. in the 29 water buffaloes we sampled, He [36] reported 58/304 (19.1%) positive for T. buffeli in Hubei province, south China. More extensive studies are necessary to determine the epidemiology of theileriosis in water buffaloes.\nWe found both goats and sheep were infected with T. luwenshuni (Table 2) using the pan-Theileria FRET-PCR and sequencing. This is a highly pathogenic organism that is known to occur in China where it is transmitted by Haemaphysalis qinghaiensis and may cause significant economic losses. While PCR assays have been described which detect and differentiate T. luwenshuni from T. uilenbergi\n[37, 38], they do not enable detection of all Theirleria spp. in all species and are thus not as versatile as our pan-Theileria FRET-PCR. Yin et al.\n[37] found the prevalence of T. luwenshuni varied from 0% to 85% in 4 provinces and we also found a wide range of positive values (4.1% to 67.4%). Such variability is probably due to tick prevalence rates, geoclimatic factors and livestock management systems. Further research using sensitive detection methods will be important in determining the best mechanisms to control infections.\nT. ovis was first identified in China in 2011 by PCR and sequencing and an infection rate of 78% was found in Xinjiang but no positive animals were found in twelve other provinces [26, 27, 39]. Our study confirms the presence of the organism in China although T. ovis is considered benign and economically unimportant as it might only cause signs in some animals which are stressed [40].\nIt is well recognized that exotic breeds are more susceptible to disease following infection with Theileria spp. than local stocks. Recent studies, however, have indicated that different breeds of animals might have similar susceptibilities to infections with Theileria spp. although some breeds are more capable of controlling the pathogenic effects of the organism [41].\nGenerally, B. p. taurus breeds are more susceptible to theileriosis than B. p. indicus breeds which might be associated with innate or immune mechanisms and/or general resistance to ticks. These factors have been mainly investigated with T. parva infections and thus it is interesting that our pan-Theileria FRET-PCR testing showed that in China it is the B. p. indicus breeds that are more likely to be positive than the B. p. taurus breeds and that the former generally had higher copy numbers indicating heavier infections. While our sample sizes were small and we cannot exclude the possibility of sample bias, our findings might be due to host genetic factors relating to infections with less pathogenic Theileria spp. or other factors such as differences in tick control and husbandry practices on different farms. Further studies taking these factors into account are needed to more precisely investigate the relationships between infections with benign Theileria spp. and the genetic background of the host.","In summary, our study has described the development and testing of a FRET-PCR which can detect recognized Theileria spp. The pan-Theileria FRET-PCR should be a useful diagnostic tool as it will enable diagnostic laboratories to detect infections in all domestic species with a single test."],"string":"[\n \"Theileria spp. are tick-transmitted, intracellular protozoan parasites infecting leukocytes and erythrocytes of wild and domestic large and small ruminants. Several Theileria spp., transmitted by ixodid ticks of the genera Rhipicephalus, Hyalomma, Amblyomma and Haemaphysalis, have been described in cattle, water buffaloes, sheep and goats in different geographical zones of the world [1–5]. Theileriosis is primarily limited to tropical and sub-tropical areas of the world, with infections mainly reported in Africa and the Middle East but also in southern Europe and northern Asia [6–11]. Infections by Theileria spp. can cause fever, anemia and hemoglobinuria and, in severe cases, death although many species are benign. Animals recovered from acute or primary infections usually remain persistently infected and may act as reservoirs of infecting ticks [12, 13].\\nWhile there have been numerous reports of theileriosis in various animal species in China since 1958 [14–27], many have been reported in Chinese and some were based on microscopic detection of parasites which can be difficult with low parasitemia and does not allow ready differentiation of species. Serological studies, although sensitive and easy to perform, are not specific as there is cross reactivity between Theileria spp. Although molecular studies have been performed, these have been to detect Theileria of specific domestic animal species, for example sheep and goats [27]. There have been no highly sensitive and specific molecular methods described which enable studies on various animals from widely divergent areas of China where different Theileria spp. might occur. To address this problem, we developed and validated a highly sensitive genus-specific Theileria FRET-qPCR that detects the recognized Theileria spp. of domestic animals and investigated the molecular prevalence of Theileria in cattle, water buffaloes, goats and sheep from nine provinces in China.\",\n \" Animals and blood collection Between 2007 and 2013, whole blood samples (around 6 ml) were collected in EDTA from apparently healthy cattle (n = 1,235), water buffaloes (29), goats (270) and sheep (111) from 9 provinces/municipality of China (Table 1). The Bos primigenius (p.) taurus studied (n = 975) were Holsteins, Simmentals, Bohai blacks, Luxis and Wannans while the Bos. p. indicus (n = 260) were the Yunlings, Minnans, and Leiqiongs (Table 1). The water buffaloes, goats and sheep in the study were bred in China and were indigenous breeds. Gender information was available for cattle from Yunnan province. After collection, the blood samples were frozen at -20°C and shipped on ice (over 2 days) to Yangzhou University where they were frozen at -80°C until thawed at room temperature for DNA extraction as described below. This study was reviewed and approved by the Institutional Animal Care and Use Committee of Yangzhou University and animal owners gave written permissions for blood collection.Table 1\\nMolecular prevalence of\\nTheileria\\nspp. in cattle, water buffalo, goat and sheep\\nAnimal speciesSubspecies /breedProvinceCityCoordinate of city\\nTheileriapositivitypositive /total\\n%\\nCattle (n = 1235)\\nBos p. taurus\\nSimmentalInner MongoliaChifeng42.17°N, 118.58°E19/13214.4%Bohai blackShandongBinzhou37.22°N, 118.02°E4/666.1%LuxiShandongJining35.23°N, 116.33°E40/40100%HolsteinJiangsuYancheng33.22°N, 120.08°E72/32122.4%HolsteinJiangsuYangzhou32.23°N, 119.26°E17/14411.8%HolsteinShanghaiShanghai31.14°N, 121.29°E9/2553.5%WannanAnhuiWuhu31.19°N, 118.22°E17/17100%\\nBos p. indicus\\nYunlingYunnanKunming25.04°N, 102.42°E124/16177.0%MinnanFujianPutian24.26°N, 119.01°E4/2516.0%LeiqiongHainanHaikou20.02°N, 110.20°E74/74100%Water buffalo (n = 29)HaiziJiangsuYancheng33.22°N, 120.08°E0/290%Goat (n = 270)XinjiangXinjiangUrumqi43.45°N, 87.36°E4/984.1%Yangtse River Delta WhiteJiangsuYangzhou32.23°N, 119.26°E116/17267.4%Sheep (n = 111)WurankeInner MongoliaXilingol43.57°N, 116.03°E36/7250.0%Sishui FurShandongJining35.23°N, 116.33°E23/3959.0%\\n\\nMolecular prevalence of\\nTheileria\\nspp. in cattle, water buffalo, goat and sheep\\n\\nBetween 2007 and 2013, whole blood samples (around 6 ml) were collected in EDTA from apparently healthy cattle (n = 1,235), water buffaloes (29), goats (270) and sheep (111) from 9 provinces/municipality of China (Table 1). The Bos primigenius (p.) taurus studied (n = 975) were Holsteins, Simmentals, Bohai blacks, Luxis and Wannans while the Bos. p. indicus (n = 260) were the Yunlings, Minnans, and Leiqiongs (Table 1). The water buffaloes, goats and sheep in the study were bred in China and were indigenous breeds. Gender information was available for cattle from Yunnan province. After collection, the blood samples were frozen at -20°C and shipped on ice (over 2 days) to Yangzhou University where they were frozen at -80°C until thawed at room temperature for DNA extraction as described below. This study was reviewed and approved by the Institutional Animal Care and Use Committee of Yangzhou University and animal owners gave written permissions for blood collection.Table 1\\nMolecular prevalence of\\nTheileria\\nspp. in cattle, water buffalo, goat and sheep\\nAnimal speciesSubspecies /breedProvinceCityCoordinate of city\\nTheileriapositivitypositive /total\\n%\\nCattle (n = 1235)\\nBos p. taurus\\nSimmentalInner MongoliaChifeng42.17°N, 118.58°E19/13214.4%Bohai blackShandongBinzhou37.22°N, 118.02°E4/666.1%LuxiShandongJining35.23°N, 116.33°E40/40100%HolsteinJiangsuYancheng33.22°N, 120.08°E72/32122.4%HolsteinJiangsuYangzhou32.23°N, 119.26°E17/14411.8%HolsteinShanghaiShanghai31.14°N, 121.29°E9/2553.5%WannanAnhuiWuhu31.19°N, 118.22°E17/17100%\\nBos p. indicus\\nYunlingYunnanKunming25.04°N, 102.42°E124/16177.0%MinnanFujianPutian24.26°N, 119.01°E4/2516.0%LeiqiongHainanHaikou20.02°N, 110.20°E74/74100%Water buffalo (n = 29)HaiziJiangsuYancheng33.22°N, 120.08°E0/290%Goat (n = 270)XinjiangXinjiangUrumqi43.45°N, 87.36°E4/984.1%Yangtse River Delta WhiteJiangsuYangzhou32.23°N, 119.26°E116/17267.4%Sheep (n = 111)WurankeInner MongoliaXilingol43.57°N, 116.03°E36/7250.0%Sishui FurShandongJining35.23°N, 116.33°E23/3959.0%\\n\\nMolecular prevalence of\\nTheileria\\nspp. in cattle, water buffalo, goat and sheep\\n\\n DNA extraction DNA was extracted from whole blood samples using a standard phenol-chloroform method previously described [28]. Two ml whole blood was used to extract DNA which was resuspended into 200 μl 1 × T10E0.1 buffer. The concentration of the extracted DNA was established with a Microscale Ultraviolet Spectrophotometer. Negative controls consisting of sterile molecular grade water were used to detect cross- contamination during DNA extraction and processing. The HMBS-based FRET-PCR was performed to verify if the extracted DNAs from blood samples were appropriate for molecular detection of tick-borne pathogens [29, 30].\\nDNA was extracted from whole blood samples using a standard phenol-chloroform method previously described [28]. Two ml whole blood was used to extract DNA which was resuspended into 200 μl 1 × T10E0.1 buffer. The concentration of the extracted DNA was established with a Microscale Ultraviolet Spectrophotometer. Negative controls consisting of sterile molecular grade water were used to detect cross- contamination during DNA extraction and processing. The HMBS-based FRET-PCR was performed to verify if the extracted DNAs from blood samples were appropriate for molecular detection of tick-borne pathogens [29, 30].\\n Theileriaspp.FRET-qPCR Primers and probes The 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\\nAlignment of oligonucleotides for\\nTheileria\\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\\n\\nAlignment of oligonucleotides for\\nTheileria\\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\\nThe 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\\nAlignment of oligonucleotides for\\nTheileria\\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\\n\\nAlignment of oligonucleotides for\\nTheileria\\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\\n Thermal cycling The Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\\nThe Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\\n Specificity PCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\\nPCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\\n Sensitivity For use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\\nFor use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\\n Primers and probes The 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\\nAlignment of oligonucleotides for\\nTheileria\\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\\n\\nAlignment of oligonucleotides for\\nTheileria\\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\\nThe 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\\nAlignment of oligonucleotides for\\nTheileria\\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\\n\\nAlignment of oligonucleotides for\\nTheileria\\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\\n Thermal cycling The Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\\nThe Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\\n Specificity PCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\\nPCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\\n Sensitivity For use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\\nFor use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\\n Identification of Theileriaspp. by PCR and sequencing The amplicon of the pan-Theileria FRET-qPCR we established has a sequence which is highly conserved among the different Theileria species. To differentiate Theileria spp. in a positive reaction, we used a standard PCR to amplify a highly polymorphic region of the 18S rRNA gene (591–594 nucleotides for different Theileria spp.) and sequenced the products (GenScript, Nanjing, Jiangsu, China). For the PCR we designed a forward primer (5′-CCTGAGAAACGGCTACCACATCT-3′) that amplified all Theileria species and used a previously described reverse primer (5′-GGACTACGACGGTATCTGATCG-3′) that also amplified all species [31].\\nThe amplicon of the pan-Theileria FRET-qPCR we established has a sequence which is highly conserved among the different Theileria species. To differentiate Theileria spp. in a positive reaction, we used a standard PCR to amplify a highly polymorphic region of the 18S rRNA gene (591–594 nucleotides for different Theileria spp.) and sequenced the products (GenScript, Nanjing, Jiangsu, China). For the PCR we designed a forward primer (5′-CCTGAGAAACGGCTACCACATCT-3′) that amplified all Theileria species and used a previously described reverse primer (5′-GGACTACGACGGTATCTGATCG-3′) that also amplified all species [31].\\n Statistical analysis Differences in positivity of Theileria spp. were analyzed by Chi-squared Test while numbers of copies of the Theileria 18S rRNA gene determined in the Theileria FRET-qPCR were log10-transformed and analyzed using the Student’s T-test. Differences of P < 0.05 were considered statistically significant.\\nDifferences in positivity of Theileria spp. were analyzed by Chi-squared Test while numbers of copies of the Theileria 18S rRNA gene determined in the Theileria FRET-qPCR were log10-transformed and analyzed using the Student’s T-test. Differences of P < 0.05 were considered statistically significant.\",\n \"Between 2007 and 2013, whole blood samples (around 6 ml) were collected in EDTA from apparently healthy cattle (n = 1,235), water buffaloes (29), goats (270) and sheep (111) from 9 provinces/municipality of China (Table 1). The Bos primigenius (p.) taurus studied (n = 975) were Holsteins, Simmentals, Bohai blacks, Luxis and Wannans while the Bos. p. indicus (n = 260) were the Yunlings, Minnans, and Leiqiongs (Table 1). The water buffaloes, goats and sheep in the study were bred in China and were indigenous breeds. Gender information was available for cattle from Yunnan province. After collection, the blood samples were frozen at -20°C and shipped on ice (over 2 days) to Yangzhou University where they were frozen at -80°C until thawed at room temperature for DNA extraction as described below. This study was reviewed and approved by the Institutional Animal Care and Use Committee of Yangzhou University and animal owners gave written permissions for blood collection.Table 1\\nMolecular prevalence of\\nTheileria\\nspp. in cattle, water buffalo, goat and sheep\\nAnimal speciesSubspecies /breedProvinceCityCoordinate of city\\nTheileriapositivitypositive /total\\n%\\nCattle (n = 1235)\\nBos p. taurus\\nSimmentalInner MongoliaChifeng42.17°N, 118.58°E19/13214.4%Bohai blackShandongBinzhou37.22°N, 118.02°E4/666.1%LuxiShandongJining35.23°N, 116.33°E40/40100%HolsteinJiangsuYancheng33.22°N, 120.08°E72/32122.4%HolsteinJiangsuYangzhou32.23°N, 119.26°E17/14411.8%HolsteinShanghaiShanghai31.14°N, 121.29°E9/2553.5%WannanAnhuiWuhu31.19°N, 118.22°E17/17100%\\nBos p. indicus\\nYunlingYunnanKunming25.04°N, 102.42°E124/16177.0%MinnanFujianPutian24.26°N, 119.01°E4/2516.0%LeiqiongHainanHaikou20.02°N, 110.20°E74/74100%Water buffalo (n = 29)HaiziJiangsuYancheng33.22°N, 120.08°E0/290%Goat (n = 270)XinjiangXinjiangUrumqi43.45°N, 87.36°E4/984.1%Yangtse River Delta WhiteJiangsuYangzhou32.23°N, 119.26°E116/17267.4%Sheep (n = 111)WurankeInner MongoliaXilingol43.57°N, 116.03°E36/7250.0%Sishui FurShandongJining35.23°N, 116.33°E23/3959.0%\\n\\nMolecular prevalence of\\nTheileria\\nspp. in cattle, water buffalo, goat and sheep\\n\",\n \"DNA was extracted from whole blood samples using a standard phenol-chloroform method previously described [28]. Two ml whole blood was used to extract DNA which was resuspended into 200 μl 1 × T10E0.1 buffer. The concentration of the extracted DNA was established with a Microscale Ultraviolet Spectrophotometer. Negative controls consisting of sterile molecular grade water were used to detect cross- contamination during DNA extraction and processing. The HMBS-based FRET-PCR was performed to verify if the extracted DNAs from blood samples were appropriate for molecular detection of tick-borne pathogens [29, 30].\",\n \" Primers and probes The 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\\nAlignment of oligonucleotides for\\nTheileria\\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\\n\\nAlignment of oligonucleotides for\\nTheileria\\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\\nThe 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\\nAlignment of oligonucleotides for\\nTheileria\\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\\n\\nAlignment of oligonucleotides for\\nTheileria\\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\\n Thermal cycling The Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\\nThe Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\\n Specificity PCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\\nPCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\\n Sensitivity For use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\\nFor use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\",\n \"The 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\\nAlignment of oligonucleotides for\\nTheileria\\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\\n\\nAlignment of oligonucleotides for\\nTheileria\\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\",\n \"The Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\",\n \"PCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\",\n \"For use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\",\n \"The amplicon of the pan-Theileria FRET-qPCR we established has a sequence which is highly conserved among the different Theileria species. To differentiate Theileria spp. in a positive reaction, we used a standard PCR to amplify a highly polymorphic region of the 18S rRNA gene (591–594 nucleotides for different Theileria spp.) and sequenced the products (GenScript, Nanjing, Jiangsu, China). For the PCR we designed a forward primer (5′-CCTGAGAAACGGCTACCACATCT-3′) that amplified all Theileria species and used a previously described reverse primer (5′-GGACTACGACGGTATCTGATCG-3′) that also amplified all species [31].\",\n \"Differences in positivity of Theileria spp. were analyzed by Chi-squared Test while numbers of copies of the Theileria 18S rRNA gene determined in the Theileria FRET-qPCR were log10-transformed and analyzed using the Student’s T-test. Differences of P < 0.05 were considered statistically significant.\",\n \" Development of the pan-TheileriaFRET-PCR Comparison of the sequences in the highly conserved region of the Theileria spp. we used showed the region is highly conserved, but is substantially different from those in closely related protozoan species (Figure 1). The two primers and two probes we chose for the pan-Theileria FRET-qPCR had 0–4 nucleotide mismatches with the Theileria spp. in GenBank, but had 25, 23, 22, 8, 16 and 15 mismatches with B. bovis, B. divergens, B. bigemina, C. felis, H. americanum and T. gondii, respectively (Figure 1). The specificity of the pan-Theileria FRET-PCR was further confirmed when it gave positive reactions with the T. orientalis control, but gave negative reactions with DNAs of B. canis, C. felis, H. americanum and T. gondii. The pan-Theileria FRET-qPCR had a specific melting curve (Tm 57.5°C) with Theileria spp. DNA. Using the gel-purified PCR products as quantitative standards, we determined the detection limit of the pan-Theileria FRET-qPCR was 2 copies of the Theileria 18S rRNA gene per reaction for T. orientalis, T. sergenti and T. luwenshuni, T. buffeli and T. ovis.\\nComparison of the sequences in the highly conserved region of the Theileria spp. we used showed the region is highly conserved, but is substantially different from those in closely related protozoan species (Figure 1). The two primers and two probes we chose for the pan-Theileria FRET-qPCR had 0–4 nucleotide mismatches with the Theileria spp. in GenBank, but had 25, 23, 22, 8, 16 and 15 mismatches with B. bovis, B. divergens, B. bigemina, C. felis, H. americanum and T. gondii, respectively (Figure 1). The specificity of the pan-Theileria FRET-PCR was further confirmed when it gave positive reactions with the T. orientalis control, but gave negative reactions with DNAs of B. canis, C. felis, H. americanum and T. gondii. The pan-Theileria FRET-qPCR had a specific melting curve (Tm 57.5°C) with Theileria spp. DNA. Using the gel-purified PCR products as quantitative standards, we determined the detection limit of the pan-Theileria FRET-qPCR was 2 copies of the Theileria 18S rRNA gene per reaction for T. orientalis, T. sergenti and T. luwenshuni, T. buffeli and T. ovis.\\n Prevalence of Theileriaspp. DNA in ruminants Animals positive for Theileria were found in each of the nine provinces sampled with several animals of each species being positive at each location, except in the case of water buffaloes which were all negative in the one site they were studied. The overall prevalences of Theileria spp. DNA in sheep (53.2%; 59/111) and goats (44.4%; 120/270) were significantly higher than in cattle (30.8%; 380/1,235) (two-tailed Chi-squared Test, P < 10-4). The pan-Theileria FRET-PCR showed that sheep had an average of 102.4 copies of Theileria 18S rRNA/ml whole blood which was significantly lower than the 104.3 copies in cattle and 105.8 copies in goats (Student’s t Test, P < 10-4). While the prevalence of Theileria spp. DNA varied greatly from 3.5% (9/255) in Holsteins from Shanghai to 100% in Luxi cattle from Shandong (40/40) and Leiqiong cattle from Hainan (74/74), the prevalence did not differ significantly in sheep from Inner Mongolia and Shandong (Table 1, Figure 2).Figure 2\\nSites in China where samples ruminants were tested for\\nTheileria\\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\\n\\nSites in China where samples ruminants were tested for\\nTheileria\\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\\nSequencing of 87 randomly selected amplicons (52 from cattle, 14 from goats and 21 from sheep) from Theileria DNA positive samples showed that T. orientalis/ T. sergenti/T. buffeli group [29] were present in cattle while T. luwenshuni was found in goats in Jiangsu province and T. ovis and T. luwenshuni in sheep from Inner Mongolia and Jiangsu province, respectively.\\nAnimals positive for Theileria were found in each of the nine provinces sampled with several animals of each species being positive at each location, except in the case of water buffaloes which were all negative in the one site they were studied. The overall prevalences of Theileria spp. DNA in sheep (53.2%; 59/111) and goats (44.4%; 120/270) were significantly higher than in cattle (30.8%; 380/1,235) (two-tailed Chi-squared Test, P < 10-4). The pan-Theileria FRET-PCR showed that sheep had an average of 102.4 copies of Theileria 18S rRNA/ml whole blood which was significantly lower than the 104.3 copies in cattle and 105.8 copies in goats (Student’s t Test, P < 10-4). While the prevalence of Theileria spp. DNA varied greatly from 3.5% (9/255) in Holsteins from Shanghai to 100% in Luxi cattle from Shandong (40/40) and Leiqiong cattle from Hainan (74/74), the prevalence did not differ significantly in sheep from Inner Mongolia and Shandong (Table 1, Figure 2).Figure 2\\nSites in China where samples ruminants were tested for\\nTheileria\\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\\n\\nSites in China where samples ruminants were tested for\\nTheileria\\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\\nSequencing of 87 randomly selected amplicons (52 from cattle, 14 from goats and 21 from sheep) from Theileria DNA positive samples showed that T. orientalis/ T. sergenti/T. buffeli group [29] were present in cattle while T. luwenshuni was found in goats in Jiangsu province and T. ovis and T. luwenshuni in sheep from Inner Mongolia and Jiangsu province, respectively.\\n Factors associated with the occurrence of theileriosis in cattle When we analyzed factors that might be associated with the prevalence of Theileria spp. DNA in cattle we found that Bos p. indicus animals had significantly higher positivity (77.7% vs. 18.3%; P < 10-4) and copy number of the Theileria 18S rRNA gene (104.81 vs. 103.73 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. Similarly, Bos p. indicus cattle were more likely to be positive (65.2% vs. 13.7%; P < 10-4) and have higher copy numbers of the Theileria 18S rRNA gene (104.88 vs. 103.00 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. The cattle from southern China had significantly higher Theileria 18S rRNA gene copy numbers (104.39 vs. 103.87 copies/per ml whole blood; P = 0.02) than those from northern China but this difference in the prevalence was not significant (31.8% vs. 26.5%). In cattle from Yunnan province where gender information was available, female animals were more commonly positive (76.7% vs. 41.3%; P < 10-4) and copy numbers (105.02 vs. 102.93Theileria/per ml whole blood; P < 10-4) than males.\\nWhen we analyzed factors that might be associated with the prevalence of Theileria spp. DNA in cattle we found that Bos p. indicus animals had significantly higher positivity (77.7% vs. 18.3%; P < 10-4) and copy number of the Theileria 18S rRNA gene (104.81 vs. 103.73 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. Similarly, Bos p. indicus cattle were more likely to be positive (65.2% vs. 13.7%; P < 10-4) and have higher copy numbers of the Theileria 18S rRNA gene (104.88 vs. 103.00 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. The cattle from southern China had significantly higher Theileria 18S rRNA gene copy numbers (104.39 vs. 103.87 copies/per ml whole blood; P = 0.02) than those from northern China but this difference in the prevalence was not significant (31.8% vs. 26.5%). In cattle from Yunnan province where gender information was available, female animals were more commonly positive (76.7% vs. 41.3%; P < 10-4) and copy numbers (105.02 vs. 102.93Theileria/per ml whole blood; P < 10-4) than males.\\n Gene accession numbers The Theileria rRNA nucleotide sequences obtained in this study that were not identical to existing entries in GenBank were deposited with the following gene accession numbers: KJ850933 and KJ850938 (T. sergenti); KJ850936 and KJ850940 (T. buffeli); KJ850934, KJ850937, KJ850943, KJ850939 and KJ850941 (T. orientalis); KJ850942 (T. ovis); KJ850935 and KM016463 (T. luwenshuni). The sequences obtained were very similar (0–4 nucleotide mismatches) to Theileria spp. sequences deposited by other laboratories in China, USA, France, Australia and Iran (Table 2).Table 2\\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\\nIsolates identified in this studyHighly similar sequences in GenBank\\nTheileriaspp.Gene accession #Source/originGene accession #Source/originMismatches\\nT. orientalis\\nKJ850934Simmental cattle, Inner MongoliaAP011948Cattle, Shintoku of Japan0/547KJ850939Luxi cattle, ShandongHM538220Cattle, Suizhou of China0/547KJ850937Yunling cattle, YunnanAB520956Cattle, New South Wales of Australia2/491KJ850941Yunling cattle, YunnanAB520955Cattle, Raymond of Australia0/509KJ850943Holstein cattle, JiangsuAB520956Cattle, New South Wales of Australia0/549\\nT. sergenti\\nKJ850933Australian Holstein cattle, JiangsuJQ723015Cattle, Hunan of China0/541KJ850938Yunling cattle, YunnanJQ723015Cattle, Hunan of China0/492\\nT. buffeli\\nKJ850936Leiqiong cattle, HainanHM538196Cattle, Hubei of China;1/508KJ850940Wannan cattle, AnhuiAY661513Cattle, USA0/545\\nT. ovis\\nKJ850942Wuranke sheep, Inner MongoliaFJ603460Sheep, Xinjiang of China;0/529\\nT. luwenshuni\\nKJ850935from Sishui-Fur sheep, ShandongKC769996, JX469518, JF719831Sheep, China0/549KM016463Yangtse River Delta White goat, JiangsuKC769997Goat, Beijing of China0/571\\n\\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\\n\\nThe Theileria rRNA nucleotide sequences obtained in this study that were not identical to existing entries in GenBank were deposited with the following gene accession numbers: KJ850933 and KJ850938 (T. sergenti); KJ850936 and KJ850940 (T. buffeli); KJ850934, KJ850937, KJ850943, KJ850939 and KJ850941 (T. orientalis); KJ850942 (T. ovis); KJ850935 and KM016463 (T. luwenshuni). The sequences obtained were very similar (0–4 nucleotide mismatches) to Theileria spp. sequences deposited by other laboratories in China, USA, France, Australia and Iran (Table 2).Table 2\\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\\nIsolates identified in this studyHighly similar sequences in GenBank\\nTheileriaspp.Gene accession #Source/originGene accession #Source/originMismatches\\nT. orientalis\\nKJ850934Simmental cattle, Inner MongoliaAP011948Cattle, Shintoku of Japan0/547KJ850939Luxi cattle, ShandongHM538220Cattle, Suizhou of China0/547KJ850937Yunling cattle, YunnanAB520956Cattle, New South Wales of Australia2/491KJ850941Yunling cattle, YunnanAB520955Cattle, Raymond of Australia0/509KJ850943Holstein cattle, JiangsuAB520956Cattle, New South Wales of Australia0/549\\nT. sergenti\\nKJ850933Australian Holstein cattle, JiangsuJQ723015Cattle, Hunan of China0/541KJ850938Yunling cattle, YunnanJQ723015Cattle, Hunan of China0/492\\nT. buffeli\\nKJ850936Leiqiong cattle, HainanHM538196Cattle, Hubei of China;1/508KJ850940Wannan cattle, AnhuiAY661513Cattle, USA0/545\\nT. ovis\\nKJ850942Wuranke sheep, Inner MongoliaFJ603460Sheep, Xinjiang of China;0/529\\nT. luwenshuni\\nKJ850935from Sishui-Fur sheep, ShandongKC769996, JX469518, JF719831Sheep, China0/549KM016463Yangtse River Delta White goat, JiangsuKC769997Goat, Beijing of China0/571\\n\\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\\n\",\n \"Comparison of the sequences in the highly conserved region of the Theileria spp. we used showed the region is highly conserved, but is substantially different from those in closely related protozoan species (Figure 1). The two primers and two probes we chose for the pan-Theileria FRET-qPCR had 0–4 nucleotide mismatches with the Theileria spp. in GenBank, but had 25, 23, 22, 8, 16 and 15 mismatches with B. bovis, B. divergens, B. bigemina, C. felis, H. americanum and T. gondii, respectively (Figure 1). The specificity of the pan-Theileria FRET-PCR was further confirmed when it gave positive reactions with the T. orientalis control, but gave negative reactions with DNAs of B. canis, C. felis, H. americanum and T. gondii. The pan-Theileria FRET-qPCR had a specific melting curve (Tm 57.5°C) with Theileria spp. DNA. Using the gel-purified PCR products as quantitative standards, we determined the detection limit of the pan-Theileria FRET-qPCR was 2 copies of the Theileria 18S rRNA gene per reaction for T. orientalis, T. sergenti and T. luwenshuni, T. buffeli and T. ovis.\",\n \"Animals positive for Theileria were found in each of the nine provinces sampled with several animals of each species being positive at each location, except in the case of water buffaloes which were all negative in the one site they were studied. The overall prevalences of Theileria spp. DNA in sheep (53.2%; 59/111) and goats (44.4%; 120/270) were significantly higher than in cattle (30.8%; 380/1,235) (two-tailed Chi-squared Test, P < 10-4). The pan-Theileria FRET-PCR showed that sheep had an average of 102.4 copies of Theileria 18S rRNA/ml whole blood which was significantly lower than the 104.3 copies in cattle and 105.8 copies in goats (Student’s t Test, P < 10-4). While the prevalence of Theileria spp. DNA varied greatly from 3.5% (9/255) in Holsteins from Shanghai to 100% in Luxi cattle from Shandong (40/40) and Leiqiong cattle from Hainan (74/74), the prevalence did not differ significantly in sheep from Inner Mongolia and Shandong (Table 1, Figure 2).Figure 2\\nSites in China where samples ruminants were tested for\\nTheileria\\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\\n\\nSites in China where samples ruminants were tested for\\nTheileria\\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\\nSequencing of 87 randomly selected amplicons (52 from cattle, 14 from goats and 21 from sheep) from Theileria DNA positive samples showed that T. orientalis/ T. sergenti/T. buffeli group [29] were present in cattle while T. luwenshuni was found in goats in Jiangsu province and T. ovis and T. luwenshuni in sheep from Inner Mongolia and Jiangsu province, respectively.\",\n \"When we analyzed factors that might be associated with the prevalence of Theileria spp. DNA in cattle we found that Bos p. indicus animals had significantly higher positivity (77.7% vs. 18.3%; P < 10-4) and copy number of the Theileria 18S rRNA gene (104.81 vs. 103.73 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. Similarly, Bos p. indicus cattle were more likely to be positive (65.2% vs. 13.7%; P < 10-4) and have higher copy numbers of the Theileria 18S rRNA gene (104.88 vs. 103.00 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. The cattle from southern China had significantly higher Theileria 18S rRNA gene copy numbers (104.39 vs. 103.87 copies/per ml whole blood; P = 0.02) than those from northern China but this difference in the prevalence was not significant (31.8% vs. 26.5%). In cattle from Yunnan province where gender information was available, female animals were more commonly positive (76.7% vs. 41.3%; P < 10-4) and copy numbers (105.02 vs. 102.93Theileria/per ml whole blood; P < 10-4) than males.\",\n \"The Theileria rRNA nucleotide sequences obtained in this study that were not identical to existing entries in GenBank were deposited with the following gene accession numbers: KJ850933 and KJ850938 (T. sergenti); KJ850936 and KJ850940 (T. buffeli); KJ850934, KJ850937, KJ850943, KJ850939 and KJ850941 (T. orientalis); KJ850942 (T. ovis); KJ850935 and KM016463 (T. luwenshuni). The sequences obtained were very similar (0–4 nucleotide mismatches) to Theileria spp. sequences deposited by other laboratories in China, USA, France, Australia and Iran (Table 2).Table 2\\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\\nIsolates identified in this studyHighly similar sequences in GenBank\\nTheileriaspp.Gene accession #Source/originGene accession #Source/originMismatches\\nT. orientalis\\nKJ850934Simmental cattle, Inner MongoliaAP011948Cattle, Shintoku of Japan0/547KJ850939Luxi cattle, ShandongHM538220Cattle, Suizhou of China0/547KJ850937Yunling cattle, YunnanAB520956Cattle, New South Wales of Australia2/491KJ850941Yunling cattle, YunnanAB520955Cattle, Raymond of Australia0/509KJ850943Holstein cattle, JiangsuAB520956Cattle, New South Wales of Australia0/549\\nT. sergenti\\nKJ850933Australian Holstein cattle, JiangsuJQ723015Cattle, Hunan of China0/541KJ850938Yunling cattle, YunnanJQ723015Cattle, Hunan of China0/492\\nT. buffeli\\nKJ850936Leiqiong cattle, HainanHM538196Cattle, Hubei of China;1/508KJ850940Wannan cattle, AnhuiAY661513Cattle, USA0/545\\nT. ovis\\nKJ850942Wuranke sheep, Inner MongoliaFJ603460Sheep, Xinjiang of China;0/529\\nT. luwenshuni\\nKJ850935from Sishui-Fur sheep, ShandongKC769996, JX469518, JF719831Sheep, China0/549KM016463Yangtse River Delta White goat, JiangsuKC769997Goat, Beijing of China0/571\\n\\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\\n\",\n \"By systematically aligning the 18S rRNA sequences of representative Theileria spp. and other related protozoa, we identified a highly conserved region to place primers and probes for a pan-Theileria FRET-qPCR. The primers and probes we designed enabled us to amplify all our reference Theileria species with a detection limit of at least 2 Theileria 18S rRNA gene copies per PCR system. None of the other protozoan species tested gave reaction products. To the best of our knowledge, this is the first FRET-qPCR which specifically detects all Theileria species.\\nOur data indicate infections with Theileria spp. are very widespread and common in cattle in China. We found positive animals in each province where we tested and an overall average of 30.77% of animals being positive. This relatively high level of positivity is similar to that obtained in the only other molecular survey for Theileria in China which showed 13.46% being positive in the northeast [32]. Sequencing data showed cattle are infected with the T. orientalis/T. sergenti/T. buffeli group which are generally recognized to be benign species [33] although some strains might cause economic losses [34].\\nAlthough we sequenced relatively few positive amplicons, we found no evidence of the more pathogenic strains, T. annulata and T. parva, which is not unexpected in the case of T. parva which has only been reported from Africa [8]. T. annulata, however, has been reported in H. asiaticum in northwestern China [35]. Our failure to demonstrate the organism in our study might be because T. annulata has an uneven distribution in China.\\nAlthough we found no evidence of Theileria spp. in the 29 water buffaloes we sampled, He [36] reported 58/304 (19.1%) positive for T. buffeli in Hubei province, south China. More extensive studies are necessary to determine the epidemiology of theileriosis in water buffaloes.\\nWe found both goats and sheep were infected with T. luwenshuni (Table 2) using the pan-Theileria FRET-PCR and sequencing. This is a highly pathogenic organism that is known to occur in China where it is transmitted by Haemaphysalis qinghaiensis and may cause significant economic losses. While PCR assays have been described which detect and differentiate T. luwenshuni from T. uilenbergi\\n[37, 38], they do not enable detection of all Theirleria spp. in all species and are thus not as versatile as our pan-Theileria FRET-PCR. Yin et al.\\n[37] found the prevalence of T. luwenshuni varied from 0% to 85% in 4 provinces and we also found a wide range of positive values (4.1% to 67.4%). Such variability is probably due to tick prevalence rates, geoclimatic factors and livestock management systems. Further research using sensitive detection methods will be important in determining the best mechanisms to control infections.\\nT. ovis was first identified in China in 2011 by PCR and sequencing and an infection rate of 78% was found in Xinjiang but no positive animals were found in twelve other provinces [26, 27, 39]. Our study confirms the presence of the organism in China although T. ovis is considered benign and economically unimportant as it might only cause signs in some animals which are stressed [40].\\nIt is well recognized that exotic breeds are more susceptible to disease following infection with Theileria spp. than local stocks. Recent studies, however, have indicated that different breeds of animals might have similar susceptibilities to infections with Theileria spp. although some breeds are more capable of controlling the pathogenic effects of the organism [41].\\nGenerally, B. p. taurus breeds are more susceptible to theileriosis than B. p. indicus breeds which might be associated with innate or immune mechanisms and/or general resistance to ticks. These factors have been mainly investigated with T. parva infections and thus it is interesting that our pan-Theileria FRET-PCR testing showed that in China it is the B. p. indicus breeds that are more likely to be positive than the B. p. taurus breeds and that the former generally had higher copy numbers indicating heavier infections. While our sample sizes were small and we cannot exclude the possibility of sample bias, our findings might be due to host genetic factors relating to infections with less pathogenic Theileria spp. or other factors such as differences in tick control and husbandry practices on different farms. Further studies taking these factors into account are needed to more precisely investigate the relationships between infections with benign Theileria spp. and the genetic background of the host.\",\n \"In summary, our study has described the development and testing of a FRET-PCR which can detect recognized Theileria spp. The pan-Theileria FRET-PCR should be a useful diagnostic tool as it will enable diagnostic laboratories to detect infections in all domestic species with a single test.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,null,null,null,null,"results",null,null,null,null,"discussion","conclusions"],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["Theileria spp","FRET-qPCR","Prevalence","Ruminants"],"string":"[\n \"Theileria spp\",\n \"FRET-qPCR\",\n \"Prevalence\",\n \"Ruminants\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Background:\nTheileria spp. are tick-transmitted, intracellular protozoan parasites infecting leukocytes and erythrocytes of wild and domestic large and small ruminants. Several Theileria spp., transmitted by ixodid ticks of the genera Rhipicephalus, Hyalomma, Amblyomma and Haemaphysalis, have been described in cattle, water buffaloes, sheep and goats in different geographical zones of the world [1–5]. Theileriosis is primarily limited to tropical and sub-tropical areas of the world, with infections mainly reported in Africa and the Middle East but also in southern Europe and northern Asia [6–11]. Infections by Theileria spp. can cause fever, anemia and hemoglobinuria and, in severe cases, death although many species are benign. Animals recovered from acute or primary infections usually remain persistently infected and may act as reservoirs of infecting ticks [12, 13].\nWhile there have been numerous reports of theileriosis in various animal species in China since 1958 [14–27], many have been reported in Chinese and some were based on microscopic detection of parasites which can be difficult with low parasitemia and does not allow ready differentiation of species. Serological studies, although sensitive and easy to perform, are not specific as there is cross reactivity between Theileria spp. Although molecular studies have been performed, these have been to detect Theileria of specific domestic animal species, for example sheep and goats [27]. There have been no highly sensitive and specific molecular methods described which enable studies on various animals from widely divergent areas of China where different Theileria spp. might occur. To address this problem, we developed and validated a highly sensitive genus-specific Theileria FRET-qPCR that detects the recognized Theileria spp. of domestic animals and investigated the molecular prevalence of Theileria in cattle, water buffaloes, goats and sheep from nine provinces in China.\n\nMethods:\n Animals and blood collection Between 2007 and 2013, whole blood samples (around 6 ml) were collected in EDTA from apparently healthy cattle (n = 1,235), water buffaloes (29), goats (270) and sheep (111) from 9 provinces/municipality of China (Table 1). The Bos primigenius (p.) taurus studied (n = 975) were Holsteins, Simmentals, Bohai blacks, Luxis and Wannans while the Bos. p. indicus (n = 260) were the Yunlings, Minnans, and Leiqiongs (Table 1). The water buffaloes, goats and sheep in the study were bred in China and were indigenous breeds. Gender information was available for cattle from Yunnan province. After collection, the blood samples were frozen at -20°C and shipped on ice (over 2 days) to Yangzhou University where they were frozen at -80°C until thawed at room temperature for DNA extraction as described below. This study was reviewed and approved by the Institutional Animal Care and Use Committee of Yangzhou University and animal owners gave written permissions for blood collection.Table 1\nMolecular prevalence of\nTheileria\nspp. in cattle, water buffalo, goat and sheep\nAnimal speciesSubspecies /breedProvinceCityCoordinate of city\nTheileriapositivitypositive /total\n%\nCattle (n = 1235)\nBos p. taurus\nSimmentalInner MongoliaChifeng42.17°N, 118.58°E19/13214.4%Bohai blackShandongBinzhou37.22°N, 118.02°E4/666.1%LuxiShandongJining35.23°N, 116.33°E40/40100%HolsteinJiangsuYancheng33.22°N, 120.08°E72/32122.4%HolsteinJiangsuYangzhou32.23°N, 119.26°E17/14411.8%HolsteinShanghaiShanghai31.14°N, 121.29°E9/2553.5%WannanAnhuiWuhu31.19°N, 118.22°E17/17100%\nBos p. indicus\nYunlingYunnanKunming25.04°N, 102.42°E124/16177.0%MinnanFujianPutian24.26°N, 119.01°E4/2516.0%LeiqiongHainanHaikou20.02°N, 110.20°E74/74100%Water buffalo (n = 29)HaiziJiangsuYancheng33.22°N, 120.08°E0/290%Goat (n = 270)XinjiangXinjiangUrumqi43.45°N, 87.36°E4/984.1%Yangtse River Delta WhiteJiangsuYangzhou32.23°N, 119.26°E116/17267.4%Sheep (n = 111)WurankeInner MongoliaXilingol43.57°N, 116.03°E36/7250.0%Sishui FurShandongJining35.23°N, 116.33°E23/3959.0%\n\nMolecular prevalence of\nTheileria\nspp. in cattle, water buffalo, goat and sheep\n\nBetween 2007 and 2013, whole blood samples (around 6 ml) were collected in EDTA from apparently healthy cattle (n = 1,235), water buffaloes (29), goats (270) and sheep (111) from 9 provinces/municipality of China (Table 1). The Bos primigenius (p.) taurus studied (n = 975) were Holsteins, Simmentals, Bohai blacks, Luxis and Wannans while the Bos. p. indicus (n = 260) were the Yunlings, Minnans, and Leiqiongs (Table 1). The water buffaloes, goats and sheep in the study were bred in China and were indigenous breeds. Gender information was available for cattle from Yunnan province. After collection, the blood samples were frozen at -20°C and shipped on ice (over 2 days) to Yangzhou University where they were frozen at -80°C until thawed at room temperature for DNA extraction as described below. This study was reviewed and approved by the Institutional Animal Care and Use Committee of Yangzhou University and animal owners gave written permissions for blood collection.Table 1\nMolecular prevalence of\nTheileria\nspp. in cattle, water buffalo, goat and sheep\nAnimal speciesSubspecies /breedProvinceCityCoordinate of city\nTheileriapositivitypositive /total\n%\nCattle (n = 1235)\nBos p. taurus\nSimmentalInner MongoliaChifeng42.17°N, 118.58°E19/13214.4%Bohai blackShandongBinzhou37.22°N, 118.02°E4/666.1%LuxiShandongJining35.23°N, 116.33°E40/40100%HolsteinJiangsuYancheng33.22°N, 120.08°E72/32122.4%HolsteinJiangsuYangzhou32.23°N, 119.26°E17/14411.8%HolsteinShanghaiShanghai31.14°N, 121.29°E9/2553.5%WannanAnhuiWuhu31.19°N, 118.22°E17/17100%\nBos p. indicus\nYunlingYunnanKunming25.04°N, 102.42°E124/16177.0%MinnanFujianPutian24.26°N, 119.01°E4/2516.0%LeiqiongHainanHaikou20.02°N, 110.20°E74/74100%Water buffalo (n = 29)HaiziJiangsuYancheng33.22°N, 120.08°E0/290%Goat (n = 270)XinjiangXinjiangUrumqi43.45°N, 87.36°E4/984.1%Yangtse River Delta WhiteJiangsuYangzhou32.23°N, 119.26°E116/17267.4%Sheep (n = 111)WurankeInner MongoliaXilingol43.57°N, 116.03°E36/7250.0%Sishui FurShandongJining35.23°N, 116.33°E23/3959.0%\n\nMolecular prevalence of\nTheileria\nspp. in cattle, water buffalo, goat and sheep\n\n DNA extraction DNA was extracted from whole blood samples using a standard phenol-chloroform method previously described [28]. Two ml whole blood was used to extract DNA which was resuspended into 200 μl 1 × T10E0.1 buffer. The concentration of the extracted DNA was established with a Microscale Ultraviolet Spectrophotometer. Negative controls consisting of sterile molecular grade water were used to detect cross- contamination during DNA extraction and processing. The HMBS-based FRET-PCR was performed to verify if the extracted DNAs from blood samples were appropriate for molecular detection of tick-borne pathogens [29, 30].\nDNA was extracted from whole blood samples using a standard phenol-chloroform method previously described [28]. Two ml whole blood was used to extract DNA which was resuspended into 200 μl 1 × T10E0.1 buffer. The concentration of the extracted DNA was established with a Microscale Ultraviolet Spectrophotometer. Negative controls consisting of sterile molecular grade water were used to detect cross- contamination during DNA extraction and processing. The HMBS-based FRET-PCR was performed to verify if the extracted DNAs from blood samples were appropriate for molecular detection of tick-borne pathogens [29, 30].\n Theileriaspp.FRET-qPCR Primers and probes The 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\nThe 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n Thermal cycling The Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\nThe Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\n Specificity PCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\nPCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\n Sensitivity For use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\nFor use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\n Primers and probes The 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\nThe 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n Thermal cycling The Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\nThe Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\n Specificity PCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\nPCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\n Sensitivity For use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\nFor use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\n Identification of Theileriaspp. by PCR and sequencing The amplicon of the pan-Theileria FRET-qPCR we established has a sequence which is highly conserved among the different Theileria species. To differentiate Theileria spp. in a positive reaction, we used a standard PCR to amplify a highly polymorphic region of the 18S rRNA gene (591–594 nucleotides for different Theileria spp.) and sequenced the products (GenScript, Nanjing, Jiangsu, China). For the PCR we designed a forward primer (5′-CCTGAGAAACGGCTACCACATCT-3′) that amplified all Theileria species and used a previously described reverse primer (5′-GGACTACGACGGTATCTGATCG-3′) that also amplified all species [31].\nThe amplicon of the pan-Theileria FRET-qPCR we established has a sequence which is highly conserved among the different Theileria species. To differentiate Theileria spp. in a positive reaction, we used a standard PCR to amplify a highly polymorphic region of the 18S rRNA gene (591–594 nucleotides for different Theileria spp.) and sequenced the products (GenScript, Nanjing, Jiangsu, China). For the PCR we designed a forward primer (5′-CCTGAGAAACGGCTACCACATCT-3′) that amplified all Theileria species and used a previously described reverse primer (5′-GGACTACGACGGTATCTGATCG-3′) that also amplified all species [31].\n Statistical analysis Differences in positivity of Theileria spp. were analyzed by Chi-squared Test while numbers of copies of the Theileria 18S rRNA gene determined in the Theileria FRET-qPCR were log10-transformed and analyzed using the Student’s T-test. Differences of P < 0.05 were considered statistically significant.\nDifferences in positivity of Theileria spp. were analyzed by Chi-squared Test while numbers of copies of the Theileria 18S rRNA gene determined in the Theileria FRET-qPCR were log10-transformed and analyzed using the Student’s T-test. Differences of P < 0.05 were considered statistically significant.\n\nAnimals and blood collection:\nBetween 2007 and 2013, whole blood samples (around 6 ml) were collected in EDTA from apparently healthy cattle (n = 1,235), water buffaloes (29), goats (270) and sheep (111) from 9 provinces/municipality of China (Table 1). The Bos primigenius (p.) taurus studied (n = 975) were Holsteins, Simmentals, Bohai blacks, Luxis and Wannans while the Bos. p. indicus (n = 260) were the Yunlings, Minnans, and Leiqiongs (Table 1). The water buffaloes, goats and sheep in the study were bred in China and were indigenous breeds. Gender information was available for cattle from Yunnan province. After collection, the blood samples were frozen at -20°C and shipped on ice (over 2 days) to Yangzhou University where they were frozen at -80°C until thawed at room temperature for DNA extraction as described below. This study was reviewed and approved by the Institutional Animal Care and Use Committee of Yangzhou University and animal owners gave written permissions for blood collection.Table 1\nMolecular prevalence of\nTheileria\nspp. in cattle, water buffalo, goat and sheep\nAnimal speciesSubspecies /breedProvinceCityCoordinate of city\nTheileriapositivitypositive /total\n%\nCattle (n = 1235)\nBos p. taurus\nSimmentalInner MongoliaChifeng42.17°N, 118.58°E19/13214.4%Bohai blackShandongBinzhou37.22°N, 118.02°E4/666.1%LuxiShandongJining35.23°N, 116.33°E40/40100%HolsteinJiangsuYancheng33.22°N, 120.08°E72/32122.4%HolsteinJiangsuYangzhou32.23°N, 119.26°E17/14411.8%HolsteinShanghaiShanghai31.14°N, 121.29°E9/2553.5%WannanAnhuiWuhu31.19°N, 118.22°E17/17100%\nBos p. indicus\nYunlingYunnanKunming25.04°N, 102.42°E124/16177.0%MinnanFujianPutian24.26°N, 119.01°E4/2516.0%LeiqiongHainanHaikou20.02°N, 110.20°E74/74100%Water buffalo (n = 29)HaiziJiangsuYancheng33.22°N, 120.08°E0/290%Goat (n = 270)XinjiangXinjiangUrumqi43.45°N, 87.36°E4/984.1%Yangtse River Delta WhiteJiangsuYangzhou32.23°N, 119.26°E116/17267.4%Sheep (n = 111)WurankeInner MongoliaXilingol43.57°N, 116.03°E36/7250.0%Sishui FurShandongJining35.23°N, 116.33°E23/3959.0%\n\nMolecular prevalence of\nTheileria\nspp. in cattle, water buffalo, goat and sheep\n\n\nDNA extraction:\nDNA was extracted from whole blood samples using a standard phenol-chloroform method previously described [28]. Two ml whole blood was used to extract DNA which was resuspended into 200 μl 1 × T10E0.1 buffer. The concentration of the extracted DNA was established with a Microscale Ultraviolet Spectrophotometer. Negative controls consisting of sterile molecular grade water were used to detect cross- contamination during DNA extraction and processing. The HMBS-based FRET-PCR was performed to verify if the extracted DNAs from blood samples were appropriate for molecular detection of tick-borne pathogens [29, 30].\n\nTheileriaspp.FRET-qPCR:\n Primers and probes The 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\nThe 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n Thermal cycling The Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\nThe Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\n Specificity PCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\nPCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\n Sensitivity For use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\nFor use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\n\nPrimers and probes:\nThe 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.Figure 1\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n\nAlignment of oligonucleotides for\nTheileria\nPCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.\n\nThermal cycling:\nThe Theileria FRET-PCR was performed in a LightCycler 480®II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.\n\nSpecificity:\nPCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).\n\nSensitivity:\nFor use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.\n\nIdentification of Theileriaspp. by PCR and sequencing:\nThe amplicon of the pan-Theileria FRET-qPCR we established has a sequence which is highly conserved among the different Theileria species. To differentiate Theileria spp. in a positive reaction, we used a standard PCR to amplify a highly polymorphic region of the 18S rRNA gene (591–594 nucleotides for different Theileria spp.) and sequenced the products (GenScript, Nanjing, Jiangsu, China). For the PCR we designed a forward primer (5′-CCTGAGAAACGGCTACCACATCT-3′) that amplified all Theileria species and used a previously described reverse primer (5′-GGACTACGACGGTATCTGATCG-3′) that also amplified all species [31].\n\nStatistical analysis:\nDifferences in positivity of Theileria spp. were analyzed by Chi-squared Test while numbers of copies of the Theileria 18S rRNA gene determined in the Theileria FRET-qPCR were log10-transformed and analyzed using the Student’s T-test. Differences of P < 0.05 were considered statistically significant.\n\nResults:\n Development of the pan-TheileriaFRET-PCR Comparison of the sequences in the highly conserved region of the Theileria spp. we used showed the region is highly conserved, but is substantially different from those in closely related protozoan species (Figure 1). The two primers and two probes we chose for the pan-Theileria FRET-qPCR had 0–4 nucleotide mismatches with the Theileria spp. in GenBank, but had 25, 23, 22, 8, 16 and 15 mismatches with B. bovis, B. divergens, B. bigemina, C. felis, H. americanum and T. gondii, respectively (Figure 1). The specificity of the pan-Theileria FRET-PCR was further confirmed when it gave positive reactions with the T. orientalis control, but gave negative reactions with DNAs of B. canis, C. felis, H. americanum and T. gondii. The pan-Theileria FRET-qPCR had a specific melting curve (Tm 57.5°C) with Theileria spp. DNA. Using the gel-purified PCR products as quantitative standards, we determined the detection limit of the pan-Theileria FRET-qPCR was 2 copies of the Theileria 18S rRNA gene per reaction for T. orientalis, T. sergenti and T. luwenshuni, T. buffeli and T. ovis.\nComparison of the sequences in the highly conserved region of the Theileria spp. we used showed the region is highly conserved, but is substantially different from those in closely related protozoan species (Figure 1). The two primers and two probes we chose for the pan-Theileria FRET-qPCR had 0–4 nucleotide mismatches with the Theileria spp. in GenBank, but had 25, 23, 22, 8, 16 and 15 mismatches with B. bovis, B. divergens, B. bigemina, C. felis, H. americanum and T. gondii, respectively (Figure 1). The specificity of the pan-Theileria FRET-PCR was further confirmed when it gave positive reactions with the T. orientalis control, but gave negative reactions with DNAs of B. canis, C. felis, H. americanum and T. gondii. The pan-Theileria FRET-qPCR had a specific melting curve (Tm 57.5°C) with Theileria spp. DNA. Using the gel-purified PCR products as quantitative standards, we determined the detection limit of the pan-Theileria FRET-qPCR was 2 copies of the Theileria 18S rRNA gene per reaction for T. orientalis, T. sergenti and T. luwenshuni, T. buffeli and T. ovis.\n Prevalence of Theileriaspp. DNA in ruminants Animals positive for Theileria were found in each of the nine provinces sampled with several animals of each species being positive at each location, except in the case of water buffaloes which were all negative in the one site they were studied. The overall prevalences of Theileria spp. DNA in sheep (53.2%; 59/111) and goats (44.4%; 120/270) were significantly higher than in cattle (30.8%; 380/1,235) (two-tailed Chi-squared Test, P < 10-4). The pan-Theileria FRET-PCR showed that sheep had an average of 102.4 copies of Theileria 18S rRNA/ml whole blood which was significantly lower than the 104.3 copies in cattle and 105.8 copies in goats (Student’s t Test, P < 10-4). While the prevalence of Theileria spp. DNA varied greatly from 3.5% (9/255) in Holsteins from Shanghai to 100% in Luxi cattle from Shandong (40/40) and Leiqiong cattle from Hainan (74/74), the prevalence did not differ significantly in sheep from Inner Mongolia and Shandong (Table 1, Figure 2).Figure 2\nSites in China where samples ruminants were tested for\nTheileria\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\n\nSites in China where samples ruminants were tested for\nTheileria\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\nSequencing of 87 randomly selected amplicons (52 from cattle, 14 from goats and 21 from sheep) from Theileria DNA positive samples showed that T. orientalis/ T. sergenti/T. buffeli group [29] were present in cattle while T. luwenshuni was found in goats in Jiangsu province and T. ovis and T. luwenshuni in sheep from Inner Mongolia and Jiangsu province, respectively.\nAnimals positive for Theileria were found in each of the nine provinces sampled with several animals of each species being positive at each location, except in the case of water buffaloes which were all negative in the one site they were studied. The overall prevalences of Theileria spp. DNA in sheep (53.2%; 59/111) and goats (44.4%; 120/270) were significantly higher than in cattle (30.8%; 380/1,235) (two-tailed Chi-squared Test, P < 10-4). The pan-Theileria FRET-PCR showed that sheep had an average of 102.4 copies of Theileria 18S rRNA/ml whole blood which was significantly lower than the 104.3 copies in cattle and 105.8 copies in goats (Student’s t Test, P < 10-4). While the prevalence of Theileria spp. DNA varied greatly from 3.5% (9/255) in Holsteins from Shanghai to 100% in Luxi cattle from Shandong (40/40) and Leiqiong cattle from Hainan (74/74), the prevalence did not differ significantly in sheep from Inner Mongolia and Shandong (Table 1, Figure 2).Figure 2\nSites in China where samples ruminants were tested for\nTheileria\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\n\nSites in China where samples ruminants were tested for\nTheileria\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\nSequencing of 87 randomly selected amplicons (52 from cattle, 14 from goats and 21 from sheep) from Theileria DNA positive samples showed that T. orientalis/ T. sergenti/T. buffeli group [29] were present in cattle while T. luwenshuni was found in goats in Jiangsu province and T. ovis and T. luwenshuni in sheep from Inner Mongolia and Jiangsu province, respectively.\n Factors associated with the occurrence of theileriosis in cattle When we analyzed factors that might be associated with the prevalence of Theileria spp. DNA in cattle we found that Bos p. indicus animals had significantly higher positivity (77.7% vs. 18.3%; P < 10-4) and copy number of the Theileria 18S rRNA gene (104.81 vs. 103.73 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. Similarly, Bos p. indicus cattle were more likely to be positive (65.2% vs. 13.7%; P < 10-4) and have higher copy numbers of the Theileria 18S rRNA gene (104.88 vs. 103.00 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. The cattle from southern China had significantly higher Theileria 18S rRNA gene copy numbers (104.39 vs. 103.87 copies/per ml whole blood; P = 0.02) than those from northern China but this difference in the prevalence was not significant (31.8% vs. 26.5%). In cattle from Yunnan province where gender information was available, female animals were more commonly positive (76.7% vs. 41.3%; P < 10-4) and copy numbers (105.02 vs. 102.93Theileria/per ml whole blood; P < 10-4) than males.\nWhen we analyzed factors that might be associated with the prevalence of Theileria spp. DNA in cattle we found that Bos p. indicus animals had significantly higher positivity (77.7% vs. 18.3%; P < 10-4) and copy number of the Theileria 18S rRNA gene (104.81 vs. 103.73 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. Similarly, Bos p. indicus cattle were more likely to be positive (65.2% vs. 13.7%; P < 10-4) and have higher copy numbers of the Theileria 18S rRNA gene (104.88 vs. 103.00 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. The cattle from southern China had significantly higher Theileria 18S rRNA gene copy numbers (104.39 vs. 103.87 copies/per ml whole blood; P = 0.02) than those from northern China but this difference in the prevalence was not significant (31.8% vs. 26.5%). In cattle from Yunnan province where gender information was available, female animals were more commonly positive (76.7% vs. 41.3%; P < 10-4) and copy numbers (105.02 vs. 102.93Theileria/per ml whole blood; P < 10-4) than males.\n Gene accession numbers The Theileria rRNA nucleotide sequences obtained in this study that were not identical to existing entries in GenBank were deposited with the following gene accession numbers: KJ850933 and KJ850938 (T. sergenti); KJ850936 and KJ850940 (T. buffeli); KJ850934, KJ850937, KJ850943, KJ850939 and KJ850941 (T. orientalis); KJ850942 (T. ovis); KJ850935 and KM016463 (T. luwenshuni). The sequences obtained were very similar (0–4 nucleotide mismatches) to Theileria spp. sequences deposited by other laboratories in China, USA, France, Australia and Iran (Table 2).Table 2\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\nIsolates identified in this studyHighly similar sequences in GenBank\nTheileriaspp.Gene accession #Source/originGene accession #Source/originMismatches\nT. orientalis\nKJ850934Simmental cattle, Inner MongoliaAP011948Cattle, Shintoku of Japan0/547KJ850939Luxi cattle, ShandongHM538220Cattle, Suizhou of China0/547KJ850937Yunling cattle, YunnanAB520956Cattle, New South Wales of Australia2/491KJ850941Yunling cattle, YunnanAB520955Cattle, Raymond of Australia0/509KJ850943Holstein cattle, JiangsuAB520956Cattle, New South Wales of Australia0/549\nT. sergenti\nKJ850933Australian Holstein cattle, JiangsuJQ723015Cattle, Hunan of China0/541KJ850938Yunling cattle, YunnanJQ723015Cattle, Hunan of China0/492\nT. buffeli\nKJ850936Leiqiong cattle, HainanHM538196Cattle, Hubei of China;1/508KJ850940Wannan cattle, AnhuiAY661513Cattle, USA0/545\nT. ovis\nKJ850942Wuranke sheep, Inner MongoliaFJ603460Sheep, Xinjiang of China;0/529\nT. luwenshuni\nKJ850935from Sishui-Fur sheep, ShandongKC769996, JX469518, JF719831Sheep, China0/549KM016463Yangtse River Delta White goat, JiangsuKC769997Goat, Beijing of China0/571\n\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\n\nThe Theileria rRNA nucleotide sequences obtained in this study that were not identical to existing entries in GenBank were deposited with the following gene accession numbers: KJ850933 and KJ850938 (T. sergenti); KJ850936 and KJ850940 (T. buffeli); KJ850934, KJ850937, KJ850943, KJ850939 and KJ850941 (T. orientalis); KJ850942 (T. ovis); KJ850935 and KM016463 (T. luwenshuni). The sequences obtained were very similar (0–4 nucleotide mismatches) to Theileria spp. sequences deposited by other laboratories in China, USA, France, Australia and Iran (Table 2).Table 2\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\nIsolates identified in this studyHighly similar sequences in GenBank\nTheileriaspp.Gene accession #Source/originGene accession #Source/originMismatches\nT. orientalis\nKJ850934Simmental cattle, Inner MongoliaAP011948Cattle, Shintoku of Japan0/547KJ850939Luxi cattle, ShandongHM538220Cattle, Suizhou of China0/547KJ850937Yunling cattle, YunnanAB520956Cattle, New South Wales of Australia2/491KJ850941Yunling cattle, YunnanAB520955Cattle, Raymond of Australia0/509KJ850943Holstein cattle, JiangsuAB520956Cattle, New South Wales of Australia0/549\nT. sergenti\nKJ850933Australian Holstein cattle, JiangsuJQ723015Cattle, Hunan of China0/541KJ850938Yunling cattle, YunnanJQ723015Cattle, Hunan of China0/492\nT. buffeli\nKJ850936Leiqiong cattle, HainanHM538196Cattle, Hubei of China;1/508KJ850940Wannan cattle, AnhuiAY661513Cattle, USA0/545\nT. ovis\nKJ850942Wuranke sheep, Inner MongoliaFJ603460Sheep, Xinjiang of China;0/529\nT. luwenshuni\nKJ850935from Sishui-Fur sheep, ShandongKC769996, JX469518, JF719831Sheep, China0/549KM016463Yangtse River Delta White goat, JiangsuKC769997Goat, Beijing of China0/571\n\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\n\n\nDevelopment of the pan-TheileriaFRET-PCR:\nComparison of the sequences in the highly conserved region of the Theileria spp. we used showed the region is highly conserved, but is substantially different from those in closely related protozoan species (Figure 1). The two primers and two probes we chose for the pan-Theileria FRET-qPCR had 0–4 nucleotide mismatches with the Theileria spp. in GenBank, but had 25, 23, 22, 8, 16 and 15 mismatches with B. bovis, B. divergens, B. bigemina, C. felis, H. americanum and T. gondii, respectively (Figure 1). The specificity of the pan-Theileria FRET-PCR was further confirmed when it gave positive reactions with the T. orientalis control, but gave negative reactions with DNAs of B. canis, C. felis, H. americanum and T. gondii. The pan-Theileria FRET-qPCR had a specific melting curve (Tm 57.5°C) with Theileria spp. DNA. Using the gel-purified PCR products as quantitative standards, we determined the detection limit of the pan-Theileria FRET-qPCR was 2 copies of the Theileria 18S rRNA gene per reaction for T. orientalis, T. sergenti and T. luwenshuni, T. buffeli and T. ovis.\n\nPrevalence of Theileriaspp. DNA in ruminants:\nAnimals positive for Theileria were found in each of the nine provinces sampled with several animals of each species being positive at each location, except in the case of water buffaloes which were all negative in the one site they were studied. The overall prevalences of Theileria spp. DNA in sheep (53.2%; 59/111) and goats (44.4%; 120/270) were significantly higher than in cattle (30.8%; 380/1,235) (two-tailed Chi-squared Test, P < 10-4). The pan-Theileria FRET-PCR showed that sheep had an average of 102.4 copies of Theileria 18S rRNA/ml whole blood which was significantly lower than the 104.3 copies in cattle and 105.8 copies in goats (Student’s t Test, P < 10-4). While the prevalence of Theileria spp. DNA varied greatly from 3.5% (9/255) in Holsteins from Shanghai to 100% in Luxi cattle from Shandong (40/40) and Leiqiong cattle from Hainan (74/74), the prevalence did not differ significantly in sheep from Inner Mongolia and Shandong (Table 1, Figure 2).Figure 2\nSites in China where samples ruminants were tested for\nTheileria\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\n\nSites in China where samples ruminants were tested for\nTheileria\nspp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.\nSequencing of 87 randomly selected amplicons (52 from cattle, 14 from goats and 21 from sheep) from Theileria DNA positive samples showed that T. orientalis/ T. sergenti/T. buffeli group [29] were present in cattle while T. luwenshuni was found in goats in Jiangsu province and T. ovis and T. luwenshuni in sheep from Inner Mongolia and Jiangsu province, respectively.\n\nFactors associated with the occurrence of theileriosis in cattle:\nWhen we analyzed factors that might be associated with the prevalence of Theileria spp. DNA in cattle we found that Bos p. indicus animals had significantly higher positivity (77.7% vs. 18.3%; P < 10-4) and copy number of the Theileria 18S rRNA gene (104.81 vs. 103.73 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. Similarly, Bos p. indicus cattle were more likely to be positive (65.2% vs. 13.7%; P < 10-4) and have higher copy numbers of the Theileria 18S rRNA gene (104.88 vs. 103.00 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. The cattle from southern China had significantly higher Theileria 18S rRNA gene copy numbers (104.39 vs. 103.87 copies/per ml whole blood; P = 0.02) than those from northern China but this difference in the prevalence was not significant (31.8% vs. 26.5%). In cattle from Yunnan province where gender information was available, female animals were more commonly positive (76.7% vs. 41.3%; P < 10-4) and copy numbers (105.02 vs. 102.93Theileria/per ml whole blood; P < 10-4) than males.\n\nGene accession numbers:\nThe Theileria rRNA nucleotide sequences obtained in this study that were not identical to existing entries in GenBank were deposited with the following gene accession numbers: KJ850933 and KJ850938 (T. sergenti); KJ850936 and KJ850940 (T. buffeli); KJ850934, KJ850937, KJ850943, KJ850939 and KJ850941 (T. orientalis); KJ850942 (T. ovis); KJ850935 and KM016463 (T. luwenshuni). The sequences obtained were very similar (0–4 nucleotide mismatches) to Theileria spp. sequences deposited by other laboratories in China, USA, France, Australia and Iran (Table 2).Table 2\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\nIsolates identified in this studyHighly similar sequences in GenBank\nTheileriaspp.Gene accession #Source/originGene accession #Source/originMismatches\nT. orientalis\nKJ850934Simmental cattle, Inner MongoliaAP011948Cattle, Shintoku of Japan0/547KJ850939Luxi cattle, ShandongHM538220Cattle, Suizhou of China0/547KJ850937Yunling cattle, YunnanAB520956Cattle, New South Wales of Australia2/491KJ850941Yunling cattle, YunnanAB520955Cattle, Raymond of Australia0/509KJ850943Holstein cattle, JiangsuAB520956Cattle, New South Wales of Australia0/549\nT. sergenti\nKJ850933Australian Holstein cattle, JiangsuJQ723015Cattle, Hunan of China0/541KJ850938Yunling cattle, YunnanJQ723015Cattle, Hunan of China0/492\nT. buffeli\nKJ850936Leiqiong cattle, HainanHM538196Cattle, Hubei of China;1/508KJ850940Wannan cattle, AnhuiAY661513Cattle, USA0/545\nT. ovis\nKJ850942Wuranke sheep, Inner MongoliaFJ603460Sheep, Xinjiang of China;0/529\nT. luwenshuni\nKJ850935from Sishui-Fur sheep, ShandongKC769996, JX469518, JF719831Sheep, China0/549KM016463Yangtse River Delta White goat, JiangsuKC769997Goat, Beijing of China0/571\n\nComparison of isolates identified in this study and similar sequences in GenBank by BLASTN\n\n\nDiscussion:\nBy systematically aligning the 18S rRNA sequences of representative Theileria spp. and other related protozoa, we identified a highly conserved region to place primers and probes for a pan-Theileria FRET-qPCR. The primers and probes we designed enabled us to amplify all our reference Theileria species with a detection limit of at least 2 Theileria 18S rRNA gene copies per PCR system. None of the other protozoan species tested gave reaction products. To the best of our knowledge, this is the first FRET-qPCR which specifically detects all Theileria species.\nOur data indicate infections with Theileria spp. are very widespread and common in cattle in China. We found positive animals in each province where we tested and an overall average of 30.77% of animals being positive. This relatively high level of positivity is similar to that obtained in the only other molecular survey for Theileria in China which showed 13.46% being positive in the northeast [32]. Sequencing data showed cattle are infected with the T. orientalis/T. sergenti/T. buffeli group which are generally recognized to be benign species [33] although some strains might cause economic losses [34].\nAlthough we sequenced relatively few positive amplicons, we found no evidence of the more pathogenic strains, T. annulata and T. parva, which is not unexpected in the case of T. parva which has only been reported from Africa [8]. T. annulata, however, has been reported in H. asiaticum in northwestern China [35]. Our failure to demonstrate the organism in our study might be because T. annulata has an uneven distribution in China.\nAlthough we found no evidence of Theileria spp. in the 29 water buffaloes we sampled, He [36] reported 58/304 (19.1%) positive for T. buffeli in Hubei province, south China. More extensive studies are necessary to determine the epidemiology of theileriosis in water buffaloes.\nWe found both goats and sheep were infected with T. luwenshuni (Table 2) using the pan-Theileria FRET-PCR and sequencing. This is a highly pathogenic organism that is known to occur in China where it is transmitted by Haemaphysalis qinghaiensis and may cause significant economic losses. While PCR assays have been described which detect and differentiate T. luwenshuni from T. uilenbergi\n[37, 38], they do not enable detection of all Theirleria spp. in all species and are thus not as versatile as our pan-Theileria FRET-PCR. Yin et al.\n[37] found the prevalence of T. luwenshuni varied from 0% to 85% in 4 provinces and we also found a wide range of positive values (4.1% to 67.4%). Such variability is probably due to tick prevalence rates, geoclimatic factors and livestock management systems. Further research using sensitive detection methods will be important in determining the best mechanisms to control infections.\nT. ovis was first identified in China in 2011 by PCR and sequencing and an infection rate of 78% was found in Xinjiang but no positive animals were found in twelve other provinces [26, 27, 39]. Our study confirms the presence of the organism in China although T. ovis is considered benign and economically unimportant as it might only cause signs in some animals which are stressed [40].\nIt is well recognized that exotic breeds are more susceptible to disease following infection with Theileria spp. than local stocks. Recent studies, however, have indicated that different breeds of animals might have similar susceptibilities to infections with Theileria spp. although some breeds are more capable of controlling the pathogenic effects of the organism [41].\nGenerally, B. p. taurus breeds are more susceptible to theileriosis than B. p. indicus breeds which might be associated with innate or immune mechanisms and/or general resistance to ticks. These factors have been mainly investigated with T. parva infections and thus it is interesting that our pan-Theileria FRET-PCR testing showed that in China it is the B. p. indicus breeds that are more likely to be positive than the B. p. taurus breeds and that the former generally had higher copy numbers indicating heavier infections. While our sample sizes were small and we cannot exclude the possibility of sample bias, our findings might be due to host genetic factors relating to infections with less pathogenic Theileria spp. or other factors such as differences in tick control and husbandry practices on different farms. Further studies taking these factors into account are needed to more precisely investigate the relationships between infections with benign Theileria spp. and the genetic background of the host.\n\nConclusion:\nIn summary, our study has described the development and testing of a FRET-PCR which can detect recognized Theileria spp. The pan-Theileria FRET-PCR should be a useful diagnostic tool as it will enable diagnostic laboratories to detect infections in all domestic species with a single test.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nTheileria spp. are tick transmitted protozoa that can infect large and small ruminants causing disease and economic losses. Diagnosis of infections is often challenging, as parasites can be difficult to detect and identify microscopically and serology is unreliable. While there are PCR assays which can identify certain Theileria spp., there is no one PCR that has been designed to identify all recognized species that occur in ruminants and which will greatly simplify the laboratory diagnoses of infections.\n\nMethods:\nPrimers and probes for a genus-specific pan-Theileria FRET-qPCR were selected by comparing sequences of recognized Theileria spp. in GenBank and the test validated using reference organisms. The assay was also tested on whole blood samples from large and small ruminants from nine provinces in China.\n\nResults:\nThe pan-Theileria FRET-qPCR detected all recognized species but none of the closely related protozoa. In whole blood samples from animals in China, Theileria spp. DNA was detected in 53.2% of the sheep tested (59/111), 44.4% of the goats (120/270) and 30.8% of the cattle (380/1,235). Water buffaloes (n = 29) were negative. Sequencing of some of the PCR products showed cattle in China were infected with T. orientalis/T. sergenti/T. buffeli group while T. ovis and T. luwenshuni were found in sheep and T. luwenshuni in goats. The prevalence of Theileria DNA was significantly higher in Bos p. indicus than in Bos p. taurus (77.7% vs. 18.3%) and copy numbers were also significantly higher (10(4.88) vs. 10(3.00) Theileria 18S rRNA gene copies/per ml whole blood).\n\nConclusions:\nThe pan-Theileria FRET-qPCR can detect all recognized Theileria spp. of ruminants in a single reaction. Large and small ruminants in China are commonly infected with a variety of Theileria spp."},"background_conclusion_text":{"kind":"string","value":"Background:\nTheileria spp. are tick-transmitted, intracellular protozoan parasites infecting leukocytes and erythrocytes of wild and domestic large and small ruminants. Several Theileria spp., transmitted by ixodid ticks of the genera Rhipicephalus, Hyalomma, Amblyomma and Haemaphysalis, have been described in cattle, water buffaloes, sheep and goats in different geographical zones of the world [1–5]. Theileriosis is primarily limited to tropical and sub-tropical areas of the world, with infections mainly reported in Africa and the Middle East but also in southern Europe and northern Asia [6–11]. Infections by Theileria spp. can cause fever, anemia and hemoglobinuria and, in severe cases, death although many species are benign. Animals recovered from acute or primary infections usually remain persistently infected and may act as reservoirs of infecting ticks [12, 13].\nWhile there have been numerous reports of theileriosis in various animal species in China since 1958 [14–27], many have been reported in Chinese and some were based on microscopic detection of parasites which can be difficult with low parasitemia and does not allow ready differentiation of species. Serological studies, although sensitive and easy to perform, are not specific as there is cross reactivity between Theileria spp. Although molecular studies have been performed, these have been to detect Theileria of specific domestic animal species, for example sheep and goats [27]. There have been no highly sensitive and specific molecular methods described which enable studies on various animals from widely divergent areas of China where different Theileria spp. might occur. To address this problem, we developed and validated a highly sensitive genus-specific Theileria FRET-qPCR that detects the recognized Theileria spp. of domestic animals and investigated the molecular prevalence of Theileria in cattle, water buffaloes, goats and sheep from nine provinces in China.\n\nConclusion:\nIn summary, our study has described the development and testing of a FRET-PCR which can detect recognized Theileria spp. The pan-Theileria FRET-PCR should be a useful diagnostic tool as it will enable diagnostic laboratories to detect infections in all domestic species with a single test."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nTheileria spp. are tick transmitted protozoa that can infect large and small ruminants causing disease and economic losses. Diagnosis of infections is often challenging, as parasites can be difficult to detect and identify microscopically and serology is unreliable. While there are PCR assays which can identify certain Theileria spp., there is no one PCR that has been designed to identify all recognized species that occur in ruminants and which will greatly simplify the laboratory diagnoses of infections.\n\nMethods:\nPrimers and probes for a genus-specific pan-Theileria FRET-qPCR were selected by comparing sequences of recognized Theileria spp. in GenBank and the test validated using reference organisms. The assay was also tested on whole blood samples from large and small ruminants from nine provinces in China.\n\nResults:\nThe pan-Theileria FRET-qPCR detected all recognized species but none of the closely related protozoa. In whole blood samples from animals in China, Theileria spp. DNA was detected in 53.2% of the sheep tested (59/111), 44.4% of the goats (120/270) and 30.8% of the cattle (380/1,235). Water buffaloes (n = 29) were negative. Sequencing of some of the PCR products showed cattle in China were infected with T. orientalis/T. sergenti/T. buffeli group while T. ovis and T. luwenshuni were found in sheep and T. luwenshuni in goats. The prevalence of Theileria DNA was significantly higher in Bos p. indicus than in Bos p. taurus (77.7% vs. 18.3%) and copy numbers were also significantly higher (10(4.88) vs. 10(3.00) Theileria 18S rRNA gene copies/per ml whole blood).\n\nConclusions:\nThe pan-Theileria FRET-qPCR can detect all recognized Theileria spp. of ruminants in a single reaction. Large and small ruminants in China are commonly infected with a variety of Theileria spp."},"whole_article_text_length":{"kind":"number","value":15204,"string":"15,204"},"whole_article_abstract_length":{"kind":"number","value":353,"string":"353"},"other_sections_lengths":{"kind":"list like","value":[338,401,111,2379,660,203,141,176,109,57,226,385,250,283],"string":"[\n 338,\n 401,\n 111,\n 2379,\n 660,\n 203,\n 141,\n 176,\n 109,\n 57,\n 226,\n 385,\n 250,\n 283\n]","isConversational":false},"num_sections":{"kind":"number","value":18,"string":"18"},"most_frequent_words":{"kind":"list like","value":["theileria","mismatches","pcr","species","spp","theileria spp","sec","cattle","gene","probes"],"string":"[\n \"theileria\",\n \"mismatches\",\n \"pcr\",\n \"species\",\n \"spp\",\n \"theileria spp\",\n \"sec\",\n \"cattle\",\n \"gene\",\n \"probes\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["pathogenic theileria spp","tick borne pathogens","occurrence theileriosis cattle","theileria spp widespread","prevalence theileria cattle"],"string":"[\n \"pathogenic theileria spp\",\n \"tick borne pathogens\",\n \"occurrence theileriosis cattle\",\n \"theileria spp widespread\",\n \"prevalence theileria cattle\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Theileria spp | FRET-qPCR | Prevalence | Ruminants [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Theileria spp | FRET-qPCR | Prevalence | Ruminants [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Theileria spp | FRET-qPCR | Prevalence | Ruminants [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Theileria spp | FRET-qPCR | Prevalence | Ruminants [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Theileria spp | FRET-qPCR | Prevalence | Ruminants [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Theileria spp | FRET-qPCR | Prevalence | Ruminants [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | DNA, Protozoan | Molecular Sequence Data | Polymerase Chain Reaction | Prevalence | Ruminants | Sensitivity and Specificity | Theileria | Theileriasis [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | DNA, Protozoan | Molecular Sequence Data | Polymerase Chain Reaction | Prevalence | Ruminants | Sensitivity and Specificity | Theileria | Theileriasis [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | DNA, Protozoan | Molecular Sequence Data | Polymerase Chain Reaction | Prevalence | Ruminants | Sensitivity and Specificity | Theileria | Theileriasis [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | DNA, Protozoan | Molecular Sequence Data | Polymerase Chain Reaction | Prevalence | Ruminants | Sensitivity and Specificity | Theileria | Theileriasis [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | DNA, Protozoan | Molecular Sequence Data | Polymerase Chain Reaction | Prevalence | Ruminants | Sensitivity and Specificity | Theileria | Theileriasis [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Animals | DNA, Protozoan | Molecular Sequence Data | Polymerase Chain Reaction | Prevalence | Ruminants | Sensitivity and Specificity | Theileria | Theileriasis [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] pathogenic theileria spp | tick borne pathogens | occurrence theileriosis cattle | theileria spp widespread | prevalence theileria cattle [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] pathogenic theileria spp | tick borne pathogens | occurrence theileriosis cattle | theileria spp widespread | prevalence theileria cattle [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] pathogenic theileria spp | tick borne pathogens | occurrence theileriosis cattle | theileria spp widespread | prevalence theileria cattle [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] pathogenic theileria spp | tick borne pathogens | occurrence theileriosis cattle | theileria spp widespread | prevalence theileria cattle [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] pathogenic theileria spp | tick borne pathogens | occurrence theileriosis cattle | theileria spp widespread | prevalence theileria cattle [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] pathogenic theileria spp | tick borne pathogens | occurrence theileriosis cattle | theileria spp widespread | prevalence theileria cattle [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] theileria | mismatches | pcr | species | spp | theileria spp | sec | cattle | gene | probes [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] theileria | mismatches | pcr | species | spp | theileria spp | sec | cattle | gene | probes [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] theileria | mismatches | pcr | species | spp | theileria spp | sec | cattle | gene | probes [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] theileria | mismatches | pcr | species | spp | theileria spp | sec | cattle | gene | probes [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] theileria | mismatches | pcr | species | spp | theileria spp | sec | cattle | gene | probes [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] theileria | mismatches | pcr | species | spp | theileria spp | sec | cattle | gene | probes [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] specific | theileria | studies | sensitive | domestic | infections | spp | theileria spp | infecting | sheep goats [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] mismatches | sec | species | probes | theileria | pcr | primer | probe | oligonucleotides | primers [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] cattle | theileria | vs | sheep | 10 | china0 | pan | pan theileria fret | pan theileria | sequences [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] diagnostic | detect | useful diagnostic tool | laboratories detect infections | summary study | summary study described | summary study described development | development testing | development testing fret | laboratories detect infections domestic [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] theileria | mismatches | cattle | sec | pcr | species | spp | theileria spp | 10 | fret [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] theileria | mismatches | cattle | sec | pcr | species | spp | theileria spp | 10 | fret [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| PCR | Theileria | PCR [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] pan-Theileria FRET-qPCR | Theileria ||| GenBank ||| nine | China [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| China | Theileria ||| 53.2% | 59/111 | 44.4% | 120/270 | 30.8% | 380/1,235 ||| 29 ||| PCR | China | T. | T. ovis | T. | T. ||| Theileria | Bos | Bos | 77.7% | 18.3% | 10(3.00 ||| 18S [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Theileria ||| ||| China | Theileria [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| PCR | Theileria | PCR ||| pan-Theileria FRET-qPCR | Theileria ||| GenBank ||| nine | China ||| ||| China | Theileria ||| 53.2% | 59/111 | 44.4% | 120/270 | 30.8% | 380/1,235 ||| 29 ||| PCR | China | T. | T. ovis | T. | T. ||| Theileria | Bos | Bos | 77.7% | 18.3% | 10(3.00 ||| 18S ||| Theileria ||| ||| China | Theileria [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| PCR | Theileria | PCR ||| pan-Theileria FRET-qPCR | Theileria ||| GenBank ||| nine | China ||| ||| China | Theileria ||| 53.2% | 59/111 | 44.4% | 120/270 | 30.8% | 380/1,235 ||| 29 ||| PCR | China | T. | T. ovis | T. | T. ||| Theileria | Bos | Bos | 77.7% | 18.3% | 10(3.00 ||| 18S ||| Theileria ||| ||| China | Theileria [SUMMARY]"}}},{"rowIdx":75,"cells":{"title":{"kind":"string","value":"Preterm birth by vacuum extraction and neonatal outcome: a population-based cohort study."},"pmid":{"kind":"string","value":"24450413"},"background_abstract":{"kind":"string","value":"Very few studies have investigated the neonatal outcomes after vacuum extraction delivery (VE) in the preterm period and the results of these studies are inconclusive. The objective of this study was to describe the use of VE for preterm delivery in Sweden and to compare rates of neonatal complications after preterm delivery by VE to those found after cesarean section during labor (CS) or unassisted vaginal delivery (VD)."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Data was obtained from Swedish national registers. In a population-based cohort from 1999 to 2010, all live-born, singleton preterm infants in a non-breech presentation at birth, born after onset of labor (either spontaneously, by induction, or by rupture of membranes) by VD, CS, or VE were included, leaving a study population of 40,764 infants. Logistic regression analyses were used to calculate adjusted odds ratios (AOR), using unassisted vaginal delivery as reference group."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"VE was used in 5.7% of the preterm deliveries, with lower rates in earlier gestations. Overall, intracranial hemorrhage (ICH) occurred in 1.51%, extracranial hemorrhage (ECH) in 0.64%, and brachial plexus injury in 0.13% of infants. Infants delivered by VE had higher risks for ICH (AOR = 1.84 (95% CI: 1.09-3.12)), ECH (AOR = 4.48 (95% CI: 2.84-7.07)) and brachial plexus injury (AOR = 6.21 (95% CI: 2.22-17.4)), while infants delivered by CS during labor had no increased risk for these complications, as compared to VD."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"While rates of neonatal complications after VE are generally low, higher odds ratios for intra- and extracranial hemorrhages and brachial plexus injuries after VE, compared with other modes of delivery, support a continued cautious use of VE for preterm delivery."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Birth Injuries","Brachial Plexus","Cesarean Section","Cohort Studies","Female","Gestational Age","Humans","Infant, Newborn","Infant, Premature","Intracranial Hemorrhage, Traumatic","Labor, Obstetric","Parturition","Pregnancy","Premature Birth","Registries","Scalp","Seizures","Sweden","Vacuum Extraction, Obstetrical","Young Adult"],"string":"[\n \"Adult\",\n \"Birth Injuries\",\n \"Brachial Plexus\",\n \"Cesarean Section\",\n \"Cohort Studies\",\n \"Female\",\n \"Gestational Age\",\n \"Humans\",\n \"Infant, Newborn\",\n \"Infant, Premature\",\n \"Intracranial Hemorrhage, Traumatic\",\n \"Labor, Obstetric\",\n \"Parturition\",\n \"Pregnancy\",\n \"Premature Birth\",\n \"Registries\",\n \"Scalp\",\n \"Seizures\",\n \"Sweden\",\n \"Vacuum Extraction, Obstetrical\",\n \"Young Adult\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"3900732"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Preterm birth is common\n[1] but still, the optimal mode of delivery of preterm infants is not known. Although neonatal outcomes in preterm infants delivered vaginally or by cesarean section (CS)\n[2-5] have been compared, there is no evidence to provide clear guidance on the method of choice\n[6]. Given the widespread assumption that assisted vaginal delivery could be harmful for fragile infants that are underweight and preterm, very few studies have addressed the use of vacuum extraction (VE) for preterm birth.\nDelivery by VE is a common obstetrical procedure, and in many countries it has replaced the use of forceps. VE is used to terminate a protracted second stage of labor and as an intervention for fetal or maternal distress. VE requires vertex presentation, a fully dilated cervix and ruptured membranes\n[7]. A cesarean section, on the other hand, can be performed at any stage of labor and does not require prerequisites of this kind.\nMost clinical guidelines do not recommend VE before 34 gestational weeks\n[8-10]. These recommendations are not based on results of randomized controlled trials, but rely on the observation that preterm infants are more likely than term infants to develop ICH, and on extrapolations from studies of term infants showing that VE is associated with an increased risk of ICH and other neonatal complications\n[11-17]. Only three studies have previously investigated the use and outcomes of VE in preterm births. The first was undertaken over 40 years ago and showed increased mortality and morbidity among preterm infants delivered by VE as compared with term infants delivered by VE\n[18]. The second study compared neonatal morbidity in preterm infants delivered vaginally with (n = 61) or without VE (n = 122), and found no differences in neonatal morbidity between the two groups\n[19]. The last study compared VE and forceps for preterm delivery (n = 64)\n[20]; the neonatal outcomes were similar in both groups. The available data are clearly untimely and hampered by limitations in power and, therefore, current knowledge on safety of preterm vacuum-assisted birth is unsatisfactory.\nThe aim of this study was to 1) describe the use of VE and compare it to rates of CS during labor in preterm deliveries in Sweden from 1999–2010, 2) characterize the distribution of perinatal risk factors associated with each mode of delivery, and 3) compare rates of neonatal intra- and extracranial hemorrhages, as well as occurrence of brachial plexus injury after preterm delivery by VE or CS during labor, using unassisted vaginal birth as a reference."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"This study was based on data from national data bases held by the Swedish National Board of Health and Welfare. The national registration number, assigned to each Swedish resident at birth, was used for individual record linkage. We used two registers: The Swedish Medical Birth Register (SMBR) that covers 99% of all births in Sweden, and The Swedish National Inpatient Register (IPR) that covers all public inpatient care. The SMBR includes prospectively collected information on maternal characteristics, reproductive history, and complications during pregnancy, delivery, and the neonatal period. The IPR includes data on each hospital admission and discharge.\n Study population During the period of 1999–2010, there were 75,296 (6.2%) preterm births in Sweden. We excluded deliveries by CS before the onset of labor (n = 17,306), forceps (n = 257), or performed with both VE and CS (n = 125). We also excluded stillbirths (fetal deaths occurring before labor or intra partum) (n = 1,839), multiple births (n = 11,088), and births in breech presentation (n = 3,917). Thus, the final study population was restricted to all live-born, preterm singleton infants with a non-breech presentation at birth, delivered after a spontaneous or induced onset of labor followed by CS, vacuum extraction (VE), or by unassisted vaginal delivery (VD) before gestational week 37 + 0 days (N = 40,764). CS during labor was defined as abdominal delivery after the onset of labor, either spontaneously, by rupture of membranes, or by induction.\nA number of independent variables were collected; the maternal anthropometrics included: age, height, and body mass index (BMI). BMI was calculated from measured height and weight obtained at the first antenatal care visit, which occurred before the 15th week of gestation in more than 95% of the pregnancies. BMI was categorized into underweight (below 18.5 kg/m2), normal (18.5–24.9 kg/m2), overweight (25–29.9 kg/m2), obese (>29.9 kg/m2), or missing. Parity was categorized as primi- or multiparity. Information on complications during pregnancy and delivery were coded according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards). The following pregnancy complications were included: diabetes – both gestational and types 1 and 2 (O24.0-9) preeclampsia- both hypertension, preeclampsia, and eclampsia (O10.0-O15.9). Labor-related risk factors or covariates includedepidural analgesia (EA; yes/no), and induction of labor (yes/no). Indications for operative delivery were classified into four major groups: prolonged labor (O62.0-2, O63.0-9), signs of fetal distress (O68.0-O68.1-9), preeclampsia, and a non-occipitoanterior presentation of the fetus (all presentations except occipitoanterior and breech, registered at birth). Gestational age (GA) for preterm infants was divided into three periods according to the World Health Organization: extremely preterm (before 28 weeks), very preterm (28–31 weeks) and moderately preterm (32–36 weeks). Furthermore, we also divided the preterm gestational period according to guidelines on instrumental delivery into either: less than 34 weeks (VE not recommended), and 34–36 weeks (VE may be used). GA was recorded in completed weeks, and was based on routine ultrasound dating performed at 17 to 18 postmenstrual weeks in 97-98% of all pregnant women. Infant birthweight was categorized as less than 1,500 grams, 1,500-2,000 grams, 2,001-2,500 grams, 2,501-3,000 grams, and 3,001-4,000 grams.\nDuring the period of 1999–2010, there were 75,296 (6.2%) preterm births in Sweden. We excluded deliveries by CS before the onset of labor (n = 17,306), forceps (n = 257), or performed with both VE and CS (n = 125). We also excluded stillbirths (fetal deaths occurring before labor or intra partum) (n = 1,839), multiple births (n = 11,088), and births in breech presentation (n = 3,917). Thus, the final study population was restricted to all live-born, preterm singleton infants with a non-breech presentation at birth, delivered after a spontaneous or induced onset of labor followed by CS, vacuum extraction (VE), or by unassisted vaginal delivery (VD) before gestational week 37 + 0 days (N = 40,764). CS during labor was defined as abdominal delivery after the onset of labor, either spontaneously, by rupture of membranes, or by induction.\nA number of independent variables were collected; the maternal anthropometrics included: age, height, and body mass index (BMI). BMI was calculated from measured height and weight obtained at the first antenatal care visit, which occurred before the 15th week of gestation in more than 95% of the pregnancies. BMI was categorized into underweight (below 18.5 kg/m2), normal (18.5–24.9 kg/m2), overweight (25–29.9 kg/m2), obese (>29.9 kg/m2), or missing. Parity was categorized as primi- or multiparity. Information on complications during pregnancy and delivery were coded according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards). The following pregnancy complications were included: diabetes – both gestational and types 1 and 2 (O24.0-9) preeclampsia- both hypertension, preeclampsia, and eclampsia (O10.0-O15.9). Labor-related risk factors or covariates includedepidural analgesia (EA; yes/no), and induction of labor (yes/no). Indications for operative delivery were classified into four major groups: prolonged labor (O62.0-2, O63.0-9), signs of fetal distress (O68.0-O68.1-9), preeclampsia, and a non-occipitoanterior presentation of the fetus (all presentations except occipitoanterior and breech, registered at birth). Gestational age (GA) for preterm infants was divided into three periods according to the World Health Organization: extremely preterm (before 28 weeks), very preterm (28–31 weeks) and moderately preterm (32–36 weeks). Furthermore, we also divided the preterm gestational period according to guidelines on instrumental delivery into either: less than 34 weeks (VE not recommended), and 34–36 weeks (VE may be used). GA was recorded in completed weeks, and was based on routine ultrasound dating performed at 17 to 18 postmenstrual weeks in 97-98% of all pregnant women. Infant birthweight was categorized as less than 1,500 grams, 1,500-2,000 grams, 2,001-2,500 grams, 2,501-3,000 grams, and 3,001-4,000 grams.\n Outcome variables Neonatal diagnoses were classified according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards), and identified/collected in the SMBR or in the IPR. The following neonatal outcomes (ICD codes) were assessed: Intracranial laceration and hemorrhage due to birth injury (P10), intracranial non-traumatic hemorrhage of fetus and newborn (P52), convulsions of newborn (P90), other disturbances of cerebral status of newborn (P 91), subgaleal hematoma (P12.2), cephalhematoma (P12.0), and brachial plexus injury (P14.0-3). The definitions of outcomes are described in detail in Table \n1.\nNeonatal outcomes studied in 40 764 preterm infants\nNeonatal diagnoses of intracranial hemorrhages in preterm infants were mainly based on imaging of the brain using ultrasonography; however, some assessments of the brain at term-equivalent age were alternatively performed with CT and/or MRI. Imaging of the brain was performed on clinical indications only in cases born moderately or late preterm, whereas all very preterm infants (born before 32 weeks of gestation) were screened for intracranial lesions, even in asymptomatic infants. A diagnosis of convulsions included infants with clinical signs of convulsions and/or convulsions verified by EEG.\nStatistical analysis was performed using proportions and odds ratios (OR) with a 95% confidence interval (CI) for neonatal complications in relation to mode of delivery, using unassisted VD as the reference group (SPSS 20.0 for Windows software package). Three models were used to assess the relationship between the different modes of delivery and the risk for neonatal complications: one crude, and two adjusted (Models 1 and 2). The included covariates have been shown previously to be related to instrumental deliveries, and were related to the outcomes in cross tabulations. In Model 1, we adjusted for the following confounders or covariates: maternal age, height, BMI, and parity, as well as infant year of birth, birthweight and GA. In Model 2, we added the indication for operative delivery and preeclampsia. The year of birth was entered as a continuous variable in accordance with a linear secular trend, and all other variables were entered as categories. Furthermore, a separate logistic regression analysis was performed to investigate severe ICH in relation to mode of delivery. Here, intraventricular hemorrhages grades 1–2 were excluded and the analysis was adjusted for GA only. We also conducted separated analyses on potential relationships between sex and ICH in relation to mode of delivery. Missing data were entered as a separate category in the analyses. The study was approved by the Regional Ethical Review Board in Stockholm, Dnr 2008/1322-31.\nNeonatal diagnoses were classified according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards), and identified/collected in the SMBR or in the IPR. The following neonatal outcomes (ICD codes) were assessed: Intracranial laceration and hemorrhage due to birth injury (P10), intracranial non-traumatic hemorrhage of fetus and newborn (P52), convulsions of newborn (P90), other disturbances of cerebral status of newborn (P 91), subgaleal hematoma (P12.2), cephalhematoma (P12.0), and brachial plexus injury (P14.0-3). The definitions of outcomes are described in detail in Table \n1.\nNeonatal outcomes studied in 40 764 preterm infants\nNeonatal diagnoses of intracranial hemorrhages in preterm infants were mainly based on imaging of the brain using ultrasonography; however, some assessments of the brain at term-equivalent age were alternatively performed with CT and/or MRI. Imaging of the brain was performed on clinical indications only in cases born moderately or late preterm, whereas all very preterm infants (born before 32 weeks of gestation) were screened for intracranial lesions, even in asymptomatic infants. A diagnosis of convulsions included infants with clinical signs of convulsions and/or convulsions verified by EEG.\nStatistical analysis was performed using proportions and odds ratios (OR) with a 95% confidence interval (CI) for neonatal complications in relation to mode of delivery, using unassisted VD as the reference group (SPSS 20.0 for Windows software package). Three models were used to assess the relationship between the different modes of delivery and the risk for neonatal complications: one crude, and two adjusted (Models 1 and 2). The included covariates have been shown previously to be related to instrumental deliveries, and were related to the outcomes in cross tabulations. In Model 1, we adjusted for the following confounders or covariates: maternal age, height, BMI, and parity, as well as infant year of birth, birthweight and GA. In Model 2, we added the indication for operative delivery and preeclampsia. The year of birth was entered as a continuous variable in accordance with a linear secular trend, and all other variables were entered as categories. Furthermore, a separate logistic regression analysis was performed to investigate severe ICH in relation to mode of delivery. Here, intraventricular hemorrhages grades 1–2 were excluded and the analysis was adjusted for GA only. We also conducted separated analyses on potential relationships between sex and ICH in relation to mode of delivery. Missing data were entered as a separate category in the analyses. The study was approved by the Regional Ethical Review Board in Stockholm, Dnr 2008/1322-31."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Use of VE in relation to gestational age Among the 40,764 (54% of all) preterm deliveries included in this study, 2,319 (5.7%) preterm infants were delivered by VE, 5,505 (13.5%) by CS during labor, and 32,940 (80.2%) by VD. The rate of VE deliveries increased gradually with gestational age, Figure \n1.\nMode of delivery in relation to gestational age. Figure\n1 shows rates (%) of different modes of delivery in relation to gestational age (in completed weeks). The blue, dotted line represents unassisted vaginal deliveries, the red, dashed line represents cesarean sections performed after onset of labor, and the green line represents the vacuum extraction deliveries.\nAmong the 40,764 (54% of all) preterm deliveries included in this study, 2,319 (5.7%) preterm infants were delivered by VE, 5,505 (13.5%) by CS during labor, and 32,940 (80.2%) by VD. The rate of VE deliveries increased gradually with gestational age, Figure \n1.\nMode of delivery in relation to gestational age. Figure\n1 shows rates (%) of different modes of delivery in relation to gestational age (in completed weeks). The blue, dotted line represents unassisted vaginal deliveries, the red, dashed line represents cesarean sections performed after onset of labor, and the green line represents the vacuum extraction deliveries.\n Distribution of risk factors or covariates in relation to mode of delivery Table \n2 shows maternal and perinatal characteristics of the study population in relation to mode of delivery. The VE rate decreased with maternal height and 80% of the women who delivered by VE were primiparae, compared with 48% of those who underwent CS during labor (not in table). More than 45% of the women who delivered by VE had received epidural analgesia during labor compared with 22% of women with VD, and 12% with CS during labor (not in table). Given the association between GA and VE, infants delivered with VE had higher birthweights.\nMaternal, pregnancy, delivery, and infant characteristics by mode of delivery\nPopulation based cohort consisting of 40 764 preterm deliveries.\nCS = caesarean section, BMI = Body Mass Index (weight in kilograms/height in meters2), EA = Epidural Analgesia.\nThe most common indication for VE was fetal distress (42%), followed by prolonged labor (25%). Having a non-occipitoanterior position (25%) or fetal distress (17%) were the most common indications for CS during labor, while only 3% in this group had a diagnosis of prolonged labor.\nTable \n2 shows maternal and perinatal characteristics of the study population in relation to mode of delivery. The VE rate decreased with maternal height and 80% of the women who delivered by VE were primiparae, compared with 48% of those who underwent CS during labor (not in table). More than 45% of the women who delivered by VE had received epidural analgesia during labor compared with 22% of women with VD, and 12% with CS during labor (not in table). Given the association between GA and VE, infants delivered with VE had higher birthweights.\nMaternal, pregnancy, delivery, and infant characteristics by mode of delivery\nPopulation based cohort consisting of 40 764 preterm deliveries.\nCS = caesarean section, BMI = Body Mass Index (weight in kilograms/height in meters2), EA = Epidural Analgesia.\nThe most common indication for VE was fetal distress (42%), followed by prolonged labor (25%). Having a non-occipitoanterior position (25%) or fetal distress (17%) were the most common indications for CS during labor, while only 3% in this group had a diagnosis of prolonged labor.\n Neonatal outcome in relation to gestational age The proportion of preterm infants diagnosed with an ICH varied more than hundred-fold in relation to GA. It decreased from 21.5% among preterm infants born at 22–28 weeks of GA to 0.1% among those born after 36 weeks of gestation. The rates of neonatal convulsions among preterm infants decreased from 2.0% at 22–28 weeks to 0.25% among those born after 36 weeks of gestation, Figure \n2. The proportion of preterm infants diagnosed with other disturbances of cerebral status (encephalopathy) decreased with GA, while proportion of infants with brachial plexus injuries or ECH increased slightly with GA, Figure \n3.\nProportion (%) preterm infants diagnosed with intracranial hemorrhage (ICH) or convulsions by gestational age. Figure\n2 shows the proportions (%) of preterm infants diagnosed with ICH and neonatal convulsions in relation to gestational age (in completed weeks). The blue, dotted line represents intracranial hemorrhage (ICH) and the red, dashed line represents neonatal convulsions.\nProportion (%) preterm infants diagnosed with extracranial hemorrhage (ECH), encephalopathy and brachial plexus injury by gestational age. Figure\n3 shows the proportions (%) of ECH, encephalopathy (ICD-code P91: other disturbances of cerebral status of newborn), and brachial plexus injury in relation to gestational age (in completed weeks). The blue, dotted line represents extracranial hemorrhage (ECH), the red, dashed line represents encephalopathy and the green line represents brachial plexus injury.\nThe proportion of preterm infants diagnosed with an ICH varied more than hundred-fold in relation to GA. It decreased from 21.5% among preterm infants born at 22–28 weeks of GA to 0.1% among those born after 36 weeks of gestation. The rates of neonatal convulsions among preterm infants decreased from 2.0% at 22–28 weeks to 0.25% among those born after 36 weeks of gestation, Figure \n2. The proportion of preterm infants diagnosed with other disturbances of cerebral status (encephalopathy) decreased with GA, while proportion of infants with brachial plexus injuries or ECH increased slightly with GA, Figure \n3.\nProportion (%) preterm infants diagnosed with intracranial hemorrhage (ICH) or convulsions by gestational age. Figure\n2 shows the proportions (%) of preterm infants diagnosed with ICH and neonatal convulsions in relation to gestational age (in completed weeks). The blue, dotted line represents intracranial hemorrhage (ICH) and the red, dashed line represents neonatal convulsions.\nProportion (%) preterm infants diagnosed with extracranial hemorrhage (ECH), encephalopathy and brachial plexus injury by gestational age. Figure\n3 shows the proportions (%) of ECH, encephalopathy (ICD-code P91: other disturbances of cerebral status of newborn), and brachial plexus injury in relation to gestational age (in completed weeks). The blue, dotted line represents extracranial hemorrhage (ECH), the red, dashed line represents encephalopathy and the green line represents brachial plexus injury.\n Neonatal outcome in relation to mode of delivery To report outcome in relation to mode of delivery, the study cohort was divided according to the current guidelines on the use of VE as either preterm births occurring between 34–36 weeks of gestation (VE may be used), or those occurring before 34 gestational weeks (VE not recommended). In our cohort, 33,202 (81.4%) of all preterm births occurred at 34–36 gestational weeks, and 7,562 (18.6%) before 34 weeks of GA.\nIn Table \n3, neonatal outcomes before and after 34 + 0 weeks of gestation are presented in relation to mode of delivery. Overall, seven preterm infants were classified as having an ICH due to birth injury, corresponding to a rate of 0.02%; and 612 infants were diagnosed with non-traumatic ICH, corresponding to a rate of 1.5%. Diagnoses of neonatal convulsions and other disturbances of neonatal cerebral status were rare, especially in infants at less than 34 weeks of GA, and occurred more frequently after VE and CS than after VD. Cephalic hematoma was the most frequent complication after VE of preterm infants (n = 72 or 3.1%), occurring much more often after VE than after CS (0.16%) and VD (0.49%). Subgaleal hemorrhage was less frequent, with a total of only18 cases. More than two thirds of those cases occurred in the VE group.\nNeonatal outcomes in preterm infants by mode of delivery and gestational age\nTable \n4 shows rates (per 1000), crude and adjusted odds ratios for the outcomes by mode of delivery, and uses infants born by VD as the reference group. After adjustments, preterm infants born by VE had almost doubled OR for ICH, while those born by CS had no increased risk. Preterm infants delivered by VE also had 4.5 times higher OR for extracranial hemorrhages, while those delivered by CS had no increased risk. A total of 540 (88.2%) of the ICH diagnoses consisted of intraventricular bleedings (ICD-codes 52.0-52.3), the type of ICH most frequently seen in preterm infants. Of these, 438 (71.6%) were graded as mild or moderate (grades 1–2). After excluding grades 1–2 of intraventricular hemorrhages from the analyses, the OR for ICH was still significantly increased for infants delivered by VE (OR 2.58, CI 1.27-5.24), but not for those delivered by CS during labor (OR 1.15, CI 0.78-1.70) after adjustment for GA. There was no difference in ICH rates between preterm boys and girls delivered by VE.\nLogistic regression (odds ratios) for intra– and extracranial hemorrhages, convulsions and other cerebral complications, and brachial plexus injury in preterm infants exposed to different modes of delivery\nCS = cesarean section, VE = vacuum extraction.\nModel 1 is adjusted for year of birth, gestational age, parity, maternal age, height, BMI, and infant birthweight.\nModel 2 is also adjusted for indications for operative delivery.\nThe ORs for convulsions were almost doubled in both the VE and CS groups after adjustments for the variables in Model 1. However, further adjustment for indication for operative delivery decreased the odds and made the associations statistically insignificant. Other disturbances of the neonatal cerebral status were significantly increased (two to three times higher) both among infants delivered by VE, and by CS during labor, although the OR was higher in the VE group.\nA total of 53 infants were diagnosed with brachial plexus injury. Of these, 14 were born by VE, corresponding to a rate in the VE group of 0.6% and an OR of 6.21 (95% CI: 2.22-17.4) in the fully-adjusted model. In contrast, infants delivered by CS had no increased risk for this complication. Among infants with brachial plexus injury, there were 11 cases of shoulder dystocia (ICD-code O66.0), of which five occurred in the VE group.\nTo report outcome in relation to mode of delivery, the study cohort was divided according to the current guidelines on the use of VE as either preterm births occurring between 34–36 weeks of gestation (VE may be used), or those occurring before 34 gestational weeks (VE not recommended). In our cohort, 33,202 (81.4%) of all preterm births occurred at 34–36 gestational weeks, and 7,562 (18.6%) before 34 weeks of GA.\nIn Table \n3, neonatal outcomes before and after 34 + 0 weeks of gestation are presented in relation to mode of delivery. Overall, seven preterm infants were classified as having an ICH due to birth injury, corresponding to a rate of 0.02%; and 612 infants were diagnosed with non-traumatic ICH, corresponding to a rate of 1.5%. Diagnoses of neonatal convulsions and other disturbances of neonatal cerebral status were rare, especially in infants at less than 34 weeks of GA, and occurred more frequently after VE and CS than after VD. Cephalic hematoma was the most frequent complication after VE of preterm infants (n = 72 or 3.1%), occurring much more often after VE than after CS (0.16%) and VD (0.49%). Subgaleal hemorrhage was less frequent, with a total of only18 cases. More than two thirds of those cases occurred in the VE group.\nNeonatal outcomes in preterm infants by mode of delivery and gestational age\nTable \n4 shows rates (per 1000), crude and adjusted odds ratios for the outcomes by mode of delivery, and uses infants born by VD as the reference group. After adjustments, preterm infants born by VE had almost doubled OR for ICH, while those born by CS had no increased risk. Preterm infants delivered by VE also had 4.5 times higher OR for extracranial hemorrhages, while those delivered by CS had no increased risk. A total of 540 (88.2%) of the ICH diagnoses consisted of intraventricular bleedings (ICD-codes 52.0-52.3), the type of ICH most frequently seen in preterm infants. Of these, 438 (71.6%) were graded as mild or moderate (grades 1–2). After excluding grades 1–2 of intraventricular hemorrhages from the analyses, the OR for ICH was still significantly increased for infants delivered by VE (OR 2.58, CI 1.27-5.24), but not for those delivered by CS during labor (OR 1.15, CI 0.78-1.70) after adjustment for GA. There was no difference in ICH rates between preterm boys and girls delivered by VE.\nLogistic regression (odds ratios) for intra– and extracranial hemorrhages, convulsions and other cerebral complications, and brachial plexus injury in preterm infants exposed to different modes of delivery\nCS = cesarean section, VE = vacuum extraction.\nModel 1 is adjusted for year of birth, gestational age, parity, maternal age, height, BMI, and infant birthweight.\nModel 2 is also adjusted for indications for operative delivery.\nThe ORs for convulsions were almost doubled in both the VE and CS groups after adjustments for the variables in Model 1. However, further adjustment for indication for operative delivery decreased the odds and made the associations statistically insignificant. Other disturbances of the neonatal cerebral status were significantly increased (two to three times higher) both among infants delivered by VE, and by CS during labor, although the OR was higher in the VE group.\nA total of 53 infants were diagnosed with brachial plexus injury. Of these, 14 were born by VE, corresponding to a rate in the VE group of 0.6% and an OR of 6.21 (95% CI: 2.22-17.4) in the fully-adjusted model. In contrast, infants delivered by CS had no increased risk for this complication. Among infants with brachial plexus injury, there were 11 cases of shoulder dystocia (ICD-code O66.0), of which five occurred in the VE group."},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"The rates of serious birth injuries and complications are generally low, but preterm infants delivered by VE have higher odds ratios for intra- and extracranial hemorrhages and brachial plexus injuries than those delivered by CS during labor or by unassisted vaginal delivery. We therefore support a continued conservative/cautious use of VE in preterm deliveries. Furthermore, the possible causal relationship between mode of delivery and ICH needs to be further investigated."},"other_sections_titles":{"kind":"list like","value":["Background","Study population","Outcome variables","Use of VE in relation to gestational age","Distribution of risk factors or covariates in relation to mode of delivery","Neonatal outcome in relation to gestational age","Neonatal outcome in relation to mode of delivery","Abbreviations","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Study population\",\n \"Outcome variables\",\n \"Use of VE in relation to gestational age\",\n \"Distribution of risk factors or covariates in relation to mode of delivery\",\n \"Neonatal outcome in relation to gestational age\",\n \"Neonatal outcome in relation to mode of delivery\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Preterm birth is common\n[1] but still, the optimal mode of delivery of preterm infants is not known. Although neonatal outcomes in preterm infants delivered vaginally or by cesarean section (CS)\n[2-5] have been compared, there is no evidence to provide clear guidance on the method of choice\n[6]. Given the widespread assumption that assisted vaginal delivery could be harmful for fragile infants that are underweight and preterm, very few studies have addressed the use of vacuum extraction (VE) for preterm birth.\nDelivery by VE is a common obstetrical procedure, and in many countries it has replaced the use of forceps. VE is used to terminate a protracted second stage of labor and as an intervention for fetal or maternal distress. VE requires vertex presentation, a fully dilated cervix and ruptured membranes\n[7]. A cesarean section, on the other hand, can be performed at any stage of labor and does not require prerequisites of this kind.\nMost clinical guidelines do not recommend VE before 34 gestational weeks\n[8-10]. These recommendations are not based on results of randomized controlled trials, but rely on the observation that preterm infants are more likely than term infants to develop ICH, and on extrapolations from studies of term infants showing that VE is associated with an increased risk of ICH and other neonatal complications\n[11-17]. Only three studies have previously investigated the use and outcomes of VE in preterm births. The first was undertaken over 40 years ago and showed increased mortality and morbidity among preterm infants delivered by VE as compared with term infants delivered by VE\n[18]. The second study compared neonatal morbidity in preterm infants delivered vaginally with (n = 61) or without VE (n = 122), and found no differences in neonatal morbidity between the two groups\n[19]. The last study compared VE and forceps for preterm delivery (n = 64)\n[20]; the neonatal outcomes were similar in both groups. The available data are clearly untimely and hampered by limitations in power and, therefore, current knowledge on safety of preterm vacuum-assisted birth is unsatisfactory.\nThe aim of this study was to 1) describe the use of VE and compare it to rates of CS during labor in preterm deliveries in Sweden from 1999–2010, 2) characterize the distribution of perinatal risk factors associated with each mode of delivery, and 3) compare rates of neonatal intra- and extracranial hemorrhages, as well as occurrence of brachial plexus injury after preterm delivery by VE or CS during labor, using unassisted vaginal birth as a reference.","During the period of 1999–2010, there were 75,296 (6.2%) preterm births in Sweden. We excluded deliveries by CS before the onset of labor (n = 17,306), forceps (n = 257), or performed with both VE and CS (n = 125). We also excluded stillbirths (fetal deaths occurring before labor or intra partum) (n = 1,839), multiple births (n = 11,088), and births in breech presentation (n = 3,917). Thus, the final study population was restricted to all live-born, preterm singleton infants with a non-breech presentation at birth, delivered after a spontaneous or induced onset of labor followed by CS, vacuum extraction (VE), or by unassisted vaginal delivery (VD) before gestational week 37 + 0 days (N = 40,764). CS during labor was defined as abdominal delivery after the onset of labor, either spontaneously, by rupture of membranes, or by induction.\nA number of independent variables were collected; the maternal anthropometrics included: age, height, and body mass index (BMI). BMI was calculated from measured height and weight obtained at the first antenatal care visit, which occurred before the 15th week of gestation in more than 95% of the pregnancies. BMI was categorized into underweight (below 18.5 kg/m2), normal (18.5–24.9 kg/m2), overweight (25–29.9 kg/m2), obese (>29.9 kg/m2), or missing. Parity was categorized as primi- or multiparity. Information on complications during pregnancy and delivery were coded according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards). The following pregnancy complications were included: diabetes – both gestational and types 1 and 2 (O24.0-9) preeclampsia- both hypertension, preeclampsia, and eclampsia (O10.0-O15.9). Labor-related risk factors or covariates includedepidural analgesia (EA; yes/no), and induction of labor (yes/no). Indications for operative delivery were classified into four major groups: prolonged labor (O62.0-2, O63.0-9), signs of fetal distress (O68.0-O68.1-9), preeclampsia, and a non-occipitoanterior presentation of the fetus (all presentations except occipitoanterior and breech, registered at birth). Gestational age (GA) for preterm infants was divided into three periods according to the World Health Organization: extremely preterm (before 28 weeks), very preterm (28–31 weeks) and moderately preterm (32–36 weeks). Furthermore, we also divided the preterm gestational period according to guidelines on instrumental delivery into either: less than 34 weeks (VE not recommended), and 34–36 weeks (VE may be used). GA was recorded in completed weeks, and was based on routine ultrasound dating performed at 17 to 18 postmenstrual weeks in 97-98% of all pregnant women. Infant birthweight was categorized as less than 1,500 grams, 1,500-2,000 grams, 2,001-2,500 grams, 2,501-3,000 grams, and 3,001-4,000 grams.","Neonatal diagnoses were classified according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards), and identified/collected in the SMBR or in the IPR. The following neonatal outcomes (ICD codes) were assessed: Intracranial laceration and hemorrhage due to birth injury (P10), intracranial non-traumatic hemorrhage of fetus and newborn (P52), convulsions of newborn (P90), other disturbances of cerebral status of newborn (P 91), subgaleal hematoma (P12.2), cephalhematoma (P12.0), and brachial plexus injury (P14.0-3). The definitions of outcomes are described in detail in Table \n1.\nNeonatal outcomes studied in 40 764 preterm infants\nNeonatal diagnoses of intracranial hemorrhages in preterm infants were mainly based on imaging of the brain using ultrasonography; however, some assessments of the brain at term-equivalent age were alternatively performed with CT and/or MRI. Imaging of the brain was performed on clinical indications only in cases born moderately or late preterm, whereas all very preterm infants (born before 32 weeks of gestation) were screened for intracranial lesions, even in asymptomatic infants. A diagnosis of convulsions included infants with clinical signs of convulsions and/or convulsions verified by EEG.\nStatistical analysis was performed using proportions and odds ratios (OR) with a 95% confidence interval (CI) for neonatal complications in relation to mode of delivery, using unassisted VD as the reference group (SPSS 20.0 for Windows software package). Three models were used to assess the relationship between the different modes of delivery and the risk for neonatal complications: one crude, and two adjusted (Models 1 and 2). The included covariates have been shown previously to be related to instrumental deliveries, and were related to the outcomes in cross tabulations. In Model 1, we adjusted for the following confounders or covariates: maternal age, height, BMI, and parity, as well as infant year of birth, birthweight and GA. In Model 2, we added the indication for operative delivery and preeclampsia. The year of birth was entered as a continuous variable in accordance with a linear secular trend, and all other variables were entered as categories. Furthermore, a separate logistic regression analysis was performed to investigate severe ICH in relation to mode of delivery. Here, intraventricular hemorrhages grades 1–2 were excluded and the analysis was adjusted for GA only. We also conducted separated analyses on potential relationships between sex and ICH in relation to mode of delivery. Missing data were entered as a separate category in the analyses. The study was approved by the Regional Ethical Review Board in Stockholm, Dnr 2008/1322-31.","Among the 40,764 (54% of all) preterm deliveries included in this study, 2,319 (5.7%) preterm infants were delivered by VE, 5,505 (13.5%) by CS during labor, and 32,940 (80.2%) by VD. The rate of VE deliveries increased gradually with gestational age, Figure \n1.\nMode of delivery in relation to gestational age. Figure\n1 shows rates (%) of different modes of delivery in relation to gestational age (in completed weeks). The blue, dotted line represents unassisted vaginal deliveries, the red, dashed line represents cesarean sections performed after onset of labor, and the green line represents the vacuum extraction deliveries.","Table \n2 shows maternal and perinatal characteristics of the study population in relation to mode of delivery. The VE rate decreased with maternal height and 80% of the women who delivered by VE were primiparae, compared with 48% of those who underwent CS during labor (not in table). More than 45% of the women who delivered by VE had received epidural analgesia during labor compared with 22% of women with VD, and 12% with CS during labor (not in table). Given the association between GA and VE, infants delivered with VE had higher birthweights.\nMaternal, pregnancy, delivery, and infant characteristics by mode of delivery\nPopulation based cohort consisting of 40 764 preterm deliveries.\nCS = caesarean section, BMI = Body Mass Index (weight in kilograms/height in meters2), EA = Epidural Analgesia.\nThe most common indication for VE was fetal distress (42%), followed by prolonged labor (25%). Having a non-occipitoanterior position (25%) or fetal distress (17%) were the most common indications for CS during labor, while only 3% in this group had a diagnosis of prolonged labor.","The proportion of preterm infants diagnosed with an ICH varied more than hundred-fold in relation to GA. It decreased from 21.5% among preterm infants born at 22–28 weeks of GA to 0.1% among those born after 36 weeks of gestation. The rates of neonatal convulsions among preterm infants decreased from 2.0% at 22–28 weeks to 0.25% among those born after 36 weeks of gestation, Figure \n2. The proportion of preterm infants diagnosed with other disturbances of cerebral status (encephalopathy) decreased with GA, while proportion of infants with brachial plexus injuries or ECH increased slightly with GA, Figure \n3.\nProportion (%) preterm infants diagnosed with intracranial hemorrhage (ICH) or convulsions by gestational age. Figure\n2 shows the proportions (%) of preterm infants diagnosed with ICH and neonatal convulsions in relation to gestational age (in completed weeks). The blue, dotted line represents intracranial hemorrhage (ICH) and the red, dashed line represents neonatal convulsions.\nProportion (%) preterm infants diagnosed with extracranial hemorrhage (ECH), encephalopathy and brachial plexus injury by gestational age. Figure\n3 shows the proportions (%) of ECH, encephalopathy (ICD-code P91: other disturbances of cerebral status of newborn), and brachial plexus injury in relation to gestational age (in completed weeks). The blue, dotted line represents extracranial hemorrhage (ECH), the red, dashed line represents encephalopathy and the green line represents brachial plexus injury.","To report outcome in relation to mode of delivery, the study cohort was divided according to the current guidelines on the use of VE as either preterm births occurring between 34–36 weeks of gestation (VE may be used), or those occurring before 34 gestational weeks (VE not recommended). In our cohort, 33,202 (81.4%) of all preterm births occurred at 34–36 gestational weeks, and 7,562 (18.6%) before 34 weeks of GA.\nIn Table \n3, neonatal outcomes before and after 34 + 0 weeks of gestation are presented in relation to mode of delivery. Overall, seven preterm infants were classified as having an ICH due to birth injury, corresponding to a rate of 0.02%; and 612 infants were diagnosed with non-traumatic ICH, corresponding to a rate of 1.5%. Diagnoses of neonatal convulsions and other disturbances of neonatal cerebral status were rare, especially in infants at less than 34 weeks of GA, and occurred more frequently after VE and CS than after VD. Cephalic hematoma was the most frequent complication after VE of preterm infants (n = 72 or 3.1%), occurring much more often after VE than after CS (0.16%) and VD (0.49%). Subgaleal hemorrhage was less frequent, with a total of only18 cases. More than two thirds of those cases occurred in the VE group.\nNeonatal outcomes in preterm infants by mode of delivery and gestational age\nTable \n4 shows rates (per 1000), crude and adjusted odds ratios for the outcomes by mode of delivery, and uses infants born by VD as the reference group. After adjustments, preterm infants born by VE had almost doubled OR for ICH, while those born by CS had no increased risk. Preterm infants delivered by VE also had 4.5 times higher OR for extracranial hemorrhages, while those delivered by CS had no increased risk. A total of 540 (88.2%) of the ICH diagnoses consisted of intraventricular bleedings (ICD-codes 52.0-52.3), the type of ICH most frequently seen in preterm infants. Of these, 438 (71.6%) were graded as mild or moderate (grades 1–2). After excluding grades 1–2 of intraventricular hemorrhages from the analyses, the OR for ICH was still significantly increased for infants delivered by VE (OR 2.58, CI 1.27-5.24), but not for those delivered by CS during labor (OR 1.15, CI 0.78-1.70) after adjustment for GA. There was no difference in ICH rates between preterm boys and girls delivered by VE.\nLogistic regression (odds ratios) for intra– and extracranial hemorrhages, convulsions and other cerebral complications, and brachial plexus injury in preterm infants exposed to different modes of delivery\nCS = cesarean section, VE = vacuum extraction.\nModel 1 is adjusted for year of birth, gestational age, parity, maternal age, height, BMI, and infant birthweight.\nModel 2 is also adjusted for indications for operative delivery.\nThe ORs for convulsions were almost doubled in both the VE and CS groups after adjustments for the variables in Model 1. However, further adjustment for indication for operative delivery decreased the odds and made the associations statistically insignificant. Other disturbances of the neonatal cerebral status were significantly increased (two to three times higher) both among infants delivered by VE, and by CS during labor, although the OR was higher in the VE group.\nA total of 53 infants were diagnosed with brachial plexus injury. Of these, 14 were born by VE, corresponding to a rate in the VE group of 0.6% and an OR of 6.21 (95% CI: 2.22-17.4) in the fully-adjusted model. In contrast, infants delivered by CS had no increased risk for this complication. Among infants with brachial plexus injury, there were 11 cases of shoulder dystocia (ICD-code O66.0), of which five occurred in the VE group.","BMI: Body mass index; CI: Confidence interval; CS: Cesarean section; CT: Computerized tomography; EA: Epidural analgesia; ECH: Extracranial hemorrhage; EEG: Electroencephalography; GA: Gestational age; ICH: Intracranial hemorrhage; LGA: Large for gestational age; OR: Odds ratio; SGA: Small for gestational age; VD: Vaginal delivery; VE: Vacuum extraction.","The authors declare that they have no competing interest.","CE had the idea for the study, designed it and carried out the statistical analysis. KÅ participated in the design and wrote the first draft of the manuscript together with CE. MN contributed to the interpretation of the results and writing of the manuscript. All authors approved the final version of the submitted article.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2393/14/42/prepub\n"],"string":"[\n \"Preterm birth is common\\n[1] but still, the optimal mode of delivery of preterm infants is not known. Although neonatal outcomes in preterm infants delivered vaginally or by cesarean section (CS)\\n[2-5] have been compared, there is no evidence to provide clear guidance on the method of choice\\n[6]. Given the widespread assumption that assisted vaginal delivery could be harmful for fragile infants that are underweight and preterm, very few studies have addressed the use of vacuum extraction (VE) for preterm birth.\\nDelivery by VE is a common obstetrical procedure, and in many countries it has replaced the use of forceps. VE is used to terminate a protracted second stage of labor and as an intervention for fetal or maternal distress. VE requires vertex presentation, a fully dilated cervix and ruptured membranes\\n[7]. A cesarean section, on the other hand, can be performed at any stage of labor and does not require prerequisites of this kind.\\nMost clinical guidelines do not recommend VE before 34 gestational weeks\\n[8-10]. These recommendations are not based on results of randomized controlled trials, but rely on the observation that preterm infants are more likely than term infants to develop ICH, and on extrapolations from studies of term infants showing that VE is associated with an increased risk of ICH and other neonatal complications\\n[11-17]. Only three studies have previously investigated the use and outcomes of VE in preterm births. The first was undertaken over 40 years ago and showed increased mortality and morbidity among preterm infants delivered by VE as compared with term infants delivered by VE\\n[18]. The second study compared neonatal morbidity in preterm infants delivered vaginally with (n = 61) or without VE (n = 122), and found no differences in neonatal morbidity between the two groups\\n[19]. The last study compared VE and forceps for preterm delivery (n = 64)\\n[20]; the neonatal outcomes were similar in both groups. The available data are clearly untimely and hampered by limitations in power and, therefore, current knowledge on safety of preterm vacuum-assisted birth is unsatisfactory.\\nThe aim of this study was to 1) describe the use of VE and compare it to rates of CS during labor in preterm deliveries in Sweden from 1999–2010, 2) characterize the distribution of perinatal risk factors associated with each mode of delivery, and 3) compare rates of neonatal intra- and extracranial hemorrhages, as well as occurrence of brachial plexus injury after preterm delivery by VE or CS during labor, using unassisted vaginal birth as a reference.\",\n \"During the period of 1999–2010, there were 75,296 (6.2%) preterm births in Sweden. We excluded deliveries by CS before the onset of labor (n = 17,306), forceps (n = 257), or performed with both VE and CS (n = 125). We also excluded stillbirths (fetal deaths occurring before labor or intra partum) (n = 1,839), multiple births (n = 11,088), and births in breech presentation (n = 3,917). Thus, the final study population was restricted to all live-born, preterm singleton infants with a non-breech presentation at birth, delivered after a spontaneous or induced onset of labor followed by CS, vacuum extraction (VE), or by unassisted vaginal delivery (VD) before gestational week 37 + 0 days (N = 40,764). CS during labor was defined as abdominal delivery after the onset of labor, either spontaneously, by rupture of membranes, or by induction.\\nA number of independent variables were collected; the maternal anthropometrics included: age, height, and body mass index (BMI). BMI was calculated from measured height and weight obtained at the first antenatal care visit, which occurred before the 15th week of gestation in more than 95% of the pregnancies. BMI was categorized into underweight (below 18.5 kg/m2), normal (18.5–24.9 kg/m2), overweight (25–29.9 kg/m2), obese (>29.9 kg/m2), or missing. Parity was categorized as primi- or multiparity. Information on complications during pregnancy and delivery were coded according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards). The following pregnancy complications were included: diabetes – both gestational and types 1 and 2 (O24.0-9) preeclampsia- both hypertension, preeclampsia, and eclampsia (O10.0-O15.9). Labor-related risk factors or covariates includedepidural analgesia (EA; yes/no), and induction of labor (yes/no). Indications for operative delivery were classified into four major groups: prolonged labor (O62.0-2, O63.0-9), signs of fetal distress (O68.0-O68.1-9), preeclampsia, and a non-occipitoanterior presentation of the fetus (all presentations except occipitoanterior and breech, registered at birth). Gestational age (GA) for preterm infants was divided into three periods according to the World Health Organization: extremely preterm (before 28 weeks), very preterm (28–31 weeks) and moderately preterm (32–36 weeks). Furthermore, we also divided the preterm gestational period according to guidelines on instrumental delivery into either: less than 34 weeks (VE not recommended), and 34–36 weeks (VE may be used). GA was recorded in completed weeks, and was based on routine ultrasound dating performed at 17 to 18 postmenstrual weeks in 97-98% of all pregnant women. Infant birthweight was categorized as less than 1,500 grams, 1,500-2,000 grams, 2,001-2,500 grams, 2,501-3,000 grams, and 3,001-4,000 grams.\",\n \"Neonatal diagnoses were classified according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards), and identified/collected in the SMBR or in the IPR. The following neonatal outcomes (ICD codes) were assessed: Intracranial laceration and hemorrhage due to birth injury (P10), intracranial non-traumatic hemorrhage of fetus and newborn (P52), convulsions of newborn (P90), other disturbances of cerebral status of newborn (P 91), subgaleal hematoma (P12.2), cephalhematoma (P12.0), and brachial plexus injury (P14.0-3). The definitions of outcomes are described in detail in Table \\n1.\\nNeonatal outcomes studied in 40 764 preterm infants\\nNeonatal diagnoses of intracranial hemorrhages in preterm infants were mainly based on imaging of the brain using ultrasonography; however, some assessments of the brain at term-equivalent age were alternatively performed with CT and/or MRI. Imaging of the brain was performed on clinical indications only in cases born moderately or late preterm, whereas all very preterm infants (born before 32 weeks of gestation) were screened for intracranial lesions, even in asymptomatic infants. A diagnosis of convulsions included infants with clinical signs of convulsions and/or convulsions verified by EEG.\\nStatistical analysis was performed using proportions and odds ratios (OR) with a 95% confidence interval (CI) for neonatal complications in relation to mode of delivery, using unassisted VD as the reference group (SPSS 20.0 for Windows software package). Three models were used to assess the relationship between the different modes of delivery and the risk for neonatal complications: one crude, and two adjusted (Models 1 and 2). The included covariates have been shown previously to be related to instrumental deliveries, and were related to the outcomes in cross tabulations. In Model 1, we adjusted for the following confounders or covariates: maternal age, height, BMI, and parity, as well as infant year of birth, birthweight and GA. In Model 2, we added the indication for operative delivery and preeclampsia. The year of birth was entered as a continuous variable in accordance with a linear secular trend, and all other variables were entered as categories. Furthermore, a separate logistic regression analysis was performed to investigate severe ICH in relation to mode of delivery. Here, intraventricular hemorrhages grades 1–2 were excluded and the analysis was adjusted for GA only. We also conducted separated analyses on potential relationships between sex and ICH in relation to mode of delivery. Missing data were entered as a separate category in the analyses. The study was approved by the Regional Ethical Review Board in Stockholm, Dnr 2008/1322-31.\",\n \"Among the 40,764 (54% of all) preterm deliveries included in this study, 2,319 (5.7%) preterm infants were delivered by VE, 5,505 (13.5%) by CS during labor, and 32,940 (80.2%) by VD. The rate of VE deliveries increased gradually with gestational age, Figure \\n1.\\nMode of delivery in relation to gestational age. Figure\\n1 shows rates (%) of different modes of delivery in relation to gestational age (in completed weeks). The blue, dotted line represents unassisted vaginal deliveries, the red, dashed line represents cesarean sections performed after onset of labor, and the green line represents the vacuum extraction deliveries.\",\n \"Table \\n2 shows maternal and perinatal characteristics of the study population in relation to mode of delivery. The VE rate decreased with maternal height and 80% of the women who delivered by VE were primiparae, compared with 48% of those who underwent CS during labor (not in table). More than 45% of the women who delivered by VE had received epidural analgesia during labor compared with 22% of women with VD, and 12% with CS during labor (not in table). Given the association between GA and VE, infants delivered with VE had higher birthweights.\\nMaternal, pregnancy, delivery, and infant characteristics by mode of delivery\\nPopulation based cohort consisting of 40 764 preterm deliveries.\\nCS = caesarean section, BMI = Body Mass Index (weight in kilograms/height in meters2), EA = Epidural Analgesia.\\nThe most common indication for VE was fetal distress (42%), followed by prolonged labor (25%). Having a non-occipitoanterior position (25%) or fetal distress (17%) were the most common indications for CS during labor, while only 3% in this group had a diagnosis of prolonged labor.\",\n \"The proportion of preterm infants diagnosed with an ICH varied more than hundred-fold in relation to GA. It decreased from 21.5% among preterm infants born at 22–28 weeks of GA to 0.1% among those born after 36 weeks of gestation. The rates of neonatal convulsions among preterm infants decreased from 2.0% at 22–28 weeks to 0.25% among those born after 36 weeks of gestation, Figure \\n2. The proportion of preterm infants diagnosed with other disturbances of cerebral status (encephalopathy) decreased with GA, while proportion of infants with brachial plexus injuries or ECH increased slightly with GA, Figure \\n3.\\nProportion (%) preterm infants diagnosed with intracranial hemorrhage (ICH) or convulsions by gestational age. Figure\\n2 shows the proportions (%) of preterm infants diagnosed with ICH and neonatal convulsions in relation to gestational age (in completed weeks). The blue, dotted line represents intracranial hemorrhage (ICH) and the red, dashed line represents neonatal convulsions.\\nProportion (%) preterm infants diagnosed with extracranial hemorrhage (ECH), encephalopathy and brachial plexus injury by gestational age. Figure\\n3 shows the proportions (%) of ECH, encephalopathy (ICD-code P91: other disturbances of cerebral status of newborn), and brachial plexus injury in relation to gestational age (in completed weeks). The blue, dotted line represents extracranial hemorrhage (ECH), the red, dashed line represents encephalopathy and the green line represents brachial plexus injury.\",\n \"To report outcome in relation to mode of delivery, the study cohort was divided according to the current guidelines on the use of VE as either preterm births occurring between 34–36 weeks of gestation (VE may be used), or those occurring before 34 gestational weeks (VE not recommended). In our cohort, 33,202 (81.4%) of all preterm births occurred at 34–36 gestational weeks, and 7,562 (18.6%) before 34 weeks of GA.\\nIn Table \\n3, neonatal outcomes before and after 34 + 0 weeks of gestation are presented in relation to mode of delivery. Overall, seven preterm infants were classified as having an ICH due to birth injury, corresponding to a rate of 0.02%; and 612 infants were diagnosed with non-traumatic ICH, corresponding to a rate of 1.5%. Diagnoses of neonatal convulsions and other disturbances of neonatal cerebral status were rare, especially in infants at less than 34 weeks of GA, and occurred more frequently after VE and CS than after VD. Cephalic hematoma was the most frequent complication after VE of preterm infants (n = 72 or 3.1%), occurring much more often after VE than after CS (0.16%) and VD (0.49%). Subgaleal hemorrhage was less frequent, with a total of only18 cases. More than two thirds of those cases occurred in the VE group.\\nNeonatal outcomes in preterm infants by mode of delivery and gestational age\\nTable \\n4 shows rates (per 1000), crude and adjusted odds ratios for the outcomes by mode of delivery, and uses infants born by VD as the reference group. After adjustments, preterm infants born by VE had almost doubled OR for ICH, while those born by CS had no increased risk. Preterm infants delivered by VE also had 4.5 times higher OR for extracranial hemorrhages, while those delivered by CS had no increased risk. A total of 540 (88.2%) of the ICH diagnoses consisted of intraventricular bleedings (ICD-codes 52.0-52.3), the type of ICH most frequently seen in preterm infants. Of these, 438 (71.6%) were graded as mild or moderate (grades 1–2). After excluding grades 1–2 of intraventricular hemorrhages from the analyses, the OR for ICH was still significantly increased for infants delivered by VE (OR 2.58, CI 1.27-5.24), but not for those delivered by CS during labor (OR 1.15, CI 0.78-1.70) after adjustment for GA. There was no difference in ICH rates between preterm boys and girls delivered by VE.\\nLogistic regression (odds ratios) for intra– and extracranial hemorrhages, convulsions and other cerebral complications, and brachial plexus injury in preterm infants exposed to different modes of delivery\\nCS = cesarean section, VE = vacuum extraction.\\nModel 1 is adjusted for year of birth, gestational age, parity, maternal age, height, BMI, and infant birthweight.\\nModel 2 is also adjusted for indications for operative delivery.\\nThe ORs for convulsions were almost doubled in both the VE and CS groups after adjustments for the variables in Model 1. However, further adjustment for indication for operative delivery decreased the odds and made the associations statistically insignificant. Other disturbances of the neonatal cerebral status were significantly increased (two to three times higher) both among infants delivered by VE, and by CS during labor, although the OR was higher in the VE group.\\nA total of 53 infants were diagnosed with brachial plexus injury. Of these, 14 were born by VE, corresponding to a rate in the VE group of 0.6% and an OR of 6.21 (95% CI: 2.22-17.4) in the fully-adjusted model. In contrast, infants delivered by CS had no increased risk for this complication. Among infants with brachial plexus injury, there were 11 cases of shoulder dystocia (ICD-code O66.0), of which five occurred in the VE group.\",\n \"BMI: Body mass index; CI: Confidence interval; CS: Cesarean section; CT: Computerized tomography; EA: Epidural analgesia; ECH: Extracranial hemorrhage; EEG: Electroencephalography; GA: Gestational age; ICH: Intracranial hemorrhage; LGA: Large for gestational age; OR: Odds ratio; SGA: Small for gestational age; VD: Vaginal delivery; VE: Vacuum extraction.\",\n \"The authors declare that they have no competing interest.\",\n \"CE had the idea for the study, designed it and carried out the statistical analysis. KÅ participated in the design and wrote the first draft of the manuscript together with CE. MN contributed to the interpretation of the results and writing of the manuscript. All authors approved the final version of the submitted article.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2393/14/42/prepub\\n\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Study population","Outcome variables","Results","Use of VE in relation to gestational age","Distribution of risk factors or covariates in relation to mode of delivery","Neonatal outcome in relation to gestational age","Neonatal outcome in relation to mode of delivery","Discussion","Conclusion","Abbreviations","Competing interests","Authors’ contributions","Pre-publication history"],"string":"[\n \"Background\",\n \"Methods\",\n \"Study population\",\n \"Outcome variables\",\n \"Results\",\n \"Use of VE in relation to gestational age\",\n \"Distribution of risk factors or covariates in relation to mode of delivery\",\n \"Neonatal outcome in relation to gestational age\",\n \"Neonatal outcome in relation to mode of delivery\",\n \"Discussion\",\n \"Conclusion\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\",\n \"Pre-publication history\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Preterm birth is common\n[1] but still, the optimal mode of delivery of preterm infants is not known. Although neonatal outcomes in preterm infants delivered vaginally or by cesarean section (CS)\n[2-5] have been compared, there is no evidence to provide clear guidance on the method of choice\n[6]. Given the widespread assumption that assisted vaginal delivery could be harmful for fragile infants that are underweight and preterm, very few studies have addressed the use of vacuum extraction (VE) for preterm birth.\nDelivery by VE is a common obstetrical procedure, and in many countries it has replaced the use of forceps. VE is used to terminate a protracted second stage of labor and as an intervention for fetal or maternal distress. VE requires vertex presentation, a fully dilated cervix and ruptured membranes\n[7]. A cesarean section, on the other hand, can be performed at any stage of labor and does not require prerequisites of this kind.\nMost clinical guidelines do not recommend VE before 34 gestational weeks\n[8-10]. These recommendations are not based on results of randomized controlled trials, but rely on the observation that preterm infants are more likely than term infants to develop ICH, and on extrapolations from studies of term infants showing that VE is associated with an increased risk of ICH and other neonatal complications\n[11-17]. Only three studies have previously investigated the use and outcomes of VE in preterm births. The first was undertaken over 40 years ago and showed increased mortality and morbidity among preterm infants delivered by VE as compared with term infants delivered by VE\n[18]. The second study compared neonatal morbidity in preterm infants delivered vaginally with (n = 61) or without VE (n = 122), and found no differences in neonatal morbidity between the two groups\n[19]. The last study compared VE and forceps for preterm delivery (n = 64)\n[20]; the neonatal outcomes were similar in both groups. The available data are clearly untimely and hampered by limitations in power and, therefore, current knowledge on safety of preterm vacuum-assisted birth is unsatisfactory.\nThe aim of this study was to 1) describe the use of VE and compare it to rates of CS during labor in preterm deliveries in Sweden from 1999–2010, 2) characterize the distribution of perinatal risk factors associated with each mode of delivery, and 3) compare rates of neonatal intra- and extracranial hemorrhages, as well as occurrence of brachial plexus injury after preterm delivery by VE or CS during labor, using unassisted vaginal birth as a reference.","This study was based on data from national data bases held by the Swedish National Board of Health and Welfare. The national registration number, assigned to each Swedish resident at birth, was used for individual record linkage. We used two registers: The Swedish Medical Birth Register (SMBR) that covers 99% of all births in Sweden, and The Swedish National Inpatient Register (IPR) that covers all public inpatient care. The SMBR includes prospectively collected information on maternal characteristics, reproductive history, and complications during pregnancy, delivery, and the neonatal period. The IPR includes data on each hospital admission and discharge.\n Study population During the period of 1999–2010, there were 75,296 (6.2%) preterm births in Sweden. We excluded deliveries by CS before the onset of labor (n = 17,306), forceps (n = 257), or performed with both VE and CS (n = 125). We also excluded stillbirths (fetal deaths occurring before labor or intra partum) (n = 1,839), multiple births (n = 11,088), and births in breech presentation (n = 3,917). Thus, the final study population was restricted to all live-born, preterm singleton infants with a non-breech presentation at birth, delivered after a spontaneous or induced onset of labor followed by CS, vacuum extraction (VE), or by unassisted vaginal delivery (VD) before gestational week 37 + 0 days (N = 40,764). CS during labor was defined as abdominal delivery after the onset of labor, either spontaneously, by rupture of membranes, or by induction.\nA number of independent variables were collected; the maternal anthropometrics included: age, height, and body mass index (BMI). BMI was calculated from measured height and weight obtained at the first antenatal care visit, which occurred before the 15th week of gestation in more than 95% of the pregnancies. BMI was categorized into underweight (below 18.5 kg/m2), normal (18.5–24.9 kg/m2), overweight (25–29.9 kg/m2), obese (>29.9 kg/m2), or missing. Parity was categorized as primi- or multiparity. Information on complications during pregnancy and delivery were coded according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards). The following pregnancy complications were included: diabetes – both gestational and types 1 and 2 (O24.0-9) preeclampsia- both hypertension, preeclampsia, and eclampsia (O10.0-O15.9). Labor-related risk factors or covariates includedepidural analgesia (EA; yes/no), and induction of labor (yes/no). Indications for operative delivery were classified into four major groups: prolonged labor (O62.0-2, O63.0-9), signs of fetal distress (O68.0-O68.1-9), preeclampsia, and a non-occipitoanterior presentation of the fetus (all presentations except occipitoanterior and breech, registered at birth). Gestational age (GA) for preterm infants was divided into three periods according to the World Health Organization: extremely preterm (before 28 weeks), very preterm (28–31 weeks) and moderately preterm (32–36 weeks). Furthermore, we also divided the preterm gestational period according to guidelines on instrumental delivery into either: less than 34 weeks (VE not recommended), and 34–36 weeks (VE may be used). GA was recorded in completed weeks, and was based on routine ultrasound dating performed at 17 to 18 postmenstrual weeks in 97-98% of all pregnant women. Infant birthweight was categorized as less than 1,500 grams, 1,500-2,000 grams, 2,001-2,500 grams, 2,501-3,000 grams, and 3,001-4,000 grams.\nDuring the period of 1999–2010, there were 75,296 (6.2%) preterm births in Sweden. We excluded deliveries by CS before the onset of labor (n = 17,306), forceps (n = 257), or performed with both VE and CS (n = 125). We also excluded stillbirths (fetal deaths occurring before labor or intra partum) (n = 1,839), multiple births (n = 11,088), and births in breech presentation (n = 3,917). Thus, the final study population was restricted to all live-born, preterm singleton infants with a non-breech presentation at birth, delivered after a spontaneous or induced onset of labor followed by CS, vacuum extraction (VE), or by unassisted vaginal delivery (VD) before gestational week 37 + 0 days (N = 40,764). CS during labor was defined as abdominal delivery after the onset of labor, either spontaneously, by rupture of membranes, or by induction.\nA number of independent variables were collected; the maternal anthropometrics included: age, height, and body mass index (BMI). BMI was calculated from measured height and weight obtained at the first antenatal care visit, which occurred before the 15th week of gestation in more than 95% of the pregnancies. BMI was categorized into underweight (below 18.5 kg/m2), normal (18.5–24.9 kg/m2), overweight (25–29.9 kg/m2), obese (>29.9 kg/m2), or missing. Parity was categorized as primi- or multiparity. Information on complications during pregnancy and delivery were coded according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards). The following pregnancy complications were included: diabetes – both gestational and types 1 and 2 (O24.0-9) preeclampsia- both hypertension, preeclampsia, and eclampsia (O10.0-O15.9). Labor-related risk factors or covariates includedepidural analgesia (EA; yes/no), and induction of labor (yes/no). Indications for operative delivery were classified into four major groups: prolonged labor (O62.0-2, O63.0-9), signs of fetal distress (O68.0-O68.1-9), preeclampsia, and a non-occipitoanterior presentation of the fetus (all presentations except occipitoanterior and breech, registered at birth). Gestational age (GA) for preterm infants was divided into three periods according to the World Health Organization: extremely preterm (before 28 weeks), very preterm (28–31 weeks) and moderately preterm (32–36 weeks). Furthermore, we also divided the preterm gestational period according to guidelines on instrumental delivery into either: less than 34 weeks (VE not recommended), and 34–36 weeks (VE may be used). GA was recorded in completed weeks, and was based on routine ultrasound dating performed at 17 to 18 postmenstrual weeks in 97-98% of all pregnant women. Infant birthweight was categorized as less than 1,500 grams, 1,500-2,000 grams, 2,001-2,500 grams, 2,501-3,000 grams, and 3,001-4,000 grams.\n Outcome variables Neonatal diagnoses were classified according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards), and identified/collected in the SMBR or in the IPR. The following neonatal outcomes (ICD codes) were assessed: Intracranial laceration and hemorrhage due to birth injury (P10), intracranial non-traumatic hemorrhage of fetus and newborn (P52), convulsions of newborn (P90), other disturbances of cerebral status of newborn (P 91), subgaleal hematoma (P12.2), cephalhematoma (P12.0), and brachial plexus injury (P14.0-3). The definitions of outcomes are described in detail in Table \n1.\nNeonatal outcomes studied in 40 764 preterm infants\nNeonatal diagnoses of intracranial hemorrhages in preterm infants were mainly based on imaging of the brain using ultrasonography; however, some assessments of the brain at term-equivalent age were alternatively performed with CT and/or MRI. Imaging of the brain was performed on clinical indications only in cases born moderately or late preterm, whereas all very preterm infants (born before 32 weeks of gestation) were screened for intracranial lesions, even in asymptomatic infants. A diagnosis of convulsions included infants with clinical signs of convulsions and/or convulsions verified by EEG.\nStatistical analysis was performed using proportions and odds ratios (OR) with a 95% confidence interval (CI) for neonatal complications in relation to mode of delivery, using unassisted VD as the reference group (SPSS 20.0 for Windows software package). Three models were used to assess the relationship between the different modes of delivery and the risk for neonatal complications: one crude, and two adjusted (Models 1 and 2). The included covariates have been shown previously to be related to instrumental deliveries, and were related to the outcomes in cross tabulations. In Model 1, we adjusted for the following confounders or covariates: maternal age, height, BMI, and parity, as well as infant year of birth, birthweight and GA. In Model 2, we added the indication for operative delivery and preeclampsia. The year of birth was entered as a continuous variable in accordance with a linear secular trend, and all other variables were entered as categories. Furthermore, a separate logistic regression analysis was performed to investigate severe ICH in relation to mode of delivery. Here, intraventricular hemorrhages grades 1–2 were excluded and the analysis was adjusted for GA only. We also conducted separated analyses on potential relationships between sex and ICH in relation to mode of delivery. Missing data were entered as a separate category in the analyses. The study was approved by the Regional Ethical Review Board in Stockholm, Dnr 2008/1322-31.\nNeonatal diagnoses were classified according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards), and identified/collected in the SMBR or in the IPR. The following neonatal outcomes (ICD codes) were assessed: Intracranial laceration and hemorrhage due to birth injury (P10), intracranial non-traumatic hemorrhage of fetus and newborn (P52), convulsions of newborn (P90), other disturbances of cerebral status of newborn (P 91), subgaleal hematoma (P12.2), cephalhematoma (P12.0), and brachial plexus injury (P14.0-3). The definitions of outcomes are described in detail in Table \n1.\nNeonatal outcomes studied in 40 764 preterm infants\nNeonatal diagnoses of intracranial hemorrhages in preterm infants were mainly based on imaging of the brain using ultrasonography; however, some assessments of the brain at term-equivalent age were alternatively performed with CT and/or MRI. Imaging of the brain was performed on clinical indications only in cases born moderately or late preterm, whereas all very preterm infants (born before 32 weeks of gestation) were screened for intracranial lesions, even in asymptomatic infants. A diagnosis of convulsions included infants with clinical signs of convulsions and/or convulsions verified by EEG.\nStatistical analysis was performed using proportions and odds ratios (OR) with a 95% confidence interval (CI) for neonatal complications in relation to mode of delivery, using unassisted VD as the reference group (SPSS 20.0 for Windows software package). Three models were used to assess the relationship between the different modes of delivery and the risk for neonatal complications: one crude, and two adjusted (Models 1 and 2). The included covariates have been shown previously to be related to instrumental deliveries, and were related to the outcomes in cross tabulations. In Model 1, we adjusted for the following confounders or covariates: maternal age, height, BMI, and parity, as well as infant year of birth, birthweight and GA. In Model 2, we added the indication for operative delivery and preeclampsia. The year of birth was entered as a continuous variable in accordance with a linear secular trend, and all other variables were entered as categories. Furthermore, a separate logistic regression analysis was performed to investigate severe ICH in relation to mode of delivery. Here, intraventricular hemorrhages grades 1–2 were excluded and the analysis was adjusted for GA only. We also conducted separated analyses on potential relationships between sex and ICH in relation to mode of delivery. Missing data were entered as a separate category in the analyses. The study was approved by the Regional Ethical Review Board in Stockholm, Dnr 2008/1322-31.","During the period of 1999–2010, there were 75,296 (6.2%) preterm births in Sweden. We excluded deliveries by CS before the onset of labor (n = 17,306), forceps (n = 257), or performed with both VE and CS (n = 125). We also excluded stillbirths (fetal deaths occurring before labor or intra partum) (n = 1,839), multiple births (n = 11,088), and births in breech presentation (n = 3,917). Thus, the final study population was restricted to all live-born, preterm singleton infants with a non-breech presentation at birth, delivered after a spontaneous or induced onset of labor followed by CS, vacuum extraction (VE), or by unassisted vaginal delivery (VD) before gestational week 37 + 0 days (N = 40,764). CS during labor was defined as abdominal delivery after the onset of labor, either spontaneously, by rupture of membranes, or by induction.\nA number of independent variables were collected; the maternal anthropometrics included: age, height, and body mass index (BMI). BMI was calculated from measured height and weight obtained at the first antenatal care visit, which occurred before the 15th week of gestation in more than 95% of the pregnancies. BMI was categorized into underweight (below 18.5 kg/m2), normal (18.5–24.9 kg/m2), overweight (25–29.9 kg/m2), obese (>29.9 kg/m2), or missing. Parity was categorized as primi- or multiparity. Information on complications during pregnancy and delivery were coded according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards). The following pregnancy complications were included: diabetes – both gestational and types 1 and 2 (O24.0-9) preeclampsia- both hypertension, preeclampsia, and eclampsia (O10.0-O15.9). Labor-related risk factors or covariates includedepidural analgesia (EA; yes/no), and induction of labor (yes/no). Indications for operative delivery were classified into four major groups: prolonged labor (O62.0-2, O63.0-9), signs of fetal distress (O68.0-O68.1-9), preeclampsia, and a non-occipitoanterior presentation of the fetus (all presentations except occipitoanterior and breech, registered at birth). Gestational age (GA) for preterm infants was divided into three periods according to the World Health Organization: extremely preterm (before 28 weeks), very preterm (28–31 weeks) and moderately preterm (32–36 weeks). Furthermore, we also divided the preterm gestational period according to guidelines on instrumental delivery into either: less than 34 weeks (VE not recommended), and 34–36 weeks (VE may be used). GA was recorded in completed weeks, and was based on routine ultrasound dating performed at 17 to 18 postmenstrual weeks in 97-98% of all pregnant women. Infant birthweight was categorized as less than 1,500 grams, 1,500-2,000 grams, 2,001-2,500 grams, 2,501-3,000 grams, and 3,001-4,000 grams.","Neonatal diagnoses were classified according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards), and identified/collected in the SMBR or in the IPR. The following neonatal outcomes (ICD codes) were assessed: Intracranial laceration and hemorrhage due to birth injury (P10), intracranial non-traumatic hemorrhage of fetus and newborn (P52), convulsions of newborn (P90), other disturbances of cerebral status of newborn (P 91), subgaleal hematoma (P12.2), cephalhematoma (P12.0), and brachial plexus injury (P14.0-3). The definitions of outcomes are described in detail in Table \n1.\nNeonatal outcomes studied in 40 764 preterm infants\nNeonatal diagnoses of intracranial hemorrhages in preterm infants were mainly based on imaging of the brain using ultrasonography; however, some assessments of the brain at term-equivalent age were alternatively performed with CT and/or MRI. Imaging of the brain was performed on clinical indications only in cases born moderately or late preterm, whereas all very preterm infants (born before 32 weeks of gestation) were screened for intracranial lesions, even in asymptomatic infants. A diagnosis of convulsions included infants with clinical signs of convulsions and/or convulsions verified by EEG.\nStatistical analysis was performed using proportions and odds ratios (OR) with a 95% confidence interval (CI) for neonatal complications in relation to mode of delivery, using unassisted VD as the reference group (SPSS 20.0 for Windows software package). Three models were used to assess the relationship between the different modes of delivery and the risk for neonatal complications: one crude, and two adjusted (Models 1 and 2). The included covariates have been shown previously to be related to instrumental deliveries, and were related to the outcomes in cross tabulations. In Model 1, we adjusted for the following confounders or covariates: maternal age, height, BMI, and parity, as well as infant year of birth, birthweight and GA. In Model 2, we added the indication for operative delivery and preeclampsia. The year of birth was entered as a continuous variable in accordance with a linear secular trend, and all other variables were entered as categories. Furthermore, a separate logistic regression analysis was performed to investigate severe ICH in relation to mode of delivery. Here, intraventricular hemorrhages grades 1–2 were excluded and the analysis was adjusted for GA only. We also conducted separated analyses on potential relationships between sex and ICH in relation to mode of delivery. Missing data were entered as a separate category in the analyses. The study was approved by the Regional Ethical Review Board in Stockholm, Dnr 2008/1322-31."," Use of VE in relation to gestational age Among the 40,764 (54% of all) preterm deliveries included in this study, 2,319 (5.7%) preterm infants were delivered by VE, 5,505 (13.5%) by CS during labor, and 32,940 (80.2%) by VD. The rate of VE deliveries increased gradually with gestational age, Figure \n1.\nMode of delivery in relation to gestational age. Figure\n1 shows rates (%) of different modes of delivery in relation to gestational age (in completed weeks). The blue, dotted line represents unassisted vaginal deliveries, the red, dashed line represents cesarean sections performed after onset of labor, and the green line represents the vacuum extraction deliveries.\nAmong the 40,764 (54% of all) preterm deliveries included in this study, 2,319 (5.7%) preterm infants were delivered by VE, 5,505 (13.5%) by CS during labor, and 32,940 (80.2%) by VD. The rate of VE deliveries increased gradually with gestational age, Figure \n1.\nMode of delivery in relation to gestational age. Figure\n1 shows rates (%) of different modes of delivery in relation to gestational age (in completed weeks). The blue, dotted line represents unassisted vaginal deliveries, the red, dashed line represents cesarean sections performed after onset of labor, and the green line represents the vacuum extraction deliveries.\n Distribution of risk factors or covariates in relation to mode of delivery Table \n2 shows maternal and perinatal characteristics of the study population in relation to mode of delivery. The VE rate decreased with maternal height and 80% of the women who delivered by VE were primiparae, compared with 48% of those who underwent CS during labor (not in table). More than 45% of the women who delivered by VE had received epidural analgesia during labor compared with 22% of women with VD, and 12% with CS during labor (not in table). Given the association between GA and VE, infants delivered with VE had higher birthweights.\nMaternal, pregnancy, delivery, and infant characteristics by mode of delivery\nPopulation based cohort consisting of 40 764 preterm deliveries.\nCS = caesarean section, BMI = Body Mass Index (weight in kilograms/height in meters2), EA = Epidural Analgesia.\nThe most common indication for VE was fetal distress (42%), followed by prolonged labor (25%). Having a non-occipitoanterior position (25%) or fetal distress (17%) were the most common indications for CS during labor, while only 3% in this group had a diagnosis of prolonged labor.\nTable \n2 shows maternal and perinatal characteristics of the study population in relation to mode of delivery. The VE rate decreased with maternal height and 80% of the women who delivered by VE were primiparae, compared with 48% of those who underwent CS during labor (not in table). More than 45% of the women who delivered by VE had received epidural analgesia during labor compared with 22% of women with VD, and 12% with CS during labor (not in table). Given the association between GA and VE, infants delivered with VE had higher birthweights.\nMaternal, pregnancy, delivery, and infant characteristics by mode of delivery\nPopulation based cohort consisting of 40 764 preterm deliveries.\nCS = caesarean section, BMI = Body Mass Index (weight in kilograms/height in meters2), EA = Epidural Analgesia.\nThe most common indication for VE was fetal distress (42%), followed by prolonged labor (25%). Having a non-occipitoanterior position (25%) or fetal distress (17%) were the most common indications for CS during labor, while only 3% in this group had a diagnosis of prolonged labor.\n Neonatal outcome in relation to gestational age The proportion of preterm infants diagnosed with an ICH varied more than hundred-fold in relation to GA. It decreased from 21.5% among preterm infants born at 22–28 weeks of GA to 0.1% among those born after 36 weeks of gestation. The rates of neonatal convulsions among preterm infants decreased from 2.0% at 22–28 weeks to 0.25% among those born after 36 weeks of gestation, Figure \n2. The proportion of preterm infants diagnosed with other disturbances of cerebral status (encephalopathy) decreased with GA, while proportion of infants with brachial plexus injuries or ECH increased slightly with GA, Figure \n3.\nProportion (%) preterm infants diagnosed with intracranial hemorrhage (ICH) or convulsions by gestational age. Figure\n2 shows the proportions (%) of preterm infants diagnosed with ICH and neonatal convulsions in relation to gestational age (in completed weeks). The blue, dotted line represents intracranial hemorrhage (ICH) and the red, dashed line represents neonatal convulsions.\nProportion (%) preterm infants diagnosed with extracranial hemorrhage (ECH), encephalopathy and brachial plexus injury by gestational age. Figure\n3 shows the proportions (%) of ECH, encephalopathy (ICD-code P91: other disturbances of cerebral status of newborn), and brachial plexus injury in relation to gestational age (in completed weeks). The blue, dotted line represents extracranial hemorrhage (ECH), the red, dashed line represents encephalopathy and the green line represents brachial plexus injury.\nThe proportion of preterm infants diagnosed with an ICH varied more than hundred-fold in relation to GA. It decreased from 21.5% among preterm infants born at 22–28 weeks of GA to 0.1% among those born after 36 weeks of gestation. The rates of neonatal convulsions among preterm infants decreased from 2.0% at 22–28 weeks to 0.25% among those born after 36 weeks of gestation, Figure \n2. The proportion of preterm infants diagnosed with other disturbances of cerebral status (encephalopathy) decreased with GA, while proportion of infants with brachial plexus injuries or ECH increased slightly with GA, Figure \n3.\nProportion (%) preterm infants diagnosed with intracranial hemorrhage (ICH) or convulsions by gestational age. Figure\n2 shows the proportions (%) of preterm infants diagnosed with ICH and neonatal convulsions in relation to gestational age (in completed weeks). The blue, dotted line represents intracranial hemorrhage (ICH) and the red, dashed line represents neonatal convulsions.\nProportion (%) preterm infants diagnosed with extracranial hemorrhage (ECH), encephalopathy and brachial plexus injury by gestational age. Figure\n3 shows the proportions (%) of ECH, encephalopathy (ICD-code P91: other disturbances of cerebral status of newborn), and brachial plexus injury in relation to gestational age (in completed weeks). The blue, dotted line represents extracranial hemorrhage (ECH), the red, dashed line represents encephalopathy and the green line represents brachial plexus injury.\n Neonatal outcome in relation to mode of delivery To report outcome in relation to mode of delivery, the study cohort was divided according to the current guidelines on the use of VE as either preterm births occurring between 34–36 weeks of gestation (VE may be used), or those occurring before 34 gestational weeks (VE not recommended). In our cohort, 33,202 (81.4%) of all preterm births occurred at 34–36 gestational weeks, and 7,562 (18.6%) before 34 weeks of GA.\nIn Table \n3, neonatal outcomes before and after 34 + 0 weeks of gestation are presented in relation to mode of delivery. Overall, seven preterm infants were classified as having an ICH due to birth injury, corresponding to a rate of 0.02%; and 612 infants were diagnosed with non-traumatic ICH, corresponding to a rate of 1.5%. Diagnoses of neonatal convulsions and other disturbances of neonatal cerebral status were rare, especially in infants at less than 34 weeks of GA, and occurred more frequently after VE and CS than after VD. Cephalic hematoma was the most frequent complication after VE of preterm infants (n = 72 or 3.1%), occurring much more often after VE than after CS (0.16%) and VD (0.49%). Subgaleal hemorrhage was less frequent, with a total of only18 cases. More than two thirds of those cases occurred in the VE group.\nNeonatal outcomes in preterm infants by mode of delivery and gestational age\nTable \n4 shows rates (per 1000), crude and adjusted odds ratios for the outcomes by mode of delivery, and uses infants born by VD as the reference group. After adjustments, preterm infants born by VE had almost doubled OR for ICH, while those born by CS had no increased risk. Preterm infants delivered by VE also had 4.5 times higher OR for extracranial hemorrhages, while those delivered by CS had no increased risk. A total of 540 (88.2%) of the ICH diagnoses consisted of intraventricular bleedings (ICD-codes 52.0-52.3), the type of ICH most frequently seen in preterm infants. Of these, 438 (71.6%) were graded as mild or moderate (grades 1–2). After excluding grades 1–2 of intraventricular hemorrhages from the analyses, the OR for ICH was still significantly increased for infants delivered by VE (OR 2.58, CI 1.27-5.24), but not for those delivered by CS during labor (OR 1.15, CI 0.78-1.70) after adjustment for GA. There was no difference in ICH rates between preterm boys and girls delivered by VE.\nLogistic regression (odds ratios) for intra– and extracranial hemorrhages, convulsions and other cerebral complications, and brachial plexus injury in preterm infants exposed to different modes of delivery\nCS = cesarean section, VE = vacuum extraction.\nModel 1 is adjusted for year of birth, gestational age, parity, maternal age, height, BMI, and infant birthweight.\nModel 2 is also adjusted for indications for operative delivery.\nThe ORs for convulsions were almost doubled in both the VE and CS groups after adjustments for the variables in Model 1. However, further adjustment for indication for operative delivery decreased the odds and made the associations statistically insignificant. Other disturbances of the neonatal cerebral status were significantly increased (two to three times higher) both among infants delivered by VE, and by CS during labor, although the OR was higher in the VE group.\nA total of 53 infants were diagnosed with brachial plexus injury. Of these, 14 were born by VE, corresponding to a rate in the VE group of 0.6% and an OR of 6.21 (95% CI: 2.22-17.4) in the fully-adjusted model. In contrast, infants delivered by CS had no increased risk for this complication. Among infants with brachial plexus injury, there were 11 cases of shoulder dystocia (ICD-code O66.0), of which five occurred in the VE group.\nTo report outcome in relation to mode of delivery, the study cohort was divided according to the current guidelines on the use of VE as either preterm births occurring between 34–36 weeks of gestation (VE may be used), or those occurring before 34 gestational weeks (VE not recommended). In our cohort, 33,202 (81.4%) of all preterm births occurred at 34–36 gestational weeks, and 7,562 (18.6%) before 34 weeks of GA.\nIn Table \n3, neonatal outcomes before and after 34 + 0 weeks of gestation are presented in relation to mode of delivery. Overall, seven preterm infants were classified as having an ICH due to birth injury, corresponding to a rate of 0.02%; and 612 infants were diagnosed with non-traumatic ICH, corresponding to a rate of 1.5%. Diagnoses of neonatal convulsions and other disturbances of neonatal cerebral status were rare, especially in infants at less than 34 weeks of GA, and occurred more frequently after VE and CS than after VD. Cephalic hematoma was the most frequent complication after VE of preterm infants (n = 72 or 3.1%), occurring much more often after VE than after CS (0.16%) and VD (0.49%). Subgaleal hemorrhage was less frequent, with a total of only18 cases. More than two thirds of those cases occurred in the VE group.\nNeonatal outcomes in preterm infants by mode of delivery and gestational age\nTable \n4 shows rates (per 1000), crude and adjusted odds ratios for the outcomes by mode of delivery, and uses infants born by VD as the reference group. After adjustments, preterm infants born by VE had almost doubled OR for ICH, while those born by CS had no increased risk. Preterm infants delivered by VE also had 4.5 times higher OR for extracranial hemorrhages, while those delivered by CS had no increased risk. A total of 540 (88.2%) of the ICH diagnoses consisted of intraventricular bleedings (ICD-codes 52.0-52.3), the type of ICH most frequently seen in preterm infants. Of these, 438 (71.6%) were graded as mild or moderate (grades 1–2). After excluding grades 1–2 of intraventricular hemorrhages from the analyses, the OR for ICH was still significantly increased for infants delivered by VE (OR 2.58, CI 1.27-5.24), but not for those delivered by CS during labor (OR 1.15, CI 0.78-1.70) after adjustment for GA. There was no difference in ICH rates between preterm boys and girls delivered by VE.\nLogistic regression (odds ratios) for intra– and extracranial hemorrhages, convulsions and other cerebral complications, and brachial plexus injury in preterm infants exposed to different modes of delivery\nCS = cesarean section, VE = vacuum extraction.\nModel 1 is adjusted for year of birth, gestational age, parity, maternal age, height, BMI, and infant birthweight.\nModel 2 is also adjusted for indications for operative delivery.\nThe ORs for convulsions were almost doubled in both the VE and CS groups after adjustments for the variables in Model 1. However, further adjustment for indication for operative delivery decreased the odds and made the associations statistically insignificant. Other disturbances of the neonatal cerebral status were significantly increased (two to three times higher) both among infants delivered by VE, and by CS during labor, although the OR was higher in the VE group.\nA total of 53 infants were diagnosed with brachial plexus injury. Of these, 14 were born by VE, corresponding to a rate in the VE group of 0.6% and an OR of 6.21 (95% CI: 2.22-17.4) in the fully-adjusted model. In contrast, infants delivered by CS had no increased risk for this complication. Among infants with brachial plexus injury, there were 11 cases of shoulder dystocia (ICD-code O66.0), of which five occurred in the VE group.","Among the 40,764 (54% of all) preterm deliveries included in this study, 2,319 (5.7%) preterm infants were delivered by VE, 5,505 (13.5%) by CS during labor, and 32,940 (80.2%) by VD. The rate of VE deliveries increased gradually with gestational age, Figure \n1.\nMode of delivery in relation to gestational age. Figure\n1 shows rates (%) of different modes of delivery in relation to gestational age (in completed weeks). The blue, dotted line represents unassisted vaginal deliveries, the red, dashed line represents cesarean sections performed after onset of labor, and the green line represents the vacuum extraction deliveries.","Table \n2 shows maternal and perinatal characteristics of the study population in relation to mode of delivery. The VE rate decreased with maternal height and 80% of the women who delivered by VE were primiparae, compared with 48% of those who underwent CS during labor (not in table). More than 45% of the women who delivered by VE had received epidural analgesia during labor compared with 22% of women with VD, and 12% with CS during labor (not in table). Given the association between GA and VE, infants delivered with VE had higher birthweights.\nMaternal, pregnancy, delivery, and infant characteristics by mode of delivery\nPopulation based cohort consisting of 40 764 preterm deliveries.\nCS = caesarean section, BMI = Body Mass Index (weight in kilograms/height in meters2), EA = Epidural Analgesia.\nThe most common indication for VE was fetal distress (42%), followed by prolonged labor (25%). Having a non-occipitoanterior position (25%) or fetal distress (17%) were the most common indications for CS during labor, while only 3% in this group had a diagnosis of prolonged labor.","The proportion of preterm infants diagnosed with an ICH varied more than hundred-fold in relation to GA. It decreased from 21.5% among preterm infants born at 22–28 weeks of GA to 0.1% among those born after 36 weeks of gestation. The rates of neonatal convulsions among preterm infants decreased from 2.0% at 22–28 weeks to 0.25% among those born after 36 weeks of gestation, Figure \n2. The proportion of preterm infants diagnosed with other disturbances of cerebral status (encephalopathy) decreased with GA, while proportion of infants with brachial plexus injuries or ECH increased slightly with GA, Figure \n3.\nProportion (%) preterm infants diagnosed with intracranial hemorrhage (ICH) or convulsions by gestational age. Figure\n2 shows the proportions (%) of preterm infants diagnosed with ICH and neonatal convulsions in relation to gestational age (in completed weeks). The blue, dotted line represents intracranial hemorrhage (ICH) and the red, dashed line represents neonatal convulsions.\nProportion (%) preterm infants diagnosed with extracranial hemorrhage (ECH), encephalopathy and brachial plexus injury by gestational age. Figure\n3 shows the proportions (%) of ECH, encephalopathy (ICD-code P91: other disturbances of cerebral status of newborn), and brachial plexus injury in relation to gestational age (in completed weeks). The blue, dotted line represents extracranial hemorrhage (ECH), the red, dashed line represents encephalopathy and the green line represents brachial plexus injury.","To report outcome in relation to mode of delivery, the study cohort was divided according to the current guidelines on the use of VE as either preterm births occurring between 34–36 weeks of gestation (VE may be used), or those occurring before 34 gestational weeks (VE not recommended). In our cohort, 33,202 (81.4%) of all preterm births occurred at 34–36 gestational weeks, and 7,562 (18.6%) before 34 weeks of GA.\nIn Table \n3, neonatal outcomes before and after 34 + 0 weeks of gestation are presented in relation to mode of delivery. Overall, seven preterm infants were classified as having an ICH due to birth injury, corresponding to a rate of 0.02%; and 612 infants were diagnosed with non-traumatic ICH, corresponding to a rate of 1.5%. Diagnoses of neonatal convulsions and other disturbances of neonatal cerebral status were rare, especially in infants at less than 34 weeks of GA, and occurred more frequently after VE and CS than after VD. Cephalic hematoma was the most frequent complication after VE of preterm infants (n = 72 or 3.1%), occurring much more often after VE than after CS (0.16%) and VD (0.49%). Subgaleal hemorrhage was less frequent, with a total of only18 cases. More than two thirds of those cases occurred in the VE group.\nNeonatal outcomes in preterm infants by mode of delivery and gestational age\nTable \n4 shows rates (per 1000), crude and adjusted odds ratios for the outcomes by mode of delivery, and uses infants born by VD as the reference group. After adjustments, preterm infants born by VE had almost doubled OR for ICH, while those born by CS had no increased risk. Preterm infants delivered by VE also had 4.5 times higher OR for extracranial hemorrhages, while those delivered by CS had no increased risk. A total of 540 (88.2%) of the ICH diagnoses consisted of intraventricular bleedings (ICD-codes 52.0-52.3), the type of ICH most frequently seen in preterm infants. Of these, 438 (71.6%) were graded as mild or moderate (grades 1–2). After excluding grades 1–2 of intraventricular hemorrhages from the analyses, the OR for ICH was still significantly increased for infants delivered by VE (OR 2.58, CI 1.27-5.24), but not for those delivered by CS during labor (OR 1.15, CI 0.78-1.70) after adjustment for GA. There was no difference in ICH rates between preterm boys and girls delivered by VE.\nLogistic regression (odds ratios) for intra– and extracranial hemorrhages, convulsions and other cerebral complications, and brachial plexus injury in preterm infants exposed to different modes of delivery\nCS = cesarean section, VE = vacuum extraction.\nModel 1 is adjusted for year of birth, gestational age, parity, maternal age, height, BMI, and infant birthweight.\nModel 2 is also adjusted for indications for operative delivery.\nThe ORs for convulsions were almost doubled in both the VE and CS groups after adjustments for the variables in Model 1. However, further adjustment for indication for operative delivery decreased the odds and made the associations statistically insignificant. Other disturbances of the neonatal cerebral status were significantly increased (two to three times higher) both among infants delivered by VE, and by CS during labor, although the OR was higher in the VE group.\nA total of 53 infants were diagnosed with brachial plexus injury. Of these, 14 were born by VE, corresponding to a rate in the VE group of 0.6% and an OR of 6.21 (95% CI: 2.22-17.4) in the fully-adjusted model. In contrast, infants delivered by CS had no increased risk for this complication. Among infants with brachial plexus injury, there were 11 cases of shoulder dystocia (ICD-code O66.0), of which five occurred in the VE group.","In this large cohort study of singleton, non-breech preterm births after onset of labor, we identify three clinically important findings related to mode of delivery: First, VE was used in 5.7% of preterm births, and despite recommendations of no use, 3.3% of preterm infants born before 34 gestational weeks were delivered by VE. Secondly, VE for preterm birth was used more frequently in shorter mothers, primiparae and among women treated with EA as pain relief during labor. Finally, and adjusting for potential confounders and covariates, preterm infants delivered by VE had almost doubled OR for ICH, four times higher OR for extracranial hemorrhage, as well as a 6-fold risk for brachial plexus palsy compared with those delivered by VD. Exclusion of intraventricular hemorrhage grades I-II (the most common form of ICH in preterm infants) from the analysis increased the OR for ICH after VE, indicating that severe bleedings were more common among preterm infants delivered by VE.\nAlthough VE was related to significantly increased rates of ICH, it is not clear whether the extraction as such causes the injury, or if there is an underlying common pathway for both VE-assisted delivery and ICH, i.e., that the relationship is confounded by indication. Since the ORs for ICH were significantly higher in the VE group compared with both the CS and unassisted VD groups, whereas the ORs for other disturbances of cerebral status were slightly higher in both the VE and CS groups as compared with VD, different mechanisms may be involved in the development of these two complications. The forces by vacuum extraction could lead to significant vertical stress, which might be avoided with CS. In a case report of MRI findings after birth injuries among infants delivered by VE\n[21], it was suggested that vertical traction on the skull and brain may produce tentorial lacerations and rupture of intracranial veins. Another explanation for our findings of different outcomes after VE and CS could be that infants delivered by VE may have been exposed to contractions for a longer time than those delivered by CS. A protective effect of CS is indicated by lower ORs for ICH; however, the exposure to contractions as the sole explanation for the increased risks for ICH after VE is less likely, as the VD group – presumably the group exposed to the largest forces of labor – exhibited significantly lower odds for hemorrhagic complications compared with infants delivered with VE.\nDuring the study period, the overall rate of ICH increased from 6% originally, up to 12% at the end of the period, most likely reflecting the increased access and use of ultrasonography among Swedish neonatologists in recent years. Improved ultrasound technology and image resolution may also have contributed to this development. Finally, we cannot exclude a contribution from misclassification: a large but normal choroid plexus could have been classified as a small subependymal hemorrhage by less experienced investigators. The finding that the diagnosis of small subependymal hemorrhage (without intraventricular extension; P52.0) increased most compared to other types of ICH during the study period (from 0.5% to 1.2%), supports these interpretations.\nThe overrepresentation of subgaleal hemorrhage and cephalhematoma after VE is less surprising, since earlier studies have stated the relation between these diagnoses and the use of VE. The risk of subgaleal hemorrhage seems to be unrelated to GA, as this study demonstrates rates similar to those in previous studies of infants born at term\n[12,13].\nThe present study showed that preterm infants delivered by VE had a 6- to 7-fold risk increase for injury to the plexus brachialis compared with unassisted VD. This injury is usually associated with large macrosomic infants and shoulder dystocia\n[22] and not to preterm birth. Our result emphasizes the importance of gentle maneuvers and avoiding application of excessive pressure or traction on the brachial plexus also when delivering the preterm infant, especially by VE.\nThe major strengths of this study were the large study population covering all preterm deliveries in Sweden during a period of twelve years, and the high quality of the registers, making it possible to analyze rare diagnoses and unusual events such as ICH in preterm infants delivered by VE. We were able to include data on risk factors, potential confounders, and outcomes collected independently from one another and without involving the study subjects, thus minimizing various types of bias (e.g., selection and recall bias). Moreover, antenatal and obstetric care is free of charge in Sweden, management routines as well as GA determinations are standardized, and 99% of births are delivered in public hospitals. This minimizes the risk for confounding by unmeasured socio-demographic factors. Another advantage was the inclusion of the main indications for VE and CS, enabling us to address the question of confounding by indication.\nA major limitation of this study is that the registers do not contain detailed information about many important factors during the VE deliveries. For instance the registry does not provide specific information about the type of VE instrument used, level, position, and attitude of the fetal head in the pelvis when applying VE, location of placement of the vacuum cup, traction work, skill of the obstetrician, pressure exposure (duration and force), and cup detachments. The register does not provide information about confounders such as use of oxytocin and application of fundal pressure.\nThere is a general recommendation not to use VE before a GA of 34 weeks. According to the Royal College of Obstetricians and Gynecologists, there is insufficient evidence to establish the safety on VE deliveries in gestations between 34 weeks + 0 days and 36 weeks + 0 days\n[9]. Our results show that the use of VE is related to rare, but serious complications also between gestational weeks 34–36.","The rates of serious birth injuries and complications are generally low, but preterm infants delivered by VE have higher odds ratios for intra- and extracranial hemorrhages and brachial plexus injuries than those delivered by CS during labor or by unassisted vaginal delivery. We therefore support a continued conservative/cautious use of VE in preterm deliveries. Furthermore, the possible causal relationship between mode of delivery and ICH needs to be further investigated.","BMI: Body mass index; CI: Confidence interval; CS: Cesarean section; CT: Computerized tomography; EA: Epidural analgesia; ECH: Extracranial hemorrhage; EEG: Electroencephalography; GA: Gestational age; ICH: Intracranial hemorrhage; LGA: Large for gestational age; OR: Odds ratio; SGA: Small for gestational age; VD: Vaginal delivery; VE: Vacuum extraction.","The authors declare that they have no competing interest.","CE had the idea for the study, designed it and carried out the statistical analysis. KÅ participated in the design and wrote the first draft of the manuscript together with CE. MN contributed to the interpretation of the results and writing of the manuscript. All authors approved the final version of the submitted article.","The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2393/14/42/prepub\n"],"string":"[\n \"Preterm birth is common\\n[1] but still, the optimal mode of delivery of preterm infants is not known. Although neonatal outcomes in preterm infants delivered vaginally or by cesarean section (CS)\\n[2-5] have been compared, there is no evidence to provide clear guidance on the method of choice\\n[6]. Given the widespread assumption that assisted vaginal delivery could be harmful for fragile infants that are underweight and preterm, very few studies have addressed the use of vacuum extraction (VE) for preterm birth.\\nDelivery by VE is a common obstetrical procedure, and in many countries it has replaced the use of forceps. VE is used to terminate a protracted second stage of labor and as an intervention for fetal or maternal distress. VE requires vertex presentation, a fully dilated cervix and ruptured membranes\\n[7]. A cesarean section, on the other hand, can be performed at any stage of labor and does not require prerequisites of this kind.\\nMost clinical guidelines do not recommend VE before 34 gestational weeks\\n[8-10]. These recommendations are not based on results of randomized controlled trials, but rely on the observation that preterm infants are more likely than term infants to develop ICH, and on extrapolations from studies of term infants showing that VE is associated with an increased risk of ICH and other neonatal complications\\n[11-17]. Only three studies have previously investigated the use and outcomes of VE in preterm births. The first was undertaken over 40 years ago and showed increased mortality and morbidity among preterm infants delivered by VE as compared with term infants delivered by VE\\n[18]. The second study compared neonatal morbidity in preterm infants delivered vaginally with (n = 61) or without VE (n = 122), and found no differences in neonatal morbidity between the two groups\\n[19]. The last study compared VE and forceps for preterm delivery (n = 64)\\n[20]; the neonatal outcomes were similar in both groups. The available data are clearly untimely and hampered by limitations in power and, therefore, current knowledge on safety of preterm vacuum-assisted birth is unsatisfactory.\\nThe aim of this study was to 1) describe the use of VE and compare it to rates of CS during labor in preterm deliveries in Sweden from 1999–2010, 2) characterize the distribution of perinatal risk factors associated with each mode of delivery, and 3) compare rates of neonatal intra- and extracranial hemorrhages, as well as occurrence of brachial plexus injury after preterm delivery by VE or CS during labor, using unassisted vaginal birth as a reference.\",\n \"This study was based on data from national data bases held by the Swedish National Board of Health and Welfare. The national registration number, assigned to each Swedish resident at birth, was used for individual record linkage. We used two registers: The Swedish Medical Birth Register (SMBR) that covers 99% of all births in Sweden, and The Swedish National Inpatient Register (IPR) that covers all public inpatient care. The SMBR includes prospectively collected information on maternal characteristics, reproductive history, and complications during pregnancy, delivery, and the neonatal period. The IPR includes data on each hospital admission and discharge.\\n Study population During the period of 1999–2010, there were 75,296 (6.2%) preterm births in Sweden. We excluded deliveries by CS before the onset of labor (n = 17,306), forceps (n = 257), or performed with both VE and CS (n = 125). We also excluded stillbirths (fetal deaths occurring before labor or intra partum) (n = 1,839), multiple births (n = 11,088), and births in breech presentation (n = 3,917). Thus, the final study population was restricted to all live-born, preterm singleton infants with a non-breech presentation at birth, delivered after a spontaneous or induced onset of labor followed by CS, vacuum extraction (VE), or by unassisted vaginal delivery (VD) before gestational week 37 + 0 days (N = 40,764). CS during labor was defined as abdominal delivery after the onset of labor, either spontaneously, by rupture of membranes, or by induction.\\nA number of independent variables were collected; the maternal anthropometrics included: age, height, and body mass index (BMI). BMI was calculated from measured height and weight obtained at the first antenatal care visit, which occurred before the 15th week of gestation in more than 95% of the pregnancies. BMI was categorized into underweight (below 18.5 kg/m2), normal (18.5–24.9 kg/m2), overweight (25–29.9 kg/m2), obese (>29.9 kg/m2), or missing. Parity was categorized as primi- or multiparity. Information on complications during pregnancy and delivery were coded according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards). The following pregnancy complications were included: diabetes – both gestational and types 1 and 2 (O24.0-9) preeclampsia- both hypertension, preeclampsia, and eclampsia (O10.0-O15.9). Labor-related risk factors or covariates includedepidural analgesia (EA; yes/no), and induction of labor (yes/no). Indications for operative delivery were classified into four major groups: prolonged labor (O62.0-2, O63.0-9), signs of fetal distress (O68.0-O68.1-9), preeclampsia, and a non-occipitoanterior presentation of the fetus (all presentations except occipitoanterior and breech, registered at birth). Gestational age (GA) for preterm infants was divided into three periods according to the World Health Organization: extremely preterm (before 28 weeks), very preterm (28–31 weeks) and moderately preterm (32–36 weeks). Furthermore, we also divided the preterm gestational period according to guidelines on instrumental delivery into either: less than 34 weeks (VE not recommended), and 34–36 weeks (VE may be used). GA was recorded in completed weeks, and was based on routine ultrasound dating performed at 17 to 18 postmenstrual weeks in 97-98% of all pregnant women. Infant birthweight was categorized as less than 1,500 grams, 1,500-2,000 grams, 2,001-2,500 grams, 2,501-3,000 grams, and 3,001-4,000 grams.\\nDuring the period of 1999–2010, there were 75,296 (6.2%) preterm births in Sweden. We excluded deliveries by CS before the onset of labor (n = 17,306), forceps (n = 257), or performed with both VE and CS (n = 125). We also excluded stillbirths (fetal deaths occurring before labor or intra partum) (n = 1,839), multiple births (n = 11,088), and births in breech presentation (n = 3,917). Thus, the final study population was restricted to all live-born, preterm singleton infants with a non-breech presentation at birth, delivered after a spontaneous or induced onset of labor followed by CS, vacuum extraction (VE), or by unassisted vaginal delivery (VD) before gestational week 37 + 0 days (N = 40,764). CS during labor was defined as abdominal delivery after the onset of labor, either spontaneously, by rupture of membranes, or by induction.\\nA number of independent variables were collected; the maternal anthropometrics included: age, height, and body mass index (BMI). BMI was calculated from measured height and weight obtained at the first antenatal care visit, which occurred before the 15th week of gestation in more than 95% of the pregnancies. BMI was categorized into underweight (below 18.5 kg/m2), normal (18.5–24.9 kg/m2), overweight (25–29.9 kg/m2), obese (>29.9 kg/m2), or missing. Parity was categorized as primi- or multiparity. Information on complications during pregnancy and delivery were coded according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards). The following pregnancy complications were included: diabetes – both gestational and types 1 and 2 (O24.0-9) preeclampsia- both hypertension, preeclampsia, and eclampsia (O10.0-O15.9). Labor-related risk factors or covariates includedepidural analgesia (EA; yes/no), and induction of labor (yes/no). Indications for operative delivery were classified into four major groups: prolonged labor (O62.0-2, O63.0-9), signs of fetal distress (O68.0-O68.1-9), preeclampsia, and a non-occipitoanterior presentation of the fetus (all presentations except occipitoanterior and breech, registered at birth). Gestational age (GA) for preterm infants was divided into three periods according to the World Health Organization: extremely preterm (before 28 weeks), very preterm (28–31 weeks) and moderately preterm (32–36 weeks). Furthermore, we also divided the preterm gestational period according to guidelines on instrumental delivery into either: less than 34 weeks (VE not recommended), and 34–36 weeks (VE may be used). GA was recorded in completed weeks, and was based on routine ultrasound dating performed at 17 to 18 postmenstrual weeks in 97-98% of all pregnant women. Infant birthweight was categorized as less than 1,500 grams, 1,500-2,000 grams, 2,001-2,500 grams, 2,501-3,000 grams, and 3,001-4,000 grams.\\n Outcome variables Neonatal diagnoses were classified according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards), and identified/collected in the SMBR or in the IPR. The following neonatal outcomes (ICD codes) were assessed: Intracranial laceration and hemorrhage due to birth injury (P10), intracranial non-traumatic hemorrhage of fetus and newborn (P52), convulsions of newborn (P90), other disturbances of cerebral status of newborn (P 91), subgaleal hematoma (P12.2), cephalhematoma (P12.0), and brachial plexus injury (P14.0-3). The definitions of outcomes are described in detail in Table \\n1.\\nNeonatal outcomes studied in 40 764 preterm infants\\nNeonatal diagnoses of intracranial hemorrhages in preterm infants were mainly based on imaging of the brain using ultrasonography; however, some assessments of the brain at term-equivalent age were alternatively performed with CT and/or MRI. Imaging of the brain was performed on clinical indications only in cases born moderately or late preterm, whereas all very preterm infants (born before 32 weeks of gestation) were screened for intracranial lesions, even in asymptomatic infants. A diagnosis of convulsions included infants with clinical signs of convulsions and/or convulsions verified by EEG.\\nStatistical analysis was performed using proportions and odds ratios (OR) with a 95% confidence interval (CI) for neonatal complications in relation to mode of delivery, using unassisted VD as the reference group (SPSS 20.0 for Windows software package). Three models were used to assess the relationship between the different modes of delivery and the risk for neonatal complications: one crude, and two adjusted (Models 1 and 2). The included covariates have been shown previously to be related to instrumental deliveries, and were related to the outcomes in cross tabulations. In Model 1, we adjusted for the following confounders or covariates: maternal age, height, BMI, and parity, as well as infant year of birth, birthweight and GA. In Model 2, we added the indication for operative delivery and preeclampsia. The year of birth was entered as a continuous variable in accordance with a linear secular trend, and all other variables were entered as categories. Furthermore, a separate logistic regression analysis was performed to investigate severe ICH in relation to mode of delivery. Here, intraventricular hemorrhages grades 1–2 were excluded and the analysis was adjusted for GA only. We also conducted separated analyses on potential relationships between sex and ICH in relation to mode of delivery. Missing data were entered as a separate category in the analyses. The study was approved by the Regional Ethical Review Board in Stockholm, Dnr 2008/1322-31.\\nNeonatal diagnoses were classified according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards), and identified/collected in the SMBR or in the IPR. The following neonatal outcomes (ICD codes) were assessed: Intracranial laceration and hemorrhage due to birth injury (P10), intracranial non-traumatic hemorrhage of fetus and newborn (P52), convulsions of newborn (P90), other disturbances of cerebral status of newborn (P 91), subgaleal hematoma (P12.2), cephalhematoma (P12.0), and brachial plexus injury (P14.0-3). The definitions of outcomes are described in detail in Table \\n1.\\nNeonatal outcomes studied in 40 764 preterm infants\\nNeonatal diagnoses of intracranial hemorrhages in preterm infants were mainly based on imaging of the brain using ultrasonography; however, some assessments of the brain at term-equivalent age were alternatively performed with CT and/or MRI. Imaging of the brain was performed on clinical indications only in cases born moderately or late preterm, whereas all very preterm infants (born before 32 weeks of gestation) were screened for intracranial lesions, even in asymptomatic infants. A diagnosis of convulsions included infants with clinical signs of convulsions and/or convulsions verified by EEG.\\nStatistical analysis was performed using proportions and odds ratios (OR) with a 95% confidence interval (CI) for neonatal complications in relation to mode of delivery, using unassisted VD as the reference group (SPSS 20.0 for Windows software package). Three models were used to assess the relationship between the different modes of delivery and the risk for neonatal complications: one crude, and two adjusted (Models 1 and 2). The included covariates have been shown previously to be related to instrumental deliveries, and were related to the outcomes in cross tabulations. In Model 1, we adjusted for the following confounders or covariates: maternal age, height, BMI, and parity, as well as infant year of birth, birthweight and GA. In Model 2, we added the indication for operative delivery and preeclampsia. The year of birth was entered as a continuous variable in accordance with a linear secular trend, and all other variables were entered as categories. Furthermore, a separate logistic regression analysis was performed to investigate severe ICH in relation to mode of delivery. Here, intraventricular hemorrhages grades 1–2 were excluded and the analysis was adjusted for GA only. We also conducted separated analyses on potential relationships between sex and ICH in relation to mode of delivery. Missing data were entered as a separate category in the analyses. The study was approved by the Regional Ethical Review Board in Stockholm, Dnr 2008/1322-31.\",\n \"During the period of 1999–2010, there were 75,296 (6.2%) preterm births in Sweden. We excluded deliveries by CS before the onset of labor (n = 17,306), forceps (n = 257), or performed with both VE and CS (n = 125). We also excluded stillbirths (fetal deaths occurring before labor or intra partum) (n = 1,839), multiple births (n = 11,088), and births in breech presentation (n = 3,917). Thus, the final study population was restricted to all live-born, preterm singleton infants with a non-breech presentation at birth, delivered after a spontaneous or induced onset of labor followed by CS, vacuum extraction (VE), or by unassisted vaginal delivery (VD) before gestational week 37 + 0 days (N = 40,764). CS during labor was defined as abdominal delivery after the onset of labor, either spontaneously, by rupture of membranes, or by induction.\\nA number of independent variables were collected; the maternal anthropometrics included: age, height, and body mass index (BMI). BMI was calculated from measured height and weight obtained at the first antenatal care visit, which occurred before the 15th week of gestation in more than 95% of the pregnancies. BMI was categorized into underweight (below 18.5 kg/m2), normal (18.5–24.9 kg/m2), overweight (25–29.9 kg/m2), obese (>29.9 kg/m2), or missing. Parity was categorized as primi- or multiparity. Information on complications during pregnancy and delivery were coded according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards). The following pregnancy complications were included: diabetes – both gestational and types 1 and 2 (O24.0-9) preeclampsia- both hypertension, preeclampsia, and eclampsia (O10.0-O15.9). Labor-related risk factors or covariates includedepidural analgesia (EA; yes/no), and induction of labor (yes/no). Indications for operative delivery were classified into four major groups: prolonged labor (O62.0-2, O63.0-9), signs of fetal distress (O68.0-O68.1-9), preeclampsia, and a non-occipitoanterior presentation of the fetus (all presentations except occipitoanterior and breech, registered at birth). Gestational age (GA) for preterm infants was divided into three periods according to the World Health Organization: extremely preterm (before 28 weeks), very preterm (28–31 weeks) and moderately preterm (32–36 weeks). Furthermore, we also divided the preterm gestational period according to guidelines on instrumental delivery into either: less than 34 weeks (VE not recommended), and 34–36 weeks (VE may be used). GA was recorded in completed weeks, and was based on routine ultrasound dating performed at 17 to 18 postmenstrual weeks in 97-98% of all pregnant women. Infant birthweight was categorized as less than 1,500 grams, 1,500-2,000 grams, 2,001-2,500 grams, 2,501-3,000 grams, and 3,001-4,000 grams.\",\n \"Neonatal diagnoses were classified according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards), and identified/collected in the SMBR or in the IPR. The following neonatal outcomes (ICD codes) were assessed: Intracranial laceration and hemorrhage due to birth injury (P10), intracranial non-traumatic hemorrhage of fetus and newborn (P52), convulsions of newborn (P90), other disturbances of cerebral status of newborn (P 91), subgaleal hematoma (P12.2), cephalhematoma (P12.0), and brachial plexus injury (P14.0-3). The definitions of outcomes are described in detail in Table \\n1.\\nNeonatal outcomes studied in 40 764 preterm infants\\nNeonatal diagnoses of intracranial hemorrhages in preterm infants were mainly based on imaging of the brain using ultrasonography; however, some assessments of the brain at term-equivalent age were alternatively performed with CT and/or MRI. Imaging of the brain was performed on clinical indications only in cases born moderately or late preterm, whereas all very preterm infants (born before 32 weeks of gestation) were screened for intracranial lesions, even in asymptomatic infants. A diagnosis of convulsions included infants with clinical signs of convulsions and/or convulsions verified by EEG.\\nStatistical analysis was performed using proportions and odds ratios (OR) with a 95% confidence interval (CI) for neonatal complications in relation to mode of delivery, using unassisted VD as the reference group (SPSS 20.0 for Windows software package). Three models were used to assess the relationship between the different modes of delivery and the risk for neonatal complications: one crude, and two adjusted (Models 1 and 2). The included covariates have been shown previously to be related to instrumental deliveries, and were related to the outcomes in cross tabulations. In Model 1, we adjusted for the following confounders or covariates: maternal age, height, BMI, and parity, as well as infant year of birth, birthweight and GA. In Model 2, we added the indication for operative delivery and preeclampsia. The year of birth was entered as a continuous variable in accordance with a linear secular trend, and all other variables were entered as categories. Furthermore, a separate logistic regression analysis was performed to investigate severe ICH in relation to mode of delivery. Here, intraventricular hemorrhages grades 1–2 were excluded and the analysis was adjusted for GA only. We also conducted separated analyses on potential relationships between sex and ICH in relation to mode of delivery. Missing data were entered as a separate category in the analyses. The study was approved by the Regional Ethical Review Board in Stockholm, Dnr 2008/1322-31.\",\n \" Use of VE in relation to gestational age Among the 40,764 (54% of all) preterm deliveries included in this study, 2,319 (5.7%) preterm infants were delivered by VE, 5,505 (13.5%) by CS during labor, and 32,940 (80.2%) by VD. The rate of VE deliveries increased gradually with gestational age, Figure \\n1.\\nMode of delivery in relation to gestational age. Figure\\n1 shows rates (%) of different modes of delivery in relation to gestational age (in completed weeks). The blue, dotted line represents unassisted vaginal deliveries, the red, dashed line represents cesarean sections performed after onset of labor, and the green line represents the vacuum extraction deliveries.\\nAmong the 40,764 (54% of all) preterm deliveries included in this study, 2,319 (5.7%) preterm infants were delivered by VE, 5,505 (13.5%) by CS during labor, and 32,940 (80.2%) by VD. The rate of VE deliveries increased gradually with gestational age, Figure \\n1.\\nMode of delivery in relation to gestational age. Figure\\n1 shows rates (%) of different modes of delivery in relation to gestational age (in completed weeks). The blue, dotted line represents unassisted vaginal deliveries, the red, dashed line represents cesarean sections performed after onset of labor, and the green line represents the vacuum extraction deliveries.\\n Distribution of risk factors or covariates in relation to mode of delivery Table \\n2 shows maternal and perinatal characteristics of the study population in relation to mode of delivery. The VE rate decreased with maternal height and 80% of the women who delivered by VE were primiparae, compared with 48% of those who underwent CS during labor (not in table). More than 45% of the women who delivered by VE had received epidural analgesia during labor compared with 22% of women with VD, and 12% with CS during labor (not in table). Given the association between GA and VE, infants delivered with VE had higher birthweights.\\nMaternal, pregnancy, delivery, and infant characteristics by mode of delivery\\nPopulation based cohort consisting of 40 764 preterm deliveries.\\nCS = caesarean section, BMI = Body Mass Index (weight in kilograms/height in meters2), EA = Epidural Analgesia.\\nThe most common indication for VE was fetal distress (42%), followed by prolonged labor (25%). Having a non-occipitoanterior position (25%) or fetal distress (17%) were the most common indications for CS during labor, while only 3% in this group had a diagnosis of prolonged labor.\\nTable \\n2 shows maternal and perinatal characteristics of the study population in relation to mode of delivery. The VE rate decreased with maternal height and 80% of the women who delivered by VE were primiparae, compared with 48% of those who underwent CS during labor (not in table). More than 45% of the women who delivered by VE had received epidural analgesia during labor compared with 22% of women with VD, and 12% with CS during labor (not in table). Given the association between GA and VE, infants delivered with VE had higher birthweights.\\nMaternal, pregnancy, delivery, and infant characteristics by mode of delivery\\nPopulation based cohort consisting of 40 764 preterm deliveries.\\nCS = caesarean section, BMI = Body Mass Index (weight in kilograms/height in meters2), EA = Epidural Analgesia.\\nThe most common indication for VE was fetal distress (42%), followed by prolonged labor (25%). Having a non-occipitoanterior position (25%) or fetal distress (17%) were the most common indications for CS during labor, while only 3% in this group had a diagnosis of prolonged labor.\\n Neonatal outcome in relation to gestational age The proportion of preterm infants diagnosed with an ICH varied more than hundred-fold in relation to GA. It decreased from 21.5% among preterm infants born at 22–28 weeks of GA to 0.1% among those born after 36 weeks of gestation. The rates of neonatal convulsions among preterm infants decreased from 2.0% at 22–28 weeks to 0.25% among those born after 36 weeks of gestation, Figure \\n2. The proportion of preterm infants diagnosed with other disturbances of cerebral status (encephalopathy) decreased with GA, while proportion of infants with brachial plexus injuries or ECH increased slightly with GA, Figure \\n3.\\nProportion (%) preterm infants diagnosed with intracranial hemorrhage (ICH) or convulsions by gestational age. Figure\\n2 shows the proportions (%) of preterm infants diagnosed with ICH and neonatal convulsions in relation to gestational age (in completed weeks). The blue, dotted line represents intracranial hemorrhage (ICH) and the red, dashed line represents neonatal convulsions.\\nProportion (%) preterm infants diagnosed with extracranial hemorrhage (ECH), encephalopathy and brachial plexus injury by gestational age. Figure\\n3 shows the proportions (%) of ECH, encephalopathy (ICD-code P91: other disturbances of cerebral status of newborn), and brachial plexus injury in relation to gestational age (in completed weeks). The blue, dotted line represents extracranial hemorrhage (ECH), the red, dashed line represents encephalopathy and the green line represents brachial plexus injury.\\nThe proportion of preterm infants diagnosed with an ICH varied more than hundred-fold in relation to GA. It decreased from 21.5% among preterm infants born at 22–28 weeks of GA to 0.1% among those born after 36 weeks of gestation. The rates of neonatal convulsions among preterm infants decreased from 2.0% at 22–28 weeks to 0.25% among those born after 36 weeks of gestation, Figure \\n2. The proportion of preterm infants diagnosed with other disturbances of cerebral status (encephalopathy) decreased with GA, while proportion of infants with brachial plexus injuries or ECH increased slightly with GA, Figure \\n3.\\nProportion (%) preterm infants diagnosed with intracranial hemorrhage (ICH) or convulsions by gestational age. Figure\\n2 shows the proportions (%) of preterm infants diagnosed with ICH and neonatal convulsions in relation to gestational age (in completed weeks). The blue, dotted line represents intracranial hemorrhage (ICH) and the red, dashed line represents neonatal convulsions.\\nProportion (%) preterm infants diagnosed with extracranial hemorrhage (ECH), encephalopathy and brachial plexus injury by gestational age. Figure\\n3 shows the proportions (%) of ECH, encephalopathy (ICD-code P91: other disturbances of cerebral status of newborn), and brachial plexus injury in relation to gestational age (in completed weeks). The blue, dotted line represents extracranial hemorrhage (ECH), the red, dashed line represents encephalopathy and the green line represents brachial plexus injury.\\n Neonatal outcome in relation to mode of delivery To report outcome in relation to mode of delivery, the study cohort was divided according to the current guidelines on the use of VE as either preterm births occurring between 34–36 weeks of gestation (VE may be used), or those occurring before 34 gestational weeks (VE not recommended). In our cohort, 33,202 (81.4%) of all preterm births occurred at 34–36 gestational weeks, and 7,562 (18.6%) before 34 weeks of GA.\\nIn Table \\n3, neonatal outcomes before and after 34 + 0 weeks of gestation are presented in relation to mode of delivery. Overall, seven preterm infants were classified as having an ICH due to birth injury, corresponding to a rate of 0.02%; and 612 infants were diagnosed with non-traumatic ICH, corresponding to a rate of 1.5%. Diagnoses of neonatal convulsions and other disturbances of neonatal cerebral status were rare, especially in infants at less than 34 weeks of GA, and occurred more frequently after VE and CS than after VD. Cephalic hematoma was the most frequent complication after VE of preterm infants (n = 72 or 3.1%), occurring much more often after VE than after CS (0.16%) and VD (0.49%). Subgaleal hemorrhage was less frequent, with a total of only18 cases. More than two thirds of those cases occurred in the VE group.\\nNeonatal outcomes in preterm infants by mode of delivery and gestational age\\nTable \\n4 shows rates (per 1000), crude and adjusted odds ratios for the outcomes by mode of delivery, and uses infants born by VD as the reference group. After adjustments, preterm infants born by VE had almost doubled OR for ICH, while those born by CS had no increased risk. Preterm infants delivered by VE also had 4.5 times higher OR for extracranial hemorrhages, while those delivered by CS had no increased risk. A total of 540 (88.2%) of the ICH diagnoses consisted of intraventricular bleedings (ICD-codes 52.0-52.3), the type of ICH most frequently seen in preterm infants. Of these, 438 (71.6%) were graded as mild or moderate (grades 1–2). After excluding grades 1–2 of intraventricular hemorrhages from the analyses, the OR for ICH was still significantly increased for infants delivered by VE (OR 2.58, CI 1.27-5.24), but not for those delivered by CS during labor (OR 1.15, CI 0.78-1.70) after adjustment for GA. There was no difference in ICH rates between preterm boys and girls delivered by VE.\\nLogistic regression (odds ratios) for intra– and extracranial hemorrhages, convulsions and other cerebral complications, and brachial plexus injury in preterm infants exposed to different modes of delivery\\nCS = cesarean section, VE = vacuum extraction.\\nModel 1 is adjusted for year of birth, gestational age, parity, maternal age, height, BMI, and infant birthweight.\\nModel 2 is also adjusted for indications for operative delivery.\\nThe ORs for convulsions were almost doubled in both the VE and CS groups after adjustments for the variables in Model 1. However, further adjustment for indication for operative delivery decreased the odds and made the associations statistically insignificant. Other disturbances of the neonatal cerebral status were significantly increased (two to three times higher) both among infants delivered by VE, and by CS during labor, although the OR was higher in the VE group.\\nA total of 53 infants were diagnosed with brachial plexus injury. Of these, 14 were born by VE, corresponding to a rate in the VE group of 0.6% and an OR of 6.21 (95% CI: 2.22-17.4) in the fully-adjusted model. In contrast, infants delivered by CS had no increased risk for this complication. Among infants with brachial plexus injury, there were 11 cases of shoulder dystocia (ICD-code O66.0), of which five occurred in the VE group.\\nTo report outcome in relation to mode of delivery, the study cohort was divided according to the current guidelines on the use of VE as either preterm births occurring between 34–36 weeks of gestation (VE may be used), or those occurring before 34 gestational weeks (VE not recommended). In our cohort, 33,202 (81.4%) of all preterm births occurred at 34–36 gestational weeks, and 7,562 (18.6%) before 34 weeks of GA.\\nIn Table \\n3, neonatal outcomes before and after 34 + 0 weeks of gestation are presented in relation to mode of delivery. Overall, seven preterm infants were classified as having an ICH due to birth injury, corresponding to a rate of 0.02%; and 612 infants were diagnosed with non-traumatic ICH, corresponding to a rate of 1.5%. Diagnoses of neonatal convulsions and other disturbances of neonatal cerebral status were rare, especially in infants at less than 34 weeks of GA, and occurred more frequently after VE and CS than after VD. Cephalic hematoma was the most frequent complication after VE of preterm infants (n = 72 or 3.1%), occurring much more often after VE than after CS (0.16%) and VD (0.49%). Subgaleal hemorrhage was less frequent, with a total of only18 cases. More than two thirds of those cases occurred in the VE group.\\nNeonatal outcomes in preterm infants by mode of delivery and gestational age\\nTable \\n4 shows rates (per 1000), crude and adjusted odds ratios for the outcomes by mode of delivery, and uses infants born by VD as the reference group. After adjustments, preterm infants born by VE had almost doubled OR for ICH, while those born by CS had no increased risk. Preterm infants delivered by VE also had 4.5 times higher OR for extracranial hemorrhages, while those delivered by CS had no increased risk. A total of 540 (88.2%) of the ICH diagnoses consisted of intraventricular bleedings (ICD-codes 52.0-52.3), the type of ICH most frequently seen in preterm infants. Of these, 438 (71.6%) were graded as mild or moderate (grades 1–2). After excluding grades 1–2 of intraventricular hemorrhages from the analyses, the OR for ICH was still significantly increased for infants delivered by VE (OR 2.58, CI 1.27-5.24), but not for those delivered by CS during labor (OR 1.15, CI 0.78-1.70) after adjustment for GA. There was no difference in ICH rates between preterm boys and girls delivered by VE.\\nLogistic regression (odds ratios) for intra– and extracranial hemorrhages, convulsions and other cerebral complications, and brachial plexus injury in preterm infants exposed to different modes of delivery\\nCS = cesarean section, VE = vacuum extraction.\\nModel 1 is adjusted for year of birth, gestational age, parity, maternal age, height, BMI, and infant birthweight.\\nModel 2 is also adjusted for indications for operative delivery.\\nThe ORs for convulsions were almost doubled in both the VE and CS groups after adjustments for the variables in Model 1. However, further adjustment for indication for operative delivery decreased the odds and made the associations statistically insignificant. Other disturbances of the neonatal cerebral status were significantly increased (two to three times higher) both among infants delivered by VE, and by CS during labor, although the OR was higher in the VE group.\\nA total of 53 infants were diagnosed with brachial plexus injury. Of these, 14 were born by VE, corresponding to a rate in the VE group of 0.6% and an OR of 6.21 (95% CI: 2.22-17.4) in the fully-adjusted model. In contrast, infants delivered by CS had no increased risk for this complication. Among infants with brachial plexus injury, there were 11 cases of shoulder dystocia (ICD-code O66.0), of which five occurred in the VE group.\",\n \"Among the 40,764 (54% of all) preterm deliveries included in this study, 2,319 (5.7%) preterm infants were delivered by VE, 5,505 (13.5%) by CS during labor, and 32,940 (80.2%) by VD. The rate of VE deliveries increased gradually with gestational age, Figure \\n1.\\nMode of delivery in relation to gestational age. Figure\\n1 shows rates (%) of different modes of delivery in relation to gestational age (in completed weeks). The blue, dotted line represents unassisted vaginal deliveries, the red, dashed line represents cesarean sections performed after onset of labor, and the green line represents the vacuum extraction deliveries.\",\n \"Table \\n2 shows maternal and perinatal characteristics of the study population in relation to mode of delivery. The VE rate decreased with maternal height and 80% of the women who delivered by VE were primiparae, compared with 48% of those who underwent CS during labor (not in table). More than 45% of the women who delivered by VE had received epidural analgesia during labor compared with 22% of women with VD, and 12% with CS during labor (not in table). Given the association between GA and VE, infants delivered with VE had higher birthweights.\\nMaternal, pregnancy, delivery, and infant characteristics by mode of delivery\\nPopulation based cohort consisting of 40 764 preterm deliveries.\\nCS = caesarean section, BMI = Body Mass Index (weight in kilograms/height in meters2), EA = Epidural Analgesia.\\nThe most common indication for VE was fetal distress (42%), followed by prolonged labor (25%). Having a non-occipitoanterior position (25%) or fetal distress (17%) were the most common indications for CS during labor, while only 3% in this group had a diagnosis of prolonged labor.\",\n \"The proportion of preterm infants diagnosed with an ICH varied more than hundred-fold in relation to GA. It decreased from 21.5% among preterm infants born at 22–28 weeks of GA to 0.1% among those born after 36 weeks of gestation. The rates of neonatal convulsions among preterm infants decreased from 2.0% at 22–28 weeks to 0.25% among those born after 36 weeks of gestation, Figure \\n2. The proportion of preterm infants diagnosed with other disturbances of cerebral status (encephalopathy) decreased with GA, while proportion of infants with brachial plexus injuries or ECH increased slightly with GA, Figure \\n3.\\nProportion (%) preterm infants diagnosed with intracranial hemorrhage (ICH) or convulsions by gestational age. Figure\\n2 shows the proportions (%) of preterm infants diagnosed with ICH and neonatal convulsions in relation to gestational age (in completed weeks). The blue, dotted line represents intracranial hemorrhage (ICH) and the red, dashed line represents neonatal convulsions.\\nProportion (%) preterm infants diagnosed with extracranial hemorrhage (ECH), encephalopathy and brachial plexus injury by gestational age. Figure\\n3 shows the proportions (%) of ECH, encephalopathy (ICD-code P91: other disturbances of cerebral status of newborn), and brachial plexus injury in relation to gestational age (in completed weeks). The blue, dotted line represents extracranial hemorrhage (ECH), the red, dashed line represents encephalopathy and the green line represents brachial plexus injury.\",\n \"To report outcome in relation to mode of delivery, the study cohort was divided according to the current guidelines on the use of VE as either preterm births occurring between 34–36 weeks of gestation (VE may be used), or those occurring before 34 gestational weeks (VE not recommended). In our cohort, 33,202 (81.4%) of all preterm births occurred at 34–36 gestational weeks, and 7,562 (18.6%) before 34 weeks of GA.\\nIn Table \\n3, neonatal outcomes before and after 34 + 0 weeks of gestation are presented in relation to mode of delivery. Overall, seven preterm infants were classified as having an ICH due to birth injury, corresponding to a rate of 0.02%; and 612 infants were diagnosed with non-traumatic ICH, corresponding to a rate of 1.5%. Diagnoses of neonatal convulsions and other disturbances of neonatal cerebral status were rare, especially in infants at less than 34 weeks of GA, and occurred more frequently after VE and CS than after VD. Cephalic hematoma was the most frequent complication after VE of preterm infants (n = 72 or 3.1%), occurring much more often after VE than after CS (0.16%) and VD (0.49%). Subgaleal hemorrhage was less frequent, with a total of only18 cases. More than two thirds of those cases occurred in the VE group.\\nNeonatal outcomes in preterm infants by mode of delivery and gestational age\\nTable \\n4 shows rates (per 1000), crude and adjusted odds ratios for the outcomes by mode of delivery, and uses infants born by VD as the reference group. After adjustments, preterm infants born by VE had almost doubled OR for ICH, while those born by CS had no increased risk. Preterm infants delivered by VE also had 4.5 times higher OR for extracranial hemorrhages, while those delivered by CS had no increased risk. A total of 540 (88.2%) of the ICH diagnoses consisted of intraventricular bleedings (ICD-codes 52.0-52.3), the type of ICH most frequently seen in preterm infants. Of these, 438 (71.6%) were graded as mild or moderate (grades 1–2). After excluding grades 1–2 of intraventricular hemorrhages from the analyses, the OR for ICH was still significantly increased for infants delivered by VE (OR 2.58, CI 1.27-5.24), but not for those delivered by CS during labor (OR 1.15, CI 0.78-1.70) after adjustment for GA. There was no difference in ICH rates between preterm boys and girls delivered by VE.\\nLogistic regression (odds ratios) for intra– and extracranial hemorrhages, convulsions and other cerebral complications, and brachial plexus injury in preterm infants exposed to different modes of delivery\\nCS = cesarean section, VE = vacuum extraction.\\nModel 1 is adjusted for year of birth, gestational age, parity, maternal age, height, BMI, and infant birthweight.\\nModel 2 is also adjusted for indications for operative delivery.\\nThe ORs for convulsions were almost doubled in both the VE and CS groups after adjustments for the variables in Model 1. However, further adjustment for indication for operative delivery decreased the odds and made the associations statistically insignificant. Other disturbances of the neonatal cerebral status were significantly increased (two to three times higher) both among infants delivered by VE, and by CS during labor, although the OR was higher in the VE group.\\nA total of 53 infants were diagnosed with brachial plexus injury. Of these, 14 were born by VE, corresponding to a rate in the VE group of 0.6% and an OR of 6.21 (95% CI: 2.22-17.4) in the fully-adjusted model. In contrast, infants delivered by CS had no increased risk for this complication. Among infants with brachial plexus injury, there were 11 cases of shoulder dystocia (ICD-code O66.0), of which five occurred in the VE group.\",\n \"In this large cohort study of singleton, non-breech preterm births after onset of labor, we identify three clinically important findings related to mode of delivery: First, VE was used in 5.7% of preterm births, and despite recommendations of no use, 3.3% of preterm infants born before 34 gestational weeks were delivered by VE. Secondly, VE for preterm birth was used more frequently in shorter mothers, primiparae and among women treated with EA as pain relief during labor. Finally, and adjusting for potential confounders and covariates, preterm infants delivered by VE had almost doubled OR for ICH, four times higher OR for extracranial hemorrhage, as well as a 6-fold risk for brachial plexus palsy compared with those delivered by VD. Exclusion of intraventricular hemorrhage grades I-II (the most common form of ICH in preterm infants) from the analysis increased the OR for ICH after VE, indicating that severe bleedings were more common among preterm infants delivered by VE.\\nAlthough VE was related to significantly increased rates of ICH, it is not clear whether the extraction as such causes the injury, or if there is an underlying common pathway for both VE-assisted delivery and ICH, i.e., that the relationship is confounded by indication. Since the ORs for ICH were significantly higher in the VE group compared with both the CS and unassisted VD groups, whereas the ORs for other disturbances of cerebral status were slightly higher in both the VE and CS groups as compared with VD, different mechanisms may be involved in the development of these two complications. The forces by vacuum extraction could lead to significant vertical stress, which might be avoided with CS. In a case report of MRI findings after birth injuries among infants delivered by VE\\n[21], it was suggested that vertical traction on the skull and brain may produce tentorial lacerations and rupture of intracranial veins. Another explanation for our findings of different outcomes after VE and CS could be that infants delivered by VE may have been exposed to contractions for a longer time than those delivered by CS. A protective effect of CS is indicated by lower ORs for ICH; however, the exposure to contractions as the sole explanation for the increased risks for ICH after VE is less likely, as the VD group – presumably the group exposed to the largest forces of labor – exhibited significantly lower odds for hemorrhagic complications compared with infants delivered with VE.\\nDuring the study period, the overall rate of ICH increased from 6% originally, up to 12% at the end of the period, most likely reflecting the increased access and use of ultrasonography among Swedish neonatologists in recent years. Improved ultrasound technology and image resolution may also have contributed to this development. Finally, we cannot exclude a contribution from misclassification: a large but normal choroid plexus could have been classified as a small subependymal hemorrhage by less experienced investigators. The finding that the diagnosis of small subependymal hemorrhage (without intraventricular extension; P52.0) increased most compared to other types of ICH during the study period (from 0.5% to 1.2%), supports these interpretations.\\nThe overrepresentation of subgaleal hemorrhage and cephalhematoma after VE is less surprising, since earlier studies have stated the relation between these diagnoses and the use of VE. The risk of subgaleal hemorrhage seems to be unrelated to GA, as this study demonstrates rates similar to those in previous studies of infants born at term\\n[12,13].\\nThe present study showed that preterm infants delivered by VE had a 6- to 7-fold risk increase for injury to the plexus brachialis compared with unassisted VD. This injury is usually associated with large macrosomic infants and shoulder dystocia\\n[22] and not to preterm birth. Our result emphasizes the importance of gentle maneuvers and avoiding application of excessive pressure or traction on the brachial plexus also when delivering the preterm infant, especially by VE.\\nThe major strengths of this study were the large study population covering all preterm deliveries in Sweden during a period of twelve years, and the high quality of the registers, making it possible to analyze rare diagnoses and unusual events such as ICH in preterm infants delivered by VE. We were able to include data on risk factors, potential confounders, and outcomes collected independently from one another and without involving the study subjects, thus minimizing various types of bias (e.g., selection and recall bias). Moreover, antenatal and obstetric care is free of charge in Sweden, management routines as well as GA determinations are standardized, and 99% of births are delivered in public hospitals. This minimizes the risk for confounding by unmeasured socio-demographic factors. Another advantage was the inclusion of the main indications for VE and CS, enabling us to address the question of confounding by indication.\\nA major limitation of this study is that the registers do not contain detailed information about many important factors during the VE deliveries. For instance the registry does not provide specific information about the type of VE instrument used, level, position, and attitude of the fetal head in the pelvis when applying VE, location of placement of the vacuum cup, traction work, skill of the obstetrician, pressure exposure (duration and force), and cup detachments. The register does not provide information about confounders such as use of oxytocin and application of fundal pressure.\\nThere is a general recommendation not to use VE before a GA of 34 weeks. According to the Royal College of Obstetricians and Gynecologists, there is insufficient evidence to establish the safety on VE deliveries in gestations between 34 weeks + 0 days and 36 weeks + 0 days\\n[9]. Our results show that the use of VE is related to rare, but serious complications also between gestational weeks 34–36.\",\n \"The rates of serious birth injuries and complications are generally low, but preterm infants delivered by VE have higher odds ratios for intra- and extracranial hemorrhages and brachial plexus injuries than those delivered by CS during labor or by unassisted vaginal delivery. We therefore support a continued conservative/cautious use of VE in preterm deliveries. Furthermore, the possible causal relationship between mode of delivery and ICH needs to be further investigated.\",\n \"BMI: Body mass index; CI: Confidence interval; CS: Cesarean section; CT: Computerized tomography; EA: Epidural analgesia; ECH: Extracranial hemorrhage; EEG: Electroencephalography; GA: Gestational age; ICH: Intracranial hemorrhage; LGA: Large for gestational age; OR: Odds ratio; SGA: Small for gestational age; VD: Vaginal delivery; VE: Vacuum extraction.\",\n \"The authors declare that they have no competing interest.\",\n \"CE had the idea for the study, designed it and carried out the statistical analysis. KÅ participated in the design and wrote the first draft of the manuscript together with CE. MN contributed to the interpretation of the results and writing of the manuscript. All authors approved the final version of the submitted article.\",\n \"The pre-publication history for this paper can be accessed here:\\n\\nhttp://www.biomedcentral.com/1471-2393/14/42/prepub\\n\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,"results",null,null,null,null,"discussion","conclusions",null,null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null,\n null\n]","isConversational":false},"keywords":{"kind":"list like","value":["Mode of delivery","Preterm delivery","Intracranial hemorrhage","Extracranial hemorrhage","Brachial plexus injury"],"string":"[\n \"Mode of delivery\",\n \"Preterm delivery\",\n \"Intracranial hemorrhage\",\n \"Extracranial hemorrhage\",\n \"Brachial plexus injury\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Background:\nPreterm birth is common\n[1] but still, the optimal mode of delivery of preterm infants is not known. Although neonatal outcomes in preterm infants delivered vaginally or by cesarean section (CS)\n[2-5] have been compared, there is no evidence to provide clear guidance on the method of choice\n[6]. Given the widespread assumption that assisted vaginal delivery could be harmful for fragile infants that are underweight and preterm, very few studies have addressed the use of vacuum extraction (VE) for preterm birth.\nDelivery by VE is a common obstetrical procedure, and in many countries it has replaced the use of forceps. VE is used to terminate a protracted second stage of labor and as an intervention for fetal or maternal distress. VE requires vertex presentation, a fully dilated cervix and ruptured membranes\n[7]. A cesarean section, on the other hand, can be performed at any stage of labor and does not require prerequisites of this kind.\nMost clinical guidelines do not recommend VE before 34 gestational weeks\n[8-10]. These recommendations are not based on results of randomized controlled trials, but rely on the observation that preterm infants are more likely than term infants to develop ICH, and on extrapolations from studies of term infants showing that VE is associated with an increased risk of ICH and other neonatal complications\n[11-17]. Only three studies have previously investigated the use and outcomes of VE in preterm births. The first was undertaken over 40 years ago and showed increased mortality and morbidity among preterm infants delivered by VE as compared with term infants delivered by VE\n[18]. The second study compared neonatal morbidity in preterm infants delivered vaginally with (n = 61) or without VE (n = 122), and found no differences in neonatal morbidity between the two groups\n[19]. The last study compared VE and forceps for preterm delivery (n = 64)\n[20]; the neonatal outcomes were similar in both groups. The available data are clearly untimely and hampered by limitations in power and, therefore, current knowledge on safety of preterm vacuum-assisted birth is unsatisfactory.\nThe aim of this study was to 1) describe the use of VE and compare it to rates of CS during labor in preterm deliveries in Sweden from 1999–2010, 2) characterize the distribution of perinatal risk factors associated with each mode of delivery, and 3) compare rates of neonatal intra- and extracranial hemorrhages, as well as occurrence of brachial plexus injury after preterm delivery by VE or CS during labor, using unassisted vaginal birth as a reference.\n\nMethods:\nThis study was based on data from national data bases held by the Swedish National Board of Health and Welfare. The national registration number, assigned to each Swedish resident at birth, was used for individual record linkage. We used two registers: The Swedish Medical Birth Register (SMBR) that covers 99% of all births in Sweden, and The Swedish National Inpatient Register (IPR) that covers all public inpatient care. The SMBR includes prospectively collected information on maternal characteristics, reproductive history, and complications during pregnancy, delivery, and the neonatal period. The IPR includes data on each hospital admission and discharge.\n Study population During the period of 1999–2010, there were 75,296 (6.2%) preterm births in Sweden. We excluded deliveries by CS before the onset of labor (n = 17,306), forceps (n = 257), or performed with both VE and CS (n = 125). We also excluded stillbirths (fetal deaths occurring before labor or intra partum) (n = 1,839), multiple births (n = 11,088), and births in breech presentation (n = 3,917). Thus, the final study population was restricted to all live-born, preterm singleton infants with a non-breech presentation at birth, delivered after a spontaneous or induced onset of labor followed by CS, vacuum extraction (VE), or by unassisted vaginal delivery (VD) before gestational week 37 + 0 days (N = 40,764). CS during labor was defined as abdominal delivery after the onset of labor, either spontaneously, by rupture of membranes, or by induction.\nA number of independent variables were collected; the maternal anthropometrics included: age, height, and body mass index (BMI). BMI was calculated from measured height and weight obtained at the first antenatal care visit, which occurred before the 15th week of gestation in more than 95% of the pregnancies. BMI was categorized into underweight (below 18.5 kg/m2), normal (18.5–24.9 kg/m2), overweight (25–29.9 kg/m2), obese (>29.9 kg/m2), or missing. Parity was categorized as primi- or multiparity. Information on complications during pregnancy and delivery were coded according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards). The following pregnancy complications were included: diabetes – both gestational and types 1 and 2 (O24.0-9) preeclampsia- both hypertension, preeclampsia, and eclampsia (O10.0-O15.9). Labor-related risk factors or covariates includedepidural analgesia (EA; yes/no), and induction of labor (yes/no). Indications for operative delivery were classified into four major groups: prolonged labor (O62.0-2, O63.0-9), signs of fetal distress (O68.0-O68.1-9), preeclampsia, and a non-occipitoanterior presentation of the fetus (all presentations except occipitoanterior and breech, registered at birth). Gestational age (GA) for preterm infants was divided into three periods according to the World Health Organization: extremely preterm (before 28 weeks), very preterm (28–31 weeks) and moderately preterm (32–36 weeks). Furthermore, we also divided the preterm gestational period according to guidelines on instrumental delivery into either: less than 34 weeks (VE not recommended), and 34–36 weeks (VE may be used). GA was recorded in completed weeks, and was based on routine ultrasound dating performed at 17 to 18 postmenstrual weeks in 97-98% of all pregnant women. Infant birthweight was categorized as less than 1,500 grams, 1,500-2,000 grams, 2,001-2,500 grams, 2,501-3,000 grams, and 3,001-4,000 grams.\nDuring the period of 1999–2010, there were 75,296 (6.2%) preterm births in Sweden. We excluded deliveries by CS before the onset of labor (n = 17,306), forceps (n = 257), or performed with both VE and CS (n = 125). We also excluded stillbirths (fetal deaths occurring before labor or intra partum) (n = 1,839), multiple births (n = 11,088), and births in breech presentation (n = 3,917). Thus, the final study population was restricted to all live-born, preterm singleton infants with a non-breech presentation at birth, delivered after a spontaneous or induced onset of labor followed by CS, vacuum extraction (VE), or by unassisted vaginal delivery (VD) before gestational week 37 + 0 days (N = 40,764). CS during labor was defined as abdominal delivery after the onset of labor, either spontaneously, by rupture of membranes, or by induction.\nA number of independent variables were collected; the maternal anthropometrics included: age, height, and body mass index (BMI). BMI was calculated from measured height and weight obtained at the first antenatal care visit, which occurred before the 15th week of gestation in more than 95% of the pregnancies. BMI was categorized into underweight (below 18.5 kg/m2), normal (18.5–24.9 kg/m2), overweight (25–29.9 kg/m2), obese (>29.9 kg/m2), or missing. Parity was categorized as primi- or multiparity. Information on complications during pregnancy and delivery were coded according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards). The following pregnancy complications were included: diabetes – both gestational and types 1 and 2 (O24.0-9) preeclampsia- both hypertension, preeclampsia, and eclampsia (O10.0-O15.9). Labor-related risk factors or covariates includedepidural analgesia (EA; yes/no), and induction of labor (yes/no). Indications for operative delivery were classified into four major groups: prolonged labor (O62.0-2, O63.0-9), signs of fetal distress (O68.0-O68.1-9), preeclampsia, and a non-occipitoanterior presentation of the fetus (all presentations except occipitoanterior and breech, registered at birth). Gestational age (GA) for preterm infants was divided into three periods according to the World Health Organization: extremely preterm (before 28 weeks), very preterm (28–31 weeks) and moderately preterm (32–36 weeks). Furthermore, we also divided the preterm gestational period according to guidelines on instrumental delivery into either: less than 34 weeks (VE not recommended), and 34–36 weeks (VE may be used). GA was recorded in completed weeks, and was based on routine ultrasound dating performed at 17 to 18 postmenstrual weeks in 97-98% of all pregnant women. Infant birthweight was categorized as less than 1,500 grams, 1,500-2,000 grams, 2,001-2,500 grams, 2,501-3,000 grams, and 3,001-4,000 grams.\n Outcome variables Neonatal diagnoses were classified according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards), and identified/collected in the SMBR or in the IPR. The following neonatal outcomes (ICD codes) were assessed: Intracranial laceration and hemorrhage due to birth injury (P10), intracranial non-traumatic hemorrhage of fetus and newborn (P52), convulsions of newborn (P90), other disturbances of cerebral status of newborn (P 91), subgaleal hematoma (P12.2), cephalhematoma (P12.0), and brachial plexus injury (P14.0-3). The definitions of outcomes are described in detail in Table \n1.\nNeonatal outcomes studied in 40 764 preterm infants\nNeonatal diagnoses of intracranial hemorrhages in preterm infants were mainly based on imaging of the brain using ultrasonography; however, some assessments of the brain at term-equivalent age were alternatively performed with CT and/or MRI. Imaging of the brain was performed on clinical indications only in cases born moderately or late preterm, whereas all very preterm infants (born before 32 weeks of gestation) were screened for intracranial lesions, even in asymptomatic infants. A diagnosis of convulsions included infants with clinical signs of convulsions and/or convulsions verified by EEG.\nStatistical analysis was performed using proportions and odds ratios (OR) with a 95% confidence interval (CI) for neonatal complications in relation to mode of delivery, using unassisted VD as the reference group (SPSS 20.0 for Windows software package). Three models were used to assess the relationship between the different modes of delivery and the risk for neonatal complications: one crude, and two adjusted (Models 1 and 2). The included covariates have been shown previously to be related to instrumental deliveries, and were related to the outcomes in cross tabulations. In Model 1, we adjusted for the following confounders or covariates: maternal age, height, BMI, and parity, as well as infant year of birth, birthweight and GA. In Model 2, we added the indication for operative delivery and preeclampsia. The year of birth was entered as a continuous variable in accordance with a linear secular trend, and all other variables were entered as categories. Furthermore, a separate logistic regression analysis was performed to investigate severe ICH in relation to mode of delivery. Here, intraventricular hemorrhages grades 1–2 were excluded and the analysis was adjusted for GA only. We also conducted separated analyses on potential relationships between sex and ICH in relation to mode of delivery. Missing data were entered as a separate category in the analyses. The study was approved by the Regional Ethical Review Board in Stockholm, Dnr 2008/1322-31.\nNeonatal diagnoses were classified according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards), and identified/collected in the SMBR or in the IPR. The following neonatal outcomes (ICD codes) were assessed: Intracranial laceration and hemorrhage due to birth injury (P10), intracranial non-traumatic hemorrhage of fetus and newborn (P52), convulsions of newborn (P90), other disturbances of cerebral status of newborn (P 91), subgaleal hematoma (P12.2), cephalhematoma (P12.0), and brachial plexus injury (P14.0-3). The definitions of outcomes are described in detail in Table \n1.\nNeonatal outcomes studied in 40 764 preterm infants\nNeonatal diagnoses of intracranial hemorrhages in preterm infants were mainly based on imaging of the brain using ultrasonography; however, some assessments of the brain at term-equivalent age were alternatively performed with CT and/or MRI. Imaging of the brain was performed on clinical indications only in cases born moderately or late preterm, whereas all very preterm infants (born before 32 weeks of gestation) were screened for intracranial lesions, even in asymptomatic infants. A diagnosis of convulsions included infants with clinical signs of convulsions and/or convulsions verified by EEG.\nStatistical analysis was performed using proportions and odds ratios (OR) with a 95% confidence interval (CI) for neonatal complications in relation to mode of delivery, using unassisted VD as the reference group (SPSS 20.0 for Windows software package). Three models were used to assess the relationship between the different modes of delivery and the risk for neonatal complications: one crude, and two adjusted (Models 1 and 2). The included covariates have been shown previously to be related to instrumental deliveries, and were related to the outcomes in cross tabulations. In Model 1, we adjusted for the following confounders or covariates: maternal age, height, BMI, and parity, as well as infant year of birth, birthweight and GA. In Model 2, we added the indication for operative delivery and preeclampsia. The year of birth was entered as a continuous variable in accordance with a linear secular trend, and all other variables were entered as categories. Furthermore, a separate logistic regression analysis was performed to investigate severe ICH in relation to mode of delivery. Here, intraventricular hemorrhages grades 1–2 were excluded and the analysis was adjusted for GA only. We also conducted separated analyses on potential relationships between sex and ICH in relation to mode of delivery. Missing data were entered as a separate category in the analyses. The study was approved by the Regional Ethical Review Board in Stockholm, Dnr 2008/1322-31.\n\nStudy population:\nDuring the period of 1999–2010, there were 75,296 (6.2%) preterm births in Sweden. We excluded deliveries by CS before the onset of labor (n = 17,306), forceps (n = 257), or performed with both VE and CS (n = 125). We also excluded stillbirths (fetal deaths occurring before labor or intra partum) (n = 1,839), multiple births (n = 11,088), and births in breech presentation (n = 3,917). Thus, the final study population was restricted to all live-born, preterm singleton infants with a non-breech presentation at birth, delivered after a spontaneous or induced onset of labor followed by CS, vacuum extraction (VE), or by unassisted vaginal delivery (VD) before gestational week 37 + 0 days (N = 40,764). CS during labor was defined as abdominal delivery after the onset of labor, either spontaneously, by rupture of membranes, or by induction.\nA number of independent variables were collected; the maternal anthropometrics included: age, height, and body mass index (BMI). BMI was calculated from measured height and weight obtained at the first antenatal care visit, which occurred before the 15th week of gestation in more than 95% of the pregnancies. BMI was categorized into underweight (below 18.5 kg/m2), normal (18.5–24.9 kg/m2), overweight (25–29.9 kg/m2), obese (>29.9 kg/m2), or missing. Parity was categorized as primi- or multiparity. Information on complications during pregnancy and delivery were coded according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards). The following pregnancy complications were included: diabetes – both gestational and types 1 and 2 (O24.0-9) preeclampsia- both hypertension, preeclampsia, and eclampsia (O10.0-O15.9). Labor-related risk factors or covariates includedepidural analgesia (EA; yes/no), and induction of labor (yes/no). Indications for operative delivery were classified into four major groups: prolonged labor (O62.0-2, O63.0-9), signs of fetal distress (O68.0-O68.1-9), preeclampsia, and a non-occipitoanterior presentation of the fetus (all presentations except occipitoanterior and breech, registered at birth). Gestational age (GA) for preterm infants was divided into three periods according to the World Health Organization: extremely preterm (before 28 weeks), very preterm (28–31 weeks) and moderately preterm (32–36 weeks). Furthermore, we also divided the preterm gestational period according to guidelines on instrumental delivery into either: less than 34 weeks (VE not recommended), and 34–36 weeks (VE may be used). GA was recorded in completed weeks, and was based on routine ultrasound dating performed at 17 to 18 postmenstrual weeks in 97-98% of all pregnant women. Infant birthweight was categorized as less than 1,500 grams, 1,500-2,000 grams, 2,001-2,500 grams, 2,501-3,000 grams, and 3,001-4,000 grams.\n\nOutcome variables:\nNeonatal diagnoses were classified according to the International Classification of Diseases (ICD) Tenth Revision (1997 and onwards), and identified/collected in the SMBR or in the IPR. The following neonatal outcomes (ICD codes) were assessed: Intracranial laceration and hemorrhage due to birth injury (P10), intracranial non-traumatic hemorrhage of fetus and newborn (P52), convulsions of newborn (P90), other disturbances of cerebral status of newborn (P 91), subgaleal hematoma (P12.2), cephalhematoma (P12.0), and brachial plexus injury (P14.0-3). The definitions of outcomes are described in detail in Table \n1.\nNeonatal outcomes studied in 40 764 preterm infants\nNeonatal diagnoses of intracranial hemorrhages in preterm infants were mainly based on imaging of the brain using ultrasonography; however, some assessments of the brain at term-equivalent age were alternatively performed with CT and/or MRI. Imaging of the brain was performed on clinical indications only in cases born moderately or late preterm, whereas all very preterm infants (born before 32 weeks of gestation) were screened for intracranial lesions, even in asymptomatic infants. A diagnosis of convulsions included infants with clinical signs of convulsions and/or convulsions verified by EEG.\nStatistical analysis was performed using proportions and odds ratios (OR) with a 95% confidence interval (CI) for neonatal complications in relation to mode of delivery, using unassisted VD as the reference group (SPSS 20.0 for Windows software package). Three models were used to assess the relationship between the different modes of delivery and the risk for neonatal complications: one crude, and two adjusted (Models 1 and 2). The included covariates have been shown previously to be related to instrumental deliveries, and were related to the outcomes in cross tabulations. In Model 1, we adjusted for the following confounders or covariates: maternal age, height, BMI, and parity, as well as infant year of birth, birthweight and GA. In Model 2, we added the indication for operative delivery and preeclampsia. The year of birth was entered as a continuous variable in accordance with a linear secular trend, and all other variables were entered as categories. Furthermore, a separate logistic regression analysis was performed to investigate severe ICH in relation to mode of delivery. Here, intraventricular hemorrhages grades 1–2 were excluded and the analysis was adjusted for GA only. We also conducted separated analyses on potential relationships between sex and ICH in relation to mode of delivery. Missing data were entered as a separate category in the analyses. The study was approved by the Regional Ethical Review Board in Stockholm, Dnr 2008/1322-31.\n\nResults:\n Use of VE in relation to gestational age Among the 40,764 (54% of all) preterm deliveries included in this study, 2,319 (5.7%) preterm infants were delivered by VE, 5,505 (13.5%) by CS during labor, and 32,940 (80.2%) by VD. The rate of VE deliveries increased gradually with gestational age, Figure \n1.\nMode of delivery in relation to gestational age. Figure\n1 shows rates (%) of different modes of delivery in relation to gestational age (in completed weeks). The blue, dotted line represents unassisted vaginal deliveries, the red, dashed line represents cesarean sections performed after onset of labor, and the green line represents the vacuum extraction deliveries.\nAmong the 40,764 (54% of all) preterm deliveries included in this study, 2,319 (5.7%) preterm infants were delivered by VE, 5,505 (13.5%) by CS during labor, and 32,940 (80.2%) by VD. The rate of VE deliveries increased gradually with gestational age, Figure \n1.\nMode of delivery in relation to gestational age. Figure\n1 shows rates (%) of different modes of delivery in relation to gestational age (in completed weeks). The blue, dotted line represents unassisted vaginal deliveries, the red, dashed line represents cesarean sections performed after onset of labor, and the green line represents the vacuum extraction deliveries.\n Distribution of risk factors or covariates in relation to mode of delivery Table \n2 shows maternal and perinatal characteristics of the study population in relation to mode of delivery. The VE rate decreased with maternal height and 80% of the women who delivered by VE were primiparae, compared with 48% of those who underwent CS during labor (not in table). More than 45% of the women who delivered by VE had received epidural analgesia during labor compared with 22% of women with VD, and 12% with CS during labor (not in table). Given the association between GA and VE, infants delivered with VE had higher birthweights.\nMaternal, pregnancy, delivery, and infant characteristics by mode of delivery\nPopulation based cohort consisting of 40 764 preterm deliveries.\nCS = caesarean section, BMI = Body Mass Index (weight in kilograms/height in meters2), EA = Epidural Analgesia.\nThe most common indication for VE was fetal distress (42%), followed by prolonged labor (25%). Having a non-occipitoanterior position (25%) or fetal distress (17%) were the most common indications for CS during labor, while only 3% in this group had a diagnosis of prolonged labor.\nTable \n2 shows maternal and perinatal characteristics of the study population in relation to mode of delivery. The VE rate decreased with maternal height and 80% of the women who delivered by VE were primiparae, compared with 48% of those who underwent CS during labor (not in table). More than 45% of the women who delivered by VE had received epidural analgesia during labor compared with 22% of women with VD, and 12% with CS during labor (not in table). Given the association between GA and VE, infants delivered with VE had higher birthweights.\nMaternal, pregnancy, delivery, and infant characteristics by mode of delivery\nPopulation based cohort consisting of 40 764 preterm deliveries.\nCS = caesarean section, BMI = Body Mass Index (weight in kilograms/height in meters2), EA = Epidural Analgesia.\nThe most common indication for VE was fetal distress (42%), followed by prolonged labor (25%). Having a non-occipitoanterior position (25%) or fetal distress (17%) were the most common indications for CS during labor, while only 3% in this group had a diagnosis of prolonged labor.\n Neonatal outcome in relation to gestational age The proportion of preterm infants diagnosed with an ICH varied more than hundred-fold in relation to GA. It decreased from 21.5% among preterm infants born at 22–28 weeks of GA to 0.1% among those born after 36 weeks of gestation. The rates of neonatal convulsions among preterm infants decreased from 2.0% at 22–28 weeks to 0.25% among those born after 36 weeks of gestation, Figure \n2. The proportion of preterm infants diagnosed with other disturbances of cerebral status (encephalopathy) decreased with GA, while proportion of infants with brachial plexus injuries or ECH increased slightly with GA, Figure \n3.\nProportion (%) preterm infants diagnosed with intracranial hemorrhage (ICH) or convulsions by gestational age. Figure\n2 shows the proportions (%) of preterm infants diagnosed with ICH and neonatal convulsions in relation to gestational age (in completed weeks). The blue, dotted line represents intracranial hemorrhage (ICH) and the red, dashed line represents neonatal convulsions.\nProportion (%) preterm infants diagnosed with extracranial hemorrhage (ECH), encephalopathy and brachial plexus injury by gestational age. Figure\n3 shows the proportions (%) of ECH, encephalopathy (ICD-code P91: other disturbances of cerebral status of newborn), and brachial plexus injury in relation to gestational age (in completed weeks). The blue, dotted line represents extracranial hemorrhage (ECH), the red, dashed line represents encephalopathy and the green line represents brachial plexus injury.\nThe proportion of preterm infants diagnosed with an ICH varied more than hundred-fold in relation to GA. It decreased from 21.5% among preterm infants born at 22–28 weeks of GA to 0.1% among those born after 36 weeks of gestation. The rates of neonatal convulsions among preterm infants decreased from 2.0% at 22–28 weeks to 0.25% among those born after 36 weeks of gestation, Figure \n2. The proportion of preterm infants diagnosed with other disturbances of cerebral status (encephalopathy) decreased with GA, while proportion of infants with brachial plexus injuries or ECH increased slightly with GA, Figure \n3.\nProportion (%) preterm infants diagnosed with intracranial hemorrhage (ICH) or convulsions by gestational age. Figure\n2 shows the proportions (%) of preterm infants diagnosed with ICH and neonatal convulsions in relation to gestational age (in completed weeks). The blue, dotted line represents intracranial hemorrhage (ICH) and the red, dashed line represents neonatal convulsions.\nProportion (%) preterm infants diagnosed with extracranial hemorrhage (ECH), encephalopathy and brachial plexus injury by gestational age. Figure\n3 shows the proportions (%) of ECH, encephalopathy (ICD-code P91: other disturbances of cerebral status of newborn), and brachial plexus injury in relation to gestational age (in completed weeks). The blue, dotted line represents extracranial hemorrhage (ECH), the red, dashed line represents encephalopathy and the green line represents brachial plexus injury.\n Neonatal outcome in relation to mode of delivery To report outcome in relation to mode of delivery, the study cohort was divided according to the current guidelines on the use of VE as either preterm births occurring between 34–36 weeks of gestation (VE may be used), or those occurring before 34 gestational weeks (VE not recommended). In our cohort, 33,202 (81.4%) of all preterm births occurred at 34–36 gestational weeks, and 7,562 (18.6%) before 34 weeks of GA.\nIn Table \n3, neonatal outcomes before and after 34 + 0 weeks of gestation are presented in relation to mode of delivery. Overall, seven preterm infants were classified as having an ICH due to birth injury, corresponding to a rate of 0.02%; and 612 infants were diagnosed with non-traumatic ICH, corresponding to a rate of 1.5%. Diagnoses of neonatal convulsions and other disturbances of neonatal cerebral status were rare, especially in infants at less than 34 weeks of GA, and occurred more frequently after VE and CS than after VD. Cephalic hematoma was the most frequent complication after VE of preterm infants (n = 72 or 3.1%), occurring much more often after VE than after CS (0.16%) and VD (0.49%). Subgaleal hemorrhage was less frequent, with a total of only18 cases. More than two thirds of those cases occurred in the VE group.\nNeonatal outcomes in preterm infants by mode of delivery and gestational age\nTable \n4 shows rates (per 1000), crude and adjusted odds ratios for the outcomes by mode of delivery, and uses infants born by VD as the reference group. After adjustments, preterm infants born by VE had almost doubled OR for ICH, while those born by CS had no increased risk. Preterm infants delivered by VE also had 4.5 times higher OR for extracranial hemorrhages, while those delivered by CS had no increased risk. A total of 540 (88.2%) of the ICH diagnoses consisted of intraventricular bleedings (ICD-codes 52.0-52.3), the type of ICH most frequently seen in preterm infants. Of these, 438 (71.6%) were graded as mild or moderate (grades 1–2). After excluding grades 1–2 of intraventricular hemorrhages from the analyses, the OR for ICH was still significantly increased for infants delivered by VE (OR 2.58, CI 1.27-5.24), but not for those delivered by CS during labor (OR 1.15, CI 0.78-1.70) after adjustment for GA. There was no difference in ICH rates between preterm boys and girls delivered by VE.\nLogistic regression (odds ratios) for intra– and extracranial hemorrhages, convulsions and other cerebral complications, and brachial plexus injury in preterm infants exposed to different modes of delivery\nCS = cesarean section, VE = vacuum extraction.\nModel 1 is adjusted for year of birth, gestational age, parity, maternal age, height, BMI, and infant birthweight.\nModel 2 is also adjusted for indications for operative delivery.\nThe ORs for convulsions were almost doubled in both the VE and CS groups after adjustments for the variables in Model 1. However, further adjustment for indication for operative delivery decreased the odds and made the associations statistically insignificant. Other disturbances of the neonatal cerebral status were significantly increased (two to three times higher) both among infants delivered by VE, and by CS during labor, although the OR was higher in the VE group.\nA total of 53 infants were diagnosed with brachial plexus injury. Of these, 14 were born by VE, corresponding to a rate in the VE group of 0.6% and an OR of 6.21 (95% CI: 2.22-17.4) in the fully-adjusted model. In contrast, infants delivered by CS had no increased risk for this complication. Among infants with brachial plexus injury, there were 11 cases of shoulder dystocia (ICD-code O66.0), of which five occurred in the VE group.\nTo report outcome in relation to mode of delivery, the study cohort was divided according to the current guidelines on the use of VE as either preterm births occurring between 34–36 weeks of gestation (VE may be used), or those occurring before 34 gestational weeks (VE not recommended). In our cohort, 33,202 (81.4%) of all preterm births occurred at 34–36 gestational weeks, and 7,562 (18.6%) before 34 weeks of GA.\nIn Table \n3, neonatal outcomes before and after 34 + 0 weeks of gestation are presented in relation to mode of delivery. Overall, seven preterm infants were classified as having an ICH due to birth injury, corresponding to a rate of 0.02%; and 612 infants were diagnosed with non-traumatic ICH, corresponding to a rate of 1.5%. Diagnoses of neonatal convulsions and other disturbances of neonatal cerebral status were rare, especially in infants at less than 34 weeks of GA, and occurred more frequently after VE and CS than after VD. Cephalic hematoma was the most frequent complication after VE of preterm infants (n = 72 or 3.1%), occurring much more often after VE than after CS (0.16%) and VD (0.49%). Subgaleal hemorrhage was less frequent, with a total of only18 cases. More than two thirds of those cases occurred in the VE group.\nNeonatal outcomes in preterm infants by mode of delivery and gestational age\nTable \n4 shows rates (per 1000), crude and adjusted odds ratios for the outcomes by mode of delivery, and uses infants born by VD as the reference group. After adjustments, preterm infants born by VE had almost doubled OR for ICH, while those born by CS had no increased risk. Preterm infants delivered by VE also had 4.5 times higher OR for extracranial hemorrhages, while those delivered by CS had no increased risk. A total of 540 (88.2%) of the ICH diagnoses consisted of intraventricular bleedings (ICD-codes 52.0-52.3), the type of ICH most frequently seen in preterm infants. Of these, 438 (71.6%) were graded as mild or moderate (grades 1–2). After excluding grades 1–2 of intraventricular hemorrhages from the analyses, the OR for ICH was still significantly increased for infants delivered by VE (OR 2.58, CI 1.27-5.24), but not for those delivered by CS during labor (OR 1.15, CI 0.78-1.70) after adjustment for GA. There was no difference in ICH rates between preterm boys and girls delivered by VE.\nLogistic regression (odds ratios) for intra– and extracranial hemorrhages, convulsions and other cerebral complications, and brachial plexus injury in preterm infants exposed to different modes of delivery\nCS = cesarean section, VE = vacuum extraction.\nModel 1 is adjusted for year of birth, gestational age, parity, maternal age, height, BMI, and infant birthweight.\nModel 2 is also adjusted for indications for operative delivery.\nThe ORs for convulsions were almost doubled in both the VE and CS groups after adjustments for the variables in Model 1. However, further adjustment for indication for operative delivery decreased the odds and made the associations statistically insignificant. Other disturbances of the neonatal cerebral status were significantly increased (two to three times higher) both among infants delivered by VE, and by CS during labor, although the OR was higher in the VE group.\nA total of 53 infants were diagnosed with brachial plexus injury. Of these, 14 were born by VE, corresponding to a rate in the VE group of 0.6% and an OR of 6.21 (95% CI: 2.22-17.4) in the fully-adjusted model. In contrast, infants delivered by CS had no increased risk for this complication. Among infants with brachial plexus injury, there were 11 cases of shoulder dystocia (ICD-code O66.0), of which five occurred in the VE group.\n\nUse of VE in relation to gestational age:\nAmong the 40,764 (54% of all) preterm deliveries included in this study, 2,319 (5.7%) preterm infants were delivered by VE, 5,505 (13.5%) by CS during labor, and 32,940 (80.2%) by VD. The rate of VE deliveries increased gradually with gestational age, Figure \n1.\nMode of delivery in relation to gestational age. Figure\n1 shows rates (%) of different modes of delivery in relation to gestational age (in completed weeks). The blue, dotted line represents unassisted vaginal deliveries, the red, dashed line represents cesarean sections performed after onset of labor, and the green line represents the vacuum extraction deliveries.\n\nDistribution of risk factors or covariates in relation to mode of delivery:\nTable \n2 shows maternal and perinatal characteristics of the study population in relation to mode of delivery. The VE rate decreased with maternal height and 80% of the women who delivered by VE were primiparae, compared with 48% of those who underwent CS during labor (not in table). More than 45% of the women who delivered by VE had received epidural analgesia during labor compared with 22% of women with VD, and 12% with CS during labor (not in table). Given the association between GA and VE, infants delivered with VE had higher birthweights.\nMaternal, pregnancy, delivery, and infant characteristics by mode of delivery\nPopulation based cohort consisting of 40 764 preterm deliveries.\nCS = caesarean section, BMI = Body Mass Index (weight in kilograms/height in meters2), EA = Epidural Analgesia.\nThe most common indication for VE was fetal distress (42%), followed by prolonged labor (25%). Having a non-occipitoanterior position (25%) or fetal distress (17%) were the most common indications for CS during labor, while only 3% in this group had a diagnosis of prolonged labor.\n\nNeonatal outcome in relation to gestational age:\nThe proportion of preterm infants diagnosed with an ICH varied more than hundred-fold in relation to GA. It decreased from 21.5% among preterm infants born at 22–28 weeks of GA to 0.1% among those born after 36 weeks of gestation. The rates of neonatal convulsions among preterm infants decreased from 2.0% at 22–28 weeks to 0.25% among those born after 36 weeks of gestation, Figure \n2. The proportion of preterm infants diagnosed with other disturbances of cerebral status (encephalopathy) decreased with GA, while proportion of infants with brachial plexus injuries or ECH increased slightly with GA, Figure \n3.\nProportion (%) preterm infants diagnosed with intracranial hemorrhage (ICH) or convulsions by gestational age. Figure\n2 shows the proportions (%) of preterm infants diagnosed with ICH and neonatal convulsions in relation to gestational age (in completed weeks). The blue, dotted line represents intracranial hemorrhage (ICH) and the red, dashed line represents neonatal convulsions.\nProportion (%) preterm infants diagnosed with extracranial hemorrhage (ECH), encephalopathy and brachial plexus injury by gestational age. Figure\n3 shows the proportions (%) of ECH, encephalopathy (ICD-code P91: other disturbances of cerebral status of newborn), and brachial plexus injury in relation to gestational age (in completed weeks). The blue, dotted line represents extracranial hemorrhage (ECH), the red, dashed line represents encephalopathy and the green line represents brachial plexus injury.\n\nNeonatal outcome in relation to mode of delivery:\nTo report outcome in relation to mode of delivery, the study cohort was divided according to the current guidelines on the use of VE as either preterm births occurring between 34–36 weeks of gestation (VE may be used), or those occurring before 34 gestational weeks (VE not recommended). In our cohort, 33,202 (81.4%) of all preterm births occurred at 34–36 gestational weeks, and 7,562 (18.6%) before 34 weeks of GA.\nIn Table \n3, neonatal outcomes before and after 34 + 0 weeks of gestation are presented in relation to mode of delivery. Overall, seven preterm infants were classified as having an ICH due to birth injury, corresponding to a rate of 0.02%; and 612 infants were diagnosed with non-traumatic ICH, corresponding to a rate of 1.5%. Diagnoses of neonatal convulsions and other disturbances of neonatal cerebral status were rare, especially in infants at less than 34 weeks of GA, and occurred more frequently after VE and CS than after VD. Cephalic hematoma was the most frequent complication after VE of preterm infants (n = 72 or 3.1%), occurring much more often after VE than after CS (0.16%) and VD (0.49%). Subgaleal hemorrhage was less frequent, with a total of only18 cases. More than two thirds of those cases occurred in the VE group.\nNeonatal outcomes in preterm infants by mode of delivery and gestational age\nTable \n4 shows rates (per 1000), crude and adjusted odds ratios for the outcomes by mode of delivery, and uses infants born by VD as the reference group. After adjustments, preterm infants born by VE had almost doubled OR for ICH, while those born by CS had no increased risk. Preterm infants delivered by VE also had 4.5 times higher OR for extracranial hemorrhages, while those delivered by CS had no increased risk. A total of 540 (88.2%) of the ICH diagnoses consisted of intraventricular bleedings (ICD-codes 52.0-52.3), the type of ICH most frequently seen in preterm infants. Of these, 438 (71.6%) were graded as mild or moderate (grades 1–2). After excluding grades 1–2 of intraventricular hemorrhages from the analyses, the OR for ICH was still significantly increased for infants delivered by VE (OR 2.58, CI 1.27-5.24), but not for those delivered by CS during labor (OR 1.15, CI 0.78-1.70) after adjustment for GA. There was no difference in ICH rates between preterm boys and girls delivered by VE.\nLogistic regression (odds ratios) for intra– and extracranial hemorrhages, convulsions and other cerebral complications, and brachial plexus injury in preterm infants exposed to different modes of delivery\nCS = cesarean section, VE = vacuum extraction.\nModel 1 is adjusted for year of birth, gestational age, parity, maternal age, height, BMI, and infant birthweight.\nModel 2 is also adjusted for indications for operative delivery.\nThe ORs for convulsions were almost doubled in both the VE and CS groups after adjustments for the variables in Model 1. However, further adjustment for indication for operative delivery decreased the odds and made the associations statistically insignificant. Other disturbances of the neonatal cerebral status were significantly increased (two to three times higher) both among infants delivered by VE, and by CS during labor, although the OR was higher in the VE group.\nA total of 53 infants were diagnosed with brachial plexus injury. Of these, 14 were born by VE, corresponding to a rate in the VE group of 0.6% and an OR of 6.21 (95% CI: 2.22-17.4) in the fully-adjusted model. In contrast, infants delivered by CS had no increased risk for this complication. Among infants with brachial plexus injury, there were 11 cases of shoulder dystocia (ICD-code O66.0), of which five occurred in the VE group.\n\nDiscussion:\nIn this large cohort study of singleton, non-breech preterm births after onset of labor, we identify three clinically important findings related to mode of delivery: First, VE was used in 5.7% of preterm births, and despite recommendations of no use, 3.3% of preterm infants born before 34 gestational weeks were delivered by VE. Secondly, VE for preterm birth was used more frequently in shorter mothers, primiparae and among women treated with EA as pain relief during labor. Finally, and adjusting for potential confounders and covariates, preterm infants delivered by VE had almost doubled OR for ICH, four times higher OR for extracranial hemorrhage, as well as a 6-fold risk for brachial plexus palsy compared with those delivered by VD. Exclusion of intraventricular hemorrhage grades I-II (the most common form of ICH in preterm infants) from the analysis increased the OR for ICH after VE, indicating that severe bleedings were more common among preterm infants delivered by VE.\nAlthough VE was related to significantly increased rates of ICH, it is not clear whether the extraction as such causes the injury, or if there is an underlying common pathway for both VE-assisted delivery and ICH, i.e., that the relationship is confounded by indication. Since the ORs for ICH were significantly higher in the VE group compared with both the CS and unassisted VD groups, whereas the ORs for other disturbances of cerebral status were slightly higher in both the VE and CS groups as compared with VD, different mechanisms may be involved in the development of these two complications. The forces by vacuum extraction could lead to significant vertical stress, which might be avoided with CS. In a case report of MRI findings after birth injuries among infants delivered by VE\n[21], it was suggested that vertical traction on the skull and brain may produce tentorial lacerations and rupture of intracranial veins. Another explanation for our findings of different outcomes after VE and CS could be that infants delivered by VE may have been exposed to contractions for a longer time than those delivered by CS. A protective effect of CS is indicated by lower ORs for ICH; however, the exposure to contractions as the sole explanation for the increased risks for ICH after VE is less likely, as the VD group – presumably the group exposed to the largest forces of labor – exhibited significantly lower odds for hemorrhagic complications compared with infants delivered with VE.\nDuring the study period, the overall rate of ICH increased from 6% originally, up to 12% at the end of the period, most likely reflecting the increased access and use of ultrasonography among Swedish neonatologists in recent years. Improved ultrasound technology and image resolution may also have contributed to this development. Finally, we cannot exclude a contribution from misclassification: a large but normal choroid plexus could have been classified as a small subependymal hemorrhage by less experienced investigators. The finding that the diagnosis of small subependymal hemorrhage (without intraventricular extension; P52.0) increased most compared to other types of ICH during the study period (from 0.5% to 1.2%), supports these interpretations.\nThe overrepresentation of subgaleal hemorrhage and cephalhematoma after VE is less surprising, since earlier studies have stated the relation between these diagnoses and the use of VE. The risk of subgaleal hemorrhage seems to be unrelated to GA, as this study demonstrates rates similar to those in previous studies of infants born at term\n[12,13].\nThe present study showed that preterm infants delivered by VE had a 6- to 7-fold risk increase for injury to the plexus brachialis compared with unassisted VD. This injury is usually associated with large macrosomic infants and shoulder dystocia\n[22] and not to preterm birth. Our result emphasizes the importance of gentle maneuvers and avoiding application of excessive pressure or traction on the brachial plexus also when delivering the preterm infant, especially by VE.\nThe major strengths of this study were the large study population covering all preterm deliveries in Sweden during a period of twelve years, and the high quality of the registers, making it possible to analyze rare diagnoses and unusual events such as ICH in preterm infants delivered by VE. We were able to include data on risk factors, potential confounders, and outcomes collected independently from one another and without involving the study subjects, thus minimizing various types of bias (e.g., selection and recall bias). Moreover, antenatal and obstetric care is free of charge in Sweden, management routines as well as GA determinations are standardized, and 99% of births are delivered in public hospitals. This minimizes the risk for confounding by unmeasured socio-demographic factors. Another advantage was the inclusion of the main indications for VE and CS, enabling us to address the question of confounding by indication.\nA major limitation of this study is that the registers do not contain detailed information about many important factors during the VE deliveries. For instance the registry does not provide specific information about the type of VE instrument used, level, position, and attitude of the fetal head in the pelvis when applying VE, location of placement of the vacuum cup, traction work, skill of the obstetrician, pressure exposure (duration and force), and cup detachments. The register does not provide information about confounders such as use of oxytocin and application of fundal pressure.\nThere is a general recommendation not to use VE before a GA of 34 weeks. According to the Royal College of Obstetricians and Gynecologists, there is insufficient evidence to establish the safety on VE deliveries in gestations between 34 weeks + 0 days and 36 weeks + 0 days\n[9]. Our results show that the use of VE is related to rare, but serious complications also between gestational weeks 34–36.\n\nConclusion:\nThe rates of serious birth injuries and complications are generally low, but preterm infants delivered by VE have higher odds ratios for intra- and extracranial hemorrhages and brachial plexus injuries than those delivered by CS during labor or by unassisted vaginal delivery. We therefore support a continued conservative/cautious use of VE in preterm deliveries. Furthermore, the possible causal relationship between mode of delivery and ICH needs to be further investigated.\n\nAbbreviations:\nBMI: Body mass index; CI: Confidence interval; CS: Cesarean section; CT: Computerized tomography; EA: Epidural analgesia; ECH: Extracranial hemorrhage; EEG: Electroencephalography; GA: Gestational age; ICH: Intracranial hemorrhage; LGA: Large for gestational age; OR: Odds ratio; SGA: Small for gestational age; VD: Vaginal delivery; VE: Vacuum extraction.\n\nCompeting interests:\nThe authors declare that they have no competing interest.\n\nAuthors’ contributions:\nCE had the idea for the study, designed it and carried out the statistical analysis. KÅ participated in the design and wrote the first draft of the manuscript together with CE. MN contributed to the interpretation of the results and writing of the manuscript. All authors approved the final version of the submitted article.\n\nPre-publication history:\nThe pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1471-2393/14/42/prepub\n\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nVery few studies have investigated the neonatal outcomes after vacuum extraction delivery (VE) in the preterm period and the results of these studies are inconclusive. The objective of this study was to describe the use of VE for preterm delivery in Sweden and to compare rates of neonatal complications after preterm delivery by VE to those found after cesarean section during labor (CS) or unassisted vaginal delivery (VD).\n\nMethods:\nData was obtained from Swedish national registers. In a population-based cohort from 1999 to 2010, all live-born, singleton preterm infants in a non-breech presentation at birth, born after onset of labor (either spontaneously, by induction, or by rupture of membranes) by VD, CS, or VE were included, leaving a study population of 40,764 infants. Logistic regression analyses were used to calculate adjusted odds ratios (AOR), using unassisted vaginal delivery as reference group.\n\nResults:\nVE was used in 5.7% of the preterm deliveries, with lower rates in earlier gestations. Overall, intracranial hemorrhage (ICH) occurred in 1.51%, extracranial hemorrhage (ECH) in 0.64%, and brachial plexus injury in 0.13% of infants. Infants delivered by VE had higher risks for ICH (AOR = 1.84 (95% CI: 1.09-3.12)), ECH (AOR = 4.48 (95% CI: 2.84-7.07)) and brachial plexus injury (AOR = 6.21 (95% CI: 2.22-17.4)), while infants delivered by CS during labor had no increased risk for these complications, as compared to VD.\n\nConclusions:\nWhile rates of neonatal complications after VE are generally low, higher odds ratios for intra- and extracranial hemorrhages and brachial plexus injuries after VE, compared with other modes of delivery, support a continued cautious use of VE for preterm delivery."},"background_conclusion_text":{"kind":"string","value":"Background:\nPreterm birth is common\n[1] but still, the optimal mode of delivery of preterm infants is not known. Although neonatal outcomes in preterm infants delivered vaginally or by cesarean section (CS)\n[2-5] have been compared, there is no evidence to provide clear guidance on the method of choice\n[6]. Given the widespread assumption that assisted vaginal delivery could be harmful for fragile infants that are underweight and preterm, very few studies have addressed the use of vacuum extraction (VE) for preterm birth.\nDelivery by VE is a common obstetrical procedure, and in many countries it has replaced the use of forceps. VE is used to terminate a protracted second stage of labor and as an intervention for fetal or maternal distress. VE requires vertex presentation, a fully dilated cervix and ruptured membranes\n[7]. A cesarean section, on the other hand, can be performed at any stage of labor and does not require prerequisites of this kind.\nMost clinical guidelines do not recommend VE before 34 gestational weeks\n[8-10]. These recommendations are not based on results of randomized controlled trials, but rely on the observation that preterm infants are more likely than term infants to develop ICH, and on extrapolations from studies of term infants showing that VE is associated with an increased risk of ICH and other neonatal complications\n[11-17]. Only three studies have previously investigated the use and outcomes of VE in preterm births. The first was undertaken over 40 years ago and showed increased mortality and morbidity among preterm infants delivered by VE as compared with term infants delivered by VE\n[18]. The second study compared neonatal morbidity in preterm infants delivered vaginally with (n = 61) or without VE (n = 122), and found no differences in neonatal morbidity between the two groups\n[19]. The last study compared VE and forceps for preterm delivery (n = 64)\n[20]; the neonatal outcomes were similar in both groups. The available data are clearly untimely and hampered by limitations in power and, therefore, current knowledge on safety of preterm vacuum-assisted birth is unsatisfactory.\nThe aim of this study was to 1) describe the use of VE and compare it to rates of CS during labor in preterm deliveries in Sweden from 1999–2010, 2) characterize the distribution of perinatal risk factors associated with each mode of delivery, and 3) compare rates of neonatal intra- and extracranial hemorrhages, as well as occurrence of brachial plexus injury after preterm delivery by VE or CS during labor, using unassisted vaginal birth as a reference.\n\nConclusion:\nThe rates of serious birth injuries and complications are generally low, but preterm infants delivered by VE have higher odds ratios for intra- and extracranial hemorrhages and brachial plexus injuries than those delivered by CS during labor or by unassisted vaginal delivery. We therefore support a continued conservative/cautious use of VE in preterm deliveries. Furthermore, the possible causal relationship between mode of delivery and ICH needs to be further investigated."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nVery few studies have investigated the neonatal outcomes after vacuum extraction delivery (VE) in the preterm period and the results of these studies are inconclusive. The objective of this study was to describe the use of VE for preterm delivery in Sweden and to compare rates of neonatal complications after preterm delivery by VE to those found after cesarean section during labor (CS) or unassisted vaginal delivery (VD).\n\nMethods:\nData was obtained from Swedish national registers. In a population-based cohort from 1999 to 2010, all live-born, singleton preterm infants in a non-breech presentation at birth, born after onset of labor (either spontaneously, by induction, or by rupture of membranes) by VD, CS, or VE were included, leaving a study population of 40,764 infants. Logistic regression analyses were used to calculate adjusted odds ratios (AOR), using unassisted vaginal delivery as reference group.\n\nResults:\nVE was used in 5.7% of the preterm deliveries, with lower rates in earlier gestations. Overall, intracranial hemorrhage (ICH) occurred in 1.51%, extracranial hemorrhage (ECH) in 0.64%, and brachial plexus injury in 0.13% of infants. Infants delivered by VE had higher risks for ICH (AOR = 1.84 (95% CI: 1.09-3.12)), ECH (AOR = 4.48 (95% CI: 2.84-7.07)) and brachial plexus injury (AOR = 6.21 (95% CI: 2.22-17.4)), while infants delivered by CS during labor had no increased risk for these complications, as compared to VD.\n\nConclusions:\nWhile rates of neonatal complications after VE are generally low, higher odds ratios for intra- and extracranial hemorrhages and brachial plexus injuries after VE, compared with other modes of delivery, support a continued cautious use of VE for preterm delivery."},"whole_article_text_length":{"kind":"number","value":9636,"string":"9,636"},"whole_article_abstract_length":{"kind":"number","value":359,"string":"359"},"other_sections_lengths":{"kind":"list like","value":[509,607,499,130,233,282,759,74,10,59,16],"string":"[\n 509,\n 607,\n 499,\n 130,\n 233,\n 282,\n 759,\n 74,\n 10,\n 59,\n 16\n]","isConversational":false},"num_sections":{"kind":"number","value":15,"string":"15"},"most_frequent_words":{"kind":"list like","value":["ve","preterm","infants","delivery","weeks","preterm infants","cs","labor","gestational","ich"],"string":"[\n \"ve\",\n \"preterm\",\n \"infants\",\n \"delivery\",\n \"weeks\",\n \"preterm infants\",\n \"cs\",\n \"labor\",\n \"gestational\",\n \"ich\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["infants mode delivery","vaginal delivery ve","preterm infants delivered","preterm birth delivery","vacuum assisted birth"],"string":"[\n \"infants mode delivery\",\n \"vaginal delivery ve\",\n \"preterm infants delivered\",\n \"preterm birth delivery\",\n \"vacuum assisted birth\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Mode of delivery | Preterm delivery | Intracranial hemorrhage | Extracranial hemorrhage | Brachial plexus injury [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Mode of delivery | Preterm delivery | Intracranial hemorrhage | Extracranial hemorrhage | Brachial plexus injury [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Mode of delivery | Preterm delivery | Intracranial hemorrhage | Extracranial hemorrhage | Brachial plexus injury [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Mode of delivery | Preterm delivery | Intracranial hemorrhage | Extracranial hemorrhage | Brachial plexus injury [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Mode of delivery | Preterm delivery | Intracranial hemorrhage | Extracranial hemorrhage | Brachial plexus injury [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Mode of delivery | Preterm delivery | Intracranial hemorrhage | Extracranial hemorrhage | Brachial plexus injury [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Birth Injuries | Brachial Plexus | Cesarean Section | Cohort Studies | Female | Gestational Age | Humans | Infant, Newborn | Infant, Premature | Intracranial Hemorrhage, Traumatic | Labor, Obstetric | Parturition | Pregnancy | Premature Birth | Registries | Scalp | Seizures | Sweden | Vacuum Extraction, Obstetrical | Young Adult [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Birth Injuries | Brachial Plexus | Cesarean Section | Cohort Studies | Female | Gestational Age | Humans | Infant, Newborn | Infant, Premature | Intracranial Hemorrhage, Traumatic | Labor, Obstetric | Parturition | Pregnancy | Premature Birth | Registries | Scalp | Seizures | Sweden | Vacuum Extraction, Obstetrical | Young Adult [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Birth Injuries | Brachial Plexus | Cesarean Section | Cohort Studies | Female | Gestational Age | Humans | Infant, Newborn | Infant, Premature | Intracranial Hemorrhage, Traumatic | Labor, Obstetric | Parturition | Pregnancy | Premature Birth | Registries | Scalp | Seizures | Sweden | Vacuum Extraction, Obstetrical | Young Adult [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Birth Injuries | Brachial Plexus | Cesarean Section | Cohort Studies | Female | Gestational Age | Humans | Infant, Newborn | Infant, Premature | Intracranial Hemorrhage, Traumatic | Labor, Obstetric | Parturition | Pregnancy | Premature Birth | Registries | Scalp | Seizures | Sweden | Vacuum Extraction, Obstetrical | Young Adult [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Birth Injuries | Brachial Plexus | Cesarean Section | Cohort Studies | Female | Gestational Age | Humans | Infant, Newborn | Infant, Premature | Intracranial Hemorrhage, Traumatic | Labor, Obstetric | Parturition | Pregnancy | Premature Birth | Registries | Scalp | Seizures | Sweden | Vacuum Extraction, Obstetrical | Young Adult [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Birth Injuries | Brachial Plexus | Cesarean Section | Cohort Studies | Female | Gestational Age | Humans | Infant, Newborn | Infant, Premature | Intracranial Hemorrhage, Traumatic | Labor, Obstetric | Parturition | Pregnancy | Premature Birth | Registries | Scalp | Seizures | Sweden | Vacuum Extraction, Obstetrical | Young Adult [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] infants mode delivery | vaginal delivery ve | preterm infants delivered | preterm birth delivery | vacuum assisted birth [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] infants mode delivery | vaginal delivery ve | preterm infants delivered | preterm birth delivery | vacuum assisted birth [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] infants mode delivery | vaginal delivery ve | preterm infants delivered | preterm birth delivery | vacuum assisted birth [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] infants mode delivery | vaginal delivery ve | preterm infants delivered | preterm birth delivery | vacuum assisted birth [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] infants mode delivery | vaginal delivery ve | preterm infants delivered | preterm birth delivery | vacuum assisted birth [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] infants mode delivery | vaginal delivery ve | preterm infants delivered | preterm birth delivery | vacuum assisted birth [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ve | preterm | infants | delivery | weeks | preterm infants | cs | labor | gestational | ich [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] ve | preterm | infants | delivery | weeks | preterm infants | cs | labor | gestational | ich [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ve | preterm | infants | delivery | weeks | preterm infants | cs | labor | gestational | ich [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ve | preterm | infants | delivery | weeks | preterm infants | cs | labor | gestational | ich [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ve | preterm | infants | delivery | weeks | preterm infants | cs | labor | gestational | ich [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ve | preterm | infants | delivery | weeks | preterm infants | cs | labor | gestational | ich [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ve | preterm | infants | neonatal | morbidity | term infants | compared | studies | use | delivery [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] preterm | delivery | grams | neonatal | labor | weeks | performed | m2 | kg | kg m2 [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ve | infants | preterm | preterm infants | cs | weeks | represents | line | line represents | gestational [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] injuries | low preterm | injuries delivered cs labor | deliveries furthermore possible causal | plexus injuries delivered cs | plexus injuries delivered | ve preterm deliveries furthermore | ve preterm deliveries | injuries complications | injuries complications generally [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ve | preterm | infants | delivery | labor | cs | preterm infants | weeks | gestational | delivered [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ve | preterm | infants | delivery | labor | cs | preterm infants | weeks | gestational | delivered [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] VE ||| VE | Sweden | VE | CS [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Swedish ||| 1999 | 2010 | VD | CS | VE | 40,764 ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] VE | 5.7% ||| 1.51% | 0.64% | 0.13% ||| VE | 1.84 | 95% | CI | 1.09-3.12 | ECH | 4.48 | 95% | CI | 2.84-7.07 | 6.21 | 95% | CI | 2.22 | CS | VD [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] VE | VE | VE [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] VE ||| VE | Sweden | VE | CS ||| Swedish ||| 1999 | 2010 | VD | CS | VE | 40,764 ||| ||| VE | 5.7% ||| 1.51% | 0.64% | 0.13% ||| VE | 1.84 | 95% | CI | 1.09-3.12 | ECH | 4.48 | 95% | CI | 2.84-7.07 | 6.21 | 95% | CI | 2.22 | CS | VD ||| VE | VE | VE [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] VE ||| VE | Sweden | VE | CS ||| Swedish ||| 1999 | 2010 | VD | CS | VE | 40,764 ||| ||| VE | 5.7% ||| 1.51% | 0.64% | 0.13% ||| VE | 1.84 | 95% | CI | 1.09-3.12 | ECH | 4.48 | 95% | CI | 2.84-7.07 | 6.21 | 95% | CI | 2.22 | CS | VD ||| VE | VE | VE [SUMMARY]"}}},{"rowIdx":76,"cells":{"title":{"kind":"string","value":"Positive detection of exfoliated colon cancer cells on linear stapler cartridges was associated with depth of tumor invasion and preoperative bowel preparation in colon cancer."},"pmid":{"kind":"string","value":"27577701"},"background_abstract":{"kind":"string","value":"The aim of this study was to investigate exfoliated cancer cells (ECCs) on linear stapler cartridges used for anastomotic sites in colon cancer."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We prospectively analyzed ECCs on linear stapler cartridges used for anastomosis in 100 colon cancer patients who underwent colectomy. Having completed the functional end-to-end anastomosis, the linear stapler cartridges were irrigated with saline, which was collected for cytological examination and cytological diagnoses were made by board-certified pathologists based on Papanicolaou staining."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The detection rate of ECCs on the linear stapler cartridges was 20 %. Positive detection of ECCs was significantly associated with depth of tumor invasion (p = 0.012) and preoperative bowel preparation (p = 0.003). There were no marked differences between ECC-positive and ECC-negative groups in terms of the operation methods, tumor location, histopathological classification, and surgical margins."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Since ECCs were identified on the cartridge of the linear stapler used for anastomosis, preoperative mechanical bowel preparation using polyethylene glycol solution and cleansing at anastomotic sites using tumoricidal agents before anastomosis may be necessary to decrease ECCs in advanced colon cancer."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Aged","Aged, 80 and over","Anastomosis, Surgical","Colectomy","Colon","Colonic Neoplasms","Enema","Female","Humans","Laxatives","Male","Margins of Excision","Middle Aged","Neoplasm Invasiveness","Neoplasm Staging","Polyethylene Glycols","Preoperative Care","Prospective Studies","Surgical Staplers","Surgical Stapling"],"string":"[\n \"Aged\",\n \"Aged, 80 and over\",\n \"Anastomosis, Surgical\",\n \"Colectomy\",\n \"Colon\",\n \"Colonic Neoplasms\",\n \"Enema\",\n \"Female\",\n \"Humans\",\n \"Laxatives\",\n \"Male\",\n \"Margins of Excision\",\n \"Middle Aged\",\n \"Neoplasm Invasiveness\",\n \"Neoplasm Staging\",\n \"Polyethylene Glycols\",\n \"Preoperative Care\",\n \"Prospective Studies\",\n \"Surgical Staplers\",\n \"Surgical Stapling\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"5006528"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"The cause of suture-line recurrence following curative colorectal cancer surgery is believed to be the presence of exfoliated cancer cells (ECCs) at the anastomotic site [1]. Many studies of suture-line recurrence have been conducted in rectal cancer surgery; however, a greater margin is used for colon cancer than for rectal cancer cases, so that suture-line recurrence is less common in colon cancer patients [2]. As a result, few studies have reported on the presence of ECCs in the colonic lumen of colon cancer patients. However, even in patients with colon cancer, the incidence of suture-line recurrence has been reported to be 0.8–5.9 % [2, 3]. Reconstruction following colon cancer surgery commonly involves the fabrication of a functional end-to-end anastomosis [4] using a linear stapler, as a convenient procedure [5]. In the present study, ECCs in the colonic lumen of colon cancer patients were examined based on the cytological examination of the solutions washing the linear stapler used for functional end-to-end anastomosis, and we further analyzed the relationship between positive detection of ECCs and clinicopathological factors and prognosis."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Cytological analysis The cytological analysis results were as follows: class I, n = 17; class II, n = 61; class III, n = 2; class IV, n = 7; and class V, n = 13 (Table 1). Based on the pathological diagnosis, all the patients were assigned to the positive group (n = 20) and the negative group (n = 80).Table 1Cytology from a cartridge of a staplerClass I17 cases (17 %)Class II61 (61 %)Class III2 (2 %)Class IV7 (7 %)Class V13 (13 %)\nCytology from a cartridge of a stapler\nThe cytological analysis results were as follows: class I, n = 17; class II, n = 61; class III, n = 2; class IV, n = 7; and class V, n = 13 (Table 1). Based on the pathological diagnosis, all the patients were assigned to the positive group (n = 20) and the negative group (n = 80).Table 1Cytology from a cartridge of a staplerClass I17 cases (17 %)Class II61 (61 %)Class III2 (2 %)Class IV7 (7 %)Class V13 (13 %)\nCytology from a cartridge of a stapler\n The relationship between the status of ECCs and clinicopathological factors All positive cases had advanced cancers that had invaded beyond the submucosal layer (T2–T4), while all of the early cancers that had invaded the submucosal layer (Tis, T1) were in the negative group (p = 0.012) (Table 2). Regarding preoperative bowel preparation, the detection rate of ECCs in cases where full-laxative mechanical cleansing with PEG was performed was significantly lower than that in cases where full-laxative mechanical cleansing was not possible (p = 0.003) (Table 2). The two groups did not differ significantly in terms of the operation method, tumor location, histopathological classification, and margin from tumor (Table 2).Table 2Comparison of clinicopathological factors in the ECC-positive and ECC-negative groupsECC-positive groupECC-negative group\np value(n = 20)(n = 80)Age67.3 ± 12.0 years old72.0 ± 9.6 years oldN.SGender M843N.S F1237Operation method Laparoscopic surgery10 (50 %)51 (63.8 %)N.S Open surgery10 (50 %)29 (36.2 %)Tumor location Cecum4 (20 %)13 (16.3 %)N.S Ascending colon9 (45 %)32 (40 %) Transverse colon4 (20 %)24 (30 %) Descending colon0 (0 %)2 (2.5 %) Sigmoid colon3 (15 %)9 (11.2 %)Depth of tumor invasion pTis0 (0 %)8 (10 %)0.012 pT10 (0 %)16 (20 %)(Tis, T1 vs. T2–T4) pT22 (10 %)5 (6.2 %) pT316 (80 %)31 (38.8 %) pT42 (10 %)20 (25 %)Tumor size (mean ± SE)58.6 ± 16.7 mm49.2 ± 32.6 mm0.0239Lymph node metastasis pN(−)1348N.S pN(+)732Distant metastasis pM0112N.S pM11968cStage 007N.S I218 II1021 III721 IV113Histopathological classification Well838N.S Moderately933 Poorly16 Others23Margin from tumor (mean ± SE) Oral side16.7 ± 2.5 cm14.6 ± 1.1 cmN.S Anal side12.3 ± 1.5 cm12.8 ± 1.0 cmN.SPreoperative bowel preparation PEG8 (40 %)60 (75 %)0.003 Others12 (60 %)20 (25 %)\nComparison of clinicopathological factors in the ECC-positive and ECC-negative groups\nAll positive cases had advanced cancers that had invaded beyond the submucosal layer (T2–T4), while all of the early cancers that had invaded the submucosal layer (Tis, T1) were in the negative group (p = 0.012) (Table 2). Regarding preoperative bowel preparation, the detection rate of ECCs in cases where full-laxative mechanical cleansing with PEG was performed was significantly lower than that in cases where full-laxative mechanical cleansing was not possible (p = 0.003) (Table 2). The two groups did not differ significantly in terms of the operation method, tumor location, histopathological classification, and margin from tumor (Table 2).Table 2Comparison of clinicopathological factors in the ECC-positive and ECC-negative groupsECC-positive groupECC-negative group\np value(n = 20)(n = 80)Age67.3 ± 12.0 years old72.0 ± 9.6 years oldN.SGender M843N.S F1237Operation method Laparoscopic surgery10 (50 %)51 (63.8 %)N.S Open surgery10 (50 %)29 (36.2 %)Tumor location Cecum4 (20 %)13 (16.3 %)N.S Ascending colon9 (45 %)32 (40 %) Transverse colon4 (20 %)24 (30 %) Descending colon0 (0 %)2 (2.5 %) Sigmoid colon3 (15 %)9 (11.2 %)Depth of tumor invasion pTis0 (0 %)8 (10 %)0.012 pT10 (0 %)16 (20 %)(Tis, T1 vs. T2–T4) pT22 (10 %)5 (6.2 %) pT316 (80 %)31 (38.8 %) pT42 (10 %)20 (25 %)Tumor size (mean ± SE)58.6 ± 16.7 mm49.2 ± 32.6 mm0.0239Lymph node metastasis pN(−)1348N.S pN(+)732Distant metastasis pM0112N.S pM11968cStage 007N.S I218 II1021 III721 IV113Histopathological classification Well838N.S Moderately933 Poorly16 Others23Margin from tumor (mean ± SE) Oral side16.7 ± 2.5 cm14.6 ± 1.1 cmN.S Anal side12.3 ± 1.5 cm12.8 ± 1.0 cmN.SPreoperative bowel preparation PEG8 (40 %)60 (75 %)0.003 Others12 (60 %)20 (25 %)\nComparison of clinicopathological factors in the ECC-positive and ECC-negative groups\n Follow-up outcome Among all the cases, 81 were followed by colonoscopy after surgery. Follow-up colonoscopy was performed in 74 patients (74 %) (observation range 3.9–121.4 months; median observation period 64.1 months), and none were found to have developed suture-line recurrence.\nAmong all the cases, 81 were followed by colonoscopy after surgery. Follow-up colonoscopy was performed in 74 patients (74 %) (observation range 3.9–121.4 months; median observation period 64.1 months), and none were found to have developed suture-line recurrence."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"We found ECCs on the cartridge of the linear stapler used for anastomosis in 20 % of the colon cancer cases we analyzed. Most of the positive ECCs were identified in advanced colon cancer without PEG. Therefore, preoperative mechanical bowel preparation using PEG and cleansing at anastomotic sites using tumoricidal agents before anastomosis may contribute to decrease ECCs in advanced colon cancer."},"other_sections_titles":{"kind":"list like","value":["Patients","Preoperative bowel preparation","Cytology procedures","Evaluation of data","Statistical analysis","Cytological analysis","The relationship between the status of ECCs and clinicopathological factors","Follow-up outcome"],"string":"[\n \"Patients\",\n \"Preoperative bowel preparation\",\n \"Cytology procedures\",\n \"Evaluation of data\",\n \"Statistical analysis\",\n \"Cytological analysis\",\n \"The relationship between the status of ECCs and clinicopathological factors\",\n \"Follow-up outcome\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["The study subjects were 100 consecutive patients who underwent laparoscopic (n = 61) or open (n = 39) colectomy for colon cancer at our hospital, in whom a functional end-to-end anastomosis using a linear stapler was performed. After completing the anastomosis in each case, the cartridge of the linear stapler was washed with saline, which was collected for cytological examination. Of the 100 patients examined, the localization of tumor was as follows: cecum 17 cases, ascending colon 41, transverse colon 28, descending colon 2, and sigmoid colon 12. The depth of tumor invasion was pTis 8 cases, pT1 16, pT2 7, pT3 47, and pT4 22. The histological diagnosis was well-differentiated adenocarcinoma 46 cases, moderately 42, poorly 7, and others 5. All patients were followed up 5 years or more after surgery, or until metastasis was developed or death. Written informed consent was obtained from all patients, after an explanation of the design and aim of this study.","Full-laxative cleansing via polyethylene glycol solution (PEG) was routinely used; however, for patients with stenosis, other preparation methods (sodium picosulfate or enema) were employed.","The procedure for the cytology of the washing samples collected from the stapler cartridges was as follows. After tumor resection, the intestinal lumen, distal and proximal to the anastomotic site, was cleaned five times using cotton balls soaked in either 5 % povidone-iodine or 0.025 % benzalkonium chloride. Afterwards, a functional end-to-end anastomosis was performed. The stapler cartridge (GIA 80-3.8, Covidien, MA, USA) used for anastomosis was washed using 100 ml of physiological saline, and the washing samples were promptly subjected to cytological analysis.","Cytological diagnoses were made by board-certified pathologists based on Papanicolaou staining. Classes I, II, and III were included in the negative group, while classes IV and V were included in the positive group. Clinicopathological factors, including age, gender, operation methods (laparoscopic or open surgery), tumor location, depth of tumor invasion, tumor size, lymph node metastasis, distant metastasis, stage classification, histopathological classification, margin from tumor, and preoperative bowel preparation, and clinical outcome were compared between the two groups.","In the present study, the χ2 test (Fisher’s exact test) was used to compare frequencies between the two groups, and the Mann-Whitney U test was used to compare intergroup differences. The level of significance was set at p < 0.05.","The cytological analysis results were as follows: class I, n = 17; class II, n = 61; class III, n = 2; class IV, n = 7; and class V, n = 13 (Table 1). Based on the pathological diagnosis, all the patients were assigned to the positive group (n = 20) and the negative group (n = 80).Table 1Cytology from a cartridge of a staplerClass I17 cases (17 %)Class II61 (61 %)Class III2 (2 %)Class IV7 (7 %)Class V13 (13 %)\nCytology from a cartridge of a stapler","All positive cases had advanced cancers that had invaded beyond the submucosal layer (T2–T4), while all of the early cancers that had invaded the submucosal layer (Tis, T1) were in the negative group (p = 0.012) (Table 2). Regarding preoperative bowel preparation, the detection rate of ECCs in cases where full-laxative mechanical cleansing with PEG was performed was significantly lower than that in cases where full-laxative mechanical cleansing was not possible (p = 0.003) (Table 2). The two groups did not differ significantly in terms of the operation method, tumor location, histopathological classification, and margin from tumor (Table 2).Table 2Comparison of clinicopathological factors in the ECC-positive and ECC-negative groupsECC-positive groupECC-negative group\np value(n = 20)(n = 80)Age67.3 ± 12.0 years old72.0 ± 9.6 years oldN.SGender M843N.S F1237Operation method Laparoscopic surgery10 (50 %)51 (63.8 %)N.S Open surgery10 (50 %)29 (36.2 %)Tumor location Cecum4 (20 %)13 (16.3 %)N.S Ascending colon9 (45 %)32 (40 %) Transverse colon4 (20 %)24 (30 %) Descending colon0 (0 %)2 (2.5 %) Sigmoid colon3 (15 %)9 (11.2 %)Depth of tumor invasion pTis0 (0 %)8 (10 %)0.012 pT10 (0 %)16 (20 %)(Tis, T1 vs. T2–T4) pT22 (10 %)5 (6.2 %) pT316 (80 %)31 (38.8 %) pT42 (10 %)20 (25 %)Tumor size (mean ± SE)58.6 ± 16.7 mm49.2 ± 32.6 mm0.0239Lymph node metastasis pN(−)1348N.S pN(+)732Distant metastasis pM0112N.S pM11968cStage 007N.S I218 II1021 III721 IV113Histopathological classification Well838N.S Moderately933 Poorly16 Others23Margin from tumor (mean ± SE) Oral side16.7 ± 2.5 cm14.6 ± 1.1 cmN.S Anal side12.3 ± 1.5 cm12.8 ± 1.0 cmN.SPreoperative bowel preparation PEG8 (40 %)60 (75 %)0.003 Others12 (60 %)20 (25 %)\nComparison of clinicopathological factors in the ECC-positive and ECC-negative groups","Among all the cases, 81 were followed by colonoscopy after surgery. Follow-up colonoscopy was performed in 74 patients (74 %) (observation range 3.9–121.4 months; median observation period 64.1 months), and none were found to have developed suture-line recurrence."],"string":"[\n \"The study subjects were 100 consecutive patients who underwent laparoscopic (n = 61) or open (n = 39) colectomy for colon cancer at our hospital, in whom a functional end-to-end anastomosis using a linear stapler was performed. After completing the anastomosis in each case, the cartridge of the linear stapler was washed with saline, which was collected for cytological examination. Of the 100 patients examined, the localization of tumor was as follows: cecum 17 cases, ascending colon 41, transverse colon 28, descending colon 2, and sigmoid colon 12. The depth of tumor invasion was pTis 8 cases, pT1 16, pT2 7, pT3 47, and pT4 22. The histological diagnosis was well-differentiated adenocarcinoma 46 cases, moderately 42, poorly 7, and others 5. All patients were followed up 5 years or more after surgery, or until metastasis was developed or death. Written informed consent was obtained from all patients, after an explanation of the design and aim of this study.\",\n \"Full-laxative cleansing via polyethylene glycol solution (PEG) was routinely used; however, for patients with stenosis, other preparation methods (sodium picosulfate or enema) were employed.\",\n \"The procedure for the cytology of the washing samples collected from the stapler cartridges was as follows. After tumor resection, the intestinal lumen, distal and proximal to the anastomotic site, was cleaned five times using cotton balls soaked in either 5 % povidone-iodine or 0.025 % benzalkonium chloride. Afterwards, a functional end-to-end anastomosis was performed. The stapler cartridge (GIA 80-3.8, Covidien, MA, USA) used for anastomosis was washed using 100 ml of physiological saline, and the washing samples were promptly subjected to cytological analysis.\",\n \"Cytological diagnoses were made by board-certified pathologists based on Papanicolaou staining. Classes I, II, and III were included in the negative group, while classes IV and V were included in the positive group. Clinicopathological factors, including age, gender, operation methods (laparoscopic or open surgery), tumor location, depth of tumor invasion, tumor size, lymph node metastasis, distant metastasis, stage classification, histopathological classification, margin from tumor, and preoperative bowel preparation, and clinical outcome were compared between the two groups.\",\n \"In the present study, the χ2 test (Fisher’s exact test) was used to compare frequencies between the two groups, and the Mann-Whitney U test was used to compare intergroup differences. The level of significance was set at p < 0.05.\",\n \"The cytological analysis results were as follows: class I, n = 17; class II, n = 61; class III, n = 2; class IV, n = 7; and class V, n = 13 (Table 1). Based on the pathological diagnosis, all the patients were assigned to the positive group (n = 20) and the negative group (n = 80).Table 1Cytology from a cartridge of a staplerClass I17 cases (17 %)Class II61 (61 %)Class III2 (2 %)Class IV7 (7 %)Class V13 (13 %)\\nCytology from a cartridge of a stapler\",\n \"All positive cases had advanced cancers that had invaded beyond the submucosal layer (T2–T4), while all of the early cancers that had invaded the submucosal layer (Tis, T1) were in the negative group (p = 0.012) (Table 2). Regarding preoperative bowel preparation, the detection rate of ECCs in cases where full-laxative mechanical cleansing with PEG was performed was significantly lower than that in cases where full-laxative mechanical cleansing was not possible (p = 0.003) (Table 2). The two groups did not differ significantly in terms of the operation method, tumor location, histopathological classification, and margin from tumor (Table 2).Table 2Comparison of clinicopathological factors in the ECC-positive and ECC-negative groupsECC-positive groupECC-negative group\\np value(n = 20)(n = 80)Age67.3 ± 12.0 years old72.0 ± 9.6 years oldN.SGender M843N.S F1237Operation method Laparoscopic surgery10 (50 %)51 (63.8 %)N.S Open surgery10 (50 %)29 (36.2 %)Tumor location Cecum4 (20 %)13 (16.3 %)N.S Ascending colon9 (45 %)32 (40 %) Transverse colon4 (20 %)24 (30 %) Descending colon0 (0 %)2 (2.5 %) Sigmoid colon3 (15 %)9 (11.2 %)Depth of tumor invasion pTis0 (0 %)8 (10 %)0.012 pT10 (0 %)16 (20 %)(Tis, T1 vs. T2–T4) pT22 (10 %)5 (6.2 %) pT316 (80 %)31 (38.8 %) pT42 (10 %)20 (25 %)Tumor size (mean ± SE)58.6 ± 16.7 mm49.2 ± 32.6 mm0.0239Lymph node metastasis pN(−)1348N.S pN(+)732Distant metastasis pM0112N.S pM11968cStage 007N.S I218 II1021 III721 IV113Histopathological classification Well838N.S Moderately933 Poorly16 Others23Margin from tumor (mean ± SE) Oral side16.7 ± 2.5 cm14.6 ± 1.1 cmN.S Anal side12.3 ± 1.5 cm12.8 ± 1.0 cmN.SPreoperative bowel preparation PEG8 (40 %)60 (75 %)0.003 Others12 (60 %)20 (25 %)\\nComparison of clinicopathological factors in the ECC-positive and ECC-negative groups\",\n \"Among all the cases, 81 were followed by colonoscopy after surgery. Follow-up colonoscopy was performed in 74 patients (74 %) (observation range 3.9–121.4 months; median observation period 64.1 months), and none were found to have developed suture-line recurrence.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Patients","Preoperative bowel preparation","Cytology procedures","Evaluation of data","Statistical analysis","Results","Cytological analysis","The relationship between the status of ECCs and clinicopathological factors","Follow-up outcome","Discussion","Conclusions"],"string":"[\n \"Background\",\n \"Methods\",\n \"Patients\",\n \"Preoperative bowel preparation\",\n \"Cytology procedures\",\n \"Evaluation of data\",\n \"Statistical analysis\",\n \"Results\",\n \"Cytological analysis\",\n \"The relationship between the status of ECCs and clinicopathological factors\",\n \"Follow-up outcome\",\n \"Discussion\",\n \"Conclusions\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["The cause of suture-line recurrence following curative colorectal cancer surgery is believed to be the presence of exfoliated cancer cells (ECCs) at the anastomotic site [1]. Many studies of suture-line recurrence have been conducted in rectal cancer surgery; however, a greater margin is used for colon cancer than for rectal cancer cases, so that suture-line recurrence is less common in colon cancer patients [2]. As a result, few studies have reported on the presence of ECCs in the colonic lumen of colon cancer patients. However, even in patients with colon cancer, the incidence of suture-line recurrence has been reported to be 0.8–5.9 % [2, 3]. Reconstruction following colon cancer surgery commonly involves the fabrication of a functional end-to-end anastomosis [4] using a linear stapler, as a convenient procedure [5]. In the present study, ECCs in the colonic lumen of colon cancer patients were examined based on the cytological examination of the solutions washing the linear stapler used for functional end-to-end anastomosis, and we further analyzed the relationship between positive detection of ECCs and clinicopathological factors and prognosis."," Patients The study subjects were 100 consecutive patients who underwent laparoscopic (n = 61) or open (n = 39) colectomy for colon cancer at our hospital, in whom a functional end-to-end anastomosis using a linear stapler was performed. After completing the anastomosis in each case, the cartridge of the linear stapler was washed with saline, which was collected for cytological examination. Of the 100 patients examined, the localization of tumor was as follows: cecum 17 cases, ascending colon 41, transverse colon 28, descending colon 2, and sigmoid colon 12. The depth of tumor invasion was pTis 8 cases, pT1 16, pT2 7, pT3 47, and pT4 22. The histological diagnosis was well-differentiated adenocarcinoma 46 cases, moderately 42, poorly 7, and others 5. All patients were followed up 5 years or more after surgery, or until metastasis was developed or death. Written informed consent was obtained from all patients, after an explanation of the design and aim of this study.\nThe study subjects were 100 consecutive patients who underwent laparoscopic (n = 61) or open (n = 39) colectomy for colon cancer at our hospital, in whom a functional end-to-end anastomosis using a linear stapler was performed. After completing the anastomosis in each case, the cartridge of the linear stapler was washed with saline, which was collected for cytological examination. Of the 100 patients examined, the localization of tumor was as follows: cecum 17 cases, ascending colon 41, transverse colon 28, descending colon 2, and sigmoid colon 12. The depth of tumor invasion was pTis 8 cases, pT1 16, pT2 7, pT3 47, and pT4 22. The histological diagnosis was well-differentiated adenocarcinoma 46 cases, moderately 42, poorly 7, and others 5. All patients were followed up 5 years or more after surgery, or until metastasis was developed or death. Written informed consent was obtained from all patients, after an explanation of the design and aim of this study.\n Preoperative bowel preparation Full-laxative cleansing via polyethylene glycol solution (PEG) was routinely used; however, for patients with stenosis, other preparation methods (sodium picosulfate or enema) were employed.\nFull-laxative cleansing via polyethylene glycol solution (PEG) was routinely used; however, for patients with stenosis, other preparation methods (sodium picosulfate or enema) were employed.\n Cytology procedures The procedure for the cytology of the washing samples collected from the stapler cartridges was as follows. After tumor resection, the intestinal lumen, distal and proximal to the anastomotic site, was cleaned five times using cotton balls soaked in either 5 % povidone-iodine or 0.025 % benzalkonium chloride. Afterwards, a functional end-to-end anastomosis was performed. The stapler cartridge (GIA 80-3.8, Covidien, MA, USA) used for anastomosis was washed using 100 ml of physiological saline, and the washing samples were promptly subjected to cytological analysis.\nThe procedure for the cytology of the washing samples collected from the stapler cartridges was as follows. After tumor resection, the intestinal lumen, distal and proximal to the anastomotic site, was cleaned five times using cotton balls soaked in either 5 % povidone-iodine or 0.025 % benzalkonium chloride. Afterwards, a functional end-to-end anastomosis was performed. The stapler cartridge (GIA 80-3.8, Covidien, MA, USA) used for anastomosis was washed using 100 ml of physiological saline, and the washing samples were promptly subjected to cytological analysis.\n Evaluation of data Cytological diagnoses were made by board-certified pathologists based on Papanicolaou staining. Classes I, II, and III were included in the negative group, while classes IV and V were included in the positive group. Clinicopathological factors, including age, gender, operation methods (laparoscopic or open surgery), tumor location, depth of tumor invasion, tumor size, lymph node metastasis, distant metastasis, stage classification, histopathological classification, margin from tumor, and preoperative bowel preparation, and clinical outcome were compared between the two groups.\nCytological diagnoses were made by board-certified pathologists based on Papanicolaou staining. Classes I, II, and III were included in the negative group, while classes IV and V were included in the positive group. Clinicopathological factors, including age, gender, operation methods (laparoscopic or open surgery), tumor location, depth of tumor invasion, tumor size, lymph node metastasis, distant metastasis, stage classification, histopathological classification, margin from tumor, and preoperative bowel preparation, and clinical outcome were compared between the two groups.\n Statistical analysis In the present study, the χ2 test (Fisher’s exact test) was used to compare frequencies between the two groups, and the Mann-Whitney U test was used to compare intergroup differences. The level of significance was set at p < 0.05.\nIn the present study, the χ2 test (Fisher’s exact test) was used to compare frequencies between the two groups, and the Mann-Whitney U test was used to compare intergroup differences. The level of significance was set at p < 0.05.","The study subjects were 100 consecutive patients who underwent laparoscopic (n = 61) or open (n = 39) colectomy for colon cancer at our hospital, in whom a functional end-to-end anastomosis using a linear stapler was performed. After completing the anastomosis in each case, the cartridge of the linear stapler was washed with saline, which was collected for cytological examination. Of the 100 patients examined, the localization of tumor was as follows: cecum 17 cases, ascending colon 41, transverse colon 28, descending colon 2, and sigmoid colon 12. The depth of tumor invasion was pTis 8 cases, pT1 16, pT2 7, pT3 47, and pT4 22. The histological diagnosis was well-differentiated adenocarcinoma 46 cases, moderately 42, poorly 7, and others 5. All patients were followed up 5 years or more after surgery, or until metastasis was developed or death. Written informed consent was obtained from all patients, after an explanation of the design and aim of this study.","Full-laxative cleansing via polyethylene glycol solution (PEG) was routinely used; however, for patients with stenosis, other preparation methods (sodium picosulfate or enema) were employed.","The procedure for the cytology of the washing samples collected from the stapler cartridges was as follows. After tumor resection, the intestinal lumen, distal and proximal to the anastomotic site, was cleaned five times using cotton balls soaked in either 5 % povidone-iodine or 0.025 % benzalkonium chloride. Afterwards, a functional end-to-end anastomosis was performed. The stapler cartridge (GIA 80-3.8, Covidien, MA, USA) used for anastomosis was washed using 100 ml of physiological saline, and the washing samples were promptly subjected to cytological analysis.","Cytological diagnoses were made by board-certified pathologists based on Papanicolaou staining. Classes I, II, and III were included in the negative group, while classes IV and V were included in the positive group. Clinicopathological factors, including age, gender, operation methods (laparoscopic or open surgery), tumor location, depth of tumor invasion, tumor size, lymph node metastasis, distant metastasis, stage classification, histopathological classification, margin from tumor, and preoperative bowel preparation, and clinical outcome were compared between the two groups.","In the present study, the χ2 test (Fisher’s exact test) was used to compare frequencies between the two groups, and the Mann-Whitney U test was used to compare intergroup differences. The level of significance was set at p < 0.05."," Cytological analysis The cytological analysis results were as follows: class I, n = 17; class II, n = 61; class III, n = 2; class IV, n = 7; and class V, n = 13 (Table 1). Based on the pathological diagnosis, all the patients were assigned to the positive group (n = 20) and the negative group (n = 80).Table 1Cytology from a cartridge of a staplerClass I17 cases (17 %)Class II61 (61 %)Class III2 (2 %)Class IV7 (7 %)Class V13 (13 %)\nCytology from a cartridge of a stapler\nThe cytological analysis results were as follows: class I, n = 17; class II, n = 61; class III, n = 2; class IV, n = 7; and class V, n = 13 (Table 1). Based on the pathological diagnosis, all the patients were assigned to the positive group (n = 20) and the negative group (n = 80).Table 1Cytology from a cartridge of a staplerClass I17 cases (17 %)Class II61 (61 %)Class III2 (2 %)Class IV7 (7 %)Class V13 (13 %)\nCytology from a cartridge of a stapler\n The relationship between the status of ECCs and clinicopathological factors All positive cases had advanced cancers that had invaded beyond the submucosal layer (T2–T4), while all of the early cancers that had invaded the submucosal layer (Tis, T1) were in the negative group (p = 0.012) (Table 2). Regarding preoperative bowel preparation, the detection rate of ECCs in cases where full-laxative mechanical cleansing with PEG was performed was significantly lower than that in cases where full-laxative mechanical cleansing was not possible (p = 0.003) (Table 2). The two groups did not differ significantly in terms of the operation method, tumor location, histopathological classification, and margin from tumor (Table 2).Table 2Comparison of clinicopathological factors in the ECC-positive and ECC-negative groupsECC-positive groupECC-negative group\np value(n = 20)(n = 80)Age67.3 ± 12.0 years old72.0 ± 9.6 years oldN.SGender M843N.S F1237Operation method Laparoscopic surgery10 (50 %)51 (63.8 %)N.S Open surgery10 (50 %)29 (36.2 %)Tumor location Cecum4 (20 %)13 (16.3 %)N.S Ascending colon9 (45 %)32 (40 %) Transverse colon4 (20 %)24 (30 %) Descending colon0 (0 %)2 (2.5 %) Sigmoid colon3 (15 %)9 (11.2 %)Depth of tumor invasion pTis0 (0 %)8 (10 %)0.012 pT10 (0 %)16 (20 %)(Tis, T1 vs. T2–T4) pT22 (10 %)5 (6.2 %) pT316 (80 %)31 (38.8 %) pT42 (10 %)20 (25 %)Tumor size (mean ± SE)58.6 ± 16.7 mm49.2 ± 32.6 mm0.0239Lymph node metastasis pN(−)1348N.S pN(+)732Distant metastasis pM0112N.S pM11968cStage 007N.S I218 II1021 III721 IV113Histopathological classification Well838N.S Moderately933 Poorly16 Others23Margin from tumor (mean ± SE) Oral side16.7 ± 2.5 cm14.6 ± 1.1 cmN.S Anal side12.3 ± 1.5 cm12.8 ± 1.0 cmN.SPreoperative bowel preparation PEG8 (40 %)60 (75 %)0.003 Others12 (60 %)20 (25 %)\nComparison of clinicopathological factors in the ECC-positive and ECC-negative groups\nAll positive cases had advanced cancers that had invaded beyond the submucosal layer (T2–T4), while all of the early cancers that had invaded the submucosal layer (Tis, T1) were in the negative group (p = 0.012) (Table 2). Regarding preoperative bowel preparation, the detection rate of ECCs in cases where full-laxative mechanical cleansing with PEG was performed was significantly lower than that in cases where full-laxative mechanical cleansing was not possible (p = 0.003) (Table 2). The two groups did not differ significantly in terms of the operation method, tumor location, histopathological classification, and margin from tumor (Table 2).Table 2Comparison of clinicopathological factors in the ECC-positive and ECC-negative groupsECC-positive groupECC-negative group\np value(n = 20)(n = 80)Age67.3 ± 12.0 years old72.0 ± 9.6 years oldN.SGender M843N.S F1237Operation method Laparoscopic surgery10 (50 %)51 (63.8 %)N.S Open surgery10 (50 %)29 (36.2 %)Tumor location Cecum4 (20 %)13 (16.3 %)N.S Ascending colon9 (45 %)32 (40 %) Transverse colon4 (20 %)24 (30 %) Descending colon0 (0 %)2 (2.5 %) Sigmoid colon3 (15 %)9 (11.2 %)Depth of tumor invasion pTis0 (0 %)8 (10 %)0.012 pT10 (0 %)16 (20 %)(Tis, T1 vs. T2–T4) pT22 (10 %)5 (6.2 %) pT316 (80 %)31 (38.8 %) pT42 (10 %)20 (25 %)Tumor size (mean ± SE)58.6 ± 16.7 mm49.2 ± 32.6 mm0.0239Lymph node metastasis pN(−)1348N.S pN(+)732Distant metastasis pM0112N.S pM11968cStage 007N.S I218 II1021 III721 IV113Histopathological classification Well838N.S Moderately933 Poorly16 Others23Margin from tumor (mean ± SE) Oral side16.7 ± 2.5 cm14.6 ± 1.1 cmN.S Anal side12.3 ± 1.5 cm12.8 ± 1.0 cmN.SPreoperative bowel preparation PEG8 (40 %)60 (75 %)0.003 Others12 (60 %)20 (25 %)\nComparison of clinicopathological factors in the ECC-positive and ECC-negative groups\n Follow-up outcome Among all the cases, 81 were followed by colonoscopy after surgery. Follow-up colonoscopy was performed in 74 patients (74 %) (observation range 3.9–121.4 months; median observation period 64.1 months), and none were found to have developed suture-line recurrence.\nAmong all the cases, 81 were followed by colonoscopy after surgery. Follow-up colonoscopy was performed in 74 patients (74 %) (observation range 3.9–121.4 months; median observation period 64.1 months), and none were found to have developed suture-line recurrence.","The cytological analysis results were as follows: class I, n = 17; class II, n = 61; class III, n = 2; class IV, n = 7; and class V, n = 13 (Table 1). Based on the pathological diagnosis, all the patients were assigned to the positive group (n = 20) and the negative group (n = 80).Table 1Cytology from a cartridge of a staplerClass I17 cases (17 %)Class II61 (61 %)Class III2 (2 %)Class IV7 (7 %)Class V13 (13 %)\nCytology from a cartridge of a stapler","All positive cases had advanced cancers that had invaded beyond the submucosal layer (T2–T4), while all of the early cancers that had invaded the submucosal layer (Tis, T1) were in the negative group (p = 0.012) (Table 2). Regarding preoperative bowel preparation, the detection rate of ECCs in cases where full-laxative mechanical cleansing with PEG was performed was significantly lower than that in cases where full-laxative mechanical cleansing was not possible (p = 0.003) (Table 2). The two groups did not differ significantly in terms of the operation method, tumor location, histopathological classification, and margin from tumor (Table 2).Table 2Comparison of clinicopathological factors in the ECC-positive and ECC-negative groupsECC-positive groupECC-negative group\np value(n = 20)(n = 80)Age67.3 ± 12.0 years old72.0 ± 9.6 years oldN.SGender M843N.S F1237Operation method Laparoscopic surgery10 (50 %)51 (63.8 %)N.S Open surgery10 (50 %)29 (36.2 %)Tumor location Cecum4 (20 %)13 (16.3 %)N.S Ascending colon9 (45 %)32 (40 %) Transverse colon4 (20 %)24 (30 %) Descending colon0 (0 %)2 (2.5 %) Sigmoid colon3 (15 %)9 (11.2 %)Depth of tumor invasion pTis0 (0 %)8 (10 %)0.012 pT10 (0 %)16 (20 %)(Tis, T1 vs. T2–T4) pT22 (10 %)5 (6.2 %) pT316 (80 %)31 (38.8 %) pT42 (10 %)20 (25 %)Tumor size (mean ± SE)58.6 ± 16.7 mm49.2 ± 32.6 mm0.0239Lymph node metastasis pN(−)1348N.S pN(+)732Distant metastasis pM0112N.S pM11968cStage 007N.S I218 II1021 III721 IV113Histopathological classification Well838N.S Moderately933 Poorly16 Others23Margin from tumor (mean ± SE) Oral side16.7 ± 2.5 cm14.6 ± 1.1 cmN.S Anal side12.3 ± 1.5 cm12.8 ± 1.0 cmN.SPreoperative bowel preparation PEG8 (40 %)60 (75 %)0.003 Others12 (60 %)20 (25 %)\nComparison of clinicopathological factors in the ECC-positive and ECC-negative groups","Among all the cases, 81 were followed by colonoscopy after surgery. Follow-up colonoscopy was performed in 74 patients (74 %) (observation range 3.9–121.4 months; median observation period 64.1 months), and none were found to have developed suture-line recurrence.","We found that ECC-positive cases were recognized at 20 % on the stapler cartridges used for anastomosis in patients with colon cancer. According to the relationship between the status of ECCs and clinicopathological factors, the presence of ECCs was associated with depth of tumor invasion and preoperative bowel preparation. Our study included 86 patients with right-sided colon cancer in 100 colon cancer patients. Hasegawa et al. [6] reported that 2 (11.1 %) and 10 (55.6 %) of 18 patients, who underwent right hemicolectomy for right-sided colon cancer, had ECCs at the terminal ileum and distal colon anastomosis sites, respectively. They demonstrated that surgical bowel occlusion on both sides of the tumor before resection improved a decrease in the number of ECCs. Recently, Maeda et al. [7] have shown that ECCs were detected at distal colon and proximal colon of the tumor within each 3 cm in almost patients with sigmoid cancer, and ECCs decreased with distance from the tumor. They also suggested that bowel ligatures could decrease ECCs, leading to prevent local recurrence. These findings indicated that the no-touch isolation technique might influence the number of ECCs as previous reports suggested [8, 9]. In the present study, we performed colectomy according to the no-touch isolation technique. Consequently, the positive rate of ECCs was 20 %. The difference of positive rate might be explained by the operative technique in addition to other factors, including cleansing methods and bowel preparation methods.\nMost studies of ECCs in the colonic lumen of colorectal cancer patients have involved rectal cancer patients. The incidence of suture-line recurrence has been reported to be 11–18 % [10, 11] in rectal cancer, while that in colon cancer has been reported to be 0.8–5.9 % [2, 3]. The possibility that ECCs can be implanted into freshly cut tissues was first postulated by Sir Ryall in 1907 [12]. This hypothesis was later advanced to explain some cases of suture-line recurrence following resection of colorectal cancer. In order to demonstrate the potential involvement of ECCs in the colonic lumen with suture-line and local recurrences in an animal model, Fermor et al. [13] injected ECCs collected from the colonic lumen of 17 colorectal cancer patients into the caudal vein of immunocompromised mice and showed that six mice developed lung metastases. Several cases of suture-line recurrence following resection of colon cancer, especially sigmoid colon, have been reported in Japan [14–17]. They discussed that implantation was considered to be the most important factor in terms of the cause of recurrence at anastomotic sites. The frequency of suture-line recurrence in colon cancer was very low though ECCs were frequently detected in colonic lumen. Indeed, we detected ECCs in 20 % of the colon cancer cases we analyzed. However, no suture-line recurrence was observed in our series of colon cancer for 5-year observation after colectomy. The cell viability of ECCs was investigated to understand etiological mechanism of anastomotic recurrence. Rosenberg et al. [18] reported the presence of no viable cells, as assessed by trypan blue exclusion. However, Umpleby et al. [19], using trypan blue exclusion and hydrolysis of fluorescein diacetate, showed that 70 % of patients with colorectal cancer had viable cells exfoliated into the bowel lumen. In general, other factors, which are associated with the potential of implantation, must be required even if ECCs still have viability.\nIn the present study, we found that the positive rate of ECCs was 11.8 % in patients with PEG, while it was 37.5 % in patients without PEG. Mechanical bowel preparation using PEG may be effective for decreasing the number of ECCs. Previous reports [6, 7] also suggested same findings. Regarding mechanical bowel preparation using PEG, Slim et al. [20] have described that anastomotic leakage was significantly found after mechanical bowel preparation (odds ratio 1.75; p = 0.032) and concluded that mechanical bowel preparation using PEG should be omitted before elective colorectal surgery. Therefore, mechanical bowel preparation is routinely not performed in patients undergoing elective colon cancer because of employing “enhanced recovery after surgery” (ERAS) protocol widely. Recently, clinical practice guidelines [21] from the USA recommend the use of both mechanical bowel preparation and oral antibiotic bowel preparation. Many surgeons still conduct mechanical bowel preparation using PEG in the USA [22] and Japan [23]. Mechanical bowel preparation using PEG might contribute to decrease positive ECCs in advanced colorectal cancer, leading to decrease the possibility of anastomotic recurrence in addition to prevention of anastomotic leakage and surgical site infection.\nIn a previous study [5], the outcome of reconstruction following colon cancer surgery was compared between hand-sewn and stapled anastomosis. While the safety was comparable, it was radiographically confirmed that suture leakage was more common with hand-sewn anastomoses. Hence, functional end-to-end anastomosis has primarily been performed using a stapler. In the present study, the washing samples of the cartridge of the linear stapler used for functional end-to-end anastomosis were examined cytologically. The rationale for using the washing samples of the cartridge in this study was that they would better reflect the presence of ECCs than the washing solutions of the colonic lumen. In addition, the presence of ECCs on the cartridge was considered to be a cause of suture-line recurrence. Prior to the present study, ECCs had been cytologically identified in the physiological saline used to irrigate the colonic lumen. In the present study, 5 % povidone-iodine was first used as a tumoricidal agent to irrigate the colonic lumen prior to anastomosis [24, 25]. However, since povidone-iodine should not be used in the abdominal cavity due to severe damage of normal mucosa, we have employed 0.025 % benzalkonium chloride, as an alternative. Therefore, the ECC detection rate might be lower in the present study than that reported by Umpleby, et al. [19] who used normal saline for bowel irrigation.\nOur results indicated that the detection rate of ECCs, which could be detected at anastomotic sites even on the stapler cartridges, might be increased in advanced colon cancer with more than T2 and in elective colon cancer patients without mechanical bowel preparation using PEG, leading to increase the possibility of incidence of suture-line recurrence. To prevent the possibility of incidence of suture-line recurrence, we should conduct preoperative mechanical bowel preparation using PEG, perform surgical operation by the no-touch isolation technique, and irrigate the colonic lumen prior to anastomosis using 0.025 % benzalkonium chloride as a tumoricidal agent.","We found ECCs on the cartridge of the linear stapler used for anastomosis in 20 % of the colon cancer cases we analyzed. Most of the positive ECCs were identified in advanced colon cancer without PEG. Therefore, preoperative mechanical bowel preparation using PEG and cleansing at anastomotic sites using tumoricidal agents before anastomosis may contribute to decrease ECCs in advanced colon cancer."],"string":"[\n \"The cause of suture-line recurrence following curative colorectal cancer surgery is believed to be the presence of exfoliated cancer cells (ECCs) at the anastomotic site [1]. Many studies of suture-line recurrence have been conducted in rectal cancer surgery; however, a greater margin is used for colon cancer than for rectal cancer cases, so that suture-line recurrence is less common in colon cancer patients [2]. As a result, few studies have reported on the presence of ECCs in the colonic lumen of colon cancer patients. However, even in patients with colon cancer, the incidence of suture-line recurrence has been reported to be 0.8–5.9 % [2, 3]. Reconstruction following colon cancer surgery commonly involves the fabrication of a functional end-to-end anastomosis [4] using a linear stapler, as a convenient procedure [5]. In the present study, ECCs in the colonic lumen of colon cancer patients were examined based on the cytological examination of the solutions washing the linear stapler used for functional end-to-end anastomosis, and we further analyzed the relationship between positive detection of ECCs and clinicopathological factors and prognosis.\",\n \" Patients The study subjects were 100 consecutive patients who underwent laparoscopic (n = 61) or open (n = 39) colectomy for colon cancer at our hospital, in whom a functional end-to-end anastomosis using a linear stapler was performed. After completing the anastomosis in each case, the cartridge of the linear stapler was washed with saline, which was collected for cytological examination. Of the 100 patients examined, the localization of tumor was as follows: cecum 17 cases, ascending colon 41, transverse colon 28, descending colon 2, and sigmoid colon 12. The depth of tumor invasion was pTis 8 cases, pT1 16, pT2 7, pT3 47, and pT4 22. The histological diagnosis was well-differentiated adenocarcinoma 46 cases, moderately 42, poorly 7, and others 5. All patients were followed up 5 years or more after surgery, or until metastasis was developed or death. Written informed consent was obtained from all patients, after an explanation of the design and aim of this study.\\nThe study subjects were 100 consecutive patients who underwent laparoscopic (n = 61) or open (n = 39) colectomy for colon cancer at our hospital, in whom a functional end-to-end anastomosis using a linear stapler was performed. After completing the anastomosis in each case, the cartridge of the linear stapler was washed with saline, which was collected for cytological examination. Of the 100 patients examined, the localization of tumor was as follows: cecum 17 cases, ascending colon 41, transverse colon 28, descending colon 2, and sigmoid colon 12. The depth of tumor invasion was pTis 8 cases, pT1 16, pT2 7, pT3 47, and pT4 22. The histological diagnosis was well-differentiated adenocarcinoma 46 cases, moderately 42, poorly 7, and others 5. All patients were followed up 5 years or more after surgery, or until metastasis was developed or death. Written informed consent was obtained from all patients, after an explanation of the design and aim of this study.\\n Preoperative bowel preparation Full-laxative cleansing via polyethylene glycol solution (PEG) was routinely used; however, for patients with stenosis, other preparation methods (sodium picosulfate or enema) were employed.\\nFull-laxative cleansing via polyethylene glycol solution (PEG) was routinely used; however, for patients with stenosis, other preparation methods (sodium picosulfate or enema) were employed.\\n Cytology procedures The procedure for the cytology of the washing samples collected from the stapler cartridges was as follows. After tumor resection, the intestinal lumen, distal and proximal to the anastomotic site, was cleaned five times using cotton balls soaked in either 5 % povidone-iodine or 0.025 % benzalkonium chloride. Afterwards, a functional end-to-end anastomosis was performed. The stapler cartridge (GIA 80-3.8, Covidien, MA, USA) used for anastomosis was washed using 100 ml of physiological saline, and the washing samples were promptly subjected to cytological analysis.\\nThe procedure for the cytology of the washing samples collected from the stapler cartridges was as follows. After tumor resection, the intestinal lumen, distal and proximal to the anastomotic site, was cleaned five times using cotton balls soaked in either 5 % povidone-iodine or 0.025 % benzalkonium chloride. Afterwards, a functional end-to-end anastomosis was performed. The stapler cartridge (GIA 80-3.8, Covidien, MA, USA) used for anastomosis was washed using 100 ml of physiological saline, and the washing samples were promptly subjected to cytological analysis.\\n Evaluation of data Cytological diagnoses were made by board-certified pathologists based on Papanicolaou staining. Classes I, II, and III were included in the negative group, while classes IV and V were included in the positive group. Clinicopathological factors, including age, gender, operation methods (laparoscopic or open surgery), tumor location, depth of tumor invasion, tumor size, lymph node metastasis, distant metastasis, stage classification, histopathological classification, margin from tumor, and preoperative bowel preparation, and clinical outcome were compared between the two groups.\\nCytological diagnoses were made by board-certified pathologists based on Papanicolaou staining. Classes I, II, and III were included in the negative group, while classes IV and V were included in the positive group. Clinicopathological factors, including age, gender, operation methods (laparoscopic or open surgery), tumor location, depth of tumor invasion, tumor size, lymph node metastasis, distant metastasis, stage classification, histopathological classification, margin from tumor, and preoperative bowel preparation, and clinical outcome were compared between the two groups.\\n Statistical analysis In the present study, the χ2 test (Fisher’s exact test) was used to compare frequencies between the two groups, and the Mann-Whitney U test was used to compare intergroup differences. The level of significance was set at p < 0.05.\\nIn the present study, the χ2 test (Fisher’s exact test) was used to compare frequencies between the two groups, and the Mann-Whitney U test was used to compare intergroup differences. The level of significance was set at p < 0.05.\",\n \"The study subjects were 100 consecutive patients who underwent laparoscopic (n = 61) or open (n = 39) colectomy for colon cancer at our hospital, in whom a functional end-to-end anastomosis using a linear stapler was performed. After completing the anastomosis in each case, the cartridge of the linear stapler was washed with saline, which was collected for cytological examination. Of the 100 patients examined, the localization of tumor was as follows: cecum 17 cases, ascending colon 41, transverse colon 28, descending colon 2, and sigmoid colon 12. The depth of tumor invasion was pTis 8 cases, pT1 16, pT2 7, pT3 47, and pT4 22. The histological diagnosis was well-differentiated adenocarcinoma 46 cases, moderately 42, poorly 7, and others 5. All patients were followed up 5 years or more after surgery, or until metastasis was developed or death. Written informed consent was obtained from all patients, after an explanation of the design and aim of this study.\",\n \"Full-laxative cleansing via polyethylene glycol solution (PEG) was routinely used; however, for patients with stenosis, other preparation methods (sodium picosulfate or enema) were employed.\",\n \"The procedure for the cytology of the washing samples collected from the stapler cartridges was as follows. After tumor resection, the intestinal lumen, distal and proximal to the anastomotic site, was cleaned five times using cotton balls soaked in either 5 % povidone-iodine or 0.025 % benzalkonium chloride. Afterwards, a functional end-to-end anastomosis was performed. The stapler cartridge (GIA 80-3.8, Covidien, MA, USA) used for anastomosis was washed using 100 ml of physiological saline, and the washing samples were promptly subjected to cytological analysis.\",\n \"Cytological diagnoses were made by board-certified pathologists based on Papanicolaou staining. Classes I, II, and III were included in the negative group, while classes IV and V were included in the positive group. Clinicopathological factors, including age, gender, operation methods (laparoscopic or open surgery), tumor location, depth of tumor invasion, tumor size, lymph node metastasis, distant metastasis, stage classification, histopathological classification, margin from tumor, and preoperative bowel preparation, and clinical outcome were compared between the two groups.\",\n \"In the present study, the χ2 test (Fisher’s exact test) was used to compare frequencies between the two groups, and the Mann-Whitney U test was used to compare intergroup differences. The level of significance was set at p < 0.05.\",\n \" Cytological analysis The cytological analysis results were as follows: class I, n = 17; class II, n = 61; class III, n = 2; class IV, n = 7; and class V, n = 13 (Table 1). Based on the pathological diagnosis, all the patients were assigned to the positive group (n = 20) and the negative group (n = 80).Table 1Cytology from a cartridge of a staplerClass I17 cases (17 %)Class II61 (61 %)Class III2 (2 %)Class IV7 (7 %)Class V13 (13 %)\\nCytology from a cartridge of a stapler\\nThe cytological analysis results were as follows: class I, n = 17; class II, n = 61; class III, n = 2; class IV, n = 7; and class V, n = 13 (Table 1). Based on the pathological diagnosis, all the patients were assigned to the positive group (n = 20) and the negative group (n = 80).Table 1Cytology from a cartridge of a staplerClass I17 cases (17 %)Class II61 (61 %)Class III2 (2 %)Class IV7 (7 %)Class V13 (13 %)\\nCytology from a cartridge of a stapler\\n The relationship between the status of ECCs and clinicopathological factors All positive cases had advanced cancers that had invaded beyond the submucosal layer (T2–T4), while all of the early cancers that had invaded the submucosal layer (Tis, T1) were in the negative group (p = 0.012) (Table 2). Regarding preoperative bowel preparation, the detection rate of ECCs in cases where full-laxative mechanical cleansing with PEG was performed was significantly lower than that in cases where full-laxative mechanical cleansing was not possible (p = 0.003) (Table 2). The two groups did not differ significantly in terms of the operation method, tumor location, histopathological classification, and margin from tumor (Table 2).Table 2Comparison of clinicopathological factors in the ECC-positive and ECC-negative groupsECC-positive groupECC-negative group\\np value(n = 20)(n = 80)Age67.3 ± 12.0 years old72.0 ± 9.6 years oldN.SGender M843N.S F1237Operation method Laparoscopic surgery10 (50 %)51 (63.8 %)N.S Open surgery10 (50 %)29 (36.2 %)Tumor location Cecum4 (20 %)13 (16.3 %)N.S Ascending colon9 (45 %)32 (40 %) Transverse colon4 (20 %)24 (30 %) Descending colon0 (0 %)2 (2.5 %) Sigmoid colon3 (15 %)9 (11.2 %)Depth of tumor invasion pTis0 (0 %)8 (10 %)0.012 pT10 (0 %)16 (20 %)(Tis, T1 vs. T2–T4) pT22 (10 %)5 (6.2 %) pT316 (80 %)31 (38.8 %) pT42 (10 %)20 (25 %)Tumor size (mean ± SE)58.6 ± 16.7 mm49.2 ± 32.6 mm0.0239Lymph node metastasis pN(−)1348N.S pN(+)732Distant metastasis pM0112N.S pM11968cStage 007N.S I218 II1021 III721 IV113Histopathological classification Well838N.S Moderately933 Poorly16 Others23Margin from tumor (mean ± SE) Oral side16.7 ± 2.5 cm14.6 ± 1.1 cmN.S Anal side12.3 ± 1.5 cm12.8 ± 1.0 cmN.SPreoperative bowel preparation PEG8 (40 %)60 (75 %)0.003 Others12 (60 %)20 (25 %)\\nComparison of clinicopathological factors in the ECC-positive and ECC-negative groups\\nAll positive cases had advanced cancers that had invaded beyond the submucosal layer (T2–T4), while all of the early cancers that had invaded the submucosal layer (Tis, T1) were in the negative group (p = 0.012) (Table 2). Regarding preoperative bowel preparation, the detection rate of ECCs in cases where full-laxative mechanical cleansing with PEG was performed was significantly lower than that in cases where full-laxative mechanical cleansing was not possible (p = 0.003) (Table 2). The two groups did not differ significantly in terms of the operation method, tumor location, histopathological classification, and margin from tumor (Table 2).Table 2Comparison of clinicopathological factors in the ECC-positive and ECC-negative groupsECC-positive groupECC-negative group\\np value(n = 20)(n = 80)Age67.3 ± 12.0 years old72.0 ± 9.6 years oldN.SGender M843N.S F1237Operation method Laparoscopic surgery10 (50 %)51 (63.8 %)N.S Open surgery10 (50 %)29 (36.2 %)Tumor location Cecum4 (20 %)13 (16.3 %)N.S Ascending colon9 (45 %)32 (40 %) Transverse colon4 (20 %)24 (30 %) Descending colon0 (0 %)2 (2.5 %) Sigmoid colon3 (15 %)9 (11.2 %)Depth of tumor invasion pTis0 (0 %)8 (10 %)0.012 pT10 (0 %)16 (20 %)(Tis, T1 vs. T2–T4) pT22 (10 %)5 (6.2 %) pT316 (80 %)31 (38.8 %) pT42 (10 %)20 (25 %)Tumor size (mean ± SE)58.6 ± 16.7 mm49.2 ± 32.6 mm0.0239Lymph node metastasis pN(−)1348N.S pN(+)732Distant metastasis pM0112N.S pM11968cStage 007N.S I218 II1021 III721 IV113Histopathological classification Well838N.S Moderately933 Poorly16 Others23Margin from tumor (mean ± SE) Oral side16.7 ± 2.5 cm14.6 ± 1.1 cmN.S Anal side12.3 ± 1.5 cm12.8 ± 1.0 cmN.SPreoperative bowel preparation PEG8 (40 %)60 (75 %)0.003 Others12 (60 %)20 (25 %)\\nComparison of clinicopathological factors in the ECC-positive and ECC-negative groups\\n Follow-up outcome Among all the cases, 81 were followed by colonoscopy after surgery. Follow-up colonoscopy was performed in 74 patients (74 %) (observation range 3.9–121.4 months; median observation period 64.1 months), and none were found to have developed suture-line recurrence.\\nAmong all the cases, 81 were followed by colonoscopy after surgery. Follow-up colonoscopy was performed in 74 patients (74 %) (observation range 3.9–121.4 months; median observation period 64.1 months), and none were found to have developed suture-line recurrence.\",\n \"The cytological analysis results were as follows: class I, n = 17; class II, n = 61; class III, n = 2; class IV, n = 7; and class V, n = 13 (Table 1). Based on the pathological diagnosis, all the patients were assigned to the positive group (n = 20) and the negative group (n = 80).Table 1Cytology from a cartridge of a staplerClass I17 cases (17 %)Class II61 (61 %)Class III2 (2 %)Class IV7 (7 %)Class V13 (13 %)\\nCytology from a cartridge of a stapler\",\n \"All positive cases had advanced cancers that had invaded beyond the submucosal layer (T2–T4), while all of the early cancers that had invaded the submucosal layer (Tis, T1) were in the negative group (p = 0.012) (Table 2). Regarding preoperative bowel preparation, the detection rate of ECCs in cases where full-laxative mechanical cleansing with PEG was performed was significantly lower than that in cases where full-laxative mechanical cleansing was not possible (p = 0.003) (Table 2). The two groups did not differ significantly in terms of the operation method, tumor location, histopathological classification, and margin from tumor (Table 2).Table 2Comparison of clinicopathological factors in the ECC-positive and ECC-negative groupsECC-positive groupECC-negative group\\np value(n = 20)(n = 80)Age67.3 ± 12.0 years old72.0 ± 9.6 years oldN.SGender M843N.S F1237Operation method Laparoscopic surgery10 (50 %)51 (63.8 %)N.S Open surgery10 (50 %)29 (36.2 %)Tumor location Cecum4 (20 %)13 (16.3 %)N.S Ascending colon9 (45 %)32 (40 %) Transverse colon4 (20 %)24 (30 %) Descending colon0 (0 %)2 (2.5 %) Sigmoid colon3 (15 %)9 (11.2 %)Depth of tumor invasion pTis0 (0 %)8 (10 %)0.012 pT10 (0 %)16 (20 %)(Tis, T1 vs. T2–T4) pT22 (10 %)5 (6.2 %) pT316 (80 %)31 (38.8 %) pT42 (10 %)20 (25 %)Tumor size (mean ± SE)58.6 ± 16.7 mm49.2 ± 32.6 mm0.0239Lymph node metastasis pN(−)1348N.S pN(+)732Distant metastasis pM0112N.S pM11968cStage 007N.S I218 II1021 III721 IV113Histopathological classification Well838N.S Moderately933 Poorly16 Others23Margin from tumor (mean ± SE) Oral side16.7 ± 2.5 cm14.6 ± 1.1 cmN.S Anal side12.3 ± 1.5 cm12.8 ± 1.0 cmN.SPreoperative bowel preparation PEG8 (40 %)60 (75 %)0.003 Others12 (60 %)20 (25 %)\\nComparison of clinicopathological factors in the ECC-positive and ECC-negative groups\",\n \"Among all the cases, 81 were followed by colonoscopy after surgery. Follow-up colonoscopy was performed in 74 patients (74 %) (observation range 3.9–121.4 months; median observation period 64.1 months), and none were found to have developed suture-line recurrence.\",\n \"We found that ECC-positive cases were recognized at 20 % on the stapler cartridges used for anastomosis in patients with colon cancer. According to the relationship between the status of ECCs and clinicopathological factors, the presence of ECCs was associated with depth of tumor invasion and preoperative bowel preparation. Our study included 86 patients with right-sided colon cancer in 100 colon cancer patients. Hasegawa et al. [6] reported that 2 (11.1 %) and 10 (55.6 %) of 18 patients, who underwent right hemicolectomy for right-sided colon cancer, had ECCs at the terminal ileum and distal colon anastomosis sites, respectively. They demonstrated that surgical bowel occlusion on both sides of the tumor before resection improved a decrease in the number of ECCs. Recently, Maeda et al. [7] have shown that ECCs were detected at distal colon and proximal colon of the tumor within each 3 cm in almost patients with sigmoid cancer, and ECCs decreased with distance from the tumor. They also suggested that bowel ligatures could decrease ECCs, leading to prevent local recurrence. These findings indicated that the no-touch isolation technique might influence the number of ECCs as previous reports suggested [8, 9]. In the present study, we performed colectomy according to the no-touch isolation technique. Consequently, the positive rate of ECCs was 20 %. The difference of positive rate might be explained by the operative technique in addition to other factors, including cleansing methods and bowel preparation methods.\\nMost studies of ECCs in the colonic lumen of colorectal cancer patients have involved rectal cancer patients. The incidence of suture-line recurrence has been reported to be 11–18 % [10, 11] in rectal cancer, while that in colon cancer has been reported to be 0.8–5.9 % [2, 3]. The possibility that ECCs can be implanted into freshly cut tissues was first postulated by Sir Ryall in 1907 [12]. This hypothesis was later advanced to explain some cases of suture-line recurrence following resection of colorectal cancer. In order to demonstrate the potential involvement of ECCs in the colonic lumen with suture-line and local recurrences in an animal model, Fermor et al. [13] injected ECCs collected from the colonic lumen of 17 colorectal cancer patients into the caudal vein of immunocompromised mice and showed that six mice developed lung metastases. Several cases of suture-line recurrence following resection of colon cancer, especially sigmoid colon, have been reported in Japan [14–17]. They discussed that implantation was considered to be the most important factor in terms of the cause of recurrence at anastomotic sites. The frequency of suture-line recurrence in colon cancer was very low though ECCs were frequently detected in colonic lumen. Indeed, we detected ECCs in 20 % of the colon cancer cases we analyzed. However, no suture-line recurrence was observed in our series of colon cancer for 5-year observation after colectomy. The cell viability of ECCs was investigated to understand etiological mechanism of anastomotic recurrence. Rosenberg et al. [18] reported the presence of no viable cells, as assessed by trypan blue exclusion. However, Umpleby et al. [19], using trypan blue exclusion and hydrolysis of fluorescein diacetate, showed that 70 % of patients with colorectal cancer had viable cells exfoliated into the bowel lumen. In general, other factors, which are associated with the potential of implantation, must be required even if ECCs still have viability.\\nIn the present study, we found that the positive rate of ECCs was 11.8 % in patients with PEG, while it was 37.5 % in patients without PEG. Mechanical bowel preparation using PEG may be effective for decreasing the number of ECCs. Previous reports [6, 7] also suggested same findings. Regarding mechanical bowel preparation using PEG, Slim et al. [20] have described that anastomotic leakage was significantly found after mechanical bowel preparation (odds ratio 1.75; p = 0.032) and concluded that mechanical bowel preparation using PEG should be omitted before elective colorectal surgery. Therefore, mechanical bowel preparation is routinely not performed in patients undergoing elective colon cancer because of employing “enhanced recovery after surgery” (ERAS) protocol widely. Recently, clinical practice guidelines [21] from the USA recommend the use of both mechanical bowel preparation and oral antibiotic bowel preparation. Many surgeons still conduct mechanical bowel preparation using PEG in the USA [22] and Japan [23]. Mechanical bowel preparation using PEG might contribute to decrease positive ECCs in advanced colorectal cancer, leading to decrease the possibility of anastomotic recurrence in addition to prevention of anastomotic leakage and surgical site infection.\\nIn a previous study [5], the outcome of reconstruction following colon cancer surgery was compared between hand-sewn and stapled anastomosis. While the safety was comparable, it was radiographically confirmed that suture leakage was more common with hand-sewn anastomoses. Hence, functional end-to-end anastomosis has primarily been performed using a stapler. In the present study, the washing samples of the cartridge of the linear stapler used for functional end-to-end anastomosis were examined cytologically. The rationale for using the washing samples of the cartridge in this study was that they would better reflect the presence of ECCs than the washing solutions of the colonic lumen. In addition, the presence of ECCs on the cartridge was considered to be a cause of suture-line recurrence. Prior to the present study, ECCs had been cytologically identified in the physiological saline used to irrigate the colonic lumen. In the present study, 5 % povidone-iodine was first used as a tumoricidal agent to irrigate the colonic lumen prior to anastomosis [24, 25]. However, since povidone-iodine should not be used in the abdominal cavity due to severe damage of normal mucosa, we have employed 0.025 % benzalkonium chloride, as an alternative. Therefore, the ECC detection rate might be lower in the present study than that reported by Umpleby, et al. [19] who used normal saline for bowel irrigation.\\nOur results indicated that the detection rate of ECCs, which could be detected at anastomotic sites even on the stapler cartridges, might be increased in advanced colon cancer with more than T2 and in elective colon cancer patients without mechanical bowel preparation using PEG, leading to increase the possibility of incidence of suture-line recurrence. To prevent the possibility of incidence of suture-line recurrence, we should conduct preoperative mechanical bowel preparation using PEG, perform surgical operation by the no-touch isolation technique, and irrigate the colonic lumen prior to anastomosis using 0.025 % benzalkonium chloride as a tumoricidal agent.\",\n \"We found ECCs on the cartridge of the linear stapler used for anastomosis in 20 % of the colon cancer cases we analyzed. Most of the positive ECCs were identified in advanced colon cancer without PEG. Therefore, preoperative mechanical bowel preparation using PEG and cleansing at anastomotic sites using tumoricidal agents before anastomosis may contribute to decrease ECCs in advanced colon cancer.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["introduction","materials|methods",null,null,null,null,null,"results",null,null,null,"discussion","conclusion"],"string":"[\n \"introduction\",\n \"materials|methods\",\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n \"discussion\",\n \"conclusion\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["Exfoliated cancer cell","Colon cancer","Functional end-to-end anastomosis","Suture-line recurrence"],"string":"[\n \"Exfoliated cancer cell\",\n \"Colon cancer\",\n \"Functional end-to-end anastomosis\",\n \"Suture-line recurrence\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Background:\nThe cause of suture-line recurrence following curative colorectal cancer surgery is believed to be the presence of exfoliated cancer cells (ECCs) at the anastomotic site [1]. Many studies of suture-line recurrence have been conducted in rectal cancer surgery; however, a greater margin is used for colon cancer than for rectal cancer cases, so that suture-line recurrence is less common in colon cancer patients [2]. As a result, few studies have reported on the presence of ECCs in the colonic lumen of colon cancer patients. However, even in patients with colon cancer, the incidence of suture-line recurrence has been reported to be 0.8–5.9 % [2, 3]. Reconstruction following colon cancer surgery commonly involves the fabrication of a functional end-to-end anastomosis [4] using a linear stapler, as a convenient procedure [5]. In the present study, ECCs in the colonic lumen of colon cancer patients were examined based on the cytological examination of the solutions washing the linear stapler used for functional end-to-end anastomosis, and we further analyzed the relationship between positive detection of ECCs and clinicopathological factors and prognosis.\n\nMethods:\n Patients The study subjects were 100 consecutive patients who underwent laparoscopic (n = 61) or open (n = 39) colectomy for colon cancer at our hospital, in whom a functional end-to-end anastomosis using a linear stapler was performed. After completing the anastomosis in each case, the cartridge of the linear stapler was washed with saline, which was collected for cytological examination. Of the 100 patients examined, the localization of tumor was as follows: cecum 17 cases, ascending colon 41, transverse colon 28, descending colon 2, and sigmoid colon 12. The depth of tumor invasion was pTis 8 cases, pT1 16, pT2 7, pT3 47, and pT4 22. The histological diagnosis was well-differentiated adenocarcinoma 46 cases, moderately 42, poorly 7, and others 5. All patients were followed up 5 years or more after surgery, or until metastasis was developed or death. Written informed consent was obtained from all patients, after an explanation of the design and aim of this study.\nThe study subjects were 100 consecutive patients who underwent laparoscopic (n = 61) or open (n = 39) colectomy for colon cancer at our hospital, in whom a functional end-to-end anastomosis using a linear stapler was performed. After completing the anastomosis in each case, the cartridge of the linear stapler was washed with saline, which was collected for cytological examination. Of the 100 patients examined, the localization of tumor was as follows: cecum 17 cases, ascending colon 41, transverse colon 28, descending colon 2, and sigmoid colon 12. The depth of tumor invasion was pTis 8 cases, pT1 16, pT2 7, pT3 47, and pT4 22. The histological diagnosis was well-differentiated adenocarcinoma 46 cases, moderately 42, poorly 7, and others 5. All patients were followed up 5 years or more after surgery, or until metastasis was developed or death. Written informed consent was obtained from all patients, after an explanation of the design and aim of this study.\n Preoperative bowel preparation Full-laxative cleansing via polyethylene glycol solution (PEG) was routinely used; however, for patients with stenosis, other preparation methods (sodium picosulfate or enema) were employed.\nFull-laxative cleansing via polyethylene glycol solution (PEG) was routinely used; however, for patients with stenosis, other preparation methods (sodium picosulfate or enema) were employed.\n Cytology procedures The procedure for the cytology of the washing samples collected from the stapler cartridges was as follows. After tumor resection, the intestinal lumen, distal and proximal to the anastomotic site, was cleaned five times using cotton balls soaked in either 5 % povidone-iodine or 0.025 % benzalkonium chloride. Afterwards, a functional end-to-end anastomosis was performed. The stapler cartridge (GIA 80-3.8, Covidien, MA, USA) used for anastomosis was washed using 100 ml of physiological saline, and the washing samples were promptly subjected to cytological analysis.\nThe procedure for the cytology of the washing samples collected from the stapler cartridges was as follows. After tumor resection, the intestinal lumen, distal and proximal to the anastomotic site, was cleaned five times using cotton balls soaked in either 5 % povidone-iodine or 0.025 % benzalkonium chloride. Afterwards, a functional end-to-end anastomosis was performed. The stapler cartridge (GIA 80-3.8, Covidien, MA, USA) used for anastomosis was washed using 100 ml of physiological saline, and the washing samples were promptly subjected to cytological analysis.\n Evaluation of data Cytological diagnoses were made by board-certified pathologists based on Papanicolaou staining. Classes I, II, and III were included in the negative group, while classes IV and V were included in the positive group. Clinicopathological factors, including age, gender, operation methods (laparoscopic or open surgery), tumor location, depth of tumor invasion, tumor size, lymph node metastasis, distant metastasis, stage classification, histopathological classification, margin from tumor, and preoperative bowel preparation, and clinical outcome were compared between the two groups.\nCytological diagnoses were made by board-certified pathologists based on Papanicolaou staining. Classes I, II, and III were included in the negative group, while classes IV and V were included in the positive group. Clinicopathological factors, including age, gender, operation methods (laparoscopic or open surgery), tumor location, depth of tumor invasion, tumor size, lymph node metastasis, distant metastasis, stage classification, histopathological classification, margin from tumor, and preoperative bowel preparation, and clinical outcome were compared between the two groups.\n Statistical analysis In the present study, the χ2 test (Fisher’s exact test) was used to compare frequencies between the two groups, and the Mann-Whitney U test was used to compare intergroup differences. The level of significance was set at p < 0.05.\nIn the present study, the χ2 test (Fisher’s exact test) was used to compare frequencies between the two groups, and the Mann-Whitney U test was used to compare intergroup differences. The level of significance was set at p < 0.05.\n\nPatients:\nThe study subjects were 100 consecutive patients who underwent laparoscopic (n = 61) or open (n = 39) colectomy for colon cancer at our hospital, in whom a functional end-to-end anastomosis using a linear stapler was performed. After completing the anastomosis in each case, the cartridge of the linear stapler was washed with saline, which was collected for cytological examination. Of the 100 patients examined, the localization of tumor was as follows: cecum 17 cases, ascending colon 41, transverse colon 28, descending colon 2, and sigmoid colon 12. The depth of tumor invasion was pTis 8 cases, pT1 16, pT2 7, pT3 47, and pT4 22. The histological diagnosis was well-differentiated adenocarcinoma 46 cases, moderately 42, poorly 7, and others 5. All patients were followed up 5 years or more after surgery, or until metastasis was developed or death. Written informed consent was obtained from all patients, after an explanation of the design and aim of this study.\n\nPreoperative bowel preparation:\nFull-laxative cleansing via polyethylene glycol solution (PEG) was routinely used; however, for patients with stenosis, other preparation methods (sodium picosulfate or enema) were employed.\n\nCytology procedures:\nThe procedure for the cytology of the washing samples collected from the stapler cartridges was as follows. After tumor resection, the intestinal lumen, distal and proximal to the anastomotic site, was cleaned five times using cotton balls soaked in either 5 % povidone-iodine or 0.025 % benzalkonium chloride. Afterwards, a functional end-to-end anastomosis was performed. The stapler cartridge (GIA 80-3.8, Covidien, MA, USA) used for anastomosis was washed using 100 ml of physiological saline, and the washing samples were promptly subjected to cytological analysis.\n\nEvaluation of data:\nCytological diagnoses were made by board-certified pathologists based on Papanicolaou staining. Classes I, II, and III were included in the negative group, while classes IV and V were included in the positive group. Clinicopathological factors, including age, gender, operation methods (laparoscopic or open surgery), tumor location, depth of tumor invasion, tumor size, lymph node metastasis, distant metastasis, stage classification, histopathological classification, margin from tumor, and preoperative bowel preparation, and clinical outcome were compared between the two groups.\n\nStatistical analysis:\nIn the present study, the χ2 test (Fisher’s exact test) was used to compare frequencies between the two groups, and the Mann-Whitney U test was used to compare intergroup differences. The level of significance was set at p < 0.05.\n\nResults:\n Cytological analysis The cytological analysis results were as follows: class I, n = 17; class II, n = 61; class III, n = 2; class IV, n = 7; and class V, n = 13 (Table 1). Based on the pathological diagnosis, all the patients were assigned to the positive group (n = 20) and the negative group (n = 80).Table 1Cytology from a cartridge of a staplerClass I17 cases (17 %)Class II61 (61 %)Class III2 (2 %)Class IV7 (7 %)Class V13 (13 %)\nCytology from a cartridge of a stapler\nThe cytological analysis results were as follows: class I, n = 17; class II, n = 61; class III, n = 2; class IV, n = 7; and class V, n = 13 (Table 1). Based on the pathological diagnosis, all the patients were assigned to the positive group (n = 20) and the negative group (n = 80).Table 1Cytology from a cartridge of a staplerClass I17 cases (17 %)Class II61 (61 %)Class III2 (2 %)Class IV7 (7 %)Class V13 (13 %)\nCytology from a cartridge of a stapler\n The relationship between the status of ECCs and clinicopathological factors All positive cases had advanced cancers that had invaded beyond the submucosal layer (T2–T4), while all of the early cancers that had invaded the submucosal layer (Tis, T1) were in the negative group (p = 0.012) (Table 2). Regarding preoperative bowel preparation, the detection rate of ECCs in cases where full-laxative mechanical cleansing with PEG was performed was significantly lower than that in cases where full-laxative mechanical cleansing was not possible (p = 0.003) (Table 2). The two groups did not differ significantly in terms of the operation method, tumor location, histopathological classification, and margin from tumor (Table 2).Table 2Comparison of clinicopathological factors in the ECC-positive and ECC-negative groupsECC-positive groupECC-negative group\np value(n = 20)(n = 80)Age67.3 ± 12.0 years old72.0 ± 9.6 years oldN.SGender M843N.S F1237Operation method Laparoscopic surgery10 (50 %)51 (63.8 %)N.S Open surgery10 (50 %)29 (36.2 %)Tumor location Cecum4 (20 %)13 (16.3 %)N.S Ascending colon9 (45 %)32 (40 %) Transverse colon4 (20 %)24 (30 %) Descending colon0 (0 %)2 (2.5 %) Sigmoid colon3 (15 %)9 (11.2 %)Depth of tumor invasion pTis0 (0 %)8 (10 %)0.012 pT10 (0 %)16 (20 %)(Tis, T1 vs. T2–T4) pT22 (10 %)5 (6.2 %) pT316 (80 %)31 (38.8 %) pT42 (10 %)20 (25 %)Tumor size (mean ± SE)58.6 ± 16.7 mm49.2 ± 32.6 mm0.0239Lymph node metastasis pN(−)1348N.S pN(+)732Distant metastasis pM0112N.S pM11968cStage 007N.S I218 II1021 III721 IV113Histopathological classification Well838N.S Moderately933 Poorly16 Others23Margin from tumor (mean ± SE) Oral side16.7 ± 2.5 cm14.6 ± 1.1 cmN.S Anal side12.3 ± 1.5 cm12.8 ± 1.0 cmN.SPreoperative bowel preparation PEG8 (40 %)60 (75 %)0.003 Others12 (60 %)20 (25 %)\nComparison of clinicopathological factors in the ECC-positive and ECC-negative groups\nAll positive cases had advanced cancers that had invaded beyond the submucosal layer (T2–T4), while all of the early cancers that had invaded the submucosal layer (Tis, T1) were in the negative group (p = 0.012) (Table 2). Regarding preoperative bowel preparation, the detection rate of ECCs in cases where full-laxative mechanical cleansing with PEG was performed was significantly lower than that in cases where full-laxative mechanical cleansing was not possible (p = 0.003) (Table 2). The two groups did not differ significantly in terms of the operation method, tumor location, histopathological classification, and margin from tumor (Table 2).Table 2Comparison of clinicopathological factors in the ECC-positive and ECC-negative groupsECC-positive groupECC-negative group\np value(n = 20)(n = 80)Age67.3 ± 12.0 years old72.0 ± 9.6 years oldN.SGender M843N.S F1237Operation method Laparoscopic surgery10 (50 %)51 (63.8 %)N.S Open surgery10 (50 %)29 (36.2 %)Tumor location Cecum4 (20 %)13 (16.3 %)N.S Ascending colon9 (45 %)32 (40 %) Transverse colon4 (20 %)24 (30 %) Descending colon0 (0 %)2 (2.5 %) Sigmoid colon3 (15 %)9 (11.2 %)Depth of tumor invasion pTis0 (0 %)8 (10 %)0.012 pT10 (0 %)16 (20 %)(Tis, T1 vs. T2–T4) pT22 (10 %)5 (6.2 %) pT316 (80 %)31 (38.8 %) pT42 (10 %)20 (25 %)Tumor size (mean ± SE)58.6 ± 16.7 mm49.2 ± 32.6 mm0.0239Lymph node metastasis pN(−)1348N.S pN(+)732Distant metastasis pM0112N.S pM11968cStage 007N.S I218 II1021 III721 IV113Histopathological classification Well838N.S Moderately933 Poorly16 Others23Margin from tumor (mean ± SE) Oral side16.7 ± 2.5 cm14.6 ± 1.1 cmN.S Anal side12.3 ± 1.5 cm12.8 ± 1.0 cmN.SPreoperative bowel preparation PEG8 (40 %)60 (75 %)0.003 Others12 (60 %)20 (25 %)\nComparison of clinicopathological factors in the ECC-positive and ECC-negative groups\n Follow-up outcome Among all the cases, 81 were followed by colonoscopy after surgery. Follow-up colonoscopy was performed in 74 patients (74 %) (observation range 3.9–121.4 months; median observation period 64.1 months), and none were found to have developed suture-line recurrence.\nAmong all the cases, 81 were followed by colonoscopy after surgery. Follow-up colonoscopy was performed in 74 patients (74 %) (observation range 3.9–121.4 months; median observation period 64.1 months), and none were found to have developed suture-line recurrence.\n\nCytological analysis:\nThe cytological analysis results were as follows: class I, n = 17; class II, n = 61; class III, n = 2; class IV, n = 7; and class V, n = 13 (Table 1). Based on the pathological diagnosis, all the patients were assigned to the positive group (n = 20) and the negative group (n = 80).Table 1Cytology from a cartridge of a staplerClass I17 cases (17 %)Class II61 (61 %)Class III2 (2 %)Class IV7 (7 %)Class V13 (13 %)\nCytology from a cartridge of a stapler\n\nThe relationship between the status of ECCs and clinicopathological factors:\nAll positive cases had advanced cancers that had invaded beyond the submucosal layer (T2–T4), while all of the early cancers that had invaded the submucosal layer (Tis, T1) were in the negative group (p = 0.012) (Table 2). Regarding preoperative bowel preparation, the detection rate of ECCs in cases where full-laxative mechanical cleansing with PEG was performed was significantly lower than that in cases where full-laxative mechanical cleansing was not possible (p = 0.003) (Table 2). The two groups did not differ significantly in terms of the operation method, tumor location, histopathological classification, and margin from tumor (Table 2).Table 2Comparison of clinicopathological factors in the ECC-positive and ECC-negative groupsECC-positive groupECC-negative group\np value(n = 20)(n = 80)Age67.3 ± 12.0 years old72.0 ± 9.6 years oldN.SGender M843N.S F1237Operation method Laparoscopic surgery10 (50 %)51 (63.8 %)N.S Open surgery10 (50 %)29 (36.2 %)Tumor location Cecum4 (20 %)13 (16.3 %)N.S Ascending colon9 (45 %)32 (40 %) Transverse colon4 (20 %)24 (30 %) Descending colon0 (0 %)2 (2.5 %) Sigmoid colon3 (15 %)9 (11.2 %)Depth of tumor invasion pTis0 (0 %)8 (10 %)0.012 pT10 (0 %)16 (20 %)(Tis, T1 vs. T2–T4) pT22 (10 %)5 (6.2 %) pT316 (80 %)31 (38.8 %) pT42 (10 %)20 (25 %)Tumor size (mean ± SE)58.6 ± 16.7 mm49.2 ± 32.6 mm0.0239Lymph node metastasis pN(−)1348N.S pN(+)732Distant metastasis pM0112N.S pM11968cStage 007N.S I218 II1021 III721 IV113Histopathological classification Well838N.S Moderately933 Poorly16 Others23Margin from tumor (mean ± SE) Oral side16.7 ± 2.5 cm14.6 ± 1.1 cmN.S Anal side12.3 ± 1.5 cm12.8 ± 1.0 cmN.SPreoperative bowel preparation PEG8 (40 %)60 (75 %)0.003 Others12 (60 %)20 (25 %)\nComparison of clinicopathological factors in the ECC-positive and ECC-negative groups\n\nFollow-up outcome:\nAmong all the cases, 81 were followed by colonoscopy after surgery. Follow-up colonoscopy was performed in 74 patients (74 %) (observation range 3.9–121.4 months; median observation period 64.1 months), and none were found to have developed suture-line recurrence.\n\nDiscussion:\nWe found that ECC-positive cases were recognized at 20 % on the stapler cartridges used for anastomosis in patients with colon cancer. According to the relationship between the status of ECCs and clinicopathological factors, the presence of ECCs was associated with depth of tumor invasion and preoperative bowel preparation. Our study included 86 patients with right-sided colon cancer in 100 colon cancer patients. Hasegawa et al. [6] reported that 2 (11.1 %) and 10 (55.6 %) of 18 patients, who underwent right hemicolectomy for right-sided colon cancer, had ECCs at the terminal ileum and distal colon anastomosis sites, respectively. They demonstrated that surgical bowel occlusion on both sides of the tumor before resection improved a decrease in the number of ECCs. Recently, Maeda et al. [7] have shown that ECCs were detected at distal colon and proximal colon of the tumor within each 3 cm in almost patients with sigmoid cancer, and ECCs decreased with distance from the tumor. They also suggested that bowel ligatures could decrease ECCs, leading to prevent local recurrence. These findings indicated that the no-touch isolation technique might influence the number of ECCs as previous reports suggested [8, 9]. In the present study, we performed colectomy according to the no-touch isolation technique. Consequently, the positive rate of ECCs was 20 %. The difference of positive rate might be explained by the operative technique in addition to other factors, including cleansing methods and bowel preparation methods.\nMost studies of ECCs in the colonic lumen of colorectal cancer patients have involved rectal cancer patients. The incidence of suture-line recurrence has been reported to be 11–18 % [10, 11] in rectal cancer, while that in colon cancer has been reported to be 0.8–5.9 % [2, 3]. The possibility that ECCs can be implanted into freshly cut tissues was first postulated by Sir Ryall in 1907 [12]. This hypothesis was later advanced to explain some cases of suture-line recurrence following resection of colorectal cancer. In order to demonstrate the potential involvement of ECCs in the colonic lumen with suture-line and local recurrences in an animal model, Fermor et al. [13] injected ECCs collected from the colonic lumen of 17 colorectal cancer patients into the caudal vein of immunocompromised mice and showed that six mice developed lung metastases. Several cases of suture-line recurrence following resection of colon cancer, especially sigmoid colon, have been reported in Japan [14–17]. They discussed that implantation was considered to be the most important factor in terms of the cause of recurrence at anastomotic sites. The frequency of suture-line recurrence in colon cancer was very low though ECCs were frequently detected in colonic lumen. Indeed, we detected ECCs in 20 % of the colon cancer cases we analyzed. However, no suture-line recurrence was observed in our series of colon cancer for 5-year observation after colectomy. The cell viability of ECCs was investigated to understand etiological mechanism of anastomotic recurrence. Rosenberg et al. [18] reported the presence of no viable cells, as assessed by trypan blue exclusion. However, Umpleby et al. [19], using trypan blue exclusion and hydrolysis of fluorescein diacetate, showed that 70 % of patients with colorectal cancer had viable cells exfoliated into the bowel lumen. In general, other factors, which are associated with the potential of implantation, must be required even if ECCs still have viability.\nIn the present study, we found that the positive rate of ECCs was 11.8 % in patients with PEG, while it was 37.5 % in patients without PEG. Mechanical bowel preparation using PEG may be effective for decreasing the number of ECCs. Previous reports [6, 7] also suggested same findings. Regarding mechanical bowel preparation using PEG, Slim et al. [20] have described that anastomotic leakage was significantly found after mechanical bowel preparation (odds ratio 1.75; p = 0.032) and concluded that mechanical bowel preparation using PEG should be omitted before elective colorectal surgery. Therefore, mechanical bowel preparation is routinely not performed in patients undergoing elective colon cancer because of employing “enhanced recovery after surgery” (ERAS) protocol widely. Recently, clinical practice guidelines [21] from the USA recommend the use of both mechanical bowel preparation and oral antibiotic bowel preparation. Many surgeons still conduct mechanical bowel preparation using PEG in the USA [22] and Japan [23]. Mechanical bowel preparation using PEG might contribute to decrease positive ECCs in advanced colorectal cancer, leading to decrease the possibility of anastomotic recurrence in addition to prevention of anastomotic leakage and surgical site infection.\nIn a previous study [5], the outcome of reconstruction following colon cancer surgery was compared between hand-sewn and stapled anastomosis. While the safety was comparable, it was radiographically confirmed that suture leakage was more common with hand-sewn anastomoses. Hence, functional end-to-end anastomosis has primarily been performed using a stapler. In the present study, the washing samples of the cartridge of the linear stapler used for functional end-to-end anastomosis were examined cytologically. The rationale for using the washing samples of the cartridge in this study was that they would better reflect the presence of ECCs than the washing solutions of the colonic lumen. In addition, the presence of ECCs on the cartridge was considered to be a cause of suture-line recurrence. Prior to the present study, ECCs had been cytologically identified in the physiological saline used to irrigate the colonic lumen. In the present study, 5 % povidone-iodine was first used as a tumoricidal agent to irrigate the colonic lumen prior to anastomosis [24, 25]. However, since povidone-iodine should not be used in the abdominal cavity due to severe damage of normal mucosa, we have employed 0.025 % benzalkonium chloride, as an alternative. Therefore, the ECC detection rate might be lower in the present study than that reported by Umpleby, et al. [19] who used normal saline for bowel irrigation.\nOur results indicated that the detection rate of ECCs, which could be detected at anastomotic sites even on the stapler cartridges, might be increased in advanced colon cancer with more than T2 and in elective colon cancer patients without mechanical bowel preparation using PEG, leading to increase the possibility of incidence of suture-line recurrence. To prevent the possibility of incidence of suture-line recurrence, we should conduct preoperative mechanical bowel preparation using PEG, perform surgical operation by the no-touch isolation technique, and irrigate the colonic lumen prior to anastomosis using 0.025 % benzalkonium chloride as a tumoricidal agent.\n\nConclusions:\nWe found ECCs on the cartridge of the linear stapler used for anastomosis in 20 % of the colon cancer cases we analyzed. Most of the positive ECCs were identified in advanced colon cancer without PEG. Therefore, preoperative mechanical bowel preparation using PEG and cleansing at anastomotic sites using tumoricidal agents before anastomosis may contribute to decrease ECCs in advanced colon cancer.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe aim of this study was to investigate exfoliated cancer cells (ECCs) on linear stapler cartridges used for anastomotic sites in colon cancer.\n\nMethods:\nWe prospectively analyzed ECCs on linear stapler cartridges used for anastomosis in 100 colon cancer patients who underwent colectomy. Having completed the functional end-to-end anastomosis, the linear stapler cartridges were irrigated with saline, which was collected for cytological examination and cytological diagnoses were made by board-certified pathologists based on Papanicolaou staining.\n\nResults:\nThe detection rate of ECCs on the linear stapler cartridges was 20 %. Positive detection of ECCs was significantly associated with depth of tumor invasion (p = 0.012) and preoperative bowel preparation (p = 0.003). There were no marked differences between ECC-positive and ECC-negative groups in terms of the operation methods, tumor location, histopathological classification, and surgical margins.\n\nConclusions:\nSince ECCs were identified on the cartridge of the linear stapler used for anastomosis, preoperative mechanical bowel preparation using polyethylene glycol solution and cleansing at anastomotic sites using tumoricidal agents before anastomosis may be necessary to decrease ECCs in advanced colon cancer."},"background_conclusion_text":{"kind":"string","value":"Background:\nThe cause of suture-line recurrence following curative colorectal cancer surgery is believed to be the presence of exfoliated cancer cells (ECCs) at the anastomotic site [1]. Many studies of suture-line recurrence have been conducted in rectal cancer surgery; however, a greater margin is used for colon cancer than for rectal cancer cases, so that suture-line recurrence is less common in colon cancer patients [2]. As a result, few studies have reported on the presence of ECCs in the colonic lumen of colon cancer patients. However, even in patients with colon cancer, the incidence of suture-line recurrence has been reported to be 0.8–5.9 % [2, 3]. Reconstruction following colon cancer surgery commonly involves the fabrication of a functional end-to-end anastomosis [4] using a linear stapler, as a convenient procedure [5]. In the present study, ECCs in the colonic lumen of colon cancer patients were examined based on the cytological examination of the solutions washing the linear stapler used for functional end-to-end anastomosis, and we further analyzed the relationship between positive detection of ECCs and clinicopathological factors and prognosis.\n\nConclusions:\nWe found ECCs on the cartridge of the linear stapler used for anastomosis in 20 % of the colon cancer cases we analyzed. Most of the positive ECCs were identified in advanced colon cancer without PEG. Therefore, preoperative mechanical bowel preparation using PEG and cleansing at anastomotic sites using tumoricidal agents before anastomosis may contribute to decrease ECCs in advanced colon cancer."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nThe aim of this study was to investigate exfoliated cancer cells (ECCs) on linear stapler cartridges used for anastomotic sites in colon cancer.\n\nMethods:\nWe prospectively analyzed ECCs on linear stapler cartridges used for anastomosis in 100 colon cancer patients who underwent colectomy. Having completed the functional end-to-end anastomosis, the linear stapler cartridges were irrigated with saline, which was collected for cytological examination and cytological diagnoses were made by board-certified pathologists based on Papanicolaou staining.\n\nResults:\nThe detection rate of ECCs on the linear stapler cartridges was 20 %. Positive detection of ECCs was significantly associated with depth of tumor invasion (p = 0.012) and preoperative bowel preparation (p = 0.003). There were no marked differences between ECC-positive and ECC-negative groups in terms of the operation methods, tumor location, histopathological classification, and surgical margins.\n\nConclusions:\nSince ECCs were identified on the cartridge of the linear stapler used for anastomosis, preoperative mechanical bowel preparation using polyethylene glycol solution and cleansing at anastomotic sites using tumoricidal agents before anastomosis may be necessary to decrease ECCs in advanced colon cancer."},"whole_article_text_length":{"kind":"number","value":5232,"string":"5,232"},"whole_article_abstract_length":{"kind":"number","value":223,"string":"223"},"other_sections_lengths":{"kind":"list like","value":[198,34,109,100,51,141,496,54],"string":"[\n 198,\n 34,\n 109,\n 100,\n 51,\n 141,\n 496,\n 54\n]","isConversational":false},"num_sections":{"kind":"number","value":13,"string":"13"},"most_frequent_words":{"kind":"list like","value":["tumor","colon","patients","cancer","eccs","cases","bowel","preparation","class","20"],"string":"[\n \"tumor\",\n \"colon\",\n \"patients\",\n \"cancer\",\n \"eccs\",\n \"cases\",\n \"bowel\",\n \"preparation\",\n \"class\",\n \"20\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["tumor resection intestinal","colectomy colon cancer","colorectal cancer surgery","recurrence colon cancer","colon cancer rectal"],"string":"[\n \"tumor resection intestinal\",\n \"colectomy colon cancer\",\n \"colorectal cancer surgery\",\n \"recurrence colon cancer\",\n \"colon cancer rectal\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Exfoliated cancer cell | Colon cancer | Functional end-to-end anastomosis | Suture-line recurrence [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Exfoliated cancer cell | Colon cancer | Functional end-to-end anastomosis | Suture-line recurrence [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Exfoliated cancer cell | Colon cancer | Functional end-to-end anastomosis | Suture-line recurrence [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Exfoliated cancer cell | Colon cancer | Functional end-to-end anastomosis | Suture-line recurrence [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Exfoliated cancer cell | Colon cancer | Functional end-to-end anastomosis | Suture-line recurrence [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Anastomosis, Surgical | Colectomy | Colon | Colonic Neoplasms | Enema | Female | Humans | Laxatives | Male | Margins of Excision | Middle Aged | Neoplasm Invasiveness | Neoplasm Staging | Polyethylene Glycols | Preoperative Care | Prospective Studies | Surgical Staplers | Surgical Stapling [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Anastomosis, Surgical | Colectomy | Colon | Colonic Neoplasms | Enema | Female | Humans | Laxatives | Male | Margins of Excision | Middle Aged | Neoplasm Invasiveness | Neoplasm Staging | Polyethylene Glycols | Preoperative Care | Prospective Studies | Surgical Staplers | Surgical Stapling [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Anastomosis, Surgical | Colectomy | Colon | Colonic Neoplasms | Enema | Female | Humans | Laxatives | Male | Margins of Excision | Middle Aged | Neoplasm Invasiveness | Neoplasm Staging | Polyethylene Glycols | Preoperative Care | Prospective Studies | Surgical Staplers | Surgical Stapling [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Anastomosis, Surgical | Colectomy | Colon | Colonic Neoplasms | Enema | Female | Humans | Laxatives | Male | Margins of Excision | Middle Aged | Neoplasm Invasiveness | Neoplasm Staging | Polyethylene Glycols | Preoperative Care | Prospective Studies | Surgical Staplers | Surgical Stapling [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Aged | Aged, 80 and over | Anastomosis, Surgical | Colectomy | Colon | Colonic Neoplasms | Enema | Female | Humans | Laxatives | Male | Margins of Excision | Middle Aged | Neoplasm Invasiveness | Neoplasm Staging | Polyethylene Glycols | Preoperative Care | Prospective Studies | Surgical Staplers | Surgical Stapling [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] tumor resection intestinal | colectomy colon cancer | colorectal cancer surgery | recurrence colon cancer | colon cancer rectal [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] tumor resection intestinal | colectomy colon cancer | colorectal cancer surgery | recurrence colon cancer | colon cancer rectal [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] tumor resection intestinal | colectomy colon cancer | colorectal cancer surgery | recurrence colon cancer | colon cancer rectal [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] tumor resection intestinal | colectomy colon cancer | colorectal cancer surgery | recurrence colon cancer | colon cancer rectal [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] tumor resection intestinal | colectomy colon cancer | colorectal cancer surgery | recurrence colon cancer | colon cancer rectal [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] tumor | colon | patients | cancer | eccs | cases | bowel | preparation | class | 20 [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] tumor | colon | patients | cancer | eccs | cases | bowel | preparation | class | 20 [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] tumor | colon | patients | cancer | eccs | cases | bowel | preparation | class | 20 [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] tumor | colon | patients | cancer | eccs | cases | bowel | preparation | class | 20 [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] tumor | colon | patients | cancer | eccs | cases | bowel | preparation | class | 20 [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] cancer | colon | colon cancer | suture line | line | suture line recurrence | line recurrence | recurrence | suture | colon cancer patients [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] class | table | 20 | tumor | negative | ecc | group | positive | 10 | cases [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] colon cancer | colon | eccs | cancer | advanced colon | advanced colon cancer | advanced | anastomosis | peg | bowel preparation peg cleansing [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] colon | class | cancer | tumor | patients | eccs | colon cancer | anastomosis | cases | end [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] colon | class | cancer | tumor | patients | eccs | colon cancer | anastomosis | cases | end [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] linear [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 20 % ||| 0.012 | 0.003 ||| ECC [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] linear ||| linear | 100 ||| Papanicolaou ||| ||| 20 % ||| 0.012 | 0.003 ||| ECC ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] linear ||| linear | 100 ||| Papanicolaou ||| ||| 20 % ||| 0.012 | 0.003 ||| ECC ||| [SUMMARY]"}}},{"rowIdx":77,"cells":{"title":{"kind":"string","value":"Association between Endocrine Disorders and Severe COVID-19 Disease in Pediatric Patients."},"pmid":{"kind":"string","value":"35417912"},"background_abstract":{"kind":"string","value":"Though severe illness due to COVID-19 is uncommon in children, there is an urgent need to better determine the risk factors for disease severity in youth. This study aims to determine the impact a preexisting endocrine diagnosis has on severity of COVID-19 presentation in youth."},"background_abstract_label":{"kind":"string","value":"INTRODUCTION"},"methods_abstract":{"kind":"string","value":"The cross-sectional chart review study included all patients less than 25 years old with a positive SARS-CoV-2 PCR at St. Louis Children's Hospital between March 2020 and February 2021. Electronic medical record data for analysis included patient demographics, BMI percentile, inpatient hospitalization or admission to the pediatric intensive care unit (PICU), and the presence of a preexisting endocrine diagnosis such as diabetes mellitus (type 1 and type 2), adrenal insufficiency, and hypothyroidism. Two outcome measures were analyzed in multivariate analysis: inpatient admission and PICU admission. Adjusted odds ratios with a 95% CI were calculated using binary logistic regression, along with p values after Wald χ2 analysis."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"390 patients were included in the study. Mean age was 123.1 (±82.2) months old. 50.3% of patients were hospitalized, and 12.1% of patients were admitted to intensive care. Preexisting diagnoses of diabetes mellitus, obesity, and hypothyroidism were associated with an increased risk of hospital and ICU admission, independent of socioeconomic status."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"This study provides evidence that unvaccinated youth with a preexisting diagnosis of obesity, hypothyroidism, or diabetes mellitus infected with COVID-19 are more likely to have a more severe clinical presentation requiring inpatient hospital admission and/or intensive care."},"conclusions_abstract_label":{"kind":"string","value":"DISCUSSION/CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adolescent","Adult","Aged, 80 and over","COVID-19","Child","Cross-Sectional Studies","Diabetes Mellitus, Type 1","Hospitalization","Humans","Hypothyroidism","Obesity","Retrospective Studies","Risk Factors","SARS-CoV-2"],"string":"[\n \"Adolescent\",\n \"Adult\",\n \"Aged, 80 and over\",\n \"COVID-19\",\n \"Child\",\n \"Cross-Sectional Studies\",\n \"Diabetes Mellitus, Type 1\",\n \"Hospitalization\",\n \"Humans\",\n \"Hypothyroidism\",\n \"Obesity\",\n \"Retrospective Studies\",\n \"Risk Factors\",\n \"SARS-CoV-2\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"9393781"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"More than 12 million children in the USA have been diagnosed with COVID-19 since the start of the pandemic, representing approximately 18.9% of cumulative cases [1]. Though most children with COVID-19 do not develop severe disease or require specific therapy, children with certain medical conditions may be at increased risk for severe disease, and therefore may be eligible for therapeutic strategies to prevent disease progression [2]. Recent studies have looked at potential risk factors for severe COVID-19 presentation to guide these recommendations and have shown that diabetes mellitus and obesity are endocrine disorders that pose a significant risk for severe disease in both children and adults with COVID-19 [3, 4]. The impact of other preexisting endocrine disorders such as hypothyroidism and adrenal insufficiency on the clinical presentation of COVID-19 in youth has not been evaluated. Such studies may inform practitioners about potential patient groups at increased risk for developing severe infection and guide pediatric-specific recommendations for the treatment of COVID-19. Furthermore, it may help guide prioritization strategies for vaccination and therapeutic interventions in limited resource settings. This study aimed to determine if preexisting endocrine disorders such as adrenal insufficiency, obesity, overweight, diabetes mellitus, and hypothyroidism in youth are associated with higher rates of severe COVID-19."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"As shown in Figure 1, 540 patients had a positive SARS-CoV-2 PCR test at St. Louis Children's Hospital between March 2020 and February 2021. 150 patients were excluded from the analysis: 132 patients were missing BMI data, 11 were admitted to SLCH for either trauma or emergent surgery, and 7 were excluded due to missing socioeconomic data.\nThe remaining 390 patients were included in this study. Population characteristics are shown in Table 1. Mean age was 123.1 months old (SD = 82.2), and 194 (49.7%) of the patients were male. Mean ADI was 63.63 (SD = 25.4) The most prevalent endocrine condition among the patients studied was obesity, followed by overweight and diabetes mellitus (both type 1 and type 2). The mean HbA1c of those with diabetes was 11.0% (SD = 3.2.). 158 (40.5%) patients were admitted, with 47 (12.1%) patients admitted to intensive care. Of the 13 patients with a preexisting diagnosis of hypothyroidism, 1 had central hypothyroidism and the rest had primary hypothyroidism. Only 2 of the patients were biochemically hypothyroid at the time of positive COVID-19 testing, and all others were euthyroid. Eleven of the 13 patients were hospitalized, and the two nonadmitted were euthyroid. Of the patients with endocrinopathies included in the study, 4 patients had coexisting hypothyroidism and adrenal insufficiency, and all 4 of these patients were admitted to the hospital. In addition, one patient had coexisting hypothyroidism and diabetes mellitus, and 2 patients had coexisting diabetes and adrenal insufficiency. National percentile socioeconomic status, diabetes mellitus, obesity, and hypothyroidism were associated with an increased odds of inpatient admission (p < 0.05). As shown in Table 2 and Figure 2, after controlling for age, SES, weight, hypothyroidism, and adrenal insufficiency in the multivariate model, children with diabetes had 8.96 times increased odds of being admitted than kids without diabetes. As shown in Table 3 and Figure 3, adrenal insufficiency, diabetes mellitus, hypothyroidism, and obesity were associated with an increased odds of PICU admission (p < 0.05). While not statistically significant when considering p values, adrenal insufficiency had an adjusted odds ratio of 1.692 (95% CI: 0.934–15.350), suggesting a possible association between adrenal insufficiency and an increased odds of intensive care."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Statement of Ethics","Funding Sources","Author Contributions"],"string":"[\n \"Statement of Ethics\",\n \"Funding Sources\",\n \"Author Contributions\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["This retrospective cohort study was conducted at Washington University School of Medicine and the Saint Louis Children's Hospital (SLCH) in Saint Louis, MO, USA. The study was approved by the University's Human Research Protection Office Committee, approval number 202101185. Chart review was performed on all patients less than 25 years old that had a positive SARS-CoV-2 PCR at St. Louis Children's Hospital between March 2020 and February 2021. Written informed consent was not required.","Funding was obtained through the National Center for Advancing Translational Sciences through the National Institutes of Health (<UL1 TR002345> [to <William Powderly>]). Funding was used to support the CRTC, which N.R.B.'s work was funded by.","Nicholas R. Banull: formal analysis, investigation, methodology, validation, visualization, writing − original draft, and writing − review & editing. Ana Maria Arbelaez: formal analysis, investigation, methodology, validation, and writing − review & editing. Patrick J. Reich: formal analysis, investigation, methodology, data collection, validation, and writing − review & editing. Dorina Kallogeri: methodology, validation, writing − review & editing, and formal analysis. Carine Anka, Jennifer May, Hope Shimony, and Kathleen Wharton: data collection, investigation, methodology, validation, and writing − review & editing."],"string":"[\n \"This retrospective cohort study was conducted at Washington University School of Medicine and the Saint Louis Children's Hospital (SLCH) in Saint Louis, MO, USA. The study was approved by the University's Human Research Protection Office Committee, approval number 202101185. Chart review was performed on all patients less than 25 years old that had a positive SARS-CoV-2 PCR at St. Louis Children's Hospital between March 2020 and February 2021. Written informed consent was not required.\",\n \"Funding was obtained through the National Center for Advancing Translational Sciences through the National Institutes of Health (<UL1 TR002345> [to <William Powderly>]). Funding was used to support the CRTC, which N.R.B.'s work was funded by.\",\n \"Nicholas R. Banull: formal analysis, investigation, methodology, validation, visualization, writing − original draft, and writing − review & editing. Ana Maria Arbelaez: formal analysis, investigation, methodology, validation, and writing − review & editing. Patrick J. Reich: formal analysis, investigation, methodology, data collection, validation, and writing − review & editing. Dorina Kallogeri: methodology, validation, writing − review & editing, and formal analysis. Carine Anka, Jennifer May, Hope Shimony, and Kathleen Wharton: data collection, investigation, methodology, validation, and writing − review & editing.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null],"string":"[\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Introduction","Materials and Methods","Results","Discussion/Conclusion","Statement of Ethics","Conflict of Interest Statement","Funding Sources","Author Contributions","Data Availability Statement"],"string":"[\n \"Introduction\",\n \"Materials and Methods\",\n \"Results\",\n \"Discussion/Conclusion\",\n \"Statement of Ethics\",\n \"Conflict of Interest Statement\",\n \"Funding Sources\",\n \"Author Contributions\",\n \"Data Availability Statement\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["More than 12 million children in the USA have been diagnosed with COVID-19 since the start of the pandemic, representing approximately 18.9% of cumulative cases [1]. Though most children with COVID-19 do not develop severe disease or require specific therapy, children with certain medical conditions may be at increased risk for severe disease, and therefore may be eligible for therapeutic strategies to prevent disease progression [2]. Recent studies have looked at potential risk factors for severe COVID-19 presentation to guide these recommendations and have shown that diabetes mellitus and obesity are endocrine disorders that pose a significant risk for severe disease in both children and adults with COVID-19 [3, 4]. The impact of other preexisting endocrine disorders such as hypothyroidism and adrenal insufficiency on the clinical presentation of COVID-19 in youth has not been evaluated. Such studies may inform practitioners about potential patient groups at increased risk for developing severe infection and guide pediatric-specific recommendations for the treatment of COVID-19. Furthermore, it may help guide prioritization strategies for vaccination and therapeutic interventions in limited resource settings. This study aimed to determine if preexisting endocrine disorders such as adrenal insufficiency, obesity, overweight, diabetes mellitus, and hypothyroidism in youth are associated with higher rates of severe COVID-19.","This retrospective cohort study was conducted at Washington University School of Medicine and Saint Louis Children's Hospital (SLCH), a 400-bed pediatric hospital with a 40-bed pediatric intensive care unit (PICU), in Saint Louis, MO, USA. The study was approved by the university's Human Research Protection Office Committee. Chart review was performed on all patients less than 25 years old who had a positive SARS-CoV-2 PCR at St. Louis Children's Hospital between March 2020 and February 2021.\nAfter chart review, data from the electronic medical record (EPIC) was collected for analysis. These measures included: age, sex, patient's address, BMI percentile, inpatient hospitalization or admission to the PICU, and the history of a preexisting endocrine diagnosis such as diabetes mellitus (type 1 and type 2), adrenal insufficiency, and hypothyroidism. The diagnosis of endocrinopathy in all patients included in this study preceded infection with COVID-19. Patients with preexisting primary or central hypothyroidism, regardless of whether they presented to the hospital biochemically euthyroid, were classified as having a diagnosis of hypothyroidism in this study. Patients were classified as having a history of adrenal insufficiency if they had a preexisting diagnosis of primary or central adrenal insufficiency or if they had a history of chronic use of high dose oral steroids that could cause adrenal suppression and be at risk for disturbed stress response during infectious stress [5]. Patients were classified as normal weight, overweight, or obese based on the Centers for Disease Control and Prevention definition [6]. Obese patients were defined as having a BMI percentile greater than 95, and overweight patients were defined as having a BMI percentile greater than 85 but less than 95. BMI percentiles were calculated using the PediTools Electronic Growth Chart Calculators with the 2000 CDC growth chart used for reference [7, 8]. To provide continuity from childhood to adulthood, we used the age 20 reference to calculate BMI percentiles when individuals were 20 years of age or older, as others have done [9, 10, 11]. Socioeconomic status was calculated using the patient's address and the Neighborhood Atlas® software from the University of Wisconsin [12]. This software uses the 2018 American Community Survey to generate a National Percentile Area Deprivation Index (ADI), which allows for an objective comparison of socioeconomic status across the USA. Severity of COVID-19 disease was determined by the need for inpatient admission and PICU admission, which is similar to the definitions in other literature [4].\nDescriptive statistics were performed on all variables. Mean, standard deviations, and confidence intervals were calculated. To examine the association between patients admitted to inpatient care, or PICU, with a preexisting endocrine disorder, binary logistic regression was performed. Two outcome measures were analyzed in multivariate analysis: inpatient admission and PICU admission. ADI score, obesity, diabetes mellitus, adrenal insufficiency, age, and hypothyroidism were included in binary logistic regression due to either statistical significance or clinical importance. Adjusted odds ratios with 95% CI were calculated, along with p values after Wald χ2 analysis. Results were interpreted as statistically significant if p < 0.05 in two-tailed tests. Analyses were performed with statistical package SPSS© Version 27 for Mac. Graphics were produced using Adobe Illustrator and PRISM.","As shown in Figure 1, 540 patients had a positive SARS-CoV-2 PCR test at St. Louis Children's Hospital between March 2020 and February 2021. 150 patients were excluded from the analysis: 132 patients were missing BMI data, 11 were admitted to SLCH for either trauma or emergent surgery, and 7 were excluded due to missing socioeconomic data.\nThe remaining 390 patients were included in this study. Population characteristics are shown in Table 1. Mean age was 123.1 months old (SD = 82.2), and 194 (49.7%) of the patients were male. Mean ADI was 63.63 (SD = 25.4) The most prevalent endocrine condition among the patients studied was obesity, followed by overweight and diabetes mellitus (both type 1 and type 2). The mean HbA1c of those with diabetes was 11.0% (SD = 3.2.). 158 (40.5%) patients were admitted, with 47 (12.1%) patients admitted to intensive care. Of the 13 patients with a preexisting diagnosis of hypothyroidism, 1 had central hypothyroidism and the rest had primary hypothyroidism. Only 2 of the patients were biochemically hypothyroid at the time of positive COVID-19 testing, and all others were euthyroid. Eleven of the 13 patients were hospitalized, and the two nonadmitted were euthyroid. Of the patients with endocrinopathies included in the study, 4 patients had coexisting hypothyroidism and adrenal insufficiency, and all 4 of these patients were admitted to the hospital. In addition, one patient had coexisting hypothyroidism and diabetes mellitus, and 2 patients had coexisting diabetes and adrenal insufficiency. National percentile socioeconomic status, diabetes mellitus, obesity, and hypothyroidism were associated with an increased odds of inpatient admission (p < 0.05). As shown in Table 2 and Figure 2, after controlling for age, SES, weight, hypothyroidism, and adrenal insufficiency in the multivariate model, children with diabetes had 8.96 times increased odds of being admitted than kids without diabetes. As shown in Table 3 and Figure 3, adrenal insufficiency, diabetes mellitus, hypothyroidism, and obesity were associated with an increased odds of PICU admission (p < 0.05). While not statistically significant when considering p values, adrenal insufficiency had an adjusted odds ratio of 1.692 (95% CI: 0.934–15.350), suggesting a possible association between adrenal insufficiency and an increased odds of intensive care.","Throughout 2020 and 2021, COVID-19 was a leading cause of death in the USA, with a high prevalence of disease in the pediatric population [1, 13]. COVID-19 hospitalization rates in adolescents increased 5-fold over the summer of 2021 with unvaccinated patients making up most admissions [14]. With this increasing number of hospitalized patients, there is an urgent need for a better understanding of predictors of disease severity to determine populations at risk that will benefit from preventative and therapeutic interventions to decrease hospitalization and mortality rates. This study supports the established association between obesity and poorly controlled diabetes mellitus, with severity of COVID-19 presentation in adults [15, 16], and it further demonstrates that pediatric patients with COVID-19 who had a preexisting diagnosis of obesity, hypothyroidism, or diabetes mellitus were more likely to have a severe clinical presentation requiring inpatient hospital admission. This association was independent of socioeconomic status. Furthermore, it demonstrates that pediatric patients with diabetes mellitus, hypothyroidism, and obesity were more likely to require intensive care. Though prior studies have discussed the association between severe COVID-19 disease and preexisting health conditions, many were based on billing code data, and the prevalence of endocrine disorders, such as obesity and diabetes, was not representative of those in the US pediatric population [17, 18, 19]; hence, this study provides evidence that preexisting endocrinopathies are risk factors for predisposing youth to severe COVID-19.\nObesity rates in children and adolescents have been increasing over the past few decades, with a current prevalence of approximately 19.3% in the general population [18]. This trend has been driven by socioeconomic status, with obesity prevalence being higher in low-income communities [20]. The prevalence of obesity in this study population was 23.6%, which is similar to national disease rates [18]. This study confirms prior data demonstrating that obesity is a risk factor for COVID-19 disease severity [4]. Children with obesity, but not overweight, were twice as likely to be admitted to the hospital with COVID-19. However, this association was present independent of socioeconomic status/residence in areas with an increased area deprivation index, age, or sex. Similarly, increased rates of inpatient admission due to other infectious diseases, such as H1N1 and bacterial infections, have been observed among obese populations [21, 22]. Chronic inflammation and altered immunity seen in patients with obesity may explain this relationship [23]. Furthermore, this data emphasizes the importance of strategies to mitigate COVID-19 in pediatric communities with higher rates of obesity regardless of socioeconomic status.\nIncreased risk for comorbidities and serious infections have been documented in patients with either type 1 or type 2 diabetes mellitus [24]. This study confirmed findings by Kompaniyets et al. [4] that showed increased hospitalization and ICU admission rates in children with diabetes mellitus and COVID-19. This association could be attributed to poor glycemic control, given that patients with diabetes mellitus in our study had an HbA1c higher than the target HbA1c recommended by the American Diabetes Association [25]. Even though some studies have demonstrated a clear relationship between poor glycemic control and lower socioeconomic status, our study failed to demonstrate that disease severity among patients with diabetes mellitus was dependent on socioeconomic status [26, 27]. This data emphasizes the importance of identifying and applying interventions that help children and young adults with diabetes mellitus reduce their risk for severe COVID-19 disease.\nThere is a paucity of data related to the association of underlying hypothyroidism with COVID-19 disease severity. Two prior studies in adults concluded that hypothyroidism does not predispose patients to a higher risk of hospitalization, ICU admission, or death [28, 29]. However, this has not previously been evaluated in COVID-19 pediatric cohort studies to date. This study showed that youth with hypothyroidism and COVID-19 were 6.9 times more likely to be admitted to a hospital and 11 times more likely to require intensive care than the general population. This data suggests that preexisting thyroid disease will likely make patients more vulnerable to a more severe COVID-19 clinical presentation. The multivariate model in this study accounted for the coexistence of hypothyroidism among patients with diabetes mellitus. Therefore, this data suggests that hypothyroidism is an independent risk factor for disease severity among pediatric patients. The increased risk of inpatient and PICU admission was independent of age, sex, BMI, or SES. Because thyroid hormone regulates multiple organ systems, different mechanisms have been speculated to be involved in the relationship between SARS-CoV-2 and thyroid illness [30]. Though the results from this study suggest that pediatric patients with hypothyroidism are at an increased risk of developing a more severe COVID-19 clinical presentation, it is unclear if preexisting thyroid disease increases their susceptibility to COVID-19 infection.\nPatients with adrenal suppression may be at higher risk of presenting with an adrenal crisis during an acute illness, such as COVID-19 [31]. However, there is currently no clear evidence that adrenal insufficiency patients are more likely to develop a severe course of disease. A study of adult COVID-19 patients early in the pandemic did not demonstrate an association between severe disease and adrenal insufficiency [32]. An abstract presented at the Endocrine Society suggested that youth with adrenal insufficiency and COVID-19 had higher rates of mortality and a more severe clinical presentation [33]. Though our study did not support that claim, prompt stress dosing (as used by the patients in this study) is key to prevent adrenal crises during COVID-19 illness. Further studies with larger sample size are needed to better understand the potential association between history of adrenal insufficiency and COVID-19 disease severity.\nLower socioeconomic status is often associated with limited healthcare access, higher rates of diabetes mellitus and cardiovascular disease, increased utilization of health services, and early death [34]. Likewise, lower socioeconomic status may also lead to increased exposure to COVID-19, placing much of the burden of the pandemic on the most disadvantaged communities [26]. In this study, we used ADI scores as a measure of socioeconomic status. ADI is based on census data that has been validated, allowing for an objective ranking of residence by degree of social deprivation. Patients in this study resided in neighborhoods that were more disadvantaged than the national average. Though ADI percentiles were higher in patients that were hospitalized or required intensive care, ADI was not a predictor of disease severity. This suggests that severe disease presentation is driven by BMI, diabetes mellitus, and hypothyroidism and not by socioeconomic status. Given the lower SES of the entire study population, findings from this study highlight the critical need for improved healthcare delivery and disease prevention strategies.\nThe findings in this study are subject to several limitations. As shown in Figure 1, 150 patients from the original sample were excluded from analysis for missing BMI data, admissions primarily for reasons other than COVID-19, and missing ADI scores. Though the exclusion of these patients may have limited the sample size, it prevented the overestimation of COVID-19 hospitalization rates. Endocrine disorders were present in a small number of patients, leading to wide 95% confidence intervals in multivariable analysis. However, the lower 95% confidence intervals for diabetes mellitus, hypothyroidism, and obesity were well above 1, suggesting that our conclusions may be generalizable and that those patients are still at elevated risk for a more severe clinical COVID-19 presentation. Despite the small number of patients with endocrinopathies, diabetes mellitus, adrenal insufficiency, and hypothyroidism had slightly higher prevalence in our study than reported rates in the general population [17, 18, 19]. This may be because SLCH is a tertiary medical center, where many patients with a higher prevalence of comorbidities are transferred from regional hospitals to receive a higher level of care. In this study, few patients had coexisting multiple endocrine disorders. While the binary logistic regression analysis accounts for these co-diagnoses, further statistical analyses in this study are limited due to the small sample size of this cohort. Thus, additional studies of this subset of patients are necessary. Endocrine conditions in this study were not subclassified by etiology; therefore, specific associations between the etiology of endocrinopathies (type 1 DM vs. type 2, or central vs. primary hypothyroidism, primary vs. secondary adrenal insufficiency, etc.) and COVID-19 disease severity may have been missed. Finally, data presented in this study predates COVID-19 vaccinations in youth; therefore, associations between endocrine disorders and COVID-19 severity presented in this study may not extrapolate to a vaccinated population.\nThis study highlights that presence of preexisting diagnosis of hypothyroidism, diabetes mellitus, and obesity predisposes unvaccinated children and adolescents to a more severe clinical presentation of COVID-19. Data from this study suggest that practitioners should consider patients with these endocrinopathies for available therapeutic and preventive strategies that decrease disease progression. Further studies are needed to analyze these relationships in the vaccinated population.","This retrospective cohort study was conducted at Washington University School of Medicine and the Saint Louis Children's Hospital (SLCH) in Saint Louis, MO, USA. The study was approved by the University's Human Research Protection Office Committee, approval number 202101185. Chart review was performed on all patients less than 25 years old that had a positive SARS-CoV-2 PCR at St. Louis Children's Hospital between March 2020 and February 2021. Written informed consent was not required.","The authors have no conflicts of interest to declare.","Funding was obtained through the National Center for Advancing Translational Sciences through the National Institutes of Health (<UL1 TR002345> [to <William Powderly>]). Funding was used to support the CRTC, which N.R.B.'s work was funded by.","Nicholas R. Banull: formal analysis, investigation, methodology, validation, visualization, writing − original draft, and writing − review & editing. Ana Maria Arbelaez: formal analysis, investigation, methodology, validation, and writing − review & editing. Patrick J. Reich: formal analysis, investigation, methodology, data collection, validation, and writing − review & editing. Dorina Kallogeri: methodology, validation, writing − review & editing, and formal analysis. Carine Anka, Jennifer May, Hope Shimony, and Kathleen Wharton: data collection, investigation, methodology, validation, and writing − review & editing.","Requests for de-identified data can be made by contacting the corresponding author (A.M.A.)."],"string":"[\n \"More than 12 million children in the USA have been diagnosed with COVID-19 since the start of the pandemic, representing approximately 18.9% of cumulative cases [1]. Though most children with COVID-19 do not develop severe disease or require specific therapy, children with certain medical conditions may be at increased risk for severe disease, and therefore may be eligible for therapeutic strategies to prevent disease progression [2]. Recent studies have looked at potential risk factors for severe COVID-19 presentation to guide these recommendations and have shown that diabetes mellitus and obesity are endocrine disorders that pose a significant risk for severe disease in both children and adults with COVID-19 [3, 4]. The impact of other preexisting endocrine disorders such as hypothyroidism and adrenal insufficiency on the clinical presentation of COVID-19 in youth has not been evaluated. Such studies may inform practitioners about potential patient groups at increased risk for developing severe infection and guide pediatric-specific recommendations for the treatment of COVID-19. Furthermore, it may help guide prioritization strategies for vaccination and therapeutic interventions in limited resource settings. This study aimed to determine if preexisting endocrine disorders such as adrenal insufficiency, obesity, overweight, diabetes mellitus, and hypothyroidism in youth are associated with higher rates of severe COVID-19.\",\n \"This retrospective cohort study was conducted at Washington University School of Medicine and Saint Louis Children's Hospital (SLCH), a 400-bed pediatric hospital with a 40-bed pediatric intensive care unit (PICU), in Saint Louis, MO, USA. The study was approved by the university's Human Research Protection Office Committee. Chart review was performed on all patients less than 25 years old who had a positive SARS-CoV-2 PCR at St. Louis Children's Hospital between March 2020 and February 2021.\\nAfter chart review, data from the electronic medical record (EPIC) was collected for analysis. These measures included: age, sex, patient's address, BMI percentile, inpatient hospitalization or admission to the PICU, and the history of a preexisting endocrine diagnosis such as diabetes mellitus (type 1 and type 2), adrenal insufficiency, and hypothyroidism. The diagnosis of endocrinopathy in all patients included in this study preceded infection with COVID-19. Patients with preexisting primary or central hypothyroidism, regardless of whether they presented to the hospital biochemically euthyroid, were classified as having a diagnosis of hypothyroidism in this study. Patients were classified as having a history of adrenal insufficiency if they had a preexisting diagnosis of primary or central adrenal insufficiency or if they had a history of chronic use of high dose oral steroids that could cause adrenal suppression and be at risk for disturbed stress response during infectious stress [5]. Patients were classified as normal weight, overweight, or obese based on the Centers for Disease Control and Prevention definition [6]. Obese patients were defined as having a BMI percentile greater than 95, and overweight patients were defined as having a BMI percentile greater than 85 but less than 95. BMI percentiles were calculated using the PediTools Electronic Growth Chart Calculators with the 2000 CDC growth chart used for reference [7, 8]. To provide continuity from childhood to adulthood, we used the age 20 reference to calculate BMI percentiles when individuals were 20 years of age or older, as others have done [9, 10, 11]. Socioeconomic status was calculated using the patient's address and the Neighborhood Atlas® software from the University of Wisconsin [12]. This software uses the 2018 American Community Survey to generate a National Percentile Area Deprivation Index (ADI), which allows for an objective comparison of socioeconomic status across the USA. Severity of COVID-19 disease was determined by the need for inpatient admission and PICU admission, which is similar to the definitions in other literature [4].\\nDescriptive statistics were performed on all variables. Mean, standard deviations, and confidence intervals were calculated. To examine the association between patients admitted to inpatient care, or PICU, with a preexisting endocrine disorder, binary logistic regression was performed. Two outcome measures were analyzed in multivariate analysis: inpatient admission and PICU admission. ADI score, obesity, diabetes mellitus, adrenal insufficiency, age, and hypothyroidism were included in binary logistic regression due to either statistical significance or clinical importance. Adjusted odds ratios with 95% CI were calculated, along with p values after Wald χ2 analysis. Results were interpreted as statistically significant if p < 0.05 in two-tailed tests. Analyses were performed with statistical package SPSS© Version 27 for Mac. Graphics were produced using Adobe Illustrator and PRISM.\",\n \"As shown in Figure 1, 540 patients had a positive SARS-CoV-2 PCR test at St. Louis Children's Hospital between March 2020 and February 2021. 150 patients were excluded from the analysis: 132 patients were missing BMI data, 11 were admitted to SLCH for either trauma or emergent surgery, and 7 were excluded due to missing socioeconomic data.\\nThe remaining 390 patients were included in this study. Population characteristics are shown in Table 1. Mean age was 123.1 months old (SD = 82.2), and 194 (49.7%) of the patients were male. Mean ADI was 63.63 (SD = 25.4) The most prevalent endocrine condition among the patients studied was obesity, followed by overweight and diabetes mellitus (both type 1 and type 2). The mean HbA1c of those with diabetes was 11.0% (SD = 3.2.). 158 (40.5%) patients were admitted, with 47 (12.1%) patients admitted to intensive care. Of the 13 patients with a preexisting diagnosis of hypothyroidism, 1 had central hypothyroidism and the rest had primary hypothyroidism. Only 2 of the patients were biochemically hypothyroid at the time of positive COVID-19 testing, and all others were euthyroid. Eleven of the 13 patients were hospitalized, and the two nonadmitted were euthyroid. Of the patients with endocrinopathies included in the study, 4 patients had coexisting hypothyroidism and adrenal insufficiency, and all 4 of these patients were admitted to the hospital. In addition, one patient had coexisting hypothyroidism and diabetes mellitus, and 2 patients had coexisting diabetes and adrenal insufficiency. National percentile socioeconomic status, diabetes mellitus, obesity, and hypothyroidism were associated with an increased odds of inpatient admission (p < 0.05). As shown in Table 2 and Figure 2, after controlling for age, SES, weight, hypothyroidism, and adrenal insufficiency in the multivariate model, children with diabetes had 8.96 times increased odds of being admitted than kids without diabetes. As shown in Table 3 and Figure 3, adrenal insufficiency, diabetes mellitus, hypothyroidism, and obesity were associated with an increased odds of PICU admission (p < 0.05). While not statistically significant when considering p values, adrenal insufficiency had an adjusted odds ratio of 1.692 (95% CI: 0.934–15.350), suggesting a possible association between adrenal insufficiency and an increased odds of intensive care.\",\n \"Throughout 2020 and 2021, COVID-19 was a leading cause of death in the USA, with a high prevalence of disease in the pediatric population [1, 13]. COVID-19 hospitalization rates in adolescents increased 5-fold over the summer of 2021 with unvaccinated patients making up most admissions [14]. With this increasing number of hospitalized patients, there is an urgent need for a better understanding of predictors of disease severity to determine populations at risk that will benefit from preventative and therapeutic interventions to decrease hospitalization and mortality rates. This study supports the established association between obesity and poorly controlled diabetes mellitus, with severity of COVID-19 presentation in adults [15, 16], and it further demonstrates that pediatric patients with COVID-19 who had a preexisting diagnosis of obesity, hypothyroidism, or diabetes mellitus were more likely to have a severe clinical presentation requiring inpatient hospital admission. This association was independent of socioeconomic status. Furthermore, it demonstrates that pediatric patients with diabetes mellitus, hypothyroidism, and obesity were more likely to require intensive care. Though prior studies have discussed the association between severe COVID-19 disease and preexisting health conditions, many were based on billing code data, and the prevalence of endocrine disorders, such as obesity and diabetes, was not representative of those in the US pediatric population [17, 18, 19]; hence, this study provides evidence that preexisting endocrinopathies are risk factors for predisposing youth to severe COVID-19.\\nObesity rates in children and adolescents have been increasing over the past few decades, with a current prevalence of approximately 19.3% in the general population [18]. This trend has been driven by socioeconomic status, with obesity prevalence being higher in low-income communities [20]. The prevalence of obesity in this study population was 23.6%, which is similar to national disease rates [18]. This study confirms prior data demonstrating that obesity is a risk factor for COVID-19 disease severity [4]. Children with obesity, but not overweight, were twice as likely to be admitted to the hospital with COVID-19. However, this association was present independent of socioeconomic status/residence in areas with an increased area deprivation index, age, or sex. Similarly, increased rates of inpatient admission due to other infectious diseases, such as H1N1 and bacterial infections, have been observed among obese populations [21, 22]. Chronic inflammation and altered immunity seen in patients with obesity may explain this relationship [23]. Furthermore, this data emphasizes the importance of strategies to mitigate COVID-19 in pediatric communities with higher rates of obesity regardless of socioeconomic status.\\nIncreased risk for comorbidities and serious infections have been documented in patients with either type 1 or type 2 diabetes mellitus [24]. This study confirmed findings by Kompaniyets et al. [4] that showed increased hospitalization and ICU admission rates in children with diabetes mellitus and COVID-19. This association could be attributed to poor glycemic control, given that patients with diabetes mellitus in our study had an HbA1c higher than the target HbA1c recommended by the American Diabetes Association [25]. Even though some studies have demonstrated a clear relationship between poor glycemic control and lower socioeconomic status, our study failed to demonstrate that disease severity among patients with diabetes mellitus was dependent on socioeconomic status [26, 27]. This data emphasizes the importance of identifying and applying interventions that help children and young adults with diabetes mellitus reduce their risk for severe COVID-19 disease.\\nThere is a paucity of data related to the association of underlying hypothyroidism with COVID-19 disease severity. Two prior studies in adults concluded that hypothyroidism does not predispose patients to a higher risk of hospitalization, ICU admission, or death [28, 29]. However, this has not previously been evaluated in COVID-19 pediatric cohort studies to date. This study showed that youth with hypothyroidism and COVID-19 were 6.9 times more likely to be admitted to a hospital and 11 times more likely to require intensive care than the general population. This data suggests that preexisting thyroid disease will likely make patients more vulnerable to a more severe COVID-19 clinical presentation. The multivariate model in this study accounted for the coexistence of hypothyroidism among patients with diabetes mellitus. Therefore, this data suggests that hypothyroidism is an independent risk factor for disease severity among pediatric patients. The increased risk of inpatient and PICU admission was independent of age, sex, BMI, or SES. Because thyroid hormone regulates multiple organ systems, different mechanisms have been speculated to be involved in the relationship between SARS-CoV-2 and thyroid illness [30]. Though the results from this study suggest that pediatric patients with hypothyroidism are at an increased risk of developing a more severe COVID-19 clinical presentation, it is unclear if preexisting thyroid disease increases their susceptibility to COVID-19 infection.\\nPatients with adrenal suppression may be at higher risk of presenting with an adrenal crisis during an acute illness, such as COVID-19 [31]. However, there is currently no clear evidence that adrenal insufficiency patients are more likely to develop a severe course of disease. A study of adult COVID-19 patients early in the pandemic did not demonstrate an association between severe disease and adrenal insufficiency [32]. An abstract presented at the Endocrine Society suggested that youth with adrenal insufficiency and COVID-19 had higher rates of mortality and a more severe clinical presentation [33]. Though our study did not support that claim, prompt stress dosing (as used by the patients in this study) is key to prevent adrenal crises during COVID-19 illness. Further studies with larger sample size are needed to better understand the potential association between history of adrenal insufficiency and COVID-19 disease severity.\\nLower socioeconomic status is often associated with limited healthcare access, higher rates of diabetes mellitus and cardiovascular disease, increased utilization of health services, and early death [34]. Likewise, lower socioeconomic status may also lead to increased exposure to COVID-19, placing much of the burden of the pandemic on the most disadvantaged communities [26]. In this study, we used ADI scores as a measure of socioeconomic status. ADI is based on census data that has been validated, allowing for an objective ranking of residence by degree of social deprivation. Patients in this study resided in neighborhoods that were more disadvantaged than the national average. Though ADI percentiles were higher in patients that were hospitalized or required intensive care, ADI was not a predictor of disease severity. This suggests that severe disease presentation is driven by BMI, diabetes mellitus, and hypothyroidism and not by socioeconomic status. Given the lower SES of the entire study population, findings from this study highlight the critical need for improved healthcare delivery and disease prevention strategies.\\nThe findings in this study are subject to several limitations. As shown in Figure 1, 150 patients from the original sample were excluded from analysis for missing BMI data, admissions primarily for reasons other than COVID-19, and missing ADI scores. Though the exclusion of these patients may have limited the sample size, it prevented the overestimation of COVID-19 hospitalization rates. Endocrine disorders were present in a small number of patients, leading to wide 95% confidence intervals in multivariable analysis. However, the lower 95% confidence intervals for diabetes mellitus, hypothyroidism, and obesity were well above 1, suggesting that our conclusions may be generalizable and that those patients are still at elevated risk for a more severe clinical COVID-19 presentation. Despite the small number of patients with endocrinopathies, diabetes mellitus, adrenal insufficiency, and hypothyroidism had slightly higher prevalence in our study than reported rates in the general population [17, 18, 19]. This may be because SLCH is a tertiary medical center, where many patients with a higher prevalence of comorbidities are transferred from regional hospitals to receive a higher level of care. In this study, few patients had coexisting multiple endocrine disorders. While the binary logistic regression analysis accounts for these co-diagnoses, further statistical analyses in this study are limited due to the small sample size of this cohort. Thus, additional studies of this subset of patients are necessary. Endocrine conditions in this study were not subclassified by etiology; therefore, specific associations between the etiology of endocrinopathies (type 1 DM vs. type 2, or central vs. primary hypothyroidism, primary vs. secondary adrenal insufficiency, etc.) and COVID-19 disease severity may have been missed. Finally, data presented in this study predates COVID-19 vaccinations in youth; therefore, associations between endocrine disorders and COVID-19 severity presented in this study may not extrapolate to a vaccinated population.\\nThis study highlights that presence of preexisting diagnosis of hypothyroidism, diabetes mellitus, and obesity predisposes unvaccinated children and adolescents to a more severe clinical presentation of COVID-19. Data from this study suggest that practitioners should consider patients with these endocrinopathies for available therapeutic and preventive strategies that decrease disease progression. Further studies are needed to analyze these relationships in the vaccinated population.\",\n \"This retrospective cohort study was conducted at Washington University School of Medicine and the Saint Louis Children's Hospital (SLCH) in Saint Louis, MO, USA. The study was approved by the University's Human Research Protection Office Committee, approval number 202101185. Chart review was performed on all patients less than 25 years old that had a positive SARS-CoV-2 PCR at St. Louis Children's Hospital between March 2020 and February 2021. Written informed consent was not required.\",\n \"The authors have no conflicts of interest to declare.\",\n \"Funding was obtained through the National Center for Advancing Translational Sciences through the National Institutes of Health (<UL1 TR002345> [to <William Powderly>]). Funding was used to support the CRTC, which N.R.B.'s work was funded by.\",\n \"Nicholas R. Banull: formal analysis, investigation, methodology, validation, visualization, writing − original draft, and writing − review & editing. Ana Maria Arbelaez: formal analysis, investigation, methodology, validation, and writing − review & editing. Patrick J. Reich: formal analysis, investigation, methodology, data collection, validation, and writing − review & editing. Dorina Kallogeri: methodology, validation, writing − review & editing, and formal analysis. Carine Anka, Jennifer May, Hope Shimony, and Kathleen Wharton: data collection, investigation, methodology, validation, and writing − review & editing.\",\n \"Requests for de-identified data can be made by contacting the corresponding author (A.M.A.).\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["intro","materials|methods","results","discussion|conclusions",null,"COI-statement",null,null,"data-availability"],"string":"[\n \"intro\",\n \"materials|methods\",\n \"results\",\n \"discussion|conclusions\",\n null,\n \"COI-statement\",\n null,\n null,\n \"data-availability\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["COVID-19","Obesity","Diabetes","Adrenal insufficiency","Hypothyroidism"],"string":"[\n \"COVID-19\",\n \"Obesity\",\n \"Diabetes\",\n \"Adrenal insufficiency\",\n \"Hypothyroidism\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Introduction:\nMore than 12 million children in the USA have been diagnosed with COVID-19 since the start of the pandemic, representing approximately 18.9% of cumulative cases [1]. Though most children with COVID-19 do not develop severe disease or require specific therapy, children with certain medical conditions may be at increased risk for severe disease, and therefore may be eligible for therapeutic strategies to prevent disease progression [2]. Recent studies have looked at potential risk factors for severe COVID-19 presentation to guide these recommendations and have shown that diabetes mellitus and obesity are endocrine disorders that pose a significant risk for severe disease in both children and adults with COVID-19 [3, 4]. The impact of other preexisting endocrine disorders such as hypothyroidism and adrenal insufficiency on the clinical presentation of COVID-19 in youth has not been evaluated. Such studies may inform practitioners about potential patient groups at increased risk for developing severe infection and guide pediatric-specific recommendations for the treatment of COVID-19. Furthermore, it may help guide prioritization strategies for vaccination and therapeutic interventions in limited resource settings. This study aimed to determine if preexisting endocrine disorders such as adrenal insufficiency, obesity, overweight, diabetes mellitus, and hypothyroidism in youth are associated with higher rates of severe COVID-19.\n\nMaterials and Methods:\nThis retrospective cohort study was conducted at Washington University School of Medicine and Saint Louis Children's Hospital (SLCH), a 400-bed pediatric hospital with a 40-bed pediatric intensive care unit (PICU), in Saint Louis, MO, USA. The study was approved by the university's Human Research Protection Office Committee. Chart review was performed on all patients less than 25 years old who had a positive SARS-CoV-2 PCR at St. Louis Children's Hospital between March 2020 and February 2021.\nAfter chart review, data from the electronic medical record (EPIC) was collected for analysis. These measures included: age, sex, patient's address, BMI percentile, inpatient hospitalization or admission to the PICU, and the history of a preexisting endocrine diagnosis such as diabetes mellitus (type 1 and type 2), adrenal insufficiency, and hypothyroidism. The diagnosis of endocrinopathy in all patients included in this study preceded infection with COVID-19. Patients with preexisting primary or central hypothyroidism, regardless of whether they presented to the hospital biochemically euthyroid, were classified as having a diagnosis of hypothyroidism in this study. Patients were classified as having a history of adrenal insufficiency if they had a preexisting diagnosis of primary or central adrenal insufficiency or if they had a history of chronic use of high dose oral steroids that could cause adrenal suppression and be at risk for disturbed stress response during infectious stress [5]. Patients were classified as normal weight, overweight, or obese based on the Centers for Disease Control and Prevention definition [6]. Obese patients were defined as having a BMI percentile greater than 95, and overweight patients were defined as having a BMI percentile greater than 85 but less than 95. BMI percentiles were calculated using the PediTools Electronic Growth Chart Calculators with the 2000 CDC growth chart used for reference [7, 8]. To provide continuity from childhood to adulthood, we used the age 20 reference to calculate BMI percentiles when individuals were 20 years of age or older, as others have done [9, 10, 11]. Socioeconomic status was calculated using the patient's address and the Neighborhood Atlas® software from the University of Wisconsin [12]. This software uses the 2018 American Community Survey to generate a National Percentile Area Deprivation Index (ADI), which allows for an objective comparison of socioeconomic status across the USA. Severity of COVID-19 disease was determined by the need for inpatient admission and PICU admission, which is similar to the definitions in other literature [4].\nDescriptive statistics were performed on all variables. Mean, standard deviations, and confidence intervals were calculated. To examine the association between patients admitted to inpatient care, or PICU, with a preexisting endocrine disorder, binary logistic regression was performed. Two outcome measures were analyzed in multivariate analysis: inpatient admission and PICU admission. ADI score, obesity, diabetes mellitus, adrenal insufficiency, age, and hypothyroidism were included in binary logistic regression due to either statistical significance or clinical importance. Adjusted odds ratios with 95% CI were calculated, along with p values after Wald χ2 analysis. Results were interpreted as statistically significant if p < 0.05 in two-tailed tests. Analyses were performed with statistical package SPSS© Version 27 for Mac. Graphics were produced using Adobe Illustrator and PRISM.\n\nResults:\nAs shown in Figure 1, 540 patients had a positive SARS-CoV-2 PCR test at St. Louis Children's Hospital between March 2020 and February 2021. 150 patients were excluded from the analysis: 132 patients were missing BMI data, 11 were admitted to SLCH for either trauma or emergent surgery, and 7 were excluded due to missing socioeconomic data.\nThe remaining 390 patients were included in this study. Population characteristics are shown in Table 1. Mean age was 123.1 months old (SD = 82.2), and 194 (49.7%) of the patients were male. Mean ADI was 63.63 (SD = 25.4) The most prevalent endocrine condition among the patients studied was obesity, followed by overweight and diabetes mellitus (both type 1 and type 2). The mean HbA1c of those with diabetes was 11.0% (SD = 3.2.). 158 (40.5%) patients were admitted, with 47 (12.1%) patients admitted to intensive care. Of the 13 patients with a preexisting diagnosis of hypothyroidism, 1 had central hypothyroidism and the rest had primary hypothyroidism. Only 2 of the patients were biochemically hypothyroid at the time of positive COVID-19 testing, and all others were euthyroid. Eleven of the 13 patients were hospitalized, and the two nonadmitted were euthyroid. Of the patients with endocrinopathies included in the study, 4 patients had coexisting hypothyroidism and adrenal insufficiency, and all 4 of these patients were admitted to the hospital. In addition, one patient had coexisting hypothyroidism and diabetes mellitus, and 2 patients had coexisting diabetes and adrenal insufficiency. National percentile socioeconomic status, diabetes mellitus, obesity, and hypothyroidism were associated with an increased odds of inpatient admission (p < 0.05). As shown in Table 2 and Figure 2, after controlling for age, SES, weight, hypothyroidism, and adrenal insufficiency in the multivariate model, children with diabetes had 8.96 times increased odds of being admitted than kids without diabetes. As shown in Table 3 and Figure 3, adrenal insufficiency, diabetes mellitus, hypothyroidism, and obesity were associated with an increased odds of PICU admission (p < 0.05). While not statistically significant when considering p values, adrenal insufficiency had an adjusted odds ratio of 1.692 (95% CI: 0.934–15.350), suggesting a possible association between adrenal insufficiency and an increased odds of intensive care.\n\nDiscussion/Conclusion:\nThroughout 2020 and 2021, COVID-19 was a leading cause of death in the USA, with a high prevalence of disease in the pediatric population [1, 13]. COVID-19 hospitalization rates in adolescents increased 5-fold over the summer of 2021 with unvaccinated patients making up most admissions [14]. With this increasing number of hospitalized patients, there is an urgent need for a better understanding of predictors of disease severity to determine populations at risk that will benefit from preventative and therapeutic interventions to decrease hospitalization and mortality rates. This study supports the established association between obesity and poorly controlled diabetes mellitus, with severity of COVID-19 presentation in adults [15, 16], and it further demonstrates that pediatric patients with COVID-19 who had a preexisting diagnosis of obesity, hypothyroidism, or diabetes mellitus were more likely to have a severe clinical presentation requiring inpatient hospital admission. This association was independent of socioeconomic status. Furthermore, it demonstrates that pediatric patients with diabetes mellitus, hypothyroidism, and obesity were more likely to require intensive care. Though prior studies have discussed the association between severe COVID-19 disease and preexisting health conditions, many were based on billing code data, and the prevalence of endocrine disorders, such as obesity and diabetes, was not representative of those in the US pediatric population [17, 18, 19]; hence, this study provides evidence that preexisting endocrinopathies are risk factors for predisposing youth to severe COVID-19.\nObesity rates in children and adolescents have been increasing over the past few decades, with a current prevalence of approximately 19.3% in the general population [18]. This trend has been driven by socioeconomic status, with obesity prevalence being higher in low-income communities [20]. The prevalence of obesity in this study population was 23.6%, which is similar to national disease rates [18]. This study confirms prior data demonstrating that obesity is a risk factor for COVID-19 disease severity [4]. Children with obesity, but not overweight, were twice as likely to be admitted to the hospital with COVID-19. However, this association was present independent of socioeconomic status/residence in areas with an increased area deprivation index, age, or sex. Similarly, increased rates of inpatient admission due to other infectious diseases, such as H1N1 and bacterial infections, have been observed among obese populations [21, 22]. Chronic inflammation and altered immunity seen in patients with obesity may explain this relationship [23]. Furthermore, this data emphasizes the importance of strategies to mitigate COVID-19 in pediatric communities with higher rates of obesity regardless of socioeconomic status.\nIncreased risk for comorbidities and serious infections have been documented in patients with either type 1 or type 2 diabetes mellitus [24]. This study confirmed findings by Kompaniyets et al. [4] that showed increased hospitalization and ICU admission rates in children with diabetes mellitus and COVID-19. This association could be attributed to poor glycemic control, given that patients with diabetes mellitus in our study had an HbA1c higher than the target HbA1c recommended by the American Diabetes Association [25]. Even though some studies have demonstrated a clear relationship between poor glycemic control and lower socioeconomic status, our study failed to demonstrate that disease severity among patients with diabetes mellitus was dependent on socioeconomic status [26, 27]. This data emphasizes the importance of identifying and applying interventions that help children and young adults with diabetes mellitus reduce their risk for severe COVID-19 disease.\nThere is a paucity of data related to the association of underlying hypothyroidism with COVID-19 disease severity. Two prior studies in adults concluded that hypothyroidism does not predispose patients to a higher risk of hospitalization, ICU admission, or death [28, 29]. However, this has not previously been evaluated in COVID-19 pediatric cohort studies to date. This study showed that youth with hypothyroidism and COVID-19 were 6.9 times more likely to be admitted to a hospital and 11 times more likely to require intensive care than the general population. This data suggests that preexisting thyroid disease will likely make patients more vulnerable to a more severe COVID-19 clinical presentation. The multivariate model in this study accounted for the coexistence of hypothyroidism among patients with diabetes mellitus. Therefore, this data suggests that hypothyroidism is an independent risk factor for disease severity among pediatric patients. The increased risk of inpatient and PICU admission was independent of age, sex, BMI, or SES. Because thyroid hormone regulates multiple organ systems, different mechanisms have been speculated to be involved in the relationship between SARS-CoV-2 and thyroid illness [30]. Though the results from this study suggest that pediatric patients with hypothyroidism are at an increased risk of developing a more severe COVID-19 clinical presentation, it is unclear if preexisting thyroid disease increases their susceptibility to COVID-19 infection.\nPatients with adrenal suppression may be at higher risk of presenting with an adrenal crisis during an acute illness, such as COVID-19 [31]. However, there is currently no clear evidence that adrenal insufficiency patients are more likely to develop a severe course of disease. A study of adult COVID-19 patients early in the pandemic did not demonstrate an association between severe disease and adrenal insufficiency [32]. An abstract presented at the Endocrine Society suggested that youth with adrenal insufficiency and COVID-19 had higher rates of mortality and a more severe clinical presentation [33]. Though our study did not support that claim, prompt stress dosing (as used by the patients in this study) is key to prevent adrenal crises during COVID-19 illness. Further studies with larger sample size are needed to better understand the potential association between history of adrenal insufficiency and COVID-19 disease severity.\nLower socioeconomic status is often associated with limited healthcare access, higher rates of diabetes mellitus and cardiovascular disease, increased utilization of health services, and early death [34]. Likewise, lower socioeconomic status may also lead to increased exposure to COVID-19, placing much of the burden of the pandemic on the most disadvantaged communities [26]. In this study, we used ADI scores as a measure of socioeconomic status. ADI is based on census data that has been validated, allowing for an objective ranking of residence by degree of social deprivation. Patients in this study resided in neighborhoods that were more disadvantaged than the national average. Though ADI percentiles were higher in patients that were hospitalized or required intensive care, ADI was not a predictor of disease severity. This suggests that severe disease presentation is driven by BMI, diabetes mellitus, and hypothyroidism and not by socioeconomic status. Given the lower SES of the entire study population, findings from this study highlight the critical need for improved healthcare delivery and disease prevention strategies.\nThe findings in this study are subject to several limitations. As shown in Figure 1, 150 patients from the original sample were excluded from analysis for missing BMI data, admissions primarily for reasons other than COVID-19, and missing ADI scores. Though the exclusion of these patients may have limited the sample size, it prevented the overestimation of COVID-19 hospitalization rates. Endocrine disorders were present in a small number of patients, leading to wide 95% confidence intervals in multivariable analysis. However, the lower 95% confidence intervals for diabetes mellitus, hypothyroidism, and obesity were well above 1, suggesting that our conclusions may be generalizable and that those patients are still at elevated risk for a more severe clinical COVID-19 presentation. Despite the small number of patients with endocrinopathies, diabetes mellitus, adrenal insufficiency, and hypothyroidism had slightly higher prevalence in our study than reported rates in the general population [17, 18, 19]. This may be because SLCH is a tertiary medical center, where many patients with a higher prevalence of comorbidities are transferred from regional hospitals to receive a higher level of care. In this study, few patients had coexisting multiple endocrine disorders. While the binary logistic regression analysis accounts for these co-diagnoses, further statistical analyses in this study are limited due to the small sample size of this cohort. Thus, additional studies of this subset of patients are necessary. Endocrine conditions in this study were not subclassified by etiology; therefore, specific associations between the etiology of endocrinopathies (type 1 DM vs. type 2, or central vs. primary hypothyroidism, primary vs. secondary adrenal insufficiency, etc.) and COVID-19 disease severity may have been missed. Finally, data presented in this study predates COVID-19 vaccinations in youth; therefore, associations between endocrine disorders and COVID-19 severity presented in this study may not extrapolate to a vaccinated population.\nThis study highlights that presence of preexisting diagnosis of hypothyroidism, diabetes mellitus, and obesity predisposes unvaccinated children and adolescents to a more severe clinical presentation of COVID-19. Data from this study suggest that practitioners should consider patients with these endocrinopathies for available therapeutic and preventive strategies that decrease disease progression. Further studies are needed to analyze these relationships in the vaccinated population.\n\nStatement of Ethics:\nThis retrospective cohort study was conducted at Washington University School of Medicine and the Saint Louis Children's Hospital (SLCH) in Saint Louis, MO, USA. The study was approved by the University's Human Research Protection Office Committee, approval number 202101185. Chart review was performed on all patients less than 25 years old that had a positive SARS-CoV-2 PCR at St. Louis Children's Hospital between March 2020 and February 2021. Written informed consent was not required.\n\nConflict of Interest Statement:\nThe authors have no conflicts of interest to declare.\n\nFunding Sources:\nFunding was obtained through the National Center for Advancing Translational Sciences through the National Institutes of Health (<UL1 TR002345> [to <William Powderly>]). Funding was used to support the CRTC, which N.R.B.'s work was funded by.\n\nAuthor Contributions:\nNicholas R. Banull: formal analysis, investigation, methodology, validation, visualization, writing − original draft, and writing − review & editing. Ana Maria Arbelaez: formal analysis, investigation, methodology, validation, and writing − review & editing. Patrick J. Reich: formal analysis, investigation, methodology, data collection, validation, and writing − review & editing. Dorina Kallogeri: methodology, validation, writing − review & editing, and formal analysis. Carine Anka, Jennifer May, Hope Shimony, and Kathleen Wharton: data collection, investigation, methodology, validation, and writing − review & editing.\n\nData Availability Statement:\nRequests for de-identified data can be made by contacting the corresponding author (A.M.A.).\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThough severe illness due to COVID-19 is uncommon in children, there is an urgent need to better determine the risk factors for disease severity in youth. This study aims to determine the impact a preexisting endocrine diagnosis has on severity of COVID-19 presentation in youth.\n\nMethods:\nThe cross-sectional chart review study included all patients less than 25 years old with a positive SARS-CoV-2 PCR at St. Louis Children's Hospital between March 2020 and February 2021. Electronic medical record data for analysis included patient demographics, BMI percentile, inpatient hospitalization or admission to the pediatric intensive care unit (PICU), and the presence of a preexisting endocrine diagnosis such as diabetes mellitus (type 1 and type 2), adrenal insufficiency, and hypothyroidism. Two outcome measures were analyzed in multivariate analysis: inpatient admission and PICU admission. Adjusted odds ratios with a 95% CI were calculated using binary logistic regression, along with p values after Wald χ2 analysis.\n\nResults:\n390 patients were included in the study. Mean age was 123.1 (±82.2) months old. 50.3% of patients were hospitalized, and 12.1% of patients were admitted to intensive care. Preexisting diagnoses of diabetes mellitus, obesity, and hypothyroidism were associated with an increased risk of hospital and ICU admission, independent of socioeconomic status.\n\nConclusions:\nThis study provides evidence that unvaccinated youth with a preexisting diagnosis of obesity, hypothyroidism, or diabetes mellitus infected with COVID-19 are more likely to have a more severe clinical presentation requiring inpatient hospital admission and/or intensive care."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":3287,"string":"3,287"},"whole_article_abstract_length":{"kind":"number","value":293,"string":"293"},"other_sections_lengths":{"kind":"list like","value":[88,47,116],"string":"[\n 88,\n 47,\n 116\n]","isConversational":false},"num_sections":{"kind":"number","value":9,"string":"9"},"most_frequent_words":{"kind":"list like","value":["patients","19","covid","covid 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over | COVID-19 | Child | Cross-Sectional Studies | Diabetes Mellitus, Type 1 | Hospitalization | Humans | Hypothyroidism | Obesity | Retrospective Studies | Risk Factors | SARS-CoV-2 [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hypothyroidism covid 19 | endocrine disorders covid | covid 19 pediatric | covid 19 obesity | youth hypothyroidism covid [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] hypothyroidism covid 19 | endocrine disorders covid | covid 19 pediatric | covid 19 obesity | youth hypothyroidism covid [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hypothyroidism covid 19 | endocrine disorders covid | covid 19 pediatric | covid 19 obesity | 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1999-2004)."},"pmid":{"kind":"string","value":"19165386"},"background_abstract":{"kind":"string","value":"The current, continuous National Health and Nutrition Examination Survey (NHANES) has included blood mercury (BHg) and fish/shellfish consumption since it began in 1999. NHANES 1999-2004 data form the basis for these analyses."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We performed univariate and bivariate analyses to determine the distribution of BHg and fish consumption in the population and to investigate differences by geography, race/ethnicity, and income. We used multivariate analysis (regression) to determine the strongest predictors of BHg among geography, demographic factors, and fish consumption."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Elevated BHg occurred more commonly among women of childbearing age living in coastal areas of the United States (approximately one in six women). Regionally, exposures differ across the United States: Northeast > South and West > Midwest. Asian women and women with higher income ate more fish and had higher BHg. Time-trend analyses identified reduced BHg and reduced intake of Hg in the upper percentiles without an overall reduction of fish consumption."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"BHg is associated with income, ethnicity, residence (census region and coastal proximity). From 1999 through 2004, BHg decreased without a concomitant decrease in fish consumption. Data are consistent with a shift over this time period in fish species in women's diets."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Animals","Diet","Ethnicity","Female","Humans","Mercury","Nutrition Surveys","Seafood","United States"],"string":"[\n \"Adult\",\n \"Animals\",\n \"Diet\",\n \"Ethnicity\",\n \"Female\",\n \"Humans\",\n \"Mercury\",\n \"Nutrition Surveys\",\n \"Seafood\",\n \"United States\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"2627864"},"background_title":{"kind":"null"},"background_text":{"kind":"null"},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Table 1 shows the distributions of estimates of the number of women with BHg concentrations > 3.5 μg/L and > 5.8 μg/L, by region. Analyses indicate that between 1999 and 2004, the Northeast had the highest percentage of women with BHg concentrations above the 3.5 μg/L level of concern (> 19%), whereas the South had the largest estimated number of women (1.21 million) with ≥ 3.5 μg/L BHg because of elevated population in that region. Geometric means (Figure 1) show similar trends, with the highest BHg concentrations in the Northeast, followed by the West, South, and Midwest census regions. In the Northeast, the highest 5% of BHg concentrations exceeded 8.2 μg/L. [For full distributions, see Supplemental Material, Table 2 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] When we included coastal regions in this analysis, additional spatial heterogeneity in BHg (Figure 2A) and estimated 30-day Hg intake (Figure 2B) was apparent, with elevated exposures in all coastal areas relative to their neighboring inland regions except in the Great Lakes. In the coastal areas, the highest 5% of BHg concentrations exceeded 7.2 μg/L, with the Atlantic Coast exceeding 10.9 μg/L. [For the full distributions, see Supplemental Material, Table 4 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] Fish species eaten by survey participants varied by region (Figure 3), with respondents in coastal regions reporting higher frequency of consumption of fish containing higher levels of Hg. BHg concentrations were strongly associated with the frequency of fish consumption (Figure 4). BHg increased with monthly estimated consumption of fish and shellfish over the range of never/rarely to 4 or more times per week. In multiple regression modeling, women from the Atlantic (p < 0.01), Pacific (p < 0.0001), and Gulf (p < 0.0001) coasts had higher BHg concentrations compared with women from the inland West, whereas women from the inland Northeast and inland Midwest had significantly lower BHg levels (p < 0.0001). [For the full regression results, see Supplemental Material, Table 11 (http://www.ehponline.org/members/2008/11674/suppl.pdf).]\nAnalysis of temporal trends through simple regression modeling showed no statistically significant difference among the three sets of study years (1999–2000–2001–2002, and 2003–2004) for BHg (p = 0.07), estimated 30-day Hg intake (p = 0.11), or reported frequency of seafood consumption (p = 0.69). However, in multiple regression modeling, adjusting for covariates including coastal/non-coastal residence, the years 1999–2000 had significantly higher BHg levels (p < 0.0001) compared with 2003–2004, and 2001–2002 had significantly lower BHg levels (p < 0.01) [Supplemental Material, Table 11 (http://www.ehponline.org/members/2008/11674/suppl.pdf)]. Although the analyses did not support the conclusion that there was a general downward trend in BHg concentrations over the 6-year study period, there was a decline in the upper percentiles reflecting the most highly exposed women with BHg concentrations greater than established levels of concern [Supplemental Material, Table 9 (http://www.ehponline.org/members/2008/11674/suppl.pdf)]. In addition, the percentage of examinees with BHg values ≥ 3.5 μg/L and ≥ 5.8 μg/L was much greater in 1999–2000 compared to 2001–2002 and 2003–2004 (Figure 5). We found no consistent trend in fish consumption across the study years. We observed a decrease in the 90th percentile of 30-day estimated intake of Hg through seafood consumption across the study years even though there was no similar decrease in the 90th percentile of 30-day estimated consumption of grams of fish and shellfish (Figure 6). This suggests a shift in consumption to seafood containing less Hg. We did not observe a similar pattern at the mean, suggesting that this shift in seafood consumption occurred mainly with the highest fish and shellfish consumers [Supplemental Material, Table 9 (http://www.ehponline.org/members/2008/11674/suppl.pdf)]. The RfD for Hg intake is 0.1 μg Hg/kg body weight (μg/kgbw) per day, or 3.0 μg Hg/kgbw per month (30 days).\nResults also showed that self-selected ethnic identity was associated with total BHg concentrations, estimated 30-day Hg intake, and frequency of either finfish or shellfish consumption. For example, BHg levels, reported frequency of seafood consumption, and 30-day Hg intakes were highest among women who designated themselves as being in the “other” category (mostly people whose ancestry is Asian, Native American, Pacific Islands, and the Caribbean Islands). [See also Supplemental Material, Tables 5–7 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] Table 2 presents the percentages of women by race/ethnicity that had ≥ 3.5 and ≥ 5.8 μg/L BHg.\nWe identified statistically significant relationships between higher income and, respectively, increasing BHg concentration (p < 0.0001), estimated 30-day intake of Hg (p = 0.008), and 30-day frequency of finfish and shellfish consumption (p < 0.0001) through bivariate regressions. [For the distributions of blood total Hg, estimated Hg intake, and frequency of finfish and shellfish consumption by annual income, see Supplemental Material, Tables 6–8 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] In addition, women from families reporting incomes of ≥ $75,000 (the reference category) had statistically higher BHg levels than did women from families with incomes of ≤ $55,000 (p < 0.01). In all cases, BHg concentrations were also significantly associated with age and estimated 30-day Hg intake (p < 0.0001). Table 3 presents the percentage of women by annual income with ≥ 3.5 and ≥ 5.8 μg/L BHg.\nIn multiple regression modeling, after adjusting for other factors related to BHg, both race/ethnicity and income remained statistically significant predictors of BHg levels observed in this study [Supplemental Material, Table 11 (http://www.ehponline.org/members/2008/11674/suppl.pdf)]. Non-Hispanic blacks (p < 0.0001) and women grouped in the “other” racial category (p = 0.002) had significantly higher BHg concentrations than did non-Hispanic whites."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"Significant geographic differences in BHg concentrations occurred within the United States: We found highest exposures in coastal areas and the Northeast census region. In the Northeast, 19% of women had BHg concentrations ≥ 3.5 μg/L. The highest 5% of BHg concentrations exceeded 8.2 μg/L in the Northeast and 7.2 μg/L in coastal areas, concentrations more than twice the 3.5 μg/L level of concern. BHg levels were predicted by the quantity and type of fish consumed. Over the 6-year period (1999–2004), the frequency of elevated BHg levels among women of childbearing age declined without a significant change in quantities of fish and shellfish consumed. This pattern suggests a more discerning series of choices in type of fish eaten rather than an overall reduction in fish consumption. Within all geographic regions, women at highest risk of elevated Hg exposures were more affluent and more likely to be of Asian or island ethnicity."},"other_sections_titles":{"kind":"list like","value":["Methodology for data analysis","Methodology for defining coastal and non-coastal areas","Regional and coastal variation in BHg concentrations and in fish consumption","Ethnic group variation on fish intake and BHg concentrations","Income differences in association with fish intake and BHg concentrations","Interactions between income and ethnic group","Time trends in Hg exposure absent changes in total fish consumption","Changes in MeHg exposure over time"],"string":"[\n \"Methodology for data analysis\",\n \"Methodology for defining coastal and non-coastal areas\",\n \"Regional and coastal variation in BHg concentrations and in fish consumption\",\n \"Ethnic group variation on fish intake and BHg concentrations\",\n \"Income differences in association with fish intake and BHg concentrations\",\n \"Interactions between income and ethnic group\",\n \"Time trends in Hg exposure absent changes in total fish consumption\",\n \"Changes in MeHg exposure over time\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["We evaluated data for examinees who participated in NHANES during survey years 1999–2004 to assess the statistical association between seafood consumption and BHg by region of residence, race/ethnicity, and annual income. Further, we examined time trends for both BHg and fish consumption. NHANES is an annual survey conducted by the NCHS. The data include BHg levels, 24-hr dietary recall, and 30-day finfish and shellfish consumption frequency for women 16–49 years of age who reside in the United States. The NHANES sampling frame includes all 50 states. The documentation and publicly available data for NHANES can be found online [Centers for Disease Control and Prevention (CDC) 2006b]. The regional data are not publicly available but can be accessed by special request to the National Center for Health Statistics (NCHS) through its Research Data Center. Procedures for submitting a proposal in order to access data that are not publicly available can be found online (CDC 2006c). We performed all analyses using SAS, version 9.1 (SAS Institute Inc., Cary, NC).\nFollowing NHANES analytic guidelines (CDC 2006a), we used SAS procedures that accurately incorporate the stratification and multistage sampling of NHANES: Proc SurveyMeans, Proc SurveyReg, and Proc SurveyFreq (SAS Institute Inc.). The weights provided by NCHS compensate for the oversampling of various subpopulations and adjust for nonresponse bias. We used weights for estimating statistics for coastal and non-coastal regions to retain these adjustments. Because we considered the variables of interest (BHg and fish consumption) to be related to some of the factors that were oversampled, we retained these adjustments to minimize the bias in the estimates. For example, NHANES over-sampled Mexican Americans, who also have lower BHg than do other racial/ethnic groups. If the weights were not used to estimate the distribution of BHg, the results would be biased low. We recognize that some bias may remain within the estimates because the weights were not specifically created for the geographic regions of coastal and noncoastal; however, these subdivisions (e.g., coastal, noncoastal, Pacific, Atlantic) are based on counties, the primary sampling units of NHANES (CDC 2006a). All multivariate analyses were done unweighted because factors associated with the dependent variables and for which over-sampling was based were included as covariates and thus adjusted for in the modeling.\nIn order to estimate long-term Hg intake, we combined data collected through the 24-hr dietary recall and the 30-day fish frequency questionnaire to estimate 30-day Hg intake [for specific methodologies, see Mahaffey and Rice (1997); Mahaffey et al. (2004)]. If we found statistically significant differences in amount of fish consumed per meal from the 24-hr dietary recall by either coastal status (participants who lived in a county that bordered the Pacific or Atlantic Oceans, the Gulf of Mexico, or the Great Lakes vs. those who did not) or data release (1999–2000–2001–2002, or 2003–2004), we calculated separate averages. We generated statistically representative estimates from these data using the statistical weights provided by NCHS and following the relevant analytical guidelines published by NCHS (CDC 2006a).","Fish consumption is generally believed to be a major contributor to BHg concentration. We hypothesized that patterns of fish and shellfish consumption would vary between U.S. residents who live on or near the coast (within ~ 25–50 miles) and those who live inland. We further hypothesized that fish consumption patterns, and thus BHg concentrations, may also vary by specific coast (e.g., residents near the Atlantic Coast may have different BHg concentrations than those on the coast of the Gulf of Mexico) and specific inland region (e.g., West vs. Midwest). To test these hypotheses, we categorized NHANES respondents as living in either a coastal or a noncoastal county and further categorized them by eight regions: Atlantic Coast, Northeast, Great Lakes, Midwest, South, Gulf of Mexico, West, and Pacific Coast.\nThe geographic unit used by NHANES is a county or county equivalent (CDC 2006a); therefore, we limited our definitions of coastal and noncoastal to follow county boundaries. We defined all counties that bordered the Pacific or Atlantic Oceans, the Gulf of Mexico, or any of the Great Lakes as coastal. Additionally, we defined counties that bordered estuaries and bays as coastal, as well as counties whose center point was within approximately 25 miles of any coast even if not directly bordering a coast. [For the list of counties, see Supplemental Material, Table 13 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] We then defined the four coastal regions based on nearest body of water; for example, counties in California, Oregon, Washington, Alaska, and Hawaii that we defined as coastal were categorized as Pacific Coast. We separated noncoastal counties into four inland regions using the U.S. Census regions; for example, noncoastal counties in California, Oregon, Washington, and Alaska along with the entire states of Idaho, Montana, Wyoming, Colorado, New Mexico, Arizona, Utah, and Nevada became the West region (we classified all of Hawaii as coastal). We also designated the entire state of Florida as coastal, split between the Atlantic Coast and the Gulf of Mexico. We designated Miami-Dade County as Atlantic Coast, and Monroe County as Gulf of Mexico. These subdivisions run the risk of small sample sizes; however, the definition of coastal was sufficiently broad to avoid single primary sampling units.","Comparisons of the distribution of BHg data with reference values aimed at protecting the fetal nervous system have been made using national-level data (Mahaffey et al. 2004). NHANES data are often used to make population estimates through application of weighting factors to variables of interest such as BHg. Population estimates for U.S. Census regions and their distribution of BHg concentrations indicate that women living in the Northeast had BHg concentrations exceeding levels of concern more often than did women living in the South and West. The lowest Hg exposures were reported among women living in the Midwest.\nBecause NHANES was not designed to provide population estimates for coastal and noncoastal areas, unbiased estimates for the number of women having BHg concentrations ≥ 3.5 μg/L and ≥ 5.8 μg/L cannot be developed comparing coastal- and noncoastal-residing women. Although the following are not population estimates, they are statistics for a geographic region: Women living in coastal areas were at greater risk of having BHg concentrations ≥ 3.5 μg/L (16.25% for coastal and 5.99% for noncoastal residents) and ≥ 5.8 μg/L (8.11% for coastal and 2.06% for noncoastal residents). Women living near the coastal areas had approximately three to four times greater risk of exceeding acceptable levels of Hg exposure than did noncoastal-dwelling women. There may be some bias in these results due to the weighting issues (see “Materials and Methods”); however, we do not believe that this bias is a major factor underlying these great differences.\nMeHg exposures exceeding health-based standards, including U.S. EPA’s RfD (Rice et al. 2003), occurred more commonly among women living in coastal areas. These health-based standards were based on avoiding MeHg-associated delays and deficits in neurologic development of children after in utero exposure to MeHg (Mergler et al. 2007; Rice et al. 2003). At higher exposures to MeHg, including the highest concentrations reported during these survey years, the women themselves may risk adverse neuropsychological and neurobehavioral outcomes (Mergler et al. 2007).\nWithin the United States, people living in coastal areas consume more fish and shellfish than do those living in noncoastal areas and consume fish with higher Hg concentrations. Reports from New York City (McKelvey et al. 2007) and Florida (Denger et al. 1994; Karouna-Renier et al. 2008) support our identification of higher Hg exposures in U.S. coastal areas. This is part of a worldwide pattern. An overall pattern of higher BHg levels has also been reported among people living on U.S. islands [Hawaii (Sato et al. 2006)] and territories [e.g., Puerto Rico (Ortiz-Roque and López-Rivera 2004)]. A similar pattern has been repeated in other islands [Bermuda (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004), Fiji (Kumar et al. 2006), Seychelles (Myers et al. 2007), and Tahiti (Chateau-Degat 2005; Dewailly et al. 2008)] compared with inland populations. Among these island populations, BHg concentrations at the upper end of the distribution fall into the range of 50 μg/L (~ 250 nmol/L) and higher (Chateau-Degat 2005). In Bermuda, cord BHg concentrations as high as 160 nmol/L (~ 35 μg/L) have been reported (arithmetic mean, 41.3 ± 4.7 nmol/L or 8.0 ± 1.0 μg/L) (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004).\nHigher BHg concentrations in the U.S. Northeast found in this study reflect, in part, more frequent fish and shellfish consumption. Additional variability may be a function of differences in Hg concentrations among species and geographic regions (Sunderland 2007). For example, recent information on “hot spots” for Hg in wildlife tissues (Evers et al. 2007) could be associated with higher Hg concentrations for locally obtained fish. One limitation of the present analysis was the use of a fish-species–specific mean Hg concentration (i.e., nondistributional values) to estimate individual exposure. Although most fish consumed by the U.S. population is not locally obtained (i.e., commercially obtained from diverse regions and countries) (Sunderland 2007), analytical results showing geographic differences in the distribution of BHg could reflect higher Hg concentrations in locally obtained fish within the Northeast states.","Ethnic origins were associated with Hg exposures with those designated as “other” (i.e., Asian, Pacific and Caribbean Islander, Native American, Alaska Native, multiracial, and unknown race) having higher BHg concentrations. From additional studies, people of Asian descent whose food choices are influenced by Asian dietary patterns (Kudo et al. 2000; Sechena et al. 2003) tended to consume fish more frequently, in greater variety, and in greater quantity than did non-Asians. The ethnic diversity of the U.S. population is well known. As of 1997, 61% of the Asian population living in the United States was foreign-born (Council of Economic Advisors 1999). By comparison with overall U.S. data, higher BHg concentrations among Asians and islanders were reported for Taiwan (Hsu et al. 2007), Cambodia (Agusa et al. 2007), Fiji (Kumar et al. 2006), and Tahiti (Dewailly et al. 2008).\nWithin the United States, fish and shellfish consumption, predicting Hg exposure described previously, varies widely, in part a reflection of ethnicity. For example, Asian countries [e.g., Cambodia (Agusa et al. 2007), Taiwan (Hsu et al. 2007; Soong et al. 1991), Japan (Murata et al. 2007; Sakamoto et al. 2007)], island nations [e.g., Bermuda (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004), Seychelles (Myers et al. 2007), Tahiti (Chateau-Degat 2005), Taiwan (Hsu et al. 2007; Soong et al. 1991), Japan (Murata et al. 2007; Sakamoto et al. 2007)], and some European countries [e.g., Spain (Falcó et al. 2006; Herreros et al. 2008) and the Faroe Islands (Weihe et al. 1996)] have reported fish/shellfish consumption levels greater than average worldwide consumption (World Health Organization 2008).","In contrast to some other environmental exposures [e.g., higher blood lead concentrations] (Mahaffey et al. 1982), BHg concentrations increased with income. This is consistent with other studies in which women from higher income groups were at greater risk of MeHg exposure, as were women living in urban areas (Hightower and Moore 2003; Saint-Phard and Van Dorsten 2006).","A more complex association between income and racial/ethnic group may also exist. According to the 1990 U.S. Census (U.S. Census Bureau 2008), the median family income of Japanese-American families exceeded that of non-Hispanic white families. By contrast, the income of Cambodian-American families was lower than that of black families. We could not address whether there is an interaction between belonging to the category designated as “other” and higher income within the NHANES data on BHg levels available at this time, because of sample size limitations.","Our analysis of 30-day Hg intake indicated that there was no consistent trend in fish consumption by women of childbearing age over the 6-year period between 1999 and 2004. Our evaluation of NHANES fish intake data indicated no differences in the mean frequency or amount of particular fish and shellfish species consumed. However, the estimated 30-day Hg intake decreased at the 90th percentile and higher, whereas total fish consumption did not, which suggests a shift in fish species consumed. The BHg data indicated a reduction of the higher end of the distribution of BHg between the first 2-year interval (the 1999 and 2000 examinees) compared with the subsequent 4-year interval (the 2001–2004 examinees).\nThe basis for these differences could possibly reflect spillover from the federal fish advisory program (U.S. EPA 2008b) in terms of total fish and shellfish consumption. A recent analysis of a nationally representative study specifically addressing fish-consumption patterns did not support this suggestion (Bradbury 2007).\nThe four fish species listed in the federal advisory [swordfish, shark, tilefish, and king mackerel (U.S. EPA 2008b)] were rarely reported by the 5,465 women in this analysis. It is clear that these four fish species contributed little to Hg exposure in this general population of U.S. women. Individual states with higher Hg exposures [e.g., Hawaii (Sato et al., 2006), Florida (Karouna-Renier et al. 2008)] and greater fish consumption [Florida (Denger et al. 1994)] have substantially broader fish consumption advisories (e.g., Hawaii and Florida) aimed at reducing Hg exposure from high-Hg–containing species obtained locally (Florida Department of Health 2007; U.S. EPA 2008b). Despite the federal advisory’s emphasis on four species of highly contaminated fish (U.S. EPA 2008b) and the states’ emphasis on game-fish, the most commonly consumed finfish in the United States is tuna. Interpretation of Hg exposure from tuna was complicated by the specific wording of the dietary questions asked of the NHANES examinees, which did not differentiate between light or skipjack tuna and albacore tuna. The latter contains approximately three times more Hg than does the former: 0.38 μg/g for frozen and fresh tuna, 0.35 μg/g for canned albacore, and 0.12 μg/g for canned light tuna (Mahaffey et al. 2008).","During the past decade, the U.S. EPA initiated substantial interventions aimed to reduce Hg releases and exposures (U.S. EPA 2008a) and issued advisories to limit consumption of high-Hg fish (U.S. EPA 2008b). Because of worldwide atmospheric distribution and subsequent deposition of Hg, local conditions and locally caught fish are not the main contributors to Hg intake for most people (Sunderland 2007). Although there are economic indications that consumption of some species of fish may have decreased in response to these advisories (Shimshack et al. 2007), Hg exposures may not follow a similar time trend despite regulatory efforts to reduce Hg exposures. A recent analysis of a nationally representative study specifically addressing fish-consumption patterns did not support this suggestion (Bradbury 2007). Our analysis of NHANES data calculating 30-day Hg intake indicated that there was no consistent trend in fish consumption by women of childbearing age over the 6-year period between 1999 and 2004."],"string":"[\n \"We evaluated data for examinees who participated in NHANES during survey years 1999–2004 to assess the statistical association between seafood consumption and BHg by region of residence, race/ethnicity, and annual income. Further, we examined time trends for both BHg and fish consumption. NHANES is an annual survey conducted by the NCHS. The data include BHg levels, 24-hr dietary recall, and 30-day finfish and shellfish consumption frequency for women 16–49 years of age who reside in the United States. The NHANES sampling frame includes all 50 states. The documentation and publicly available data for NHANES can be found online [Centers for Disease Control and Prevention (CDC) 2006b]. The regional data are not publicly available but can be accessed by special request to the National Center for Health Statistics (NCHS) through its Research Data Center. Procedures for submitting a proposal in order to access data that are not publicly available can be found online (CDC 2006c). We performed all analyses using SAS, version 9.1 (SAS Institute Inc., Cary, NC).\\nFollowing NHANES analytic guidelines (CDC 2006a), we used SAS procedures that accurately incorporate the stratification and multistage sampling of NHANES: Proc SurveyMeans, Proc SurveyReg, and Proc SurveyFreq (SAS Institute Inc.). The weights provided by NCHS compensate for the oversampling of various subpopulations and adjust for nonresponse bias. We used weights for estimating statistics for coastal and non-coastal regions to retain these adjustments. Because we considered the variables of interest (BHg and fish consumption) to be related to some of the factors that were oversampled, we retained these adjustments to minimize the bias in the estimates. For example, NHANES over-sampled Mexican Americans, who also have lower BHg than do other racial/ethnic groups. If the weights were not used to estimate the distribution of BHg, the results would be biased low. We recognize that some bias may remain within the estimates because the weights were not specifically created for the geographic regions of coastal and noncoastal; however, these subdivisions (e.g., coastal, noncoastal, Pacific, Atlantic) are based on counties, the primary sampling units of NHANES (CDC 2006a). All multivariate analyses were done unweighted because factors associated with the dependent variables and for which over-sampling was based were included as covariates and thus adjusted for in the modeling.\\nIn order to estimate long-term Hg intake, we combined data collected through the 24-hr dietary recall and the 30-day fish frequency questionnaire to estimate 30-day Hg intake [for specific methodologies, see Mahaffey and Rice (1997); Mahaffey et al. (2004)]. If we found statistically significant differences in amount of fish consumed per meal from the 24-hr dietary recall by either coastal status (participants who lived in a county that bordered the Pacific or Atlantic Oceans, the Gulf of Mexico, or the Great Lakes vs. those who did not) or data release (1999–2000–2001–2002, or 2003–2004), we calculated separate averages. We generated statistically representative estimates from these data using the statistical weights provided by NCHS and following the relevant analytical guidelines published by NCHS (CDC 2006a).\",\n \"Fish consumption is generally believed to be a major contributor to BHg concentration. We hypothesized that patterns of fish and shellfish consumption would vary between U.S. residents who live on or near the coast (within ~ 25–50 miles) and those who live inland. We further hypothesized that fish consumption patterns, and thus BHg concentrations, may also vary by specific coast (e.g., residents near the Atlantic Coast may have different BHg concentrations than those on the coast of the Gulf of Mexico) and specific inland region (e.g., West vs. Midwest). To test these hypotheses, we categorized NHANES respondents as living in either a coastal or a noncoastal county and further categorized them by eight regions: Atlantic Coast, Northeast, Great Lakes, Midwest, South, Gulf of Mexico, West, and Pacific Coast.\\nThe geographic unit used by NHANES is a county or county equivalent (CDC 2006a); therefore, we limited our definitions of coastal and noncoastal to follow county boundaries. We defined all counties that bordered the Pacific or Atlantic Oceans, the Gulf of Mexico, or any of the Great Lakes as coastal. Additionally, we defined counties that bordered estuaries and bays as coastal, as well as counties whose center point was within approximately 25 miles of any coast even if not directly bordering a coast. [For the list of counties, see Supplemental Material, Table 13 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] We then defined the four coastal regions based on nearest body of water; for example, counties in California, Oregon, Washington, Alaska, and Hawaii that we defined as coastal were categorized as Pacific Coast. We separated noncoastal counties into four inland regions using the U.S. Census regions; for example, noncoastal counties in California, Oregon, Washington, and Alaska along with the entire states of Idaho, Montana, Wyoming, Colorado, New Mexico, Arizona, Utah, and Nevada became the West region (we classified all of Hawaii as coastal). We also designated the entire state of Florida as coastal, split between the Atlantic Coast and the Gulf of Mexico. We designated Miami-Dade County as Atlantic Coast, and Monroe County as Gulf of Mexico. These subdivisions run the risk of small sample sizes; however, the definition of coastal was sufficiently broad to avoid single primary sampling units.\",\n \"Comparisons of the distribution of BHg data with reference values aimed at protecting the fetal nervous system have been made using national-level data (Mahaffey et al. 2004). NHANES data are often used to make population estimates through application of weighting factors to variables of interest such as BHg. Population estimates for U.S. Census regions and their distribution of BHg concentrations indicate that women living in the Northeast had BHg concentrations exceeding levels of concern more often than did women living in the South and West. The lowest Hg exposures were reported among women living in the Midwest.\\nBecause NHANES was not designed to provide population estimates for coastal and noncoastal areas, unbiased estimates for the number of women having BHg concentrations ≥ 3.5 μg/L and ≥ 5.8 μg/L cannot be developed comparing coastal- and noncoastal-residing women. Although the following are not population estimates, they are statistics for a geographic region: Women living in coastal areas were at greater risk of having BHg concentrations ≥ 3.5 μg/L (16.25% for coastal and 5.99% for noncoastal residents) and ≥ 5.8 μg/L (8.11% for coastal and 2.06% for noncoastal residents). Women living near the coastal areas had approximately three to four times greater risk of exceeding acceptable levels of Hg exposure than did noncoastal-dwelling women. There may be some bias in these results due to the weighting issues (see “Materials and Methods”); however, we do not believe that this bias is a major factor underlying these great differences.\\nMeHg exposures exceeding health-based standards, including U.S. EPA’s RfD (Rice et al. 2003), occurred more commonly among women living in coastal areas. These health-based standards were based on avoiding MeHg-associated delays and deficits in neurologic development of children after in utero exposure to MeHg (Mergler et al. 2007; Rice et al. 2003). At higher exposures to MeHg, including the highest concentrations reported during these survey years, the women themselves may risk adverse neuropsychological and neurobehavioral outcomes (Mergler et al. 2007).\\nWithin the United States, people living in coastal areas consume more fish and shellfish than do those living in noncoastal areas and consume fish with higher Hg concentrations. Reports from New York City (McKelvey et al. 2007) and Florida (Denger et al. 1994; Karouna-Renier et al. 2008) support our identification of higher Hg exposures in U.S. coastal areas. This is part of a worldwide pattern. An overall pattern of higher BHg levels has also been reported among people living on U.S. islands [Hawaii (Sato et al. 2006)] and territories [e.g., Puerto Rico (Ortiz-Roque and López-Rivera 2004)]. A similar pattern has been repeated in other islands [Bermuda (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004), Fiji (Kumar et al. 2006), Seychelles (Myers et al. 2007), and Tahiti (Chateau-Degat 2005; Dewailly et al. 2008)] compared with inland populations. Among these island populations, BHg concentrations at the upper end of the distribution fall into the range of 50 μg/L (~ 250 nmol/L) and higher (Chateau-Degat 2005). In Bermuda, cord BHg concentrations as high as 160 nmol/L (~ 35 μg/L) have been reported (arithmetic mean, 41.3 ± 4.7 nmol/L or 8.0 ± 1.0 μg/L) (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004).\\nHigher BHg concentrations in the U.S. Northeast found in this study reflect, in part, more frequent fish and shellfish consumption. Additional variability may be a function of differences in Hg concentrations among species and geographic regions (Sunderland 2007). For example, recent information on “hot spots” for Hg in wildlife tissues (Evers et al. 2007) could be associated with higher Hg concentrations for locally obtained fish. One limitation of the present analysis was the use of a fish-species–specific mean Hg concentration (i.e., nondistributional values) to estimate individual exposure. Although most fish consumed by the U.S. population is not locally obtained (i.e., commercially obtained from diverse regions and countries) (Sunderland 2007), analytical results showing geographic differences in the distribution of BHg could reflect higher Hg concentrations in locally obtained fish within the Northeast states.\",\n \"Ethnic origins were associated with Hg exposures with those designated as “other” (i.e., Asian, Pacific and Caribbean Islander, Native American, Alaska Native, multiracial, and unknown race) having higher BHg concentrations. From additional studies, people of Asian descent whose food choices are influenced by Asian dietary patterns (Kudo et al. 2000; Sechena et al. 2003) tended to consume fish more frequently, in greater variety, and in greater quantity than did non-Asians. The ethnic diversity of the U.S. population is well known. As of 1997, 61% of the Asian population living in the United States was foreign-born (Council of Economic Advisors 1999). By comparison with overall U.S. data, higher BHg concentrations among Asians and islanders were reported for Taiwan (Hsu et al. 2007), Cambodia (Agusa et al. 2007), Fiji (Kumar et al. 2006), and Tahiti (Dewailly et al. 2008).\\nWithin the United States, fish and shellfish consumption, predicting Hg exposure described previously, varies widely, in part a reflection of ethnicity. For example, Asian countries [e.g., Cambodia (Agusa et al. 2007), Taiwan (Hsu et al. 2007; Soong et al. 1991), Japan (Murata et al. 2007; Sakamoto et al. 2007)], island nations [e.g., Bermuda (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004), Seychelles (Myers et al. 2007), Tahiti (Chateau-Degat 2005), Taiwan (Hsu et al. 2007; Soong et al. 1991), Japan (Murata et al. 2007; Sakamoto et al. 2007)], and some European countries [e.g., Spain (Falcó et al. 2006; Herreros et al. 2008) and the Faroe Islands (Weihe et al. 1996)] have reported fish/shellfish consumption levels greater than average worldwide consumption (World Health Organization 2008).\",\n \"In contrast to some other environmental exposures [e.g., higher blood lead concentrations] (Mahaffey et al. 1982), BHg concentrations increased with income. This is consistent with other studies in which women from higher income groups were at greater risk of MeHg exposure, as were women living in urban areas (Hightower and Moore 2003; Saint-Phard and Van Dorsten 2006).\",\n \"A more complex association between income and racial/ethnic group may also exist. According to the 1990 U.S. Census (U.S. Census Bureau 2008), the median family income of Japanese-American families exceeded that of non-Hispanic white families. By contrast, the income of Cambodian-American families was lower than that of black families. We could not address whether there is an interaction between belonging to the category designated as “other” and higher income within the NHANES data on BHg levels available at this time, because of sample size limitations.\",\n \"Our analysis of 30-day Hg intake indicated that there was no consistent trend in fish consumption by women of childbearing age over the 6-year period between 1999 and 2004. Our evaluation of NHANES fish intake data indicated no differences in the mean frequency or amount of particular fish and shellfish species consumed. However, the estimated 30-day Hg intake decreased at the 90th percentile and higher, whereas total fish consumption did not, which suggests a shift in fish species consumed. The BHg data indicated a reduction of the higher end of the distribution of BHg between the first 2-year interval (the 1999 and 2000 examinees) compared with the subsequent 4-year interval (the 2001–2004 examinees).\\nThe basis for these differences could possibly reflect spillover from the federal fish advisory program (U.S. EPA 2008b) in terms of total fish and shellfish consumption. A recent analysis of a nationally representative study specifically addressing fish-consumption patterns did not support this suggestion (Bradbury 2007).\\nThe four fish species listed in the federal advisory [swordfish, shark, tilefish, and king mackerel (U.S. EPA 2008b)] were rarely reported by the 5,465 women in this analysis. It is clear that these four fish species contributed little to Hg exposure in this general population of U.S. women. Individual states with higher Hg exposures [e.g., Hawaii (Sato et al., 2006), Florida (Karouna-Renier et al. 2008)] and greater fish consumption [Florida (Denger et al. 1994)] have substantially broader fish consumption advisories (e.g., Hawaii and Florida) aimed at reducing Hg exposure from high-Hg–containing species obtained locally (Florida Department of Health 2007; U.S. EPA 2008b). Despite the federal advisory’s emphasis on four species of highly contaminated fish (U.S. EPA 2008b) and the states’ emphasis on game-fish, the most commonly consumed finfish in the United States is tuna. Interpretation of Hg exposure from tuna was complicated by the specific wording of the dietary questions asked of the NHANES examinees, which did not differentiate between light or skipjack tuna and albacore tuna. The latter contains approximately three times more Hg than does the former: 0.38 μg/g for frozen and fresh tuna, 0.35 μg/g for canned albacore, and 0.12 μg/g for canned light tuna (Mahaffey et al. 2008).\",\n \"During the past decade, the U.S. EPA initiated substantial interventions aimed to reduce Hg releases and exposures (U.S. EPA 2008a) and issued advisories to limit consumption of high-Hg fish (U.S. EPA 2008b). Because of worldwide atmospheric distribution and subsequent deposition of Hg, local conditions and locally caught fish are not the main contributors to Hg intake for most people (Sunderland 2007). Although there are economic indications that consumption of some species of fish may have decreased in response to these advisories (Shimshack et al. 2007), Hg exposures may not follow a similar time trend despite regulatory efforts to reduce Hg exposures. A recent analysis of a nationally representative study specifically addressing fish-consumption patterns did not support this suggestion (Bradbury 2007). Our analysis of NHANES data calculating 30-day Hg intake indicated that there was no consistent trend in fish consumption by women of childbearing age over the 6-year period between 1999 and 2004.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Materials and Methods","Methodology for data analysis","Methodology for defining coastal and non-coastal areas","Results","Discussion","Regional and coastal variation in BHg concentrations and in fish consumption","Ethnic group variation on fish intake and BHg concentrations","Income differences in association with fish intake and BHg concentrations","Interactions between income and ethnic group","Time trends in Hg exposure absent changes in total fish consumption","Changes in MeHg exposure over time","Conclusions"],"string":"[\n \"Materials and Methods\",\n \"Methodology for data analysis\",\n \"Methodology for defining coastal and non-coastal areas\",\n \"Results\",\n \"Discussion\",\n \"Regional and coastal variation in BHg concentrations and in fish consumption\",\n \"Ethnic group variation on fish intake and BHg concentrations\",\n \"Income differences in association with fish intake and BHg concentrations\",\n \"Interactions between income and ethnic group\",\n \"Time trends in Hg exposure absent changes in total fish consumption\",\n \"Changes in MeHg exposure over time\",\n \"Conclusions\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":[" Methodology for data analysis We evaluated data for examinees who participated in NHANES during survey years 1999–2004 to assess the statistical association between seafood consumption and BHg by region of residence, race/ethnicity, and annual income. Further, we examined time trends for both BHg and fish consumption. NHANES is an annual survey conducted by the NCHS. The data include BHg levels, 24-hr dietary recall, and 30-day finfish and shellfish consumption frequency for women 16–49 years of age who reside in the United States. The NHANES sampling frame includes all 50 states. The documentation and publicly available data for NHANES can be found online [Centers for Disease Control and Prevention (CDC) 2006b]. The regional data are not publicly available but can be accessed by special request to the National Center for Health Statistics (NCHS) through its Research Data Center. Procedures for submitting a proposal in order to access data that are not publicly available can be found online (CDC 2006c). We performed all analyses using SAS, version 9.1 (SAS Institute Inc., Cary, NC).\nFollowing NHANES analytic guidelines (CDC 2006a), we used SAS procedures that accurately incorporate the stratification and multistage sampling of NHANES: Proc SurveyMeans, Proc SurveyReg, and Proc SurveyFreq (SAS Institute Inc.). The weights provided by NCHS compensate for the oversampling of various subpopulations and adjust for nonresponse bias. We used weights for estimating statistics for coastal and non-coastal regions to retain these adjustments. Because we considered the variables of interest (BHg and fish consumption) to be related to some of the factors that were oversampled, we retained these adjustments to minimize the bias in the estimates. For example, NHANES over-sampled Mexican Americans, who also have lower BHg than do other racial/ethnic groups. If the weights were not used to estimate the distribution of BHg, the results would be biased low. We recognize that some bias may remain within the estimates because the weights were not specifically created for the geographic regions of coastal and noncoastal; however, these subdivisions (e.g., coastal, noncoastal, Pacific, Atlantic) are based on counties, the primary sampling units of NHANES (CDC 2006a). All multivariate analyses were done unweighted because factors associated with the dependent variables and for which over-sampling was based were included as covariates and thus adjusted for in the modeling.\nIn order to estimate long-term Hg intake, we combined data collected through the 24-hr dietary recall and the 30-day fish frequency questionnaire to estimate 30-day Hg intake [for specific methodologies, see Mahaffey and Rice (1997); Mahaffey et al. (2004)]. If we found statistically significant differences in amount of fish consumed per meal from the 24-hr dietary recall by either coastal status (participants who lived in a county that bordered the Pacific or Atlantic Oceans, the Gulf of Mexico, or the Great Lakes vs. those who did not) or data release (1999–2000–2001–2002, or 2003–2004), we calculated separate averages. We generated statistically representative estimates from these data using the statistical weights provided by NCHS and following the relevant analytical guidelines published by NCHS (CDC 2006a).\nWe evaluated data for examinees who participated in NHANES during survey years 1999–2004 to assess the statistical association between seafood consumption and BHg by region of residence, race/ethnicity, and annual income. Further, we examined time trends for both BHg and fish consumption. NHANES is an annual survey conducted by the NCHS. The data include BHg levels, 24-hr dietary recall, and 30-day finfish and shellfish consumption frequency for women 16–49 years of age who reside in the United States. The NHANES sampling frame includes all 50 states. The documentation and publicly available data for NHANES can be found online [Centers for Disease Control and Prevention (CDC) 2006b]. The regional data are not publicly available but can be accessed by special request to the National Center for Health Statistics (NCHS) through its Research Data Center. Procedures for submitting a proposal in order to access data that are not publicly available can be found online (CDC 2006c). We performed all analyses using SAS, version 9.1 (SAS Institute Inc., Cary, NC).\nFollowing NHANES analytic guidelines (CDC 2006a), we used SAS procedures that accurately incorporate the stratification and multistage sampling of NHANES: Proc SurveyMeans, Proc SurveyReg, and Proc SurveyFreq (SAS Institute Inc.). The weights provided by NCHS compensate for the oversampling of various subpopulations and adjust for nonresponse bias. We used weights for estimating statistics for coastal and non-coastal regions to retain these adjustments. Because we considered the variables of interest (BHg and fish consumption) to be related to some of the factors that were oversampled, we retained these adjustments to minimize the bias in the estimates. For example, NHANES over-sampled Mexican Americans, who also have lower BHg than do other racial/ethnic groups. If the weights were not used to estimate the distribution of BHg, the results would be biased low. We recognize that some bias may remain within the estimates because the weights were not specifically created for the geographic regions of coastal and noncoastal; however, these subdivisions (e.g., coastal, noncoastal, Pacific, Atlantic) are based on counties, the primary sampling units of NHANES (CDC 2006a). All multivariate analyses were done unweighted because factors associated with the dependent variables and for which over-sampling was based were included as covariates and thus adjusted for in the modeling.\nIn order to estimate long-term Hg intake, we combined data collected through the 24-hr dietary recall and the 30-day fish frequency questionnaire to estimate 30-day Hg intake [for specific methodologies, see Mahaffey and Rice (1997); Mahaffey et al. (2004)]. If we found statistically significant differences in amount of fish consumed per meal from the 24-hr dietary recall by either coastal status (participants who lived in a county that bordered the Pacific or Atlantic Oceans, the Gulf of Mexico, or the Great Lakes vs. those who did not) or data release (1999–2000–2001–2002, or 2003–2004), we calculated separate averages. We generated statistically representative estimates from these data using the statistical weights provided by NCHS and following the relevant analytical guidelines published by NCHS (CDC 2006a).\n Methodology for defining coastal and non-coastal areas Fish consumption is generally believed to be a major contributor to BHg concentration. We hypothesized that patterns of fish and shellfish consumption would vary between U.S. residents who live on or near the coast (within ~ 25–50 miles) and those who live inland. We further hypothesized that fish consumption patterns, and thus BHg concentrations, may also vary by specific coast (e.g., residents near the Atlantic Coast may have different BHg concentrations than those on the coast of the Gulf of Mexico) and specific inland region (e.g., West vs. Midwest). To test these hypotheses, we categorized NHANES respondents as living in either a coastal or a noncoastal county and further categorized them by eight regions: Atlantic Coast, Northeast, Great Lakes, Midwest, South, Gulf of Mexico, West, and Pacific Coast.\nThe geographic unit used by NHANES is a county or county equivalent (CDC 2006a); therefore, we limited our definitions of coastal and noncoastal to follow county boundaries. We defined all counties that bordered the Pacific or Atlantic Oceans, the Gulf of Mexico, or any of the Great Lakes as coastal. Additionally, we defined counties that bordered estuaries and bays as coastal, as well as counties whose center point was within approximately 25 miles of any coast even if not directly bordering a coast. [For the list of counties, see Supplemental Material, Table 13 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] We then defined the four coastal regions based on nearest body of water; for example, counties in California, Oregon, Washington, Alaska, and Hawaii that we defined as coastal were categorized as Pacific Coast. We separated noncoastal counties into four inland regions using the U.S. Census regions; for example, noncoastal counties in California, Oregon, Washington, and Alaska along with the entire states of Idaho, Montana, Wyoming, Colorado, New Mexico, Arizona, Utah, and Nevada became the West region (we classified all of Hawaii as coastal). We also designated the entire state of Florida as coastal, split between the Atlantic Coast and the Gulf of Mexico. We designated Miami-Dade County as Atlantic Coast, and Monroe County as Gulf of Mexico. These subdivisions run the risk of small sample sizes; however, the definition of coastal was sufficiently broad to avoid single primary sampling units.\nFish consumption is generally believed to be a major contributor to BHg concentration. We hypothesized that patterns of fish and shellfish consumption would vary between U.S. residents who live on or near the coast (within ~ 25–50 miles) and those who live inland. We further hypothesized that fish consumption patterns, and thus BHg concentrations, may also vary by specific coast (e.g., residents near the Atlantic Coast may have different BHg concentrations than those on the coast of the Gulf of Mexico) and specific inland region (e.g., West vs. Midwest). To test these hypotheses, we categorized NHANES respondents as living in either a coastal or a noncoastal county and further categorized them by eight regions: Atlantic Coast, Northeast, Great Lakes, Midwest, South, Gulf of Mexico, West, and Pacific Coast.\nThe geographic unit used by NHANES is a county or county equivalent (CDC 2006a); therefore, we limited our definitions of coastal and noncoastal to follow county boundaries. We defined all counties that bordered the Pacific or Atlantic Oceans, the Gulf of Mexico, or any of the Great Lakes as coastal. Additionally, we defined counties that bordered estuaries and bays as coastal, as well as counties whose center point was within approximately 25 miles of any coast even if not directly bordering a coast. [For the list of counties, see Supplemental Material, Table 13 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] We then defined the four coastal regions based on nearest body of water; for example, counties in California, Oregon, Washington, Alaska, and Hawaii that we defined as coastal were categorized as Pacific Coast. We separated noncoastal counties into four inland regions using the U.S. Census regions; for example, noncoastal counties in California, Oregon, Washington, and Alaska along with the entire states of Idaho, Montana, Wyoming, Colorado, New Mexico, Arizona, Utah, and Nevada became the West region (we classified all of Hawaii as coastal). We also designated the entire state of Florida as coastal, split between the Atlantic Coast and the Gulf of Mexico. We designated Miami-Dade County as Atlantic Coast, and Monroe County as Gulf of Mexico. These subdivisions run the risk of small sample sizes; however, the definition of coastal was sufficiently broad to avoid single primary sampling units.","We evaluated data for examinees who participated in NHANES during survey years 1999–2004 to assess the statistical association between seafood consumption and BHg by region of residence, race/ethnicity, and annual income. Further, we examined time trends for both BHg and fish consumption. NHANES is an annual survey conducted by the NCHS. The data include BHg levels, 24-hr dietary recall, and 30-day finfish and shellfish consumption frequency for women 16–49 years of age who reside in the United States. The NHANES sampling frame includes all 50 states. The documentation and publicly available data for NHANES can be found online [Centers for Disease Control and Prevention (CDC) 2006b]. The regional data are not publicly available but can be accessed by special request to the National Center for Health Statistics (NCHS) through its Research Data Center. Procedures for submitting a proposal in order to access data that are not publicly available can be found online (CDC 2006c). We performed all analyses using SAS, version 9.1 (SAS Institute Inc., Cary, NC).\nFollowing NHANES analytic guidelines (CDC 2006a), we used SAS procedures that accurately incorporate the stratification and multistage sampling of NHANES: Proc SurveyMeans, Proc SurveyReg, and Proc SurveyFreq (SAS Institute Inc.). The weights provided by NCHS compensate for the oversampling of various subpopulations and adjust for nonresponse bias. We used weights for estimating statistics for coastal and non-coastal regions to retain these adjustments. Because we considered the variables of interest (BHg and fish consumption) to be related to some of the factors that were oversampled, we retained these adjustments to minimize the bias in the estimates. For example, NHANES over-sampled Mexican Americans, who also have lower BHg than do other racial/ethnic groups. If the weights were not used to estimate the distribution of BHg, the results would be biased low. We recognize that some bias may remain within the estimates because the weights were not specifically created for the geographic regions of coastal and noncoastal; however, these subdivisions (e.g., coastal, noncoastal, Pacific, Atlantic) are based on counties, the primary sampling units of NHANES (CDC 2006a). All multivariate analyses were done unweighted because factors associated with the dependent variables and for which over-sampling was based were included as covariates and thus adjusted for in the modeling.\nIn order to estimate long-term Hg intake, we combined data collected through the 24-hr dietary recall and the 30-day fish frequency questionnaire to estimate 30-day Hg intake [for specific methodologies, see Mahaffey and Rice (1997); Mahaffey et al. (2004)]. If we found statistically significant differences in amount of fish consumed per meal from the 24-hr dietary recall by either coastal status (participants who lived in a county that bordered the Pacific or Atlantic Oceans, the Gulf of Mexico, or the Great Lakes vs. those who did not) or data release (1999–2000–2001–2002, or 2003–2004), we calculated separate averages. We generated statistically representative estimates from these data using the statistical weights provided by NCHS and following the relevant analytical guidelines published by NCHS (CDC 2006a).","Fish consumption is generally believed to be a major contributor to BHg concentration. We hypothesized that patterns of fish and shellfish consumption would vary between U.S. residents who live on or near the coast (within ~ 25–50 miles) and those who live inland. We further hypothesized that fish consumption patterns, and thus BHg concentrations, may also vary by specific coast (e.g., residents near the Atlantic Coast may have different BHg concentrations than those on the coast of the Gulf of Mexico) and specific inland region (e.g., West vs. Midwest). To test these hypotheses, we categorized NHANES respondents as living in either a coastal or a noncoastal county and further categorized them by eight regions: Atlantic Coast, Northeast, Great Lakes, Midwest, South, Gulf of Mexico, West, and Pacific Coast.\nThe geographic unit used by NHANES is a county or county equivalent (CDC 2006a); therefore, we limited our definitions of coastal and noncoastal to follow county boundaries. We defined all counties that bordered the Pacific or Atlantic Oceans, the Gulf of Mexico, or any of the Great Lakes as coastal. Additionally, we defined counties that bordered estuaries and bays as coastal, as well as counties whose center point was within approximately 25 miles of any coast even if not directly bordering a coast. [For the list of counties, see Supplemental Material, Table 13 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] We then defined the four coastal regions based on nearest body of water; for example, counties in California, Oregon, Washington, Alaska, and Hawaii that we defined as coastal were categorized as Pacific Coast. We separated noncoastal counties into four inland regions using the U.S. Census regions; for example, noncoastal counties in California, Oregon, Washington, and Alaska along with the entire states of Idaho, Montana, Wyoming, Colorado, New Mexico, Arizona, Utah, and Nevada became the West region (we classified all of Hawaii as coastal). We also designated the entire state of Florida as coastal, split between the Atlantic Coast and the Gulf of Mexico. We designated Miami-Dade County as Atlantic Coast, and Monroe County as Gulf of Mexico. These subdivisions run the risk of small sample sizes; however, the definition of coastal was sufficiently broad to avoid single primary sampling units.","Table 1 shows the distributions of estimates of the number of women with BHg concentrations > 3.5 μg/L and > 5.8 μg/L, by region. Analyses indicate that between 1999 and 2004, the Northeast had the highest percentage of women with BHg concentrations above the 3.5 μg/L level of concern (> 19%), whereas the South had the largest estimated number of women (1.21 million) with ≥ 3.5 μg/L BHg because of elevated population in that region. Geometric means (Figure 1) show similar trends, with the highest BHg concentrations in the Northeast, followed by the West, South, and Midwest census regions. In the Northeast, the highest 5% of BHg concentrations exceeded 8.2 μg/L. [For full distributions, see Supplemental Material, Table 2 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] When we included coastal regions in this analysis, additional spatial heterogeneity in BHg (Figure 2A) and estimated 30-day Hg intake (Figure 2B) was apparent, with elevated exposures in all coastal areas relative to their neighboring inland regions except in the Great Lakes. In the coastal areas, the highest 5% of BHg concentrations exceeded 7.2 μg/L, with the Atlantic Coast exceeding 10.9 μg/L. [For the full distributions, see Supplemental Material, Table 4 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] Fish species eaten by survey participants varied by region (Figure 3), with respondents in coastal regions reporting higher frequency of consumption of fish containing higher levels of Hg. BHg concentrations were strongly associated with the frequency of fish consumption (Figure 4). BHg increased with monthly estimated consumption of fish and shellfish over the range of never/rarely to 4 or more times per week. In multiple regression modeling, women from the Atlantic (p < 0.01), Pacific (p < 0.0001), and Gulf (p < 0.0001) coasts had higher BHg concentrations compared with women from the inland West, whereas women from the inland Northeast and inland Midwest had significantly lower BHg levels (p < 0.0001). [For the full regression results, see Supplemental Material, Table 11 (http://www.ehponline.org/members/2008/11674/suppl.pdf).]\nAnalysis of temporal trends through simple regression modeling showed no statistically significant difference among the three sets of study years (1999–2000–2001–2002, and 2003–2004) for BHg (p = 0.07), estimated 30-day Hg intake (p = 0.11), or reported frequency of seafood consumption (p = 0.69). However, in multiple regression modeling, adjusting for covariates including coastal/non-coastal residence, the years 1999–2000 had significantly higher BHg levels (p < 0.0001) compared with 2003–2004, and 2001–2002 had significantly lower BHg levels (p < 0.01) [Supplemental Material, Table 11 (http://www.ehponline.org/members/2008/11674/suppl.pdf)]. Although the analyses did not support the conclusion that there was a general downward trend in BHg concentrations over the 6-year study period, there was a decline in the upper percentiles reflecting the most highly exposed women with BHg concentrations greater than established levels of concern [Supplemental Material, Table 9 (http://www.ehponline.org/members/2008/11674/suppl.pdf)]. In addition, the percentage of examinees with BHg values ≥ 3.5 μg/L and ≥ 5.8 μg/L was much greater in 1999–2000 compared to 2001–2002 and 2003–2004 (Figure 5). We found no consistent trend in fish consumption across the study years. We observed a decrease in the 90th percentile of 30-day estimated intake of Hg through seafood consumption across the study years even though there was no similar decrease in the 90th percentile of 30-day estimated consumption of grams of fish and shellfish (Figure 6). This suggests a shift in consumption to seafood containing less Hg. We did not observe a similar pattern at the mean, suggesting that this shift in seafood consumption occurred mainly with the highest fish and shellfish consumers [Supplemental Material, Table 9 (http://www.ehponline.org/members/2008/11674/suppl.pdf)]. The RfD for Hg intake is 0.1 μg Hg/kg body weight (μg/kgbw) per day, or 3.0 μg Hg/kgbw per month (30 days).\nResults also showed that self-selected ethnic identity was associated with total BHg concentrations, estimated 30-day Hg intake, and frequency of either finfish or shellfish consumption. For example, BHg levels, reported frequency of seafood consumption, and 30-day Hg intakes were highest among women who designated themselves as being in the “other” category (mostly people whose ancestry is Asian, Native American, Pacific Islands, and the Caribbean Islands). [See also Supplemental Material, Tables 5–7 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] Table 2 presents the percentages of women by race/ethnicity that had ≥ 3.5 and ≥ 5.8 μg/L BHg.\nWe identified statistically significant relationships between higher income and, respectively, increasing BHg concentration (p < 0.0001), estimated 30-day intake of Hg (p = 0.008), and 30-day frequency of finfish and shellfish consumption (p < 0.0001) through bivariate regressions. [For the distributions of blood total Hg, estimated Hg intake, and frequency of finfish and shellfish consumption by annual income, see Supplemental Material, Tables 6–8 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] In addition, women from families reporting incomes of ≥ $75,000 (the reference category) had statistically higher BHg levels than did women from families with incomes of ≤ $55,000 (p < 0.01). In all cases, BHg concentrations were also significantly associated with age and estimated 30-day Hg intake (p < 0.0001). Table 3 presents the percentage of women by annual income with ≥ 3.5 and ≥ 5.8 μg/L BHg.\nIn multiple regression modeling, after adjusting for other factors related to BHg, both race/ethnicity and income remained statistically significant predictors of BHg levels observed in this study [Supplemental Material, Table 11 (http://www.ehponline.org/members/2008/11674/suppl.pdf)]. Non-Hispanic blacks (p < 0.0001) and women grouped in the “other” racial category (p = 0.002) had significantly higher BHg concentrations than did non-Hispanic whites."," Regional and coastal variation in BHg concentrations and in fish consumption Comparisons of the distribution of BHg data with reference values aimed at protecting the fetal nervous system have been made using national-level data (Mahaffey et al. 2004). NHANES data are often used to make population estimates through application of weighting factors to variables of interest such as BHg. Population estimates for U.S. Census regions and their distribution of BHg concentrations indicate that women living in the Northeast had BHg concentrations exceeding levels of concern more often than did women living in the South and West. The lowest Hg exposures were reported among women living in the Midwest.\nBecause NHANES was not designed to provide population estimates for coastal and noncoastal areas, unbiased estimates for the number of women having BHg concentrations ≥ 3.5 μg/L and ≥ 5.8 μg/L cannot be developed comparing coastal- and noncoastal-residing women. Although the following are not population estimates, they are statistics for a geographic region: Women living in coastal areas were at greater risk of having BHg concentrations ≥ 3.5 μg/L (16.25% for coastal and 5.99% for noncoastal residents) and ≥ 5.8 μg/L (8.11% for coastal and 2.06% for noncoastal residents). Women living near the coastal areas had approximately three to four times greater risk of exceeding acceptable levels of Hg exposure than did noncoastal-dwelling women. There may be some bias in these results due to the weighting issues (see “Materials and Methods”); however, we do not believe that this bias is a major factor underlying these great differences.\nMeHg exposures exceeding health-based standards, including U.S. EPA’s RfD (Rice et al. 2003), occurred more commonly among women living in coastal areas. These health-based standards were based on avoiding MeHg-associated delays and deficits in neurologic development of children after in utero exposure to MeHg (Mergler et al. 2007; Rice et al. 2003). At higher exposures to MeHg, including the highest concentrations reported during these survey years, the women themselves may risk adverse neuropsychological and neurobehavioral outcomes (Mergler et al. 2007).\nWithin the United States, people living in coastal areas consume more fish and shellfish than do those living in noncoastal areas and consume fish with higher Hg concentrations. Reports from New York City (McKelvey et al. 2007) and Florida (Denger et al. 1994; Karouna-Renier et al. 2008) support our identification of higher Hg exposures in U.S. coastal areas. This is part of a worldwide pattern. An overall pattern of higher BHg levels has also been reported among people living on U.S. islands [Hawaii (Sato et al. 2006)] and territories [e.g., Puerto Rico (Ortiz-Roque and López-Rivera 2004)]. A similar pattern has been repeated in other islands [Bermuda (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004), Fiji (Kumar et al. 2006), Seychelles (Myers et al. 2007), and Tahiti (Chateau-Degat 2005; Dewailly et al. 2008)] compared with inland populations. Among these island populations, BHg concentrations at the upper end of the distribution fall into the range of 50 μg/L (~ 250 nmol/L) and higher (Chateau-Degat 2005). In Bermuda, cord BHg concentrations as high as 160 nmol/L (~ 35 μg/L) have been reported (arithmetic mean, 41.3 ± 4.7 nmol/L or 8.0 ± 1.0 μg/L) (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004).\nHigher BHg concentrations in the U.S. Northeast found in this study reflect, in part, more frequent fish and shellfish consumption. Additional variability may be a function of differences in Hg concentrations among species and geographic regions (Sunderland 2007). For example, recent information on “hot spots” for Hg in wildlife tissues (Evers et al. 2007) could be associated with higher Hg concentrations for locally obtained fish. One limitation of the present analysis was the use of a fish-species–specific mean Hg concentration (i.e., nondistributional values) to estimate individual exposure. Although most fish consumed by the U.S. population is not locally obtained (i.e., commercially obtained from diverse regions and countries) (Sunderland 2007), analytical results showing geographic differences in the distribution of BHg could reflect higher Hg concentrations in locally obtained fish within the Northeast states.\nComparisons of the distribution of BHg data with reference values aimed at protecting the fetal nervous system have been made using national-level data (Mahaffey et al. 2004). NHANES data are often used to make population estimates through application of weighting factors to variables of interest such as BHg. Population estimates for U.S. Census regions and their distribution of BHg concentrations indicate that women living in the Northeast had BHg concentrations exceeding levels of concern more often than did women living in the South and West. The lowest Hg exposures were reported among women living in the Midwest.\nBecause NHANES was not designed to provide population estimates for coastal and noncoastal areas, unbiased estimates for the number of women having BHg concentrations ≥ 3.5 μg/L and ≥ 5.8 μg/L cannot be developed comparing coastal- and noncoastal-residing women. Although the following are not population estimates, they are statistics for a geographic region: Women living in coastal areas were at greater risk of having BHg concentrations ≥ 3.5 μg/L (16.25% for coastal and 5.99% for noncoastal residents) and ≥ 5.8 μg/L (8.11% for coastal and 2.06% for noncoastal residents). Women living near the coastal areas had approximately three to four times greater risk of exceeding acceptable levels of Hg exposure than did noncoastal-dwelling women. There may be some bias in these results due to the weighting issues (see “Materials and Methods”); however, we do not believe that this bias is a major factor underlying these great differences.\nMeHg exposures exceeding health-based standards, including U.S. EPA’s RfD (Rice et al. 2003), occurred more commonly among women living in coastal areas. These health-based standards were based on avoiding MeHg-associated delays and deficits in neurologic development of children after in utero exposure to MeHg (Mergler et al. 2007; Rice et al. 2003). At higher exposures to MeHg, including the highest concentrations reported during these survey years, the women themselves may risk adverse neuropsychological and neurobehavioral outcomes (Mergler et al. 2007).\nWithin the United States, people living in coastal areas consume more fish and shellfish than do those living in noncoastal areas and consume fish with higher Hg concentrations. Reports from New York City (McKelvey et al. 2007) and Florida (Denger et al. 1994; Karouna-Renier et al. 2008) support our identification of higher Hg exposures in U.S. coastal areas. This is part of a worldwide pattern. An overall pattern of higher BHg levels has also been reported among people living on U.S. islands [Hawaii (Sato et al. 2006)] and territories [e.g., Puerto Rico (Ortiz-Roque and López-Rivera 2004)]. A similar pattern has been repeated in other islands [Bermuda (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004), Fiji (Kumar et al. 2006), Seychelles (Myers et al. 2007), and Tahiti (Chateau-Degat 2005; Dewailly et al. 2008)] compared with inland populations. Among these island populations, BHg concentrations at the upper end of the distribution fall into the range of 50 μg/L (~ 250 nmol/L) and higher (Chateau-Degat 2005). In Bermuda, cord BHg concentrations as high as 160 nmol/L (~ 35 μg/L) have been reported (arithmetic mean, 41.3 ± 4.7 nmol/L or 8.0 ± 1.0 μg/L) (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004).\nHigher BHg concentrations in the U.S. Northeast found in this study reflect, in part, more frequent fish and shellfish consumption. Additional variability may be a function of differences in Hg concentrations among species and geographic regions (Sunderland 2007). For example, recent information on “hot spots” for Hg in wildlife tissues (Evers et al. 2007) could be associated with higher Hg concentrations for locally obtained fish. One limitation of the present analysis was the use of a fish-species–specific mean Hg concentration (i.e., nondistributional values) to estimate individual exposure. Although most fish consumed by the U.S. population is not locally obtained (i.e., commercially obtained from diverse regions and countries) (Sunderland 2007), analytical results showing geographic differences in the distribution of BHg could reflect higher Hg concentrations in locally obtained fish within the Northeast states.\n Ethnic group variation on fish intake and BHg concentrations Ethnic origins were associated with Hg exposures with those designated as “other” (i.e., Asian, Pacific and Caribbean Islander, Native American, Alaska Native, multiracial, and unknown race) having higher BHg concentrations. From additional studies, people of Asian descent whose food choices are influenced by Asian dietary patterns (Kudo et al. 2000; Sechena et al. 2003) tended to consume fish more frequently, in greater variety, and in greater quantity than did non-Asians. The ethnic diversity of the U.S. population is well known. As of 1997, 61% of the Asian population living in the United States was foreign-born (Council of Economic Advisors 1999). By comparison with overall U.S. data, higher BHg concentrations among Asians and islanders were reported for Taiwan (Hsu et al. 2007), Cambodia (Agusa et al. 2007), Fiji (Kumar et al. 2006), and Tahiti (Dewailly et al. 2008).\nWithin the United States, fish and shellfish consumption, predicting Hg exposure described previously, varies widely, in part a reflection of ethnicity. For example, Asian countries [e.g., Cambodia (Agusa et al. 2007), Taiwan (Hsu et al. 2007; Soong et al. 1991), Japan (Murata et al. 2007; Sakamoto et al. 2007)], island nations [e.g., Bermuda (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004), Seychelles (Myers et al. 2007), Tahiti (Chateau-Degat 2005), Taiwan (Hsu et al. 2007; Soong et al. 1991), Japan (Murata et al. 2007; Sakamoto et al. 2007)], and some European countries [e.g., Spain (Falcó et al. 2006; Herreros et al. 2008) and the Faroe Islands (Weihe et al. 1996)] have reported fish/shellfish consumption levels greater than average worldwide consumption (World Health Organization 2008).\nEthnic origins were associated with Hg exposures with those designated as “other” (i.e., Asian, Pacific and Caribbean Islander, Native American, Alaska Native, multiracial, and unknown race) having higher BHg concentrations. From additional studies, people of Asian descent whose food choices are influenced by Asian dietary patterns (Kudo et al. 2000; Sechena et al. 2003) tended to consume fish more frequently, in greater variety, and in greater quantity than did non-Asians. The ethnic diversity of the U.S. population is well known. As of 1997, 61% of the Asian population living in the United States was foreign-born (Council of Economic Advisors 1999). By comparison with overall U.S. data, higher BHg concentrations among Asians and islanders were reported for Taiwan (Hsu et al. 2007), Cambodia (Agusa et al. 2007), Fiji (Kumar et al. 2006), and Tahiti (Dewailly et al. 2008).\nWithin the United States, fish and shellfish consumption, predicting Hg exposure described previously, varies widely, in part a reflection of ethnicity. For example, Asian countries [e.g., Cambodia (Agusa et al. 2007), Taiwan (Hsu et al. 2007; Soong et al. 1991), Japan (Murata et al. 2007; Sakamoto et al. 2007)], island nations [e.g., Bermuda (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004), Seychelles (Myers et al. 2007), Tahiti (Chateau-Degat 2005), Taiwan (Hsu et al. 2007; Soong et al. 1991), Japan (Murata et al. 2007; Sakamoto et al. 2007)], and some European countries [e.g., Spain (Falcó et al. 2006; Herreros et al. 2008) and the Faroe Islands (Weihe et al. 1996)] have reported fish/shellfish consumption levels greater than average worldwide consumption (World Health Organization 2008).\n Income differences in association with fish intake and BHg concentrations In contrast to some other environmental exposures [e.g., higher blood lead concentrations] (Mahaffey et al. 1982), BHg concentrations increased with income. This is consistent with other studies in which women from higher income groups were at greater risk of MeHg exposure, as were women living in urban areas (Hightower and Moore 2003; Saint-Phard and Van Dorsten 2006).\nIn contrast to some other environmental exposures [e.g., higher blood lead concentrations] (Mahaffey et al. 1982), BHg concentrations increased with income. This is consistent with other studies in which women from higher income groups were at greater risk of MeHg exposure, as were women living in urban areas (Hightower and Moore 2003; Saint-Phard and Van Dorsten 2006).\n Interactions between income and ethnic group A more complex association between income and racial/ethnic group may also exist. According to the 1990 U.S. Census (U.S. Census Bureau 2008), the median family income of Japanese-American families exceeded that of non-Hispanic white families. By contrast, the income of Cambodian-American families was lower than that of black families. We could not address whether there is an interaction between belonging to the category designated as “other” and higher income within the NHANES data on BHg levels available at this time, because of sample size limitations.\nA more complex association between income and racial/ethnic group may also exist. According to the 1990 U.S. Census (U.S. Census Bureau 2008), the median family income of Japanese-American families exceeded that of non-Hispanic white families. By contrast, the income of Cambodian-American families was lower than that of black families. We could not address whether there is an interaction between belonging to the category designated as “other” and higher income within the NHANES data on BHg levels available at this time, because of sample size limitations.\n Time trends in Hg exposure absent changes in total fish consumption Our analysis of 30-day Hg intake indicated that there was no consistent trend in fish consumption by women of childbearing age over the 6-year period between 1999 and 2004. Our evaluation of NHANES fish intake data indicated no differences in the mean frequency or amount of particular fish and shellfish species consumed. However, the estimated 30-day Hg intake decreased at the 90th percentile and higher, whereas total fish consumption did not, which suggests a shift in fish species consumed. The BHg data indicated a reduction of the higher end of the distribution of BHg between the first 2-year interval (the 1999 and 2000 examinees) compared with the subsequent 4-year interval (the 2001–2004 examinees).\nThe basis for these differences could possibly reflect spillover from the federal fish advisory program (U.S. EPA 2008b) in terms of total fish and shellfish consumption. A recent analysis of a nationally representative study specifically addressing fish-consumption patterns did not support this suggestion (Bradbury 2007).\nThe four fish species listed in the federal advisory [swordfish, shark, tilefish, and king mackerel (U.S. EPA 2008b)] were rarely reported by the 5,465 women in this analysis. It is clear that these four fish species contributed little to Hg exposure in this general population of U.S. women. Individual states with higher Hg exposures [e.g., Hawaii (Sato et al., 2006), Florida (Karouna-Renier et al. 2008)] and greater fish consumption [Florida (Denger et al. 1994)] have substantially broader fish consumption advisories (e.g., Hawaii and Florida) aimed at reducing Hg exposure from high-Hg–containing species obtained locally (Florida Department of Health 2007; U.S. EPA 2008b). Despite the federal advisory’s emphasis on four species of highly contaminated fish (U.S. EPA 2008b) and the states’ emphasis on game-fish, the most commonly consumed finfish in the United States is tuna. Interpretation of Hg exposure from tuna was complicated by the specific wording of the dietary questions asked of the NHANES examinees, which did not differentiate between light or skipjack tuna and albacore tuna. The latter contains approximately three times more Hg than does the former: 0.38 μg/g for frozen and fresh tuna, 0.35 μg/g for canned albacore, and 0.12 μg/g for canned light tuna (Mahaffey et al. 2008).\nOur analysis of 30-day Hg intake indicated that there was no consistent trend in fish consumption by women of childbearing age over the 6-year period between 1999 and 2004. Our evaluation of NHANES fish intake data indicated no differences in the mean frequency or amount of particular fish and shellfish species consumed. However, the estimated 30-day Hg intake decreased at the 90th percentile and higher, whereas total fish consumption did not, which suggests a shift in fish species consumed. The BHg data indicated a reduction of the higher end of the distribution of BHg between the first 2-year interval (the 1999 and 2000 examinees) compared with the subsequent 4-year interval (the 2001–2004 examinees).\nThe basis for these differences could possibly reflect spillover from the federal fish advisory program (U.S. EPA 2008b) in terms of total fish and shellfish consumption. A recent analysis of a nationally representative study specifically addressing fish-consumption patterns did not support this suggestion (Bradbury 2007).\nThe four fish species listed in the federal advisory [swordfish, shark, tilefish, and king mackerel (U.S. EPA 2008b)] were rarely reported by the 5,465 women in this analysis. It is clear that these four fish species contributed little to Hg exposure in this general population of U.S. women. Individual states with higher Hg exposures [e.g., Hawaii (Sato et al., 2006), Florida (Karouna-Renier et al. 2008)] and greater fish consumption [Florida (Denger et al. 1994)] have substantially broader fish consumption advisories (e.g., Hawaii and Florida) aimed at reducing Hg exposure from high-Hg–containing species obtained locally (Florida Department of Health 2007; U.S. EPA 2008b). Despite the federal advisory’s emphasis on four species of highly contaminated fish (U.S. EPA 2008b) and the states’ emphasis on game-fish, the most commonly consumed finfish in the United States is tuna. Interpretation of Hg exposure from tuna was complicated by the specific wording of the dietary questions asked of the NHANES examinees, which did not differentiate between light or skipjack tuna and albacore tuna. The latter contains approximately three times more Hg than does the former: 0.38 μg/g for frozen and fresh tuna, 0.35 μg/g for canned albacore, and 0.12 μg/g for canned light tuna (Mahaffey et al. 2008).\n Changes in MeHg exposure over time During the past decade, the U.S. EPA initiated substantial interventions aimed to reduce Hg releases and exposures (U.S. EPA 2008a) and issued advisories to limit consumption of high-Hg fish (U.S. EPA 2008b). Because of worldwide atmospheric distribution and subsequent deposition of Hg, local conditions and locally caught fish are not the main contributors to Hg intake for most people (Sunderland 2007). Although there are economic indications that consumption of some species of fish may have decreased in response to these advisories (Shimshack et al. 2007), Hg exposures may not follow a similar time trend despite regulatory efforts to reduce Hg exposures. A recent analysis of a nationally representative study specifically addressing fish-consumption patterns did not support this suggestion (Bradbury 2007). Our analysis of NHANES data calculating 30-day Hg intake indicated that there was no consistent trend in fish consumption by women of childbearing age over the 6-year period between 1999 and 2004.\nDuring the past decade, the U.S. EPA initiated substantial interventions aimed to reduce Hg releases and exposures (U.S. EPA 2008a) and issued advisories to limit consumption of high-Hg fish (U.S. EPA 2008b). Because of worldwide atmospheric distribution and subsequent deposition of Hg, local conditions and locally caught fish are not the main contributors to Hg intake for most people (Sunderland 2007). Although there are economic indications that consumption of some species of fish may have decreased in response to these advisories (Shimshack et al. 2007), Hg exposures may not follow a similar time trend despite regulatory efforts to reduce Hg exposures. A recent analysis of a nationally representative study specifically addressing fish-consumption patterns did not support this suggestion (Bradbury 2007). Our analysis of NHANES data calculating 30-day Hg intake indicated that there was no consistent trend in fish consumption by women of childbearing age over the 6-year period between 1999 and 2004.","Comparisons of the distribution of BHg data with reference values aimed at protecting the fetal nervous system have been made using national-level data (Mahaffey et al. 2004). NHANES data are often used to make population estimates through application of weighting factors to variables of interest such as BHg. Population estimates for U.S. Census regions and their distribution of BHg concentrations indicate that women living in the Northeast had BHg concentrations exceeding levels of concern more often than did women living in the South and West. The lowest Hg exposures were reported among women living in the Midwest.\nBecause NHANES was not designed to provide population estimates for coastal and noncoastal areas, unbiased estimates for the number of women having BHg concentrations ≥ 3.5 μg/L and ≥ 5.8 μg/L cannot be developed comparing coastal- and noncoastal-residing women. Although the following are not population estimates, they are statistics for a geographic region: Women living in coastal areas were at greater risk of having BHg concentrations ≥ 3.5 μg/L (16.25% for coastal and 5.99% for noncoastal residents) and ≥ 5.8 μg/L (8.11% for coastal and 2.06% for noncoastal residents). Women living near the coastal areas had approximately three to four times greater risk of exceeding acceptable levels of Hg exposure than did noncoastal-dwelling women. There may be some bias in these results due to the weighting issues (see “Materials and Methods”); however, we do not believe that this bias is a major factor underlying these great differences.\nMeHg exposures exceeding health-based standards, including U.S. EPA’s RfD (Rice et al. 2003), occurred more commonly among women living in coastal areas. These health-based standards were based on avoiding MeHg-associated delays and deficits in neurologic development of children after in utero exposure to MeHg (Mergler et al. 2007; Rice et al. 2003). At higher exposures to MeHg, including the highest concentrations reported during these survey years, the women themselves may risk adverse neuropsychological and neurobehavioral outcomes (Mergler et al. 2007).\nWithin the United States, people living in coastal areas consume more fish and shellfish than do those living in noncoastal areas and consume fish with higher Hg concentrations. Reports from New York City (McKelvey et al. 2007) and Florida (Denger et al. 1994; Karouna-Renier et al. 2008) support our identification of higher Hg exposures in U.S. coastal areas. This is part of a worldwide pattern. An overall pattern of higher BHg levels has also been reported among people living on U.S. islands [Hawaii (Sato et al. 2006)] and territories [e.g., Puerto Rico (Ortiz-Roque and López-Rivera 2004)]. A similar pattern has been repeated in other islands [Bermuda (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004), Fiji (Kumar et al. 2006), Seychelles (Myers et al. 2007), and Tahiti (Chateau-Degat 2005; Dewailly et al. 2008)] compared with inland populations. Among these island populations, BHg concentrations at the upper end of the distribution fall into the range of 50 μg/L (~ 250 nmol/L) and higher (Chateau-Degat 2005). In Bermuda, cord BHg concentrations as high as 160 nmol/L (~ 35 μg/L) have been reported (arithmetic mean, 41.3 ± 4.7 nmol/L or 8.0 ± 1.0 μg/L) (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004).\nHigher BHg concentrations in the U.S. Northeast found in this study reflect, in part, more frequent fish and shellfish consumption. Additional variability may be a function of differences in Hg concentrations among species and geographic regions (Sunderland 2007). For example, recent information on “hot spots” for Hg in wildlife tissues (Evers et al. 2007) could be associated with higher Hg concentrations for locally obtained fish. One limitation of the present analysis was the use of a fish-species–specific mean Hg concentration (i.e., nondistributional values) to estimate individual exposure. Although most fish consumed by the U.S. population is not locally obtained (i.e., commercially obtained from diverse regions and countries) (Sunderland 2007), analytical results showing geographic differences in the distribution of BHg could reflect higher Hg concentrations in locally obtained fish within the Northeast states.","Ethnic origins were associated with Hg exposures with those designated as “other” (i.e., Asian, Pacific and Caribbean Islander, Native American, Alaska Native, multiracial, and unknown race) having higher BHg concentrations. From additional studies, people of Asian descent whose food choices are influenced by Asian dietary patterns (Kudo et al. 2000; Sechena et al. 2003) tended to consume fish more frequently, in greater variety, and in greater quantity than did non-Asians. The ethnic diversity of the U.S. population is well known. As of 1997, 61% of the Asian population living in the United States was foreign-born (Council of Economic Advisors 1999). By comparison with overall U.S. data, higher BHg concentrations among Asians and islanders were reported for Taiwan (Hsu et al. 2007), Cambodia (Agusa et al. 2007), Fiji (Kumar et al. 2006), and Tahiti (Dewailly et al. 2008).\nWithin the United States, fish and shellfish consumption, predicting Hg exposure described previously, varies widely, in part a reflection of ethnicity. For example, Asian countries [e.g., Cambodia (Agusa et al. 2007), Taiwan (Hsu et al. 2007; Soong et al. 1991), Japan (Murata et al. 2007; Sakamoto et al. 2007)], island nations [e.g., Bermuda (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004), Seychelles (Myers et al. 2007), Tahiti (Chateau-Degat 2005), Taiwan (Hsu et al. 2007; Soong et al. 1991), Japan (Murata et al. 2007; Sakamoto et al. 2007)], and some European countries [e.g., Spain (Falcó et al. 2006; Herreros et al. 2008) and the Faroe Islands (Weihe et al. 1996)] have reported fish/shellfish consumption levels greater than average worldwide consumption (World Health Organization 2008).","In contrast to some other environmental exposures [e.g., higher blood lead concentrations] (Mahaffey et al. 1982), BHg concentrations increased with income. This is consistent with other studies in which women from higher income groups were at greater risk of MeHg exposure, as were women living in urban areas (Hightower and Moore 2003; Saint-Phard and Van Dorsten 2006).","A more complex association between income and racial/ethnic group may also exist. According to the 1990 U.S. Census (U.S. Census Bureau 2008), the median family income of Japanese-American families exceeded that of non-Hispanic white families. By contrast, the income of Cambodian-American families was lower than that of black families. We could not address whether there is an interaction between belonging to the category designated as “other” and higher income within the NHANES data on BHg levels available at this time, because of sample size limitations.","Our analysis of 30-day Hg intake indicated that there was no consistent trend in fish consumption by women of childbearing age over the 6-year period between 1999 and 2004. Our evaluation of NHANES fish intake data indicated no differences in the mean frequency or amount of particular fish and shellfish species consumed. However, the estimated 30-day Hg intake decreased at the 90th percentile and higher, whereas total fish consumption did not, which suggests a shift in fish species consumed. The BHg data indicated a reduction of the higher end of the distribution of BHg between the first 2-year interval (the 1999 and 2000 examinees) compared with the subsequent 4-year interval (the 2001–2004 examinees).\nThe basis for these differences could possibly reflect spillover from the federal fish advisory program (U.S. EPA 2008b) in terms of total fish and shellfish consumption. A recent analysis of a nationally representative study specifically addressing fish-consumption patterns did not support this suggestion (Bradbury 2007).\nThe four fish species listed in the federal advisory [swordfish, shark, tilefish, and king mackerel (U.S. EPA 2008b)] were rarely reported by the 5,465 women in this analysis. It is clear that these four fish species contributed little to Hg exposure in this general population of U.S. women. Individual states with higher Hg exposures [e.g., Hawaii (Sato et al., 2006), Florida (Karouna-Renier et al. 2008)] and greater fish consumption [Florida (Denger et al. 1994)] have substantially broader fish consumption advisories (e.g., Hawaii and Florida) aimed at reducing Hg exposure from high-Hg–containing species obtained locally (Florida Department of Health 2007; U.S. EPA 2008b). Despite the federal advisory’s emphasis on four species of highly contaminated fish (U.S. EPA 2008b) and the states’ emphasis on game-fish, the most commonly consumed finfish in the United States is tuna. Interpretation of Hg exposure from tuna was complicated by the specific wording of the dietary questions asked of the NHANES examinees, which did not differentiate between light or skipjack tuna and albacore tuna. The latter contains approximately three times more Hg than does the former: 0.38 μg/g for frozen and fresh tuna, 0.35 μg/g for canned albacore, and 0.12 μg/g for canned light tuna (Mahaffey et al. 2008).","During the past decade, the U.S. EPA initiated substantial interventions aimed to reduce Hg releases and exposures (U.S. EPA 2008a) and issued advisories to limit consumption of high-Hg fish (U.S. EPA 2008b). Because of worldwide atmospheric distribution and subsequent deposition of Hg, local conditions and locally caught fish are not the main contributors to Hg intake for most people (Sunderland 2007). Although there are economic indications that consumption of some species of fish may have decreased in response to these advisories (Shimshack et al. 2007), Hg exposures may not follow a similar time trend despite regulatory efforts to reduce Hg exposures. A recent analysis of a nationally representative study specifically addressing fish-consumption patterns did not support this suggestion (Bradbury 2007). Our analysis of NHANES data calculating 30-day Hg intake indicated that there was no consistent trend in fish consumption by women of childbearing age over the 6-year period between 1999 and 2004.","Significant geographic differences in BHg concentrations occurred within the United States: We found highest exposures in coastal areas and the Northeast census region. In the Northeast, 19% of women had BHg concentrations ≥ 3.5 μg/L. The highest 5% of BHg concentrations exceeded 8.2 μg/L in the Northeast and 7.2 μg/L in coastal areas, concentrations more than twice the 3.5 μg/L level of concern. BHg levels were predicted by the quantity and type of fish consumed. Over the 6-year period (1999–2004), the frequency of elevated BHg levels among women of childbearing age declined without a significant change in quantities of fish and shellfish consumed. This pattern suggests a more discerning series of choices in type of fish eaten rather than an overall reduction in fish consumption. Within all geographic regions, women at highest risk of elevated Hg exposures were more affluent and more likely to be of Asian or island ethnicity."],"string":"[\n \" Methodology for data analysis We evaluated data for examinees who participated in NHANES during survey years 1999–2004 to assess the statistical association between seafood consumption and BHg by region of residence, race/ethnicity, and annual income. Further, we examined time trends for both BHg and fish consumption. NHANES is an annual survey conducted by the NCHS. The data include BHg levels, 24-hr dietary recall, and 30-day finfish and shellfish consumption frequency for women 16–49 years of age who reside in the United States. The NHANES sampling frame includes all 50 states. The documentation and publicly available data for NHANES can be found online [Centers for Disease Control and Prevention (CDC) 2006b]. The regional data are not publicly available but can be accessed by special request to the National Center for Health Statistics (NCHS) through its Research Data Center. Procedures for submitting a proposal in order to access data that are not publicly available can be found online (CDC 2006c). We performed all analyses using SAS, version 9.1 (SAS Institute Inc., Cary, NC).\\nFollowing NHANES analytic guidelines (CDC 2006a), we used SAS procedures that accurately incorporate the stratification and multistage sampling of NHANES: Proc SurveyMeans, Proc SurveyReg, and Proc SurveyFreq (SAS Institute Inc.). The weights provided by NCHS compensate for the oversampling of various subpopulations and adjust for nonresponse bias. We used weights for estimating statistics for coastal and non-coastal regions to retain these adjustments. Because we considered the variables of interest (BHg and fish consumption) to be related to some of the factors that were oversampled, we retained these adjustments to minimize the bias in the estimates. For example, NHANES over-sampled Mexican Americans, who also have lower BHg than do other racial/ethnic groups. If the weights were not used to estimate the distribution of BHg, the results would be biased low. We recognize that some bias may remain within the estimates because the weights were not specifically created for the geographic regions of coastal and noncoastal; however, these subdivisions (e.g., coastal, noncoastal, Pacific, Atlantic) are based on counties, the primary sampling units of NHANES (CDC 2006a). All multivariate analyses were done unweighted because factors associated with the dependent variables and for which over-sampling was based were included as covariates and thus adjusted for in the modeling.\\nIn order to estimate long-term Hg intake, we combined data collected through the 24-hr dietary recall and the 30-day fish frequency questionnaire to estimate 30-day Hg intake [for specific methodologies, see Mahaffey and Rice (1997); Mahaffey et al. (2004)]. If we found statistically significant differences in amount of fish consumed per meal from the 24-hr dietary recall by either coastal status (participants who lived in a county that bordered the Pacific or Atlantic Oceans, the Gulf of Mexico, or the Great Lakes vs. those who did not) or data release (1999–2000–2001–2002, or 2003–2004), we calculated separate averages. We generated statistically representative estimates from these data using the statistical weights provided by NCHS and following the relevant analytical guidelines published by NCHS (CDC 2006a).\\nWe evaluated data for examinees who participated in NHANES during survey years 1999–2004 to assess the statistical association between seafood consumption and BHg by region of residence, race/ethnicity, and annual income. Further, we examined time trends for both BHg and fish consumption. NHANES is an annual survey conducted by the NCHS. The data include BHg levels, 24-hr dietary recall, and 30-day finfish and shellfish consumption frequency for women 16–49 years of age who reside in the United States. The NHANES sampling frame includes all 50 states. The documentation and publicly available data for NHANES can be found online [Centers for Disease Control and Prevention (CDC) 2006b]. The regional data are not publicly available but can be accessed by special request to the National Center for Health Statistics (NCHS) through its Research Data Center. Procedures for submitting a proposal in order to access data that are not publicly available can be found online (CDC 2006c). We performed all analyses using SAS, version 9.1 (SAS Institute Inc., Cary, NC).\\nFollowing NHANES analytic guidelines (CDC 2006a), we used SAS procedures that accurately incorporate the stratification and multistage sampling of NHANES: Proc SurveyMeans, Proc SurveyReg, and Proc SurveyFreq (SAS Institute Inc.). The weights provided by NCHS compensate for the oversampling of various subpopulations and adjust for nonresponse bias. We used weights for estimating statistics for coastal and non-coastal regions to retain these adjustments. Because we considered the variables of interest (BHg and fish consumption) to be related to some of the factors that were oversampled, we retained these adjustments to minimize the bias in the estimates. For example, NHANES over-sampled Mexican Americans, who also have lower BHg than do other racial/ethnic groups. If the weights were not used to estimate the distribution of BHg, the results would be biased low. We recognize that some bias may remain within the estimates because the weights were not specifically created for the geographic regions of coastal and noncoastal; however, these subdivisions (e.g., coastal, noncoastal, Pacific, Atlantic) are based on counties, the primary sampling units of NHANES (CDC 2006a). All multivariate analyses were done unweighted because factors associated with the dependent variables and for which over-sampling was based were included as covariates and thus adjusted for in the modeling.\\nIn order to estimate long-term Hg intake, we combined data collected through the 24-hr dietary recall and the 30-day fish frequency questionnaire to estimate 30-day Hg intake [for specific methodologies, see Mahaffey and Rice (1997); Mahaffey et al. (2004)]. If we found statistically significant differences in amount of fish consumed per meal from the 24-hr dietary recall by either coastal status (participants who lived in a county that bordered the Pacific or Atlantic Oceans, the Gulf of Mexico, or the Great Lakes vs. those who did not) or data release (1999–2000–2001–2002, or 2003–2004), we calculated separate averages. We generated statistically representative estimates from these data using the statistical weights provided by NCHS and following the relevant analytical guidelines published by NCHS (CDC 2006a).\\n Methodology for defining coastal and non-coastal areas Fish consumption is generally believed to be a major contributor to BHg concentration. We hypothesized that patterns of fish and shellfish consumption would vary between U.S. residents who live on or near the coast (within ~ 25–50 miles) and those who live inland. We further hypothesized that fish consumption patterns, and thus BHg concentrations, may also vary by specific coast (e.g., residents near the Atlantic Coast may have different BHg concentrations than those on the coast of the Gulf of Mexico) and specific inland region (e.g., West vs. Midwest). To test these hypotheses, we categorized NHANES respondents as living in either a coastal or a noncoastal county and further categorized them by eight regions: Atlantic Coast, Northeast, Great Lakes, Midwest, South, Gulf of Mexico, West, and Pacific Coast.\\nThe geographic unit used by NHANES is a county or county equivalent (CDC 2006a); therefore, we limited our definitions of coastal and noncoastal to follow county boundaries. We defined all counties that bordered the Pacific or Atlantic Oceans, the Gulf of Mexico, or any of the Great Lakes as coastal. Additionally, we defined counties that bordered estuaries and bays as coastal, as well as counties whose center point was within approximately 25 miles of any coast even if not directly bordering a coast. [For the list of counties, see Supplemental Material, Table 13 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] We then defined the four coastal regions based on nearest body of water; for example, counties in California, Oregon, Washington, Alaska, and Hawaii that we defined as coastal were categorized as Pacific Coast. We separated noncoastal counties into four inland regions using the U.S. Census regions; for example, noncoastal counties in California, Oregon, Washington, and Alaska along with the entire states of Idaho, Montana, Wyoming, Colorado, New Mexico, Arizona, Utah, and Nevada became the West region (we classified all of Hawaii as coastal). We also designated the entire state of Florida as coastal, split between the Atlantic Coast and the Gulf of Mexico. We designated Miami-Dade County as Atlantic Coast, and Monroe County as Gulf of Mexico. These subdivisions run the risk of small sample sizes; however, the definition of coastal was sufficiently broad to avoid single primary sampling units.\\nFish consumption is generally believed to be a major contributor to BHg concentration. We hypothesized that patterns of fish and shellfish consumption would vary between U.S. residents who live on or near the coast (within ~ 25–50 miles) and those who live inland. We further hypothesized that fish consumption patterns, and thus BHg concentrations, may also vary by specific coast (e.g., residents near the Atlantic Coast may have different BHg concentrations than those on the coast of the Gulf of Mexico) and specific inland region (e.g., West vs. Midwest). To test these hypotheses, we categorized NHANES respondents as living in either a coastal or a noncoastal county and further categorized them by eight regions: Atlantic Coast, Northeast, Great Lakes, Midwest, South, Gulf of Mexico, West, and Pacific Coast.\\nThe geographic unit used by NHANES is a county or county equivalent (CDC 2006a); therefore, we limited our definitions of coastal and noncoastal to follow county boundaries. We defined all counties that bordered the Pacific or Atlantic Oceans, the Gulf of Mexico, or any of the Great Lakes as coastal. Additionally, we defined counties that bordered estuaries and bays as coastal, as well as counties whose center point was within approximately 25 miles of any coast even if not directly bordering a coast. [For the list of counties, see Supplemental Material, Table 13 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] We then defined the four coastal regions based on nearest body of water; for example, counties in California, Oregon, Washington, Alaska, and Hawaii that we defined as coastal were categorized as Pacific Coast. We separated noncoastal counties into four inland regions using the U.S. Census regions; for example, noncoastal counties in California, Oregon, Washington, and Alaska along with the entire states of Idaho, Montana, Wyoming, Colorado, New Mexico, Arizona, Utah, and Nevada became the West region (we classified all of Hawaii as coastal). We also designated the entire state of Florida as coastal, split between the Atlantic Coast and the Gulf of Mexico. We designated Miami-Dade County as Atlantic Coast, and Monroe County as Gulf of Mexico. These subdivisions run the risk of small sample sizes; however, the definition of coastal was sufficiently broad to avoid single primary sampling units.\",\n \"We evaluated data for examinees who participated in NHANES during survey years 1999–2004 to assess the statistical association between seafood consumption and BHg by region of residence, race/ethnicity, and annual income. Further, we examined time trends for both BHg and fish consumption. NHANES is an annual survey conducted by the NCHS. The data include BHg levels, 24-hr dietary recall, and 30-day finfish and shellfish consumption frequency for women 16–49 years of age who reside in the United States. The NHANES sampling frame includes all 50 states. The documentation and publicly available data for NHANES can be found online [Centers for Disease Control and Prevention (CDC) 2006b]. The regional data are not publicly available but can be accessed by special request to the National Center for Health Statistics (NCHS) through its Research Data Center. Procedures for submitting a proposal in order to access data that are not publicly available can be found online (CDC 2006c). We performed all analyses using SAS, version 9.1 (SAS Institute Inc., Cary, NC).\\nFollowing NHANES analytic guidelines (CDC 2006a), we used SAS procedures that accurately incorporate the stratification and multistage sampling of NHANES: Proc SurveyMeans, Proc SurveyReg, and Proc SurveyFreq (SAS Institute Inc.). The weights provided by NCHS compensate for the oversampling of various subpopulations and adjust for nonresponse bias. We used weights for estimating statistics for coastal and non-coastal regions to retain these adjustments. Because we considered the variables of interest (BHg and fish consumption) to be related to some of the factors that were oversampled, we retained these adjustments to minimize the bias in the estimates. For example, NHANES over-sampled Mexican Americans, who also have lower BHg than do other racial/ethnic groups. If the weights were not used to estimate the distribution of BHg, the results would be biased low. We recognize that some bias may remain within the estimates because the weights were not specifically created for the geographic regions of coastal and noncoastal; however, these subdivisions (e.g., coastal, noncoastal, Pacific, Atlantic) are based on counties, the primary sampling units of NHANES (CDC 2006a). All multivariate analyses were done unweighted because factors associated with the dependent variables and for which over-sampling was based were included as covariates and thus adjusted for in the modeling.\\nIn order to estimate long-term Hg intake, we combined data collected through the 24-hr dietary recall and the 30-day fish frequency questionnaire to estimate 30-day Hg intake [for specific methodologies, see Mahaffey and Rice (1997); Mahaffey et al. (2004)]. If we found statistically significant differences in amount of fish consumed per meal from the 24-hr dietary recall by either coastal status (participants who lived in a county that bordered the Pacific or Atlantic Oceans, the Gulf of Mexico, or the Great Lakes vs. those who did not) or data release (1999–2000–2001–2002, or 2003–2004), we calculated separate averages. We generated statistically representative estimates from these data using the statistical weights provided by NCHS and following the relevant analytical guidelines published by NCHS (CDC 2006a).\",\n \"Fish consumption is generally believed to be a major contributor to BHg concentration. We hypothesized that patterns of fish and shellfish consumption would vary between U.S. residents who live on or near the coast (within ~ 25–50 miles) and those who live inland. We further hypothesized that fish consumption patterns, and thus BHg concentrations, may also vary by specific coast (e.g., residents near the Atlantic Coast may have different BHg concentrations than those on the coast of the Gulf of Mexico) and specific inland region (e.g., West vs. Midwest). To test these hypotheses, we categorized NHANES respondents as living in either a coastal or a noncoastal county and further categorized them by eight regions: Atlantic Coast, Northeast, Great Lakes, Midwest, South, Gulf of Mexico, West, and Pacific Coast.\\nThe geographic unit used by NHANES is a county or county equivalent (CDC 2006a); therefore, we limited our definitions of coastal and noncoastal to follow county boundaries. We defined all counties that bordered the Pacific or Atlantic Oceans, the Gulf of Mexico, or any of the Great Lakes as coastal. Additionally, we defined counties that bordered estuaries and bays as coastal, as well as counties whose center point was within approximately 25 miles of any coast even if not directly bordering a coast. [For the list of counties, see Supplemental Material, Table 13 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] We then defined the four coastal regions based on nearest body of water; for example, counties in California, Oregon, Washington, Alaska, and Hawaii that we defined as coastal were categorized as Pacific Coast. We separated noncoastal counties into four inland regions using the U.S. Census regions; for example, noncoastal counties in California, Oregon, Washington, and Alaska along with the entire states of Idaho, Montana, Wyoming, Colorado, New Mexico, Arizona, Utah, and Nevada became the West region (we classified all of Hawaii as coastal). We also designated the entire state of Florida as coastal, split between the Atlantic Coast and the Gulf of Mexico. We designated Miami-Dade County as Atlantic Coast, and Monroe County as Gulf of Mexico. These subdivisions run the risk of small sample sizes; however, the definition of coastal was sufficiently broad to avoid single primary sampling units.\",\n \"Table 1 shows the distributions of estimates of the number of women with BHg concentrations > 3.5 μg/L and > 5.8 μg/L, by region. Analyses indicate that between 1999 and 2004, the Northeast had the highest percentage of women with BHg concentrations above the 3.5 μg/L level of concern (> 19%), whereas the South had the largest estimated number of women (1.21 million) with ≥ 3.5 μg/L BHg because of elevated population in that region. Geometric means (Figure 1) show similar trends, with the highest BHg concentrations in the Northeast, followed by the West, South, and Midwest census regions. In the Northeast, the highest 5% of BHg concentrations exceeded 8.2 μg/L. [For full distributions, see Supplemental Material, Table 2 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] When we included coastal regions in this analysis, additional spatial heterogeneity in BHg (Figure 2A) and estimated 30-day Hg intake (Figure 2B) was apparent, with elevated exposures in all coastal areas relative to their neighboring inland regions except in the Great Lakes. In the coastal areas, the highest 5% of BHg concentrations exceeded 7.2 μg/L, with the Atlantic Coast exceeding 10.9 μg/L. [For the full distributions, see Supplemental Material, Table 4 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] Fish species eaten by survey participants varied by region (Figure 3), with respondents in coastal regions reporting higher frequency of consumption of fish containing higher levels of Hg. BHg concentrations were strongly associated with the frequency of fish consumption (Figure 4). BHg increased with monthly estimated consumption of fish and shellfish over the range of never/rarely to 4 or more times per week. In multiple regression modeling, women from the Atlantic (p < 0.01), Pacific (p < 0.0001), and Gulf (p < 0.0001) coasts had higher BHg concentrations compared with women from the inland West, whereas women from the inland Northeast and inland Midwest had significantly lower BHg levels (p < 0.0001). [For the full regression results, see Supplemental Material, Table 11 (http://www.ehponline.org/members/2008/11674/suppl.pdf).]\\nAnalysis of temporal trends through simple regression modeling showed no statistically significant difference among the three sets of study years (1999–2000–2001–2002, and 2003–2004) for BHg (p = 0.07), estimated 30-day Hg intake (p = 0.11), or reported frequency of seafood consumption (p = 0.69). However, in multiple regression modeling, adjusting for covariates including coastal/non-coastal residence, the years 1999–2000 had significantly higher BHg levels (p < 0.0001) compared with 2003–2004, and 2001–2002 had significantly lower BHg levels (p < 0.01) [Supplemental Material, Table 11 (http://www.ehponline.org/members/2008/11674/suppl.pdf)]. Although the analyses did not support the conclusion that there was a general downward trend in BHg concentrations over the 6-year study period, there was a decline in the upper percentiles reflecting the most highly exposed women with BHg concentrations greater than established levels of concern [Supplemental Material, Table 9 (http://www.ehponline.org/members/2008/11674/suppl.pdf)]. In addition, the percentage of examinees with BHg values ≥ 3.5 μg/L and ≥ 5.8 μg/L was much greater in 1999–2000 compared to 2001–2002 and 2003–2004 (Figure 5). We found no consistent trend in fish consumption across the study years. We observed a decrease in the 90th percentile of 30-day estimated intake of Hg through seafood consumption across the study years even though there was no similar decrease in the 90th percentile of 30-day estimated consumption of grams of fish and shellfish (Figure 6). This suggests a shift in consumption to seafood containing less Hg. We did not observe a similar pattern at the mean, suggesting that this shift in seafood consumption occurred mainly with the highest fish and shellfish consumers [Supplemental Material, Table 9 (http://www.ehponline.org/members/2008/11674/suppl.pdf)]. The RfD for Hg intake is 0.1 μg Hg/kg body weight (μg/kgbw) per day, or 3.0 μg Hg/kgbw per month (30 days).\\nResults also showed that self-selected ethnic identity was associated with total BHg concentrations, estimated 30-day Hg intake, and frequency of either finfish or shellfish consumption. For example, BHg levels, reported frequency of seafood consumption, and 30-day Hg intakes were highest among women who designated themselves as being in the “other” category (mostly people whose ancestry is Asian, Native American, Pacific Islands, and the Caribbean Islands). [See also Supplemental Material, Tables 5–7 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] Table 2 presents the percentages of women by race/ethnicity that had ≥ 3.5 and ≥ 5.8 μg/L BHg.\\nWe identified statistically significant relationships between higher income and, respectively, increasing BHg concentration (p < 0.0001), estimated 30-day intake of Hg (p = 0.008), and 30-day frequency of finfish and shellfish consumption (p < 0.0001) through bivariate regressions. [For the distributions of blood total Hg, estimated Hg intake, and frequency of finfish and shellfish consumption by annual income, see Supplemental Material, Tables 6–8 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] In addition, women from families reporting incomes of ≥ $75,000 (the reference category) had statistically higher BHg levels than did women from families with incomes of ≤ $55,000 (p < 0.01). In all cases, BHg concentrations were also significantly associated with age and estimated 30-day Hg intake (p < 0.0001). Table 3 presents the percentage of women by annual income with ≥ 3.5 and ≥ 5.8 μg/L BHg.\\nIn multiple regression modeling, after adjusting for other factors related to BHg, both race/ethnicity and income remained statistically significant predictors of BHg levels observed in this study [Supplemental Material, Table 11 (http://www.ehponline.org/members/2008/11674/suppl.pdf)]. Non-Hispanic blacks (p < 0.0001) and women grouped in the “other” racial category (p = 0.002) had significantly higher BHg concentrations than did non-Hispanic whites.\",\n \" Regional and coastal variation in BHg concentrations and in fish consumption Comparisons of the distribution of BHg data with reference values aimed at protecting the fetal nervous system have been made using national-level data (Mahaffey et al. 2004). NHANES data are often used to make population estimates through application of weighting factors to variables of interest such as BHg. Population estimates for U.S. Census regions and their distribution of BHg concentrations indicate that women living in the Northeast had BHg concentrations exceeding levels of concern more often than did women living in the South and West. The lowest Hg exposures were reported among women living in the Midwest.\\nBecause NHANES was not designed to provide population estimates for coastal and noncoastal areas, unbiased estimates for the number of women having BHg concentrations ≥ 3.5 μg/L and ≥ 5.8 μg/L cannot be developed comparing coastal- and noncoastal-residing women. Although the following are not population estimates, they are statistics for a geographic region: Women living in coastal areas were at greater risk of having BHg concentrations ≥ 3.5 μg/L (16.25% for coastal and 5.99% for noncoastal residents) and ≥ 5.8 μg/L (8.11% for coastal and 2.06% for noncoastal residents). Women living near the coastal areas had approximately three to four times greater risk of exceeding acceptable levels of Hg exposure than did noncoastal-dwelling women. There may be some bias in these results due to the weighting issues (see “Materials and Methods”); however, we do not believe that this bias is a major factor underlying these great differences.\\nMeHg exposures exceeding health-based standards, including U.S. EPA’s RfD (Rice et al. 2003), occurred more commonly among women living in coastal areas. These health-based standards were based on avoiding MeHg-associated delays and deficits in neurologic development of children after in utero exposure to MeHg (Mergler et al. 2007; Rice et al. 2003). At higher exposures to MeHg, including the highest concentrations reported during these survey years, the women themselves may risk adverse neuropsychological and neurobehavioral outcomes (Mergler et al. 2007).\\nWithin the United States, people living in coastal areas consume more fish and shellfish than do those living in noncoastal areas and consume fish with higher Hg concentrations. Reports from New York City (McKelvey et al. 2007) and Florida (Denger et al. 1994; Karouna-Renier et al. 2008) support our identification of higher Hg exposures in U.S. coastal areas. This is part of a worldwide pattern. An overall pattern of higher BHg levels has also been reported among people living on U.S. islands [Hawaii (Sato et al. 2006)] and territories [e.g., Puerto Rico (Ortiz-Roque and López-Rivera 2004)]. A similar pattern has been repeated in other islands [Bermuda (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004), Fiji (Kumar et al. 2006), Seychelles (Myers et al. 2007), and Tahiti (Chateau-Degat 2005; Dewailly et al. 2008)] compared with inland populations. Among these island populations, BHg concentrations at the upper end of the distribution fall into the range of 50 μg/L (~ 250 nmol/L) and higher (Chateau-Degat 2005). In Bermuda, cord BHg concentrations as high as 160 nmol/L (~ 35 μg/L) have been reported (arithmetic mean, 41.3 ± 4.7 nmol/L or 8.0 ± 1.0 μg/L) (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004).\\nHigher BHg concentrations in the U.S. Northeast found in this study reflect, in part, more frequent fish and shellfish consumption. Additional variability may be a function of differences in Hg concentrations among species and geographic regions (Sunderland 2007). For example, recent information on “hot spots” for Hg in wildlife tissues (Evers et al. 2007) could be associated with higher Hg concentrations for locally obtained fish. One limitation of the present analysis was the use of a fish-species–specific mean Hg concentration (i.e., nondistributional values) to estimate individual exposure. Although most fish consumed by the U.S. population is not locally obtained (i.e., commercially obtained from diverse regions and countries) (Sunderland 2007), analytical results showing geographic differences in the distribution of BHg could reflect higher Hg concentrations in locally obtained fish within the Northeast states.\\nComparisons of the distribution of BHg data with reference values aimed at protecting the fetal nervous system have been made using national-level data (Mahaffey et al. 2004). NHANES data are often used to make population estimates through application of weighting factors to variables of interest such as BHg. Population estimates for U.S. Census regions and their distribution of BHg concentrations indicate that women living in the Northeast had BHg concentrations exceeding levels of concern more often than did women living in the South and West. The lowest Hg exposures were reported among women living in the Midwest.\\nBecause NHANES was not designed to provide population estimates for coastal and noncoastal areas, unbiased estimates for the number of women having BHg concentrations ≥ 3.5 μg/L and ≥ 5.8 μg/L cannot be developed comparing coastal- and noncoastal-residing women. Although the following are not population estimates, they are statistics for a geographic region: Women living in coastal areas were at greater risk of having BHg concentrations ≥ 3.5 μg/L (16.25% for coastal and 5.99% for noncoastal residents) and ≥ 5.8 μg/L (8.11% for coastal and 2.06% for noncoastal residents). Women living near the coastal areas had approximately three to four times greater risk of exceeding acceptable levels of Hg exposure than did noncoastal-dwelling women. There may be some bias in these results due to the weighting issues (see “Materials and Methods”); however, we do not believe that this bias is a major factor underlying these great differences.\\nMeHg exposures exceeding health-based standards, including U.S. EPA’s RfD (Rice et al. 2003), occurred more commonly among women living in coastal areas. These health-based standards were based on avoiding MeHg-associated delays and deficits in neurologic development of children after in utero exposure to MeHg (Mergler et al. 2007; Rice et al. 2003). At higher exposures to MeHg, including the highest concentrations reported during these survey years, the women themselves may risk adverse neuropsychological and neurobehavioral outcomes (Mergler et al. 2007).\\nWithin the United States, people living in coastal areas consume more fish and shellfish than do those living in noncoastal areas and consume fish with higher Hg concentrations. Reports from New York City (McKelvey et al. 2007) and Florida (Denger et al. 1994; Karouna-Renier et al. 2008) support our identification of higher Hg exposures in U.S. coastal areas. This is part of a worldwide pattern. An overall pattern of higher BHg levels has also been reported among people living on U.S. islands [Hawaii (Sato et al. 2006)] and territories [e.g., Puerto Rico (Ortiz-Roque and López-Rivera 2004)]. A similar pattern has been repeated in other islands [Bermuda (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004), Fiji (Kumar et al. 2006), Seychelles (Myers et al. 2007), and Tahiti (Chateau-Degat 2005; Dewailly et al. 2008)] compared with inland populations. Among these island populations, BHg concentrations at the upper end of the distribution fall into the range of 50 μg/L (~ 250 nmol/L) and higher (Chateau-Degat 2005). In Bermuda, cord BHg concentrations as high as 160 nmol/L (~ 35 μg/L) have been reported (arithmetic mean, 41.3 ± 4.7 nmol/L or 8.0 ± 1.0 μg/L) (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004).\\nHigher BHg concentrations in the U.S. Northeast found in this study reflect, in part, more frequent fish and shellfish consumption. Additional variability may be a function of differences in Hg concentrations among species and geographic regions (Sunderland 2007). For example, recent information on “hot spots” for Hg in wildlife tissues (Evers et al. 2007) could be associated with higher Hg concentrations for locally obtained fish. One limitation of the present analysis was the use of a fish-species–specific mean Hg concentration (i.e., nondistributional values) to estimate individual exposure. Although most fish consumed by the U.S. population is not locally obtained (i.e., commercially obtained from diverse regions and countries) (Sunderland 2007), analytical results showing geographic differences in the distribution of BHg could reflect higher Hg concentrations in locally obtained fish within the Northeast states.\\n Ethnic group variation on fish intake and BHg concentrations Ethnic origins were associated with Hg exposures with those designated as “other” (i.e., Asian, Pacific and Caribbean Islander, Native American, Alaska Native, multiracial, and unknown race) having higher BHg concentrations. From additional studies, people of Asian descent whose food choices are influenced by Asian dietary patterns (Kudo et al. 2000; Sechena et al. 2003) tended to consume fish more frequently, in greater variety, and in greater quantity than did non-Asians. The ethnic diversity of the U.S. population is well known. As of 1997, 61% of the Asian population living in the United States was foreign-born (Council of Economic Advisors 1999). By comparison with overall U.S. data, higher BHg concentrations among Asians and islanders were reported for Taiwan (Hsu et al. 2007), Cambodia (Agusa et al. 2007), Fiji (Kumar et al. 2006), and Tahiti (Dewailly et al. 2008).\\nWithin the United States, fish and shellfish consumption, predicting Hg exposure described previously, varies widely, in part a reflection of ethnicity. For example, Asian countries [e.g., Cambodia (Agusa et al. 2007), Taiwan (Hsu et al. 2007; Soong et al. 1991), Japan (Murata et al. 2007; Sakamoto et al. 2007)], island nations [e.g., Bermuda (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004), Seychelles (Myers et al. 2007), Tahiti (Chateau-Degat 2005), Taiwan (Hsu et al. 2007; Soong et al. 1991), Japan (Murata et al. 2007; Sakamoto et al. 2007)], and some European countries [e.g., Spain (Falcó et al. 2006; Herreros et al. 2008) and the Faroe Islands (Weihe et al. 1996)] have reported fish/shellfish consumption levels greater than average worldwide consumption (World Health Organization 2008).\\nEthnic origins were associated with Hg exposures with those designated as “other” (i.e., Asian, Pacific and Caribbean Islander, Native American, Alaska Native, multiracial, and unknown race) having higher BHg concentrations. From additional studies, people of Asian descent whose food choices are influenced by Asian dietary patterns (Kudo et al. 2000; Sechena et al. 2003) tended to consume fish more frequently, in greater variety, and in greater quantity than did non-Asians. The ethnic diversity of the U.S. population is well known. As of 1997, 61% of the Asian population living in the United States was foreign-born (Council of Economic Advisors 1999). By comparison with overall U.S. data, higher BHg concentrations among Asians and islanders were reported for Taiwan (Hsu et al. 2007), Cambodia (Agusa et al. 2007), Fiji (Kumar et al. 2006), and Tahiti (Dewailly et al. 2008).\\nWithin the United States, fish and shellfish consumption, predicting Hg exposure described previously, varies widely, in part a reflection of ethnicity. For example, Asian countries [e.g., Cambodia (Agusa et al. 2007), Taiwan (Hsu et al. 2007; Soong et al. 1991), Japan (Murata et al. 2007; Sakamoto et al. 2007)], island nations [e.g., Bermuda (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004), Seychelles (Myers et al. 2007), Tahiti (Chateau-Degat 2005), Taiwan (Hsu et al. 2007; Soong et al. 1991), Japan (Murata et al. 2007; Sakamoto et al. 2007)], and some European countries [e.g., Spain (Falcó et al. 2006; Herreros et al. 2008) and the Faroe Islands (Weihe et al. 1996)] have reported fish/shellfish consumption levels greater than average worldwide consumption (World Health Organization 2008).\\n Income differences in association with fish intake and BHg concentrations In contrast to some other environmental exposures [e.g., higher blood lead concentrations] (Mahaffey et al. 1982), BHg concentrations increased with income. This is consistent with other studies in which women from higher income groups were at greater risk of MeHg exposure, as were women living in urban areas (Hightower and Moore 2003; Saint-Phard and Van Dorsten 2006).\\nIn contrast to some other environmental exposures [e.g., higher blood lead concentrations] (Mahaffey et al. 1982), BHg concentrations increased with income. This is consistent with other studies in which women from higher income groups were at greater risk of MeHg exposure, as were women living in urban areas (Hightower and Moore 2003; Saint-Phard and Van Dorsten 2006).\\n Interactions between income and ethnic group A more complex association between income and racial/ethnic group may also exist. According to the 1990 U.S. Census (U.S. Census Bureau 2008), the median family income of Japanese-American families exceeded that of non-Hispanic white families. By contrast, the income of Cambodian-American families was lower than that of black families. We could not address whether there is an interaction between belonging to the category designated as “other” and higher income within the NHANES data on BHg levels available at this time, because of sample size limitations.\\nA more complex association between income and racial/ethnic group may also exist. According to the 1990 U.S. Census (U.S. Census Bureau 2008), the median family income of Japanese-American families exceeded that of non-Hispanic white families. By contrast, the income of Cambodian-American families was lower than that of black families. We could not address whether there is an interaction between belonging to the category designated as “other” and higher income within the NHANES data on BHg levels available at this time, because of sample size limitations.\\n Time trends in Hg exposure absent changes in total fish consumption Our analysis of 30-day Hg intake indicated that there was no consistent trend in fish consumption by women of childbearing age over the 6-year period between 1999 and 2004. Our evaluation of NHANES fish intake data indicated no differences in the mean frequency or amount of particular fish and shellfish species consumed. However, the estimated 30-day Hg intake decreased at the 90th percentile and higher, whereas total fish consumption did not, which suggests a shift in fish species consumed. The BHg data indicated a reduction of the higher end of the distribution of BHg between the first 2-year interval (the 1999 and 2000 examinees) compared with the subsequent 4-year interval (the 2001–2004 examinees).\\nThe basis for these differences could possibly reflect spillover from the federal fish advisory program (U.S. EPA 2008b) in terms of total fish and shellfish consumption. A recent analysis of a nationally representative study specifically addressing fish-consumption patterns did not support this suggestion (Bradbury 2007).\\nThe four fish species listed in the federal advisory [swordfish, shark, tilefish, and king mackerel (U.S. EPA 2008b)] were rarely reported by the 5,465 women in this analysis. It is clear that these four fish species contributed little to Hg exposure in this general population of U.S. women. Individual states with higher Hg exposures [e.g., Hawaii (Sato et al., 2006), Florida (Karouna-Renier et al. 2008)] and greater fish consumption [Florida (Denger et al. 1994)] have substantially broader fish consumption advisories (e.g., Hawaii and Florida) aimed at reducing Hg exposure from high-Hg–containing species obtained locally (Florida Department of Health 2007; U.S. EPA 2008b). Despite the federal advisory’s emphasis on four species of highly contaminated fish (U.S. EPA 2008b) and the states’ emphasis on game-fish, the most commonly consumed finfish in the United States is tuna. Interpretation of Hg exposure from tuna was complicated by the specific wording of the dietary questions asked of the NHANES examinees, which did not differentiate between light or skipjack tuna and albacore tuna. The latter contains approximately three times more Hg than does the former: 0.38 μg/g for frozen and fresh tuna, 0.35 μg/g for canned albacore, and 0.12 μg/g for canned light tuna (Mahaffey et al. 2008).\\nOur analysis of 30-day Hg intake indicated that there was no consistent trend in fish consumption by women of childbearing age over the 6-year period between 1999 and 2004. Our evaluation of NHANES fish intake data indicated no differences in the mean frequency or amount of particular fish and shellfish species consumed. However, the estimated 30-day Hg intake decreased at the 90th percentile and higher, whereas total fish consumption did not, which suggests a shift in fish species consumed. The BHg data indicated a reduction of the higher end of the distribution of BHg between the first 2-year interval (the 1999 and 2000 examinees) compared with the subsequent 4-year interval (the 2001–2004 examinees).\\nThe basis for these differences could possibly reflect spillover from the federal fish advisory program (U.S. EPA 2008b) in terms of total fish and shellfish consumption. A recent analysis of a nationally representative study specifically addressing fish-consumption patterns did not support this suggestion (Bradbury 2007).\\nThe four fish species listed in the federal advisory [swordfish, shark, tilefish, and king mackerel (U.S. EPA 2008b)] were rarely reported by the 5,465 women in this analysis. It is clear that these four fish species contributed little to Hg exposure in this general population of U.S. women. Individual states with higher Hg exposures [e.g., Hawaii (Sato et al., 2006), Florida (Karouna-Renier et al. 2008)] and greater fish consumption [Florida (Denger et al. 1994)] have substantially broader fish consumption advisories (e.g., Hawaii and Florida) aimed at reducing Hg exposure from high-Hg–containing species obtained locally (Florida Department of Health 2007; U.S. EPA 2008b). Despite the federal advisory’s emphasis on four species of highly contaminated fish (U.S. EPA 2008b) and the states’ emphasis on game-fish, the most commonly consumed finfish in the United States is tuna. Interpretation of Hg exposure from tuna was complicated by the specific wording of the dietary questions asked of the NHANES examinees, which did not differentiate between light or skipjack tuna and albacore tuna. The latter contains approximately three times more Hg than does the former: 0.38 μg/g for frozen and fresh tuna, 0.35 μg/g for canned albacore, and 0.12 μg/g for canned light tuna (Mahaffey et al. 2008).\\n Changes in MeHg exposure over time During the past decade, the U.S. EPA initiated substantial interventions aimed to reduce Hg releases and exposures (U.S. EPA 2008a) and issued advisories to limit consumption of high-Hg fish (U.S. EPA 2008b). Because of worldwide atmospheric distribution and subsequent deposition of Hg, local conditions and locally caught fish are not the main contributors to Hg intake for most people (Sunderland 2007). Although there are economic indications that consumption of some species of fish may have decreased in response to these advisories (Shimshack et al. 2007), Hg exposures may not follow a similar time trend despite regulatory efforts to reduce Hg exposures. A recent analysis of a nationally representative study specifically addressing fish-consumption patterns did not support this suggestion (Bradbury 2007). Our analysis of NHANES data calculating 30-day Hg intake indicated that there was no consistent trend in fish consumption by women of childbearing age over the 6-year period between 1999 and 2004.\\nDuring the past decade, the U.S. EPA initiated substantial interventions aimed to reduce Hg releases and exposures (U.S. EPA 2008a) and issued advisories to limit consumption of high-Hg fish (U.S. EPA 2008b). Because of worldwide atmospheric distribution and subsequent deposition of Hg, local conditions and locally caught fish are not the main contributors to Hg intake for most people (Sunderland 2007). Although there are economic indications that consumption of some species of fish may have decreased in response to these advisories (Shimshack et al. 2007), Hg exposures may not follow a similar time trend despite regulatory efforts to reduce Hg exposures. A recent analysis of a nationally representative study specifically addressing fish-consumption patterns did not support this suggestion (Bradbury 2007). Our analysis of NHANES data calculating 30-day Hg intake indicated that there was no consistent trend in fish consumption by women of childbearing age over the 6-year period between 1999 and 2004.\",\n \"Comparisons of the distribution of BHg data with reference values aimed at protecting the fetal nervous system have been made using national-level data (Mahaffey et al. 2004). NHANES data are often used to make population estimates through application of weighting factors to variables of interest such as BHg. Population estimates for U.S. Census regions and their distribution of BHg concentrations indicate that women living in the Northeast had BHg concentrations exceeding levels of concern more often than did women living in the South and West. The lowest Hg exposures were reported among women living in the Midwest.\\nBecause NHANES was not designed to provide population estimates for coastal and noncoastal areas, unbiased estimates for the number of women having BHg concentrations ≥ 3.5 μg/L and ≥ 5.8 μg/L cannot be developed comparing coastal- and noncoastal-residing women. Although the following are not population estimates, they are statistics for a geographic region: Women living in coastal areas were at greater risk of having BHg concentrations ≥ 3.5 μg/L (16.25% for coastal and 5.99% for noncoastal residents) and ≥ 5.8 μg/L (8.11% for coastal and 2.06% for noncoastal residents). Women living near the coastal areas had approximately three to four times greater risk of exceeding acceptable levels of Hg exposure than did noncoastal-dwelling women. There may be some bias in these results due to the weighting issues (see “Materials and Methods”); however, we do not believe that this bias is a major factor underlying these great differences.\\nMeHg exposures exceeding health-based standards, including U.S. EPA’s RfD (Rice et al. 2003), occurred more commonly among women living in coastal areas. These health-based standards were based on avoiding MeHg-associated delays and deficits in neurologic development of children after in utero exposure to MeHg (Mergler et al. 2007; Rice et al. 2003). At higher exposures to MeHg, including the highest concentrations reported during these survey years, the women themselves may risk adverse neuropsychological and neurobehavioral outcomes (Mergler et al. 2007).\\nWithin the United States, people living in coastal areas consume more fish and shellfish than do those living in noncoastal areas and consume fish with higher Hg concentrations. Reports from New York City (McKelvey et al. 2007) and Florida (Denger et al. 1994; Karouna-Renier et al. 2008) support our identification of higher Hg exposures in U.S. coastal areas. This is part of a worldwide pattern. An overall pattern of higher BHg levels has also been reported among people living on U.S. islands [Hawaii (Sato et al. 2006)] and territories [e.g., Puerto Rico (Ortiz-Roque and López-Rivera 2004)]. A similar pattern has been repeated in other islands [Bermuda (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004), Fiji (Kumar et al. 2006), Seychelles (Myers et al. 2007), and Tahiti (Chateau-Degat 2005; Dewailly et al. 2008)] compared with inland populations. Among these island populations, BHg concentrations at the upper end of the distribution fall into the range of 50 μg/L (~ 250 nmol/L) and higher (Chateau-Degat 2005). In Bermuda, cord BHg concentrations as high as 160 nmol/L (~ 35 μg/L) have been reported (arithmetic mean, 41.3 ± 4.7 nmol/L or 8.0 ± 1.0 μg/L) (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004).\\nHigher BHg concentrations in the U.S. Northeast found in this study reflect, in part, more frequent fish and shellfish consumption. Additional variability may be a function of differences in Hg concentrations among species and geographic regions (Sunderland 2007). For example, recent information on “hot spots” for Hg in wildlife tissues (Evers et al. 2007) could be associated with higher Hg concentrations for locally obtained fish. One limitation of the present analysis was the use of a fish-species–specific mean Hg concentration (i.e., nondistributional values) to estimate individual exposure. Although most fish consumed by the U.S. population is not locally obtained (i.e., commercially obtained from diverse regions and countries) (Sunderland 2007), analytical results showing geographic differences in the distribution of BHg could reflect higher Hg concentrations in locally obtained fish within the Northeast states.\",\n \"Ethnic origins were associated with Hg exposures with those designated as “other” (i.e., Asian, Pacific and Caribbean Islander, Native American, Alaska Native, multiracial, and unknown race) having higher BHg concentrations. From additional studies, people of Asian descent whose food choices are influenced by Asian dietary patterns (Kudo et al. 2000; Sechena et al. 2003) tended to consume fish more frequently, in greater variety, and in greater quantity than did non-Asians. The ethnic diversity of the U.S. population is well known. As of 1997, 61% of the Asian population living in the United States was foreign-born (Council of Economic Advisors 1999). By comparison with overall U.S. data, higher BHg concentrations among Asians and islanders were reported for Taiwan (Hsu et al. 2007), Cambodia (Agusa et al. 2007), Fiji (Kumar et al. 2006), and Tahiti (Dewailly et al. 2008).\\nWithin the United States, fish and shellfish consumption, predicting Hg exposure described previously, varies widely, in part a reflection of ethnicity. For example, Asian countries [e.g., Cambodia (Agusa et al. 2007), Taiwan (Hsu et al. 2007; Soong et al. 1991), Japan (Murata et al. 2007; Sakamoto et al. 2007)], island nations [e.g., Bermuda (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004), Seychelles (Myers et al. 2007), Tahiti (Chateau-Degat 2005), Taiwan (Hsu et al. 2007; Soong et al. 1991), Japan (Murata et al. 2007; Sakamoto et al. 2007)], and some European countries [e.g., Spain (Falcó et al. 2006; Herreros et al. 2008) and the Faroe Islands (Weihe et al. 1996)] have reported fish/shellfish consumption levels greater than average worldwide consumption (World Health Organization 2008).\",\n \"In contrast to some other environmental exposures [e.g., higher blood lead concentrations] (Mahaffey et al. 1982), BHg concentrations increased with income. This is consistent with other studies in which women from higher income groups were at greater risk of MeHg exposure, as were women living in urban areas (Hightower and Moore 2003; Saint-Phard and Van Dorsten 2006).\",\n \"A more complex association between income and racial/ethnic group may also exist. According to the 1990 U.S. Census (U.S. Census Bureau 2008), the median family income of Japanese-American families exceeded that of non-Hispanic white families. By contrast, the income of Cambodian-American families was lower than that of black families. We could not address whether there is an interaction between belonging to the category designated as “other” and higher income within the NHANES data on BHg levels available at this time, because of sample size limitations.\",\n \"Our analysis of 30-day Hg intake indicated that there was no consistent trend in fish consumption by women of childbearing age over the 6-year period between 1999 and 2004. Our evaluation of NHANES fish intake data indicated no differences in the mean frequency or amount of particular fish and shellfish species consumed. However, the estimated 30-day Hg intake decreased at the 90th percentile and higher, whereas total fish consumption did not, which suggests a shift in fish species consumed. The BHg data indicated a reduction of the higher end of the distribution of BHg between the first 2-year interval (the 1999 and 2000 examinees) compared with the subsequent 4-year interval (the 2001–2004 examinees).\\nThe basis for these differences could possibly reflect spillover from the federal fish advisory program (U.S. EPA 2008b) in terms of total fish and shellfish consumption. A recent analysis of a nationally representative study specifically addressing fish-consumption patterns did not support this suggestion (Bradbury 2007).\\nThe four fish species listed in the federal advisory [swordfish, shark, tilefish, and king mackerel (U.S. EPA 2008b)] were rarely reported by the 5,465 women in this analysis. It is clear that these four fish species contributed little to Hg exposure in this general population of U.S. women. Individual states with higher Hg exposures [e.g., Hawaii (Sato et al., 2006), Florida (Karouna-Renier et al. 2008)] and greater fish consumption [Florida (Denger et al. 1994)] have substantially broader fish consumption advisories (e.g., Hawaii and Florida) aimed at reducing Hg exposure from high-Hg–containing species obtained locally (Florida Department of Health 2007; U.S. EPA 2008b). Despite the federal advisory’s emphasis on four species of highly contaminated fish (U.S. EPA 2008b) and the states’ emphasis on game-fish, the most commonly consumed finfish in the United States is tuna. Interpretation of Hg exposure from tuna was complicated by the specific wording of the dietary questions asked of the NHANES examinees, which did not differentiate between light or skipjack tuna and albacore tuna. The latter contains approximately three times more Hg than does the former: 0.38 μg/g for frozen and fresh tuna, 0.35 μg/g for canned albacore, and 0.12 μg/g for canned light tuna (Mahaffey et al. 2008).\",\n \"During the past decade, the U.S. EPA initiated substantial interventions aimed to reduce Hg releases and exposures (U.S. EPA 2008a) and issued advisories to limit consumption of high-Hg fish (U.S. EPA 2008b). Because of worldwide atmospheric distribution and subsequent deposition of Hg, local conditions and locally caught fish are not the main contributors to Hg intake for most people (Sunderland 2007). Although there are economic indications that consumption of some species of fish may have decreased in response to these advisories (Shimshack et al. 2007), Hg exposures may not follow a similar time trend despite regulatory efforts to reduce Hg exposures. A recent analysis of a nationally representative study specifically addressing fish-consumption patterns did not support this suggestion (Bradbury 2007). Our analysis of NHANES data calculating 30-day Hg intake indicated that there was no consistent trend in fish consumption by women of childbearing age over the 6-year period between 1999 and 2004.\",\n \"Significant geographic differences in BHg concentrations occurred within the United States: We found highest exposures in coastal areas and the Northeast census region. In the Northeast, 19% of women had BHg concentrations ≥ 3.5 μg/L. The highest 5% of BHg concentrations exceeded 8.2 μg/L in the Northeast and 7.2 μg/L in coastal areas, concentrations more than twice the 3.5 μg/L level of concern. BHg levels were predicted by the quantity and type of fish consumed. Over the 6-year period (1999–2004), the frequency of elevated BHg levels among women of childbearing age declined without a significant change in quantities of fish and shellfish consumed. This pattern suggests a more discerning series of choices in type of fish eaten rather than an overall reduction in fish consumption. Within all geographic regions, women at highest risk of elevated Hg exposures were more affluent and more likely to be of Asian or island ethnicity.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["materials|methods",null,null,"results","discussion",null,null,null,null,null,null,"conclusions"],"string":"[\n \"materials|methods\",\n null,\n null,\n \"results\",\n \"discussion\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"conclusions\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["blood","coastal","fish","mercury","NHANES","regional"],"string":"[\n \"blood\",\n \"coastal\",\n \"fish\",\n \"mercury\",\n \"NHANES\",\n \"regional\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Materials and Methods:\n Methodology for data analysis We evaluated data for examinees who participated in NHANES during survey years 1999–2004 to assess the statistical association between seafood consumption and BHg by region of residence, race/ethnicity, and annual income. Further, we examined time trends for both BHg and fish consumption. NHANES is an annual survey conducted by the NCHS. The data include BHg levels, 24-hr dietary recall, and 30-day finfish and shellfish consumption frequency for women 16–49 years of age who reside in the United States. The NHANES sampling frame includes all 50 states. The documentation and publicly available data for NHANES can be found online [Centers for Disease Control and Prevention (CDC) 2006b]. The regional data are not publicly available but can be accessed by special request to the National Center for Health Statistics (NCHS) through its Research Data Center. Procedures for submitting a proposal in order to access data that are not publicly available can be found online (CDC 2006c). We performed all analyses using SAS, version 9.1 (SAS Institute Inc., Cary, NC).\nFollowing NHANES analytic guidelines (CDC 2006a), we used SAS procedures that accurately incorporate the stratification and multistage sampling of NHANES: Proc SurveyMeans, Proc SurveyReg, and Proc SurveyFreq (SAS Institute Inc.). The weights provided by NCHS compensate for the oversampling of various subpopulations and adjust for nonresponse bias. We used weights for estimating statistics for coastal and non-coastal regions to retain these adjustments. Because we considered the variables of interest (BHg and fish consumption) to be related to some of the factors that were oversampled, we retained these adjustments to minimize the bias in the estimates. For example, NHANES over-sampled Mexican Americans, who also have lower BHg than do other racial/ethnic groups. If the weights were not used to estimate the distribution of BHg, the results would be biased low. We recognize that some bias may remain within the estimates because the weights were not specifically created for the geographic regions of coastal and noncoastal; however, these subdivisions (e.g., coastal, noncoastal, Pacific, Atlantic) are based on counties, the primary sampling units of NHANES (CDC 2006a). All multivariate analyses were done unweighted because factors associated with the dependent variables and for which over-sampling was based were included as covariates and thus adjusted for in the modeling.\nIn order to estimate long-term Hg intake, we combined data collected through the 24-hr dietary recall and the 30-day fish frequency questionnaire to estimate 30-day Hg intake [for specific methodologies, see Mahaffey and Rice (1997); Mahaffey et al. (2004)]. If we found statistically significant differences in amount of fish consumed per meal from the 24-hr dietary recall by either coastal status (participants who lived in a county that bordered the Pacific or Atlantic Oceans, the Gulf of Mexico, or the Great Lakes vs. those who did not) or data release (1999–2000–2001–2002, or 2003–2004), we calculated separate averages. We generated statistically representative estimates from these data using the statistical weights provided by NCHS and following the relevant analytical guidelines published by NCHS (CDC 2006a).\nWe evaluated data for examinees who participated in NHANES during survey years 1999–2004 to assess the statistical association between seafood consumption and BHg by region of residence, race/ethnicity, and annual income. Further, we examined time trends for both BHg and fish consumption. NHANES is an annual survey conducted by the NCHS. The data include BHg levels, 24-hr dietary recall, and 30-day finfish and shellfish consumption frequency for women 16–49 years of age who reside in the United States. The NHANES sampling frame includes all 50 states. The documentation and publicly available data for NHANES can be found online [Centers for Disease Control and Prevention (CDC) 2006b]. The regional data are not publicly available but can be accessed by special request to the National Center for Health Statistics (NCHS) through its Research Data Center. Procedures for submitting a proposal in order to access data that are not publicly available can be found online (CDC 2006c). We performed all analyses using SAS, version 9.1 (SAS Institute Inc., Cary, NC).\nFollowing NHANES analytic guidelines (CDC 2006a), we used SAS procedures that accurately incorporate the stratification and multistage sampling of NHANES: Proc SurveyMeans, Proc SurveyReg, and Proc SurveyFreq (SAS Institute Inc.). The weights provided by NCHS compensate for the oversampling of various subpopulations and adjust for nonresponse bias. We used weights for estimating statistics for coastal and non-coastal regions to retain these adjustments. Because we considered the variables of interest (BHg and fish consumption) to be related to some of the factors that were oversampled, we retained these adjustments to minimize the bias in the estimates. For example, NHANES over-sampled Mexican Americans, who also have lower BHg than do other racial/ethnic groups. If the weights were not used to estimate the distribution of BHg, the results would be biased low. We recognize that some bias may remain within the estimates because the weights were not specifically created for the geographic regions of coastal and noncoastal; however, these subdivisions (e.g., coastal, noncoastal, Pacific, Atlantic) are based on counties, the primary sampling units of NHANES (CDC 2006a). All multivariate analyses were done unweighted because factors associated with the dependent variables and for which over-sampling was based were included as covariates and thus adjusted for in the modeling.\nIn order to estimate long-term Hg intake, we combined data collected through the 24-hr dietary recall and the 30-day fish frequency questionnaire to estimate 30-day Hg intake [for specific methodologies, see Mahaffey and Rice (1997); Mahaffey et al. (2004)]. If we found statistically significant differences in amount of fish consumed per meal from the 24-hr dietary recall by either coastal status (participants who lived in a county that bordered the Pacific or Atlantic Oceans, the Gulf of Mexico, or the Great Lakes vs. those who did not) or data release (1999–2000–2001–2002, or 2003–2004), we calculated separate averages. We generated statistically representative estimates from these data using the statistical weights provided by NCHS and following the relevant analytical guidelines published by NCHS (CDC 2006a).\n Methodology for defining coastal and non-coastal areas Fish consumption is generally believed to be a major contributor to BHg concentration. We hypothesized that patterns of fish and shellfish consumption would vary between U.S. residents who live on or near the coast (within ~ 25–50 miles) and those who live inland. We further hypothesized that fish consumption patterns, and thus BHg concentrations, may also vary by specific coast (e.g., residents near the Atlantic Coast may have different BHg concentrations than those on the coast of the Gulf of Mexico) and specific inland region (e.g., West vs. Midwest). To test these hypotheses, we categorized NHANES respondents as living in either a coastal or a noncoastal county and further categorized them by eight regions: Atlantic Coast, Northeast, Great Lakes, Midwest, South, Gulf of Mexico, West, and Pacific Coast.\nThe geographic unit used by NHANES is a county or county equivalent (CDC 2006a); therefore, we limited our definitions of coastal and noncoastal to follow county boundaries. We defined all counties that bordered the Pacific or Atlantic Oceans, the Gulf of Mexico, or any of the Great Lakes as coastal. Additionally, we defined counties that bordered estuaries and bays as coastal, as well as counties whose center point was within approximately 25 miles of any coast even if not directly bordering a coast. [For the list of counties, see Supplemental Material, Table 13 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] We then defined the four coastal regions based on nearest body of water; for example, counties in California, Oregon, Washington, Alaska, and Hawaii that we defined as coastal were categorized as Pacific Coast. We separated noncoastal counties into four inland regions using the U.S. Census regions; for example, noncoastal counties in California, Oregon, Washington, and Alaska along with the entire states of Idaho, Montana, Wyoming, Colorado, New Mexico, Arizona, Utah, and Nevada became the West region (we classified all of Hawaii as coastal). We also designated the entire state of Florida as coastal, split between the Atlantic Coast and the Gulf of Mexico. We designated Miami-Dade County as Atlantic Coast, and Monroe County as Gulf of Mexico. These subdivisions run the risk of small sample sizes; however, the definition of coastal was sufficiently broad to avoid single primary sampling units.\nFish consumption is generally believed to be a major contributor to BHg concentration. We hypothesized that patterns of fish and shellfish consumption would vary between U.S. residents who live on or near the coast (within ~ 25–50 miles) and those who live inland. We further hypothesized that fish consumption patterns, and thus BHg concentrations, may also vary by specific coast (e.g., residents near the Atlantic Coast may have different BHg concentrations than those on the coast of the Gulf of Mexico) and specific inland region (e.g., West vs. Midwest). To test these hypotheses, we categorized NHANES respondents as living in either a coastal or a noncoastal county and further categorized them by eight regions: Atlantic Coast, Northeast, Great Lakes, Midwest, South, Gulf of Mexico, West, and Pacific Coast.\nThe geographic unit used by NHANES is a county or county equivalent (CDC 2006a); therefore, we limited our definitions of coastal and noncoastal to follow county boundaries. We defined all counties that bordered the Pacific or Atlantic Oceans, the Gulf of Mexico, or any of the Great Lakes as coastal. Additionally, we defined counties that bordered estuaries and bays as coastal, as well as counties whose center point was within approximately 25 miles of any coast even if not directly bordering a coast. [For the list of counties, see Supplemental Material, Table 13 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] We then defined the four coastal regions based on nearest body of water; for example, counties in California, Oregon, Washington, Alaska, and Hawaii that we defined as coastal were categorized as Pacific Coast. We separated noncoastal counties into four inland regions using the U.S. Census regions; for example, noncoastal counties in California, Oregon, Washington, and Alaska along with the entire states of Idaho, Montana, Wyoming, Colorado, New Mexico, Arizona, Utah, and Nevada became the West region (we classified all of Hawaii as coastal). We also designated the entire state of Florida as coastal, split between the Atlantic Coast and the Gulf of Mexico. We designated Miami-Dade County as Atlantic Coast, and Monroe County as Gulf of Mexico. These subdivisions run the risk of small sample sizes; however, the definition of coastal was sufficiently broad to avoid single primary sampling units.\n\nMethodology for data analysis:\nWe evaluated data for examinees who participated in NHANES during survey years 1999–2004 to assess the statistical association between seafood consumption and BHg by region of residence, race/ethnicity, and annual income. Further, we examined time trends for both BHg and fish consumption. NHANES is an annual survey conducted by the NCHS. The data include BHg levels, 24-hr dietary recall, and 30-day finfish and shellfish consumption frequency for women 16–49 years of age who reside in the United States. The NHANES sampling frame includes all 50 states. The documentation and publicly available data for NHANES can be found online [Centers for Disease Control and Prevention (CDC) 2006b]. The regional data are not publicly available but can be accessed by special request to the National Center for Health Statistics (NCHS) through its Research Data Center. Procedures for submitting a proposal in order to access data that are not publicly available can be found online (CDC 2006c). We performed all analyses using SAS, version 9.1 (SAS Institute Inc., Cary, NC).\nFollowing NHANES analytic guidelines (CDC 2006a), we used SAS procedures that accurately incorporate the stratification and multistage sampling of NHANES: Proc SurveyMeans, Proc SurveyReg, and Proc SurveyFreq (SAS Institute Inc.). The weights provided by NCHS compensate for the oversampling of various subpopulations and adjust for nonresponse bias. We used weights for estimating statistics for coastal and non-coastal regions to retain these adjustments. Because we considered the variables of interest (BHg and fish consumption) to be related to some of the factors that were oversampled, we retained these adjustments to minimize the bias in the estimates. For example, NHANES over-sampled Mexican Americans, who also have lower BHg than do other racial/ethnic groups. If the weights were not used to estimate the distribution of BHg, the results would be biased low. We recognize that some bias may remain within the estimates because the weights were not specifically created for the geographic regions of coastal and noncoastal; however, these subdivisions (e.g., coastal, noncoastal, Pacific, Atlantic) are based on counties, the primary sampling units of NHANES (CDC 2006a). All multivariate analyses were done unweighted because factors associated with the dependent variables and for which over-sampling was based were included as covariates and thus adjusted for in the modeling.\nIn order to estimate long-term Hg intake, we combined data collected through the 24-hr dietary recall and the 30-day fish frequency questionnaire to estimate 30-day Hg intake [for specific methodologies, see Mahaffey and Rice (1997); Mahaffey et al. (2004)]. If we found statistically significant differences in amount of fish consumed per meal from the 24-hr dietary recall by either coastal status (participants who lived in a county that bordered the Pacific or Atlantic Oceans, the Gulf of Mexico, or the Great Lakes vs. those who did not) or data release (1999–2000–2001–2002, or 2003–2004), we calculated separate averages. We generated statistically representative estimates from these data using the statistical weights provided by NCHS and following the relevant analytical guidelines published by NCHS (CDC 2006a).\n\nMethodology for defining coastal and non-coastal areas:\nFish consumption is generally believed to be a major contributor to BHg concentration. We hypothesized that patterns of fish and shellfish consumption would vary between U.S. residents who live on or near the coast (within ~ 25–50 miles) and those who live inland. We further hypothesized that fish consumption patterns, and thus BHg concentrations, may also vary by specific coast (e.g., residents near the Atlantic Coast may have different BHg concentrations than those on the coast of the Gulf of Mexico) and specific inland region (e.g., West vs. Midwest). To test these hypotheses, we categorized NHANES respondents as living in either a coastal or a noncoastal county and further categorized them by eight regions: Atlantic Coast, Northeast, Great Lakes, Midwest, South, Gulf of Mexico, West, and Pacific Coast.\nThe geographic unit used by NHANES is a county or county equivalent (CDC 2006a); therefore, we limited our definitions of coastal and noncoastal to follow county boundaries. We defined all counties that bordered the Pacific or Atlantic Oceans, the Gulf of Mexico, or any of the Great Lakes as coastal. Additionally, we defined counties that bordered estuaries and bays as coastal, as well as counties whose center point was within approximately 25 miles of any coast even if not directly bordering a coast. [For the list of counties, see Supplemental Material, Table 13 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] We then defined the four coastal regions based on nearest body of water; for example, counties in California, Oregon, Washington, Alaska, and Hawaii that we defined as coastal were categorized as Pacific Coast. We separated noncoastal counties into four inland regions using the U.S. Census regions; for example, noncoastal counties in California, Oregon, Washington, and Alaska along with the entire states of Idaho, Montana, Wyoming, Colorado, New Mexico, Arizona, Utah, and Nevada became the West region (we classified all of Hawaii as coastal). We also designated the entire state of Florida as coastal, split between the Atlantic Coast and the Gulf of Mexico. We designated Miami-Dade County as Atlantic Coast, and Monroe County as Gulf of Mexico. These subdivisions run the risk of small sample sizes; however, the definition of coastal was sufficiently broad to avoid single primary sampling units.\n\nResults:\nTable 1 shows the distributions of estimates of the number of women with BHg concentrations > 3.5 μg/L and > 5.8 μg/L, by region. Analyses indicate that between 1999 and 2004, the Northeast had the highest percentage of women with BHg concentrations above the 3.5 μg/L level of concern (> 19%), whereas the South had the largest estimated number of women (1.21 million) with ≥ 3.5 μg/L BHg because of elevated population in that region. Geometric means (Figure 1) show similar trends, with the highest BHg concentrations in the Northeast, followed by the West, South, and Midwest census regions. In the Northeast, the highest 5% of BHg concentrations exceeded 8.2 μg/L. [For full distributions, see Supplemental Material, Table 2 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] When we included coastal regions in this analysis, additional spatial heterogeneity in BHg (Figure 2A) and estimated 30-day Hg intake (Figure 2B) was apparent, with elevated exposures in all coastal areas relative to their neighboring inland regions except in the Great Lakes. In the coastal areas, the highest 5% of BHg concentrations exceeded 7.2 μg/L, with the Atlantic Coast exceeding 10.9 μg/L. [For the full distributions, see Supplemental Material, Table 4 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] Fish species eaten by survey participants varied by region (Figure 3), with respondents in coastal regions reporting higher frequency of consumption of fish containing higher levels of Hg. BHg concentrations were strongly associated with the frequency of fish consumption (Figure 4). BHg increased with monthly estimated consumption of fish and shellfish over the range of never/rarely to 4 or more times per week. In multiple regression modeling, women from the Atlantic (p < 0.01), Pacific (p < 0.0001), and Gulf (p < 0.0001) coasts had higher BHg concentrations compared with women from the inland West, whereas women from the inland Northeast and inland Midwest had significantly lower BHg levels (p < 0.0001). [For the full regression results, see Supplemental Material, Table 11 (http://www.ehponline.org/members/2008/11674/suppl.pdf).]\nAnalysis of temporal trends through simple regression modeling showed no statistically significant difference among the three sets of study years (1999–2000–2001–2002, and 2003–2004) for BHg (p = 0.07), estimated 30-day Hg intake (p = 0.11), or reported frequency of seafood consumption (p = 0.69). However, in multiple regression modeling, adjusting for covariates including coastal/non-coastal residence, the years 1999–2000 had significantly higher BHg levels (p < 0.0001) compared with 2003–2004, and 2001–2002 had significantly lower BHg levels (p < 0.01) [Supplemental Material, Table 11 (http://www.ehponline.org/members/2008/11674/suppl.pdf)]. Although the analyses did not support the conclusion that there was a general downward trend in BHg concentrations over the 6-year study period, there was a decline in the upper percentiles reflecting the most highly exposed women with BHg concentrations greater than established levels of concern [Supplemental Material, Table 9 (http://www.ehponline.org/members/2008/11674/suppl.pdf)]. In addition, the percentage of examinees with BHg values ≥ 3.5 μg/L and ≥ 5.8 μg/L was much greater in 1999–2000 compared to 2001–2002 and 2003–2004 (Figure 5). We found no consistent trend in fish consumption across the study years. We observed a decrease in the 90th percentile of 30-day estimated intake of Hg through seafood consumption across the study years even though there was no similar decrease in the 90th percentile of 30-day estimated consumption of grams of fish and shellfish (Figure 6). This suggests a shift in consumption to seafood containing less Hg. We did not observe a similar pattern at the mean, suggesting that this shift in seafood consumption occurred mainly with the highest fish and shellfish consumers [Supplemental Material, Table 9 (http://www.ehponline.org/members/2008/11674/suppl.pdf)]. The RfD for Hg intake is 0.1 μg Hg/kg body weight (μg/kgbw) per day, or 3.0 μg Hg/kgbw per month (30 days).\nResults also showed that self-selected ethnic identity was associated with total BHg concentrations, estimated 30-day Hg intake, and frequency of either finfish or shellfish consumption. For example, BHg levels, reported frequency of seafood consumption, and 30-day Hg intakes were highest among women who designated themselves as being in the “other” category (mostly people whose ancestry is Asian, Native American, Pacific Islands, and the Caribbean Islands). [See also Supplemental Material, Tables 5–7 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] Table 2 presents the percentages of women by race/ethnicity that had ≥ 3.5 and ≥ 5.8 μg/L BHg.\nWe identified statistically significant relationships between higher income and, respectively, increasing BHg concentration (p < 0.0001), estimated 30-day intake of Hg (p = 0.008), and 30-day frequency of finfish and shellfish consumption (p < 0.0001) through bivariate regressions. [For the distributions of blood total Hg, estimated Hg intake, and frequency of finfish and shellfish consumption by annual income, see Supplemental Material, Tables 6–8 (http://www.ehponline.org/members/2008/11674/suppl.pdf).] In addition, women from families reporting incomes of ≥ $75,000 (the reference category) had statistically higher BHg levels than did women from families with incomes of ≤ $55,000 (p < 0.01). In all cases, BHg concentrations were also significantly associated with age and estimated 30-day Hg intake (p < 0.0001). Table 3 presents the percentage of women by annual income with ≥ 3.5 and ≥ 5.8 μg/L BHg.\nIn multiple regression modeling, after adjusting for other factors related to BHg, both race/ethnicity and income remained statistically significant predictors of BHg levels observed in this study [Supplemental Material, Table 11 (http://www.ehponline.org/members/2008/11674/suppl.pdf)]. Non-Hispanic blacks (p < 0.0001) and women grouped in the “other” racial category (p = 0.002) had significantly higher BHg concentrations than did non-Hispanic whites.\n\nDiscussion:\n Regional and coastal variation in BHg concentrations and in fish consumption Comparisons of the distribution of BHg data with reference values aimed at protecting the fetal nervous system have been made using national-level data (Mahaffey et al. 2004). NHANES data are often used to make population estimates through application of weighting factors to variables of interest such as BHg. Population estimates for U.S. Census regions and their distribution of BHg concentrations indicate that women living in the Northeast had BHg concentrations exceeding levels of concern more often than did women living in the South and West. The lowest Hg exposures were reported among women living in the Midwest.\nBecause NHANES was not designed to provide population estimates for coastal and noncoastal areas, unbiased estimates for the number of women having BHg concentrations ≥ 3.5 μg/L and ≥ 5.8 μg/L cannot be developed comparing coastal- and noncoastal-residing women. Although the following are not population estimates, they are statistics for a geographic region: Women living in coastal areas were at greater risk of having BHg concentrations ≥ 3.5 μg/L (16.25% for coastal and 5.99% for noncoastal residents) and ≥ 5.8 μg/L (8.11% for coastal and 2.06% for noncoastal residents). Women living near the coastal areas had approximately three to four times greater risk of exceeding acceptable levels of Hg exposure than did noncoastal-dwelling women. There may be some bias in these results due to the weighting issues (see “Materials and Methods”); however, we do not believe that this bias is a major factor underlying these great differences.\nMeHg exposures exceeding health-based standards, including U.S. EPA’s RfD (Rice et al. 2003), occurred more commonly among women living in coastal areas. These health-based standards were based on avoiding MeHg-associated delays and deficits in neurologic development of children after in utero exposure to MeHg (Mergler et al. 2007; Rice et al. 2003). At higher exposures to MeHg, including the highest concentrations reported during these survey years, the women themselves may risk adverse neuropsychological and neurobehavioral outcomes (Mergler et al. 2007).\nWithin the United States, people living in coastal areas consume more fish and shellfish than do those living in noncoastal areas and consume fish with higher Hg concentrations. Reports from New York City (McKelvey et al. 2007) and Florida (Denger et al. 1994; Karouna-Renier et al. 2008) support our identification of higher Hg exposures in U.S. coastal areas. This is part of a worldwide pattern. An overall pattern of higher BHg levels has also been reported among people living on U.S. islands [Hawaii (Sato et al. 2006)] and territories [e.g., Puerto Rico (Ortiz-Roque and López-Rivera 2004)]. A similar pattern has been repeated in other islands [Bermuda (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004), Fiji (Kumar et al. 2006), Seychelles (Myers et al. 2007), and Tahiti (Chateau-Degat 2005; Dewailly et al. 2008)] compared with inland populations. Among these island populations, BHg concentrations at the upper end of the distribution fall into the range of 50 μg/L (~ 250 nmol/L) and higher (Chateau-Degat 2005). In Bermuda, cord BHg concentrations as high as 160 nmol/L (~ 35 μg/L) have been reported (arithmetic mean, 41.3 ± 4.7 nmol/L or 8.0 ± 1.0 μg/L) (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004).\nHigher BHg concentrations in the U.S. Northeast found in this study reflect, in part, more frequent fish and shellfish consumption. Additional variability may be a function of differences in Hg concentrations among species and geographic regions (Sunderland 2007). For example, recent information on “hot spots” for Hg in wildlife tissues (Evers et al. 2007) could be associated with higher Hg concentrations for locally obtained fish. One limitation of the present analysis was the use of a fish-species–specific mean Hg concentration (i.e., nondistributional values) to estimate individual exposure. Although most fish consumed by the U.S. population is not locally obtained (i.e., commercially obtained from diverse regions and countries) (Sunderland 2007), analytical results showing geographic differences in the distribution of BHg could reflect higher Hg concentrations in locally obtained fish within the Northeast states.\nComparisons of the distribution of BHg data with reference values aimed at protecting the fetal nervous system have been made using national-level data (Mahaffey et al. 2004). NHANES data are often used to make population estimates through application of weighting factors to variables of interest such as BHg. Population estimates for U.S. Census regions and their distribution of BHg concentrations indicate that women living in the Northeast had BHg concentrations exceeding levels of concern more often than did women living in the South and West. The lowest Hg exposures were reported among women living in the Midwest.\nBecause NHANES was not designed to provide population estimates for coastal and noncoastal areas, unbiased estimates for the number of women having BHg concentrations ≥ 3.5 μg/L and ≥ 5.8 μg/L cannot be developed comparing coastal- and noncoastal-residing women. Although the following are not population estimates, they are statistics for a geographic region: Women living in coastal areas were at greater risk of having BHg concentrations ≥ 3.5 μg/L (16.25% for coastal and 5.99% for noncoastal residents) and ≥ 5.8 μg/L (8.11% for coastal and 2.06% for noncoastal residents). Women living near the coastal areas had approximately three to four times greater risk of exceeding acceptable levels of Hg exposure than did noncoastal-dwelling women. There may be some bias in these results due to the weighting issues (see “Materials and Methods”); however, we do not believe that this bias is a major factor underlying these great differences.\nMeHg exposures exceeding health-based standards, including U.S. EPA’s RfD (Rice et al. 2003), occurred more commonly among women living in coastal areas. These health-based standards were based on avoiding MeHg-associated delays and deficits in neurologic development of children after in utero exposure to MeHg (Mergler et al. 2007; Rice et al. 2003). At higher exposures to MeHg, including the highest concentrations reported during these survey years, the women themselves may risk adverse neuropsychological and neurobehavioral outcomes (Mergler et al. 2007).\nWithin the United States, people living in coastal areas consume more fish and shellfish than do those living in noncoastal areas and consume fish with higher Hg concentrations. Reports from New York City (McKelvey et al. 2007) and Florida (Denger et al. 1994; Karouna-Renier et al. 2008) support our identification of higher Hg exposures in U.S. coastal areas. This is part of a worldwide pattern. An overall pattern of higher BHg levels has also been reported among people living on U.S. islands [Hawaii (Sato et al. 2006)] and territories [e.g., Puerto Rico (Ortiz-Roque and López-Rivera 2004)]. A similar pattern has been repeated in other islands [Bermuda (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004), Fiji (Kumar et al. 2006), Seychelles (Myers et al. 2007), and Tahiti (Chateau-Degat 2005; Dewailly et al. 2008)] compared with inland populations. Among these island populations, BHg concentrations at the upper end of the distribution fall into the range of 50 μg/L (~ 250 nmol/L) and higher (Chateau-Degat 2005). In Bermuda, cord BHg concentrations as high as 160 nmol/L (~ 35 μg/L) have been reported (arithmetic mean, 41.3 ± 4.7 nmol/L or 8.0 ± 1.0 μg/L) (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004).\nHigher BHg concentrations in the U.S. Northeast found in this study reflect, in part, more frequent fish and shellfish consumption. Additional variability may be a function of differences in Hg concentrations among species and geographic regions (Sunderland 2007). For example, recent information on “hot spots” for Hg in wildlife tissues (Evers et al. 2007) could be associated with higher Hg concentrations for locally obtained fish. One limitation of the present analysis was the use of a fish-species–specific mean Hg concentration (i.e., nondistributional values) to estimate individual exposure. Although most fish consumed by the U.S. population is not locally obtained (i.e., commercially obtained from diverse regions and countries) (Sunderland 2007), analytical results showing geographic differences in the distribution of BHg could reflect higher Hg concentrations in locally obtained fish within the Northeast states.\n Ethnic group variation on fish intake and BHg concentrations Ethnic origins were associated with Hg exposures with those designated as “other” (i.e., Asian, Pacific and Caribbean Islander, Native American, Alaska Native, multiracial, and unknown race) having higher BHg concentrations. From additional studies, people of Asian descent whose food choices are influenced by Asian dietary patterns (Kudo et al. 2000; Sechena et al. 2003) tended to consume fish more frequently, in greater variety, and in greater quantity than did non-Asians. The ethnic diversity of the U.S. population is well known. As of 1997, 61% of the Asian population living in the United States was foreign-born (Council of Economic Advisors 1999). By comparison with overall U.S. data, higher BHg concentrations among Asians and islanders were reported for Taiwan (Hsu et al. 2007), Cambodia (Agusa et al. 2007), Fiji (Kumar et al. 2006), and Tahiti (Dewailly et al. 2008).\nWithin the United States, fish and shellfish consumption, predicting Hg exposure described previously, varies widely, in part a reflection of ethnicity. For example, Asian countries [e.g., Cambodia (Agusa et al. 2007), Taiwan (Hsu et al. 2007; Soong et al. 1991), Japan (Murata et al. 2007; Sakamoto et al. 2007)], island nations [e.g., Bermuda (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004), Seychelles (Myers et al. 2007), Tahiti (Chateau-Degat 2005), Taiwan (Hsu et al. 2007; Soong et al. 1991), Japan (Murata et al. 2007; Sakamoto et al. 2007)], and some European countries [e.g., Spain (Falcó et al. 2006; Herreros et al. 2008) and the Faroe Islands (Weihe et al. 1996)] have reported fish/shellfish consumption levels greater than average worldwide consumption (World Health Organization 2008).\nEthnic origins were associated with Hg exposures with those designated as “other” (i.e., Asian, Pacific and Caribbean Islander, Native American, Alaska Native, multiracial, and unknown race) having higher BHg concentrations. From additional studies, people of Asian descent whose food choices are influenced by Asian dietary patterns (Kudo et al. 2000; Sechena et al. 2003) tended to consume fish more frequently, in greater variety, and in greater quantity than did non-Asians. The ethnic diversity of the U.S. population is well known. As of 1997, 61% of the Asian population living in the United States was foreign-born (Council of Economic Advisors 1999). By comparison with overall U.S. data, higher BHg concentrations among Asians and islanders were reported for Taiwan (Hsu et al. 2007), Cambodia (Agusa et al. 2007), Fiji (Kumar et al. 2006), and Tahiti (Dewailly et al. 2008).\nWithin the United States, fish and shellfish consumption, predicting Hg exposure described previously, varies widely, in part a reflection of ethnicity. For example, Asian countries [e.g., Cambodia (Agusa et al. 2007), Taiwan (Hsu et al. 2007; Soong et al. 1991), Japan (Murata et al. 2007; Sakamoto et al. 2007)], island nations [e.g., Bermuda (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004), Seychelles (Myers et al. 2007), Tahiti (Chateau-Degat 2005), Taiwan (Hsu et al. 2007; Soong et al. 1991), Japan (Murata et al. 2007; Sakamoto et al. 2007)], and some European countries [e.g., Spain (Falcó et al. 2006; Herreros et al. 2008) and the Faroe Islands (Weihe et al. 1996)] have reported fish/shellfish consumption levels greater than average worldwide consumption (World Health Organization 2008).\n Income differences in association with fish intake and BHg concentrations In contrast to some other environmental exposures [e.g., higher blood lead concentrations] (Mahaffey et al. 1982), BHg concentrations increased with income. This is consistent with other studies in which women from higher income groups were at greater risk of MeHg exposure, as were women living in urban areas (Hightower and Moore 2003; Saint-Phard and Van Dorsten 2006).\nIn contrast to some other environmental exposures [e.g., higher blood lead concentrations] (Mahaffey et al. 1982), BHg concentrations increased with income. This is consistent with other studies in which women from higher income groups were at greater risk of MeHg exposure, as were women living in urban areas (Hightower and Moore 2003; Saint-Phard and Van Dorsten 2006).\n Interactions between income and ethnic group A more complex association between income and racial/ethnic group may also exist. According to the 1990 U.S. Census (U.S. Census Bureau 2008), the median family income of Japanese-American families exceeded that of non-Hispanic white families. By contrast, the income of Cambodian-American families was lower than that of black families. We could not address whether there is an interaction between belonging to the category designated as “other” and higher income within the NHANES data on BHg levels available at this time, because of sample size limitations.\nA more complex association between income and racial/ethnic group may also exist. According to the 1990 U.S. Census (U.S. Census Bureau 2008), the median family income of Japanese-American families exceeded that of non-Hispanic white families. By contrast, the income of Cambodian-American families was lower than that of black families. We could not address whether there is an interaction between belonging to the category designated as “other” and higher income within the NHANES data on BHg levels available at this time, because of sample size limitations.\n Time trends in Hg exposure absent changes in total fish consumption Our analysis of 30-day Hg intake indicated that there was no consistent trend in fish consumption by women of childbearing age over the 6-year period between 1999 and 2004. Our evaluation of NHANES fish intake data indicated no differences in the mean frequency or amount of particular fish and shellfish species consumed. However, the estimated 30-day Hg intake decreased at the 90th percentile and higher, whereas total fish consumption did not, which suggests a shift in fish species consumed. The BHg data indicated a reduction of the higher end of the distribution of BHg between the first 2-year interval (the 1999 and 2000 examinees) compared with the subsequent 4-year interval (the 2001–2004 examinees).\nThe basis for these differences could possibly reflect spillover from the federal fish advisory program (U.S. EPA 2008b) in terms of total fish and shellfish consumption. A recent analysis of a nationally representative study specifically addressing fish-consumption patterns did not support this suggestion (Bradbury 2007).\nThe four fish species listed in the federal advisory [swordfish, shark, tilefish, and king mackerel (U.S. EPA 2008b)] were rarely reported by the 5,465 women in this analysis. It is clear that these four fish species contributed little to Hg exposure in this general population of U.S. women. Individual states with higher Hg exposures [e.g., Hawaii (Sato et al., 2006), Florida (Karouna-Renier et al. 2008)] and greater fish consumption [Florida (Denger et al. 1994)] have substantially broader fish consumption advisories (e.g., Hawaii and Florida) aimed at reducing Hg exposure from high-Hg–containing species obtained locally (Florida Department of Health 2007; U.S. EPA 2008b). Despite the federal advisory’s emphasis on four species of highly contaminated fish (U.S. EPA 2008b) and the states’ emphasis on game-fish, the most commonly consumed finfish in the United States is tuna. Interpretation of Hg exposure from tuna was complicated by the specific wording of the dietary questions asked of the NHANES examinees, which did not differentiate between light or skipjack tuna and albacore tuna. The latter contains approximately three times more Hg than does the former: 0.38 μg/g for frozen and fresh tuna, 0.35 μg/g for canned albacore, and 0.12 μg/g for canned light tuna (Mahaffey et al. 2008).\nOur analysis of 30-day Hg intake indicated that there was no consistent trend in fish consumption by women of childbearing age over the 6-year period between 1999 and 2004. Our evaluation of NHANES fish intake data indicated no differences in the mean frequency or amount of particular fish and shellfish species consumed. However, the estimated 30-day Hg intake decreased at the 90th percentile and higher, whereas total fish consumption did not, which suggests a shift in fish species consumed. The BHg data indicated a reduction of the higher end of the distribution of BHg between the first 2-year interval (the 1999 and 2000 examinees) compared with the subsequent 4-year interval (the 2001–2004 examinees).\nThe basis for these differences could possibly reflect spillover from the federal fish advisory program (U.S. EPA 2008b) in terms of total fish and shellfish consumption. A recent analysis of a nationally representative study specifically addressing fish-consumption patterns did not support this suggestion (Bradbury 2007).\nThe four fish species listed in the federal advisory [swordfish, shark, tilefish, and king mackerel (U.S. EPA 2008b)] were rarely reported by the 5,465 women in this analysis. It is clear that these four fish species contributed little to Hg exposure in this general population of U.S. women. Individual states with higher Hg exposures [e.g., Hawaii (Sato et al., 2006), Florida (Karouna-Renier et al. 2008)] and greater fish consumption [Florida (Denger et al. 1994)] have substantially broader fish consumption advisories (e.g., Hawaii and Florida) aimed at reducing Hg exposure from high-Hg–containing species obtained locally (Florida Department of Health 2007; U.S. EPA 2008b). Despite the federal advisory’s emphasis on four species of highly contaminated fish (U.S. EPA 2008b) and the states’ emphasis on game-fish, the most commonly consumed finfish in the United States is tuna. Interpretation of Hg exposure from tuna was complicated by the specific wording of the dietary questions asked of the NHANES examinees, which did not differentiate between light or skipjack tuna and albacore tuna. The latter contains approximately three times more Hg than does the former: 0.38 μg/g for frozen and fresh tuna, 0.35 μg/g for canned albacore, and 0.12 μg/g for canned light tuna (Mahaffey et al. 2008).\n Changes in MeHg exposure over time During the past decade, the U.S. EPA initiated substantial interventions aimed to reduce Hg releases and exposures (U.S. EPA 2008a) and issued advisories to limit consumption of high-Hg fish (U.S. EPA 2008b). Because of worldwide atmospheric distribution and subsequent deposition of Hg, local conditions and locally caught fish are not the main contributors to Hg intake for most people (Sunderland 2007). Although there are economic indications that consumption of some species of fish may have decreased in response to these advisories (Shimshack et al. 2007), Hg exposures may not follow a similar time trend despite regulatory efforts to reduce Hg exposures. A recent analysis of a nationally representative study specifically addressing fish-consumption patterns did not support this suggestion (Bradbury 2007). Our analysis of NHANES data calculating 30-day Hg intake indicated that there was no consistent trend in fish consumption by women of childbearing age over the 6-year period between 1999 and 2004.\nDuring the past decade, the U.S. EPA initiated substantial interventions aimed to reduce Hg releases and exposures (U.S. EPA 2008a) and issued advisories to limit consumption of high-Hg fish (U.S. EPA 2008b). Because of worldwide atmospheric distribution and subsequent deposition of Hg, local conditions and locally caught fish are not the main contributors to Hg intake for most people (Sunderland 2007). Although there are economic indications that consumption of some species of fish may have decreased in response to these advisories (Shimshack et al. 2007), Hg exposures may not follow a similar time trend despite regulatory efforts to reduce Hg exposures. A recent analysis of a nationally representative study specifically addressing fish-consumption patterns did not support this suggestion (Bradbury 2007). Our analysis of NHANES data calculating 30-day Hg intake indicated that there was no consistent trend in fish consumption by women of childbearing age over the 6-year period between 1999 and 2004.\n\nRegional and coastal variation in BHg concentrations and in fish consumption:\nComparisons of the distribution of BHg data with reference values aimed at protecting the fetal nervous system have been made using national-level data (Mahaffey et al. 2004). NHANES data are often used to make population estimates through application of weighting factors to variables of interest such as BHg. Population estimates for U.S. Census regions and their distribution of BHg concentrations indicate that women living in the Northeast had BHg concentrations exceeding levels of concern more often than did women living in the South and West. The lowest Hg exposures were reported among women living in the Midwest.\nBecause NHANES was not designed to provide population estimates for coastal and noncoastal areas, unbiased estimates for the number of women having BHg concentrations ≥ 3.5 μg/L and ≥ 5.8 μg/L cannot be developed comparing coastal- and noncoastal-residing women. Although the following are not population estimates, they are statistics for a geographic region: Women living in coastal areas were at greater risk of having BHg concentrations ≥ 3.5 μg/L (16.25% for coastal and 5.99% for noncoastal residents) and ≥ 5.8 μg/L (8.11% for coastal and 2.06% for noncoastal residents). Women living near the coastal areas had approximately three to four times greater risk of exceeding acceptable levels of Hg exposure than did noncoastal-dwelling women. There may be some bias in these results due to the weighting issues (see “Materials and Methods”); however, we do not believe that this bias is a major factor underlying these great differences.\nMeHg exposures exceeding health-based standards, including U.S. EPA’s RfD (Rice et al. 2003), occurred more commonly among women living in coastal areas. These health-based standards were based on avoiding MeHg-associated delays and deficits in neurologic development of children after in utero exposure to MeHg (Mergler et al. 2007; Rice et al. 2003). At higher exposures to MeHg, including the highest concentrations reported during these survey years, the women themselves may risk adverse neuropsychological and neurobehavioral outcomes (Mergler et al. 2007).\nWithin the United States, people living in coastal areas consume more fish and shellfish than do those living in noncoastal areas and consume fish with higher Hg concentrations. Reports from New York City (McKelvey et al. 2007) and Florida (Denger et al. 1994; Karouna-Renier et al. 2008) support our identification of higher Hg exposures in U.S. coastal areas. This is part of a worldwide pattern. An overall pattern of higher BHg levels has also been reported among people living on U.S. islands [Hawaii (Sato et al. 2006)] and territories [e.g., Puerto Rico (Ortiz-Roque and López-Rivera 2004)]. A similar pattern has been repeated in other islands [Bermuda (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004), Fiji (Kumar et al. 2006), Seychelles (Myers et al. 2007), and Tahiti (Chateau-Degat 2005; Dewailly et al. 2008)] compared with inland populations. Among these island populations, BHg concentrations at the upper end of the distribution fall into the range of 50 μg/L (~ 250 nmol/L) and higher (Chateau-Degat 2005). In Bermuda, cord BHg concentrations as high as 160 nmol/L (~ 35 μg/L) have been reported (arithmetic mean, 41.3 ± 4.7 nmol/L or 8.0 ± 1.0 μg/L) (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004).\nHigher BHg concentrations in the U.S. Northeast found in this study reflect, in part, more frequent fish and shellfish consumption. Additional variability may be a function of differences in Hg concentrations among species and geographic regions (Sunderland 2007). For example, recent information on “hot spots” for Hg in wildlife tissues (Evers et al. 2007) could be associated with higher Hg concentrations for locally obtained fish. One limitation of the present analysis was the use of a fish-species–specific mean Hg concentration (i.e., nondistributional values) to estimate individual exposure. Although most fish consumed by the U.S. population is not locally obtained (i.e., commercially obtained from diverse regions and countries) (Sunderland 2007), analytical results showing geographic differences in the distribution of BHg could reflect higher Hg concentrations in locally obtained fish within the Northeast states.\n\nEthnic group variation on fish intake and BHg concentrations:\nEthnic origins were associated with Hg exposures with those designated as “other” (i.e., Asian, Pacific and Caribbean Islander, Native American, Alaska Native, multiracial, and unknown race) having higher BHg concentrations. From additional studies, people of Asian descent whose food choices are influenced by Asian dietary patterns (Kudo et al. 2000; Sechena et al. 2003) tended to consume fish more frequently, in greater variety, and in greater quantity than did non-Asians. The ethnic diversity of the U.S. population is well known. As of 1997, 61% of the Asian population living in the United States was foreign-born (Council of Economic Advisors 1999). By comparison with overall U.S. data, higher BHg concentrations among Asians and islanders were reported for Taiwan (Hsu et al. 2007), Cambodia (Agusa et al. 2007), Fiji (Kumar et al. 2006), and Tahiti (Dewailly et al. 2008).\nWithin the United States, fish and shellfish consumption, predicting Hg exposure described previously, varies widely, in part a reflection of ethnicity. For example, Asian countries [e.g., Cambodia (Agusa et al. 2007), Taiwan (Hsu et al. 2007; Soong et al. 1991), Japan (Murata et al. 2007; Sakamoto et al. 2007)], island nations [e.g., Bermuda (Dewailly and Pereg 2004; see also Bermuda Biological Stations for Research 2004), Seychelles (Myers et al. 2007), Tahiti (Chateau-Degat 2005), Taiwan (Hsu et al. 2007; Soong et al. 1991), Japan (Murata et al. 2007; Sakamoto et al. 2007)], and some European countries [e.g., Spain (Falcó et al. 2006; Herreros et al. 2008) and the Faroe Islands (Weihe et al. 1996)] have reported fish/shellfish consumption levels greater than average worldwide consumption (World Health Organization 2008).\n\nIncome differences in association with fish intake and BHg concentrations:\nIn contrast to some other environmental exposures [e.g., higher blood lead concentrations] (Mahaffey et al. 1982), BHg concentrations increased with income. This is consistent with other studies in which women from higher income groups were at greater risk of MeHg exposure, as were women living in urban areas (Hightower and Moore 2003; Saint-Phard and Van Dorsten 2006).\n\nInteractions between income and ethnic group:\nA more complex association between income and racial/ethnic group may also exist. According to the 1990 U.S. Census (U.S. Census Bureau 2008), the median family income of Japanese-American families exceeded that of non-Hispanic white families. By contrast, the income of Cambodian-American families was lower than that of black families. We could not address whether there is an interaction between belonging to the category designated as “other” and higher income within the NHANES data on BHg levels available at this time, because of sample size limitations.\n\nTime trends in Hg exposure absent changes in total fish consumption:\nOur analysis of 30-day Hg intake indicated that there was no consistent trend in fish consumption by women of childbearing age over the 6-year period between 1999 and 2004. Our evaluation of NHANES fish intake data indicated no differences in the mean frequency or amount of particular fish and shellfish species consumed. However, the estimated 30-day Hg intake decreased at the 90th percentile and higher, whereas total fish consumption did not, which suggests a shift in fish species consumed. The BHg data indicated a reduction of the higher end of the distribution of BHg between the first 2-year interval (the 1999 and 2000 examinees) compared with the subsequent 4-year interval (the 2001–2004 examinees).\nThe basis for these differences could possibly reflect spillover from the federal fish advisory program (U.S. EPA 2008b) in terms of total fish and shellfish consumption. A recent analysis of a nationally representative study specifically addressing fish-consumption patterns did not support this suggestion (Bradbury 2007).\nThe four fish species listed in the federal advisory [swordfish, shark, tilefish, and king mackerel (U.S. EPA 2008b)] were rarely reported by the 5,465 women in this analysis. It is clear that these four fish species contributed little to Hg exposure in this general population of U.S. women. Individual states with higher Hg exposures [e.g., Hawaii (Sato et al., 2006), Florida (Karouna-Renier et al. 2008)] and greater fish consumption [Florida (Denger et al. 1994)] have substantially broader fish consumption advisories (e.g., Hawaii and Florida) aimed at reducing Hg exposure from high-Hg–containing species obtained locally (Florida Department of Health 2007; U.S. EPA 2008b). Despite the federal advisory’s emphasis on four species of highly contaminated fish (U.S. EPA 2008b) and the states’ emphasis on game-fish, the most commonly consumed finfish in the United States is tuna. Interpretation of Hg exposure from tuna was complicated by the specific wording of the dietary questions asked of the NHANES examinees, which did not differentiate between light or skipjack tuna and albacore tuna. The latter contains approximately three times more Hg than does the former: 0.38 μg/g for frozen and fresh tuna, 0.35 μg/g for canned albacore, and 0.12 μg/g for canned light tuna (Mahaffey et al. 2008).\n\nChanges in MeHg exposure over time:\nDuring the past decade, the U.S. EPA initiated substantial interventions aimed to reduce Hg releases and exposures (U.S. EPA 2008a) and issued advisories to limit consumption of high-Hg fish (U.S. EPA 2008b). Because of worldwide atmospheric distribution and subsequent deposition of Hg, local conditions and locally caught fish are not the main contributors to Hg intake for most people (Sunderland 2007). Although there are economic indications that consumption of some species of fish may have decreased in response to these advisories (Shimshack et al. 2007), Hg exposures may not follow a similar time trend despite regulatory efforts to reduce Hg exposures. A recent analysis of a nationally representative study specifically addressing fish-consumption patterns did not support this suggestion (Bradbury 2007). Our analysis of NHANES data calculating 30-day Hg intake indicated that there was no consistent trend in fish consumption by women of childbearing age over the 6-year period between 1999 and 2004.\n\nConclusions:\nSignificant geographic differences in BHg concentrations occurred within the United States: We found highest exposures in coastal areas and the Northeast census region. In the Northeast, 19% of women had BHg concentrations ≥ 3.5 μg/L. The highest 5% of BHg concentrations exceeded 8.2 μg/L in the Northeast and 7.2 μg/L in coastal areas, concentrations more than twice the 3.5 μg/L level of concern. BHg levels were predicted by the quantity and type of fish consumed. Over the 6-year period (1999–2004), the frequency of elevated BHg levels among women of childbearing age declined without a significant change in quantities of fish and shellfish consumed. This pattern suggests a more discerning series of choices in type of fish eaten rather than an overall reduction in fish consumption. Within all geographic regions, women at highest risk of elevated Hg exposures were more affluent and more likely to be of Asian or island ethnicity.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe current, continuous National Health and Nutrition Examination Survey (NHANES) has included blood mercury (BHg) and fish/shellfish consumption since it began in 1999. NHANES 1999-2004 data form the basis for these analyses.\n\nMethods:\nWe performed univariate and bivariate analyses to determine the distribution of BHg and fish consumption in the population and to investigate differences by geography, race/ethnicity, and income. We used multivariate analysis (regression) to determine the strongest predictors of BHg among geography, demographic factors, and fish consumption.\n\nResults:\nElevated BHg occurred more commonly among women of childbearing age living in coastal areas of the United States (approximately one in six women). Regionally, exposures differ across the United States: Northeast > South and West > Midwest. Asian women and women with higher income ate more fish and had higher BHg. Time-trend analyses identified reduced BHg and reduced intake of Hg in the upper percentiles without an overall reduction of fish consumption.\n\nConclusions:\nBHg is associated with income, ethnicity, residence (census region and coastal proximity). From 1999 through 2004, BHg decreased without a concomitant decrease in fish consumption. Data are consistent with a shift over this time period in fish species in women's diets."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":10715,"string":"10,715"},"whole_article_abstract_length":{"kind":"number","value":246,"string":"246"},"other_sections_lengths":{"kind":"list like","value":[603,435,842,381,72,104,452,179],"string":"[\n 603,\n 435,\n 842,\n 381,\n 72,\n 104,\n 452,\n 179\n]","isConversational":false},"num_sections":{"kind":"number","value":12,"string":"12"},"most_frequent_words":{"kind":"list like","value":["fish","bhg","hg","coastal","consumption","concentrations","women","2007","bhg concentrations","higher"],"string":"[\n \"fish\",\n \"bhg\",\n \"hg\",\n \"coastal\",\n \"consumption\",\n \"concentrations\",\n \"women\",\n \"2007\",\n \"bhg concentrations\",\n \"higher\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["fish consumption 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[SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":79,"cells":{"title":{"kind":"string","value":"Willingness to work in rural areas and the role of intrinsic versus extrinsic professional motivations - a survey of medical students in Ghana."},"pmid":{"kind":"string","value":"21827698"},"background_abstract":{"kind":"string","value":"Retaining health workers in rural areas is challenging for a number of reasons, ranging from personal preferences to difficult work conditions and low remuneration. This paper assesses the influence of intrinsic and extrinsic motivation on willingness to accept postings to deprived areas among medical students in Ghana."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"A computer-based survey involving 302 fourth year medical students was conducted from May-August 2009. Logistic regression was used to assess the association between students' willingness to accept rural postings and their professional motivations, rural exposure and family parental professional and educational status (PPES)."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Over 85% of students were born in urban areas and 57% came from affluent backgrounds. Nearly two-thirds of students reported strong intrinsic motivation to study medicine. After controlling for demographic characteristics and rural exposure, motivational factors did not influence willingness to practice in rural areas. High family PPES was consistently associated with lower willingness to work in rural areas."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Although most Ghanaian medical students are motivated to study medicine by the desire to help others, this does not translate into willingness to work in rural areas. Efforts should be made to build on intrinsic motivation during medical training and in designing rural postings, as well as favour lower PPES students for admission."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Choice Behavior","Data Collection","Female","Ghana","Humans","Logistic Models","Male","Motivation","Professional Practice Location","Rural Health Services","Students, Medical","Young Adult"],"string":"[\n \"Choice Behavior\",\n \"Data Collection\",\n \"Female\",\n \"Ghana\",\n \"Humans\",\n \"Logistic Models\",\n \"Male\",\n \"Motivation\",\n \"Professional Practice Location\",\n \"Rural Health Services\",\n \"Students, Medical\",\n \"Young Adult\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"3170278"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"The World Health Organization estimates that more than 4 million health workers are needed to fill the health workforce gap globally. This includes 2.4 million physicians, nurses and midwives. Fifty-seven countries are defined as having a critical shortage of health staff; of these, 36 are in sub-Saharan Africa. Of the total world's health work force of 59.2 million, Africa has only 3% of the world's health workers in spite of having 25% of the global burden of disease [1,2]. The shortage of health staff cripples the health delivery system. It is also a threat to provision of essential, life-saving interventions such as childhood immunizations, provision of safe water, safe pregnancy and childbirth services for mothers as well as access to treatment for AIDS, tuberculosis and malaria. Health workers are critical to the global preparedness for and response to threats posed by emerging and epidemic-prone diseases. Different interventions have been tried to address these shortages [3-5].\nRetaining health staff in rural areas has proven extremely difficult as young professionals increasingly prefer urban postings and health systems do not reward (and through neglect often penalize) rural service [6, 7. 8]. For example, Wilson and colleagues, 2009, Dovlo, 2003 and Kruk and colleagues, 2010, found that rural exposure, poor working conditions, low job satisfaction, political and ethnic problems, and sometimes, civil strife and poor security in most underserved areas, predispose new graduates to select cities [9-11]. Qualitative research has also shown the importance of health care providers' personal characteristics and value systems, such as religious beliefs and sociopolitical convictions, to their motivation towards rural practice. Emigration of skilled professionals to high-income countries is another barrier to adequate staffing of health facilities. A study in Ghana in 2006 on trainee physicians and nurses revealed that the majority had considered emigrating. More physicians (68%) than nurses (57%) considered emigration. These findings imply that achieving improvements in the health status of people living in low-income countries, and particularly, in rural areas, will be extremely difficult and the attainment of the United Nations Millennium Development Goals 4, 5, and 6 by 2015 [12,13], in Ghana is unlikely.\nWhile previous research has looked at incentives and working conditions to promote uptake of rural posts, few studies have focused on motivation crowding and its effect on willingness to accept postings to rural area. Motivation crowding [14] is the conflict between external factors (extrinsic), such as monetary incentives or punishments, and the underlying desire or willingness to work (intrinsic) in areas needed most. Students may have a mix of extrinsic and intrinsic motivations for studying medicine. Extrinsic factors may either undermine or strengthen intrinsic motivation, led by the belief that medicine has the imperative to help others, as enshrined in the Hippocratic Oath [15-17]. Current monetary incentives, which favour urban practice, may crowd-out the intrinsic desire to give back to society by working in underserved areas [18,19]. This could have debilitating effects on health worker retention in rural areas [20-22]. To tackle the maldistribution of human resources for health (HRH), understanding the factors that crowd-out the intrinsic motivation of medical students and their willingness to accept postings to rural underserved area is integral. This paper analyzes the effect of extrinsic versus intrinsic motivational factors on stated willingness to accept postings to rural underserved areas in Ghana."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Study site Ghana is located in the west coast of Africa with an estimated population of 23 million [23]. It is mainly an agrarian economy with estimated per capita income of USD 1500. More than two-thirds of the economy is rural, with the cities of Kumasi and Accra having the highest population densities due to brisk economic activities, and relatively strong economic and cultural infrastructure [24]. These attributes make the two main cities destinations for rural-urban migration and a highly heterogeneous socio-cultural context. There are four medical schools in Ghana: the University of Ghana (UG), Kwame Nkrumah University of Science and Technology (KNUST), University for Development Studies (UDS), and University of Cape Coast (UCC).\nIn Ghana, medical education consists of three years of basic science/para-clinical studies, three years of clinical training at a teaching hospital, and a two-year rotating housemanship. The study was conducted with two public universities in Ghana: University of Ghana (UG) in Accra and Kwame Nkrumah University of Science and Technology (KNUST) in Kumasi. These universities were selected because all the fourth year medical students in the public universities had their clinical training at either UG or KNUST at the time of the study. All 310 fourth year medical students in the country were invited to participate in the study; no sampling was conducted. Fourth-year medical students were selected because they had completed the BSc. Human Biology and had also been exposed to field work, but had not yet made their final decisions about rural or urban practice.\nGhana is located in the west coast of Africa with an estimated population of 23 million [23]. It is mainly an agrarian economy with estimated per capita income of USD 1500. More than two-thirds of the economy is rural, with the cities of Kumasi and Accra having the highest population densities due to brisk economic activities, and relatively strong economic and cultural infrastructure [24]. These attributes make the two main cities destinations for rural-urban migration and a highly heterogeneous socio-cultural context. There are four medical schools in Ghana: the University of Ghana (UG), Kwame Nkrumah University of Science and Technology (KNUST), University for Development Studies (UDS), and University of Cape Coast (UCC).\nIn Ghana, medical education consists of three years of basic science/para-clinical studies, three years of clinical training at a teaching hospital, and a two-year rotating housemanship. The study was conducted with two public universities in Ghana: University of Ghana (UG) in Accra and Kwame Nkrumah University of Science and Technology (KNUST) in Kumasi. These universities were selected because all the fourth year medical students in the public universities had their clinical training at either UG or KNUST at the time of the study. All 310 fourth year medical students in the country were invited to participate in the study; no sampling was conducted. Fourth-year medical students were selected because they had completed the BSc. Human Biology and had also been exposed to field work, but had not yet made their final decisions about rural or urban practice.\n Data collection Data collection was preceded by discussions with the heads of medical training institutions, who informed the content of the questionnaire and provided access to the student population. The data collection instruments were developed after seven focus group discussions of 6-8 participants in each group facilitated by trained social scientists were held with third and fifth year medical students at UG and KNUST. The themes for the focus group discussion were motivation, willingness to work in deprived areas, experience in the field, and the influence of background characteristics on willingness to work in deprived areas. The survey instrument, which included semi-structured questions as well as a discrete choice experiment, were then pretested and finalized for the study. The questionnaires were administered electronically with Sawtooth Software SSI Web CAPI [25], in computer laboratories in UG and KNUST.\nData collection was preceded by discussions with the heads of medical training institutions, who informed the content of the questionnaire and provided access to the student population. The data collection instruments were developed after seven focus group discussions of 6-8 participants in each group facilitated by trained social scientists were held with third and fifth year medical students at UG and KNUST. The themes for the focus group discussion were motivation, willingness to work in deprived areas, experience in the field, and the influence of background characteristics on willingness to work in deprived areas. The survey instrument, which included semi-structured questions as well as a discrete choice experiment, were then pretested and finalized for the study. The questionnaires were administered electronically with Sawtooth Software SSI Web CAPI [25], in computer laboratories in UG and KNUST.\n Ethical considerations The study received ethics approval from the Ghana Health Service Ethical Review Committee; the UG Medical School; the KNUST Committee on Human Research, Publications, and Ethics; and the University of Michigan Institutional Review Board. All respondents voluntarily participated after the intent and the design of the study had been explained to them and signing informed consent forms.\nThe study received ethics approval from the Ghana Health Service Ethical Review Committee; the UG Medical School; the KNUST Committee on Human Research, Publications, and Ethics; and the University of Michigan Institutional Review Board. All respondents voluntarily participated after the intent and the design of the study had been explained to them and signing informed consent forms.\n Statistical Analysis The study used STATA v10.1 for statistical analyses [26]. The main outcome of interest was the willingness to work in a deprived area after graduation. Students were asked to rate how likely they were to work in a deprived area on a scale from 1-4, where 1 represented \"I will definitely not work in a deprived area;\" 2 \"I am unlikely to work in a deprived area;\" 3 \"I am likely to work in a deprived area;\" and 4 \"I will definitely work in a deprived area.\" We collapsed this response set to a dichotomous willing (groups 3 or 4) versus unwilling (groups 1 or 2) to practice in a deprived area. We defined deprived area as \"a rural area that is distant from the big cities with few social amenities such as schools, roads, pipe-borne water, etc.\"--as per Ministry of Health definition.\nPredictors of interest included motivation (intrinsic and extrinsic), demographic characteristics, and rural exposure. Students were asked to identify the top three factors that motivated them to study medicine from a list of twelve factors identified as important by the focus group discussions. Five intrinsic motivations included: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one. Seven extrinsic motivation factors included: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities. Respondents were coded as having \"strong\" intrinsic or extrinsic motivation if two or more of their motivational factors were intrinsic or extrinsic. Thus, \"strong\" intrinsic and extrinsic motivation groups were mutually exclusive.\nDemographic factors included: sex, age, ethnicity (Akan vs. non-Akan), partnership status (in a relationship vs. not in a relationship), and parental parental professional and educational status (PPES). The Akan peoples (including the Asante, Fante, Kwahu, Akuapem, Bono and others) are the largest ethnic group, representing approximately half of Ghanaians; we grouped all other smaller ethnic groups (Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa) together as \"non-Akans\" for this analysis. High parental professional and educational status (PPES) was defined as having a mother and/or father who is a University-trained professional (e.g. doctor, lawyer, engineer, accountant, technical, etc) and low PPES was defined as neither mother nor father is a University-trained professional. Rural (an area with a population less than 5000) exposure factors included: birth location (urban vs. rural), location of pre-medical studies (urban/vs. rural), having ever lived in rural area (from the age of 5 onwards), exposure to rural service in medical training (for a minimum of 6 months).\nBivariate associations and 95% confidence intervals were estimated using logistic regression. In model 1, the influence of strong intrinsic/extrinsic motivation on willingness to accept postings to rural area were assessed. Socio-demographic factors were added to the regression in Model 2, and rural exposure factors were further added in Model 3.\nThe study used STATA v10.1 for statistical analyses [26]. The main outcome of interest was the willingness to work in a deprived area after graduation. Students were asked to rate how likely they were to work in a deprived area on a scale from 1-4, where 1 represented \"I will definitely not work in a deprived area;\" 2 \"I am unlikely to work in a deprived area;\" 3 \"I am likely to work in a deprived area;\" and 4 \"I will definitely work in a deprived area.\" We collapsed this response set to a dichotomous willing (groups 3 or 4) versus unwilling (groups 1 or 2) to practice in a deprived area. We defined deprived area as \"a rural area that is distant from the big cities with few social amenities such as schools, roads, pipe-borne water, etc.\"--as per Ministry of Health definition.\nPredictors of interest included motivation (intrinsic and extrinsic), demographic characteristics, and rural exposure. Students were asked to identify the top three factors that motivated them to study medicine from a list of twelve factors identified as important by the focus group discussions. Five intrinsic motivations included: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one. Seven extrinsic motivation factors included: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities. Respondents were coded as having \"strong\" intrinsic or extrinsic motivation if two or more of their motivational factors were intrinsic or extrinsic. Thus, \"strong\" intrinsic and extrinsic motivation groups were mutually exclusive.\nDemographic factors included: sex, age, ethnicity (Akan vs. non-Akan), partnership status (in a relationship vs. not in a relationship), and parental parental professional and educational status (PPES). The Akan peoples (including the Asante, Fante, Kwahu, Akuapem, Bono and others) are the largest ethnic group, representing approximately half of Ghanaians; we grouped all other smaller ethnic groups (Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa) together as \"non-Akans\" for this analysis. High parental professional and educational status (PPES) was defined as having a mother and/or father who is a University-trained professional (e.g. doctor, lawyer, engineer, accountant, technical, etc) and low PPES was defined as neither mother nor father is a University-trained professional. Rural (an area with a population less than 5000) exposure factors included: birth location (urban vs. rural), location of pre-medical studies (urban/vs. rural), having ever lived in rural area (from the age of 5 onwards), exposure to rural service in medical training (for a minimum of 6 months).\nBivariate associations and 95% confidence intervals were estimated using logistic regression. In model 1, the influence of strong intrinsic/extrinsic motivation on willingness to accept postings to rural area were assessed. Socio-demographic factors were added to the regression in Model 2, and rural exposure factors were further added in Model 3."},"results_title":{"kind":"null"},"results_text":{"kind":"null"},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"The pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6920/11/56/prepub\n"},"other_sections_titles":{"kind":"list like","value":["Background","Study site","Data collection","Ethical considerations","Statistical Analysis","Results","Socio-demographic characteristics","Intrinsic and extrinsic motivation and likelihood of working in underserved area","Regression analysis of motivations and the willingness to accept postings to rural underserved area after graduation","Discussion","Conclusions"],"string":"[\n \"Background\",\n \"Study site\",\n \"Data collection\",\n \"Ethical considerations\",\n \"Statistical Analysis\",\n \"Results\",\n \"Socio-demographic characteristics\",\n \"Intrinsic and extrinsic motivation and likelihood of working in underserved area\",\n \"Regression analysis of motivations and the willingness to accept postings to rural underserved area after graduation\",\n \"Discussion\",\n \"Conclusions\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["The World Health Organization estimates that more than 4 million health workers are needed to fill the health workforce gap globally. This includes 2.4 million physicians, nurses and midwives. Fifty-seven countries are defined as having a critical shortage of health staff; of these, 36 are in sub-Saharan Africa. Of the total world's health work force of 59.2 million, Africa has only 3% of the world's health workers in spite of having 25% of the global burden of disease [1,2]. The shortage of health staff cripples the health delivery system. It is also a threat to provision of essential, life-saving interventions such as childhood immunizations, provision of safe water, safe pregnancy and childbirth services for mothers as well as access to treatment for AIDS, tuberculosis and malaria. Health workers are critical to the global preparedness for and response to threats posed by emerging and epidemic-prone diseases. Different interventions have been tried to address these shortages [3-5].\nRetaining health staff in rural areas has proven extremely difficult as young professionals increasingly prefer urban postings and health systems do not reward (and through neglect often penalize) rural service [6, 7. 8]. For example, Wilson and colleagues, 2009, Dovlo, 2003 and Kruk and colleagues, 2010, found that rural exposure, poor working conditions, low job satisfaction, political and ethnic problems, and sometimes, civil strife and poor security in most underserved areas, predispose new graduates to select cities [9-11]. Qualitative research has also shown the importance of health care providers' personal characteristics and value systems, such as religious beliefs and sociopolitical convictions, to their motivation towards rural practice. Emigration of skilled professionals to high-income countries is another barrier to adequate staffing of health facilities. A study in Ghana in 2006 on trainee physicians and nurses revealed that the majority had considered emigrating. More physicians (68%) than nurses (57%) considered emigration. These findings imply that achieving improvements in the health status of people living in low-income countries, and particularly, in rural areas, will be extremely difficult and the attainment of the United Nations Millennium Development Goals 4, 5, and 6 by 2015 [12,13], in Ghana is unlikely.\nWhile previous research has looked at incentives and working conditions to promote uptake of rural posts, few studies have focused on motivation crowding and its effect on willingness to accept postings to rural area. Motivation crowding [14] is the conflict between external factors (extrinsic), such as monetary incentives or punishments, and the underlying desire or willingness to work (intrinsic) in areas needed most. Students may have a mix of extrinsic and intrinsic motivations for studying medicine. Extrinsic factors may either undermine or strengthen intrinsic motivation, led by the belief that medicine has the imperative to help others, as enshrined in the Hippocratic Oath [15-17]. Current monetary incentives, which favour urban practice, may crowd-out the intrinsic desire to give back to society by working in underserved areas [18,19]. This could have debilitating effects on health worker retention in rural areas [20-22]. To tackle the maldistribution of human resources for health (HRH), understanding the factors that crowd-out the intrinsic motivation of medical students and their willingness to accept postings to rural underserved area is integral. This paper analyzes the effect of extrinsic versus intrinsic motivational factors on stated willingness to accept postings to rural underserved areas in Ghana.","Ghana is located in the west coast of Africa with an estimated population of 23 million [23]. It is mainly an agrarian economy with estimated per capita income of USD 1500. More than two-thirds of the economy is rural, with the cities of Kumasi and Accra having the highest population densities due to brisk economic activities, and relatively strong economic and cultural infrastructure [24]. These attributes make the two main cities destinations for rural-urban migration and a highly heterogeneous socio-cultural context. There are four medical schools in Ghana: the University of Ghana (UG), Kwame Nkrumah University of Science and Technology (KNUST), University for Development Studies (UDS), and University of Cape Coast (UCC).\nIn Ghana, medical education consists of three years of basic science/para-clinical studies, three years of clinical training at a teaching hospital, and a two-year rotating housemanship. The study was conducted with two public universities in Ghana: University of Ghana (UG) in Accra and Kwame Nkrumah University of Science and Technology (KNUST) in Kumasi. These universities were selected because all the fourth year medical students in the public universities had their clinical training at either UG or KNUST at the time of the study. All 310 fourth year medical students in the country were invited to participate in the study; no sampling was conducted. Fourth-year medical students were selected because they had completed the BSc. Human Biology and had also been exposed to field work, but had not yet made their final decisions about rural or urban practice.","Data collection was preceded by discussions with the heads of medical training institutions, who informed the content of the questionnaire and provided access to the student population. The data collection instruments were developed after seven focus group discussions of 6-8 participants in each group facilitated by trained social scientists were held with third and fifth year medical students at UG and KNUST. The themes for the focus group discussion were motivation, willingness to work in deprived areas, experience in the field, and the influence of background characteristics on willingness to work in deprived areas. The survey instrument, which included semi-structured questions as well as a discrete choice experiment, were then pretested and finalized for the study. The questionnaires were administered electronically with Sawtooth Software SSI Web CAPI [25], in computer laboratories in UG and KNUST.","The study received ethics approval from the Ghana Health Service Ethical Review Committee; the UG Medical School; the KNUST Committee on Human Research, Publications, and Ethics; and the University of Michigan Institutional Review Board. All respondents voluntarily participated after the intent and the design of the study had been explained to them and signing informed consent forms.","The study used STATA v10.1 for statistical analyses [26]. The main outcome of interest was the willingness to work in a deprived area after graduation. Students were asked to rate how likely they were to work in a deprived area on a scale from 1-4, where 1 represented \"I will definitely not work in a deprived area;\" 2 \"I am unlikely to work in a deprived area;\" 3 \"I am likely to work in a deprived area;\" and 4 \"I will definitely work in a deprived area.\" We collapsed this response set to a dichotomous willing (groups 3 or 4) versus unwilling (groups 1 or 2) to practice in a deprived area. We defined deprived area as \"a rural area that is distant from the big cities with few social amenities such as schools, roads, pipe-borne water, etc.\"--as per Ministry of Health definition.\nPredictors of interest included motivation (intrinsic and extrinsic), demographic characteristics, and rural exposure. Students were asked to identify the top three factors that motivated them to study medicine from a list of twelve factors identified as important by the focus group discussions. Five intrinsic motivations included: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one. Seven extrinsic motivation factors included: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities. Respondents were coded as having \"strong\" intrinsic or extrinsic motivation if two or more of their motivational factors were intrinsic or extrinsic. Thus, \"strong\" intrinsic and extrinsic motivation groups were mutually exclusive.\nDemographic factors included: sex, age, ethnicity (Akan vs. non-Akan), partnership status (in a relationship vs. not in a relationship), and parental parental professional and educational status (PPES). The Akan peoples (including the Asante, Fante, Kwahu, Akuapem, Bono and others) are the largest ethnic group, representing approximately half of Ghanaians; we grouped all other smaller ethnic groups (Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa) together as \"non-Akans\" for this analysis. High parental professional and educational status (PPES) was defined as having a mother and/or father who is a University-trained professional (e.g. doctor, lawyer, engineer, accountant, technical, etc) and low PPES was defined as neither mother nor father is a University-trained professional. Rural (an area with a population less than 5000) exposure factors included: birth location (urban vs. rural), location of pre-medical studies (urban/vs. rural), having ever lived in rural area (from the age of 5 onwards), exposure to rural service in medical training (for a minimum of 6 months).\nBivariate associations and 95% confidence intervals were estimated using logistic regression. In model 1, the influence of strong intrinsic/extrinsic motivation on willingness to accept postings to rural area were assessed. Socio-demographic factors were added to the regression in Model 2, and rural exposure factors were further added in Model 3."," Socio-demographic characteristics Of the 310 eligible medical students, 307 participated in the survey (99%). Of these, five survey files were corrupted by viruses or lost due to computer malfunction; thus the analysis was conducted with 302 total records. The socio-demographic characteristics of respondents are presented in Table 1. Of the 302 respondents recruited for the study, the majority were male (183 or 60.6%), with a mean age of 22.9 (STD 1.4). Most respondents were born in or around urban areas (264 or 87.4%) and had never lived in rural underserved area (75.8%). In terms of socioeconomic status, 173 (57.3%) students came from high PPES families. About half of the respondents (142 or 47%) were exposed to rural service.\nDemographic characteristics and rural exposure of respondents (N = 302)\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High Family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low Family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents; rural area defined as a place with less than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies\nOf the 310 eligible medical students, 307 participated in the survey (99%). Of these, five survey files were corrupted by viruses or lost due to computer malfunction; thus the analysis was conducted with 302 total records. The socio-demographic characteristics of respondents are presented in Table 1. Of the 302 respondents recruited for the study, the majority were male (183 or 60.6%), with a mean age of 22.9 (STD 1.4). Most respondents were born in or around urban areas (264 or 87.4%) and had never lived in rural underserved area (75.8%). In terms of socioeconomic status, 173 (57.3%) students came from high PPES families. About half of the respondents (142 or 47%) were exposed to rural service.\nDemographic characteristics and rural exposure of respondents (N = 302)\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High Family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low Family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents; rural area defined as a place with less than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies\n Intrinsic and extrinsic motivation and likelihood of working in underserved area The intensities of current motivational factors are presented in Table 2. Overall, 158 (55.4%) of students stated that they were likely to or definitely would work in an underserved area. More than 6 in 10 respondents (181 or 63.5%) had strong intrinsic motivation. A higher proportion of respondents who had strong intrinsic motivation indicated willingness to work in a rural area, compared to those with weak intrinsic motivation (χ2 = 6.96, p = 0.008). These results were reversed for those with strong extrinsic motivation (χ2 = 6.12, p = 0.013).\nIntrinsic versus extrinsic motivations to study medicine predict reported likelihood to work in an under-served area, 285 Ghanaian Medical Students\n1. Excludes 17 missing values for willingness to work rural and/or current motivation.\n2. Intrinsic motivation includes: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one; Pearson χ2(1) = 6.9592, p = 0.008.\n3. Extrinsic motivation includes: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities; Pearson χ2(1) = 6.1208, p = 0.013.\nThe intensities of current motivational factors are presented in Table 2. Overall, 158 (55.4%) of students stated that they were likely to or definitely would work in an underserved area. More than 6 in 10 respondents (181 or 63.5%) had strong intrinsic motivation. A higher proportion of respondents who had strong intrinsic motivation indicated willingness to work in a rural area, compared to those with weak intrinsic motivation (χ2 = 6.96, p = 0.008). These results were reversed for those with strong extrinsic motivation (χ2 = 6.12, p = 0.013).\nIntrinsic versus extrinsic motivations to study medicine predict reported likelihood to work in an under-served area, 285 Ghanaian Medical Students\n1. Excludes 17 missing values for willingness to work rural and/or current motivation.\n2. Intrinsic motivation includes: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one; Pearson χ2(1) = 6.9592, p = 0.008.\n3. Extrinsic motivation includes: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities; Pearson χ2(1) = 6.1208, p = 0.013.\n Regression analysis of motivations and the willingness to accept postings to rural underserved area after graduation We present the multivariate logistic regression results for the strength of intrinsic motivation and willingness to work in a rural underserved area after graduation in Table 3. In the unadjusted model (Model 1) there was a significant association between strong intrinsic motivation and willingness to work in rural underserved area (1.92[95% CI 1.18-3.13]). In Model 2, with demographic factors, female gender and high PPES were associated with reduced willingness to practice in a deprived area (0.50[95% CI 0.29-0.88]) and (0.42[95% CI 0.24, 0.71]), respectively, while age was associated with greater willingness to practice in a rural area (1.23[95% CI 1.00-1.52]). Rural exposure factors were not significant when added to the model with intrinsic motivation and demographic factors (Model 3). In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\nMultivariate logistic regression results for strength of intrinsic motivation and willingness to work in a rural underserved area after graduation, Ghanaian medical students\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies\nIn Table 4 the influence of strong extrinsic motivation on willingness of students to work in rural underserved area is presented. In Model 1, having a strong extrinsic motivation reduced the odds of being willing to accept a job in an underserved area to (0.54[95% CI 0.33-0.88]). In the model adjusting for demographics, Model 2, female gender and high PPES were associated with reduced willingness to practice in underserved areas (0.50[95% CI 0.28-0.87] and (0.42[95% CI 0.24-0.71]) respectively, while age was associated with greater willingness to practice in a rural area (1.24[95% CI 1.00-1.53]). Rural exposure factors in model 3 did not influence the outcome of willingness to work in rural underserved area. In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\nMultivariate logistic regression results for strength of extrinsic motivation and willingness to work in a rural underserved area after graduation\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies\nWe present the multivariate logistic regression results for the strength of intrinsic motivation and willingness to work in a rural underserved area after graduation in Table 3. In the unadjusted model (Model 1) there was a significant association between strong intrinsic motivation and willingness to work in rural underserved area (1.92[95% CI 1.18-3.13]). In Model 2, with demographic factors, female gender and high PPES were associated with reduced willingness to practice in a deprived area (0.50[95% CI 0.29-0.88]) and (0.42[95% CI 0.24, 0.71]), respectively, while age was associated with greater willingness to practice in a rural area (1.23[95% CI 1.00-1.52]). Rural exposure factors were not significant when added to the model with intrinsic motivation and demographic factors (Model 3). In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\nMultivariate logistic regression results for strength of intrinsic motivation and willingness to work in a rural underserved area after graduation, Ghanaian medical students\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies\nIn Table 4 the influence of strong extrinsic motivation on willingness of students to work in rural underserved area is presented. In Model 1, having a strong extrinsic motivation reduced the odds of being willing to accept a job in an underserved area to (0.54[95% CI 0.33-0.88]). In the model adjusting for demographics, Model 2, female gender and high PPES were associated with reduced willingness to practice in underserved areas (0.50[95% CI 0.28-0.87] and (0.42[95% CI 0.24-0.71]) respectively, while age was associated with greater willingness to practice in a rural area (1.24[95% CI 1.00-1.53]). Rural exposure factors in model 3 did not influence the outcome of willingness to work in rural underserved area. In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\nMultivariate logistic regression results for strength of extrinsic motivation and willingness to work in a rural underserved area after graduation\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies","Of the 310 eligible medical students, 307 participated in the survey (99%). Of these, five survey files were corrupted by viruses or lost due to computer malfunction; thus the analysis was conducted with 302 total records. The socio-demographic characteristics of respondents are presented in Table 1. Of the 302 respondents recruited for the study, the majority were male (183 or 60.6%), with a mean age of 22.9 (STD 1.4). Most respondents were born in or around urban areas (264 or 87.4%) and had never lived in rural underserved area (75.8%). In terms of socioeconomic status, 173 (57.3%) students came from high PPES families. About half of the respondents (142 or 47%) were exposed to rural service.\nDemographic characteristics and rural exposure of respondents (N = 302)\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High Family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low Family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents; rural area defined as a place with less than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies","The intensities of current motivational factors are presented in Table 2. Overall, 158 (55.4%) of students stated that they were likely to or definitely would work in an underserved area. More than 6 in 10 respondents (181 or 63.5%) had strong intrinsic motivation. A higher proportion of respondents who had strong intrinsic motivation indicated willingness to work in a rural area, compared to those with weak intrinsic motivation (χ2 = 6.96, p = 0.008). These results were reversed for those with strong extrinsic motivation (χ2 = 6.12, p = 0.013).\nIntrinsic versus extrinsic motivations to study medicine predict reported likelihood to work in an under-served area, 285 Ghanaian Medical Students\n1. Excludes 17 missing values for willingness to work rural and/or current motivation.\n2. Intrinsic motivation includes: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one; Pearson χ2(1) = 6.9592, p = 0.008.\n3. Extrinsic motivation includes: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities; Pearson χ2(1) = 6.1208, p = 0.013.","We present the multivariate logistic regression results for the strength of intrinsic motivation and willingness to work in a rural underserved area after graduation in Table 3. In the unadjusted model (Model 1) there was a significant association between strong intrinsic motivation and willingness to work in rural underserved area (1.92[95% CI 1.18-3.13]). In Model 2, with demographic factors, female gender and high PPES were associated with reduced willingness to practice in a deprived area (0.50[95% CI 0.29-0.88]) and (0.42[95% CI 0.24, 0.71]), respectively, while age was associated with greater willingness to practice in a rural area (1.23[95% CI 1.00-1.52]). Rural exposure factors were not significant when added to the model with intrinsic motivation and demographic factors (Model 3). In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\nMultivariate logistic regression results for strength of intrinsic motivation and willingness to work in a rural underserved area after graduation, Ghanaian medical students\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies\nIn Table 4 the influence of strong extrinsic motivation on willingness of students to work in rural underserved area is presented. In Model 1, having a strong extrinsic motivation reduced the odds of being willing to accept a job in an underserved area to (0.54[95% CI 0.33-0.88]). In the model adjusting for demographics, Model 2, female gender and high PPES were associated with reduced willingness to practice in underserved areas (0.50[95% CI 0.28-0.87] and (0.42[95% CI 0.24-0.71]) respectively, while age was associated with greater willingness to practice in a rural area (1.24[95% CI 1.00-1.53]). Rural exposure factors in model 3 did not influence the outcome of willingness to work in rural underserved area. In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\nMultivariate logistic regression results for strength of extrinsic motivation and willingness to work in a rural underserved area after graduation\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies","We found that twice as many students reported high intrinsic motivation compared to high extrinsic motivation to study and practice medicine. This may reflect the underlying altruistic motivation for many students entering a profession focused on serving others [12,13]. There may also be an element of social desirability bias in the students' responses as intrinsic motivation may be thought to be more socially acceptable than extrinsic motivation. Nonetheless, we found that high extrinsic motivation was associated with low self-reported likelihood of rural practice and that the converse was true for high intrinsic motivation [2,3]. Interestingly, this association lost statistical significance at the 95% confidence level in models with demographic and rural exposure confounders, whereas socioeconomic status (PPES) retained a highly influential role, as discussed below.\nIn this study, rural origin did not influence students' willingness to practice in rural areas after controlling for intrinsic/extrinsic motivation and demographic characteristics. This is in contrast with studies which have found rural origin to be an important motivator for rural practice [8,17,23]. Our findings highlight the heterogeneity of trends in motivation dynamics for rural practice and the importance of locally-relevant data for decision making. High PPES, measured using parental education and profession, was consistently associated with lack of willingness to work in rural areas. This is concerning as nearly 6 in 10 medical students in this cohort were from high PPES backgrounds--which is typical for Ghanaian medical schools [7]. These findings suggest that admission policies that favour well-to do applicants may be reducing the pool of students willing to consider rural practice. Female gender was also strongly associated with reduced interest in rural practice for women even after controlling for extrinsic/intrinsic motivation and rural exposure variables. This is consistent with similar studies among health staff which revealed that women are less likely to accept positions in remote areas due to varying family reasons; they would like to live where their husbands jobs are, have difficulties convincing their husbands to follow them to rural areas and want their children to have better education in the urban areas [10,11,22]. The studies further explained that female doctors rarely live in the same village as their assigned post and have higher overall absentee rates in rural practice [19,20]. With increasing representation of female healthcare professionals in many places in sub-Saharan Africa, [10,11], it is likely that the supply of health staff to rural underserved areas will remain a major setback if professional motivations are designed to attract more female students to rural practice. Although our study showed a lower proportion of female medical students in Ghana compared to other areas, they are likely to become a more important cadre in the coming years [11,19]. More research is urgently needed to determine how female healthcare professionals' motivations towards rural practice can be better engaged by policy-makers.\nThe limitations of this study include the possibility of social desirability bias in responses on motivation and likelihood of rural practice, as noted above. Despite the fact that study participants were assured of anonymity, confidentiality, in responding to the questions, some social desirability bias is likely. For this reason, we selected a measure of high intrinsic and extrinsic motivation for use in the regression models. Research comparing students stated intentions with their actual career choices during internship is urgently needed as few studies on matched follow-ups are available. In addition, most the students participating in the study were young and had not yet tasted the rigors of working in a rural area, which may affect their job preferences. Thus the findings of this study may not be applicable to practicing physicians. Finally, these findings are only generalizable to students in the current medical education system. The findings may be different if selection criteria for medical school admission change.\nThe major strengths of this study are its high response rate of 99% and that its ability to capture an entire population of young medical students who are one of the targets for addressing the rural-urban health staff recruitment imbalance. Surveying practicing physicians would have missed out those who had migrated.\nThis study has several implications. First, the majority of students profess high intrinsic motivation for rural service. More research is needed to determine the potency of this motivation source in real-life decision making and how to best engage it via HRH policy. It is possible that emphasizing the community service aspect of medical practice and elevating the status of rural primary care in under-graduate and post-graduate training may help narrow the gap between motivation and eventual career choice in favour of rural areas. In addition, well-supervised and supported rural placements in which students experience the rewards of rural practice may help to persuade students who are largely unfamiliar with rural life. However, the success of these rural rotations is likely to depend heavily on having adequate local infrastructure and mentorship [13,23].\nSecond, admission criteria may need to be reconsidered in light of the strong relationship between high PPES and lack of interest in rural practice. For example, medical school admission slots might be reserved for qualified students from poorer families. These students may not need to come from rural areas as we found that none of the rural exposure factors were significant after controlling for motivation and demographics.\nThird, our results suggest that programmes to promote and support rural practice after graduation may have some success. Our focus groups and discrete choice experiment suggested that students may be willing to commit to short-term placements of 2 years or less in rural areas [9]. The Ministry of Health may want to consider the possibility of short contracts that rotate physicians in and out of difficult to staff rural areas.","The majority of students still claim high intrinsic motivation and therefore it is important to appeal and build on this in medical school curricula and in designing rural postings. However, extrinsic motivation and, perhaps most importantly, PPES, will likely continue to be an important factor in deciding on job postings. Future research should explore how motivations could be directly supported, how motivations are formed, the influence of contextual factors on motivation among medical students, and motivation crowding among practicing health professionals in rural underserved areas. Qualitative work may be especially informative in this effort. Our research also suggests that increasing efforts to recruit medical students from low PPES backgrounds may be the most effective current pathway to increasing the yield of physicians willing to practice in underserved areas."],"string":"[\n \"The World Health Organization estimates that more than 4 million health workers are needed to fill the health workforce gap globally. This includes 2.4 million physicians, nurses and midwives. Fifty-seven countries are defined as having a critical shortage of health staff; of these, 36 are in sub-Saharan Africa. Of the total world's health work force of 59.2 million, Africa has only 3% of the world's health workers in spite of having 25% of the global burden of disease [1,2]. The shortage of health staff cripples the health delivery system. It is also a threat to provision of essential, life-saving interventions such as childhood immunizations, provision of safe water, safe pregnancy and childbirth services for mothers as well as access to treatment for AIDS, tuberculosis and malaria. Health workers are critical to the global preparedness for and response to threats posed by emerging and epidemic-prone diseases. Different interventions have been tried to address these shortages [3-5].\\nRetaining health staff in rural areas has proven extremely difficult as young professionals increasingly prefer urban postings and health systems do not reward (and through neglect often penalize) rural service [6, 7. 8]. For example, Wilson and colleagues, 2009, Dovlo, 2003 and Kruk and colleagues, 2010, found that rural exposure, poor working conditions, low job satisfaction, political and ethnic problems, and sometimes, civil strife and poor security in most underserved areas, predispose new graduates to select cities [9-11]. Qualitative research has also shown the importance of health care providers' personal characteristics and value systems, such as religious beliefs and sociopolitical convictions, to their motivation towards rural practice. Emigration of skilled professionals to high-income countries is another barrier to adequate staffing of health facilities. A study in Ghana in 2006 on trainee physicians and nurses revealed that the majority had considered emigrating. More physicians (68%) than nurses (57%) considered emigration. These findings imply that achieving improvements in the health status of people living in low-income countries, and particularly, in rural areas, will be extremely difficult and the attainment of the United Nations Millennium Development Goals 4, 5, and 6 by 2015 [12,13], in Ghana is unlikely.\\nWhile previous research has looked at incentives and working conditions to promote uptake of rural posts, few studies have focused on motivation crowding and its effect on willingness to accept postings to rural area. Motivation crowding [14] is the conflict between external factors (extrinsic), such as monetary incentives or punishments, and the underlying desire or willingness to work (intrinsic) in areas needed most. Students may have a mix of extrinsic and intrinsic motivations for studying medicine. Extrinsic factors may either undermine or strengthen intrinsic motivation, led by the belief that medicine has the imperative to help others, as enshrined in the Hippocratic Oath [15-17]. Current monetary incentives, which favour urban practice, may crowd-out the intrinsic desire to give back to society by working in underserved areas [18,19]. This could have debilitating effects on health worker retention in rural areas [20-22]. To tackle the maldistribution of human resources for health (HRH), understanding the factors that crowd-out the intrinsic motivation of medical students and their willingness to accept postings to rural underserved area is integral. This paper analyzes the effect of extrinsic versus intrinsic motivational factors on stated willingness to accept postings to rural underserved areas in Ghana.\",\n \"Ghana is located in the west coast of Africa with an estimated population of 23 million [23]. It is mainly an agrarian economy with estimated per capita income of USD 1500. More than two-thirds of the economy is rural, with the cities of Kumasi and Accra having the highest population densities due to brisk economic activities, and relatively strong economic and cultural infrastructure [24]. These attributes make the two main cities destinations for rural-urban migration and a highly heterogeneous socio-cultural context. There are four medical schools in Ghana: the University of Ghana (UG), Kwame Nkrumah University of Science and Technology (KNUST), University for Development Studies (UDS), and University of Cape Coast (UCC).\\nIn Ghana, medical education consists of three years of basic science/para-clinical studies, three years of clinical training at a teaching hospital, and a two-year rotating housemanship. The study was conducted with two public universities in Ghana: University of Ghana (UG) in Accra and Kwame Nkrumah University of Science and Technology (KNUST) in Kumasi. These universities were selected because all the fourth year medical students in the public universities had their clinical training at either UG or KNUST at the time of the study. All 310 fourth year medical students in the country were invited to participate in the study; no sampling was conducted. Fourth-year medical students were selected because they had completed the BSc. Human Biology and had also been exposed to field work, but had not yet made their final decisions about rural or urban practice.\",\n \"Data collection was preceded by discussions with the heads of medical training institutions, who informed the content of the questionnaire and provided access to the student population. The data collection instruments were developed after seven focus group discussions of 6-8 participants in each group facilitated by trained social scientists were held with third and fifth year medical students at UG and KNUST. The themes for the focus group discussion were motivation, willingness to work in deprived areas, experience in the field, and the influence of background characteristics on willingness to work in deprived areas. The survey instrument, which included semi-structured questions as well as a discrete choice experiment, were then pretested and finalized for the study. The questionnaires were administered electronically with Sawtooth Software SSI Web CAPI [25], in computer laboratories in UG and KNUST.\",\n \"The study received ethics approval from the Ghana Health Service Ethical Review Committee; the UG Medical School; the KNUST Committee on Human Research, Publications, and Ethics; and the University of Michigan Institutional Review Board. All respondents voluntarily participated after the intent and the design of the study had been explained to them and signing informed consent forms.\",\n \"The study used STATA v10.1 for statistical analyses [26]. The main outcome of interest was the willingness to work in a deprived area after graduation. Students were asked to rate how likely they were to work in a deprived area on a scale from 1-4, where 1 represented \\\"I will definitely not work in a deprived area;\\\" 2 \\\"I am unlikely to work in a deprived area;\\\" 3 \\\"I am likely to work in a deprived area;\\\" and 4 \\\"I will definitely work in a deprived area.\\\" We collapsed this response set to a dichotomous willing (groups 3 or 4) versus unwilling (groups 1 or 2) to practice in a deprived area. We defined deprived area as \\\"a rural area that is distant from the big cities with few social amenities such as schools, roads, pipe-borne water, etc.\\\"--as per Ministry of Health definition.\\nPredictors of interest included motivation (intrinsic and extrinsic), demographic characteristics, and rural exposure. Students were asked to identify the top three factors that motivated them to study medicine from a list of twelve factors identified as important by the focus group discussions. Five intrinsic motivations included: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one. Seven extrinsic motivation factors included: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities. Respondents were coded as having \\\"strong\\\" intrinsic or extrinsic motivation if two or more of their motivational factors were intrinsic or extrinsic. Thus, \\\"strong\\\" intrinsic and extrinsic motivation groups were mutually exclusive.\\nDemographic factors included: sex, age, ethnicity (Akan vs. non-Akan), partnership status (in a relationship vs. not in a relationship), and parental parental professional and educational status (PPES). The Akan peoples (including the Asante, Fante, Kwahu, Akuapem, Bono and others) are the largest ethnic group, representing approximately half of Ghanaians; we grouped all other smaller ethnic groups (Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa) together as \\\"non-Akans\\\" for this analysis. High parental professional and educational status (PPES) was defined as having a mother and/or father who is a University-trained professional (e.g. doctor, lawyer, engineer, accountant, technical, etc) and low PPES was defined as neither mother nor father is a University-trained professional. Rural (an area with a population less than 5000) exposure factors included: birth location (urban vs. rural), location of pre-medical studies (urban/vs. rural), having ever lived in rural area (from the age of 5 onwards), exposure to rural service in medical training (for a minimum of 6 months).\\nBivariate associations and 95% confidence intervals were estimated using logistic regression. In model 1, the influence of strong intrinsic/extrinsic motivation on willingness to accept postings to rural area were assessed. Socio-demographic factors were added to the regression in Model 2, and rural exposure factors were further added in Model 3.\",\n \" Socio-demographic characteristics Of the 310 eligible medical students, 307 participated in the survey (99%). Of these, five survey files were corrupted by viruses or lost due to computer malfunction; thus the analysis was conducted with 302 total records. The socio-demographic characteristics of respondents are presented in Table 1. Of the 302 respondents recruited for the study, the majority were male (183 or 60.6%), with a mean age of 22.9 (STD 1.4). Most respondents were born in or around urban areas (264 or 87.4%) and had never lived in rural underserved area (75.8%). In terms of socioeconomic status, 173 (57.3%) students came from high PPES families. About half of the respondents (142 or 47%) were exposed to rural service.\\nDemographic characteristics and rural exposure of respondents (N = 302)\\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\\n2. High Family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low Family PPES: Neither mother nor father is a University-trained Professional\\n3. Urban area defined as a place with more than 5,000 residents; rural area defined as a place with less than 5,000 residents\\n4. From age five on\\n5. Participated in outreach or service in a deprived area during medical studies\\nOf the 310 eligible medical students, 307 participated in the survey (99%). Of these, five survey files were corrupted by viruses or lost due to computer malfunction; thus the analysis was conducted with 302 total records. The socio-demographic characteristics of respondents are presented in Table 1. Of the 302 respondents recruited for the study, the majority were male (183 or 60.6%), with a mean age of 22.9 (STD 1.4). Most respondents were born in or around urban areas (264 or 87.4%) and had never lived in rural underserved area (75.8%). In terms of socioeconomic status, 173 (57.3%) students came from high PPES families. About half of the respondents (142 or 47%) were exposed to rural service.\\nDemographic characteristics and rural exposure of respondents (N = 302)\\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\\n2. High Family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low Family PPES: Neither mother nor father is a University-trained Professional\\n3. Urban area defined as a place with more than 5,000 residents; rural area defined as a place with less than 5,000 residents\\n4. From age five on\\n5. Participated in outreach or service in a deprived area during medical studies\\n Intrinsic and extrinsic motivation and likelihood of working in underserved area The intensities of current motivational factors are presented in Table 2. Overall, 158 (55.4%) of students stated that they were likely to or definitely would work in an underserved area. More than 6 in 10 respondents (181 or 63.5%) had strong intrinsic motivation. A higher proportion of respondents who had strong intrinsic motivation indicated willingness to work in a rural area, compared to those with weak intrinsic motivation (χ2 = 6.96, p = 0.008). These results were reversed for those with strong extrinsic motivation (χ2 = 6.12, p = 0.013).\\nIntrinsic versus extrinsic motivations to study medicine predict reported likelihood to work in an under-served area, 285 Ghanaian Medical Students\\n1. Excludes 17 missing values for willingness to work rural and/or current motivation.\\n2. Intrinsic motivation includes: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one; Pearson χ2(1) = 6.9592, p = 0.008.\\n3. Extrinsic motivation includes: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities; Pearson χ2(1) = 6.1208, p = 0.013.\\nThe intensities of current motivational factors are presented in Table 2. Overall, 158 (55.4%) of students stated that they were likely to or definitely would work in an underserved area. More than 6 in 10 respondents (181 or 63.5%) had strong intrinsic motivation. A higher proportion of respondents who had strong intrinsic motivation indicated willingness to work in a rural area, compared to those with weak intrinsic motivation (χ2 = 6.96, p = 0.008). These results were reversed for those with strong extrinsic motivation (χ2 = 6.12, p = 0.013).\\nIntrinsic versus extrinsic motivations to study medicine predict reported likelihood to work in an under-served area, 285 Ghanaian Medical Students\\n1. Excludes 17 missing values for willingness to work rural and/or current motivation.\\n2. Intrinsic motivation includes: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one; Pearson χ2(1) = 6.9592, p = 0.008.\\n3. Extrinsic motivation includes: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities; Pearson χ2(1) = 6.1208, p = 0.013.\\n Regression analysis of motivations and the willingness to accept postings to rural underserved area after graduation We present the multivariate logistic regression results for the strength of intrinsic motivation and willingness to work in a rural underserved area after graduation in Table 3. In the unadjusted model (Model 1) there was a significant association between strong intrinsic motivation and willingness to work in rural underserved area (1.92[95% CI 1.18-3.13]). In Model 2, with demographic factors, female gender and high PPES were associated with reduced willingness to practice in a deprived area (0.50[95% CI 0.29-0.88]) and (0.42[95% CI 0.24, 0.71]), respectively, while age was associated with greater willingness to practice in a rural area (1.23[95% CI 1.00-1.52]). Rural exposure factors were not significant when added to the model with intrinsic motivation and demographic factors (Model 3). In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\\nMultivariate logistic regression results for strength of intrinsic motivation and willingness to work in a rural underserved area after graduation, Ghanaian medical students\\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\\n3. Urban area defined as a place with more than 5,000 residents\\n4. From age five on\\n5. Participated in outreach or service in a deprived area during medical studies\\nIn Table 4 the influence of strong extrinsic motivation on willingness of students to work in rural underserved area is presented. In Model 1, having a strong extrinsic motivation reduced the odds of being willing to accept a job in an underserved area to (0.54[95% CI 0.33-0.88]). In the model adjusting for demographics, Model 2, female gender and high PPES were associated with reduced willingness to practice in underserved areas (0.50[95% CI 0.28-0.87] and (0.42[95% CI 0.24-0.71]) respectively, while age was associated with greater willingness to practice in a rural area (1.24[95% CI 1.00-1.53]). Rural exposure factors in model 3 did not influence the outcome of willingness to work in rural underserved area. In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\\nMultivariate logistic regression results for strength of extrinsic motivation and willingness to work in a rural underserved area after graduation\\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\\n3. Urban area defined as a place with more than 5,000 residents\\n4. From age five on\\n5. Participated in outreach or service in a deprived area during medical studies\\nWe present the multivariate logistic regression results for the strength of intrinsic motivation and willingness to work in a rural underserved area after graduation in Table 3. In the unadjusted model (Model 1) there was a significant association between strong intrinsic motivation and willingness to work in rural underserved area (1.92[95% CI 1.18-3.13]). In Model 2, with demographic factors, female gender and high PPES were associated with reduced willingness to practice in a deprived area (0.50[95% CI 0.29-0.88]) and (0.42[95% CI 0.24, 0.71]), respectively, while age was associated with greater willingness to practice in a rural area (1.23[95% CI 1.00-1.52]). Rural exposure factors were not significant when added to the model with intrinsic motivation and demographic factors (Model 3). In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\\nMultivariate logistic regression results for strength of intrinsic motivation and willingness to work in a rural underserved area after graduation, Ghanaian medical students\\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\\n3. Urban area defined as a place with more than 5,000 residents\\n4. From age five on\\n5. Participated in outreach or service in a deprived area during medical studies\\nIn Table 4 the influence of strong extrinsic motivation on willingness of students to work in rural underserved area is presented. In Model 1, having a strong extrinsic motivation reduced the odds of being willing to accept a job in an underserved area to (0.54[95% CI 0.33-0.88]). In the model adjusting for demographics, Model 2, female gender and high PPES were associated with reduced willingness to practice in underserved areas (0.50[95% CI 0.28-0.87] and (0.42[95% CI 0.24-0.71]) respectively, while age was associated with greater willingness to practice in a rural area (1.24[95% CI 1.00-1.53]). Rural exposure factors in model 3 did not influence the outcome of willingness to work in rural underserved area. In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\\nMultivariate logistic regression results for strength of extrinsic motivation and willingness to work in a rural underserved area after graduation\\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\\n3. Urban area defined as a place with more than 5,000 residents\\n4. From age five on\\n5. Participated in outreach or service in a deprived area during medical studies\",\n \"Of the 310 eligible medical students, 307 participated in the survey (99%). Of these, five survey files were corrupted by viruses or lost due to computer malfunction; thus the analysis was conducted with 302 total records. The socio-demographic characteristics of respondents are presented in Table 1. Of the 302 respondents recruited for the study, the majority were male (183 or 60.6%), with a mean age of 22.9 (STD 1.4). Most respondents were born in or around urban areas (264 or 87.4%) and had never lived in rural underserved area (75.8%). In terms of socioeconomic status, 173 (57.3%) students came from high PPES families. About half of the respondents (142 or 47%) were exposed to rural service.\\nDemographic characteristics and rural exposure of respondents (N = 302)\\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\\n2. High Family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low Family PPES: Neither mother nor father is a University-trained Professional\\n3. Urban area defined as a place with more than 5,000 residents; rural area defined as a place with less than 5,000 residents\\n4. From age five on\\n5. Participated in outreach or service in a deprived area during medical studies\",\n \"The intensities of current motivational factors are presented in Table 2. Overall, 158 (55.4%) of students stated that they were likely to or definitely would work in an underserved area. More than 6 in 10 respondents (181 or 63.5%) had strong intrinsic motivation. A higher proportion of respondents who had strong intrinsic motivation indicated willingness to work in a rural area, compared to those with weak intrinsic motivation (χ2 = 6.96, p = 0.008). These results were reversed for those with strong extrinsic motivation (χ2 = 6.12, p = 0.013).\\nIntrinsic versus extrinsic motivations to study medicine predict reported likelihood to work in an under-served area, 285 Ghanaian Medical Students\\n1. Excludes 17 missing values for willingness to work rural and/or current motivation.\\n2. Intrinsic motivation includes: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one; Pearson χ2(1) = 6.9592, p = 0.008.\\n3. Extrinsic motivation includes: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities; Pearson χ2(1) = 6.1208, p = 0.013.\",\n \"We present the multivariate logistic regression results for the strength of intrinsic motivation and willingness to work in a rural underserved area after graduation in Table 3. In the unadjusted model (Model 1) there was a significant association between strong intrinsic motivation and willingness to work in rural underserved area (1.92[95% CI 1.18-3.13]). In Model 2, with demographic factors, female gender and high PPES were associated with reduced willingness to practice in a deprived area (0.50[95% CI 0.29-0.88]) and (0.42[95% CI 0.24, 0.71]), respectively, while age was associated with greater willingness to practice in a rural area (1.23[95% CI 1.00-1.52]). Rural exposure factors were not significant when added to the model with intrinsic motivation and demographic factors (Model 3). In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\\nMultivariate logistic regression results for strength of intrinsic motivation and willingness to work in a rural underserved area after graduation, Ghanaian medical students\\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\\n3. Urban area defined as a place with more than 5,000 residents\\n4. From age five on\\n5. Participated in outreach or service in a deprived area during medical studies\\nIn Table 4 the influence of strong extrinsic motivation on willingness of students to work in rural underserved area is presented. In Model 1, having a strong extrinsic motivation reduced the odds of being willing to accept a job in an underserved area to (0.54[95% CI 0.33-0.88]). In the model adjusting for demographics, Model 2, female gender and high PPES were associated with reduced willingness to practice in underserved areas (0.50[95% CI 0.28-0.87] and (0.42[95% CI 0.24-0.71]) respectively, while age was associated with greater willingness to practice in a rural area (1.24[95% CI 1.00-1.53]). Rural exposure factors in model 3 did not influence the outcome of willingness to work in rural underserved area. In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\\nMultivariate logistic regression results for strength of extrinsic motivation and willingness to work in a rural underserved area after graduation\\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\\n3. Urban area defined as a place with more than 5,000 residents\\n4. From age five on\\n5. Participated in outreach or service in a deprived area during medical studies\",\n \"We found that twice as many students reported high intrinsic motivation compared to high extrinsic motivation to study and practice medicine. This may reflect the underlying altruistic motivation for many students entering a profession focused on serving others [12,13]. There may also be an element of social desirability bias in the students' responses as intrinsic motivation may be thought to be more socially acceptable than extrinsic motivation. Nonetheless, we found that high extrinsic motivation was associated with low self-reported likelihood of rural practice and that the converse was true for high intrinsic motivation [2,3]. Interestingly, this association lost statistical significance at the 95% confidence level in models with demographic and rural exposure confounders, whereas socioeconomic status (PPES) retained a highly influential role, as discussed below.\\nIn this study, rural origin did not influence students' willingness to practice in rural areas after controlling for intrinsic/extrinsic motivation and demographic characteristics. This is in contrast with studies which have found rural origin to be an important motivator for rural practice [8,17,23]. Our findings highlight the heterogeneity of trends in motivation dynamics for rural practice and the importance of locally-relevant data for decision making. High PPES, measured using parental education and profession, was consistently associated with lack of willingness to work in rural areas. This is concerning as nearly 6 in 10 medical students in this cohort were from high PPES backgrounds--which is typical for Ghanaian medical schools [7]. These findings suggest that admission policies that favour well-to do applicants may be reducing the pool of students willing to consider rural practice. Female gender was also strongly associated with reduced interest in rural practice for women even after controlling for extrinsic/intrinsic motivation and rural exposure variables. This is consistent with similar studies among health staff which revealed that women are less likely to accept positions in remote areas due to varying family reasons; they would like to live where their husbands jobs are, have difficulties convincing their husbands to follow them to rural areas and want their children to have better education in the urban areas [10,11,22]. The studies further explained that female doctors rarely live in the same village as their assigned post and have higher overall absentee rates in rural practice [19,20]. With increasing representation of female healthcare professionals in many places in sub-Saharan Africa, [10,11], it is likely that the supply of health staff to rural underserved areas will remain a major setback if professional motivations are designed to attract more female students to rural practice. Although our study showed a lower proportion of female medical students in Ghana compared to other areas, they are likely to become a more important cadre in the coming years [11,19]. More research is urgently needed to determine how female healthcare professionals' motivations towards rural practice can be better engaged by policy-makers.\\nThe limitations of this study include the possibility of social desirability bias in responses on motivation and likelihood of rural practice, as noted above. Despite the fact that study participants were assured of anonymity, confidentiality, in responding to the questions, some social desirability bias is likely. For this reason, we selected a measure of high intrinsic and extrinsic motivation for use in the regression models. Research comparing students stated intentions with their actual career choices during internship is urgently needed as few studies on matched follow-ups are available. In addition, most the students participating in the study were young and had not yet tasted the rigors of working in a rural area, which may affect their job preferences. Thus the findings of this study may not be applicable to practicing physicians. Finally, these findings are only generalizable to students in the current medical education system. The findings may be different if selection criteria for medical school admission change.\\nThe major strengths of this study are its high response rate of 99% and that its ability to capture an entire population of young medical students who are one of the targets for addressing the rural-urban health staff recruitment imbalance. Surveying practicing physicians would have missed out those who had migrated.\\nThis study has several implications. First, the majority of students profess high intrinsic motivation for rural service. More research is needed to determine the potency of this motivation source in real-life decision making and how to best engage it via HRH policy. It is possible that emphasizing the community service aspect of medical practice and elevating the status of rural primary care in under-graduate and post-graduate training may help narrow the gap between motivation and eventual career choice in favour of rural areas. In addition, well-supervised and supported rural placements in which students experience the rewards of rural practice may help to persuade students who are largely unfamiliar with rural life. However, the success of these rural rotations is likely to depend heavily on having adequate local infrastructure and mentorship [13,23].\\nSecond, admission criteria may need to be reconsidered in light of the strong relationship between high PPES and lack of interest in rural practice. For example, medical school admission slots might be reserved for qualified students from poorer families. These students may not need to come from rural areas as we found that none of the rural exposure factors were significant after controlling for motivation and demographics.\\nThird, our results suggest that programmes to promote and support rural practice after graduation may have some success. Our focus groups and discrete choice experiment suggested that students may be willing to commit to short-term placements of 2 years or less in rural areas [9]. The Ministry of Health may want to consider the possibility of short contracts that rotate physicians in and out of difficult to staff rural areas.\",\n \"The majority of students still claim high intrinsic motivation and therefore it is important to appeal and build on this in medical school curricula and in designing rural postings. However, extrinsic motivation and, perhaps most importantly, PPES, will likely continue to be an important factor in deciding on job postings. Future research should explore how motivations could be directly supported, how motivations are formed, the influence of contextual factors on motivation among medical students, and motivation crowding among practicing health professionals in rural underserved areas. Qualitative work may be especially informative in this effort. Our research also suggests that increasing efforts to recruit medical students from low PPES backgrounds may be the most effective current pathway to increasing the yield of physicians willing to practice in underserved areas.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Study site","Data collection","Ethical considerations","Statistical Analysis","Results","Socio-demographic characteristics","Intrinsic and extrinsic motivation and likelihood of working in underserved area","Regression analysis of motivations and the willingness to accept postings to rural underserved area after graduation","Discussion","Conclusions"],"string":"[\n \"Background\",\n \"Methods\",\n \"Study site\",\n \"Data collection\",\n \"Ethical considerations\",\n \"Statistical Analysis\",\n \"Results\",\n \"Socio-demographic characteristics\",\n \"Intrinsic and extrinsic motivation and likelihood of working in underserved area\",\n \"Regression analysis of motivations and the willingness to accept postings to rural underserved area after graduation\",\n \"Discussion\",\n \"Conclusions\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["The World Health Organization estimates that more than 4 million health workers are needed to fill the health workforce gap globally. This includes 2.4 million physicians, nurses and midwives. Fifty-seven countries are defined as having a critical shortage of health staff; of these, 36 are in sub-Saharan Africa. Of the total world's health work force of 59.2 million, Africa has only 3% of the world's health workers in spite of having 25% of the global burden of disease [1,2]. The shortage of health staff cripples the health delivery system. It is also a threat to provision of essential, life-saving interventions such as childhood immunizations, provision of safe water, safe pregnancy and childbirth services for mothers as well as access to treatment for AIDS, tuberculosis and malaria. Health workers are critical to the global preparedness for and response to threats posed by emerging and epidemic-prone diseases. Different interventions have been tried to address these shortages [3-5].\nRetaining health staff in rural areas has proven extremely difficult as young professionals increasingly prefer urban postings and health systems do not reward (and through neglect often penalize) rural service [6, 7. 8]. For example, Wilson and colleagues, 2009, Dovlo, 2003 and Kruk and colleagues, 2010, found that rural exposure, poor working conditions, low job satisfaction, political and ethnic problems, and sometimes, civil strife and poor security in most underserved areas, predispose new graduates to select cities [9-11]. Qualitative research has also shown the importance of health care providers' personal characteristics and value systems, such as religious beliefs and sociopolitical convictions, to their motivation towards rural practice. Emigration of skilled professionals to high-income countries is another barrier to adequate staffing of health facilities. A study in Ghana in 2006 on trainee physicians and nurses revealed that the majority had considered emigrating. More physicians (68%) than nurses (57%) considered emigration. These findings imply that achieving improvements in the health status of people living in low-income countries, and particularly, in rural areas, will be extremely difficult and the attainment of the United Nations Millennium Development Goals 4, 5, and 6 by 2015 [12,13], in Ghana is unlikely.\nWhile previous research has looked at incentives and working conditions to promote uptake of rural posts, few studies have focused on motivation crowding and its effect on willingness to accept postings to rural area. Motivation crowding [14] is the conflict between external factors (extrinsic), such as monetary incentives or punishments, and the underlying desire or willingness to work (intrinsic) in areas needed most. Students may have a mix of extrinsic and intrinsic motivations for studying medicine. Extrinsic factors may either undermine or strengthen intrinsic motivation, led by the belief that medicine has the imperative to help others, as enshrined in the Hippocratic Oath [15-17]. Current monetary incentives, which favour urban practice, may crowd-out the intrinsic desire to give back to society by working in underserved areas [18,19]. This could have debilitating effects on health worker retention in rural areas [20-22]. To tackle the maldistribution of human resources for health (HRH), understanding the factors that crowd-out the intrinsic motivation of medical students and their willingness to accept postings to rural underserved area is integral. This paper analyzes the effect of extrinsic versus intrinsic motivational factors on stated willingness to accept postings to rural underserved areas in Ghana."," Study site Ghana is located in the west coast of Africa with an estimated population of 23 million [23]. It is mainly an agrarian economy with estimated per capita income of USD 1500. More than two-thirds of the economy is rural, with the cities of Kumasi and Accra having the highest population densities due to brisk economic activities, and relatively strong economic and cultural infrastructure [24]. These attributes make the two main cities destinations for rural-urban migration and a highly heterogeneous socio-cultural context. There are four medical schools in Ghana: the University of Ghana (UG), Kwame Nkrumah University of Science and Technology (KNUST), University for Development Studies (UDS), and University of Cape Coast (UCC).\nIn Ghana, medical education consists of three years of basic science/para-clinical studies, three years of clinical training at a teaching hospital, and a two-year rotating housemanship. The study was conducted with two public universities in Ghana: University of Ghana (UG) in Accra and Kwame Nkrumah University of Science and Technology (KNUST) in Kumasi. These universities were selected because all the fourth year medical students in the public universities had their clinical training at either UG or KNUST at the time of the study. All 310 fourth year medical students in the country were invited to participate in the study; no sampling was conducted. Fourth-year medical students were selected because they had completed the BSc. Human Biology and had also been exposed to field work, but had not yet made their final decisions about rural or urban practice.\nGhana is located in the west coast of Africa with an estimated population of 23 million [23]. It is mainly an agrarian economy with estimated per capita income of USD 1500. More than two-thirds of the economy is rural, with the cities of Kumasi and Accra having the highest population densities due to brisk economic activities, and relatively strong economic and cultural infrastructure [24]. These attributes make the two main cities destinations for rural-urban migration and a highly heterogeneous socio-cultural context. There are four medical schools in Ghana: the University of Ghana (UG), Kwame Nkrumah University of Science and Technology (KNUST), University for Development Studies (UDS), and University of Cape Coast (UCC).\nIn Ghana, medical education consists of three years of basic science/para-clinical studies, three years of clinical training at a teaching hospital, and a two-year rotating housemanship. The study was conducted with two public universities in Ghana: University of Ghana (UG) in Accra and Kwame Nkrumah University of Science and Technology (KNUST) in Kumasi. These universities were selected because all the fourth year medical students in the public universities had their clinical training at either UG or KNUST at the time of the study. All 310 fourth year medical students in the country were invited to participate in the study; no sampling was conducted. Fourth-year medical students were selected because they had completed the BSc. Human Biology and had also been exposed to field work, but had not yet made their final decisions about rural or urban practice.\n Data collection Data collection was preceded by discussions with the heads of medical training institutions, who informed the content of the questionnaire and provided access to the student population. The data collection instruments were developed after seven focus group discussions of 6-8 participants in each group facilitated by trained social scientists were held with third and fifth year medical students at UG and KNUST. The themes for the focus group discussion were motivation, willingness to work in deprived areas, experience in the field, and the influence of background characteristics on willingness to work in deprived areas. The survey instrument, which included semi-structured questions as well as a discrete choice experiment, were then pretested and finalized for the study. The questionnaires were administered electronically with Sawtooth Software SSI Web CAPI [25], in computer laboratories in UG and KNUST.\nData collection was preceded by discussions with the heads of medical training institutions, who informed the content of the questionnaire and provided access to the student population. The data collection instruments were developed after seven focus group discussions of 6-8 participants in each group facilitated by trained social scientists were held with third and fifth year medical students at UG and KNUST. The themes for the focus group discussion were motivation, willingness to work in deprived areas, experience in the field, and the influence of background characteristics on willingness to work in deprived areas. The survey instrument, which included semi-structured questions as well as a discrete choice experiment, were then pretested and finalized for the study. The questionnaires were administered electronically with Sawtooth Software SSI Web CAPI [25], in computer laboratories in UG and KNUST.\n Ethical considerations The study received ethics approval from the Ghana Health Service Ethical Review Committee; the UG Medical School; the KNUST Committee on Human Research, Publications, and Ethics; and the University of Michigan Institutional Review Board. All respondents voluntarily participated after the intent and the design of the study had been explained to them and signing informed consent forms.\nThe study received ethics approval from the Ghana Health Service Ethical Review Committee; the UG Medical School; the KNUST Committee on Human Research, Publications, and Ethics; and the University of Michigan Institutional Review Board. All respondents voluntarily participated after the intent and the design of the study had been explained to them and signing informed consent forms.\n Statistical Analysis The study used STATA v10.1 for statistical analyses [26]. The main outcome of interest was the willingness to work in a deprived area after graduation. Students were asked to rate how likely they were to work in a deprived area on a scale from 1-4, where 1 represented \"I will definitely not work in a deprived area;\" 2 \"I am unlikely to work in a deprived area;\" 3 \"I am likely to work in a deprived area;\" and 4 \"I will definitely work in a deprived area.\" We collapsed this response set to a dichotomous willing (groups 3 or 4) versus unwilling (groups 1 or 2) to practice in a deprived area. We defined deprived area as \"a rural area that is distant from the big cities with few social amenities such as schools, roads, pipe-borne water, etc.\"--as per Ministry of Health definition.\nPredictors of interest included motivation (intrinsic and extrinsic), demographic characteristics, and rural exposure. Students were asked to identify the top three factors that motivated them to study medicine from a list of twelve factors identified as important by the focus group discussions. Five intrinsic motivations included: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one. Seven extrinsic motivation factors included: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities. Respondents were coded as having \"strong\" intrinsic or extrinsic motivation if two or more of their motivational factors were intrinsic or extrinsic. Thus, \"strong\" intrinsic and extrinsic motivation groups were mutually exclusive.\nDemographic factors included: sex, age, ethnicity (Akan vs. non-Akan), partnership status (in a relationship vs. not in a relationship), and parental parental professional and educational status (PPES). The Akan peoples (including the Asante, Fante, Kwahu, Akuapem, Bono and others) are the largest ethnic group, representing approximately half of Ghanaians; we grouped all other smaller ethnic groups (Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa) together as \"non-Akans\" for this analysis. High parental professional and educational status (PPES) was defined as having a mother and/or father who is a University-trained professional (e.g. doctor, lawyer, engineer, accountant, technical, etc) and low PPES was defined as neither mother nor father is a University-trained professional. Rural (an area with a population less than 5000) exposure factors included: birth location (urban vs. rural), location of pre-medical studies (urban/vs. rural), having ever lived in rural area (from the age of 5 onwards), exposure to rural service in medical training (for a minimum of 6 months).\nBivariate associations and 95% confidence intervals were estimated using logistic regression. In model 1, the influence of strong intrinsic/extrinsic motivation on willingness to accept postings to rural area were assessed. Socio-demographic factors were added to the regression in Model 2, and rural exposure factors were further added in Model 3.\nThe study used STATA v10.1 for statistical analyses [26]. The main outcome of interest was the willingness to work in a deprived area after graduation. Students were asked to rate how likely they were to work in a deprived area on a scale from 1-4, where 1 represented \"I will definitely not work in a deprived area;\" 2 \"I am unlikely to work in a deprived area;\" 3 \"I am likely to work in a deprived area;\" and 4 \"I will definitely work in a deprived area.\" We collapsed this response set to a dichotomous willing (groups 3 or 4) versus unwilling (groups 1 or 2) to practice in a deprived area. We defined deprived area as \"a rural area that is distant from the big cities with few social amenities such as schools, roads, pipe-borne water, etc.\"--as per Ministry of Health definition.\nPredictors of interest included motivation (intrinsic and extrinsic), demographic characteristics, and rural exposure. Students were asked to identify the top three factors that motivated them to study medicine from a list of twelve factors identified as important by the focus group discussions. Five intrinsic motivations included: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one. Seven extrinsic motivation factors included: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities. Respondents were coded as having \"strong\" intrinsic or extrinsic motivation if two or more of their motivational factors were intrinsic or extrinsic. Thus, \"strong\" intrinsic and extrinsic motivation groups were mutually exclusive.\nDemographic factors included: sex, age, ethnicity (Akan vs. non-Akan), partnership status (in a relationship vs. not in a relationship), and parental parental professional and educational status (PPES). The Akan peoples (including the Asante, Fante, Kwahu, Akuapem, Bono and others) are the largest ethnic group, representing approximately half of Ghanaians; we grouped all other smaller ethnic groups (Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa) together as \"non-Akans\" for this analysis. High parental professional and educational status (PPES) was defined as having a mother and/or father who is a University-trained professional (e.g. doctor, lawyer, engineer, accountant, technical, etc) and low PPES was defined as neither mother nor father is a University-trained professional. Rural (an area with a population less than 5000) exposure factors included: birth location (urban vs. rural), location of pre-medical studies (urban/vs. rural), having ever lived in rural area (from the age of 5 onwards), exposure to rural service in medical training (for a minimum of 6 months).\nBivariate associations and 95% confidence intervals were estimated using logistic regression. In model 1, the influence of strong intrinsic/extrinsic motivation on willingness to accept postings to rural area were assessed. Socio-demographic factors were added to the regression in Model 2, and rural exposure factors were further added in Model 3.","Ghana is located in the west coast of Africa with an estimated population of 23 million [23]. It is mainly an agrarian economy with estimated per capita income of USD 1500. More than two-thirds of the economy is rural, with the cities of Kumasi and Accra having the highest population densities due to brisk economic activities, and relatively strong economic and cultural infrastructure [24]. These attributes make the two main cities destinations for rural-urban migration and a highly heterogeneous socio-cultural context. There are four medical schools in Ghana: the University of Ghana (UG), Kwame Nkrumah University of Science and Technology (KNUST), University for Development Studies (UDS), and University of Cape Coast (UCC).\nIn Ghana, medical education consists of three years of basic science/para-clinical studies, three years of clinical training at a teaching hospital, and a two-year rotating housemanship. The study was conducted with two public universities in Ghana: University of Ghana (UG) in Accra and Kwame Nkrumah University of Science and Technology (KNUST) in Kumasi. These universities were selected because all the fourth year medical students in the public universities had their clinical training at either UG or KNUST at the time of the study. All 310 fourth year medical students in the country were invited to participate in the study; no sampling was conducted. Fourth-year medical students were selected because they had completed the BSc. Human Biology and had also been exposed to field work, but had not yet made their final decisions about rural or urban practice.","Data collection was preceded by discussions with the heads of medical training institutions, who informed the content of the questionnaire and provided access to the student population. The data collection instruments were developed after seven focus group discussions of 6-8 participants in each group facilitated by trained social scientists were held with third and fifth year medical students at UG and KNUST. The themes for the focus group discussion were motivation, willingness to work in deprived areas, experience in the field, and the influence of background characteristics on willingness to work in deprived areas. The survey instrument, which included semi-structured questions as well as a discrete choice experiment, were then pretested and finalized for the study. The questionnaires were administered electronically with Sawtooth Software SSI Web CAPI [25], in computer laboratories in UG and KNUST.","The study received ethics approval from the Ghana Health Service Ethical Review Committee; the UG Medical School; the KNUST Committee on Human Research, Publications, and Ethics; and the University of Michigan Institutional Review Board. All respondents voluntarily participated after the intent and the design of the study had been explained to them and signing informed consent forms.","The study used STATA v10.1 for statistical analyses [26]. The main outcome of interest was the willingness to work in a deprived area after graduation. Students were asked to rate how likely they were to work in a deprived area on a scale from 1-4, where 1 represented \"I will definitely not work in a deprived area;\" 2 \"I am unlikely to work in a deprived area;\" 3 \"I am likely to work in a deprived area;\" and 4 \"I will definitely work in a deprived area.\" We collapsed this response set to a dichotomous willing (groups 3 or 4) versus unwilling (groups 1 or 2) to practice in a deprived area. We defined deprived area as \"a rural area that is distant from the big cities with few social amenities such as schools, roads, pipe-borne water, etc.\"--as per Ministry of Health definition.\nPredictors of interest included motivation (intrinsic and extrinsic), demographic characteristics, and rural exposure. Students were asked to identify the top three factors that motivated them to study medicine from a list of twelve factors identified as important by the focus group discussions. Five intrinsic motivations included: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one. Seven extrinsic motivation factors included: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities. Respondents were coded as having \"strong\" intrinsic or extrinsic motivation if two or more of their motivational factors were intrinsic or extrinsic. Thus, \"strong\" intrinsic and extrinsic motivation groups were mutually exclusive.\nDemographic factors included: sex, age, ethnicity (Akan vs. non-Akan), partnership status (in a relationship vs. not in a relationship), and parental parental professional and educational status (PPES). The Akan peoples (including the Asante, Fante, Kwahu, Akuapem, Bono and others) are the largest ethnic group, representing approximately half of Ghanaians; we grouped all other smaller ethnic groups (Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa) together as \"non-Akans\" for this analysis. High parental professional and educational status (PPES) was defined as having a mother and/or father who is a University-trained professional (e.g. doctor, lawyer, engineer, accountant, technical, etc) and low PPES was defined as neither mother nor father is a University-trained professional. Rural (an area with a population less than 5000) exposure factors included: birth location (urban vs. rural), location of pre-medical studies (urban/vs. rural), having ever lived in rural area (from the age of 5 onwards), exposure to rural service in medical training (for a minimum of 6 months).\nBivariate associations and 95% confidence intervals were estimated using logistic regression. In model 1, the influence of strong intrinsic/extrinsic motivation on willingness to accept postings to rural area were assessed. Socio-demographic factors were added to the regression in Model 2, and rural exposure factors were further added in Model 3."," Socio-demographic characteristics Of the 310 eligible medical students, 307 participated in the survey (99%). Of these, five survey files were corrupted by viruses or lost due to computer malfunction; thus the analysis was conducted with 302 total records. The socio-demographic characteristics of respondents are presented in Table 1. Of the 302 respondents recruited for the study, the majority were male (183 or 60.6%), with a mean age of 22.9 (STD 1.4). Most respondents were born in or around urban areas (264 or 87.4%) and had never lived in rural underserved area (75.8%). In terms of socioeconomic status, 173 (57.3%) students came from high PPES families. About half of the respondents (142 or 47%) were exposed to rural service.\nDemographic characteristics and rural exposure of respondents (N = 302)\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High Family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low Family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents; rural area defined as a place with less than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies\nOf the 310 eligible medical students, 307 participated in the survey (99%). Of these, five survey files were corrupted by viruses or lost due to computer malfunction; thus the analysis was conducted with 302 total records. The socio-demographic characteristics of respondents are presented in Table 1. Of the 302 respondents recruited for the study, the majority were male (183 or 60.6%), with a mean age of 22.9 (STD 1.4). Most respondents were born in or around urban areas (264 or 87.4%) and had never lived in rural underserved area (75.8%). In terms of socioeconomic status, 173 (57.3%) students came from high PPES families. About half of the respondents (142 or 47%) were exposed to rural service.\nDemographic characteristics and rural exposure of respondents (N = 302)\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High Family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low Family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents; rural area defined as a place with less than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies\n Intrinsic and extrinsic motivation and likelihood of working in underserved area The intensities of current motivational factors are presented in Table 2. Overall, 158 (55.4%) of students stated that they were likely to or definitely would work in an underserved area. More than 6 in 10 respondents (181 or 63.5%) had strong intrinsic motivation. A higher proportion of respondents who had strong intrinsic motivation indicated willingness to work in a rural area, compared to those with weak intrinsic motivation (χ2 = 6.96, p = 0.008). These results were reversed for those with strong extrinsic motivation (χ2 = 6.12, p = 0.013).\nIntrinsic versus extrinsic motivations to study medicine predict reported likelihood to work in an under-served area, 285 Ghanaian Medical Students\n1. Excludes 17 missing values for willingness to work rural and/or current motivation.\n2. Intrinsic motivation includes: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one; Pearson χ2(1) = 6.9592, p = 0.008.\n3. Extrinsic motivation includes: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities; Pearson χ2(1) = 6.1208, p = 0.013.\nThe intensities of current motivational factors are presented in Table 2. Overall, 158 (55.4%) of students stated that they were likely to or definitely would work in an underserved area. More than 6 in 10 respondents (181 or 63.5%) had strong intrinsic motivation. A higher proportion of respondents who had strong intrinsic motivation indicated willingness to work in a rural area, compared to those with weak intrinsic motivation (χ2 = 6.96, p = 0.008). These results were reversed for those with strong extrinsic motivation (χ2 = 6.12, p = 0.013).\nIntrinsic versus extrinsic motivations to study medicine predict reported likelihood to work in an under-served area, 285 Ghanaian Medical Students\n1. Excludes 17 missing values for willingness to work rural and/or current motivation.\n2. Intrinsic motivation includes: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one; Pearson χ2(1) = 6.9592, p = 0.008.\n3. Extrinsic motivation includes: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities; Pearson χ2(1) = 6.1208, p = 0.013.\n Regression analysis of motivations and the willingness to accept postings to rural underserved area after graduation We present the multivariate logistic regression results for the strength of intrinsic motivation and willingness to work in a rural underserved area after graduation in Table 3. In the unadjusted model (Model 1) there was a significant association between strong intrinsic motivation and willingness to work in rural underserved area (1.92[95% CI 1.18-3.13]). In Model 2, with demographic factors, female gender and high PPES were associated with reduced willingness to practice in a deprived area (0.50[95% CI 0.29-0.88]) and (0.42[95% CI 0.24, 0.71]), respectively, while age was associated with greater willingness to practice in a rural area (1.23[95% CI 1.00-1.52]). Rural exposure factors were not significant when added to the model with intrinsic motivation and demographic factors (Model 3). In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\nMultivariate logistic regression results for strength of intrinsic motivation and willingness to work in a rural underserved area after graduation, Ghanaian medical students\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies\nIn Table 4 the influence of strong extrinsic motivation on willingness of students to work in rural underserved area is presented. In Model 1, having a strong extrinsic motivation reduced the odds of being willing to accept a job in an underserved area to (0.54[95% CI 0.33-0.88]). In the model adjusting for demographics, Model 2, female gender and high PPES were associated with reduced willingness to practice in underserved areas (0.50[95% CI 0.28-0.87] and (0.42[95% CI 0.24-0.71]) respectively, while age was associated with greater willingness to practice in a rural area (1.24[95% CI 1.00-1.53]). Rural exposure factors in model 3 did not influence the outcome of willingness to work in rural underserved area. In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\nMultivariate logistic regression results for strength of extrinsic motivation and willingness to work in a rural underserved area after graduation\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies\nWe present the multivariate logistic regression results for the strength of intrinsic motivation and willingness to work in a rural underserved area after graduation in Table 3. In the unadjusted model (Model 1) there was a significant association between strong intrinsic motivation and willingness to work in rural underserved area (1.92[95% CI 1.18-3.13]). In Model 2, with demographic factors, female gender and high PPES were associated with reduced willingness to practice in a deprived area (0.50[95% CI 0.29-0.88]) and (0.42[95% CI 0.24, 0.71]), respectively, while age was associated with greater willingness to practice in a rural area (1.23[95% CI 1.00-1.52]). Rural exposure factors were not significant when added to the model with intrinsic motivation and demographic factors (Model 3). In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\nMultivariate logistic regression results for strength of intrinsic motivation and willingness to work in a rural underserved area after graduation, Ghanaian medical students\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies\nIn Table 4 the influence of strong extrinsic motivation on willingness of students to work in rural underserved area is presented. In Model 1, having a strong extrinsic motivation reduced the odds of being willing to accept a job in an underserved area to (0.54[95% CI 0.33-0.88]). In the model adjusting for demographics, Model 2, female gender and high PPES were associated with reduced willingness to practice in underserved areas (0.50[95% CI 0.28-0.87] and (0.42[95% CI 0.24-0.71]) respectively, while age was associated with greater willingness to practice in a rural area (1.24[95% CI 1.00-1.53]). Rural exposure factors in model 3 did not influence the outcome of willingness to work in rural underserved area. In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\nMultivariate logistic regression results for strength of extrinsic motivation and willingness to work in a rural underserved area after graduation\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies","Of the 310 eligible medical students, 307 participated in the survey (99%). Of these, five survey files were corrupted by viruses or lost due to computer malfunction; thus the analysis was conducted with 302 total records. The socio-demographic characteristics of respondents are presented in Table 1. Of the 302 respondents recruited for the study, the majority were male (183 or 60.6%), with a mean age of 22.9 (STD 1.4). Most respondents were born in or around urban areas (264 or 87.4%) and had never lived in rural underserved area (75.8%). In terms of socioeconomic status, 173 (57.3%) students came from high PPES families. About half of the respondents (142 or 47%) were exposed to rural service.\nDemographic characteristics and rural exposure of respondents (N = 302)\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High Family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low Family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents; rural area defined as a place with less than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies","The intensities of current motivational factors are presented in Table 2. Overall, 158 (55.4%) of students stated that they were likely to or definitely would work in an underserved area. More than 6 in 10 respondents (181 or 63.5%) had strong intrinsic motivation. A higher proportion of respondents who had strong intrinsic motivation indicated willingness to work in a rural area, compared to those with weak intrinsic motivation (χ2 = 6.96, p = 0.008). These results were reversed for those with strong extrinsic motivation (χ2 = 6.12, p = 0.013).\nIntrinsic versus extrinsic motivations to study medicine predict reported likelihood to work in an under-served area, 285 Ghanaian Medical Students\n1. Excludes 17 missing values for willingness to work rural and/or current motivation.\n2. Intrinsic motivation includes: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one; Pearson χ2(1) = 6.9592, p = 0.008.\n3. Extrinsic motivation includes: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities; Pearson χ2(1) = 6.1208, p = 0.013.","We present the multivariate logistic regression results for the strength of intrinsic motivation and willingness to work in a rural underserved area after graduation in Table 3. In the unadjusted model (Model 1) there was a significant association between strong intrinsic motivation and willingness to work in rural underserved area (1.92[95% CI 1.18-3.13]). In Model 2, with demographic factors, female gender and high PPES were associated with reduced willingness to practice in a deprived area (0.50[95% CI 0.29-0.88]) and (0.42[95% CI 0.24, 0.71]), respectively, while age was associated with greater willingness to practice in a rural area (1.23[95% CI 1.00-1.52]). Rural exposure factors were not significant when added to the model with intrinsic motivation and demographic factors (Model 3). In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\nMultivariate logistic regression results for strength of intrinsic motivation and willingness to work in a rural underserved area after graduation, Ghanaian medical students\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies\nIn Table 4 the influence of strong extrinsic motivation on willingness of students to work in rural underserved area is presented. In Model 1, having a strong extrinsic motivation reduced the odds of being willing to accept a job in an underserved area to (0.54[95% CI 0.33-0.88]). In the model adjusting for demographics, Model 2, female gender and high PPES were associated with reduced willingness to practice in underserved areas (0.50[95% CI 0.28-0.87] and (0.42[95% CI 0.24-0.71]) respectively, while age was associated with greater willingness to practice in a rural area (1.24[95% CI 1.00-1.53]). Rural exposure factors in model 3 did not influence the outcome of willingness to work in rural underserved area. In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\nMultivariate logistic regression results for strength of extrinsic motivation and willingness to work in a rural underserved area after graduation\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies","We found that twice as many students reported high intrinsic motivation compared to high extrinsic motivation to study and practice medicine. This may reflect the underlying altruistic motivation for many students entering a profession focused on serving others [12,13]. There may also be an element of social desirability bias in the students' responses as intrinsic motivation may be thought to be more socially acceptable than extrinsic motivation. Nonetheless, we found that high extrinsic motivation was associated with low self-reported likelihood of rural practice and that the converse was true for high intrinsic motivation [2,3]. Interestingly, this association lost statistical significance at the 95% confidence level in models with demographic and rural exposure confounders, whereas socioeconomic status (PPES) retained a highly influential role, as discussed below.\nIn this study, rural origin did not influence students' willingness to practice in rural areas after controlling for intrinsic/extrinsic motivation and demographic characteristics. This is in contrast with studies which have found rural origin to be an important motivator for rural practice [8,17,23]. Our findings highlight the heterogeneity of trends in motivation dynamics for rural practice and the importance of locally-relevant data for decision making. High PPES, measured using parental education and profession, was consistently associated with lack of willingness to work in rural areas. This is concerning as nearly 6 in 10 medical students in this cohort were from high PPES backgrounds--which is typical for Ghanaian medical schools [7]. These findings suggest that admission policies that favour well-to do applicants may be reducing the pool of students willing to consider rural practice. Female gender was also strongly associated with reduced interest in rural practice for women even after controlling for extrinsic/intrinsic motivation and rural exposure variables. This is consistent with similar studies among health staff which revealed that women are less likely to accept positions in remote areas due to varying family reasons; they would like to live where their husbands jobs are, have difficulties convincing their husbands to follow them to rural areas and want their children to have better education in the urban areas [10,11,22]. The studies further explained that female doctors rarely live in the same village as their assigned post and have higher overall absentee rates in rural practice [19,20]. With increasing representation of female healthcare professionals in many places in sub-Saharan Africa, [10,11], it is likely that the supply of health staff to rural underserved areas will remain a major setback if professional motivations are designed to attract more female students to rural practice. Although our study showed a lower proportion of female medical students in Ghana compared to other areas, they are likely to become a more important cadre in the coming years [11,19]. More research is urgently needed to determine how female healthcare professionals' motivations towards rural practice can be better engaged by policy-makers.\nThe limitations of this study include the possibility of social desirability bias in responses on motivation and likelihood of rural practice, as noted above. Despite the fact that study participants were assured of anonymity, confidentiality, in responding to the questions, some social desirability bias is likely. For this reason, we selected a measure of high intrinsic and extrinsic motivation for use in the regression models. Research comparing students stated intentions with their actual career choices during internship is urgently needed as few studies on matched follow-ups are available. In addition, most the students participating in the study were young and had not yet tasted the rigors of working in a rural area, which may affect their job preferences. Thus the findings of this study may not be applicable to practicing physicians. Finally, these findings are only generalizable to students in the current medical education system. The findings may be different if selection criteria for medical school admission change.\nThe major strengths of this study are its high response rate of 99% and that its ability to capture an entire population of young medical students who are one of the targets for addressing the rural-urban health staff recruitment imbalance. Surveying practicing physicians would have missed out those who had migrated.\nThis study has several implications. First, the majority of students profess high intrinsic motivation for rural service. More research is needed to determine the potency of this motivation source in real-life decision making and how to best engage it via HRH policy. It is possible that emphasizing the community service aspect of medical practice and elevating the status of rural primary care in under-graduate and post-graduate training may help narrow the gap between motivation and eventual career choice in favour of rural areas. In addition, well-supervised and supported rural placements in which students experience the rewards of rural practice may help to persuade students who are largely unfamiliar with rural life. However, the success of these rural rotations is likely to depend heavily on having adequate local infrastructure and mentorship [13,23].\nSecond, admission criteria may need to be reconsidered in light of the strong relationship between high PPES and lack of interest in rural practice. For example, medical school admission slots might be reserved for qualified students from poorer families. These students may not need to come from rural areas as we found that none of the rural exposure factors were significant after controlling for motivation and demographics.\nThird, our results suggest that programmes to promote and support rural practice after graduation may have some success. Our focus groups and discrete choice experiment suggested that students may be willing to commit to short-term placements of 2 years or less in rural areas [9]. The Ministry of Health may want to consider the possibility of short contracts that rotate physicians in and out of difficult to staff rural areas.","The majority of students still claim high intrinsic motivation and therefore it is important to appeal and build on this in medical school curricula and in designing rural postings. However, extrinsic motivation and, perhaps most importantly, PPES, will likely continue to be an important factor in deciding on job postings. Future research should explore how motivations could be directly supported, how motivations are formed, the influence of contextual factors on motivation among medical students, and motivation crowding among practicing health professionals in rural underserved areas. Qualitative work may be especially informative in this effort. Our research also suggests that increasing efforts to recruit medical students from low PPES backgrounds may be the most effective current pathway to increasing the yield of physicians willing to practice in underserved areas."],"string":"[\n \"The World Health Organization estimates that more than 4 million health workers are needed to fill the health workforce gap globally. This includes 2.4 million physicians, nurses and midwives. Fifty-seven countries are defined as having a critical shortage of health staff; of these, 36 are in sub-Saharan Africa. Of the total world's health work force of 59.2 million, Africa has only 3% of the world's health workers in spite of having 25% of the global burden of disease [1,2]. The shortage of health staff cripples the health delivery system. It is also a threat to provision of essential, life-saving interventions such as childhood immunizations, provision of safe water, safe pregnancy and childbirth services for mothers as well as access to treatment for AIDS, tuberculosis and malaria. Health workers are critical to the global preparedness for and response to threats posed by emerging and epidemic-prone diseases. Different interventions have been tried to address these shortages [3-5].\\nRetaining health staff in rural areas has proven extremely difficult as young professionals increasingly prefer urban postings and health systems do not reward (and through neglect often penalize) rural service [6, 7. 8]. For example, Wilson and colleagues, 2009, Dovlo, 2003 and Kruk and colleagues, 2010, found that rural exposure, poor working conditions, low job satisfaction, political and ethnic problems, and sometimes, civil strife and poor security in most underserved areas, predispose new graduates to select cities [9-11]. Qualitative research has also shown the importance of health care providers' personal characteristics and value systems, such as religious beliefs and sociopolitical convictions, to their motivation towards rural practice. Emigration of skilled professionals to high-income countries is another barrier to adequate staffing of health facilities. A study in Ghana in 2006 on trainee physicians and nurses revealed that the majority had considered emigrating. More physicians (68%) than nurses (57%) considered emigration. These findings imply that achieving improvements in the health status of people living in low-income countries, and particularly, in rural areas, will be extremely difficult and the attainment of the United Nations Millennium Development Goals 4, 5, and 6 by 2015 [12,13], in Ghana is unlikely.\\nWhile previous research has looked at incentives and working conditions to promote uptake of rural posts, few studies have focused on motivation crowding and its effect on willingness to accept postings to rural area. Motivation crowding [14] is the conflict between external factors (extrinsic), such as monetary incentives or punishments, and the underlying desire or willingness to work (intrinsic) in areas needed most. Students may have a mix of extrinsic and intrinsic motivations for studying medicine. Extrinsic factors may either undermine or strengthen intrinsic motivation, led by the belief that medicine has the imperative to help others, as enshrined in the Hippocratic Oath [15-17]. Current monetary incentives, which favour urban practice, may crowd-out the intrinsic desire to give back to society by working in underserved areas [18,19]. This could have debilitating effects on health worker retention in rural areas [20-22]. To tackle the maldistribution of human resources for health (HRH), understanding the factors that crowd-out the intrinsic motivation of medical students and their willingness to accept postings to rural underserved area is integral. This paper analyzes the effect of extrinsic versus intrinsic motivational factors on stated willingness to accept postings to rural underserved areas in Ghana.\",\n \" Study site Ghana is located in the west coast of Africa with an estimated population of 23 million [23]. It is mainly an agrarian economy with estimated per capita income of USD 1500. More than two-thirds of the economy is rural, with the cities of Kumasi and Accra having the highest population densities due to brisk economic activities, and relatively strong economic and cultural infrastructure [24]. These attributes make the two main cities destinations for rural-urban migration and a highly heterogeneous socio-cultural context. There are four medical schools in Ghana: the University of Ghana (UG), Kwame Nkrumah University of Science and Technology (KNUST), University for Development Studies (UDS), and University of Cape Coast (UCC).\\nIn Ghana, medical education consists of three years of basic science/para-clinical studies, three years of clinical training at a teaching hospital, and a two-year rotating housemanship. The study was conducted with two public universities in Ghana: University of Ghana (UG) in Accra and Kwame Nkrumah University of Science and Technology (KNUST) in Kumasi. These universities were selected because all the fourth year medical students in the public universities had their clinical training at either UG or KNUST at the time of the study. All 310 fourth year medical students in the country were invited to participate in the study; no sampling was conducted. Fourth-year medical students were selected because they had completed the BSc. Human Biology and had also been exposed to field work, but had not yet made their final decisions about rural or urban practice.\\nGhana is located in the west coast of Africa with an estimated population of 23 million [23]. It is mainly an agrarian economy with estimated per capita income of USD 1500. More than two-thirds of the economy is rural, with the cities of Kumasi and Accra having the highest population densities due to brisk economic activities, and relatively strong economic and cultural infrastructure [24]. These attributes make the two main cities destinations for rural-urban migration and a highly heterogeneous socio-cultural context. There are four medical schools in Ghana: the University of Ghana (UG), Kwame Nkrumah University of Science and Technology (KNUST), University for Development Studies (UDS), and University of Cape Coast (UCC).\\nIn Ghana, medical education consists of three years of basic science/para-clinical studies, three years of clinical training at a teaching hospital, and a two-year rotating housemanship. The study was conducted with two public universities in Ghana: University of Ghana (UG) in Accra and Kwame Nkrumah University of Science and Technology (KNUST) in Kumasi. These universities were selected because all the fourth year medical students in the public universities had their clinical training at either UG or KNUST at the time of the study. All 310 fourth year medical students in the country were invited to participate in the study; no sampling was conducted. Fourth-year medical students were selected because they had completed the BSc. Human Biology and had also been exposed to field work, but had not yet made their final decisions about rural or urban practice.\\n Data collection Data collection was preceded by discussions with the heads of medical training institutions, who informed the content of the questionnaire and provided access to the student population. The data collection instruments were developed after seven focus group discussions of 6-8 participants in each group facilitated by trained social scientists were held with third and fifth year medical students at UG and KNUST. The themes for the focus group discussion were motivation, willingness to work in deprived areas, experience in the field, and the influence of background characteristics on willingness to work in deprived areas. The survey instrument, which included semi-structured questions as well as a discrete choice experiment, were then pretested and finalized for the study. The questionnaires were administered electronically with Sawtooth Software SSI Web CAPI [25], in computer laboratories in UG and KNUST.\\nData collection was preceded by discussions with the heads of medical training institutions, who informed the content of the questionnaire and provided access to the student population. The data collection instruments were developed after seven focus group discussions of 6-8 participants in each group facilitated by trained social scientists were held with third and fifth year medical students at UG and KNUST. The themes for the focus group discussion were motivation, willingness to work in deprived areas, experience in the field, and the influence of background characteristics on willingness to work in deprived areas. The survey instrument, which included semi-structured questions as well as a discrete choice experiment, were then pretested and finalized for the study. The questionnaires were administered electronically with Sawtooth Software SSI Web CAPI [25], in computer laboratories in UG and KNUST.\\n Ethical considerations The study received ethics approval from the Ghana Health Service Ethical Review Committee; the UG Medical School; the KNUST Committee on Human Research, Publications, and Ethics; and the University of Michigan Institutional Review Board. All respondents voluntarily participated after the intent and the design of the study had been explained to them and signing informed consent forms.\\nThe study received ethics approval from the Ghana Health Service Ethical Review Committee; the UG Medical School; the KNUST Committee on Human Research, Publications, and Ethics; and the University of Michigan Institutional Review Board. All respondents voluntarily participated after the intent and the design of the study had been explained to them and signing informed consent forms.\\n Statistical Analysis The study used STATA v10.1 for statistical analyses [26]. The main outcome of interest was the willingness to work in a deprived area after graduation. Students were asked to rate how likely they were to work in a deprived area on a scale from 1-4, where 1 represented \\\"I will definitely not work in a deprived area;\\\" 2 \\\"I am unlikely to work in a deprived area;\\\" 3 \\\"I am likely to work in a deprived area;\\\" and 4 \\\"I will definitely work in a deprived area.\\\" We collapsed this response set to a dichotomous willing (groups 3 or 4) versus unwilling (groups 1 or 2) to practice in a deprived area. We defined deprived area as \\\"a rural area that is distant from the big cities with few social amenities such as schools, roads, pipe-borne water, etc.\\\"--as per Ministry of Health definition.\\nPredictors of interest included motivation (intrinsic and extrinsic), demographic characteristics, and rural exposure. Students were asked to identify the top three factors that motivated them to study medicine from a list of twelve factors identified as important by the focus group discussions. Five intrinsic motivations included: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one. Seven extrinsic motivation factors included: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities. Respondents were coded as having \\\"strong\\\" intrinsic or extrinsic motivation if two or more of their motivational factors were intrinsic or extrinsic. Thus, \\\"strong\\\" intrinsic and extrinsic motivation groups were mutually exclusive.\\nDemographic factors included: sex, age, ethnicity (Akan vs. non-Akan), partnership status (in a relationship vs. not in a relationship), and parental parental professional and educational status (PPES). The Akan peoples (including the Asante, Fante, Kwahu, Akuapem, Bono and others) are the largest ethnic group, representing approximately half of Ghanaians; we grouped all other smaller ethnic groups (Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa) together as \\\"non-Akans\\\" for this analysis. High parental professional and educational status (PPES) was defined as having a mother and/or father who is a University-trained professional (e.g. doctor, lawyer, engineer, accountant, technical, etc) and low PPES was defined as neither mother nor father is a University-trained professional. Rural (an area with a population less than 5000) exposure factors included: birth location (urban vs. rural), location of pre-medical studies (urban/vs. rural), having ever lived in rural area (from the age of 5 onwards), exposure to rural service in medical training (for a minimum of 6 months).\\nBivariate associations and 95% confidence intervals were estimated using logistic regression. In model 1, the influence of strong intrinsic/extrinsic motivation on willingness to accept postings to rural area were assessed. Socio-demographic factors were added to the regression in Model 2, and rural exposure factors were further added in Model 3.\\nThe study used STATA v10.1 for statistical analyses [26]. The main outcome of interest was the willingness to work in a deprived area after graduation. Students were asked to rate how likely they were to work in a deprived area on a scale from 1-4, where 1 represented \\\"I will definitely not work in a deprived area;\\\" 2 \\\"I am unlikely to work in a deprived area;\\\" 3 \\\"I am likely to work in a deprived area;\\\" and 4 \\\"I will definitely work in a deprived area.\\\" We collapsed this response set to a dichotomous willing (groups 3 or 4) versus unwilling (groups 1 or 2) to practice in a deprived area. We defined deprived area as \\\"a rural area that is distant from the big cities with few social amenities such as schools, roads, pipe-borne water, etc.\\\"--as per Ministry of Health definition.\\nPredictors of interest included motivation (intrinsic and extrinsic), demographic characteristics, and rural exposure. Students were asked to identify the top three factors that motivated them to study medicine from a list of twelve factors identified as important by the focus group discussions. Five intrinsic motivations included: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one. Seven extrinsic motivation factors included: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities. Respondents were coded as having \\\"strong\\\" intrinsic or extrinsic motivation if two or more of their motivational factors were intrinsic or extrinsic. Thus, \\\"strong\\\" intrinsic and extrinsic motivation groups were mutually exclusive.\\nDemographic factors included: sex, age, ethnicity (Akan vs. non-Akan), partnership status (in a relationship vs. not in a relationship), and parental parental professional and educational status (PPES). The Akan peoples (including the Asante, Fante, Kwahu, Akuapem, Bono and others) are the largest ethnic group, representing approximately half of Ghanaians; we grouped all other smaller ethnic groups (Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa) together as \\\"non-Akans\\\" for this analysis. High parental professional and educational status (PPES) was defined as having a mother and/or father who is a University-trained professional (e.g. doctor, lawyer, engineer, accountant, technical, etc) and low PPES was defined as neither mother nor father is a University-trained professional. Rural (an area with a population less than 5000) exposure factors included: birth location (urban vs. rural), location of pre-medical studies (urban/vs. rural), having ever lived in rural area (from the age of 5 onwards), exposure to rural service in medical training (for a minimum of 6 months).\\nBivariate associations and 95% confidence intervals were estimated using logistic regression. In model 1, the influence of strong intrinsic/extrinsic motivation on willingness to accept postings to rural area were assessed. Socio-demographic factors were added to the regression in Model 2, and rural exposure factors were further added in Model 3.\",\n \"Ghana is located in the west coast of Africa with an estimated population of 23 million [23]. It is mainly an agrarian economy with estimated per capita income of USD 1500. More than two-thirds of the economy is rural, with the cities of Kumasi and Accra having the highest population densities due to brisk economic activities, and relatively strong economic and cultural infrastructure [24]. These attributes make the two main cities destinations for rural-urban migration and a highly heterogeneous socio-cultural context. There are four medical schools in Ghana: the University of Ghana (UG), Kwame Nkrumah University of Science and Technology (KNUST), University for Development Studies (UDS), and University of Cape Coast (UCC).\\nIn Ghana, medical education consists of three years of basic science/para-clinical studies, three years of clinical training at a teaching hospital, and a two-year rotating housemanship. The study was conducted with two public universities in Ghana: University of Ghana (UG) in Accra and Kwame Nkrumah University of Science and Technology (KNUST) in Kumasi. These universities were selected because all the fourth year medical students in the public universities had their clinical training at either UG or KNUST at the time of the study. All 310 fourth year medical students in the country were invited to participate in the study; no sampling was conducted. Fourth-year medical students were selected because they had completed the BSc. Human Biology and had also been exposed to field work, but had not yet made their final decisions about rural or urban practice.\",\n \"Data collection was preceded by discussions with the heads of medical training institutions, who informed the content of the questionnaire and provided access to the student population. The data collection instruments were developed after seven focus group discussions of 6-8 participants in each group facilitated by trained social scientists were held with third and fifth year medical students at UG and KNUST. The themes for the focus group discussion were motivation, willingness to work in deprived areas, experience in the field, and the influence of background characteristics on willingness to work in deprived areas. The survey instrument, which included semi-structured questions as well as a discrete choice experiment, were then pretested and finalized for the study. The questionnaires were administered electronically with Sawtooth Software SSI Web CAPI [25], in computer laboratories in UG and KNUST.\",\n \"The study received ethics approval from the Ghana Health Service Ethical Review Committee; the UG Medical School; the KNUST Committee on Human Research, Publications, and Ethics; and the University of Michigan Institutional Review Board. All respondents voluntarily participated after the intent and the design of the study had been explained to them and signing informed consent forms.\",\n \"The study used STATA v10.1 for statistical analyses [26]. The main outcome of interest was the willingness to work in a deprived area after graduation. Students were asked to rate how likely they were to work in a deprived area on a scale from 1-4, where 1 represented \\\"I will definitely not work in a deprived area;\\\" 2 \\\"I am unlikely to work in a deprived area;\\\" 3 \\\"I am likely to work in a deprived area;\\\" and 4 \\\"I will definitely work in a deprived area.\\\" We collapsed this response set to a dichotomous willing (groups 3 or 4) versus unwilling (groups 1 or 2) to practice in a deprived area. We defined deprived area as \\\"a rural area that is distant from the big cities with few social amenities such as schools, roads, pipe-borne water, etc.\\\"--as per Ministry of Health definition.\\nPredictors of interest included motivation (intrinsic and extrinsic), demographic characteristics, and rural exposure. Students were asked to identify the top three factors that motivated them to study medicine from a list of twelve factors identified as important by the focus group discussions. Five intrinsic motivations included: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one. Seven extrinsic motivation factors included: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities. Respondents were coded as having \\\"strong\\\" intrinsic or extrinsic motivation if two or more of their motivational factors were intrinsic or extrinsic. Thus, \\\"strong\\\" intrinsic and extrinsic motivation groups were mutually exclusive.\\nDemographic factors included: sex, age, ethnicity (Akan vs. non-Akan), partnership status (in a relationship vs. not in a relationship), and parental parental professional and educational status (PPES). The Akan peoples (including the Asante, Fante, Kwahu, Akuapem, Bono and others) are the largest ethnic group, representing approximately half of Ghanaians; we grouped all other smaller ethnic groups (Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa) together as \\\"non-Akans\\\" for this analysis. High parental professional and educational status (PPES) was defined as having a mother and/or father who is a University-trained professional (e.g. doctor, lawyer, engineer, accountant, technical, etc) and low PPES was defined as neither mother nor father is a University-trained professional. Rural (an area with a population less than 5000) exposure factors included: birth location (urban vs. rural), location of pre-medical studies (urban/vs. rural), having ever lived in rural area (from the age of 5 onwards), exposure to rural service in medical training (for a minimum of 6 months).\\nBivariate associations and 95% confidence intervals were estimated using logistic regression. In model 1, the influence of strong intrinsic/extrinsic motivation on willingness to accept postings to rural area were assessed. Socio-demographic factors were added to the regression in Model 2, and rural exposure factors were further added in Model 3.\",\n \" Socio-demographic characteristics Of the 310 eligible medical students, 307 participated in the survey (99%). Of these, five survey files were corrupted by viruses or lost due to computer malfunction; thus the analysis was conducted with 302 total records. The socio-demographic characteristics of respondents are presented in Table 1. Of the 302 respondents recruited for the study, the majority were male (183 or 60.6%), with a mean age of 22.9 (STD 1.4). Most respondents were born in or around urban areas (264 or 87.4%) and had never lived in rural underserved area (75.8%). In terms of socioeconomic status, 173 (57.3%) students came from high PPES families. About half of the respondents (142 or 47%) were exposed to rural service.\\nDemographic characteristics and rural exposure of respondents (N = 302)\\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\\n2. High Family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low Family PPES: Neither mother nor father is a University-trained Professional\\n3. Urban area defined as a place with more than 5,000 residents; rural area defined as a place with less than 5,000 residents\\n4. From age five on\\n5. Participated in outreach or service in a deprived area during medical studies\\nOf the 310 eligible medical students, 307 participated in the survey (99%). Of these, five survey files were corrupted by viruses or lost due to computer malfunction; thus the analysis was conducted with 302 total records. The socio-demographic characteristics of respondents are presented in Table 1. Of the 302 respondents recruited for the study, the majority were male (183 or 60.6%), with a mean age of 22.9 (STD 1.4). Most respondents were born in or around urban areas (264 or 87.4%) and had never lived in rural underserved area (75.8%). In terms of socioeconomic status, 173 (57.3%) students came from high PPES families. About half of the respondents (142 or 47%) were exposed to rural service.\\nDemographic characteristics and rural exposure of respondents (N = 302)\\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\\n2. High Family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low Family PPES: Neither mother nor father is a University-trained Professional\\n3. Urban area defined as a place with more than 5,000 residents; rural area defined as a place with less than 5,000 residents\\n4. From age five on\\n5. Participated in outreach or service in a deprived area during medical studies\\n Intrinsic and extrinsic motivation and likelihood of working in underserved area The intensities of current motivational factors are presented in Table 2. Overall, 158 (55.4%) of students stated that they were likely to or definitely would work in an underserved area. More than 6 in 10 respondents (181 or 63.5%) had strong intrinsic motivation. A higher proportion of respondents who had strong intrinsic motivation indicated willingness to work in a rural area, compared to those with weak intrinsic motivation (χ2 = 6.96, p = 0.008). These results were reversed for those with strong extrinsic motivation (χ2 = 6.12, p = 0.013).\\nIntrinsic versus extrinsic motivations to study medicine predict reported likelihood to work in an under-served area, 285 Ghanaian Medical Students\\n1. Excludes 17 missing values for willingness to work rural and/or current motivation.\\n2. Intrinsic motivation includes: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one; Pearson χ2(1) = 6.9592, p = 0.008.\\n3. Extrinsic motivation includes: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities; Pearson χ2(1) = 6.1208, p = 0.013.\\nThe intensities of current motivational factors are presented in Table 2. Overall, 158 (55.4%) of students stated that they were likely to or definitely would work in an underserved area. More than 6 in 10 respondents (181 or 63.5%) had strong intrinsic motivation. A higher proportion of respondents who had strong intrinsic motivation indicated willingness to work in a rural area, compared to those with weak intrinsic motivation (χ2 = 6.96, p = 0.008). These results were reversed for those with strong extrinsic motivation (χ2 = 6.12, p = 0.013).\\nIntrinsic versus extrinsic motivations to study medicine predict reported likelihood to work in an under-served area, 285 Ghanaian Medical Students\\n1. Excludes 17 missing values for willingness to work rural and/or current motivation.\\n2. Intrinsic motivation includes: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one; Pearson χ2(1) = 6.9592, p = 0.008.\\n3. Extrinsic motivation includes: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities; Pearson χ2(1) = 6.1208, p = 0.013.\\n Regression analysis of motivations and the willingness to accept postings to rural underserved area after graduation We present the multivariate logistic regression results for the strength of intrinsic motivation and willingness to work in a rural underserved area after graduation in Table 3. In the unadjusted model (Model 1) there was a significant association between strong intrinsic motivation and willingness to work in rural underserved area (1.92[95% CI 1.18-3.13]). In Model 2, with demographic factors, female gender and high PPES were associated with reduced willingness to practice in a deprived area (0.50[95% CI 0.29-0.88]) and (0.42[95% CI 0.24, 0.71]), respectively, while age was associated with greater willingness to practice in a rural area (1.23[95% CI 1.00-1.52]). Rural exposure factors were not significant when added to the model with intrinsic motivation and demographic factors (Model 3). In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\\nMultivariate logistic regression results for strength of intrinsic motivation and willingness to work in a rural underserved area after graduation, Ghanaian medical students\\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\\n3. Urban area defined as a place with more than 5,000 residents\\n4. From age five on\\n5. Participated in outreach or service in a deprived area during medical studies\\nIn Table 4 the influence of strong extrinsic motivation on willingness of students to work in rural underserved area is presented. In Model 1, having a strong extrinsic motivation reduced the odds of being willing to accept a job in an underserved area to (0.54[95% CI 0.33-0.88]). In the model adjusting for demographics, Model 2, female gender and high PPES were associated with reduced willingness to practice in underserved areas (0.50[95% CI 0.28-0.87] and (0.42[95% CI 0.24-0.71]) respectively, while age was associated with greater willingness to practice in a rural area (1.24[95% CI 1.00-1.53]). Rural exposure factors in model 3 did not influence the outcome of willingness to work in rural underserved area. In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\\nMultivariate logistic regression results for strength of extrinsic motivation and willingness to work in a rural underserved area after graduation\\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\\n3. Urban area defined as a place with more than 5,000 residents\\n4. From age five on\\n5. Participated in outreach or service in a deprived area during medical studies\\nWe present the multivariate logistic regression results for the strength of intrinsic motivation and willingness to work in a rural underserved area after graduation in Table 3. In the unadjusted model (Model 1) there was a significant association between strong intrinsic motivation and willingness to work in rural underserved area (1.92[95% CI 1.18-3.13]). In Model 2, with demographic factors, female gender and high PPES were associated with reduced willingness to practice in a deprived area (0.50[95% CI 0.29-0.88]) and (0.42[95% CI 0.24, 0.71]), respectively, while age was associated with greater willingness to practice in a rural area (1.23[95% CI 1.00-1.52]). Rural exposure factors were not significant when added to the model with intrinsic motivation and demographic factors (Model 3). In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\\nMultivariate logistic regression results for strength of intrinsic motivation and willingness to work in a rural underserved area after graduation, Ghanaian medical students\\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\\n3. Urban area defined as a place with more than 5,000 residents\\n4. From age five on\\n5. Participated in outreach or service in a deprived area during medical studies\\nIn Table 4 the influence of strong extrinsic motivation on willingness of students to work in rural underserved area is presented. In Model 1, having a strong extrinsic motivation reduced the odds of being willing to accept a job in an underserved area to (0.54[95% CI 0.33-0.88]). In the model adjusting for demographics, Model 2, female gender and high PPES were associated with reduced willingness to practice in underserved areas (0.50[95% CI 0.28-0.87] and (0.42[95% CI 0.24-0.71]) respectively, while age was associated with greater willingness to practice in a rural area (1.24[95% CI 1.00-1.53]). Rural exposure factors in model 3 did not influence the outcome of willingness to work in rural underserved area. In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\\nMultivariate logistic regression results for strength of extrinsic motivation and willingness to work in a rural underserved area after graduation\\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\\n3. Urban area defined as a place with more than 5,000 residents\\n4. From age five on\\n5. Participated in outreach or service in a deprived area during medical studies\",\n \"Of the 310 eligible medical students, 307 participated in the survey (99%). Of these, five survey files were corrupted by viruses or lost due to computer malfunction; thus the analysis was conducted with 302 total records. The socio-demographic characteristics of respondents are presented in Table 1. Of the 302 respondents recruited for the study, the majority were male (183 or 60.6%), with a mean age of 22.9 (STD 1.4). Most respondents were born in or around urban areas (264 or 87.4%) and had never lived in rural underserved area (75.8%). In terms of socioeconomic status, 173 (57.3%) students came from high PPES families. About half of the respondents (142 or 47%) were exposed to rural service.\\nDemographic characteristics and rural exposure of respondents (N = 302)\\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\\n2. High Family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low Family PPES: Neither mother nor father is a University-trained Professional\\n3. Urban area defined as a place with more than 5,000 residents; rural area defined as a place with less than 5,000 residents\\n4. From age five on\\n5. Participated in outreach or service in a deprived area during medical studies\",\n \"The intensities of current motivational factors are presented in Table 2. Overall, 158 (55.4%) of students stated that they were likely to or definitely would work in an underserved area. More than 6 in 10 respondents (181 or 63.5%) had strong intrinsic motivation. A higher proportion of respondents who had strong intrinsic motivation indicated willingness to work in a rural area, compared to those with weak intrinsic motivation (χ2 = 6.96, p = 0.008). These results were reversed for those with strong extrinsic motivation (χ2 = 6.12, p = 0.013).\\nIntrinsic versus extrinsic motivations to study medicine predict reported likelihood to work in an under-served area, 285 Ghanaian Medical Students\\n1. Excludes 17 missing values for willingness to work rural and/or current motivation.\\n2. Intrinsic motivation includes: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one; Pearson χ2(1) = 6.9592, p = 0.008.\\n3. Extrinsic motivation includes: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities; Pearson χ2(1) = 6.1208, p = 0.013.\",\n \"We present the multivariate logistic regression results for the strength of intrinsic motivation and willingness to work in a rural underserved area after graduation in Table 3. In the unadjusted model (Model 1) there was a significant association between strong intrinsic motivation and willingness to work in rural underserved area (1.92[95% CI 1.18-3.13]). In Model 2, with demographic factors, female gender and high PPES were associated with reduced willingness to practice in a deprived area (0.50[95% CI 0.29-0.88]) and (0.42[95% CI 0.24, 0.71]), respectively, while age was associated with greater willingness to practice in a rural area (1.23[95% CI 1.00-1.52]). Rural exposure factors were not significant when added to the model with intrinsic motivation and demographic factors (Model 3). In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\\nMultivariate logistic regression results for strength of intrinsic motivation and willingness to work in a rural underserved area after graduation, Ghanaian medical students\\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\\n3. Urban area defined as a place with more than 5,000 residents\\n4. From age five on\\n5. Participated in outreach or service in a deprived area during medical studies\\nIn Table 4 the influence of strong extrinsic motivation on willingness of students to work in rural underserved area is presented. In Model 1, having a strong extrinsic motivation reduced the odds of being willing to accept a job in an underserved area to (0.54[95% CI 0.33-0.88]). In the model adjusting for demographics, Model 2, female gender and high PPES were associated with reduced willingness to practice in underserved areas (0.50[95% CI 0.28-0.87] and (0.42[95% CI 0.24-0.71]) respectively, while age was associated with greater willingness to practice in a rural area (1.24[95% CI 1.00-1.53]). Rural exposure factors in model 3 did not influence the outcome of willingness to work in rural underserved area. In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\\nMultivariate logistic regression results for strength of extrinsic motivation and willingness to work in a rural underserved area after graduation\\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\\n3. Urban area defined as a place with more than 5,000 residents\\n4. From age five on\\n5. Participated in outreach or service in a deprived area during medical studies\",\n \"We found that twice as many students reported high intrinsic motivation compared to high extrinsic motivation to study and practice medicine. This may reflect the underlying altruistic motivation for many students entering a profession focused on serving others [12,13]. There may also be an element of social desirability bias in the students' responses as intrinsic motivation may be thought to be more socially acceptable than extrinsic motivation. Nonetheless, we found that high extrinsic motivation was associated with low self-reported likelihood of rural practice and that the converse was true for high intrinsic motivation [2,3]. Interestingly, this association lost statistical significance at the 95% confidence level in models with demographic and rural exposure confounders, whereas socioeconomic status (PPES) retained a highly influential role, as discussed below.\\nIn this study, rural origin did not influence students' willingness to practice in rural areas after controlling for intrinsic/extrinsic motivation and demographic characteristics. This is in contrast with studies which have found rural origin to be an important motivator for rural practice [8,17,23]. Our findings highlight the heterogeneity of trends in motivation dynamics for rural practice and the importance of locally-relevant data for decision making. High PPES, measured using parental education and profession, was consistently associated with lack of willingness to work in rural areas. This is concerning as nearly 6 in 10 medical students in this cohort were from high PPES backgrounds--which is typical for Ghanaian medical schools [7]. These findings suggest that admission policies that favour well-to do applicants may be reducing the pool of students willing to consider rural practice. Female gender was also strongly associated with reduced interest in rural practice for women even after controlling for extrinsic/intrinsic motivation and rural exposure variables. This is consistent with similar studies among health staff which revealed that women are less likely to accept positions in remote areas due to varying family reasons; they would like to live where their husbands jobs are, have difficulties convincing their husbands to follow them to rural areas and want their children to have better education in the urban areas [10,11,22]. The studies further explained that female doctors rarely live in the same village as their assigned post and have higher overall absentee rates in rural practice [19,20]. With increasing representation of female healthcare professionals in many places in sub-Saharan Africa, [10,11], it is likely that the supply of health staff to rural underserved areas will remain a major setback if professional motivations are designed to attract more female students to rural practice. Although our study showed a lower proportion of female medical students in Ghana compared to other areas, they are likely to become a more important cadre in the coming years [11,19]. More research is urgently needed to determine how female healthcare professionals' motivations towards rural practice can be better engaged by policy-makers.\\nThe limitations of this study include the possibility of social desirability bias in responses on motivation and likelihood of rural practice, as noted above. Despite the fact that study participants were assured of anonymity, confidentiality, in responding to the questions, some social desirability bias is likely. For this reason, we selected a measure of high intrinsic and extrinsic motivation for use in the regression models. Research comparing students stated intentions with their actual career choices during internship is urgently needed as few studies on matched follow-ups are available. In addition, most the students participating in the study were young and had not yet tasted the rigors of working in a rural area, which may affect their job preferences. Thus the findings of this study may not be applicable to practicing physicians. Finally, these findings are only generalizable to students in the current medical education system. The findings may be different if selection criteria for medical school admission change.\\nThe major strengths of this study are its high response rate of 99% and that its ability to capture an entire population of young medical students who are one of the targets for addressing the rural-urban health staff recruitment imbalance. Surveying practicing physicians would have missed out those who had migrated.\\nThis study has several implications. First, the majority of students profess high intrinsic motivation for rural service. More research is needed to determine the potency of this motivation source in real-life decision making and how to best engage it via HRH policy. It is possible that emphasizing the community service aspect of medical practice and elevating the status of rural primary care in under-graduate and post-graduate training may help narrow the gap between motivation and eventual career choice in favour of rural areas. In addition, well-supervised and supported rural placements in which students experience the rewards of rural practice may help to persuade students who are largely unfamiliar with rural life. However, the success of these rural rotations is likely to depend heavily on having adequate local infrastructure and mentorship [13,23].\\nSecond, admission criteria may need to be reconsidered in light of the strong relationship between high PPES and lack of interest in rural practice. For example, medical school admission slots might be reserved for qualified students from poorer families. These students may not need to come from rural areas as we found that none of the rural exposure factors were significant after controlling for motivation and demographics.\\nThird, our results suggest that programmes to promote and support rural practice after graduation may have some success. Our focus groups and discrete choice experiment suggested that students may be willing to commit to short-term placements of 2 years or less in rural areas [9]. The Ministry of Health may want to consider the possibility of short contracts that rotate physicians in and out of difficult to staff rural areas.\",\n \"The majority of students still claim high intrinsic motivation and therefore it is important to appeal and build on this in medical school curricula and in designing rural postings. However, extrinsic motivation and, perhaps most importantly, PPES, will likely continue to be an important factor in deciding on job postings. Future research should explore how motivations could be directly supported, how motivations are formed, the influence of contextual factors on motivation among medical students, and motivation crowding among practicing health professionals in rural underserved areas. Qualitative work may be especially informative in this effort. Our research also suggests that increasing efforts to recruit medical students from low PPES backgrounds may be the most effective current pathway to increasing the yield of physicians willing to practice in underserved areas.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"keywords":{"kind":"list like","value":["Health Manpower","Motivation","Rural Health Services","Ghana"],"string":"[\n \"Health Manpower\",\n \"Motivation\",\n \"Rural Health Services\",\n \"Ghana\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Background:\nThe World Health Organization estimates that more than 4 million health workers are needed to fill the health workforce gap globally. This includes 2.4 million physicians, nurses and midwives. Fifty-seven countries are defined as having a critical shortage of health staff; of these, 36 are in sub-Saharan Africa. Of the total world's health work force of 59.2 million, Africa has only 3% of the world's health workers in spite of having 25% of the global burden of disease [1,2]. The shortage of health staff cripples the health delivery system. It is also a threat to provision of essential, life-saving interventions such as childhood immunizations, provision of safe water, safe pregnancy and childbirth services for mothers as well as access to treatment for AIDS, tuberculosis and malaria. Health workers are critical to the global preparedness for and response to threats posed by emerging and epidemic-prone diseases. Different interventions have been tried to address these shortages [3-5].\nRetaining health staff in rural areas has proven extremely difficult as young professionals increasingly prefer urban postings and health systems do not reward (and through neglect often penalize) rural service [6, 7. 8]. For example, Wilson and colleagues, 2009, Dovlo, 2003 and Kruk and colleagues, 2010, found that rural exposure, poor working conditions, low job satisfaction, political and ethnic problems, and sometimes, civil strife and poor security in most underserved areas, predispose new graduates to select cities [9-11]. Qualitative research has also shown the importance of health care providers' personal characteristics and value systems, such as religious beliefs and sociopolitical convictions, to their motivation towards rural practice. Emigration of skilled professionals to high-income countries is another barrier to adequate staffing of health facilities. A study in Ghana in 2006 on trainee physicians and nurses revealed that the majority had considered emigrating. More physicians (68%) than nurses (57%) considered emigration. These findings imply that achieving improvements in the health status of people living in low-income countries, and particularly, in rural areas, will be extremely difficult and the attainment of the United Nations Millennium Development Goals 4, 5, and 6 by 2015 [12,13], in Ghana is unlikely.\nWhile previous research has looked at incentives and working conditions to promote uptake of rural posts, few studies have focused on motivation crowding and its effect on willingness to accept postings to rural area. Motivation crowding [14] is the conflict between external factors (extrinsic), such as monetary incentives or punishments, and the underlying desire or willingness to work (intrinsic) in areas needed most. Students may have a mix of extrinsic and intrinsic motivations for studying medicine. Extrinsic factors may either undermine or strengthen intrinsic motivation, led by the belief that medicine has the imperative to help others, as enshrined in the Hippocratic Oath [15-17]. Current monetary incentives, which favour urban practice, may crowd-out the intrinsic desire to give back to society by working in underserved areas [18,19]. This could have debilitating effects on health worker retention in rural areas [20-22]. To tackle the maldistribution of human resources for health (HRH), understanding the factors that crowd-out the intrinsic motivation of medical students and their willingness to accept postings to rural underserved area is integral. This paper analyzes the effect of extrinsic versus intrinsic motivational factors on stated willingness to accept postings to rural underserved areas in Ghana.\n\nMethods:\n Study site Ghana is located in the west coast of Africa with an estimated population of 23 million [23]. It is mainly an agrarian economy with estimated per capita income of USD 1500. More than two-thirds of the economy is rural, with the cities of Kumasi and Accra having the highest population densities due to brisk economic activities, and relatively strong economic and cultural infrastructure [24]. These attributes make the two main cities destinations for rural-urban migration and a highly heterogeneous socio-cultural context. There are four medical schools in Ghana: the University of Ghana (UG), Kwame Nkrumah University of Science and Technology (KNUST), University for Development Studies (UDS), and University of Cape Coast (UCC).\nIn Ghana, medical education consists of three years of basic science/para-clinical studies, three years of clinical training at a teaching hospital, and a two-year rotating housemanship. The study was conducted with two public universities in Ghana: University of Ghana (UG) in Accra and Kwame Nkrumah University of Science and Technology (KNUST) in Kumasi. These universities were selected because all the fourth year medical students in the public universities had their clinical training at either UG or KNUST at the time of the study. All 310 fourth year medical students in the country were invited to participate in the study; no sampling was conducted. Fourth-year medical students were selected because they had completed the BSc. Human Biology and had also been exposed to field work, but had not yet made their final decisions about rural or urban practice.\nGhana is located in the west coast of Africa with an estimated population of 23 million [23]. It is mainly an agrarian economy with estimated per capita income of USD 1500. More than two-thirds of the economy is rural, with the cities of Kumasi and Accra having the highest population densities due to brisk economic activities, and relatively strong economic and cultural infrastructure [24]. These attributes make the two main cities destinations for rural-urban migration and a highly heterogeneous socio-cultural context. There are four medical schools in Ghana: the University of Ghana (UG), Kwame Nkrumah University of Science and Technology (KNUST), University for Development Studies (UDS), and University of Cape Coast (UCC).\nIn Ghana, medical education consists of three years of basic science/para-clinical studies, three years of clinical training at a teaching hospital, and a two-year rotating housemanship. The study was conducted with two public universities in Ghana: University of Ghana (UG) in Accra and Kwame Nkrumah University of Science and Technology (KNUST) in Kumasi. These universities were selected because all the fourth year medical students in the public universities had their clinical training at either UG or KNUST at the time of the study. All 310 fourth year medical students in the country were invited to participate in the study; no sampling was conducted. Fourth-year medical students were selected because they had completed the BSc. Human Biology and had also been exposed to field work, but had not yet made their final decisions about rural or urban practice.\n Data collection Data collection was preceded by discussions with the heads of medical training institutions, who informed the content of the questionnaire and provided access to the student population. The data collection instruments were developed after seven focus group discussions of 6-8 participants in each group facilitated by trained social scientists were held with third and fifth year medical students at UG and KNUST. The themes for the focus group discussion were motivation, willingness to work in deprived areas, experience in the field, and the influence of background characteristics on willingness to work in deprived areas. The survey instrument, which included semi-structured questions as well as a discrete choice experiment, were then pretested and finalized for the study. The questionnaires were administered electronically with Sawtooth Software SSI Web CAPI [25], in computer laboratories in UG and KNUST.\nData collection was preceded by discussions with the heads of medical training institutions, who informed the content of the questionnaire and provided access to the student population. The data collection instruments were developed after seven focus group discussions of 6-8 participants in each group facilitated by trained social scientists were held with third and fifth year medical students at UG and KNUST. The themes for the focus group discussion were motivation, willingness to work in deprived areas, experience in the field, and the influence of background characteristics on willingness to work in deprived areas. The survey instrument, which included semi-structured questions as well as a discrete choice experiment, were then pretested and finalized for the study. The questionnaires were administered electronically with Sawtooth Software SSI Web CAPI [25], in computer laboratories in UG and KNUST.\n Ethical considerations The study received ethics approval from the Ghana Health Service Ethical Review Committee; the UG Medical School; the KNUST Committee on Human Research, Publications, and Ethics; and the University of Michigan Institutional Review Board. All respondents voluntarily participated after the intent and the design of the study had been explained to them and signing informed consent forms.\nThe study received ethics approval from the Ghana Health Service Ethical Review Committee; the UG Medical School; the KNUST Committee on Human Research, Publications, and Ethics; and the University of Michigan Institutional Review Board. All respondents voluntarily participated after the intent and the design of the study had been explained to them and signing informed consent forms.\n Statistical Analysis The study used STATA v10.1 for statistical analyses [26]. The main outcome of interest was the willingness to work in a deprived area after graduation. Students were asked to rate how likely they were to work in a deprived area on a scale from 1-4, where 1 represented \"I will definitely not work in a deprived area;\" 2 \"I am unlikely to work in a deprived area;\" 3 \"I am likely to work in a deprived area;\" and 4 \"I will definitely work in a deprived area.\" We collapsed this response set to a dichotomous willing (groups 3 or 4) versus unwilling (groups 1 or 2) to practice in a deprived area. We defined deprived area as \"a rural area that is distant from the big cities with few social amenities such as schools, roads, pipe-borne water, etc.\"--as per Ministry of Health definition.\nPredictors of interest included motivation (intrinsic and extrinsic), demographic characteristics, and rural exposure. Students were asked to identify the top three factors that motivated them to study medicine from a list of twelve factors identified as important by the focus group discussions. Five intrinsic motivations included: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one. Seven extrinsic motivation factors included: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities. Respondents were coded as having \"strong\" intrinsic or extrinsic motivation if two or more of their motivational factors were intrinsic or extrinsic. Thus, \"strong\" intrinsic and extrinsic motivation groups were mutually exclusive.\nDemographic factors included: sex, age, ethnicity (Akan vs. non-Akan), partnership status (in a relationship vs. not in a relationship), and parental parental professional and educational status (PPES). The Akan peoples (including the Asante, Fante, Kwahu, Akuapem, Bono and others) are the largest ethnic group, representing approximately half of Ghanaians; we grouped all other smaller ethnic groups (Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa) together as \"non-Akans\" for this analysis. High parental professional and educational status (PPES) was defined as having a mother and/or father who is a University-trained professional (e.g. doctor, lawyer, engineer, accountant, technical, etc) and low PPES was defined as neither mother nor father is a University-trained professional. Rural (an area with a population less than 5000) exposure factors included: birth location (urban vs. rural), location of pre-medical studies (urban/vs. rural), having ever lived in rural area (from the age of 5 onwards), exposure to rural service in medical training (for a minimum of 6 months).\nBivariate associations and 95% confidence intervals were estimated using logistic regression. In model 1, the influence of strong intrinsic/extrinsic motivation on willingness to accept postings to rural area were assessed. Socio-demographic factors were added to the regression in Model 2, and rural exposure factors were further added in Model 3.\nThe study used STATA v10.1 for statistical analyses [26]. The main outcome of interest was the willingness to work in a deprived area after graduation. Students were asked to rate how likely they were to work in a deprived area on a scale from 1-4, where 1 represented \"I will definitely not work in a deprived area;\" 2 \"I am unlikely to work in a deprived area;\" 3 \"I am likely to work in a deprived area;\" and 4 \"I will definitely work in a deprived area.\" We collapsed this response set to a dichotomous willing (groups 3 or 4) versus unwilling (groups 1 or 2) to practice in a deprived area. We defined deprived area as \"a rural area that is distant from the big cities with few social amenities such as schools, roads, pipe-borne water, etc.\"--as per Ministry of Health definition.\nPredictors of interest included motivation (intrinsic and extrinsic), demographic characteristics, and rural exposure. Students were asked to identify the top three factors that motivated them to study medicine from a list of twelve factors identified as important by the focus group discussions. Five intrinsic motivations included: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one. Seven extrinsic motivation factors included: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities. Respondents were coded as having \"strong\" intrinsic or extrinsic motivation if two or more of their motivational factors were intrinsic or extrinsic. Thus, \"strong\" intrinsic and extrinsic motivation groups were mutually exclusive.\nDemographic factors included: sex, age, ethnicity (Akan vs. non-Akan), partnership status (in a relationship vs. not in a relationship), and parental parental professional and educational status (PPES). The Akan peoples (including the Asante, Fante, Kwahu, Akuapem, Bono and others) are the largest ethnic group, representing approximately half of Ghanaians; we grouped all other smaller ethnic groups (Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa) together as \"non-Akans\" for this analysis. High parental professional and educational status (PPES) was defined as having a mother and/or father who is a University-trained professional (e.g. doctor, lawyer, engineer, accountant, technical, etc) and low PPES was defined as neither mother nor father is a University-trained professional. Rural (an area with a population less than 5000) exposure factors included: birth location (urban vs. rural), location of pre-medical studies (urban/vs. rural), having ever lived in rural area (from the age of 5 onwards), exposure to rural service in medical training (for a minimum of 6 months).\nBivariate associations and 95% confidence intervals were estimated using logistic regression. In model 1, the influence of strong intrinsic/extrinsic motivation on willingness to accept postings to rural area were assessed. Socio-demographic factors were added to the regression in Model 2, and rural exposure factors were further added in Model 3.\n\nStudy site:\nGhana is located in the west coast of Africa with an estimated population of 23 million [23]. It is mainly an agrarian economy with estimated per capita income of USD 1500. More than two-thirds of the economy is rural, with the cities of Kumasi and Accra having the highest population densities due to brisk economic activities, and relatively strong economic and cultural infrastructure [24]. These attributes make the two main cities destinations for rural-urban migration and a highly heterogeneous socio-cultural context. There are four medical schools in Ghana: the University of Ghana (UG), Kwame Nkrumah University of Science and Technology (KNUST), University for Development Studies (UDS), and University of Cape Coast (UCC).\nIn Ghana, medical education consists of three years of basic science/para-clinical studies, three years of clinical training at a teaching hospital, and a two-year rotating housemanship. The study was conducted with two public universities in Ghana: University of Ghana (UG) in Accra and Kwame Nkrumah University of Science and Technology (KNUST) in Kumasi. These universities were selected because all the fourth year medical students in the public universities had their clinical training at either UG or KNUST at the time of the study. All 310 fourth year medical students in the country were invited to participate in the study; no sampling was conducted. Fourth-year medical students were selected because they had completed the BSc. Human Biology and had also been exposed to field work, but had not yet made their final decisions about rural or urban practice.\n\nData collection:\nData collection was preceded by discussions with the heads of medical training institutions, who informed the content of the questionnaire and provided access to the student population. The data collection instruments were developed after seven focus group discussions of 6-8 participants in each group facilitated by trained social scientists were held with third and fifth year medical students at UG and KNUST. The themes for the focus group discussion were motivation, willingness to work in deprived areas, experience in the field, and the influence of background characteristics on willingness to work in deprived areas. The survey instrument, which included semi-structured questions as well as a discrete choice experiment, were then pretested and finalized for the study. The questionnaires were administered electronically with Sawtooth Software SSI Web CAPI [25], in computer laboratories in UG and KNUST.\n\nEthical considerations:\nThe study received ethics approval from the Ghana Health Service Ethical Review Committee; the UG Medical School; the KNUST Committee on Human Research, Publications, and Ethics; and the University of Michigan Institutional Review Board. All respondents voluntarily participated after the intent and the design of the study had been explained to them and signing informed consent forms.\n\nStatistical Analysis:\nThe study used STATA v10.1 for statistical analyses [26]. The main outcome of interest was the willingness to work in a deprived area after graduation. Students were asked to rate how likely they were to work in a deprived area on a scale from 1-4, where 1 represented \"I will definitely not work in a deprived area;\" 2 \"I am unlikely to work in a deprived area;\" 3 \"I am likely to work in a deprived area;\" and 4 \"I will definitely work in a deprived area.\" We collapsed this response set to a dichotomous willing (groups 3 or 4) versus unwilling (groups 1 or 2) to practice in a deprived area. We defined deprived area as \"a rural area that is distant from the big cities with few social amenities such as schools, roads, pipe-borne water, etc.\"--as per Ministry of Health definition.\nPredictors of interest included motivation (intrinsic and extrinsic), demographic characteristics, and rural exposure. Students were asked to identify the top three factors that motivated them to study medicine from a list of twelve factors identified as important by the focus group discussions. Five intrinsic motivations included: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one. Seven extrinsic motivation factors included: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities. Respondents were coded as having \"strong\" intrinsic or extrinsic motivation if two or more of their motivational factors were intrinsic or extrinsic. Thus, \"strong\" intrinsic and extrinsic motivation groups were mutually exclusive.\nDemographic factors included: sex, age, ethnicity (Akan vs. non-Akan), partnership status (in a relationship vs. not in a relationship), and parental parental professional and educational status (PPES). The Akan peoples (including the Asante, Fante, Kwahu, Akuapem, Bono and others) are the largest ethnic group, representing approximately half of Ghanaians; we grouped all other smaller ethnic groups (Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa) together as \"non-Akans\" for this analysis. High parental professional and educational status (PPES) was defined as having a mother and/or father who is a University-trained professional (e.g. doctor, lawyer, engineer, accountant, technical, etc) and low PPES was defined as neither mother nor father is a University-trained professional. Rural (an area with a population less than 5000) exposure factors included: birth location (urban vs. rural), location of pre-medical studies (urban/vs. rural), having ever lived in rural area (from the age of 5 onwards), exposure to rural service in medical training (for a minimum of 6 months).\nBivariate associations and 95% confidence intervals were estimated using logistic regression. In model 1, the influence of strong intrinsic/extrinsic motivation on willingness to accept postings to rural area were assessed. Socio-demographic factors were added to the regression in Model 2, and rural exposure factors were further added in Model 3.\n\nResults:\n Socio-demographic characteristics Of the 310 eligible medical students, 307 participated in the survey (99%). Of these, five survey files were corrupted by viruses or lost due to computer malfunction; thus the analysis was conducted with 302 total records. The socio-demographic characteristics of respondents are presented in Table 1. Of the 302 respondents recruited for the study, the majority were male (183 or 60.6%), with a mean age of 22.9 (STD 1.4). Most respondents were born in or around urban areas (264 or 87.4%) and had never lived in rural underserved area (75.8%). In terms of socioeconomic status, 173 (57.3%) students came from high PPES families. About half of the respondents (142 or 47%) were exposed to rural service.\nDemographic characteristics and rural exposure of respondents (N = 302)\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High Family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low Family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents; rural area defined as a place with less than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies\nOf the 310 eligible medical students, 307 participated in the survey (99%). Of these, five survey files were corrupted by viruses or lost due to computer malfunction; thus the analysis was conducted with 302 total records. The socio-demographic characteristics of respondents are presented in Table 1. Of the 302 respondents recruited for the study, the majority were male (183 or 60.6%), with a mean age of 22.9 (STD 1.4). Most respondents were born in or around urban areas (264 or 87.4%) and had never lived in rural underserved area (75.8%). In terms of socioeconomic status, 173 (57.3%) students came from high PPES families. About half of the respondents (142 or 47%) were exposed to rural service.\nDemographic characteristics and rural exposure of respondents (N = 302)\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High Family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low Family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents; rural area defined as a place with less than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies\n Intrinsic and extrinsic motivation and likelihood of working in underserved area The intensities of current motivational factors are presented in Table 2. Overall, 158 (55.4%) of students stated that they were likely to or definitely would work in an underserved area. More than 6 in 10 respondents (181 or 63.5%) had strong intrinsic motivation. A higher proportion of respondents who had strong intrinsic motivation indicated willingness to work in a rural area, compared to those with weak intrinsic motivation (χ2 = 6.96, p = 0.008). These results were reversed for those with strong extrinsic motivation (χ2 = 6.12, p = 0.013).\nIntrinsic versus extrinsic motivations to study medicine predict reported likelihood to work in an under-served area, 285 Ghanaian Medical Students\n1. Excludes 17 missing values for willingness to work rural and/or current motivation.\n2. Intrinsic motivation includes: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one; Pearson χ2(1) = 6.9592, p = 0.008.\n3. Extrinsic motivation includes: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities; Pearson χ2(1) = 6.1208, p = 0.013.\nThe intensities of current motivational factors are presented in Table 2. Overall, 158 (55.4%) of students stated that they were likely to or definitely would work in an underserved area. More than 6 in 10 respondents (181 or 63.5%) had strong intrinsic motivation. A higher proportion of respondents who had strong intrinsic motivation indicated willingness to work in a rural area, compared to those with weak intrinsic motivation (χ2 = 6.96, p = 0.008). These results were reversed for those with strong extrinsic motivation (χ2 = 6.12, p = 0.013).\nIntrinsic versus extrinsic motivations to study medicine predict reported likelihood to work in an under-served area, 285 Ghanaian Medical Students\n1. Excludes 17 missing values for willingness to work rural and/or current motivation.\n2. Intrinsic motivation includes: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one; Pearson χ2(1) = 6.9592, p = 0.008.\n3. Extrinsic motivation includes: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities; Pearson χ2(1) = 6.1208, p = 0.013.\n Regression analysis of motivations and the willingness to accept postings to rural underserved area after graduation We present the multivariate logistic regression results for the strength of intrinsic motivation and willingness to work in a rural underserved area after graduation in Table 3. In the unadjusted model (Model 1) there was a significant association between strong intrinsic motivation and willingness to work in rural underserved area (1.92[95% CI 1.18-3.13]). In Model 2, with demographic factors, female gender and high PPES were associated with reduced willingness to practice in a deprived area (0.50[95% CI 0.29-0.88]) and (0.42[95% CI 0.24, 0.71]), respectively, while age was associated with greater willingness to practice in a rural area (1.23[95% CI 1.00-1.52]). Rural exposure factors were not significant when added to the model with intrinsic motivation and demographic factors (Model 3). In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\nMultivariate logistic regression results for strength of intrinsic motivation and willingness to work in a rural underserved area after graduation, Ghanaian medical students\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies\nIn Table 4 the influence of strong extrinsic motivation on willingness of students to work in rural underserved area is presented. In Model 1, having a strong extrinsic motivation reduced the odds of being willing to accept a job in an underserved area to (0.54[95% CI 0.33-0.88]). In the model adjusting for demographics, Model 2, female gender and high PPES were associated with reduced willingness to practice in underserved areas (0.50[95% CI 0.28-0.87] and (0.42[95% CI 0.24-0.71]) respectively, while age was associated with greater willingness to practice in a rural area (1.24[95% CI 1.00-1.53]). Rural exposure factors in model 3 did not influence the outcome of willingness to work in rural underserved area. In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\nMultivariate logistic regression results for strength of extrinsic motivation and willingness to work in a rural underserved area after graduation\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies\nWe present the multivariate logistic regression results for the strength of intrinsic motivation and willingness to work in a rural underserved area after graduation in Table 3. In the unadjusted model (Model 1) there was a significant association between strong intrinsic motivation and willingness to work in rural underserved area (1.92[95% CI 1.18-3.13]). In Model 2, with demographic factors, female gender and high PPES were associated with reduced willingness to practice in a deprived area (0.50[95% CI 0.29-0.88]) and (0.42[95% CI 0.24, 0.71]), respectively, while age was associated with greater willingness to practice in a rural area (1.23[95% CI 1.00-1.52]). Rural exposure factors were not significant when added to the model with intrinsic motivation and demographic factors (Model 3). In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\nMultivariate logistic regression results for strength of intrinsic motivation and willingness to work in a rural underserved area after graduation, Ghanaian medical students\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies\nIn Table 4 the influence of strong extrinsic motivation on willingness of students to work in rural underserved area is presented. In Model 1, having a strong extrinsic motivation reduced the odds of being willing to accept a job in an underserved area to (0.54[95% CI 0.33-0.88]). In the model adjusting for demographics, Model 2, female gender and high PPES were associated with reduced willingness to practice in underserved areas (0.50[95% CI 0.28-0.87] and (0.42[95% CI 0.24-0.71]) respectively, while age was associated with greater willingness to practice in a rural area (1.24[95% CI 1.00-1.53]). Rural exposure factors in model 3 did not influence the outcome of willingness to work in rural underserved area. In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\nMultivariate logistic regression results for strength of extrinsic motivation and willingness to work in a rural underserved area after graduation\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies\n\nSocio-demographic characteristics:\nOf the 310 eligible medical students, 307 participated in the survey (99%). Of these, five survey files were corrupted by viruses or lost due to computer malfunction; thus the analysis was conducted with 302 total records. The socio-demographic characteristics of respondents are presented in Table 1. Of the 302 respondents recruited for the study, the majority were male (183 or 60.6%), with a mean age of 22.9 (STD 1.4). Most respondents were born in or around urban areas (264 or 87.4%) and had never lived in rural underserved area (75.8%). In terms of socioeconomic status, 173 (57.3%) students came from high PPES families. About half of the respondents (142 or 47%) were exposed to rural service.\nDemographic characteristics and rural exposure of respondents (N = 302)\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High Family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low Family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents; rural area defined as a place with less than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies\n\nIntrinsic and extrinsic motivation and likelihood of working in underserved area:\nThe intensities of current motivational factors are presented in Table 2. Overall, 158 (55.4%) of students stated that they were likely to or definitely would work in an underserved area. More than 6 in 10 respondents (181 or 63.5%) had strong intrinsic motivation. A higher proportion of respondents who had strong intrinsic motivation indicated willingness to work in a rural area, compared to those with weak intrinsic motivation (χ2 = 6.96, p = 0.008). These results were reversed for those with strong extrinsic motivation (χ2 = 6.12, p = 0.013).\nIntrinsic versus extrinsic motivations to study medicine predict reported likelihood to work in an under-served area, 285 Ghanaian Medical Students\n1. Excludes 17 missing values for willingness to work rural and/or current motivation.\n2. Intrinsic motivation includes: desire to help others, desire to give back to their home community or country, interest in medicine as a subject matter, inspiration by a role model, and loss of a loved one; Pearson χ2(1) = 6.9592, p = 0.008.\n3. Extrinsic motivation includes: income of physicians, job security and lifestyle, social status/prestige, proposed by parents, opportunities to travel and work internationally, ability to use new cutting edge technologies, and research opportunities; Pearson χ2(1) = 6.1208, p = 0.013.\n\nRegression analysis of motivations and the willingness to accept postings to rural underserved area after graduation:\nWe present the multivariate logistic regression results for the strength of intrinsic motivation and willingness to work in a rural underserved area after graduation in Table 3. In the unadjusted model (Model 1) there was a significant association between strong intrinsic motivation and willingness to work in rural underserved area (1.92[95% CI 1.18-3.13]). In Model 2, with demographic factors, female gender and high PPES were associated with reduced willingness to practice in a deprived area (0.50[95% CI 0.29-0.88]) and (0.42[95% CI 0.24, 0.71]), respectively, while age was associated with greater willingness to practice in a rural area (1.23[95% CI 1.00-1.52]). Rural exposure factors were not significant when added to the model with intrinsic motivation and demographic factors (Model 3). In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\nMultivariate logistic regression results for strength of intrinsic motivation and willingness to work in a rural underserved area after graduation, Ghanaian medical students\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies\nIn Table 4 the influence of strong extrinsic motivation on willingness of students to work in rural underserved area is presented. In Model 1, having a strong extrinsic motivation reduced the odds of being willing to accept a job in an underserved area to (0.54[95% CI 0.33-0.88]). In the model adjusting for demographics, Model 2, female gender and high PPES were associated with reduced willingness to practice in underserved areas (0.50[95% CI 0.28-0.87] and (0.42[95% CI 0.24-0.71]) respectively, while age was associated with greater willingness to practice in a rural area (1.24[95% CI 1.00-1.53]). Rural exposure factors in model 3 did not influence the outcome of willingness to work in rural underserved area. In the adjusted models 2 and 3, motivation was no longer a significant predictor of willingness to practice in a deprived area.\nMultivariate logistic regression results for strength of extrinsic motivation and willingness to work in a rural underserved area after graduation\n1. Akan includes Asante, Fante, Kwahu, Akuapim, Bono, etc; Non-Akan includes Ga/Dangme, Ewe, Guan, Mole-Dagbani, Grussi, Gruma, and Hausa peoples\n2. High family PPES: Mother and/or father is a University-trained Professional (e.g. doctor, lawyer, engineer, accountant, technical, etc); Low family PPES: Neither mother nor father is a University-trained Professional\n3. Urban area defined as a place with more than 5,000 residents\n4. From age five on\n5. Participated in outreach or service in a deprived area during medical studies\n\nDiscussion:\nWe found that twice as many students reported high intrinsic motivation compared to high extrinsic motivation to study and practice medicine. This may reflect the underlying altruistic motivation for many students entering a profession focused on serving others [12,13]. There may also be an element of social desirability bias in the students' responses as intrinsic motivation may be thought to be more socially acceptable than extrinsic motivation. Nonetheless, we found that high extrinsic motivation was associated with low self-reported likelihood of rural practice and that the converse was true for high intrinsic motivation [2,3]. Interestingly, this association lost statistical significance at the 95% confidence level in models with demographic and rural exposure confounders, whereas socioeconomic status (PPES) retained a highly influential role, as discussed below.\nIn this study, rural origin did not influence students' willingness to practice in rural areas after controlling for intrinsic/extrinsic motivation and demographic characteristics. This is in contrast with studies which have found rural origin to be an important motivator for rural practice [8,17,23]. Our findings highlight the heterogeneity of trends in motivation dynamics for rural practice and the importance of locally-relevant data for decision making. High PPES, measured using parental education and profession, was consistently associated with lack of willingness to work in rural areas. This is concerning as nearly 6 in 10 medical students in this cohort were from high PPES backgrounds--which is typical for Ghanaian medical schools [7]. These findings suggest that admission policies that favour well-to do applicants may be reducing the pool of students willing to consider rural practice. Female gender was also strongly associated with reduced interest in rural practice for women even after controlling for extrinsic/intrinsic motivation and rural exposure variables. This is consistent with similar studies among health staff which revealed that women are less likely to accept positions in remote areas due to varying family reasons; they would like to live where their husbands jobs are, have difficulties convincing their husbands to follow them to rural areas and want their children to have better education in the urban areas [10,11,22]. The studies further explained that female doctors rarely live in the same village as their assigned post and have higher overall absentee rates in rural practice [19,20]. With increasing representation of female healthcare professionals in many places in sub-Saharan Africa, [10,11], it is likely that the supply of health staff to rural underserved areas will remain a major setback if professional motivations are designed to attract more female students to rural practice. Although our study showed a lower proportion of female medical students in Ghana compared to other areas, they are likely to become a more important cadre in the coming years [11,19]. More research is urgently needed to determine how female healthcare professionals' motivations towards rural practice can be better engaged by policy-makers.\nThe limitations of this study include the possibility of social desirability bias in responses on motivation and likelihood of rural practice, as noted above. Despite the fact that study participants were assured of anonymity, confidentiality, in responding to the questions, some social desirability bias is likely. For this reason, we selected a measure of high intrinsic and extrinsic motivation for use in the regression models. Research comparing students stated intentions with their actual career choices during internship is urgently needed as few studies on matched follow-ups are available. In addition, most the students participating in the study were young and had not yet tasted the rigors of working in a rural area, which may affect their job preferences. Thus the findings of this study may not be applicable to practicing physicians. Finally, these findings are only generalizable to students in the current medical education system. The findings may be different if selection criteria for medical school admission change.\nThe major strengths of this study are its high response rate of 99% and that its ability to capture an entire population of young medical students who are one of the targets for addressing the rural-urban health staff recruitment imbalance. Surveying practicing physicians would have missed out those who had migrated.\nThis study has several implications. First, the majority of students profess high intrinsic motivation for rural service. More research is needed to determine the potency of this motivation source in real-life decision making and how to best engage it via HRH policy. It is possible that emphasizing the community service aspect of medical practice and elevating the status of rural primary care in under-graduate and post-graduate training may help narrow the gap between motivation and eventual career choice in favour of rural areas. In addition, well-supervised and supported rural placements in which students experience the rewards of rural practice may help to persuade students who are largely unfamiliar with rural life. However, the success of these rural rotations is likely to depend heavily on having adequate local infrastructure and mentorship [13,23].\nSecond, admission criteria may need to be reconsidered in light of the strong relationship between high PPES and lack of interest in rural practice. For example, medical school admission slots might be reserved for qualified students from poorer families. These students may not need to come from rural areas as we found that none of the rural exposure factors were significant after controlling for motivation and demographics.\nThird, our results suggest that programmes to promote and support rural practice after graduation may have some success. Our focus groups and discrete choice experiment suggested that students may be willing to commit to short-term placements of 2 years or less in rural areas [9]. The Ministry of Health may want to consider the possibility of short contracts that rotate physicians in and out of difficult to staff rural areas.\n\nConclusions:\nThe majority of students still claim high intrinsic motivation and therefore it is important to appeal and build on this in medical school curricula and in designing rural postings. However, extrinsic motivation and, perhaps most importantly, PPES, will likely continue to be an important factor in deciding on job postings. Future research should explore how motivations could be directly supported, how motivations are formed, the influence of contextual factors on motivation among medical students, and motivation crowding among practicing health professionals in rural underserved areas. Qualitative work may be especially informative in this effort. Our research also suggests that increasing efforts to recruit medical students from low PPES backgrounds may be the most effective current pathway to increasing the yield of physicians willing to practice in underserved areas.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nRetaining health workers in rural areas is challenging for a number of reasons, ranging from personal preferences to difficult work conditions and low remuneration. This paper assesses the influence of intrinsic and extrinsic motivation on willingness to accept postings to deprived areas among medical students in Ghana.\n\nMethods:\nA computer-based survey involving 302 fourth year medical students was conducted from May-August 2009. Logistic regression was used to assess the association between students' willingness to accept rural postings and their professional motivations, rural exposure and family parental professional and educational status (PPES).\n\nResults:\nOver 85% of students were born in urban areas and 57% came from affluent backgrounds. Nearly two-thirds of students reported strong intrinsic motivation to study medicine. After controlling for demographic characteristics and rural exposure, motivational factors did not influence willingness to practice in rural areas. High family PPES was consistently associated with lower willingness to work in rural areas.\n\nConclusions:\nAlthough most Ghanaian medical students are motivated to study medicine by the desire to help others, this does not translate into willingness to work in rural areas. Efforts should be made to build on intrinsic motivation during medical training and in designing rural postings, as well as favour lower PPES students for admission."},"background_conclusion_text":{"kind":"string","value":"Background:\nThe World Health Organization estimates that more than 4 million health workers are needed to fill the health workforce gap globally. This includes 2.4 million physicians, nurses and midwives. Fifty-seven countries are defined as having a critical shortage of health staff; of these, 36 are in sub-Saharan Africa. Of the total world's health work force of 59.2 million, Africa has only 3% of the world's health workers in spite of having 25% of the global burden of disease [1,2]. The shortage of health staff cripples the health delivery system. It is also a threat to provision of essential, life-saving interventions such as childhood immunizations, provision of safe water, safe pregnancy and childbirth services for mothers as well as access to treatment for AIDS, tuberculosis and malaria. Health workers are critical to the global preparedness for and response to threats posed by emerging and epidemic-prone diseases. Different interventions have been tried to address these shortages [3-5].\nRetaining health staff in rural areas has proven extremely difficult as young professionals increasingly prefer urban postings and health systems do not reward (and through neglect often penalize) rural service [6, 7. 8]. For example, Wilson and colleagues, 2009, Dovlo, 2003 and Kruk and colleagues, 2010, found that rural exposure, poor working conditions, low job satisfaction, political and ethnic problems, and sometimes, civil strife and poor security in most underserved areas, predispose new graduates to select cities [9-11]. Qualitative research has also shown the importance of health care providers' personal characteristics and value systems, such as religious beliefs and sociopolitical convictions, to their motivation towards rural practice. Emigration of skilled professionals to high-income countries is another barrier to adequate staffing of health facilities. A study in Ghana in 2006 on trainee physicians and nurses revealed that the majority had considered emigrating. More physicians (68%) than nurses (57%) considered emigration. These findings imply that achieving improvements in the health status of people living in low-income countries, and particularly, in rural areas, will be extremely difficult and the attainment of the United Nations Millennium Development Goals 4, 5, and 6 by 2015 [12,13], in Ghana is unlikely.\nWhile previous research has looked at incentives and working conditions to promote uptake of rural posts, few studies have focused on motivation crowding and its effect on willingness to accept postings to rural area. Motivation crowding [14] is the conflict between external factors (extrinsic), such as monetary incentives or punishments, and the underlying desire or willingness to work (intrinsic) in areas needed most. Students may have a mix of extrinsic and intrinsic motivations for studying medicine. Extrinsic factors may either undermine or strengthen intrinsic motivation, led by the belief that medicine has the imperative to help others, as enshrined in the Hippocratic Oath [15-17]. Current monetary incentives, which favour urban practice, may crowd-out the intrinsic desire to give back to society by working in underserved areas [18,19]. This could have debilitating effects on health worker retention in rural areas [20-22]. To tackle the maldistribution of human resources for health (HRH), understanding the factors that crowd-out the intrinsic motivation of medical students and their willingness to accept postings to rural underserved area is integral. This paper analyzes the effect of extrinsic versus intrinsic motivational factors on stated willingness to accept postings to rural underserved areas in Ghana.\n\nConclusions:\nThe pre-publication history for this paper can be accessed here:\n\nhttp://www.biomedcentral.com/1472-6920/11/56/prepub\n"},"background_conclusion_abstract":{"kind":"string","value":"Background:\nRetaining health workers in rural areas is challenging for a number of reasons, ranging from personal preferences to difficult work conditions and low remuneration. This paper assesses the influence of intrinsic and extrinsic motivation on willingness to accept postings to deprived areas among medical students in Ghana.\n\nMethods:\nA computer-based survey involving 302 fourth year medical students was conducted from May-August 2009. Logistic regression was used to assess the association between students' willingness to accept rural postings and their professional motivations, rural exposure and family parental professional and educational status (PPES).\n\nResults:\nOver 85% of students were born in urban areas and 57% came from affluent backgrounds. Nearly two-thirds of students reported strong intrinsic motivation to study medicine. After controlling for demographic characteristics and rural exposure, motivational factors did not influence willingness to practice in rural areas. High family PPES was consistently associated with lower willingness to work in rural areas.\n\nConclusions:\nAlthough most Ghanaian medical students are motivated to study medicine by the desire to help others, this does not translate into willingness to work in rural areas. Efforts should be made to build on intrinsic motivation during medical training and in designing rural postings, as well as favour lower PPES students for admission."},"whole_article_text_length":{"kind":"number","value":9073,"string":"9,073"},"whole_article_abstract_length":{"kind":"number","value":243,"string":"243"},"other_sections_lengths":{"kind":"list like","value":[670,303,153,64,642,2418,297,257,635,1072,140],"string":"[\n 670,\n 303,\n 153,\n 64,\n 642,\n 2418,\n 297,\n 257,\n 635,\n 1072,\n 140\n]","isConversational":false},"num_sections":{"kind":"number","value":12,"string":"12"},"most_frequent_words":{"kind":"list like","value":["rural","area","motivation","work","willingness","intrinsic","medical","students","extrinsic","deprived"],"string":"[\n \"rural\",\n \"area\",\n \"motivation\",\n \"work\",\n \"willingness\",\n \"intrinsic\",\n \"medical\",\n \"students\",\n \"extrinsic\",\n \"deprived\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["adequate staffing health","shortage health","health professionals rural","supply health staff","fill health workforce"],"string":"[\n \"adequate staffing health\",\n \"shortage health\",\n \"health professionals rural\",\n \"supply health staff\",\n \"fill health workforce\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"null"},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Health Manpower | Motivation | Rural Health Services | Ghana [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Health 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Collection | Female | Ghana | Humans | Logistic Models | Male | Motivation | Professional Practice Location | Rural Health Services | Students, Medical | Young Adult [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"null"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Choice Behavior | Data Collection | Female | Ghana | Humans | Logistic Models | Male | Motivation | Professional Practice Location | Rural Health Services | Students, Medical | Young Adult [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Choice Behavior | Data Collection | Female | Ghana | Humans | Logistic Models | Male | Motivation | Professional Practice Location | Rural Health Services | Students, Medical | Young Adult [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Choice Behavior | Data Collection | Female | Ghana | Humans | Logistic Models | Male | Motivation | Professional Practice Location | Rural Health Services | Students, Medical | Young Adult [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] adequate staffing health | shortage health | health professionals rural | supply health staff | fill health workforce [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] adequate staffing health | shortage health | health professionals rural | supply health staff | fill health workforce [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"null"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] adequate staffing health | shortage health | health professionals rural | supply health staff | fill health workforce [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] adequate staffing health | shortage health | health professionals rural | supply health staff | fill health workforce [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] adequate staffing health | shortage health | health professionals rural | supply health staff | fill health workforce [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] rural | area | motivation | work | willingness | intrinsic | medical | students | extrinsic | deprived [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] rural | area | motivation | work | willingness | intrinsic | medical | students | extrinsic | deprived [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"null"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] rural | area | motivation | work | willingness | intrinsic | medical | students | extrinsic | deprived 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increasing | important | postings | underserved areas | students | medical | motivations | ppes | underserved [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] rural | area | motivation | intrinsic | work | willingness | students | medical | deprived | university [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] rural | area | motivation | intrinsic | work | willingness | students | medical | deprived | university [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Ghana [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 302 fourth year | May-August 2009 ||| PPES [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"null"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Ghanaian ||| PPES [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Ghana ||| 302 fourth year | May-August 2009 ||| PPES ||| Over 85% | 57% ||| Nearly two-thirds ||| ||| PPES ||| Ghanaian ||| PPES [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| Ghana ||| 302 fourth year | May-August 2009 ||| PPES ||| Over 85% | 57% ||| Nearly two-thirds ||| ||| PPES ||| Ghanaian ||| PPES [SUMMARY]"}}},{"rowIdx":80,"cells":{"title":{"kind":"string","value":"Evaluation of the influence of genetic variants in Cereblon gene on the response to the treatment of erythema nodosum leprosum with thalidomide."},"pmid":{"kind":"string","value":"36383784"},"background_abstract":{"kind":"string","value":"Erythema nodosum leprosum (ENL) is an acute and systemic inflammatory reaction of leprosy characterised by painful nodules and involvement of various organs. Thalidomide is an immunomodulatory and anti-inflammatory drug currently used to treat this condition. Cereblon (CRBN) protein is the primary target of thalidomide, and it has been pointed out as necessary for the efficacy of this drug in others therapeutics settings."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"A total of 103 ENL patients in treatment with thalidomide were included in this study. DNA samples were obtained from saliva and molecular analysis of CRBN gene were performed to investigate the variants rs1620675, rs1672770 and rs4183. Different genotypes of CRBN variants were evaluated in relation to their influence on the dose of thalidomide and on the occurrence of adverse effects."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"No association was found between CRBN variants and thalidomide dose variation. However, the genotypes of rs1672770 showed association with gastrointestinal effects (p = 0.040). Moreover, the haplotype DEL/C/T (rs4183/rs1672770/rs1620675) was also associated with gastrointestinal adverse effects (p = 0.015)."},"results_abstract_label":{"kind":"string","value":"FINDINGS"},"conclusions_abstract":{"kind":"string","value":"Our results show that CRBN variants affect the treatment of ENH with thalidomide, especially on the adverse effects related to the drug."},"conclusions_abstract_label":{"kind":"string","value":"MAIN CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Humans","Erythema Nodosum","Thalidomide","Leprosy, Lepromatous","Leprostatic Agents","Leprosy, Multibacillary"],"string":"[\n \"Humans\",\n \"Erythema Nodosum\",\n \"Thalidomide\",\n \"Leprosy, Lepromatous\",\n \"Leprostatic Agents\",\n \"Leprosy, Multibacillary\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"9668341"},"background_title":{"kind":"null"},"background_text":{"kind":"null"},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"In total, 103 ENL patients were included, being 82 (79.6%) male and 79 (76.7%) presenting LL (Table I). Forty-six (44.7%) patients were using MDT and two of these used an alternative MDT regimen. The mean time of treatment with thalidomide was 226 days.\n\nTABLE IClinical and demographic characteristics of ENL patients (n = 103)Characteristic n (%)Male 82 (79.6)MDT for leprosy46 (44.7)MDT time in months median (P25/P75)*4.3 (1.5/10.9)Other medications81 (78.6)Thalidomide dose median (P25/P75)100 (100/200)Days of consultation mean (min/max)108.19 (0/721)Patient origin\nNortheast Region89 (86.4)North Region14 (13.6)Leprosy classification\nBorderline-lepromatous 24 (23.3)Lepromatous-leprosy 79 (76.7)Adverse effects\nNeurological\na\n\n31 (30.1)Grastrointestinal\nb\n\n18 (17.5)Musculoskeletal\nc\n\n29 (28.2)Ocular\nd\n\n19 (18.4)Oedema16 (15.6)Dermatological\ne\n\n2 (1.9)Fever11 (10.7)\na: paresthesias, dizziness, tremor, neuritis and headache; b: diarrhoea, vomiting, gastric fullness and constipation; c: myalgia, arthralgia and weakness; d: decreased visual acuity and eye irritation; e: pruritus, dry skin and hair loss; *: time of multidrug therapy (MDT) when patients developed erythema nodosum leprosum (ENL) (n = 40).\n\n\na: paresthesias, dizziness, tremor, neuritis and headache; b: diarrhoea, vomiting, gastric fullness and constipation; c: myalgia, arthralgia and weakness; d: decreased visual acuity and eye irritation; e: pruritus, dry skin and hair loss; *: time of multidrug therapy (MDT) when patients developed erythema nodosum leprosum (ENL) (n = 40).\nThe genotypic distributions were in Hardy-Weinberg equilibrium and the allelic and genotypic frequencies of the polymorphisms are shown in Table II. A LD [rs4183/rs1672770 (r2 = 0.513), rs4183/rs1620675 (r2 = 0.943), rs1672770/rs1620675 (r2 = 0.567)] between the variants studied was identified (see Supplementary Table) and four haplotypes were identified in the sample (Table III). For all single nucleotide polymorphisms (SNPs), there was no association of the thalidomide dose with time of treatment (Table IV). In addition, no association was found between haplotypes and thalidomide dose.\n\nTABLE IIGenotypic and allelic frequencies of CRBN polymorphisms in ENL patients on treatment with thalidomidePolymorphismAlleles/genotypesFrequency n (%)rs1620675AA31 (30.1)AC53 (5.5)CC19 (18.4)A115 (55.8)C91 (44.2)\n\nrs1672770*AA31 (30.1)AG52 (50.5)GG17 (16.5)A114 (55.3)G86 (44.7)rs4183INS/INS19 (18.4)INS/DEL56 (54.4)DEL/DEL28 (27.2)INS94 (45.6)DEL115 (54.4)DEL: deletion; ENL: erythema nodosum leprosum; INS: insertion; MDT: multidrug therapy; *: n = 100.\n\nDEL: deletion; ENL: erythema nodosum leprosum; INS: insertion; MDT: multidrug therapy; *: n = 100.\n\nTABLE IIIHaplotype frequencies of CRBN\nHaplotypen(%)INS/C/T31.5INS/T/G9244.7DEL/C/T8340.3DEL/T/T2813.6Haplotypes in the following order: rs4183/rs1672770/rs1620675. DEL: deletion; CRBN: cereblon gene; INS: insertion.\n\nHaplotypes in the following order: rs4183/rs1672770/rs1620675. DEL: deletion; CRBN: cereblon gene; INS: insertion.\n\nTABLE IVAnalysis of interaction between CRBN genotype and time related to estimated thalidomide dose using the GEE model in ENL treatmentPolymorphism Interactionp-valuers1620675rs16206750.079Concomitant MDT for leprosy0.271Other medications0.205Region0.138Time0.001rs1672770rs1620675*Time0.392rs16727700.229Concomitant MDT for leprosy0.296Other medications0.071Region0.085Time0.001rs1672770*Time0.273rs4183rs41830.098Concomitant MDT for leprosy0.293Other medications0.208Region0.117Time0.001rs4183*Time0.669Dependent variable: thalidomide dose. GEE model: region, MDT, other medications, thalidomide dose, genotype, time, genotype*Time). CRBN: cereblon; ENL: erythema nodosum leprosum; GEE: generalised estimating equation model; MDT: multidrug therapy.\n\nDependent variable: thalidomide dose. GEE model: region, MDT, other medications, thalidomide dose, genotype, time, genotype*Time). CRBN: cereblon; ENL: erythema nodosum leprosum; GEE: generalised estimating equation model; MDT: multidrug therapy.\nIn regard to the relationship of CRBN variants and adverse effects of the use of thalidomide, it was found associations between the genotypes of rs1672770 (p = 0.040) and gastrointestinal effects, which include diarrhoea, vomiting, nausea, constipation and inappetence (Tables V and VI). Haplotype analysis was performed with all SNPs because only one combination of SNPs showed r2>0.8 (rs4183/rs1620675 with r2 = 0.943). The three polymorphisms were maintained in the analysis because when combining rs4183/rs1672770 or rs1672770/rs1620675, it showed r2<0.8. The analysis of the association between haplotypes and adverse effects also showed an association of gastrointestinal adverse effects (p = 0.015) with haplotype DEL/C/T (rs4183/rs1672770/rs1620675) (Tables VII and VIII). These effects were present in eight patients (9.6%) who carried this haplotype. \n\nTABLE VAnalysis of the association of CRBN variants on the occurrence of gastrointestinal adverse effects\na\n\nPolymorphismOR\nb\n\np-value*OR\nb\n\np-value**rs1672770\n\n\n\nAAREF-REF-AG0.325 (0.111-0.951)0.0400.040 (0.111-0.948)0.040GG0.131 (0.015-1.115)0.0630.127(0.015-1.100)0.061Dependent variable: gastrointestinal. CRBN: cereblon gene; OR: odds ratio; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence); b: logistic regression (OR and 95% confidence interval); *: model rs1672770; **: model sex, rs1672770.\n\nDependent variable: gastrointestinal. CRBN: cereblon gene; OR: odds ratio; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence); b: logistic regression (OR and 95% confidence interval); *: model rs1672770; **: model sex, rs1672770.\n\nTABLE VIFrequency of gastrointestinal adverse effects\na\n according to CRBN genotypesPolymorphismGenotypeAbsence (n) (% within effect)Presence (n) (% within effect)rs1672770AA23 (67.6)11 (32.4)AG45 (86.5)7 (13.5)GG16 (94.1)1 (5.9)\nCRBN: cereblon gene; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence).\n\n\nCRBN: cereblon gene; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence).\n\nTABLE VIIAnalysis of the association of CRBN haplotypes on the occurrence of gastrointestinal adverse effects\na\n\nHaplotypeOR\nb\n\np-value*OR\nb\n\np-value**INS/T/GREF-REF-DEL/C/T0.339 (0.142-0.812)0.0150.333 (0.138-0.805)0.015DEL/T/T1.061 (0.398-2.827)0.9061.022 (0.371-2.818)0.966Dependent variable: gastrointestinal. DEL: deletion; CRBN: cereblon gene; INS: insertion; OR: odds ratio; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence); b: logistic regression (OR and 95% confidence interval); *: model haplotypes; **: model sex, haplotypes.\n\nDependent variable: gastrointestinal. DEL: deletion; CRBN: cereblon gene; INS: insertion; OR: odds ratio; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence); b: logistic regression (OR and 95% confidence interval); *: model haplotypes; **: model sex, haplotypes.\n\nTABLE VIIIFrequency of gastrointestinal adverse effects\na\n according to CRBN haplotypesHaplotypesAbsence (n) (% within effect)Presence (n) (% within effect)INS/T/G70 (76.1)22 (23.9)DEL/C/T75 (90.4)8 (9.6)DEL/T/T21 (75)7 (25)DEL: deletion; CRBN: cereblon gene; INS: insertion; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence).\n\nDEL: deletion; CRBN: cereblon gene; INS: insertion; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence)."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":[],"string":"[]","isConversational":false},"other_sections_texts":{"kind":"list like","value":[],"string":"[]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[],"string":"[]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["SUBJECTS AND METHODS","RESULTS","DISCUSSION"],"string":"[\n \"SUBJECTS AND METHODS\",\n \"RESULTS\",\n \"DISCUSSION\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["\nSample - The sample consisted of 103 ENL patients who were selected from National Reference Center of Sanitary Dermatology Dona Libânia in Fortaleza (state of Ceará, Brazil), Humanized Reference Center of Sanitary Dermatology in Imperatriz and Aquiles Lisboa Hospital in São Luís (Maranhão State) in northeast Brazil, and from the Dermatology Ambulatory of the University of São Paulo in Monte Negro (state of Rondônia, Brazil) in north Brazil. \nThe patients used thalidomide at different doses and had a follow-up of up to six visits (average of 3.6 months). Data from up to six consultations annotated in the patient’s medical record were analysed with the collection of clinical and demographic information, including sex, age and region of origin, history of leprosy (moment of diagnosis and treatment used) and history of ENL (diagnosis, treatment, adverse effects, history of relapse and dose of medications used).\n\nGenetic analyses - DNA was extracted from saliva samples using the Oragene DNA Extraction Kit (DNA Genotek, Ottawa, Canada), according to the manufacturer’s instructions. A pair of primer was designed to amplify a fragment of 682 base pairs containing the region encompassing the three studied CRBN variants: forward 5’-TGTGGTCTTGGCAACCAGCAATTT-3’ and reverse 5’-ACTGCCGTTCATGCTTGTTTCCT-3’. This region was amplified by polymerase chain reaction (PCR). The fragment obtained was visualised on a 2% agarose gel, purified and sequenced using the same primers.\nSequences were visualised and analysed using CodonCodeAligner®, version 3.0.1 (CodonCode Corporation, Dedham, USA). The hg19 sequence deposited in GenBank was used as the reference sequence. When there was doubt about the variant, sequencing was repeated for confirmation.\n\nStatistical analyses - Chi-square test was used to evaluate Hardy-Weinberg equilibrium for all polymorphisms. Generalised estimating equations method (GEE) was used to evaluate the influence of CRBN variants on thalidomide dose. This method is a repeated measures analysis focused on average changes in response over time and on the impact of covariates on these changes. GEE can model the average response of variables as a linear function of covariates of interest through a transformation or link function and can be used in studies where the data is asymmetric or the data distribution is difficult to verify due to the small-size sample.\n24\n\n,\n\n25\n The covariates inserted in the model were place of origin of the patient, concomitant use of multidrug therapy (MDT) for leprosy and use of other medications and other treatments for ENL.\nThe evaluation of the association of CRBN variants and haplotypes in the occurrence of adverse effects due to thalidomide treatment was based on clinical data using logistic regression. Models with and without correction by gender were used. The INS/C/T (rs4183/rs1672770/rs1620675) haplotype was removed from the association analyses because it presented few events and disturbed the analyses. Peripheral polyneuropathy, although is an adverse effect common to the use of thalidomide, has not been evaluated because it is difficult to distinguish it from polyneuropathy caused by ENL and leprosy. All statistical analyses were performed with SPSS version 20 (www.spss.com).\nMLocus tool was used to calculate linkage disequilibrium (LD) for the variants,\n26\n and haplotypes were inferred using the Bayesian algorithm of the phase 2.1.1 program.\n27\n\n,\n\n28\n\n\n\nData availability - The data analysed during the current study are not publicly available to maintain patient confidentiality. Moreover, this type of request has not been previously approved by participants nor the human research committee. This data could, however, be available (anonymously) from the corresponding author on reasonable request.\n\nEthics - All participants were informed about the research objectives and signed an informed consent form. This study was approved by the Ethics Committee of the Hospital de Clínicas of Porto Alegre under number CAAE 21184413.0.0000.5327. All research was performed in accordance with Brazilian regulations and informed consent was obtained from all participants. This research has been performed in accordance with the Declaration of Helsinki.","In total, 103 ENL patients were included, being 82 (79.6%) male and 79 (76.7%) presenting LL (Table I). Forty-six (44.7%) patients were using MDT and two of these used an alternative MDT regimen. The mean time of treatment with thalidomide was 226 days.\n\nTABLE IClinical and demographic characteristics of ENL patients (n = 103)Characteristic n (%)Male 82 (79.6)MDT for leprosy46 (44.7)MDT time in months median (P25/P75)*4.3 (1.5/10.9)Other medications81 (78.6)Thalidomide dose median (P25/P75)100 (100/200)Days of consultation mean (min/max)108.19 (0/721)Patient origin\nNortheast Region89 (86.4)North Region14 (13.6)Leprosy classification\nBorderline-lepromatous 24 (23.3)Lepromatous-leprosy 79 (76.7)Adverse effects\nNeurological\na\n\n31 (30.1)Grastrointestinal\nb\n\n18 (17.5)Musculoskeletal\nc\n\n29 (28.2)Ocular\nd\n\n19 (18.4)Oedema16 (15.6)Dermatological\ne\n\n2 (1.9)Fever11 (10.7)\na: paresthesias, dizziness, tremor, neuritis and headache; b: diarrhoea, vomiting, gastric fullness and constipation; c: myalgia, arthralgia and weakness; d: decreased visual acuity and eye irritation; e: pruritus, dry skin and hair loss; *: time of multidrug therapy (MDT) when patients developed erythema nodosum leprosum (ENL) (n = 40).\n\n\na: paresthesias, dizziness, tremor, neuritis and headache; b: diarrhoea, vomiting, gastric fullness and constipation; c: myalgia, arthralgia and weakness; d: decreased visual acuity and eye irritation; e: pruritus, dry skin and hair loss; *: time of multidrug therapy (MDT) when patients developed erythema nodosum leprosum (ENL) (n = 40).\nThe genotypic distributions were in Hardy-Weinberg equilibrium and the allelic and genotypic frequencies of the polymorphisms are shown in Table II. A LD [rs4183/rs1672770 (r2 = 0.513), rs4183/rs1620675 (r2 = 0.943), rs1672770/rs1620675 (r2 = 0.567)] between the variants studied was identified (see Supplementary Table) and four haplotypes were identified in the sample (Table III). For all single nucleotide polymorphisms (SNPs), there was no association of the thalidomide dose with time of treatment (Table IV). In addition, no association was found between haplotypes and thalidomide dose.\n\nTABLE IIGenotypic and allelic frequencies of CRBN polymorphisms in ENL patients on treatment with thalidomidePolymorphismAlleles/genotypesFrequency n (%)rs1620675AA31 (30.1)AC53 (5.5)CC19 (18.4)A115 (55.8)C91 (44.2)\n\nrs1672770*AA31 (30.1)AG52 (50.5)GG17 (16.5)A114 (55.3)G86 (44.7)rs4183INS/INS19 (18.4)INS/DEL56 (54.4)DEL/DEL28 (27.2)INS94 (45.6)DEL115 (54.4)DEL: deletion; ENL: erythema nodosum leprosum; INS: insertion; MDT: multidrug therapy; *: n = 100.\n\nDEL: deletion; ENL: erythema nodosum leprosum; INS: insertion; MDT: multidrug therapy; *: n = 100.\n\nTABLE IIIHaplotype frequencies of CRBN\nHaplotypen(%)INS/C/T31.5INS/T/G9244.7DEL/C/T8340.3DEL/T/T2813.6Haplotypes in the following order: rs4183/rs1672770/rs1620675. DEL: deletion; CRBN: cereblon gene; INS: insertion.\n\nHaplotypes in the following order: rs4183/rs1672770/rs1620675. DEL: deletion; CRBN: cereblon gene; INS: insertion.\n\nTABLE IVAnalysis of interaction between CRBN genotype and time related to estimated thalidomide dose using the GEE model in ENL treatmentPolymorphism Interactionp-valuers1620675rs16206750.079Concomitant MDT for leprosy0.271Other medications0.205Region0.138Time0.001rs1672770rs1620675*Time0.392rs16727700.229Concomitant MDT for leprosy0.296Other medications0.071Region0.085Time0.001rs1672770*Time0.273rs4183rs41830.098Concomitant MDT for leprosy0.293Other medications0.208Region0.117Time0.001rs4183*Time0.669Dependent variable: thalidomide dose. GEE model: region, MDT, other medications, thalidomide dose, genotype, time, genotype*Time). CRBN: cereblon; ENL: erythema nodosum leprosum; GEE: generalised estimating equation model; MDT: multidrug therapy.\n\nDependent variable: thalidomide dose. GEE model: region, MDT, other medications, thalidomide dose, genotype, time, genotype*Time). CRBN: cereblon; ENL: erythema nodosum leprosum; GEE: generalised estimating equation model; MDT: multidrug therapy.\nIn regard to the relationship of CRBN variants and adverse effects of the use of thalidomide, it was found associations between the genotypes of rs1672770 (p = 0.040) and gastrointestinal effects, which include diarrhoea, vomiting, nausea, constipation and inappetence (Tables V and VI). Haplotype analysis was performed with all SNPs because only one combination of SNPs showed r2>0.8 (rs4183/rs1620675 with r2 = 0.943). The three polymorphisms were maintained in the analysis because when combining rs4183/rs1672770 or rs1672770/rs1620675, it showed r2<0.8. The analysis of the association between haplotypes and adverse effects also showed an association of gastrointestinal adverse effects (p = 0.015) with haplotype DEL/C/T (rs4183/rs1672770/rs1620675) (Tables VII and VIII). These effects were present in eight patients (9.6%) who carried this haplotype. \n\nTABLE VAnalysis of the association of CRBN variants on the occurrence of gastrointestinal adverse effects\na\n\nPolymorphismOR\nb\n\np-value*OR\nb\n\np-value**rs1672770\n\n\n\nAAREF-REF-AG0.325 (0.111-0.951)0.0400.040 (0.111-0.948)0.040GG0.131 (0.015-1.115)0.0630.127(0.015-1.100)0.061Dependent variable: gastrointestinal. CRBN: cereblon gene; OR: odds ratio; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence); b: logistic regression (OR and 95% confidence interval); *: model rs1672770; **: model sex, rs1672770.\n\nDependent variable: gastrointestinal. CRBN: cereblon gene; OR: odds ratio; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence); b: logistic regression (OR and 95% confidence interval); *: model rs1672770; **: model sex, rs1672770.\n\nTABLE VIFrequency of gastrointestinal adverse effects\na\n according to CRBN genotypesPolymorphismGenotypeAbsence (n) (% within effect)Presence (n) (% within effect)rs1672770AA23 (67.6)11 (32.4)AG45 (86.5)7 (13.5)GG16 (94.1)1 (5.9)\nCRBN: cereblon gene; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence).\n\n\nCRBN: cereblon gene; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence).\n\nTABLE VIIAnalysis of the association of CRBN haplotypes on the occurrence of gastrointestinal adverse effects\na\n\nHaplotypeOR\nb\n\np-value*OR\nb\n\np-value**INS/T/GREF-REF-DEL/C/T0.339 (0.142-0.812)0.0150.333 (0.138-0.805)0.015DEL/T/T1.061 (0.398-2.827)0.9061.022 (0.371-2.818)0.966Dependent variable: gastrointestinal. DEL: deletion; CRBN: cereblon gene; INS: insertion; OR: odds ratio; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence); b: logistic regression (OR and 95% confidence interval); *: model haplotypes; **: model sex, haplotypes.\n\nDependent variable: gastrointestinal. DEL: deletion; CRBN: cereblon gene; INS: insertion; OR: odds ratio; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence); b: logistic regression (OR and 95% confidence interval); *: model haplotypes; **: model sex, haplotypes.\n\nTABLE VIIIFrequency of gastrointestinal adverse effects\na\n according to CRBN haplotypesHaplotypesAbsence (n) (% within effect)Presence (n) (% within effect)INS/T/G70 (76.1)22 (23.9)DEL/C/T75 (90.4)8 (9.6)DEL/T/T21 (75)7 (25)DEL: deletion; CRBN: cereblon gene; INS: insertion; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence).\n\nDEL: deletion; CRBN: cereblon gene; INS: insertion; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence).","This study aimed to identify genetic variants in CRBN gene that might influence in response to the treatment of ENL with thalidomide. We identified that the SNP rs1672770 and the haplotype DEL/C/T (rs4183/rs1672770/rs1620675) were associated with the manifestation of adverse gastrointestinal effects.\nCRBN acts as a substrate receptor as part of the E3 ubiquitin ligase complex (CRL4CRBN), which controls the expression of target proteins by their ubiquitination and degradation. It is necessary for the teratogenic effect of thalidomide and also important for the antiproliferative effect of thalidomide and other IMiDs in MM.\n29\n It is postulated that when thalidomide binds to CRBN, it modifies its function causing teratogenic effects by preventing the degradation proteins and/or by creating neosubtrates for ubiquitination and proteasomal degradation, which play a crucial role in embryonic development.\n30\n\n,\n\n31\n In the case of MM, thalidomide binding to CRBN promotes recruitment of the neosubstrates Ikaros (IKZF1) and Aiolos (IKZF3) to the ubiquitin-ligase complex, resulting in increased ubiquitination and degradation of these transcription factors in T cells and MM cells.\n31\n\n-\n\n33\n In addition, some studies have associated low CRBN mRNA expression with poorer clinical response to IMiDs, suggesting a potential role of CRBN as a predictive biomarker for treatment response.\n17\n\n,\n\n31\n\n,\n\n32\n\n,\n\n34\n Since the 1990s, it is known that one of the main effects of thalidomide is to decrease the TNF mRNA half-life, which explains some of its therapeutic effects. Using CRBN knockdown, it has been shown that the inhibitory effect of IMiDs on TNF-α production was also impaired in the silencing of CRBN.\n16\n\n,\n\n35\n\n\n\nCRBN is composed of 11 exons extending over 30 Kb. Thalidomide binds to CRBN in a region of 104 amino acids (339-442) located in the C-terminal portion, encoded by exons 9, 10 and 11.\n13\n This gene is extremely conserved and a few polymorphisms was found in the coding region.\n23\n\n,\n\n36\n Studies performed with MM showed that variants in non-coding regions of CRBN were associated with response to thalidomide and others IMIDs therapy.\n20\n\n,\n\n21\n\n,\n\n37\n\n\nThe genetic variants evaluated in the present study were identified in a previous study as possible splicing sites.\n23\n The association of these SNPs and the dose of thalidomide could indicate that polymorphisms in these regions could interfere in the expression of the CRBN gene or in the activity of CRBN, being able to modulate the response to treatment with thalidomide. However, in this study, we were unable to identify an association between these variants and the dose variation of thalidomide over time.\nWe found an association between the genetic variant rs1672770 (p = 0.040) and the manifestation of gastrointestinal adverse effects. There was also an association of gastrointestinal adverse effects (p = 0.015) with haplotype DEL/C/T (rs4183/rs1672770/rs1620675). These adverse gastrointestinal effects consisted of diarrhoea, vomiting, nausea and constipation. The most common symptom was constipation, a commonly reported side effect of thalidomide.\n22\n\n,\n\n38\n\n,\n\n39\n The association of adverse effects with the polymorphisms studied may also be related to differences in CRBN expression. Mlak et al.\n22\n found an association between CRBN variants (rs6768972, rs16727) and the risk of polyneuropathy and gastrointestinal disorders in patients with MM treated with thalidomide-based regimens. These variants are located in the CRBN promoter region. Thus, they could influence the expression or activity of CRBN.\n23\n In the haplotype analysis, only one combination of SNPs showed r2>0.8 (rs4183/rs1620675 with r2 = 0.943), as can be observed in the Supplementary Table. Taking into account this result, our small sample size, and that this is the first study to evaluate CRBN haplotypes, we decided to maintain the logistic regression analysis considering all SNPs. Further studies should be performed in order to elucidate and confirm the haplotypes and associations found here.\nIn this study, 46 patients were using MDT during the treatment for ENL and only two patients received an alternative MDT regimen. For this reason, they were not divided into a subgroup during the analyses. In addition, the concomitant medications that patients used during treatment for ENL can be diverse. That is why they were not listed in a table. MDT and other drugs were used as covariates in the GEE analysis to avoid bias in the use of these drugs. However, it was not possible to use this correction in the other analyses. The epidemiological data of this study were in agreement with the data found in other studies.\n2\n\n,\n\n40\n Most of the individuals with ENL had LL and were male. These frequencies corroborate that men may be more affected by LL and that the bacillary index is a risk factor for the development of the reaction as demonstrated in other studies.\n2\n\n,\n\n41\n\n,\n\n42\n In addition, many patients were on MDT during treatment for ENL, confirming that the reaction manifests mainly in the first year of illness, during MDT.\n43\n\n,\n\n44\n\n\nSome limitations should be considered during the interpretation of our study. It was not possible to obtain samples of skin lesions from these patients. Thus, the DNA samples analysed in this work were obtained from saliva because it presents a methodology that is easier to obtain. However, this does not influence the results presented because it is about the analysis of genetic variants and not the gene expression. Clearly, further studies could focus on the impact of the CRBN expression in the skin lesions before and after the treatment. In addition, the use of clofazimine and in MDT for leprosy, because of its anti-inflammatory effect, and the other drugs may interfere with both the dose reduction of drugs and in the manifestation of adverse effects.\n2\n\n,\n\n3\n This is also a retrospective study carried out with clinical data of patients, thalidomide dose, and manifestation of adverse effects obtained through the analysis of medical records of up to six consultations. The lack of standardization in the description of this information or even the absence of a description of the adverse effects cannot be ruled out. Furthermore, as ENL is a chronic condition and its treatment is very long, in this study, the mean treatment time during the collection period was 226 days, but some patients had been in treatment for years and remained on thalidomide treatment after the data collection period. Thus, in some cases, it was not possible to completely monitor the treatment of patients. Peripheral neuropathy, one of the main adverse effects of thalidomide,\n3\n\n,\n\n4\n was not evaluated in this study due to its retrospective nature, making it difficult to differentiate neuropathy due to the disease itself or due to the use of thalidomide. Accordingly, the evaluation of the association of variants on the onset of peripheral neuropathy should be performed by a prospective study to reduce potential biases. Another limitation was that this study was carried out with individuals from different regions of the country and the heterogeneous genetic background of the Brazilian population might have been underestimated in the analysis and interpretation of the results. In addition, the limited sample size used in this study can become difficult the identification of minor effects from the genetic variants in different outcomes.\nOn basis of the results of this study, we identified some genetic variants of CRBN were associated with adverse gastrointestinal effects. These results indicate that such variants could impact the protein and influence the outcome of treatment with thalidomide. This also indicates that CRBN may also be necessary for the action of thalidomide in ENL, as already described in MM.\n29\n Clearly, more studies evaluating the impact the variants in CRBN, as well as associations with ENL treatment, must be performed in order to confirm this hypothesis. ENL is a chronic and difficult-to-control condition. Thalidomide is an effective drug, but has restrictions on its use due to peripheral neuropathy and its teratogenicity.\n7\n\n,\n\n8\n Therefore, it is important to identify useful biomarkers in predicting treatment response to limit its use to patients who will benefit most from treatment. To our knowledge, this is the first study to evaluate the association of genetic variants of CRBN with thalidomide treatment in ENL patients. There are still many gaps to be filled in our knowledge of the mechanism of action of thalidomide and on how CRBN participates in this process. Thus, this study shows that evaluation of CRBN and its expression may help to understand the action of thalidomide in ENL and perhaps, in the future, be a useful biomarker in ENL."],"string":"[\n \"\\nSample - The sample consisted of 103 ENL patients who were selected from National Reference Center of Sanitary Dermatology Dona Libânia in Fortaleza (state of Ceará, Brazil), Humanized Reference Center of Sanitary Dermatology in Imperatriz and Aquiles Lisboa Hospital in São Luís (Maranhão State) in northeast Brazil, and from the Dermatology Ambulatory of the University of São Paulo in Monte Negro (state of Rondônia, Brazil) in north Brazil. \\nThe patients used thalidomide at different doses and had a follow-up of up to six visits (average of 3.6 months). Data from up to six consultations annotated in the patient’s medical record were analysed with the collection of clinical and demographic information, including sex, age and region of origin, history of leprosy (moment of diagnosis and treatment used) and history of ENL (diagnosis, treatment, adverse effects, history of relapse and dose of medications used).\\n\\nGenetic analyses - DNA was extracted from saliva samples using the Oragene DNA Extraction Kit (DNA Genotek, Ottawa, Canada), according to the manufacturer’s instructions. A pair of primer was designed to amplify a fragment of 682 base pairs containing the region encompassing the three studied CRBN variants: forward 5’-TGTGGTCTTGGCAACCAGCAATTT-3’ and reverse 5’-ACTGCCGTTCATGCTTGTTTCCT-3’. This region was amplified by polymerase chain reaction (PCR). The fragment obtained was visualised on a 2% agarose gel, purified and sequenced using the same primers.\\nSequences were visualised and analysed using CodonCodeAligner®, version 3.0.1 (CodonCode Corporation, Dedham, USA). The hg19 sequence deposited in GenBank was used as the reference sequence. When there was doubt about the variant, sequencing was repeated for confirmation.\\n\\nStatistical analyses - Chi-square test was used to evaluate Hardy-Weinberg equilibrium for all polymorphisms. Generalised estimating equations method (GEE) was used to evaluate the influence of CRBN variants on thalidomide dose. This method is a repeated measures analysis focused on average changes in response over time and on the impact of covariates on these changes. GEE can model the average response of variables as a linear function of covariates of interest through a transformation or link function and can be used in studies where the data is asymmetric or the data distribution is difficult to verify due to the small-size sample.\\n24\\n\\n,\\n\\n25\\n The covariates inserted in the model were place of origin of the patient, concomitant use of multidrug therapy (MDT) for leprosy and use of other medications and other treatments for ENL.\\nThe evaluation of the association of CRBN variants and haplotypes in the occurrence of adverse effects due to thalidomide treatment was based on clinical data using logistic regression. Models with and without correction by gender were used. The INS/C/T (rs4183/rs1672770/rs1620675) haplotype was removed from the association analyses because it presented few events and disturbed the analyses. Peripheral polyneuropathy, although is an adverse effect common to the use of thalidomide, has not been evaluated because it is difficult to distinguish it from polyneuropathy caused by ENL and leprosy. All statistical analyses were performed with SPSS version 20 (www.spss.com).\\nMLocus tool was used to calculate linkage disequilibrium (LD) for the variants,\\n26\\n and haplotypes were inferred using the Bayesian algorithm of the phase 2.1.1 program.\\n27\\n\\n,\\n\\n28\\n\\n\\n\\nData availability - The data analysed during the current study are not publicly available to maintain patient confidentiality. Moreover, this type of request has not been previously approved by participants nor the human research committee. This data could, however, be available (anonymously) from the corresponding author on reasonable request.\\n\\nEthics - All participants were informed about the research objectives and signed an informed consent form. This study was approved by the Ethics Committee of the Hospital de Clínicas of Porto Alegre under number CAAE 21184413.0.0000.5327. All research was performed in accordance with Brazilian regulations and informed consent was obtained from all participants. This research has been performed in accordance with the Declaration of Helsinki.\",\n \"In total, 103 ENL patients were included, being 82 (79.6%) male and 79 (76.7%) presenting LL (Table I). Forty-six (44.7%) patients were using MDT and two of these used an alternative MDT regimen. The mean time of treatment with thalidomide was 226 days.\\n\\nTABLE IClinical and demographic characteristics of ENL patients (n = 103)Characteristic n (%)Male 82 (79.6)MDT for leprosy46 (44.7)MDT time in months median (P25/P75)*4.3 (1.5/10.9)Other medications81 (78.6)Thalidomide dose median (P25/P75)100 (100/200)Days of consultation mean (min/max)108.19 (0/721)Patient origin\\nNortheast Region89 (86.4)North Region14 (13.6)Leprosy classification\\nBorderline-lepromatous 24 (23.3)Lepromatous-leprosy 79 (76.7)Adverse effects\\nNeurological\\na\\n\\n31 (30.1)Grastrointestinal\\nb\\n\\n18 (17.5)Musculoskeletal\\nc\\n\\n29 (28.2)Ocular\\nd\\n\\n19 (18.4)Oedema16 (15.6)Dermatological\\ne\\n\\n2 (1.9)Fever11 (10.7)\\na: paresthesias, dizziness, tremor, neuritis and headache; b: diarrhoea, vomiting, gastric fullness and constipation; c: myalgia, arthralgia and weakness; d: decreased visual acuity and eye irritation; e: pruritus, dry skin and hair loss; *: time of multidrug therapy (MDT) when patients developed erythema nodosum leprosum (ENL) (n = 40).\\n\\n\\na: paresthesias, dizziness, tremor, neuritis and headache; b: diarrhoea, vomiting, gastric fullness and constipation; c: myalgia, arthralgia and weakness; d: decreased visual acuity and eye irritation; e: pruritus, dry skin and hair loss; *: time of multidrug therapy (MDT) when patients developed erythema nodosum leprosum (ENL) (n = 40).\\nThe genotypic distributions were in Hardy-Weinberg equilibrium and the allelic and genotypic frequencies of the polymorphisms are shown in Table II. A LD [rs4183/rs1672770 (r2 = 0.513), rs4183/rs1620675 (r2 = 0.943), rs1672770/rs1620675 (r2 = 0.567)] between the variants studied was identified (see Supplementary Table) and four haplotypes were identified in the sample (Table III). For all single nucleotide polymorphisms (SNPs), there was no association of the thalidomide dose with time of treatment (Table IV). In addition, no association was found between haplotypes and thalidomide dose.\\n\\nTABLE IIGenotypic and allelic frequencies of CRBN polymorphisms in ENL patients on treatment with thalidomidePolymorphismAlleles/genotypesFrequency n (%)rs1620675AA31 (30.1)AC53 (5.5)CC19 (18.4)A115 (55.8)C91 (44.2)\\n\\nrs1672770*AA31 (30.1)AG52 (50.5)GG17 (16.5)A114 (55.3)G86 (44.7)rs4183INS/INS19 (18.4)INS/DEL56 (54.4)DEL/DEL28 (27.2)INS94 (45.6)DEL115 (54.4)DEL: deletion; ENL: erythema nodosum leprosum; INS: insertion; MDT: multidrug therapy; *: n = 100.\\n\\nDEL: deletion; ENL: erythema nodosum leprosum; INS: insertion; MDT: multidrug therapy; *: n = 100.\\n\\nTABLE IIIHaplotype frequencies of CRBN\\nHaplotypen(%)INS/C/T31.5INS/T/G9244.7DEL/C/T8340.3DEL/T/T2813.6Haplotypes in the following order: rs4183/rs1672770/rs1620675. DEL: deletion; CRBN: cereblon gene; INS: insertion.\\n\\nHaplotypes in the following order: rs4183/rs1672770/rs1620675. DEL: deletion; CRBN: cereblon gene; INS: insertion.\\n\\nTABLE IVAnalysis of interaction between CRBN genotype and time related to estimated thalidomide dose using the GEE model in ENL treatmentPolymorphism Interactionp-valuers1620675rs16206750.079Concomitant MDT for leprosy0.271Other medications0.205Region0.138Time0.001rs1672770rs1620675*Time0.392rs16727700.229Concomitant MDT for leprosy0.296Other medications0.071Region0.085Time0.001rs1672770*Time0.273rs4183rs41830.098Concomitant MDT for leprosy0.293Other medications0.208Region0.117Time0.001rs4183*Time0.669Dependent variable: thalidomide dose. GEE model: region, MDT, other medications, thalidomide dose, genotype, time, genotype*Time). CRBN: cereblon; ENL: erythema nodosum leprosum; GEE: generalised estimating equation model; MDT: multidrug therapy.\\n\\nDependent variable: thalidomide dose. GEE model: region, MDT, other medications, thalidomide dose, genotype, time, genotype*Time). CRBN: cereblon; ENL: erythema nodosum leprosum; GEE: generalised estimating equation model; MDT: multidrug therapy.\\nIn regard to the relationship of CRBN variants and adverse effects of the use of thalidomide, it was found associations between the genotypes of rs1672770 (p = 0.040) and gastrointestinal effects, which include diarrhoea, vomiting, nausea, constipation and inappetence (Tables V and VI). Haplotype analysis was performed with all SNPs because only one combination of SNPs showed r2>0.8 (rs4183/rs1620675 with r2 = 0.943). The three polymorphisms were maintained in the analysis because when combining rs4183/rs1672770 or rs1672770/rs1620675, it showed r2<0.8. The analysis of the association between haplotypes and adverse effects also showed an association of gastrointestinal adverse effects (p = 0.015) with haplotype DEL/C/T (rs4183/rs1672770/rs1620675) (Tables VII and VIII). These effects were present in eight patients (9.6%) who carried this haplotype. \\n\\nTABLE VAnalysis of the association of CRBN variants on the occurrence of gastrointestinal adverse effects\\na\\n\\nPolymorphismOR\\nb\\n\\np-value*OR\\nb\\n\\np-value**rs1672770\\n\\n\\n\\nAAREF-REF-AG0.325 (0.111-0.951)0.0400.040 (0.111-0.948)0.040GG0.131 (0.015-1.115)0.0630.127(0.015-1.100)0.061Dependent variable: gastrointestinal. CRBN: cereblon gene; OR: odds ratio; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence); b: logistic regression (OR and 95% confidence interval); *: model rs1672770; **: model sex, rs1672770.\\n\\nDependent variable: gastrointestinal. CRBN: cereblon gene; OR: odds ratio; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence); b: logistic regression (OR and 95% confidence interval); *: model rs1672770; **: model sex, rs1672770.\\n\\nTABLE VIFrequency of gastrointestinal adverse effects\\na\\n according to CRBN genotypesPolymorphismGenotypeAbsence (n) (% within effect)Presence (n) (% within effect)rs1672770AA23 (67.6)11 (32.4)AG45 (86.5)7 (13.5)GG16 (94.1)1 (5.9)\\nCRBN: cereblon gene; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence).\\n\\n\\nCRBN: cereblon gene; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence).\\n\\nTABLE VIIAnalysis of the association of CRBN haplotypes on the occurrence of gastrointestinal adverse effects\\na\\n\\nHaplotypeOR\\nb\\n\\np-value*OR\\nb\\n\\np-value**INS/T/GREF-REF-DEL/C/T0.339 (0.142-0.812)0.0150.333 (0.138-0.805)0.015DEL/T/T1.061 (0.398-2.827)0.9061.022 (0.371-2.818)0.966Dependent variable: gastrointestinal. DEL: deletion; CRBN: cereblon gene; INS: insertion; OR: odds ratio; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence); b: logistic regression (OR and 95% confidence interval); *: model haplotypes; **: model sex, haplotypes.\\n\\nDependent variable: gastrointestinal. DEL: deletion; CRBN: cereblon gene; INS: insertion; OR: odds ratio; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence); b: logistic regression (OR and 95% confidence interval); *: model haplotypes; **: model sex, haplotypes.\\n\\nTABLE VIIIFrequency of gastrointestinal adverse effects\\na\\n according to CRBN haplotypesHaplotypesAbsence (n) (% within effect)Presence (n) (% within effect)INS/T/G70 (76.1)22 (23.9)DEL/C/T75 (90.4)8 (9.6)DEL/T/T21 (75)7 (25)DEL: deletion; CRBN: cereblon gene; INS: insertion; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence).\\n\\nDEL: deletion; CRBN: cereblon gene; INS: insertion; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence).\",\n \"This study aimed to identify genetic variants in CRBN gene that might influence in response to the treatment of ENL with thalidomide. We identified that the SNP rs1672770 and the haplotype DEL/C/T (rs4183/rs1672770/rs1620675) were associated with the manifestation of adverse gastrointestinal effects.\\nCRBN acts as a substrate receptor as part of the E3 ubiquitin ligase complex (CRL4CRBN), which controls the expression of target proteins by their ubiquitination and degradation. It is necessary for the teratogenic effect of thalidomide and also important for the antiproliferative effect of thalidomide and other IMiDs in MM.\\n29\\n It is postulated that when thalidomide binds to CRBN, it modifies its function causing teratogenic effects by preventing the degradation proteins and/or by creating neosubtrates for ubiquitination and proteasomal degradation, which play a crucial role in embryonic development.\\n30\\n\\n,\\n\\n31\\n In the case of MM, thalidomide binding to CRBN promotes recruitment of the neosubstrates Ikaros (IKZF1) and Aiolos (IKZF3) to the ubiquitin-ligase complex, resulting in increased ubiquitination and degradation of these transcription factors in T cells and MM cells.\\n31\\n\\n-\\n\\n33\\n In addition, some studies have associated low CRBN mRNA expression with poorer clinical response to IMiDs, suggesting a potential role of CRBN as a predictive biomarker for treatment response.\\n17\\n\\n,\\n\\n31\\n\\n,\\n\\n32\\n\\n,\\n\\n34\\n Since the 1990s, it is known that one of the main effects of thalidomide is to decrease the TNF mRNA half-life, which explains some of its therapeutic effects. Using CRBN knockdown, it has been shown that the inhibitory effect of IMiDs on TNF-α production was also impaired in the silencing of CRBN.\\n16\\n\\n,\\n\\n35\\n\\n\\n\\nCRBN is composed of 11 exons extending over 30 Kb. Thalidomide binds to CRBN in a region of 104 amino acids (339-442) located in the C-terminal portion, encoded by exons 9, 10 and 11.\\n13\\n This gene is extremely conserved and a few polymorphisms was found in the coding region.\\n23\\n\\n,\\n\\n36\\n Studies performed with MM showed that variants in non-coding regions of CRBN were associated with response to thalidomide and others IMIDs therapy.\\n20\\n\\n,\\n\\n21\\n\\n,\\n\\n37\\n\\n\\nThe genetic variants evaluated in the present study were identified in a previous study as possible splicing sites.\\n23\\n The association of these SNPs and the dose of thalidomide could indicate that polymorphisms in these regions could interfere in the expression of the CRBN gene or in the activity of CRBN, being able to modulate the response to treatment with thalidomide. However, in this study, we were unable to identify an association between these variants and the dose variation of thalidomide over time.\\nWe found an association between the genetic variant rs1672770 (p = 0.040) and the manifestation of gastrointestinal adverse effects. There was also an association of gastrointestinal adverse effects (p = 0.015) with haplotype DEL/C/T (rs4183/rs1672770/rs1620675). These adverse gastrointestinal effects consisted of diarrhoea, vomiting, nausea and constipation. The most common symptom was constipation, a commonly reported side effect of thalidomide.\\n22\\n\\n,\\n\\n38\\n\\n,\\n\\n39\\n The association of adverse effects with the polymorphisms studied may also be related to differences in CRBN expression. Mlak et al.\\n22\\n found an association between CRBN variants (rs6768972, rs16727) and the risk of polyneuropathy and gastrointestinal disorders in patients with MM treated with thalidomide-based regimens. These variants are located in the CRBN promoter region. Thus, they could influence the expression or activity of CRBN.\\n23\\n In the haplotype analysis, only one combination of SNPs showed r2>0.8 (rs4183/rs1620675 with r2 = 0.943), as can be observed in the Supplementary Table. Taking into account this result, our small sample size, and that this is the first study to evaluate CRBN haplotypes, we decided to maintain the logistic regression analysis considering all SNPs. Further studies should be performed in order to elucidate and confirm the haplotypes and associations found here.\\nIn this study, 46 patients were using MDT during the treatment for ENL and only two patients received an alternative MDT regimen. For this reason, they were not divided into a subgroup during the analyses. In addition, the concomitant medications that patients used during treatment for ENL can be diverse. That is why they were not listed in a table. MDT and other drugs were used as covariates in the GEE analysis to avoid bias in the use of these drugs. However, it was not possible to use this correction in the other analyses. The epidemiological data of this study were in agreement with the data found in other studies.\\n2\\n\\n,\\n\\n40\\n Most of the individuals with ENL had LL and were male. These frequencies corroborate that men may be more affected by LL and that the bacillary index is a risk factor for the development of the reaction as demonstrated in other studies.\\n2\\n\\n,\\n\\n41\\n\\n,\\n\\n42\\n In addition, many patients were on MDT during treatment for ENL, confirming that the reaction manifests mainly in the first year of illness, during MDT.\\n43\\n\\n,\\n\\n44\\n\\n\\nSome limitations should be considered during the interpretation of our study. It was not possible to obtain samples of skin lesions from these patients. Thus, the DNA samples analysed in this work were obtained from saliva because it presents a methodology that is easier to obtain. However, this does not influence the results presented because it is about the analysis of genetic variants and not the gene expression. Clearly, further studies could focus on the impact of the CRBN expression in the skin lesions before and after the treatment. In addition, the use of clofazimine and in MDT for leprosy, because of its anti-inflammatory effect, and the other drugs may interfere with both the dose reduction of drugs and in the manifestation of adverse effects.\\n2\\n\\n,\\n\\n3\\n This is also a retrospective study carried out with clinical data of patients, thalidomide dose, and manifestation of adverse effects obtained through the analysis of medical records of up to six consultations. The lack of standardization in the description of this information or even the absence of a description of the adverse effects cannot be ruled out. Furthermore, as ENL is a chronic condition and its treatment is very long, in this study, the mean treatment time during the collection period was 226 days, but some patients had been in treatment for years and remained on thalidomide treatment after the data collection period. Thus, in some cases, it was not possible to completely monitor the treatment of patients. Peripheral neuropathy, one of the main adverse effects of thalidomide,\\n3\\n\\n,\\n\\n4\\n was not evaluated in this study due to its retrospective nature, making it difficult to differentiate neuropathy due to the disease itself or due to the use of thalidomide. Accordingly, the evaluation of the association of variants on the onset of peripheral neuropathy should be performed by a prospective study to reduce potential biases. Another limitation was that this study was carried out with individuals from different regions of the country and the heterogeneous genetic background of the Brazilian population might have been underestimated in the analysis and interpretation of the results. In addition, the limited sample size used in this study can become difficult the identification of minor effects from the genetic variants in different outcomes.\\nOn basis of the results of this study, we identified some genetic variants of CRBN were associated with adverse gastrointestinal effects. These results indicate that such variants could impact the protein and influence the outcome of treatment with thalidomide. This also indicates that CRBN may also be necessary for the action of thalidomide in ENL, as already described in MM.\\n29\\n Clearly, more studies evaluating the impact the variants in CRBN, as well as associations with ENL treatment, must be performed in order to confirm this hypothesis. ENL is a chronic and difficult-to-control condition. Thalidomide is an effective drug, but has restrictions on its use due to peripheral neuropathy and its teratogenicity.\\n7\\n\\n,\\n\\n8\\n Therefore, it is important to identify useful biomarkers in predicting treatment response to limit its use to patients who will benefit most from treatment. To our knowledge, this is the first study to evaluate the association of genetic variants of CRBN with thalidomide treatment in ENL patients. There are still many gaps to be filled in our knowledge of the mechanism of action of thalidomide and on how CRBN participates in this process. Thus, this study shows that evaluation of CRBN and its expression may help to understand the action of thalidomide in ENL and perhaps, in the future, be a useful biomarker in ENL.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["materials|methods","results","discussion"],"string":"[\n \"materials|methods\",\n \"results\",\n \"discussion\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["CRBN","leprosy","pharmacogenomics","pharmacogenetics","personalised medicine"],"string":"[\n \"CRBN\",\n \"leprosy\",\n \"pharmacogenomics\",\n \"pharmacogenetics\",\n \"personalised medicine\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"SUBJECTS AND METHODS:\n\nSample - The sample consisted of 103 ENL patients who were selected from National Reference Center of Sanitary Dermatology Dona Libânia in Fortaleza (state of Ceará, Brazil), Humanized Reference Center of Sanitary Dermatology in Imperatriz and Aquiles Lisboa Hospital in São Luís (Maranhão State) in northeast Brazil, and from the Dermatology Ambulatory of the University of São Paulo in Monte Negro (state of Rondônia, Brazil) in north Brazil. \nThe patients used thalidomide at different doses and had a follow-up of up to six visits (average of 3.6 months). Data from up to six consultations annotated in the patient’s medical record were analysed with the collection of clinical and demographic information, including sex, age and region of origin, history of leprosy (moment of diagnosis and treatment used) and history of ENL (diagnosis, treatment, adverse effects, history of relapse and dose of medications used).\n\nGenetic analyses - DNA was extracted from saliva samples using the Oragene DNA Extraction Kit (DNA Genotek, Ottawa, Canada), according to the manufacturer’s instructions. A pair of primer was designed to amplify a fragment of 682 base pairs containing the region encompassing the three studied CRBN variants: forward 5’-TGTGGTCTTGGCAACCAGCAATTT-3’ and reverse 5’-ACTGCCGTTCATGCTTGTTTCCT-3’. This region was amplified by polymerase chain reaction (PCR). The fragment obtained was visualised on a 2% agarose gel, purified and sequenced using the same primers.\nSequences were visualised and analysed using CodonCodeAligner®, version 3.0.1 (CodonCode Corporation, Dedham, USA). The hg19 sequence deposited in GenBank was used as the reference sequence. When there was doubt about the variant, sequencing was repeated for confirmation.\n\nStatistical analyses - Chi-square test was used to evaluate Hardy-Weinberg equilibrium for all polymorphisms. Generalised estimating equations method (GEE) was used to evaluate the influence of CRBN variants on thalidomide dose. This method is a repeated measures analysis focused on average changes in response over time and on the impact of covariates on these changes. GEE can model the average response of variables as a linear function of covariates of interest through a transformation or link function and can be used in studies where the data is asymmetric or the data distribution is difficult to verify due to the small-size sample.\n24\n\n,\n\n25\n The covariates inserted in the model were place of origin of the patient, concomitant use of multidrug therapy (MDT) for leprosy and use of other medications and other treatments for ENL.\nThe evaluation of the association of CRBN variants and haplotypes in the occurrence of adverse effects due to thalidomide treatment was based on clinical data using logistic regression. Models with and without correction by gender were used. The INS/C/T (rs4183/rs1672770/rs1620675) haplotype was removed from the association analyses because it presented few events and disturbed the analyses. Peripheral polyneuropathy, although is an adverse effect common to the use of thalidomide, has not been evaluated because it is difficult to distinguish it from polyneuropathy caused by ENL and leprosy. All statistical analyses were performed with SPSS version 20 (www.spss.com).\nMLocus tool was used to calculate linkage disequilibrium (LD) for the variants,\n26\n and haplotypes were inferred using the Bayesian algorithm of the phase 2.1.1 program.\n27\n\n,\n\n28\n\n\n\nData availability - The data analysed during the current study are not publicly available to maintain patient confidentiality. Moreover, this type of request has not been previously approved by participants nor the human research committee. This data could, however, be available (anonymously) from the corresponding author on reasonable request.\n\nEthics - All participants were informed about the research objectives and signed an informed consent form. This study was approved by the Ethics Committee of the Hospital de Clínicas of Porto Alegre under number CAAE 21184413.0.0000.5327. All research was performed in accordance with Brazilian regulations and informed consent was obtained from all participants. This research has been performed in accordance with the Declaration of Helsinki.\n\nRESULTS:\nIn total, 103 ENL patients were included, being 82 (79.6%) male and 79 (76.7%) presenting LL (Table I). Forty-six (44.7%) patients were using MDT and two of these used an alternative MDT regimen. The mean time of treatment with thalidomide was 226 days.\n\nTABLE IClinical and demographic characteristics of ENL patients (n = 103)Characteristic n (%)Male 82 (79.6)MDT for leprosy46 (44.7)MDT time in months median (P25/P75)*4.3 (1.5/10.9)Other medications81 (78.6)Thalidomide dose median (P25/P75)100 (100/200)Days of consultation mean (min/max)108.19 (0/721)Patient origin\nNortheast Region89 (86.4)North Region14 (13.6)Leprosy classification\nBorderline-lepromatous 24 (23.3)Lepromatous-leprosy 79 (76.7)Adverse effects\nNeurological\na\n\n31 (30.1)Grastrointestinal\nb\n\n18 (17.5)Musculoskeletal\nc\n\n29 (28.2)Ocular\nd\n\n19 (18.4)Oedema16 (15.6)Dermatological\ne\n\n2 (1.9)Fever11 (10.7)\na: paresthesias, dizziness, tremor, neuritis and headache; b: diarrhoea, vomiting, gastric fullness and constipation; c: myalgia, arthralgia and weakness; d: decreased visual acuity and eye irritation; e: pruritus, dry skin and hair loss; *: time of multidrug therapy (MDT) when patients developed erythema nodosum leprosum (ENL) (n = 40).\n\n\na: paresthesias, dizziness, tremor, neuritis and headache; b: diarrhoea, vomiting, gastric fullness and constipation; c: myalgia, arthralgia and weakness; d: decreased visual acuity and eye irritation; e: pruritus, dry skin and hair loss; *: time of multidrug therapy (MDT) when patients developed erythema nodosum leprosum (ENL) (n = 40).\nThe genotypic distributions were in Hardy-Weinberg equilibrium and the allelic and genotypic frequencies of the polymorphisms are shown in Table II. A LD [rs4183/rs1672770 (r2 = 0.513), rs4183/rs1620675 (r2 = 0.943), rs1672770/rs1620675 (r2 = 0.567)] between the variants studied was identified (see Supplementary Table) and four haplotypes were identified in the sample (Table III). For all single nucleotide polymorphisms (SNPs), there was no association of the thalidomide dose with time of treatment (Table IV). In addition, no association was found between haplotypes and thalidomide dose.\n\nTABLE IIGenotypic and allelic frequencies of CRBN polymorphisms in ENL patients on treatment with thalidomidePolymorphismAlleles/genotypesFrequency n (%)rs1620675AA31 (30.1)AC53 (5.5)CC19 (18.4)A115 (55.8)C91 (44.2)\n\nrs1672770*AA31 (30.1)AG52 (50.5)GG17 (16.5)A114 (55.3)G86 (44.7)rs4183INS/INS19 (18.4)INS/DEL56 (54.4)DEL/DEL28 (27.2)INS94 (45.6)DEL115 (54.4)DEL: deletion; ENL: erythema nodosum leprosum; INS: insertion; MDT: multidrug therapy; *: n = 100.\n\nDEL: deletion; ENL: erythema nodosum leprosum; INS: insertion; MDT: multidrug therapy; *: n = 100.\n\nTABLE IIIHaplotype frequencies of CRBN\nHaplotypen(%)INS/C/T31.5INS/T/G9244.7DEL/C/T8340.3DEL/T/T2813.6Haplotypes in the following order: rs4183/rs1672770/rs1620675. DEL: deletion; CRBN: cereblon gene; INS: insertion.\n\nHaplotypes in the following order: rs4183/rs1672770/rs1620675. DEL: deletion; CRBN: cereblon gene; INS: insertion.\n\nTABLE IVAnalysis of interaction between CRBN genotype and time related to estimated thalidomide dose using the GEE model in ENL treatmentPolymorphism Interactionp-valuers1620675rs16206750.079Concomitant MDT for leprosy0.271Other medications0.205Region0.138Time0.001rs1672770rs1620675*Time0.392rs16727700.229Concomitant MDT for leprosy0.296Other medications0.071Region0.085Time0.001rs1672770*Time0.273rs4183rs41830.098Concomitant MDT for leprosy0.293Other medications0.208Region0.117Time0.001rs4183*Time0.669Dependent variable: thalidomide dose. GEE model: region, MDT, other medications, thalidomide dose, genotype, time, genotype*Time). CRBN: cereblon; ENL: erythema nodosum leprosum; GEE: generalised estimating equation model; MDT: multidrug therapy.\n\nDependent variable: thalidomide dose. GEE model: region, MDT, other medications, thalidomide dose, genotype, time, genotype*Time). CRBN: cereblon; ENL: erythema nodosum leprosum; GEE: generalised estimating equation model; MDT: multidrug therapy.\nIn regard to the relationship of CRBN variants and adverse effects of the use of thalidomide, it was found associations between the genotypes of rs1672770 (p = 0.040) and gastrointestinal effects, which include diarrhoea, vomiting, nausea, constipation and inappetence (Tables V and VI). Haplotype analysis was performed with all SNPs because only one combination of SNPs showed r2>0.8 (rs4183/rs1620675 with r2 = 0.943). The three polymorphisms were maintained in the analysis because when combining rs4183/rs1672770 or rs1672770/rs1620675, it showed r2<0.8. The analysis of the association between haplotypes and adverse effects also showed an association of gastrointestinal adverse effects (p = 0.015) with haplotype DEL/C/T (rs4183/rs1672770/rs1620675) (Tables VII and VIII). These effects were present in eight patients (9.6%) who carried this haplotype. \n\nTABLE VAnalysis of the association of CRBN variants on the occurrence of gastrointestinal adverse effects\na\n\nPolymorphismOR\nb\n\np-value*OR\nb\n\np-value**rs1672770\n\n\n\nAAREF-REF-AG0.325 (0.111-0.951)0.0400.040 (0.111-0.948)0.040GG0.131 (0.015-1.115)0.0630.127(0.015-1.100)0.061Dependent variable: gastrointestinal. CRBN: cereblon gene; OR: odds ratio; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence); b: logistic regression (OR and 95% confidence interval); *: model rs1672770; **: model sex, rs1672770.\n\nDependent variable: gastrointestinal. CRBN: cereblon gene; OR: odds ratio; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence); b: logistic regression (OR and 95% confidence interval); *: model rs1672770; **: model sex, rs1672770.\n\nTABLE VIFrequency of gastrointestinal adverse effects\na\n according to CRBN genotypesPolymorphismGenotypeAbsence (n) (% within effect)Presence (n) (% within effect)rs1672770AA23 (67.6)11 (32.4)AG45 (86.5)7 (13.5)GG16 (94.1)1 (5.9)\nCRBN: cereblon gene; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence).\n\n\nCRBN: cereblon gene; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence).\n\nTABLE VIIAnalysis of the association of CRBN haplotypes on the occurrence of gastrointestinal adverse effects\na\n\nHaplotypeOR\nb\n\np-value*OR\nb\n\np-value**INS/T/GREF-REF-DEL/C/T0.339 (0.142-0.812)0.0150.333 (0.138-0.805)0.015DEL/T/T1.061 (0.398-2.827)0.9061.022 (0.371-2.818)0.966Dependent variable: gastrointestinal. DEL: deletion; CRBN: cereblon gene; INS: insertion; OR: odds ratio; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence); b: logistic regression (OR and 95% confidence interval); *: model haplotypes; **: model sex, haplotypes.\n\nDependent variable: gastrointestinal. DEL: deletion; CRBN: cereblon gene; INS: insertion; OR: odds ratio; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence); b: logistic regression (OR and 95% confidence interval); *: model haplotypes; **: model sex, haplotypes.\n\nTABLE VIIIFrequency of gastrointestinal adverse effects\na\n according to CRBN haplotypesHaplotypesAbsence (n) (% within effect)Presence (n) (% within effect)INS/T/G70 (76.1)22 (23.9)DEL/C/T75 (90.4)8 (9.6)DEL/T/T21 (75)7 (25)DEL: deletion; CRBN: cereblon gene; INS: insertion; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence).\n\nDEL: deletion; CRBN: cereblon gene; INS: insertion; a: gastrointestinal adverse effects (diarrhoea, vomiting, nausea, constipation and inappetence).\n\nDISCUSSION:\nThis study aimed to identify genetic variants in CRBN gene that might influence in response to the treatment of ENL with thalidomide. We identified that the SNP rs1672770 and the haplotype DEL/C/T (rs4183/rs1672770/rs1620675) were associated with the manifestation of adverse gastrointestinal effects.\nCRBN acts as a substrate receptor as part of the E3 ubiquitin ligase complex (CRL4CRBN), which controls the expression of target proteins by their ubiquitination and degradation. It is necessary for the teratogenic effect of thalidomide and also important for the antiproliferative effect of thalidomide and other IMiDs in MM.\n29\n It is postulated that when thalidomide binds to CRBN, it modifies its function causing teratogenic effects by preventing the degradation proteins and/or by creating neosubtrates for ubiquitination and proteasomal degradation, which play a crucial role in embryonic development.\n30\n\n,\n\n31\n In the case of MM, thalidomide binding to CRBN promotes recruitment of the neosubstrates Ikaros (IKZF1) and Aiolos (IKZF3) to the ubiquitin-ligase complex, resulting in increased ubiquitination and degradation of these transcription factors in T cells and MM cells.\n31\n\n-\n\n33\n In addition, some studies have associated low CRBN mRNA expression with poorer clinical response to IMiDs, suggesting a potential role of CRBN as a predictive biomarker for treatment response.\n17\n\n,\n\n31\n\n,\n\n32\n\n,\n\n34\n Since the 1990s, it is known that one of the main effects of thalidomide is to decrease the TNF mRNA half-life, which explains some of its therapeutic effects. Using CRBN knockdown, it has been shown that the inhibitory effect of IMiDs on TNF-α production was also impaired in the silencing of CRBN.\n16\n\n,\n\n35\n\n\n\nCRBN is composed of 11 exons extending over 30 Kb. Thalidomide binds to CRBN in a region of 104 amino acids (339-442) located in the C-terminal portion, encoded by exons 9, 10 and 11.\n13\n This gene is extremely conserved and a few polymorphisms was found in the coding region.\n23\n\n,\n\n36\n Studies performed with MM showed that variants in non-coding regions of CRBN were associated with response to thalidomide and others IMIDs therapy.\n20\n\n,\n\n21\n\n,\n\n37\n\n\nThe genetic variants evaluated in the present study were identified in a previous study as possible splicing sites.\n23\n The association of these SNPs and the dose of thalidomide could indicate that polymorphisms in these regions could interfere in the expression of the CRBN gene or in the activity of CRBN, being able to modulate the response to treatment with thalidomide. However, in this study, we were unable to identify an association between these variants and the dose variation of thalidomide over time.\nWe found an association between the genetic variant rs1672770 (p = 0.040) and the manifestation of gastrointestinal adverse effects. There was also an association of gastrointestinal adverse effects (p = 0.015) with haplotype DEL/C/T (rs4183/rs1672770/rs1620675). These adverse gastrointestinal effects consisted of diarrhoea, vomiting, nausea and constipation. The most common symptom was constipation, a commonly reported side effect of thalidomide.\n22\n\n,\n\n38\n\n,\n\n39\n The association of adverse effects with the polymorphisms studied may also be related to differences in CRBN expression. Mlak et al.\n22\n found an association between CRBN variants (rs6768972, rs16727) and the risk of polyneuropathy and gastrointestinal disorders in patients with MM treated with thalidomide-based regimens. These variants are located in the CRBN promoter region. Thus, they could influence the expression or activity of CRBN.\n23\n In the haplotype analysis, only one combination of SNPs showed r2>0.8 (rs4183/rs1620675 with r2 = 0.943), as can be observed in the Supplementary Table. Taking into account this result, our small sample size, and that this is the first study to evaluate CRBN haplotypes, we decided to maintain the logistic regression analysis considering all SNPs. Further studies should be performed in order to elucidate and confirm the haplotypes and associations found here.\nIn this study, 46 patients were using MDT during the treatment for ENL and only two patients received an alternative MDT regimen. For this reason, they were not divided into a subgroup during the analyses. In addition, the concomitant medications that patients used during treatment for ENL can be diverse. That is why they were not listed in a table. MDT and other drugs were used as covariates in the GEE analysis to avoid bias in the use of these drugs. However, it was not possible to use this correction in the other analyses. The epidemiological data of this study were in agreement with the data found in other studies.\n2\n\n,\n\n40\n Most of the individuals with ENL had LL and were male. These frequencies corroborate that men may be more affected by LL and that the bacillary index is a risk factor for the development of the reaction as demonstrated in other studies.\n2\n\n,\n\n41\n\n,\n\n42\n In addition, many patients were on MDT during treatment for ENL, confirming that the reaction manifests mainly in the first year of illness, during MDT.\n43\n\n,\n\n44\n\n\nSome limitations should be considered during the interpretation of our study. It was not possible to obtain samples of skin lesions from these patients. Thus, the DNA samples analysed in this work were obtained from saliva because it presents a methodology that is easier to obtain. However, this does not influence the results presented because it is about the analysis of genetic variants and not the gene expression. Clearly, further studies could focus on the impact of the CRBN expression in the skin lesions before and after the treatment. In addition, the use of clofazimine and in MDT for leprosy, because of its anti-inflammatory effect, and the other drugs may interfere with both the dose reduction of drugs and in the manifestation of adverse effects.\n2\n\n,\n\n3\n This is also a retrospective study carried out with clinical data of patients, thalidomide dose, and manifestation of adverse effects obtained through the analysis of medical records of up to six consultations. The lack of standardization in the description of this information or even the absence of a description of the adverse effects cannot be ruled out. Furthermore, as ENL is a chronic condition and its treatment is very long, in this study, the mean treatment time during the collection period was 226 days, but some patients had been in treatment for years and remained on thalidomide treatment after the data collection period. Thus, in some cases, it was not possible to completely monitor the treatment of patients. Peripheral neuropathy, one of the main adverse effects of thalidomide,\n3\n\n,\n\n4\n was not evaluated in this study due to its retrospective nature, making it difficult to differentiate neuropathy due to the disease itself or due to the use of thalidomide. Accordingly, the evaluation of the association of variants on the onset of peripheral neuropathy should be performed by a prospective study to reduce potential biases. Another limitation was that this study was carried out with individuals from different regions of the country and the heterogeneous genetic background of the Brazilian population might have been underestimated in the analysis and interpretation of the results. In addition, the limited sample size used in this study can become difficult the identification of minor effects from the genetic variants in different outcomes.\nOn basis of the results of this study, we identified some genetic variants of CRBN were associated with adverse gastrointestinal effects. These results indicate that such variants could impact the protein and influence the outcome of treatment with thalidomide. This also indicates that CRBN may also be necessary for the action of thalidomide in ENL, as already described in MM.\n29\n Clearly, more studies evaluating the impact the variants in CRBN, as well as associations with ENL treatment, must be performed in order to confirm this hypothesis. ENL is a chronic and difficult-to-control condition. Thalidomide is an effective drug, but has restrictions on its use due to peripheral neuropathy and its teratogenicity.\n7\n\n,\n\n8\n Therefore, it is important to identify useful biomarkers in predicting treatment response to limit its use to patients who will benefit most from treatment. To our knowledge, this is the first study to evaluate the association of genetic variants of CRBN with thalidomide treatment in ENL patients. There are still many gaps to be filled in our knowledge of the mechanism of action of thalidomide and on how CRBN participates in this process. Thus, this study shows that evaluation of CRBN and its expression may help to understand the action of thalidomide in ENL and perhaps, in the future, be a useful biomarker in ENL.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nErythema nodosum leprosum (ENL) is an acute and systemic inflammatory reaction of leprosy characterised by painful nodules and involvement of various organs. Thalidomide is an immunomodulatory and anti-inflammatory drug currently used to treat this condition. Cereblon (CRBN) protein is the primary target of thalidomide, and it has been pointed out as necessary for the efficacy of this drug in others therapeutics settings.\n\nMethods:\nA total of 103 ENL patients in treatment with thalidomide were included in this study. DNA samples were obtained from saliva and molecular analysis of CRBN gene were performed to investigate the variants rs1620675, rs1672770 and rs4183. Different genotypes of CRBN variants were evaluated in relation to their influence on the dose of thalidomide and on the occurrence of adverse effects.\n\nResults:\nNo association was found between CRBN variants and thalidomide dose variation. However, the genotypes of rs1672770 showed association with gastrointestinal effects (p = 0.040). Moreover, the haplotype DEL/C/T (rs4183/rs1672770/rs1620675) was also associated with gastrointestinal adverse effects (p = 0.015).\n\nConclusions:\nOur results show that CRBN variants affect the treatment of ENH with thalidomide, especially on the adverse effects related to the drug."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":3934,"string":"3,934"},"whole_article_abstract_length":{"kind":"number","value":234,"string":"234"},"other_sections_lengths":{"kind":"list like","value":[],"string":"[]","isConversational":false},"num_sections":{"kind":"number","value":3,"string":"3"},"most_frequent_words":{"kind":"list like","value":["crbn","thalidomide","effects","adverse","enl","adverse effects","gastrointestinal","treatment","mdt","variants"],"string":"[\n \"crbn\",\n \"thalidomide\",\n \"effects\",\n \"adverse\",\n \"enl\",\n \"adverse effects\",\n \"gastrointestinal\",\n \"treatment\",\n \"mdt\",\n \"variants\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["snps dose thalidomide","use thalidomide found","patients thalidomide different","thalidomide dose genotype","brazil patients thalidomide"],"string":"[\n \"snps dose thalidomide\",\n \"use thalidomide found\",\n \"patients thalidomide different\",\n \"thalidomide dose genotype\",\n \"brazil patients thalidomide\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"null"},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"null"},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keywords_background_abstract_prompt":{"kind":"null"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] CRBN | leprosy | pharmacogenomics | pharmacogenetics | personalised medicine [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"null"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] CRBN | leprosy | pharmacogenomics | pharmacogenetics | personalised medicine [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_mesh_background_abstract_prompt":{"kind":"null"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Erythema Nodosum | Thalidomide | Leprosy, Lepromatous | Leprostatic Agents | Leprosy, Multibacillary [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"null"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Humans | Erythema Nodosum | Thalidomide | Leprosy, Lepromatous | Leprostatic Agents | Leprosy, Multibacillary [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_keybert_background_abstract_prompt":{"kind":"null"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] snps dose thalidomide | use thalidomide found | patients thalidomide different | thalidomide dose genotype | brazil patients thalidomide [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"null"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] snps dose thalidomide | use thalidomide found | patients thalidomide different | thalidomide dose genotype | brazil patients thalidomide [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_most_frequent_background_abstract_prompt":{"kind":"null"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] crbn | thalidomide | effects | adverse | enl | adverse effects | gastrointestinal | treatment | mdt | variants [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] crbn | thalidomide | effects | adverse | enl | adverse effects | gastrointestinal | treatment | mdt | variants [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"null"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] gastrointestinal | crbn cereblon | cereblon | crbn | effects | crbn cereblon gene | cereblon gene | gastrointestinal adverse | table | gastrointestinal adverse effects [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] crbn | thalidomide | effects | adverse | study | enl | gastrointestinal | treatment | adverse effects | variants [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"null"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] CRBN ||| rs1672770 | 0.040 ||| DEL/C/T | 0.015 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ENL ||| ||| Cereblon | CRBN ||| 103 | ENL ||| CRBN | rs1672770 | rs4183 ||| CRBN ||| ||| CRBN ||| rs1672770 | 0.040 ||| DEL/C/T | 0.015 ||| CRBN | ENH [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":81,"cells":{"title":{"kind":"string","value":"Objective parallel-forms reliability assessment of 3 dimension real time body posture screening tests."},"pmid":{"kind":"string","value":"25189936"},"background_abstract":{"kind":"string","value":"Screening tests play a significant role in rapid and reliable assessment of normal individual development in the entire population of children and adolescents. Body posture screening tests carried out at schools reveal that 50-60% of children and adolescents demonstrate body posture abnormalities, with 10% of this group at risk for progressive spinal deformities. This necessitates the search for effective and economically feasible forms of screening diagnosis. The aim of this study was to assess the reliability of clinical evaluation of body posture compared to objective assessment with the Zebris CMS-10 system (Zebris Medical GmbH)."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"The study enrolled 13-15-year-old pupils attending a junior secondary school (mean age 14.2 years). The study group consisted of 138 participants, including 71 girls and 67 boys, who underwent a clinical evaluation of the body posture and an examination with the Zebris CMS 10 system."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Statistically significant discrepancies between the clinical and objective evaluation were noted with regard to lumbar lordosis in boys (n = 67) and thoracic kyphosis in girls (n = 71). No statistically significant differences in both groups were noted for pelvic rotation and trunk position in the frontal plane."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"1. The finding of significant discrepancies between the results of assessment in the sagittal plane obtained in the clinical examination and Zebris CMS-10-based assessment suggests that clinical evaluation should be used to provide a general estimation of accentuation or reduction of spinal curvatures in the sagittal plane.2. The clinical evaluation of posture is reliable with regard to assessment in the frontal plane.3. The Zebris CMS-10 system makes the clinical examination significantly more objective with regard to assessment of the physiological curvatures and may be used to make screening tests more objective with regard to detecting postural defects."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adolescent","Female","Humans","Male","Physical Examination","Posture","Reproducibility of Results","Spinal Curvatures"],"string":"[\n \"Adolescent\",\n \"Female\",\n \"Humans\",\n \"Male\",\n \"Physical Examination\",\n \"Posture\",\n \"Reproducibility of Results\",\n \"Spinal Curvatures\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"4169808"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Human body posture is a motor habit associated with daily activity with an underlying morphological and functional basis\n[1]. It reflects the psychophysical status of the individual and is an index of mechanical efficiency of the kinaesthetic sense, muscle balance and musculomotor co-ordination\n[2]. Normal human posture in the vertical position relies on the spine and its position against the head and pelvis\n[3, 4]. The spatial relations among and between bony structures and articulations are stabilised by a system of fasciae, ligaments and muscles, while the central nervous system is the superior controller of body posture\n[5, 6]. Body posture variability depends on age, sex and environmental factors influencing its development during body growth\n[7, 8]. The following conditions are regarded as postural defects: abnormal shape of the physiological spinal curvatures, asymmetrical positioning of the shoulder or pelvic girdle, disturbance of the knee joint axis and abnormal shape of the foot arches. Screening studies of postural defects carried out at schools reveal that 50-60% of children and adolescents demonstrate body posture abnormalities, with 10% of this group at risk for scoliosis or other progressive spinal deformities\n[9–12]. An alarmingly high percentage of these defects are attributable to poor motor activity of children and adolescents, rapid changes taking place in the body during individual development and excessive time spent in the seated position\n[13]. An early and reliable detection programme for the population of children and adolescents combined with prophylactic measures to prevent the persistent spinal and trunk deformities is an appropriate strategy that can also mininise the medical and financial outcome of the more complex process of future treatment of postural defects and scolioses that might be necessary. The findings of a clinical evaluation of body posture and trunk asymmetry in a child depend on the experience of the examiner, compliance of the child and availability of bedside diagnostic equipment. Screening tests rely mainly on clinical evaluation since screening is supposed to be available to the entire population of children and adolescents. The easy availability and simple procedure used also need to guarantee a high reliability of diagnoses of postural defects. Non-invasive methods that will make diagnosis easier and more comprehensive are being sought to ensure more objective measurements. The Zebris CMS-10, a system for assessing body posture in three planes, offers a non-invasive method for evaluating the spatial positioning of selected topographic reference points in the frontal, sagittal and transverse planes, thus supplying objective data to support a clinical evaluation. The Zebris CMS 10 system demonstrates a high degree of test-retest reliability, intertester reliability and intratester reliability\n[14, 15]. The inclinometer method demonstrates a high degree of intertester reliability and intratester reliability\n[16, 17]. A variation of up to 1.5° was allowed using this technique. Measurements were repeated several times in each participant until two consecutive attempts by two independent examiners yielded the same angle values (including the admissible variation of 1.5°), thus complying with the principles of intertester and alternate-forms reliability. The aim of this study was to assess the reliability of clinical evaluation of body posture compared to objective assessment with the Zebris CMS-10 system."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"The methodology was approved by the Ethics Committee of the Rehabilitation Hospital for Children, Olsztyn, Poland. The study enrolled 13-15-year-old pupils attending a junior secondary school. The mean age was 14.2 (±0.6) years. The study group consisted of 138 participants, including 71 girls (mean age 14.1 ± 0.4 years, mean height 160.3 ± 3.4 cm, mean body weight 64.8 ± 3.9 kg) and 67 boys (mean age 14.4 ± 0.8 years, mean height 166.6 ± 2.9 cm, mean body weight 68.1 ± 3.6 kg). The exclusion criteria were a diagnosis of scoliosis and/or status post spinal surgery and/or feeling any pain. The screening test was carried out with the participants in a free standing position, involving specialists in rehabilitation as examiners and a Zebris CMS 10 system. The objective of the examination was not revealed to the examiners. In the first part, reference skeletal landmarks were marked on the body according to the principles of palpation anatomy. Trunk positioning was evaluated clinically in the sagittal and frontal planes. The findings were recorded in the study protocol (Additional file\n1). Thoracic kyphosis and lumbar lordosis were evaluated in the sagittal plane with a Saunders inclinometer. Pelvic rotation was also evaluated in the sagittal plane. A Saunders inclinometer was placed in the cervicothoracic junction with the long arm pointing downwards from the spinous process at the apex of the curve and in the lumbosacral junction with the long arm pointing upwards from the spinous process at the apex of the curve (Figures \n1,\n2). The respective reference ranges assumed for kyphosis and lordosis were 30-40° and 25-35°\n[18]. The symmetry of position of the shoulder and pelvic girdles was evaluated in the frontal plane.Figure 1\nMarking of anatomical skeletal reference landmarks.\nFigure 2\nClinical examination with Saunders inclinometer. Reference marker is a belt attached below iliac spines.\n\nMarking of anatomical skeletal reference landmarks.\n\n\nClinical examination with Saunders inclinometer. Reference marker is a belt attached below iliac spines.\nFollowing the clinical evaluation, the same postural parameters were assessed with a Zebris CMS-10 device (Zebris Medical GmbH). The Zebris CMS 10 uses WinSpine software in the Microsoft Windows XP environment. Measurement error is defined by the manufacturer at 1.96 degrees and 2.2 millimeters for all parameters. Measurement sensitivity is 0.2 millimeters and 0.5 degrees. The software includes a data base of projects, patients and individual measurements. The core component of the testing system is a measuring device, an ultrasound point indicator probe and a reference marker\n[19]. The testing device is placed on an adjustable-height arm. The point indicator probe, which is placed directly onto skeletal reference landmarks on the patient’s body, has two ultrasound markers with their central points aligned with the tip of the probe. The skeletal reference landmarks are all thoracic and lumbar spinous processes of the spine. The software precisely calculates the position of the probe. A reference marker in the form of a belt is attached laterally below the posterior superior iliac spines and anterior superior iliac spines so as not to cover the measurement sites. The reference marker is used to eliminate changes of position during the examination. The testing unit was composed of a platform with built-in levels, a Zebris CMS-10 system and a computer. A transverse valve was mounted at one-third of the length of the platform in order to immobilise the Zebris CMS-10 device. Owing to this, the device could be placed in a fixed position and random movements during use were eliminated. A transverse red line was marked permanently at 80 cm from the transverse valve. One side of a 25 × 25 cm square was drawn on this line. The square was contoured with black lines. The lateral sides of the square were used to indicate where the examinees should place their feet in the standing position. The examinees were also instructed to place their feet in front of the red transverse line (Figure \n3). The device was calibrated against the ground before each examination.Figure 3\nExamination with Zebris CMS-10. Ultrasound probe recording the position of marked skeletal landmarks.\n\nExamination with Zebris CMS-10. Ultrasound probe recording the position of marked skeletal landmarks.\nFor statistical analysis of the clinical vs. Zebris-based assessment, physiological spinal curvatures in the sagittal plane were assigned a value of 0 and accentuation or reduction of the curvatures in the sagittal plane below 30° or above 40° for thoracic kyphosis and below 25° or above 35° for lumbar lordosis was assigned a value of 1. A symmetrical position of the pelvis was assigned a value of - 0, and pelvic rotation, a value of 1. Pelvic rotation was assessed manually as a deficit of rotation of the iliac bone relative to the sacral bone on the left and right side of the body. In the frontal plane, symmetry of the acromions and of the pelvis was assigned a value of - 0, and an asymmetry greater than 1 cm in the vertical dimension, a value of 1. The statistical analysis was conducted in Statistica 7 software package, version 10.1 and based on the calculation of means, percentages and the Chi2 test statistics (empirical and expected), and Cramer’s V statistic, which reflects the strength of association of two parameters. The level of significance was set at p < 0.05.\nThe parents of the children in the study group had provided written consent for their children to participate in the study. All the patients gave their written consent prior to their inclusion in the study.\nThe study, funded from a scientific grant, was conducted in the years 2011–2014."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Cramer’s V values confirmed a significant correlation between the parameters in the case of lordosis and a clear correlation in the case of kyphosis. Thus, the study demonstrated that the Zebris CMS-10 system for three-dimensional analysis of body posture contributed a statistically significant adjustment to the clinical evaluation of the spine in the sagittal view; Cramer’s V was 0.514 for the evaluation of thoracic kyphosis in girls and 0.433 in the evaluation of lumbar lordosis in boys (Figures \n4,\n5,\n6,\n7,\n8,\n9).Figure 4\nResults of evaluation of thoracic kyphosis in the sagittal plane in boys.\nFigure 5\nResults of evaluation of lumbar lordosis in the sagittal plane in boys.\nFigure 6\nResults of evaluation of pelvic rotation in the sagittal plane in boys.\nFigure 7\nResults of evaluation of thoracic kyphosis in the sagittal plane in girls.\nFigure 8\nResults of evaluation of lumbar lordosis in the sagittal plane in girls.\nFigure 9\nResults of evaluation of pelvic rotation in the sagittal plane in girls.\n\n\nResults of evaluation of thoracic kyphosis in the sagittal plane in boys.\n\n\nResults of evaluation of lumbar lordosis in the sagittal plane in boys.\n\n\nResults of evaluation of pelvic rotation in the sagittal plane in boys.\n\n\nResults of evaluation of thoracic kyphosis in the sagittal plane in girls.\n\n\nResults of evaluation of lumbar lordosis in the sagittal plane in girls.\n\n\nResults of evaluation of pelvic rotation in the sagittal plane in girls.\n\nNo statistically significant differences were found with regard to the accuracy of evaluation of pelvic rotation, indicating a similar degree of precision of both techniques, with a Cramer’s V of 0.112 in boys and 0.042 in girls. Similarly, no significant differences were revealed in the frontal plane, also suggesting a similar precision (Tables \n1 and\n2). These results show that clinical and device-assisted topography is characterised by a similar degree of accuracy. Minor differences were noted with regard to trunk asymmetry in the frontal plane. The differences were not statistically significant (Table \n1). Clinical evaluation is thus a reliable method for assessing trunk asymmetry in the frontal plane and does not need to be confirmed by measurement system-based assessment.Table 1\nTrunk assessment in boys, sagittal and frontal planes\nTrunk assessmentCMS -10 evaluationClinical evaluationCalculated Chi\n2\nSignificance level p < 0.05Degrees of freedomTabular Chi\n2\nHypothesis acceptedCramer’s VKyphosis12.80.0525.9alternative0.309Accentuated2117Reduced2510Normal2140Total6767Lordosis25.10.0525.9alternative0.433Accentuated339Reduced1715Normal1743Total6767Pelvis1.70.0513.7null0.112Rotated4942Not rotated1825Total6767Asymmetry5.880.0525.9null0.173Shoulder girdle4825Inferior scapular angles4513Pelvic obliqueness3728Total13066Table 2\nTrunk assessment in girls, sagittal and frontal planes\nTrunk assessmentCMS -10 evaluationClinical evaluationCalculated Chi\n2\nSignificance level p < 0.05Degrees of freedomTabular Chi\n2\nHypothesis acceptedCramer’s VKyphosis37.50.0525.9alternative0.514Accentuated438Reduced613Normal2250Total7171Lordosis20.10.0525.9alternative0.377Accentuated237Reduced2212Normal2652Total7171Pelvis0.30.0513.7null0.042Rotated3740Not rotated3431Total7171Asymmetry3.90.0525.9null0.151Shoulder girdle5224Inferior scapular angles4218Pelvic obliqueness1817Total11259\n\nTrunk assessment in boys, sagittal and frontal planes\n\n\nTrunk assessment in girls, sagittal and frontal planes\n"},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"\n\n\nThe finding of significant discrepancies between the results of assessment in the sagittal plane obtained in the clinical examination and Zebris CMS-10-based assessment suggests that clinical evaluation should be used to provide a general estimation of accentuation or reduction of spinal curvatures in the sagittal plane.\n\n\nThe clinical evaluation of posture is reliable with regard to assessment in the frontal plane.\n\n\nThe Zebris CMS-10 system makes the clinical examination significantly more objective with regard to assessment of the physiological curvatures and may be used to make screening tests more objective with regard to detecting postural defects.\n\n\n\nThe finding of significant discrepancies between the results of assessment in the sagittal plane obtained in the clinical examination and Zebris CMS-10-based assessment suggests that clinical evaluation should be used to provide a general estimation of accentuation or reduction of spinal curvatures in the sagittal plane.\nThe clinical evaluation of posture is reliable with regard to assessment in the frontal plane.\nThe Zebris CMS-10 system makes the clinical examination significantly more objective with regard to assessment of the physiological curvatures and may be used to make screening tests more objective with regard to detecting postural defects."},"other_sections_titles":{"kind":"list like","value":["Background",""],"string":"[\n \"Background\",\n \"\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Human body posture is a motor habit associated with daily activity with an underlying morphological and functional basis\n[1]. It reflects the psychophysical status of the individual and is an index of mechanical efficiency of the kinaesthetic sense, muscle balance and musculomotor co-ordination\n[2]. Normal human posture in the vertical position relies on the spine and its position against the head and pelvis\n[3, 4]. The spatial relations among and between bony structures and articulations are stabilised by a system of fasciae, ligaments and muscles, while the central nervous system is the superior controller of body posture\n[5, 6]. Body posture variability depends on age, sex and environmental factors influencing its development during body growth\n[7, 8]. The following conditions are regarded as postural defects: abnormal shape of the physiological spinal curvatures, asymmetrical positioning of the shoulder or pelvic girdle, disturbance of the knee joint axis and abnormal shape of the foot arches. Screening studies of postural defects carried out at schools reveal that 50-60% of children and adolescents demonstrate body posture abnormalities, with 10% of this group at risk for scoliosis or other progressive spinal deformities\n[9–12]. An alarmingly high percentage of these defects are attributable to poor motor activity of children and adolescents, rapid changes taking place in the body during individual development and excessive time spent in the seated position\n[13]. An early and reliable detection programme for the population of children and adolescents combined with prophylactic measures to prevent the persistent spinal and trunk deformities is an appropriate strategy that can also mininise the medical and financial outcome of the more complex process of future treatment of postural defects and scolioses that might be necessary. The findings of a clinical evaluation of body posture and trunk asymmetry in a child depend on the experience of the examiner, compliance of the child and availability of bedside diagnostic equipment. Screening tests rely mainly on clinical evaluation since screening is supposed to be available to the entire population of children and adolescents. The easy availability and simple procedure used also need to guarantee a high reliability of diagnoses of postural defects. Non-invasive methods that will make diagnosis easier and more comprehensive are being sought to ensure more objective measurements. The Zebris CMS-10, a system for assessing body posture in three planes, offers a non-invasive method for evaluating the spatial positioning of selected topographic reference points in the frontal, sagittal and transverse planes, thus supplying objective data to support a clinical evaluation. The Zebris CMS 10 system demonstrates a high degree of test-retest reliability, intertester reliability and intratester reliability\n[14, 15]. The inclinometer method demonstrates a high degree of intertester reliability and intratester reliability\n[16, 17]. A variation of up to 1.5° was allowed using this technique. Measurements were repeated several times in each participant until two consecutive attempts by two independent examiners yielded the same angle values (including the admissible variation of 1.5°), thus complying with the principles of intertester and alternate-forms reliability. The aim of this study was to assess the reliability of clinical evaluation of body posture compared to objective assessment with the Zebris CMS-10 system.","Additional file 1:\nSample examination protocol.\n(DOCX 14 KB)"],"string":"[\n \"Human body posture is a motor habit associated with daily activity with an underlying morphological and functional basis\\n[1]. It reflects the psychophysical status of the individual and is an index of mechanical efficiency of the kinaesthetic sense, muscle balance and musculomotor co-ordination\\n[2]. Normal human posture in the vertical position relies on the spine and its position against the head and pelvis\\n[3, 4]. The spatial relations among and between bony structures and articulations are stabilised by a system of fasciae, ligaments and muscles, while the central nervous system is the superior controller of body posture\\n[5, 6]. Body posture variability depends on age, sex and environmental factors influencing its development during body growth\\n[7, 8]. The following conditions are regarded as postural defects: abnormal shape of the physiological spinal curvatures, asymmetrical positioning of the shoulder or pelvic girdle, disturbance of the knee joint axis and abnormal shape of the foot arches. Screening studies of postural defects carried out at schools reveal that 50-60% of children and adolescents demonstrate body posture abnormalities, with 10% of this group at risk for scoliosis or other progressive spinal deformities\\n[9–12]. An alarmingly high percentage of these defects are attributable to poor motor activity of children and adolescents, rapid changes taking place in the body during individual development and excessive time spent in the seated position\\n[13]. An early and reliable detection programme for the population of children and adolescents combined with prophylactic measures to prevent the persistent spinal and trunk deformities is an appropriate strategy that can also mininise the medical and financial outcome of the more complex process of future treatment of postural defects and scolioses that might be necessary. The findings of a clinical evaluation of body posture and trunk asymmetry in a child depend on the experience of the examiner, compliance of the child and availability of bedside diagnostic equipment. Screening tests rely mainly on clinical evaluation since screening is supposed to be available to the entire population of children and adolescents. The easy availability and simple procedure used also need to guarantee a high reliability of diagnoses of postural defects. Non-invasive methods that will make diagnosis easier and more comprehensive are being sought to ensure more objective measurements. The Zebris CMS-10, a system for assessing body posture in three planes, offers a non-invasive method for evaluating the spatial positioning of selected topographic reference points in the frontal, sagittal and transverse planes, thus supplying objective data to support a clinical evaluation. The Zebris CMS 10 system demonstrates a high degree of test-retest reliability, intertester reliability and intratester reliability\\n[14, 15]. The inclinometer method demonstrates a high degree of intertester reliability and intratester reliability\\n[16, 17]. A variation of up to 1.5° was allowed using this technique. Measurements were repeated several times in each participant until two consecutive attempts by two independent examiners yielded the same angle values (including the admissible variation of 1.5°), thus complying with the principles of intertester and alternate-forms reliability. The aim of this study was to assess the reliability of clinical evaluation of body posture compared to objective assessment with the Zebris CMS-10 system.\",\n \"Additional file 1:\\nSample examination protocol.\\n(DOCX 14 KB)\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null],"string":"[\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Results","Discussion","Conclusions","Electronic supplementary material",""],"string":"[\n \"Background\",\n \"Methods\",\n \"Results\",\n \"Discussion\",\n \"Conclusions\",\n \"Electronic supplementary material\",\n \"\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Human body posture is a motor habit associated with daily activity with an underlying morphological and functional basis\n[1]. It reflects the psychophysical status of the individual and is an index of mechanical efficiency of the kinaesthetic sense, muscle balance and musculomotor co-ordination\n[2]. Normal human posture in the vertical position relies on the spine and its position against the head and pelvis\n[3, 4]. The spatial relations among and between bony structures and articulations are stabilised by a system of fasciae, ligaments and muscles, while the central nervous system is the superior controller of body posture\n[5, 6]. Body posture variability depends on age, sex and environmental factors influencing its development during body growth\n[7, 8]. The following conditions are regarded as postural defects: abnormal shape of the physiological spinal curvatures, asymmetrical positioning of the shoulder or pelvic girdle, disturbance of the knee joint axis and abnormal shape of the foot arches. Screening studies of postural defects carried out at schools reveal that 50-60% of children and adolescents demonstrate body posture abnormalities, with 10% of this group at risk for scoliosis or other progressive spinal deformities\n[9–12]. An alarmingly high percentage of these defects are attributable to poor motor activity of children and adolescents, rapid changes taking place in the body during individual development and excessive time spent in the seated position\n[13]. An early and reliable detection programme for the population of children and adolescents combined with prophylactic measures to prevent the persistent spinal and trunk deformities is an appropriate strategy that can also mininise the medical and financial outcome of the more complex process of future treatment of postural defects and scolioses that might be necessary. The findings of a clinical evaluation of body posture and trunk asymmetry in a child depend on the experience of the examiner, compliance of the child and availability of bedside diagnostic equipment. Screening tests rely mainly on clinical evaluation since screening is supposed to be available to the entire population of children and adolescents. The easy availability and simple procedure used also need to guarantee a high reliability of diagnoses of postural defects. Non-invasive methods that will make diagnosis easier and more comprehensive are being sought to ensure more objective measurements. The Zebris CMS-10, a system for assessing body posture in three planes, offers a non-invasive method for evaluating the spatial positioning of selected topographic reference points in the frontal, sagittal and transverse planes, thus supplying objective data to support a clinical evaluation. The Zebris CMS 10 system demonstrates a high degree of test-retest reliability, intertester reliability and intratester reliability\n[14, 15]. The inclinometer method demonstrates a high degree of intertester reliability and intratester reliability\n[16, 17]. A variation of up to 1.5° was allowed using this technique. Measurements were repeated several times in each participant until two consecutive attempts by two independent examiners yielded the same angle values (including the admissible variation of 1.5°), thus complying with the principles of intertester and alternate-forms reliability. The aim of this study was to assess the reliability of clinical evaluation of body posture compared to objective assessment with the Zebris CMS-10 system.","The methodology was approved by the Ethics Committee of the Rehabilitation Hospital for Children, Olsztyn, Poland. The study enrolled 13-15-year-old pupils attending a junior secondary school. The mean age was 14.2 (±0.6) years. The study group consisted of 138 participants, including 71 girls (mean age 14.1 ± 0.4 years, mean height 160.3 ± 3.4 cm, mean body weight 64.8 ± 3.9 kg) and 67 boys (mean age 14.4 ± 0.8 years, mean height 166.6 ± 2.9 cm, mean body weight 68.1 ± 3.6 kg). The exclusion criteria were a diagnosis of scoliosis and/or status post spinal surgery and/or feeling any pain. The screening test was carried out with the participants in a free standing position, involving specialists in rehabilitation as examiners and a Zebris CMS 10 system. The objective of the examination was not revealed to the examiners. In the first part, reference skeletal landmarks were marked on the body according to the principles of palpation anatomy. Trunk positioning was evaluated clinically in the sagittal and frontal planes. The findings were recorded in the study protocol (Additional file\n1). Thoracic kyphosis and lumbar lordosis were evaluated in the sagittal plane with a Saunders inclinometer. Pelvic rotation was also evaluated in the sagittal plane. A Saunders inclinometer was placed in the cervicothoracic junction with the long arm pointing downwards from the spinous process at the apex of the curve and in the lumbosacral junction with the long arm pointing upwards from the spinous process at the apex of the curve (Figures \n1,\n2). The respective reference ranges assumed for kyphosis and lordosis were 30-40° and 25-35°\n[18]. The symmetry of position of the shoulder and pelvic girdles was evaluated in the frontal plane.Figure 1\nMarking of anatomical skeletal reference landmarks.\nFigure 2\nClinical examination with Saunders inclinometer. Reference marker is a belt attached below iliac spines.\n\nMarking of anatomical skeletal reference landmarks.\n\n\nClinical examination with Saunders inclinometer. Reference marker is a belt attached below iliac spines.\nFollowing the clinical evaluation, the same postural parameters were assessed with a Zebris CMS-10 device (Zebris Medical GmbH). The Zebris CMS 10 uses WinSpine software in the Microsoft Windows XP environment. Measurement error is defined by the manufacturer at 1.96 degrees and 2.2 millimeters for all parameters. Measurement sensitivity is 0.2 millimeters and 0.5 degrees. The software includes a data base of projects, patients and individual measurements. The core component of the testing system is a measuring device, an ultrasound point indicator probe and a reference marker\n[19]. The testing device is placed on an adjustable-height arm. The point indicator probe, which is placed directly onto skeletal reference landmarks on the patient’s body, has two ultrasound markers with their central points aligned with the tip of the probe. The skeletal reference landmarks are all thoracic and lumbar spinous processes of the spine. The software precisely calculates the position of the probe. A reference marker in the form of a belt is attached laterally below the posterior superior iliac spines and anterior superior iliac spines so as not to cover the measurement sites. The reference marker is used to eliminate changes of position during the examination. The testing unit was composed of a platform with built-in levels, a Zebris CMS-10 system and a computer. A transverse valve was mounted at one-third of the length of the platform in order to immobilise the Zebris CMS-10 device. Owing to this, the device could be placed in a fixed position and random movements during use were eliminated. A transverse red line was marked permanently at 80 cm from the transverse valve. One side of a 25 × 25 cm square was drawn on this line. The square was contoured with black lines. The lateral sides of the square were used to indicate where the examinees should place their feet in the standing position. The examinees were also instructed to place their feet in front of the red transverse line (Figure \n3). The device was calibrated against the ground before each examination.Figure 3\nExamination with Zebris CMS-10. Ultrasound probe recording the position of marked skeletal landmarks.\n\nExamination with Zebris CMS-10. Ultrasound probe recording the position of marked skeletal landmarks.\nFor statistical analysis of the clinical vs. Zebris-based assessment, physiological spinal curvatures in the sagittal plane were assigned a value of 0 and accentuation or reduction of the curvatures in the sagittal plane below 30° or above 40° for thoracic kyphosis and below 25° or above 35° for lumbar lordosis was assigned a value of 1. A symmetrical position of the pelvis was assigned a value of - 0, and pelvic rotation, a value of 1. Pelvic rotation was assessed manually as a deficit of rotation of the iliac bone relative to the sacral bone on the left and right side of the body. In the frontal plane, symmetry of the acromions and of the pelvis was assigned a value of - 0, and an asymmetry greater than 1 cm in the vertical dimension, a value of 1. The statistical analysis was conducted in Statistica 7 software package, version 10.1 and based on the calculation of means, percentages and the Chi2 test statistics (empirical and expected), and Cramer’s V statistic, which reflects the strength of association of two parameters. The level of significance was set at p < 0.05.\nThe parents of the children in the study group had provided written consent for their children to participate in the study. All the patients gave their written consent prior to their inclusion in the study.\nThe study, funded from a scientific grant, was conducted in the years 2011–2014.","Cramer’s V values confirmed a significant correlation between the parameters in the case of lordosis and a clear correlation in the case of kyphosis. Thus, the study demonstrated that the Zebris CMS-10 system for three-dimensional analysis of body posture contributed a statistically significant adjustment to the clinical evaluation of the spine in the sagittal view; Cramer’s V was 0.514 for the evaluation of thoracic kyphosis in girls and 0.433 in the evaluation of lumbar lordosis in boys (Figures \n4,\n5,\n6,\n7,\n8,\n9).Figure 4\nResults of evaluation of thoracic kyphosis in the sagittal plane in boys.\nFigure 5\nResults of evaluation of lumbar lordosis in the sagittal plane in boys.\nFigure 6\nResults of evaluation of pelvic rotation in the sagittal plane in boys.\nFigure 7\nResults of evaluation of thoracic kyphosis in the sagittal plane in girls.\nFigure 8\nResults of evaluation of lumbar lordosis in the sagittal plane in girls.\nFigure 9\nResults of evaluation of pelvic rotation in the sagittal plane in girls.\n\n\nResults of evaluation of thoracic kyphosis in the sagittal plane in boys.\n\n\nResults of evaluation of lumbar lordosis in the sagittal plane in boys.\n\n\nResults of evaluation of pelvic rotation in the sagittal plane in boys.\n\n\nResults of evaluation of thoracic kyphosis in the sagittal plane in girls.\n\n\nResults of evaluation of lumbar lordosis in the sagittal plane in girls.\n\n\nResults of evaluation of pelvic rotation in the sagittal plane in girls.\n\nNo statistically significant differences were found with regard to the accuracy of evaluation of pelvic rotation, indicating a similar degree of precision of both techniques, with a Cramer’s V of 0.112 in boys and 0.042 in girls. Similarly, no significant differences were revealed in the frontal plane, also suggesting a similar precision (Tables \n1 and\n2). These results show that clinical and device-assisted topography is characterised by a similar degree of accuracy. Minor differences were noted with regard to trunk asymmetry in the frontal plane. The differences were not statistically significant (Table \n1). Clinical evaluation is thus a reliable method for assessing trunk asymmetry in the frontal plane and does not need to be confirmed by measurement system-based assessment.Table 1\nTrunk assessment in boys, sagittal and frontal planes\nTrunk assessmentCMS -10 evaluationClinical evaluationCalculated Chi\n2\nSignificance level p < 0.05Degrees of freedomTabular Chi\n2\nHypothesis acceptedCramer’s VKyphosis12.80.0525.9alternative0.309Accentuated2117Reduced2510Normal2140Total6767Lordosis25.10.0525.9alternative0.433Accentuated339Reduced1715Normal1743Total6767Pelvis1.70.0513.7null0.112Rotated4942Not rotated1825Total6767Asymmetry5.880.0525.9null0.173Shoulder girdle4825Inferior scapular angles4513Pelvic obliqueness3728Total13066Table 2\nTrunk assessment in girls, sagittal and frontal planes\nTrunk assessmentCMS -10 evaluationClinical evaluationCalculated Chi\n2\nSignificance level p < 0.05Degrees of freedomTabular Chi\n2\nHypothesis acceptedCramer’s VKyphosis37.50.0525.9alternative0.514Accentuated438Reduced613Normal2250Total7171Lordosis20.10.0525.9alternative0.377Accentuated237Reduced2212Normal2652Total7171Pelvis0.30.0513.7null0.042Rotated3740Not rotated3431Total7171Asymmetry3.90.0525.9null0.151Shoulder girdle5224Inferior scapular angles4218Pelvic obliqueness1817Total11259\n\nTrunk assessment in boys, sagittal and frontal planes\n\n\nTrunk assessment in girls, sagittal and frontal planes\n","Screening tests play a significant role in assessment of normal individual development in the population of children and adolescents. Accurate screening allows for selecting children and adolescents at risk for the development of postural defects or spinal and trunk deformities in order to refer them to appropriate specialists\n[20]. A clinical examination is the simplest and also the most common form of postural assessment.\nIn order to make the findings of clinical assessment more objective, measuring devices were gradually introduced in the 20th and 21st centuries, beginning with the Moire method, a photostereometric technique first used by Takasaki in 1970\n[21], followed by raster plots projected onto the object being assessed in raster photogrammetry for massive screening tests\n[22–24]. Modern devices for three-dimensional motion analysis (Metercom system) use the Saunders digital inclinometer and an anthropostereometric technique\n[15]. Techniques of video capturing of body posture are also available. Importantly, studies comparing postural parameters assessed using different devices do not reveal statistically significant differences in either device-to-device or device-to-clinical examination comparisons\n[25].\nNew achievements in objective assessment methods to support clinical examinations based on a mathematical system of three-dimensional body posture analysis were revealed by American and German centres as early as the late 1990’s and in the first decade of this century\n[26]. German studies show that the Zebris CMS-10 is a precise device that produces a detailed analysis of the trunk position based on anatomical skeletal reference landmarks in static positions with an option to expand sequences of functional movement\n[27]. Only static positions were analysed in the present study.\nThe present results confirm that the most difficult aspect of assessment of clinical deformities of the spine is the analysis of pathological spinal curvatures in the sagittal plane, while pelvic rotation and frontal positioning of the trunk are relatively easy to assess clinically. Similar results were obtained by Bibrowicz & Skolimowski\n[22]. Kyphosis and lordosis are subject to considerable interindividual variability and there are also no standards to define reference ranges for angle values in relation to sex and age in adolescents. The present study confirmed discrepancies between the populations of boys and girls. Abnormal spinal curvatures are one of many problems of adolescence\n[23]. The development and monitoring of a correct posture during the development of a child and adolescent is a prolonged process that depends on one’s somatic structure and the pace of individual development\n[3, 8]. The results for the frontal plane showed less discrepancy between the clinical examination and Zebris CMS-10-based assessment. Skolimowski et al. presented similar findings using other research tools\n[22].\nInternational scientific societies emphasise the need to verify clinical and scientific research to make it more objective. The terminological system proposed by SRS (Scoliosis Research Society) in 1994 reflects the three-dimensional nature of scoliosis and other spinal deformities\n[26]. The terminology serves the goal of promoting systematic descriptions of deformities and rationalising and facilitating examinations in clinical practice\n[28]. Consensus statements published by SOSORT (Society on Scoliosis Orthopeaedic and Rehabilitation Treatment) systematise the level of reliability of diagnostic and research procedures employed in the diagnosis of postural defects\n[2, 10–12].\nOur study shows that the Zebris CMS-10 system provides a detailed analysis of the position of set skeletal reference landmarks, thus representing a valuable adjunct to the clinical examination to increase the intrinsic value of screening tests.","\n\n\nThe finding of significant discrepancies between the results of assessment in the sagittal plane obtained in the clinical examination and Zebris CMS-10-based assessment suggests that clinical evaluation should be used to provide a general estimation of accentuation or reduction of spinal curvatures in the sagittal plane.\n\n\nThe clinical evaluation of posture is reliable with regard to assessment in the frontal plane.\n\n\nThe Zebris CMS-10 system makes the clinical examination significantly more objective with regard to assessment of the physiological curvatures and may be used to make screening tests more objective with regard to detecting postural defects.\n\n\n\nThe finding of significant discrepancies between the results of assessment in the sagittal plane obtained in the clinical examination and Zebris CMS-10-based assessment suggests that clinical evaluation should be used to provide a general estimation of accentuation or reduction of spinal curvatures in the sagittal plane.\nThe clinical evaluation of posture is reliable with regard to assessment in the frontal plane.\nThe Zebris CMS-10 system makes the clinical examination significantly more objective with regard to assessment of the physiological curvatures and may be used to make screening tests more objective with regard to detecting postural defects."," Additional file 1:\nSample examination protocol.\n(DOCX 14 KB)\nAdditional file 1:\nSample examination protocol.\n(DOCX 14 KB)","Additional file 1:\nSample examination protocol.\n(DOCX 14 KB)"],"string":"[\n \"Human body posture is a motor habit associated with daily activity with an underlying morphological and functional basis\\n[1]. It reflects the psychophysical status of the individual and is an index of mechanical efficiency of the kinaesthetic sense, muscle balance and musculomotor co-ordination\\n[2]. Normal human posture in the vertical position relies on the spine and its position against the head and pelvis\\n[3, 4]. The spatial relations among and between bony structures and articulations are stabilised by a system of fasciae, ligaments and muscles, while the central nervous system is the superior controller of body posture\\n[5, 6]. Body posture variability depends on age, sex and environmental factors influencing its development during body growth\\n[7, 8]. The following conditions are regarded as postural defects: abnormal shape of the physiological spinal curvatures, asymmetrical positioning of the shoulder or pelvic girdle, disturbance of the knee joint axis and abnormal shape of the foot arches. Screening studies of postural defects carried out at schools reveal that 50-60% of children and adolescents demonstrate body posture abnormalities, with 10% of this group at risk for scoliosis or other progressive spinal deformities\\n[9–12]. An alarmingly high percentage of these defects are attributable to poor motor activity of children and adolescents, rapid changes taking place in the body during individual development and excessive time spent in the seated position\\n[13]. An early and reliable detection programme for the population of children and adolescents combined with prophylactic measures to prevent the persistent spinal and trunk deformities is an appropriate strategy that can also mininise the medical and financial outcome of the more complex process of future treatment of postural defects and scolioses that might be necessary. The findings of a clinical evaluation of body posture and trunk asymmetry in a child depend on the experience of the examiner, compliance of the child and availability of bedside diagnostic equipment. Screening tests rely mainly on clinical evaluation since screening is supposed to be available to the entire population of children and adolescents. The easy availability and simple procedure used also need to guarantee a high reliability of diagnoses of postural defects. Non-invasive methods that will make diagnosis easier and more comprehensive are being sought to ensure more objective measurements. The Zebris CMS-10, a system for assessing body posture in three planes, offers a non-invasive method for evaluating the spatial positioning of selected topographic reference points in the frontal, sagittal and transverse planes, thus supplying objective data to support a clinical evaluation. The Zebris CMS 10 system demonstrates a high degree of test-retest reliability, intertester reliability and intratester reliability\\n[14, 15]. The inclinometer method demonstrates a high degree of intertester reliability and intratester reliability\\n[16, 17]. A variation of up to 1.5° was allowed using this technique. Measurements were repeated several times in each participant until two consecutive attempts by two independent examiners yielded the same angle values (including the admissible variation of 1.5°), thus complying with the principles of intertester and alternate-forms reliability. The aim of this study was to assess the reliability of clinical evaluation of body posture compared to objective assessment with the Zebris CMS-10 system.\",\n \"The methodology was approved by the Ethics Committee of the Rehabilitation Hospital for Children, Olsztyn, Poland. The study enrolled 13-15-year-old pupils attending a junior secondary school. The mean age was 14.2 (±0.6) years. The study group consisted of 138 participants, including 71 girls (mean age 14.1 ± 0.4 years, mean height 160.3 ± 3.4 cm, mean body weight 64.8 ± 3.9 kg) and 67 boys (mean age 14.4 ± 0.8 years, mean height 166.6 ± 2.9 cm, mean body weight 68.1 ± 3.6 kg). The exclusion criteria were a diagnosis of scoliosis and/or status post spinal surgery and/or feeling any pain. The screening test was carried out with the participants in a free standing position, involving specialists in rehabilitation as examiners and a Zebris CMS 10 system. The objective of the examination was not revealed to the examiners. In the first part, reference skeletal landmarks were marked on the body according to the principles of palpation anatomy. Trunk positioning was evaluated clinically in the sagittal and frontal planes. The findings were recorded in the study protocol (Additional file\\n1). Thoracic kyphosis and lumbar lordosis were evaluated in the sagittal plane with a Saunders inclinometer. Pelvic rotation was also evaluated in the sagittal plane. A Saunders inclinometer was placed in the cervicothoracic junction with the long arm pointing downwards from the spinous process at the apex of the curve and in the lumbosacral junction with the long arm pointing upwards from the spinous process at the apex of the curve (Figures \\n1,\\n2). The respective reference ranges assumed for kyphosis and lordosis were 30-40° and 25-35°\\n[18]. The symmetry of position of the shoulder and pelvic girdles was evaluated in the frontal plane.Figure 1\\nMarking of anatomical skeletal reference landmarks.\\nFigure 2\\nClinical examination with Saunders inclinometer. Reference marker is a belt attached below iliac spines.\\n\\nMarking of anatomical skeletal reference landmarks.\\n\\n\\nClinical examination with Saunders inclinometer. Reference marker is a belt attached below iliac spines.\\nFollowing the clinical evaluation, the same postural parameters were assessed with a Zebris CMS-10 device (Zebris Medical GmbH). The Zebris CMS 10 uses WinSpine software in the Microsoft Windows XP environment. Measurement error is defined by the manufacturer at 1.96 degrees and 2.2 millimeters for all parameters. Measurement sensitivity is 0.2 millimeters and 0.5 degrees. The software includes a data base of projects, patients and individual measurements. The core component of the testing system is a measuring device, an ultrasound point indicator probe and a reference marker\\n[19]. The testing device is placed on an adjustable-height arm. The point indicator probe, which is placed directly onto skeletal reference landmarks on the patient’s body, has two ultrasound markers with their central points aligned with the tip of the probe. The skeletal reference landmarks are all thoracic and lumbar spinous processes of the spine. The software precisely calculates the position of the probe. A reference marker in the form of a belt is attached laterally below the posterior superior iliac spines and anterior superior iliac spines so as not to cover the measurement sites. The reference marker is used to eliminate changes of position during the examination. The testing unit was composed of a platform with built-in levels, a Zebris CMS-10 system and a computer. A transverse valve was mounted at one-third of the length of the platform in order to immobilise the Zebris CMS-10 device. Owing to this, the device could be placed in a fixed position and random movements during use were eliminated. A transverse red line was marked permanently at 80 cm from the transverse valve. One side of a 25 × 25 cm square was drawn on this line. The square was contoured with black lines. The lateral sides of the square were used to indicate where the examinees should place their feet in the standing position. The examinees were also instructed to place their feet in front of the red transverse line (Figure \\n3). The device was calibrated against the ground before each examination.Figure 3\\nExamination with Zebris CMS-10. Ultrasound probe recording the position of marked skeletal landmarks.\\n\\nExamination with Zebris CMS-10. Ultrasound probe recording the position of marked skeletal landmarks.\\nFor statistical analysis of the clinical vs. Zebris-based assessment, physiological spinal curvatures in the sagittal plane were assigned a value of 0 and accentuation or reduction of the curvatures in the sagittal plane below 30° or above 40° for thoracic kyphosis and below 25° or above 35° for lumbar lordosis was assigned a value of 1. A symmetrical position of the pelvis was assigned a value of - 0, and pelvic rotation, a value of 1. Pelvic rotation was assessed manually as a deficit of rotation of the iliac bone relative to the sacral bone on the left and right side of the body. In the frontal plane, symmetry of the acromions and of the pelvis was assigned a value of - 0, and an asymmetry greater than 1 cm in the vertical dimension, a value of 1. The statistical analysis was conducted in Statistica 7 software package, version 10.1 and based on the calculation of means, percentages and the Chi2 test statistics (empirical and expected), and Cramer’s V statistic, which reflects the strength of association of two parameters. The level of significance was set at p < 0.05.\\nThe parents of the children in the study group had provided written consent for their children to participate in the study. All the patients gave their written consent prior to their inclusion in the study.\\nThe study, funded from a scientific grant, was conducted in the years 2011–2014.\",\n \"Cramer’s V values confirmed a significant correlation between the parameters in the case of lordosis and a clear correlation in the case of kyphosis. Thus, the study demonstrated that the Zebris CMS-10 system for three-dimensional analysis of body posture contributed a statistically significant adjustment to the clinical evaluation of the spine in the sagittal view; Cramer’s V was 0.514 for the evaluation of thoracic kyphosis in girls and 0.433 in the evaluation of lumbar lordosis in boys (Figures \\n4,\\n5,\\n6,\\n7,\\n8,\\n9).Figure 4\\nResults of evaluation of thoracic kyphosis in the sagittal plane in boys.\\nFigure 5\\nResults of evaluation of lumbar lordosis in the sagittal plane in boys.\\nFigure 6\\nResults of evaluation of pelvic rotation in the sagittal plane in boys.\\nFigure 7\\nResults of evaluation of thoracic kyphosis in the sagittal plane in girls.\\nFigure 8\\nResults of evaluation of lumbar lordosis in the sagittal plane in girls.\\nFigure 9\\nResults of evaluation of pelvic rotation in the sagittal plane in girls.\\n\\n\\nResults of evaluation of thoracic kyphosis in the sagittal plane in boys.\\n\\n\\nResults of evaluation of lumbar lordosis in the sagittal plane in boys.\\n\\n\\nResults of evaluation of pelvic rotation in the sagittal plane in boys.\\n\\n\\nResults of evaluation of thoracic kyphosis in the sagittal plane in girls.\\n\\n\\nResults of evaluation of lumbar lordosis in the sagittal plane in girls.\\n\\n\\nResults of evaluation of pelvic rotation in the sagittal plane in girls.\\n\\nNo statistically significant differences were found with regard to the accuracy of evaluation of pelvic rotation, indicating a similar degree of precision of both techniques, with a Cramer’s V of 0.112 in boys and 0.042 in girls. Similarly, no significant differences were revealed in the frontal plane, also suggesting a similar precision (Tables \\n1 and\\n2). These results show that clinical and device-assisted topography is characterised by a similar degree of accuracy. Minor differences were noted with regard to trunk asymmetry in the frontal plane. The differences were not statistically significant (Table \\n1). Clinical evaluation is thus a reliable method for assessing trunk asymmetry in the frontal plane and does not need to be confirmed by measurement system-based assessment.Table 1\\nTrunk assessment in boys, sagittal and frontal planes\\nTrunk assessmentCMS -10 evaluationClinical evaluationCalculated Chi\\n2\\nSignificance level p < 0.05Degrees of freedomTabular Chi\\n2\\nHypothesis acceptedCramer’s VKyphosis12.80.0525.9alternative0.309Accentuated2117Reduced2510Normal2140Total6767Lordosis25.10.0525.9alternative0.433Accentuated339Reduced1715Normal1743Total6767Pelvis1.70.0513.7null0.112Rotated4942Not rotated1825Total6767Asymmetry5.880.0525.9null0.173Shoulder girdle4825Inferior scapular angles4513Pelvic obliqueness3728Total13066Table 2\\nTrunk assessment in girls, sagittal and frontal planes\\nTrunk assessmentCMS -10 evaluationClinical evaluationCalculated Chi\\n2\\nSignificance level p < 0.05Degrees of freedomTabular Chi\\n2\\nHypothesis acceptedCramer’s VKyphosis37.50.0525.9alternative0.514Accentuated438Reduced613Normal2250Total7171Lordosis20.10.0525.9alternative0.377Accentuated237Reduced2212Normal2652Total7171Pelvis0.30.0513.7null0.042Rotated3740Not rotated3431Total7171Asymmetry3.90.0525.9null0.151Shoulder girdle5224Inferior scapular angles4218Pelvic obliqueness1817Total11259\\n\\nTrunk assessment in boys, sagittal and frontal planes\\n\\n\\nTrunk assessment in girls, sagittal and frontal planes\\n\",\n \"Screening tests play a significant role in assessment of normal individual development in the population of children and adolescents. Accurate screening allows for selecting children and adolescents at risk for the development of postural defects or spinal and trunk deformities in order to refer them to appropriate specialists\\n[20]. A clinical examination is the simplest and also the most common form of postural assessment.\\nIn order to make the findings of clinical assessment more objective, measuring devices were gradually introduced in the 20th and 21st centuries, beginning with the Moire method, a photostereometric technique first used by Takasaki in 1970\\n[21], followed by raster plots projected onto the object being assessed in raster photogrammetry for massive screening tests\\n[22–24]. Modern devices for three-dimensional motion analysis (Metercom system) use the Saunders digital inclinometer and an anthropostereometric technique\\n[15]. Techniques of video capturing of body posture are also available. Importantly, studies comparing postural parameters assessed using different devices do not reveal statistically significant differences in either device-to-device or device-to-clinical examination comparisons\\n[25].\\nNew achievements in objective assessment methods to support clinical examinations based on a mathematical system of three-dimensional body posture analysis were revealed by American and German centres as early as the late 1990’s and in the first decade of this century\\n[26]. German studies show that the Zebris CMS-10 is a precise device that produces a detailed analysis of the trunk position based on anatomical skeletal reference landmarks in static positions with an option to expand sequences of functional movement\\n[27]. Only static positions were analysed in the present study.\\nThe present results confirm that the most difficult aspect of assessment of clinical deformities of the spine is the analysis of pathological spinal curvatures in the sagittal plane, while pelvic rotation and frontal positioning of the trunk are relatively easy to assess clinically. Similar results were obtained by Bibrowicz & Skolimowski\\n[22]. Kyphosis and lordosis are subject to considerable interindividual variability and there are also no standards to define reference ranges for angle values in relation to sex and age in adolescents. The present study confirmed discrepancies between the populations of boys and girls. Abnormal spinal curvatures are one of many problems of adolescence\\n[23]. The development and monitoring of a correct posture during the development of a child and adolescent is a prolonged process that depends on one’s somatic structure and the pace of individual development\\n[3, 8]. The results for the frontal plane showed less discrepancy between the clinical examination and Zebris CMS-10-based assessment. Skolimowski et al. presented similar findings using other research tools\\n[22].\\nInternational scientific societies emphasise the need to verify clinical and scientific research to make it more objective. The terminological system proposed by SRS (Scoliosis Research Society) in 1994 reflects the three-dimensional nature of scoliosis and other spinal deformities\\n[26]. The terminology serves the goal of promoting systematic descriptions of deformities and rationalising and facilitating examinations in clinical practice\\n[28]. Consensus statements published by SOSORT (Society on Scoliosis Orthopeaedic and Rehabilitation Treatment) systematise the level of reliability of diagnostic and research procedures employed in the diagnosis of postural defects\\n[2, 10–12].\\nOur study shows that the Zebris CMS-10 system provides a detailed analysis of the position of set skeletal reference landmarks, thus representing a valuable adjunct to the clinical examination to increase the intrinsic value of screening tests.\",\n \"\\n\\n\\nThe finding of significant discrepancies between the results of assessment in the sagittal plane obtained in the clinical examination and Zebris CMS-10-based assessment suggests that clinical evaluation should be used to provide a general estimation of accentuation or reduction of spinal curvatures in the sagittal plane.\\n\\n\\nThe clinical evaluation of posture is reliable with regard to assessment in the frontal plane.\\n\\n\\nThe Zebris CMS-10 system makes the clinical examination significantly more objective with regard to assessment of the physiological curvatures and may be used to make screening tests more objective with regard to detecting postural defects.\\n\\n\\n\\nThe finding of significant discrepancies between the results of assessment in the sagittal plane obtained in the clinical examination and Zebris CMS-10-based assessment suggests that clinical evaluation should be used to provide a general estimation of accentuation or reduction of spinal curvatures in the sagittal plane.\\nThe clinical evaluation of posture is reliable with regard to assessment in the frontal plane.\\nThe Zebris CMS-10 system makes the clinical examination significantly more objective with regard to assessment of the physiological curvatures and may be used to make screening tests more objective with regard to detecting postural defects.\",\n \" Additional file 1:\\nSample examination protocol.\\n(DOCX 14 KB)\\nAdditional file 1:\\nSample examination protocol.\\n(DOCX 14 KB)\",\n \"Additional file 1:\\nSample examination protocol.\\n(DOCX 14 KB)\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":[null,"methods","results","discussion","conclusions","supplementary-material",null],"string":"[\n null,\n \"methods\",\n \"results\",\n \"discussion\",\n \"conclusions\",\n \"supplementary-material\",\n null\n]","isConversational":false},"keywords":{"kind":"list like","value":["Postural defects","Spinal deformities","Screening tests","Topography"],"string":"[\n \"Postural defects\",\n \"Spinal deformities\",\n \"Screening tests\",\n \"Topography\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Background:\nHuman body posture is a motor habit associated with daily activity with an underlying morphological and functional basis\n[1]. It reflects the psychophysical status of the individual and is an index of mechanical efficiency of the kinaesthetic sense, muscle balance and musculomotor co-ordination\n[2]. Normal human posture in the vertical position relies on the spine and its position against the head and pelvis\n[3, 4]. The spatial relations among and between bony structures and articulations are stabilised by a system of fasciae, ligaments and muscles, while the central nervous system is the superior controller of body posture\n[5, 6]. Body posture variability depends on age, sex and environmental factors influencing its development during body growth\n[7, 8]. The following conditions are regarded as postural defects: abnormal shape of the physiological spinal curvatures, asymmetrical positioning of the shoulder or pelvic girdle, disturbance of the knee joint axis and abnormal shape of the foot arches. Screening studies of postural defects carried out at schools reveal that 50-60% of children and adolescents demonstrate body posture abnormalities, with 10% of this group at risk for scoliosis or other progressive spinal deformities\n[9–12]. An alarmingly high percentage of these defects are attributable to poor motor activity of children and adolescents, rapid changes taking place in the body during individual development and excessive time spent in the seated position\n[13]. An early and reliable detection programme for the population of children and adolescents combined with prophylactic measures to prevent the persistent spinal and trunk deformities is an appropriate strategy that can also mininise the medical and financial outcome of the more complex process of future treatment of postural defects and scolioses that might be necessary. The findings of a clinical evaluation of body posture and trunk asymmetry in a child depend on the experience of the examiner, compliance of the child and availability of bedside diagnostic equipment. Screening tests rely mainly on clinical evaluation since screening is supposed to be available to the entire population of children and adolescents. The easy availability and simple procedure used also need to guarantee a high reliability of diagnoses of postural defects. Non-invasive methods that will make diagnosis easier and more comprehensive are being sought to ensure more objective measurements. The Zebris CMS-10, a system for assessing body posture in three planes, offers a non-invasive method for evaluating the spatial positioning of selected topographic reference points in the frontal, sagittal and transverse planes, thus supplying objective data to support a clinical evaluation. The Zebris CMS 10 system demonstrates a high degree of test-retest reliability, intertester reliability and intratester reliability\n[14, 15]. The inclinometer method demonstrates a high degree of intertester reliability and intratester reliability\n[16, 17]. A variation of up to 1.5° was allowed using this technique. Measurements were repeated several times in each participant until two consecutive attempts by two independent examiners yielded the same angle values (including the admissible variation of 1.5°), thus complying with the principles of intertester and alternate-forms reliability. The aim of this study was to assess the reliability of clinical evaluation of body posture compared to objective assessment with the Zebris CMS-10 system.\n\nMethods:\nThe methodology was approved by the Ethics Committee of the Rehabilitation Hospital for Children, Olsztyn, Poland. The study enrolled 13-15-year-old pupils attending a junior secondary school. The mean age was 14.2 (±0.6) years. The study group consisted of 138 participants, including 71 girls (mean age 14.1 ± 0.4 years, mean height 160.3 ± 3.4 cm, mean body weight 64.8 ± 3.9 kg) and 67 boys (mean age 14.4 ± 0.8 years, mean height 166.6 ± 2.9 cm, mean body weight 68.1 ± 3.6 kg). The exclusion criteria were a diagnosis of scoliosis and/or status post spinal surgery and/or feeling any pain. The screening test was carried out with the participants in a free standing position, involving specialists in rehabilitation as examiners and a Zebris CMS 10 system. The objective of the examination was not revealed to the examiners. In the first part, reference skeletal landmarks were marked on the body according to the principles of palpation anatomy. Trunk positioning was evaluated clinically in the sagittal and frontal planes. The findings were recorded in the study protocol (Additional file\n1). Thoracic kyphosis and lumbar lordosis were evaluated in the sagittal plane with a Saunders inclinometer. Pelvic rotation was also evaluated in the sagittal plane. A Saunders inclinometer was placed in the cervicothoracic junction with the long arm pointing downwards from the spinous process at the apex of the curve and in the lumbosacral junction with the long arm pointing upwards from the spinous process at the apex of the curve (Figures \n1,\n2). The respective reference ranges assumed for kyphosis and lordosis were 30-40° and 25-35°\n[18]. The symmetry of position of the shoulder and pelvic girdles was evaluated in the frontal plane.Figure 1\nMarking of anatomical skeletal reference landmarks.\nFigure 2\nClinical examination with Saunders inclinometer. Reference marker is a belt attached below iliac spines.\n\nMarking of anatomical skeletal reference landmarks.\n\n\nClinical examination with Saunders inclinometer. Reference marker is a belt attached below iliac spines.\nFollowing the clinical evaluation, the same postural parameters were assessed with a Zebris CMS-10 device (Zebris Medical GmbH). The Zebris CMS 10 uses WinSpine software in the Microsoft Windows XP environment. Measurement error is defined by the manufacturer at 1.96 degrees and 2.2 millimeters for all parameters. Measurement sensitivity is 0.2 millimeters and 0.5 degrees. The software includes a data base of projects, patients and individual measurements. The core component of the testing system is a measuring device, an ultrasound point indicator probe and a reference marker\n[19]. The testing device is placed on an adjustable-height arm. The point indicator probe, which is placed directly onto skeletal reference landmarks on the patient’s body, has two ultrasound markers with their central points aligned with the tip of the probe. The skeletal reference landmarks are all thoracic and lumbar spinous processes of the spine. The software precisely calculates the position of the probe. A reference marker in the form of a belt is attached laterally below the posterior superior iliac spines and anterior superior iliac spines so as not to cover the measurement sites. The reference marker is used to eliminate changes of position during the examination. The testing unit was composed of a platform with built-in levels, a Zebris CMS-10 system and a computer. A transverse valve was mounted at one-third of the length of the platform in order to immobilise the Zebris CMS-10 device. Owing to this, the device could be placed in a fixed position and random movements during use were eliminated. A transverse red line was marked permanently at 80 cm from the transverse valve. One side of a 25 × 25 cm square was drawn on this line. The square was contoured with black lines. The lateral sides of the square were used to indicate where the examinees should place their feet in the standing position. The examinees were also instructed to place their feet in front of the red transverse line (Figure \n3). The device was calibrated against the ground before each examination.Figure 3\nExamination with Zebris CMS-10. Ultrasound probe recording the position of marked skeletal landmarks.\n\nExamination with Zebris CMS-10. Ultrasound probe recording the position of marked skeletal landmarks.\nFor statistical analysis of the clinical vs. Zebris-based assessment, physiological spinal curvatures in the sagittal plane were assigned a value of 0 and accentuation or reduction of the curvatures in the sagittal plane below 30° or above 40° for thoracic kyphosis and below 25° or above 35° for lumbar lordosis was assigned a value of 1. A symmetrical position of the pelvis was assigned a value of - 0, and pelvic rotation, a value of 1. Pelvic rotation was assessed manually as a deficit of rotation of the iliac bone relative to the sacral bone on the left and right side of the body. In the frontal plane, symmetry of the acromions and of the pelvis was assigned a value of - 0, and an asymmetry greater than 1 cm in the vertical dimension, a value of 1. The statistical analysis was conducted in Statistica 7 software package, version 10.1 and based on the calculation of means, percentages and the Chi2 test statistics (empirical and expected), and Cramer’s V statistic, which reflects the strength of association of two parameters. The level of significance was set at p < 0.05.\nThe parents of the children in the study group had provided written consent for their children to participate in the study. All the patients gave their written consent prior to their inclusion in the study.\nThe study, funded from a scientific grant, was conducted in the years 2011–2014.\n\nResults:\nCramer’s V values confirmed a significant correlation between the parameters in the case of lordosis and a clear correlation in the case of kyphosis. Thus, the study demonstrated that the Zebris CMS-10 system for three-dimensional analysis of body posture contributed a statistically significant adjustment to the clinical evaluation of the spine in the sagittal view; Cramer’s V was 0.514 for the evaluation of thoracic kyphosis in girls and 0.433 in the evaluation of lumbar lordosis in boys (Figures \n4,\n5,\n6,\n7,\n8,\n9).Figure 4\nResults of evaluation of thoracic kyphosis in the sagittal plane in boys.\nFigure 5\nResults of evaluation of lumbar lordosis in the sagittal plane in boys.\nFigure 6\nResults of evaluation of pelvic rotation in the sagittal plane in boys.\nFigure 7\nResults of evaluation of thoracic kyphosis in the sagittal plane in girls.\nFigure 8\nResults of evaluation of lumbar lordosis in the sagittal plane in girls.\nFigure 9\nResults of evaluation of pelvic rotation in the sagittal plane in girls.\n\n\nResults of evaluation of thoracic kyphosis in the sagittal plane in boys.\n\n\nResults of evaluation of lumbar lordosis in the sagittal plane in boys.\n\n\nResults of evaluation of pelvic rotation in the sagittal plane in boys.\n\n\nResults of evaluation of thoracic kyphosis in the sagittal plane in girls.\n\n\nResults of evaluation of lumbar lordosis in the sagittal plane in girls.\n\n\nResults of evaluation of pelvic rotation in the sagittal plane in girls.\n\nNo statistically significant differences were found with regard to the accuracy of evaluation of pelvic rotation, indicating a similar degree of precision of both techniques, with a Cramer’s V of 0.112 in boys and 0.042 in girls. Similarly, no significant differences were revealed in the frontal plane, also suggesting a similar precision (Tables \n1 and\n2). These results show that clinical and device-assisted topography is characterised by a similar degree of accuracy. Minor differences were noted with regard to trunk asymmetry in the frontal plane. The differences were not statistically significant (Table \n1). Clinical evaluation is thus a reliable method for assessing trunk asymmetry in the frontal plane and does not need to be confirmed by measurement system-based assessment.Table 1\nTrunk assessment in boys, sagittal and frontal planes\nTrunk assessmentCMS -10 evaluationClinical evaluationCalculated Chi\n2\nSignificance level p < 0.05Degrees of freedomTabular Chi\n2\nHypothesis acceptedCramer’s VKyphosis12.80.0525.9alternative0.309Accentuated2117Reduced2510Normal2140Total6767Lordosis25.10.0525.9alternative0.433Accentuated339Reduced1715Normal1743Total6767Pelvis1.70.0513.7null0.112Rotated4942Not rotated1825Total6767Asymmetry5.880.0525.9null0.173Shoulder girdle4825Inferior scapular angles4513Pelvic obliqueness3728Total13066Table 2\nTrunk assessment in girls, sagittal and frontal planes\nTrunk assessmentCMS -10 evaluationClinical evaluationCalculated Chi\n2\nSignificance level p < 0.05Degrees of freedomTabular Chi\n2\nHypothesis acceptedCramer’s VKyphosis37.50.0525.9alternative0.514Accentuated438Reduced613Normal2250Total7171Lordosis20.10.0525.9alternative0.377Accentuated237Reduced2212Normal2652Total7171Pelvis0.30.0513.7null0.042Rotated3740Not rotated3431Total7171Asymmetry3.90.0525.9null0.151Shoulder girdle5224Inferior scapular angles4218Pelvic obliqueness1817Total11259\n\nTrunk assessment in boys, sagittal and frontal planes\n\n\nTrunk assessment in girls, sagittal and frontal planes\n\n\nDiscussion:\nScreening tests play a significant role in assessment of normal individual development in the population of children and adolescents. Accurate screening allows for selecting children and adolescents at risk for the development of postural defects or spinal and trunk deformities in order to refer them to appropriate specialists\n[20]. A clinical examination is the simplest and also the most common form of postural assessment.\nIn order to make the findings of clinical assessment more objective, measuring devices were gradually introduced in the 20th and 21st centuries, beginning with the Moire method, a photostereometric technique first used by Takasaki in 1970\n[21], followed by raster plots projected onto the object being assessed in raster photogrammetry for massive screening tests\n[22–24]. Modern devices for three-dimensional motion analysis (Metercom system) use the Saunders digital inclinometer and an anthropostereometric technique\n[15]. Techniques of video capturing of body posture are also available. Importantly, studies comparing postural parameters assessed using different devices do not reveal statistically significant differences in either device-to-device or device-to-clinical examination comparisons\n[25].\nNew achievements in objective assessment methods to support clinical examinations based on a mathematical system of three-dimensional body posture analysis were revealed by American and German centres as early as the late 1990’s and in the first decade of this century\n[26]. German studies show that the Zebris CMS-10 is a precise device that produces a detailed analysis of the trunk position based on anatomical skeletal reference landmarks in static positions with an option to expand sequences of functional movement\n[27]. Only static positions were analysed in the present study.\nThe present results confirm that the most difficult aspect of assessment of clinical deformities of the spine is the analysis of pathological spinal curvatures in the sagittal plane, while pelvic rotation and frontal positioning of the trunk are relatively easy to assess clinically. Similar results were obtained by Bibrowicz & Skolimowski\n[22]. Kyphosis and lordosis are subject to considerable interindividual variability and there are also no standards to define reference ranges for angle values in relation to sex and age in adolescents. The present study confirmed discrepancies between the populations of boys and girls. Abnormal spinal curvatures are one of many problems of adolescence\n[23]. The development and monitoring of a correct posture during the development of a child and adolescent is a prolonged process that depends on one’s somatic structure and the pace of individual development\n[3, 8]. The results for the frontal plane showed less discrepancy between the clinical examination and Zebris CMS-10-based assessment. Skolimowski et al. presented similar findings using other research tools\n[22].\nInternational scientific societies emphasise the need to verify clinical and scientific research to make it more objective. The terminological system proposed by SRS (Scoliosis Research Society) in 1994 reflects the three-dimensional nature of scoliosis and other spinal deformities\n[26]. The terminology serves the goal of promoting systematic descriptions of deformities and rationalising and facilitating examinations in clinical practice\n[28]. Consensus statements published by SOSORT (Society on Scoliosis Orthopeaedic and Rehabilitation Treatment) systematise the level of reliability of diagnostic and research procedures employed in the diagnosis of postural defects\n[2, 10–12].\nOur study shows that the Zebris CMS-10 system provides a detailed analysis of the position of set skeletal reference landmarks, thus representing a valuable adjunct to the clinical examination to increase the intrinsic value of screening tests.\n\nConclusions:\n\n\n\nThe finding of significant discrepancies between the results of assessment in the sagittal plane obtained in the clinical examination and Zebris CMS-10-based assessment suggests that clinical evaluation should be used to provide a general estimation of accentuation or reduction of spinal curvatures in the sagittal plane.\n\n\nThe clinical evaluation of posture is reliable with regard to assessment in the frontal plane.\n\n\nThe Zebris CMS-10 system makes the clinical examination significantly more objective with regard to assessment of the physiological curvatures and may be used to make screening tests more objective with regard to detecting postural defects.\n\n\n\nThe finding of significant discrepancies between the results of assessment in the sagittal plane obtained in the clinical examination and Zebris CMS-10-based assessment suggests that clinical evaluation should be used to provide a general estimation of accentuation or reduction of spinal curvatures in the sagittal plane.\nThe clinical evaluation of posture is reliable with regard to assessment in the frontal plane.\nThe Zebris CMS-10 system makes the clinical examination significantly more objective with regard to assessment of the physiological curvatures and may be used to make screening tests more objective with regard to detecting postural defects.\n\nElectronic supplementary material:\n Additional file 1:\nSample examination protocol.\n(DOCX 14 KB)\nAdditional file 1:\nSample examination protocol.\n(DOCX 14 KB)\n\n:\nAdditional file 1:\nSample examination protocol.\n(DOCX 14 KB)\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nScreening tests play a significant role in rapid and reliable assessment of normal individual development in the entire population of children and adolescents. Body posture screening tests carried out at schools reveal that 50-60% of children and adolescents demonstrate body posture abnormalities, with 10% of this group at risk for progressive spinal deformities. This necessitates the search for effective and economically feasible forms of screening diagnosis. The aim of this study was to assess the reliability of clinical evaluation of body posture compared to objective assessment with the Zebris CMS-10 system (Zebris Medical GmbH).\n\nMethods:\nThe study enrolled 13-15-year-old pupils attending a junior secondary school (mean age 14.2 years). The study group consisted of 138 participants, including 71 girls and 67 boys, who underwent a clinical evaluation of the body posture and an examination with the Zebris CMS 10 system.\n\nResults:\nStatistically significant discrepancies between the clinical and objective evaluation were noted with regard to lumbar lordosis in boys (n = 67) and thoracic kyphosis in girls (n = 71). No statistically significant differences in both groups were noted for pelvic rotation and trunk position in the frontal plane.\n\nConclusions:\n1. The finding of significant discrepancies between the results of assessment in the sagittal plane obtained in the clinical examination and Zebris CMS-10-based assessment suggests that clinical evaluation should be used to provide a general estimation of accentuation or reduction of spinal curvatures in the sagittal plane.2. The clinical evaluation of posture is reliable with regard to assessment in the frontal plane.3. The Zebris CMS-10 system makes the clinical examination significantly more objective with regard to assessment of the physiological curvatures and may be used to make screening tests more objective with regard to detecting postural defects."},"background_conclusion_text":{"kind":"string","value":"Background:\nHuman body posture is a motor habit associated with daily activity with an underlying morphological and functional basis\n[1]. It reflects the psychophysical status of the individual and is an index of mechanical efficiency of the kinaesthetic sense, muscle balance and musculomotor co-ordination\n[2]. Normal human posture in the vertical position relies on the spine and its position against the head and pelvis\n[3, 4]. The spatial relations among and between bony structures and articulations are stabilised by a system of fasciae, ligaments and muscles, while the central nervous system is the superior controller of body posture\n[5, 6]. Body posture variability depends on age, sex and environmental factors influencing its development during body growth\n[7, 8]. The following conditions are regarded as postural defects: abnormal shape of the physiological spinal curvatures, asymmetrical positioning of the shoulder or pelvic girdle, disturbance of the knee joint axis and abnormal shape of the foot arches. Screening studies of postural defects carried out at schools reveal that 50-60% of children and adolescents demonstrate body posture abnormalities, with 10% of this group at risk for scoliosis or other progressive spinal deformities\n[9–12]. An alarmingly high percentage of these defects are attributable to poor motor activity of children and adolescents, rapid changes taking place in the body during individual development and excessive time spent in the seated position\n[13]. An early and reliable detection programme for the population of children and adolescents combined with prophylactic measures to prevent the persistent spinal and trunk deformities is an appropriate strategy that can also mininise the medical and financial outcome of the more complex process of future treatment of postural defects and scolioses that might be necessary. The findings of a clinical evaluation of body posture and trunk asymmetry in a child depend on the experience of the examiner, compliance of the child and availability of bedside diagnostic equipment. Screening tests rely mainly on clinical evaluation since screening is supposed to be available to the entire population of children and adolescents. The easy availability and simple procedure used also need to guarantee a high reliability of diagnoses of postural defects. Non-invasive methods that will make diagnosis easier and more comprehensive are being sought to ensure more objective measurements. The Zebris CMS-10, a system for assessing body posture in three planes, offers a non-invasive method for evaluating the spatial positioning of selected topographic reference points in the frontal, sagittal and transverse planes, thus supplying objective data to support a clinical evaluation. The Zebris CMS 10 system demonstrates a high degree of test-retest reliability, intertester reliability and intratester reliability\n[14, 15]. The inclinometer method demonstrates a high degree of intertester reliability and intratester reliability\n[16, 17]. A variation of up to 1.5° was allowed using this technique. Measurements were repeated several times in each participant until two consecutive attempts by two independent examiners yielded the same angle values (including the admissible variation of 1.5°), thus complying with the principles of intertester and alternate-forms reliability. The aim of this study was to assess the reliability of clinical evaluation of body posture compared to objective assessment with the Zebris CMS-10 system.\n\nConclusions:\n\n\n\nThe finding of significant discrepancies between the results of assessment in the sagittal plane obtained in the clinical examination and Zebris CMS-10-based assessment suggests that clinical evaluation should be used to provide a general estimation of accentuation or reduction of spinal curvatures in the sagittal plane.\n\n\nThe clinical evaluation of posture is reliable with regard to assessment in the frontal plane.\n\n\nThe Zebris CMS-10 system makes the clinical examination significantly more objective with regard to assessment of the physiological curvatures and may be used to make screening tests more objective with regard to detecting postural defects.\n\n\n\nThe finding of significant discrepancies between the results of assessment in the sagittal plane obtained in the clinical examination and Zebris CMS-10-based assessment suggests that clinical evaluation should be used to provide a general estimation of accentuation or reduction of spinal curvatures in the sagittal plane.\nThe clinical evaluation of posture is reliable with regard to assessment in the frontal plane.\nThe Zebris CMS-10 system makes the clinical examination significantly more objective with regard to assessment of the physiological curvatures and may be used to make screening tests more objective with regard to detecting postural defects."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nScreening tests play a significant role in rapid and reliable assessment of normal individual development in the entire population of children and adolescents. Body posture screening tests carried out at schools reveal that 50-60% of children and adolescents demonstrate body posture abnormalities, with 10% of this group at risk for progressive spinal deformities. This necessitates the search for effective and economically feasible forms of screening diagnosis. The aim of this study was to assess the reliability of clinical evaluation of body posture compared to objective assessment with the Zebris CMS-10 system (Zebris Medical GmbH).\n\nMethods:\nThe study enrolled 13-15-year-old pupils attending a junior secondary school (mean age 14.2 years). The study group consisted of 138 participants, including 71 girls and 67 boys, who underwent a clinical evaluation of the body posture and an examination with the Zebris CMS 10 system.\n\nResults:\nStatistically significant discrepancies between the clinical and objective evaluation were noted with regard to lumbar lordosis in boys (n = 67) and thoracic kyphosis in girls (n = 71). No statistically significant differences in both groups were noted for pelvic rotation and trunk position in the frontal plane.\n\nConclusions:\n1. The finding of significant discrepancies between the results of assessment in the sagittal plane obtained in the clinical examination and Zebris CMS-10-based assessment suggests that clinical evaluation should be used to provide a general estimation of accentuation or reduction of spinal curvatures in the sagittal plane.2. The clinical evaluation of posture is reliable with regard to assessment in the frontal plane.3. The Zebris CMS-10 system makes the clinical examination significantly more objective with regard to assessment of the physiological curvatures and may be used to make screening tests more objective with regard to detecting postural defects."},"whole_article_text_length":{"kind":"number","value":3178,"string":"3,178"},"whole_article_abstract_length":{"kind":"number","value":334,"string":"334"},"other_sections_lengths":{"kind":"list like","value":[604,15],"string":"[\n 604,\n 15\n]","isConversational":false},"num_sections":{"kind":"number","value":7,"string":"7"},"most_frequent_words":{"kind":"list like","value":["plane","clinical","sagittal","evaluation","10","sagittal plane","assessment","zebris","results","examination"],"string":"[\n \"plane\",\n \"clinical\",\n \"sagittal\",\n \"evaluation\",\n \"10\",\n \"sagittal plane\",\n \"assessment\",\n \"zebris\",\n \"results\",\n \"examination\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["body posture 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[SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] examination | clinical | plane | examination protocol | file sample examination protocol | file sample examination | file sample | sample | sample examination | sample examination protocol [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 50-60% | 10% ||| ||| Zebris | Zebris Medical GmbH [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 13 | 15-year-old | age 14.2 years ||| 138 | 71 | 67 | Zebris | 10 [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 67 | 71 ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] 1 ||| Zebris ||| ||| Zebris [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 50-60% | 10% ||| ||| Zebris | Zebris Medical GmbH ||| 13 | 15-year-old | age 14.2 years ||| 138 | 71 | 67 | Zebris | 10 ||| 67 | 71 ||| ||| 1 ||| Zebris ||| ||| Zebris [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 50-60% | 10% ||| ||| Zebris | Zebris Medical GmbH ||| 13 | 15-year-old | age 14.2 years ||| 138 | 71 | 67 | Zebris | 10 ||| 67 | 71 ||| ||| 1 ||| Zebris ||| ||| Zebris [SUMMARY]"}}},{"rowIdx":82,"cells":{"title":{"kind":"string","value":"Accelerative effect of topical "},"pmid":{"kind":"string","value":"33378625"},"background_abstract":{"kind":"string","value":"Zataria multiflora Boiss (Lamiaceae) essential oil (ZME) is believed to be a bactericide herbal medicine and might alleviate negative effects of infection."},"background_abstract_label":{"kind":"string","value":"CONTEXT"},"methods_abstract":{"kind":"string","value":"A full-thickness excisional skin wound was surgically created in each mouse and inoculated with 5 × 107 suspension containing Pseudomonas aeruginosa and Staphylococcus aureus. The BALB/c mice (n = 72) were divided into four groups: (1) negative control that received base ointment (NCG), (2) positive control that daily received Mupirocin® (MG), (3) therapeutic ointment containing 2% ZMEO and (4) therapeutic ointment containing 4% ZMEO, for 21 days. Wound contraction, total bacterial count, histopathological parameters, antioxidant activity, qRT-PCR analysis for expression of IL-1β, TNF-α, VEGF, IGF-1, TGF-β, IL-10, and FGF-2 mRNA levels were assessed on days 3, 7, and 14 following the wounding."},"methods_abstract_label":{"kind":"string","value":"MATERIALS AND METHODS"},"results_abstract":{"kind":"string","value":"Topical administration of ZMEO significantly decreased the total bacterial count and wound area and also expression of IL-1β and TNF-α compared to the control groups (p < 0.05) in all days. This could also increase significantly the expression of TGF-β, IL-10 IGF-1, FGF-2, and VEGF, and also angiogenesis, fibroblasts, fibrocytes, epithelialization ratio, and collagen deposition and improve antioxidant status compared to the control group (p < 0.05)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"ZMEO accelerated the healing process of infected wounds by shortening the inflammatory factors and increasing proliferative phase. Applying ZMEO only and/or in combination with chemical agents for the treatment of wound healing could be suggested."},"conclusions_abstract_label":{"kind":"string","value":"DISCUSSION AND CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Administration, Topical","Animals","Collagen","Inflammation","Lamiaceae","Mice","Mice, Inbred BALB C","Neovascularization, Pathologic","Oils, Volatile","Surgical Wound Infection","Wound Healing"],"string":"[\n \"Administration, Topical\",\n \"Animals\",\n \"Collagen\",\n \"Inflammation\",\n \"Lamiaceae\",\n \"Mice\",\n \"Mice, Inbred BALB C\",\n \"Neovascularization, Pathologic\",\n \"Oils, Volatile\",\n \"Surgical Wound Infection\",\n \"Wound Healing\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"7782911"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Wound infections are important challenges that cause the suffering of patients and economic losses (Vittorazzi et al. 2016; Daemi et al. 2019a; Farghali et al. 2019). Skin is a regenerative organ which acts as a barrier between the body and the external environment (Hassan et al. 2014). A wound is defined as a disturbance in anatomic structure of skin and its functional integrity (Farghali et al. 2019). Skin prevents the penetration of bacteria and fungi that are important challenges that induce mortality and morbidity. Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic bacteria that induce infection in patients (Cardona and Wilson 2015). The bacteria are commonly detected in the upper and deepest region of wound bed (Serra et al. 2015). Colonization of S. aureus and P. aeruginosa in the wound site postpones the wound healing process (Farahpour et al. 2020; Khezri et al. 2020). The wound healing process comprises several phases, including coagulation, inflammation, epithelialization, granulation tissue formation, and tissue remodelling (Daemi et al. 2019a, 2019b; Farahpour et al. 2020). Inflammatory phase occurs after activation of inflammatory chemokines (Farahpour et al. 2020). Interleukin-1β (IL-1β) is significantly synthesized 12–24 h after infliction of the wound, and its level returns to basal level after the proliferative stage is completed (Fahey and Doyle 2019). Tumour necrosis factor-α (TNF-α) is significantly synthesized by macrophages and T lymphocytes and its level increases under inflammation and infection conditions (Xue and Falcon 2019). Following the induction of inflammation, proliferative phase occurs in which many genes are involved. Insulin-like growth factor 1 (IGF-1) promotes the production of keratinocyte and fibroblast proliferation and improves the re-epithelialization (Daemi et al. 2019b). Decreased endothelial insulin/IGF-1 signal delays in the wound healing process (Aghdam et al. 2012). Vascular endothelial growth factor (VEGF) induces angiogenesis and stimulates cell migration and proliferation (Farahpour et al. 2020). Fibroblast growth factor-2 (FGF-2) is a protein that participates in the wound healing process (Souto et al. 2020). Interleukin-10 (IL-10) reduces the production of pro-inflammatory cytokine, but tumour growth factor-β (TGF-β) has roles in increasing the proliferative phase improving the proliferation and differentiation of fibroblasts, collagen production, and wound contraction (Khezri et al. 2020). On the other hand, oxidative stress increases under wound disorders and increases the production of reactive oxygen species (ROS) in the site of wound (Ustuner et al. 2019). Applying antibacterial and antioxidant agents shortens the inflammatory phase, promotes the proliferative phase and accelerates the wound healing process (Bardaa et al. 2016; Vittorazzi et al. 2016; Daemi et al. 2019a, 2019b).\nZataria multiflora Boiss (Lamiaceae) (ZM) has antibacterial activity against Pseudomonas spp. (Barkhori-Mehni et al. 2017). Its antibacterial activity is attributed to its phenolic compounds that destroy the cell wall in bacteria (Barkhori-Mehni et al. 2017). It demonstrates antioxidant properties due to its compounds such as thymol and carvacrol (Mojaddar Langroodi et al. 2019). Seemingly, ZM can accelerate the wound healing process due to its antibacterial and antioxidant features, but no studies have been conducted yet to evaluate the Zataria multiflora essential oil (ZME) on the wound healing process. Therefore, this study was conducted to evaluate the effect of an ointment prepared from ZME on the healing process of infected wound model by S. aureus and P. aeruginosa by assessing wound contraction, total bacterial count, histopathological and immunohistochemical, antioxidant activity, and qRT-PCR analyses."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":" Composition of the Zataria multiflora essential oil The analysis of Zataria multiflora essential oil with GC-FID and GC-mass spectrometry (MS) identified 29 compounds which accounted for 99.6% of the total essential oil composition (Table 1). It contained thymol (52.90%), p-cymene (9.10), γ-terpinene (8.10%) and carvacrol (6.80%) as major constituents (Figure 1).\nGC-MS chromatogram of the essential oil of Zataria multiflora.\nChemical constituents of Zataria multiflora essential oil.\nRI-Cal: retention indices calculated based on C6–C24\nn-alkenes from a DB-5 column.\nRI-Cal: retention indices retrieved from literature (Adams 2007).\nBold values show major compounds in the essential oil.\nThe analysis of Zataria multiflora essential oil with GC-FID and GC-mass spectrometry (MS) identified 29 compounds which accounted for 99.6% of the total essential oil composition (Table 1). It contained thymol (52.90%), p-cymene (9.10), γ-terpinene (8.10%) and carvacrol (6.80%) as major constituents (Figure 1).\nGC-MS chromatogram of the essential oil of Zataria multiflora.\nChemical constituents of Zataria multiflora essential oil.\nRI-Cal: retention indices calculated based on C6–C24\nn-alkenes from a DB-5 column.\nRI-Cal: retention indices retrieved from literature (Adams 2007).\nBold values show major compounds in the essential oil.\n Wound area The findings for the effects of ZMEO on the percentage of wound contraction are shown in Figure 2. The results implied that topical administration of therapeutic ointments significantly (p < 0.05) increased wound contraction rate on days 7 and 14 compared to the NCG group (Figure 2(A,B)). The comparison between treatment groups revealed that a higher wound contraction rate was observed in the 4% ZMEO-treated group compared to the 2% ZMEO-treated and MG groups (p < 0.05).\nThe effects of topical administration of ZMEO on (B) wound contraction rate and (C) tissue bacterial count (CFU/g) in different days. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\nThe findings for the effects of ZMEO on the percentage of wound contraction are shown in Figure 2. The results implied that topical administration of therapeutic ointments significantly (p < 0.05) increased wound contraction rate on days 7 and 14 compared to the NCG group (Figure 2(A,B)). The comparison between treatment groups revealed that a higher wound contraction rate was observed in the 4% ZMEO-treated group compared to the 2% ZMEO-treated and MG groups (p < 0.05).\nThe effects of topical administration of ZMEO on (B) wound contraction rate and (C) tissue bacterial count (CFU/g) in different days. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\n The bacteriological count The results concerning the effects of ZMEO on tissue bacteria counts are represented in Figure 2(C). The results showed that topical administration of therapeutic ointments significantly decreased bacterial colonization in all the days compared to the NCG group (p < 0.05). The comparison between treatment groups demonstrated that a higher level of ZMEO (4%) significantly decreased the total bacterial count compared to that of a lower level of it (2%) and MG groups (p < 0.05).\nThe results concerning the effects of ZMEO on tissue bacteria counts are represented in Figure 2(C). The results showed that topical administration of therapeutic ointments significantly decreased bacterial colonization in all the days compared to the NCG group (p < 0.05). The comparison between treatment groups demonstrated that a higher level of ZMEO (4%) significantly decreased the total bacterial count compared to that of a lower level of it (2%) and MG groups (p < 0.05).\n Histopathological parameters The results for histopathological parameters are represented in Table 2. The results showed that edoema score was significantly higher in the NCG group. It was observed to be significantly lower in the animals treated with the 4% ZMEO-treated group and also in 2% ZMEO and MG-treated groups. The findings implied that the lowest edoema was observed in 4% ZMEO in day 14; meanwhile, there was no observed edoema in the 4% ZMEO group on day 14. The results showed that immune cells were significantly lower in ZMEO groups in day 3 compared to other groups (p < 0.05), yet it decreased on days 7 and 14 in the ZMEO groups compared to other groups (p < 0.05). It means that the application of the ZMEO decreases immune cells in days 7 and 14, and increased it on day 3. Angiogenesis (Figure 3) and fibroblast infiltration were significantly higher in all treated groups compared to the NCG group and the highest level was observed in level of the 4%-ZMEO group. The results for collagen condensation (Figure 4) and re-epithelialization (Figure 5) revealed that parameters were significantly higher in the ZMEO groups, which confirms the results.\nImmunohistochemical staining for angiogenesis:; (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See elevated angiogenesis in ZMEO-treated group (arrows) on day 7 after wound induction. CD 31 staining, 400×.\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. Note well-formed collagen deposition in cross sections from ZMEO-treated animals on day 7 after wound induction (first row), which is significantly increased on day 14 after injury (second row). The third row represents the software analyze for collagen intensity. The cross sections from animals in ZMEO-treated groups exhibited condense collagen deposition versus NCG and MG groups. Masson-trichrome staining and fluorescent staining for collagen, 400× magnification.\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See well re-epithelialization in ZMEO-treated animals. The re-epithelialization initiated on day 7 after wound induction in ZMEO-treated animals. However, the cross sections from NCG and MG groups are not representing epithelialization. Note well-organized dermis and complete epithelialization with well-formed papillae in ZMEO-treated animals in comparison to NCG and MG groups. Masson-trichrome staining, 100×.\nThe effects of ZMEO on histopathological parameters in different days.\nSuperscripts (a–d) indicate significant differences in same day at p < 0.05.\nThe results for histopathological parameters are represented in Table 2. The results showed that edoema score was significantly higher in the NCG group. It was observed to be significantly lower in the animals treated with the 4% ZMEO-treated group and also in 2% ZMEO and MG-treated groups. The findings implied that the lowest edoema was observed in 4% ZMEO in day 14; meanwhile, there was no observed edoema in the 4% ZMEO group on day 14. The results showed that immune cells were significantly lower in ZMEO groups in day 3 compared to other groups (p < 0.05), yet it decreased on days 7 and 14 in the ZMEO groups compared to other groups (p < 0.05). It means that the application of the ZMEO decreases immune cells in days 7 and 14, and increased it on day 3. Angiogenesis (Figure 3) and fibroblast infiltration were significantly higher in all treated groups compared to the NCG group and the highest level was observed in level of the 4%-ZMEO group. The results for collagen condensation (Figure 4) and re-epithelialization (Figure 5) revealed that parameters were significantly higher in the ZMEO groups, which confirms the results.\nImmunohistochemical staining for angiogenesis:; (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See elevated angiogenesis in ZMEO-treated group (arrows) on day 7 after wound induction. CD 31 staining, 400×.\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. Note well-formed collagen deposition in cross sections from ZMEO-treated animals on day 7 after wound induction (first row), which is significantly increased on day 14 after injury (second row). The third row represents the software analyze for collagen intensity. The cross sections from animals in ZMEO-treated groups exhibited condense collagen deposition versus NCG and MG groups. Masson-trichrome staining and fluorescent staining for collagen, 400× magnification.\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See well re-epithelialization in ZMEO-treated animals. The re-epithelialization initiated on day 7 after wound induction in ZMEO-treated animals. However, the cross sections from NCG and MG groups are not representing epithelialization. Note well-organized dermis and complete epithelialization with well-formed papillae in ZMEO-treated animals in comparison to NCG and MG groups. Masson-trichrome staining, 100×.\nThe effects of ZMEO on histopathological parameters in different days.\nSuperscripts (a–d) indicate significant differences in same day at p < 0.05.\n Molecular analyses The results for gene expression are presented in Figure 6. Gene expression of IL-1β and TNF-α was significantly (p < 0.05) lower on all sampling days at treated groups compared to the NCG group. The greatest decrease was observed in the 4% ZMEO-treated animals. Gene expressions of IGF-1 and VEGF was significantly (p < 0.05) higher at all treated groups on day 7 after the wounding, but the levels significantly decreased on day 14. The expression of IL-10 level increased on day 7 whereas it decreased on day 14 compared to the NCG group (p < 0.05). Further analysis of gene expressions of TGF-β and FGF-2 indicate that its level does not have significant difference (p > 0.05) on day 3, yet its level significantly (p < 0.05) increased in all treated groups, particularly at 4% ZMEO-treated animals on day 7. The amount of TGF-β and FGF-2 gene expression levels also were significantly (p < 0.05) reduced on day 14 compared to the NCG group.\nThe effects of topical application of ZMEO on gene expression. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\nThe results for gene expression are presented in Figure 6. Gene expression of IL-1β and TNF-α was significantly (p < 0.05) lower on all sampling days at treated groups compared to the NCG group. The greatest decrease was observed in the 4% ZMEO-treated animals. Gene expressions of IGF-1 and VEGF was significantly (p < 0.05) higher at all treated groups on day 7 after the wounding, but the levels significantly decreased on day 14. The expression of IL-10 level increased on day 7 whereas it decreased on day 14 compared to the NCG group (p < 0.05). Further analysis of gene expressions of TGF-β and FGF-2 indicate that its level does not have significant difference (p > 0.05) on day 3, yet its level significantly (p < 0.05) increased in all treated groups, particularly at 4% ZMEO-treated animals on day 7. The amount of TGF-β and FGF-2 gene expression levels also were significantly (p < 0.05) reduced on day 14 compared to the NCG group.\nThe effects of topical application of ZMEO on gene expression. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\n Antioxidant status Antioxidant status in different groups is represented in Figure 7. Using the 2% and 4%-ZMEO significantly (p < 0.05) increased TAC and TTP levels compared to the NCG group in all of the days after the wounding. Interestingly, the highest increase was observed in the 4% ZMEO-treated animals. Further analysis of MDA level indicates significant (p < 0.05) decrease in all of the treated groups compared to the NCG group in all of the days after the wounding. The highest decrease was observed in the 4% ZMEO-treated group.\nThe effects of topical application of ZMEO on antioxidant properties. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\nAntioxidant status in different groups is represented in Figure 7. Using the 2% and 4%-ZMEO significantly (p < 0.05) increased TAC and TTP levels compared to the NCG group in all of the days after the wounding. Interestingly, the highest increase was observed in the 4% ZMEO-treated animals. Further analysis of MDA level indicates significant (p < 0.05) decrease in all of the treated groups compared to the NCG group in all of the days after the wounding. The highest decrease was observed in the 4% ZMEO-treated group.\nThe effects of topical application of ZMEO on antioxidant properties. Superscripts (a–d) indicate significant differences in same day at p < 0.05."},"conclusions_title":{"kind":"string","value":"Conclusion"},"conclusions_text":{"kind":"string","value":"The topical application of ZMEO decreased the inflammation and the total bacterial count, which progresses the wound healing process towards the proliferative phase. It increases the expression of proliferative genes, helps to alleviate the inflammation phase and accelerates the proliferative phase. Antioxidant activity of ZMEO accelerated the wound healing process. All the above-mentioned factors hasten the wound healing process, as seen for the wound area. Accordingly, applying ZMEO only and/or in combination with chemical agents for the treatment of wound healing could be suggested."},"other_sections_titles":{"kind":"list like","value":["Materials","Essential oil analysis and identification","Animals","Induction of wound","Wound area","The investigation of the bacteriological count in the wound area","Histological analysis","Immunohistochemical staining (IHC) for angiogenesis (CD31)","Fluorescent staining for collagen","RNA isolation and cDNA synthesis","Antioxidant capacity","Statistical analysis","Composition of the Zataria multiflora essential oil","Wound area","The bacteriological count","Histopathological parameters","Molecular analyses","Antioxidant status"],"string":"[\n \"Materials\",\n \"Essential oil analysis and identification\",\n \"Animals\",\n \"Induction of wound\",\n \"Wound area\",\n \"The investigation of the bacteriological count in the wound area\",\n \"Histological analysis\",\n \"Immunohistochemical staining (IHC) for angiogenesis (CD31)\",\n \"Fluorescent staining for collagen\",\n \"RNA isolation and cDNA synthesis\",\n \"Antioxidant capacity\",\n \"Statistical analysis\",\n \"Composition of the Zataria multiflora essential oil\",\n \"Wound area\",\n \"The bacteriological count\",\n \"Histopathological parameters\",\n \"Molecular analyses\",\n \"Antioxidant status\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Zataria multiflora essential oil was purchased from Barij Essence Company (Kashan-Iran) and approved by the same herbarium company (No.1203). Commercial base ointment and mupirocin were prepared from Pars Daruo. Ltd., Tehran, Iran. The commercial base ointment contained 90% soft paraffin, 5% hard paraffin and 5% lanolin. Rodent feeds were prepared from Javaneh Khorasan Company (Khorasan, Iran).","Gas chromatography (GC) analysis was employed in order to identify the compounds. The components of the essential oil were recognized using calculation of their retention indices under temperature-programmed conditions for n-alkanes (C6–C24) and the oil on DB-5 column.","Seventy-two BALB/c mice weighing 25 ± 3 g were prepared. The mice had free access to water and pelleted feed for rodents (Javaneh Khorasan Company, Khorasan, Iran). The mice were kept in an ambient temperature of 23 ± 3 °C, at a constant air humidity, and a natural day–night cycle. This study was approved by the Animal Research Committee of the Urmia Islamic Azad University with ethical No. IAU-UB 11044.","To induce the wound, the mice were primarily anesthetized by intraperitoneal administration route using 50 mg/kg ketamine and 5 mg/kg xylazine. Following the induction of anaesthesia, dorsal region of each mouse was shaven for surgical actions. A circular wound model was induced in a size of 7 mm using a surgical biopsy punch and per wound was inoculated by an aliquot of 5 × 107 suspension containing S. aureus (ATCC 26313) and P. aeruginosa (ATCC 27853) in 50 μL phosphate-buffered saline (Farahpour et al. 2020; Khezri et al. 2019). Subsequently, animals were divided into four groups (n = 18), as (group I) negative control group (NCG) that was administered commercial base ointment, (group II) positive control group that were treated with Mupirocin® (MG), and (groups III and IV) treated groups with therapeutic ointments containing 2 g and 4 g ZME mixed in NCG (2% and 4% ZMEO), respectively. Concerning the colonization of the bacteria, 24 h after the induction of the wounds, ointments were applied on wounds, once a day. In addition, according to the tissue sampling on days 3, 7, and 14 after the wounding, the animals in each group were divided into three subgroups (n = 6).","The rate of wound closure was estimated as reported previously (Nejati et al. 2015; Khezri et al. 2019). In order to calculate the percentage of wound closure rate, a transparent paper was placed on it and it was calculated based on the following formula:\nPercentage of wound closure = [(Wound area on Day 0 − Wound area on Day X)/Wound area on Day 0] × 100.","To calculate the bacteriological count, the granulated tissues were excised and 0.1 g of the sample was crushed, minced, and homogenized in a sterile mortar containing 10 mL of sterile saline. It was the diluted in tubes containing 9 mL of sterile saline, was cultured on plate count agar (Merck KGaA, Darmstadt, Germany), incubated at 37 °C for 48 h, calculated as CFU/g of granulation tissue (Farahpour et al. 2020; Khezri et al. 2019).","Concerning the evaluation of the histological parameters, the mice were euthanized by a special CO2 device and the granulation tissue were excised in along to 1 to 2 mm from surrounding normal skin. The samples were fixed in neutral-buffered formalin 10%, routinely processed, and paraffin wax was embedded, sectioned at 5 µm, and stained with Masson’s trichrome and then examined under light microscopy (Olympus CX31RBSF attached cameraman) as reported by Farahpour et al. (2018). Cellular infiltration, edoema and collagen deposition were assessed. An image pro-insight software was utilised for evaluating the collagen deposition. Morphometric lens (Olympus, Germany) was used for assessing the epithelial thickness. edoema was assessed as a 5-score as reported previously (Nejati et al. 2015).","To assess the IHC, it was conducted as reported by Farahpour et al. (2018). The IHC was conducted based on manufacturer’s protocol (Biocare, Yorba Linda, CA). The tissue sections were rinsed gently in the washing buffer and placed in a buffer bath. A DAB chromogen was employed for assessing the tissue sections and then incubated for 5 min. The sections were then dipped in weak ammonia (0.037 M/L) for 10 times, rinsed with distilled water and cover slipped. Brown stains were considered as positive immunohistochemical.","To wash the slides, acetic acid 1% was used for several minutes. The slides were stained using acridine-orange. Phosphate buffer was utilized to remove staining. The slides were the washed in distilled water, mounted with buffer drop and analyzed with a fluorescent microscope. The bunds with red and yellowish red colour were considered as collagen bunds (Farahpour et al. 2018).","The wound tissues were isolated as previously reported and RNA was extracted applying a standard TRIZOL procedure as reported by Farahpour et al. (2018), and assessed by spectrophotometer (260 nm and 260/280 = 1.8–2.0). To prepare the cDNA, it was put in a reaction being mixed with 1 µg RNA, oligo (dT) primer (1 µL), 5 × reaction buffer (4 µL), RNAse inhibitor (1 µL), 10 mM dNTP mix (2 µL) and M-MuLV Reverse Transcriptase (1 µL) as described by producer protocol (Fermentas, GmbH, Germany). Time and temperature were as reported by Farahpour et al. (2018). The used primers included IL-1β, forward (5′-AAC AAA CCC TGC AGT GGT TCG-3′) and reverse (5′-AGCTGCTTCAGACACTTGCAC-3′); TGF-β, forward (5-CCAAACGCCGAAGACTTATCC-3′) and reverse (5′-CTTATTACCGATGGGATGGGATAGCCC-3′); IL-10, forward (5′-GAAGCTCCCTCAGCGAGGACA-3′) and reverse (5′-TTGGGCCAGTGAGTGAAAGGG-3′), TNF-α, forward (5′-GAAGCTCCCTCAGCGAGGACA-3′) and reverse (5′-TTGGGCCAGTGAGTGAAAGGG-3′), FGF-2, forward (5′-GGAACCCGGCGGGACACGGAC-3′) and reverse (5′-CCGCTGTGGCAGCTCTTG GGG-3′); VEGF, forward (5′-GCTCCGTAGTAGCCGTGGTCT-3′) and reverse (5′-GGAACCCGGCGGGACACGGAC-3′), and IGF-1, forward (5′-TAGGTGGTTGATGAATGGT-3′) and reverse (5′-GAAAGGGCAGGGCTAAT-3′).","To assess the antioxidant capacity, the wound granulation tissue was homogenized in ice-cold KCl (150 mM) and the mixture was then centrifuged at 3000 g for 10 min. The supernatant was used to evaluate the total antioxidant capacity (TAC), malondialdehyde (MDA) and total tissue thiol molecules (TTM) content. The Lowry method was employed for evaluating the protein content of the samples (Daemi et al. 2019a) and MDA content was used for assessing the lipid peroxidation rate. To assess the TTM, the collected granulation tissue sample was homogenized and the supernatant was added to Tris-EDTA as reported by Daemi et al. (2019a).","The results were reported as mean ± standard deviation and analyzed by Graph Pad Prism Software. One-way ANOVA was utilized to analyze the results. Dunnett’s test for pair-wise comparisons was used for evaluating the effect of treatments and p < 0.05 was considered to be of significance.","The analysis of Zataria multiflora essential oil with GC-FID and GC-mass spectrometry (MS) identified 29 compounds which accounted for 99.6% of the total essential oil composition (Table 1). It contained thymol (52.90%), p-cymene (9.10), γ-terpinene (8.10%) and carvacrol (6.80%) as major constituents (Figure 1).\nGC-MS chromatogram of the essential oil of Zataria multiflora.\nChemical constituents of Zataria multiflora essential oil.\nRI-Cal: retention indices calculated based on C6–C24\nn-alkenes from a DB-5 column.\nRI-Cal: retention indices retrieved from literature (Adams 2007).\nBold values show major compounds in the essential oil.","The findings for the effects of ZMEO on the percentage of wound contraction are shown in Figure 2. The results implied that topical administration of therapeutic ointments significantly (p < 0.05) increased wound contraction rate on days 7 and 14 compared to the NCG group (Figure 2(A,B)). The comparison between treatment groups revealed that a higher wound contraction rate was observed in the 4% ZMEO-treated group compared to the 2% ZMEO-treated and MG groups (p < 0.05).\nThe effects of topical administration of ZMEO on (B) wound contraction rate and (C) tissue bacterial count (CFU/g) in different days. Superscripts (a–d) indicate significant differences in same day at p < 0.05.","The results concerning the effects of ZMEO on tissue bacteria counts are represented in Figure 2(C). The results showed that topical administration of therapeutic ointments significantly decreased bacterial colonization in all the days compared to the NCG group (p < 0.05). The comparison between treatment groups demonstrated that a higher level of ZMEO (4%) significantly decreased the total bacterial count compared to that of a lower level of it (2%) and MG groups (p < 0.05).","The results for histopathological parameters are represented in Table 2. The results showed that edoema score was significantly higher in the NCG group. It was observed to be significantly lower in the animals treated with the 4% ZMEO-treated group and also in 2% ZMEO and MG-treated groups. The findings implied that the lowest edoema was observed in 4% ZMEO in day 14; meanwhile, there was no observed edoema in the 4% ZMEO group on day 14. The results showed that immune cells were significantly lower in ZMEO groups in day 3 compared to other groups (p < 0.05), yet it decreased on days 7 and 14 in the ZMEO groups compared to other groups (p < 0.05). It means that the application of the ZMEO decreases immune cells in days 7 and 14, and increased it on day 3. Angiogenesis (Figure 3) and fibroblast infiltration were significantly higher in all treated groups compared to the NCG group and the highest level was observed in level of the 4%-ZMEO group. The results for collagen condensation (Figure 4) and re-epithelialization (Figure 5) revealed that parameters were significantly higher in the ZMEO groups, which confirms the results.\nImmunohistochemical staining for angiogenesis:; (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See elevated angiogenesis in ZMEO-treated group (arrows) on day 7 after wound induction. CD 31 staining, 400×.\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. Note well-formed collagen deposition in cross sections from ZMEO-treated animals on day 7 after wound induction (first row), which is significantly increased on day 14 after injury (second row). The third row represents the software analyze for collagen intensity. The cross sections from animals in ZMEO-treated groups exhibited condense collagen deposition versus NCG and MG groups. Masson-trichrome staining and fluorescent staining for collagen, 400× magnification.\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See well re-epithelialization in ZMEO-treated animals. The re-epithelialization initiated on day 7 after wound induction in ZMEO-treated animals. However, the cross sections from NCG and MG groups are not representing epithelialization. Note well-organized dermis and complete epithelialization with well-formed papillae in ZMEO-treated animals in comparison to NCG and MG groups. Masson-trichrome staining, 100×.\nThe effects of ZMEO on histopathological parameters in different days.\nSuperscripts (a–d) indicate significant differences in same day at p < 0.05.","The results for gene expression are presented in Figure 6. Gene expression of IL-1β and TNF-α was significantly (p < 0.05) lower on all sampling days at treated groups compared to the NCG group. The greatest decrease was observed in the 4% ZMEO-treated animals. Gene expressions of IGF-1 and VEGF was significantly (p < 0.05) higher at all treated groups on day 7 after the wounding, but the levels significantly decreased on day 14. The expression of IL-10 level increased on day 7 whereas it decreased on day 14 compared to the NCG group (p < 0.05). Further analysis of gene expressions of TGF-β and FGF-2 indicate that its level does not have significant difference (p > 0.05) on day 3, yet its level significantly (p < 0.05) increased in all treated groups, particularly at 4% ZMEO-treated animals on day 7. The amount of TGF-β and FGF-2 gene expression levels also were significantly (p < 0.05) reduced on day 14 compared to the NCG group.\nThe effects of topical application of ZMEO on gene expression. Superscripts (a–d) indicate significant differences in same day at p < 0.05.","Antioxidant status in different groups is represented in Figure 7. Using the 2% and 4%-ZMEO significantly (p < 0.05) increased TAC and TTP levels compared to the NCG group in all of the days after the wounding. Interestingly, the highest increase was observed in the 4% ZMEO-treated animals. Further analysis of MDA level indicates significant (p < 0.05) decrease in all of the treated groups compared to the NCG group in all of the days after the wounding. The highest decrease was observed in the 4% ZMEO-treated group.\nThe effects of topical application of ZMEO on antioxidant properties. Superscripts (a–d) indicate significant differences in same day at p < 0.05."],"string":"[\n \"Zataria multiflora essential oil was purchased from Barij Essence Company (Kashan-Iran) and approved by the same herbarium company (No.1203). Commercial base ointment and mupirocin were prepared from Pars Daruo. Ltd., Tehran, Iran. The commercial base ointment contained 90% soft paraffin, 5% hard paraffin and 5% lanolin. Rodent feeds were prepared from Javaneh Khorasan Company (Khorasan, Iran).\",\n \"Gas chromatography (GC) analysis was employed in order to identify the compounds. The components of the essential oil were recognized using calculation of their retention indices under temperature-programmed conditions for n-alkanes (C6–C24) and the oil on DB-5 column.\",\n \"Seventy-two BALB/c mice weighing 25 ± 3 g were prepared. The mice had free access to water and pelleted feed for rodents (Javaneh Khorasan Company, Khorasan, Iran). The mice were kept in an ambient temperature of 23 ± 3 °C, at a constant air humidity, and a natural day–night cycle. This study was approved by the Animal Research Committee of the Urmia Islamic Azad University with ethical No. IAU-UB 11044.\",\n \"To induce the wound, the mice were primarily anesthetized by intraperitoneal administration route using 50 mg/kg ketamine and 5 mg/kg xylazine. Following the induction of anaesthesia, dorsal region of each mouse was shaven for surgical actions. A circular wound model was induced in a size of 7 mm using a surgical biopsy punch and per wound was inoculated by an aliquot of 5 × 107 suspension containing S. aureus (ATCC 26313) and P. aeruginosa (ATCC 27853) in 50 μL phosphate-buffered saline (Farahpour et al. 2020; Khezri et al. 2019). Subsequently, animals were divided into four groups (n = 18), as (group I) negative control group (NCG) that was administered commercial base ointment, (group II) positive control group that were treated with Mupirocin® (MG), and (groups III and IV) treated groups with therapeutic ointments containing 2 g and 4 g ZME mixed in NCG (2% and 4% ZMEO), respectively. Concerning the colonization of the bacteria, 24 h after the induction of the wounds, ointments were applied on wounds, once a day. In addition, according to the tissue sampling on days 3, 7, and 14 after the wounding, the animals in each group were divided into three subgroups (n = 6).\",\n \"The rate of wound closure was estimated as reported previously (Nejati et al. 2015; Khezri et al. 2019). In order to calculate the percentage of wound closure rate, a transparent paper was placed on it and it was calculated based on the following formula:\\nPercentage of wound closure = [(Wound area on Day 0 − Wound area on Day X)/Wound area on Day 0] × 100.\",\n \"To calculate the bacteriological count, the granulated tissues were excised and 0.1 g of the sample was crushed, minced, and homogenized in a sterile mortar containing 10 mL of sterile saline. It was the diluted in tubes containing 9 mL of sterile saline, was cultured on plate count agar (Merck KGaA, Darmstadt, Germany), incubated at 37 °C for 48 h, calculated as CFU/g of granulation tissue (Farahpour et al. 2020; Khezri et al. 2019).\",\n \"Concerning the evaluation of the histological parameters, the mice were euthanized by a special CO2 device and the granulation tissue were excised in along to 1 to 2 mm from surrounding normal skin. The samples were fixed in neutral-buffered formalin 10%, routinely processed, and paraffin wax was embedded, sectioned at 5 µm, and stained with Masson’s trichrome and then examined under light microscopy (Olympus CX31RBSF attached cameraman) as reported by Farahpour et al. (2018). Cellular infiltration, edoema and collagen deposition were assessed. An image pro-insight software was utilised for evaluating the collagen deposition. Morphometric lens (Olympus, Germany) was used for assessing the epithelial thickness. edoema was assessed as a 5-score as reported previously (Nejati et al. 2015).\",\n \"To assess the IHC, it was conducted as reported by Farahpour et al. (2018). The IHC was conducted based on manufacturer’s protocol (Biocare, Yorba Linda, CA). The tissue sections were rinsed gently in the washing buffer and placed in a buffer bath. A DAB chromogen was employed for assessing the tissue sections and then incubated for 5 min. The sections were then dipped in weak ammonia (0.037 M/L) for 10 times, rinsed with distilled water and cover slipped. Brown stains were considered as positive immunohistochemical.\",\n \"To wash the slides, acetic acid 1% was used for several minutes. The slides were stained using acridine-orange. Phosphate buffer was utilized to remove staining. The slides were the washed in distilled water, mounted with buffer drop and analyzed with a fluorescent microscope. The bunds with red and yellowish red colour were considered as collagen bunds (Farahpour et al. 2018).\",\n \"The wound tissues were isolated as previously reported and RNA was extracted applying a standard TRIZOL procedure as reported by Farahpour et al. (2018), and assessed by spectrophotometer (260 nm and 260/280 = 1.8–2.0). To prepare the cDNA, it was put in a reaction being mixed with 1 µg RNA, oligo (dT) primer (1 µL), 5 × reaction buffer (4 µL), RNAse inhibitor (1 µL), 10 mM dNTP mix (2 µL) and M-MuLV Reverse Transcriptase (1 µL) as described by producer protocol (Fermentas, GmbH, Germany). Time and temperature were as reported by Farahpour et al. (2018). The used primers included IL-1β, forward (5′-AAC AAA CCC TGC AGT GGT TCG-3′) and reverse (5′-AGCTGCTTCAGACACTTGCAC-3′); TGF-β, forward (5-CCAAACGCCGAAGACTTATCC-3′) and reverse (5′-CTTATTACCGATGGGATGGGATAGCCC-3′); IL-10, forward (5′-GAAGCTCCCTCAGCGAGGACA-3′) and reverse (5′-TTGGGCCAGTGAGTGAAAGGG-3′), TNF-α, forward (5′-GAAGCTCCCTCAGCGAGGACA-3′) and reverse (5′-TTGGGCCAGTGAGTGAAAGGG-3′), FGF-2, forward (5′-GGAACCCGGCGGGACACGGAC-3′) and reverse (5′-CCGCTGTGGCAGCTCTTG GGG-3′); VEGF, forward (5′-GCTCCGTAGTAGCCGTGGTCT-3′) and reverse (5′-GGAACCCGGCGGGACACGGAC-3′), and IGF-1, forward (5′-TAGGTGGTTGATGAATGGT-3′) and reverse (5′-GAAAGGGCAGGGCTAAT-3′).\",\n \"To assess the antioxidant capacity, the wound granulation tissue was homogenized in ice-cold KCl (150 mM) and the mixture was then centrifuged at 3000 g for 10 min. The supernatant was used to evaluate the total antioxidant capacity (TAC), malondialdehyde (MDA) and total tissue thiol molecules (TTM) content. The Lowry method was employed for evaluating the protein content of the samples (Daemi et al. 2019a) and MDA content was used for assessing the lipid peroxidation rate. To assess the TTM, the collected granulation tissue sample was homogenized and the supernatant was added to Tris-EDTA as reported by Daemi et al. (2019a).\",\n \"The results were reported as mean ± standard deviation and analyzed by Graph Pad Prism Software. One-way ANOVA was utilized to analyze the results. Dunnett’s test for pair-wise comparisons was used for evaluating the effect of treatments and p < 0.05 was considered to be of significance.\",\n \"The analysis of Zataria multiflora essential oil with GC-FID and GC-mass spectrometry (MS) identified 29 compounds which accounted for 99.6% of the total essential oil composition (Table 1). It contained thymol (52.90%), p-cymene (9.10), γ-terpinene (8.10%) and carvacrol (6.80%) as major constituents (Figure 1).\\nGC-MS chromatogram of the essential oil of Zataria multiflora.\\nChemical constituents of Zataria multiflora essential oil.\\nRI-Cal: retention indices calculated based on C6–C24\\nn-alkenes from a DB-5 column.\\nRI-Cal: retention indices retrieved from literature (Adams 2007).\\nBold values show major compounds in the essential oil.\",\n \"The findings for the effects of ZMEO on the percentage of wound contraction are shown in Figure 2. The results implied that topical administration of therapeutic ointments significantly (p < 0.05) increased wound contraction rate on days 7 and 14 compared to the NCG group (Figure 2(A,B)). The comparison between treatment groups revealed that a higher wound contraction rate was observed in the 4% ZMEO-treated group compared to the 2% ZMEO-treated and MG groups (p < 0.05).\\nThe effects of topical administration of ZMEO on (B) wound contraction rate and (C) tissue bacterial count (CFU/g) in different days. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\",\n \"The results concerning the effects of ZMEO on tissue bacteria counts are represented in Figure 2(C). The results showed that topical administration of therapeutic ointments significantly decreased bacterial colonization in all the days compared to the NCG group (p < 0.05). The comparison between treatment groups demonstrated that a higher level of ZMEO (4%) significantly decreased the total bacterial count compared to that of a lower level of it (2%) and MG groups (p < 0.05).\",\n \"The results for histopathological parameters are represented in Table 2. The results showed that edoema score was significantly higher in the NCG group. It was observed to be significantly lower in the animals treated with the 4% ZMEO-treated group and also in 2% ZMEO and MG-treated groups. The findings implied that the lowest edoema was observed in 4% ZMEO in day 14; meanwhile, there was no observed edoema in the 4% ZMEO group on day 14. The results showed that immune cells were significantly lower in ZMEO groups in day 3 compared to other groups (p < 0.05), yet it decreased on days 7 and 14 in the ZMEO groups compared to other groups (p < 0.05). It means that the application of the ZMEO decreases immune cells in days 7 and 14, and increased it on day 3. Angiogenesis (Figure 3) and fibroblast infiltration were significantly higher in all treated groups compared to the NCG group and the highest level was observed in level of the 4%-ZMEO group. The results for collagen condensation (Figure 4) and re-epithelialization (Figure 5) revealed that parameters were significantly higher in the ZMEO groups, which confirms the results.\\nImmunohistochemical staining for angiogenesis:; (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See elevated angiogenesis in ZMEO-treated group (arrows) on day 7 after wound induction. CD 31 staining, 400×.\\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. Note well-formed collagen deposition in cross sections from ZMEO-treated animals on day 7 after wound induction (first row), which is significantly increased on day 14 after injury (second row). The third row represents the software analyze for collagen intensity. The cross sections from animals in ZMEO-treated groups exhibited condense collagen deposition versus NCG and MG groups. Masson-trichrome staining and fluorescent staining for collagen, 400× magnification.\\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See well re-epithelialization in ZMEO-treated animals. The re-epithelialization initiated on day 7 after wound induction in ZMEO-treated animals. However, the cross sections from NCG and MG groups are not representing epithelialization. Note well-organized dermis and complete epithelialization with well-formed papillae in ZMEO-treated animals in comparison to NCG and MG groups. Masson-trichrome staining, 100×.\\nThe effects of ZMEO on histopathological parameters in different days.\\nSuperscripts (a–d) indicate significant differences in same day at p < 0.05.\",\n \"The results for gene expression are presented in Figure 6. Gene expression of IL-1β and TNF-α was significantly (p < 0.05) lower on all sampling days at treated groups compared to the NCG group. The greatest decrease was observed in the 4% ZMEO-treated animals. Gene expressions of IGF-1 and VEGF was significantly (p < 0.05) higher at all treated groups on day 7 after the wounding, but the levels significantly decreased on day 14. The expression of IL-10 level increased on day 7 whereas it decreased on day 14 compared to the NCG group (p < 0.05). Further analysis of gene expressions of TGF-β and FGF-2 indicate that its level does not have significant difference (p > 0.05) on day 3, yet its level significantly (p < 0.05) increased in all treated groups, particularly at 4% ZMEO-treated animals on day 7. The amount of TGF-β and FGF-2 gene expression levels also were significantly (p < 0.05) reduced on day 14 compared to the NCG group.\\nThe effects of topical application of ZMEO on gene expression. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\",\n \"Antioxidant status in different groups is represented in Figure 7. Using the 2% and 4%-ZMEO significantly (p < 0.05) increased TAC and TTP levels compared to the NCG group in all of the days after the wounding. Interestingly, the highest increase was observed in the 4% ZMEO-treated animals. Further analysis of MDA level indicates significant (p < 0.05) decrease in all of the treated groups compared to the NCG group in all of the days after the wounding. The highest decrease was observed in the 4% ZMEO-treated group.\\nThe effects of topical application of ZMEO on antioxidant properties. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Introduction","Materials and methods","Materials","Essential oil analysis and identification","Animals","Induction of wound","Wound area","The investigation of the bacteriological count in the wound area","Histological analysis","Immunohistochemical staining (IHC) for angiogenesis (CD31)","Fluorescent staining for collagen","RNA isolation and cDNA synthesis","Antioxidant capacity","Statistical analysis","Results","Composition of the Zataria multiflora essential oil","Wound area","The bacteriological count","Histopathological parameters","Molecular analyses","Antioxidant status","Discussion","Conclusion"],"string":"[\n \"Introduction\",\n \"Materials and methods\",\n \"Materials\",\n \"Essential oil analysis and identification\",\n \"Animals\",\n \"Induction of wound\",\n \"Wound area\",\n \"The investigation of the bacteriological count in the wound area\",\n \"Histological analysis\",\n \"Immunohistochemical staining (IHC) for angiogenesis (CD31)\",\n \"Fluorescent staining for collagen\",\n \"RNA isolation and cDNA synthesis\",\n \"Antioxidant capacity\",\n \"Statistical analysis\",\n \"Results\",\n \"Composition of the Zataria multiflora essential oil\",\n \"Wound area\",\n \"The bacteriological count\",\n \"Histopathological parameters\",\n \"Molecular analyses\",\n \"Antioxidant status\",\n \"Discussion\",\n \"Conclusion\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Wound infections are important challenges that cause the suffering of patients and economic losses (Vittorazzi et al. 2016; Daemi et al. 2019a; Farghali et al. 2019). Skin is a regenerative organ which acts as a barrier between the body and the external environment (Hassan et al. 2014). A wound is defined as a disturbance in anatomic structure of skin and its functional integrity (Farghali et al. 2019). Skin prevents the penetration of bacteria and fungi that are important challenges that induce mortality and morbidity. Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic bacteria that induce infection in patients (Cardona and Wilson 2015). The bacteria are commonly detected in the upper and deepest region of wound bed (Serra et al. 2015). Colonization of S. aureus and P. aeruginosa in the wound site postpones the wound healing process (Farahpour et al. 2020; Khezri et al. 2020). The wound healing process comprises several phases, including coagulation, inflammation, epithelialization, granulation tissue formation, and tissue remodelling (Daemi et al. 2019a, 2019b; Farahpour et al. 2020). Inflammatory phase occurs after activation of inflammatory chemokines (Farahpour et al. 2020). Interleukin-1β (IL-1β) is significantly synthesized 12–24 h after infliction of the wound, and its level returns to basal level after the proliferative stage is completed (Fahey and Doyle 2019). Tumour necrosis factor-α (TNF-α) is significantly synthesized by macrophages and T lymphocytes and its level increases under inflammation and infection conditions (Xue and Falcon 2019). Following the induction of inflammation, proliferative phase occurs in which many genes are involved. Insulin-like growth factor 1 (IGF-1) promotes the production of keratinocyte and fibroblast proliferation and improves the re-epithelialization (Daemi et al. 2019b). Decreased endothelial insulin/IGF-1 signal delays in the wound healing process (Aghdam et al. 2012). Vascular endothelial growth factor (VEGF) induces angiogenesis and stimulates cell migration and proliferation (Farahpour et al. 2020). Fibroblast growth factor-2 (FGF-2) is a protein that participates in the wound healing process (Souto et al. 2020). Interleukin-10 (IL-10) reduces the production of pro-inflammatory cytokine, but tumour growth factor-β (TGF-β) has roles in increasing the proliferative phase improving the proliferation and differentiation of fibroblasts, collagen production, and wound contraction (Khezri et al. 2020). On the other hand, oxidative stress increases under wound disorders and increases the production of reactive oxygen species (ROS) in the site of wound (Ustuner et al. 2019). Applying antibacterial and antioxidant agents shortens the inflammatory phase, promotes the proliferative phase and accelerates the wound healing process (Bardaa et al. 2016; Vittorazzi et al. 2016; Daemi et al. 2019a, 2019b).\nZataria multiflora Boiss (Lamiaceae) (ZM) has antibacterial activity against Pseudomonas spp. (Barkhori-Mehni et al. 2017). Its antibacterial activity is attributed to its phenolic compounds that destroy the cell wall in bacteria (Barkhori-Mehni et al. 2017). It demonstrates antioxidant properties due to its compounds such as thymol and carvacrol (Mojaddar Langroodi et al. 2019). Seemingly, ZM can accelerate the wound healing process due to its antibacterial and antioxidant features, but no studies have been conducted yet to evaluate the Zataria multiflora essential oil (ZME) on the wound healing process. Therefore, this study was conducted to evaluate the effect of an ointment prepared from ZME on the healing process of infected wound model by S. aureus and P. aeruginosa by assessing wound contraction, total bacterial count, histopathological and immunohistochemical, antioxidant activity, and qRT-PCR analyses."," Materials Zataria multiflora essential oil was purchased from Barij Essence Company (Kashan-Iran) and approved by the same herbarium company (No.1203). Commercial base ointment and mupirocin were prepared from Pars Daruo. Ltd., Tehran, Iran. The commercial base ointment contained 90% soft paraffin, 5% hard paraffin and 5% lanolin. Rodent feeds were prepared from Javaneh Khorasan Company (Khorasan, Iran).\nZataria multiflora essential oil was purchased from Barij Essence Company (Kashan-Iran) and approved by the same herbarium company (No.1203). Commercial base ointment and mupirocin were prepared from Pars Daruo. Ltd., Tehran, Iran. The commercial base ointment contained 90% soft paraffin, 5% hard paraffin and 5% lanolin. Rodent feeds were prepared from Javaneh Khorasan Company (Khorasan, Iran).\n Essential oil analysis and identification Gas chromatography (GC) analysis was employed in order to identify the compounds. The components of the essential oil were recognized using calculation of their retention indices under temperature-programmed conditions for n-alkanes (C6–C24) and the oil on DB-5 column.\nGas chromatography (GC) analysis was employed in order to identify the compounds. The components of the essential oil were recognized using calculation of their retention indices under temperature-programmed conditions for n-alkanes (C6–C24) and the oil on DB-5 column.\n Animals Seventy-two BALB/c mice weighing 25 ± 3 g were prepared. The mice had free access to water and pelleted feed for rodents (Javaneh Khorasan Company, Khorasan, Iran). The mice were kept in an ambient temperature of 23 ± 3 °C, at a constant air humidity, and a natural day–night cycle. This study was approved by the Animal Research Committee of the Urmia Islamic Azad University with ethical No. IAU-UB 11044.\nSeventy-two BALB/c mice weighing 25 ± 3 g were prepared. The mice had free access to water and pelleted feed for rodents (Javaneh Khorasan Company, Khorasan, Iran). The mice were kept in an ambient temperature of 23 ± 3 °C, at a constant air humidity, and a natural day–night cycle. This study was approved by the Animal Research Committee of the Urmia Islamic Azad University with ethical No. IAU-UB 11044.\n Induction of wound To induce the wound, the mice were primarily anesthetized by intraperitoneal administration route using 50 mg/kg ketamine and 5 mg/kg xylazine. Following the induction of anaesthesia, dorsal region of each mouse was shaven for surgical actions. A circular wound model was induced in a size of 7 mm using a surgical biopsy punch and per wound was inoculated by an aliquot of 5 × 107 suspension containing S. aureus (ATCC 26313) and P. aeruginosa (ATCC 27853) in 50 μL phosphate-buffered saline (Farahpour et al. 2020; Khezri et al. 2019). Subsequently, animals were divided into four groups (n = 18), as (group I) negative control group (NCG) that was administered commercial base ointment, (group II) positive control group that were treated with Mupirocin® (MG), and (groups III and IV) treated groups with therapeutic ointments containing 2 g and 4 g ZME mixed in NCG (2% and 4% ZMEO), respectively. Concerning the colonization of the bacteria, 24 h after the induction of the wounds, ointments were applied on wounds, once a day. In addition, according to the tissue sampling on days 3, 7, and 14 after the wounding, the animals in each group were divided into three subgroups (n = 6).\nTo induce the wound, the mice were primarily anesthetized by intraperitoneal administration route using 50 mg/kg ketamine and 5 mg/kg xylazine. Following the induction of anaesthesia, dorsal region of each mouse was shaven for surgical actions. A circular wound model was induced in a size of 7 mm using a surgical biopsy punch and per wound was inoculated by an aliquot of 5 × 107 suspension containing S. aureus (ATCC 26313) and P. aeruginosa (ATCC 27853) in 50 μL phosphate-buffered saline (Farahpour et al. 2020; Khezri et al. 2019). Subsequently, animals were divided into four groups (n = 18), as (group I) negative control group (NCG) that was administered commercial base ointment, (group II) positive control group that were treated with Mupirocin® (MG), and (groups III and IV) treated groups with therapeutic ointments containing 2 g and 4 g ZME mixed in NCG (2% and 4% ZMEO), respectively. Concerning the colonization of the bacteria, 24 h after the induction of the wounds, ointments were applied on wounds, once a day. In addition, according to the tissue sampling on days 3, 7, and 14 after the wounding, the animals in each group were divided into three subgroups (n = 6).\n Wound area The rate of wound closure was estimated as reported previously (Nejati et al. 2015; Khezri et al. 2019). In order to calculate the percentage of wound closure rate, a transparent paper was placed on it and it was calculated based on the following formula:\nPercentage of wound closure = [(Wound area on Day 0 − Wound area on Day X)/Wound area on Day 0] × 100.\nThe rate of wound closure was estimated as reported previously (Nejati et al. 2015; Khezri et al. 2019). In order to calculate the percentage of wound closure rate, a transparent paper was placed on it and it was calculated based on the following formula:\nPercentage of wound closure = [(Wound area on Day 0 − Wound area on Day X)/Wound area on Day 0] × 100.\n The investigation of the bacteriological count in the wound area To calculate the bacteriological count, the granulated tissues were excised and 0.1 g of the sample was crushed, minced, and homogenized in a sterile mortar containing 10 mL of sterile saline. It was the diluted in tubes containing 9 mL of sterile saline, was cultured on plate count agar (Merck KGaA, Darmstadt, Germany), incubated at 37 °C for 48 h, calculated as CFU/g of granulation tissue (Farahpour et al. 2020; Khezri et al. 2019).\nTo calculate the bacteriological count, the granulated tissues were excised and 0.1 g of the sample was crushed, minced, and homogenized in a sterile mortar containing 10 mL of sterile saline. It was the diluted in tubes containing 9 mL of sterile saline, was cultured on plate count agar (Merck KGaA, Darmstadt, Germany), incubated at 37 °C for 48 h, calculated as CFU/g of granulation tissue (Farahpour et al. 2020; Khezri et al. 2019).\n Histological analysis Concerning the evaluation of the histological parameters, the mice were euthanized by a special CO2 device and the granulation tissue were excised in along to 1 to 2 mm from surrounding normal skin. The samples were fixed in neutral-buffered formalin 10%, routinely processed, and paraffin wax was embedded, sectioned at 5 µm, and stained with Masson’s trichrome and then examined under light microscopy (Olympus CX31RBSF attached cameraman) as reported by Farahpour et al. (2018). Cellular infiltration, edoema and collagen deposition were assessed. An image pro-insight software was utilised for evaluating the collagen deposition. Morphometric lens (Olympus, Germany) was used for assessing the epithelial thickness. edoema was assessed as a 5-score as reported previously (Nejati et al. 2015).\nConcerning the evaluation of the histological parameters, the mice were euthanized by a special CO2 device and the granulation tissue were excised in along to 1 to 2 mm from surrounding normal skin. The samples were fixed in neutral-buffered formalin 10%, routinely processed, and paraffin wax was embedded, sectioned at 5 µm, and stained with Masson’s trichrome and then examined under light microscopy (Olympus CX31RBSF attached cameraman) as reported by Farahpour et al. (2018). Cellular infiltration, edoema and collagen deposition were assessed. An image pro-insight software was utilised for evaluating the collagen deposition. Morphometric lens (Olympus, Germany) was used for assessing the epithelial thickness. edoema was assessed as a 5-score as reported previously (Nejati et al. 2015).\n Immunohistochemical staining (IHC) for angiogenesis (CD31) To assess the IHC, it was conducted as reported by Farahpour et al. (2018). The IHC was conducted based on manufacturer’s protocol (Biocare, Yorba Linda, CA). The tissue sections were rinsed gently in the washing buffer and placed in a buffer bath. A DAB chromogen was employed for assessing the tissue sections and then incubated for 5 min. The sections were then dipped in weak ammonia (0.037 M/L) for 10 times, rinsed with distilled water and cover slipped. Brown stains were considered as positive immunohistochemical.\nTo assess the IHC, it was conducted as reported by Farahpour et al. (2018). The IHC was conducted based on manufacturer’s protocol (Biocare, Yorba Linda, CA). The tissue sections were rinsed gently in the washing buffer and placed in a buffer bath. A DAB chromogen was employed for assessing the tissue sections and then incubated for 5 min. The sections were then dipped in weak ammonia (0.037 M/L) for 10 times, rinsed with distilled water and cover slipped. Brown stains were considered as positive immunohistochemical.\n Fluorescent staining for collagen To wash the slides, acetic acid 1% was used for several minutes. The slides were stained using acridine-orange. Phosphate buffer was utilized to remove staining. The slides were the washed in distilled water, mounted with buffer drop and analyzed with a fluorescent microscope. The bunds with red and yellowish red colour were considered as collagen bunds (Farahpour et al. 2018).\nTo wash the slides, acetic acid 1% was used for several minutes. The slides were stained using acridine-orange. Phosphate buffer was utilized to remove staining. The slides were the washed in distilled water, mounted with buffer drop and analyzed with a fluorescent microscope. The bunds with red and yellowish red colour were considered as collagen bunds (Farahpour et al. 2018).\n RNA isolation and cDNA synthesis The wound tissues were isolated as previously reported and RNA was extracted applying a standard TRIZOL procedure as reported by Farahpour et al. (2018), and assessed by spectrophotometer (260 nm and 260/280 = 1.8–2.0). To prepare the cDNA, it was put in a reaction being mixed with 1 µg RNA, oligo (dT) primer (1 µL), 5 × reaction buffer (4 µL), RNAse inhibitor (1 µL), 10 mM dNTP mix (2 µL) and M-MuLV Reverse Transcriptase (1 µL) as described by producer protocol (Fermentas, GmbH, Germany). Time and temperature were as reported by Farahpour et al. (2018). The used primers included IL-1β, forward (5′-AAC AAA CCC TGC AGT GGT TCG-3′) and reverse (5′-AGCTGCTTCAGACACTTGCAC-3′); TGF-β, forward (5-CCAAACGCCGAAGACTTATCC-3′) and reverse (5′-CTTATTACCGATGGGATGGGATAGCCC-3′); IL-10, forward (5′-GAAGCTCCCTCAGCGAGGACA-3′) and reverse (5′-TTGGGCCAGTGAGTGAAAGGG-3′), TNF-α, forward (5′-GAAGCTCCCTCAGCGAGGACA-3′) and reverse (5′-TTGGGCCAGTGAGTGAAAGGG-3′), FGF-2, forward (5′-GGAACCCGGCGGGACACGGAC-3′) and reverse (5′-CCGCTGTGGCAGCTCTTG GGG-3′); VEGF, forward (5′-GCTCCGTAGTAGCCGTGGTCT-3′) and reverse (5′-GGAACCCGGCGGGACACGGAC-3′), and IGF-1, forward (5′-TAGGTGGTTGATGAATGGT-3′) and reverse (5′-GAAAGGGCAGGGCTAAT-3′).\nThe wound tissues were isolated as previously reported and RNA was extracted applying a standard TRIZOL procedure as reported by Farahpour et al. (2018), and assessed by spectrophotometer (260 nm and 260/280 = 1.8–2.0). To prepare the cDNA, it was put in a reaction being mixed with 1 µg RNA, oligo (dT) primer (1 µL), 5 × reaction buffer (4 µL), RNAse inhibitor (1 µL), 10 mM dNTP mix (2 µL) and M-MuLV Reverse Transcriptase (1 µL) as described by producer protocol (Fermentas, GmbH, Germany). Time and temperature were as reported by Farahpour et al. (2018). The used primers included IL-1β, forward (5′-AAC AAA CCC TGC AGT GGT TCG-3′) and reverse (5′-AGCTGCTTCAGACACTTGCAC-3′); TGF-β, forward (5-CCAAACGCCGAAGACTTATCC-3′) and reverse (5′-CTTATTACCGATGGGATGGGATAGCCC-3′); IL-10, forward (5′-GAAGCTCCCTCAGCGAGGACA-3′) and reverse (5′-TTGGGCCAGTGAGTGAAAGGG-3′), TNF-α, forward (5′-GAAGCTCCCTCAGCGAGGACA-3′) and reverse (5′-TTGGGCCAGTGAGTGAAAGGG-3′), FGF-2, forward (5′-GGAACCCGGCGGGACACGGAC-3′) and reverse (5′-CCGCTGTGGCAGCTCTTG GGG-3′); VEGF, forward (5′-GCTCCGTAGTAGCCGTGGTCT-3′) and reverse (5′-GGAACCCGGCGGGACACGGAC-3′), and IGF-1, forward (5′-TAGGTGGTTGATGAATGGT-3′) and reverse (5′-GAAAGGGCAGGGCTAAT-3′).\n Antioxidant capacity To assess the antioxidant capacity, the wound granulation tissue was homogenized in ice-cold KCl (150 mM) and the mixture was then centrifuged at 3000 g for 10 min. The supernatant was used to evaluate the total antioxidant capacity (TAC), malondialdehyde (MDA) and total tissue thiol molecules (TTM) content. The Lowry method was employed for evaluating the protein content of the samples (Daemi et al. 2019a) and MDA content was used for assessing the lipid peroxidation rate. To assess the TTM, the collected granulation tissue sample was homogenized and the supernatant was added to Tris-EDTA as reported by Daemi et al. (2019a).\nTo assess the antioxidant capacity, the wound granulation tissue was homogenized in ice-cold KCl (150 mM) and the mixture was then centrifuged at 3000 g for 10 min. The supernatant was used to evaluate the total antioxidant capacity (TAC), malondialdehyde (MDA) and total tissue thiol molecules (TTM) content. The Lowry method was employed for evaluating the protein content of the samples (Daemi et al. 2019a) and MDA content was used for assessing the lipid peroxidation rate. To assess the TTM, the collected granulation tissue sample was homogenized and the supernatant was added to Tris-EDTA as reported by Daemi et al. (2019a).\n Statistical analysis The results were reported as mean ± standard deviation and analyzed by Graph Pad Prism Software. One-way ANOVA was utilized to analyze the results. Dunnett’s test for pair-wise comparisons was used for evaluating the effect of treatments and p < 0.05 was considered to be of significance.\nThe results were reported as mean ± standard deviation and analyzed by Graph Pad Prism Software. One-way ANOVA was utilized to analyze the results. Dunnett’s test for pair-wise comparisons was used for evaluating the effect of treatments and p < 0.05 was considered to be of significance.","Zataria multiflora essential oil was purchased from Barij Essence Company (Kashan-Iran) and approved by the same herbarium company (No.1203). Commercial base ointment and mupirocin were prepared from Pars Daruo. Ltd., Tehran, Iran. The commercial base ointment contained 90% soft paraffin, 5% hard paraffin and 5% lanolin. Rodent feeds were prepared from Javaneh Khorasan Company (Khorasan, Iran).","Gas chromatography (GC) analysis was employed in order to identify the compounds. The components of the essential oil were recognized using calculation of their retention indices under temperature-programmed conditions for n-alkanes (C6–C24) and the oil on DB-5 column.","Seventy-two BALB/c mice weighing 25 ± 3 g were prepared. The mice had free access to water and pelleted feed for rodents (Javaneh Khorasan Company, Khorasan, Iran). The mice were kept in an ambient temperature of 23 ± 3 °C, at a constant air humidity, and a natural day–night cycle. This study was approved by the Animal Research Committee of the Urmia Islamic Azad University with ethical No. IAU-UB 11044.","To induce the wound, the mice were primarily anesthetized by intraperitoneal administration route using 50 mg/kg ketamine and 5 mg/kg xylazine. Following the induction of anaesthesia, dorsal region of each mouse was shaven for surgical actions. A circular wound model was induced in a size of 7 mm using a surgical biopsy punch and per wound was inoculated by an aliquot of 5 × 107 suspension containing S. aureus (ATCC 26313) and P. aeruginosa (ATCC 27853) in 50 μL phosphate-buffered saline (Farahpour et al. 2020; Khezri et al. 2019). Subsequently, animals were divided into four groups (n = 18), as (group I) negative control group (NCG) that was administered commercial base ointment, (group II) positive control group that were treated with Mupirocin® (MG), and (groups III and IV) treated groups with therapeutic ointments containing 2 g and 4 g ZME mixed in NCG (2% and 4% ZMEO), respectively. Concerning the colonization of the bacteria, 24 h after the induction of the wounds, ointments were applied on wounds, once a day. In addition, according to the tissue sampling on days 3, 7, and 14 after the wounding, the animals in each group were divided into three subgroups (n = 6).","The rate of wound closure was estimated as reported previously (Nejati et al. 2015; Khezri et al. 2019). In order to calculate the percentage of wound closure rate, a transparent paper was placed on it and it was calculated based on the following formula:\nPercentage of wound closure = [(Wound area on Day 0 − Wound area on Day X)/Wound area on Day 0] × 100.","To calculate the bacteriological count, the granulated tissues were excised and 0.1 g of the sample was crushed, minced, and homogenized in a sterile mortar containing 10 mL of sterile saline. It was the diluted in tubes containing 9 mL of sterile saline, was cultured on plate count agar (Merck KGaA, Darmstadt, Germany), incubated at 37 °C for 48 h, calculated as CFU/g of granulation tissue (Farahpour et al. 2020; Khezri et al. 2019).","Concerning the evaluation of the histological parameters, the mice were euthanized by a special CO2 device and the granulation tissue were excised in along to 1 to 2 mm from surrounding normal skin. The samples were fixed in neutral-buffered formalin 10%, routinely processed, and paraffin wax was embedded, sectioned at 5 µm, and stained with Masson’s trichrome and then examined under light microscopy (Olympus CX31RBSF attached cameraman) as reported by Farahpour et al. (2018). Cellular infiltration, edoema and collagen deposition were assessed. An image pro-insight software was utilised for evaluating the collagen deposition. Morphometric lens (Olympus, Germany) was used for assessing the epithelial thickness. edoema was assessed as a 5-score as reported previously (Nejati et al. 2015).","To assess the IHC, it was conducted as reported by Farahpour et al. (2018). The IHC was conducted based on manufacturer’s protocol (Biocare, Yorba Linda, CA). The tissue sections were rinsed gently in the washing buffer and placed in a buffer bath. A DAB chromogen was employed for assessing the tissue sections and then incubated for 5 min. The sections were then dipped in weak ammonia (0.037 M/L) for 10 times, rinsed with distilled water and cover slipped. Brown stains were considered as positive immunohistochemical.","To wash the slides, acetic acid 1% was used for several minutes. The slides were stained using acridine-orange. Phosphate buffer was utilized to remove staining. The slides were the washed in distilled water, mounted with buffer drop and analyzed with a fluorescent microscope. The bunds with red and yellowish red colour were considered as collagen bunds (Farahpour et al. 2018).","The wound tissues were isolated as previously reported and RNA was extracted applying a standard TRIZOL procedure as reported by Farahpour et al. (2018), and assessed by spectrophotometer (260 nm and 260/280 = 1.8–2.0). To prepare the cDNA, it was put in a reaction being mixed with 1 µg RNA, oligo (dT) primer (1 µL), 5 × reaction buffer (4 µL), RNAse inhibitor (1 µL), 10 mM dNTP mix (2 µL) and M-MuLV Reverse Transcriptase (1 µL) as described by producer protocol (Fermentas, GmbH, Germany). Time and temperature were as reported by Farahpour et al. (2018). The used primers included IL-1β, forward (5′-AAC AAA CCC TGC AGT GGT TCG-3′) and reverse (5′-AGCTGCTTCAGACACTTGCAC-3′); TGF-β, forward (5-CCAAACGCCGAAGACTTATCC-3′) and reverse (5′-CTTATTACCGATGGGATGGGATAGCCC-3′); IL-10, forward (5′-GAAGCTCCCTCAGCGAGGACA-3′) and reverse (5′-TTGGGCCAGTGAGTGAAAGGG-3′), TNF-α, forward (5′-GAAGCTCCCTCAGCGAGGACA-3′) and reverse (5′-TTGGGCCAGTGAGTGAAAGGG-3′), FGF-2, forward (5′-GGAACCCGGCGGGACACGGAC-3′) and reverse (5′-CCGCTGTGGCAGCTCTTG GGG-3′); VEGF, forward (5′-GCTCCGTAGTAGCCGTGGTCT-3′) and reverse (5′-GGAACCCGGCGGGACACGGAC-3′), and IGF-1, forward (5′-TAGGTGGTTGATGAATGGT-3′) and reverse (5′-GAAAGGGCAGGGCTAAT-3′).","To assess the antioxidant capacity, the wound granulation tissue was homogenized in ice-cold KCl (150 mM) and the mixture was then centrifuged at 3000 g for 10 min. The supernatant was used to evaluate the total antioxidant capacity (TAC), malondialdehyde (MDA) and total tissue thiol molecules (TTM) content. The Lowry method was employed for evaluating the protein content of the samples (Daemi et al. 2019a) and MDA content was used for assessing the lipid peroxidation rate. To assess the TTM, the collected granulation tissue sample was homogenized and the supernatant was added to Tris-EDTA as reported by Daemi et al. (2019a).","The results were reported as mean ± standard deviation and analyzed by Graph Pad Prism Software. One-way ANOVA was utilized to analyze the results. Dunnett’s test for pair-wise comparisons was used for evaluating the effect of treatments and p < 0.05 was considered to be of significance."," Composition of the Zataria multiflora essential oil The analysis of Zataria multiflora essential oil with GC-FID and GC-mass spectrometry (MS) identified 29 compounds which accounted for 99.6% of the total essential oil composition (Table 1). It contained thymol (52.90%), p-cymene (9.10), γ-terpinene (8.10%) and carvacrol (6.80%) as major constituents (Figure 1).\nGC-MS chromatogram of the essential oil of Zataria multiflora.\nChemical constituents of Zataria multiflora essential oil.\nRI-Cal: retention indices calculated based on C6–C24\nn-alkenes from a DB-5 column.\nRI-Cal: retention indices retrieved from literature (Adams 2007).\nBold values show major compounds in the essential oil.\nThe analysis of Zataria multiflora essential oil with GC-FID and GC-mass spectrometry (MS) identified 29 compounds which accounted for 99.6% of the total essential oil composition (Table 1). It contained thymol (52.90%), p-cymene (9.10), γ-terpinene (8.10%) and carvacrol (6.80%) as major constituents (Figure 1).\nGC-MS chromatogram of the essential oil of Zataria multiflora.\nChemical constituents of Zataria multiflora essential oil.\nRI-Cal: retention indices calculated based on C6–C24\nn-alkenes from a DB-5 column.\nRI-Cal: retention indices retrieved from literature (Adams 2007).\nBold values show major compounds in the essential oil.\n Wound area The findings for the effects of ZMEO on the percentage of wound contraction are shown in Figure 2. The results implied that topical administration of therapeutic ointments significantly (p < 0.05) increased wound contraction rate on days 7 and 14 compared to the NCG group (Figure 2(A,B)). The comparison between treatment groups revealed that a higher wound contraction rate was observed in the 4% ZMEO-treated group compared to the 2% ZMEO-treated and MG groups (p < 0.05).\nThe effects of topical administration of ZMEO on (B) wound contraction rate and (C) tissue bacterial count (CFU/g) in different days. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\nThe findings for the effects of ZMEO on the percentage of wound contraction are shown in Figure 2. The results implied that topical administration of therapeutic ointments significantly (p < 0.05) increased wound contraction rate on days 7 and 14 compared to the NCG group (Figure 2(A,B)). The comparison between treatment groups revealed that a higher wound contraction rate was observed in the 4% ZMEO-treated group compared to the 2% ZMEO-treated and MG groups (p < 0.05).\nThe effects of topical administration of ZMEO on (B) wound contraction rate and (C) tissue bacterial count (CFU/g) in different days. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\n The bacteriological count The results concerning the effects of ZMEO on tissue bacteria counts are represented in Figure 2(C). The results showed that topical administration of therapeutic ointments significantly decreased bacterial colonization in all the days compared to the NCG group (p < 0.05). The comparison between treatment groups demonstrated that a higher level of ZMEO (4%) significantly decreased the total bacterial count compared to that of a lower level of it (2%) and MG groups (p < 0.05).\nThe results concerning the effects of ZMEO on tissue bacteria counts are represented in Figure 2(C). The results showed that topical administration of therapeutic ointments significantly decreased bacterial colonization in all the days compared to the NCG group (p < 0.05). The comparison between treatment groups demonstrated that a higher level of ZMEO (4%) significantly decreased the total bacterial count compared to that of a lower level of it (2%) and MG groups (p < 0.05).\n Histopathological parameters The results for histopathological parameters are represented in Table 2. The results showed that edoema score was significantly higher in the NCG group. It was observed to be significantly lower in the animals treated with the 4% ZMEO-treated group and also in 2% ZMEO and MG-treated groups. The findings implied that the lowest edoema was observed in 4% ZMEO in day 14; meanwhile, there was no observed edoema in the 4% ZMEO group on day 14. The results showed that immune cells were significantly lower in ZMEO groups in day 3 compared to other groups (p < 0.05), yet it decreased on days 7 and 14 in the ZMEO groups compared to other groups (p < 0.05). It means that the application of the ZMEO decreases immune cells in days 7 and 14, and increased it on day 3. Angiogenesis (Figure 3) and fibroblast infiltration were significantly higher in all treated groups compared to the NCG group and the highest level was observed in level of the 4%-ZMEO group. The results for collagen condensation (Figure 4) and re-epithelialization (Figure 5) revealed that parameters were significantly higher in the ZMEO groups, which confirms the results.\nImmunohistochemical staining for angiogenesis:; (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See elevated angiogenesis in ZMEO-treated group (arrows) on day 7 after wound induction. CD 31 staining, 400×.\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. Note well-formed collagen deposition in cross sections from ZMEO-treated animals on day 7 after wound induction (first row), which is significantly increased on day 14 after injury (second row). The third row represents the software analyze for collagen intensity. The cross sections from animals in ZMEO-treated groups exhibited condense collagen deposition versus NCG and MG groups. Masson-trichrome staining and fluorescent staining for collagen, 400× magnification.\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See well re-epithelialization in ZMEO-treated animals. The re-epithelialization initiated on day 7 after wound induction in ZMEO-treated animals. However, the cross sections from NCG and MG groups are not representing epithelialization. Note well-organized dermis and complete epithelialization with well-formed papillae in ZMEO-treated animals in comparison to NCG and MG groups. Masson-trichrome staining, 100×.\nThe effects of ZMEO on histopathological parameters in different days.\nSuperscripts (a–d) indicate significant differences in same day at p < 0.05.\nThe results for histopathological parameters are represented in Table 2. The results showed that edoema score was significantly higher in the NCG group. It was observed to be significantly lower in the animals treated with the 4% ZMEO-treated group and also in 2% ZMEO and MG-treated groups. The findings implied that the lowest edoema was observed in 4% ZMEO in day 14; meanwhile, there was no observed edoema in the 4% ZMEO group on day 14. The results showed that immune cells were significantly lower in ZMEO groups in day 3 compared to other groups (p < 0.05), yet it decreased on days 7 and 14 in the ZMEO groups compared to other groups (p < 0.05). It means that the application of the ZMEO decreases immune cells in days 7 and 14, and increased it on day 3. Angiogenesis (Figure 3) and fibroblast infiltration were significantly higher in all treated groups compared to the NCG group and the highest level was observed in level of the 4%-ZMEO group. The results for collagen condensation (Figure 4) and re-epithelialization (Figure 5) revealed that parameters were significantly higher in the ZMEO groups, which confirms the results.\nImmunohistochemical staining for angiogenesis:; (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See elevated angiogenesis in ZMEO-treated group (arrows) on day 7 after wound induction. CD 31 staining, 400×.\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. Note well-formed collagen deposition in cross sections from ZMEO-treated animals on day 7 after wound induction (first row), which is significantly increased on day 14 after injury (second row). The third row represents the software analyze for collagen intensity. The cross sections from animals in ZMEO-treated groups exhibited condense collagen deposition versus NCG and MG groups. Masson-trichrome staining and fluorescent staining for collagen, 400× magnification.\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See well re-epithelialization in ZMEO-treated animals. The re-epithelialization initiated on day 7 after wound induction in ZMEO-treated animals. However, the cross sections from NCG and MG groups are not representing epithelialization. Note well-organized dermis and complete epithelialization with well-formed papillae in ZMEO-treated animals in comparison to NCG and MG groups. Masson-trichrome staining, 100×.\nThe effects of ZMEO on histopathological parameters in different days.\nSuperscripts (a–d) indicate significant differences in same day at p < 0.05.\n Molecular analyses The results for gene expression are presented in Figure 6. Gene expression of IL-1β and TNF-α was significantly (p < 0.05) lower on all sampling days at treated groups compared to the NCG group. The greatest decrease was observed in the 4% ZMEO-treated animals. Gene expressions of IGF-1 and VEGF was significantly (p < 0.05) higher at all treated groups on day 7 after the wounding, but the levels significantly decreased on day 14. The expression of IL-10 level increased on day 7 whereas it decreased on day 14 compared to the NCG group (p < 0.05). Further analysis of gene expressions of TGF-β and FGF-2 indicate that its level does not have significant difference (p > 0.05) on day 3, yet its level significantly (p < 0.05) increased in all treated groups, particularly at 4% ZMEO-treated animals on day 7. The amount of TGF-β and FGF-2 gene expression levels also were significantly (p < 0.05) reduced on day 14 compared to the NCG group.\nThe effects of topical application of ZMEO on gene expression. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\nThe results for gene expression are presented in Figure 6. Gene expression of IL-1β and TNF-α was significantly (p < 0.05) lower on all sampling days at treated groups compared to the NCG group. The greatest decrease was observed in the 4% ZMEO-treated animals. Gene expressions of IGF-1 and VEGF was significantly (p < 0.05) higher at all treated groups on day 7 after the wounding, but the levels significantly decreased on day 14. The expression of IL-10 level increased on day 7 whereas it decreased on day 14 compared to the NCG group (p < 0.05). Further analysis of gene expressions of TGF-β and FGF-2 indicate that its level does not have significant difference (p > 0.05) on day 3, yet its level significantly (p < 0.05) increased in all treated groups, particularly at 4% ZMEO-treated animals on day 7. The amount of TGF-β and FGF-2 gene expression levels also were significantly (p < 0.05) reduced on day 14 compared to the NCG group.\nThe effects of topical application of ZMEO on gene expression. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\n Antioxidant status Antioxidant status in different groups is represented in Figure 7. Using the 2% and 4%-ZMEO significantly (p < 0.05) increased TAC and TTP levels compared to the NCG group in all of the days after the wounding. Interestingly, the highest increase was observed in the 4% ZMEO-treated animals. Further analysis of MDA level indicates significant (p < 0.05) decrease in all of the treated groups compared to the NCG group in all of the days after the wounding. The highest decrease was observed in the 4% ZMEO-treated group.\nThe effects of topical application of ZMEO on antioxidant properties. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\nAntioxidant status in different groups is represented in Figure 7. Using the 2% and 4%-ZMEO significantly (p < 0.05) increased TAC and TTP levels compared to the NCG group in all of the days after the wounding. Interestingly, the highest increase was observed in the 4% ZMEO-treated animals. Further analysis of MDA level indicates significant (p < 0.05) decrease in all of the treated groups compared to the NCG group in all of the days after the wounding. The highest decrease was observed in the 4% ZMEO-treated group.\nThe effects of topical application of ZMEO on antioxidant properties. Superscripts (a–d) indicate significant differences in same day at p < 0.05.","The analysis of Zataria multiflora essential oil with GC-FID and GC-mass spectrometry (MS) identified 29 compounds which accounted for 99.6% of the total essential oil composition (Table 1). It contained thymol (52.90%), p-cymene (9.10), γ-terpinene (8.10%) and carvacrol (6.80%) as major constituents (Figure 1).\nGC-MS chromatogram of the essential oil of Zataria multiflora.\nChemical constituents of Zataria multiflora essential oil.\nRI-Cal: retention indices calculated based on C6–C24\nn-alkenes from a DB-5 column.\nRI-Cal: retention indices retrieved from literature (Adams 2007).\nBold values show major compounds in the essential oil.","The findings for the effects of ZMEO on the percentage of wound contraction are shown in Figure 2. The results implied that topical administration of therapeutic ointments significantly (p < 0.05) increased wound contraction rate on days 7 and 14 compared to the NCG group (Figure 2(A,B)). The comparison between treatment groups revealed that a higher wound contraction rate was observed in the 4% ZMEO-treated group compared to the 2% ZMEO-treated and MG groups (p < 0.05).\nThe effects of topical administration of ZMEO on (B) wound contraction rate and (C) tissue bacterial count (CFU/g) in different days. Superscripts (a–d) indicate significant differences in same day at p < 0.05.","The results concerning the effects of ZMEO on tissue bacteria counts are represented in Figure 2(C). The results showed that topical administration of therapeutic ointments significantly decreased bacterial colonization in all the days compared to the NCG group (p < 0.05). The comparison between treatment groups demonstrated that a higher level of ZMEO (4%) significantly decreased the total bacterial count compared to that of a lower level of it (2%) and MG groups (p < 0.05).","The results for histopathological parameters are represented in Table 2. The results showed that edoema score was significantly higher in the NCG group. It was observed to be significantly lower in the animals treated with the 4% ZMEO-treated group and also in 2% ZMEO and MG-treated groups. The findings implied that the lowest edoema was observed in 4% ZMEO in day 14; meanwhile, there was no observed edoema in the 4% ZMEO group on day 14. The results showed that immune cells were significantly lower in ZMEO groups in day 3 compared to other groups (p < 0.05), yet it decreased on days 7 and 14 in the ZMEO groups compared to other groups (p < 0.05). It means that the application of the ZMEO decreases immune cells in days 7 and 14, and increased it on day 3. Angiogenesis (Figure 3) and fibroblast infiltration were significantly higher in all treated groups compared to the NCG group and the highest level was observed in level of the 4%-ZMEO group. The results for collagen condensation (Figure 4) and re-epithelialization (Figure 5) revealed that parameters were significantly higher in the ZMEO groups, which confirms the results.\nImmunohistochemical staining for angiogenesis:; (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See elevated angiogenesis in ZMEO-treated group (arrows) on day 7 after wound induction. CD 31 staining, 400×.\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. Note well-formed collagen deposition in cross sections from ZMEO-treated animals on day 7 after wound induction (first row), which is significantly increased on day 14 after injury (second row). The third row represents the software analyze for collagen intensity. The cross sections from animals in ZMEO-treated groups exhibited condense collagen deposition versus NCG and MG groups. Masson-trichrome staining and fluorescent staining for collagen, 400× magnification.\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See well re-epithelialization in ZMEO-treated animals. The re-epithelialization initiated on day 7 after wound induction in ZMEO-treated animals. However, the cross sections from NCG and MG groups are not representing epithelialization. Note well-organized dermis and complete epithelialization with well-formed papillae in ZMEO-treated animals in comparison to NCG and MG groups. Masson-trichrome staining, 100×.\nThe effects of ZMEO on histopathological parameters in different days.\nSuperscripts (a–d) indicate significant differences in same day at p < 0.05.","The results for gene expression are presented in Figure 6. Gene expression of IL-1β and TNF-α was significantly (p < 0.05) lower on all sampling days at treated groups compared to the NCG group. The greatest decrease was observed in the 4% ZMEO-treated animals. Gene expressions of IGF-1 and VEGF was significantly (p < 0.05) higher at all treated groups on day 7 after the wounding, but the levels significantly decreased on day 14. The expression of IL-10 level increased on day 7 whereas it decreased on day 14 compared to the NCG group (p < 0.05). Further analysis of gene expressions of TGF-β and FGF-2 indicate that its level does not have significant difference (p > 0.05) on day 3, yet its level significantly (p < 0.05) increased in all treated groups, particularly at 4% ZMEO-treated animals on day 7. The amount of TGF-β and FGF-2 gene expression levels also were significantly (p < 0.05) reduced on day 14 compared to the NCG group.\nThe effects of topical application of ZMEO on gene expression. Superscripts (a–d) indicate significant differences in same day at p < 0.05.","Antioxidant status in different groups is represented in Figure 7. Using the 2% and 4%-ZMEO significantly (p < 0.05) increased TAC and TTP levels compared to the NCG group in all of the days after the wounding. Interestingly, the highest increase was observed in the 4% ZMEO-treated animals. Further analysis of MDA level indicates significant (p < 0.05) decrease in all of the treated groups compared to the NCG group in all of the days after the wounding. The highest decrease was observed in the 4% ZMEO-treated group.\nThe effects of topical application of ZMEO on antioxidant properties. Superscripts (a–d) indicate significant differences in same day at p < 0.05.","Wounds infected by pathogenic bacteria such as S. aureus, and P. aeruginosa are believed to be serious challenges for regarding their management and treatment (Manzuoerh et al. 2019). Different agents are employed for the treatment of wounds. Active compounds of the essential oils can have pivotal roles in their treatment. The findings indicated that thymol (52.90%), p-cymene (9.10), γ-terpinene (8.10%) and carvacrol (6.80%) were the major constituents in the essential oil. Similar to our results, previous studies demonstrated that the major compounds of Z. multiflora essential oil were thymol, carvacrol and p-cymene (Aida et al. 2015), thymol, c-terpinene, p-cymene, and carvacrol (Saei-Dehkordi et al. 2010), carvacrol (Misaghi and Basti 2007), thymol, carvacrol, and p-cymene (Sharififar et al. 2007). The results showed that ZMEO accelerated wound healing compared to the NCG group and synthetic agent of mupirocin. Herein, the results are described based on the mechanisms and active compounds, step-by-step.\nInflammation is the first step in the wound healing process, in which different factors and genes are involved. Inflammation is triggered once the injury induces to living tissues by live organisms, such as bacteria and/or physical injury and faulted immune response. Inflammation removes foreign substances in order to improve the wound healing process (Garrett et al. 2010). It promotes macrophages, neutrophils and immune cells for initiation of inflammation (Oguntibeju 2018). IL-1β and TNF-α are commonly produced by immune cells, such as macrophages and mast cells (Thacker et al. 2007). The results showed that the topical application of ZMEO decreased immune cells and the expressions of IL-1β and TNF-α in the first 3 days. The results implied a positive relation between the number of immune cells with the expression of IL-1β and TNF-α in the treatments. Meaning that immune cells increase the expression of IL-1β and TNF-α in the first 3 days. Increased inflammation delays the wound healing process. The findings revealed that topical administration of ZMEO decreased inflammation on days 7 and 14, and acted as an antibacterial and anti-inflammation agent. Antibacterial and anti-inflammatory activity of ZMEO is attributed to its compounds. The essence contains thymol and carvacrol that are of antibacterial and anti-inflammatory properties. It was reported that the administration of carvacrol significantly reduced TNF-α levels in pleural lavage (Guimarães et al. 2012). The anti-inflammatory activity of thymol leads to prevention of the phosphorylation of extracellular signal-regulated protein kinase, c-Jun N-terminal kinase, and nuclear factor-κB (Liang et al. 2014). Carvacrol prevented leukocyte migration, and decreased edoema, and showed anti-inflammatory effects (Fachini-Queiroz et al. 2012). The results indicated that immune cells decreased and expression of IL-1β and TNF-α also decreased in ZMEO groups. The findings showed that the total bacterial count significantly decreased in ZMEO groups compared to the NCG group. Mupirocin is used as a bactericide agent for the treatment of infected wounds. The results convey that ZMEO, particularly at a higher level, have better antibacterial activity compared to mupirocin. Higher total bacterial count increases the inflammation and delays the wound healing process. It means that topical administration of ZMEO decreases the inflammation with its antibacterial activity. It was reported that the antibacterial activity of Zataria multiflora is in view of its compounds (Shafiee et al. 1999; Barkhori-Mehni et al. 2017). In essence, ZMEO decreased the inflammation and inflammatory-associated parameters. Higher levels of ZMEO provide higher doses of active compounds that act as anti-inflammatory factors. Decreased inflammation leads the wound healing process towards the proliferative phase.\nOur findings implied that the topical application of ZMEO improved the proliferative phase. The results showed that the genes expression of VEGF, FGF-2 and IGF-1 increased in ZMEO groups. IGF-1 increases the transport of glucose during the wound healing process and improves the proliferative phase (Daemi et al. 2019b). VEGF induces angiogenesis and stimulates cell migration and proliferation (Norton & Popel 2016; Farahpour et al. 2020; Gharaboghaz et al. 2020). FGF-2, a protein that participates in the wound healing process (Souto et al. 2020), together with other growth factors, such as VEGF, are reported to increase the angiogenesis and indirectly support the cellular nutrient, oxygen and energy supplements (Harding et al. 2002). So far, studies have not investigated the effect of ZMEO on genes expression of VEGF, FGF-2 and IGF-1. It means that VEGF improves angiogenesis, and vessel number was significantly higher in ZMEO groups. VEGF improved angiogenesis and number of vessels. Other genes involved were TGF-β, and IL-10 whose expressions increased on day 7. IL-10 is a factor that reduces the production of pro-inflammatory cytokine, but TGF-β leads the phase towards the proliferative phase by improving the proliferation and differentiation of fibroblasts, collagen production, and wound contraction (Khezri et al. 2020). Seemingly, both genes are of greatly important roles in decreasing the inflammation and passing to the proliferative phase. The IHC results indicated improved collagen deposition. The biosynthesis of collagen during the proliferation stage has pivotal roles in dermal maturation in which some factors are involved (Yakaew et al. 2016; Daemi et al. 2019a). The results showed that the topical application of ZMEO increased fibroblast, which is due to its effect on IGF-1. It was reported that IGF-1 increases fibroblast cells in in vitro assessment (Lisa et al. 2011). Improving cellular proliferation and differentiation are essential for shortening the healing time (Oryan et al. 2015; Karimzadeh and Farahpour 2017). Proliferative effect of ZMEO is partly owing to its antioxidant activity. The topical application of ZMEO improved antioxidant activity. Previous studies have reported antioxidant activity of Zataria multiflora essential oil (Fatemi et al. 2012; Karimian et al. 2012; Kavoosi and Teixeira da Silva 2012). Oxidative stress in the wound area increases the damage of proteins, nucleotides and lipid levels (Cano Sanchez et al. 2018; Farahpour et al. 2020; Gharaboghaz et al. 2020) and decreases the antioxidant activity. This fact is associated with increased changes in the antioxidant profile, which are contributed to the increase in the levels of MDA content (Sharma et al. 2012; Bardaa et al. 2016; Vittorazzi et al. 2016).","The topical application of ZMEO decreased the inflammation and the total bacterial count, which progresses the wound healing process towards the proliferative phase. It increases the expression of proliferative genes, helps to alleviate the inflammation phase and accelerates the proliferative phase. Antioxidant activity of ZMEO accelerated the wound healing process. All the above-mentioned factors hasten the wound healing process, as seen for the wound area. Accordingly, applying ZMEO only and/or in combination with chemical agents for the treatment of wound healing could be suggested."],"string":"[\n \"Wound infections are important challenges that cause the suffering of patients and economic losses (Vittorazzi et al. 2016; Daemi et al. 2019a; Farghali et al. 2019). Skin is a regenerative organ which acts as a barrier between the body and the external environment (Hassan et al. 2014). A wound is defined as a disturbance in anatomic structure of skin and its functional integrity (Farghali et al. 2019). Skin prevents the penetration of bacteria and fungi that are important challenges that induce mortality and morbidity. Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic bacteria that induce infection in patients (Cardona and Wilson 2015). The bacteria are commonly detected in the upper and deepest region of wound bed (Serra et al. 2015). Colonization of S. aureus and P. aeruginosa in the wound site postpones the wound healing process (Farahpour et al. 2020; Khezri et al. 2020). The wound healing process comprises several phases, including coagulation, inflammation, epithelialization, granulation tissue formation, and tissue remodelling (Daemi et al. 2019a, 2019b; Farahpour et al. 2020). Inflammatory phase occurs after activation of inflammatory chemokines (Farahpour et al. 2020). Interleukin-1β (IL-1β) is significantly synthesized 12–24 h after infliction of the wound, and its level returns to basal level after the proliferative stage is completed (Fahey and Doyle 2019). Tumour necrosis factor-α (TNF-α) is significantly synthesized by macrophages and T lymphocytes and its level increases under inflammation and infection conditions (Xue and Falcon 2019). Following the induction of inflammation, proliferative phase occurs in which many genes are involved. Insulin-like growth factor 1 (IGF-1) promotes the production of keratinocyte and fibroblast proliferation and improves the re-epithelialization (Daemi et al. 2019b). Decreased endothelial insulin/IGF-1 signal delays in the wound healing process (Aghdam et al. 2012). Vascular endothelial growth factor (VEGF) induces angiogenesis and stimulates cell migration and proliferation (Farahpour et al. 2020). Fibroblast growth factor-2 (FGF-2) is a protein that participates in the wound healing process (Souto et al. 2020). Interleukin-10 (IL-10) reduces the production of pro-inflammatory cytokine, but tumour growth factor-β (TGF-β) has roles in increasing the proliferative phase improving the proliferation and differentiation of fibroblasts, collagen production, and wound contraction (Khezri et al. 2020). On the other hand, oxidative stress increases under wound disorders and increases the production of reactive oxygen species (ROS) in the site of wound (Ustuner et al. 2019). Applying antibacterial and antioxidant agents shortens the inflammatory phase, promotes the proliferative phase and accelerates the wound healing process (Bardaa et al. 2016; Vittorazzi et al. 2016; Daemi et al. 2019a, 2019b).\\nZataria multiflora Boiss (Lamiaceae) (ZM) has antibacterial activity against Pseudomonas spp. (Barkhori-Mehni et al. 2017). Its antibacterial activity is attributed to its phenolic compounds that destroy the cell wall in bacteria (Barkhori-Mehni et al. 2017). It demonstrates antioxidant properties due to its compounds such as thymol and carvacrol (Mojaddar Langroodi et al. 2019). Seemingly, ZM can accelerate the wound healing process due to its antibacterial and antioxidant features, but no studies have been conducted yet to evaluate the Zataria multiflora essential oil (ZME) on the wound healing process. Therefore, this study was conducted to evaluate the effect of an ointment prepared from ZME on the healing process of infected wound model by S. aureus and P. aeruginosa by assessing wound contraction, total bacterial count, histopathological and immunohistochemical, antioxidant activity, and qRT-PCR analyses.\",\n \" Materials Zataria multiflora essential oil was purchased from Barij Essence Company (Kashan-Iran) and approved by the same herbarium company (No.1203). Commercial base ointment and mupirocin were prepared from Pars Daruo. Ltd., Tehran, Iran. The commercial base ointment contained 90% soft paraffin, 5% hard paraffin and 5% lanolin. Rodent feeds were prepared from Javaneh Khorasan Company (Khorasan, Iran).\\nZataria multiflora essential oil was purchased from Barij Essence Company (Kashan-Iran) and approved by the same herbarium company (No.1203). Commercial base ointment and mupirocin were prepared from Pars Daruo. Ltd., Tehran, Iran. The commercial base ointment contained 90% soft paraffin, 5% hard paraffin and 5% lanolin. Rodent feeds were prepared from Javaneh Khorasan Company (Khorasan, Iran).\\n Essential oil analysis and identification Gas chromatography (GC) analysis was employed in order to identify the compounds. The components of the essential oil were recognized using calculation of their retention indices under temperature-programmed conditions for n-alkanes (C6–C24) and the oil on DB-5 column.\\nGas chromatography (GC) analysis was employed in order to identify the compounds. The components of the essential oil were recognized using calculation of their retention indices under temperature-programmed conditions for n-alkanes (C6–C24) and the oil on DB-5 column.\\n Animals Seventy-two BALB/c mice weighing 25 ± 3 g were prepared. The mice had free access to water and pelleted feed for rodents (Javaneh Khorasan Company, Khorasan, Iran). The mice were kept in an ambient temperature of 23 ± 3 °C, at a constant air humidity, and a natural day–night cycle. This study was approved by the Animal Research Committee of the Urmia Islamic Azad University with ethical No. IAU-UB 11044.\\nSeventy-two BALB/c mice weighing 25 ± 3 g were prepared. The mice had free access to water and pelleted feed for rodents (Javaneh Khorasan Company, Khorasan, Iran). The mice were kept in an ambient temperature of 23 ± 3 °C, at a constant air humidity, and a natural day–night cycle. This study was approved by the Animal Research Committee of the Urmia Islamic Azad University with ethical No. IAU-UB 11044.\\n Induction of wound To induce the wound, the mice were primarily anesthetized by intraperitoneal administration route using 50 mg/kg ketamine and 5 mg/kg xylazine. Following the induction of anaesthesia, dorsal region of each mouse was shaven for surgical actions. A circular wound model was induced in a size of 7 mm using a surgical biopsy punch and per wound was inoculated by an aliquot of 5 × 107 suspension containing S. aureus (ATCC 26313) and P. aeruginosa (ATCC 27853) in 50 μL phosphate-buffered saline (Farahpour et al. 2020; Khezri et al. 2019). Subsequently, animals were divided into four groups (n = 18), as (group I) negative control group (NCG) that was administered commercial base ointment, (group II) positive control group that were treated with Mupirocin® (MG), and (groups III and IV) treated groups with therapeutic ointments containing 2 g and 4 g ZME mixed in NCG (2% and 4% ZMEO), respectively. Concerning the colonization of the bacteria, 24 h after the induction of the wounds, ointments were applied on wounds, once a day. In addition, according to the tissue sampling on days 3, 7, and 14 after the wounding, the animals in each group were divided into three subgroups (n = 6).\\nTo induce the wound, the mice were primarily anesthetized by intraperitoneal administration route using 50 mg/kg ketamine and 5 mg/kg xylazine. Following the induction of anaesthesia, dorsal region of each mouse was shaven for surgical actions. A circular wound model was induced in a size of 7 mm using a surgical biopsy punch and per wound was inoculated by an aliquot of 5 × 107 suspension containing S. aureus (ATCC 26313) and P. aeruginosa (ATCC 27853) in 50 μL phosphate-buffered saline (Farahpour et al. 2020; Khezri et al. 2019). Subsequently, animals were divided into four groups (n = 18), as (group I) negative control group (NCG) that was administered commercial base ointment, (group II) positive control group that were treated with Mupirocin® (MG), and (groups III and IV) treated groups with therapeutic ointments containing 2 g and 4 g ZME mixed in NCG (2% and 4% ZMEO), respectively. Concerning the colonization of the bacteria, 24 h after the induction of the wounds, ointments were applied on wounds, once a day. In addition, according to the tissue sampling on days 3, 7, and 14 after the wounding, the animals in each group were divided into three subgroups (n = 6).\\n Wound area The rate of wound closure was estimated as reported previously (Nejati et al. 2015; Khezri et al. 2019). In order to calculate the percentage of wound closure rate, a transparent paper was placed on it and it was calculated based on the following formula:\\nPercentage of wound closure = [(Wound area on Day 0 − Wound area on Day X)/Wound area on Day 0] × 100.\\nThe rate of wound closure was estimated as reported previously (Nejati et al. 2015; Khezri et al. 2019). In order to calculate the percentage of wound closure rate, a transparent paper was placed on it and it was calculated based on the following formula:\\nPercentage of wound closure = [(Wound area on Day 0 − Wound area on Day X)/Wound area on Day 0] × 100.\\n The investigation of the bacteriological count in the wound area To calculate the bacteriological count, the granulated tissues were excised and 0.1 g of the sample was crushed, minced, and homogenized in a sterile mortar containing 10 mL of sterile saline. It was the diluted in tubes containing 9 mL of sterile saline, was cultured on plate count agar (Merck KGaA, Darmstadt, Germany), incubated at 37 °C for 48 h, calculated as CFU/g of granulation tissue (Farahpour et al. 2020; Khezri et al. 2019).\\nTo calculate the bacteriological count, the granulated tissues were excised and 0.1 g of the sample was crushed, minced, and homogenized in a sterile mortar containing 10 mL of sterile saline. It was the diluted in tubes containing 9 mL of sterile saline, was cultured on plate count agar (Merck KGaA, Darmstadt, Germany), incubated at 37 °C for 48 h, calculated as CFU/g of granulation tissue (Farahpour et al. 2020; Khezri et al. 2019).\\n Histological analysis Concerning the evaluation of the histological parameters, the mice were euthanized by a special CO2 device and the granulation tissue were excised in along to 1 to 2 mm from surrounding normal skin. The samples were fixed in neutral-buffered formalin 10%, routinely processed, and paraffin wax was embedded, sectioned at 5 µm, and stained with Masson’s trichrome and then examined under light microscopy (Olympus CX31RBSF attached cameraman) as reported by Farahpour et al. (2018). Cellular infiltration, edoema and collagen deposition were assessed. An image pro-insight software was utilised for evaluating the collagen deposition. Morphometric lens (Olympus, Germany) was used for assessing the epithelial thickness. edoema was assessed as a 5-score as reported previously (Nejati et al. 2015).\\nConcerning the evaluation of the histological parameters, the mice were euthanized by a special CO2 device and the granulation tissue were excised in along to 1 to 2 mm from surrounding normal skin. The samples were fixed in neutral-buffered formalin 10%, routinely processed, and paraffin wax was embedded, sectioned at 5 µm, and stained with Masson’s trichrome and then examined under light microscopy (Olympus CX31RBSF attached cameraman) as reported by Farahpour et al. (2018). Cellular infiltration, edoema and collagen deposition were assessed. An image pro-insight software was utilised for evaluating the collagen deposition. Morphometric lens (Olympus, Germany) was used for assessing the epithelial thickness. edoema was assessed as a 5-score as reported previously (Nejati et al. 2015).\\n Immunohistochemical staining (IHC) for angiogenesis (CD31) To assess the IHC, it was conducted as reported by Farahpour et al. (2018). The IHC was conducted based on manufacturer’s protocol (Biocare, Yorba Linda, CA). The tissue sections were rinsed gently in the washing buffer and placed in a buffer bath. A DAB chromogen was employed for assessing the tissue sections and then incubated for 5 min. The sections were then dipped in weak ammonia (0.037 M/L) for 10 times, rinsed with distilled water and cover slipped. Brown stains were considered as positive immunohistochemical.\\nTo assess the IHC, it was conducted as reported by Farahpour et al. (2018). The IHC was conducted based on manufacturer’s protocol (Biocare, Yorba Linda, CA). The tissue sections were rinsed gently in the washing buffer and placed in a buffer bath. A DAB chromogen was employed for assessing the tissue sections and then incubated for 5 min. The sections were then dipped in weak ammonia (0.037 M/L) for 10 times, rinsed with distilled water and cover slipped. Brown stains were considered as positive immunohistochemical.\\n Fluorescent staining for collagen To wash the slides, acetic acid 1% was used for several minutes. The slides were stained using acridine-orange. Phosphate buffer was utilized to remove staining. The slides were the washed in distilled water, mounted with buffer drop and analyzed with a fluorescent microscope. The bunds with red and yellowish red colour were considered as collagen bunds (Farahpour et al. 2018).\\nTo wash the slides, acetic acid 1% was used for several minutes. The slides were stained using acridine-orange. Phosphate buffer was utilized to remove staining. The slides were the washed in distilled water, mounted with buffer drop and analyzed with a fluorescent microscope. The bunds with red and yellowish red colour were considered as collagen bunds (Farahpour et al. 2018).\\n RNA isolation and cDNA synthesis The wound tissues were isolated as previously reported and RNA was extracted applying a standard TRIZOL procedure as reported by Farahpour et al. (2018), and assessed by spectrophotometer (260 nm and 260/280 = 1.8–2.0). To prepare the cDNA, it was put in a reaction being mixed with 1 µg RNA, oligo (dT) primer (1 µL), 5 × reaction buffer (4 µL), RNAse inhibitor (1 µL), 10 mM dNTP mix (2 µL) and M-MuLV Reverse Transcriptase (1 µL) as described by producer protocol (Fermentas, GmbH, Germany). Time and temperature were as reported by Farahpour et al. (2018). The used primers included IL-1β, forward (5′-AAC AAA CCC TGC AGT GGT TCG-3′) and reverse (5′-AGCTGCTTCAGACACTTGCAC-3′); TGF-β, forward (5-CCAAACGCCGAAGACTTATCC-3′) and reverse (5′-CTTATTACCGATGGGATGGGATAGCCC-3′); IL-10, forward (5′-GAAGCTCCCTCAGCGAGGACA-3′) and reverse (5′-TTGGGCCAGTGAGTGAAAGGG-3′), TNF-α, forward (5′-GAAGCTCCCTCAGCGAGGACA-3′) and reverse (5′-TTGGGCCAGTGAGTGAAAGGG-3′), FGF-2, forward (5′-GGAACCCGGCGGGACACGGAC-3′) and reverse (5′-CCGCTGTGGCAGCTCTTG GGG-3′); VEGF, forward (5′-GCTCCGTAGTAGCCGTGGTCT-3′) and reverse (5′-GGAACCCGGCGGGACACGGAC-3′), and IGF-1, forward (5′-TAGGTGGTTGATGAATGGT-3′) and reverse (5′-GAAAGGGCAGGGCTAAT-3′).\\nThe wound tissues were isolated as previously reported and RNA was extracted applying a standard TRIZOL procedure as reported by Farahpour et al. (2018), and assessed by spectrophotometer (260 nm and 260/280 = 1.8–2.0). To prepare the cDNA, it was put in a reaction being mixed with 1 µg RNA, oligo (dT) primer (1 µL), 5 × reaction buffer (4 µL), RNAse inhibitor (1 µL), 10 mM dNTP mix (2 µL) and M-MuLV Reverse Transcriptase (1 µL) as described by producer protocol (Fermentas, GmbH, Germany). Time and temperature were as reported by Farahpour et al. (2018). The used primers included IL-1β, forward (5′-AAC AAA CCC TGC AGT GGT TCG-3′) and reverse (5′-AGCTGCTTCAGACACTTGCAC-3′); TGF-β, forward (5-CCAAACGCCGAAGACTTATCC-3′) and reverse (5′-CTTATTACCGATGGGATGGGATAGCCC-3′); IL-10, forward (5′-GAAGCTCCCTCAGCGAGGACA-3′) and reverse (5′-TTGGGCCAGTGAGTGAAAGGG-3′), TNF-α, forward (5′-GAAGCTCCCTCAGCGAGGACA-3′) and reverse (5′-TTGGGCCAGTGAGTGAAAGGG-3′), FGF-2, forward (5′-GGAACCCGGCGGGACACGGAC-3′) and reverse (5′-CCGCTGTGGCAGCTCTTG GGG-3′); VEGF, forward (5′-GCTCCGTAGTAGCCGTGGTCT-3′) and reverse (5′-GGAACCCGGCGGGACACGGAC-3′), and IGF-1, forward (5′-TAGGTGGTTGATGAATGGT-3′) and reverse (5′-GAAAGGGCAGGGCTAAT-3′).\\n Antioxidant capacity To assess the antioxidant capacity, the wound granulation tissue was homogenized in ice-cold KCl (150 mM) and the mixture was then centrifuged at 3000 g for 10 min. The supernatant was used to evaluate the total antioxidant capacity (TAC), malondialdehyde (MDA) and total tissue thiol molecules (TTM) content. The Lowry method was employed for evaluating the protein content of the samples (Daemi et al. 2019a) and MDA content was used for assessing the lipid peroxidation rate. To assess the TTM, the collected granulation tissue sample was homogenized and the supernatant was added to Tris-EDTA as reported by Daemi et al. (2019a).\\nTo assess the antioxidant capacity, the wound granulation tissue was homogenized in ice-cold KCl (150 mM) and the mixture was then centrifuged at 3000 g for 10 min. The supernatant was used to evaluate the total antioxidant capacity (TAC), malondialdehyde (MDA) and total tissue thiol molecules (TTM) content. The Lowry method was employed for evaluating the protein content of the samples (Daemi et al. 2019a) and MDA content was used for assessing the lipid peroxidation rate. To assess the TTM, the collected granulation tissue sample was homogenized and the supernatant was added to Tris-EDTA as reported by Daemi et al. (2019a).\\n Statistical analysis The results were reported as mean ± standard deviation and analyzed by Graph Pad Prism Software. One-way ANOVA was utilized to analyze the results. Dunnett’s test for pair-wise comparisons was used for evaluating the effect of treatments and p < 0.05 was considered to be of significance.\\nThe results were reported as mean ± standard deviation and analyzed by Graph Pad Prism Software. One-way ANOVA was utilized to analyze the results. Dunnett’s test for pair-wise comparisons was used for evaluating the effect of treatments and p < 0.05 was considered to be of significance.\",\n \"Zataria multiflora essential oil was purchased from Barij Essence Company (Kashan-Iran) and approved by the same herbarium company (No.1203). Commercial base ointment and mupirocin were prepared from Pars Daruo. Ltd., Tehran, Iran. The commercial base ointment contained 90% soft paraffin, 5% hard paraffin and 5% lanolin. Rodent feeds were prepared from Javaneh Khorasan Company (Khorasan, Iran).\",\n \"Gas chromatography (GC) analysis was employed in order to identify the compounds. The components of the essential oil were recognized using calculation of their retention indices under temperature-programmed conditions for n-alkanes (C6–C24) and the oil on DB-5 column.\",\n \"Seventy-two BALB/c mice weighing 25 ± 3 g were prepared. The mice had free access to water and pelleted feed for rodents (Javaneh Khorasan Company, Khorasan, Iran). The mice were kept in an ambient temperature of 23 ± 3 °C, at a constant air humidity, and a natural day–night cycle. This study was approved by the Animal Research Committee of the Urmia Islamic Azad University with ethical No. IAU-UB 11044.\",\n \"To induce the wound, the mice were primarily anesthetized by intraperitoneal administration route using 50 mg/kg ketamine and 5 mg/kg xylazine. Following the induction of anaesthesia, dorsal region of each mouse was shaven for surgical actions. A circular wound model was induced in a size of 7 mm using a surgical biopsy punch and per wound was inoculated by an aliquot of 5 × 107 suspension containing S. aureus (ATCC 26313) and P. aeruginosa (ATCC 27853) in 50 μL phosphate-buffered saline (Farahpour et al. 2020; Khezri et al. 2019). Subsequently, animals were divided into four groups (n = 18), as (group I) negative control group (NCG) that was administered commercial base ointment, (group II) positive control group that were treated with Mupirocin® (MG), and (groups III and IV) treated groups with therapeutic ointments containing 2 g and 4 g ZME mixed in NCG (2% and 4% ZMEO), respectively. Concerning the colonization of the bacteria, 24 h after the induction of the wounds, ointments were applied on wounds, once a day. In addition, according to the tissue sampling on days 3, 7, and 14 after the wounding, the animals in each group were divided into three subgroups (n = 6).\",\n \"The rate of wound closure was estimated as reported previously (Nejati et al. 2015; Khezri et al. 2019). In order to calculate the percentage of wound closure rate, a transparent paper was placed on it and it was calculated based on the following formula:\\nPercentage of wound closure = [(Wound area on Day 0 − Wound area on Day X)/Wound area on Day 0] × 100.\",\n \"To calculate the bacteriological count, the granulated tissues were excised and 0.1 g of the sample was crushed, minced, and homogenized in a sterile mortar containing 10 mL of sterile saline. It was the diluted in tubes containing 9 mL of sterile saline, was cultured on plate count agar (Merck KGaA, Darmstadt, Germany), incubated at 37 °C for 48 h, calculated as CFU/g of granulation tissue (Farahpour et al. 2020; Khezri et al. 2019).\",\n \"Concerning the evaluation of the histological parameters, the mice were euthanized by a special CO2 device and the granulation tissue were excised in along to 1 to 2 mm from surrounding normal skin. The samples were fixed in neutral-buffered formalin 10%, routinely processed, and paraffin wax was embedded, sectioned at 5 µm, and stained with Masson’s trichrome and then examined under light microscopy (Olympus CX31RBSF attached cameraman) as reported by Farahpour et al. (2018). Cellular infiltration, edoema and collagen deposition were assessed. An image pro-insight software was utilised for evaluating the collagen deposition. Morphometric lens (Olympus, Germany) was used for assessing the epithelial thickness. edoema was assessed as a 5-score as reported previously (Nejati et al. 2015).\",\n \"To assess the IHC, it was conducted as reported by Farahpour et al. (2018). The IHC was conducted based on manufacturer’s protocol (Biocare, Yorba Linda, CA). The tissue sections were rinsed gently in the washing buffer and placed in a buffer bath. A DAB chromogen was employed for assessing the tissue sections and then incubated for 5 min. The sections were then dipped in weak ammonia (0.037 M/L) for 10 times, rinsed with distilled water and cover slipped. Brown stains were considered as positive immunohistochemical.\",\n \"To wash the slides, acetic acid 1% was used for several minutes. The slides were stained using acridine-orange. Phosphate buffer was utilized to remove staining. The slides were the washed in distilled water, mounted with buffer drop and analyzed with a fluorescent microscope. The bunds with red and yellowish red colour were considered as collagen bunds (Farahpour et al. 2018).\",\n \"The wound tissues were isolated as previously reported and RNA was extracted applying a standard TRIZOL procedure as reported by Farahpour et al. (2018), and assessed by spectrophotometer (260 nm and 260/280 = 1.8–2.0). To prepare the cDNA, it was put in a reaction being mixed with 1 µg RNA, oligo (dT) primer (1 µL), 5 × reaction buffer (4 µL), RNAse inhibitor (1 µL), 10 mM dNTP mix (2 µL) and M-MuLV Reverse Transcriptase (1 µL) as described by producer protocol (Fermentas, GmbH, Germany). Time and temperature were as reported by Farahpour et al. (2018). The used primers included IL-1β, forward (5′-AAC AAA CCC TGC AGT GGT TCG-3′) and reverse (5′-AGCTGCTTCAGACACTTGCAC-3′); TGF-β, forward (5-CCAAACGCCGAAGACTTATCC-3′) and reverse (5′-CTTATTACCGATGGGATGGGATAGCCC-3′); IL-10, forward (5′-GAAGCTCCCTCAGCGAGGACA-3′) and reverse (5′-TTGGGCCAGTGAGTGAAAGGG-3′), TNF-α, forward (5′-GAAGCTCCCTCAGCGAGGACA-3′) and reverse (5′-TTGGGCCAGTGAGTGAAAGGG-3′), FGF-2, forward (5′-GGAACCCGGCGGGACACGGAC-3′) and reverse (5′-CCGCTGTGGCAGCTCTTG GGG-3′); VEGF, forward (5′-GCTCCGTAGTAGCCGTGGTCT-3′) and reverse (5′-GGAACCCGGCGGGACACGGAC-3′), and IGF-1, forward (5′-TAGGTGGTTGATGAATGGT-3′) and reverse (5′-GAAAGGGCAGGGCTAAT-3′).\",\n \"To assess the antioxidant capacity, the wound granulation tissue was homogenized in ice-cold KCl (150 mM) and the mixture was then centrifuged at 3000 g for 10 min. The supernatant was used to evaluate the total antioxidant capacity (TAC), malondialdehyde (MDA) and total tissue thiol molecules (TTM) content. The Lowry method was employed for evaluating the protein content of the samples (Daemi et al. 2019a) and MDA content was used for assessing the lipid peroxidation rate. To assess the TTM, the collected granulation tissue sample was homogenized and the supernatant was added to Tris-EDTA as reported by Daemi et al. (2019a).\",\n \"The results were reported as mean ± standard deviation and analyzed by Graph Pad Prism Software. One-way ANOVA was utilized to analyze the results. Dunnett’s test for pair-wise comparisons was used for evaluating the effect of treatments and p < 0.05 was considered to be of significance.\",\n \" Composition of the Zataria multiflora essential oil The analysis of Zataria multiflora essential oil with GC-FID and GC-mass spectrometry (MS) identified 29 compounds which accounted for 99.6% of the total essential oil composition (Table 1). It contained thymol (52.90%), p-cymene (9.10), γ-terpinene (8.10%) and carvacrol (6.80%) as major constituents (Figure 1).\\nGC-MS chromatogram of the essential oil of Zataria multiflora.\\nChemical constituents of Zataria multiflora essential oil.\\nRI-Cal: retention indices calculated based on C6–C24\\nn-alkenes from a DB-5 column.\\nRI-Cal: retention indices retrieved from literature (Adams 2007).\\nBold values show major compounds in the essential oil.\\nThe analysis of Zataria multiflora essential oil with GC-FID and GC-mass spectrometry (MS) identified 29 compounds which accounted for 99.6% of the total essential oil composition (Table 1). It contained thymol (52.90%), p-cymene (9.10), γ-terpinene (8.10%) and carvacrol (6.80%) as major constituents (Figure 1).\\nGC-MS chromatogram of the essential oil of Zataria multiflora.\\nChemical constituents of Zataria multiflora essential oil.\\nRI-Cal: retention indices calculated based on C6–C24\\nn-alkenes from a DB-5 column.\\nRI-Cal: retention indices retrieved from literature (Adams 2007).\\nBold values show major compounds in the essential oil.\\n Wound area The findings for the effects of ZMEO on the percentage of wound contraction are shown in Figure 2. The results implied that topical administration of therapeutic ointments significantly (p < 0.05) increased wound contraction rate on days 7 and 14 compared to the NCG group (Figure 2(A,B)). The comparison between treatment groups revealed that a higher wound contraction rate was observed in the 4% ZMEO-treated group compared to the 2% ZMEO-treated and MG groups (p < 0.05).\\nThe effects of topical administration of ZMEO on (B) wound contraction rate and (C) tissue bacterial count (CFU/g) in different days. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\\nThe findings for the effects of ZMEO on the percentage of wound contraction are shown in Figure 2. The results implied that topical administration of therapeutic ointments significantly (p < 0.05) increased wound contraction rate on days 7 and 14 compared to the NCG group (Figure 2(A,B)). The comparison between treatment groups revealed that a higher wound contraction rate was observed in the 4% ZMEO-treated group compared to the 2% ZMEO-treated and MG groups (p < 0.05).\\nThe effects of topical administration of ZMEO on (B) wound contraction rate and (C) tissue bacterial count (CFU/g) in different days. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\\n The bacteriological count The results concerning the effects of ZMEO on tissue bacteria counts are represented in Figure 2(C). The results showed that topical administration of therapeutic ointments significantly decreased bacterial colonization in all the days compared to the NCG group (p < 0.05). The comparison between treatment groups demonstrated that a higher level of ZMEO (4%) significantly decreased the total bacterial count compared to that of a lower level of it (2%) and MG groups (p < 0.05).\\nThe results concerning the effects of ZMEO on tissue bacteria counts are represented in Figure 2(C). The results showed that topical administration of therapeutic ointments significantly decreased bacterial colonization in all the days compared to the NCG group (p < 0.05). The comparison between treatment groups demonstrated that a higher level of ZMEO (4%) significantly decreased the total bacterial count compared to that of a lower level of it (2%) and MG groups (p < 0.05).\\n Histopathological parameters The results for histopathological parameters are represented in Table 2. The results showed that edoema score was significantly higher in the NCG group. It was observed to be significantly lower in the animals treated with the 4% ZMEO-treated group and also in 2% ZMEO and MG-treated groups. The findings implied that the lowest edoema was observed in 4% ZMEO in day 14; meanwhile, there was no observed edoema in the 4% ZMEO group on day 14. The results showed that immune cells were significantly lower in ZMEO groups in day 3 compared to other groups (p < 0.05), yet it decreased on days 7 and 14 in the ZMEO groups compared to other groups (p < 0.05). It means that the application of the ZMEO decreases immune cells in days 7 and 14, and increased it on day 3. Angiogenesis (Figure 3) and fibroblast infiltration were significantly higher in all treated groups compared to the NCG group and the highest level was observed in level of the 4%-ZMEO group. The results for collagen condensation (Figure 4) and re-epithelialization (Figure 5) revealed that parameters were significantly higher in the ZMEO groups, which confirms the results.\\nImmunohistochemical staining for angiogenesis:; (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See elevated angiogenesis in ZMEO-treated group (arrows) on day 7 after wound induction. CD 31 staining, 400×.\\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. Note well-formed collagen deposition in cross sections from ZMEO-treated animals on day 7 after wound induction (first row), which is significantly increased on day 14 after injury (second row). The third row represents the software analyze for collagen intensity. The cross sections from animals in ZMEO-treated groups exhibited condense collagen deposition versus NCG and MG groups. Masson-trichrome staining and fluorescent staining for collagen, 400× magnification.\\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See well re-epithelialization in ZMEO-treated animals. The re-epithelialization initiated on day 7 after wound induction in ZMEO-treated animals. However, the cross sections from NCG and MG groups are not representing epithelialization. Note well-organized dermis and complete epithelialization with well-formed papillae in ZMEO-treated animals in comparison to NCG and MG groups. Masson-trichrome staining, 100×.\\nThe effects of ZMEO on histopathological parameters in different days.\\nSuperscripts (a–d) indicate significant differences in same day at p < 0.05.\\nThe results for histopathological parameters are represented in Table 2. The results showed that edoema score was significantly higher in the NCG group. It was observed to be significantly lower in the animals treated with the 4% ZMEO-treated group and also in 2% ZMEO and MG-treated groups. The findings implied that the lowest edoema was observed in 4% ZMEO in day 14; meanwhile, there was no observed edoema in the 4% ZMEO group on day 14. The results showed that immune cells were significantly lower in ZMEO groups in day 3 compared to other groups (p < 0.05), yet it decreased on days 7 and 14 in the ZMEO groups compared to other groups (p < 0.05). It means that the application of the ZMEO decreases immune cells in days 7 and 14, and increased it on day 3. Angiogenesis (Figure 3) and fibroblast infiltration were significantly higher in all treated groups compared to the NCG group and the highest level was observed in level of the 4%-ZMEO group. The results for collagen condensation (Figure 4) and re-epithelialization (Figure 5) revealed that parameters were significantly higher in the ZMEO groups, which confirms the results.\\nImmunohistochemical staining for angiogenesis:; (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See elevated angiogenesis in ZMEO-treated group (arrows) on day 7 after wound induction. CD 31 staining, 400×.\\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. Note well-formed collagen deposition in cross sections from ZMEO-treated animals on day 7 after wound induction (first row), which is significantly increased on day 14 after injury (second row). The third row represents the software analyze for collagen intensity. The cross sections from animals in ZMEO-treated groups exhibited condense collagen deposition versus NCG and MG groups. Masson-trichrome staining and fluorescent staining for collagen, 400× magnification.\\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See well re-epithelialization in ZMEO-treated animals. The re-epithelialization initiated on day 7 after wound induction in ZMEO-treated animals. However, the cross sections from NCG and MG groups are not representing epithelialization. Note well-organized dermis and complete epithelialization with well-formed papillae in ZMEO-treated animals in comparison to NCG and MG groups. Masson-trichrome staining, 100×.\\nThe effects of ZMEO on histopathological parameters in different days.\\nSuperscripts (a–d) indicate significant differences in same day at p < 0.05.\\n Molecular analyses The results for gene expression are presented in Figure 6. Gene expression of IL-1β and TNF-α was significantly (p < 0.05) lower on all sampling days at treated groups compared to the NCG group. The greatest decrease was observed in the 4% ZMEO-treated animals. Gene expressions of IGF-1 and VEGF was significantly (p < 0.05) higher at all treated groups on day 7 after the wounding, but the levels significantly decreased on day 14. The expression of IL-10 level increased on day 7 whereas it decreased on day 14 compared to the NCG group (p < 0.05). Further analysis of gene expressions of TGF-β and FGF-2 indicate that its level does not have significant difference (p > 0.05) on day 3, yet its level significantly (p < 0.05) increased in all treated groups, particularly at 4% ZMEO-treated animals on day 7. The amount of TGF-β and FGF-2 gene expression levels also were significantly (p < 0.05) reduced on day 14 compared to the NCG group.\\nThe effects of topical application of ZMEO on gene expression. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\\nThe results for gene expression are presented in Figure 6. Gene expression of IL-1β and TNF-α was significantly (p < 0.05) lower on all sampling days at treated groups compared to the NCG group. The greatest decrease was observed in the 4% ZMEO-treated animals. Gene expressions of IGF-1 and VEGF was significantly (p < 0.05) higher at all treated groups on day 7 after the wounding, but the levels significantly decreased on day 14. The expression of IL-10 level increased on day 7 whereas it decreased on day 14 compared to the NCG group (p < 0.05). Further analysis of gene expressions of TGF-β and FGF-2 indicate that its level does not have significant difference (p > 0.05) on day 3, yet its level significantly (p < 0.05) increased in all treated groups, particularly at 4% ZMEO-treated animals on day 7. The amount of TGF-β and FGF-2 gene expression levels also were significantly (p < 0.05) reduced on day 14 compared to the NCG group.\\nThe effects of topical application of ZMEO on gene expression. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\\n Antioxidant status Antioxidant status in different groups is represented in Figure 7. Using the 2% and 4%-ZMEO significantly (p < 0.05) increased TAC and TTP levels compared to the NCG group in all of the days after the wounding. Interestingly, the highest increase was observed in the 4% ZMEO-treated animals. Further analysis of MDA level indicates significant (p < 0.05) decrease in all of the treated groups compared to the NCG group in all of the days after the wounding. The highest decrease was observed in the 4% ZMEO-treated group.\\nThe effects of topical application of ZMEO on antioxidant properties. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\\nAntioxidant status in different groups is represented in Figure 7. Using the 2% and 4%-ZMEO significantly (p < 0.05) increased TAC and TTP levels compared to the NCG group in all of the days after the wounding. Interestingly, the highest increase was observed in the 4% ZMEO-treated animals. Further analysis of MDA level indicates significant (p < 0.05) decrease in all of the treated groups compared to the NCG group in all of the days after the wounding. The highest decrease was observed in the 4% ZMEO-treated group.\\nThe effects of topical application of ZMEO on antioxidant properties. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\",\n \"The analysis of Zataria multiflora essential oil with GC-FID and GC-mass spectrometry (MS) identified 29 compounds which accounted for 99.6% of the total essential oil composition (Table 1). It contained thymol (52.90%), p-cymene (9.10), γ-terpinene (8.10%) and carvacrol (6.80%) as major constituents (Figure 1).\\nGC-MS chromatogram of the essential oil of Zataria multiflora.\\nChemical constituents of Zataria multiflora essential oil.\\nRI-Cal: retention indices calculated based on C6–C24\\nn-alkenes from a DB-5 column.\\nRI-Cal: retention indices retrieved from literature (Adams 2007).\\nBold values show major compounds in the essential oil.\",\n \"The findings for the effects of ZMEO on the percentage of wound contraction are shown in Figure 2. The results implied that topical administration of therapeutic ointments significantly (p < 0.05) increased wound contraction rate on days 7 and 14 compared to the NCG group (Figure 2(A,B)). The comparison between treatment groups revealed that a higher wound contraction rate was observed in the 4% ZMEO-treated group compared to the 2% ZMEO-treated and MG groups (p < 0.05).\\nThe effects of topical administration of ZMEO on (B) wound contraction rate and (C) tissue bacterial count (CFU/g) in different days. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\",\n \"The results concerning the effects of ZMEO on tissue bacteria counts are represented in Figure 2(C). The results showed that topical administration of therapeutic ointments significantly decreased bacterial colonization in all the days compared to the NCG group (p < 0.05). The comparison between treatment groups demonstrated that a higher level of ZMEO (4%) significantly decreased the total bacterial count compared to that of a lower level of it (2%) and MG groups (p < 0.05).\",\n \"The results for histopathological parameters are represented in Table 2. The results showed that edoema score was significantly higher in the NCG group. It was observed to be significantly lower in the animals treated with the 4% ZMEO-treated group and also in 2% ZMEO and MG-treated groups. The findings implied that the lowest edoema was observed in 4% ZMEO in day 14; meanwhile, there was no observed edoema in the 4% ZMEO group on day 14. The results showed that immune cells were significantly lower in ZMEO groups in day 3 compared to other groups (p < 0.05), yet it decreased on days 7 and 14 in the ZMEO groups compared to other groups (p < 0.05). It means that the application of the ZMEO decreases immune cells in days 7 and 14, and increased it on day 3. Angiogenesis (Figure 3) and fibroblast infiltration were significantly higher in all treated groups compared to the NCG group and the highest level was observed in level of the 4%-ZMEO group. The results for collagen condensation (Figure 4) and re-epithelialization (Figure 5) revealed that parameters were significantly higher in the ZMEO groups, which confirms the results.\\nImmunohistochemical staining for angiogenesis:; (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See elevated angiogenesis in ZMEO-treated group (arrows) on day 7 after wound induction. CD 31 staining, 400×.\\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. Note well-formed collagen deposition in cross sections from ZMEO-treated animals on day 7 after wound induction (first row), which is significantly increased on day 14 after injury (second row). The third row represents the software analyze for collagen intensity. The cross sections from animals in ZMEO-treated groups exhibited condense collagen deposition versus NCG and MG groups. Masson-trichrome staining and fluorescent staining for collagen, 400× magnification.\\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See well re-epithelialization in ZMEO-treated animals. The re-epithelialization initiated on day 7 after wound induction in ZMEO-treated animals. However, the cross sections from NCG and MG groups are not representing epithelialization. Note well-organized dermis and complete epithelialization with well-formed papillae in ZMEO-treated animals in comparison to NCG and MG groups. Masson-trichrome staining, 100×.\\nThe effects of ZMEO on histopathological parameters in different days.\\nSuperscripts (a–d) indicate significant differences in same day at p < 0.05.\",\n \"The results for gene expression are presented in Figure 6. Gene expression of IL-1β and TNF-α was significantly (p < 0.05) lower on all sampling days at treated groups compared to the NCG group. The greatest decrease was observed in the 4% ZMEO-treated animals. Gene expressions of IGF-1 and VEGF was significantly (p < 0.05) higher at all treated groups on day 7 after the wounding, but the levels significantly decreased on day 14. The expression of IL-10 level increased on day 7 whereas it decreased on day 14 compared to the NCG group (p < 0.05). Further analysis of gene expressions of TGF-β and FGF-2 indicate that its level does not have significant difference (p > 0.05) on day 3, yet its level significantly (p < 0.05) increased in all treated groups, particularly at 4% ZMEO-treated animals on day 7. The amount of TGF-β and FGF-2 gene expression levels also were significantly (p < 0.05) reduced on day 14 compared to the NCG group.\\nThe effects of topical application of ZMEO on gene expression. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\",\n \"Antioxidant status in different groups is represented in Figure 7. Using the 2% and 4%-ZMEO significantly (p < 0.05) increased TAC and TTP levels compared to the NCG group in all of the days after the wounding. Interestingly, the highest increase was observed in the 4% ZMEO-treated animals. Further analysis of MDA level indicates significant (p < 0.05) decrease in all of the treated groups compared to the NCG group in all of the days after the wounding. The highest decrease was observed in the 4% ZMEO-treated group.\\nThe effects of topical application of ZMEO on antioxidant properties. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\",\n \"Wounds infected by pathogenic bacteria such as S. aureus, and P. aeruginosa are believed to be serious challenges for regarding their management and treatment (Manzuoerh et al. 2019). Different agents are employed for the treatment of wounds. Active compounds of the essential oils can have pivotal roles in their treatment. The findings indicated that thymol (52.90%), p-cymene (9.10), γ-terpinene (8.10%) and carvacrol (6.80%) were the major constituents in the essential oil. Similar to our results, previous studies demonstrated that the major compounds of Z. multiflora essential oil were thymol, carvacrol and p-cymene (Aida et al. 2015), thymol, c-terpinene, p-cymene, and carvacrol (Saei-Dehkordi et al. 2010), carvacrol (Misaghi and Basti 2007), thymol, carvacrol, and p-cymene (Sharififar et al. 2007). The results showed that ZMEO accelerated wound healing compared to the NCG group and synthetic agent of mupirocin. Herein, the results are described based on the mechanisms and active compounds, step-by-step.\\nInflammation is the first step in the wound healing process, in which different factors and genes are involved. Inflammation is triggered once the injury induces to living tissues by live organisms, such as bacteria and/or physical injury and faulted immune response. Inflammation removes foreign substances in order to improve the wound healing process (Garrett et al. 2010). It promotes macrophages, neutrophils and immune cells for initiation of inflammation (Oguntibeju 2018). IL-1β and TNF-α are commonly produced by immune cells, such as macrophages and mast cells (Thacker et al. 2007). The results showed that the topical application of ZMEO decreased immune cells and the expressions of IL-1β and TNF-α in the first 3 days. The results implied a positive relation between the number of immune cells with the expression of IL-1β and TNF-α in the treatments. Meaning that immune cells increase the expression of IL-1β and TNF-α in the first 3 days. Increased inflammation delays the wound healing process. The findings revealed that topical administration of ZMEO decreased inflammation on days 7 and 14, and acted as an antibacterial and anti-inflammation agent. Antibacterial and anti-inflammatory activity of ZMEO is attributed to its compounds. The essence contains thymol and carvacrol that are of antibacterial and anti-inflammatory properties. It was reported that the administration of carvacrol significantly reduced TNF-α levels in pleural lavage (Guimarães et al. 2012). The anti-inflammatory activity of thymol leads to prevention of the phosphorylation of extracellular signal-regulated protein kinase, c-Jun N-terminal kinase, and nuclear factor-κB (Liang et al. 2014). Carvacrol prevented leukocyte migration, and decreased edoema, and showed anti-inflammatory effects (Fachini-Queiroz et al. 2012). The results indicated that immune cells decreased and expression of IL-1β and TNF-α also decreased in ZMEO groups. The findings showed that the total bacterial count significantly decreased in ZMEO groups compared to the NCG group. Mupirocin is used as a bactericide agent for the treatment of infected wounds. The results convey that ZMEO, particularly at a higher level, have better antibacterial activity compared to mupirocin. Higher total bacterial count increases the inflammation and delays the wound healing process. It means that topical administration of ZMEO decreases the inflammation with its antibacterial activity. It was reported that the antibacterial activity of Zataria multiflora is in view of its compounds (Shafiee et al. 1999; Barkhori-Mehni et al. 2017). In essence, ZMEO decreased the inflammation and inflammatory-associated parameters. Higher levels of ZMEO provide higher doses of active compounds that act as anti-inflammatory factors. Decreased inflammation leads the wound healing process towards the proliferative phase.\\nOur findings implied that the topical application of ZMEO improved the proliferative phase. The results showed that the genes expression of VEGF, FGF-2 and IGF-1 increased in ZMEO groups. IGF-1 increases the transport of glucose during the wound healing process and improves the proliferative phase (Daemi et al. 2019b). VEGF induces angiogenesis and stimulates cell migration and proliferation (Norton & Popel 2016; Farahpour et al. 2020; Gharaboghaz et al. 2020). FGF-2, a protein that participates in the wound healing process (Souto et al. 2020), together with other growth factors, such as VEGF, are reported to increase the angiogenesis and indirectly support the cellular nutrient, oxygen and energy supplements (Harding et al. 2002). So far, studies have not investigated the effect of ZMEO on genes expression of VEGF, FGF-2 and IGF-1. It means that VEGF improves angiogenesis, and vessel number was significantly higher in ZMEO groups. VEGF improved angiogenesis and number of vessels. Other genes involved were TGF-β, and IL-10 whose expressions increased on day 7. IL-10 is a factor that reduces the production of pro-inflammatory cytokine, but TGF-β leads the phase towards the proliferative phase by improving the proliferation and differentiation of fibroblasts, collagen production, and wound contraction (Khezri et al. 2020). Seemingly, both genes are of greatly important roles in decreasing the inflammation and passing to the proliferative phase. The IHC results indicated improved collagen deposition. The biosynthesis of collagen during the proliferation stage has pivotal roles in dermal maturation in which some factors are involved (Yakaew et al. 2016; Daemi et al. 2019a). The results showed that the topical application of ZMEO increased fibroblast, which is due to its effect on IGF-1. It was reported that IGF-1 increases fibroblast cells in in vitro assessment (Lisa et al. 2011). Improving cellular proliferation and differentiation are essential for shortening the healing time (Oryan et al. 2015; Karimzadeh and Farahpour 2017). Proliferative effect of ZMEO is partly owing to its antioxidant activity. The topical application of ZMEO improved antioxidant activity. Previous studies have reported antioxidant activity of Zataria multiflora essential oil (Fatemi et al. 2012; Karimian et al. 2012; Kavoosi and Teixeira da Silva 2012). Oxidative stress in the wound area increases the damage of proteins, nucleotides and lipid levels (Cano Sanchez et al. 2018; Farahpour et al. 2020; Gharaboghaz et al. 2020) and decreases the antioxidant activity. This fact is associated with increased changes in the antioxidant profile, which are contributed to the increase in the levels of MDA content (Sharma et al. 2012; Bardaa et al. 2016; Vittorazzi et al. 2016).\",\n \"The topical application of ZMEO decreased the inflammation and the total bacterial count, which progresses the wound healing process towards the proliferative phase. It increases the expression of proliferative genes, helps to alleviate the inflammation phase and accelerates the proliferative phase. Antioxidant activity of ZMEO accelerated the wound healing process. All the above-mentioned factors hasten the wound healing process, as seen for the wound area. Accordingly, applying ZMEO only and/or in combination with chemical agents for the treatment of wound healing could be suggested.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["intro","materials",null,null,null,null,null,null,null,null,null,null,null,null,"results",null,null,null,null,null,null,"discussion","conclusions"],"string":"[\n \"intro\",\n \"materials\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["Infected wound healing","inflammatory cytokines","antimicrobial properties","thymol"],"string":"[\n \"Infected wound healing\",\n \"inflammatory cytokines\",\n \"antimicrobial properties\",\n \"thymol\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Introduction:\nWound infections are important challenges that cause the suffering of patients and economic losses (Vittorazzi et al. 2016; Daemi et al. 2019a; Farghali et al. 2019). Skin is a regenerative organ which acts as a barrier between the body and the external environment (Hassan et al. 2014). A wound is defined as a disturbance in anatomic structure of skin and its functional integrity (Farghali et al. 2019). Skin prevents the penetration of bacteria and fungi that are important challenges that induce mortality and morbidity. Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic bacteria that induce infection in patients (Cardona and Wilson 2015). The bacteria are commonly detected in the upper and deepest region of wound bed (Serra et al. 2015). Colonization of S. aureus and P. aeruginosa in the wound site postpones the wound healing process (Farahpour et al. 2020; Khezri et al. 2020). The wound healing process comprises several phases, including coagulation, inflammation, epithelialization, granulation tissue formation, and tissue remodelling (Daemi et al. 2019a, 2019b; Farahpour et al. 2020). Inflammatory phase occurs after activation of inflammatory chemokines (Farahpour et al. 2020). Interleukin-1β (IL-1β) is significantly synthesized 12–24 h after infliction of the wound, and its level returns to basal level after the proliferative stage is completed (Fahey and Doyle 2019). Tumour necrosis factor-α (TNF-α) is significantly synthesized by macrophages and T lymphocytes and its level increases under inflammation and infection conditions (Xue and Falcon 2019). Following the induction of inflammation, proliferative phase occurs in which many genes are involved. Insulin-like growth factor 1 (IGF-1) promotes the production of keratinocyte and fibroblast proliferation and improves the re-epithelialization (Daemi et al. 2019b). Decreased endothelial insulin/IGF-1 signal delays in the wound healing process (Aghdam et al. 2012). Vascular endothelial growth factor (VEGF) induces angiogenesis and stimulates cell migration and proliferation (Farahpour et al. 2020). Fibroblast growth factor-2 (FGF-2) is a protein that participates in the wound healing process (Souto et al. 2020). Interleukin-10 (IL-10) reduces the production of pro-inflammatory cytokine, but tumour growth factor-β (TGF-β) has roles in increasing the proliferative phase improving the proliferation and differentiation of fibroblasts, collagen production, and wound contraction (Khezri et al. 2020). On the other hand, oxidative stress increases under wound disorders and increases the production of reactive oxygen species (ROS) in the site of wound (Ustuner et al. 2019). Applying antibacterial and antioxidant agents shortens the inflammatory phase, promotes the proliferative phase and accelerates the wound healing process (Bardaa et al. 2016; Vittorazzi et al. 2016; Daemi et al. 2019a, 2019b).\nZataria multiflora Boiss (Lamiaceae) (ZM) has antibacterial activity against Pseudomonas spp. (Barkhori-Mehni et al. 2017). Its antibacterial activity is attributed to its phenolic compounds that destroy the cell wall in bacteria (Barkhori-Mehni et al. 2017). It demonstrates antioxidant properties due to its compounds such as thymol and carvacrol (Mojaddar Langroodi et al. 2019). Seemingly, ZM can accelerate the wound healing process due to its antibacterial and antioxidant features, but no studies have been conducted yet to evaluate the Zataria multiflora essential oil (ZME) on the wound healing process. Therefore, this study was conducted to evaluate the effect of an ointment prepared from ZME on the healing process of infected wound model by S. aureus and P. aeruginosa by assessing wound contraction, total bacterial count, histopathological and immunohistochemical, antioxidant activity, and qRT-PCR analyses.\n\nMaterials and methods:\n Materials Zataria multiflora essential oil was purchased from Barij Essence Company (Kashan-Iran) and approved by the same herbarium company (No.1203). Commercial base ointment and mupirocin were prepared from Pars Daruo. Ltd., Tehran, Iran. The commercial base ointment contained 90% soft paraffin, 5% hard paraffin and 5% lanolin. Rodent feeds were prepared from Javaneh Khorasan Company (Khorasan, Iran).\nZataria multiflora essential oil was purchased from Barij Essence Company (Kashan-Iran) and approved by the same herbarium company (No.1203). Commercial base ointment and mupirocin were prepared from Pars Daruo. Ltd., Tehran, Iran. The commercial base ointment contained 90% soft paraffin, 5% hard paraffin and 5% lanolin. Rodent feeds were prepared from Javaneh Khorasan Company (Khorasan, Iran).\n Essential oil analysis and identification Gas chromatography (GC) analysis was employed in order to identify the compounds. The components of the essential oil were recognized using calculation of their retention indices under temperature-programmed conditions for n-alkanes (C6–C24) and the oil on DB-5 column.\nGas chromatography (GC) analysis was employed in order to identify the compounds. The components of the essential oil were recognized using calculation of their retention indices under temperature-programmed conditions for n-alkanes (C6–C24) and the oil on DB-5 column.\n Animals Seventy-two BALB/c mice weighing 25 ± 3 g were prepared. The mice had free access to water and pelleted feed for rodents (Javaneh Khorasan Company, Khorasan, Iran). The mice were kept in an ambient temperature of 23 ± 3 °C, at a constant air humidity, and a natural day–night cycle. This study was approved by the Animal Research Committee of the Urmia Islamic Azad University with ethical No. IAU-UB 11044.\nSeventy-two BALB/c mice weighing 25 ± 3 g were prepared. The mice had free access to water and pelleted feed for rodents (Javaneh Khorasan Company, Khorasan, Iran). The mice were kept in an ambient temperature of 23 ± 3 °C, at a constant air humidity, and a natural day–night cycle. This study was approved by the Animal Research Committee of the Urmia Islamic Azad University with ethical No. IAU-UB 11044.\n Induction of wound To induce the wound, the mice were primarily anesthetized by intraperitoneal administration route using 50 mg/kg ketamine and 5 mg/kg xylazine. Following the induction of anaesthesia, dorsal region of each mouse was shaven for surgical actions. A circular wound model was induced in a size of 7 mm using a surgical biopsy punch and per wound was inoculated by an aliquot of 5 × 107 suspension containing S. aureus (ATCC 26313) and P. aeruginosa (ATCC 27853) in 50 μL phosphate-buffered saline (Farahpour et al. 2020; Khezri et al. 2019). Subsequently, animals were divided into four groups (n = 18), as (group I) negative control group (NCG) that was administered commercial base ointment, (group II) positive control group that were treated with Mupirocin® (MG), and (groups III and IV) treated groups with therapeutic ointments containing 2 g and 4 g ZME mixed in NCG (2% and 4% ZMEO), respectively. Concerning the colonization of the bacteria, 24 h after the induction of the wounds, ointments were applied on wounds, once a day. In addition, according to the tissue sampling on days 3, 7, and 14 after the wounding, the animals in each group were divided into three subgroups (n = 6).\nTo induce the wound, the mice were primarily anesthetized by intraperitoneal administration route using 50 mg/kg ketamine and 5 mg/kg xylazine. Following the induction of anaesthesia, dorsal region of each mouse was shaven for surgical actions. A circular wound model was induced in a size of 7 mm using a surgical biopsy punch and per wound was inoculated by an aliquot of 5 × 107 suspension containing S. aureus (ATCC 26313) and P. aeruginosa (ATCC 27853) in 50 μL phosphate-buffered saline (Farahpour et al. 2020; Khezri et al. 2019). Subsequently, animals were divided into four groups (n = 18), as (group I) negative control group (NCG) that was administered commercial base ointment, (group II) positive control group that were treated with Mupirocin® (MG), and (groups III and IV) treated groups with therapeutic ointments containing 2 g and 4 g ZME mixed in NCG (2% and 4% ZMEO), respectively. Concerning the colonization of the bacteria, 24 h after the induction of the wounds, ointments were applied on wounds, once a day. In addition, according to the tissue sampling on days 3, 7, and 14 after the wounding, the animals in each group were divided into three subgroups (n = 6).\n Wound area The rate of wound closure was estimated as reported previously (Nejati et al. 2015; Khezri et al. 2019). In order to calculate the percentage of wound closure rate, a transparent paper was placed on it and it was calculated based on the following formula:\nPercentage of wound closure = [(Wound area on Day 0 − Wound area on Day X)/Wound area on Day 0] × 100.\nThe rate of wound closure was estimated as reported previously (Nejati et al. 2015; Khezri et al. 2019). In order to calculate the percentage of wound closure rate, a transparent paper was placed on it and it was calculated based on the following formula:\nPercentage of wound closure = [(Wound area on Day 0 − Wound area on Day X)/Wound area on Day 0] × 100.\n The investigation of the bacteriological count in the wound area To calculate the bacteriological count, the granulated tissues were excised and 0.1 g of the sample was crushed, minced, and homogenized in a sterile mortar containing 10 mL of sterile saline. It was the diluted in tubes containing 9 mL of sterile saline, was cultured on plate count agar (Merck KGaA, Darmstadt, Germany), incubated at 37 °C for 48 h, calculated as CFU/g of granulation tissue (Farahpour et al. 2020; Khezri et al. 2019).\nTo calculate the bacteriological count, the granulated tissues were excised and 0.1 g of the sample was crushed, minced, and homogenized in a sterile mortar containing 10 mL of sterile saline. It was the diluted in tubes containing 9 mL of sterile saline, was cultured on plate count agar (Merck KGaA, Darmstadt, Germany), incubated at 37 °C for 48 h, calculated as CFU/g of granulation tissue (Farahpour et al. 2020; Khezri et al. 2019).\n Histological analysis Concerning the evaluation of the histological parameters, the mice were euthanized by a special CO2 device and the granulation tissue were excised in along to 1 to 2 mm from surrounding normal skin. The samples were fixed in neutral-buffered formalin 10%, routinely processed, and paraffin wax was embedded, sectioned at 5 µm, and stained with Masson’s trichrome and then examined under light microscopy (Olympus CX31RBSF attached cameraman) as reported by Farahpour et al. (2018). Cellular infiltration, edoema and collagen deposition were assessed. An image pro-insight software was utilised for evaluating the collagen deposition. Morphometric lens (Olympus, Germany) was used for assessing the epithelial thickness. edoema was assessed as a 5-score as reported previously (Nejati et al. 2015).\nConcerning the evaluation of the histological parameters, the mice were euthanized by a special CO2 device and the granulation tissue were excised in along to 1 to 2 mm from surrounding normal skin. The samples were fixed in neutral-buffered formalin 10%, routinely processed, and paraffin wax was embedded, sectioned at 5 µm, and stained with Masson’s trichrome and then examined under light microscopy (Olympus CX31RBSF attached cameraman) as reported by Farahpour et al. (2018). Cellular infiltration, edoema and collagen deposition were assessed. An image pro-insight software was utilised for evaluating the collagen deposition. Morphometric lens (Olympus, Germany) was used for assessing the epithelial thickness. edoema was assessed as a 5-score as reported previously (Nejati et al. 2015).\n Immunohistochemical staining (IHC) for angiogenesis (CD31) To assess the IHC, it was conducted as reported by Farahpour et al. (2018). The IHC was conducted based on manufacturer’s protocol (Biocare, Yorba Linda, CA). The tissue sections were rinsed gently in the washing buffer and placed in a buffer bath. A DAB chromogen was employed for assessing the tissue sections and then incubated for 5 min. The sections were then dipped in weak ammonia (0.037 M/L) for 10 times, rinsed with distilled water and cover slipped. Brown stains were considered as positive immunohistochemical.\nTo assess the IHC, it was conducted as reported by Farahpour et al. (2018). The IHC was conducted based on manufacturer’s protocol (Biocare, Yorba Linda, CA). The tissue sections were rinsed gently in the washing buffer and placed in a buffer bath. A DAB chromogen was employed for assessing the tissue sections and then incubated for 5 min. The sections were then dipped in weak ammonia (0.037 M/L) for 10 times, rinsed with distilled water and cover slipped. Brown stains were considered as positive immunohistochemical.\n Fluorescent staining for collagen To wash the slides, acetic acid 1% was used for several minutes. The slides were stained using acridine-orange. Phosphate buffer was utilized to remove staining. The slides were the washed in distilled water, mounted with buffer drop and analyzed with a fluorescent microscope. The bunds with red and yellowish red colour were considered as collagen bunds (Farahpour et al. 2018).\nTo wash the slides, acetic acid 1% was used for several minutes. The slides were stained using acridine-orange. Phosphate buffer was utilized to remove staining. The slides were the washed in distilled water, mounted with buffer drop and analyzed with a fluorescent microscope. The bunds with red and yellowish red colour were considered as collagen bunds (Farahpour et al. 2018).\n RNA isolation and cDNA synthesis The wound tissues were isolated as previously reported and RNA was extracted applying a standard TRIZOL procedure as reported by Farahpour et al. (2018), and assessed by spectrophotometer (260 nm and 260/280 = 1.8–2.0). To prepare the cDNA, it was put in a reaction being mixed with 1 µg RNA, oligo (dT) primer (1 µL), 5 × reaction buffer (4 µL), RNAse inhibitor (1 µL), 10 mM dNTP mix (2 µL) and M-MuLV Reverse Transcriptase (1 µL) as described by producer protocol (Fermentas, GmbH, Germany). Time and temperature were as reported by Farahpour et al. (2018). The used primers included IL-1β, forward (5′-AAC AAA CCC TGC AGT GGT TCG-3′) and reverse (5′-AGCTGCTTCAGACACTTGCAC-3′); TGF-β, forward (5-CCAAACGCCGAAGACTTATCC-3′) and reverse (5′-CTTATTACCGATGGGATGGGATAGCCC-3′); IL-10, forward (5′-GAAGCTCCCTCAGCGAGGACA-3′) and reverse (5′-TTGGGCCAGTGAGTGAAAGGG-3′), TNF-α, forward (5′-GAAGCTCCCTCAGCGAGGACA-3′) and reverse (5′-TTGGGCCAGTGAGTGAAAGGG-3′), FGF-2, forward (5′-GGAACCCGGCGGGACACGGAC-3′) and reverse (5′-CCGCTGTGGCAGCTCTTG GGG-3′); VEGF, forward (5′-GCTCCGTAGTAGCCGTGGTCT-3′) and reverse (5′-GGAACCCGGCGGGACACGGAC-3′), and IGF-1, forward (5′-TAGGTGGTTGATGAATGGT-3′) and reverse (5′-GAAAGGGCAGGGCTAAT-3′).\nThe wound tissues were isolated as previously reported and RNA was extracted applying a standard TRIZOL procedure as reported by Farahpour et al. (2018), and assessed by spectrophotometer (260 nm and 260/280 = 1.8–2.0). To prepare the cDNA, it was put in a reaction being mixed with 1 µg RNA, oligo (dT) primer (1 µL), 5 × reaction buffer (4 µL), RNAse inhibitor (1 µL), 10 mM dNTP mix (2 µL) and M-MuLV Reverse Transcriptase (1 µL) as described by producer protocol (Fermentas, GmbH, Germany). Time and temperature were as reported by Farahpour et al. (2018). The used primers included IL-1β, forward (5′-AAC AAA CCC TGC AGT GGT TCG-3′) and reverse (5′-AGCTGCTTCAGACACTTGCAC-3′); TGF-β, forward (5-CCAAACGCCGAAGACTTATCC-3′) and reverse (5′-CTTATTACCGATGGGATGGGATAGCCC-3′); IL-10, forward (5′-GAAGCTCCCTCAGCGAGGACA-3′) and reverse (5′-TTGGGCCAGTGAGTGAAAGGG-3′), TNF-α, forward (5′-GAAGCTCCCTCAGCGAGGACA-3′) and reverse (5′-TTGGGCCAGTGAGTGAAAGGG-3′), FGF-2, forward (5′-GGAACCCGGCGGGACACGGAC-3′) and reverse (5′-CCGCTGTGGCAGCTCTTG GGG-3′); VEGF, forward (5′-GCTCCGTAGTAGCCGTGGTCT-3′) and reverse (5′-GGAACCCGGCGGGACACGGAC-3′), and IGF-1, forward (5′-TAGGTGGTTGATGAATGGT-3′) and reverse (5′-GAAAGGGCAGGGCTAAT-3′).\n Antioxidant capacity To assess the antioxidant capacity, the wound granulation tissue was homogenized in ice-cold KCl (150 mM) and the mixture was then centrifuged at 3000 g for 10 min. The supernatant was used to evaluate the total antioxidant capacity (TAC), malondialdehyde (MDA) and total tissue thiol molecules (TTM) content. The Lowry method was employed for evaluating the protein content of the samples (Daemi et al. 2019a) and MDA content was used for assessing the lipid peroxidation rate. To assess the TTM, the collected granulation tissue sample was homogenized and the supernatant was added to Tris-EDTA as reported by Daemi et al. (2019a).\nTo assess the antioxidant capacity, the wound granulation tissue was homogenized in ice-cold KCl (150 mM) and the mixture was then centrifuged at 3000 g for 10 min. The supernatant was used to evaluate the total antioxidant capacity (TAC), malondialdehyde (MDA) and total tissue thiol molecules (TTM) content. The Lowry method was employed for evaluating the protein content of the samples (Daemi et al. 2019a) and MDA content was used for assessing the lipid peroxidation rate. To assess the TTM, the collected granulation tissue sample was homogenized and the supernatant was added to Tris-EDTA as reported by Daemi et al. (2019a).\n Statistical analysis The results were reported as mean ± standard deviation and analyzed by Graph Pad Prism Software. One-way ANOVA was utilized to analyze the results. Dunnett’s test for pair-wise comparisons was used for evaluating the effect of treatments and p < 0.05 was considered to be of significance.\nThe results were reported as mean ± standard deviation and analyzed by Graph Pad Prism Software. One-way ANOVA was utilized to analyze the results. Dunnett’s test for pair-wise comparisons was used for evaluating the effect of treatments and p < 0.05 was considered to be of significance.\n\nMaterials:\nZataria multiflora essential oil was purchased from Barij Essence Company (Kashan-Iran) and approved by the same herbarium company (No.1203). Commercial base ointment and mupirocin were prepared from Pars Daruo. Ltd., Tehran, Iran. The commercial base ointment contained 90% soft paraffin, 5% hard paraffin and 5% lanolin. Rodent feeds were prepared from Javaneh Khorasan Company (Khorasan, Iran).\n\nEssential oil analysis and identification:\nGas chromatography (GC) analysis was employed in order to identify the compounds. The components of the essential oil were recognized using calculation of their retention indices under temperature-programmed conditions for n-alkanes (C6–C24) and the oil on DB-5 column.\n\nAnimals:\nSeventy-two BALB/c mice weighing 25 ± 3 g were prepared. The mice had free access to water and pelleted feed for rodents (Javaneh Khorasan Company, Khorasan, Iran). The mice were kept in an ambient temperature of 23 ± 3 °C, at a constant air humidity, and a natural day–night cycle. This study was approved by the Animal Research Committee of the Urmia Islamic Azad University with ethical No. IAU-UB 11044.\n\nInduction of wound:\nTo induce the wound, the mice were primarily anesthetized by intraperitoneal administration route using 50 mg/kg ketamine and 5 mg/kg xylazine. Following the induction of anaesthesia, dorsal region of each mouse was shaven for surgical actions. A circular wound model was induced in a size of 7 mm using a surgical biopsy punch and per wound was inoculated by an aliquot of 5 × 107 suspension containing S. aureus (ATCC 26313) and P. aeruginosa (ATCC 27853) in 50 μL phosphate-buffered saline (Farahpour et al. 2020; Khezri et al. 2019). Subsequently, animals were divided into four groups (n = 18), as (group I) negative control group (NCG) that was administered commercial base ointment, (group II) positive control group that were treated with Mupirocin® (MG), and (groups III and IV) treated groups with therapeutic ointments containing 2 g and 4 g ZME mixed in NCG (2% and 4% ZMEO), respectively. Concerning the colonization of the bacteria, 24 h after the induction of the wounds, ointments were applied on wounds, once a day. In addition, according to the tissue sampling on days 3, 7, and 14 after the wounding, the animals in each group were divided into three subgroups (n = 6).\n\nWound area:\nThe rate of wound closure was estimated as reported previously (Nejati et al. 2015; Khezri et al. 2019). In order to calculate the percentage of wound closure rate, a transparent paper was placed on it and it was calculated based on the following formula:\nPercentage of wound closure = [(Wound area on Day 0 − Wound area on Day X)/Wound area on Day 0] × 100.\n\nThe investigation of the bacteriological count in the wound area:\nTo calculate the bacteriological count, the granulated tissues were excised and 0.1 g of the sample was crushed, minced, and homogenized in a sterile mortar containing 10 mL of sterile saline. It was the diluted in tubes containing 9 mL of sterile saline, was cultured on plate count agar (Merck KGaA, Darmstadt, Germany), incubated at 37 °C for 48 h, calculated as CFU/g of granulation tissue (Farahpour et al. 2020; Khezri et al. 2019).\n\nHistological analysis:\nConcerning the evaluation of the histological parameters, the mice were euthanized by a special CO2 device and the granulation tissue were excised in along to 1 to 2 mm from surrounding normal skin. The samples were fixed in neutral-buffered formalin 10%, routinely processed, and paraffin wax was embedded, sectioned at 5 µm, and stained with Masson’s trichrome and then examined under light microscopy (Olympus CX31RBSF attached cameraman) as reported by Farahpour et al. (2018). Cellular infiltration, edoema and collagen deposition were assessed. An image pro-insight software was utilised for evaluating the collagen deposition. Morphometric lens (Olympus, Germany) was used for assessing the epithelial thickness. edoema was assessed as a 5-score as reported previously (Nejati et al. 2015).\n\nImmunohistochemical staining (IHC) for angiogenesis (CD31):\nTo assess the IHC, it was conducted as reported by Farahpour et al. (2018). The IHC was conducted based on manufacturer’s protocol (Biocare, Yorba Linda, CA). The tissue sections were rinsed gently in the washing buffer and placed in a buffer bath. A DAB chromogen was employed for assessing the tissue sections and then incubated for 5 min. The sections were then dipped in weak ammonia (0.037 M/L) for 10 times, rinsed with distilled water and cover slipped. Brown stains were considered as positive immunohistochemical.\n\nFluorescent staining for collagen:\nTo wash the slides, acetic acid 1% was used for several minutes. The slides were stained using acridine-orange. Phosphate buffer was utilized to remove staining. The slides were the washed in distilled water, mounted with buffer drop and analyzed with a fluorescent microscope. The bunds with red and yellowish red colour were considered as collagen bunds (Farahpour et al. 2018).\n\nRNA isolation and cDNA synthesis:\nThe wound tissues were isolated as previously reported and RNA was extracted applying a standard TRIZOL procedure as reported by Farahpour et al. (2018), and assessed by spectrophotometer (260 nm and 260/280 = 1.8–2.0). To prepare the cDNA, it was put in a reaction being mixed with 1 µg RNA, oligo (dT) primer (1 µL), 5 × reaction buffer (4 µL), RNAse inhibitor (1 µL), 10 mM dNTP mix (2 µL) and M-MuLV Reverse Transcriptase (1 µL) as described by producer protocol (Fermentas, GmbH, Germany). Time and temperature were as reported by Farahpour et al. (2018). The used primers included IL-1β, forward (5′-AAC AAA CCC TGC AGT GGT TCG-3′) and reverse (5′-AGCTGCTTCAGACACTTGCAC-3′); TGF-β, forward (5-CCAAACGCCGAAGACTTATCC-3′) and reverse (5′-CTTATTACCGATGGGATGGGATAGCCC-3′); IL-10, forward (5′-GAAGCTCCCTCAGCGAGGACA-3′) and reverse (5′-TTGGGCCAGTGAGTGAAAGGG-3′), TNF-α, forward (5′-GAAGCTCCCTCAGCGAGGACA-3′) and reverse (5′-TTGGGCCAGTGAGTGAAAGGG-3′), FGF-2, forward (5′-GGAACCCGGCGGGACACGGAC-3′) and reverse (5′-CCGCTGTGGCAGCTCTTG GGG-3′); VEGF, forward (5′-GCTCCGTAGTAGCCGTGGTCT-3′) and reverse (5′-GGAACCCGGCGGGACACGGAC-3′), and IGF-1, forward (5′-TAGGTGGTTGATGAATGGT-3′) and reverse (5′-GAAAGGGCAGGGCTAAT-3′).\n\nAntioxidant capacity:\nTo assess the antioxidant capacity, the wound granulation tissue was homogenized in ice-cold KCl (150 mM) and the mixture was then centrifuged at 3000 g for 10 min. The supernatant was used to evaluate the total antioxidant capacity (TAC), malondialdehyde (MDA) and total tissue thiol molecules (TTM) content. The Lowry method was employed for evaluating the protein content of the samples (Daemi et al. 2019a) and MDA content was used for assessing the lipid peroxidation rate. To assess the TTM, the collected granulation tissue sample was homogenized and the supernatant was added to Tris-EDTA as reported by Daemi et al. (2019a).\n\nStatistical analysis:\nThe results were reported as mean ± standard deviation and analyzed by Graph Pad Prism Software. One-way ANOVA was utilized to analyze the results. Dunnett’s test for pair-wise comparisons was used for evaluating the effect of treatments and p < 0.05 was considered to be of significance.\n\nResults:\n Composition of the Zataria multiflora essential oil The analysis of Zataria multiflora essential oil with GC-FID and GC-mass spectrometry (MS) identified 29 compounds which accounted for 99.6% of the total essential oil composition (Table 1). It contained thymol (52.90%), p-cymene (9.10), γ-terpinene (8.10%) and carvacrol (6.80%) as major constituents (Figure 1).\nGC-MS chromatogram of the essential oil of Zataria multiflora.\nChemical constituents of Zataria multiflora essential oil.\nRI-Cal: retention indices calculated based on C6–C24\nn-alkenes from a DB-5 column.\nRI-Cal: retention indices retrieved from literature (Adams 2007).\nBold values show major compounds in the essential oil.\nThe analysis of Zataria multiflora essential oil with GC-FID and GC-mass spectrometry (MS) identified 29 compounds which accounted for 99.6% of the total essential oil composition (Table 1). It contained thymol (52.90%), p-cymene (9.10), γ-terpinene (8.10%) and carvacrol (6.80%) as major constituents (Figure 1).\nGC-MS chromatogram of the essential oil of Zataria multiflora.\nChemical constituents of Zataria multiflora essential oil.\nRI-Cal: retention indices calculated based on C6–C24\nn-alkenes from a DB-5 column.\nRI-Cal: retention indices retrieved from literature (Adams 2007).\nBold values show major compounds in the essential oil.\n Wound area The findings for the effects of ZMEO on the percentage of wound contraction are shown in Figure 2. The results implied that topical administration of therapeutic ointments significantly (p < 0.05) increased wound contraction rate on days 7 and 14 compared to the NCG group (Figure 2(A,B)). The comparison between treatment groups revealed that a higher wound contraction rate was observed in the 4% ZMEO-treated group compared to the 2% ZMEO-treated and MG groups (p < 0.05).\nThe effects of topical administration of ZMEO on (B) wound contraction rate and (C) tissue bacterial count (CFU/g) in different days. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\nThe findings for the effects of ZMEO on the percentage of wound contraction are shown in Figure 2. The results implied that topical administration of therapeutic ointments significantly (p < 0.05) increased wound contraction rate on days 7 and 14 compared to the NCG group (Figure 2(A,B)). The comparison between treatment groups revealed that a higher wound contraction rate was observed in the 4% ZMEO-treated group compared to the 2% ZMEO-treated and MG groups (p < 0.05).\nThe effects of topical administration of ZMEO on (B) wound contraction rate and (C) tissue bacterial count (CFU/g) in different days. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\n The bacteriological count The results concerning the effects of ZMEO on tissue bacteria counts are represented in Figure 2(C). The results showed that topical administration of therapeutic ointments significantly decreased bacterial colonization in all the days compared to the NCG group (p < 0.05). The comparison between treatment groups demonstrated that a higher level of ZMEO (4%) significantly decreased the total bacterial count compared to that of a lower level of it (2%) and MG groups (p < 0.05).\nThe results concerning the effects of ZMEO on tissue bacteria counts are represented in Figure 2(C). The results showed that topical administration of therapeutic ointments significantly decreased bacterial colonization in all the days compared to the NCG group (p < 0.05). The comparison between treatment groups demonstrated that a higher level of ZMEO (4%) significantly decreased the total bacterial count compared to that of a lower level of it (2%) and MG groups (p < 0.05).\n Histopathological parameters The results for histopathological parameters are represented in Table 2. The results showed that edoema score was significantly higher in the NCG group. It was observed to be significantly lower in the animals treated with the 4% ZMEO-treated group and also in 2% ZMEO and MG-treated groups. The findings implied that the lowest edoema was observed in 4% ZMEO in day 14; meanwhile, there was no observed edoema in the 4% ZMEO group on day 14. The results showed that immune cells were significantly lower in ZMEO groups in day 3 compared to other groups (p < 0.05), yet it decreased on days 7 and 14 in the ZMEO groups compared to other groups (p < 0.05). It means that the application of the ZMEO decreases immune cells in days 7 and 14, and increased it on day 3. Angiogenesis (Figure 3) and fibroblast infiltration were significantly higher in all treated groups compared to the NCG group and the highest level was observed in level of the 4%-ZMEO group. The results for collagen condensation (Figure 4) and re-epithelialization (Figure 5) revealed that parameters were significantly higher in the ZMEO groups, which confirms the results.\nImmunohistochemical staining for angiogenesis:; (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See elevated angiogenesis in ZMEO-treated group (arrows) on day 7 after wound induction. CD 31 staining, 400×.\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. Note well-formed collagen deposition in cross sections from ZMEO-treated animals on day 7 after wound induction (first row), which is significantly increased on day 14 after injury (second row). The third row represents the software analyze for collagen intensity. The cross sections from animals in ZMEO-treated groups exhibited condense collagen deposition versus NCG and MG groups. Masson-trichrome staining and fluorescent staining for collagen, 400× magnification.\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See well re-epithelialization in ZMEO-treated animals. The re-epithelialization initiated on day 7 after wound induction in ZMEO-treated animals. However, the cross sections from NCG and MG groups are not representing epithelialization. Note well-organized dermis and complete epithelialization with well-formed papillae in ZMEO-treated animals in comparison to NCG and MG groups. Masson-trichrome staining, 100×.\nThe effects of ZMEO on histopathological parameters in different days.\nSuperscripts (a–d) indicate significant differences in same day at p < 0.05.\nThe results for histopathological parameters are represented in Table 2. The results showed that edoema score was significantly higher in the NCG group. It was observed to be significantly lower in the animals treated with the 4% ZMEO-treated group and also in 2% ZMEO and MG-treated groups. The findings implied that the lowest edoema was observed in 4% ZMEO in day 14; meanwhile, there was no observed edoema in the 4% ZMEO group on day 14. The results showed that immune cells were significantly lower in ZMEO groups in day 3 compared to other groups (p < 0.05), yet it decreased on days 7 and 14 in the ZMEO groups compared to other groups (p < 0.05). It means that the application of the ZMEO decreases immune cells in days 7 and 14, and increased it on day 3. Angiogenesis (Figure 3) and fibroblast infiltration were significantly higher in all treated groups compared to the NCG group and the highest level was observed in level of the 4%-ZMEO group. The results for collagen condensation (Figure 4) and re-epithelialization (Figure 5) revealed that parameters were significantly higher in the ZMEO groups, which confirms the results.\nImmunohistochemical staining for angiogenesis:; (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See elevated angiogenesis in ZMEO-treated group (arrows) on day 7 after wound induction. CD 31 staining, 400×.\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. Note well-formed collagen deposition in cross sections from ZMEO-treated animals on day 7 after wound induction (first row), which is significantly increased on day 14 after injury (second row). The third row represents the software analyze for collagen intensity. The cross sections from animals in ZMEO-treated groups exhibited condense collagen deposition versus NCG and MG groups. Masson-trichrome staining and fluorescent staining for collagen, 400× magnification.\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See well re-epithelialization in ZMEO-treated animals. The re-epithelialization initiated on day 7 after wound induction in ZMEO-treated animals. However, the cross sections from NCG and MG groups are not representing epithelialization. Note well-organized dermis and complete epithelialization with well-formed papillae in ZMEO-treated animals in comparison to NCG and MG groups. Masson-trichrome staining, 100×.\nThe effects of ZMEO on histopathological parameters in different days.\nSuperscripts (a–d) indicate significant differences in same day at p < 0.05.\n Molecular analyses The results for gene expression are presented in Figure 6. Gene expression of IL-1β and TNF-α was significantly (p < 0.05) lower on all sampling days at treated groups compared to the NCG group. The greatest decrease was observed in the 4% ZMEO-treated animals. Gene expressions of IGF-1 and VEGF was significantly (p < 0.05) higher at all treated groups on day 7 after the wounding, but the levels significantly decreased on day 14. The expression of IL-10 level increased on day 7 whereas it decreased on day 14 compared to the NCG group (p < 0.05). Further analysis of gene expressions of TGF-β and FGF-2 indicate that its level does not have significant difference (p > 0.05) on day 3, yet its level significantly (p < 0.05) increased in all treated groups, particularly at 4% ZMEO-treated animals on day 7. The amount of TGF-β and FGF-2 gene expression levels also were significantly (p < 0.05) reduced on day 14 compared to the NCG group.\nThe effects of topical application of ZMEO on gene expression. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\nThe results for gene expression are presented in Figure 6. Gene expression of IL-1β and TNF-α was significantly (p < 0.05) lower on all sampling days at treated groups compared to the NCG group. The greatest decrease was observed in the 4% ZMEO-treated animals. Gene expressions of IGF-1 and VEGF was significantly (p < 0.05) higher at all treated groups on day 7 after the wounding, but the levels significantly decreased on day 14. The expression of IL-10 level increased on day 7 whereas it decreased on day 14 compared to the NCG group (p < 0.05). Further analysis of gene expressions of TGF-β and FGF-2 indicate that its level does not have significant difference (p > 0.05) on day 3, yet its level significantly (p < 0.05) increased in all treated groups, particularly at 4% ZMEO-treated animals on day 7. The amount of TGF-β and FGF-2 gene expression levels also were significantly (p < 0.05) reduced on day 14 compared to the NCG group.\nThe effects of topical application of ZMEO on gene expression. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\n Antioxidant status Antioxidant status in different groups is represented in Figure 7. Using the 2% and 4%-ZMEO significantly (p < 0.05) increased TAC and TTP levels compared to the NCG group in all of the days after the wounding. Interestingly, the highest increase was observed in the 4% ZMEO-treated animals. Further analysis of MDA level indicates significant (p < 0.05) decrease in all of the treated groups compared to the NCG group in all of the days after the wounding. The highest decrease was observed in the 4% ZMEO-treated group.\nThe effects of topical application of ZMEO on antioxidant properties. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\nAntioxidant status in different groups is represented in Figure 7. Using the 2% and 4%-ZMEO significantly (p < 0.05) increased TAC and TTP levels compared to the NCG group in all of the days after the wounding. Interestingly, the highest increase was observed in the 4% ZMEO-treated animals. Further analysis of MDA level indicates significant (p < 0.05) decrease in all of the treated groups compared to the NCG group in all of the days after the wounding. The highest decrease was observed in the 4% ZMEO-treated group.\nThe effects of topical application of ZMEO on antioxidant properties. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\n\nComposition of the Zataria multiflora essential oil:\nThe analysis of Zataria multiflora essential oil with GC-FID and GC-mass spectrometry (MS) identified 29 compounds which accounted for 99.6% of the total essential oil composition (Table 1). It contained thymol (52.90%), p-cymene (9.10), γ-terpinene (8.10%) and carvacrol (6.80%) as major constituents (Figure 1).\nGC-MS chromatogram of the essential oil of Zataria multiflora.\nChemical constituents of Zataria multiflora essential oil.\nRI-Cal: retention indices calculated based on C6–C24\nn-alkenes from a DB-5 column.\nRI-Cal: retention indices retrieved from literature (Adams 2007).\nBold values show major compounds in the essential oil.\n\nWound area:\nThe findings for the effects of ZMEO on the percentage of wound contraction are shown in Figure 2. The results implied that topical administration of therapeutic ointments significantly (p < 0.05) increased wound contraction rate on days 7 and 14 compared to the NCG group (Figure 2(A,B)). The comparison between treatment groups revealed that a higher wound contraction rate was observed in the 4% ZMEO-treated group compared to the 2% ZMEO-treated and MG groups (p < 0.05).\nThe effects of topical administration of ZMEO on (B) wound contraction rate and (C) tissue bacterial count (CFU/g) in different days. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\n\nThe bacteriological count:\nThe results concerning the effects of ZMEO on tissue bacteria counts are represented in Figure 2(C). The results showed that topical administration of therapeutic ointments significantly decreased bacterial colonization in all the days compared to the NCG group (p < 0.05). The comparison between treatment groups demonstrated that a higher level of ZMEO (4%) significantly decreased the total bacterial count compared to that of a lower level of it (2%) and MG groups (p < 0.05).\n\nHistopathological parameters:\nThe results for histopathological parameters are represented in Table 2. The results showed that edoema score was significantly higher in the NCG group. It was observed to be significantly lower in the animals treated with the 4% ZMEO-treated group and also in 2% ZMEO and MG-treated groups. The findings implied that the lowest edoema was observed in 4% ZMEO in day 14; meanwhile, there was no observed edoema in the 4% ZMEO group on day 14. The results showed that immune cells were significantly lower in ZMEO groups in day 3 compared to other groups (p < 0.05), yet it decreased on days 7 and 14 in the ZMEO groups compared to other groups (p < 0.05). It means that the application of the ZMEO decreases immune cells in days 7 and 14, and increased it on day 3. Angiogenesis (Figure 3) and fibroblast infiltration were significantly higher in all treated groups compared to the NCG group and the highest level was observed in level of the 4%-ZMEO group. The results for collagen condensation (Figure 4) and re-epithelialization (Figure 5) revealed that parameters were significantly higher in the ZMEO groups, which confirms the results.\nImmunohistochemical staining for angiogenesis:; (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See elevated angiogenesis in ZMEO-treated group (arrows) on day 7 after wound induction. CD 31 staining, 400×.\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. Note well-formed collagen deposition in cross sections from ZMEO-treated animals on day 7 after wound induction (first row), which is significantly increased on day 14 after injury (second row). The third row represents the software analyze for collagen intensity. The cross sections from animals in ZMEO-treated groups exhibited condense collagen deposition versus NCG and MG groups. Masson-trichrome staining and fluorescent staining for collagen, 400× magnification.\nCross section from wound area: (A) NCG group, (B) MG group, (C and D) 2 and 4% ZMEO-treated groups. See well re-epithelialization in ZMEO-treated animals. The re-epithelialization initiated on day 7 after wound induction in ZMEO-treated animals. However, the cross sections from NCG and MG groups are not representing epithelialization. Note well-organized dermis and complete epithelialization with well-formed papillae in ZMEO-treated animals in comparison to NCG and MG groups. Masson-trichrome staining, 100×.\nThe effects of ZMEO on histopathological parameters in different days.\nSuperscripts (a–d) indicate significant differences in same day at p < 0.05.\n\nMolecular analyses:\nThe results for gene expression are presented in Figure 6. Gene expression of IL-1β and TNF-α was significantly (p < 0.05) lower on all sampling days at treated groups compared to the NCG group. The greatest decrease was observed in the 4% ZMEO-treated animals. Gene expressions of IGF-1 and VEGF was significantly (p < 0.05) higher at all treated groups on day 7 after the wounding, but the levels significantly decreased on day 14. The expression of IL-10 level increased on day 7 whereas it decreased on day 14 compared to the NCG group (p < 0.05). Further analysis of gene expressions of TGF-β and FGF-2 indicate that its level does not have significant difference (p > 0.05) on day 3, yet its level significantly (p < 0.05) increased in all treated groups, particularly at 4% ZMEO-treated animals on day 7. The amount of TGF-β and FGF-2 gene expression levels also were significantly (p < 0.05) reduced on day 14 compared to the NCG group.\nThe effects of topical application of ZMEO on gene expression. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\n\nAntioxidant status:\nAntioxidant status in different groups is represented in Figure 7. Using the 2% and 4%-ZMEO significantly (p < 0.05) increased TAC and TTP levels compared to the NCG group in all of the days after the wounding. Interestingly, the highest increase was observed in the 4% ZMEO-treated animals. Further analysis of MDA level indicates significant (p < 0.05) decrease in all of the treated groups compared to the NCG group in all of the days after the wounding. The highest decrease was observed in the 4% ZMEO-treated group.\nThe effects of topical application of ZMEO on antioxidant properties. Superscripts (a–d) indicate significant differences in same day at p < 0.05.\n\nDiscussion:\nWounds infected by pathogenic bacteria such as S. aureus, and P. aeruginosa are believed to be serious challenges for regarding their management and treatment (Manzuoerh et al. 2019). Different agents are employed for the treatment of wounds. Active compounds of the essential oils can have pivotal roles in their treatment. The findings indicated that thymol (52.90%), p-cymene (9.10), γ-terpinene (8.10%) and carvacrol (6.80%) were the major constituents in the essential oil. Similar to our results, previous studies demonstrated that the major compounds of Z. multiflora essential oil were thymol, carvacrol and p-cymene (Aida et al. 2015), thymol, c-terpinene, p-cymene, and carvacrol (Saei-Dehkordi et al. 2010), carvacrol (Misaghi and Basti 2007), thymol, carvacrol, and p-cymene (Sharififar et al. 2007). The results showed that ZMEO accelerated wound healing compared to the NCG group and synthetic agent of mupirocin. Herein, the results are described based on the mechanisms and active compounds, step-by-step.\nInflammation is the first step in the wound healing process, in which different factors and genes are involved. Inflammation is triggered once the injury induces to living tissues by live organisms, such as bacteria and/or physical injury and faulted immune response. Inflammation removes foreign substances in order to improve the wound healing process (Garrett et al. 2010). It promotes macrophages, neutrophils and immune cells for initiation of inflammation (Oguntibeju 2018). IL-1β and TNF-α are commonly produced by immune cells, such as macrophages and mast cells (Thacker et al. 2007). The results showed that the topical application of ZMEO decreased immune cells and the expressions of IL-1β and TNF-α in the first 3 days. The results implied a positive relation between the number of immune cells with the expression of IL-1β and TNF-α in the treatments. Meaning that immune cells increase the expression of IL-1β and TNF-α in the first 3 days. Increased inflammation delays the wound healing process. The findings revealed that topical administration of ZMEO decreased inflammation on days 7 and 14, and acted as an antibacterial and anti-inflammation agent. Antibacterial and anti-inflammatory activity of ZMEO is attributed to its compounds. The essence contains thymol and carvacrol that are of antibacterial and anti-inflammatory properties. It was reported that the administration of carvacrol significantly reduced TNF-α levels in pleural lavage (Guimarães et al. 2012). The anti-inflammatory activity of thymol leads to prevention of the phosphorylation of extracellular signal-regulated protein kinase, c-Jun N-terminal kinase, and nuclear factor-κB (Liang et al. 2014). Carvacrol prevented leukocyte migration, and decreased edoema, and showed anti-inflammatory effects (Fachini-Queiroz et al. 2012). The results indicated that immune cells decreased and expression of IL-1β and TNF-α also decreased in ZMEO groups. The findings showed that the total bacterial count significantly decreased in ZMEO groups compared to the NCG group. Mupirocin is used as a bactericide agent for the treatment of infected wounds. The results convey that ZMEO, particularly at a higher level, have better antibacterial activity compared to mupirocin. Higher total bacterial count increases the inflammation and delays the wound healing process. It means that topical administration of ZMEO decreases the inflammation with its antibacterial activity. It was reported that the antibacterial activity of Zataria multiflora is in view of its compounds (Shafiee et al. 1999; Barkhori-Mehni et al. 2017). In essence, ZMEO decreased the inflammation and inflammatory-associated parameters. Higher levels of ZMEO provide higher doses of active compounds that act as anti-inflammatory factors. Decreased inflammation leads the wound healing process towards the proliferative phase.\nOur findings implied that the topical application of ZMEO improved the proliferative phase. The results showed that the genes expression of VEGF, FGF-2 and IGF-1 increased in ZMEO groups. IGF-1 increases the transport of glucose during the wound healing process and improves the proliferative phase (Daemi et al. 2019b). VEGF induces angiogenesis and stimulates cell migration and proliferation (Norton & Popel 2016; Farahpour et al. 2020; Gharaboghaz et al. 2020). FGF-2, a protein that participates in the wound healing process (Souto et al. 2020), together with other growth factors, such as VEGF, are reported to increase the angiogenesis and indirectly support the cellular nutrient, oxygen and energy supplements (Harding et al. 2002). So far, studies have not investigated the effect of ZMEO on genes expression of VEGF, FGF-2 and IGF-1. It means that VEGF improves angiogenesis, and vessel number was significantly higher in ZMEO groups. VEGF improved angiogenesis and number of vessels. Other genes involved were TGF-β, and IL-10 whose expressions increased on day 7. IL-10 is a factor that reduces the production of pro-inflammatory cytokine, but TGF-β leads the phase towards the proliferative phase by improving the proliferation and differentiation of fibroblasts, collagen production, and wound contraction (Khezri et al. 2020). Seemingly, both genes are of greatly important roles in decreasing the inflammation and passing to the proliferative phase. The IHC results indicated improved collagen deposition. The biosynthesis of collagen during the proliferation stage has pivotal roles in dermal maturation in which some factors are involved (Yakaew et al. 2016; Daemi et al. 2019a). The results showed that the topical application of ZMEO increased fibroblast, which is due to its effect on IGF-1. It was reported that IGF-1 increases fibroblast cells in in vitro assessment (Lisa et al. 2011). Improving cellular proliferation and differentiation are essential for shortening the healing time (Oryan et al. 2015; Karimzadeh and Farahpour 2017). Proliferative effect of ZMEO is partly owing to its antioxidant activity. The topical application of ZMEO improved antioxidant activity. Previous studies have reported antioxidant activity of Zataria multiflora essential oil (Fatemi et al. 2012; Karimian et al. 2012; Kavoosi and Teixeira da Silva 2012). Oxidative stress in the wound area increases the damage of proteins, nucleotides and lipid levels (Cano Sanchez et al. 2018; Farahpour et al. 2020; Gharaboghaz et al. 2020) and decreases the antioxidant activity. This fact is associated with increased changes in the antioxidant profile, which are contributed to the increase in the levels of MDA content (Sharma et al. 2012; Bardaa et al. 2016; Vittorazzi et al. 2016).\n\nConclusion:\nThe topical application of ZMEO decreased the inflammation and the total bacterial count, which progresses the wound healing process towards the proliferative phase. It increases the expression of proliferative genes, helps to alleviate the inflammation phase and accelerates the proliferative phase. Antioxidant activity of ZMEO accelerated the wound healing process. All the above-mentioned factors hasten the wound healing process, as seen for the wound area. Accordingly, applying ZMEO only and/or in combination with chemical agents for the treatment of wound healing could be suggested.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nZataria multiflora Boiss (Lamiaceae) essential oil (ZME) is believed to be a bactericide herbal medicine and might alleviate negative effects of infection.\n\nMethods:\nA full-thickness excisional skin wound was surgically created in each mouse and inoculated with 5 × 107 suspension containing Pseudomonas aeruginosa and Staphylococcus aureus. The BALB/c mice (n = 72) were divided into four groups: (1) negative control that received base ointment (NCG), (2) positive control that daily received Mupirocin® (MG), (3) therapeutic ointment containing 2% ZMEO and (4) therapeutic ointment containing 4% ZMEO, for 21 days. Wound contraction, total bacterial count, histopathological parameters, antioxidant activity, qRT-PCR analysis for expression of IL-1β, TNF-α, VEGF, IGF-1, TGF-β, IL-10, and FGF-2 mRNA levels were assessed on days 3, 7, and 14 following the wounding.\n\nResults:\nTopical administration of ZMEO significantly decreased the total bacterial count and wound area and also expression of IL-1β and TNF-α compared to the control groups (p < 0.05) in all days. This could also increase significantly the expression of TGF-β, IL-10 IGF-1, FGF-2, and VEGF, and also angiogenesis, fibroblasts, fibrocytes, epithelialization ratio, and collagen deposition and improve antioxidant status compared to the control group (p < 0.05).\n\nConclusions:\nZMEO accelerated the healing process of infected wounds by shortening the inflammatory factors and increasing proliferative phase. Applying ZMEO only and/or in combination with chemical agents for the treatment of wound healing could be suggested."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nWound infections are important challenges that cause the suffering of patients and economic losses (Vittorazzi et al. 2016; Daemi et al. 2019a; Farghali et al. 2019). Skin is a regenerative organ which acts as a barrier between the body and the external environment (Hassan et al. 2014). A wound is defined as a disturbance in anatomic structure of skin and its functional integrity (Farghali et al. 2019). Skin prevents the penetration of bacteria and fungi that are important challenges that induce mortality and morbidity. Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic bacteria that induce infection in patients (Cardona and Wilson 2015). The bacteria are commonly detected in the upper and deepest region of wound bed (Serra et al. 2015). Colonization of S. aureus and P. aeruginosa in the wound site postpones the wound healing process (Farahpour et al. 2020; Khezri et al. 2020). The wound healing process comprises several phases, including coagulation, inflammation, epithelialization, granulation tissue formation, and tissue remodelling (Daemi et al. 2019a, 2019b; Farahpour et al. 2020). Inflammatory phase occurs after activation of inflammatory chemokines (Farahpour et al. 2020). Interleukin-1β (IL-1β) is significantly synthesized 12–24 h after infliction of the wound, and its level returns to basal level after the proliferative stage is completed (Fahey and Doyle 2019). Tumour necrosis factor-α (TNF-α) is significantly synthesized by macrophages and T lymphocytes and its level increases under inflammation and infection conditions (Xue and Falcon 2019). Following the induction of inflammation, proliferative phase occurs in which many genes are involved. Insulin-like growth factor 1 (IGF-1) promotes the production of keratinocyte and fibroblast proliferation and improves the re-epithelialization (Daemi et al. 2019b). Decreased endothelial insulin/IGF-1 signal delays in the wound healing process (Aghdam et al. 2012). Vascular endothelial growth factor (VEGF) induces angiogenesis and stimulates cell migration and proliferation (Farahpour et al. 2020). Fibroblast growth factor-2 (FGF-2) is a protein that participates in the wound healing process (Souto et al. 2020). Interleukin-10 (IL-10) reduces the production of pro-inflammatory cytokine, but tumour growth factor-β (TGF-β) has roles in increasing the proliferative phase improving the proliferation and differentiation of fibroblasts, collagen production, and wound contraction (Khezri et al. 2020). On the other hand, oxidative stress increases under wound disorders and increases the production of reactive oxygen species (ROS) in the site of wound (Ustuner et al. 2019). Applying antibacterial and antioxidant agents shortens the inflammatory phase, promotes the proliferative phase and accelerates the wound healing process (Bardaa et al. 2016; Vittorazzi et al. 2016; Daemi et al. 2019a, 2019b).\nZataria multiflora Boiss (Lamiaceae) (ZM) has antibacterial activity against Pseudomonas spp. (Barkhori-Mehni et al. 2017). Its antibacterial activity is attributed to its phenolic compounds that destroy the cell wall in bacteria (Barkhori-Mehni et al. 2017). It demonstrates antioxidant properties due to its compounds such as thymol and carvacrol (Mojaddar Langroodi et al. 2019). Seemingly, ZM can accelerate the wound healing process due to its antibacterial and antioxidant features, but no studies have been conducted yet to evaluate the Zataria multiflora essential oil (ZME) on the wound healing process. Therefore, this study was conducted to evaluate the effect of an ointment prepared from ZME on the healing process of infected wound model by S. aureus and P. aeruginosa by assessing wound contraction, total bacterial count, histopathological and immunohistochemical, antioxidant activity, and qRT-PCR analyses.\n\nConclusion:\nThe topical application of ZMEO decreased the inflammation and the total bacterial count, which progresses the wound healing process towards the proliferative phase. It increases the expression of proliferative genes, helps to alleviate the inflammation phase and accelerates the proliferative phase. Antioxidant activity of ZMEO accelerated the wound healing process. All the above-mentioned factors hasten the wound healing process, as seen for the wound area. Accordingly, applying ZMEO only and/or in combination with chemical agents for the treatment of wound healing could be suggested."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nZataria multiflora Boiss (Lamiaceae) essential oil (ZME) is believed to be a bactericide herbal medicine and might alleviate negative effects of infection.\n\nMethods:\nA full-thickness excisional skin wound was surgically created in each mouse and inoculated with 5 × 107 suspension containing Pseudomonas aeruginosa and Staphylococcus aureus. The BALB/c mice (n = 72) were divided into four groups: (1) negative control that received base ointment (NCG), (2) positive control that daily received Mupirocin® (MG), (3) therapeutic ointment containing 2% ZMEO and (4) therapeutic ointment containing 4% ZMEO, for 21 days. Wound contraction, total bacterial count, histopathological parameters, antioxidant activity, qRT-PCR analysis for expression of IL-1β, TNF-α, VEGF, IGF-1, TGF-β, IL-10, and FGF-2 mRNA levels were assessed on days 3, 7, and 14 following the wounding.\n\nResults:\nTopical administration of ZMEO significantly decreased the total bacterial count and wound area and also expression of IL-1β and TNF-α compared to the control groups (p < 0.05) in all days. This could also increase significantly the expression of TGF-β, IL-10 IGF-1, FGF-2, and VEGF, and also angiogenesis, fibroblasts, fibrocytes, epithelialization ratio, and collagen deposition and improve antioxidant status compared to the control group (p < 0.05).\n\nConclusions:\nZMEO accelerated the healing process of infected wounds by shortening the inflammatory factors and increasing proliferative phase. Applying ZMEO only and/or in combination with chemical agents for the treatment of wound healing could be suggested."},"whole_article_text_length":{"kind":"number","value":10623,"string":"10,623"},"whole_article_abstract_length":{"kind":"number","value":321,"string":"321"},"other_sections_lengths":{"kind":"list like","value":[76,50,96,267,82,100,152,108,74,247,131,59,144,149,94,543,242,140],"string":"[\n 76,\n 50,\n 96,\n 267,\n 82,\n 100,\n 152,\n 108,\n 74,\n 247,\n 131,\n 59,\n 144,\n 149,\n 94,\n 543,\n 242,\n 140\n]","isConversational":false},"num_sections":{"kind":"number","value":23,"string":"23"},"most_frequent_words":{"kind":"list like","value":["zmeo","wound","groups","group","treated","day","05","ncg","significantly","zmeo treated"],"string":"[\n \"zmeo\",\n \"wound\",\n \"groups\",\n \"group\",\n \"treated\",\n \"day\",\n \"05\",\n \"ncg\",\n \"significantly\",\n \"zmeo treated\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["bacteriological count wound","aeruginosa wound site","wound infections important","wounds infected pathogenic","wound model aureus"],"string":"[\n \"bacteriological count wound\",\n \"aeruginosa wound site\",\n \"wound infections important\",\n \"wounds infected pathogenic\",\n \"wound model aureus\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Infected wound healing | inflammatory cytokines | antimicrobial properties | thymol [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Infected wound healing | inflammatory cytokines | antimicrobial properties | thymol [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Infected wound healing | inflammatory cytokines | antimicrobial properties | thymol [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Infected wound healing | inflammatory cytokines | antimicrobial properties | thymol [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Infected wound healing | inflammatory cytokines | antimicrobial properties | thymol [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Administration, Topical | Animals | Collagen | Inflammation | Lamiaceae | Mice | Mice, Inbred BALB C | Neovascularization, Pathologic | Oils, Volatile | Surgical Wound Infection | Wound Healing [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Administration, Topical | Animals | Collagen | Inflammation | Lamiaceae | Mice | Mice, Inbred BALB C | Neovascularization, Pathologic | Oils, Volatile | Surgical Wound Infection | Wound Healing [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Administration, Topical | Animals | Collagen | Inflammation | Lamiaceae | Mice | Mice, Inbred BALB C | Neovascularization, Pathologic | Oils, Volatile | Surgical Wound Infection | Wound Healing [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Administration, Topical | Animals | Collagen | Inflammation | Lamiaceae | Mice | Mice, Inbred BALB C | Neovascularization, Pathologic | Oils, Volatile | Surgical Wound Infection | Wound Healing [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Administration, Topical | Animals | Collagen | Inflammation | Lamiaceae | Mice | Mice, Inbred BALB C | Neovascularization, Pathologic | Oils, Volatile | Surgical Wound Infection | Wound Healing [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] bacteriological count wound | aeruginosa wound site | wound infections important | wounds infected pathogenic | wound model aureus [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] bacteriological count wound | aeruginosa wound site | wound infections important | wounds infected pathogenic | wound model aureus [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] bacteriological count wound | aeruginosa wound site | wound infections important | wounds infected pathogenic | wound model aureus [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] bacteriological count wound | aeruginosa wound site | wound infections important | wounds infected pathogenic | wound model aureus [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] bacteriological count wound | aeruginosa wound site | wound infections important | wounds infected pathogenic | wound model aureus [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] zmeo | wound | groups | group | treated | day | 05 | ncg | significantly | zmeo treated [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] zmeo | wound | groups | group | treated | day | 05 | ncg | significantly | zmeo treated [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] zmeo | wound | groups | group | treated | day | 05 | ncg | significantly | zmeo treated [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] zmeo | wound | groups | group | treated | day | 05 | ncg | significantly | zmeo treated [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] zmeo | wound | groups | group | treated | day | 05 | ncg | significantly | zmeo treated [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] wound | process | healing | healing process | wound healing process | wound healing | 2020 | factor | phase | growth factor [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] zmeo | treated | groups | group | zmeo treated | 05 | day | significantly | ncg | compared [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] healing | wound healing | wound | process | phase | wound healing process | proliferative | healing process | inflammation | proliferative phase [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] zmeo | wound | group | groups | treated | 05 | day | zmeo treated | significantly | ncg [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] zmeo | wound | group | groups | treated | 05 | day | zmeo treated | significantly | ncg [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Boiss | Lamiaceae [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ZMEO | IL-1β | TNF | 0.05 | all days ||| FGF-2 | VEGF | 0.05 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ZMEO ||| ZMEO [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Boiss | Lamiaceae ||| 5 | 107 | Staphylococcus ||| 72 | four | 1 | NCG | 2 | daily | Mupirocin® | 3 | 2% | ZMEO | 4 | 4% | ZMEO | 21 days ||| IL-1β | TNF | VEGF | IGF-1 | IL-10 | FGF-2 | days 3, 7 | 14 ||| ZMEO | IL-1β | TNF | 0.05 | all days ||| FGF-2 | VEGF | 0.05 ||| ||| ZMEO [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Boiss | Lamiaceae ||| 5 | 107 | Staphylococcus ||| 72 | four | 1 | NCG | 2 | daily | Mupirocin® | 3 | 2% | ZMEO | 4 | 4% | ZMEO | 21 days ||| IL-1β | TNF | VEGF | IGF-1 | IL-10 | FGF-2 | days 3, 7 | 14 ||| ZMEO | IL-1β | TNF | 0.05 | all days ||| FGF-2 | VEGF | 0.05 ||| ||| ZMEO [SUMMARY]"}}},{"rowIdx":83,"cells":{"title":{"kind":"string","value":"Submicroscopic malaria infection during pregnancy and the impact of intermittent preventive treatment."},"pmid":{"kind":"string","value":"25023697"},"background_abstract":{"kind":"string","value":"Malaria during pregnancy results in adverse outcomes for mothers and infants. Intermittent preventive treatment (IPT) with sulphadoxine-pyrimethamine (SP) is the primary intervention aimed at reducing malaria infection during pregnancy. Although submicroscopic infection is common during pregnancy and at delivery, its impact throughout pregnancy on the development of placental malaria and adverse pregnancy outcomes has not been clearly established."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Quantitative PCR was used to detect submicroscopic infections in pregnant women enrolled in an observational study in Blantyre, Malawi to determine their effect on maternal, foetal and placental outcomes. The ability of SP to treat and prevent submicroscopic infections was also assessed."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"2,681 samples from 448 women were analysed and 95 submicroscopic infections were detected in 68 women, a rate of 0.6 episodes per person-year of follow-up. Submicroscopic infections were most often detected at enrolment. The majority of women with submicroscopic infections did not have a microscopically detectable infection detected during pregnancy. Submicroscopic infection was associated with placental malaria even after controlling for microscopically detectable infection and was associated with decreased maternal haemoglobin at the time of detection. However, submicroscopic infection was not associated with adverse maternal or foetal outcomes at delivery. One-third of women with evidence of placental malaria did not have documented peripheral infection during pregnancy. SP was moderately effective in treating submicroscopic infections, but did not prevent the development of new submicroscopic infections in the month after administration."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Submicroscopic malaria infection is common and occurs early in pregnancy. SP-IPT can clear some submicroscopic infections but does not prevent new infections after administration. To effectively control pregnancy-associated malaria, new interventions are required to target women prior to their first antenatal care visit and to effectively treat and prevent all malaria infections."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Antimalarials","Artemether, Lumefantrine Drug Combination","Artemisinins","Asymptomatic Diseases","DNA, Protozoan","Drug Administration Schedule","Drug Combinations","Drug Resistance","Erythrocytes","Ethanolamines","False Negative Reactions","Female","Fetal Diseases","Fluorenes","Follow-Up Studies","Hemeproteins","Humans","Infant, Newborn","Infectious Disease Transmission, Vertical","Malaria","Malawi","Parasitemia","Placenta","Plasmodium","Polymerase Chain Reaction","Pregnancy","Pregnancy Complications, Infectious","Pregnancy Outcome","Pregnancy Trimesters","Prevalence","Pyrimethamine","Quinine","Sulfadoxine"],"string":"[\n \"Adult\",\n \"Antimalarials\",\n \"Artemether, Lumefantrine Drug Combination\",\n \"Artemisinins\",\n \"Asymptomatic Diseases\",\n \"DNA, Protozoan\",\n \"Drug Administration Schedule\",\n \"Drug Combinations\",\n \"Drug Resistance\",\n \"Erythrocytes\",\n \"Ethanolamines\",\n \"False Negative Reactions\",\n \"Female\",\n \"Fetal Diseases\",\n \"Fluorenes\",\n \"Follow-Up Studies\",\n \"Hemeproteins\",\n \"Humans\",\n \"Infant, Newborn\",\n \"Infectious Disease Transmission, Vertical\",\n \"Malaria\",\n \"Malawi\",\n \"Parasitemia\",\n \"Placenta\",\n \"Plasmodium\",\n \"Polymerase Chain Reaction\",\n \"Pregnancy\",\n \"Pregnancy Complications, Infectious\",\n \"Pregnancy Outcome\",\n \"Pregnancy Trimesters\",\n \"Prevalence\",\n \"Pyrimethamine\",\n \"Quinine\",\n \"Sulfadoxine\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"4110536"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Each year, 25 million pregnant women in sub-Saharan Africa are at risk for malaria infection. Malaria during pregnancy is associated with maternal anaemia and infant low birth weight. Malaria during pregnancy is estimated to result in 100,000 infant deaths annually in Africa\n[1].\nMalaria infections during pregnancy may be identified by microscopic examination of a blood smear or may be submicroscopic, detectable only by more sensitive molecular methods. Studies in a variety of transmission settings have shown that submicroscopic infections can be up to five times more common than microscopic infections during pregnancy\n[2-7]. While the associations between microscopically detectable malaria during pregnancy and maternal anaemia at delivery and low birth weight have been supported in most studies\n[1], the impact of submicroscopic infection during pregnancy on these adverse outcomes has not been systematically assessed.\nBecause malaria infections during pregnancy are often asymptomatic, a key intervention to decrease the burden of malaria in pregnancy is intermittent preventive treatment (IPT), providing anti-malarial chemotherapy at routine intervals during pregnancy. Currently, the recommended anti-malarial chemotherapy for IPT in the majority of Africa is sulphadoxine-pyrimethamine (SP), which is intended to cure current infections and to provide a period of post-treatment prophylaxis to prevent future infections.\nIn much of sub-Saharan Africa, increasing resistance to SP\n[8] is generating concerns that SP-IPT efficacy will be compromised\n[9]. Understanding the relative importance of the treatment and prophylactic effects of SP-IPT and the differential impact of resistance on these effects may have implications for the selection of a new drug to replace SP for IPT.\nIn the context of an observational study of a large cohort of pregnant women in Malawi, the effect of submicroscopic infections on maternal and foetal outcomes was examined. All women received SP-IPT according to the national policy. At each visit a blood smear was examined and women were treated for malaria infections detected by microscopy. The primary analysis of infections detected solely by microscopy showed an association between infection at enrolment in antenatal care and placental malaria, but only infections detected at the time of delivery were associated with adverse pregnancy outcomes\n[10]. Because higher rates of submicroscopic infection than microscopically detectable infection were anticipated and submicroscopic infections were not treated, it was hypothesized that submicroscopic infections would be associated with placental malaria, maternal anaemia and infant low birth weight. It was further hypothesized that, due to high rates of SP resistance, SP-IPT would fail to clear and prevent submicroscopic infections."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Study population Four-hundred and fifty pregnant women were enrolled in an observational cohort study of malaria during pregnancy in Blantyre, Malawi between June 2009 and June 2010. All women were in their first or second pregnancy and were less than or equal to 28 weeks gestational age based on clinical assessment at enrolment. Women were followed monthly during pregnancy and encouraged to come to the clinic if they had intercurrent illness. At each encounter, peripheral blood smears and dried blood spots on filter paper were collected. At delivery, these same specimens and placental blood and tissue samples were collected. After quickening, women received SP-IPT up to three times separated by at least four weeks. Women with malaria detectable by blood smear at routine or sick visits were treated for malaria in accordance with the national guidelines (quinine in the first trimester and artemether-lumefantrine in the second and third trimesters). Details are described by Kalilani-Phiri et al.[10].\nFour-hundred and fifty pregnant women were enrolled in an observational cohort study of malaria during pregnancy in Blantyre, Malawi between June 2009 and June 2010. All women were in their first or second pregnancy and were less than or equal to 28 weeks gestational age based on clinical assessment at enrolment. Women were followed monthly during pregnancy and encouraged to come to the clinic if they had intercurrent illness. At each encounter, peripheral blood smears and dried blood spots on filter paper were collected. At delivery, these same specimens and placental blood and tissue samples were collected. After quickening, women received SP-IPT up to three times separated by at least four weeks. Women with malaria detectable by blood smear at routine or sick visits were treated for malaria in accordance with the national guidelines (quinine in the first trimester and artemether-lumefantrine in the second and third trimesters). Details are described by Kalilani-Phiri et al.[10].\n Laboratory procedures Microscopy Peripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\nPeripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\n Haemoglobin Haemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\nHaemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\n Placental biopsies Placental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\nPlacental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\n Molecular detection DNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\n[11].\nDNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\n[11].\n Data analysis Peripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\nPeripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\n Ethical considerations Ethical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\nEthical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\n Microscopy Peripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\nPeripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\n Haemoglobin Haemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\nHaemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\n Placental biopsies Placental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\nPlacental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\n Molecular detection DNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\n[11].\nDNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\n[11].\n Data analysis Peripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\nPeripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\n Ethical considerations Ethical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\nEthical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Two thousand six hundred and eighty-one peripheral blood samples from 448 women enrolled in the study were screened for submicroscopic infection. Two women were not included in the analysis because they did not have any filter papers available for molecular analysis. Ninety-five incident submicroscopic infections were detected in 68 women. Pregnant women had 0.6 episodes of submicroscopic malaria per person-year of follow-up. As previously published, women in this cohort also experienced 0.6 episodes of microscopically detected infection per person-year of follow-up\n[10]. Thus, the overall rate of malaria infection was 1.2 episodes per person year of follow up.\nAmong women with submicroscopic infections, the mean number of infections was 1.4 (95% CI 1.2-1.6, range 1–5). Forty-nine women (72%) with submicroscopic infections never had an infection detected by microscopy during pregnancy. Submicroscopic infection was not associated with first versus second pregnancy, age, bed net use, or malaria treatment during pregnancy prior to enrolment, but was associated with enrolment at an earlier gestational age, more frequent visits, and lack of secondary school attendance (Table \n1). Gestational age at enrolment was inversely correlated with number of visits.Among 311 placentas with both histological and molecular data available for evaluation, 104 (33%) had evidence of placental malaria (Figure \n1). Thirty-three (11%) had both parasites and haemozoin detected microscopically. Three (1%) had no haemozoin but did have parasites detected by histology and confirmed by qPCR. Thirty-eight (12%) had haemozoin without histological or molecular evidence of active parasite infection. Thirty (10%) had submicroscopic infection with no histological evidence of parasites or haemozoin.\nEnrolment characteristics of women with and without submicroscopic infection detected during pregnancy irrespective of the presence of microscopic infection\nCI: confidence interval.\nFrequency of histological and molecular findings of placental malaria.\nHistological results were not available for six placentas; all had parasites detected by qPCR.\n Timing and clinical presentation of submicroscopic infection Among women with at least one submicroscopic infection, 69% had submicroscopic infection detected at enrolment (47/68). Submicroscopic infections were detected in each trimester and at delivery. Based on estimated gestational age at enrolment, submicroscopic infection was detected in 12% (6/50) of first trimester, 6% (63/1,050) of second trimester, and 1.4% (19/1,372) of third trimester samples prior to delivery. These infections occurred in six, fifty-six, and eighteen women, respectively. Twelve women had infection in more than one trimester. After excluding the submicroscopic infections present at enrolment, the prevalence of submicroscopic infection was higher in the second trimester than the third trimester [22/650 (3.4%) versus 19/1,372 (1.4%) of samples, p = 0.003].\nNine of 308 women (3%) with peripheral samples obtained at delivery had submicroscopic infection. Two women with submicroscopic infection at delivery also had submicroscopic infection at their last antepartum visit, thus infection at delivery was not considered a new submicroscopic infection.\nSubmicroscopic infection was not associated with documented fever (p = 0.4) or reported fever within 48 hours prior to the visit (p = 0.2). Three women reported cough, sneezing and body pains at a visit during which submicroscopic infection was detected. Otherwise, no women reported any symptoms of illness during visits at which submicroscopic infection was detected. However, submicroscopic infection was associated with a 0.6 g/dL decrease in mean haemoglobin at the time of detection (11.5 g/dL vs 12.1 g/dL, p = 0.001). This decrease remained significant after adjusting for gestational age and repeated measurements.\nAmong women with at least one submicroscopic infection, 69% had submicroscopic infection detected at enrolment (47/68). Submicroscopic infections were detected in each trimester and at delivery. Based on estimated gestational age at enrolment, submicroscopic infection was detected in 12% (6/50) of first trimester, 6% (63/1,050) of second trimester, and 1.4% (19/1,372) of third trimester samples prior to delivery. These infections occurred in six, fifty-six, and eighteen women, respectively. Twelve women had infection in more than one trimester. After excluding the submicroscopic infections present at enrolment, the prevalence of submicroscopic infection was higher in the second trimester than the third trimester [22/650 (3.4%) versus 19/1,372 (1.4%) of samples, p = 0.003].\nNine of 308 women (3%) with peripheral samples obtained at delivery had submicroscopic infection. Two women with submicroscopic infection at delivery also had submicroscopic infection at their last antepartum visit, thus infection at delivery was not considered a new submicroscopic infection.\nSubmicroscopic infection was not associated with documented fever (p = 0.4) or reported fever within 48 hours prior to the visit (p = 0.2). Three women reported cough, sneezing and body pains at a visit during which submicroscopic infection was detected. Otherwise, no women reported any symptoms of illness during visits at which submicroscopic infection was detected. However, submicroscopic infection was associated with a 0.6 g/dL decrease in mean haemoglobin at the time of detection (11.5 g/dL vs 12.1 g/dL, p = 0.001). This decrease remained significant after adjusting for gestational age and repeated measurements.\n Impact of submicroscopic infection Women who had submicroscopic infections were more likely to have placental malaria than those without infections. After controlling for microscopically detectable infection, women with submicroscopic infection were more than seven-fold more likely to have placental malaria than women without any documented infection during pregnancy (Table \n2). Each additional episode of submicroscopic infection increased the risk of placental malaria nearly five-fold (OR 4.9, 95% CI 2.7-9.1, p < 0.001). All women with submicroscopic infection detected in peripheral blood at delivery had placental malaria. Thirty-five percent (39/110) of women who had evidence of placental malaria did not have any microscopic or submicroscopic peripheral infections detected during pregnancy or at delivery.\nAssociation of submicroscopic infection during pregnancy and placental malaria\nCI: confidence interval.\nAmong women without microscopic infection during pregnancy, submicroscopic infection detected at enrolment was more strongly associated with placental malaria than submicroscopic infection detected after enrolment. After excluding infections detected at enrolment, third trimester infections were more strongly associated with placental malaria than second trimester infections (Table \n3).\nTiming of submicroscopic infection during pregnancy and odds of placental malaria among women without microscopically detected infection\n*Four women did not have molecular data available from their enrolment visit.\nCI: confidence interval.\nSubmicroscopic infection during pregnancy, at delivery or in the placenta was not associated with adverse maternal or foetal outcomes. The overall rates of these adverse outcomes were: 18% low birth weight, 13% small for gestational age, 15% preterm delivery and 11% maternal anaemia at delivery. The prevalence of these outcomes was the same in women who did and did not have submicroscopic infection in their peripheral blood or placenta (Table \n4).\nMaternal and foetal outcomes in singleton, live births from women with only submicroscopic infection compared to women without infection during pregnancy, at delivery or in the placenta\nCI: confidence interval.\n[Hb]: haemoglobin concentration.\nWomen who had submicroscopic infections were more likely to have placental malaria than those without infections. After controlling for microscopically detectable infection, women with submicroscopic infection were more than seven-fold more likely to have placental malaria than women without any documented infection during pregnancy (Table \n2). Each additional episode of submicroscopic infection increased the risk of placental malaria nearly five-fold (OR 4.9, 95% CI 2.7-9.1, p < 0.001). All women with submicroscopic infection detected in peripheral blood at delivery had placental malaria. Thirty-five percent (39/110) of women who had evidence of placental malaria did not have any microscopic or submicroscopic peripheral infections detected during pregnancy or at delivery.\nAssociation of submicroscopic infection during pregnancy and placental malaria\nCI: confidence interval.\nAmong women without microscopic infection during pregnancy, submicroscopic infection detected at enrolment was more strongly associated with placental malaria than submicroscopic infection detected after enrolment. After excluding infections detected at enrolment, third trimester infections were more strongly associated with placental malaria than second trimester infections (Table \n3).\nTiming of submicroscopic infection during pregnancy and odds of placental malaria among women without microscopically detected infection\n*Four women did not have molecular data available from their enrolment visit.\nCI: confidence interval.\nSubmicroscopic infection during pregnancy, at delivery or in the placenta was not associated with adverse maternal or foetal outcomes. The overall rates of these adverse outcomes were: 18% low birth weight, 13% small for gestational age, 15% preterm delivery and 11% maternal anaemia at delivery. The prevalence of these outcomes was the same in women who did and did not have submicroscopic infection in their peripheral blood or placenta (Table \n4).\nMaternal and foetal outcomes in singleton, live births from women with only submicroscopic infection compared to women without infection during pregnancy, at delivery or in the placenta\nCI: confidence interval.\n[Hb]: haemoglobin concentration.\n Effect of SP-IPT on submicroscopic infections The ability of SP to clear submicroscopic infections was evaluated by examining women who had submicroscopic infection and a subsequent visit within 28 days (N = 79 visits). Women who received a dose of SP when they had a submicroscopic infection were less likely to have an infection at their next visit within 28 days than women with submicroscopic infections who did not receive SP (23.9 vs 48.5%; p = 0.02).\nThe prophylactic efficacy of SP was assessed by comparing the rates of malaria infection within 28 days in women who did and did not receive SP when they had no malaria infection. A total of 1,779 visits were included in the analysis. Women who did and did not receive SP had the same rate of parasitaemia within the next 28 days (2.0 vs 2.2%; p = 0.83) (Figure \n2).\nSP-IPT treatment of submicroscopic infection and prevention of any infection.\nThe ability of SP to clear submicroscopic infections was evaluated by examining women who had submicroscopic infection and a subsequent visit within 28 days (N = 79 visits). Women who received a dose of SP when they had a submicroscopic infection were less likely to have an infection at their next visit within 28 days than women with submicroscopic infections who did not receive SP (23.9 vs 48.5%; p = 0.02).\nThe prophylactic efficacy of SP was assessed by comparing the rates of malaria infection within 28 days in women who did and did not receive SP when they had no malaria infection. A total of 1,779 visits were included in the analysis. Women who did and did not receive SP had the same rate of parasitaemia within the next 28 days (2.0 vs 2.2%; p = 0.83) (Figure \n2).\nSP-IPT treatment of submicroscopic infection and prevention of any infection."},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"Submicroscopic malaria infection is common, occurs early in pregnancy and is associated with placental malaria. SP-IPT appears to treat some submicroscopic infections but does not prevent new infections. To effectively control pregnancy-associated malaria, new interventions are required to target women prior to their first antenatal care visit and to effectively treat and prevent all malaria infections."},"other_sections_titles":{"kind":"list like","value":["Background","Study population","Laboratory procedures","Microscopy","Haemoglobin","Placental biopsies","Molecular detection","Data analysis","Ethical considerations","Timing and clinical presentation of submicroscopic infection","Impact of submicroscopic infection","Effect of SP-IPT on submicroscopic infections","Abbreviations","Competing interests","Authors’ contributions"],"string":"[\n \"Background\",\n \"Study population\",\n \"Laboratory procedures\",\n \"Microscopy\",\n \"Haemoglobin\",\n \"Placental biopsies\",\n \"Molecular detection\",\n \"Data analysis\",\n \"Ethical considerations\",\n \"Timing and clinical presentation of submicroscopic infection\",\n \"Impact of submicroscopic infection\",\n \"Effect of SP-IPT on submicroscopic infections\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Each year, 25 million pregnant women in sub-Saharan Africa are at risk for malaria infection. Malaria during pregnancy is associated with maternal anaemia and infant low birth weight. Malaria during pregnancy is estimated to result in 100,000 infant deaths annually in Africa\n[1].\nMalaria infections during pregnancy may be identified by microscopic examination of a blood smear or may be submicroscopic, detectable only by more sensitive molecular methods. Studies in a variety of transmission settings have shown that submicroscopic infections can be up to five times more common than microscopic infections during pregnancy\n[2-7]. While the associations between microscopically detectable malaria during pregnancy and maternal anaemia at delivery and low birth weight have been supported in most studies\n[1], the impact of submicroscopic infection during pregnancy on these adverse outcomes has not been systematically assessed.\nBecause malaria infections during pregnancy are often asymptomatic, a key intervention to decrease the burden of malaria in pregnancy is intermittent preventive treatment (IPT), providing anti-malarial chemotherapy at routine intervals during pregnancy. Currently, the recommended anti-malarial chemotherapy for IPT in the majority of Africa is sulphadoxine-pyrimethamine (SP), which is intended to cure current infections and to provide a period of post-treatment prophylaxis to prevent future infections.\nIn much of sub-Saharan Africa, increasing resistance to SP\n[8] is generating concerns that SP-IPT efficacy will be compromised\n[9]. Understanding the relative importance of the treatment and prophylactic effects of SP-IPT and the differential impact of resistance on these effects may have implications for the selection of a new drug to replace SP for IPT.\nIn the context of an observational study of a large cohort of pregnant women in Malawi, the effect of submicroscopic infections on maternal and foetal outcomes was examined. All women received SP-IPT according to the national policy. At each visit a blood smear was examined and women were treated for malaria infections detected by microscopy. The primary analysis of infections detected solely by microscopy showed an association between infection at enrolment in antenatal care and placental malaria, but only infections detected at the time of delivery were associated with adverse pregnancy outcomes\n[10]. Because higher rates of submicroscopic infection than microscopically detectable infection were anticipated and submicroscopic infections were not treated, it was hypothesized that submicroscopic infections would be associated with placental malaria, maternal anaemia and infant low birth weight. It was further hypothesized that, due to high rates of SP resistance, SP-IPT would fail to clear and prevent submicroscopic infections.","Four-hundred and fifty pregnant women were enrolled in an observational cohort study of malaria during pregnancy in Blantyre, Malawi between June 2009 and June 2010. All women were in their first or second pregnancy and were less than or equal to 28 weeks gestational age based on clinical assessment at enrolment. Women were followed monthly during pregnancy and encouraged to come to the clinic if they had intercurrent illness. At each encounter, peripheral blood smears and dried blood spots on filter paper were collected. At delivery, these same specimens and placental blood and tissue samples were collected. After quickening, women received SP-IPT up to three times separated by at least four weeks. Women with malaria detectable by blood smear at routine or sick visits were treated for malaria in accordance with the national guidelines (quinine in the first trimester and artemether-lumefantrine in the second and third trimesters). Details are described by Kalilani-Phiri et al.[10]."," Microscopy Peripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\nPeripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\n Haemoglobin Haemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\nHaemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\n Placental biopsies Placental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\nPlacental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\n Molecular detection DNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\n[11].\nDNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\n[11].\n Data analysis Peripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\nPeripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\n Ethical considerations Ethical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\nEthical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.","Peripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.","Haemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.","Placental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.","DNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\n[11].","Peripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.","Ethical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.","Among women with at least one submicroscopic infection, 69% had submicroscopic infection detected at enrolment (47/68). Submicroscopic infections were detected in each trimester and at delivery. Based on estimated gestational age at enrolment, submicroscopic infection was detected in 12% (6/50) of first trimester, 6% (63/1,050) of second trimester, and 1.4% (19/1,372) of third trimester samples prior to delivery. These infections occurred in six, fifty-six, and eighteen women, respectively. Twelve women had infection in more than one trimester. After excluding the submicroscopic infections present at enrolment, the prevalence of submicroscopic infection was higher in the second trimester than the third trimester [22/650 (3.4%) versus 19/1,372 (1.4%) of samples, p = 0.003].\nNine of 308 women (3%) with peripheral samples obtained at delivery had submicroscopic infection. Two women with submicroscopic infection at delivery also had submicroscopic infection at their last antepartum visit, thus infection at delivery was not considered a new submicroscopic infection.\nSubmicroscopic infection was not associated with documented fever (p = 0.4) or reported fever within 48 hours prior to the visit (p = 0.2). Three women reported cough, sneezing and body pains at a visit during which submicroscopic infection was detected. Otherwise, no women reported any symptoms of illness during visits at which submicroscopic infection was detected. However, submicroscopic infection was associated with a 0.6 g/dL decrease in mean haemoglobin at the time of detection (11.5 g/dL vs 12.1 g/dL, p = 0.001). This decrease remained significant after adjusting for gestational age and repeated measurements.","Women who had submicroscopic infections were more likely to have placental malaria than those without infections. After controlling for microscopically detectable infection, women with submicroscopic infection were more than seven-fold more likely to have placental malaria than women without any documented infection during pregnancy (Table \n2). Each additional episode of submicroscopic infection increased the risk of placental malaria nearly five-fold (OR 4.9, 95% CI 2.7-9.1, p < 0.001). All women with submicroscopic infection detected in peripheral blood at delivery had placental malaria. Thirty-five percent (39/110) of women who had evidence of placental malaria did not have any microscopic or submicroscopic peripheral infections detected during pregnancy or at delivery.\nAssociation of submicroscopic infection during pregnancy and placental malaria\nCI: confidence interval.\nAmong women without microscopic infection during pregnancy, submicroscopic infection detected at enrolment was more strongly associated with placental malaria than submicroscopic infection detected after enrolment. After excluding infections detected at enrolment, third trimester infections were more strongly associated with placental malaria than second trimester infections (Table \n3).\nTiming of submicroscopic infection during pregnancy and odds of placental malaria among women without microscopically detected infection\n*Four women did not have molecular data available from their enrolment visit.\nCI: confidence interval.\nSubmicroscopic infection during pregnancy, at delivery or in the placenta was not associated with adverse maternal or foetal outcomes. The overall rates of these adverse outcomes were: 18% low birth weight, 13% small for gestational age, 15% preterm delivery and 11% maternal anaemia at delivery. The prevalence of these outcomes was the same in women who did and did not have submicroscopic infection in their peripheral blood or placenta (Table \n4).\nMaternal and foetal outcomes in singleton, live births from women with only submicroscopic infection compared to women without infection during pregnancy, at delivery or in the placenta\nCI: confidence interval.\n[Hb]: haemoglobin concentration.","The ability of SP to clear submicroscopic infections was evaluated by examining women who had submicroscopic infection and a subsequent visit within 28 days (N = 79 visits). Women who received a dose of SP when they had a submicroscopic infection were less likely to have an infection at their next visit within 28 days than women with submicroscopic infections who did not receive SP (23.9 vs 48.5%; p = 0.02).\nThe prophylactic efficacy of SP was assessed by comparing the rates of malaria infection within 28 days in women who did and did not receive SP when they had no malaria infection. A total of 1,779 visits were included in the analysis. Women who did and did not receive SP had the same rate of parasitaemia within the next 28 days (2.0 vs 2.2%; p = 0.83) (Figure \n2).\nSP-IPT treatment of submicroscopic infection and prevention of any infection.","IPT: Intermittent preventive treatment; SP: Sulphadoxine-pyrimethamine; SP-IPT: Intermittent preventive treatment with sulphadoxine-pyrimethamine; PCR: Polymerase chain reaction; qPCR: Quantitative polymerase chain reaction.","The authors have declared that they have no financial disclosures or competing interests.","LKP, TET and MKL conceived of and designed the field study. LKP, TET, MKL, PCT, MM, PW, and GM conducted the clinical study. LMC and MKL designed the experiments. KM, SK and AM completed the histopathology. LMC, SB, RM, SJ, and KBS conducted the experiments. LMC and MKL performed the analysis and led the writing of the manuscript. All authors read and approved the final manuscript."],"string":"[\n \"Each year, 25 million pregnant women in sub-Saharan Africa are at risk for malaria infection. Malaria during pregnancy is associated with maternal anaemia and infant low birth weight. Malaria during pregnancy is estimated to result in 100,000 infant deaths annually in Africa\\n[1].\\nMalaria infections during pregnancy may be identified by microscopic examination of a blood smear or may be submicroscopic, detectable only by more sensitive molecular methods. Studies in a variety of transmission settings have shown that submicroscopic infections can be up to five times more common than microscopic infections during pregnancy\\n[2-7]. While the associations between microscopically detectable malaria during pregnancy and maternal anaemia at delivery and low birth weight have been supported in most studies\\n[1], the impact of submicroscopic infection during pregnancy on these adverse outcomes has not been systematically assessed.\\nBecause malaria infections during pregnancy are often asymptomatic, a key intervention to decrease the burden of malaria in pregnancy is intermittent preventive treatment (IPT), providing anti-malarial chemotherapy at routine intervals during pregnancy. Currently, the recommended anti-malarial chemotherapy for IPT in the majority of Africa is sulphadoxine-pyrimethamine (SP), which is intended to cure current infections and to provide a period of post-treatment prophylaxis to prevent future infections.\\nIn much of sub-Saharan Africa, increasing resistance to SP\\n[8] is generating concerns that SP-IPT efficacy will be compromised\\n[9]. Understanding the relative importance of the treatment and prophylactic effects of SP-IPT and the differential impact of resistance on these effects may have implications for the selection of a new drug to replace SP for IPT.\\nIn the context of an observational study of a large cohort of pregnant women in Malawi, the effect of submicroscopic infections on maternal and foetal outcomes was examined. All women received SP-IPT according to the national policy. At each visit a blood smear was examined and women were treated for malaria infections detected by microscopy. The primary analysis of infections detected solely by microscopy showed an association between infection at enrolment in antenatal care and placental malaria, but only infections detected at the time of delivery were associated with adverse pregnancy outcomes\\n[10]. Because higher rates of submicroscopic infection than microscopically detectable infection were anticipated and submicroscopic infections were not treated, it was hypothesized that submicroscopic infections would be associated with placental malaria, maternal anaemia and infant low birth weight. It was further hypothesized that, due to high rates of SP resistance, SP-IPT would fail to clear and prevent submicroscopic infections.\",\n \"Four-hundred and fifty pregnant women were enrolled in an observational cohort study of malaria during pregnancy in Blantyre, Malawi between June 2009 and June 2010. All women were in their first or second pregnancy and were less than or equal to 28 weeks gestational age based on clinical assessment at enrolment. Women were followed monthly during pregnancy and encouraged to come to the clinic if they had intercurrent illness. At each encounter, peripheral blood smears and dried blood spots on filter paper were collected. At delivery, these same specimens and placental blood and tissue samples were collected. After quickening, women received SP-IPT up to three times separated by at least four weeks. Women with malaria detectable by blood smear at routine or sick visits were treated for malaria in accordance with the national guidelines (quinine in the first trimester and artemether-lumefantrine in the second and third trimesters). Details are described by Kalilani-Phiri et al.[10].\",\n \" Microscopy Peripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\\nPeripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\\n Haemoglobin Haemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\\nHaemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\\n Placental biopsies Placental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\\nPlacental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\\n Molecular detection DNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\\n[11].\\nDNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\\n[11].\\n Data analysis Peripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\\nPeripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\\n Ethical considerations Ethical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\\nEthical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\",\n \"Peripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\",\n \"Haemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\",\n \"Placental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\",\n \"DNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\\n[11].\",\n \"Peripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\",\n \"Ethical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\",\n \"Among women with at least one submicroscopic infection, 69% had submicroscopic infection detected at enrolment (47/68). Submicroscopic infections were detected in each trimester and at delivery. Based on estimated gestational age at enrolment, submicroscopic infection was detected in 12% (6/50) of first trimester, 6% (63/1,050) of second trimester, and 1.4% (19/1,372) of third trimester samples prior to delivery. These infections occurred in six, fifty-six, and eighteen women, respectively. Twelve women had infection in more than one trimester. After excluding the submicroscopic infections present at enrolment, the prevalence of submicroscopic infection was higher in the second trimester than the third trimester [22/650 (3.4%) versus 19/1,372 (1.4%) of samples, p = 0.003].\\nNine of 308 women (3%) with peripheral samples obtained at delivery had submicroscopic infection. Two women with submicroscopic infection at delivery also had submicroscopic infection at their last antepartum visit, thus infection at delivery was not considered a new submicroscopic infection.\\nSubmicroscopic infection was not associated with documented fever (p = 0.4) or reported fever within 48 hours prior to the visit (p = 0.2). Three women reported cough, sneezing and body pains at a visit during which submicroscopic infection was detected. Otherwise, no women reported any symptoms of illness during visits at which submicroscopic infection was detected. However, submicroscopic infection was associated with a 0.6 g/dL decrease in mean haemoglobin at the time of detection (11.5 g/dL vs 12.1 g/dL, p = 0.001). This decrease remained significant after adjusting for gestational age and repeated measurements.\",\n \"Women who had submicroscopic infections were more likely to have placental malaria than those without infections. After controlling for microscopically detectable infection, women with submicroscopic infection were more than seven-fold more likely to have placental malaria than women without any documented infection during pregnancy (Table \\n2). Each additional episode of submicroscopic infection increased the risk of placental malaria nearly five-fold (OR 4.9, 95% CI 2.7-9.1, p < 0.001). All women with submicroscopic infection detected in peripheral blood at delivery had placental malaria. Thirty-five percent (39/110) of women who had evidence of placental malaria did not have any microscopic or submicroscopic peripheral infections detected during pregnancy or at delivery.\\nAssociation of submicroscopic infection during pregnancy and placental malaria\\nCI: confidence interval.\\nAmong women without microscopic infection during pregnancy, submicroscopic infection detected at enrolment was more strongly associated with placental malaria than submicroscopic infection detected after enrolment. After excluding infections detected at enrolment, third trimester infections were more strongly associated with placental malaria than second trimester infections (Table \\n3).\\nTiming of submicroscopic infection during pregnancy and odds of placental malaria among women without microscopically detected infection\\n*Four women did not have molecular data available from their enrolment visit.\\nCI: confidence interval.\\nSubmicroscopic infection during pregnancy, at delivery or in the placenta was not associated with adverse maternal or foetal outcomes. The overall rates of these adverse outcomes were: 18% low birth weight, 13% small for gestational age, 15% preterm delivery and 11% maternal anaemia at delivery. The prevalence of these outcomes was the same in women who did and did not have submicroscopic infection in their peripheral blood or placenta (Table \\n4).\\nMaternal and foetal outcomes in singleton, live births from women with only submicroscopic infection compared to women without infection during pregnancy, at delivery or in the placenta\\nCI: confidence interval.\\n[Hb]: haemoglobin concentration.\",\n \"The ability of SP to clear submicroscopic infections was evaluated by examining women who had submicroscopic infection and a subsequent visit within 28 days (N = 79 visits). Women who received a dose of SP when they had a submicroscopic infection were less likely to have an infection at their next visit within 28 days than women with submicroscopic infections who did not receive SP (23.9 vs 48.5%; p = 0.02).\\nThe prophylactic efficacy of SP was assessed by comparing the rates of malaria infection within 28 days in women who did and did not receive SP when they had no malaria infection. A total of 1,779 visits were included in the analysis. Women who did and did not receive SP had the same rate of parasitaemia within the next 28 days (2.0 vs 2.2%; p = 0.83) (Figure \\n2).\\nSP-IPT treatment of submicroscopic infection and prevention of any infection.\",\n \"IPT: Intermittent preventive treatment; SP: Sulphadoxine-pyrimethamine; SP-IPT: Intermittent preventive treatment with sulphadoxine-pyrimethamine; PCR: Polymerase chain reaction; qPCR: Quantitative polymerase chain reaction.\",\n \"The authors have declared that they have no financial disclosures or competing interests.\",\n \"LKP, TET and MKL conceived of and designed the field study. LKP, TET, MKL, PCT, MM, PW, and GM conducted the clinical study. LMC and MKL designed the experiments. KM, SK and AM completed the histopathology. LMC, SB, RM, SJ, and KBS conducted the experiments. LMC and MKL performed the analysis and led the writing of the manuscript. All authors read and approved the final manuscript.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Study population","Laboratory procedures","Microscopy","Haemoglobin","Placental biopsies","Molecular detection","Data analysis","Ethical considerations","Results","Timing and clinical presentation of submicroscopic infection","Impact of submicroscopic infection","Effect of SP-IPT on submicroscopic infections","Discussion","Conclusions","Abbreviations","Competing interests","Authors’ contributions"],"string":"[\n \"Background\",\n \"Methods\",\n \"Study population\",\n \"Laboratory procedures\",\n \"Microscopy\",\n \"Haemoglobin\",\n \"Placental biopsies\",\n \"Molecular detection\",\n \"Data analysis\",\n \"Ethical considerations\",\n \"Results\",\n \"Timing and clinical presentation of submicroscopic infection\",\n \"Impact of submicroscopic infection\",\n \"Effect of SP-IPT on submicroscopic infections\",\n \"Discussion\",\n \"Conclusions\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Each year, 25 million pregnant women in sub-Saharan Africa are at risk for malaria infection. Malaria during pregnancy is associated with maternal anaemia and infant low birth weight. Malaria during pregnancy is estimated to result in 100,000 infant deaths annually in Africa\n[1].\nMalaria infections during pregnancy may be identified by microscopic examination of a blood smear or may be submicroscopic, detectable only by more sensitive molecular methods. Studies in a variety of transmission settings have shown that submicroscopic infections can be up to five times more common than microscopic infections during pregnancy\n[2-7]. While the associations between microscopically detectable malaria during pregnancy and maternal anaemia at delivery and low birth weight have been supported in most studies\n[1], the impact of submicroscopic infection during pregnancy on these adverse outcomes has not been systematically assessed.\nBecause malaria infections during pregnancy are often asymptomatic, a key intervention to decrease the burden of malaria in pregnancy is intermittent preventive treatment (IPT), providing anti-malarial chemotherapy at routine intervals during pregnancy. Currently, the recommended anti-malarial chemotherapy for IPT in the majority of Africa is sulphadoxine-pyrimethamine (SP), which is intended to cure current infections and to provide a period of post-treatment prophylaxis to prevent future infections.\nIn much of sub-Saharan Africa, increasing resistance to SP\n[8] is generating concerns that SP-IPT efficacy will be compromised\n[9]. Understanding the relative importance of the treatment and prophylactic effects of SP-IPT and the differential impact of resistance on these effects may have implications for the selection of a new drug to replace SP for IPT.\nIn the context of an observational study of a large cohort of pregnant women in Malawi, the effect of submicroscopic infections on maternal and foetal outcomes was examined. All women received SP-IPT according to the national policy. At each visit a blood smear was examined and women were treated for malaria infections detected by microscopy. The primary analysis of infections detected solely by microscopy showed an association between infection at enrolment in antenatal care and placental malaria, but only infections detected at the time of delivery were associated with adverse pregnancy outcomes\n[10]. Because higher rates of submicroscopic infection than microscopically detectable infection were anticipated and submicroscopic infections were not treated, it was hypothesized that submicroscopic infections would be associated with placental malaria, maternal anaemia and infant low birth weight. It was further hypothesized that, due to high rates of SP resistance, SP-IPT would fail to clear and prevent submicroscopic infections."," Study population Four-hundred and fifty pregnant women were enrolled in an observational cohort study of malaria during pregnancy in Blantyre, Malawi between June 2009 and June 2010. All women were in their first or second pregnancy and were less than or equal to 28 weeks gestational age based on clinical assessment at enrolment. Women were followed monthly during pregnancy and encouraged to come to the clinic if they had intercurrent illness. At each encounter, peripheral blood smears and dried blood spots on filter paper were collected. At delivery, these same specimens and placental blood and tissue samples were collected. After quickening, women received SP-IPT up to three times separated by at least four weeks. Women with malaria detectable by blood smear at routine or sick visits were treated for malaria in accordance with the national guidelines (quinine in the first trimester and artemether-lumefantrine in the second and third trimesters). Details are described by Kalilani-Phiri et al.[10].\nFour-hundred and fifty pregnant women were enrolled in an observational cohort study of malaria during pregnancy in Blantyre, Malawi between June 2009 and June 2010. All women were in their first or second pregnancy and were less than or equal to 28 weeks gestational age based on clinical assessment at enrolment. Women were followed monthly during pregnancy and encouraged to come to the clinic if they had intercurrent illness. At each encounter, peripheral blood smears and dried blood spots on filter paper were collected. At delivery, these same specimens and placental blood and tissue samples were collected. After quickening, women received SP-IPT up to three times separated by at least four weeks. Women with malaria detectable by blood smear at routine or sick visits were treated for malaria in accordance with the national guidelines (quinine in the first trimester and artemether-lumefantrine in the second and third trimesters). Details are described by Kalilani-Phiri et al.[10].\n Laboratory procedures Microscopy Peripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\nPeripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\n Haemoglobin Haemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\nHaemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\n Placental biopsies Placental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\nPlacental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\n Molecular detection DNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\n[11].\nDNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\n[11].\n Data analysis Peripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\nPeripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\n Ethical considerations Ethical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\nEthical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\n Microscopy Peripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\nPeripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\n Haemoglobin Haemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\nHaemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\n Placental biopsies Placental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\nPlacental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\n Molecular detection DNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\n[11].\nDNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\n[11].\n Data analysis Peripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\nPeripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\n Ethical considerations Ethical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\nEthical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.","Four-hundred and fifty pregnant women were enrolled in an observational cohort study of malaria during pregnancy in Blantyre, Malawi between June 2009 and June 2010. All women were in their first or second pregnancy and were less than or equal to 28 weeks gestational age based on clinical assessment at enrolment. Women were followed monthly during pregnancy and encouraged to come to the clinic if they had intercurrent illness. At each encounter, peripheral blood smears and dried blood spots on filter paper were collected. At delivery, these same specimens and placental blood and tissue samples were collected. After quickening, women received SP-IPT up to three times separated by at least four weeks. Women with malaria detectable by blood smear at routine or sick visits were treated for malaria in accordance with the national guidelines (quinine in the first trimester and artemether-lumefantrine in the second and third trimesters). Details are described by Kalilani-Phiri et al.[10]."," Microscopy Peripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\nPeripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\n Haemoglobin Haemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\nHaemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\n Placental biopsies Placental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\nPlacental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\n Molecular detection DNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\n[11].\nDNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\n[11].\n Data analysis Peripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\nPeripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\n Ethical considerations Ethical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\nEthical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.","Peripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.","Haemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.","Placental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.","DNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\n[11].","Peripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.","Ethical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.","Two thousand six hundred and eighty-one peripheral blood samples from 448 women enrolled in the study were screened for submicroscopic infection. Two women were not included in the analysis because they did not have any filter papers available for molecular analysis. Ninety-five incident submicroscopic infections were detected in 68 women. Pregnant women had 0.6 episodes of submicroscopic malaria per person-year of follow-up. As previously published, women in this cohort also experienced 0.6 episodes of microscopically detected infection per person-year of follow-up\n[10]. Thus, the overall rate of malaria infection was 1.2 episodes per person year of follow up.\nAmong women with submicroscopic infections, the mean number of infections was 1.4 (95% CI 1.2-1.6, range 1–5). Forty-nine women (72%) with submicroscopic infections never had an infection detected by microscopy during pregnancy. Submicroscopic infection was not associated with first versus second pregnancy, age, bed net use, or malaria treatment during pregnancy prior to enrolment, but was associated with enrolment at an earlier gestational age, more frequent visits, and lack of secondary school attendance (Table \n1). Gestational age at enrolment was inversely correlated with number of visits.Among 311 placentas with both histological and molecular data available for evaluation, 104 (33%) had evidence of placental malaria (Figure \n1). Thirty-three (11%) had both parasites and haemozoin detected microscopically. Three (1%) had no haemozoin but did have parasites detected by histology and confirmed by qPCR. Thirty-eight (12%) had haemozoin without histological or molecular evidence of active parasite infection. Thirty (10%) had submicroscopic infection with no histological evidence of parasites or haemozoin.\nEnrolment characteristics of women with and without submicroscopic infection detected during pregnancy irrespective of the presence of microscopic infection\nCI: confidence interval.\nFrequency of histological and molecular findings of placental malaria.\nHistological results were not available for six placentas; all had parasites detected by qPCR.\n Timing and clinical presentation of submicroscopic infection Among women with at least one submicroscopic infection, 69% had submicroscopic infection detected at enrolment (47/68). Submicroscopic infections were detected in each trimester and at delivery. Based on estimated gestational age at enrolment, submicroscopic infection was detected in 12% (6/50) of first trimester, 6% (63/1,050) of second trimester, and 1.4% (19/1,372) of third trimester samples prior to delivery. These infections occurred in six, fifty-six, and eighteen women, respectively. Twelve women had infection in more than one trimester. After excluding the submicroscopic infections present at enrolment, the prevalence of submicroscopic infection was higher in the second trimester than the third trimester [22/650 (3.4%) versus 19/1,372 (1.4%) of samples, p = 0.003].\nNine of 308 women (3%) with peripheral samples obtained at delivery had submicroscopic infection. Two women with submicroscopic infection at delivery also had submicroscopic infection at their last antepartum visit, thus infection at delivery was not considered a new submicroscopic infection.\nSubmicroscopic infection was not associated with documented fever (p = 0.4) or reported fever within 48 hours prior to the visit (p = 0.2). Three women reported cough, sneezing and body pains at a visit during which submicroscopic infection was detected. Otherwise, no women reported any symptoms of illness during visits at which submicroscopic infection was detected. However, submicroscopic infection was associated with a 0.6 g/dL decrease in mean haemoglobin at the time of detection (11.5 g/dL vs 12.1 g/dL, p = 0.001). This decrease remained significant after adjusting for gestational age and repeated measurements.\nAmong women with at least one submicroscopic infection, 69% had submicroscopic infection detected at enrolment (47/68). Submicroscopic infections were detected in each trimester and at delivery. Based on estimated gestational age at enrolment, submicroscopic infection was detected in 12% (6/50) of first trimester, 6% (63/1,050) of second trimester, and 1.4% (19/1,372) of third trimester samples prior to delivery. These infections occurred in six, fifty-six, and eighteen women, respectively. Twelve women had infection in more than one trimester. After excluding the submicroscopic infections present at enrolment, the prevalence of submicroscopic infection was higher in the second trimester than the third trimester [22/650 (3.4%) versus 19/1,372 (1.4%) of samples, p = 0.003].\nNine of 308 women (3%) with peripheral samples obtained at delivery had submicroscopic infection. Two women with submicroscopic infection at delivery also had submicroscopic infection at their last antepartum visit, thus infection at delivery was not considered a new submicroscopic infection.\nSubmicroscopic infection was not associated with documented fever (p = 0.4) or reported fever within 48 hours prior to the visit (p = 0.2). Three women reported cough, sneezing and body pains at a visit during which submicroscopic infection was detected. Otherwise, no women reported any symptoms of illness during visits at which submicroscopic infection was detected. However, submicroscopic infection was associated with a 0.6 g/dL decrease in mean haemoglobin at the time of detection (11.5 g/dL vs 12.1 g/dL, p = 0.001). This decrease remained significant after adjusting for gestational age and repeated measurements.\n Impact of submicroscopic infection Women who had submicroscopic infections were more likely to have placental malaria than those without infections. After controlling for microscopically detectable infection, women with submicroscopic infection were more than seven-fold more likely to have placental malaria than women without any documented infection during pregnancy (Table \n2). Each additional episode of submicroscopic infection increased the risk of placental malaria nearly five-fold (OR 4.9, 95% CI 2.7-9.1, p < 0.001). All women with submicroscopic infection detected in peripheral blood at delivery had placental malaria. Thirty-five percent (39/110) of women who had evidence of placental malaria did not have any microscopic or submicroscopic peripheral infections detected during pregnancy or at delivery.\nAssociation of submicroscopic infection during pregnancy and placental malaria\nCI: confidence interval.\nAmong women without microscopic infection during pregnancy, submicroscopic infection detected at enrolment was more strongly associated with placental malaria than submicroscopic infection detected after enrolment. After excluding infections detected at enrolment, third trimester infections were more strongly associated with placental malaria than second trimester infections (Table \n3).\nTiming of submicroscopic infection during pregnancy and odds of placental malaria among women without microscopically detected infection\n*Four women did not have molecular data available from their enrolment visit.\nCI: confidence interval.\nSubmicroscopic infection during pregnancy, at delivery or in the placenta was not associated with adverse maternal or foetal outcomes. The overall rates of these adverse outcomes were: 18% low birth weight, 13% small for gestational age, 15% preterm delivery and 11% maternal anaemia at delivery. The prevalence of these outcomes was the same in women who did and did not have submicroscopic infection in their peripheral blood or placenta (Table \n4).\nMaternal and foetal outcomes in singleton, live births from women with only submicroscopic infection compared to women without infection during pregnancy, at delivery or in the placenta\nCI: confidence interval.\n[Hb]: haemoglobin concentration.\nWomen who had submicroscopic infections were more likely to have placental malaria than those without infections. After controlling for microscopically detectable infection, women with submicroscopic infection were more than seven-fold more likely to have placental malaria than women without any documented infection during pregnancy (Table \n2). Each additional episode of submicroscopic infection increased the risk of placental malaria nearly five-fold (OR 4.9, 95% CI 2.7-9.1, p < 0.001). All women with submicroscopic infection detected in peripheral blood at delivery had placental malaria. Thirty-five percent (39/110) of women who had evidence of placental malaria did not have any microscopic or submicroscopic peripheral infections detected during pregnancy or at delivery.\nAssociation of submicroscopic infection during pregnancy and placental malaria\nCI: confidence interval.\nAmong women without microscopic infection during pregnancy, submicroscopic infection detected at enrolment was more strongly associated with placental malaria than submicroscopic infection detected after enrolment. After excluding infections detected at enrolment, third trimester infections were more strongly associated with placental malaria than second trimester infections (Table \n3).\nTiming of submicroscopic infection during pregnancy and odds of placental malaria among women without microscopically detected infection\n*Four women did not have molecular data available from their enrolment visit.\nCI: confidence interval.\nSubmicroscopic infection during pregnancy, at delivery or in the placenta was not associated with adverse maternal or foetal outcomes. The overall rates of these adverse outcomes were: 18% low birth weight, 13% small for gestational age, 15% preterm delivery and 11% maternal anaemia at delivery. The prevalence of these outcomes was the same in women who did and did not have submicroscopic infection in their peripheral blood or placenta (Table \n4).\nMaternal and foetal outcomes in singleton, live births from women with only submicroscopic infection compared to women without infection during pregnancy, at delivery or in the placenta\nCI: confidence interval.\n[Hb]: haemoglobin concentration.\n Effect of SP-IPT on submicroscopic infections The ability of SP to clear submicroscopic infections was evaluated by examining women who had submicroscopic infection and a subsequent visit within 28 days (N = 79 visits). Women who received a dose of SP when they had a submicroscopic infection were less likely to have an infection at their next visit within 28 days than women with submicroscopic infections who did not receive SP (23.9 vs 48.5%; p = 0.02).\nThe prophylactic efficacy of SP was assessed by comparing the rates of malaria infection within 28 days in women who did and did not receive SP when they had no malaria infection. A total of 1,779 visits were included in the analysis. Women who did and did not receive SP had the same rate of parasitaemia within the next 28 days (2.0 vs 2.2%; p = 0.83) (Figure \n2).\nSP-IPT treatment of submicroscopic infection and prevention of any infection.\nThe ability of SP to clear submicroscopic infections was evaluated by examining women who had submicroscopic infection and a subsequent visit within 28 days (N = 79 visits). Women who received a dose of SP when they had a submicroscopic infection were less likely to have an infection at their next visit within 28 days than women with submicroscopic infections who did not receive SP (23.9 vs 48.5%; p = 0.02).\nThe prophylactic efficacy of SP was assessed by comparing the rates of malaria infection within 28 days in women who did and did not receive SP when they had no malaria infection. A total of 1,779 visits were included in the analysis. Women who did and did not receive SP had the same rate of parasitaemia within the next 28 days (2.0 vs 2.2%; p = 0.83) (Figure \n2).\nSP-IPT treatment of submicroscopic infection and prevention of any infection.","Among women with at least one submicroscopic infection, 69% had submicroscopic infection detected at enrolment (47/68). Submicroscopic infections were detected in each trimester and at delivery. Based on estimated gestational age at enrolment, submicroscopic infection was detected in 12% (6/50) of first trimester, 6% (63/1,050) of second trimester, and 1.4% (19/1,372) of third trimester samples prior to delivery. These infections occurred in six, fifty-six, and eighteen women, respectively. Twelve women had infection in more than one trimester. After excluding the submicroscopic infections present at enrolment, the prevalence of submicroscopic infection was higher in the second trimester than the third trimester [22/650 (3.4%) versus 19/1,372 (1.4%) of samples, p = 0.003].\nNine of 308 women (3%) with peripheral samples obtained at delivery had submicroscopic infection. Two women with submicroscopic infection at delivery also had submicroscopic infection at their last antepartum visit, thus infection at delivery was not considered a new submicroscopic infection.\nSubmicroscopic infection was not associated with documented fever (p = 0.4) or reported fever within 48 hours prior to the visit (p = 0.2). Three women reported cough, sneezing and body pains at a visit during which submicroscopic infection was detected. Otherwise, no women reported any symptoms of illness during visits at which submicroscopic infection was detected. However, submicroscopic infection was associated with a 0.6 g/dL decrease in mean haemoglobin at the time of detection (11.5 g/dL vs 12.1 g/dL, p = 0.001). This decrease remained significant after adjusting for gestational age and repeated measurements.","Women who had submicroscopic infections were more likely to have placental malaria than those without infections. After controlling for microscopically detectable infection, women with submicroscopic infection were more than seven-fold more likely to have placental malaria than women without any documented infection during pregnancy (Table \n2). Each additional episode of submicroscopic infection increased the risk of placental malaria nearly five-fold (OR 4.9, 95% CI 2.7-9.1, p < 0.001). All women with submicroscopic infection detected in peripheral blood at delivery had placental malaria. Thirty-five percent (39/110) of women who had evidence of placental malaria did not have any microscopic or submicroscopic peripheral infections detected during pregnancy or at delivery.\nAssociation of submicroscopic infection during pregnancy and placental malaria\nCI: confidence interval.\nAmong women without microscopic infection during pregnancy, submicroscopic infection detected at enrolment was more strongly associated with placental malaria than submicroscopic infection detected after enrolment. After excluding infections detected at enrolment, third trimester infections were more strongly associated with placental malaria than second trimester infections (Table \n3).\nTiming of submicroscopic infection during pregnancy and odds of placental malaria among women without microscopically detected infection\n*Four women did not have molecular data available from their enrolment visit.\nCI: confidence interval.\nSubmicroscopic infection during pregnancy, at delivery or in the placenta was not associated with adverse maternal or foetal outcomes. The overall rates of these adverse outcomes were: 18% low birth weight, 13% small for gestational age, 15% preterm delivery and 11% maternal anaemia at delivery. The prevalence of these outcomes was the same in women who did and did not have submicroscopic infection in their peripheral blood or placenta (Table \n4).\nMaternal and foetal outcomes in singleton, live births from women with only submicroscopic infection compared to women without infection during pregnancy, at delivery or in the placenta\nCI: confidence interval.\n[Hb]: haemoglobin concentration.","The ability of SP to clear submicroscopic infections was evaluated by examining women who had submicroscopic infection and a subsequent visit within 28 days (N = 79 visits). Women who received a dose of SP when they had a submicroscopic infection were less likely to have an infection at their next visit within 28 days than women with submicroscopic infections who did not receive SP (23.9 vs 48.5%; p = 0.02).\nThe prophylactic efficacy of SP was assessed by comparing the rates of malaria infection within 28 days in women who did and did not receive SP when they had no malaria infection. A total of 1,779 visits were included in the analysis. Women who did and did not receive SP had the same rate of parasitaemia within the next 28 days (2.0 vs 2.2%; p = 0.83) (Figure \n2).\nSP-IPT treatment of submicroscopic infection and prevention of any infection.","Even with adherence to SP-IPT administration three times during pregnancy and active case detection and treatment of malaria, submicroscopic infection of the peripheral blood during pregnancy is frequent, especially at the first antenatal visit. Low-density malaria infections, detected only by molecular methods, are associated with placental malaria infection and a decrease in maternal haemoglobin levels. SP-IPT appears to treat some submicroscopic infections but does not prevent new infections. These findings have important implications for the development of further interventions for prevention of malaria pregnancy.\nThis is the first report of the effect of submicroscopic infection throughout pregnancy on maternal and foetal outcomes. Prior studies of submicroscopic malaria during pregnancy have been limited by being cross-sectional, rather than longitudinal in nature\n[2,3,6,7,12-15]. Linking antenatal infections to specific delivery and neonatal outcomes is essential to evaluating the importance of submicroscopic infections and designing strategies to optimize maternal and infant health.\nThe results are consistent with previous studies indicating that submicroscopic infection is associated with anaemia at the time of detection\n[12,15]. Two previous studies reported an association between submicroscopic infection at delivery and infant low birth weight\n[6] or maternal anaemia at the time of delivery\n[7]. However, these cross-sectional studies took place prior to, or as part of, IPT trials and did not assess previous exposure to or treatment of malaria infection during pregnancy. Thus, submicroscopic infection may have been a marker of frequent malaria exposure or lack of IPT administration prior to delivery. Due to detailed follow-up and treatment of microscopically detectable infection, the results of this study more accurately reflect the specific contribution of submicroscopic infections to clinical outcomes.\nThe lack of association between submicroscopic infection during pregnancy and adverse outcomes despite the strong association with placental malaria may have been a function of the design of the study and the clinical care the participants received. The results from the examination of the effect of microscopically detectable infections on maternal and foetal outcomes yielded similar results, as published previously\n[10]. The combination of three doses of SP-IPT and active case detection and treatment may have protected against adverse effects. It is also possible that other interventions, such as screening for anaemia or the appropriate treatment of non-malaria infections, might have improved the overall health of women, decreasing the frequency of adverse outcomes. Given the low prevalence of adverse outcomes, sample size also limited the ability to detect associations. Previous studies have demonstrated a 50% increase in rates of low birth weight\n[6] and maternal anaemia\n[7] at delivery associated with malaria during pregnancy. This study had only 15 and 7% power to detect a 50% increase in low birth weight and maternal anaemia at delivery, respectively, given the baseline rates of these outcomes in this cohort.\nThe presence of placental malaria infection in women who did not have any peripheral malaria infection detected during pregnancy was unexpected. This phenomenon may be due to transient infections that were not captured during sampling intervals. Results from this study indicate that pregnant women frequently clear submicroscopic infections spontaneously. Another source of the placental malaria infection may be infection that occurred prior to enrolment in antenatal care. The strong association between malaria at enrolment and placental infection suggests that early infection, even those that had been cleared at the time of the first antenatal visit, may have the greatest likelihood of causing placental pathology.\nThe modest efficacy of SP in clearing submicroscopic infection and the lack of prophylactic efficacy likely reflects the high level of SP resistance among malaria parasites in Malawi\n[8,16]. This is consistent with studies of SP-IPT in infants, that showed the protective effect of SP was approximately one month and the duration of this protective efficacy is inversely related to presence of in vitro markers of SP resistance\n[17,18].\nThere are several important features of this study that may limit the generalizability of the findings. Women had an average of six visits during the study and were screened with microscopy at each encounter. Thus, women received more screening and treatment when participating in the study than they would have during routine antenatal care. Despite the intensive follow-up and medical attention provided in the study, submicroscopic infection is strongly associated with placental malaria and anaemia during pregnancy. In real life settings, the association between submicroscopic malaria and adverse outcomes would likely have been more pronounced. Having few encounters during the first trimester and having samples obtained intermittently limited analysis of the timing of infection leading to placental infection. Women with submicroscopic infection did have more visits during the study, suggesting that increasing sampling frequency would increase detection of submicroscopic infection. Alternatively, women with submicroscopic infections may have symptoms leading them to present to the antenatal clinic more frequently. However, there was not an association between submicroscopic infection and fever or routine versus sick visit.\nThese results have important implications for the development of alternatives to SP-IPT for prevention of malaria pregnancy. Two possible strategies are intermittent screening and treatment or IPT with an anti-malarial more effective than SP. In one trial, intermittent screening and treatment has been shown to be equivalent to SP-IPT\n[19]. The intermittent screening and treatment arm in that study used antigen-detecting rapid diagnostic tests for screening. The parasite density threshold for detection for rapid diagnostic tests, like that for microscopy, is ten- to 100-fold higher than for qPCR\n[20,21]. Rapid diagnostic tests, like microscopy, will fail to detect infection at the submicroscopic level. Thus, a rapid diagnostic test-based screening and treatment strategy will fail to detect infections that this study demonstrates are associated with placental malaria. Within the context of the usual antenatal package of four antenatal visits in pregnancy if IPT is not provided, many microscopic and all submicroscopic infections will not be treated. Under these conditions, with prolonged and untreated episodes of low-density malaria infection, it is expected that the adverse effects of placental malaria would occur\n[22-24].\nIf IPT with a more effective anti-malarial is considered, for example as in ongoing trials of IPT with dihydroartemisinin-piperaquine\n[25], azithromycin combined with chloroquine\n[26] and mefloquine\n[27], it will be important to characterize the treatment and prophylactic effect of candidate drugs. Most drug efficacy trials examine the ability of a drug to cure the infection at the time of drug administration. However, IPT is most beneficial in high transmission settings if it cures current infections and prevents subsequent infections. The results of this study indicate that the loss of prophylactic efficacy may precede the loss of curative efficacy. Thus, in trials of alternative anti-malarials for IPT, prophylactic efficacy should be included as an outcome measure.\nThe finding that the highest rates of asymptomatic and submicroscopic malaria are detected in women at their first antenatal care visit highlights an essential shortcoming of the current policies to prevent pregnancy-associated malaria. Interventions such as the distribution of bed nets at antenatal clinics and the administration of IPT are delayed until women seek antenatal care. In Malawi, as in other countries in sub-Saharan Africa, this is typically late in the second or into the third trimester\n[28]. By the time women have their first antenatal visit, peripheral and placental infection has already been established. Innovative interventions that can target women early in pregnancy, or even prior to pregnancy, are urgently needed to further decrease the burden of malaria during pregnancy.","Submicroscopic malaria infection is common, occurs early in pregnancy and is associated with placental malaria. SP-IPT appears to treat some submicroscopic infections but does not prevent new infections. To effectively control pregnancy-associated malaria, new interventions are required to target women prior to their first antenatal care visit and to effectively treat and prevent all malaria infections.","IPT: Intermittent preventive treatment; SP: Sulphadoxine-pyrimethamine; SP-IPT: Intermittent preventive treatment with sulphadoxine-pyrimethamine; PCR: Polymerase chain reaction; qPCR: Quantitative polymerase chain reaction.","The authors have declared that they have no financial disclosures or competing interests.","LKP, TET and MKL conceived of and designed the field study. LKP, TET, MKL, PCT, MM, PW, and GM conducted the clinical study. LMC and MKL designed the experiments. KM, SK and AM completed the histopathology. LMC, SB, RM, SJ, and KBS conducted the experiments. LMC and MKL performed the analysis and led the writing of the manuscript. All authors read and approved the final manuscript."],"string":"[\n \"Each year, 25 million pregnant women in sub-Saharan Africa are at risk for malaria infection. Malaria during pregnancy is associated with maternal anaemia and infant low birth weight. Malaria during pregnancy is estimated to result in 100,000 infant deaths annually in Africa\\n[1].\\nMalaria infections during pregnancy may be identified by microscopic examination of a blood smear or may be submicroscopic, detectable only by more sensitive molecular methods. Studies in a variety of transmission settings have shown that submicroscopic infections can be up to five times more common than microscopic infections during pregnancy\\n[2-7]. While the associations between microscopically detectable malaria during pregnancy and maternal anaemia at delivery and low birth weight have been supported in most studies\\n[1], the impact of submicroscopic infection during pregnancy on these adverse outcomes has not been systematically assessed.\\nBecause malaria infections during pregnancy are often asymptomatic, a key intervention to decrease the burden of malaria in pregnancy is intermittent preventive treatment (IPT), providing anti-malarial chemotherapy at routine intervals during pregnancy. Currently, the recommended anti-malarial chemotherapy for IPT in the majority of Africa is sulphadoxine-pyrimethamine (SP), which is intended to cure current infections and to provide a period of post-treatment prophylaxis to prevent future infections.\\nIn much of sub-Saharan Africa, increasing resistance to SP\\n[8] is generating concerns that SP-IPT efficacy will be compromised\\n[9]. Understanding the relative importance of the treatment and prophylactic effects of SP-IPT and the differential impact of resistance on these effects may have implications for the selection of a new drug to replace SP for IPT.\\nIn the context of an observational study of a large cohort of pregnant women in Malawi, the effect of submicroscopic infections on maternal and foetal outcomes was examined. All women received SP-IPT according to the national policy. At each visit a blood smear was examined and women were treated for malaria infections detected by microscopy. The primary analysis of infections detected solely by microscopy showed an association between infection at enrolment in antenatal care and placental malaria, but only infections detected at the time of delivery were associated with adverse pregnancy outcomes\\n[10]. Because higher rates of submicroscopic infection than microscopically detectable infection were anticipated and submicroscopic infections were not treated, it was hypothesized that submicroscopic infections would be associated with placental malaria, maternal anaemia and infant low birth weight. It was further hypothesized that, due to high rates of SP resistance, SP-IPT would fail to clear and prevent submicroscopic infections.\",\n \" Study population Four-hundred and fifty pregnant women were enrolled in an observational cohort study of malaria during pregnancy in Blantyre, Malawi between June 2009 and June 2010. All women were in their first or second pregnancy and were less than or equal to 28 weeks gestational age based on clinical assessment at enrolment. Women were followed monthly during pregnancy and encouraged to come to the clinic if they had intercurrent illness. At each encounter, peripheral blood smears and dried blood spots on filter paper were collected. At delivery, these same specimens and placental blood and tissue samples were collected. After quickening, women received SP-IPT up to three times separated by at least four weeks. Women with malaria detectable by blood smear at routine or sick visits were treated for malaria in accordance with the national guidelines (quinine in the first trimester and artemether-lumefantrine in the second and third trimesters). Details are described by Kalilani-Phiri et al.[10].\\nFour-hundred and fifty pregnant women were enrolled in an observational cohort study of malaria during pregnancy in Blantyre, Malawi between June 2009 and June 2010. All women were in their first or second pregnancy and were less than or equal to 28 weeks gestational age based on clinical assessment at enrolment. Women were followed monthly during pregnancy and encouraged to come to the clinic if they had intercurrent illness. At each encounter, peripheral blood smears and dried blood spots on filter paper were collected. At delivery, these same specimens and placental blood and tissue samples were collected. After quickening, women received SP-IPT up to three times separated by at least four weeks. Women with malaria detectable by blood smear at routine or sick visits were treated for malaria in accordance with the national guidelines (quinine in the first trimester and artemether-lumefantrine in the second and third trimesters). Details are described by Kalilani-Phiri et al.[10].\\n Laboratory procedures Microscopy Peripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\\nPeripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\\n Haemoglobin Haemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\\nHaemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\\n Placental biopsies Placental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\\nPlacental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\\n Molecular detection DNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\\n[11].\\nDNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\\n[11].\\n Data analysis Peripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\\nPeripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\\n Ethical considerations Ethical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\\nEthical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\\n Microscopy Peripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\\nPeripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\\n Haemoglobin Haemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\\nHaemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\\n Placental biopsies Placental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\\nPlacental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\\n Molecular detection DNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\\n[11].\\nDNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\\n[11].\\n Data analysis Peripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\\nPeripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\\n Ethical considerations Ethical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\\nEthical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\",\n \"Four-hundred and fifty pregnant women were enrolled in an observational cohort study of malaria during pregnancy in Blantyre, Malawi between June 2009 and June 2010. All women were in their first or second pregnancy and were less than or equal to 28 weeks gestational age based on clinical assessment at enrolment. Women were followed monthly during pregnancy and encouraged to come to the clinic if they had intercurrent illness. At each encounter, peripheral blood smears and dried blood spots on filter paper were collected. At delivery, these same specimens and placental blood and tissue samples were collected. After quickening, women received SP-IPT up to three times separated by at least four weeks. Women with malaria detectable by blood smear at routine or sick visits were treated for malaria in accordance with the national guidelines (quinine in the first trimester and artemether-lumefantrine in the second and third trimesters). Details are described by Kalilani-Phiri et al.[10].\",\n \" Microscopy Peripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\\nPeripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\\n Haemoglobin Haemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\\nHaemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\\n Placental biopsies Placental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\\nPlacental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\\n Molecular detection DNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\\n[11].\\nDNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\\n[11].\\n Data analysis Peripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\\nPeripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\\n Ethical considerations Ethical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\\nEthical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\",\n \"Peripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\",\n \"Haemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\",\n \"Placental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\",\n \"DNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\\n[11].\",\n \"Peripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\",\n \"Ethical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\",\n \"Two thousand six hundred and eighty-one peripheral blood samples from 448 women enrolled in the study were screened for submicroscopic infection. Two women were not included in the analysis because they did not have any filter papers available for molecular analysis. Ninety-five incident submicroscopic infections were detected in 68 women. Pregnant women had 0.6 episodes of submicroscopic malaria per person-year of follow-up. As previously published, women in this cohort also experienced 0.6 episodes of microscopically detected infection per person-year of follow-up\\n[10]. Thus, the overall rate of malaria infection was 1.2 episodes per person year of follow up.\\nAmong women with submicroscopic infections, the mean number of infections was 1.4 (95% CI 1.2-1.6, range 1–5). Forty-nine women (72%) with submicroscopic infections never had an infection detected by microscopy during pregnancy. Submicroscopic infection was not associated with first versus second pregnancy, age, bed net use, or malaria treatment during pregnancy prior to enrolment, but was associated with enrolment at an earlier gestational age, more frequent visits, and lack of secondary school attendance (Table \\n1). Gestational age at enrolment was inversely correlated with number of visits.Among 311 placentas with both histological and molecular data available for evaluation, 104 (33%) had evidence of placental malaria (Figure \\n1). Thirty-three (11%) had both parasites and haemozoin detected microscopically. Three (1%) had no haemozoin but did have parasites detected by histology and confirmed by qPCR. Thirty-eight (12%) had haemozoin without histological or molecular evidence of active parasite infection. Thirty (10%) had submicroscopic infection with no histological evidence of parasites or haemozoin.\\nEnrolment characteristics of women with and without submicroscopic infection detected during pregnancy irrespective of the presence of microscopic infection\\nCI: confidence interval.\\nFrequency of histological and molecular findings of placental malaria.\\nHistological results were not available for six placentas; all had parasites detected by qPCR.\\n Timing and clinical presentation of submicroscopic infection Among women with at least one submicroscopic infection, 69% had submicroscopic infection detected at enrolment (47/68). Submicroscopic infections were detected in each trimester and at delivery. Based on estimated gestational age at enrolment, submicroscopic infection was detected in 12% (6/50) of first trimester, 6% (63/1,050) of second trimester, and 1.4% (19/1,372) of third trimester samples prior to delivery. These infections occurred in six, fifty-six, and eighteen women, respectively. Twelve women had infection in more than one trimester. After excluding the submicroscopic infections present at enrolment, the prevalence of submicroscopic infection was higher in the second trimester than the third trimester [22/650 (3.4%) versus 19/1,372 (1.4%) of samples, p = 0.003].\\nNine of 308 women (3%) with peripheral samples obtained at delivery had submicroscopic infection. Two women with submicroscopic infection at delivery also had submicroscopic infection at their last antepartum visit, thus infection at delivery was not considered a new submicroscopic infection.\\nSubmicroscopic infection was not associated with documented fever (p = 0.4) or reported fever within 48 hours prior to the visit (p = 0.2). Three women reported cough, sneezing and body pains at a visit during which submicroscopic infection was detected. Otherwise, no women reported any symptoms of illness during visits at which submicroscopic infection was detected. However, submicroscopic infection was associated with a 0.6 g/dL decrease in mean haemoglobin at the time of detection (11.5 g/dL vs 12.1 g/dL, p = 0.001). This decrease remained significant after adjusting for gestational age and repeated measurements.\\nAmong women with at least one submicroscopic infection, 69% had submicroscopic infection detected at enrolment (47/68). Submicroscopic infections were detected in each trimester and at delivery. Based on estimated gestational age at enrolment, submicroscopic infection was detected in 12% (6/50) of first trimester, 6% (63/1,050) of second trimester, and 1.4% (19/1,372) of third trimester samples prior to delivery. These infections occurred in six, fifty-six, and eighteen women, respectively. Twelve women had infection in more than one trimester. After excluding the submicroscopic infections present at enrolment, the prevalence of submicroscopic infection was higher in the second trimester than the third trimester [22/650 (3.4%) versus 19/1,372 (1.4%) of samples, p = 0.003].\\nNine of 308 women (3%) with peripheral samples obtained at delivery had submicroscopic infection. Two women with submicroscopic infection at delivery also had submicroscopic infection at their last antepartum visit, thus infection at delivery was not considered a new submicroscopic infection.\\nSubmicroscopic infection was not associated with documented fever (p = 0.4) or reported fever within 48 hours prior to the visit (p = 0.2). Three women reported cough, sneezing and body pains at a visit during which submicroscopic infection was detected. Otherwise, no women reported any symptoms of illness during visits at which submicroscopic infection was detected. However, submicroscopic infection was associated with a 0.6 g/dL decrease in mean haemoglobin at the time of detection (11.5 g/dL vs 12.1 g/dL, p = 0.001). This decrease remained significant after adjusting for gestational age and repeated measurements.\\n Impact of submicroscopic infection Women who had submicroscopic infections were more likely to have placental malaria than those without infections. After controlling for microscopically detectable infection, women with submicroscopic infection were more than seven-fold more likely to have placental malaria than women without any documented infection during pregnancy (Table \\n2). Each additional episode of submicroscopic infection increased the risk of placental malaria nearly five-fold (OR 4.9, 95% CI 2.7-9.1, p < 0.001). All women with submicroscopic infection detected in peripheral blood at delivery had placental malaria. Thirty-five percent (39/110) of women who had evidence of placental malaria did not have any microscopic or submicroscopic peripheral infections detected during pregnancy or at delivery.\\nAssociation of submicroscopic infection during pregnancy and placental malaria\\nCI: confidence interval.\\nAmong women without microscopic infection during pregnancy, submicroscopic infection detected at enrolment was more strongly associated with placental malaria than submicroscopic infection detected after enrolment. After excluding infections detected at enrolment, third trimester infections were more strongly associated with placental malaria than second trimester infections (Table \\n3).\\nTiming of submicroscopic infection during pregnancy and odds of placental malaria among women without microscopically detected infection\\n*Four women did not have molecular data available from their enrolment visit.\\nCI: confidence interval.\\nSubmicroscopic infection during pregnancy, at delivery or in the placenta was not associated with adverse maternal or foetal outcomes. The overall rates of these adverse outcomes were: 18% low birth weight, 13% small for gestational age, 15% preterm delivery and 11% maternal anaemia at delivery. The prevalence of these outcomes was the same in women who did and did not have submicroscopic infection in their peripheral blood or placenta (Table \\n4).\\nMaternal and foetal outcomes in singleton, live births from women with only submicroscopic infection compared to women without infection during pregnancy, at delivery or in the placenta\\nCI: confidence interval.\\n[Hb]: haemoglobin concentration.\\nWomen who had submicroscopic infections were more likely to have placental malaria than those without infections. After controlling for microscopically detectable infection, women with submicroscopic infection were more than seven-fold more likely to have placental malaria than women without any documented infection during pregnancy (Table \\n2). Each additional episode of submicroscopic infection increased the risk of placental malaria nearly five-fold (OR 4.9, 95% CI 2.7-9.1, p < 0.001). All women with submicroscopic infection detected in peripheral blood at delivery had placental malaria. Thirty-five percent (39/110) of women who had evidence of placental malaria did not have any microscopic or submicroscopic peripheral infections detected during pregnancy or at delivery.\\nAssociation of submicroscopic infection during pregnancy and placental malaria\\nCI: confidence interval.\\nAmong women without microscopic infection during pregnancy, submicroscopic infection detected at enrolment was more strongly associated with placental malaria than submicroscopic infection detected after enrolment. After excluding infections detected at enrolment, third trimester infections were more strongly associated with placental malaria than second trimester infections (Table \\n3).\\nTiming of submicroscopic infection during pregnancy and odds of placental malaria among women without microscopically detected infection\\n*Four women did not have molecular data available from their enrolment visit.\\nCI: confidence interval.\\nSubmicroscopic infection during pregnancy, at delivery or in the placenta was not associated with adverse maternal or foetal outcomes. The overall rates of these adverse outcomes were: 18% low birth weight, 13% small for gestational age, 15% preterm delivery and 11% maternal anaemia at delivery. The prevalence of these outcomes was the same in women who did and did not have submicroscopic infection in their peripheral blood or placenta (Table \\n4).\\nMaternal and foetal outcomes in singleton, live births from women with only submicroscopic infection compared to women without infection during pregnancy, at delivery or in the placenta\\nCI: confidence interval.\\n[Hb]: haemoglobin concentration.\\n Effect of SP-IPT on submicroscopic infections The ability of SP to clear submicroscopic infections was evaluated by examining women who had submicroscopic infection and a subsequent visit within 28 days (N = 79 visits). Women who received a dose of SP when they had a submicroscopic infection were less likely to have an infection at their next visit within 28 days than women with submicroscopic infections who did not receive SP (23.9 vs 48.5%; p = 0.02).\\nThe prophylactic efficacy of SP was assessed by comparing the rates of malaria infection within 28 days in women who did and did not receive SP when they had no malaria infection. A total of 1,779 visits were included in the analysis. Women who did and did not receive SP had the same rate of parasitaemia within the next 28 days (2.0 vs 2.2%; p = 0.83) (Figure \\n2).\\nSP-IPT treatment of submicroscopic infection and prevention of any infection.\\nThe ability of SP to clear submicroscopic infections was evaluated by examining women who had submicroscopic infection and a subsequent visit within 28 days (N = 79 visits). Women who received a dose of SP when they had a submicroscopic infection were less likely to have an infection at their next visit within 28 days than women with submicroscopic infections who did not receive SP (23.9 vs 48.5%; p = 0.02).\\nThe prophylactic efficacy of SP was assessed by comparing the rates of malaria infection within 28 days in women who did and did not receive SP when they had no malaria infection. A total of 1,779 visits were included in the analysis. Women who did and did not receive SP had the same rate of parasitaemia within the next 28 days (2.0 vs 2.2%; p = 0.83) (Figure \\n2).\\nSP-IPT treatment of submicroscopic infection and prevention of any infection.\",\n \"Among women with at least one submicroscopic infection, 69% had submicroscopic infection detected at enrolment (47/68). Submicroscopic infections were detected in each trimester and at delivery. Based on estimated gestational age at enrolment, submicroscopic infection was detected in 12% (6/50) of first trimester, 6% (63/1,050) of second trimester, and 1.4% (19/1,372) of third trimester samples prior to delivery. These infections occurred in six, fifty-six, and eighteen women, respectively. Twelve women had infection in more than one trimester. After excluding the submicroscopic infections present at enrolment, the prevalence of submicroscopic infection was higher in the second trimester than the third trimester [22/650 (3.4%) versus 19/1,372 (1.4%) of samples, p = 0.003].\\nNine of 308 women (3%) with peripheral samples obtained at delivery had submicroscopic infection. Two women with submicroscopic infection at delivery also had submicroscopic infection at their last antepartum visit, thus infection at delivery was not considered a new submicroscopic infection.\\nSubmicroscopic infection was not associated with documented fever (p = 0.4) or reported fever within 48 hours prior to the visit (p = 0.2). Three women reported cough, sneezing and body pains at a visit during which submicroscopic infection was detected. Otherwise, no women reported any symptoms of illness during visits at which submicroscopic infection was detected. However, submicroscopic infection was associated with a 0.6 g/dL decrease in mean haemoglobin at the time of detection (11.5 g/dL vs 12.1 g/dL, p = 0.001). This decrease remained significant after adjusting for gestational age and repeated measurements.\",\n \"Women who had submicroscopic infections were more likely to have placental malaria than those without infections. After controlling for microscopically detectable infection, women with submicroscopic infection were more than seven-fold more likely to have placental malaria than women without any documented infection during pregnancy (Table \\n2). Each additional episode of submicroscopic infection increased the risk of placental malaria nearly five-fold (OR 4.9, 95% CI 2.7-9.1, p < 0.001). All women with submicroscopic infection detected in peripheral blood at delivery had placental malaria. Thirty-five percent (39/110) of women who had evidence of placental malaria did not have any microscopic or submicroscopic peripheral infections detected during pregnancy or at delivery.\\nAssociation of submicroscopic infection during pregnancy and placental malaria\\nCI: confidence interval.\\nAmong women without microscopic infection during pregnancy, submicroscopic infection detected at enrolment was more strongly associated with placental malaria than submicroscopic infection detected after enrolment. After excluding infections detected at enrolment, third trimester infections were more strongly associated with placental malaria than second trimester infections (Table \\n3).\\nTiming of submicroscopic infection during pregnancy and odds of placental malaria among women without microscopically detected infection\\n*Four women did not have molecular data available from their enrolment visit.\\nCI: confidence interval.\\nSubmicroscopic infection during pregnancy, at delivery or in the placenta was not associated with adverse maternal or foetal outcomes. The overall rates of these adverse outcomes were: 18% low birth weight, 13% small for gestational age, 15% preterm delivery and 11% maternal anaemia at delivery. The prevalence of these outcomes was the same in women who did and did not have submicroscopic infection in their peripheral blood or placenta (Table \\n4).\\nMaternal and foetal outcomes in singleton, live births from women with only submicroscopic infection compared to women without infection during pregnancy, at delivery or in the placenta\\nCI: confidence interval.\\n[Hb]: haemoglobin concentration.\",\n \"The ability of SP to clear submicroscopic infections was evaluated by examining women who had submicroscopic infection and a subsequent visit within 28 days (N = 79 visits). Women who received a dose of SP when they had a submicroscopic infection were less likely to have an infection at their next visit within 28 days than women with submicroscopic infections who did not receive SP (23.9 vs 48.5%; p = 0.02).\\nThe prophylactic efficacy of SP was assessed by comparing the rates of malaria infection within 28 days in women who did and did not receive SP when they had no malaria infection. A total of 1,779 visits were included in the analysis. Women who did and did not receive SP had the same rate of parasitaemia within the next 28 days (2.0 vs 2.2%; p = 0.83) (Figure \\n2).\\nSP-IPT treatment of submicroscopic infection and prevention of any infection.\",\n \"Even with adherence to SP-IPT administration three times during pregnancy and active case detection and treatment of malaria, submicroscopic infection of the peripheral blood during pregnancy is frequent, especially at the first antenatal visit. Low-density malaria infections, detected only by molecular methods, are associated with placental malaria infection and a decrease in maternal haemoglobin levels. SP-IPT appears to treat some submicroscopic infections but does not prevent new infections. These findings have important implications for the development of further interventions for prevention of malaria pregnancy.\\nThis is the first report of the effect of submicroscopic infection throughout pregnancy on maternal and foetal outcomes. Prior studies of submicroscopic malaria during pregnancy have been limited by being cross-sectional, rather than longitudinal in nature\\n[2,3,6,7,12-15]. Linking antenatal infections to specific delivery and neonatal outcomes is essential to evaluating the importance of submicroscopic infections and designing strategies to optimize maternal and infant health.\\nThe results are consistent with previous studies indicating that submicroscopic infection is associated with anaemia at the time of detection\\n[12,15]. Two previous studies reported an association between submicroscopic infection at delivery and infant low birth weight\\n[6] or maternal anaemia at the time of delivery\\n[7]. However, these cross-sectional studies took place prior to, or as part of, IPT trials and did not assess previous exposure to or treatment of malaria infection during pregnancy. Thus, submicroscopic infection may have been a marker of frequent malaria exposure or lack of IPT administration prior to delivery. Due to detailed follow-up and treatment of microscopically detectable infection, the results of this study more accurately reflect the specific contribution of submicroscopic infections to clinical outcomes.\\nThe lack of association between submicroscopic infection during pregnancy and adverse outcomes despite the strong association with placental malaria may have been a function of the design of the study and the clinical care the participants received. The results from the examination of the effect of microscopically detectable infections on maternal and foetal outcomes yielded similar results, as published previously\\n[10]. The combination of three doses of SP-IPT and active case detection and treatment may have protected against adverse effects. It is also possible that other interventions, such as screening for anaemia or the appropriate treatment of non-malaria infections, might have improved the overall health of women, decreasing the frequency of adverse outcomes. Given the low prevalence of adverse outcomes, sample size also limited the ability to detect associations. Previous studies have demonstrated a 50% increase in rates of low birth weight\\n[6] and maternal anaemia\\n[7] at delivery associated with malaria during pregnancy. This study had only 15 and 7% power to detect a 50% increase in low birth weight and maternal anaemia at delivery, respectively, given the baseline rates of these outcomes in this cohort.\\nThe presence of placental malaria infection in women who did not have any peripheral malaria infection detected during pregnancy was unexpected. This phenomenon may be due to transient infections that were not captured during sampling intervals. Results from this study indicate that pregnant women frequently clear submicroscopic infections spontaneously. Another source of the placental malaria infection may be infection that occurred prior to enrolment in antenatal care. The strong association between malaria at enrolment and placental infection suggests that early infection, even those that had been cleared at the time of the first antenatal visit, may have the greatest likelihood of causing placental pathology.\\nThe modest efficacy of SP in clearing submicroscopic infection and the lack of prophylactic efficacy likely reflects the high level of SP resistance among malaria parasites in Malawi\\n[8,16]. This is consistent with studies of SP-IPT in infants, that showed the protective effect of SP was approximately one month and the duration of this protective efficacy is inversely related to presence of in vitro markers of SP resistance\\n[17,18].\\nThere are several important features of this study that may limit the generalizability of the findings. Women had an average of six visits during the study and were screened with microscopy at each encounter. Thus, women received more screening and treatment when participating in the study than they would have during routine antenatal care. Despite the intensive follow-up and medical attention provided in the study, submicroscopic infection is strongly associated with placental malaria and anaemia during pregnancy. In real life settings, the association between submicroscopic malaria and adverse outcomes would likely have been more pronounced. Having few encounters during the first trimester and having samples obtained intermittently limited analysis of the timing of infection leading to placental infection. Women with submicroscopic infection did have more visits during the study, suggesting that increasing sampling frequency would increase detection of submicroscopic infection. Alternatively, women with submicroscopic infections may have symptoms leading them to present to the antenatal clinic more frequently. However, there was not an association between submicroscopic infection and fever or routine versus sick visit.\\nThese results have important implications for the development of alternatives to SP-IPT for prevention of malaria pregnancy. Two possible strategies are intermittent screening and treatment or IPT with an anti-malarial more effective than SP. In one trial, intermittent screening and treatment has been shown to be equivalent to SP-IPT\\n[19]. The intermittent screening and treatment arm in that study used antigen-detecting rapid diagnostic tests for screening. The parasite density threshold for detection for rapid diagnostic tests, like that for microscopy, is ten- to 100-fold higher than for qPCR\\n[20,21]. Rapid diagnostic tests, like microscopy, will fail to detect infection at the submicroscopic level. Thus, a rapid diagnostic test-based screening and treatment strategy will fail to detect infections that this study demonstrates are associated with placental malaria. Within the context of the usual antenatal package of four antenatal visits in pregnancy if IPT is not provided, many microscopic and all submicroscopic infections will not be treated. Under these conditions, with prolonged and untreated episodes of low-density malaria infection, it is expected that the adverse effects of placental malaria would occur\\n[22-24].\\nIf IPT with a more effective anti-malarial is considered, for example as in ongoing trials of IPT with dihydroartemisinin-piperaquine\\n[25], azithromycin combined with chloroquine\\n[26] and mefloquine\\n[27], it will be important to characterize the treatment and prophylactic effect of candidate drugs. Most drug efficacy trials examine the ability of a drug to cure the infection at the time of drug administration. However, IPT is most beneficial in high transmission settings if it cures current infections and prevents subsequent infections. The results of this study indicate that the loss of prophylactic efficacy may precede the loss of curative efficacy. Thus, in trials of alternative anti-malarials for IPT, prophylactic efficacy should be included as an outcome measure.\\nThe finding that the highest rates of asymptomatic and submicroscopic malaria are detected in women at their first antenatal care visit highlights an essential shortcoming of the current policies to prevent pregnancy-associated malaria. Interventions such as the distribution of bed nets at antenatal clinics and the administration of IPT are delayed until women seek antenatal care. In Malawi, as in other countries in sub-Saharan Africa, this is typically late in the second or into the third trimester\\n[28]. By the time women have their first antenatal visit, peripheral and placental infection has already been established. Innovative interventions that can target women early in pregnancy, or even prior to pregnancy, are urgently needed to further decrease the burden of malaria during pregnancy.\",\n \"Submicroscopic malaria infection is common, occurs early in pregnancy and is associated with placental malaria. SP-IPT appears to treat some submicroscopic infections but does not prevent new infections. To effectively control pregnancy-associated malaria, new interventions are required to target women prior to their first antenatal care visit and to effectively treat and prevent all malaria infections.\",\n \"IPT: Intermittent preventive treatment; SP: Sulphadoxine-pyrimethamine; SP-IPT: Intermittent preventive treatment with sulphadoxine-pyrimethamine; PCR: Polymerase chain reaction; qPCR: Quantitative polymerase chain reaction.\",\n \"The authors have declared that they have no financial disclosures or competing interests.\",\n \"LKP, TET and MKL conceived of and designed the field study. LKP, TET, MKL, PCT, MM, PW, and GM conducted the clinical study. LMC and MKL designed the experiments. KM, SK and AM completed the histopathology. LMC, SB, RM, SJ, and KBS conducted the experiments. LMC and MKL performed the analysis and led the writing of the manuscript. All authors read and approved the final manuscript.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,null,null,null,"results",null,null,null,"discussion","conclusions",null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\",\n null,\n null,\n null\n]","isConversational":false},"keywords":{"kind":"list like","value":["Malaria in pregnancy","Submicroscopic infection","Placental malaria","Sulphadoxine-pyrimethamine intermittent preventive treatment","Sulphadoxine-pyrimethamine resistance"],"string":"[\n \"Malaria in pregnancy\",\n \"Submicroscopic infection\",\n \"Placental malaria\",\n \"Sulphadoxine-pyrimethamine intermittent preventive treatment\",\n \"Sulphadoxine-pyrimethamine resistance\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Background:\nEach year, 25 million pregnant women in sub-Saharan Africa are at risk for malaria infection. Malaria during pregnancy is associated with maternal anaemia and infant low birth weight. Malaria during pregnancy is estimated to result in 100,000 infant deaths annually in Africa\n[1].\nMalaria infections during pregnancy may be identified by microscopic examination of a blood smear or may be submicroscopic, detectable only by more sensitive molecular methods. Studies in a variety of transmission settings have shown that submicroscopic infections can be up to five times more common than microscopic infections during pregnancy\n[2-7]. While the associations between microscopically detectable malaria during pregnancy and maternal anaemia at delivery and low birth weight have been supported in most studies\n[1], the impact of submicroscopic infection during pregnancy on these adverse outcomes has not been systematically assessed.\nBecause malaria infections during pregnancy are often asymptomatic, a key intervention to decrease the burden of malaria in pregnancy is intermittent preventive treatment (IPT), providing anti-malarial chemotherapy at routine intervals during pregnancy. Currently, the recommended anti-malarial chemotherapy for IPT in the majority of Africa is sulphadoxine-pyrimethamine (SP), which is intended to cure current infections and to provide a period of post-treatment prophylaxis to prevent future infections.\nIn much of sub-Saharan Africa, increasing resistance to SP\n[8] is generating concerns that SP-IPT efficacy will be compromised\n[9]. Understanding the relative importance of the treatment and prophylactic effects of SP-IPT and the differential impact of resistance on these effects may have implications for the selection of a new drug to replace SP for IPT.\nIn the context of an observational study of a large cohort of pregnant women in Malawi, the effect of submicroscopic infections on maternal and foetal outcomes was examined. All women received SP-IPT according to the national policy. At each visit a blood smear was examined and women were treated for malaria infections detected by microscopy. The primary analysis of infections detected solely by microscopy showed an association between infection at enrolment in antenatal care and placental malaria, but only infections detected at the time of delivery were associated with adverse pregnancy outcomes\n[10]. Because higher rates of submicroscopic infection than microscopically detectable infection were anticipated and submicroscopic infections were not treated, it was hypothesized that submicroscopic infections would be associated with placental malaria, maternal anaemia and infant low birth weight. It was further hypothesized that, due to high rates of SP resistance, SP-IPT would fail to clear and prevent submicroscopic infections.\n\nMethods:\n Study population Four-hundred and fifty pregnant women were enrolled in an observational cohort study of malaria during pregnancy in Blantyre, Malawi between June 2009 and June 2010. All women were in their first or second pregnancy and were less than or equal to 28 weeks gestational age based on clinical assessment at enrolment. Women were followed monthly during pregnancy and encouraged to come to the clinic if they had intercurrent illness. At each encounter, peripheral blood smears and dried blood spots on filter paper were collected. At delivery, these same specimens and placental blood and tissue samples were collected. After quickening, women received SP-IPT up to three times separated by at least four weeks. Women with malaria detectable by blood smear at routine or sick visits were treated for malaria in accordance with the national guidelines (quinine in the first trimester and artemether-lumefantrine in the second and third trimesters). Details are described by Kalilani-Phiri et al.[10].\nFour-hundred and fifty pregnant women were enrolled in an observational cohort study of malaria during pregnancy in Blantyre, Malawi between June 2009 and June 2010. All women were in their first or second pregnancy and were less than or equal to 28 weeks gestational age based on clinical assessment at enrolment. Women were followed monthly during pregnancy and encouraged to come to the clinic if they had intercurrent illness. At each encounter, peripheral blood smears and dried blood spots on filter paper were collected. At delivery, these same specimens and placental blood and tissue samples were collected. After quickening, women received SP-IPT up to three times separated by at least four weeks. Women with malaria detectable by blood smear at routine or sick visits were treated for malaria in accordance with the national guidelines (quinine in the first trimester and artemether-lumefantrine in the second and third trimesters). Details are described by Kalilani-Phiri et al.[10].\n Laboratory procedures Microscopy Peripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\nPeripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\n Haemoglobin Haemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\nHaemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\n Placental biopsies Placental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\nPlacental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\n Molecular detection DNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\n[11].\nDNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\n[11].\n Data analysis Peripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\nPeripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\n Ethical considerations Ethical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\nEthical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\n Microscopy Peripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\nPeripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\n Haemoglobin Haemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\nHaemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\n Placental biopsies Placental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\nPlacental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\n Molecular detection DNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\n[11].\nDNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\n[11].\n Data analysis Peripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\nPeripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\n Ethical considerations Ethical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\nEthical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\n\nStudy population:\nFour-hundred and fifty pregnant women were enrolled in an observational cohort study of malaria during pregnancy in Blantyre, Malawi between June 2009 and June 2010. All women were in their first or second pregnancy and were less than or equal to 28 weeks gestational age based on clinical assessment at enrolment. Women were followed monthly during pregnancy and encouraged to come to the clinic if they had intercurrent illness. At each encounter, peripheral blood smears and dried blood spots on filter paper were collected. At delivery, these same specimens and placental blood and tissue samples were collected. After quickening, women received SP-IPT up to three times separated by at least four weeks. Women with malaria detectable by blood smear at routine or sick visits were treated for malaria in accordance with the national guidelines (quinine in the first trimester and artemether-lumefantrine in the second and third trimesters). Details are described by Kalilani-Phiri et al.[10].\n\nLaboratory procedures:\n Microscopy Peripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\nPeripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\n Haemoglobin Haemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\nHaemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\n Placental biopsies Placental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\nPlacental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\n Molecular detection DNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\n[11].\nDNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\n[11].\n Data analysis Peripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\nPeripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\n Ethical considerations Ethical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\nEthical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\n\nMicroscopy:\nPeripheral blood slides were Field’s stained and examined using a 100× oil immersion objective to detect and quantify parasitaemia using an estimated white blood cell count of 8,000 per microlitre. A diagnosis of microscopically detectable malaria infection was made when asexual stage malaria parasites were detected on a thick film. A smear was recorded as negative after examining 100 high power fields. Two microscopists read all slides; in cases of disagreement between the readings, a third expert reader adjudicated.\n\nHaemoglobin:\nHaemoglobin measurement was obtained from a finger prick blood samples using HemoCue® AB.\n\nPlacental biopsies:\nPlacental biopsies were preserved in 10% neutral buffered formalin, embedded in paraffin wax, cut into four micron thick sections, and then stained with haematoxylin and eosin. Slides were examined for presence of malaria parasites and haemozoin pigment.\n\nMolecular detection:\nDNA was extracted from frozen placental samples and dried blood spots of peripheral blood and placental blood. Quantitative real time polymerase chain reaction (qPCR) was used to detect the gene for Plasmodium falciparum lactate dehydrogenase. Extraction and qPCR protocols are described on our website\n[11].\n\nData analysis:\nPeripheral blood infections were categorized as either microscopic (smear positive, confirmed by qPCR) or submicroscopic (smear negative, but qPCR positive). An episode of submicroscopic infection was defined as a positive qPCR and a negative malaria smear obtained at the same time. Sequential episodes of submicroscopic parasitaemia were only counted once. Gestational age at enrolment was calculated based on the last menstrual period or by the fundal height if the last menstrual period was not known. Gestational age at birth was determined based on the last menstrual period and the Ballard score. Fundal height at enrolment was included in the estimate of gestational age at birth if the last menstrual period and Ballard estimates were more than two weeks’ discrepant. The first trimester was defined as conception through 13 weeks and the second trimester was from 14 through 27 weeks. Low birth weight was defined as birth weight less than 2,500 g. To further investigate low birth weight, preterm birth and small for gestational age were analysed separately. If the gestational age at delivery was less than 37 weeks, the delivery was classified as preterm. Infants were considered small for gestational age if the birth weight for gestational age, based on WHO growth curves, was less than a Z-score of −2. Fever was defined as a measured axillary temperature ≥37.5°C. Maternal anaemia was defined as haemoglobin <11.0 g/dL. Placental malaria was classified as the presence of haemozoin pigment or parasites, by either histology or qPCR. Submicroscopic placental malaria was defined as parasites detected by qPCR, but not histology. Only women with both molecular and histological placental results were included in descriptive analyses of placental malaria. However, women missing histological results were included in analysis of the presence or absence of placental malaria because detection of parasites by qPCR alone is sufficient to categorize them as having placental malaria. Data from twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. Analyses of the effects of SP were restricted to visits that occurred within 28 days after the first visit.\nData analysis was performed using STATA version 12.1 software (Stata Corp, College Station, TX, USA). Student’s t-tests or Wilcoxon rank-sum were used for comparisons of normal and non-normal distributions of continuous variables, respectively. Chi-squared and Fisher’s exact tests were used for comparisons of proportions. Odds ratios were calculated using univariate and multivariate logistic regression. Analyses of haemoglobin during pregnancy were controlled for gestational age and adjusted for repeated measurements using robust cluster variance estimation. All P-values are two-sided, and statistical significance was set at P ≤0.05.\n\nEthical considerations:\nEthical approval was obtained from the University of Malawi College of Medicine Research and Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.\n\nResults:\nTwo thousand six hundred and eighty-one peripheral blood samples from 448 women enrolled in the study were screened for submicroscopic infection. Two women were not included in the analysis because they did not have any filter papers available for molecular analysis. Ninety-five incident submicroscopic infections were detected in 68 women. Pregnant women had 0.6 episodes of submicroscopic malaria per person-year of follow-up. As previously published, women in this cohort also experienced 0.6 episodes of microscopically detected infection per person-year of follow-up\n[10]. Thus, the overall rate of malaria infection was 1.2 episodes per person year of follow up.\nAmong women with submicroscopic infections, the mean number of infections was 1.4 (95% CI 1.2-1.6, range 1–5). Forty-nine women (72%) with submicroscopic infections never had an infection detected by microscopy during pregnancy. Submicroscopic infection was not associated with first versus second pregnancy, age, bed net use, or malaria treatment during pregnancy prior to enrolment, but was associated with enrolment at an earlier gestational age, more frequent visits, and lack of secondary school attendance (Table \n1). Gestational age at enrolment was inversely correlated with number of visits.Among 311 placentas with both histological and molecular data available for evaluation, 104 (33%) had evidence of placental malaria (Figure \n1). Thirty-three (11%) had both parasites and haemozoin detected microscopically. Three (1%) had no haemozoin but did have parasites detected by histology and confirmed by qPCR. Thirty-eight (12%) had haemozoin without histological or molecular evidence of active parasite infection. Thirty (10%) had submicroscopic infection with no histological evidence of parasites or haemozoin.\nEnrolment characteristics of women with and without submicroscopic infection detected during pregnancy irrespective of the presence of microscopic infection\nCI: confidence interval.\nFrequency of histological and molecular findings of placental malaria.\nHistological results were not available for six placentas; all had parasites detected by qPCR.\n Timing and clinical presentation of submicroscopic infection Among women with at least one submicroscopic infection, 69% had submicroscopic infection detected at enrolment (47/68). Submicroscopic infections were detected in each trimester and at delivery. Based on estimated gestational age at enrolment, submicroscopic infection was detected in 12% (6/50) of first trimester, 6% (63/1,050) of second trimester, and 1.4% (19/1,372) of third trimester samples prior to delivery. These infections occurred in six, fifty-six, and eighteen women, respectively. Twelve women had infection in more than one trimester. After excluding the submicroscopic infections present at enrolment, the prevalence of submicroscopic infection was higher in the second trimester than the third trimester [22/650 (3.4%) versus 19/1,372 (1.4%) of samples, p = 0.003].\nNine of 308 women (3%) with peripheral samples obtained at delivery had submicroscopic infection. Two women with submicroscopic infection at delivery also had submicroscopic infection at their last antepartum visit, thus infection at delivery was not considered a new submicroscopic infection.\nSubmicroscopic infection was not associated with documented fever (p = 0.4) or reported fever within 48 hours prior to the visit (p = 0.2). Three women reported cough, sneezing and body pains at a visit during which submicroscopic infection was detected. Otherwise, no women reported any symptoms of illness during visits at which submicroscopic infection was detected. However, submicroscopic infection was associated with a 0.6 g/dL decrease in mean haemoglobin at the time of detection (11.5 g/dL vs 12.1 g/dL, p = 0.001). This decrease remained significant after adjusting for gestational age and repeated measurements.\nAmong women with at least one submicroscopic infection, 69% had submicroscopic infection detected at enrolment (47/68). Submicroscopic infections were detected in each trimester and at delivery. Based on estimated gestational age at enrolment, submicroscopic infection was detected in 12% (6/50) of first trimester, 6% (63/1,050) of second trimester, and 1.4% (19/1,372) of third trimester samples prior to delivery. These infections occurred in six, fifty-six, and eighteen women, respectively. Twelve women had infection in more than one trimester. After excluding the submicroscopic infections present at enrolment, the prevalence of submicroscopic infection was higher in the second trimester than the third trimester [22/650 (3.4%) versus 19/1,372 (1.4%) of samples, p = 0.003].\nNine of 308 women (3%) with peripheral samples obtained at delivery had submicroscopic infection. Two women with submicroscopic infection at delivery also had submicroscopic infection at their last antepartum visit, thus infection at delivery was not considered a new submicroscopic infection.\nSubmicroscopic infection was not associated with documented fever (p = 0.4) or reported fever within 48 hours prior to the visit (p = 0.2). Three women reported cough, sneezing and body pains at a visit during which submicroscopic infection was detected. Otherwise, no women reported any symptoms of illness during visits at which submicroscopic infection was detected. However, submicroscopic infection was associated with a 0.6 g/dL decrease in mean haemoglobin at the time of detection (11.5 g/dL vs 12.1 g/dL, p = 0.001). This decrease remained significant after adjusting for gestational age and repeated measurements.\n Impact of submicroscopic infection Women who had submicroscopic infections were more likely to have placental malaria than those without infections. After controlling for microscopically detectable infection, women with submicroscopic infection were more than seven-fold more likely to have placental malaria than women without any documented infection during pregnancy (Table \n2). Each additional episode of submicroscopic infection increased the risk of placental malaria nearly five-fold (OR 4.9, 95% CI 2.7-9.1, p < 0.001). All women with submicroscopic infection detected in peripheral blood at delivery had placental malaria. Thirty-five percent (39/110) of women who had evidence of placental malaria did not have any microscopic or submicroscopic peripheral infections detected during pregnancy or at delivery.\nAssociation of submicroscopic infection during pregnancy and placental malaria\nCI: confidence interval.\nAmong women without microscopic infection during pregnancy, submicroscopic infection detected at enrolment was more strongly associated with placental malaria than submicroscopic infection detected after enrolment. After excluding infections detected at enrolment, third trimester infections were more strongly associated with placental malaria than second trimester infections (Table \n3).\nTiming of submicroscopic infection during pregnancy and odds of placental malaria among women without microscopically detected infection\n*Four women did not have molecular data available from their enrolment visit.\nCI: confidence interval.\nSubmicroscopic infection during pregnancy, at delivery or in the placenta was not associated with adverse maternal or foetal outcomes. The overall rates of these adverse outcomes were: 18% low birth weight, 13% small for gestational age, 15% preterm delivery and 11% maternal anaemia at delivery. The prevalence of these outcomes was the same in women who did and did not have submicroscopic infection in their peripheral blood or placenta (Table \n4).\nMaternal and foetal outcomes in singleton, live births from women with only submicroscopic infection compared to women without infection during pregnancy, at delivery or in the placenta\nCI: confidence interval.\n[Hb]: haemoglobin concentration.\nWomen who had submicroscopic infections were more likely to have placental malaria than those without infections. After controlling for microscopically detectable infection, women with submicroscopic infection were more than seven-fold more likely to have placental malaria than women without any documented infection during pregnancy (Table \n2). Each additional episode of submicroscopic infection increased the risk of placental malaria nearly five-fold (OR 4.9, 95% CI 2.7-9.1, p < 0.001). All women with submicroscopic infection detected in peripheral blood at delivery had placental malaria. Thirty-five percent (39/110) of women who had evidence of placental malaria did not have any microscopic or submicroscopic peripheral infections detected during pregnancy or at delivery.\nAssociation of submicroscopic infection during pregnancy and placental malaria\nCI: confidence interval.\nAmong women without microscopic infection during pregnancy, submicroscopic infection detected at enrolment was more strongly associated with placental malaria than submicroscopic infection detected after enrolment. After excluding infections detected at enrolment, third trimester infections were more strongly associated with placental malaria than second trimester infections (Table \n3).\nTiming of submicroscopic infection during pregnancy and odds of placental malaria among women without microscopically detected infection\n*Four women did not have molecular data available from their enrolment visit.\nCI: confidence interval.\nSubmicroscopic infection during pregnancy, at delivery or in the placenta was not associated with adverse maternal or foetal outcomes. The overall rates of these adverse outcomes were: 18% low birth weight, 13% small for gestational age, 15% preterm delivery and 11% maternal anaemia at delivery. The prevalence of these outcomes was the same in women who did and did not have submicroscopic infection in their peripheral blood or placenta (Table \n4).\nMaternal and foetal outcomes in singleton, live births from women with only submicroscopic infection compared to women without infection during pregnancy, at delivery or in the placenta\nCI: confidence interval.\n[Hb]: haemoglobin concentration.\n Effect of SP-IPT on submicroscopic infections The ability of SP to clear submicroscopic infections was evaluated by examining women who had submicroscopic infection and a subsequent visit within 28 days (N = 79 visits). Women who received a dose of SP when they had a submicroscopic infection were less likely to have an infection at their next visit within 28 days than women with submicroscopic infections who did not receive SP (23.9 vs 48.5%; p = 0.02).\nThe prophylactic efficacy of SP was assessed by comparing the rates of malaria infection within 28 days in women who did and did not receive SP when they had no malaria infection. A total of 1,779 visits were included in the analysis. Women who did and did not receive SP had the same rate of parasitaemia within the next 28 days (2.0 vs 2.2%; p = 0.83) (Figure \n2).\nSP-IPT treatment of submicroscopic infection and prevention of any infection.\nThe ability of SP to clear submicroscopic infections was evaluated by examining women who had submicroscopic infection and a subsequent visit within 28 days (N = 79 visits). Women who received a dose of SP when they had a submicroscopic infection were less likely to have an infection at their next visit within 28 days than women with submicroscopic infections who did not receive SP (23.9 vs 48.5%; p = 0.02).\nThe prophylactic efficacy of SP was assessed by comparing the rates of malaria infection within 28 days in women who did and did not receive SP when they had no malaria infection. A total of 1,779 visits were included in the analysis. Women who did and did not receive SP had the same rate of parasitaemia within the next 28 days (2.0 vs 2.2%; p = 0.83) (Figure \n2).\nSP-IPT treatment of submicroscopic infection and prevention of any infection.\n\nTiming and clinical presentation of submicroscopic infection:\nAmong women with at least one submicroscopic infection, 69% had submicroscopic infection detected at enrolment (47/68). Submicroscopic infections were detected in each trimester and at delivery. Based on estimated gestational age at enrolment, submicroscopic infection was detected in 12% (6/50) of first trimester, 6% (63/1,050) of second trimester, and 1.4% (19/1,372) of third trimester samples prior to delivery. These infections occurred in six, fifty-six, and eighteen women, respectively. Twelve women had infection in more than one trimester. After excluding the submicroscopic infections present at enrolment, the prevalence of submicroscopic infection was higher in the second trimester than the third trimester [22/650 (3.4%) versus 19/1,372 (1.4%) of samples, p = 0.003].\nNine of 308 women (3%) with peripheral samples obtained at delivery had submicroscopic infection. Two women with submicroscopic infection at delivery also had submicroscopic infection at their last antepartum visit, thus infection at delivery was not considered a new submicroscopic infection.\nSubmicroscopic infection was not associated with documented fever (p = 0.4) or reported fever within 48 hours prior to the visit (p = 0.2). Three women reported cough, sneezing and body pains at a visit during which submicroscopic infection was detected. Otherwise, no women reported any symptoms of illness during visits at which submicroscopic infection was detected. However, submicroscopic infection was associated with a 0.6 g/dL decrease in mean haemoglobin at the time of detection (11.5 g/dL vs 12.1 g/dL, p = 0.001). This decrease remained significant after adjusting for gestational age and repeated measurements.\n\nImpact of submicroscopic infection:\nWomen who had submicroscopic infections were more likely to have placental malaria than those without infections. After controlling for microscopically detectable infection, women with submicroscopic infection were more than seven-fold more likely to have placental malaria than women without any documented infection during pregnancy (Table \n2). Each additional episode of submicroscopic infection increased the risk of placental malaria nearly five-fold (OR 4.9, 95% CI 2.7-9.1, p < 0.001). All women with submicroscopic infection detected in peripheral blood at delivery had placental malaria. Thirty-five percent (39/110) of women who had evidence of placental malaria did not have any microscopic or submicroscopic peripheral infections detected during pregnancy or at delivery.\nAssociation of submicroscopic infection during pregnancy and placental malaria\nCI: confidence interval.\nAmong women without microscopic infection during pregnancy, submicroscopic infection detected at enrolment was more strongly associated with placental malaria than submicroscopic infection detected after enrolment. After excluding infections detected at enrolment, third trimester infections were more strongly associated with placental malaria than second trimester infections (Table \n3).\nTiming of submicroscopic infection during pregnancy and odds of placental malaria among women without microscopically detected infection\n*Four women did not have molecular data available from their enrolment visit.\nCI: confidence interval.\nSubmicroscopic infection during pregnancy, at delivery or in the placenta was not associated with adverse maternal or foetal outcomes. The overall rates of these adverse outcomes were: 18% low birth weight, 13% small for gestational age, 15% preterm delivery and 11% maternal anaemia at delivery. The prevalence of these outcomes was the same in women who did and did not have submicroscopic infection in their peripheral blood or placenta (Table \n4).\nMaternal and foetal outcomes in singleton, live births from women with only submicroscopic infection compared to women without infection during pregnancy, at delivery or in the placenta\nCI: confidence interval.\n[Hb]: haemoglobin concentration.\n\nEffect of SP-IPT on submicroscopic infections:\nThe ability of SP to clear submicroscopic infections was evaluated by examining women who had submicroscopic infection and a subsequent visit within 28 days (N = 79 visits). Women who received a dose of SP when they had a submicroscopic infection were less likely to have an infection at their next visit within 28 days than women with submicroscopic infections who did not receive SP (23.9 vs 48.5%; p = 0.02).\nThe prophylactic efficacy of SP was assessed by comparing the rates of malaria infection within 28 days in women who did and did not receive SP when they had no malaria infection. A total of 1,779 visits were included in the analysis. Women who did and did not receive SP had the same rate of parasitaemia within the next 28 days (2.0 vs 2.2%; p = 0.83) (Figure \n2).\nSP-IPT treatment of submicroscopic infection and prevention of any infection.\n\nDiscussion:\nEven with adherence to SP-IPT administration three times during pregnancy and active case detection and treatment of malaria, submicroscopic infection of the peripheral blood during pregnancy is frequent, especially at the first antenatal visit. Low-density malaria infections, detected only by molecular methods, are associated with placental malaria infection and a decrease in maternal haemoglobin levels. SP-IPT appears to treat some submicroscopic infections but does not prevent new infections. These findings have important implications for the development of further interventions for prevention of malaria pregnancy.\nThis is the first report of the effect of submicroscopic infection throughout pregnancy on maternal and foetal outcomes. Prior studies of submicroscopic malaria during pregnancy have been limited by being cross-sectional, rather than longitudinal in nature\n[2,3,6,7,12-15]. Linking antenatal infections to specific delivery and neonatal outcomes is essential to evaluating the importance of submicroscopic infections and designing strategies to optimize maternal and infant health.\nThe results are consistent with previous studies indicating that submicroscopic infection is associated with anaemia at the time of detection\n[12,15]. Two previous studies reported an association between submicroscopic infection at delivery and infant low birth weight\n[6] or maternal anaemia at the time of delivery\n[7]. However, these cross-sectional studies took place prior to, or as part of, IPT trials and did not assess previous exposure to or treatment of malaria infection during pregnancy. Thus, submicroscopic infection may have been a marker of frequent malaria exposure or lack of IPT administration prior to delivery. Due to detailed follow-up and treatment of microscopically detectable infection, the results of this study more accurately reflect the specific contribution of submicroscopic infections to clinical outcomes.\nThe lack of association between submicroscopic infection during pregnancy and adverse outcomes despite the strong association with placental malaria may have been a function of the design of the study and the clinical care the participants received. The results from the examination of the effect of microscopically detectable infections on maternal and foetal outcomes yielded similar results, as published previously\n[10]. The combination of three doses of SP-IPT and active case detection and treatment may have protected against adverse effects. It is also possible that other interventions, such as screening for anaemia or the appropriate treatment of non-malaria infections, might have improved the overall health of women, decreasing the frequency of adverse outcomes. Given the low prevalence of adverse outcomes, sample size also limited the ability to detect associations. Previous studies have demonstrated a 50% increase in rates of low birth weight\n[6] and maternal anaemia\n[7] at delivery associated with malaria during pregnancy. This study had only 15 and 7% power to detect a 50% increase in low birth weight and maternal anaemia at delivery, respectively, given the baseline rates of these outcomes in this cohort.\nThe presence of placental malaria infection in women who did not have any peripheral malaria infection detected during pregnancy was unexpected. This phenomenon may be due to transient infections that were not captured during sampling intervals. Results from this study indicate that pregnant women frequently clear submicroscopic infections spontaneously. Another source of the placental malaria infection may be infection that occurred prior to enrolment in antenatal care. The strong association between malaria at enrolment and placental infection suggests that early infection, even those that had been cleared at the time of the first antenatal visit, may have the greatest likelihood of causing placental pathology.\nThe modest efficacy of SP in clearing submicroscopic infection and the lack of prophylactic efficacy likely reflects the high level of SP resistance among malaria parasites in Malawi\n[8,16]. This is consistent with studies of SP-IPT in infants, that showed the protective effect of SP was approximately one month and the duration of this protective efficacy is inversely related to presence of in vitro markers of SP resistance\n[17,18].\nThere are several important features of this study that may limit the generalizability of the findings. Women had an average of six visits during the study and were screened with microscopy at each encounter. Thus, women received more screening and treatment when participating in the study than they would have during routine antenatal care. Despite the intensive follow-up and medical attention provided in the study, submicroscopic infection is strongly associated with placental malaria and anaemia during pregnancy. In real life settings, the association between submicroscopic malaria and adverse outcomes would likely have been more pronounced. Having few encounters during the first trimester and having samples obtained intermittently limited analysis of the timing of infection leading to placental infection. Women with submicroscopic infection did have more visits during the study, suggesting that increasing sampling frequency would increase detection of submicroscopic infection. Alternatively, women with submicroscopic infections may have symptoms leading them to present to the antenatal clinic more frequently. However, there was not an association between submicroscopic infection and fever or routine versus sick visit.\nThese results have important implications for the development of alternatives to SP-IPT for prevention of malaria pregnancy. Two possible strategies are intermittent screening and treatment or IPT with an anti-malarial more effective than SP. In one trial, intermittent screening and treatment has been shown to be equivalent to SP-IPT\n[19]. The intermittent screening and treatment arm in that study used antigen-detecting rapid diagnostic tests for screening. The parasite density threshold for detection for rapid diagnostic tests, like that for microscopy, is ten- to 100-fold higher than for qPCR\n[20,21]. Rapid diagnostic tests, like microscopy, will fail to detect infection at the submicroscopic level. Thus, a rapid diagnostic test-based screening and treatment strategy will fail to detect infections that this study demonstrates are associated with placental malaria. Within the context of the usual antenatal package of four antenatal visits in pregnancy if IPT is not provided, many microscopic and all submicroscopic infections will not be treated. Under these conditions, with prolonged and untreated episodes of low-density malaria infection, it is expected that the adverse effects of placental malaria would occur\n[22-24].\nIf IPT with a more effective anti-malarial is considered, for example as in ongoing trials of IPT with dihydroartemisinin-piperaquine\n[25], azithromycin combined with chloroquine\n[26] and mefloquine\n[27], it will be important to characterize the treatment and prophylactic effect of candidate drugs. Most drug efficacy trials examine the ability of a drug to cure the infection at the time of drug administration. However, IPT is most beneficial in high transmission settings if it cures current infections and prevents subsequent infections. The results of this study indicate that the loss of prophylactic efficacy may precede the loss of curative efficacy. Thus, in trials of alternative anti-malarials for IPT, prophylactic efficacy should be included as an outcome measure.\nThe finding that the highest rates of asymptomatic and submicroscopic malaria are detected in women at their first antenatal care visit highlights an essential shortcoming of the current policies to prevent pregnancy-associated malaria. Interventions such as the distribution of bed nets at antenatal clinics and the administration of IPT are delayed until women seek antenatal care. In Malawi, as in other countries in sub-Saharan Africa, this is typically late in the second or into the third trimester\n[28]. By the time women have their first antenatal visit, peripheral and placental infection has already been established. Innovative interventions that can target women early in pregnancy, or even prior to pregnancy, are urgently needed to further decrease the burden of malaria during pregnancy.\n\nConclusions:\nSubmicroscopic malaria infection is common, occurs early in pregnancy and is associated with placental malaria. SP-IPT appears to treat some submicroscopic infections but does not prevent new infections. To effectively control pregnancy-associated malaria, new interventions are required to target women prior to their first antenatal care visit and to effectively treat and prevent all malaria infections.\n\nAbbreviations:\nIPT: Intermittent preventive treatment; SP: Sulphadoxine-pyrimethamine; SP-IPT: Intermittent preventive treatment with sulphadoxine-pyrimethamine; PCR: Polymerase chain reaction; qPCR: Quantitative polymerase chain reaction.\n\nCompeting interests:\nThe authors have declared that they have no financial disclosures or competing interests.\n\nAuthors’ contributions:\nLKP, TET and MKL conceived of and designed the field study. LKP, TET, MKL, PCT, MM, PW, and GM conducted the clinical study. LMC and MKL designed the experiments. KM, SK and AM completed the histopathology. LMC, SB, RM, SJ, and KBS conducted the experiments. LMC and MKL performed the analysis and led the writing of the manuscript. All authors read and approved the final manuscript.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nMalaria during pregnancy results in adverse outcomes for mothers and infants. Intermittent preventive treatment (IPT) with sulphadoxine-pyrimethamine (SP) is the primary intervention aimed at reducing malaria infection during pregnancy. Although submicroscopic infection is common during pregnancy and at delivery, its impact throughout pregnancy on the development of placental malaria and adverse pregnancy outcomes has not been clearly established.\n\nMethods:\nQuantitative PCR was used to detect submicroscopic infections in pregnant women enrolled in an observational study in Blantyre, Malawi to determine their effect on maternal, foetal and placental outcomes. The ability of SP to treat and prevent submicroscopic infections was also assessed.\n\nResults:\n2,681 samples from 448 women were analysed and 95 submicroscopic infections were detected in 68 women, a rate of 0.6 episodes per person-year of follow-up. Submicroscopic infections were most often detected at enrolment. The majority of women with submicroscopic infections did not have a microscopically detectable infection detected during pregnancy. Submicroscopic infection was associated with placental malaria even after controlling for microscopically detectable infection and was associated with decreased maternal haemoglobin at the time of detection. However, submicroscopic infection was not associated with adverse maternal or foetal outcomes at delivery. One-third of women with evidence of placental malaria did not have documented peripheral infection during pregnancy. SP was moderately effective in treating submicroscopic infections, but did not prevent the development of new submicroscopic infections in the month after administration.\n\nConclusions:\nSubmicroscopic malaria infection is common and occurs early in pregnancy. SP-IPT can clear some submicroscopic infections but does not prevent new infections after administration. To effectively control pregnancy-associated malaria, new interventions are required to target women prior to their first antenatal care visit and to effectively treat and prevent all malaria infections."},"background_conclusion_text":{"kind":"string","value":"Background:\nEach year, 25 million pregnant women in sub-Saharan Africa are at risk for malaria infection. Malaria during pregnancy is associated with maternal anaemia and infant low birth weight. Malaria during pregnancy is estimated to result in 100,000 infant deaths annually in Africa\n[1].\nMalaria infections during pregnancy may be identified by microscopic examination of a blood smear or may be submicroscopic, detectable only by more sensitive molecular methods. Studies in a variety of transmission settings have shown that submicroscopic infections can be up to five times more common than microscopic infections during pregnancy\n[2-7]. While the associations between microscopically detectable malaria during pregnancy and maternal anaemia at delivery and low birth weight have been supported in most studies\n[1], the impact of submicroscopic infection during pregnancy on these adverse outcomes has not been systematically assessed.\nBecause malaria infections during pregnancy are often asymptomatic, a key intervention to decrease the burden of malaria in pregnancy is intermittent preventive treatment (IPT), providing anti-malarial chemotherapy at routine intervals during pregnancy. Currently, the recommended anti-malarial chemotherapy for IPT in the majority of Africa is sulphadoxine-pyrimethamine (SP), which is intended to cure current infections and to provide a period of post-treatment prophylaxis to prevent future infections.\nIn much of sub-Saharan Africa, increasing resistance to SP\n[8] is generating concerns that SP-IPT efficacy will be compromised\n[9]. Understanding the relative importance of the treatment and prophylactic effects of SP-IPT and the differential impact of resistance on these effects may have implications for the selection of a new drug to replace SP for IPT.\nIn the context of an observational study of a large cohort of pregnant women in Malawi, the effect of submicroscopic infections on maternal and foetal outcomes was examined. All women received SP-IPT according to the national policy. At each visit a blood smear was examined and women were treated for malaria infections detected by microscopy. The primary analysis of infections detected solely by microscopy showed an association between infection at enrolment in antenatal care and placental malaria, but only infections detected at the time of delivery were associated with adverse pregnancy outcomes\n[10]. Because higher rates of submicroscopic infection than microscopically detectable infection were anticipated and submicroscopic infections were not treated, it was hypothesized that submicroscopic infections would be associated with placental malaria, maternal anaemia and infant low birth weight. It was further hypothesized that, due to high rates of SP resistance, SP-IPT would fail to clear and prevent submicroscopic infections.\n\nConclusions:\nSubmicroscopic malaria infection is common, occurs early in pregnancy and is associated with placental malaria. SP-IPT appears to treat some submicroscopic infections but does not prevent new infections. To effectively control pregnancy-associated malaria, new interventions are required to target women prior to their first antenatal care visit and to effectively treat and prevent all malaria infections."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nMalaria during pregnancy results in adverse outcomes for mothers and infants. Intermittent preventive treatment (IPT) with sulphadoxine-pyrimethamine (SP) is the primary intervention aimed at reducing malaria infection during pregnancy. Although submicroscopic infection is common during pregnancy and at delivery, its impact throughout pregnancy on the development of placental malaria and adverse pregnancy outcomes has not been clearly established.\n\nMethods:\nQuantitative PCR was used to detect submicroscopic infections in pregnant women enrolled in an observational study in Blantyre, Malawi to determine their effect on maternal, foetal and placental outcomes. The ability of SP to treat and prevent submicroscopic infections was also assessed.\n\nResults:\n2,681 samples from 448 women were analysed and 95 submicroscopic infections were detected in 68 women, a rate of 0.6 episodes per person-year of follow-up. Submicroscopic infections were most often detected at enrolment. The majority of women with submicroscopic infections did not have a microscopically detectable infection detected during pregnancy. Submicroscopic infection was associated with placental malaria even after controlling for microscopically detectable infection and was associated with decreased maternal haemoglobin at the time of detection. However, submicroscopic infection was not associated with adverse maternal or foetal outcomes at delivery. One-third of women with evidence of placental malaria did not have documented peripheral infection during pregnancy. SP was moderately effective in treating submicroscopic infections, but did not prevent the development of new submicroscopic infections in the month after administration.\n\nConclusions:\nSubmicroscopic malaria infection is common and occurs early in pregnancy. SP-IPT can clear some submicroscopic infections but does not prevent new infections after administration. To effectively control pregnancy-associated malaria, new interventions are required to target women prior to their first antenatal care visit and to effectively treat and prevent all malaria infections."},"whole_article_text_length":{"kind":"number","value":11226,"string":"11,226"},"whole_article_abstract_length":{"kind":"number","value":334,"string":"334"},"other_sections_lengths":{"kind":"list like","value":[486,178,1550,86,15,43,53,502,62,324,374,183,37,14,85],"string":"[\n 486,\n 178,\n 1550,\n 86,\n 15,\n 43,\n 53,\n 502,\n 62,\n 324,\n 374,\n 183,\n 37,\n 14,\n 85\n]","isConversational":false},"num_sections":{"kind":"number","value":19,"string":"19"},"most_frequent_words":{"kind":"list like","value":["infection","submicroscopic","malaria","women","placental","submicroscopic infection","placental malaria","infections","blood","age"],"string":"[\n \"infection\",\n \"submicroscopic\",\n \"malaria\",\n \"women\",\n \"placental\",\n \"submicroscopic infection\",\n \"placental malaria\",\n \"infections\",\n \"blood\",\n \"age\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["pregnancy associated malaria","detectable malaria pregnancy","study malaria pregnancy","malaria infections pregnancy","submicroscopic malaria pregnancy"],"string":"[\n \"pregnancy associated malaria\",\n \"detectable malaria pregnancy\",\n \"study malaria pregnancy\",\n \"malaria infections pregnancy\",\n \"submicroscopic malaria pregnancy\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Malaria in pregnancy | Submicroscopic infection | Placental malaria | Sulphadoxine-pyrimethamine intermittent preventive treatment | Sulphadoxine-pyrimethamine resistance [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Malaria in pregnancy | Submicroscopic infection | Placental malaria | Sulphadoxine-pyrimethamine intermittent preventive treatment | Sulphadoxine-pyrimethamine resistance [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Malaria in pregnancy | Submicroscopic infection | Placental malaria | Sulphadoxine-pyrimethamine intermittent preventive treatment | Sulphadoxine-pyrimethamine resistance [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Malaria in pregnancy | Submicroscopic infection | Placental malaria | Sulphadoxine-pyrimethamine intermittent preventive treatment | Sulphadoxine-pyrimethamine resistance [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Malaria in pregnancy | Submicroscopic infection | Placental malaria | Sulphadoxine-pyrimethamine intermittent preventive treatment | Sulphadoxine-pyrimethamine resistance [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Malaria in pregnancy | Submicroscopic infection | Placental malaria | Sulphadoxine-pyrimethamine intermittent preventive treatment | Sulphadoxine-pyrimethamine resistance [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Antimalarials | Artemether, Lumefantrine Drug Combination | Artemisinins | Asymptomatic Diseases | DNA, Protozoan | Drug Administration Schedule | Drug Combinations | Drug Resistance | Erythrocytes | Ethanolamines | False Negative Reactions | Female | Fetal Diseases | Fluorenes | Follow-Up Studies | Hemeproteins | Humans | Infant, Newborn | Infectious Disease Transmission, Vertical | Malaria | Malawi | Parasitemia | Placenta | Plasmodium | Polymerase Chain Reaction | Pregnancy | Pregnancy Complications, Infectious | Pregnancy Outcome | Pregnancy Trimesters | Prevalence | Pyrimethamine | Quinine | Sulfadoxine [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Antimalarials | Artemether, Lumefantrine Drug Combination | Artemisinins | 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Drug Resistance | Erythrocytes | Ethanolamines | False Negative Reactions | Female | Fetal Diseases | Fluorenes | Follow-Up Studies | Hemeproteins | Humans | Infant, Newborn | Infectious Disease Transmission, Vertical | Malaria | Malawi | Parasitemia | Placenta | Plasmodium | Polymerase Chain Reaction | Pregnancy | Pregnancy Complications, Infectious | Pregnancy Outcome | Pregnancy Trimesters | Prevalence | Pyrimethamine | Quinine | Sulfadoxine [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] pregnancy associated malaria | detectable malaria pregnancy | study malaria pregnancy | malaria infections pregnancy | submicroscopic malaria pregnancy [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] pregnancy associated malaria | detectable malaria pregnancy | study malaria pregnancy | malaria infections pregnancy | submicroscopic malaria pregnancy 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[SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] infection | submicroscopic | malaria | women | placental | submicroscopic infection | placental malaria | infections | blood | age [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] infection | submicroscopic | malaria | women | placental | submicroscopic infection | placental malaria | infections | blood | age [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] infection | submicroscopic | malaria | women | placental | submicroscopic infection | placental malaria | infections | blood | age [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] infection | submicroscopic | malaria | women | placental | submicroscopic infection | placental malaria | infections | blood | age [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] infection | submicroscopic | malaria | women | placental | submicroscopic infection | placental malaria | infections | blood | age [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] infection | submicroscopic | malaria | women | placental | submicroscopic infection | placental malaria | infections | blood | age [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] infections | pregnancy | malaria | sp | submicroscopic | ipt | africa | infections pregnancy | submicroscopic infections | malaria pregnancy [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] placental | malaria | gestational | age | gestational age | defined | birth | qpcr | blood | analyses [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] infection | submicroscopic | submicroscopic infection | women | detected | women submicroscopic | infections | submicroscopic infection detected | malaria | infection detected [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] effectively | treat | malaria | prevent | pregnancy associated | infections | new | associated | pregnancy | placental malaria sp [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] submicroscopic | infection | malaria | women | submicroscopic infection | placental | infections | blood | pregnancy | placental malaria [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] submicroscopic | infection | malaria | women | submicroscopic infection | placental | infections | blood | pregnancy | placental malaria [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Malaria ||| IPT ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Blantyre | Malawi ||| SP [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 2,681 | 448 | 95 | 68 | 0.6 ||| ||| ||| ||| ||| One-third ||| the month [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| SP-IPT ||| first [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Malaria ||| IPT ||| ||| Blantyre | Malawi ||| SP ||| 2,681 | 448 | 95 | 68 | 0.6 ||| ||| ||| ||| ||| One-third ||| the month ||| ||| SP-IPT ||| first [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Malaria ||| IPT ||| ||| Blantyre | Malawi ||| SP ||| 2,681 | 448 | 95 | 68 | 0.6 ||| ||| ||| ||| ||| One-third ||| the month ||| ||| SP-IPT ||| first [SUMMARY]"}}},{"rowIdx":84,"cells":{"title":{"kind":"string","value":"Effect of Transport Time on the Use of Reperfusion Therapy for Patients with Acute Ischemic Stroke in Korea."},"pmid":{"kind":"string","value":"33754510"},"background_abstract":{"kind":"string","value":"We investigated the association between geographic proximity to hospitals and the administration rate of reperfusion therapy for acute ischemic stroke."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We identified patients with acute ischemic stroke who visited the hospital within 12 hours of symptom onset from a prospective nationwide multicenter stroke registry. Reperfusion therapy was classified as intravenous thrombolysis (IVT), endovascular therapy (EVT), or combined therapy. The association between the proportion of patients who were treated with reperfusion therapy and the ground transport time was evaluated using a spline regression analysis adjusted for patient-level characteristics. We also estimated the proportion of Korean population that lived within each 30-minute incremental service area from 67 stroke centers accredited by the Korean Stroke Society."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Of 12,172 patients (mean age, 68 ± 13 years; men, 59.7%) who met the eligibility criteria, 96.5% lived within 90 minutes of ground transport time from the admitting hospital. The proportion of patients treated with IVT decreased significantly when stroke patients lived beyond 90 minutes of the transport time (P = 0.006). The proportion treated with EVT also showed a similar trend with the transport time. Based on the residential area, 98.4% of Korean population was accessible to 67 stroke centers within 90 minutes."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The use of reperfusion therapy for acute stroke decreased when patients lived beyond 90 minutes of the ground transport time from the hospital. More than 95% of the South Korean population was accessible to 67 stroke centers within 90 minutes of the ground transport time."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Administration, Intravenous","Aged","Aged, 80 and over","Combined Modality Therapy","Endovascular Procedures","Female","Fibrinolytic Agents","Humans","Ischemic Stroke","Male","Middle Aged","Registries","Republic of Korea","Thrombolytic Therapy","Time Factors","Time-to-Treatment"],"string":"[\n \"Administration, Intravenous\",\n \"Aged\",\n \"Aged, 80 and over\",\n \"Combined Modality Therapy\",\n \"Endovascular Procedures\",\n \"Female\",\n \"Fibrinolytic Agents\",\n \"Humans\",\n \"Ischemic Stroke\",\n \"Male\",\n \"Middle Aged\",\n \"Registries\",\n \"Republic of Korea\",\n \"Thrombolytic Therapy\",\n \"Time Factors\",\n \"Time-to-Treatment\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"7985286"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Geographic access to hospitals that offer acute care for patients with trauma or acute myocardial infarction can be crucial because timely treatment can improve outcomes.1234 For patients with acute ischemic stroke, access to hospitals that provide reperfusion therapy is important because the reperfusion therapy for acute ischemic stroke is also highly time-sensitive.56 Several previous studies have suggested the probability of receiving reperfusion therapies for acute ischemic stroke increased when the patients lived in close geographic proximity to hospitals that provide the therapy, however other studies did not find a significant association between the transport time from home to hospital and the use of reperfusion therapy.78910 For example, longer driving time to the treating hospital was significantly associated with longer onset-to-arrival time to the hospitals and lower odds of receiving intravenous thrombolysis (IVT) in a retrospective study of 118,683 acute stroke patients admitted to 1,489 US hospitals.10 For endovascular therapy (EVT) for acute ischemic stroke, the proportion of stroke patients receiving EVT decreased significantly when patients lived beyond a 1-hour ground transport time from hospitals that offer EVT in California during 2009 and 2010.7\nAmong patients with ST-segment elevation myocardial infarction, a wide regional variation exists in the rate of reperfusion therapy and median time from symptom onset to treatment due to differences in geography, local resource, and organization of regional health system.11 Likewise the association between geographic proximity to a hospital that offers the reperfusion therapy for acute stroke patients and the probability of receiving the therapy can vary depending on population density, geography, and systems of care.1213 Therefore, each region needs to develop an optimal geospatial modeling of acute care centers to provide maximal population access to such centers.114\nIn this study, we investigated the association between geographic proximity to a hospital that offers reperfusion therapy and the administration rate of the therapy for acute ischemic stroke in South Korea, one of the planet's most densely populated countries with a population density of 529 people per square kilometer in 2018.15"},"methods_title":{"kind":"string","value":"METHODS"},"methods_text":{"kind":"string","value":"Patients The Clinical Research Collaboration for Stroke in Korea (CRCS-K) is a web-based prospective nationwide multicenter stroke registry.1617 The registry started enrolling patients in April 2008 and the collection of each patient's home address was permitted until August 2014. From the registry, we identified patients with acute ischemic stroke who were over 18 years old and visited one of the 12 CRCS-K hospitals within 12 hours of symptom onset time. The last known normal time was used when the onset of stroke symptom was unclear. Records were excluded if address information was incorrect or if symptom onset or hospital arrival time was unavailable. We also collected the exact time of intravenous tissue plasminogen activator (tPA) administration and groin-puncture time, and calculated onset-to-door, door-to-needle, and door-to-puncture time. We limited our analyses to the addresses within the same provincial area of the treating hospital because transferring acute stroke patients across provincial line is not considered a usual practice. Reperfusion therapy was classified as either IVT alone, vs. EVT or combined IVT and EVT. For patients who were treated with combined IVT and EVT, we identified patients who had received intravenous tPA at an outside hospital and then transferred to the CRCS-K center for EVT, i.e., drip-and-ship paradigm. For an initial stroke severity measurement, we used the National Institutes of Health Stroke Scale (NIHSS) score. For the subtyping of ischemic stroke, we employed the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification with minor modification1819 and classified the subtype as large-artery atherosclerosis (LAA), small-vessel occlusion (SVO), cardioembolism (CE), or undetermined etiology (UDE)/other determined etiology (ODE).\nThe Clinical Research Collaboration for Stroke in Korea (CRCS-K) is a web-based prospective nationwide multicenter stroke registry.1617 The registry started enrolling patients in April 2008 and the collection of each patient's home address was permitted until August 2014. From the registry, we identified patients with acute ischemic stroke who were over 18 years old and visited one of the 12 CRCS-K hospitals within 12 hours of symptom onset time. The last known normal time was used when the onset of stroke symptom was unclear. Records were excluded if address information was incorrect or if symptom onset or hospital arrival time was unavailable. We also collected the exact time of intravenous tissue plasminogen activator (tPA) administration and groin-puncture time, and calculated onset-to-door, door-to-needle, and door-to-puncture time. We limited our analyses to the addresses within the same provincial area of the treating hospital because transferring acute stroke patients across provincial line is not considered a usual practice. Reperfusion therapy was classified as either IVT alone, vs. EVT or combined IVT and EVT. For patients who were treated with combined IVT and EVT, we identified patients who had received intravenous tPA at an outside hospital and then transferred to the CRCS-K center for EVT, i.e., drip-and-ship paradigm. For an initial stroke severity measurement, we used the National Institutes of Health Stroke Scale (NIHSS) score. For the subtyping of ischemic stroke, we employed the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification with minor modification1819 and classified the subtype as large-artery atherosclerosis (LAA), small-vessel occlusion (SVO), cardioembolism (CE), or undetermined etiology (UDE)/other determined etiology (ODE).\nTravel distance, ground transport time, and service area In this study, we classified the patients' residence as metropolitan (city with a population of more than 500,000), urban (city with a population of more than 50,000), or rural region according to the local government act of Korea. After geocoding of the home address and treating hospital for each patient, ground transport time was estimated based on the driving time along the best route from the patient's address to the admitting hospitals using a web-based map service which is optimized for actual traffic condition in Korea (https://map.kakao.com/). For analyses, the travel time was divided into 30-minute increments of ground transport time (i.e., 0–30, 31–60, 61–90, 90–120, and > 120 minutes). To project the findings from CRCS-K hospitals to the national level, we estimated the proportion of the Korean population that lived within each incremental service area from 67 stroke centers accredited by the Korean Stroke Society in 2018 (https://www.stroke.or.kr:4454/hospital/index2.php). To do this, we first obtained the service area from 67 stroke centers using the 30-minute incremental ground transport time threshold. Then, we overlaid these service area maps on the residential areas of 255 administrative districts of South Korea. Finally, we calculated the proportion of the population from each administrative district of South Korea within each service area based on intersection of the service area maps and residential areas. In addition, we repeated the same analysis using the whole territorial area of South Korea including both residential and nonresidential areas. Population data for each administrative district in South Korea were from the Korean National Statistical Office as of December 2019.20 ArcGIS Pro (Version 2.4.0; Esri Inc, Redlands, CA, USA) was used for all geographic analyses.\nIn this study, we classified the patients' residence as metropolitan (city with a population of more than 500,000), urban (city with a population of more than 50,000), or rural region according to the local government act of Korea. After geocoding of the home address and treating hospital for each patient, ground transport time was estimated based on the driving time along the best route from the patient's address to the admitting hospitals using a web-based map service which is optimized for actual traffic condition in Korea (https://map.kakao.com/). For analyses, the travel time was divided into 30-minute increments of ground transport time (i.e., 0–30, 31–60, 61–90, 90–120, and > 120 minutes). To project the findings from CRCS-K hospitals to the national level, we estimated the proportion of the Korean population that lived within each incremental service area from 67 stroke centers accredited by the Korean Stroke Society in 2018 (https://www.stroke.or.kr:4454/hospital/index2.php). To do this, we first obtained the service area from 67 stroke centers using the 30-minute incremental ground transport time threshold. Then, we overlaid these service area maps on the residential areas of 255 administrative districts of South Korea. Finally, we calculated the proportion of the population from each administrative district of South Korea within each service area based on intersection of the service area maps and residential areas. In addition, we repeated the same analysis using the whole territorial area of South Korea including both residential and nonresidential areas. Population data for each administrative district in South Korea were from the Korean National Statistical Office as of December 2019.20 ArcGIS Pro (Version 2.4.0; Esri Inc, Redlands, CA, USA) was used for all geographic analyses.\nStatistical analysis Baseline characteristics of patients were compared using a test for trend across the ordered groups divided into the 30-minute increments of ground transport time. Regression analyses were used to explore the relationship between ground transport time and the proportion of patients that received reperfusion therapies (either IVT alone or EVT). For the non-linear relationship, a restricted cubic spline function with 4 knots defined at the 30 minutes, 60 minutes, 90 minutes, and 120 minutes of ground transport time was used. In the regression analyses, covariates entered the regression models when they had P values less than 0.10 in the univariate analyses. The regression analyses were performed with R software using the “rms” package (version 3.6.0, R Foundation for Statistical Computing, Vienna, Austria). In all analyses, a two-tailed test with P value of less than 0.05 was considered significant.\nBaseline characteristics of patients were compared using a test for trend across the ordered groups divided into the 30-minute increments of ground transport time. Regression analyses were used to explore the relationship between ground transport time and the proportion of patients that received reperfusion therapies (either IVT alone or EVT). For the non-linear relationship, a restricted cubic spline function with 4 knots defined at the 30 minutes, 60 minutes, 90 minutes, and 120 minutes of ground transport time was used. In the regression analyses, covariates entered the regression models when they had P values less than 0.10 in the univariate analyses. The regression analyses were performed with R software using the “rms” package (version 3.6.0, R Foundation for Statistical Computing, Vienna, Austria). In all analyses, a two-tailed test with P value of less than 0.05 was considered significant.\nEthics statement This study was approved by the Institutional Review Boards of Jeju National University Hospital (approval No. JEJUNUH 2018-09-003) and all other participating centers with a waiver of informed consent of individual patients because of study subject anonymity and minimal risk to participants.\nThis study was approved by the Institutional Review Boards of Jeju National University Hospital (approval No. JEJUNUH 2018-09-003) and all other participating centers with a waiver of informed consent of individual patients because of study subject anonymity and minimal risk to participants."},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"Of 27,122 acute stroke patients who had been admitted to CRCS-K hospitals between April 2008 and August 2014, 12,172 patients met the eligibility criteria (Supplementary Fig. 1). Mean age was 68 ± 13 years, and 59.7% were men. Of 12,172 patients, 2,216 patients (18.2%) were treated with IVT alone and 1238 patients (10.2%) were treated with either EVT alone or combined IVT and EVT. The patients who received reperfusion therapy were more likely to be older, to have atrial fibrillation and higher initial NIHSS score (Supplementary Table 1). In this study, 48.2% of the patients' residence were classified as metropolitan, 41.3% as urban, and 10.5% as rural. The average estimated ground transport time from home to hospital was 23 minutes (interquartile range [IQR], 14–39), and median travel distance was 9.4 km (IQR, 4.5–25.1). In this study, 64.4% of the patients lived within 30 minutes or less to the admitting hospital, 88.7% within 60 minutes or less, 96.5% within 90 minutes or less, and 99.2% with 120 minutes or less. Ground transport times of 30, 60, 90, and 120 minutes corresponded to travel distances of 20.4, 53.5, 86.6, and 119.8 km, respectively. Patients that lived farther from admitting hospitals were more likely to be older, to have atrial fibrillation and CE subtype, and to have a greater initial NIHSS score, but were less likely to have hypertension, diabetes, hyperlipidemia, coronary artery disease and had a lower initial systolic blood pressure (Table 1). The ground transport time did not differ between patients treated with IVT only and patients untreated with reperfusion therapy (rank sum test P = 0.450). However, the ground transport time was slightly but significantly greater for patients treated with EVT compared with patients untreated with reperfusion therapy (24 minutes [14–44] vs. 23 minutes [14–39], rank sum test P = 0.015; Fig. 1 and Supplementary Fig. 2).\nData are presented as number (%) or mean ± standard deviation.\nIQR = interquartile range, NIHSS = National Institutes of Health Stroke Scale, LAA = large artery atherosclerosis, CE = cardioembolism, SVO = small-vessel occlusion, UDE = undetermined etiology, ODE = other determined etiology, LDL = low-density lipoprotein, BUN = blood urea nitrogen, IVT = intravenous thrombolysis, EVT = endovascular therapy.\nCRCS-K = The Clinical Research Collaboration for Stroke in Korea, IVT = intravenous thrombolysis, EVT = endovascular therapy.\nExact treatment time was available in 1,669 patients (83.4%) treated with IVT alone. The median door-to-needle time was 40 minutes (30–53) and the median onset-to-needle time was 124 minutes (88–170). As expected, the onset-to-needle time was positively correlated with ground transport time (Pearson coefficient 0.201, P < 0.001) since it took more time to arrive at the hospital. However, the door-to-needle was inversely associated with ground transport time (Pearson coefficient −0.108, P < 0.001). Regarding EVT, exact treatment time for patients treated with EVT was available for 1,077 patients (87.0%). The median door-to-puncture time was 107 minutes (85–135) and the median onset-to-puncture time was 240 minutes (170–340). The onset-to-puncture time was positively correlated with ground transport time (Pearson coefficient 0.192, P < 0.001). Like the door-to-needle time in IVT, the door-to-puncture showed a negative association with ground transport time in patients treated with EVT (Pearson coefficient −0.089, P < 0.001) (Supplementary Fig. 3).\nThe proportion of patients treated with IVT ranged from 17.1% to 22.1% among the groups with 30-minute increments of ground transport time, and proportion of EVT ranged from 7.5% to 12.4% (Supplementary Fig. 4). In the restricted cubic spline analyses, the proportion of patients treated with IVT decreased significantly when the patients lived beyond a 90-minute ground transport time from the hospital (unadjusted P = 0.003; adjusted P = 0.006). The proportion of stroke patients treated with EVT showed a similar trend with estimated ground transport time in the restricted cubic spline analyses, but it did not reach statistical significance in both unadjusted and adjusted analyses (unadjusted P = 0.086; adjusted P = 0.105) (Fig. 2).\n(A) IVT, unadjusted analysis (P = 0.003); (B) IVT, adjusted analysis (P = 0.006); adjusted for age, hypertension, diabetes, atrial fibrillation, stroke history, coronary artery disease, use of an antiplatelet agent, oral anticoagulants or statins before the index stroke, diastolic blood pressure, fasting blood glucose, low-density lipoprotein cholesterol, platelet count, onset-to-arrival time, initial NIHSS score, and TOAST classification. (C) EVT, unadjusted analysis (P = 0.090); (D) EVT, adjusted analysis (P = 0.100); adjusted for age, sex, hypertension, diabetes, atrial fibrillation, hyperlipidemia, smoking, coronary artery disease, use of an antiplatelet agent or oral anticoagulants before the index stroke, systolic blood pressure, hemoglobin, platelet count, blood urea nitrogen, onset-to-arrival time, initial NIHSS score, and TOAST classification.\nIVT = intravenous thrombolysis, EVT = endovascular therapy, NIHSS = National Institutes of Health Stroke Scale, TOAST = Trial of Org 10172 in Acute Stroke Treatment.\nOf 779 patients who had been treated with combined IVT-EVT, we could identify exact referral pattern in 439 patients (56.4%). Among 439 patients, 69 patients (15.7%) were treated by drip- and-ship paradigm. The use of drip-and-ship paradigm increased significantly with greater ground transport time (P < 0.001) (Supplementary Table 2).\nFor national coverage from the 67 stroke centers, only 9.3% of the total land area of South Korea was reachable within 30 minutes while 75.0% of the land was accessible within 90 minutes. In contrast, 73.0% of the residential area was reachable within 30 minutes of the ground transport time, and 98.4% of the area was accessible within 90 minutes (Fig. 3 and Table 2).\nCRCS-K = The Clinical Research Collaboration for Stroke in Korea."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Patients","Travel distance, ground transport time, and service area","Statistical analysis","Ethics statement"],"string":"[\n \"Patients\",\n \"Travel distance, ground transport time, and service area\",\n \"Statistical analysis\",\n \"Ethics statement\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["The Clinical Research Collaboration for Stroke in Korea (CRCS-K) is a web-based prospective nationwide multicenter stroke registry.1617 The registry started enrolling patients in April 2008 and the collection of each patient's home address was permitted until August 2014. From the registry, we identified patients with acute ischemic stroke who were over 18 years old and visited one of the 12 CRCS-K hospitals within 12 hours of symptom onset time. The last known normal time was used when the onset of stroke symptom was unclear. Records were excluded if address information was incorrect or if symptom onset or hospital arrival time was unavailable. We also collected the exact time of intravenous tissue plasminogen activator (tPA) administration and groin-puncture time, and calculated onset-to-door, door-to-needle, and door-to-puncture time. We limited our analyses to the addresses within the same provincial area of the treating hospital because transferring acute stroke patients across provincial line is not considered a usual practice. Reperfusion therapy was classified as either IVT alone, vs. EVT or combined IVT and EVT. For patients who were treated with combined IVT and EVT, we identified patients who had received intravenous tPA at an outside hospital and then transferred to the CRCS-K center for EVT, i.e., drip-and-ship paradigm. For an initial stroke severity measurement, we used the National Institutes of Health Stroke Scale (NIHSS) score. For the subtyping of ischemic stroke, we employed the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification with minor modification1819 and classified the subtype as large-artery atherosclerosis (LAA), small-vessel occlusion (SVO), cardioembolism (CE), or undetermined etiology (UDE)/other determined etiology (ODE).","In this study, we classified the patients' residence as metropolitan (city with a population of more than 500,000), urban (city with a population of more than 50,000), or rural region according to the local government act of Korea. After geocoding of the home address and treating hospital for each patient, ground transport time was estimated based on the driving time along the best route from the patient's address to the admitting hospitals using a web-based map service which is optimized for actual traffic condition in Korea (https://map.kakao.com/). For analyses, the travel time was divided into 30-minute increments of ground transport time (i.e., 0–30, 31–60, 61–90, 90–120, and > 120 minutes). To project the findings from CRCS-K hospitals to the national level, we estimated the proportion of the Korean population that lived within each incremental service area from 67 stroke centers accredited by the Korean Stroke Society in 2018 (https://www.stroke.or.kr:4454/hospital/index2.php). To do this, we first obtained the service area from 67 stroke centers using the 30-minute incremental ground transport time threshold. Then, we overlaid these service area maps on the residential areas of 255 administrative districts of South Korea. Finally, we calculated the proportion of the population from each administrative district of South Korea within each service area based on intersection of the service area maps and residential areas. In addition, we repeated the same analysis using the whole territorial area of South Korea including both residential and nonresidential areas. Population data for each administrative district in South Korea were from the Korean National Statistical Office as of December 2019.20 ArcGIS Pro (Version 2.4.0; Esri Inc, Redlands, CA, USA) was used for all geographic analyses.","Baseline characteristics of patients were compared using a test for trend across the ordered groups divided into the 30-minute increments of ground transport time. Regression analyses were used to explore the relationship between ground transport time and the proportion of patients that received reperfusion therapies (either IVT alone or EVT). For the non-linear relationship, a restricted cubic spline function with 4 knots defined at the 30 minutes, 60 minutes, 90 minutes, and 120 minutes of ground transport time was used. In the regression analyses, covariates entered the regression models when they had P values less than 0.10 in the univariate analyses. The regression analyses were performed with R software using the “rms” package (version 3.6.0, R Foundation for Statistical Computing, Vienna, Austria). In all analyses, a two-tailed test with P value of less than 0.05 was considered significant.","This study was approved by the Institutional Review Boards of Jeju National University Hospital (approval No. JEJUNUH 2018-09-003) and all other participating centers with a waiver of informed consent of individual patients because of study subject anonymity and minimal risk to participants."],"string":"[\n \"The Clinical Research Collaboration for Stroke in Korea (CRCS-K) is a web-based prospective nationwide multicenter stroke registry.1617 The registry started enrolling patients in April 2008 and the collection of each patient's home address was permitted until August 2014. From the registry, we identified patients with acute ischemic stroke who were over 18 years old and visited one of the 12 CRCS-K hospitals within 12 hours of symptom onset time. The last known normal time was used when the onset of stroke symptom was unclear. Records were excluded if address information was incorrect or if symptom onset or hospital arrival time was unavailable. We also collected the exact time of intravenous tissue plasminogen activator (tPA) administration and groin-puncture time, and calculated onset-to-door, door-to-needle, and door-to-puncture time. We limited our analyses to the addresses within the same provincial area of the treating hospital because transferring acute stroke patients across provincial line is not considered a usual practice. Reperfusion therapy was classified as either IVT alone, vs. EVT or combined IVT and EVT. For patients who were treated with combined IVT and EVT, we identified patients who had received intravenous tPA at an outside hospital and then transferred to the CRCS-K center for EVT, i.e., drip-and-ship paradigm. For an initial stroke severity measurement, we used the National Institutes of Health Stroke Scale (NIHSS) score. For the subtyping of ischemic stroke, we employed the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification with minor modification1819 and classified the subtype as large-artery atherosclerosis (LAA), small-vessel occlusion (SVO), cardioembolism (CE), or undetermined etiology (UDE)/other determined etiology (ODE).\",\n \"In this study, we classified the patients' residence as metropolitan (city with a population of more than 500,000), urban (city with a population of more than 50,000), or rural region according to the local government act of Korea. After geocoding of the home address and treating hospital for each patient, ground transport time was estimated based on the driving time along the best route from the patient's address to the admitting hospitals using a web-based map service which is optimized for actual traffic condition in Korea (https://map.kakao.com/). For analyses, the travel time was divided into 30-minute increments of ground transport time (i.e., 0–30, 31–60, 61–90, 90–120, and > 120 minutes). To project the findings from CRCS-K hospitals to the national level, we estimated the proportion of the Korean population that lived within each incremental service area from 67 stroke centers accredited by the Korean Stroke Society in 2018 (https://www.stroke.or.kr:4454/hospital/index2.php). To do this, we first obtained the service area from 67 stroke centers using the 30-minute incremental ground transport time threshold. Then, we overlaid these service area maps on the residential areas of 255 administrative districts of South Korea. Finally, we calculated the proportion of the population from each administrative district of South Korea within each service area based on intersection of the service area maps and residential areas. In addition, we repeated the same analysis using the whole territorial area of South Korea including both residential and nonresidential areas. Population data for each administrative district in South Korea were from the Korean National Statistical Office as of December 2019.20 ArcGIS Pro (Version 2.4.0; Esri Inc, Redlands, CA, USA) was used for all geographic analyses.\",\n \"Baseline characteristics of patients were compared using a test for trend across the ordered groups divided into the 30-minute increments of ground transport time. Regression analyses were used to explore the relationship between ground transport time and the proportion of patients that received reperfusion therapies (either IVT alone or EVT). For the non-linear relationship, a restricted cubic spline function with 4 knots defined at the 30 minutes, 60 minutes, 90 minutes, and 120 minutes of ground transport time was used. In the regression analyses, covariates entered the regression models when they had P values less than 0.10 in the univariate analyses. The regression analyses were performed with R software using the “rms” package (version 3.6.0, R Foundation for Statistical Computing, Vienna, Austria). In all analyses, a two-tailed test with P value of less than 0.05 was considered significant.\",\n \"This study was approved by the Institutional Review Boards of Jeju National University Hospital (approval No. JEJUNUH 2018-09-003) and all other participating centers with a waiver of informed consent of individual patients because of study subject anonymity and minimal risk to participants.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null],"string":"[\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","METHODS","Patients","Travel distance, ground transport time, and service area","Statistical analysis","Ethics statement","RESULTS","DISCUSSION"],"string":"[\n \"INTRODUCTION\",\n \"METHODS\",\n \"Patients\",\n \"Travel distance, ground transport time, and service area\",\n \"Statistical analysis\",\n \"Ethics statement\",\n \"RESULTS\",\n \"DISCUSSION\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Geographic access to hospitals that offer acute care for patients with trauma or acute myocardial infarction can be crucial because timely treatment can improve outcomes.1234 For patients with acute ischemic stroke, access to hospitals that provide reperfusion therapy is important because the reperfusion therapy for acute ischemic stroke is also highly time-sensitive.56 Several previous studies have suggested the probability of receiving reperfusion therapies for acute ischemic stroke increased when the patients lived in close geographic proximity to hospitals that provide the therapy, however other studies did not find a significant association between the transport time from home to hospital and the use of reperfusion therapy.78910 For example, longer driving time to the treating hospital was significantly associated with longer onset-to-arrival time to the hospitals and lower odds of receiving intravenous thrombolysis (IVT) in a retrospective study of 118,683 acute stroke patients admitted to 1,489 US hospitals.10 For endovascular therapy (EVT) for acute ischemic stroke, the proportion of stroke patients receiving EVT decreased significantly when patients lived beyond a 1-hour ground transport time from hospitals that offer EVT in California during 2009 and 2010.7\nAmong patients with ST-segment elevation myocardial infarction, a wide regional variation exists in the rate of reperfusion therapy and median time from symptom onset to treatment due to differences in geography, local resource, and organization of regional health system.11 Likewise the association between geographic proximity to a hospital that offers the reperfusion therapy for acute stroke patients and the probability of receiving the therapy can vary depending on population density, geography, and systems of care.1213 Therefore, each region needs to develop an optimal geospatial modeling of acute care centers to provide maximal population access to such centers.114\nIn this study, we investigated the association between geographic proximity to a hospital that offers reperfusion therapy and the administration rate of the therapy for acute ischemic stroke in South Korea, one of the planet's most densely populated countries with a population density of 529 people per square kilometer in 2018.15","Patients The Clinical Research Collaboration for Stroke in Korea (CRCS-K) is a web-based prospective nationwide multicenter stroke registry.1617 The registry started enrolling patients in April 2008 and the collection of each patient's home address was permitted until August 2014. From the registry, we identified patients with acute ischemic stroke who were over 18 years old and visited one of the 12 CRCS-K hospitals within 12 hours of symptom onset time. The last known normal time was used when the onset of stroke symptom was unclear. Records were excluded if address information was incorrect or if symptom onset or hospital arrival time was unavailable. We also collected the exact time of intravenous tissue plasminogen activator (tPA) administration and groin-puncture time, and calculated onset-to-door, door-to-needle, and door-to-puncture time. We limited our analyses to the addresses within the same provincial area of the treating hospital because transferring acute stroke patients across provincial line is not considered a usual practice. Reperfusion therapy was classified as either IVT alone, vs. EVT or combined IVT and EVT. For patients who were treated with combined IVT and EVT, we identified patients who had received intravenous tPA at an outside hospital and then transferred to the CRCS-K center for EVT, i.e., drip-and-ship paradigm. For an initial stroke severity measurement, we used the National Institutes of Health Stroke Scale (NIHSS) score. For the subtyping of ischemic stroke, we employed the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification with minor modification1819 and classified the subtype as large-artery atherosclerosis (LAA), small-vessel occlusion (SVO), cardioembolism (CE), or undetermined etiology (UDE)/other determined etiology (ODE).\nThe Clinical Research Collaboration for Stroke in Korea (CRCS-K) is a web-based prospective nationwide multicenter stroke registry.1617 The registry started enrolling patients in April 2008 and the collection of each patient's home address was permitted until August 2014. From the registry, we identified patients with acute ischemic stroke who were over 18 years old and visited one of the 12 CRCS-K hospitals within 12 hours of symptom onset time. The last known normal time was used when the onset of stroke symptom was unclear. Records were excluded if address information was incorrect or if symptom onset or hospital arrival time was unavailable. We also collected the exact time of intravenous tissue plasminogen activator (tPA) administration and groin-puncture time, and calculated onset-to-door, door-to-needle, and door-to-puncture time. We limited our analyses to the addresses within the same provincial area of the treating hospital because transferring acute stroke patients across provincial line is not considered a usual practice. Reperfusion therapy was classified as either IVT alone, vs. EVT or combined IVT and EVT. For patients who were treated with combined IVT and EVT, we identified patients who had received intravenous tPA at an outside hospital and then transferred to the CRCS-K center for EVT, i.e., drip-and-ship paradigm. For an initial stroke severity measurement, we used the National Institutes of Health Stroke Scale (NIHSS) score. For the subtyping of ischemic stroke, we employed the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification with minor modification1819 and classified the subtype as large-artery atherosclerosis (LAA), small-vessel occlusion (SVO), cardioembolism (CE), or undetermined etiology (UDE)/other determined etiology (ODE).\nTravel distance, ground transport time, and service area In this study, we classified the patients' residence as metropolitan (city with a population of more than 500,000), urban (city with a population of more than 50,000), or rural region according to the local government act of Korea. After geocoding of the home address and treating hospital for each patient, ground transport time was estimated based on the driving time along the best route from the patient's address to the admitting hospitals using a web-based map service which is optimized for actual traffic condition in Korea (https://map.kakao.com/). For analyses, the travel time was divided into 30-minute increments of ground transport time (i.e., 0–30, 31–60, 61–90, 90–120, and > 120 minutes). To project the findings from CRCS-K hospitals to the national level, we estimated the proportion of the Korean population that lived within each incremental service area from 67 stroke centers accredited by the Korean Stroke Society in 2018 (https://www.stroke.or.kr:4454/hospital/index2.php). To do this, we first obtained the service area from 67 stroke centers using the 30-minute incremental ground transport time threshold. Then, we overlaid these service area maps on the residential areas of 255 administrative districts of South Korea. Finally, we calculated the proportion of the population from each administrative district of South Korea within each service area based on intersection of the service area maps and residential areas. In addition, we repeated the same analysis using the whole territorial area of South Korea including both residential and nonresidential areas. Population data for each administrative district in South Korea were from the Korean National Statistical Office as of December 2019.20 ArcGIS Pro (Version 2.4.0; Esri Inc, Redlands, CA, USA) was used for all geographic analyses.\nIn this study, we classified the patients' residence as metropolitan (city with a population of more than 500,000), urban (city with a population of more than 50,000), or rural region according to the local government act of Korea. After geocoding of the home address and treating hospital for each patient, ground transport time was estimated based on the driving time along the best route from the patient's address to the admitting hospitals using a web-based map service which is optimized for actual traffic condition in Korea (https://map.kakao.com/). For analyses, the travel time was divided into 30-minute increments of ground transport time (i.e., 0–30, 31–60, 61–90, 90–120, and > 120 minutes). To project the findings from CRCS-K hospitals to the national level, we estimated the proportion of the Korean population that lived within each incremental service area from 67 stroke centers accredited by the Korean Stroke Society in 2018 (https://www.stroke.or.kr:4454/hospital/index2.php). To do this, we first obtained the service area from 67 stroke centers using the 30-minute incremental ground transport time threshold. Then, we overlaid these service area maps on the residential areas of 255 administrative districts of South Korea. Finally, we calculated the proportion of the population from each administrative district of South Korea within each service area based on intersection of the service area maps and residential areas. In addition, we repeated the same analysis using the whole territorial area of South Korea including both residential and nonresidential areas. Population data for each administrative district in South Korea were from the Korean National Statistical Office as of December 2019.20 ArcGIS Pro (Version 2.4.0; Esri Inc, Redlands, CA, USA) was used for all geographic analyses.\nStatistical analysis Baseline characteristics of patients were compared using a test for trend across the ordered groups divided into the 30-minute increments of ground transport time. Regression analyses were used to explore the relationship between ground transport time and the proportion of patients that received reperfusion therapies (either IVT alone or EVT). For the non-linear relationship, a restricted cubic spline function with 4 knots defined at the 30 minutes, 60 minutes, 90 minutes, and 120 minutes of ground transport time was used. In the regression analyses, covariates entered the regression models when they had P values less than 0.10 in the univariate analyses. The regression analyses were performed with R software using the “rms” package (version 3.6.0, R Foundation for Statistical Computing, Vienna, Austria). In all analyses, a two-tailed test with P value of less than 0.05 was considered significant.\nBaseline characteristics of patients were compared using a test for trend across the ordered groups divided into the 30-minute increments of ground transport time. Regression analyses were used to explore the relationship between ground transport time and the proportion of patients that received reperfusion therapies (either IVT alone or EVT). For the non-linear relationship, a restricted cubic spline function with 4 knots defined at the 30 minutes, 60 minutes, 90 minutes, and 120 minutes of ground transport time was used. In the regression analyses, covariates entered the regression models when they had P values less than 0.10 in the univariate analyses. The regression analyses were performed with R software using the “rms” package (version 3.6.0, R Foundation for Statistical Computing, Vienna, Austria). In all analyses, a two-tailed test with P value of less than 0.05 was considered significant.\nEthics statement This study was approved by the Institutional Review Boards of Jeju National University Hospital (approval No. JEJUNUH 2018-09-003) and all other participating centers with a waiver of informed consent of individual patients because of study subject anonymity and minimal risk to participants.\nThis study was approved by the Institutional Review Boards of Jeju National University Hospital (approval No. JEJUNUH 2018-09-003) and all other participating centers with a waiver of informed consent of individual patients because of study subject anonymity and minimal risk to participants.","The Clinical Research Collaboration for Stroke in Korea (CRCS-K) is a web-based prospective nationwide multicenter stroke registry.1617 The registry started enrolling patients in April 2008 and the collection of each patient's home address was permitted until August 2014. From the registry, we identified patients with acute ischemic stroke who were over 18 years old and visited one of the 12 CRCS-K hospitals within 12 hours of symptom onset time. The last known normal time was used when the onset of stroke symptom was unclear. Records were excluded if address information was incorrect or if symptom onset or hospital arrival time was unavailable. We also collected the exact time of intravenous tissue plasminogen activator (tPA) administration and groin-puncture time, and calculated onset-to-door, door-to-needle, and door-to-puncture time. We limited our analyses to the addresses within the same provincial area of the treating hospital because transferring acute stroke patients across provincial line is not considered a usual practice. Reperfusion therapy was classified as either IVT alone, vs. EVT or combined IVT and EVT. For patients who were treated with combined IVT and EVT, we identified patients who had received intravenous tPA at an outside hospital and then transferred to the CRCS-K center for EVT, i.e., drip-and-ship paradigm. For an initial stroke severity measurement, we used the National Institutes of Health Stroke Scale (NIHSS) score. For the subtyping of ischemic stroke, we employed the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification with minor modification1819 and classified the subtype as large-artery atherosclerosis (LAA), small-vessel occlusion (SVO), cardioembolism (CE), or undetermined etiology (UDE)/other determined etiology (ODE).","In this study, we classified the patients' residence as metropolitan (city with a population of more than 500,000), urban (city with a population of more than 50,000), or rural region according to the local government act of Korea. After geocoding of the home address and treating hospital for each patient, ground transport time was estimated based on the driving time along the best route from the patient's address to the admitting hospitals using a web-based map service which is optimized for actual traffic condition in Korea (https://map.kakao.com/). For analyses, the travel time was divided into 30-minute increments of ground transport time (i.e., 0–30, 31–60, 61–90, 90–120, and > 120 minutes). To project the findings from CRCS-K hospitals to the national level, we estimated the proportion of the Korean population that lived within each incremental service area from 67 stroke centers accredited by the Korean Stroke Society in 2018 (https://www.stroke.or.kr:4454/hospital/index2.php). To do this, we first obtained the service area from 67 stroke centers using the 30-minute incremental ground transport time threshold. Then, we overlaid these service area maps on the residential areas of 255 administrative districts of South Korea. Finally, we calculated the proportion of the population from each administrative district of South Korea within each service area based on intersection of the service area maps and residential areas. In addition, we repeated the same analysis using the whole territorial area of South Korea including both residential and nonresidential areas. Population data for each administrative district in South Korea were from the Korean National Statistical Office as of December 2019.20 ArcGIS Pro (Version 2.4.0; Esri Inc, Redlands, CA, USA) was used for all geographic analyses.","Baseline characteristics of patients were compared using a test for trend across the ordered groups divided into the 30-minute increments of ground transport time. Regression analyses were used to explore the relationship between ground transport time and the proportion of patients that received reperfusion therapies (either IVT alone or EVT). For the non-linear relationship, a restricted cubic spline function with 4 knots defined at the 30 minutes, 60 minutes, 90 minutes, and 120 minutes of ground transport time was used. In the regression analyses, covariates entered the regression models when they had P values less than 0.10 in the univariate analyses. The regression analyses were performed with R software using the “rms” package (version 3.6.0, R Foundation for Statistical Computing, Vienna, Austria). In all analyses, a two-tailed test with P value of less than 0.05 was considered significant.","This study was approved by the Institutional Review Boards of Jeju National University Hospital (approval No. JEJUNUH 2018-09-003) and all other participating centers with a waiver of informed consent of individual patients because of study subject anonymity and minimal risk to participants.","Of 27,122 acute stroke patients who had been admitted to CRCS-K hospitals between April 2008 and August 2014, 12,172 patients met the eligibility criteria (Supplementary Fig. 1). Mean age was 68 ± 13 years, and 59.7% were men. Of 12,172 patients, 2,216 patients (18.2%) were treated with IVT alone and 1238 patients (10.2%) were treated with either EVT alone or combined IVT and EVT. The patients who received reperfusion therapy were more likely to be older, to have atrial fibrillation and higher initial NIHSS score (Supplementary Table 1). In this study, 48.2% of the patients' residence were classified as metropolitan, 41.3% as urban, and 10.5% as rural. The average estimated ground transport time from home to hospital was 23 minutes (interquartile range [IQR], 14–39), and median travel distance was 9.4 km (IQR, 4.5–25.1). In this study, 64.4% of the patients lived within 30 minutes or less to the admitting hospital, 88.7% within 60 minutes or less, 96.5% within 90 minutes or less, and 99.2% with 120 minutes or less. Ground transport times of 30, 60, 90, and 120 minutes corresponded to travel distances of 20.4, 53.5, 86.6, and 119.8 km, respectively. Patients that lived farther from admitting hospitals were more likely to be older, to have atrial fibrillation and CE subtype, and to have a greater initial NIHSS score, but were less likely to have hypertension, diabetes, hyperlipidemia, coronary artery disease and had a lower initial systolic blood pressure (Table 1). The ground transport time did not differ between patients treated with IVT only and patients untreated with reperfusion therapy (rank sum test P = 0.450). However, the ground transport time was slightly but significantly greater for patients treated with EVT compared with patients untreated with reperfusion therapy (24 minutes [14–44] vs. 23 minutes [14–39], rank sum test P = 0.015; Fig. 1 and Supplementary Fig. 2).\nData are presented as number (%) or mean ± standard deviation.\nIQR = interquartile range, NIHSS = National Institutes of Health Stroke Scale, LAA = large artery atherosclerosis, CE = cardioembolism, SVO = small-vessel occlusion, UDE = undetermined etiology, ODE = other determined etiology, LDL = low-density lipoprotein, BUN = blood urea nitrogen, IVT = intravenous thrombolysis, EVT = endovascular therapy.\nCRCS-K = The Clinical Research Collaboration for Stroke in Korea, IVT = intravenous thrombolysis, EVT = endovascular therapy.\nExact treatment time was available in 1,669 patients (83.4%) treated with IVT alone. The median door-to-needle time was 40 minutes (30–53) and the median onset-to-needle time was 124 minutes (88–170). As expected, the onset-to-needle time was positively correlated with ground transport time (Pearson coefficient 0.201, P < 0.001) since it took more time to arrive at the hospital. However, the door-to-needle was inversely associated with ground transport time (Pearson coefficient −0.108, P < 0.001). Regarding EVT, exact treatment time for patients treated with EVT was available for 1,077 patients (87.0%). The median door-to-puncture time was 107 minutes (85–135) and the median onset-to-puncture time was 240 minutes (170–340). The onset-to-puncture time was positively correlated with ground transport time (Pearson coefficient 0.192, P < 0.001). Like the door-to-needle time in IVT, the door-to-puncture showed a negative association with ground transport time in patients treated with EVT (Pearson coefficient −0.089, P < 0.001) (Supplementary Fig. 3).\nThe proportion of patients treated with IVT ranged from 17.1% to 22.1% among the groups with 30-minute increments of ground transport time, and proportion of EVT ranged from 7.5% to 12.4% (Supplementary Fig. 4). In the restricted cubic spline analyses, the proportion of patients treated with IVT decreased significantly when the patients lived beyond a 90-minute ground transport time from the hospital (unadjusted P = 0.003; adjusted P = 0.006). The proportion of stroke patients treated with EVT showed a similar trend with estimated ground transport time in the restricted cubic spline analyses, but it did not reach statistical significance in both unadjusted and adjusted analyses (unadjusted P = 0.086; adjusted P = 0.105) (Fig. 2).\n(A) IVT, unadjusted analysis (P = 0.003); (B) IVT, adjusted analysis (P = 0.006); adjusted for age, hypertension, diabetes, atrial fibrillation, stroke history, coronary artery disease, use of an antiplatelet agent, oral anticoagulants or statins before the index stroke, diastolic blood pressure, fasting blood glucose, low-density lipoprotein cholesterol, platelet count, onset-to-arrival time, initial NIHSS score, and TOAST classification. (C) EVT, unadjusted analysis (P = 0.090); (D) EVT, adjusted analysis (P = 0.100); adjusted for age, sex, hypertension, diabetes, atrial fibrillation, hyperlipidemia, smoking, coronary artery disease, use of an antiplatelet agent or oral anticoagulants before the index stroke, systolic blood pressure, hemoglobin, platelet count, blood urea nitrogen, onset-to-arrival time, initial NIHSS score, and TOAST classification.\nIVT = intravenous thrombolysis, EVT = endovascular therapy, NIHSS = National Institutes of Health Stroke Scale, TOAST = Trial of Org 10172 in Acute Stroke Treatment.\nOf 779 patients who had been treated with combined IVT-EVT, we could identify exact referral pattern in 439 patients (56.4%). Among 439 patients, 69 patients (15.7%) were treated by drip- and-ship paradigm. The use of drip-and-ship paradigm increased significantly with greater ground transport time (P < 0.001) (Supplementary Table 2).\nFor national coverage from the 67 stroke centers, only 9.3% of the total land area of South Korea was reachable within 30 minutes while 75.0% of the land was accessible within 90 minutes. In contrast, 73.0% of the residential area was reachable within 30 minutes of the ground transport time, and 98.4% of the area was accessible within 90 minutes (Fig. 3 and Table 2).\nCRCS-K = The Clinical Research Collaboration for Stroke in Korea.","In this nationwide analysis, we found that more than 95% of patients with acute ischemic stroke who visited one of the 12 CRCS-K hospitals within 12 hours of symptom onset had less than 90 minutes of ground transport time. The proportion of patients treated with IVT decreased significantly when patients had more than 90 minutes of the ground transport time from the hospital. The proportion of stroke patients treated with EVT also showed a similar trend with the transport time. From a national viewpoint, more than 98% of the residential area was accessible within 90 minutes of ground transport time from 67 accredited stroke centers in South Korea.\nGeographic access to hospitals offering reperfusion therapy can be crucial to successful reperfusion therapy because the time spent on symptom recognition to decision to medical seeking and the transport time from the scene to the hospital are important contributors to prehospital delays. Living close to treating hospitals was associated with a significantly higher chance of receiving thrombolytic therapy in a study conducted in St. Louis in the US.8 Interestingly, the increased use of thrombolytic therapy was not explained by earlier arrival time in that study, which indicates the presence of other factors such as the use of ambulance transport and health-seeking behavior. Other previous research that used claims data from more than 100,000 Japanese stroke patients did not find an association between driving time and administration of tissue plasminogen activator.9 Instead, they found a significant association between the use of ambulance and the use of thrombolytic therapy regardless of driving time and population density of the area. However, a recent large study enrolling 118,683 patients from the Get With The Guidelines-Stroke Registry (GWTG-Stroke) in the US found a significant association between a longer driving time to hospital and longer onset to arrival time and lower odds of IVT.10 In our study, the proportion of patients treated with IVT decreased significantly when patients had more than 90 minutes of the ground transport time from the hospital. Our findings suggest that the distance to the hospital will not be a major barrier to the use of IVT in South Korea as more than 98% of the South Korean population lives within 90 min from 67 accredited stroke centers in this study.\nAlthough several recent clinical trials have extended the treatment time window for EVT using various perfusion imaging studies, the time interval between stroke onset and reperfusion therapy is still the most important factor affecting eligibility and outcomes for reperfusion therapy in most patients with acute ischemic stroke.212223 For the association between the use of EVT and distance from the hospital, there is not much evidence from the previous literature despite the prehospital delay due to greater transport time could be also crucial to the timely performance of this life-saving therapy for acute ischemic stroke due to large vessel occlusion. In our previous study conducted in California, 94% of the stroke patients lived within a 2-hour ground transport time to the hospital offering the treatment. However, those who lived within 1-hour ground transport time had a significantly increased chance of receiving EVT compared with those who lived beyond 1-hour ground transport time (0.9% vs. 0.2%, P < 0.001).7 In this study, the proportion of stroke patients treated with EVT also decreased when the patients lived beyond a 90-minute ground transport time, however the association did not reach statistical significance. This finding is possibly due to selective referral of the patients who were candidates for EVT from other hospitals although we could not verify this due to lack of information on referral patterns. As with IVT, the ground transport time will not be an important obstacle to receive EVT in South Korea because more than 98% of the population lives within 90 min from 67 stroke centers where most of the EVT procedures would take place.\nIn addition to personal factors such as health-seeking behavior, or recognition of stroke symptoms, various systemic factors can be associated with thrombolysis administration rates in acute ischemic stroke. Studies have found that urban location, centralized acute stroke care system, use of ambulance, neurologist staffing, and use of acute stroke protocol had a significant impact on the increased use of thrombolysis.24 According to the recent report on the national averages for acute stroke care in Korea during 2013–2014, median arrival time to the hospital was 6 h and only 50% of patients with ischemic stroke used ambulance.25 Overall, IVT was used in 10.7% and EVT in 3.6% of patients with acute ischemic stroke. IVT seems to have been used more frequently than other countries although the drip-and-ship paradigm was used only in 1%. However, significant regional disparities in the rates of reperfusion therapy were found and substantial number of acute stroke patients were treated with reperfusion therapy at low-volume hospitals.2526 In another research conducted with Korean national stroke audit data, only one-third of patients who were EVT candidates were initially taken to EVT-capable hospitals and initial routing to EVT-capable hospitals was associated with more than two-folds increased chance of receiving EVT compared with initial routing to primary stroke hospital.27 Since South Korea has a universal national health insurance system with a high population density, it is advantageous for treating acute stroke. Therefore, there are a lot of opportunities to improve the rates of reperfusion therapy for stroke patients by enhancing collaboration between stroke centers and emergency medical system, and redefining roles of comprehensive and primary stroke centers with special care for rural residence.\nAs of 2018, 81.4% of the Korean population live in the urban area according to the world bank data.28 Of 12,172 patients treated at 12 CRCS-K hospitals, 1,272 patients (10.5%) lived in rural region. Therefore, the rural population was underrepresented in our study population and the actual proportion of the stroke patients who received IVT or EVT could be lower in rural regions compared with the urban area. In this study, drip-and-ship paradigm was used in only 15.7% of patients treated with combined IVT-EVT and the use of the paradigm significantly correlated with increased ground transport time. This finding is consistent with our previous work that also reported infrequent use of the paradigm (13.2%) among 1843 patients treated with intravenous tPA at the CRCS-K hospitals.29 From a national perspective, the drip-and-ship paradigm was used in only 1.0% of acute stroke patients and only one-third of EVT candidates were initially routed to EVT-capable hospitals.2527 Additionally, interhospital transfer of patients initially routed to primary stroke hospitals to EVT-capable hospital occurred in only 17.4% in Korea. Because the stroke patients who live in the rural region would be the most probable patients who need more than 90 min of ground transportation time to stroke centers, placing EVT-capable hospitals in strategic locations readily accessible to the rural population should be considered in planning nationwide coverage for stroke systems of care. The nationwide stroke systems of care should ensure effective interhospital transfer of patients for further treatment and monitoring whenever it is needed and direct routing of patients suspected of large vessel occlusion to EVT-capable hospitals.\nThis study has several limitations. First, we used the data from 2008 to 2014 when the collection of patients' home addresses was permitted. Therefore, the current study may not reflect the exact nature of the EVT population because the use of a stentriever or aspiration catheter was not as common during most of this period. Second, we do not have information on referrals and use of ambulance which could be associated with the increased use of reperfusion therapy. Referral bias might play an important role in the estimating the proportion of EVT among the patients who lived farther from the admitting hospital. Third, various personal and systemic factors could affect the rate of reperfusion therapy and many of them were not measured in our registry. Fourth, we do not have exact information for the annual volume of IVT or EVT at 67 stroke centers, and therefore we could not estimate the accessibility of the Korean population for high-volume centers separately. Lastly, we also do not have exact information on the place where the patient had suffered the stroke that led to the current admission. Patients could have developed stroke symptoms at other places and therefore the estimation of the ground transport time between the patients' residence and the admitting hospital serves only as a proxy for geographic access to treating hospitals.\nIn conclusion, more than 95% of the patients with acute ischemic stroke who visited the hospitals within 12 hours of symptom onset lived within 90 minutes of ground transport time from the hospital in this nationwide study. The use of reperfusion therapy decreased when the patients lived beyond 90 minutes of transport time from the hospital. From a national perspective, more than 98% of the South Korean population was accessible to 67 stroke centers within 90 minutes of the ground transport time."],"string":"[\n \"Geographic access to hospitals that offer acute care for patients with trauma or acute myocardial infarction can be crucial because timely treatment can improve outcomes.1234 For patients with acute ischemic stroke, access to hospitals that provide reperfusion therapy is important because the reperfusion therapy for acute ischemic stroke is also highly time-sensitive.56 Several previous studies have suggested the probability of receiving reperfusion therapies for acute ischemic stroke increased when the patients lived in close geographic proximity to hospitals that provide the therapy, however other studies did not find a significant association between the transport time from home to hospital and the use of reperfusion therapy.78910 For example, longer driving time to the treating hospital was significantly associated with longer onset-to-arrival time to the hospitals and lower odds of receiving intravenous thrombolysis (IVT) in a retrospective study of 118,683 acute stroke patients admitted to 1,489 US hospitals.10 For endovascular therapy (EVT) for acute ischemic stroke, the proportion of stroke patients receiving EVT decreased significantly when patients lived beyond a 1-hour ground transport time from hospitals that offer EVT in California during 2009 and 2010.7\\nAmong patients with ST-segment elevation myocardial infarction, a wide regional variation exists in the rate of reperfusion therapy and median time from symptom onset to treatment due to differences in geography, local resource, and organization of regional health system.11 Likewise the association between geographic proximity to a hospital that offers the reperfusion therapy for acute stroke patients and the probability of receiving the therapy can vary depending on population density, geography, and systems of care.1213 Therefore, each region needs to develop an optimal geospatial modeling of acute care centers to provide maximal population access to such centers.114\\nIn this study, we investigated the association between geographic proximity to a hospital that offers reperfusion therapy and the administration rate of the therapy for acute ischemic stroke in South Korea, one of the planet's most densely populated countries with a population density of 529 people per square kilometer in 2018.15\",\n \"Patients The Clinical Research Collaboration for Stroke in Korea (CRCS-K) is a web-based prospective nationwide multicenter stroke registry.1617 The registry started enrolling patients in April 2008 and the collection of each patient's home address was permitted until August 2014. From the registry, we identified patients with acute ischemic stroke who were over 18 years old and visited one of the 12 CRCS-K hospitals within 12 hours of symptom onset time. The last known normal time was used when the onset of stroke symptom was unclear. Records were excluded if address information was incorrect or if symptom onset or hospital arrival time was unavailable. We also collected the exact time of intravenous tissue plasminogen activator (tPA) administration and groin-puncture time, and calculated onset-to-door, door-to-needle, and door-to-puncture time. We limited our analyses to the addresses within the same provincial area of the treating hospital because transferring acute stroke patients across provincial line is not considered a usual practice. Reperfusion therapy was classified as either IVT alone, vs. EVT or combined IVT and EVT. For patients who were treated with combined IVT and EVT, we identified patients who had received intravenous tPA at an outside hospital and then transferred to the CRCS-K center for EVT, i.e., drip-and-ship paradigm. For an initial stroke severity measurement, we used the National Institutes of Health Stroke Scale (NIHSS) score. For the subtyping of ischemic stroke, we employed the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification with minor modification1819 and classified the subtype as large-artery atherosclerosis (LAA), small-vessel occlusion (SVO), cardioembolism (CE), or undetermined etiology (UDE)/other determined etiology (ODE).\\nThe Clinical Research Collaboration for Stroke in Korea (CRCS-K) is a web-based prospective nationwide multicenter stroke registry.1617 The registry started enrolling patients in April 2008 and the collection of each patient's home address was permitted until August 2014. From the registry, we identified patients with acute ischemic stroke who were over 18 years old and visited one of the 12 CRCS-K hospitals within 12 hours of symptom onset time. The last known normal time was used when the onset of stroke symptom was unclear. Records were excluded if address information was incorrect or if symptom onset or hospital arrival time was unavailable. We also collected the exact time of intravenous tissue plasminogen activator (tPA) administration and groin-puncture time, and calculated onset-to-door, door-to-needle, and door-to-puncture time. We limited our analyses to the addresses within the same provincial area of the treating hospital because transferring acute stroke patients across provincial line is not considered a usual practice. Reperfusion therapy was classified as either IVT alone, vs. EVT or combined IVT and EVT. For patients who were treated with combined IVT and EVT, we identified patients who had received intravenous tPA at an outside hospital and then transferred to the CRCS-K center for EVT, i.e., drip-and-ship paradigm. For an initial stroke severity measurement, we used the National Institutes of Health Stroke Scale (NIHSS) score. For the subtyping of ischemic stroke, we employed the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification with minor modification1819 and classified the subtype as large-artery atherosclerosis (LAA), small-vessel occlusion (SVO), cardioembolism (CE), or undetermined etiology (UDE)/other determined etiology (ODE).\\nTravel distance, ground transport time, and service area In this study, we classified the patients' residence as metropolitan (city with a population of more than 500,000), urban (city with a population of more than 50,000), or rural region according to the local government act of Korea. After geocoding of the home address and treating hospital for each patient, ground transport time was estimated based on the driving time along the best route from the patient's address to the admitting hospitals using a web-based map service which is optimized for actual traffic condition in Korea (https://map.kakao.com/). For analyses, the travel time was divided into 30-minute increments of ground transport time (i.e., 0–30, 31–60, 61–90, 90–120, and > 120 minutes). To project the findings from CRCS-K hospitals to the national level, we estimated the proportion of the Korean population that lived within each incremental service area from 67 stroke centers accredited by the Korean Stroke Society in 2018 (https://www.stroke.or.kr:4454/hospital/index2.php). To do this, we first obtained the service area from 67 stroke centers using the 30-minute incremental ground transport time threshold. Then, we overlaid these service area maps on the residential areas of 255 administrative districts of South Korea. Finally, we calculated the proportion of the population from each administrative district of South Korea within each service area based on intersection of the service area maps and residential areas. In addition, we repeated the same analysis using the whole territorial area of South Korea including both residential and nonresidential areas. Population data for each administrative district in South Korea were from the Korean National Statistical Office as of December 2019.20 ArcGIS Pro (Version 2.4.0; Esri Inc, Redlands, CA, USA) was used for all geographic analyses.\\nIn this study, we classified the patients' residence as metropolitan (city with a population of more than 500,000), urban (city with a population of more than 50,000), or rural region according to the local government act of Korea. After geocoding of the home address and treating hospital for each patient, ground transport time was estimated based on the driving time along the best route from the patient's address to the admitting hospitals using a web-based map service which is optimized for actual traffic condition in Korea (https://map.kakao.com/). For analyses, the travel time was divided into 30-minute increments of ground transport time (i.e., 0–30, 31–60, 61–90, 90–120, and > 120 minutes). To project the findings from CRCS-K hospitals to the national level, we estimated the proportion of the Korean population that lived within each incremental service area from 67 stroke centers accredited by the Korean Stroke Society in 2018 (https://www.stroke.or.kr:4454/hospital/index2.php). To do this, we first obtained the service area from 67 stroke centers using the 30-minute incremental ground transport time threshold. Then, we overlaid these service area maps on the residential areas of 255 administrative districts of South Korea. Finally, we calculated the proportion of the population from each administrative district of South Korea within each service area based on intersection of the service area maps and residential areas. In addition, we repeated the same analysis using the whole territorial area of South Korea including both residential and nonresidential areas. Population data for each administrative district in South Korea were from the Korean National Statistical Office as of December 2019.20 ArcGIS Pro (Version 2.4.0; Esri Inc, Redlands, CA, USA) was used for all geographic analyses.\\nStatistical analysis Baseline characteristics of patients were compared using a test for trend across the ordered groups divided into the 30-minute increments of ground transport time. Regression analyses were used to explore the relationship between ground transport time and the proportion of patients that received reperfusion therapies (either IVT alone or EVT). For the non-linear relationship, a restricted cubic spline function with 4 knots defined at the 30 minutes, 60 minutes, 90 minutes, and 120 minutes of ground transport time was used. In the regression analyses, covariates entered the regression models when they had P values less than 0.10 in the univariate analyses. The regression analyses were performed with R software using the “rms” package (version 3.6.0, R Foundation for Statistical Computing, Vienna, Austria). In all analyses, a two-tailed test with P value of less than 0.05 was considered significant.\\nBaseline characteristics of patients were compared using a test for trend across the ordered groups divided into the 30-minute increments of ground transport time. Regression analyses were used to explore the relationship between ground transport time and the proportion of patients that received reperfusion therapies (either IVT alone or EVT). For the non-linear relationship, a restricted cubic spline function with 4 knots defined at the 30 minutes, 60 minutes, 90 minutes, and 120 minutes of ground transport time was used. In the regression analyses, covariates entered the regression models when they had P values less than 0.10 in the univariate analyses. The regression analyses were performed with R software using the “rms” package (version 3.6.0, R Foundation for Statistical Computing, Vienna, Austria). In all analyses, a two-tailed test with P value of less than 0.05 was considered significant.\\nEthics statement This study was approved by the Institutional Review Boards of Jeju National University Hospital (approval No. JEJUNUH 2018-09-003) and all other participating centers with a waiver of informed consent of individual patients because of study subject anonymity and minimal risk to participants.\\nThis study was approved by the Institutional Review Boards of Jeju National University Hospital (approval No. JEJUNUH 2018-09-003) and all other participating centers with a waiver of informed consent of individual patients because of study subject anonymity and minimal risk to participants.\",\n \"The Clinical Research Collaboration for Stroke in Korea (CRCS-K) is a web-based prospective nationwide multicenter stroke registry.1617 The registry started enrolling patients in April 2008 and the collection of each patient's home address was permitted until August 2014. From the registry, we identified patients with acute ischemic stroke who were over 18 years old and visited one of the 12 CRCS-K hospitals within 12 hours of symptom onset time. The last known normal time was used when the onset of stroke symptom was unclear. Records were excluded if address information was incorrect or if symptom onset or hospital arrival time was unavailable. We also collected the exact time of intravenous tissue plasminogen activator (tPA) administration and groin-puncture time, and calculated onset-to-door, door-to-needle, and door-to-puncture time. We limited our analyses to the addresses within the same provincial area of the treating hospital because transferring acute stroke patients across provincial line is not considered a usual practice. Reperfusion therapy was classified as either IVT alone, vs. EVT or combined IVT and EVT. For patients who were treated with combined IVT and EVT, we identified patients who had received intravenous tPA at an outside hospital and then transferred to the CRCS-K center for EVT, i.e., drip-and-ship paradigm. For an initial stroke severity measurement, we used the National Institutes of Health Stroke Scale (NIHSS) score. For the subtyping of ischemic stroke, we employed the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification with minor modification1819 and classified the subtype as large-artery atherosclerosis (LAA), small-vessel occlusion (SVO), cardioembolism (CE), or undetermined etiology (UDE)/other determined etiology (ODE).\",\n \"In this study, we classified the patients' residence as metropolitan (city with a population of more than 500,000), urban (city with a population of more than 50,000), or rural region according to the local government act of Korea. After geocoding of the home address and treating hospital for each patient, ground transport time was estimated based on the driving time along the best route from the patient's address to the admitting hospitals using a web-based map service which is optimized for actual traffic condition in Korea (https://map.kakao.com/). For analyses, the travel time was divided into 30-minute increments of ground transport time (i.e., 0–30, 31–60, 61–90, 90–120, and > 120 minutes). To project the findings from CRCS-K hospitals to the national level, we estimated the proportion of the Korean population that lived within each incremental service area from 67 stroke centers accredited by the Korean Stroke Society in 2018 (https://www.stroke.or.kr:4454/hospital/index2.php). To do this, we first obtained the service area from 67 stroke centers using the 30-minute incremental ground transport time threshold. Then, we overlaid these service area maps on the residential areas of 255 administrative districts of South Korea. Finally, we calculated the proportion of the population from each administrative district of South Korea within each service area based on intersection of the service area maps and residential areas. In addition, we repeated the same analysis using the whole territorial area of South Korea including both residential and nonresidential areas. Population data for each administrative district in South Korea were from the Korean National Statistical Office as of December 2019.20 ArcGIS Pro (Version 2.4.0; Esri Inc, Redlands, CA, USA) was used for all geographic analyses.\",\n \"Baseline characteristics of patients were compared using a test for trend across the ordered groups divided into the 30-minute increments of ground transport time. Regression analyses were used to explore the relationship between ground transport time and the proportion of patients that received reperfusion therapies (either IVT alone or EVT). For the non-linear relationship, a restricted cubic spline function with 4 knots defined at the 30 minutes, 60 minutes, 90 minutes, and 120 minutes of ground transport time was used. In the regression analyses, covariates entered the regression models when they had P values less than 0.10 in the univariate analyses. The regression analyses were performed with R software using the “rms” package (version 3.6.0, R Foundation for Statistical Computing, Vienna, Austria). In all analyses, a two-tailed test with P value of less than 0.05 was considered significant.\",\n \"This study was approved by the Institutional Review Boards of Jeju National University Hospital (approval No. JEJUNUH 2018-09-003) and all other participating centers with a waiver of informed consent of individual patients because of study subject anonymity and minimal risk to participants.\",\n \"Of 27,122 acute stroke patients who had been admitted to CRCS-K hospitals between April 2008 and August 2014, 12,172 patients met the eligibility criteria (Supplementary Fig. 1). Mean age was 68 ± 13 years, and 59.7% were men. Of 12,172 patients, 2,216 patients (18.2%) were treated with IVT alone and 1238 patients (10.2%) were treated with either EVT alone or combined IVT and EVT. The patients who received reperfusion therapy were more likely to be older, to have atrial fibrillation and higher initial NIHSS score (Supplementary Table 1). In this study, 48.2% of the patients' residence were classified as metropolitan, 41.3% as urban, and 10.5% as rural. The average estimated ground transport time from home to hospital was 23 minutes (interquartile range [IQR], 14–39), and median travel distance was 9.4 km (IQR, 4.5–25.1). In this study, 64.4% of the patients lived within 30 minutes or less to the admitting hospital, 88.7% within 60 minutes or less, 96.5% within 90 minutes or less, and 99.2% with 120 minutes or less. Ground transport times of 30, 60, 90, and 120 minutes corresponded to travel distances of 20.4, 53.5, 86.6, and 119.8 km, respectively. Patients that lived farther from admitting hospitals were more likely to be older, to have atrial fibrillation and CE subtype, and to have a greater initial NIHSS score, but were less likely to have hypertension, diabetes, hyperlipidemia, coronary artery disease and had a lower initial systolic blood pressure (Table 1). The ground transport time did not differ between patients treated with IVT only and patients untreated with reperfusion therapy (rank sum test P = 0.450). However, the ground transport time was slightly but significantly greater for patients treated with EVT compared with patients untreated with reperfusion therapy (24 minutes [14–44] vs. 23 minutes [14–39], rank sum test P = 0.015; Fig. 1 and Supplementary Fig. 2).\\nData are presented as number (%) or mean ± standard deviation.\\nIQR = interquartile range, NIHSS = National Institutes of Health Stroke Scale, LAA = large artery atherosclerosis, CE = cardioembolism, SVO = small-vessel occlusion, UDE = undetermined etiology, ODE = other determined etiology, LDL = low-density lipoprotein, BUN = blood urea nitrogen, IVT = intravenous thrombolysis, EVT = endovascular therapy.\\nCRCS-K = The Clinical Research Collaboration for Stroke in Korea, IVT = intravenous thrombolysis, EVT = endovascular therapy.\\nExact treatment time was available in 1,669 patients (83.4%) treated with IVT alone. The median door-to-needle time was 40 minutes (30–53) and the median onset-to-needle time was 124 minutes (88–170). As expected, the onset-to-needle time was positively correlated with ground transport time (Pearson coefficient 0.201, P < 0.001) since it took more time to arrive at the hospital. However, the door-to-needle was inversely associated with ground transport time (Pearson coefficient −0.108, P < 0.001). Regarding EVT, exact treatment time for patients treated with EVT was available for 1,077 patients (87.0%). The median door-to-puncture time was 107 minutes (85–135) and the median onset-to-puncture time was 240 minutes (170–340). The onset-to-puncture time was positively correlated with ground transport time (Pearson coefficient 0.192, P < 0.001). Like the door-to-needle time in IVT, the door-to-puncture showed a negative association with ground transport time in patients treated with EVT (Pearson coefficient −0.089, P < 0.001) (Supplementary Fig. 3).\\nThe proportion of patients treated with IVT ranged from 17.1% to 22.1% among the groups with 30-minute increments of ground transport time, and proportion of EVT ranged from 7.5% to 12.4% (Supplementary Fig. 4). In the restricted cubic spline analyses, the proportion of patients treated with IVT decreased significantly when the patients lived beyond a 90-minute ground transport time from the hospital (unadjusted P = 0.003; adjusted P = 0.006). The proportion of stroke patients treated with EVT showed a similar trend with estimated ground transport time in the restricted cubic spline analyses, but it did not reach statistical significance in both unadjusted and adjusted analyses (unadjusted P = 0.086; adjusted P = 0.105) (Fig. 2).\\n(A) IVT, unadjusted analysis (P = 0.003); (B) IVT, adjusted analysis (P = 0.006); adjusted for age, hypertension, diabetes, atrial fibrillation, stroke history, coronary artery disease, use of an antiplatelet agent, oral anticoagulants or statins before the index stroke, diastolic blood pressure, fasting blood glucose, low-density lipoprotein cholesterol, platelet count, onset-to-arrival time, initial NIHSS score, and TOAST classification. (C) EVT, unadjusted analysis (P = 0.090); (D) EVT, adjusted analysis (P = 0.100); adjusted for age, sex, hypertension, diabetes, atrial fibrillation, hyperlipidemia, smoking, coronary artery disease, use of an antiplatelet agent or oral anticoagulants before the index stroke, systolic blood pressure, hemoglobin, platelet count, blood urea nitrogen, onset-to-arrival time, initial NIHSS score, and TOAST classification.\\nIVT = intravenous thrombolysis, EVT = endovascular therapy, NIHSS = National Institutes of Health Stroke Scale, TOAST = Trial of Org 10172 in Acute Stroke Treatment.\\nOf 779 patients who had been treated with combined IVT-EVT, we could identify exact referral pattern in 439 patients (56.4%). Among 439 patients, 69 patients (15.7%) were treated by drip- and-ship paradigm. The use of drip-and-ship paradigm increased significantly with greater ground transport time (P < 0.001) (Supplementary Table 2).\\nFor national coverage from the 67 stroke centers, only 9.3% of the total land area of South Korea was reachable within 30 minutes while 75.0% of the land was accessible within 90 minutes. In contrast, 73.0% of the residential area was reachable within 30 minutes of the ground transport time, and 98.4% of the area was accessible within 90 minutes (Fig. 3 and Table 2).\\nCRCS-K = The Clinical Research Collaboration for Stroke in Korea.\",\n \"In this nationwide analysis, we found that more than 95% of patients with acute ischemic stroke who visited one of the 12 CRCS-K hospitals within 12 hours of symptom onset had less than 90 minutes of ground transport time. The proportion of patients treated with IVT decreased significantly when patients had more than 90 minutes of the ground transport time from the hospital. The proportion of stroke patients treated with EVT also showed a similar trend with the transport time. From a national viewpoint, more than 98% of the residential area was accessible within 90 minutes of ground transport time from 67 accredited stroke centers in South Korea.\\nGeographic access to hospitals offering reperfusion therapy can be crucial to successful reperfusion therapy because the time spent on symptom recognition to decision to medical seeking and the transport time from the scene to the hospital are important contributors to prehospital delays. Living close to treating hospitals was associated with a significantly higher chance of receiving thrombolytic therapy in a study conducted in St. Louis in the US.8 Interestingly, the increased use of thrombolytic therapy was not explained by earlier arrival time in that study, which indicates the presence of other factors such as the use of ambulance transport and health-seeking behavior. Other previous research that used claims data from more than 100,000 Japanese stroke patients did not find an association between driving time and administration of tissue plasminogen activator.9 Instead, they found a significant association between the use of ambulance and the use of thrombolytic therapy regardless of driving time and population density of the area. However, a recent large study enrolling 118,683 patients from the Get With The Guidelines-Stroke Registry (GWTG-Stroke) in the US found a significant association between a longer driving time to hospital and longer onset to arrival time and lower odds of IVT.10 In our study, the proportion of patients treated with IVT decreased significantly when patients had more than 90 minutes of the ground transport time from the hospital. Our findings suggest that the distance to the hospital will not be a major barrier to the use of IVT in South Korea as more than 98% of the South Korean population lives within 90 min from 67 accredited stroke centers in this study.\\nAlthough several recent clinical trials have extended the treatment time window for EVT using various perfusion imaging studies, the time interval between stroke onset and reperfusion therapy is still the most important factor affecting eligibility and outcomes for reperfusion therapy in most patients with acute ischemic stroke.212223 For the association between the use of EVT and distance from the hospital, there is not much evidence from the previous literature despite the prehospital delay due to greater transport time could be also crucial to the timely performance of this life-saving therapy for acute ischemic stroke due to large vessel occlusion. In our previous study conducted in California, 94% of the stroke patients lived within a 2-hour ground transport time to the hospital offering the treatment. However, those who lived within 1-hour ground transport time had a significantly increased chance of receiving EVT compared with those who lived beyond 1-hour ground transport time (0.9% vs. 0.2%, P < 0.001).7 In this study, the proportion of stroke patients treated with EVT also decreased when the patients lived beyond a 90-minute ground transport time, however the association did not reach statistical significance. This finding is possibly due to selective referral of the patients who were candidates for EVT from other hospitals although we could not verify this due to lack of information on referral patterns. As with IVT, the ground transport time will not be an important obstacle to receive EVT in South Korea because more than 98% of the population lives within 90 min from 67 stroke centers where most of the EVT procedures would take place.\\nIn addition to personal factors such as health-seeking behavior, or recognition of stroke symptoms, various systemic factors can be associated with thrombolysis administration rates in acute ischemic stroke. Studies have found that urban location, centralized acute stroke care system, use of ambulance, neurologist staffing, and use of acute stroke protocol had a significant impact on the increased use of thrombolysis.24 According to the recent report on the national averages for acute stroke care in Korea during 2013–2014, median arrival time to the hospital was 6 h and only 50% of patients with ischemic stroke used ambulance.25 Overall, IVT was used in 10.7% and EVT in 3.6% of patients with acute ischemic stroke. IVT seems to have been used more frequently than other countries although the drip-and-ship paradigm was used only in 1%. However, significant regional disparities in the rates of reperfusion therapy were found and substantial number of acute stroke patients were treated with reperfusion therapy at low-volume hospitals.2526 In another research conducted with Korean national stroke audit data, only one-third of patients who were EVT candidates were initially taken to EVT-capable hospitals and initial routing to EVT-capable hospitals was associated with more than two-folds increased chance of receiving EVT compared with initial routing to primary stroke hospital.27 Since South Korea has a universal national health insurance system with a high population density, it is advantageous for treating acute stroke. Therefore, there are a lot of opportunities to improve the rates of reperfusion therapy for stroke patients by enhancing collaboration between stroke centers and emergency medical system, and redefining roles of comprehensive and primary stroke centers with special care for rural residence.\\nAs of 2018, 81.4% of the Korean population live in the urban area according to the world bank data.28 Of 12,172 patients treated at 12 CRCS-K hospitals, 1,272 patients (10.5%) lived in rural region. Therefore, the rural population was underrepresented in our study population and the actual proportion of the stroke patients who received IVT or EVT could be lower in rural regions compared with the urban area. In this study, drip-and-ship paradigm was used in only 15.7% of patients treated with combined IVT-EVT and the use of the paradigm significantly correlated with increased ground transport time. This finding is consistent with our previous work that also reported infrequent use of the paradigm (13.2%) among 1843 patients treated with intravenous tPA at the CRCS-K hospitals.29 From a national perspective, the drip-and-ship paradigm was used in only 1.0% of acute stroke patients and only one-third of EVT candidates were initially routed to EVT-capable hospitals.2527 Additionally, interhospital transfer of patients initially routed to primary stroke hospitals to EVT-capable hospital occurred in only 17.4% in Korea. Because the stroke patients who live in the rural region would be the most probable patients who need more than 90 min of ground transportation time to stroke centers, placing EVT-capable hospitals in strategic locations readily accessible to the rural population should be considered in planning nationwide coverage for stroke systems of care. The nationwide stroke systems of care should ensure effective interhospital transfer of patients for further treatment and monitoring whenever it is needed and direct routing of patients suspected of large vessel occlusion to EVT-capable hospitals.\\nThis study has several limitations. First, we used the data from 2008 to 2014 when the collection of patients' home addresses was permitted. Therefore, the current study may not reflect the exact nature of the EVT population because the use of a stentriever or aspiration catheter was not as common during most of this period. Second, we do not have information on referrals and use of ambulance which could be associated with the increased use of reperfusion therapy. Referral bias might play an important role in the estimating the proportion of EVT among the patients who lived farther from the admitting hospital. Third, various personal and systemic factors could affect the rate of reperfusion therapy and many of them were not measured in our registry. Fourth, we do not have exact information for the annual volume of IVT or EVT at 67 stroke centers, and therefore we could not estimate the accessibility of the Korean population for high-volume centers separately. Lastly, we also do not have exact information on the place where the patient had suffered the stroke that led to the current admission. Patients could have developed stroke symptoms at other places and therefore the estimation of the ground transport time between the patients' residence and the admitting hospital serves only as a proxy for geographic access to treating hospitals.\\nIn conclusion, more than 95% of the patients with acute ischemic stroke who visited the hospitals within 12 hours of symptom onset lived within 90 minutes of ground transport time from the hospital in this nationwide study. The use of reperfusion therapy decreased when the patients lived beyond 90 minutes of transport time from the hospital. From a national perspective, more than 98% of the South Korean population was accessible to 67 stroke centers within 90 minutes of the ground transport time.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,null,null,null,"results","discussion"],"string":"[\n \"intro\",\n \"methods\",\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["Ischemic Stroke","Reperfusion","Thrombolysis","Endovascular Treatment","Utilization"],"string":"[\n \"Ischemic Stroke\",\n \"Reperfusion\",\n \"Thrombolysis\",\n \"Endovascular Treatment\",\n \"Utilization\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nGeographic access to hospitals that offer acute care for patients with trauma or acute myocardial infarction can be crucial because timely treatment can improve outcomes.1234 For patients with acute ischemic stroke, access to hospitals that provide reperfusion therapy is important because the reperfusion therapy for acute ischemic stroke is also highly time-sensitive.56 Several previous studies have suggested the probability of receiving reperfusion therapies for acute ischemic stroke increased when the patients lived in close geographic proximity to hospitals that provide the therapy, however other studies did not find a significant association between the transport time from home to hospital and the use of reperfusion therapy.78910 For example, longer driving time to the treating hospital was significantly associated with longer onset-to-arrival time to the hospitals and lower odds of receiving intravenous thrombolysis (IVT) in a retrospective study of 118,683 acute stroke patients admitted to 1,489 US hospitals.10 For endovascular therapy (EVT) for acute ischemic stroke, the proportion of stroke patients receiving EVT decreased significantly when patients lived beyond a 1-hour ground transport time from hospitals that offer EVT in California during 2009 and 2010.7\nAmong patients with ST-segment elevation myocardial infarction, a wide regional variation exists in the rate of reperfusion therapy and median time from symptom onset to treatment due to differences in geography, local resource, and organization of regional health system.11 Likewise the association between geographic proximity to a hospital that offers the reperfusion therapy for acute stroke patients and the probability of receiving the therapy can vary depending on population density, geography, and systems of care.1213 Therefore, each region needs to develop an optimal geospatial modeling of acute care centers to provide maximal population access to such centers.114\nIn this study, we investigated the association between geographic proximity to a hospital that offers reperfusion therapy and the administration rate of the therapy for acute ischemic stroke in South Korea, one of the planet's most densely populated countries with a population density of 529 people per square kilometer in 2018.15\n\nMETHODS:\nPatients The Clinical Research Collaboration for Stroke in Korea (CRCS-K) is a web-based prospective nationwide multicenter stroke registry.1617 The registry started enrolling patients in April 2008 and the collection of each patient's home address was permitted until August 2014. From the registry, we identified patients with acute ischemic stroke who were over 18 years old and visited one of the 12 CRCS-K hospitals within 12 hours of symptom onset time. The last known normal time was used when the onset of stroke symptom was unclear. Records were excluded if address information was incorrect or if symptom onset or hospital arrival time was unavailable. We also collected the exact time of intravenous tissue plasminogen activator (tPA) administration and groin-puncture time, and calculated onset-to-door, door-to-needle, and door-to-puncture time. We limited our analyses to the addresses within the same provincial area of the treating hospital because transferring acute stroke patients across provincial line is not considered a usual practice. Reperfusion therapy was classified as either IVT alone, vs. EVT or combined IVT and EVT. For patients who were treated with combined IVT and EVT, we identified patients who had received intravenous tPA at an outside hospital and then transferred to the CRCS-K center for EVT, i.e., drip-and-ship paradigm. For an initial stroke severity measurement, we used the National Institutes of Health Stroke Scale (NIHSS) score. For the subtyping of ischemic stroke, we employed the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification with minor modification1819 and classified the subtype as large-artery atherosclerosis (LAA), small-vessel occlusion (SVO), cardioembolism (CE), or undetermined etiology (UDE)/other determined etiology (ODE).\nThe Clinical Research Collaboration for Stroke in Korea (CRCS-K) is a web-based prospective nationwide multicenter stroke registry.1617 The registry started enrolling patients in April 2008 and the collection of each patient's home address was permitted until August 2014. From the registry, we identified patients with acute ischemic stroke who were over 18 years old and visited one of the 12 CRCS-K hospitals within 12 hours of symptom onset time. The last known normal time was used when the onset of stroke symptom was unclear. Records were excluded if address information was incorrect or if symptom onset or hospital arrival time was unavailable. We also collected the exact time of intravenous tissue plasminogen activator (tPA) administration and groin-puncture time, and calculated onset-to-door, door-to-needle, and door-to-puncture time. We limited our analyses to the addresses within the same provincial area of the treating hospital because transferring acute stroke patients across provincial line is not considered a usual practice. Reperfusion therapy was classified as either IVT alone, vs. EVT or combined IVT and EVT. For patients who were treated with combined IVT and EVT, we identified patients who had received intravenous tPA at an outside hospital and then transferred to the CRCS-K center for EVT, i.e., drip-and-ship paradigm. For an initial stroke severity measurement, we used the National Institutes of Health Stroke Scale (NIHSS) score. For the subtyping of ischemic stroke, we employed the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification with minor modification1819 and classified the subtype as large-artery atherosclerosis (LAA), small-vessel occlusion (SVO), cardioembolism (CE), or undetermined etiology (UDE)/other determined etiology (ODE).\nTravel distance, ground transport time, and service area In this study, we classified the patients' residence as metropolitan (city with a population of more than 500,000), urban (city with a population of more than 50,000), or rural region according to the local government act of Korea. After geocoding of the home address and treating hospital for each patient, ground transport time was estimated based on the driving time along the best route from the patient's address to the admitting hospitals using a web-based map service which is optimized for actual traffic condition in Korea (https://map.kakao.com/). For analyses, the travel time was divided into 30-minute increments of ground transport time (i.e., 0–30, 31–60, 61–90, 90–120, and > 120 minutes). To project the findings from CRCS-K hospitals to the national level, we estimated the proportion of the Korean population that lived within each incremental service area from 67 stroke centers accredited by the Korean Stroke Society in 2018 (https://www.stroke.or.kr:4454/hospital/index2.php). To do this, we first obtained the service area from 67 stroke centers using the 30-minute incremental ground transport time threshold. Then, we overlaid these service area maps on the residential areas of 255 administrative districts of South Korea. Finally, we calculated the proportion of the population from each administrative district of South Korea within each service area based on intersection of the service area maps and residential areas. In addition, we repeated the same analysis using the whole territorial area of South Korea including both residential and nonresidential areas. Population data for each administrative district in South Korea were from the Korean National Statistical Office as of December 2019.20 ArcGIS Pro (Version 2.4.0; Esri Inc, Redlands, CA, USA) was used for all geographic analyses.\nIn this study, we classified the patients' residence as metropolitan (city with a population of more than 500,000), urban (city with a population of more than 50,000), or rural region according to the local government act of Korea. After geocoding of the home address and treating hospital for each patient, ground transport time was estimated based on the driving time along the best route from the patient's address to the admitting hospitals using a web-based map service which is optimized for actual traffic condition in Korea (https://map.kakao.com/). For analyses, the travel time was divided into 30-minute increments of ground transport time (i.e., 0–30, 31–60, 61–90, 90–120, and > 120 minutes). To project the findings from CRCS-K hospitals to the national level, we estimated the proportion of the Korean population that lived within each incremental service area from 67 stroke centers accredited by the Korean Stroke Society in 2018 (https://www.stroke.or.kr:4454/hospital/index2.php). To do this, we first obtained the service area from 67 stroke centers using the 30-minute incremental ground transport time threshold. Then, we overlaid these service area maps on the residential areas of 255 administrative districts of South Korea. Finally, we calculated the proportion of the population from each administrative district of South Korea within each service area based on intersection of the service area maps and residential areas. In addition, we repeated the same analysis using the whole territorial area of South Korea including both residential and nonresidential areas. Population data for each administrative district in South Korea were from the Korean National Statistical Office as of December 2019.20 ArcGIS Pro (Version 2.4.0; Esri Inc, Redlands, CA, USA) was used for all geographic analyses.\nStatistical analysis Baseline characteristics of patients were compared using a test for trend across the ordered groups divided into the 30-minute increments of ground transport time. Regression analyses were used to explore the relationship between ground transport time and the proportion of patients that received reperfusion therapies (either IVT alone or EVT). For the non-linear relationship, a restricted cubic spline function with 4 knots defined at the 30 minutes, 60 minutes, 90 minutes, and 120 minutes of ground transport time was used. In the regression analyses, covariates entered the regression models when they had P values less than 0.10 in the univariate analyses. The regression analyses were performed with R software using the “rms” package (version 3.6.0, R Foundation for Statistical Computing, Vienna, Austria). In all analyses, a two-tailed test with P value of less than 0.05 was considered significant.\nBaseline characteristics of patients were compared using a test for trend across the ordered groups divided into the 30-minute increments of ground transport time. Regression analyses were used to explore the relationship between ground transport time and the proportion of patients that received reperfusion therapies (either IVT alone or EVT). For the non-linear relationship, a restricted cubic spline function with 4 knots defined at the 30 minutes, 60 minutes, 90 minutes, and 120 minutes of ground transport time was used. In the regression analyses, covariates entered the regression models when they had P values less than 0.10 in the univariate analyses. The regression analyses were performed with R software using the “rms” package (version 3.6.0, R Foundation for Statistical Computing, Vienna, Austria). In all analyses, a two-tailed test with P value of less than 0.05 was considered significant.\nEthics statement This study was approved by the Institutional Review Boards of Jeju National University Hospital (approval No. JEJUNUH 2018-09-003) and all other participating centers with a waiver of informed consent of individual patients because of study subject anonymity and minimal risk to participants.\nThis study was approved by the Institutional Review Boards of Jeju National University Hospital (approval No. JEJUNUH 2018-09-003) and all other participating centers with a waiver of informed consent of individual patients because of study subject anonymity and minimal risk to participants.\n\nPatients:\nThe Clinical Research Collaboration for Stroke in Korea (CRCS-K) is a web-based prospective nationwide multicenter stroke registry.1617 The registry started enrolling patients in April 2008 and the collection of each patient's home address was permitted until August 2014. From the registry, we identified patients with acute ischemic stroke who were over 18 years old and visited one of the 12 CRCS-K hospitals within 12 hours of symptom onset time. The last known normal time was used when the onset of stroke symptom was unclear. Records were excluded if address information was incorrect or if symptom onset or hospital arrival time was unavailable. We also collected the exact time of intravenous tissue plasminogen activator (tPA) administration and groin-puncture time, and calculated onset-to-door, door-to-needle, and door-to-puncture time. We limited our analyses to the addresses within the same provincial area of the treating hospital because transferring acute stroke patients across provincial line is not considered a usual practice. Reperfusion therapy was classified as either IVT alone, vs. EVT or combined IVT and EVT. For patients who were treated with combined IVT and EVT, we identified patients who had received intravenous tPA at an outside hospital and then transferred to the CRCS-K center for EVT, i.e., drip-and-ship paradigm. For an initial stroke severity measurement, we used the National Institutes of Health Stroke Scale (NIHSS) score. For the subtyping of ischemic stroke, we employed the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification with minor modification1819 and classified the subtype as large-artery atherosclerosis (LAA), small-vessel occlusion (SVO), cardioembolism (CE), or undetermined etiology (UDE)/other determined etiology (ODE).\n\nTravel distance, ground transport time, and service area:\nIn this study, we classified the patients' residence as metropolitan (city with a population of more than 500,000), urban (city with a population of more than 50,000), or rural region according to the local government act of Korea. After geocoding of the home address and treating hospital for each patient, ground transport time was estimated based on the driving time along the best route from the patient's address to the admitting hospitals using a web-based map service which is optimized for actual traffic condition in Korea (https://map.kakao.com/). For analyses, the travel time was divided into 30-minute increments of ground transport time (i.e., 0–30, 31–60, 61–90, 90–120, and > 120 minutes). To project the findings from CRCS-K hospitals to the national level, we estimated the proportion of the Korean population that lived within each incremental service area from 67 stroke centers accredited by the Korean Stroke Society in 2018 (https://www.stroke.or.kr:4454/hospital/index2.php). To do this, we first obtained the service area from 67 stroke centers using the 30-minute incremental ground transport time threshold. Then, we overlaid these service area maps on the residential areas of 255 administrative districts of South Korea. Finally, we calculated the proportion of the population from each administrative district of South Korea within each service area based on intersection of the service area maps and residential areas. In addition, we repeated the same analysis using the whole territorial area of South Korea including both residential and nonresidential areas. Population data for each administrative district in South Korea were from the Korean National Statistical Office as of December 2019.20 ArcGIS Pro (Version 2.4.0; Esri Inc, Redlands, CA, USA) was used for all geographic analyses.\n\nStatistical analysis:\nBaseline characteristics of patients were compared using a test for trend across the ordered groups divided into the 30-minute increments of ground transport time. Regression analyses were used to explore the relationship between ground transport time and the proportion of patients that received reperfusion therapies (either IVT alone or EVT). For the non-linear relationship, a restricted cubic spline function with 4 knots defined at the 30 minutes, 60 minutes, 90 minutes, and 120 minutes of ground transport time was used. In the regression analyses, covariates entered the regression models when they had P values less than 0.10 in the univariate analyses. The regression analyses were performed with R software using the “rms” package (version 3.6.0, R Foundation for Statistical Computing, Vienna, Austria). In all analyses, a two-tailed test with P value of less than 0.05 was considered significant.\n\nEthics statement:\nThis study was approved by the Institutional Review Boards of Jeju National University Hospital (approval No. JEJUNUH 2018-09-003) and all other participating centers with a waiver of informed consent of individual patients because of study subject anonymity and minimal risk to participants.\n\nRESULTS:\nOf 27,122 acute stroke patients who had been admitted to CRCS-K hospitals between April 2008 and August 2014, 12,172 patients met the eligibility criteria (Supplementary Fig. 1). Mean age was 68 ± 13 years, and 59.7% were men. Of 12,172 patients, 2,216 patients (18.2%) were treated with IVT alone and 1238 patients (10.2%) were treated with either EVT alone or combined IVT and EVT. The patients who received reperfusion therapy were more likely to be older, to have atrial fibrillation and higher initial NIHSS score (Supplementary Table 1). In this study, 48.2% of the patients' residence were classified as metropolitan, 41.3% as urban, and 10.5% as rural. The average estimated ground transport time from home to hospital was 23 minutes (interquartile range [IQR], 14–39), and median travel distance was 9.4 km (IQR, 4.5–25.1). In this study, 64.4% of the patients lived within 30 minutes or less to the admitting hospital, 88.7% within 60 minutes or less, 96.5% within 90 minutes or less, and 99.2% with 120 minutes or less. Ground transport times of 30, 60, 90, and 120 minutes corresponded to travel distances of 20.4, 53.5, 86.6, and 119.8 km, respectively. Patients that lived farther from admitting hospitals were more likely to be older, to have atrial fibrillation and CE subtype, and to have a greater initial NIHSS score, but were less likely to have hypertension, diabetes, hyperlipidemia, coronary artery disease and had a lower initial systolic blood pressure (Table 1). The ground transport time did not differ between patients treated with IVT only and patients untreated with reperfusion therapy (rank sum test P = 0.450). However, the ground transport time was slightly but significantly greater for patients treated with EVT compared with patients untreated with reperfusion therapy (24 minutes [14–44] vs. 23 minutes [14–39], rank sum test P = 0.015; Fig. 1 and Supplementary Fig. 2).\nData are presented as number (%) or mean ± standard deviation.\nIQR = interquartile range, NIHSS = National Institutes of Health Stroke Scale, LAA = large artery atherosclerosis, CE = cardioembolism, SVO = small-vessel occlusion, UDE = undetermined etiology, ODE = other determined etiology, LDL = low-density lipoprotein, BUN = blood urea nitrogen, IVT = intravenous thrombolysis, EVT = endovascular therapy.\nCRCS-K = The Clinical Research Collaboration for Stroke in Korea, IVT = intravenous thrombolysis, EVT = endovascular therapy.\nExact treatment time was available in 1,669 patients (83.4%) treated with IVT alone. The median door-to-needle time was 40 minutes (30–53) and the median onset-to-needle time was 124 minutes (88–170). As expected, the onset-to-needle time was positively correlated with ground transport time (Pearson coefficient 0.201, P < 0.001) since it took more time to arrive at the hospital. However, the door-to-needle was inversely associated with ground transport time (Pearson coefficient −0.108, P < 0.001). Regarding EVT, exact treatment time for patients treated with EVT was available for 1,077 patients (87.0%). The median door-to-puncture time was 107 minutes (85–135) and the median onset-to-puncture time was 240 minutes (170–340). The onset-to-puncture time was positively correlated with ground transport time (Pearson coefficient 0.192, P < 0.001). Like the door-to-needle time in IVT, the door-to-puncture showed a negative association with ground transport time in patients treated with EVT (Pearson coefficient −0.089, P < 0.001) (Supplementary Fig. 3).\nThe proportion of patients treated with IVT ranged from 17.1% to 22.1% among the groups with 30-minute increments of ground transport time, and proportion of EVT ranged from 7.5% to 12.4% (Supplementary Fig. 4). In the restricted cubic spline analyses, the proportion of patients treated with IVT decreased significantly when the patients lived beyond a 90-minute ground transport time from the hospital (unadjusted P = 0.003; adjusted P = 0.006). The proportion of stroke patients treated with EVT showed a similar trend with estimated ground transport time in the restricted cubic spline analyses, but it did not reach statistical significance in both unadjusted and adjusted analyses (unadjusted P = 0.086; adjusted P = 0.105) (Fig. 2).\n(A) IVT, unadjusted analysis (P = 0.003); (B) IVT, adjusted analysis (P = 0.006); adjusted for age, hypertension, diabetes, atrial fibrillation, stroke history, coronary artery disease, use of an antiplatelet agent, oral anticoagulants or statins before the index stroke, diastolic blood pressure, fasting blood glucose, low-density lipoprotein cholesterol, platelet count, onset-to-arrival time, initial NIHSS score, and TOAST classification. (C) EVT, unadjusted analysis (P = 0.090); (D) EVT, adjusted analysis (P = 0.100); adjusted for age, sex, hypertension, diabetes, atrial fibrillation, hyperlipidemia, smoking, coronary artery disease, use of an antiplatelet agent or oral anticoagulants before the index stroke, systolic blood pressure, hemoglobin, platelet count, blood urea nitrogen, onset-to-arrival time, initial NIHSS score, and TOAST classification.\nIVT = intravenous thrombolysis, EVT = endovascular therapy, NIHSS = National Institutes of Health Stroke Scale, TOAST = Trial of Org 10172 in Acute Stroke Treatment.\nOf 779 patients who had been treated with combined IVT-EVT, we could identify exact referral pattern in 439 patients (56.4%). Among 439 patients, 69 patients (15.7%) were treated by drip- and-ship paradigm. The use of drip-and-ship paradigm increased significantly with greater ground transport time (P < 0.001) (Supplementary Table 2).\nFor national coverage from the 67 stroke centers, only 9.3% of the total land area of South Korea was reachable within 30 minutes while 75.0% of the land was accessible within 90 minutes. In contrast, 73.0% of the residential area was reachable within 30 minutes of the ground transport time, and 98.4% of the area was accessible within 90 minutes (Fig. 3 and Table 2).\nCRCS-K = The Clinical Research Collaboration for Stroke in Korea.\n\nDISCUSSION:\nIn this nationwide analysis, we found that more than 95% of patients with acute ischemic stroke who visited one of the 12 CRCS-K hospitals within 12 hours of symptom onset had less than 90 minutes of ground transport time. The proportion of patients treated with IVT decreased significantly when patients had more than 90 minutes of the ground transport time from the hospital. The proportion of stroke patients treated with EVT also showed a similar trend with the transport time. From a national viewpoint, more than 98% of the residential area was accessible within 90 minutes of ground transport time from 67 accredited stroke centers in South Korea.\nGeographic access to hospitals offering reperfusion therapy can be crucial to successful reperfusion therapy because the time spent on symptom recognition to decision to medical seeking and the transport time from the scene to the hospital are important contributors to prehospital delays. Living close to treating hospitals was associated with a significantly higher chance of receiving thrombolytic therapy in a study conducted in St. Louis in the US.8 Interestingly, the increased use of thrombolytic therapy was not explained by earlier arrival time in that study, which indicates the presence of other factors such as the use of ambulance transport and health-seeking behavior. Other previous research that used claims data from more than 100,000 Japanese stroke patients did not find an association between driving time and administration of tissue plasminogen activator.9 Instead, they found a significant association between the use of ambulance and the use of thrombolytic therapy regardless of driving time and population density of the area. However, a recent large study enrolling 118,683 patients from the Get With The Guidelines-Stroke Registry (GWTG-Stroke) in the US found a significant association between a longer driving time to hospital and longer onset to arrival time and lower odds of IVT.10 In our study, the proportion of patients treated with IVT decreased significantly when patients had more than 90 minutes of the ground transport time from the hospital. Our findings suggest that the distance to the hospital will not be a major barrier to the use of IVT in South Korea as more than 98% of the South Korean population lives within 90 min from 67 accredited stroke centers in this study.\nAlthough several recent clinical trials have extended the treatment time window for EVT using various perfusion imaging studies, the time interval between stroke onset and reperfusion therapy is still the most important factor affecting eligibility and outcomes for reperfusion therapy in most patients with acute ischemic stroke.212223 For the association between the use of EVT and distance from the hospital, there is not much evidence from the previous literature despite the prehospital delay due to greater transport time could be also crucial to the timely performance of this life-saving therapy for acute ischemic stroke due to large vessel occlusion. In our previous study conducted in California, 94% of the stroke patients lived within a 2-hour ground transport time to the hospital offering the treatment. However, those who lived within 1-hour ground transport time had a significantly increased chance of receiving EVT compared with those who lived beyond 1-hour ground transport time (0.9% vs. 0.2%, P < 0.001).7 In this study, the proportion of stroke patients treated with EVT also decreased when the patients lived beyond a 90-minute ground transport time, however the association did not reach statistical significance. This finding is possibly due to selective referral of the patients who were candidates for EVT from other hospitals although we could not verify this due to lack of information on referral patterns. As with IVT, the ground transport time will not be an important obstacle to receive EVT in South Korea because more than 98% of the population lives within 90 min from 67 stroke centers where most of the EVT procedures would take place.\nIn addition to personal factors such as health-seeking behavior, or recognition of stroke symptoms, various systemic factors can be associated with thrombolysis administration rates in acute ischemic stroke. Studies have found that urban location, centralized acute stroke care system, use of ambulance, neurologist staffing, and use of acute stroke protocol had a significant impact on the increased use of thrombolysis.24 According to the recent report on the national averages for acute stroke care in Korea during 2013–2014, median arrival time to the hospital was 6 h and only 50% of patients with ischemic stroke used ambulance.25 Overall, IVT was used in 10.7% and EVT in 3.6% of patients with acute ischemic stroke. IVT seems to have been used more frequently than other countries although the drip-and-ship paradigm was used only in 1%. However, significant regional disparities in the rates of reperfusion therapy were found and substantial number of acute stroke patients were treated with reperfusion therapy at low-volume hospitals.2526 In another research conducted with Korean national stroke audit data, only one-third of patients who were EVT candidates were initially taken to EVT-capable hospitals and initial routing to EVT-capable hospitals was associated with more than two-folds increased chance of receiving EVT compared with initial routing to primary stroke hospital.27 Since South Korea has a universal national health insurance system with a high population density, it is advantageous for treating acute stroke. Therefore, there are a lot of opportunities to improve the rates of reperfusion therapy for stroke patients by enhancing collaboration between stroke centers and emergency medical system, and redefining roles of comprehensive and primary stroke centers with special care for rural residence.\nAs of 2018, 81.4% of the Korean population live in the urban area according to the world bank data.28 Of 12,172 patients treated at 12 CRCS-K hospitals, 1,272 patients (10.5%) lived in rural region. Therefore, the rural population was underrepresented in our study population and the actual proportion of the stroke patients who received IVT or EVT could be lower in rural regions compared with the urban area. In this study, drip-and-ship paradigm was used in only 15.7% of patients treated with combined IVT-EVT and the use of the paradigm significantly correlated with increased ground transport time. This finding is consistent with our previous work that also reported infrequent use of the paradigm (13.2%) among 1843 patients treated with intravenous tPA at the CRCS-K hospitals.29 From a national perspective, the drip-and-ship paradigm was used in only 1.0% of acute stroke patients and only one-third of EVT candidates were initially routed to EVT-capable hospitals.2527 Additionally, interhospital transfer of patients initially routed to primary stroke hospitals to EVT-capable hospital occurred in only 17.4% in Korea. Because the stroke patients who live in the rural region would be the most probable patients who need more than 90 min of ground transportation time to stroke centers, placing EVT-capable hospitals in strategic locations readily accessible to the rural population should be considered in planning nationwide coverage for stroke systems of care. The nationwide stroke systems of care should ensure effective interhospital transfer of patients for further treatment and monitoring whenever it is needed and direct routing of patients suspected of large vessel occlusion to EVT-capable hospitals.\nThis study has several limitations. First, we used the data from 2008 to 2014 when the collection of patients' home addresses was permitted. Therefore, the current study may not reflect the exact nature of the EVT population because the use of a stentriever or aspiration catheter was not as common during most of this period. Second, we do not have information on referrals and use of ambulance which could be associated with the increased use of reperfusion therapy. Referral bias might play an important role in the estimating the proportion of EVT among the patients who lived farther from the admitting hospital. Third, various personal and systemic factors could affect the rate of reperfusion therapy and many of them were not measured in our registry. Fourth, we do not have exact information for the annual volume of IVT or EVT at 67 stroke centers, and therefore we could not estimate the accessibility of the Korean population for high-volume centers separately. Lastly, we also do not have exact information on the place where the patient had suffered the stroke that led to the current admission. Patients could have developed stroke symptoms at other places and therefore the estimation of the ground transport time between the patients' residence and the admitting hospital serves only as a proxy for geographic access to treating hospitals.\nIn conclusion, more than 95% of the patients with acute ischemic stroke who visited the hospitals within 12 hours of symptom onset lived within 90 minutes of ground transport time from the hospital in this nationwide study. The use of reperfusion therapy decreased when the patients lived beyond 90 minutes of transport time from the hospital. From a national perspective, more than 98% of the South Korean population was accessible to 67 stroke centers within 90 minutes of the ground transport time.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nWe investigated the association between geographic proximity to hospitals and the administration rate of reperfusion therapy for acute ischemic stroke.\n\nMethods:\nWe identified patients with acute ischemic stroke who visited the hospital within 12 hours of symptom onset from a prospective nationwide multicenter stroke registry. Reperfusion therapy was classified as intravenous thrombolysis (IVT), endovascular therapy (EVT), or combined therapy. The association between the proportion of patients who were treated with reperfusion therapy and the ground transport time was evaluated using a spline regression analysis adjusted for patient-level characteristics. We also estimated the proportion of Korean population that lived within each 30-minute incremental service area from 67 stroke centers accredited by the Korean Stroke Society.\n\nResults:\nOf 12,172 patients (mean age, 68 ± 13 years; men, 59.7%) who met the eligibility criteria, 96.5% lived within 90 minutes of ground transport time from the admitting hospital. The proportion of patients treated with IVT decreased significantly when stroke patients lived beyond 90 minutes of the transport time (P = 0.006). The proportion treated with EVT also showed a similar trend with the transport time. Based on the residential area, 98.4% of Korean population was accessible to 67 stroke centers within 90 minutes.\n\nConclusions:\nThe use of reperfusion therapy for acute stroke decreased when patients lived beyond 90 minutes of the ground transport time from the hospital. More than 95% of the South Korean population was accessible to 67 stroke centers within 90 minutes of the ground transport time."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":5940,"string":"5,940"},"whole_article_abstract_length":{"kind":"number","value":291,"string":"291"},"other_sections_lengths":{"kind":"list like","value":[334,324,165,50],"string":"[\n 334,\n 324,\n 165,\n 50\n]","isConversational":false},"num_sections":{"kind":"number","value":8,"string":"8"},"most_frequent_words":{"kind":"list like","value":["time","patients","stroke","evt","transport","transport time","ground","ground transport","ground transport time","hospital"],"string":"[\n \"time\",\n \"patients\",\n \"stroke\",\n \"evt\",\n \"transport\",\n \"transport time\",\n \"ground\",\n \"ground transport\",\n \"ground transport time\",\n \"hospital\"\n]","isConversational":false},"keybert_topics":{"kind":"list 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ground | ivt | transport [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] stroke | time | patients | evt | transport | transport time | ground | ground transport | ground transport time | minutes [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 12 hours ||| IVT | EVT ||| ||| Korean | 30-minute | 67 | the Korean Stroke Society [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 12,172 | 68 | 13 years | 59.7% | 96.5% | 90 minutes ||| IVT | 90 minutes | 0.006 ||| EVT ||| 98.4% | Korean | 67 | 90 minutes [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| 12 hours ||| IVT | EVT ||| ||| Korean | 30-minute | 67 | the Korean Stroke Society ||| 12,172 | 68 | 13 years | 59.7% | 96.5% | 90 minutes ||| IVT | 90 minutes | 0.006 ||| EVT ||| 98.4% | Korean | 67 | 90 minutes ||| 90 minutes ||| More than 95% | South Korean | 67 | 90 minutes [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":85,"cells":{"title":{"kind":"string","value":"Medical science students' experiences of test anxiety: a phenomenological study."},"pmid":{"kind":"string","value":"35906665"},"background_abstract":{"kind":"string","value":"The studies show test anxiety is a common disorder in students that causes academic failure. There are not enough studies and specific theoretical background about test anxiety and ways to deal with it, so the purpose of this study was to do a qualitative study to fully understand the ways to deal with test anxiety in medical Sciences students."},"background_abstract_label":{"kind":"string","value":"INTRODUCTION"},"methods_abstract":{"kind":"string","value":"This is a qualitative study. The participants are the students of the last 2 years of pharmacy, medicine and dentistry at Isfahan University of Medical Sciences. Ten students were selected by purposeful sampling, and interviews continued until the data saturation stage and the lack of access to new data. The data were analyzed by seven-level Colaizzi method."},"methods_abstract_label":{"kind":"string","value":"MATERIALS AND METHODS"},"results_abstract":{"kind":"string","value":"After analyzing data, about 50 codes were extracted. These codes divided into 16 subclasses, and among them, ultimately five main themes are extracted: \"Prayer and Dialogue with God\", \"Interaction and communication with friends and relatives\", \"studying strategies\", \"Finding ways to relax and self-care\" and \"Negative strategies\" were extracted."},"results_abstract_label":{"kind":"string","value":"FINDINGS"},"conclusions_abstract":{"kind":"string","value":"The result of this study showed that students often use positive strategies to overcome the test anxiety and try to use positive strategies, but some students are advised of undesirable strategies such as misuse of authorized drugs and writing cheating that that lead to a lot of complex problems. The educational system should do its utmost effort to empower students to manage the anxiety by learning the best strategies."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Anxiety","Humans","Learning","Qualitative Research","Students, Medical","Test Anxiety"],"string":"[\n \"Anxiety\",\n \"Humans\",\n \"Learning\",\n \"Qualitative Research\",\n \"Students, Medical\",\n \"Test Anxiety\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"9336078"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"One of the concerns of the educational system in universities is the test anxiety [1]. Test anxiety is a special type of anxiety that is characterized by physical, cognitive, and behavioral symptoms when preparing for the test and performing test, It becomes a problem when high levels of anxiety interfere with preparing for and taking the test. Test anxiety is characterized by severe fear of poor performance in tests [2]. In fact, the test anxiety is an emotional experience, feelings and anxiety in situations in which a person feels that his/her performance is evaluated [3]. Test anxiety is defined as the experience of fear, apprehension, and worry before, during, or after a test that can lead to mental distraction, memory impairment, and physical symptoms such as nausea, headache, and tachycardia [4].\nThis type of anxiety occurs between the ages of 10 and 12 and increases with increasing age and maximizes in higher education [5]. According to estimates by researchers in the United States, 15% of students experience different levels of anxiety annually [6]. In the study by Tsegay et al. [7] it was reported that the rate of test anxiety in medical students was 52.3% and significantly higher in female students. Test anxiety is important because it affects test success [8]. Test anxiety can cause academic failure by reducing intrinsic motivation and existing cognitive ability [9]. Sarason [10] considers test anxiety as a self-preoccupation, which is characterized by self-defeat and doubt about his/her abilities. this anxiety often leads to negative cognitive assessment, undesirable physiological reactions and the decline of academic performance. As a result, there is a significant reverse relationship between anxiety scores and test scores [10]. Various studies have shown that test anxiety has a significant and important effect on the academic performance of students. Cassady [11] showed in his study that students with higher test anxiety had a weaker academic performance [11].\nStudents try to cope with anxiety in order to manage it. Coping is the effort to control and manage situations that seem dangerous and tense [12]. There are two main strategies for coping with anxiety that are known as problem-focused coping and emotion-focused coping strategies. The problem-focused coping strategy in which the main objective is to dominate the position and change in the source of anxiety, and emotion-focused coping strategies in which the main objective is to reduce or transform the emotional disturbance quickly [13].\nNarimany and et al. stated that students manage their anxiety through conventional methods such as: they think about good memories of the past, praying and eliminating negative thoughts and beliefs [14].\nIn previous studies, descriptive studies have focused more on estimating the extent to which positive methods have been used, and less on the overall evaluation of all coping strategies that students use. Also, in this study, an attempt has been made to study the strategies of medical students to deal with test anxiety in Eastern and Islamic countries. In Eastern countries, beliefs are different from some other countries where previous studies have been conducted, so its findings can help improve knowledge in this area. On the other hand, this study has selected medical students as the target population because these students face a high volume of difficult. They may also choose different strategies to deal with test anxiety. In this regard, the present study was designed in qualitative study in this target group and conducted with the aim of identifying strategies to deal with test anxiety in medical students."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"null"},"results_text":{"kind":"null"},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"The result of this study showed that students often use positive strategies to overcome the test anxiety and try to use positive strategies, but some students are advised of undesirable strategies such as misuse of authorized drug and writing cheating that lead to a lot of complex problems. Given that the test are an important part of academic life and that every student is always involved in a test and study evaluation, the educational system should do its utmost effort to empower students to manage anxiety by learning the best strategies."},"other_sections_titles":{"kind":"list like","value":["Findings"],"string":"[\n \"Findings\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Ten students participated in the study, of which 7 persons were females and 3 persons were males. Students were selected of pharmacy, medicine and dentistry.\nAfter analyzing the data, about 50 codes were\nextracted. These codes are divided into 16 subclasses, and among them,\nultimately, five main themes, called “prayer\nand dialogue with God”, “Interaction and communication with friends and relatives”,\n“studying strategies”, “Finding ways to relax and self-care” and “Negative\nstrategies” were extracted.\nPrayer and dialogue with God: Pray to God and trust in God led to the extraction of this theme. Participant No. 3 stated: “I am asking the mother to pray for me to take my test successfully and reduce my anxiety”. Participant No. 7 stated that “I believe in the Jafar e Tayyar prayers (it is a special continuous prayer for one’s requests from God) and at night before of test, and I would be very calm”.\nParticipant No. 1 stated that “I already have a good relationship with the Quran (Islam’s book), and I read a Quran page before the test and it calmed me”.\nThe other subtheme was trusting in God. A student on this subtheme stated that “I vow about tests that I have a lot of anxiety, and this creates peace in me”.\nParticipant No.3 said: “I trust in God and I ask him to help me, and thereby keep calm down and begin studying with greater focus”.\n\nInteraction and communication with friends and relatives: This theme was included two subthemes: “communication with the family” and “communication with friends”. Participant No. 4 about communication with the family said, “I talk to my parents over the phone at the test night in the dormitory, and they will calm me with their words”.\nOn communication with friends, Participant No. 10 stated: “I talk to friends who are very intimate and express my anxiety and this reduces my anxiety”. Participant No. 2 said, “I talk to classmates who have a joint test, and talk about the test. This will reduce my anxiety”.\n\nStudying strategies: This theme includes two subthemes: “More effort in education” and “Applying different study strategies”. For “Studying earlier” Participant No. 1 stated that “I’ve been studying for weeks before the test, this will reduce my anxiety”.\nParticipant No. 4 about using the different strategies of study, said that “I will try to study with other friends (group study) for any test that I have anxiety”. Participant No. 3 stated: “I am studying similar questions to reduce my anxiety”. Participant No. 7 said that; “Reading the summary of the important content that other friends extracting from my booklet and book reduces my anxiety”.\n\nFinding ways to relax and self-care: it was another major theme that was included subthemes: “relaxing activities”, “exercise”, and “consuming Caffeinated beverages”.\nRegarding this theme, Participant No. 5 stated that; “When I have anxiety due to the test, I try to listen to my favorite music. This will manage my thoughts and also do not sleep”. Or Participant No. 1 said that; “Walking in the open air helps me become more fluent and calm down”. Participant No. 3 noted that “I usually drink coffee or Nescafe when I have hard and difficult test, this will reduce my anxiety”.\n\n“Negative strategies” This theme has two subthemes: “drug abuse” and “rely on cheating”. Participant No. 9 about rely to the cheating said that “I am cheating to reduce the anxiety of the test, even if I do not intend to use it, this makes calm down me”. Participant No. 8 about drug abuse stated that, “I’m eating a Propranolol 10 when I having an overwhelming anxiety”. Participant No. 6 stated that, “I used the Ritalin tablet when I fear from test and anxietyed me, although it did not work as a result of my test”."],"string":"[\n \"Ten students participated in the study, of which 7 persons were females and 3 persons were males. Students were selected of pharmacy, medicine and dentistry.\\nAfter analyzing the data, about 50 codes were\\nextracted. These codes are divided into 16 subclasses, and among them,\\nultimately, five main themes, called “prayer\\nand dialogue with God”, “Interaction and communication with friends and relatives”,\\n“studying strategies”, “Finding ways to relax and self-care” and “Negative\\nstrategies” were extracted.\\nPrayer and dialogue with God: Pray to God and trust in God led to the extraction of this theme. Participant No. 3 stated: “I am asking the mother to pray for me to take my test successfully and reduce my anxiety”. Participant No. 7 stated that “I believe in the Jafar e Tayyar prayers (it is a special continuous prayer for one’s requests from God) and at night before of test, and I would be very calm”.\\nParticipant No. 1 stated that “I already have a good relationship with the Quran (Islam’s book), and I read a Quran page before the test and it calmed me”.\\nThe other subtheme was trusting in God. A student on this subtheme stated that “I vow about tests that I have a lot of anxiety, and this creates peace in me”.\\nParticipant No.3 said: “I trust in God and I ask him to help me, and thereby keep calm down and begin studying with greater focus”.\\n\\nInteraction and communication with friends and relatives: This theme was included two subthemes: “communication with the family” and “communication with friends”. Participant No. 4 about communication with the family said, “I talk to my parents over the phone at the test night in the dormitory, and they will calm me with their words”.\\nOn communication with friends, Participant No. 10 stated: “I talk to friends who are very intimate and express my anxiety and this reduces my anxiety”. Participant No. 2 said, “I talk to classmates who have a joint test, and talk about the test. This will reduce my anxiety”.\\n\\nStudying strategies: This theme includes two subthemes: “More effort in education” and “Applying different study strategies”. For “Studying earlier” Participant No. 1 stated that “I’ve been studying for weeks before the test, this will reduce my anxiety”.\\nParticipant No. 4 about using the different strategies of study, said that “I will try to study with other friends (group study) for any test that I have anxiety”. Participant No. 3 stated: “I am studying similar questions to reduce my anxiety”. Participant No. 7 said that; “Reading the summary of the important content that other friends extracting from my booklet and book reduces my anxiety”.\\n\\nFinding ways to relax and self-care: it was another major theme that was included subthemes: “relaxing activities”, “exercise”, and “consuming Caffeinated beverages”.\\nRegarding this theme, Participant No. 5 stated that; “When I have anxiety due to the test, I try to listen to my favorite music. This will manage my thoughts and also do not sleep”. Or Participant No. 1 said that; “Walking in the open air helps me become more fluent and calm down”. Participant No. 3 noted that “I usually drink coffee or Nescafe when I have hard and difficult test, this will reduce my anxiety”.\\n\\n“Negative strategies” This theme has two subthemes: “drug abuse” and “rely on cheating”. Participant No. 9 about rely to the cheating said that “I am cheating to reduce the anxiety of the test, even if I do not intend to use it, this makes calm down me”. Participant No. 8 about drug abuse stated that, “I’m eating a Propranolol 10 when I having an overwhelming anxiety”. Participant No. 6 stated that, “I used the Ritalin tablet when I fear from test and anxietyed me, although it did not work as a result of my test”.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null],"string":"[\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Introduction","Materials and methods","Findings","Discussion","Conclusions"],"string":"[\n \"Introduction\",\n \"Materials and methods\",\n \"Findings\",\n \"Discussion\",\n \"Conclusions\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["One of the concerns of the educational system in universities is the test anxiety [1]. Test anxiety is a special type of anxiety that is characterized by physical, cognitive, and behavioral symptoms when preparing for the test and performing test, It becomes a problem when high levels of anxiety interfere with preparing for and taking the test. Test anxiety is characterized by severe fear of poor performance in tests [2]. In fact, the test anxiety is an emotional experience, feelings and anxiety in situations in which a person feels that his/her performance is evaluated [3]. Test anxiety is defined as the experience of fear, apprehension, and worry before, during, or after a test that can lead to mental distraction, memory impairment, and physical symptoms such as nausea, headache, and tachycardia [4].\nThis type of anxiety occurs between the ages of 10 and 12 and increases with increasing age and maximizes in higher education [5]. According to estimates by researchers in the United States, 15% of students experience different levels of anxiety annually [6]. In the study by Tsegay et al. [7] it was reported that the rate of test anxiety in medical students was 52.3% and significantly higher in female students. Test anxiety is important because it affects test success [8]. Test anxiety can cause academic failure by reducing intrinsic motivation and existing cognitive ability [9]. Sarason [10] considers test anxiety as a self-preoccupation, which is characterized by self-defeat and doubt about his/her abilities. this anxiety often leads to negative cognitive assessment, undesirable physiological reactions and the decline of academic performance. As a result, there is a significant reverse relationship between anxiety scores and test scores [10]. Various studies have shown that test anxiety has a significant and important effect on the academic performance of students. Cassady [11] showed in his study that students with higher test anxiety had a weaker academic performance [11].\nStudents try to cope with anxiety in order to manage it. Coping is the effort to control and manage situations that seem dangerous and tense [12]. There are two main strategies for coping with anxiety that are known as problem-focused coping and emotion-focused coping strategies. The problem-focused coping strategy in which the main objective is to dominate the position and change in the source of anxiety, and emotion-focused coping strategies in which the main objective is to reduce or transform the emotional disturbance quickly [13].\nNarimany and et al. stated that students manage their anxiety through conventional methods such as: they think about good memories of the past, praying and eliminating negative thoughts and beliefs [14].\nIn previous studies, descriptive studies have focused more on estimating the extent to which positive methods have been used, and less on the overall evaluation of all coping strategies that students use. Also, in this study, an attempt has been made to study the strategies of medical students to deal with test anxiety in Eastern and Islamic countries. In Eastern countries, beliefs are different from some other countries where previous studies have been conducted, so its findings can help improve knowledge in this area. On the other hand, this study has selected medical students as the target population because these students face a high volume of difficult. They may also choose different strategies to deal with test anxiety. In this regard, the present study was designed in qualitative study in this target group and conducted with the aim of identifying strategies to deal with test anxiety in medical students.","This study is qualitative. The qualitative research method adopted for this study is interpretative phenomenological analysis (IPA), because IPA is a qualitative thematic approach developed within psychology, focusing on the subjective lived experiences of individuals. The participants were students of pharmacy, medicine and dentistry at Isfahan University of Medical Sciences. A purposeful method was used for targeted sampling. Inclusion criteria included studying in the last 2 years of dentistry, medicine and pharmacy, as well as having the desire to participate in the study. Sampling was continued until data saturation.\nThis study received ethical approval from the Institutional Review Board (IRB) to which the researchers are affiliated. All study protocols were performed in accordance with the Declaration of Helsinki. This study considered ethical considerations such as the confidentiality of the interviewees’ names and the written consent of interviewees. Interviews were conducted in 2020. Informed consent of every participant was obtained after clearly explaining the objectives as well as the significance of the study for each participant. We advised the participants about the right to participate as well as refuse or discontinue participation at any time they want and the chance to ask anything about the study. The participants were also advised that all data collected would remain confidential.\nData collection was done in a semi-structured interview. The questions were about the test anxiety manage strategies used by students. Interviews continued until data saturation and lack of access to new data. Interview questions were semi-structured and probing questions were asked. Every interview lasted roughly between 30 and 70 min. Interviews were recorded by audio tape and at the earliest opportunity verbatim transcription of interview data was done. Statements were written word-by-word and then were manually coded.\nData was analyzed by the seven-level Colaizzi method. The Colaizzi steps were performed as follows; (1) Transcribing all the participants’ descriptions. participant narratives transcribed from the audio-taped interviews. We didn’t use any software. (2) Extracting significant statements, statements that directly relate to the test anxiety. The researchers repeated all participants’ descriptions and in order to understand these concepts, it was felt by them, then extracted the sentences and vocabulary related to the phenomenon under study and gave a special meaning to each of the extracted sentences. (3) Creating formulated meanings. In this stage, each significant statement is extracted from the participant’s narratives. (4) Aggregating formulated meanings into theme clusters. We organized formulated meanings into groups of similar type. (5) Developing an exhaustive description. An exhaustive description developed through a synthesis of all theme clusters and associated formulated meanings explicated by the researchers. (6) After articulation of the symbolic representation which occurred during the interview. Researchers did an interpretative analysis of symbolic representations for test anxiety. (7) We tried to identify the fundamental structure of the test anxiety by explication’ through a rigorous analysis of the exhaustive description of it.\nIn order to ensure the accuracy of the data, rigor and trustworthiness was determined based on Guba and Lincholn criteria (1994) which include Credibility, Dependability, Confirmability and Transferability [15, 16]. Therefore, we used member checking, researcher creditability, prolonged engagement (semi-structured interview) with the participants, the use of peer debriefing. A follow-up appointment was made between the researcher and each participant for the purpose of validating the essence of the phenomenon with students.\nIn order to get rigor and trustworthiness data, we established comfortable interactions at the beginning of the interviews, which was maintained until the end of the interview. Participants were also surveyed about the codes for approval after each interview. Data, coding and themes were also reviewed by an expert in this subject.","Ten students participated in the study, of which 7 persons were females and 3 persons were males. Students were selected of pharmacy, medicine and dentistry.\nAfter analyzing the data, about 50 codes were\nextracted. These codes are divided into 16 subclasses, and among them,\nultimately, five main themes, called “prayer\nand dialogue with God”, “Interaction and communication with friends and relatives”,\n“studying strategies”, “Finding ways to relax and self-care” and “Negative\nstrategies” were extracted.\nPrayer and dialogue with God: Pray to God and trust in God led to the extraction of this theme. Participant No. 3 stated: “I am asking the mother to pray for me to take my test successfully and reduce my anxiety”. Participant No. 7 stated that “I believe in the Jafar e Tayyar prayers (it is a special continuous prayer for one’s requests from God) and at night before of test, and I would be very calm”.\nParticipant No. 1 stated that “I already have a good relationship with the Quran (Islam’s book), and I read a Quran page before the test and it calmed me”.\nThe other subtheme was trusting in God. A student on this subtheme stated that “I vow about tests that I have a lot of anxiety, and this creates peace in me”.\nParticipant No.3 said: “I trust in God and I ask him to help me, and thereby keep calm down and begin studying with greater focus”.\n\nInteraction and communication with friends and relatives: This theme was included two subthemes: “communication with the family” and “communication with friends”. Participant No. 4 about communication with the family said, “I talk to my parents over the phone at the test night in the dormitory, and they will calm me with their words”.\nOn communication with friends, Participant No. 10 stated: “I talk to friends who are very intimate and express my anxiety and this reduces my anxiety”. Participant No. 2 said, “I talk to classmates who have a joint test, and talk about the test. This will reduce my anxiety”.\n\nStudying strategies: This theme includes two subthemes: “More effort in education” and “Applying different study strategies”. For “Studying earlier” Participant No. 1 stated that “I’ve been studying for weeks before the test, this will reduce my anxiety”.\nParticipant No. 4 about using the different strategies of study, said that “I will try to study with other friends (group study) for any test that I have anxiety”. Participant No. 3 stated: “I am studying similar questions to reduce my anxiety”. Participant No. 7 said that; “Reading the summary of the important content that other friends extracting from my booklet and book reduces my anxiety”.\n\nFinding ways to relax and self-care: it was another major theme that was included subthemes: “relaxing activities”, “exercise”, and “consuming Caffeinated beverages”.\nRegarding this theme, Participant No. 5 stated that; “When I have anxiety due to the test, I try to listen to my favorite music. This will manage my thoughts and also do not sleep”. Or Participant No. 1 said that; “Walking in the open air helps me become more fluent and calm down”. Participant No. 3 noted that “I usually drink coffee or Nescafe when I have hard and difficult test, this will reduce my anxiety”.\n\n“Negative strategies” This theme has two subthemes: “drug abuse” and “rely on cheating”. Participant No. 9 about rely to the cheating said that “I am cheating to reduce the anxiety of the test, even if I do not intend to use it, this makes calm down me”. Participant No. 8 about drug abuse stated that, “I’m eating a Propranolol 10 when I having an overwhelming anxiety”. Participant No. 6 stated that, “I used the Ritalin tablet when I fear from test and anxietyed me, although it did not work as a result of my test”.","In the present study, with a qualitative approach and interviews with students, researchers tried to identify and describe the experiences of strategies for coping with the anxiety of the test among students in dentistry, medicine and pharmacy.\nOne of the main themes that students use to cope test anxiety is “prayer and dialogue with God”. Students use prayer and dialogue with God to reduce their anxiety. Among the religious and spiritual sources, the greatest source used to reduce anxiety and anxiety is “prayer”. The prayer is derived from the Latin word of precarious meaning “obtained by pray and pleasure” [17]. The other studies showed praying is an effective strategy to cope with test anxiety. Adroishi et al. [18] showed that listening to pray could significantly reduce the test anxiety in students. The study of Masomi et al. showed that listening to the Quran sound, such as the sound of music before the test, is an effective strategy for the anxiety test, and the Quran sound is more effective in reducing student test anxiety [19]. In a study conducted by Narimany et al., 44.5% and 27.5% of the students used the use of prayer as a strategy for coping with anxiety [14]. Also, training religious values [20] and spiritual training, including prayer, forgiveness, transcendental and spiritual meditation [21] have also been reported to reduce the depression and anxiety of students. Ganji et al. also reported that religious beliefs are related with anxiety levels and reduce it [22]. In Papazisis et al. research, strong religious and spiritual beliefs have positive relationships increasing, the self-confidence, and a negative relationship with depression, anxiety and anxiety as a personality trait [23].\nBased on the findings of various studies, spiritual health determines the integrity of the person, and is the only force that harmonizes the physical, psychological and social dimensions. Religious and spiritual beliefs make a person calm and play a vital role in adapting to tension [24]. Most people believe that the influence of uncontrollable positions can be controlled through relay to God [25]. Religion, spirituality, and existential health are important predictors of mental health [26, 27].\nAnother strategy for students to calm down and manage their anxiety was to communicate with those who had some kind of attachment to them. In fact, this communication with people such as parents or intimate friends has made them relaxed and helped them better control the tension of the test. Cassidy and Shaver, in their justification of the relationship between attachment style and mental health, stated that the consequence of a safe attachment process is creating a sense of safety in a person, and the consequence of an unsafe attachment process is to create fear in a person [28]. Roberts et al. in their justification of this relationship, believe that the psychological consequence of unsafe attachment styles in tension situations is anxiety and depression. The psychological consequence of a safe attachment style in such situations is mental relaxation [29]. By studying 314 surviving adults from Bam earthquake, Rahimian Bouger et al. found that there was a significant positive relationship between safe attachment style and mental health, and a significant negative relationship between avoidance and ambivalent attachment styles with mental health [30]. The study of Besharat et al. revealed that the subjects with a safe attachment style rather than an unsafe type and those with an avoidant attachment style had fewer interpersonal problems than ambivalent styles. The results of this study refer to authenticity of safe attachment to the first requirement and its transfer to subsequent generations [31]. Safford has shown that people with an unsafe attachment style are more likely to experience anxiety and depression [32].\nMorey and Taylor [33] study, which was similar to the present study in terms of quality with in-depth interviews, found that exercise and talking to friends are two important strategies for students to deal with stress. Fujii [34] reported that one of the ways to deal with English test anxiety in students is “cooperation with others” and “building confidence”.\n\nStudying strategies which were students’ strategies to cope with test anxiety, can be considered as a kind of sensitization to manage anxiety and manage of the situation. These strategies are effective and positive strategies that can help the student in a constructive way to gain more control and to manage negative thoughts, and reduce his own anxiety through such actions. Motevalli et al. [35] reported that teaching new and practical study skills helps students manage test anxiety. They found that learning some skills such as time managing for studying, properly review and summarize, how to answer multiple-choice questions, correct/incorrect and descriptive questions can control test anxiety [35]. Yusefzadeh et al. [36] reported that based on the findings of a training and evaluation program (teacher 10–35% of the final grade of the course is based on activity during the semester), the implementation of such programs significantly reduces anxiety Students are tested. Ozbiçakçi et al. [37] also believe that teaching method reduces test anxiety. But L. Hsu mentioned that further research is needed to determine best practices for alleviating student stress and anxiety [38].\n\n“Finding ways to relax and self-care” is the other main theme for coping with test anxiety. It included Relaxing activities, consuming caffeine and Soothing food. Findings of the study by Mojarrab et al. [39] showed that the use of relaxation techniques along with playing soothing music reduces nursing students’ test anxiety and also improves their clinical test scores. Morey and Taylor [33] reported that many students believed exercise would distract them from test anxiety and lead to be calm. Walking, running and yoga were among the activities that students focused on, with more emphasis on walking [33].\nIn this study, negative strategies were also used to manage the anxiety by the students, such as Unusual use of some medications. Evidences show that the consumption of stimulants and non-drug use of a variety of drugs is one of the threats to students. Several evidences suggest that consuming drug abuse among young prople, especially students, is increasing. Methylphenidate or Ritalin is one of the most widely used drugs that has recently been abused among adolescents, especially students [40]. Many students take this medicine to stay awake for several hours and to maintain their unusual focuses for a long time. During the tests, the use of such drugs, including Ritalin, increases. Ritalin is the most common prescribed psychotropic drug for children in the USA [14]. Currently, non-medical consumption of prescription stimulants, including Methylphenidate, is a growing problem among students in the USA. Statistics have shown that 7% of American students have at least once used this drug over their lifetime, and the prevalence of Methylphenidate consumption among students over the course of 1 year was 3% [13]. In this regard, several studies have reported 3–35% of Ritalin abuse among students [41–46]. The most common side effects of consuming Ritalin are insomnia, nervousness, anxiety, headache, and loss of appetite. In excessive consumption, restlessness, delirium, psychosis, hypertension, seizure, hyperthermia, and arrhythmias may occur [47].\n\nWriting cheating was one of the other negative strategies that students used to reduce their test anxiety, although they had no intention of using this cheating during the test. The student, by writing cheat, wants to create a reliance point on himself/herself and thereby calm himself down and overcome his anxiety. In fact, cheating as a reliance point of its existence is an important part of assurance, not to applying it.","The result of this study showed that students often use positive strategies to overcome the test anxiety and try to use positive strategies, but some students are advised of undesirable strategies such as misuse of authorized drug and writing cheating that lead to a lot of complex problems. Given that the test are an important part of academic life and that every student is always involved in a test and study evaluation, the educational system should do its utmost effort to empower students to manage anxiety by learning the best strategies."],"string":"[\n \"One of the concerns of the educational system in universities is the test anxiety [1]. Test anxiety is a special type of anxiety that is characterized by physical, cognitive, and behavioral symptoms when preparing for the test and performing test, It becomes a problem when high levels of anxiety interfere with preparing for and taking the test. Test anxiety is characterized by severe fear of poor performance in tests [2]. In fact, the test anxiety is an emotional experience, feelings and anxiety in situations in which a person feels that his/her performance is evaluated [3]. Test anxiety is defined as the experience of fear, apprehension, and worry before, during, or after a test that can lead to mental distraction, memory impairment, and physical symptoms such as nausea, headache, and tachycardia [4].\\nThis type of anxiety occurs between the ages of 10 and 12 and increases with increasing age and maximizes in higher education [5]. According to estimates by researchers in the United States, 15% of students experience different levels of anxiety annually [6]. In the study by Tsegay et al. [7] it was reported that the rate of test anxiety in medical students was 52.3% and significantly higher in female students. Test anxiety is important because it affects test success [8]. Test anxiety can cause academic failure by reducing intrinsic motivation and existing cognitive ability [9]. Sarason [10] considers test anxiety as a self-preoccupation, which is characterized by self-defeat and doubt about his/her abilities. this anxiety often leads to negative cognitive assessment, undesirable physiological reactions and the decline of academic performance. As a result, there is a significant reverse relationship between anxiety scores and test scores [10]. Various studies have shown that test anxiety has a significant and important effect on the academic performance of students. Cassady [11] showed in his study that students with higher test anxiety had a weaker academic performance [11].\\nStudents try to cope with anxiety in order to manage it. Coping is the effort to control and manage situations that seem dangerous and tense [12]. There are two main strategies for coping with anxiety that are known as problem-focused coping and emotion-focused coping strategies. The problem-focused coping strategy in which the main objective is to dominate the position and change in the source of anxiety, and emotion-focused coping strategies in which the main objective is to reduce or transform the emotional disturbance quickly [13].\\nNarimany and et al. stated that students manage their anxiety through conventional methods such as: they think about good memories of the past, praying and eliminating negative thoughts and beliefs [14].\\nIn previous studies, descriptive studies have focused more on estimating the extent to which positive methods have been used, and less on the overall evaluation of all coping strategies that students use. Also, in this study, an attempt has been made to study the strategies of medical students to deal with test anxiety in Eastern and Islamic countries. In Eastern countries, beliefs are different from some other countries where previous studies have been conducted, so its findings can help improve knowledge in this area. On the other hand, this study has selected medical students as the target population because these students face a high volume of difficult. They may also choose different strategies to deal with test anxiety. In this regard, the present study was designed in qualitative study in this target group and conducted with the aim of identifying strategies to deal with test anxiety in medical students.\",\n \"This study is qualitative. The qualitative research method adopted for this study is interpretative phenomenological analysis (IPA), because IPA is a qualitative thematic approach developed within psychology, focusing on the subjective lived experiences of individuals. The participants were students of pharmacy, medicine and dentistry at Isfahan University of Medical Sciences. A purposeful method was used for targeted sampling. Inclusion criteria included studying in the last 2 years of dentistry, medicine and pharmacy, as well as having the desire to participate in the study. Sampling was continued until data saturation.\\nThis study received ethical approval from the Institutional Review Board (IRB) to which the researchers are affiliated. All study protocols were performed in accordance with the Declaration of Helsinki. This study considered ethical considerations such as the confidentiality of the interviewees’ names and the written consent of interviewees. Interviews were conducted in 2020. Informed consent of every participant was obtained after clearly explaining the objectives as well as the significance of the study for each participant. We advised the participants about the right to participate as well as refuse or discontinue participation at any time they want and the chance to ask anything about the study. The participants were also advised that all data collected would remain confidential.\\nData collection was done in a semi-structured interview. The questions were about the test anxiety manage strategies used by students. Interviews continued until data saturation and lack of access to new data. Interview questions were semi-structured and probing questions were asked. Every interview lasted roughly between 30 and 70 min. Interviews were recorded by audio tape and at the earliest opportunity verbatim transcription of interview data was done. Statements were written word-by-word and then were manually coded.\\nData was analyzed by the seven-level Colaizzi method. The Colaizzi steps were performed as follows; (1) Transcribing all the participants’ descriptions. participant narratives transcribed from the audio-taped interviews. We didn’t use any software. (2) Extracting significant statements, statements that directly relate to the test anxiety. The researchers repeated all participants’ descriptions and in order to understand these concepts, it was felt by them, then extracted the sentences and vocabulary related to the phenomenon under study and gave a special meaning to each of the extracted sentences. (3) Creating formulated meanings. In this stage, each significant statement is extracted from the participant’s narratives. (4) Aggregating formulated meanings into theme clusters. We organized formulated meanings into groups of similar type. (5) Developing an exhaustive description. An exhaustive description developed through a synthesis of all theme clusters and associated formulated meanings explicated by the researchers. (6) After articulation of the symbolic representation which occurred during the interview. Researchers did an interpretative analysis of symbolic representations for test anxiety. (7) We tried to identify the fundamental structure of the test anxiety by explication’ through a rigorous analysis of the exhaustive description of it.\\nIn order to ensure the accuracy of the data, rigor and trustworthiness was determined based on Guba and Lincholn criteria (1994) which include Credibility, Dependability, Confirmability and Transferability [15, 16]. Therefore, we used member checking, researcher creditability, prolonged engagement (semi-structured interview) with the participants, the use of peer debriefing. A follow-up appointment was made between the researcher and each participant for the purpose of validating the essence of the phenomenon with students.\\nIn order to get rigor and trustworthiness data, we established comfortable interactions at the beginning of the interviews, which was maintained until the end of the interview. Participants were also surveyed about the codes for approval after each interview. Data, coding and themes were also reviewed by an expert in this subject.\",\n \"Ten students participated in the study, of which 7 persons were females and 3 persons were males. Students were selected of pharmacy, medicine and dentistry.\\nAfter analyzing the data, about 50 codes were\\nextracted. These codes are divided into 16 subclasses, and among them,\\nultimately, five main themes, called “prayer\\nand dialogue with God”, “Interaction and communication with friends and relatives”,\\n“studying strategies”, “Finding ways to relax and self-care” and “Negative\\nstrategies” were extracted.\\nPrayer and dialogue with God: Pray to God and trust in God led to the extraction of this theme. Participant No. 3 stated: “I am asking the mother to pray for me to take my test successfully and reduce my anxiety”. Participant No. 7 stated that “I believe in the Jafar e Tayyar prayers (it is a special continuous prayer for one’s requests from God) and at night before of test, and I would be very calm”.\\nParticipant No. 1 stated that “I already have a good relationship with the Quran (Islam’s book), and I read a Quran page before the test and it calmed me”.\\nThe other subtheme was trusting in God. A student on this subtheme stated that “I vow about tests that I have a lot of anxiety, and this creates peace in me”.\\nParticipant No.3 said: “I trust in God and I ask him to help me, and thereby keep calm down and begin studying with greater focus”.\\n\\nInteraction and communication with friends and relatives: This theme was included two subthemes: “communication with the family” and “communication with friends”. Participant No. 4 about communication with the family said, “I talk to my parents over the phone at the test night in the dormitory, and they will calm me with their words”.\\nOn communication with friends, Participant No. 10 stated: “I talk to friends who are very intimate and express my anxiety and this reduces my anxiety”. Participant No. 2 said, “I talk to classmates who have a joint test, and talk about the test. This will reduce my anxiety”.\\n\\nStudying strategies: This theme includes two subthemes: “More effort in education” and “Applying different study strategies”. For “Studying earlier” Participant No. 1 stated that “I’ve been studying for weeks before the test, this will reduce my anxiety”.\\nParticipant No. 4 about using the different strategies of study, said that “I will try to study with other friends (group study) for any test that I have anxiety”. Participant No. 3 stated: “I am studying similar questions to reduce my anxiety”. Participant No. 7 said that; “Reading the summary of the important content that other friends extracting from my booklet and book reduces my anxiety”.\\n\\nFinding ways to relax and self-care: it was another major theme that was included subthemes: “relaxing activities”, “exercise”, and “consuming Caffeinated beverages”.\\nRegarding this theme, Participant No. 5 stated that; “When I have anxiety due to the test, I try to listen to my favorite music. This will manage my thoughts and also do not sleep”. Or Participant No. 1 said that; “Walking in the open air helps me become more fluent and calm down”. Participant No. 3 noted that “I usually drink coffee or Nescafe when I have hard and difficult test, this will reduce my anxiety”.\\n\\n“Negative strategies” This theme has two subthemes: “drug abuse” and “rely on cheating”. Participant No. 9 about rely to the cheating said that “I am cheating to reduce the anxiety of the test, even if I do not intend to use it, this makes calm down me”. Participant No. 8 about drug abuse stated that, “I’m eating a Propranolol 10 when I having an overwhelming anxiety”. Participant No. 6 stated that, “I used the Ritalin tablet when I fear from test and anxietyed me, although it did not work as a result of my test”.\",\n \"In the present study, with a qualitative approach and interviews with students, researchers tried to identify and describe the experiences of strategies for coping with the anxiety of the test among students in dentistry, medicine and pharmacy.\\nOne of the main themes that students use to cope test anxiety is “prayer and dialogue with God”. Students use prayer and dialogue with God to reduce their anxiety. Among the religious and spiritual sources, the greatest source used to reduce anxiety and anxiety is “prayer”. The prayer is derived from the Latin word of precarious meaning “obtained by pray and pleasure” [17]. The other studies showed praying is an effective strategy to cope with test anxiety. Adroishi et al. [18] showed that listening to pray could significantly reduce the test anxiety in students. The study of Masomi et al. showed that listening to the Quran sound, such as the sound of music before the test, is an effective strategy for the anxiety test, and the Quran sound is more effective in reducing student test anxiety [19]. In a study conducted by Narimany et al., 44.5% and 27.5% of the students used the use of prayer as a strategy for coping with anxiety [14]. Also, training religious values [20] and spiritual training, including prayer, forgiveness, transcendental and spiritual meditation [21] have also been reported to reduce the depression and anxiety of students. Ganji et al. also reported that religious beliefs are related with anxiety levels and reduce it [22]. In Papazisis et al. research, strong religious and spiritual beliefs have positive relationships increasing, the self-confidence, and a negative relationship with depression, anxiety and anxiety as a personality trait [23].\\nBased on the findings of various studies, spiritual health determines the integrity of the person, and is the only force that harmonizes the physical, psychological and social dimensions. Religious and spiritual beliefs make a person calm and play a vital role in adapting to tension [24]. Most people believe that the influence of uncontrollable positions can be controlled through relay to God [25]. Religion, spirituality, and existential health are important predictors of mental health [26, 27].\\nAnother strategy for students to calm down and manage their anxiety was to communicate with those who had some kind of attachment to them. In fact, this communication with people such as parents or intimate friends has made them relaxed and helped them better control the tension of the test. Cassidy and Shaver, in their justification of the relationship between attachment style and mental health, stated that the consequence of a safe attachment process is creating a sense of safety in a person, and the consequence of an unsafe attachment process is to create fear in a person [28]. Roberts et al. in their justification of this relationship, believe that the psychological consequence of unsafe attachment styles in tension situations is anxiety and depression. The psychological consequence of a safe attachment style in such situations is mental relaxation [29]. By studying 314 surviving adults from Bam earthquake, Rahimian Bouger et al. found that there was a significant positive relationship between safe attachment style and mental health, and a significant negative relationship between avoidance and ambivalent attachment styles with mental health [30]. The study of Besharat et al. revealed that the subjects with a safe attachment style rather than an unsafe type and those with an avoidant attachment style had fewer interpersonal problems than ambivalent styles. The results of this study refer to authenticity of safe attachment to the first requirement and its transfer to subsequent generations [31]. Safford has shown that people with an unsafe attachment style are more likely to experience anxiety and depression [32].\\nMorey and Taylor [33] study, which was similar to the present study in terms of quality with in-depth interviews, found that exercise and talking to friends are two important strategies for students to deal with stress. Fujii [34] reported that one of the ways to deal with English test anxiety in students is “cooperation with others” and “building confidence”.\\n\\nStudying strategies which were students’ strategies to cope with test anxiety, can be considered as a kind of sensitization to manage anxiety and manage of the situation. These strategies are effective and positive strategies that can help the student in a constructive way to gain more control and to manage negative thoughts, and reduce his own anxiety through such actions. Motevalli et al. [35] reported that teaching new and practical study skills helps students manage test anxiety. They found that learning some skills such as time managing for studying, properly review and summarize, how to answer multiple-choice questions, correct/incorrect and descriptive questions can control test anxiety [35]. Yusefzadeh et al. [36] reported that based on the findings of a training and evaluation program (teacher 10–35% of the final grade of the course is based on activity during the semester), the implementation of such programs significantly reduces anxiety Students are tested. Ozbiçakçi et al. [37] also believe that teaching method reduces test anxiety. But L. Hsu mentioned that further research is needed to determine best practices for alleviating student stress and anxiety [38].\\n\\n“Finding ways to relax and self-care” is the other main theme for coping with test anxiety. It included Relaxing activities, consuming caffeine and Soothing food. Findings of the study by Mojarrab et al. [39] showed that the use of relaxation techniques along with playing soothing music reduces nursing students’ test anxiety and also improves their clinical test scores. Morey and Taylor [33] reported that many students believed exercise would distract them from test anxiety and lead to be calm. Walking, running and yoga were among the activities that students focused on, with more emphasis on walking [33].\\nIn this study, negative strategies were also used to manage the anxiety by the students, such as Unusual use of some medications. Evidences show that the consumption of stimulants and non-drug use of a variety of drugs is one of the threats to students. Several evidences suggest that consuming drug abuse among young prople, especially students, is increasing. Methylphenidate or Ritalin is one of the most widely used drugs that has recently been abused among adolescents, especially students [40]. Many students take this medicine to stay awake for several hours and to maintain their unusual focuses for a long time. During the tests, the use of such drugs, including Ritalin, increases. Ritalin is the most common prescribed psychotropic drug for children in the USA [14]. Currently, non-medical consumption of prescription stimulants, including Methylphenidate, is a growing problem among students in the USA. Statistics have shown that 7% of American students have at least once used this drug over their lifetime, and the prevalence of Methylphenidate consumption among students over the course of 1 year was 3% [13]. In this regard, several studies have reported 3–35% of Ritalin abuse among students [41–46]. The most common side effects of consuming Ritalin are insomnia, nervousness, anxiety, headache, and loss of appetite. In excessive consumption, restlessness, delirium, psychosis, hypertension, seizure, hyperthermia, and arrhythmias may occur [47].\\n\\nWriting cheating was one of the other negative strategies that students used to reduce their test anxiety, although they had no intention of using this cheating during the test. The student, by writing cheat, wants to create a reliance point on himself/herself and thereby calm himself down and overcome his anxiety. In fact, cheating as a reliance point of its existence is an important part of assurance, not to applying it.\",\n \"The result of this study showed that students often use positive strategies to overcome the test anxiety and try to use positive strategies, but some students are advised of undesirable strategies such as misuse of authorized drug and writing cheating that lead to a lot of complex problems. Given that the test are an important part of academic life and that every student is always involved in a test and study evaluation, the educational system should do its utmost effort to empower students to manage anxiety by learning the best strategies.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["introduction","materials|methods",null,"discussion","conclusion"],"string":"[\n \"introduction\",\n \"materials|methods\",\n null,\n \"discussion\",\n \"conclusion\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["Test anxiety","Higher education","Coping strategies","Qualitative study"],"string":"[\n \"Test anxiety\",\n \"Higher education\",\n \"Coping strategies\",\n \"Qualitative study\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Introduction:\nOne of the concerns of the educational system in universities is the test anxiety [1]. Test anxiety is a special type of anxiety that is characterized by physical, cognitive, and behavioral symptoms when preparing for the test and performing test, It becomes a problem when high levels of anxiety interfere with preparing for and taking the test. Test anxiety is characterized by severe fear of poor performance in tests [2]. In fact, the test anxiety is an emotional experience, feelings and anxiety in situations in which a person feels that his/her performance is evaluated [3]. Test anxiety is defined as the experience of fear, apprehension, and worry before, during, or after a test that can lead to mental distraction, memory impairment, and physical symptoms such as nausea, headache, and tachycardia [4].\nThis type of anxiety occurs between the ages of 10 and 12 and increases with increasing age and maximizes in higher education [5]. According to estimates by researchers in the United States, 15% of students experience different levels of anxiety annually [6]. In the study by Tsegay et al. [7] it was reported that the rate of test anxiety in medical students was 52.3% and significantly higher in female students. Test anxiety is important because it affects test success [8]. Test anxiety can cause academic failure by reducing intrinsic motivation and existing cognitive ability [9]. Sarason [10] considers test anxiety as a self-preoccupation, which is characterized by self-defeat and doubt about his/her abilities. this anxiety often leads to negative cognitive assessment, undesirable physiological reactions and the decline of academic performance. As a result, there is a significant reverse relationship between anxiety scores and test scores [10]. Various studies have shown that test anxiety has a significant and important effect on the academic performance of students. Cassady [11] showed in his study that students with higher test anxiety had a weaker academic performance [11].\nStudents try to cope with anxiety in order to manage it. Coping is the effort to control and manage situations that seem dangerous and tense [12]. There are two main strategies for coping with anxiety that are known as problem-focused coping and emotion-focused coping strategies. The problem-focused coping strategy in which the main objective is to dominate the position and change in the source of anxiety, and emotion-focused coping strategies in which the main objective is to reduce or transform the emotional disturbance quickly [13].\nNarimany and et al. stated that students manage their anxiety through conventional methods such as: they think about good memories of the past, praying and eliminating negative thoughts and beliefs [14].\nIn previous studies, descriptive studies have focused more on estimating the extent to which positive methods have been used, and less on the overall evaluation of all coping strategies that students use. Also, in this study, an attempt has been made to study the strategies of medical students to deal with test anxiety in Eastern and Islamic countries. In Eastern countries, beliefs are different from some other countries where previous studies have been conducted, so its findings can help improve knowledge in this area. On the other hand, this study has selected medical students as the target population because these students face a high volume of difficult. They may also choose different strategies to deal with test anxiety. In this regard, the present study was designed in qualitative study in this target group and conducted with the aim of identifying strategies to deal with test anxiety in medical students.\n\nMaterials and methods:\nThis study is qualitative. The qualitative research method adopted for this study is interpretative phenomenological analysis (IPA), because IPA is a qualitative thematic approach developed within psychology, focusing on the subjective lived experiences of individuals. The participants were students of pharmacy, medicine and dentistry at Isfahan University of Medical Sciences. A purposeful method was used for targeted sampling. Inclusion criteria included studying in the last 2 years of dentistry, medicine and pharmacy, as well as having the desire to participate in the study. Sampling was continued until data saturation.\nThis study received ethical approval from the Institutional Review Board (IRB) to which the researchers are affiliated. All study protocols were performed in accordance with the Declaration of Helsinki. This study considered ethical considerations such as the confidentiality of the interviewees’ names and the written consent of interviewees. Interviews were conducted in 2020. Informed consent of every participant was obtained after clearly explaining the objectives as well as the significance of the study for each participant. We advised the participants about the right to participate as well as refuse or discontinue participation at any time they want and the chance to ask anything about the study. The participants were also advised that all data collected would remain confidential.\nData collection was done in a semi-structured interview. The questions were about the test anxiety manage strategies used by students. Interviews continued until data saturation and lack of access to new data. Interview questions were semi-structured and probing questions were asked. Every interview lasted roughly between 30 and 70 min. Interviews were recorded by audio tape and at the earliest opportunity verbatim transcription of interview data was done. Statements were written word-by-word and then were manually coded.\nData was analyzed by the seven-level Colaizzi method. The Colaizzi steps were performed as follows; (1) Transcribing all the participants’ descriptions. participant narratives transcribed from the audio-taped interviews. We didn’t use any software. (2) Extracting significant statements, statements that directly relate to the test anxiety. The researchers repeated all participants’ descriptions and in order to understand these concepts, it was felt by them, then extracted the sentences and vocabulary related to the phenomenon under study and gave a special meaning to each of the extracted sentences. (3) Creating formulated meanings. In this stage, each significant statement is extracted from the participant’s narratives. (4) Aggregating formulated meanings into theme clusters. We organized formulated meanings into groups of similar type. (5) Developing an exhaustive description. An exhaustive description developed through a synthesis of all theme clusters and associated formulated meanings explicated by the researchers. (6) After articulation of the symbolic representation which occurred during the interview. Researchers did an interpretative analysis of symbolic representations for test anxiety. (7) We tried to identify the fundamental structure of the test anxiety by explication’ through a rigorous analysis of the exhaustive description of it.\nIn order to ensure the accuracy of the data, rigor and trustworthiness was determined based on Guba and Lincholn criteria (1994) which include Credibility, Dependability, Confirmability and Transferability [15, 16]. Therefore, we used member checking, researcher creditability, prolonged engagement (semi-structured interview) with the participants, the use of peer debriefing. A follow-up appointment was made between the researcher and each participant for the purpose of validating the essence of the phenomenon with students.\nIn order to get rigor and trustworthiness data, we established comfortable interactions at the beginning of the interviews, which was maintained until the end of the interview. Participants were also surveyed about the codes for approval after each interview. Data, coding and themes were also reviewed by an expert in this subject.\n\nFindings:\nTen students participated in the study, of which 7 persons were females and 3 persons were males. Students were selected of pharmacy, medicine and dentistry.\nAfter analyzing the data, about 50 codes were\nextracted. These codes are divided into 16 subclasses, and among them,\nultimately, five main themes, called “prayer\nand dialogue with God”, “Interaction and communication with friends and relatives”,\n“studying strategies”, “Finding ways to relax and self-care” and “Negative\nstrategies” were extracted.\nPrayer and dialogue with God: Pray to God and trust in God led to the extraction of this theme. Participant No. 3 stated: “I am asking the mother to pray for me to take my test successfully and reduce my anxiety”. Participant No. 7 stated that “I believe in the Jafar e Tayyar prayers (it is a special continuous prayer for one’s requests from God) and at night before of test, and I would be very calm”.\nParticipant No. 1 stated that “I already have a good relationship with the Quran (Islam’s book), and I read a Quran page before the test and it calmed me”.\nThe other subtheme was trusting in God. A student on this subtheme stated that “I vow about tests that I have a lot of anxiety, and this creates peace in me”.\nParticipant No.3 said: “I trust in God and I ask him to help me, and thereby keep calm down and begin studying with greater focus”.\n\nInteraction and communication with friends and relatives: This theme was included two subthemes: “communication with the family” and “communication with friends”. Participant No. 4 about communication with the family said, “I talk to my parents over the phone at the test night in the dormitory, and they will calm me with their words”.\nOn communication with friends, Participant No. 10 stated: “I talk to friends who are very intimate and express my anxiety and this reduces my anxiety”. Participant No. 2 said, “I talk to classmates who have a joint test, and talk about the test. This will reduce my anxiety”.\n\nStudying strategies: This theme includes two subthemes: “More effort in education” and “Applying different study strategies”. For “Studying earlier” Participant No. 1 stated that “I’ve been studying for weeks before the test, this will reduce my anxiety”.\nParticipant No. 4 about using the different strategies of study, said that “I will try to study with other friends (group study) for any test that I have anxiety”. Participant No. 3 stated: “I am studying similar questions to reduce my anxiety”. Participant No. 7 said that; “Reading the summary of the important content that other friends extracting from my booklet and book reduces my anxiety”.\n\nFinding ways to relax and self-care: it was another major theme that was included subthemes: “relaxing activities”, “exercise”, and “consuming Caffeinated beverages”.\nRegarding this theme, Participant No. 5 stated that; “When I have anxiety due to the test, I try to listen to my favorite music. This will manage my thoughts and also do not sleep”. Or Participant No. 1 said that; “Walking in the open air helps me become more fluent and calm down”. Participant No. 3 noted that “I usually drink coffee or Nescafe when I have hard and difficult test, this will reduce my anxiety”.\n\n“Negative strategies” This theme has two subthemes: “drug abuse” and “rely on cheating”. Participant No. 9 about rely to the cheating said that “I am cheating to reduce the anxiety of the test, even if I do not intend to use it, this makes calm down me”. Participant No. 8 about drug abuse stated that, “I’m eating a Propranolol 10 when I having an overwhelming anxiety”. Participant No. 6 stated that, “I used the Ritalin tablet when I fear from test and anxietyed me, although it did not work as a result of my test”.\n\nDiscussion:\nIn the present study, with a qualitative approach and interviews with students, researchers tried to identify and describe the experiences of strategies for coping with the anxiety of the test among students in dentistry, medicine and pharmacy.\nOne of the main themes that students use to cope test anxiety is “prayer and dialogue with God”. Students use prayer and dialogue with God to reduce their anxiety. Among the religious and spiritual sources, the greatest source used to reduce anxiety and anxiety is “prayer”. The prayer is derived from the Latin word of precarious meaning “obtained by pray and pleasure” [17]. The other studies showed praying is an effective strategy to cope with test anxiety. Adroishi et al. [18] showed that listening to pray could significantly reduce the test anxiety in students. The study of Masomi et al. showed that listening to the Quran sound, such as the sound of music before the test, is an effective strategy for the anxiety test, and the Quran sound is more effective in reducing student test anxiety [19]. In a study conducted by Narimany et al., 44.5% and 27.5% of the students used the use of prayer as a strategy for coping with anxiety [14]. Also, training religious values [20] and spiritual training, including prayer, forgiveness, transcendental and spiritual meditation [21] have also been reported to reduce the depression and anxiety of students. Ganji et al. also reported that religious beliefs are related with anxiety levels and reduce it [22]. In Papazisis et al. research, strong religious and spiritual beliefs have positive relationships increasing, the self-confidence, and a negative relationship with depression, anxiety and anxiety as a personality trait [23].\nBased on the findings of various studies, spiritual health determines the integrity of the person, and is the only force that harmonizes the physical, psychological and social dimensions. Religious and spiritual beliefs make a person calm and play a vital role in adapting to tension [24]. Most people believe that the influence of uncontrollable positions can be controlled through relay to God [25]. Religion, spirituality, and existential health are important predictors of mental health [26, 27].\nAnother strategy for students to calm down and manage their anxiety was to communicate with those who had some kind of attachment to them. In fact, this communication with people such as parents or intimate friends has made them relaxed and helped them better control the tension of the test. Cassidy and Shaver, in their justification of the relationship between attachment style and mental health, stated that the consequence of a safe attachment process is creating a sense of safety in a person, and the consequence of an unsafe attachment process is to create fear in a person [28]. Roberts et al. in their justification of this relationship, believe that the psychological consequence of unsafe attachment styles in tension situations is anxiety and depression. The psychological consequence of a safe attachment style in such situations is mental relaxation [29]. By studying 314 surviving adults from Bam earthquake, Rahimian Bouger et al. found that there was a significant positive relationship between safe attachment style and mental health, and a significant negative relationship between avoidance and ambivalent attachment styles with mental health [30]. The study of Besharat et al. revealed that the subjects with a safe attachment style rather than an unsafe type and those with an avoidant attachment style had fewer interpersonal problems than ambivalent styles. The results of this study refer to authenticity of safe attachment to the first requirement and its transfer to subsequent generations [31]. Safford has shown that people with an unsafe attachment style are more likely to experience anxiety and depression [32].\nMorey and Taylor [33] study, which was similar to the present study in terms of quality with in-depth interviews, found that exercise and talking to friends are two important strategies for students to deal with stress. Fujii [34] reported that one of the ways to deal with English test anxiety in students is “cooperation with others” and “building confidence”.\n\nStudying strategies which were students’ strategies to cope with test anxiety, can be considered as a kind of sensitization to manage anxiety and manage of the situation. These strategies are effective and positive strategies that can help the student in a constructive way to gain more control and to manage negative thoughts, and reduce his own anxiety through such actions. Motevalli et al. [35] reported that teaching new and practical study skills helps students manage test anxiety. They found that learning some skills such as time managing for studying, properly review and summarize, how to answer multiple-choice questions, correct/incorrect and descriptive questions can control test anxiety [35]. Yusefzadeh et al. [36] reported that based on the findings of a training and evaluation program (teacher 10–35% of the final grade of the course is based on activity during the semester), the implementation of such programs significantly reduces anxiety Students are tested. Ozbiçakçi et al. [37] also believe that teaching method reduces test anxiety. But L. Hsu mentioned that further research is needed to determine best practices for alleviating student stress and anxiety [38].\n\n“Finding ways to relax and self-care” is the other main theme for coping with test anxiety. It included Relaxing activities, consuming caffeine and Soothing food. Findings of the study by Mojarrab et al. [39] showed that the use of relaxation techniques along with playing soothing music reduces nursing students’ test anxiety and also improves their clinical test scores. Morey and Taylor [33] reported that many students believed exercise would distract them from test anxiety and lead to be calm. Walking, running and yoga were among the activities that students focused on, with more emphasis on walking [33].\nIn this study, negative strategies were also used to manage the anxiety by the students, such as Unusual use of some medications. Evidences show that the consumption of stimulants and non-drug use of a variety of drugs is one of the threats to students. Several evidences suggest that consuming drug abuse among young prople, especially students, is increasing. Methylphenidate or Ritalin is one of the most widely used drugs that has recently been abused among adolescents, especially students [40]. Many students take this medicine to stay awake for several hours and to maintain their unusual focuses for a long time. During the tests, the use of such drugs, including Ritalin, increases. Ritalin is the most common prescribed psychotropic drug for children in the USA [14]. Currently, non-medical consumption of prescription stimulants, including Methylphenidate, is a growing problem among students in the USA. Statistics have shown that 7% of American students have at least once used this drug over their lifetime, and the prevalence of Methylphenidate consumption among students over the course of 1 year was 3% [13]. In this regard, several studies have reported 3–35% of Ritalin abuse among students [41–46]. The most common side effects of consuming Ritalin are insomnia, nervousness, anxiety, headache, and loss of appetite. In excessive consumption, restlessness, delirium, psychosis, hypertension, seizure, hyperthermia, and arrhythmias may occur [47].\n\nWriting cheating was one of the other negative strategies that students used to reduce their test anxiety, although they had no intention of using this cheating during the test. The student, by writing cheat, wants to create a reliance point on himself/herself and thereby calm himself down and overcome his anxiety. In fact, cheating as a reliance point of its existence is an important part of assurance, not to applying it.\n\nConclusions:\nThe result of this study showed that students often use positive strategies to overcome the test anxiety and try to use positive strategies, but some students are advised of undesirable strategies such as misuse of authorized drug and writing cheating that lead to a lot of complex problems. Given that the test are an important part of academic life and that every student is always involved in a test and study evaluation, the educational system should do its utmost effort to empower students to manage anxiety by learning the best strategies.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe studies show test anxiety is a common disorder in students that causes academic failure. There are not enough studies and specific theoretical background about test anxiety and ways to deal with it, so the purpose of this study was to do a qualitative study to fully understand the ways to deal with test anxiety in medical Sciences students.\n\nMethods:\nThis is a qualitative study. The participants are the students of the last 2 years of pharmacy, medicine and dentistry at Isfahan University of Medical Sciences. Ten students were selected by purposeful sampling, and interviews continued until the data saturation stage and the lack of access to new data. The data were analyzed by seven-level Colaizzi method.\n\nResults:\nAfter analyzing data, about 50 codes were extracted. These codes divided into 16 subclasses, and among them, ultimately five main themes are extracted: \"Prayer and Dialogue with God\", \"Interaction and communication with friends and relatives\", \"studying strategies\", \"Finding ways to relax and self-care\" and \"Negative strategies\" were extracted.\n\nConclusions:\nThe result of this study showed that students often use positive strategies to overcome the test anxiety and try to use positive strategies, but some students are advised of undesirable strategies such as misuse of authorized drugs and writing cheating that that lead to a lot of complex problems. The educational system should do its utmost effort to empower students to manage the anxiety by learning the best strategies."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nOne of the concerns of the educational system in universities is the test anxiety [1]. Test anxiety is a special type of anxiety that is characterized by physical, cognitive, and behavioral symptoms when preparing for the test and performing test, It becomes a problem when high levels of anxiety interfere with preparing for and taking the test. Test anxiety is characterized by severe fear of poor performance in tests [2]. In fact, the test anxiety is an emotional experience, feelings and anxiety in situations in which a person feels that his/her performance is evaluated [3]. Test anxiety is defined as the experience of fear, apprehension, and worry before, during, or after a test that can lead to mental distraction, memory impairment, and physical symptoms such as nausea, headache, and tachycardia [4].\nThis type of anxiety occurs between the ages of 10 and 12 and increases with increasing age and maximizes in higher education [5]. According to estimates by researchers in the United States, 15% of students experience different levels of anxiety annually [6]. In the study by Tsegay et al. [7] it was reported that the rate of test anxiety in medical students was 52.3% and significantly higher in female students. Test anxiety is important because it affects test success [8]. Test anxiety can cause academic failure by reducing intrinsic motivation and existing cognitive ability [9]. Sarason [10] considers test anxiety as a self-preoccupation, which is characterized by self-defeat and doubt about his/her abilities. this anxiety often leads to negative cognitive assessment, undesirable physiological reactions and the decline of academic performance. As a result, there is a significant reverse relationship between anxiety scores and test scores [10]. Various studies have shown that test anxiety has a significant and important effect on the academic performance of students. Cassady [11] showed in his study that students with higher test anxiety had a weaker academic performance [11].\nStudents try to cope with anxiety in order to manage it. Coping is the effort to control and manage situations that seem dangerous and tense [12]. There are two main strategies for coping with anxiety that are known as problem-focused coping and emotion-focused coping strategies. The problem-focused coping strategy in which the main objective is to dominate the position and change in the source of anxiety, and emotion-focused coping strategies in which the main objective is to reduce or transform the emotional disturbance quickly [13].\nNarimany and et al. stated that students manage their anxiety through conventional methods such as: they think about good memories of the past, praying and eliminating negative thoughts and beliefs [14].\nIn previous studies, descriptive studies have focused more on estimating the extent to which positive methods have been used, and less on the overall evaluation of all coping strategies that students use. Also, in this study, an attempt has been made to study the strategies of medical students to deal with test anxiety in Eastern and Islamic countries. In Eastern countries, beliefs are different from some other countries where previous studies have been conducted, so its findings can help improve knowledge in this area. On the other hand, this study has selected medical students as the target population because these students face a high volume of difficult. They may also choose different strategies to deal with test anxiety. In this regard, the present study was designed in qualitative study in this target group and conducted with the aim of identifying strategies to deal with test anxiety in medical students.\n\nConclusions:\nThe result of this study showed that students often use positive strategies to overcome the test anxiety and try to use positive strategies, but some students are advised of undesirable strategies such as misuse of authorized drug and writing cheating that lead to a lot of complex problems. Given that the test are an important part of academic life and that every student is always involved in a test and study evaluation, the educational system should do its utmost effort to empower students to manage anxiety by learning the best strategies."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nThe studies show test anxiety is a common disorder in students that causes academic failure. There are not enough studies and specific theoretical background about test anxiety and ways to deal with it, so the purpose of this study was to do a qualitative study to fully understand the ways to deal with test anxiety in medical Sciences students.\n\nMethods:\nThis is a qualitative study. The participants are the students of the last 2 years of pharmacy, medicine and dentistry at Isfahan University of Medical Sciences. Ten students were selected by purposeful sampling, and interviews continued until the data saturation stage and the lack of access to new data. The data were analyzed by seven-level Colaizzi method.\n\nResults:\nAfter analyzing data, about 50 codes were extracted. These codes divided into 16 subclasses, and among them, ultimately five main themes are extracted: \"Prayer and Dialogue with God\", \"Interaction and communication with friends and relatives\", \"studying strategies\", \"Finding ways to relax and self-care\" and \"Negative strategies\" were extracted.\n\nConclusions:\nThe result of this study showed that students often use positive strategies to overcome the test anxiety and try to use positive strategies, but some students are advised of undesirable strategies such as misuse of authorized drugs and writing cheating that that lead to a lot of complex problems. The educational system should do its utmost effort to empower students to manage the anxiety by learning the best strategies."},"whole_article_text_length":{"kind":"number","value":3824,"string":"3,824"},"whole_article_abstract_length":{"kind":"number","value":283,"string":"283"},"other_sections_lengths":{"kind":"list like","value":[825],"string":"[\n 825\n]","isConversational":false},"num_sections":{"kind":"number","value":5,"string":"5"},"most_frequent_words":{"kind":"list like","value":["anxiety","test","students","test anxiety","study","strategies","participant","reduce","use","attachment"],"string":"[\n \"anxiety\",\n \"test\",\n \"students\",\n \"test anxiety\",\n \"study\",\n \"strategies\",\n \"participant\",\n \"reduce\",\n \"use\",\n \"attachment\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["study test anxiety","test anxiety cause","test anxiety characterized","student test anxiety","students test anxiety"],"string":"[\n \"study test anxiety\",\n \"test anxiety cause\",\n \"test anxiety characterized\",\n \"student test anxiety\",\n \"students test anxiety\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"null"},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Test anxiety | Higher education | Coping strategies | Qualitative study [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"null"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Test anxiety | Higher education | Coping strategies | Qualitative study [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Test anxiety | Higher education | Coping strategies | Qualitative study [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Test anxiety | Higher education | Coping strategies | Qualitative study [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Anxiety | Humans | Learning | Qualitative Research | Students, Medical | Test Anxiety [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"null"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Anxiety | Humans | Learning | Qualitative Research | Students, Medical | Test Anxiety [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Anxiety | Humans | Learning | Qualitative Research | Students, Medical | Test Anxiety [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Anxiety | Humans | Learning | Qualitative Research | Students, Medical | Test Anxiety [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] study test anxiety | test anxiety cause | test anxiety characterized | student test anxiety | students test anxiety [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"null"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] study test anxiety | test anxiety cause | test anxiety characterized | student test anxiety | students test anxiety [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] study test anxiety | test anxiety cause | test anxiety characterized | student test anxiety | students test anxiety [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] study test anxiety | test anxiety cause | test anxiety characterized | student test anxiety | students test anxiety [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] anxiety | test | students | test anxiety | study | strategies | participant | reduce | use | attachment [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"null"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] anxiety | test | students | test anxiety | study | strategies | participant | reduce | use | attachment [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] anxiety | test | students | test anxiety | study | strategies | participant | reduce | use | attachment [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] anxiety | test | students | test anxiety | study | strategies | participant | reduce | use | attachment [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] anxiety | test | test anxiety | students | coping | performance | focused | focused coping | medical students | strategies [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"null"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] use positive | use positive strategies | strategies | positive strategies | test | students | positive | overcome test anxiety try | overcome test anxiety | educational system utmost effort [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] anxiety | test | students | participant | study | strategies | test anxiety | data | interview | attachment [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] anxiety | test | students | participant | study | strategies | test anxiety | data | interview | attachment [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"null"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| the last 2 years | Isfahan University of Medical Sciences ||| Ten ||| seven ||| about 50 ||| 16 | five ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| the last 2 years | Isfahan University of Medical Sciences ||| Ten ||| seven ||| about 50 ||| 16 | five ||| ||| [SUMMARY]"}}},{"rowIdx":86,"cells":{"title":{"kind":"string","value":"Peer Reviewers in Central Asia: Publons Based Analysis."},"pmid":{"kind":"string","value":"34184435"},"background_abstract":{"kind":"string","value":"The five Central Asian republics comprise of Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan, and Uzbekistan. Their research and publication activities are gradually improving but there is limited data on how good their peer reviewing practices are."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We have use the Publons database to extract information on the reviewers registered including the number of verified review, Publons award winners, and top universities in the domain of peer reviewing. This has been analysed overall and country wise."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Of 15,764 researchers registered on Publons, only 370 (11.7%) have verified records of peer-reviewing. There are 8 Publons award winners. There is great heterogeneity in the number of active reviewers across the five countries. Kazakhstan and Uzbekistan account for more than 90% of verified reviewers. Only Kazakhstan has more than 100 active reviewers and 6 Publons award recipients. Amongst the top 20 reviewers from Central Asia, half of them are from the Nazarbayev University, Nur-Sultan, Kazakhstan. Three countries have less than 10 universities registered on Publons."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Central Asia has a good number of peer reviewers on Publons though only a minority of researchers are involved in peer reviewing. However, the heterogeneity between the nations can be best dealt with by promoting awareness and international networking including e-learning and mentoring programs."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Asia, Central","Databases, Bibliographic","Humans","Peer Review","Periodicals as Topic","Publications"],"string":"[\n \"Asia, Central\",\n \"Databases, Bibliographic\",\n \"Humans\",\n \"Peer Review\",\n \"Periodicals as Topic\",\n \"Publications\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"8239425"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Central Asia (CA) has a rich scientific temperament but has reduced visibility in the field of scientific literature. This may stem from the previous dependence on the Russian language and reduced exposure to international science.1 Things are slowly changing. Some countries are doing well and some have yet to catch up in terms of publications.2 Previous analyses have focussed on publications from these regions.234 The increasing number of manuscripts portrays growing scientific curiosity, research intent and the increasing ability to understand the intricacies and ethics of publication. Complementary to scientific publishing, another integral requirement for the development of scientific rationale and thinking is peer-reviewing.5\nPeer reviewing is an integral part of the research publication process, and often it is considered a thankless job.6 However, mainly thanks to the Publons initiative, more and more researchers are being recognised for their contributions to peer review.7 The coronavirus disease 2019 pandemic has stressed the peer review process8 and might be the refining fire that forges the next generation of peer reviewers. A peer reviewer, who is below par, may disrupt the trust that underlies this privileged gate-keeping function with some offhand remark.9 Thus, it is pertinent to have peer reviewers at par with the best in the world.\nOverall, Asia is estimated to have a good pool of upcoming reviewers.10 In the field of publishing, Kazakhstan seems to be doing well.11 A recent analysis of the top institutes in CA and the neighbouring region has shown dominance by China in various Publons ratings.4 The same Publons platform provides a unique opportunity to explore the reviewing experiences and capacities of these nations.7 Publons was initially launched to provide credit for reviewers. However, it has expanded laterally and now also includes editorial records. After being brought under Clarivate Analytics, it is now also laterally integrated with the Web of Science from which publication information is synchronized. The ‘Publons Reviewer Connect’ is an artificial intelligence based program that can help editors identify potential reviewers.12 It can also synchronize records with the ORCID database.13 Thus, it is one of the best available tools to analyse reviewer profiles, in relationship to the reviewers' publications and editorial activities. Thus, we have attempted to analyse the numbers and expertise of peer reviews as well as top universities involved in peer reviewing from various Central Asian countries using Publons."},"methods_title":{"kind":"string","value":"METHODS"},"methods_text":{"kind":"string","value":"There are 5 Central Asian republics with diverse research evaluation and publication strategies: Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan and Uzbekistan. The inbuilt search engine of Publons was used to extract data on May the 10th, 2021.\nData import First, the total number of researchers registered for each of these countries was noted. Then, the total number of reviewers with at least one verified review, with more than one verified review, the number of Publons recognized Top Reviewers, and the number of reviewers with at least one verified review in the last 12 months was extracted (Table 1). Also, the total number of reviews for each country as well as the total number of reviews for each country in the preceding 12 months was totalled. Finally, the top institutes of CA having the most number of reviews to their name, and the top 10 institutes per country were imported from Publons. Additionally, the number of Web of Science publications for the reviewers and the top institutes were also noted.\naCountry code is provided for Kazakhstan (“181”) as an example.\nFirst, the total number of researchers registered for each of these countries was noted. Then, the total number of reviewers with at least one verified review, with more than one verified review, the number of Publons recognized Top Reviewers, and the number of reviewers with at least one verified review in the last 12 months was extracted (Table 1). Also, the total number of reviews for each country as well as the total number of reviews for each country in the preceding 12 months was totalled. Finally, the top institutes of CA having the most number of reviews to their name, and the top 10 institutes per country were imported from Publons. Additionally, the number of Web of Science publications for the reviewers and the top institutes were also noted.\naCountry code is provided for Kazakhstan (“181”) as an example.\nData analysis Normality of data was assessed by the Shapiro-Wilk test. Descriptive statistics are described along with graphical presentations including bar-graphs, pie-charts and scatter-plots as relevant.\nNormality of data was assessed by the Shapiro-Wilk test. Descriptive statistics are described along with graphical presentations including bar-graphs, pie-charts and scatter-plots as relevant.\nSoftware Data was formatted into spreadsheets in MS Excel and then imported into the R statistical environment version 4.0.3. Both Excel and R packages ggpubr, dplyr, GGally, ggplot2 were used for data analysis and data visualization.\nData was formatted into spreadsheets in MS Excel and then imported into the R statistical environment version 4.0.3. Both Excel and R packages ggpubr, dplyr, GGally, ggplot2 were used for data analysis and data visualization."},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"The total number of researchers from CA registered on Publons was 15,764 and the countrywide distribution is mentioned in Table 2. However, only 370 (11.7%) have at least 1 verified review (Fig. 1). This varies amongst the countries (Fig. 2). Six reviewers from Kazakhstan and one each from Uzbekistan and Kyrgyzstan have achieved the distinction of being awarded the Publons Reviewer Award.\nThe top 20 reviewers across CA with the maximum number of verified reviews are listed in Table 3. Ten of the top 20 reviewers belong to Nazarbayev University, Nur-Sultan, Kazakhstan. Seven of the top reviewers are also active editors with a different number of editorial records to their names.\nThe top 10 universities with the maximum number of reviews per country have been listed in Table 4. However, this list is abbreviated for 3 countries that have less than 10 universities registered on Publons. Fig. 3 shows the relationship between the number of verified reviews and publication from these 29 universities across the five countries."},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":["Data import","Data analysis","Software"],"string":"[\n \"Data import\",\n \"Data analysis\",\n \"Software\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["First, the total number of researchers registered for each of these countries was noted. Then, the total number of reviewers with at least one verified review, with more than one verified review, the number of Publons recognized Top Reviewers, and the number of reviewers with at least one verified review in the last 12 months was extracted (Table 1). Also, the total number of reviews for each country as well as the total number of reviews for each country in the preceding 12 months was totalled. Finally, the top institutes of CA having the most number of reviews to their name, and the top 10 institutes per country were imported from Publons. Additionally, the number of Web of Science publications for the reviewers and the top institutes were also noted.\naCountry code is provided for Kazakhstan (“181”) as an example.","Normality of data was assessed by the Shapiro-Wilk test. Descriptive statistics are described along with graphical presentations including bar-graphs, pie-charts and scatter-plots as relevant.","Data was formatted into spreadsheets in MS Excel and then imported into the R statistical environment version 4.0.3. Both Excel and R packages ggpubr, dplyr, GGally, ggplot2 were used for data analysis and data visualization."],"string":"[\n \"First, the total number of researchers registered for each of these countries was noted. Then, the total number of reviewers with at least one verified review, with more than one verified review, the number of Publons recognized Top Reviewers, and the number of reviewers with at least one verified review in the last 12 months was extracted (Table 1). Also, the total number of reviews for each country as well as the total number of reviews for each country in the preceding 12 months was totalled. Finally, the top institutes of CA having the most number of reviews to their name, and the top 10 institutes per country were imported from Publons. Additionally, the number of Web of Science publications for the reviewers and the top institutes were also noted.\\naCountry code is provided for Kazakhstan (“181”) as an example.\",\n \"Normality of data was assessed by the Shapiro-Wilk test. Descriptive statistics are described along with graphical presentations including bar-graphs, pie-charts and scatter-plots as relevant.\",\n \"Data was formatted into spreadsheets in MS Excel and then imported into the R statistical environment version 4.0.3. Both Excel and R packages ggpubr, dplyr, GGally, ggplot2 were used for data analysis and data visualization.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null],"string":"[\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","METHODS","Data import","Data analysis","Software","RESULTS","DISCUSSION"],"string":"[\n \"INTRODUCTION\",\n \"METHODS\",\n \"Data import\",\n \"Data analysis\",\n \"Software\",\n \"RESULTS\",\n \"DISCUSSION\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Central Asia (CA) has a rich scientific temperament but has reduced visibility in the field of scientific literature. This may stem from the previous dependence on the Russian language and reduced exposure to international science.1 Things are slowly changing. Some countries are doing well and some have yet to catch up in terms of publications.2 Previous analyses have focussed on publications from these regions.234 The increasing number of manuscripts portrays growing scientific curiosity, research intent and the increasing ability to understand the intricacies and ethics of publication. Complementary to scientific publishing, another integral requirement for the development of scientific rationale and thinking is peer-reviewing.5\nPeer reviewing is an integral part of the research publication process, and often it is considered a thankless job.6 However, mainly thanks to the Publons initiative, more and more researchers are being recognised for their contributions to peer review.7 The coronavirus disease 2019 pandemic has stressed the peer review process8 and might be the refining fire that forges the next generation of peer reviewers. A peer reviewer, who is below par, may disrupt the trust that underlies this privileged gate-keeping function with some offhand remark.9 Thus, it is pertinent to have peer reviewers at par with the best in the world.\nOverall, Asia is estimated to have a good pool of upcoming reviewers.10 In the field of publishing, Kazakhstan seems to be doing well.11 A recent analysis of the top institutes in CA and the neighbouring region has shown dominance by China in various Publons ratings.4 The same Publons platform provides a unique opportunity to explore the reviewing experiences and capacities of these nations.7 Publons was initially launched to provide credit for reviewers. However, it has expanded laterally and now also includes editorial records. After being brought under Clarivate Analytics, it is now also laterally integrated with the Web of Science from which publication information is synchronized. The ‘Publons Reviewer Connect’ is an artificial intelligence based program that can help editors identify potential reviewers.12 It can also synchronize records with the ORCID database.13 Thus, it is one of the best available tools to analyse reviewer profiles, in relationship to the reviewers' publications and editorial activities. Thus, we have attempted to analyse the numbers and expertise of peer reviews as well as top universities involved in peer reviewing from various Central Asian countries using Publons.","There are 5 Central Asian republics with diverse research evaluation and publication strategies: Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan and Uzbekistan. The inbuilt search engine of Publons was used to extract data on May the 10th, 2021.\nData import First, the total number of researchers registered for each of these countries was noted. Then, the total number of reviewers with at least one verified review, with more than one verified review, the number of Publons recognized Top Reviewers, and the number of reviewers with at least one verified review in the last 12 months was extracted (Table 1). Also, the total number of reviews for each country as well as the total number of reviews for each country in the preceding 12 months was totalled. Finally, the top institutes of CA having the most number of reviews to their name, and the top 10 institutes per country were imported from Publons. Additionally, the number of Web of Science publications for the reviewers and the top institutes were also noted.\naCountry code is provided for Kazakhstan (“181”) as an example.\nFirst, the total number of researchers registered for each of these countries was noted. Then, the total number of reviewers with at least one verified review, with more than one verified review, the number of Publons recognized Top Reviewers, and the number of reviewers with at least one verified review in the last 12 months was extracted (Table 1). Also, the total number of reviews for each country as well as the total number of reviews for each country in the preceding 12 months was totalled. Finally, the top institutes of CA having the most number of reviews to their name, and the top 10 institutes per country were imported from Publons. Additionally, the number of Web of Science publications for the reviewers and the top institutes were also noted.\naCountry code is provided for Kazakhstan (“181”) as an example.\nData analysis Normality of data was assessed by the Shapiro-Wilk test. Descriptive statistics are described along with graphical presentations including bar-graphs, pie-charts and scatter-plots as relevant.\nNormality of data was assessed by the Shapiro-Wilk test. Descriptive statistics are described along with graphical presentations including bar-graphs, pie-charts and scatter-plots as relevant.\nSoftware Data was formatted into spreadsheets in MS Excel and then imported into the R statistical environment version 4.0.3. Both Excel and R packages ggpubr, dplyr, GGally, ggplot2 were used for data analysis and data visualization.\nData was formatted into spreadsheets in MS Excel and then imported into the R statistical environment version 4.0.3. Both Excel and R packages ggpubr, dplyr, GGally, ggplot2 were used for data analysis and data visualization.","First, the total number of researchers registered for each of these countries was noted. Then, the total number of reviewers with at least one verified review, with more than one verified review, the number of Publons recognized Top Reviewers, and the number of reviewers with at least one verified review in the last 12 months was extracted (Table 1). Also, the total number of reviews for each country as well as the total number of reviews for each country in the preceding 12 months was totalled. Finally, the top institutes of CA having the most number of reviews to their name, and the top 10 institutes per country were imported from Publons. Additionally, the number of Web of Science publications for the reviewers and the top institutes were also noted.\naCountry code is provided for Kazakhstan (“181”) as an example.","Normality of data was assessed by the Shapiro-Wilk test. Descriptive statistics are described along with graphical presentations including bar-graphs, pie-charts and scatter-plots as relevant.","Data was formatted into spreadsheets in MS Excel and then imported into the R statistical environment version 4.0.3. Both Excel and R packages ggpubr, dplyr, GGally, ggplot2 were used for data analysis and data visualization.","The total number of researchers from CA registered on Publons was 15,764 and the countrywide distribution is mentioned in Table 2. However, only 370 (11.7%) have at least 1 verified review (Fig. 1). This varies amongst the countries (Fig. 2). Six reviewers from Kazakhstan and one each from Uzbekistan and Kyrgyzstan have achieved the distinction of being awarded the Publons Reviewer Award.\nThe top 20 reviewers across CA with the maximum number of verified reviews are listed in Table 3. Ten of the top 20 reviewers belong to Nazarbayev University, Nur-Sultan, Kazakhstan. Seven of the top reviewers are also active editors with a different number of editorial records to their names.\nThe top 10 universities with the maximum number of reviews per country have been listed in Table 4. However, this list is abbreviated for 3 countries that have less than 10 universities registered on Publons. Fig. 3 shows the relationship between the number of verified reviews and publication from these 29 universities across the five countries.","This analysis of the reviewers from the core CA countries reveals that Kazakhstan seems to top the list while two specific countries need a major boost. The number of active reviewers as well institutes in Kazakhstan reveal its potential as a rising star in the region. Uzbekistan also seems to be doing well. These two countries represent more than 90% of researchers registered on Publons as well as those involved in active peer-reviewing.\nKazakhstan has natural resources and is a major contributor to the economy of CA.14 Similarly, Uzbekistan has been gradually improving its economy and contribution to education in the last decade. These can be helping them made rapid strides in research and publications.4 Similarly, the increase in publications can also influence knowledge of publication ethics and promote peer reviewing practices.\nLooking at the performance of their top institutes, Kazakhstan has a good ratio of peer-reviews to publications. This ratio is less for all others including Uzbekistan. Also, the eight reviewers from the region who have received recognition from Publons as top reviewers highlight the point that the potential of this region is second to none. This recognition can also help stimulate their peer to achieve such distinctions.\nPeer reviewing not only trains the mind to be analytical but also teaches many aspects of publication ethics. These include understanding plagiarism,15 authorship criteria,16 declaring potential conflicts of interest17 and also the skill of scientific writing. Thus, it has been proposed that peer-reviewing should be a part of ongoing medical education.18\nThere are different metrics to measure researcher and author impact.13 However, for peer reviewers, they are no other metrics beyond what is present in Publons: the number of journals reviewed for, and the number of verified reviews (overall and in the last 12 months). Also, Publons has an option in which Editor can recommend someone as an excellent reviewer and also, it has its mechanism to award top reviewer recognition.\nThe most pertinent question is how to mine the potential of Central Asian reviewers. The Publons Academy is an initiation that enables experienced peer reviewers to mentor novice and upcoming reviewers. There is a need to involve both experts on this initiative to devote their time for mentoring and also spread awareness amongst the CA reviewers to get registered for such mentoring. Journal editors can help organize online or offline courses on peer-reviewing for these nations. Online education has added the advantage of bringing together experts from varied regions of the world. Nevertheless, it cannot be a one-way process. The CA countries, especially the underperforming ones, will also need to pull up their bootstraps. There has been freedom of knowledge, an open platform for learning and limitless striving to yearn for perfection. The other countries can take a leaf out of the book from Kazakhstan and stress for more open networking and learning resources.\nThe leading university of Kazakhstan, the Nazarbayev University, is contributing to half the top reviewers in the region, as well as the highest proportion of verified reviews. Thus, it has an unparalleled capacity for the region and can take an initiative to bridge across to other universities in the region and disseminate their expertise.\nThere are limitations to this analysis. Firstly, some active reviewers may not be registered on Publons. All registered reviewers also might not be active on Publons. Secondly, there may be predatory journals also registered on Publons and all the peer reviews verified may not have any minimum quality assurance.19\nThere seems to be no practical way to include reviewers not on Publons, in our analysis. We presume they would be in equal proportion in each country. However, a greater proportion may be from Tajikistan and Turkmenistan possible due to reduced awareness about Publons. The CA countries have different access to the internet and social media.20 Also, Publons is evolving and trying out different strategies to exclude fake or poor reviews.12\nThus, there is heterogeneity in the distribution of peer reviewer in the CA countries. There seems to be a good number of upcoming reviewers in a couple of countries while the others may need more awareness. This region may benefit much from international networking, symposia and e-learning platforms dedicated to promoting peer-reviewing."],"string":"[\n \"Central Asia (CA) has a rich scientific temperament but has reduced visibility in the field of scientific literature. This may stem from the previous dependence on the Russian language and reduced exposure to international science.1 Things are slowly changing. Some countries are doing well and some have yet to catch up in terms of publications.2 Previous analyses have focussed on publications from these regions.234 The increasing number of manuscripts portrays growing scientific curiosity, research intent and the increasing ability to understand the intricacies and ethics of publication. Complementary to scientific publishing, another integral requirement for the development of scientific rationale and thinking is peer-reviewing.5\\nPeer reviewing is an integral part of the research publication process, and often it is considered a thankless job.6 However, mainly thanks to the Publons initiative, more and more researchers are being recognised for their contributions to peer review.7 The coronavirus disease 2019 pandemic has stressed the peer review process8 and might be the refining fire that forges the next generation of peer reviewers. A peer reviewer, who is below par, may disrupt the trust that underlies this privileged gate-keeping function with some offhand remark.9 Thus, it is pertinent to have peer reviewers at par with the best in the world.\\nOverall, Asia is estimated to have a good pool of upcoming reviewers.10 In the field of publishing, Kazakhstan seems to be doing well.11 A recent analysis of the top institutes in CA and the neighbouring region has shown dominance by China in various Publons ratings.4 The same Publons platform provides a unique opportunity to explore the reviewing experiences and capacities of these nations.7 Publons was initially launched to provide credit for reviewers. However, it has expanded laterally and now also includes editorial records. After being brought under Clarivate Analytics, it is now also laterally integrated with the Web of Science from which publication information is synchronized. The ‘Publons Reviewer Connect’ is an artificial intelligence based program that can help editors identify potential reviewers.12 It can also synchronize records with the ORCID database.13 Thus, it is one of the best available tools to analyse reviewer profiles, in relationship to the reviewers' publications and editorial activities. Thus, we have attempted to analyse the numbers and expertise of peer reviews as well as top universities involved in peer reviewing from various Central Asian countries using Publons.\",\n \"There are 5 Central Asian republics with diverse research evaluation and publication strategies: Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan and Uzbekistan. The inbuilt search engine of Publons was used to extract data on May the 10th, 2021.\\nData import First, the total number of researchers registered for each of these countries was noted. Then, the total number of reviewers with at least one verified review, with more than one verified review, the number of Publons recognized Top Reviewers, and the number of reviewers with at least one verified review in the last 12 months was extracted (Table 1). Also, the total number of reviews for each country as well as the total number of reviews for each country in the preceding 12 months was totalled. Finally, the top institutes of CA having the most number of reviews to their name, and the top 10 institutes per country were imported from Publons. Additionally, the number of Web of Science publications for the reviewers and the top institutes were also noted.\\naCountry code is provided for Kazakhstan (“181”) as an example.\\nFirst, the total number of researchers registered for each of these countries was noted. Then, the total number of reviewers with at least one verified review, with more than one verified review, the number of Publons recognized Top Reviewers, and the number of reviewers with at least one verified review in the last 12 months was extracted (Table 1). Also, the total number of reviews for each country as well as the total number of reviews for each country in the preceding 12 months was totalled. Finally, the top institutes of CA having the most number of reviews to their name, and the top 10 institutes per country were imported from Publons. Additionally, the number of Web of Science publications for the reviewers and the top institutes were also noted.\\naCountry code is provided for Kazakhstan (“181”) as an example.\\nData analysis Normality of data was assessed by the Shapiro-Wilk test. Descriptive statistics are described along with graphical presentations including bar-graphs, pie-charts and scatter-plots as relevant.\\nNormality of data was assessed by the Shapiro-Wilk test. Descriptive statistics are described along with graphical presentations including bar-graphs, pie-charts and scatter-plots as relevant.\\nSoftware Data was formatted into spreadsheets in MS Excel and then imported into the R statistical environment version 4.0.3. Both Excel and R packages ggpubr, dplyr, GGally, ggplot2 were used for data analysis and data visualization.\\nData was formatted into spreadsheets in MS Excel and then imported into the R statistical environment version 4.0.3. Both Excel and R packages ggpubr, dplyr, GGally, ggplot2 were used for data analysis and data visualization.\",\n \"First, the total number of researchers registered for each of these countries was noted. Then, the total number of reviewers with at least one verified review, with more than one verified review, the number of Publons recognized Top Reviewers, and the number of reviewers with at least one verified review in the last 12 months was extracted (Table 1). Also, the total number of reviews for each country as well as the total number of reviews for each country in the preceding 12 months was totalled. Finally, the top institutes of CA having the most number of reviews to their name, and the top 10 institutes per country were imported from Publons. Additionally, the number of Web of Science publications for the reviewers and the top institutes were also noted.\\naCountry code is provided for Kazakhstan (“181”) as an example.\",\n \"Normality of data was assessed by the Shapiro-Wilk test. Descriptive statistics are described along with graphical presentations including bar-graphs, pie-charts and scatter-plots as relevant.\",\n \"Data was formatted into spreadsheets in MS Excel and then imported into the R statistical environment version 4.0.3. Both Excel and R packages ggpubr, dplyr, GGally, ggplot2 were used for data analysis and data visualization.\",\n \"The total number of researchers from CA registered on Publons was 15,764 and the countrywide distribution is mentioned in Table 2. However, only 370 (11.7%) have at least 1 verified review (Fig. 1). This varies amongst the countries (Fig. 2). Six reviewers from Kazakhstan and one each from Uzbekistan and Kyrgyzstan have achieved the distinction of being awarded the Publons Reviewer Award.\\nThe top 20 reviewers across CA with the maximum number of verified reviews are listed in Table 3. Ten of the top 20 reviewers belong to Nazarbayev University, Nur-Sultan, Kazakhstan. Seven of the top reviewers are also active editors with a different number of editorial records to their names.\\nThe top 10 universities with the maximum number of reviews per country have been listed in Table 4. However, this list is abbreviated for 3 countries that have less than 10 universities registered on Publons. Fig. 3 shows the relationship between the number of verified reviews and publication from these 29 universities across the five countries.\",\n \"This analysis of the reviewers from the core CA countries reveals that Kazakhstan seems to top the list while two specific countries need a major boost. The number of active reviewers as well institutes in Kazakhstan reveal its potential as a rising star in the region. Uzbekistan also seems to be doing well. These two countries represent more than 90% of researchers registered on Publons as well as those involved in active peer-reviewing.\\nKazakhstan has natural resources and is a major contributor to the economy of CA.14 Similarly, Uzbekistan has been gradually improving its economy and contribution to education in the last decade. These can be helping them made rapid strides in research and publications.4 Similarly, the increase in publications can also influence knowledge of publication ethics and promote peer reviewing practices.\\nLooking at the performance of their top institutes, Kazakhstan has a good ratio of peer-reviews to publications. This ratio is less for all others including Uzbekistan. Also, the eight reviewers from the region who have received recognition from Publons as top reviewers highlight the point that the potential of this region is second to none. This recognition can also help stimulate their peer to achieve such distinctions.\\nPeer reviewing not only trains the mind to be analytical but also teaches many aspects of publication ethics. These include understanding plagiarism,15 authorship criteria,16 declaring potential conflicts of interest17 and also the skill of scientific writing. Thus, it has been proposed that peer-reviewing should be a part of ongoing medical education.18\\nThere are different metrics to measure researcher and author impact.13 However, for peer reviewers, they are no other metrics beyond what is present in Publons: the number of journals reviewed for, and the number of verified reviews (overall and in the last 12 months). Also, Publons has an option in which Editor can recommend someone as an excellent reviewer and also, it has its mechanism to award top reviewer recognition.\\nThe most pertinent question is how to mine the potential of Central Asian reviewers. The Publons Academy is an initiation that enables experienced peer reviewers to mentor novice and upcoming reviewers. There is a need to involve both experts on this initiative to devote their time for mentoring and also spread awareness amongst the CA reviewers to get registered for such mentoring. Journal editors can help organize online or offline courses on peer-reviewing for these nations. Online education has added the advantage of bringing together experts from varied regions of the world. Nevertheless, it cannot be a one-way process. The CA countries, especially the underperforming ones, will also need to pull up their bootstraps. There has been freedom of knowledge, an open platform for learning and limitless striving to yearn for perfection. The other countries can take a leaf out of the book from Kazakhstan and stress for more open networking and learning resources.\\nThe leading university of Kazakhstan, the Nazarbayev University, is contributing to half the top reviewers in the region, as well as the highest proportion of verified reviews. Thus, it has an unparalleled capacity for the region and can take an initiative to bridge across to other universities in the region and disseminate their expertise.\\nThere are limitations to this analysis. Firstly, some active reviewers may not be registered on Publons. All registered reviewers also might not be active on Publons. Secondly, there may be predatory journals also registered on Publons and all the peer reviews verified may not have any minimum quality assurance.19\\nThere seems to be no practical way to include reviewers not on Publons, in our analysis. We presume they would be in equal proportion in each country. However, a greater proportion may be from Tajikistan and Turkmenistan possible due to reduced awareness about Publons. The CA countries have different access to the internet and social media.20 Also, Publons is evolving and trying out different strategies to exclude fake or poor reviews.12\\nThus, there is heterogeneity in the distribution of peer reviewer in the CA countries. There seems to be a good number of upcoming reviewers in a couple of countries while the others may need more awareness. This region may benefit much from international networking, symposia and e-learning platforms dedicated to promoting peer-reviewing.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["intro","methods",null,null,null,"results","discussion"],"string":"[\n \"intro\",\n \"methods\",\n null,\n null,\n null,\n \"results\",\n \"discussion\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["Central Asia","Peer Review","Publications","Publons","Universities"],"string":"[\n \"Central Asia\",\n \"Peer Review\",\n \"Publications\",\n \"Publons\",\n \"Universities\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nCentral Asia (CA) has a rich scientific temperament but has reduced visibility in the field of scientific literature. This may stem from the previous dependence on the Russian language and reduced exposure to international science.1 Things are slowly changing. Some countries are doing well and some have yet to catch up in terms of publications.2 Previous analyses have focussed on publications from these regions.234 The increasing number of manuscripts portrays growing scientific curiosity, research intent and the increasing ability to understand the intricacies and ethics of publication. Complementary to scientific publishing, another integral requirement for the development of scientific rationale and thinking is peer-reviewing.5\nPeer reviewing is an integral part of the research publication process, and often it is considered a thankless job.6 However, mainly thanks to the Publons initiative, more and more researchers are being recognised for their contributions to peer review.7 The coronavirus disease 2019 pandemic has stressed the peer review process8 and might be the refining fire that forges the next generation of peer reviewers. A peer reviewer, who is below par, may disrupt the trust that underlies this privileged gate-keeping function with some offhand remark.9 Thus, it is pertinent to have peer reviewers at par with the best in the world.\nOverall, Asia is estimated to have a good pool of upcoming reviewers.10 In the field of publishing, Kazakhstan seems to be doing well.11 A recent analysis of the top institutes in CA and the neighbouring region has shown dominance by China in various Publons ratings.4 The same Publons platform provides a unique opportunity to explore the reviewing experiences and capacities of these nations.7 Publons was initially launched to provide credit for reviewers. However, it has expanded laterally and now also includes editorial records. After being brought under Clarivate Analytics, it is now also laterally integrated with the Web of Science from which publication information is synchronized. The ‘Publons Reviewer Connect’ is an artificial intelligence based program that can help editors identify potential reviewers.12 It can also synchronize records with the ORCID database.13 Thus, it is one of the best available tools to analyse reviewer profiles, in relationship to the reviewers' publications and editorial activities. Thus, we have attempted to analyse the numbers and expertise of peer reviews as well as top universities involved in peer reviewing from various Central Asian countries using Publons.\n\nMETHODS:\nThere are 5 Central Asian republics with diverse research evaluation and publication strategies: Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan and Uzbekistan. The inbuilt search engine of Publons was used to extract data on May the 10th, 2021.\nData import First, the total number of researchers registered for each of these countries was noted. Then, the total number of reviewers with at least one verified review, with more than one verified review, the number of Publons recognized Top Reviewers, and the number of reviewers with at least one verified review in the last 12 months was extracted (Table 1). Also, the total number of reviews for each country as well as the total number of reviews for each country in the preceding 12 months was totalled. Finally, the top institutes of CA having the most number of reviews to their name, and the top 10 institutes per country were imported from Publons. Additionally, the number of Web of Science publications for the reviewers and the top institutes were also noted.\naCountry code is provided for Kazakhstan (“181”) as an example.\nFirst, the total number of researchers registered for each of these countries was noted. Then, the total number of reviewers with at least one verified review, with more than one verified review, the number of Publons recognized Top Reviewers, and the number of reviewers with at least one verified review in the last 12 months was extracted (Table 1). Also, the total number of reviews for each country as well as the total number of reviews for each country in the preceding 12 months was totalled. Finally, the top institutes of CA having the most number of reviews to their name, and the top 10 institutes per country were imported from Publons. Additionally, the number of Web of Science publications for the reviewers and the top institutes were also noted.\naCountry code is provided for Kazakhstan (“181”) as an example.\nData analysis Normality of data was assessed by the Shapiro-Wilk test. Descriptive statistics are described along with graphical presentations including bar-graphs, pie-charts and scatter-plots as relevant.\nNormality of data was assessed by the Shapiro-Wilk test. Descriptive statistics are described along with graphical presentations including bar-graphs, pie-charts and scatter-plots as relevant.\nSoftware Data was formatted into spreadsheets in MS Excel and then imported into the R statistical environment version 4.0.3. Both Excel and R packages ggpubr, dplyr, GGally, ggplot2 were used for data analysis and data visualization.\nData was formatted into spreadsheets in MS Excel and then imported into the R statistical environment version 4.0.3. Both Excel and R packages ggpubr, dplyr, GGally, ggplot2 were used for data analysis and data visualization.\n\nData import:\nFirst, the total number of researchers registered for each of these countries was noted. Then, the total number of reviewers with at least one verified review, with more than one verified review, the number of Publons recognized Top Reviewers, and the number of reviewers with at least one verified review in the last 12 months was extracted (Table 1). Also, the total number of reviews for each country as well as the total number of reviews for each country in the preceding 12 months was totalled. Finally, the top institutes of CA having the most number of reviews to their name, and the top 10 institutes per country were imported from Publons. Additionally, the number of Web of Science publications for the reviewers and the top institutes were also noted.\naCountry code is provided for Kazakhstan (“181”) as an example.\n\nData analysis:\nNormality of data was assessed by the Shapiro-Wilk test. Descriptive statistics are described along with graphical presentations including bar-graphs, pie-charts and scatter-plots as relevant.\n\nSoftware:\nData was formatted into spreadsheets in MS Excel and then imported into the R statistical environment version 4.0.3. Both Excel and R packages ggpubr, dplyr, GGally, ggplot2 were used for data analysis and data visualization.\n\nRESULTS:\nThe total number of researchers from CA registered on Publons was 15,764 and the countrywide distribution is mentioned in Table 2. However, only 370 (11.7%) have at least 1 verified review (Fig. 1). This varies amongst the countries (Fig. 2). Six reviewers from Kazakhstan and one each from Uzbekistan and Kyrgyzstan have achieved the distinction of being awarded the Publons Reviewer Award.\nThe top 20 reviewers across CA with the maximum number of verified reviews are listed in Table 3. Ten of the top 20 reviewers belong to Nazarbayev University, Nur-Sultan, Kazakhstan. Seven of the top reviewers are also active editors with a different number of editorial records to their names.\nThe top 10 universities with the maximum number of reviews per country have been listed in Table 4. However, this list is abbreviated for 3 countries that have less than 10 universities registered on Publons. Fig. 3 shows the relationship between the number of verified reviews and publication from these 29 universities across the five countries.\n\nDISCUSSION:\nThis analysis of the reviewers from the core CA countries reveals that Kazakhstan seems to top the list while two specific countries need a major boost. The number of active reviewers as well institutes in Kazakhstan reveal its potential as a rising star in the region. Uzbekistan also seems to be doing well. These two countries represent more than 90% of researchers registered on Publons as well as those involved in active peer-reviewing.\nKazakhstan has natural resources and is a major contributor to the economy of CA.14 Similarly, Uzbekistan has been gradually improving its economy and contribution to education in the last decade. These can be helping them made rapid strides in research and publications.4 Similarly, the increase in publications can also influence knowledge of publication ethics and promote peer reviewing practices.\nLooking at the performance of their top institutes, Kazakhstan has a good ratio of peer-reviews to publications. This ratio is less for all others including Uzbekistan. Also, the eight reviewers from the region who have received recognition from Publons as top reviewers highlight the point that the potential of this region is second to none. This recognition can also help stimulate their peer to achieve such distinctions.\nPeer reviewing not only trains the mind to be analytical but also teaches many aspects of publication ethics. These include understanding plagiarism,15 authorship criteria,16 declaring potential conflicts of interest17 and also the skill of scientific writing. Thus, it has been proposed that peer-reviewing should be a part of ongoing medical education.18\nThere are different metrics to measure researcher and author impact.13 However, for peer reviewers, they are no other metrics beyond what is present in Publons: the number of journals reviewed for, and the number of verified reviews (overall and in the last 12 months). Also, Publons has an option in which Editor can recommend someone as an excellent reviewer and also, it has its mechanism to award top reviewer recognition.\nThe most pertinent question is how to mine the potential of Central Asian reviewers. The Publons Academy is an initiation that enables experienced peer reviewers to mentor novice and upcoming reviewers. There is a need to involve both experts on this initiative to devote their time for mentoring and also spread awareness amongst the CA reviewers to get registered for such mentoring. Journal editors can help organize online or offline courses on peer-reviewing for these nations. Online education has added the advantage of bringing together experts from varied regions of the world. Nevertheless, it cannot be a one-way process. The CA countries, especially the underperforming ones, will also need to pull up their bootstraps. There has been freedom of knowledge, an open platform for learning and limitless striving to yearn for perfection. The other countries can take a leaf out of the book from Kazakhstan and stress for more open networking and learning resources.\nThe leading university of Kazakhstan, the Nazarbayev University, is contributing to half the top reviewers in the region, as well as the highest proportion of verified reviews. Thus, it has an unparalleled capacity for the region and can take an initiative to bridge across to other universities in the region and disseminate their expertise.\nThere are limitations to this analysis. Firstly, some active reviewers may not be registered on Publons. All registered reviewers also might not be active on Publons. Secondly, there may be predatory journals also registered on Publons and all the peer reviews verified may not have any minimum quality assurance.19\nThere seems to be no practical way to include reviewers not on Publons, in our analysis. We presume they would be in equal proportion in each country. However, a greater proportion may be from Tajikistan and Turkmenistan possible due to reduced awareness about Publons. The CA countries have different access to the internet and social media.20 Also, Publons is evolving and trying out different strategies to exclude fake or poor reviews.12\nThus, there is heterogeneity in the distribution of peer reviewer in the CA countries. There seems to be a good number of upcoming reviewers in a couple of countries while the others may need more awareness. This region may benefit much from international networking, symposia and e-learning platforms dedicated to promoting peer-reviewing.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe five Central Asian republics comprise of Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan, and Uzbekistan. Their research and publication activities are gradually improving but there is limited data on how good their peer reviewing practices are.\n\nMethods:\nWe have use the Publons database to extract information on the reviewers registered including the number of verified review, Publons award winners, and top universities in the domain of peer reviewing. This has been analysed overall and country wise.\n\nResults:\nOf 15,764 researchers registered on Publons, only 370 (11.7%) have verified records of peer-reviewing. There are 8 Publons award winners. There is great heterogeneity in the number of active reviewers across the five countries. Kazakhstan and Uzbekistan account for more than 90% of verified reviewers. Only Kazakhstan has more than 100 active reviewers and 6 Publons award recipients. Amongst the top 20 reviewers from Central Asia, half of them are from the Nazarbayev University, Nur-Sultan, Kazakhstan. Three countries have less than 10 universities registered on Publons.\n\nConclusions:\nCentral Asia has a good number of peer reviewers on Publons though only a minority of researchers are involved in peer reviewing. However, the heterogeneity between the nations can be best dealt with by promoting awareness and international networking including e-learning and mentoring programs."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":2200,"string":"2,200"},"whole_article_abstract_length":{"kind":"number","value":252,"string":"252"},"other_sections_lengths":{"kind":"list like","value":[162,35,40],"string":"[\n 162,\n 35,\n 40\n]","isConversational":false},"num_sections":{"kind":"number","value":7,"string":"7"},"most_frequent_words":{"kind":"list like","value":["reviewers","number","publons","peer","reviews","data","countries","verified","kazakhstan","total number"],"string":"[\n \"reviewers\",\n \"number\",\n \"publons\",\n \"peer\",\n \"reviews\",\n \"data\",\n \"countries\",\n \"verified\",\n \"kazakhstan\",\n \"total number\"\n]","isConversational":false},"keybert_topics":{"kind":"list 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MAÏA"},"pmid":{"kind":"string","value":"34016099"},"background_abstract":{"kind":"string","value":"Malaria vector control relies upon the use of insecticide-treated nets and indoor residual spraying. However, as the emergency of insecticide resistance in malaria vectors grows, the effectiveness of these measures could be limited. Alternative tools are needed. In this context, repellents can play an important role against exophagic and exophilic mosquitoes. This study evaluated the efficacy of MAÏA®, a novel repellent ointment, in laboratory and field conditions in Burkina Faso."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"For laboratory and field assessment, 20 volunteers were enrolled and trained for nocturnal collection of mosquitoes using human landing catches (HLC). In the laboratory tests, 2 mg/sq cm of treatment (either MAIA® or 20 % DEET) were used to assess median complete protection time (CPT) against two species: Anopheles gambiae and Aedes aegypti, following WHO guidelines. For both species, two strains consisting of susceptible and local strains were used. The susceptible strains were Kisumu and Bora Bora for An. gambiae and Ae. aegypti, respectively. For the field test, the median CPT of MAÏA® was compared to that of a negative (70 % ethanol) and positive (20 % DEET) after carrying out HLCs in rural Burkina Faso in both indoor and outdoor settings."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Laboratory tests showed median Kaplan-Meier CPT of 6 h 30 min for An. gambiae (Kisumu), 5 h 30 min for An. gambiae (Goden, local strain), and 4 h for Ae. aegypti for both the local and sensitive strain. These laboratory results suggest that MAÏA® is a good repellent against the three mosquito species. During these field tests, a total of 3979 mosquitoes were caught. In this population, anophelines represented 98.5 %, with culicines (Aedes) making up the remaining 1.5 %. Among anopheline mosquitoes, 95 % belonged to the An. gambiae complex, followed by Anopheles funestus and Anopheles pharoensis. The median CPT of 20 % DEET and MAÏA® were similar (8 h) and much longer than that of the negative control (2 h)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Results from the present studies showed that MAÏA® offers high protection against anophelines biting indoors and outdoors and could play an important role in malaria prevention in Africa."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Aedes","Animals","Anopheles","Burkina Faso","DEET","Female","Humans","Insect Repellents","Malaria","Male","Ointments","Young Adult"],"string":"[\n \"Adult\",\n \"Aedes\",\n \"Animals\",\n \"Anopheles\",\n \"Burkina Faso\",\n \"DEET\",\n \"Female\",\n \"Humans\",\n \"Insect Repellents\",\n \"Malaria\",\n \"Male\",\n \"Ointments\",\n \"Young Adult\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"8139107"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Malaria is one of the deadliest diseases in many low- and middle-income countries, affecting mainly children and pregnant women in sub-Saharan Africa [1]. Long-lasting insecticide-treated nets (LLINs) have been regarded as the most effective method for controlling mosquitoes transmitting malaria parasites. Since 2000, about one billion nets have been distributed in Africa, resulting in a significant decline in malaria-related deaths on the continent between 2000 and 2015 [24].\nHowever, the massive use of insecticides in public health in addition to that in agriculture causes concern regarding insecticide resistance [57] and changing behaviour [8, 9] of the malaria vectors. For example, a study conducted in Papua New Guinea showed a shift in mosquito biting from night to earlier hours in the evening after a nationwide distribution of LLINs [10]. Similar changes in the behaviour of Anopheles funestus have been observed in Benin and Senegal after LLIN distribution achieved a high level of coverage [9, 10]. Furthermore, studies suggest that the scaling up of LLIN distribution and indoor residual spraying (IRS) have led to more outdoor biting by Anopheles gambiae sensu lato (s.l.), commonly considered endophagic mosquitoes [1113]. A recent study in the Cascades region of Burkina Faso showed a high level of insecticide resistance [14] where more than 50% of the major vector, An. gambiae s.l., were collected biting outdoors [15]. These altered patterns of outdoors, early evening and morning biting, by anophelines, combined with resistance to insecticides appear to be caused by the mass distribution of LLINs and imply the inexorable loss of efficacy of these interventions [16, 17]. A recent study highlighted that an increase in early evening biting could increase transmission not only because people are unprotected by nets, but also because there is a higher chance of malaria vectors becoming infectious [18]. The development of new vector control tools, in addition to LLINs, is therefore necessary to protect people, when they are not under a bed net.\nTopical repellents could play an important role in addressing this problem if they are effective and accepted by the population. A systematic review of repellent interventions and mathematical modeling has shown that user compliance is indeed one of the most decisive factors for the success of this intervention [19]. In sub-Saharan Africa, ointments are used primarily by mothers and children to moisturize their skins. In Burkina Faso, ointments are applied to 80% of children every evening, when mosquitoes start biting (Kadidia Ouedraogo et al., in prep). Maa Africa, a company based in Burkina Faso, has developed a mosquito repellent ointment, MAA, uniquely designed with local mothers, to be used daily within their families. The underlying idea is to leverage the existing habits of mothers to protect their families from infectious bites whenever they are not under a net. Affordability is a key criterion for the products adoption and use. MAA has been industrially produced since June 2020 in Cte dIvoire and integrates a large share of ingredients sourced in West Africa. The ointment was officially launched in August 2020 in Burkina Faso and over 50,000 units were sold in the first four months. In March 2021, over 500 points of sales (mainly general stores, pharmacies and kiosks) distributed the product in the country. If MAA proves that it is both effective and accepted by the population, it could play a key role in reducing the probability of children experiencing infectious bites during the evening and be positioned as a complementary intervention to LLINs.\nThe aim of this study is to evaluate the effectiveness of MAA in both laboratory and field conditions, especially the median complete protection time (CPT) offered by the product. Results from these evaluations are important for validating how effective this new repellent is; behavioral responses to repellent differ between wild mosquito and laboratory-reared mosquito populations [20]."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Laboratory tests Overall, under laboratory conditions the relative repellency (median CPT) was higher for both MAA and 20% DEET against An. gambiae compare to Ae. aegypti (Table1). MAA performed well in repelling the four mosquito species used in this study. The median CPTs were, respectively, 6.5h for An. gambiae (Kisumu), 5.5h for An. gambiae (Goden, local strain) and 4h for Ae. aegypti for both the local and sensitive strain. There was no significant difference between the two treatments for each of the experiment (Kisumu: 2=2.1, p value=0.14; Goden: 2=0.8, p value=0.36; Bora bora: 2=1.7, p value=0.19; Ae. aegypti (local strain): 2=0.9, p value=0. 35) indicating that both MAA and 20% DEET have equal repellency for these strains. The Kaplan-Meier curves for MAA and 20% DEET, respectively, for Kisumu, An-Goden, Bora bora and Loc-Aedes are shown in Fig.2.\n\nTable 1\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \nAn. gambiae\nKisumu\nAn. gambiae\nGoden\nAe. aegypti\nBora bora\nAe. aegypti\nLocalDEETMAADEETMAADEETMAADEETMAAMedian. CPT390390300330270240240240Lower CI368334272216252239212225Upper CI412446328444288241268255Fig. 2Kaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\n\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \nKaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\nOverall, under laboratory conditions the relative repellency (median CPT) was higher for both MAA and 20% DEET against An. gambiae compare to Ae. aegypti (Table1). MAA performed well in repelling the four mosquito species used in this study. The median CPTs were, respectively, 6.5h for An. gambiae (Kisumu), 5.5h for An. gambiae (Goden, local strain) and 4h for Ae. aegypti for both the local and sensitive strain. There was no significant difference between the two treatments for each of the experiment (Kisumu: 2=2.1, p value=0.14; Goden: 2=0.8, p value=0.36; Bora bora: 2=1.7, p value=0.19; Ae. aegypti (local strain): 2=0.9, p value=0. 35) indicating that both MAA and 20% DEET have equal repellency for these strains. The Kaplan-Meier curves for MAA and 20% DEET, respectively, for Kisumu, An-Goden, Bora bora and Loc-Aedes are shown in Fig.2.\n\nTable 1\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \nAn. gambiae\nKisumu\nAn. gambiae\nGoden\nAe. aegypti\nBora bora\nAe. aegypti\nLocalDEETMAADEETMAADEETMAADEETMAAMedian. CPT390390300330270240240240Lower CI368334272216252239212225Upper CI412446328444288241268255Fig. 2Kaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\n\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \nKaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\nField test Mosquito species composition and biting behaviours A total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\nAnopheles gambiae sensu lato (s.l.)\n6862660480\nAnopheles funestus\n121Other Anopheles193Culicines710623\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\nA total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\nAnopheles gambiae sensu lato (s.l.)\n6862660480\nAnopheles funestus\n121Other Anopheles193Culicines710623\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\nRepellency against mosquitoes Repellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\nRepellency of 20% DEET and MAA indoor and outdoor collection\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\nRepellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\nRepellency of 20% DEET and MAA indoor and outdoor collection\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\nComplete protection time The overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nAnopheles gambiae s.l.\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nAnopheles gambiae s.l\n\nIndoors \nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nThe overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nAnopheles gambiae s.l.\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nAnopheles gambiae s.l\n\nIndoors \nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nMosquito species composition and biting behaviours A total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\nAnopheles gambiae sensu lato (s.l.)\n6862660480\nAnopheles funestus\n121Other Anopheles193Culicines710623\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\nA total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\nAnopheles gambiae sensu lato (s.l.)\n6862660480\nAnopheles funestus\n121Other Anopheles193Culicines710623\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\nRepellency against mosquitoes Repellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\nRepellency of 20% DEET and MAA indoor and outdoor collection\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\nRepellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\nRepellency of 20% DEET and MAA indoor and outdoor collection\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\nComplete protection time The overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nAnopheles gambiae s.l.\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nAnopheles gambiae s.l\n\nIndoors \nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nThe overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nAnopheles gambiae s.l.\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nAnopheles gambiae s.l\n\nIndoors \nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)"},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"MAA, a novel ointment formulated with shea butter widely used in West Africa to moisturize the skin of children, has shown high repellency against laboratory-reared and wild malaria vectors. In the context of widespread vector resistance to insecticide and growing tendency of mosquitoes to bite outside houses, there is a need to add MAA ointment to the vector control tools used in sub-Saharan countries with high malaria burden."},"other_sections_titles":{"kind":"list like","value":["Background","Methods","Study area","Human volunteer preparation","Repellents","Strains of mosquitoes","Evaluation in the laboratory","Field evaluation","Side effects","Ethical clearance","Data analysis","Laboratory tests","Field test","Mosquito species composition and biting behaviours","Repellency against mosquitoes","Complete protection time"],"string":"[\n \"Background\",\n \"Methods\",\n \"Study area\",\n \"Human volunteer preparation\",\n \"Repellents\",\n \"Strains of mosquitoes\",\n \"Evaluation in the laboratory\",\n \"Field evaluation\",\n \"Side effects\",\n \"Ethical clearance\",\n \"Data analysis\",\n \"Laboratory tests\",\n \"Field test\",\n \"Mosquito species composition and biting behaviours\",\n \"Repellency against mosquitoes\",\n \"Complete protection time\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Malaria is one of the deadliest diseases in many low- and middle-income countries, affecting mainly children and pregnant women in sub-Saharan Africa [1]. Long-lasting insecticide-treated nets (LLINs) have been regarded as the most effective method for controlling mosquitoes transmitting malaria parasites. Since 2000, about one billion nets have been distributed in Africa, resulting in a significant decline in malaria-related deaths on the continent between 2000 and 2015 [24].\nHowever, the massive use of insecticides in public health in addition to that in agriculture causes concern regarding insecticide resistance [57] and changing behaviour [8, 9] of the malaria vectors. For example, a study conducted in Papua New Guinea showed a shift in mosquito biting from night to earlier hours in the evening after a nationwide distribution of LLINs [10]. Similar changes in the behaviour of Anopheles funestus have been observed in Benin and Senegal after LLIN distribution achieved a high level of coverage [9, 10]. Furthermore, studies suggest that the scaling up of LLIN distribution and indoor residual spraying (IRS) have led to more outdoor biting by Anopheles gambiae sensu lato (s.l.), commonly considered endophagic mosquitoes [1113]. A recent study in the Cascades region of Burkina Faso showed a high level of insecticide resistance [14] where more than 50% of the major vector, An. gambiae s.l., were collected biting outdoors [15]. These altered patterns of outdoors, early evening and morning biting, by anophelines, combined with resistance to insecticides appear to be caused by the mass distribution of LLINs and imply the inexorable loss of efficacy of these interventions [16, 17]. A recent study highlighted that an increase in early evening biting could increase transmission not only because people are unprotected by nets, but also because there is a higher chance of malaria vectors becoming infectious [18]. The development of new vector control tools, in addition to LLINs, is therefore necessary to protect people, when they are not under a bed net.\nTopical repellents could play an important role in addressing this problem if they are effective and accepted by the population. A systematic review of repellent interventions and mathematical modeling has shown that user compliance is indeed one of the most decisive factors for the success of this intervention [19]. In sub-Saharan Africa, ointments are used primarily by mothers and children to moisturize their skins. In Burkina Faso, ointments are applied to 80% of children every evening, when mosquitoes start biting (Kadidia Ouedraogo et al., in prep). Maa Africa, a company based in Burkina Faso, has developed a mosquito repellent ointment, MAA, uniquely designed with local mothers, to be used daily within their families. The underlying idea is to leverage the existing habits of mothers to protect their families from infectious bites whenever they are not under a net. Affordability is a key criterion for the products adoption and use. MAA has been industrially produced since June 2020 in Cte dIvoire and integrates a large share of ingredients sourced in West Africa. The ointment was officially launched in August 2020 in Burkina Faso and over 50,000 units were sold in the first four months. In March 2021, over 500 points of sales (mainly general stores, pharmacies and kiosks) distributed the product in the country. If MAA proves that it is both effective and accepted by the population, it could play a key role in reducing the probability of children experiencing infectious bites during the evening and be positioned as a complementary intervention to LLINs.\nThe aim of this study is to evaluate the effectiveness of MAA in both laboratory and field conditions, especially the median complete protection time (CPT) offered by the product. Results from these evaluations are important for validating how effective this new repellent is; behavioral responses to repellent differ between wild mosquito and laboratory-reared mosquito populations [20].","Study area Laboratory tests were conducted in May 2019 in the insectary of Centre National de Recherche et de Formation sur le Paludisme (CNRFP) in Ouagadougou, Burkina Faso. Field tests were carried out at Goden (1225N, 120W), a site located at 15km northeast of Ouagadougou, the capital city of Burkina Faso (Fig.1). Goden is a rural village with a Sudanian savanna climate and rainfall under 900 mm annually. The ~800 inhabitants mainly belong to the Mossi ethnic group, and are mostly devoted to agriculture and raising pigs, dogs, goats, and chickens within their compounds. LLINs were distributed in 2016 to ~90% of the population. Goden is known for its high density of malaria vectors due to its proximity to the Massili River. The field study was carried out during the rainy season (August to November 2019) corresponding to high vector density and high malaria transmission. A preliminary assessment of the mosquito density on the collection site was carried out using human landing catches (HLCs) before the tests started.\n\nFig. 1Study area\nStudy area\nLaboratory tests were conducted in May 2019 in the insectary of Centre National de Recherche et de Formation sur le Paludisme (CNRFP) in Ouagadougou, Burkina Faso. Field tests were carried out at Goden (1225N, 120W), a site located at 15km northeast of Ouagadougou, the capital city of Burkina Faso (Fig.1). Goden is a rural village with a Sudanian savanna climate and rainfall under 900 mm annually. The ~800 inhabitants mainly belong to the Mossi ethnic group, and are mostly devoted to agriculture and raising pigs, dogs, goats, and chickens within their compounds. LLINs were distributed in 2016 to ~90% of the population. Goden is known for its high density of malaria vectors due to its proximity to the Massili River. The field study was carried out during the rainy season (August to November 2019) corresponding to high vector density and high malaria transmission. A preliminary assessment of the mosquito density on the collection site was carried out using human landing catches (HLCs) before the tests started.\n\nFig. 1Study area\nStudy area\nHuman volunteer preparation Healthy adult male volunteers aged between 18 and 40 years were enrolled in this study. The volunteers were instructed not to use fragranced soaps, perfume, tobacco, or alcohol 12h before the start and throughout their participation. To establish the amount of repellent required for application, the surface area of the arm (for laboratory tests) or the leg (for field tests) of volunteers was determined using the following formula: Area= (Cw+ Ce) Dwe.\nHere Cw is the circumference of the wrist or ankle in cm, Ce is the elbow cubital fossa or the knee circumference in cm, and Dwe is the distance in cm between Ce and Cw [21]. The amount of ointment needed for each volunteer was determined depending on the area of their forearm or leg length. The quantity of product left in bottles was weighed using a precision weighing balance (KERN & SOHN GmbH, Balingen, Germany) to determine the amount applied by each volunteer.\nHealthy adult male volunteers aged between 18 and 40 years were enrolled in this study. The volunteers were instructed not to use fragranced soaps, perfume, tobacco, or alcohol 12h before the start and throughout their participation. To establish the amount of repellent required for application, the surface area of the arm (for laboratory tests) or the leg (for field tests) of volunteers was determined using the following formula: Area= (Cw+ Ce) Dwe.\nHere Cw is the circumference of the wrist or ankle in cm, Ce is the elbow cubital fossa or the knee circumference in cm, and Dwe is the distance in cm between Ce and Cw [21]. The amount of ointment needed for each volunteer was determined depending on the area of their forearm or leg length. The quantity of product left in bottles was weighed using a precision weighing balance (KERN & SOHN GmbH, Balingen, Germany) to determine the amount applied by each volunteer.\nRepellents MAA, a shea butter-based ointment containing 15% DEET (N, N-diethyl-3-methylbenzamide) was received from Maa Africa SAS. It was tested against an ethanolic solution of 20% DEET as positive control and a negative control of 70% ethanol. DEET is known as the standard repellent reference.\nMAA, a shea butter-based ointment containing 15% DEET (N, N-diethyl-3-methylbenzamide) was received from Maa Africa SAS. It was tested against an ethanolic solution of 20% DEET as positive control and a negative control of 70% ethanol. DEET is known as the standard repellent reference.\nStrains of mosquitoes Four strains of mosquitoes were used in the laboratory tests, including Kisumu F57 and Bora bora F58 susceptible strains of respectively An. gambiae and Aedes aegypti. In addition, local strains laboratory-colonized from rural areas of Goden, in Burkina Faso, were used: hereafter named An-Goden (An. gambiae local strain, F418) and Loc-Aedes (local Ae. aegypti, F318). These species were maintained under a 12:12h (light: dark) photoperiod. During rearing, larvae were fed on fish food while glucose was used for adults. The temperature and relative humidity in the rearing room were 2528C and 6080%, respectively. Individual mosquitoes used in these experiments were 5 to 10 days old nulliparous females starved from sugar solution for 12h before the experiment.\nFour strains of mosquitoes were used in the laboratory tests, including Kisumu F57 and Bora bora F58 susceptible strains of respectively An. gambiae and Aedes aegypti. In addition, local strains laboratory-colonized from rural areas of Goden, in Burkina Faso, were used: hereafter named An-Goden (An. gambiae local strain, F418) and Loc-Aedes (local Ae. aegypti, F318). These species were maintained under a 12:12h (light: dark) photoperiod. During rearing, larvae were fed on fish food while glucose was used for adults. The temperature and relative humidity in the rearing room were 2528C and 6080%, respectively. Individual mosquitoes used in these experiments were 5 to 10 days old nulliparous females starved from sugar solution for 12h before the experiment.\nEvaluation in the laboratory The laboratory experiments were conducted following WHO guidelines for the arm-in-cage test [21]. Cages were 454545cm screen enclosures. Two test cages were used, one for the repellent candidate and the other for the positive control. The test cages contained 200 females aged 5 to 10 days of one of the four mosquito strains: Kisumu, An-Goden, Bora bora and loc-Aedes. The experiment in the laboratory was carried out at temperatures ranging between 25 and 28C, with relative humidity between 60 and 80%.\nOverall, 2mg of ointment were applied per sq cm on the left forearm of each volunteer, a concentration estimated from the average of 5 volunteers from the laboratory asked to apply ointment on their left forearm as they would normally do in real life. In all subsequent repellent trials, volunteers were then supplied with a total volume that would achieve this concentration over the surface area of their forearms and/or legs (determined as described above). A steel spatula was used to apply the ointment on the forearm of each volunteer prior to each experiment to avoid absorbing ointment on non-targeted areas of skin. Positive control consisted of 1 ml of 20% DEET solution applied to the right forearms of volunteers.\nNegative controls and MAA test arms were prepared by first washing left forearms with odorless soap, drying and rinsing with 70% ethanol solution and then drying again. All volunteers wore latex gloves to protect their hands from mosquitoes. To assess the readiness of the mosquitoes to land, both left and right cleaned forearms of volunteers were exposed in the experimental cages for 30s (or until 10 landings of mosquito were counted). Then, for each volunteer, the right forearm was treated from wrist to elbow using 1 ml of the 20% DEET solution whilst the left forearm was treated from the wrist to elbow with MAA ointment. Thirty mins after application of the repellents, the volunteer exposed their treated forearm in the test cage for 3min. The procedure was repeated every 30min until the first bite occurred and the elapsed time to the first bite was recorded. The test was performed three times for each volunteer per mosquito species. Considering the difference in the relative periods of biting activity of each mosquito species, the tests using Ae. aegypti strains were carried out between 09:00 and 18:00, whereas those for An. gambiae were conducted between 17:00 and 05:00 [21].\nThe laboratory experiments were conducted following WHO guidelines for the arm-in-cage test [21]. Cages were 454545cm screen enclosures. Two test cages were used, one for the repellent candidate and the other for the positive control. The test cages contained 200 females aged 5 to 10 days of one of the four mosquito strains: Kisumu, An-Goden, Bora bora and loc-Aedes. The experiment in the laboratory was carried out at temperatures ranging between 25 and 28C, with relative humidity between 60 and 80%.\nOverall, 2mg of ointment were applied per sq cm on the left forearm of each volunteer, a concentration estimated from the average of 5 volunteers from the laboratory asked to apply ointment on their left forearm as they would normally do in real life. In all subsequent repellent trials, volunteers were then supplied with a total volume that would achieve this concentration over the surface area of their forearms and/or legs (determined as described above). A steel spatula was used to apply the ointment on the forearm of each volunteer prior to each experiment to avoid absorbing ointment on non-targeted areas of skin. Positive control consisted of 1 ml of 20% DEET solution applied to the right forearms of volunteers.\nNegative controls and MAA test arms were prepared by first washing left forearms with odorless soap, drying and rinsing with 70% ethanol solution and then drying again. All volunteers wore latex gloves to protect their hands from mosquitoes. To assess the readiness of the mosquitoes to land, both left and right cleaned forearms of volunteers were exposed in the experimental cages for 30s (or until 10 landings of mosquito were counted). Then, for each volunteer, the right forearm was treated from wrist to elbow using 1 ml of the 20% DEET solution whilst the left forearm was treated from the wrist to elbow with MAA ointment. Thirty mins after application of the repellents, the volunteer exposed their treated forearm in the test cage for 3min. The procedure was repeated every 30min until the first bite occurred and the elapsed time to the first bite was recorded. The test was performed three times for each volunteer per mosquito species. Considering the difference in the relative periods of biting activity of each mosquito species, the tests using Ae. aegypti strains were carried out between 09:00 and 18:00, whereas those for An. gambiae were conducted between 17:00 and 05:00 [21].\nField evaluation The lower legs of volunteers were washed with neutral soap, rinsed with 70% of ethanol solution and naturally dried. Once their legs were treated volunteers were asked to avoid rubbing, touching or wetting the repellent-treated area. Two mg of MAA per sq cm (2.40.2g per 118979.2 sq cm) and 2 ml per sq cm of 20% DEET (2 ml0.1 ml per 118979.2 sq cm, as a positive control) were applied to volunteers lower legs, from knee to ankle. A total of 20 volunteers were recruited from Goden village and trained for nocturnal mosquito collection using HLC. Each volunteer was later randomly allocated to one of the five groups (2 for MAIA, 2 for positive control, 1 for negative control) of four volunteers according to the repellent received. Each night of collection, the experiment took place at five different households, located at least 20m apart as per WHO guidelines [21], in order to avoid biases in attractiveness to the mosquitoes.\nMosquito collection started 30min following treatments. Volunteers acting as bait, sat on a chair in pairs (one indoor and one outdoor) and actively collected mosquitoes that landed on their treated lower leg using mouth aspirator and flash torch [22] for 45min, followed by a 15-mins break. Volunteers wore long-sleeved shirts, buttoned at the wrist, long trousers, closed shoes and latex gloves with a hat on their head, but with the treated lower leg to be exposed to mosquitoes by rolling up trousers to the knee. During these experiments, mosquitoes were collected simultaneously indoors and outdoors between 19:00 and 06:00. To avoid biases introduced by individual attractiveness and skills [23, 24] volunteers at the same household rotated between indoors and outdoors hourly. In each household two groups of two people rotated collecting from 18:00 to 24:00 and from 00:00 to 06:00, following the Williams balanced Latin Square design.\nCollected mosquitoes were transferred into plastic cups, covered with a piece of untreated net, with a small hole at the bottom to allow mosquitoes to be easily aspirated into them. After collection mosquitoes were brought to the entomological laboratory of CNRFP and morphologically identified using a stereo microscope and identification keys [25].\nThe lower legs of volunteers were washed with neutral soap, rinsed with 70% of ethanol solution and naturally dried. Once their legs were treated volunteers were asked to avoid rubbing, touching or wetting the repellent-treated area. Two mg of MAA per sq cm (2.40.2g per 118979.2 sq cm) and 2 ml per sq cm of 20% DEET (2 ml0.1 ml per 118979.2 sq cm, as a positive control) were applied to volunteers lower legs, from knee to ankle. A total of 20 volunteers were recruited from Goden village and trained for nocturnal mosquito collection using HLC. Each volunteer was later randomly allocated to one of the five groups (2 for MAIA, 2 for positive control, 1 for negative control) of four volunteers according to the repellent received. Each night of collection, the experiment took place at five different households, located at least 20m apart as per WHO guidelines [21], in order to avoid biases in attractiveness to the mosquitoes.\nMosquito collection started 30min following treatments. Volunteers acting as bait, sat on a chair in pairs (one indoor and one outdoor) and actively collected mosquitoes that landed on their treated lower leg using mouth aspirator and flash torch [22] for 45min, followed by a 15-mins break. Volunteers wore long-sleeved shirts, buttoned at the wrist, long trousers, closed shoes and latex gloves with a hat on their head, but with the treated lower leg to be exposed to mosquitoes by rolling up trousers to the knee. During these experiments, mosquitoes were collected simultaneously indoors and outdoors between 19:00 and 06:00. To avoid biases introduced by individual attractiveness and skills [23, 24] volunteers at the same household rotated between indoors and outdoors hourly. In each household two groups of two people rotated collecting from 18:00 to 24:00 and from 00:00 to 06:00, following the Williams balanced Latin Square design.\nCollected mosquitoes were transferred into plastic cups, covered with a piece of untreated net, with a small hole at the bottom to allow mosquitoes to be easily aspirated into them. After collection mosquitoes were brought to the entomological laboratory of CNRFP and morphologically identified using a stereo microscope and identification keys [25].\nSide effects No side effects were observed or reported by any of the volunteers throughout the period of tests both in the laboratory and in the field.\nNo side effects were observed or reported by any of the volunteers throughout the period of tests both in the laboratory and in the field.\nEthical clearance Written informed consent was obtained from all volunteers and household owners recruited in this study. The study was approved by the institutional ethic committee of CNRFP under 2019/000008/MS/SG/CNRFP/CIB.\nWritten informed consent was obtained from all volunteers and household owners recruited in this study. The study was approved by the institutional ethic committee of CNRFP under 2019/000008/MS/SG/CNRFP/CIB.\nData analysis All data were collected on standard forms and entered twice in a database by different people. Databases have been compared using Epi Info 3.5.3, and inconsistencies were verified using printed and corrected forms. The performance of the repellent was measured by calculating the repulsive efficiency and the median full protection time. A generalized linear mixed model (GLMM) was used to further analyse the effect of the location (indoors vs. outdoors) on the performance of the treatments. Variation in the average number of bites received between treatments was also assessed.\nThe median CPT is defined as the interval of time between the beginning of collection/test and the first mosquito landing. To estimate the median CPT of each treatment, a Kaplan-Meier survival analysis was performed for each vector species and strain used in the laboratory experiments and on field data through survival function from R software-version 3.5.0 (2018-04-23). However, for the field test, the analysis was performed on only An. gambiae s.l. as it was the most abundant species collected (~96% of the total collection). The analysis consisted of assessing the median CPT and the repulsive efficacy. The repulsive efficacy was calculated as a percentage of repulsion (% R) according to the formula % R = ((C T) / C) 100, where C is the number of mosquitoes collected on the treated legs of the two control treatments separately, and T is the total number of mosquito bite attempts on the volunteers legs treated with the test product [21].\nAll data were collected on standard forms and entered twice in a database by different people. Databases have been compared using Epi Info 3.5.3, and inconsistencies were verified using printed and corrected forms. The performance of the repellent was measured by calculating the repulsive efficiency and the median full protection time. A generalized linear mixed model (GLMM) was used to further analyse the effect of the location (indoors vs. outdoors) on the performance of the treatments. Variation in the average number of bites received between treatments was also assessed.\nThe median CPT is defined as the interval of time between the beginning of collection/test and the first mosquito landing. To estimate the median CPT of each treatment, a Kaplan-Meier survival analysis was performed for each vector species and strain used in the laboratory experiments and on field data through survival function from R software-version 3.5.0 (2018-04-23). However, for the field test, the analysis was performed on only An. gambiae s.l. as it was the most abundant species collected (~96% of the total collection). The analysis consisted of assessing the median CPT and the repulsive efficacy. The repulsive efficacy was calculated as a percentage of repulsion (% R) according to the formula % R = ((C T) / C) 100, where C is the number of mosquitoes collected on the treated legs of the two control treatments separately, and T is the total number of mosquito bite attempts on the volunteers legs treated with the test product [21].","Laboratory tests were conducted in May 2019 in the insectary of Centre National de Recherche et de Formation sur le Paludisme (CNRFP) in Ouagadougou, Burkina Faso. Field tests were carried out at Goden (1225N, 120W), a site located at 15km northeast of Ouagadougou, the capital city of Burkina Faso (Fig.1). Goden is a rural village with a Sudanian savanna climate and rainfall under 900 mm annually. The ~800 inhabitants mainly belong to the Mossi ethnic group, and are mostly devoted to agriculture and raising pigs, dogs, goats, and chickens within their compounds. LLINs were distributed in 2016 to ~90% of the population. Goden is known for its high density of malaria vectors due to its proximity to the Massili River. The field study was carried out during the rainy season (August to November 2019) corresponding to high vector density and high malaria transmission. A preliminary assessment of the mosquito density on the collection site was carried out using human landing catches (HLCs) before the tests started.\n\nFig. 1Study area\nStudy area","Healthy adult male volunteers aged between 18 and 40 years were enrolled in this study. The volunteers were instructed not to use fragranced soaps, perfume, tobacco, or alcohol 12h before the start and throughout their participation. To establish the amount of repellent required for application, the surface area of the arm (for laboratory tests) or the leg (for field tests) of volunteers was determined using the following formula: Area= (Cw+ Ce) Dwe.\nHere Cw is the circumference of the wrist or ankle in cm, Ce is the elbow cubital fossa or the knee circumference in cm, and Dwe is the distance in cm between Ce and Cw [21]. The amount of ointment needed for each volunteer was determined depending on the area of their forearm or leg length. The quantity of product left in bottles was weighed using a precision weighing balance (KERN & SOHN GmbH, Balingen, Germany) to determine the amount applied by each volunteer.","MAA, a shea butter-based ointment containing 15% DEET (N, N-diethyl-3-methylbenzamide) was received from Maa Africa SAS. It was tested against an ethanolic solution of 20% DEET as positive control and a negative control of 70% ethanol. DEET is known as the standard repellent reference.","Four strains of mosquitoes were used in the laboratory tests, including Kisumu F57 and Bora bora F58 susceptible strains of respectively An. gambiae and Aedes aegypti. In addition, local strains laboratory-colonized from rural areas of Goden, in Burkina Faso, were used: hereafter named An-Goden (An. gambiae local strain, F418) and Loc-Aedes (local Ae. aegypti, F318). These species were maintained under a 12:12h (light: dark) photoperiod. During rearing, larvae were fed on fish food while glucose was used for adults. The temperature and relative humidity in the rearing room were 2528C and 6080%, respectively. Individual mosquitoes used in these experiments were 5 to 10 days old nulliparous females starved from sugar solution for 12h before the experiment.","The laboratory experiments were conducted following WHO guidelines for the arm-in-cage test [21]. Cages were 454545cm screen enclosures. Two test cages were used, one for the repellent candidate and the other for the positive control. The test cages contained 200 females aged 5 to 10 days of one of the four mosquito strains: Kisumu, An-Goden, Bora bora and loc-Aedes. The experiment in the laboratory was carried out at temperatures ranging between 25 and 28C, with relative humidity between 60 and 80%.\nOverall, 2mg of ointment were applied per sq cm on the left forearm of each volunteer, a concentration estimated from the average of 5 volunteers from the laboratory asked to apply ointment on their left forearm as they would normally do in real life. In all subsequent repellent trials, volunteers were then supplied with a total volume that would achieve this concentration over the surface area of their forearms and/or legs (determined as described above). A steel spatula was used to apply the ointment on the forearm of each volunteer prior to each experiment to avoid absorbing ointment on non-targeted areas of skin. Positive control consisted of 1 ml of 20% DEET solution applied to the right forearms of volunteers.\nNegative controls and MAA test arms were prepared by first washing left forearms with odorless soap, drying and rinsing with 70% ethanol solution and then drying again. All volunteers wore latex gloves to protect their hands from mosquitoes. To assess the readiness of the mosquitoes to land, both left and right cleaned forearms of volunteers were exposed in the experimental cages for 30s (or until 10 landings of mosquito were counted). Then, for each volunteer, the right forearm was treated from wrist to elbow using 1 ml of the 20% DEET solution whilst the left forearm was treated from the wrist to elbow with MAA ointment. Thirty mins after application of the repellents, the volunteer exposed their treated forearm in the test cage for 3min. The procedure was repeated every 30min until the first bite occurred and the elapsed time to the first bite was recorded. The test was performed three times for each volunteer per mosquito species. Considering the difference in the relative periods of biting activity of each mosquito species, the tests using Ae. aegypti strains were carried out between 09:00 and 18:00, whereas those for An. gambiae were conducted between 17:00 and 05:00 [21].","The lower legs of volunteers were washed with neutral soap, rinsed with 70% of ethanol solution and naturally dried. Once their legs were treated volunteers were asked to avoid rubbing, touching or wetting the repellent-treated area. Two mg of MAA per sq cm (2.40.2g per 118979.2 sq cm) and 2 ml per sq cm of 20% DEET (2 ml0.1 ml per 118979.2 sq cm, as a positive control) were applied to volunteers lower legs, from knee to ankle. A total of 20 volunteers were recruited from Goden village and trained for nocturnal mosquito collection using HLC. Each volunteer was later randomly allocated to one of the five groups (2 for MAIA, 2 for positive control, 1 for negative control) of four volunteers according to the repellent received. Each night of collection, the experiment took place at five different households, located at least 20m apart as per WHO guidelines [21], in order to avoid biases in attractiveness to the mosquitoes.\nMosquito collection started 30min following treatments. Volunteers acting as bait, sat on a chair in pairs (one indoor and one outdoor) and actively collected mosquitoes that landed on their treated lower leg using mouth aspirator and flash torch [22] for 45min, followed by a 15-mins break. Volunteers wore long-sleeved shirts, buttoned at the wrist, long trousers, closed shoes and latex gloves with a hat on their head, but with the treated lower leg to be exposed to mosquitoes by rolling up trousers to the knee. During these experiments, mosquitoes were collected simultaneously indoors and outdoors between 19:00 and 06:00. To avoid biases introduced by individual attractiveness and skills [23, 24] volunteers at the same household rotated between indoors and outdoors hourly. In each household two groups of two people rotated collecting from 18:00 to 24:00 and from 00:00 to 06:00, following the Williams balanced Latin Square design.\nCollected mosquitoes were transferred into plastic cups, covered with a piece of untreated net, with a small hole at the bottom to allow mosquitoes to be easily aspirated into them. After collection mosquitoes were brought to the entomological laboratory of CNRFP and morphologically identified using a stereo microscope and identification keys [25].","No side effects were observed or reported by any of the volunteers throughout the period of tests both in the laboratory and in the field.","Written informed consent was obtained from all volunteers and household owners recruited in this study. The study was approved by the institutional ethic committee of CNRFP under 2019/000008/MS/SG/CNRFP/CIB.","All data were collected on standard forms and entered twice in a database by different people. Databases have been compared using Epi Info 3.5.3, and inconsistencies were verified using printed and corrected forms. The performance of the repellent was measured by calculating the repulsive efficiency and the median full protection time. A generalized linear mixed model (GLMM) was used to further analyse the effect of the location (indoors vs. outdoors) on the performance of the treatments. Variation in the average number of bites received between treatments was also assessed.\nThe median CPT is defined as the interval of time between the beginning of collection/test and the first mosquito landing. To estimate the median CPT of each treatment, a Kaplan-Meier survival analysis was performed for each vector species and strain used in the laboratory experiments and on field data through survival function from R software-version 3.5.0 (2018-04-23). However, for the field test, the analysis was performed on only An. gambiae s.l. as it was the most abundant species collected (~96% of the total collection). The analysis consisted of assessing the median CPT and the repulsive efficacy. The repulsive efficacy was calculated as a percentage of repulsion (% R) according to the formula % R = ((C T) / C) 100, where C is the number of mosquitoes collected on the treated legs of the two control treatments separately, and T is the total number of mosquito bite attempts on the volunteers legs treated with the test product [21].","Overall, under laboratory conditions the relative repellency (median CPT) was higher for both MAA and 20% DEET against An. gambiae compare to Ae. aegypti (Table1). MAA performed well in repelling the four mosquito species used in this study. The median CPTs were, respectively, 6.5h for An. gambiae (Kisumu), 5.5h for An. gambiae (Goden, local strain) and 4h for Ae. aegypti for both the local and sensitive strain. There was no significant difference between the two treatments for each of the experiment (Kisumu: 2=2.1, p value=0.14; Goden: 2=0.8, p value=0.36; Bora bora: 2=1.7, p value=0.19; Ae. aegypti (local strain): 2=0.9, p value=0. 35) indicating that both MAA and 20% DEET have equal repellency for these strains. The Kaplan-Meier curves for MAA and 20% DEET, respectively, for Kisumu, An-Goden, Bora bora and Loc-Aedes are shown in Fig.2.\n\nTable 1\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \nAn. gambiae\nKisumu\nAn. gambiae\nGoden\nAe. aegypti\nBora bora\nAe. aegypti\nLocalDEETMAADEETMAADEETMAADEETMAAMedian. CPT390390300330270240240240Lower CI368334272216252239212225Upper CI412446328444288241268255Fig. 2Kaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\n\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \nKaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers","Mosquito species composition and biting behaviours A total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\nAnopheles gambiae sensu lato (s.l.)\n6862660480\nAnopheles funestus\n121Other Anopheles193Culicines710623\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\nA total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\nAnopheles gambiae sensu lato (s.l.)\n6862660480\nAnopheles funestus\n121Other Anopheles193Culicines710623\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\nRepellency against mosquitoes Repellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\nRepellency of 20% DEET and MAA indoor and outdoor collection\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\nRepellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\nRepellency of 20% DEET and MAA indoor and outdoor collection\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\nComplete protection time The overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nAnopheles gambiae s.l.\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nAnopheles gambiae s.l\n\nIndoors \nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nThe overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nAnopheles gambiae s.l.\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nAnopheles gambiae s.l\n\nIndoors \nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)","A total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\nAnopheles gambiae sensu lato (s.l.)\n6862660480\nAnopheles funestus\n121Other Anopheles193Culicines710623\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%","Repellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\nRepellency of 20% DEET and MAA indoor and outdoor collection\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.","The overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nAnopheles gambiae s.l.\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nAnopheles gambiae s.l\n\nIndoors \nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)"],"string":"[\n \"Malaria is one of the deadliest diseases in many low- and middle-income countries, affecting mainly children and pregnant women in sub-Saharan Africa [1]. Long-lasting insecticide-treated nets (LLINs) have been regarded as the most effective method for controlling mosquitoes transmitting malaria parasites. Since 2000, about one billion nets have been distributed in Africa, resulting in a significant decline in malaria-related deaths on the continent between 2000 and 2015 [24].\\nHowever, the massive use of insecticides in public health in addition to that in agriculture causes concern regarding insecticide resistance [57] and changing behaviour [8, 9] of the malaria vectors. For example, a study conducted in Papua New Guinea showed a shift in mosquito biting from night to earlier hours in the evening after a nationwide distribution of LLINs [10]. Similar changes in the behaviour of Anopheles funestus have been observed in Benin and Senegal after LLIN distribution achieved a high level of coverage [9, 10]. Furthermore, studies suggest that the scaling up of LLIN distribution and indoor residual spraying (IRS) have led to more outdoor biting by Anopheles gambiae sensu lato (s.l.), commonly considered endophagic mosquitoes [1113]. A recent study in the Cascades region of Burkina Faso showed a high level of insecticide resistance [14] where more than 50% of the major vector, An. gambiae s.l., were collected biting outdoors [15]. These altered patterns of outdoors, early evening and morning biting, by anophelines, combined with resistance to insecticides appear to be caused by the mass distribution of LLINs and imply the inexorable loss of efficacy of these interventions [16, 17]. A recent study highlighted that an increase in early evening biting could increase transmission not only because people are unprotected by nets, but also because there is a higher chance of malaria vectors becoming infectious [18]. The development of new vector control tools, in addition to LLINs, is therefore necessary to protect people, when they are not under a bed net.\\nTopical repellents could play an important role in addressing this problem if they are effective and accepted by the population. A systematic review of repellent interventions and mathematical modeling has shown that user compliance is indeed one of the most decisive factors for the success of this intervention [19]. In sub-Saharan Africa, ointments are used primarily by mothers and children to moisturize their skins. In Burkina Faso, ointments are applied to 80% of children every evening, when mosquitoes start biting (Kadidia Ouedraogo et al., in prep). Maa Africa, a company based in Burkina Faso, has developed a mosquito repellent ointment, MAA, uniquely designed with local mothers, to be used daily within their families. The underlying idea is to leverage the existing habits of mothers to protect their families from infectious bites whenever they are not under a net. Affordability is a key criterion for the products adoption and use. MAA has been industrially produced since June 2020 in Cte dIvoire and integrates a large share of ingredients sourced in West Africa. The ointment was officially launched in August 2020 in Burkina Faso and over 50,000 units were sold in the first four months. In March 2021, over 500 points of sales (mainly general stores, pharmacies and kiosks) distributed the product in the country. If MAA proves that it is both effective and accepted by the population, it could play a key role in reducing the probability of children experiencing infectious bites during the evening and be positioned as a complementary intervention to LLINs.\\nThe aim of this study is to evaluate the effectiveness of MAA in both laboratory and field conditions, especially the median complete protection time (CPT) offered by the product. Results from these evaluations are important for validating how effective this new repellent is; behavioral responses to repellent differ between wild mosquito and laboratory-reared mosquito populations [20].\",\n \"Study area Laboratory tests were conducted in May 2019 in the insectary of Centre National de Recherche et de Formation sur le Paludisme (CNRFP) in Ouagadougou, Burkina Faso. Field tests were carried out at Goden (1225N, 120W), a site located at 15km northeast of Ouagadougou, the capital city of Burkina Faso (Fig.1). Goden is a rural village with a Sudanian savanna climate and rainfall under 900 mm annually. The ~800 inhabitants mainly belong to the Mossi ethnic group, and are mostly devoted to agriculture and raising pigs, dogs, goats, and chickens within their compounds. LLINs were distributed in 2016 to ~90% of the population. Goden is known for its high density of malaria vectors due to its proximity to the Massili River. The field study was carried out during the rainy season (August to November 2019) corresponding to high vector density and high malaria transmission. A preliminary assessment of the mosquito density on the collection site was carried out using human landing catches (HLCs) before the tests started.\\n\\nFig. 1Study area\\nStudy area\\nLaboratory tests were conducted in May 2019 in the insectary of Centre National de Recherche et de Formation sur le Paludisme (CNRFP) in Ouagadougou, Burkina Faso. Field tests were carried out at Goden (1225N, 120W), a site located at 15km northeast of Ouagadougou, the capital city of Burkina Faso (Fig.1). Goden is a rural village with a Sudanian savanna climate and rainfall under 900 mm annually. The ~800 inhabitants mainly belong to the Mossi ethnic group, and are mostly devoted to agriculture and raising pigs, dogs, goats, and chickens within their compounds. LLINs were distributed in 2016 to ~90% of the population. Goden is known for its high density of malaria vectors due to its proximity to the Massili River. The field study was carried out during the rainy season (August to November 2019) corresponding to high vector density and high malaria transmission. A preliminary assessment of the mosquito density on the collection site was carried out using human landing catches (HLCs) before the tests started.\\n\\nFig. 1Study area\\nStudy area\\nHuman volunteer preparation Healthy adult male volunteers aged between 18 and 40 years were enrolled in this study. The volunteers were instructed not to use fragranced soaps, perfume, tobacco, or alcohol 12h before the start and throughout their participation. To establish the amount of repellent required for application, the surface area of the arm (for laboratory tests) or the leg (for field tests) of volunteers was determined using the following formula: Area= (Cw+ Ce) Dwe.\\nHere Cw is the circumference of the wrist or ankle in cm, Ce is the elbow cubital fossa or the knee circumference in cm, and Dwe is the distance in cm between Ce and Cw [21]. The amount of ointment needed for each volunteer was determined depending on the area of their forearm or leg length. The quantity of product left in bottles was weighed using a precision weighing balance (KERN & SOHN GmbH, Balingen, Germany) to determine the amount applied by each volunteer.\\nHealthy adult male volunteers aged between 18 and 40 years were enrolled in this study. The volunteers were instructed not to use fragranced soaps, perfume, tobacco, or alcohol 12h before the start and throughout their participation. To establish the amount of repellent required for application, the surface area of the arm (for laboratory tests) or the leg (for field tests) of volunteers was determined using the following formula: Area= (Cw+ Ce) Dwe.\\nHere Cw is the circumference of the wrist or ankle in cm, Ce is the elbow cubital fossa or the knee circumference in cm, and Dwe is the distance in cm between Ce and Cw [21]. The amount of ointment needed for each volunteer was determined depending on the area of their forearm or leg length. The quantity of product left in bottles was weighed using a precision weighing balance (KERN & SOHN GmbH, Balingen, Germany) to determine the amount applied by each volunteer.\\nRepellents MAA, a shea butter-based ointment containing 15% DEET (N, N-diethyl-3-methylbenzamide) was received from Maa Africa SAS. It was tested against an ethanolic solution of 20% DEET as positive control and a negative control of 70% ethanol. DEET is known as the standard repellent reference.\\nMAA, a shea butter-based ointment containing 15% DEET (N, N-diethyl-3-methylbenzamide) was received from Maa Africa SAS. It was tested against an ethanolic solution of 20% DEET as positive control and a negative control of 70% ethanol. DEET is known as the standard repellent reference.\\nStrains of mosquitoes Four strains of mosquitoes were used in the laboratory tests, including Kisumu F57 and Bora bora F58 susceptible strains of respectively An. gambiae and Aedes aegypti. In addition, local strains laboratory-colonized from rural areas of Goden, in Burkina Faso, were used: hereafter named An-Goden (An. gambiae local strain, F418) and Loc-Aedes (local Ae. aegypti, F318). These species were maintained under a 12:12h (light: dark) photoperiod. During rearing, larvae were fed on fish food while glucose was used for adults. The temperature and relative humidity in the rearing room were 2528C and 6080%, respectively. Individual mosquitoes used in these experiments were 5 to 10 days old nulliparous females starved from sugar solution for 12h before the experiment.\\nFour strains of mosquitoes were used in the laboratory tests, including Kisumu F57 and Bora bora F58 susceptible strains of respectively An. gambiae and Aedes aegypti. In addition, local strains laboratory-colonized from rural areas of Goden, in Burkina Faso, were used: hereafter named An-Goden (An. gambiae local strain, F418) and Loc-Aedes (local Ae. aegypti, F318). These species were maintained under a 12:12h (light: dark) photoperiod. During rearing, larvae were fed on fish food while glucose was used for adults. The temperature and relative humidity in the rearing room were 2528C and 6080%, respectively. Individual mosquitoes used in these experiments were 5 to 10 days old nulliparous females starved from sugar solution for 12h before the experiment.\\nEvaluation in the laboratory The laboratory experiments were conducted following WHO guidelines for the arm-in-cage test [21]. Cages were 454545cm screen enclosures. Two test cages were used, one for the repellent candidate and the other for the positive control. The test cages contained 200 females aged 5 to 10 days of one of the four mosquito strains: Kisumu, An-Goden, Bora bora and loc-Aedes. The experiment in the laboratory was carried out at temperatures ranging between 25 and 28C, with relative humidity between 60 and 80%.\\nOverall, 2mg of ointment were applied per sq cm on the left forearm of each volunteer, a concentration estimated from the average of 5 volunteers from the laboratory asked to apply ointment on their left forearm as they would normally do in real life. In all subsequent repellent trials, volunteers were then supplied with a total volume that would achieve this concentration over the surface area of their forearms and/or legs (determined as described above). A steel spatula was used to apply the ointment on the forearm of each volunteer prior to each experiment to avoid absorbing ointment on non-targeted areas of skin. Positive control consisted of 1 ml of 20% DEET solution applied to the right forearms of volunteers.\\nNegative controls and MAA test arms were prepared by first washing left forearms with odorless soap, drying and rinsing with 70% ethanol solution and then drying again. All volunteers wore latex gloves to protect their hands from mosquitoes. To assess the readiness of the mosquitoes to land, both left and right cleaned forearms of volunteers were exposed in the experimental cages for 30s (or until 10 landings of mosquito were counted). Then, for each volunteer, the right forearm was treated from wrist to elbow using 1 ml of the 20% DEET solution whilst the left forearm was treated from the wrist to elbow with MAA ointment. Thirty mins after application of the repellents, the volunteer exposed their treated forearm in the test cage for 3min. The procedure was repeated every 30min until the first bite occurred and the elapsed time to the first bite was recorded. The test was performed three times for each volunteer per mosquito species. Considering the difference in the relative periods of biting activity of each mosquito species, the tests using Ae. aegypti strains were carried out between 09:00 and 18:00, whereas those for An. gambiae were conducted between 17:00 and 05:00 [21].\\nThe laboratory experiments were conducted following WHO guidelines for the arm-in-cage test [21]. Cages were 454545cm screen enclosures. Two test cages were used, one for the repellent candidate and the other for the positive control. The test cages contained 200 females aged 5 to 10 days of one of the four mosquito strains: Kisumu, An-Goden, Bora bora and loc-Aedes. The experiment in the laboratory was carried out at temperatures ranging between 25 and 28C, with relative humidity between 60 and 80%.\\nOverall, 2mg of ointment were applied per sq cm on the left forearm of each volunteer, a concentration estimated from the average of 5 volunteers from the laboratory asked to apply ointment on their left forearm as they would normally do in real life. In all subsequent repellent trials, volunteers were then supplied with a total volume that would achieve this concentration over the surface area of their forearms and/or legs (determined as described above). A steel spatula was used to apply the ointment on the forearm of each volunteer prior to each experiment to avoid absorbing ointment on non-targeted areas of skin. Positive control consisted of 1 ml of 20% DEET solution applied to the right forearms of volunteers.\\nNegative controls and MAA test arms were prepared by first washing left forearms with odorless soap, drying and rinsing with 70% ethanol solution and then drying again. All volunteers wore latex gloves to protect their hands from mosquitoes. To assess the readiness of the mosquitoes to land, both left and right cleaned forearms of volunteers were exposed in the experimental cages for 30s (or until 10 landings of mosquito were counted). Then, for each volunteer, the right forearm was treated from wrist to elbow using 1 ml of the 20% DEET solution whilst the left forearm was treated from the wrist to elbow with MAA ointment. Thirty mins after application of the repellents, the volunteer exposed their treated forearm in the test cage for 3min. The procedure was repeated every 30min until the first bite occurred and the elapsed time to the first bite was recorded. The test was performed three times for each volunteer per mosquito species. Considering the difference in the relative periods of biting activity of each mosquito species, the tests using Ae. aegypti strains were carried out between 09:00 and 18:00, whereas those for An. gambiae were conducted between 17:00 and 05:00 [21].\\nField evaluation The lower legs of volunteers were washed with neutral soap, rinsed with 70% of ethanol solution and naturally dried. Once their legs were treated volunteers were asked to avoid rubbing, touching or wetting the repellent-treated area. Two mg of MAA per sq cm (2.40.2g per 118979.2 sq cm) and 2 ml per sq cm of 20% DEET (2 ml0.1 ml per 118979.2 sq cm, as a positive control) were applied to volunteers lower legs, from knee to ankle. A total of 20 volunteers were recruited from Goden village and trained for nocturnal mosquito collection using HLC. Each volunteer was later randomly allocated to one of the five groups (2 for MAIA, 2 for positive control, 1 for negative control) of four volunteers according to the repellent received. Each night of collection, the experiment took place at five different households, located at least 20m apart as per WHO guidelines [21], in order to avoid biases in attractiveness to the mosquitoes.\\nMosquito collection started 30min following treatments. Volunteers acting as bait, sat on a chair in pairs (one indoor and one outdoor) and actively collected mosquitoes that landed on their treated lower leg using mouth aspirator and flash torch [22] for 45min, followed by a 15-mins break. Volunteers wore long-sleeved shirts, buttoned at the wrist, long trousers, closed shoes and latex gloves with a hat on their head, but with the treated lower leg to be exposed to mosquitoes by rolling up trousers to the knee. During these experiments, mosquitoes were collected simultaneously indoors and outdoors between 19:00 and 06:00. To avoid biases introduced by individual attractiveness and skills [23, 24] volunteers at the same household rotated between indoors and outdoors hourly. In each household two groups of two people rotated collecting from 18:00 to 24:00 and from 00:00 to 06:00, following the Williams balanced Latin Square design.\\nCollected mosquitoes were transferred into plastic cups, covered with a piece of untreated net, with a small hole at the bottom to allow mosquitoes to be easily aspirated into them. After collection mosquitoes were brought to the entomological laboratory of CNRFP and morphologically identified using a stereo microscope and identification keys [25].\\nThe lower legs of volunteers were washed with neutral soap, rinsed with 70% of ethanol solution and naturally dried. Once their legs were treated volunteers were asked to avoid rubbing, touching or wetting the repellent-treated area. Two mg of MAA per sq cm (2.40.2g per 118979.2 sq cm) and 2 ml per sq cm of 20% DEET (2 ml0.1 ml per 118979.2 sq cm, as a positive control) were applied to volunteers lower legs, from knee to ankle. A total of 20 volunteers were recruited from Goden village and trained for nocturnal mosquito collection using HLC. Each volunteer was later randomly allocated to one of the five groups (2 for MAIA, 2 for positive control, 1 for negative control) of four volunteers according to the repellent received. Each night of collection, the experiment took place at five different households, located at least 20m apart as per WHO guidelines [21], in order to avoid biases in attractiveness to the mosquitoes.\\nMosquito collection started 30min following treatments. Volunteers acting as bait, sat on a chair in pairs (one indoor and one outdoor) and actively collected mosquitoes that landed on their treated lower leg using mouth aspirator and flash torch [22] for 45min, followed by a 15-mins break. Volunteers wore long-sleeved shirts, buttoned at the wrist, long trousers, closed shoes and latex gloves with a hat on their head, but with the treated lower leg to be exposed to mosquitoes by rolling up trousers to the knee. During these experiments, mosquitoes were collected simultaneously indoors and outdoors between 19:00 and 06:00. To avoid biases introduced by individual attractiveness and skills [23, 24] volunteers at the same household rotated between indoors and outdoors hourly. In each household two groups of two people rotated collecting from 18:00 to 24:00 and from 00:00 to 06:00, following the Williams balanced Latin Square design.\\nCollected mosquitoes were transferred into plastic cups, covered with a piece of untreated net, with a small hole at the bottom to allow mosquitoes to be easily aspirated into them. After collection mosquitoes were brought to the entomological laboratory of CNRFP and morphologically identified using a stereo microscope and identification keys [25].\\nSide effects No side effects were observed or reported by any of the volunteers throughout the period of tests both in the laboratory and in the field.\\nNo side effects were observed or reported by any of the volunteers throughout the period of tests both in the laboratory and in the field.\\nEthical clearance Written informed consent was obtained from all volunteers and household owners recruited in this study. The study was approved by the institutional ethic committee of CNRFP under 2019/000008/MS/SG/CNRFP/CIB.\\nWritten informed consent was obtained from all volunteers and household owners recruited in this study. The study was approved by the institutional ethic committee of CNRFP under 2019/000008/MS/SG/CNRFP/CIB.\\nData analysis All data were collected on standard forms and entered twice in a database by different people. Databases have been compared using Epi Info 3.5.3, and inconsistencies were verified using printed and corrected forms. The performance of the repellent was measured by calculating the repulsive efficiency and the median full protection time. A generalized linear mixed model (GLMM) was used to further analyse the effect of the location (indoors vs. outdoors) on the performance of the treatments. Variation in the average number of bites received between treatments was also assessed.\\nThe median CPT is defined as the interval of time between the beginning of collection/test and the first mosquito landing. To estimate the median CPT of each treatment, a Kaplan-Meier survival analysis was performed for each vector species and strain used in the laboratory experiments and on field data through survival function from R software-version 3.5.0 (2018-04-23). However, for the field test, the analysis was performed on only An. gambiae s.l. as it was the most abundant species collected (~96% of the total collection). The analysis consisted of assessing the median CPT and the repulsive efficacy. The repulsive efficacy was calculated as a percentage of repulsion (% R) according to the formula % R = ((C T) / C) 100, where C is the number of mosquitoes collected on the treated legs of the two control treatments separately, and T is the total number of mosquito bite attempts on the volunteers legs treated with the test product [21].\\nAll data were collected on standard forms and entered twice in a database by different people. Databases have been compared using Epi Info 3.5.3, and inconsistencies were verified using printed and corrected forms. The performance of the repellent was measured by calculating the repulsive efficiency and the median full protection time. A generalized linear mixed model (GLMM) was used to further analyse the effect of the location (indoors vs. outdoors) on the performance of the treatments. Variation in the average number of bites received between treatments was also assessed.\\nThe median CPT is defined as the interval of time between the beginning of collection/test and the first mosquito landing. To estimate the median CPT of each treatment, a Kaplan-Meier survival analysis was performed for each vector species and strain used in the laboratory experiments and on field data through survival function from R software-version 3.5.0 (2018-04-23). However, for the field test, the analysis was performed on only An. gambiae s.l. as it was the most abundant species collected (~96% of the total collection). The analysis consisted of assessing the median CPT and the repulsive efficacy. The repulsive efficacy was calculated as a percentage of repulsion (% R) according to the formula % R = ((C T) / C) 100, where C is the number of mosquitoes collected on the treated legs of the two control treatments separately, and T is the total number of mosquito bite attempts on the volunteers legs treated with the test product [21].\",\n \"Laboratory tests were conducted in May 2019 in the insectary of Centre National de Recherche et de Formation sur le Paludisme (CNRFP) in Ouagadougou, Burkina Faso. Field tests were carried out at Goden (1225N, 120W), a site located at 15km northeast of Ouagadougou, the capital city of Burkina Faso (Fig.1). Goden is a rural village with a Sudanian savanna climate and rainfall under 900 mm annually. The ~800 inhabitants mainly belong to the Mossi ethnic group, and are mostly devoted to agriculture and raising pigs, dogs, goats, and chickens within their compounds. LLINs were distributed in 2016 to ~90% of the population. Goden is known for its high density of malaria vectors due to its proximity to the Massili River. The field study was carried out during the rainy season (August to November 2019) corresponding to high vector density and high malaria transmission. A preliminary assessment of the mosquito density on the collection site was carried out using human landing catches (HLCs) before the tests started.\\n\\nFig. 1Study area\\nStudy area\",\n \"Healthy adult male volunteers aged between 18 and 40 years were enrolled in this study. The volunteers were instructed not to use fragranced soaps, perfume, tobacco, or alcohol 12h before the start and throughout their participation. To establish the amount of repellent required for application, the surface area of the arm (for laboratory tests) or the leg (for field tests) of volunteers was determined using the following formula: Area= (Cw+ Ce) Dwe.\\nHere Cw is the circumference of the wrist or ankle in cm, Ce is the elbow cubital fossa or the knee circumference in cm, and Dwe is the distance in cm between Ce and Cw [21]. The amount of ointment needed for each volunteer was determined depending on the area of their forearm or leg length. The quantity of product left in bottles was weighed using a precision weighing balance (KERN & SOHN GmbH, Balingen, Germany) to determine the amount applied by each volunteer.\",\n \"MAA, a shea butter-based ointment containing 15% DEET (N, N-diethyl-3-methylbenzamide) was received from Maa Africa SAS. It was tested against an ethanolic solution of 20% DEET as positive control and a negative control of 70% ethanol. DEET is known as the standard repellent reference.\",\n \"Four strains of mosquitoes were used in the laboratory tests, including Kisumu F57 and Bora bora F58 susceptible strains of respectively An. gambiae and Aedes aegypti. In addition, local strains laboratory-colonized from rural areas of Goden, in Burkina Faso, were used: hereafter named An-Goden (An. gambiae local strain, F418) and Loc-Aedes (local Ae. aegypti, F318). These species were maintained under a 12:12h (light: dark) photoperiod. During rearing, larvae were fed on fish food while glucose was used for adults. The temperature and relative humidity in the rearing room were 2528C and 6080%, respectively. Individual mosquitoes used in these experiments were 5 to 10 days old nulliparous females starved from sugar solution for 12h before the experiment.\",\n \"The laboratory experiments were conducted following WHO guidelines for the arm-in-cage test [21]. Cages were 454545cm screen enclosures. Two test cages were used, one for the repellent candidate and the other for the positive control. The test cages contained 200 females aged 5 to 10 days of one of the four mosquito strains: Kisumu, An-Goden, Bora bora and loc-Aedes. The experiment in the laboratory was carried out at temperatures ranging between 25 and 28C, with relative humidity between 60 and 80%.\\nOverall, 2mg of ointment were applied per sq cm on the left forearm of each volunteer, a concentration estimated from the average of 5 volunteers from the laboratory asked to apply ointment on their left forearm as they would normally do in real life. In all subsequent repellent trials, volunteers were then supplied with a total volume that would achieve this concentration over the surface area of their forearms and/or legs (determined as described above). A steel spatula was used to apply the ointment on the forearm of each volunteer prior to each experiment to avoid absorbing ointment on non-targeted areas of skin. Positive control consisted of 1 ml of 20% DEET solution applied to the right forearms of volunteers.\\nNegative controls and MAA test arms were prepared by first washing left forearms with odorless soap, drying and rinsing with 70% ethanol solution and then drying again. All volunteers wore latex gloves to protect their hands from mosquitoes. To assess the readiness of the mosquitoes to land, both left and right cleaned forearms of volunteers were exposed in the experimental cages for 30s (or until 10 landings of mosquito were counted). Then, for each volunteer, the right forearm was treated from wrist to elbow using 1 ml of the 20% DEET solution whilst the left forearm was treated from the wrist to elbow with MAA ointment. Thirty mins after application of the repellents, the volunteer exposed their treated forearm in the test cage for 3min. The procedure was repeated every 30min until the first bite occurred and the elapsed time to the first bite was recorded. The test was performed three times for each volunteer per mosquito species. Considering the difference in the relative periods of biting activity of each mosquito species, the tests using Ae. aegypti strains were carried out between 09:00 and 18:00, whereas those for An. gambiae were conducted between 17:00 and 05:00 [21].\",\n \"The lower legs of volunteers were washed with neutral soap, rinsed with 70% of ethanol solution and naturally dried. Once their legs were treated volunteers were asked to avoid rubbing, touching or wetting the repellent-treated area. Two mg of MAA per sq cm (2.40.2g per 118979.2 sq cm) and 2 ml per sq cm of 20% DEET (2 ml0.1 ml per 118979.2 sq cm, as a positive control) were applied to volunteers lower legs, from knee to ankle. A total of 20 volunteers were recruited from Goden village and trained for nocturnal mosquito collection using HLC. Each volunteer was later randomly allocated to one of the five groups (2 for MAIA, 2 for positive control, 1 for negative control) of four volunteers according to the repellent received. Each night of collection, the experiment took place at five different households, located at least 20m apart as per WHO guidelines [21], in order to avoid biases in attractiveness to the mosquitoes.\\nMosquito collection started 30min following treatments. Volunteers acting as bait, sat on a chair in pairs (one indoor and one outdoor) and actively collected mosquitoes that landed on their treated lower leg using mouth aspirator and flash torch [22] for 45min, followed by a 15-mins break. Volunteers wore long-sleeved shirts, buttoned at the wrist, long trousers, closed shoes and latex gloves with a hat on their head, but with the treated lower leg to be exposed to mosquitoes by rolling up trousers to the knee. During these experiments, mosquitoes were collected simultaneously indoors and outdoors between 19:00 and 06:00. To avoid biases introduced by individual attractiveness and skills [23, 24] volunteers at the same household rotated between indoors and outdoors hourly. In each household two groups of two people rotated collecting from 18:00 to 24:00 and from 00:00 to 06:00, following the Williams balanced Latin Square design.\\nCollected mosquitoes were transferred into plastic cups, covered with a piece of untreated net, with a small hole at the bottom to allow mosquitoes to be easily aspirated into them. After collection mosquitoes were brought to the entomological laboratory of CNRFP and morphologically identified using a stereo microscope and identification keys [25].\",\n \"No side effects were observed or reported by any of the volunteers throughout the period of tests both in the laboratory and in the field.\",\n \"Written informed consent was obtained from all volunteers and household owners recruited in this study. The study was approved by the institutional ethic committee of CNRFP under 2019/000008/MS/SG/CNRFP/CIB.\",\n \"All data were collected on standard forms and entered twice in a database by different people. Databases have been compared using Epi Info 3.5.3, and inconsistencies were verified using printed and corrected forms. The performance of the repellent was measured by calculating the repulsive efficiency and the median full protection time. A generalized linear mixed model (GLMM) was used to further analyse the effect of the location (indoors vs. outdoors) on the performance of the treatments. Variation in the average number of bites received between treatments was also assessed.\\nThe median CPT is defined as the interval of time between the beginning of collection/test and the first mosquito landing. To estimate the median CPT of each treatment, a Kaplan-Meier survival analysis was performed for each vector species and strain used in the laboratory experiments and on field data through survival function from R software-version 3.5.0 (2018-04-23). However, for the field test, the analysis was performed on only An. gambiae s.l. as it was the most abundant species collected (~96% of the total collection). The analysis consisted of assessing the median CPT and the repulsive efficacy. The repulsive efficacy was calculated as a percentage of repulsion (% R) according to the formula % R = ((C T) / C) 100, where C is the number of mosquitoes collected on the treated legs of the two control treatments separately, and T is the total number of mosquito bite attempts on the volunteers legs treated with the test product [21].\",\n \"Overall, under laboratory conditions the relative repellency (median CPT) was higher for both MAA and 20% DEET against An. gambiae compare to Ae. aegypti (Table1). MAA performed well in repelling the four mosquito species used in this study. The median CPTs were, respectively, 6.5h for An. gambiae (Kisumu), 5.5h for An. gambiae (Goden, local strain) and 4h for Ae. aegypti for both the local and sensitive strain. There was no significant difference between the two treatments for each of the experiment (Kisumu: 2=2.1, p value=0.14; Goden: 2=0.8, p value=0.36; Bora bora: 2=1.7, p value=0.19; Ae. aegypti (local strain): 2=0.9, p value=0. 35) indicating that both MAA and 20% DEET have equal repellency for these strains. The Kaplan-Meier curves for MAA and 20% DEET, respectively, for Kisumu, An-Goden, Bora bora and Loc-Aedes are shown in Fig.2.\\n\\nTable 1\\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \\nAn. gambiae\\nKisumu\\nAn. gambiae\\nGoden\\nAe. aegypti\\nBora bora\\nAe. aegypti\\nLocalDEETMAADEETMAADEETMAADEETMAAMedian. CPT390390300330270240240240Lower CI368334272216252239212225Upper CI412446328444288241268255Fig. 2Kaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\\n\\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \\nKaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\",\n \"Mosquito species composition and biting behaviours A total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\\nAnopheles gambiae sensu lato (s.l.)\\n6862660480\\nAnopheles funestus\\n121Other Anopheles193Culicines710623\\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\\nA total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\\nAnopheles gambiae sensu lato (s.l.)\\n6862660480\\nAnopheles funestus\\n121Other Anopheles193Culicines710623\\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\\nRepellency against mosquitoes Repellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\\nRepellency of 20% DEET and MAA indoor and outdoor collection\\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\\nRepellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\\nRepellency of 20% DEET and MAA indoor and outdoor collection\\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\\nComplete protection time The overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\\nAnopheles gambiae s.l.\\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\\nAnopheles gambiae s.l\\n\\nIndoors \\nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\\nThe overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\\nAnopheles gambiae s.l.\\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\\nAnopheles gambiae s.l\\n\\nIndoors \\nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\",\n \"A total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\\nAnopheles gambiae sensu lato (s.l.)\\n6862660480\\nAnopheles funestus\\n121Other Anopheles193Culicines710623\\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\",\n \"Repellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\\nRepellency of 20% DEET and MAA indoor and outdoor collection\\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\",\n \"The overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\\nAnopheles gambiae s.l.\\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\\nAnopheles gambiae s.l\\n\\nIndoors \\nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Study area","Human volunteer preparation","Repellents","Strains of mosquitoes","Evaluation in the laboratory","Field evaluation","Side effects","Ethical clearance","Data analysis","Results","Laboratory tests","Field test","Mosquito species composition and biting behaviours","Repellency against mosquitoes","Complete protection time","Discussion","Conclusions"],"string":"[\n \"Background\",\n \"Methods\",\n \"Study area\",\n \"Human volunteer preparation\",\n \"Repellents\",\n \"Strains of mosquitoes\",\n \"Evaluation in the laboratory\",\n \"Field evaluation\",\n \"Side effects\",\n \"Ethical clearance\",\n \"Data analysis\",\n \"Results\",\n \"Laboratory tests\",\n \"Field test\",\n \"Mosquito species composition and biting behaviours\",\n \"Repellency against mosquitoes\",\n \"Complete protection time\",\n \"Discussion\",\n \"Conclusions\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Malaria is one of the deadliest diseases in many low- and middle-income countries, affecting mainly children and pregnant women in sub-Saharan Africa [1]. Long-lasting insecticide-treated nets (LLINs) have been regarded as the most effective method for controlling mosquitoes transmitting malaria parasites. Since 2000, about one billion nets have been distributed in Africa, resulting in a significant decline in malaria-related deaths on the continent between 2000 and 2015 [24].\nHowever, the massive use of insecticides in public health in addition to that in agriculture causes concern regarding insecticide resistance [57] and changing behaviour [8, 9] of the malaria vectors. For example, a study conducted in Papua New Guinea showed a shift in mosquito biting from night to earlier hours in the evening after a nationwide distribution of LLINs [10]. Similar changes in the behaviour of Anopheles funestus have been observed in Benin and Senegal after LLIN distribution achieved a high level of coverage [9, 10]. Furthermore, studies suggest that the scaling up of LLIN distribution and indoor residual spraying (IRS) have led to more outdoor biting by Anopheles gambiae sensu lato (s.l.), commonly considered endophagic mosquitoes [1113]. A recent study in the Cascades region of Burkina Faso showed a high level of insecticide resistance [14] where more than 50% of the major vector, An. gambiae s.l., were collected biting outdoors [15]. These altered patterns of outdoors, early evening and morning biting, by anophelines, combined with resistance to insecticides appear to be caused by the mass distribution of LLINs and imply the inexorable loss of efficacy of these interventions [16, 17]. A recent study highlighted that an increase in early evening biting could increase transmission not only because people are unprotected by nets, but also because there is a higher chance of malaria vectors becoming infectious [18]. The development of new vector control tools, in addition to LLINs, is therefore necessary to protect people, when they are not under a bed net.\nTopical repellents could play an important role in addressing this problem if they are effective and accepted by the population. A systematic review of repellent interventions and mathematical modeling has shown that user compliance is indeed one of the most decisive factors for the success of this intervention [19]. In sub-Saharan Africa, ointments are used primarily by mothers and children to moisturize their skins. In Burkina Faso, ointments are applied to 80% of children every evening, when mosquitoes start biting (Kadidia Ouedraogo et al., in prep). Maa Africa, a company based in Burkina Faso, has developed a mosquito repellent ointment, MAA, uniquely designed with local mothers, to be used daily within their families. The underlying idea is to leverage the existing habits of mothers to protect their families from infectious bites whenever they are not under a net. Affordability is a key criterion for the products adoption and use. MAA has been industrially produced since June 2020 in Cte dIvoire and integrates a large share of ingredients sourced in West Africa. The ointment was officially launched in August 2020 in Burkina Faso and over 50,000 units were sold in the first four months. In March 2021, over 500 points of sales (mainly general stores, pharmacies and kiosks) distributed the product in the country. If MAA proves that it is both effective and accepted by the population, it could play a key role in reducing the probability of children experiencing infectious bites during the evening and be positioned as a complementary intervention to LLINs.\nThe aim of this study is to evaluate the effectiveness of MAA in both laboratory and field conditions, especially the median complete protection time (CPT) offered by the product. Results from these evaluations are important for validating how effective this new repellent is; behavioral responses to repellent differ between wild mosquito and laboratory-reared mosquito populations [20].","Study area Laboratory tests were conducted in May 2019 in the insectary of Centre National de Recherche et de Formation sur le Paludisme (CNRFP) in Ouagadougou, Burkina Faso. Field tests were carried out at Goden (1225N, 120W), a site located at 15km northeast of Ouagadougou, the capital city of Burkina Faso (Fig.1). Goden is a rural village with a Sudanian savanna climate and rainfall under 900 mm annually. The ~800 inhabitants mainly belong to the Mossi ethnic group, and are mostly devoted to agriculture and raising pigs, dogs, goats, and chickens within their compounds. LLINs were distributed in 2016 to ~90% of the population. Goden is known for its high density of malaria vectors due to its proximity to the Massili River. The field study was carried out during the rainy season (August to November 2019) corresponding to high vector density and high malaria transmission. A preliminary assessment of the mosquito density on the collection site was carried out using human landing catches (HLCs) before the tests started.\n\nFig. 1Study area\nStudy area\nLaboratory tests were conducted in May 2019 in the insectary of Centre National de Recherche et de Formation sur le Paludisme (CNRFP) in Ouagadougou, Burkina Faso. Field tests were carried out at Goden (1225N, 120W), a site located at 15km northeast of Ouagadougou, the capital city of Burkina Faso (Fig.1). Goden is a rural village with a Sudanian savanna climate and rainfall under 900 mm annually. The ~800 inhabitants mainly belong to the Mossi ethnic group, and are mostly devoted to agriculture and raising pigs, dogs, goats, and chickens within their compounds. LLINs were distributed in 2016 to ~90% of the population. Goden is known for its high density of malaria vectors due to its proximity to the Massili River. The field study was carried out during the rainy season (August to November 2019) corresponding to high vector density and high malaria transmission. A preliminary assessment of the mosquito density on the collection site was carried out using human landing catches (HLCs) before the tests started.\n\nFig. 1Study area\nStudy area\nHuman volunteer preparation Healthy adult male volunteers aged between 18 and 40 years were enrolled in this study. The volunteers were instructed not to use fragranced soaps, perfume, tobacco, or alcohol 12h before the start and throughout their participation. To establish the amount of repellent required for application, the surface area of the arm (for laboratory tests) or the leg (for field tests) of volunteers was determined using the following formula: Area= (Cw+ Ce) Dwe.\nHere Cw is the circumference of the wrist or ankle in cm, Ce is the elbow cubital fossa or the knee circumference in cm, and Dwe is the distance in cm between Ce and Cw [21]. The amount of ointment needed for each volunteer was determined depending on the area of their forearm or leg length. The quantity of product left in bottles was weighed using a precision weighing balance (KERN & SOHN GmbH, Balingen, Germany) to determine the amount applied by each volunteer.\nHealthy adult male volunteers aged between 18 and 40 years were enrolled in this study. The volunteers were instructed not to use fragranced soaps, perfume, tobacco, or alcohol 12h before the start and throughout their participation. To establish the amount of repellent required for application, the surface area of the arm (for laboratory tests) or the leg (for field tests) of volunteers was determined using the following formula: Area= (Cw+ Ce) Dwe.\nHere Cw is the circumference of the wrist or ankle in cm, Ce is the elbow cubital fossa or the knee circumference in cm, and Dwe is the distance in cm between Ce and Cw [21]. The amount of ointment needed for each volunteer was determined depending on the area of their forearm or leg length. The quantity of product left in bottles was weighed using a precision weighing balance (KERN & SOHN GmbH, Balingen, Germany) to determine the amount applied by each volunteer.\nRepellents MAA, a shea butter-based ointment containing 15% DEET (N, N-diethyl-3-methylbenzamide) was received from Maa Africa SAS. It was tested against an ethanolic solution of 20% DEET as positive control and a negative control of 70% ethanol. DEET is known as the standard repellent reference.\nMAA, a shea butter-based ointment containing 15% DEET (N, N-diethyl-3-methylbenzamide) was received from Maa Africa SAS. It was tested against an ethanolic solution of 20% DEET as positive control and a negative control of 70% ethanol. DEET is known as the standard repellent reference.\nStrains of mosquitoes Four strains of mosquitoes were used in the laboratory tests, including Kisumu F57 and Bora bora F58 susceptible strains of respectively An. gambiae and Aedes aegypti. In addition, local strains laboratory-colonized from rural areas of Goden, in Burkina Faso, were used: hereafter named An-Goden (An. gambiae local strain, F418) and Loc-Aedes (local Ae. aegypti, F318). These species were maintained under a 12:12h (light: dark) photoperiod. During rearing, larvae were fed on fish food while glucose was used for adults. The temperature and relative humidity in the rearing room were 2528C and 6080%, respectively. Individual mosquitoes used in these experiments were 5 to 10 days old nulliparous females starved from sugar solution for 12h before the experiment.\nFour strains of mosquitoes were used in the laboratory tests, including Kisumu F57 and Bora bora F58 susceptible strains of respectively An. gambiae and Aedes aegypti. In addition, local strains laboratory-colonized from rural areas of Goden, in Burkina Faso, were used: hereafter named An-Goden (An. gambiae local strain, F418) and Loc-Aedes (local Ae. aegypti, F318). These species were maintained under a 12:12h (light: dark) photoperiod. During rearing, larvae were fed on fish food while glucose was used for adults. The temperature and relative humidity in the rearing room were 2528C and 6080%, respectively. Individual mosquitoes used in these experiments were 5 to 10 days old nulliparous females starved from sugar solution for 12h before the experiment.\nEvaluation in the laboratory The laboratory experiments were conducted following WHO guidelines for the arm-in-cage test [21]. Cages were 454545cm screen enclosures. Two test cages were used, one for the repellent candidate and the other for the positive control. The test cages contained 200 females aged 5 to 10 days of one of the four mosquito strains: Kisumu, An-Goden, Bora bora and loc-Aedes. The experiment in the laboratory was carried out at temperatures ranging between 25 and 28C, with relative humidity between 60 and 80%.\nOverall, 2mg of ointment were applied per sq cm on the left forearm of each volunteer, a concentration estimated from the average of 5 volunteers from the laboratory asked to apply ointment on their left forearm as they would normally do in real life. In all subsequent repellent trials, volunteers were then supplied with a total volume that would achieve this concentration over the surface area of their forearms and/or legs (determined as described above). A steel spatula was used to apply the ointment on the forearm of each volunteer prior to each experiment to avoid absorbing ointment on non-targeted areas of skin. Positive control consisted of 1 ml of 20% DEET solution applied to the right forearms of volunteers.\nNegative controls and MAA test arms were prepared by first washing left forearms with odorless soap, drying and rinsing with 70% ethanol solution and then drying again. All volunteers wore latex gloves to protect their hands from mosquitoes. To assess the readiness of the mosquitoes to land, both left and right cleaned forearms of volunteers were exposed in the experimental cages for 30s (or until 10 landings of mosquito were counted). Then, for each volunteer, the right forearm was treated from wrist to elbow using 1 ml of the 20% DEET solution whilst the left forearm was treated from the wrist to elbow with MAA ointment. Thirty mins after application of the repellents, the volunteer exposed their treated forearm in the test cage for 3min. The procedure was repeated every 30min until the first bite occurred and the elapsed time to the first bite was recorded. The test was performed three times for each volunteer per mosquito species. Considering the difference in the relative periods of biting activity of each mosquito species, the tests using Ae. aegypti strains were carried out between 09:00 and 18:00, whereas those for An. gambiae were conducted between 17:00 and 05:00 [21].\nThe laboratory experiments were conducted following WHO guidelines for the arm-in-cage test [21]. Cages were 454545cm screen enclosures. Two test cages were used, one for the repellent candidate and the other for the positive control. The test cages contained 200 females aged 5 to 10 days of one of the four mosquito strains: Kisumu, An-Goden, Bora bora and loc-Aedes. The experiment in the laboratory was carried out at temperatures ranging between 25 and 28C, with relative humidity between 60 and 80%.\nOverall, 2mg of ointment were applied per sq cm on the left forearm of each volunteer, a concentration estimated from the average of 5 volunteers from the laboratory asked to apply ointment on their left forearm as they would normally do in real life. In all subsequent repellent trials, volunteers were then supplied with a total volume that would achieve this concentration over the surface area of their forearms and/or legs (determined as described above). A steel spatula was used to apply the ointment on the forearm of each volunteer prior to each experiment to avoid absorbing ointment on non-targeted areas of skin. Positive control consisted of 1 ml of 20% DEET solution applied to the right forearms of volunteers.\nNegative controls and MAA test arms were prepared by first washing left forearms with odorless soap, drying and rinsing with 70% ethanol solution and then drying again. All volunteers wore latex gloves to protect their hands from mosquitoes. To assess the readiness of the mosquitoes to land, both left and right cleaned forearms of volunteers were exposed in the experimental cages for 30s (or until 10 landings of mosquito were counted). Then, for each volunteer, the right forearm was treated from wrist to elbow using 1 ml of the 20% DEET solution whilst the left forearm was treated from the wrist to elbow with MAA ointment. Thirty mins after application of the repellents, the volunteer exposed their treated forearm in the test cage for 3min. The procedure was repeated every 30min until the first bite occurred and the elapsed time to the first bite was recorded. The test was performed three times for each volunteer per mosquito species. Considering the difference in the relative periods of biting activity of each mosquito species, the tests using Ae. aegypti strains were carried out between 09:00 and 18:00, whereas those for An. gambiae were conducted between 17:00 and 05:00 [21].\nField evaluation The lower legs of volunteers were washed with neutral soap, rinsed with 70% of ethanol solution and naturally dried. Once their legs were treated volunteers were asked to avoid rubbing, touching or wetting the repellent-treated area. Two mg of MAA per sq cm (2.40.2g per 118979.2 sq cm) and 2 ml per sq cm of 20% DEET (2 ml0.1 ml per 118979.2 sq cm, as a positive control) were applied to volunteers lower legs, from knee to ankle. A total of 20 volunteers were recruited from Goden village and trained for nocturnal mosquito collection using HLC. Each volunteer was later randomly allocated to one of the five groups (2 for MAIA, 2 for positive control, 1 for negative control) of four volunteers according to the repellent received. Each night of collection, the experiment took place at five different households, located at least 20m apart as per WHO guidelines [21], in order to avoid biases in attractiveness to the mosquitoes.\nMosquito collection started 30min following treatments. Volunteers acting as bait, sat on a chair in pairs (one indoor and one outdoor) and actively collected mosquitoes that landed on their treated lower leg using mouth aspirator and flash torch [22] for 45min, followed by a 15-mins break. Volunteers wore long-sleeved shirts, buttoned at the wrist, long trousers, closed shoes and latex gloves with a hat on their head, but with the treated lower leg to be exposed to mosquitoes by rolling up trousers to the knee. During these experiments, mosquitoes were collected simultaneously indoors and outdoors between 19:00 and 06:00. To avoid biases introduced by individual attractiveness and skills [23, 24] volunteers at the same household rotated between indoors and outdoors hourly. In each household two groups of two people rotated collecting from 18:00 to 24:00 and from 00:00 to 06:00, following the Williams balanced Latin Square design.\nCollected mosquitoes were transferred into plastic cups, covered with a piece of untreated net, with a small hole at the bottom to allow mosquitoes to be easily aspirated into them. After collection mosquitoes were brought to the entomological laboratory of CNRFP and morphologically identified using a stereo microscope and identification keys [25].\nThe lower legs of volunteers were washed with neutral soap, rinsed with 70% of ethanol solution and naturally dried. Once their legs were treated volunteers were asked to avoid rubbing, touching or wetting the repellent-treated area. Two mg of MAA per sq cm (2.40.2g per 118979.2 sq cm) and 2 ml per sq cm of 20% DEET (2 ml0.1 ml per 118979.2 sq cm, as a positive control) were applied to volunteers lower legs, from knee to ankle. A total of 20 volunteers were recruited from Goden village and trained for nocturnal mosquito collection using HLC. Each volunteer was later randomly allocated to one of the five groups (2 for MAIA, 2 for positive control, 1 for negative control) of four volunteers according to the repellent received. Each night of collection, the experiment took place at five different households, located at least 20m apart as per WHO guidelines [21], in order to avoid biases in attractiveness to the mosquitoes.\nMosquito collection started 30min following treatments. Volunteers acting as bait, sat on a chair in pairs (one indoor and one outdoor) and actively collected mosquitoes that landed on their treated lower leg using mouth aspirator and flash torch [22] for 45min, followed by a 15-mins break. Volunteers wore long-sleeved shirts, buttoned at the wrist, long trousers, closed shoes and latex gloves with a hat on their head, but with the treated lower leg to be exposed to mosquitoes by rolling up trousers to the knee. During these experiments, mosquitoes were collected simultaneously indoors and outdoors between 19:00 and 06:00. To avoid biases introduced by individual attractiveness and skills [23, 24] volunteers at the same household rotated between indoors and outdoors hourly. In each household two groups of two people rotated collecting from 18:00 to 24:00 and from 00:00 to 06:00, following the Williams balanced Latin Square design.\nCollected mosquitoes were transferred into plastic cups, covered with a piece of untreated net, with a small hole at the bottom to allow mosquitoes to be easily aspirated into them. After collection mosquitoes were brought to the entomological laboratory of CNRFP and morphologically identified using a stereo microscope and identification keys [25].\nSide effects No side effects were observed or reported by any of the volunteers throughout the period of tests both in the laboratory and in the field.\nNo side effects were observed or reported by any of the volunteers throughout the period of tests both in the laboratory and in the field.\nEthical clearance Written informed consent was obtained from all volunteers and household owners recruited in this study. The study was approved by the institutional ethic committee of CNRFP under 2019/000008/MS/SG/CNRFP/CIB.\nWritten informed consent was obtained from all volunteers and household owners recruited in this study. The study was approved by the institutional ethic committee of CNRFP under 2019/000008/MS/SG/CNRFP/CIB.\nData analysis All data were collected on standard forms and entered twice in a database by different people. Databases have been compared using Epi Info 3.5.3, and inconsistencies were verified using printed and corrected forms. The performance of the repellent was measured by calculating the repulsive efficiency and the median full protection time. A generalized linear mixed model (GLMM) was used to further analyse the effect of the location (indoors vs. outdoors) on the performance of the treatments. Variation in the average number of bites received between treatments was also assessed.\nThe median CPT is defined as the interval of time between the beginning of collection/test and the first mosquito landing. To estimate the median CPT of each treatment, a Kaplan-Meier survival analysis was performed for each vector species and strain used in the laboratory experiments and on field data through survival function from R software-version 3.5.0 (2018-04-23). However, for the field test, the analysis was performed on only An. gambiae s.l. as it was the most abundant species collected (~96% of the total collection). The analysis consisted of assessing the median CPT and the repulsive efficacy. The repulsive efficacy was calculated as a percentage of repulsion (% R) according to the formula % R = ((C T) / C) 100, where C is the number of mosquitoes collected on the treated legs of the two control treatments separately, and T is the total number of mosquito bite attempts on the volunteers legs treated with the test product [21].\nAll data were collected on standard forms and entered twice in a database by different people. Databases have been compared using Epi Info 3.5.3, and inconsistencies were verified using printed and corrected forms. The performance of the repellent was measured by calculating the repulsive efficiency and the median full protection time. A generalized linear mixed model (GLMM) was used to further analyse the effect of the location (indoors vs. outdoors) on the performance of the treatments. Variation in the average number of bites received between treatments was also assessed.\nThe median CPT is defined as the interval of time between the beginning of collection/test and the first mosquito landing. To estimate the median CPT of each treatment, a Kaplan-Meier survival analysis was performed for each vector species and strain used in the laboratory experiments and on field data through survival function from R software-version 3.5.0 (2018-04-23). However, for the field test, the analysis was performed on only An. gambiae s.l. as it was the most abundant species collected (~96% of the total collection). The analysis consisted of assessing the median CPT and the repulsive efficacy. The repulsive efficacy was calculated as a percentage of repulsion (% R) according to the formula % R = ((C T) / C) 100, where C is the number of mosquitoes collected on the treated legs of the two control treatments separately, and T is the total number of mosquito bite attempts on the volunteers legs treated with the test product [21].","Laboratory tests were conducted in May 2019 in the insectary of Centre National de Recherche et de Formation sur le Paludisme (CNRFP) in Ouagadougou, Burkina Faso. Field tests were carried out at Goden (1225N, 120W), a site located at 15km northeast of Ouagadougou, the capital city of Burkina Faso (Fig.1). Goden is a rural village with a Sudanian savanna climate and rainfall under 900 mm annually. The ~800 inhabitants mainly belong to the Mossi ethnic group, and are mostly devoted to agriculture and raising pigs, dogs, goats, and chickens within their compounds. LLINs were distributed in 2016 to ~90% of the population. Goden is known for its high density of malaria vectors due to its proximity to the Massili River. The field study was carried out during the rainy season (August to November 2019) corresponding to high vector density and high malaria transmission. A preliminary assessment of the mosquito density on the collection site was carried out using human landing catches (HLCs) before the tests started.\n\nFig. 1Study area\nStudy area","Healthy adult male volunteers aged between 18 and 40 years were enrolled in this study. The volunteers were instructed not to use fragranced soaps, perfume, tobacco, or alcohol 12h before the start and throughout their participation. To establish the amount of repellent required for application, the surface area of the arm (for laboratory tests) or the leg (for field tests) of volunteers was determined using the following formula: Area= (Cw+ Ce) Dwe.\nHere Cw is the circumference of the wrist or ankle in cm, Ce is the elbow cubital fossa or the knee circumference in cm, and Dwe is the distance in cm between Ce and Cw [21]. The amount of ointment needed for each volunteer was determined depending on the area of their forearm or leg length. The quantity of product left in bottles was weighed using a precision weighing balance (KERN & SOHN GmbH, Balingen, Germany) to determine the amount applied by each volunteer.","MAA, a shea butter-based ointment containing 15% DEET (N, N-diethyl-3-methylbenzamide) was received from Maa Africa SAS. It was tested against an ethanolic solution of 20% DEET as positive control and a negative control of 70% ethanol. DEET is known as the standard repellent reference.","Four strains of mosquitoes were used in the laboratory tests, including Kisumu F57 and Bora bora F58 susceptible strains of respectively An. gambiae and Aedes aegypti. In addition, local strains laboratory-colonized from rural areas of Goden, in Burkina Faso, were used: hereafter named An-Goden (An. gambiae local strain, F418) and Loc-Aedes (local Ae. aegypti, F318). These species were maintained under a 12:12h (light: dark) photoperiod. During rearing, larvae were fed on fish food while glucose was used for adults. The temperature and relative humidity in the rearing room were 2528C and 6080%, respectively. Individual mosquitoes used in these experiments were 5 to 10 days old nulliparous females starved from sugar solution for 12h before the experiment.","The laboratory experiments were conducted following WHO guidelines for the arm-in-cage test [21]. Cages were 454545cm screen enclosures. Two test cages were used, one for the repellent candidate and the other for the positive control. The test cages contained 200 females aged 5 to 10 days of one of the four mosquito strains: Kisumu, An-Goden, Bora bora and loc-Aedes. The experiment in the laboratory was carried out at temperatures ranging between 25 and 28C, with relative humidity between 60 and 80%.\nOverall, 2mg of ointment were applied per sq cm on the left forearm of each volunteer, a concentration estimated from the average of 5 volunteers from the laboratory asked to apply ointment on their left forearm as they would normally do in real life. In all subsequent repellent trials, volunteers were then supplied with a total volume that would achieve this concentration over the surface area of their forearms and/or legs (determined as described above). A steel spatula was used to apply the ointment on the forearm of each volunteer prior to each experiment to avoid absorbing ointment on non-targeted areas of skin. Positive control consisted of 1 ml of 20% DEET solution applied to the right forearms of volunteers.\nNegative controls and MAA test arms were prepared by first washing left forearms with odorless soap, drying and rinsing with 70% ethanol solution and then drying again. All volunteers wore latex gloves to protect their hands from mosquitoes. To assess the readiness of the mosquitoes to land, both left and right cleaned forearms of volunteers were exposed in the experimental cages for 30s (or until 10 landings of mosquito were counted). Then, for each volunteer, the right forearm was treated from wrist to elbow using 1 ml of the 20% DEET solution whilst the left forearm was treated from the wrist to elbow with MAA ointment. Thirty mins after application of the repellents, the volunteer exposed their treated forearm in the test cage for 3min. The procedure was repeated every 30min until the first bite occurred and the elapsed time to the first bite was recorded. The test was performed three times for each volunteer per mosquito species. Considering the difference in the relative periods of biting activity of each mosquito species, the tests using Ae. aegypti strains were carried out between 09:00 and 18:00, whereas those for An. gambiae were conducted between 17:00 and 05:00 [21].","The lower legs of volunteers were washed with neutral soap, rinsed with 70% of ethanol solution and naturally dried. Once their legs were treated volunteers were asked to avoid rubbing, touching or wetting the repellent-treated area. Two mg of MAA per sq cm (2.40.2g per 118979.2 sq cm) and 2 ml per sq cm of 20% DEET (2 ml0.1 ml per 118979.2 sq cm, as a positive control) were applied to volunteers lower legs, from knee to ankle. A total of 20 volunteers were recruited from Goden village and trained for nocturnal mosquito collection using HLC. Each volunteer was later randomly allocated to one of the five groups (2 for MAIA, 2 for positive control, 1 for negative control) of four volunteers according to the repellent received. Each night of collection, the experiment took place at five different households, located at least 20m apart as per WHO guidelines [21], in order to avoid biases in attractiveness to the mosquitoes.\nMosquito collection started 30min following treatments. Volunteers acting as bait, sat on a chair in pairs (one indoor and one outdoor) and actively collected mosquitoes that landed on their treated lower leg using mouth aspirator and flash torch [22] for 45min, followed by a 15-mins break. Volunteers wore long-sleeved shirts, buttoned at the wrist, long trousers, closed shoes and latex gloves with a hat on their head, but with the treated lower leg to be exposed to mosquitoes by rolling up trousers to the knee. During these experiments, mosquitoes were collected simultaneously indoors and outdoors between 19:00 and 06:00. To avoid biases introduced by individual attractiveness and skills [23, 24] volunteers at the same household rotated between indoors and outdoors hourly. In each household two groups of two people rotated collecting from 18:00 to 24:00 and from 00:00 to 06:00, following the Williams balanced Latin Square design.\nCollected mosquitoes were transferred into plastic cups, covered with a piece of untreated net, with a small hole at the bottom to allow mosquitoes to be easily aspirated into them. After collection mosquitoes were brought to the entomological laboratory of CNRFP and morphologically identified using a stereo microscope and identification keys [25].","No side effects were observed or reported by any of the volunteers throughout the period of tests both in the laboratory and in the field.","Written informed consent was obtained from all volunteers and household owners recruited in this study. The study was approved by the institutional ethic committee of CNRFP under 2019/000008/MS/SG/CNRFP/CIB.","All data were collected on standard forms and entered twice in a database by different people. Databases have been compared using Epi Info 3.5.3, and inconsistencies were verified using printed and corrected forms. The performance of the repellent was measured by calculating the repulsive efficiency and the median full protection time. A generalized linear mixed model (GLMM) was used to further analyse the effect of the location (indoors vs. outdoors) on the performance of the treatments. Variation in the average number of bites received between treatments was also assessed.\nThe median CPT is defined as the interval of time between the beginning of collection/test and the first mosquito landing. To estimate the median CPT of each treatment, a Kaplan-Meier survival analysis was performed for each vector species and strain used in the laboratory experiments and on field data through survival function from R software-version 3.5.0 (2018-04-23). However, for the field test, the analysis was performed on only An. gambiae s.l. as it was the most abundant species collected (~96% of the total collection). The analysis consisted of assessing the median CPT and the repulsive efficacy. The repulsive efficacy was calculated as a percentage of repulsion (% R) according to the formula % R = ((C T) / C) 100, where C is the number of mosquitoes collected on the treated legs of the two control treatments separately, and T is the total number of mosquito bite attempts on the volunteers legs treated with the test product [21].","Laboratory tests Overall, under laboratory conditions the relative repellency (median CPT) was higher for both MAA and 20% DEET against An. gambiae compare to Ae. aegypti (Table1). MAA performed well in repelling the four mosquito species used in this study. The median CPTs were, respectively, 6.5h for An. gambiae (Kisumu), 5.5h for An. gambiae (Goden, local strain) and 4h for Ae. aegypti for both the local and sensitive strain. There was no significant difference between the two treatments for each of the experiment (Kisumu: 2=2.1, p value=0.14; Goden: 2=0.8, p value=0.36; Bora bora: 2=1.7, p value=0.19; Ae. aegypti (local strain): 2=0.9, p value=0. 35) indicating that both MAA and 20% DEET have equal repellency for these strains. The Kaplan-Meier curves for MAA and 20% DEET, respectively, for Kisumu, An-Goden, Bora bora and Loc-Aedes are shown in Fig.2.\n\nTable 1\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \nAn. gambiae\nKisumu\nAn. gambiae\nGoden\nAe. aegypti\nBora bora\nAe. aegypti\nLocalDEETMAADEETMAADEETMAADEETMAAMedian. CPT390390300330270240240240Lower CI368334272216252239212225Upper CI412446328444288241268255Fig. 2Kaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\n\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \nKaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\nOverall, under laboratory conditions the relative repellency (median CPT) was higher for both MAA and 20% DEET against An. gambiae compare to Ae. aegypti (Table1). MAA performed well in repelling the four mosquito species used in this study. The median CPTs were, respectively, 6.5h for An. gambiae (Kisumu), 5.5h for An. gambiae (Goden, local strain) and 4h for Ae. aegypti for both the local and sensitive strain. There was no significant difference between the two treatments for each of the experiment (Kisumu: 2=2.1, p value=0.14; Goden: 2=0.8, p value=0.36; Bora bora: 2=1.7, p value=0.19; Ae. aegypti (local strain): 2=0.9, p value=0. 35) indicating that both MAA and 20% DEET have equal repellency for these strains. The Kaplan-Meier curves for MAA and 20% DEET, respectively, for Kisumu, An-Goden, Bora bora and Loc-Aedes are shown in Fig.2.\n\nTable 1\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \nAn. gambiae\nKisumu\nAn. gambiae\nGoden\nAe. aegypti\nBora bora\nAe. aegypti\nLocalDEETMAADEETMAADEETMAADEETMAAMedian. CPT390390300330270240240240Lower CI368334272216252239212225Upper CI412446328444288241268255Fig. 2Kaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\n\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \nKaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\nField test Mosquito species composition and biting behaviours A total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\nAnopheles gambiae sensu lato (s.l.)\n6862660480\nAnopheles funestus\n121Other Anopheles193Culicines710623\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\nA total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\nAnopheles gambiae sensu lato (s.l.)\n6862660480\nAnopheles funestus\n121Other Anopheles193Culicines710623\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\nRepellency against mosquitoes Repellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\nRepellency of 20% DEET and MAA indoor and outdoor collection\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\nRepellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\nRepellency of 20% DEET and MAA indoor and outdoor collection\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\nComplete protection time The overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nAnopheles gambiae s.l.\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nAnopheles gambiae s.l\n\nIndoors \nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nThe overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nAnopheles gambiae s.l.\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nAnopheles gambiae s.l\n\nIndoors \nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nMosquito species composition and biting behaviours A total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\nAnopheles gambiae sensu lato (s.l.)\n6862660480\nAnopheles funestus\n121Other Anopheles193Culicines710623\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\nA total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\nAnopheles gambiae sensu lato (s.l.)\n6862660480\nAnopheles funestus\n121Other Anopheles193Culicines710623\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\nRepellency against mosquitoes Repellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\nRepellency of 20% DEET and MAA indoor and outdoor collection\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\nRepellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\nRepellency of 20% DEET and MAA indoor and outdoor collection\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\nComplete protection time The overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nAnopheles gambiae s.l.\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nAnopheles gambiae s.l\n\nIndoors \nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nThe overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nAnopheles gambiae s.l.\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nAnopheles gambiae s.l\n\nIndoors \nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)","Overall, under laboratory conditions the relative repellency (median CPT) was higher for both MAA and 20% DEET against An. gambiae compare to Ae. aegypti (Table1). MAA performed well in repelling the four mosquito species used in this study. The median CPTs were, respectively, 6.5h for An. gambiae (Kisumu), 5.5h for An. gambiae (Goden, local strain) and 4h for Ae. aegypti for both the local and sensitive strain. There was no significant difference between the two treatments for each of the experiment (Kisumu: 2=2.1, p value=0.14; Goden: 2=0.8, p value=0.36; Bora bora: 2=1.7, p value=0.19; Ae. aegypti (local strain): 2=0.9, p value=0. 35) indicating that both MAA and 20% DEET have equal repellency for these strains. The Kaplan-Meier curves for MAA and 20% DEET, respectively, for Kisumu, An-Goden, Bora bora and Loc-Aedes are shown in Fig.2.\n\nTable 1\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \nAn. gambiae\nKisumu\nAn. gambiae\nGoden\nAe. aegypti\nBora bora\nAe. aegypti\nLocalDEETMAADEETMAADEETMAADEETMAAMedian. CPT390390300330270240240240Lower CI368334272216252239212225Upper CI412446328444288241268255Fig. 2Kaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\n\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \nKaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers","Mosquito species composition and biting behaviours A total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\nAnopheles gambiae sensu lato (s.l.)\n6862660480\nAnopheles funestus\n121Other Anopheles193Culicines710623\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\nA total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\nAnopheles gambiae sensu lato (s.l.)\n6862660480\nAnopheles funestus\n121Other Anopheles193Culicines710623\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\nRepellency against mosquitoes Repellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\nRepellency of 20% DEET and MAA indoor and outdoor collection\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\nRepellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\nRepellency of 20% DEET and MAA indoor and outdoor collection\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\nComplete protection time The overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nAnopheles gambiae s.l.\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nAnopheles gambiae s.l\n\nIndoors \nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nThe overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nAnopheles gambiae s.l.\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nAnopheles gambiae s.l\n\nIndoors \nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)","A total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\nAnopheles gambiae sensu lato (s.l.)\n6862660480\nAnopheles funestus\n121Other Anopheles193Culicines710623\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%","Repellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\nRepellency of 20% DEET and MAA indoor and outdoor collection\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.","The overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nAnopheles gambiae s.l.\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nAnopheles gambiae s.l\n\nIndoors \nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)","The results of this study demonstrated that MAA, a shea butter-based ointment with 15% DEET provides high protection against mosquitoes in Goden, a rural area of Burkina Faso. Both tests under field and laboratory conditions suggested that MAA has equal repellency effect as 20% DEET ethanolic solution during the period of collection. Similar results were also found both indoors and outdoors. The percentage of repellency when using MAA varied between 100 and 80% over the major malaria vector biting period, which occurs between 18:00 and 06:00. The median CPT were also similar and estimated around 480min. Both MAA and the 20% DEET ethanolic solution were found to provide up to 90% of repellency during the first 6h after their applications. Furthermore, results suggested that using MAA, the average hourly bites received was significantly lower (less than 1 bite per hour) compared to that of both 20% DEET and negative control. However, one of the limitations is that biting activity on control arms was not checked at the end of laboratory experiments to validate consistency of mosquito avidity, but this did not affect the overall result. Overall, it can be argued that MAA could provide protection to people before they go to bed.\nPrevious studies in the same locality comparing repellency between three different repellents found that DEET, IR3535 and KBR 3023 were effective against An. gambiae s.l. and other Afrotropical vector mosquitoes [26]. In this study authors showed that protection from KBR 3023, DEET and IR3535 were still high against anophelines for up to 10h post-exposure. In contrast, results from the current study indicated that the relative repellency was 100% for ~8h. Results were similar to that from a recent study in Ethiopia comparing DEET (N, N-diethyl-1,3-methylbenzamide) and MyggA (p-methane diol) and other laboratory products (20% neem oil and 20% chinaberry oil), where the mean CPT was 8h for DEET whilst an estimated 6h for MyggA [27]. Eight hours of repellency may suffice to protect against earlier vector biting both indoors and outdoors before residents take protection from insecticide-treated nets deployed indoors.\nTo date, 11 countries across the world are classified as having a high burden of malaria (2). In these countries malaria vector control is still based on the use of insecticides, either in the form of indoor spraying or by promoting the large-scale distribution of LLINs. These strategies can effectively reduce the number of malaria cases [28], however the major challenge is the resistance of malaria vectors to different classes of insecticides and the shifts in their feeding and resting behaviours, with the tendency of biting and resting outdoors. For example, a study in the Cascades region in Burkina Faso indicate that in addition to insecticide resistance, more than 50% of the malaria vector biting were taking place outdoors. Therefore, new and supplementary methods are urgently needed to complement these tools in the perspective of malaria elimination [29]. In accordance with the spirit of locally adapted-integrated vector and disease control [30], repellents can usefully complement existing control strategies and provide an additional tool in the management of insecticide resistance. In the context of widespread resistant vectors to insecticide and the tendency of mosquitoes to bite outside houses, there is a need to add MAA ointment to vector control tools in sub-Saharan, malaria-burdened countries.\nThe originality of MAA comes from its formulation based on local butter extensively used in rural areas of West African, and which contributes to womens economic income. Promotion of use of local endogen strategies can sustain malaria control, and also improve the economic situation for African women. Additionally, it has been shown that shea butter is a source of anti-inflammatory and anti-tumour promoting compounds [31]. Another interesting compound in shea butter is cinnamic acid which is known for its antibacterial, antifungal and antiviral properties [32]. Shea butter both moisturizes and heals the skin. Clinical studies have shown it to be safe for skin [33]. MAA ointment will not only protect from mosquito-borne diseases but also against other micro-organisms.\nBesides the level of protection offered by repellents, daily compliance and appropriate use seem to be major obstacles to achieving the potential impact on malaria [34]. An efficacy study carried out in Tanzania has shown that volunteers preferred MAA ointment to a more classical 20% DEET solution [35]. Sales by Maa Africa SAS of 50,000 units in local stores in Burkina Faso between August and November 2021 further illustrate its desirability. However, more data are needed to understand who is likely to use the product and whether its usage is appropriate in terms of frequency and application in order to have an impact on mosquito-borne diseases, such as malaria.","MAA, a novel ointment formulated with shea butter widely used in West Africa to moisturize the skin of children, has shown high repellency against laboratory-reared and wild malaria vectors. In the context of widespread vector resistance to insecticide and growing tendency of mosquitoes to bite outside houses, there is a need to add MAA ointment to the vector control tools used in sub-Saharan countries with high malaria burden."],"string":"[\n \"Malaria is one of the deadliest diseases in many low- and middle-income countries, affecting mainly children and pregnant women in sub-Saharan Africa [1]. Long-lasting insecticide-treated nets (LLINs) have been regarded as the most effective method for controlling mosquitoes transmitting malaria parasites. Since 2000, about one billion nets have been distributed in Africa, resulting in a significant decline in malaria-related deaths on the continent between 2000 and 2015 [24].\\nHowever, the massive use of insecticides in public health in addition to that in agriculture causes concern regarding insecticide resistance [57] and changing behaviour [8, 9] of the malaria vectors. For example, a study conducted in Papua New Guinea showed a shift in mosquito biting from night to earlier hours in the evening after a nationwide distribution of LLINs [10]. Similar changes in the behaviour of Anopheles funestus have been observed in Benin and Senegal after LLIN distribution achieved a high level of coverage [9, 10]. Furthermore, studies suggest that the scaling up of LLIN distribution and indoor residual spraying (IRS) have led to more outdoor biting by Anopheles gambiae sensu lato (s.l.), commonly considered endophagic mosquitoes [1113]. A recent study in the Cascades region of Burkina Faso showed a high level of insecticide resistance [14] where more than 50% of the major vector, An. gambiae s.l., were collected biting outdoors [15]. These altered patterns of outdoors, early evening and morning biting, by anophelines, combined with resistance to insecticides appear to be caused by the mass distribution of LLINs and imply the inexorable loss of efficacy of these interventions [16, 17]. A recent study highlighted that an increase in early evening biting could increase transmission not only because people are unprotected by nets, but also because there is a higher chance of malaria vectors becoming infectious [18]. The development of new vector control tools, in addition to LLINs, is therefore necessary to protect people, when they are not under a bed net.\\nTopical repellents could play an important role in addressing this problem if they are effective and accepted by the population. A systematic review of repellent interventions and mathematical modeling has shown that user compliance is indeed one of the most decisive factors for the success of this intervention [19]. In sub-Saharan Africa, ointments are used primarily by mothers and children to moisturize their skins. In Burkina Faso, ointments are applied to 80% of children every evening, when mosquitoes start biting (Kadidia Ouedraogo et al., in prep). Maa Africa, a company based in Burkina Faso, has developed a mosquito repellent ointment, MAA, uniquely designed with local mothers, to be used daily within their families. The underlying idea is to leverage the existing habits of mothers to protect their families from infectious bites whenever they are not under a net. Affordability is a key criterion for the products adoption and use. MAA has been industrially produced since June 2020 in Cte dIvoire and integrates a large share of ingredients sourced in West Africa. The ointment was officially launched in August 2020 in Burkina Faso and over 50,000 units were sold in the first four months. In March 2021, over 500 points of sales (mainly general stores, pharmacies and kiosks) distributed the product in the country. If MAA proves that it is both effective and accepted by the population, it could play a key role in reducing the probability of children experiencing infectious bites during the evening and be positioned as a complementary intervention to LLINs.\\nThe aim of this study is to evaluate the effectiveness of MAA in both laboratory and field conditions, especially the median complete protection time (CPT) offered by the product. Results from these evaluations are important for validating how effective this new repellent is; behavioral responses to repellent differ between wild mosquito and laboratory-reared mosquito populations [20].\",\n \"Study area Laboratory tests were conducted in May 2019 in the insectary of Centre National de Recherche et de Formation sur le Paludisme (CNRFP) in Ouagadougou, Burkina Faso. Field tests were carried out at Goden (1225N, 120W), a site located at 15km northeast of Ouagadougou, the capital city of Burkina Faso (Fig.1). Goden is a rural village with a Sudanian savanna climate and rainfall under 900 mm annually. The ~800 inhabitants mainly belong to the Mossi ethnic group, and are mostly devoted to agriculture and raising pigs, dogs, goats, and chickens within their compounds. LLINs were distributed in 2016 to ~90% of the population. Goden is known for its high density of malaria vectors due to its proximity to the Massili River. The field study was carried out during the rainy season (August to November 2019) corresponding to high vector density and high malaria transmission. A preliminary assessment of the mosquito density on the collection site was carried out using human landing catches (HLCs) before the tests started.\\n\\nFig. 1Study area\\nStudy area\\nLaboratory tests were conducted in May 2019 in the insectary of Centre National de Recherche et de Formation sur le Paludisme (CNRFP) in Ouagadougou, Burkina Faso. Field tests were carried out at Goden (1225N, 120W), a site located at 15km northeast of Ouagadougou, the capital city of Burkina Faso (Fig.1). Goden is a rural village with a Sudanian savanna climate and rainfall under 900 mm annually. The ~800 inhabitants mainly belong to the Mossi ethnic group, and are mostly devoted to agriculture and raising pigs, dogs, goats, and chickens within their compounds. LLINs were distributed in 2016 to ~90% of the population. Goden is known for its high density of malaria vectors due to its proximity to the Massili River. The field study was carried out during the rainy season (August to November 2019) corresponding to high vector density and high malaria transmission. A preliminary assessment of the mosquito density on the collection site was carried out using human landing catches (HLCs) before the tests started.\\n\\nFig. 1Study area\\nStudy area\\nHuman volunteer preparation Healthy adult male volunteers aged between 18 and 40 years were enrolled in this study. The volunteers were instructed not to use fragranced soaps, perfume, tobacco, or alcohol 12h before the start and throughout their participation. To establish the amount of repellent required for application, the surface area of the arm (for laboratory tests) or the leg (for field tests) of volunteers was determined using the following formula: Area= (Cw+ Ce) Dwe.\\nHere Cw is the circumference of the wrist or ankle in cm, Ce is the elbow cubital fossa or the knee circumference in cm, and Dwe is the distance in cm between Ce and Cw [21]. The amount of ointment needed for each volunteer was determined depending on the area of their forearm or leg length. The quantity of product left in bottles was weighed using a precision weighing balance (KERN & SOHN GmbH, Balingen, Germany) to determine the amount applied by each volunteer.\\nHealthy adult male volunteers aged between 18 and 40 years were enrolled in this study. The volunteers were instructed not to use fragranced soaps, perfume, tobacco, or alcohol 12h before the start and throughout their participation. To establish the amount of repellent required for application, the surface area of the arm (for laboratory tests) or the leg (for field tests) of volunteers was determined using the following formula: Area= (Cw+ Ce) Dwe.\\nHere Cw is the circumference of the wrist or ankle in cm, Ce is the elbow cubital fossa or the knee circumference in cm, and Dwe is the distance in cm between Ce and Cw [21]. The amount of ointment needed for each volunteer was determined depending on the area of their forearm or leg length. The quantity of product left in bottles was weighed using a precision weighing balance (KERN & SOHN GmbH, Balingen, Germany) to determine the amount applied by each volunteer.\\nRepellents MAA, a shea butter-based ointment containing 15% DEET (N, N-diethyl-3-methylbenzamide) was received from Maa Africa SAS. It was tested against an ethanolic solution of 20% DEET as positive control and a negative control of 70% ethanol. DEET is known as the standard repellent reference.\\nMAA, a shea butter-based ointment containing 15% DEET (N, N-diethyl-3-methylbenzamide) was received from Maa Africa SAS. It was tested against an ethanolic solution of 20% DEET as positive control and a negative control of 70% ethanol. DEET is known as the standard repellent reference.\\nStrains of mosquitoes Four strains of mosquitoes were used in the laboratory tests, including Kisumu F57 and Bora bora F58 susceptible strains of respectively An. gambiae and Aedes aegypti. In addition, local strains laboratory-colonized from rural areas of Goden, in Burkina Faso, were used: hereafter named An-Goden (An. gambiae local strain, F418) and Loc-Aedes (local Ae. aegypti, F318). These species were maintained under a 12:12h (light: dark) photoperiod. During rearing, larvae were fed on fish food while glucose was used for adults. The temperature and relative humidity in the rearing room were 2528C and 6080%, respectively. Individual mosquitoes used in these experiments were 5 to 10 days old nulliparous females starved from sugar solution for 12h before the experiment.\\nFour strains of mosquitoes were used in the laboratory tests, including Kisumu F57 and Bora bora F58 susceptible strains of respectively An. gambiae and Aedes aegypti. In addition, local strains laboratory-colonized from rural areas of Goden, in Burkina Faso, were used: hereafter named An-Goden (An. gambiae local strain, F418) and Loc-Aedes (local Ae. aegypti, F318). These species were maintained under a 12:12h (light: dark) photoperiod. During rearing, larvae were fed on fish food while glucose was used for adults. The temperature and relative humidity in the rearing room were 2528C and 6080%, respectively. Individual mosquitoes used in these experiments were 5 to 10 days old nulliparous females starved from sugar solution for 12h before the experiment.\\nEvaluation in the laboratory The laboratory experiments were conducted following WHO guidelines for the arm-in-cage test [21]. Cages were 454545cm screen enclosures. Two test cages were used, one for the repellent candidate and the other for the positive control. The test cages contained 200 females aged 5 to 10 days of one of the four mosquito strains: Kisumu, An-Goden, Bora bora and loc-Aedes. The experiment in the laboratory was carried out at temperatures ranging between 25 and 28C, with relative humidity between 60 and 80%.\\nOverall, 2mg of ointment were applied per sq cm on the left forearm of each volunteer, a concentration estimated from the average of 5 volunteers from the laboratory asked to apply ointment on their left forearm as they would normally do in real life. In all subsequent repellent trials, volunteers were then supplied with a total volume that would achieve this concentration over the surface area of their forearms and/or legs (determined as described above). A steel spatula was used to apply the ointment on the forearm of each volunteer prior to each experiment to avoid absorbing ointment on non-targeted areas of skin. Positive control consisted of 1 ml of 20% DEET solution applied to the right forearms of volunteers.\\nNegative controls and MAA test arms were prepared by first washing left forearms with odorless soap, drying and rinsing with 70% ethanol solution and then drying again. All volunteers wore latex gloves to protect their hands from mosquitoes. To assess the readiness of the mosquitoes to land, both left and right cleaned forearms of volunteers were exposed in the experimental cages for 30s (or until 10 landings of mosquito were counted). Then, for each volunteer, the right forearm was treated from wrist to elbow using 1 ml of the 20% DEET solution whilst the left forearm was treated from the wrist to elbow with MAA ointment. Thirty mins after application of the repellents, the volunteer exposed their treated forearm in the test cage for 3min. The procedure was repeated every 30min until the first bite occurred and the elapsed time to the first bite was recorded. The test was performed three times for each volunteer per mosquito species. Considering the difference in the relative periods of biting activity of each mosquito species, the tests using Ae. aegypti strains were carried out between 09:00 and 18:00, whereas those for An. gambiae were conducted between 17:00 and 05:00 [21].\\nThe laboratory experiments were conducted following WHO guidelines for the arm-in-cage test [21]. Cages were 454545cm screen enclosures. Two test cages were used, one for the repellent candidate and the other for the positive control. The test cages contained 200 females aged 5 to 10 days of one of the four mosquito strains: Kisumu, An-Goden, Bora bora and loc-Aedes. The experiment in the laboratory was carried out at temperatures ranging between 25 and 28C, with relative humidity between 60 and 80%.\\nOverall, 2mg of ointment were applied per sq cm on the left forearm of each volunteer, a concentration estimated from the average of 5 volunteers from the laboratory asked to apply ointment on their left forearm as they would normally do in real life. In all subsequent repellent trials, volunteers were then supplied with a total volume that would achieve this concentration over the surface area of their forearms and/or legs (determined as described above). A steel spatula was used to apply the ointment on the forearm of each volunteer prior to each experiment to avoid absorbing ointment on non-targeted areas of skin. Positive control consisted of 1 ml of 20% DEET solution applied to the right forearms of volunteers.\\nNegative controls and MAA test arms were prepared by first washing left forearms with odorless soap, drying and rinsing with 70% ethanol solution and then drying again. All volunteers wore latex gloves to protect their hands from mosquitoes. To assess the readiness of the mosquitoes to land, both left and right cleaned forearms of volunteers were exposed in the experimental cages for 30s (or until 10 landings of mosquito were counted). Then, for each volunteer, the right forearm was treated from wrist to elbow using 1 ml of the 20% DEET solution whilst the left forearm was treated from the wrist to elbow with MAA ointment. Thirty mins after application of the repellents, the volunteer exposed their treated forearm in the test cage for 3min. The procedure was repeated every 30min until the first bite occurred and the elapsed time to the first bite was recorded. The test was performed three times for each volunteer per mosquito species. Considering the difference in the relative periods of biting activity of each mosquito species, the tests using Ae. aegypti strains were carried out between 09:00 and 18:00, whereas those for An. gambiae were conducted between 17:00 and 05:00 [21].\\nField evaluation The lower legs of volunteers were washed with neutral soap, rinsed with 70% of ethanol solution and naturally dried. Once their legs were treated volunteers were asked to avoid rubbing, touching or wetting the repellent-treated area. Two mg of MAA per sq cm (2.40.2g per 118979.2 sq cm) and 2 ml per sq cm of 20% DEET (2 ml0.1 ml per 118979.2 sq cm, as a positive control) were applied to volunteers lower legs, from knee to ankle. A total of 20 volunteers were recruited from Goden village and trained for nocturnal mosquito collection using HLC. Each volunteer was later randomly allocated to one of the five groups (2 for MAIA, 2 for positive control, 1 for negative control) of four volunteers according to the repellent received. Each night of collection, the experiment took place at five different households, located at least 20m apart as per WHO guidelines [21], in order to avoid biases in attractiveness to the mosquitoes.\\nMosquito collection started 30min following treatments. Volunteers acting as bait, sat on a chair in pairs (one indoor and one outdoor) and actively collected mosquitoes that landed on their treated lower leg using mouth aspirator and flash torch [22] for 45min, followed by a 15-mins break. Volunteers wore long-sleeved shirts, buttoned at the wrist, long trousers, closed shoes and latex gloves with a hat on their head, but with the treated lower leg to be exposed to mosquitoes by rolling up trousers to the knee. During these experiments, mosquitoes were collected simultaneously indoors and outdoors between 19:00 and 06:00. To avoid biases introduced by individual attractiveness and skills [23, 24] volunteers at the same household rotated between indoors and outdoors hourly. In each household two groups of two people rotated collecting from 18:00 to 24:00 and from 00:00 to 06:00, following the Williams balanced Latin Square design.\\nCollected mosquitoes were transferred into plastic cups, covered with a piece of untreated net, with a small hole at the bottom to allow mosquitoes to be easily aspirated into them. After collection mosquitoes were brought to the entomological laboratory of CNRFP and morphologically identified using a stereo microscope and identification keys [25].\\nThe lower legs of volunteers were washed with neutral soap, rinsed with 70% of ethanol solution and naturally dried. Once their legs were treated volunteers were asked to avoid rubbing, touching or wetting the repellent-treated area. Two mg of MAA per sq cm (2.40.2g per 118979.2 sq cm) and 2 ml per sq cm of 20% DEET (2 ml0.1 ml per 118979.2 sq cm, as a positive control) were applied to volunteers lower legs, from knee to ankle. A total of 20 volunteers were recruited from Goden village and trained for nocturnal mosquito collection using HLC. Each volunteer was later randomly allocated to one of the five groups (2 for MAIA, 2 for positive control, 1 for negative control) of four volunteers according to the repellent received. Each night of collection, the experiment took place at five different households, located at least 20m apart as per WHO guidelines [21], in order to avoid biases in attractiveness to the mosquitoes.\\nMosquito collection started 30min following treatments. Volunteers acting as bait, sat on a chair in pairs (one indoor and one outdoor) and actively collected mosquitoes that landed on their treated lower leg using mouth aspirator and flash torch [22] for 45min, followed by a 15-mins break. Volunteers wore long-sleeved shirts, buttoned at the wrist, long trousers, closed shoes and latex gloves with a hat on their head, but with the treated lower leg to be exposed to mosquitoes by rolling up trousers to the knee. During these experiments, mosquitoes were collected simultaneously indoors and outdoors between 19:00 and 06:00. To avoid biases introduced by individual attractiveness and skills [23, 24] volunteers at the same household rotated between indoors and outdoors hourly. In each household two groups of two people rotated collecting from 18:00 to 24:00 and from 00:00 to 06:00, following the Williams balanced Latin Square design.\\nCollected mosquitoes were transferred into plastic cups, covered with a piece of untreated net, with a small hole at the bottom to allow mosquitoes to be easily aspirated into them. After collection mosquitoes were brought to the entomological laboratory of CNRFP and morphologically identified using a stereo microscope and identification keys [25].\\nSide effects No side effects were observed or reported by any of the volunteers throughout the period of tests both in the laboratory and in the field.\\nNo side effects were observed or reported by any of the volunteers throughout the period of tests both in the laboratory and in the field.\\nEthical clearance Written informed consent was obtained from all volunteers and household owners recruited in this study. The study was approved by the institutional ethic committee of CNRFP under 2019/000008/MS/SG/CNRFP/CIB.\\nWritten informed consent was obtained from all volunteers and household owners recruited in this study. The study was approved by the institutional ethic committee of CNRFP under 2019/000008/MS/SG/CNRFP/CIB.\\nData analysis All data were collected on standard forms and entered twice in a database by different people. Databases have been compared using Epi Info 3.5.3, and inconsistencies were verified using printed and corrected forms. The performance of the repellent was measured by calculating the repulsive efficiency and the median full protection time. A generalized linear mixed model (GLMM) was used to further analyse the effect of the location (indoors vs. outdoors) on the performance of the treatments. Variation in the average number of bites received between treatments was also assessed.\\nThe median CPT is defined as the interval of time between the beginning of collection/test and the first mosquito landing. To estimate the median CPT of each treatment, a Kaplan-Meier survival analysis was performed for each vector species and strain used in the laboratory experiments and on field data through survival function from R software-version 3.5.0 (2018-04-23). However, for the field test, the analysis was performed on only An. gambiae s.l. as it was the most abundant species collected (~96% of the total collection). The analysis consisted of assessing the median CPT and the repulsive efficacy. The repulsive efficacy was calculated as a percentage of repulsion (% R) according to the formula % R = ((C T) / C) 100, where C is the number of mosquitoes collected on the treated legs of the two control treatments separately, and T is the total number of mosquito bite attempts on the volunteers legs treated with the test product [21].\\nAll data were collected on standard forms and entered twice in a database by different people. Databases have been compared using Epi Info 3.5.3, and inconsistencies were verified using printed and corrected forms. The performance of the repellent was measured by calculating the repulsive efficiency and the median full protection time. A generalized linear mixed model (GLMM) was used to further analyse the effect of the location (indoors vs. outdoors) on the performance of the treatments. Variation in the average number of bites received between treatments was also assessed.\\nThe median CPT is defined as the interval of time between the beginning of collection/test and the first mosquito landing. To estimate the median CPT of each treatment, a Kaplan-Meier survival analysis was performed for each vector species and strain used in the laboratory experiments and on field data through survival function from R software-version 3.5.0 (2018-04-23). However, for the field test, the analysis was performed on only An. gambiae s.l. as it was the most abundant species collected (~96% of the total collection). The analysis consisted of assessing the median CPT and the repulsive efficacy. The repulsive efficacy was calculated as a percentage of repulsion (% R) according to the formula % R = ((C T) / C) 100, where C is the number of mosquitoes collected on the treated legs of the two control treatments separately, and T is the total number of mosquito bite attempts on the volunteers legs treated with the test product [21].\",\n \"Laboratory tests were conducted in May 2019 in the insectary of Centre National de Recherche et de Formation sur le Paludisme (CNRFP) in Ouagadougou, Burkina Faso. Field tests were carried out at Goden (1225N, 120W), a site located at 15km northeast of Ouagadougou, the capital city of Burkina Faso (Fig.1). Goden is a rural village with a Sudanian savanna climate and rainfall under 900 mm annually. The ~800 inhabitants mainly belong to the Mossi ethnic group, and are mostly devoted to agriculture and raising pigs, dogs, goats, and chickens within their compounds. LLINs were distributed in 2016 to ~90% of the population. Goden is known for its high density of malaria vectors due to its proximity to the Massili River. The field study was carried out during the rainy season (August to November 2019) corresponding to high vector density and high malaria transmission. A preliminary assessment of the mosquito density on the collection site was carried out using human landing catches (HLCs) before the tests started.\\n\\nFig. 1Study area\\nStudy area\",\n \"Healthy adult male volunteers aged between 18 and 40 years were enrolled in this study. The volunteers were instructed not to use fragranced soaps, perfume, tobacco, or alcohol 12h before the start and throughout their participation. To establish the amount of repellent required for application, the surface area of the arm (for laboratory tests) or the leg (for field tests) of volunteers was determined using the following formula: Area= (Cw+ Ce) Dwe.\\nHere Cw is the circumference of the wrist or ankle in cm, Ce is the elbow cubital fossa or the knee circumference in cm, and Dwe is the distance in cm between Ce and Cw [21]. The amount of ointment needed for each volunteer was determined depending on the area of their forearm or leg length. The quantity of product left in bottles was weighed using a precision weighing balance (KERN & SOHN GmbH, Balingen, Germany) to determine the amount applied by each volunteer.\",\n \"MAA, a shea butter-based ointment containing 15% DEET (N, N-diethyl-3-methylbenzamide) was received from Maa Africa SAS. It was tested against an ethanolic solution of 20% DEET as positive control and a negative control of 70% ethanol. DEET is known as the standard repellent reference.\",\n \"Four strains of mosquitoes were used in the laboratory tests, including Kisumu F57 and Bora bora F58 susceptible strains of respectively An. gambiae and Aedes aegypti. In addition, local strains laboratory-colonized from rural areas of Goden, in Burkina Faso, were used: hereafter named An-Goden (An. gambiae local strain, F418) and Loc-Aedes (local Ae. aegypti, F318). These species were maintained under a 12:12h (light: dark) photoperiod. During rearing, larvae were fed on fish food while glucose was used for adults. The temperature and relative humidity in the rearing room were 2528C and 6080%, respectively. Individual mosquitoes used in these experiments were 5 to 10 days old nulliparous females starved from sugar solution for 12h before the experiment.\",\n \"The laboratory experiments were conducted following WHO guidelines for the arm-in-cage test [21]. Cages were 454545cm screen enclosures. Two test cages were used, one for the repellent candidate and the other for the positive control. The test cages contained 200 females aged 5 to 10 days of one of the four mosquito strains: Kisumu, An-Goden, Bora bora and loc-Aedes. The experiment in the laboratory was carried out at temperatures ranging between 25 and 28C, with relative humidity between 60 and 80%.\\nOverall, 2mg of ointment were applied per sq cm on the left forearm of each volunteer, a concentration estimated from the average of 5 volunteers from the laboratory asked to apply ointment on their left forearm as they would normally do in real life. In all subsequent repellent trials, volunteers were then supplied with a total volume that would achieve this concentration over the surface area of their forearms and/or legs (determined as described above). A steel spatula was used to apply the ointment on the forearm of each volunteer prior to each experiment to avoid absorbing ointment on non-targeted areas of skin. Positive control consisted of 1 ml of 20% DEET solution applied to the right forearms of volunteers.\\nNegative controls and MAA test arms were prepared by first washing left forearms with odorless soap, drying and rinsing with 70% ethanol solution and then drying again. All volunteers wore latex gloves to protect their hands from mosquitoes. To assess the readiness of the mosquitoes to land, both left and right cleaned forearms of volunteers were exposed in the experimental cages for 30s (or until 10 landings of mosquito were counted). Then, for each volunteer, the right forearm was treated from wrist to elbow using 1 ml of the 20% DEET solution whilst the left forearm was treated from the wrist to elbow with MAA ointment. Thirty mins after application of the repellents, the volunteer exposed their treated forearm in the test cage for 3min. The procedure was repeated every 30min until the first bite occurred and the elapsed time to the first bite was recorded. The test was performed three times for each volunteer per mosquito species. Considering the difference in the relative periods of biting activity of each mosquito species, the tests using Ae. aegypti strains were carried out between 09:00 and 18:00, whereas those for An. gambiae were conducted between 17:00 and 05:00 [21].\",\n \"The lower legs of volunteers were washed with neutral soap, rinsed with 70% of ethanol solution and naturally dried. Once their legs were treated volunteers were asked to avoid rubbing, touching or wetting the repellent-treated area. Two mg of MAA per sq cm (2.40.2g per 118979.2 sq cm) and 2 ml per sq cm of 20% DEET (2 ml0.1 ml per 118979.2 sq cm, as a positive control) were applied to volunteers lower legs, from knee to ankle. A total of 20 volunteers were recruited from Goden village and trained for nocturnal mosquito collection using HLC. Each volunteer was later randomly allocated to one of the five groups (2 for MAIA, 2 for positive control, 1 for negative control) of four volunteers according to the repellent received. Each night of collection, the experiment took place at five different households, located at least 20m apart as per WHO guidelines [21], in order to avoid biases in attractiveness to the mosquitoes.\\nMosquito collection started 30min following treatments. Volunteers acting as bait, sat on a chair in pairs (one indoor and one outdoor) and actively collected mosquitoes that landed on their treated lower leg using mouth aspirator and flash torch [22] for 45min, followed by a 15-mins break. Volunteers wore long-sleeved shirts, buttoned at the wrist, long trousers, closed shoes and latex gloves with a hat on their head, but with the treated lower leg to be exposed to mosquitoes by rolling up trousers to the knee. During these experiments, mosquitoes were collected simultaneously indoors and outdoors between 19:00 and 06:00. To avoid biases introduced by individual attractiveness and skills [23, 24] volunteers at the same household rotated between indoors and outdoors hourly. In each household two groups of two people rotated collecting from 18:00 to 24:00 and from 00:00 to 06:00, following the Williams balanced Latin Square design.\\nCollected mosquitoes were transferred into plastic cups, covered with a piece of untreated net, with a small hole at the bottom to allow mosquitoes to be easily aspirated into them. After collection mosquitoes were brought to the entomological laboratory of CNRFP and morphologically identified using a stereo microscope and identification keys [25].\",\n \"No side effects were observed or reported by any of the volunteers throughout the period of tests both in the laboratory and in the field.\",\n \"Written informed consent was obtained from all volunteers and household owners recruited in this study. The study was approved by the institutional ethic committee of CNRFP under 2019/000008/MS/SG/CNRFP/CIB.\",\n \"All data were collected on standard forms and entered twice in a database by different people. Databases have been compared using Epi Info 3.5.3, and inconsistencies were verified using printed and corrected forms. The performance of the repellent was measured by calculating the repulsive efficiency and the median full protection time. A generalized linear mixed model (GLMM) was used to further analyse the effect of the location (indoors vs. outdoors) on the performance of the treatments. Variation in the average number of bites received between treatments was also assessed.\\nThe median CPT is defined as the interval of time between the beginning of collection/test and the first mosquito landing. To estimate the median CPT of each treatment, a Kaplan-Meier survival analysis was performed for each vector species and strain used in the laboratory experiments and on field data through survival function from R software-version 3.5.0 (2018-04-23). However, for the field test, the analysis was performed on only An. gambiae s.l. as it was the most abundant species collected (~96% of the total collection). The analysis consisted of assessing the median CPT and the repulsive efficacy. The repulsive efficacy was calculated as a percentage of repulsion (% R) according to the formula % R = ((C T) / C) 100, where C is the number of mosquitoes collected on the treated legs of the two control treatments separately, and T is the total number of mosquito bite attempts on the volunteers legs treated with the test product [21].\",\n \"Laboratory tests Overall, under laboratory conditions the relative repellency (median CPT) was higher for both MAA and 20% DEET against An. gambiae compare to Ae. aegypti (Table1). MAA performed well in repelling the four mosquito species used in this study. The median CPTs were, respectively, 6.5h for An. gambiae (Kisumu), 5.5h for An. gambiae (Goden, local strain) and 4h for Ae. aegypti for both the local and sensitive strain. There was no significant difference between the two treatments for each of the experiment (Kisumu: 2=2.1, p value=0.14; Goden: 2=0.8, p value=0.36; Bora bora: 2=1.7, p value=0.19; Ae. aegypti (local strain): 2=0.9, p value=0. 35) indicating that both MAA and 20% DEET have equal repellency for these strains. The Kaplan-Meier curves for MAA and 20% DEET, respectively, for Kisumu, An-Goden, Bora bora and Loc-Aedes are shown in Fig.2.\\n\\nTable 1\\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \\nAn. gambiae\\nKisumu\\nAn. gambiae\\nGoden\\nAe. aegypti\\nBora bora\\nAe. aegypti\\nLocalDEETMAADEETMAADEETMAADEETMAAMedian. CPT390390300330270240240240Lower CI368334272216252239212225Upper CI412446328444288241268255Fig. 2Kaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\\n\\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \\nKaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\\nOverall, under laboratory conditions the relative repellency (median CPT) was higher for both MAA and 20% DEET against An. gambiae compare to Ae. aegypti (Table1). MAA performed well in repelling the four mosquito species used in this study. The median CPTs were, respectively, 6.5h for An. gambiae (Kisumu), 5.5h for An. gambiae (Goden, local strain) and 4h for Ae. aegypti for both the local and sensitive strain. There was no significant difference between the two treatments for each of the experiment (Kisumu: 2=2.1, p value=0.14; Goden: 2=0.8, p value=0.36; Bora bora: 2=1.7, p value=0.19; Ae. aegypti (local strain): 2=0.9, p value=0. 35) indicating that both MAA and 20% DEET have equal repellency for these strains. The Kaplan-Meier curves for MAA and 20% DEET, respectively, for Kisumu, An-Goden, Bora bora and Loc-Aedes are shown in Fig.2.\\n\\nTable 1\\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \\nAn. gambiae\\nKisumu\\nAn. gambiae\\nGoden\\nAe. aegypti\\nBora bora\\nAe. aegypti\\nLocalDEETMAADEETMAADEETMAADEETMAAMedian. CPT390390300330270240240240Lower CI368334272216252239212225Upper CI412446328444288241268255Fig. 2Kaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\\n\\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \\nKaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\\nField test Mosquito species composition and biting behaviours A total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\\nAnopheles gambiae sensu lato (s.l.)\\n6862660480\\nAnopheles funestus\\n121Other Anopheles193Culicines710623\\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\\nA total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\\nAnopheles gambiae sensu lato (s.l.)\\n6862660480\\nAnopheles funestus\\n121Other Anopheles193Culicines710623\\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\\nRepellency against mosquitoes Repellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\\nRepellency of 20% DEET and MAA indoor and outdoor collection\\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\\nRepellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\\nRepellency of 20% DEET and MAA indoor and outdoor collection\\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\\nComplete protection time The overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\\nAnopheles gambiae s.l.\\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\\nAnopheles gambiae s.l\\n\\nIndoors \\nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\\nThe overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\\nAnopheles gambiae s.l.\\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\\nAnopheles gambiae s.l\\n\\nIndoors \\nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\\nMosquito species composition and biting behaviours A total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\\nAnopheles gambiae sensu lato (s.l.)\\n6862660480\\nAnopheles funestus\\n121Other Anopheles193Culicines710623\\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\\nA total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\\nAnopheles gambiae sensu lato (s.l.)\\n6862660480\\nAnopheles funestus\\n121Other Anopheles193Culicines710623\\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\\nRepellency against mosquitoes Repellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\\nRepellency of 20% DEET and MAA indoor and outdoor collection\\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\\nRepellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\\nRepellency of 20% DEET and MAA indoor and outdoor collection\\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\\nComplete protection time The overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\\nAnopheles gambiae s.l.\\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\\nAnopheles gambiae s.l\\n\\nIndoors \\nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\\nThe overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\\nAnopheles gambiae s.l.\\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\\nAnopheles gambiae s.l\\n\\nIndoors \\nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\",\n \"Overall, under laboratory conditions the relative repellency (median CPT) was higher for both MAA and 20% DEET against An. gambiae compare to Ae. aegypti (Table1). MAA performed well in repelling the four mosquito species used in this study. The median CPTs were, respectively, 6.5h for An. gambiae (Kisumu), 5.5h for An. gambiae (Goden, local strain) and 4h for Ae. aegypti for both the local and sensitive strain. There was no significant difference between the two treatments for each of the experiment (Kisumu: 2=2.1, p value=0.14; Goden: 2=0.8, p value=0.36; Bora bora: 2=1.7, p value=0.19; Ae. aegypti (local strain): 2=0.9, p value=0. 35) indicating that both MAA and 20% DEET have equal repellency for these strains. The Kaplan-Meier curves for MAA and 20% DEET, respectively, for Kisumu, An-Goden, Bora bora and Loc-Aedes are shown in Fig.2.\\n\\nTable 1\\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \\nAn. gambiae\\nKisumu\\nAn. gambiae\\nGoden\\nAe. aegypti\\nBora bora\\nAe. aegypti\\nLocalDEETMAADEETMAADEETMAADEETMAAMedian. CPT390390300330270240240240Lower CI368334272216252239212225Upper CI412446328444288241268255Fig. 2Kaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\\n\\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \\nKaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\",\n \"Mosquito species composition and biting behaviours A total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\\nAnopheles gambiae sensu lato (s.l.)\\n6862660480\\nAnopheles funestus\\n121Other Anopheles193Culicines710623\\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\\nA total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\\nAnopheles gambiae sensu lato (s.l.)\\n6862660480\\nAnopheles funestus\\n121Other Anopheles193Culicines710623\\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\\nRepellency against mosquitoes Repellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\\nRepellency of 20% DEET and MAA indoor and outdoor collection\\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\\nRepellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\\nRepellency of 20% DEET and MAA indoor and outdoor collection\\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\\nComplete protection time The overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\\nAnopheles gambiae s.l.\\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\\nAnopheles gambiae s.l\\n\\nIndoors \\nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\\nThe overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\\nAnopheles gambiae s.l.\\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\\nAnopheles gambiae s.l\\n\\nIndoors \\nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\",\n \"A total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\\nAnopheles gambiae sensu lato (s.l.)\\n6862660480\\nAnopheles funestus\\n121Other Anopheles193Culicines710623\\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\",\n \"Repellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\\nRepellency of 20% DEET and MAA indoor and outdoor collection\\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\",\n \"The overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\\nAnopheles gambiae s.l.\\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\\nAnopheles gambiae s.l\\n\\nIndoors \\nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\",\n \"The results of this study demonstrated that MAA, a shea butter-based ointment with 15% DEET provides high protection against mosquitoes in Goden, a rural area of Burkina Faso. Both tests under field and laboratory conditions suggested that MAA has equal repellency effect as 20% DEET ethanolic solution during the period of collection. Similar results were also found both indoors and outdoors. The percentage of repellency when using MAA varied between 100 and 80% over the major malaria vector biting period, which occurs between 18:00 and 06:00. The median CPT were also similar and estimated around 480min. Both MAA and the 20% DEET ethanolic solution were found to provide up to 90% of repellency during the first 6h after their applications. Furthermore, results suggested that using MAA, the average hourly bites received was significantly lower (less than 1 bite per hour) compared to that of both 20% DEET and negative control. However, one of the limitations is that biting activity on control arms was not checked at the end of laboratory experiments to validate consistency of mosquito avidity, but this did not affect the overall result. Overall, it can be argued that MAA could provide protection to people before they go to bed.\\nPrevious studies in the same locality comparing repellency between three different repellents found that DEET, IR3535 and KBR 3023 were effective against An. gambiae s.l. and other Afrotropical vector mosquitoes [26]. In this study authors showed that protection from KBR 3023, DEET and IR3535 were still high against anophelines for up to 10h post-exposure. In contrast, results from the current study indicated that the relative repellency was 100% for ~8h. Results were similar to that from a recent study in Ethiopia comparing DEET (N, N-diethyl-1,3-methylbenzamide) and MyggA (p-methane diol) and other laboratory products (20% neem oil and 20% chinaberry oil), where the mean CPT was 8h for DEET whilst an estimated 6h for MyggA [27]. Eight hours of repellency may suffice to protect against earlier vector biting both indoors and outdoors before residents take protection from insecticide-treated nets deployed indoors.\\nTo date, 11 countries across the world are classified as having a high burden of malaria (2). In these countries malaria vector control is still based on the use of insecticides, either in the form of indoor spraying or by promoting the large-scale distribution of LLINs. These strategies can effectively reduce the number of malaria cases [28], however the major challenge is the resistance of malaria vectors to different classes of insecticides and the shifts in their feeding and resting behaviours, with the tendency of biting and resting outdoors. For example, a study in the Cascades region in Burkina Faso indicate that in addition to insecticide resistance, more than 50% of the malaria vector biting were taking place outdoors. Therefore, new and supplementary methods are urgently needed to complement these tools in the perspective of malaria elimination [29]. In accordance with the spirit of locally adapted-integrated vector and disease control [30], repellents can usefully complement existing control strategies and provide an additional tool in the management of insecticide resistance. In the context of widespread resistant vectors to insecticide and the tendency of mosquitoes to bite outside houses, there is a need to add MAA ointment to vector control tools in sub-Saharan, malaria-burdened countries.\\nThe originality of MAA comes from its formulation based on local butter extensively used in rural areas of West African, and which contributes to womens economic income. Promotion of use of local endogen strategies can sustain malaria control, and also improve the economic situation for African women. Additionally, it has been shown that shea butter is a source of anti-inflammatory and anti-tumour promoting compounds [31]. Another interesting compound in shea butter is cinnamic acid which is known for its antibacterial, antifungal and antiviral properties [32]. Shea butter both moisturizes and heals the skin. Clinical studies have shown it to be safe for skin [33]. MAA ointment will not only protect from mosquito-borne diseases but also against other micro-organisms.\\nBesides the level of protection offered by repellents, daily compliance and appropriate use seem to be major obstacles to achieving the potential impact on malaria [34]. An efficacy study carried out in Tanzania has shown that volunteers preferred MAA ointment to a more classical 20% DEET solution [35]. Sales by Maa Africa SAS of 50,000 units in local stores in Burkina Faso between August and November 2021 further illustrate its desirability. However, more data are needed to understand who is likely to use the product and whether its usage is appropriate in terms of frequency and application in order to have an impact on mosquito-borne diseases, such as malaria.\",\n \"MAA, a novel ointment formulated with shea butter widely used in West Africa to moisturize the skin of children, has shown high repellency against laboratory-reared and wild malaria vectors. In the context of widespread vector resistance to insecticide and growing tendency of mosquitoes to bite outside houses, there is a need to add MAA ointment to the vector control tools used in sub-Saharan countries with high malaria burden.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,"results",null,null,null,null,null,"discussion","conclusion"],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n null,\n \"discussion\",\n \"conclusion\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["Malaria","Mosquito","\nAnopheles gambiae\n","\nAedes aegypti\n","Repellent","MAA","Burkina Faso"],"string":"[\n \"Malaria\",\n \"Mosquito\",\n \"\\nAnopheles gambiae\\n\",\n \"\\nAedes aegypti\\n\",\n \"Repellent\",\n \"MAA\",\n \"Burkina Faso\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Background:\nMalaria is one of the deadliest diseases in many low- and middle-income countries, affecting mainly children and pregnant women in sub-Saharan Africa [1]. Long-lasting insecticide-treated nets (LLINs) have been regarded as the most effective method for controlling mosquitoes transmitting malaria parasites. Since 2000, about one billion nets have been distributed in Africa, resulting in a significant decline in malaria-related deaths on the continent between 2000 and 2015 [24].\nHowever, the massive use of insecticides in public health in addition to that in agriculture causes concern regarding insecticide resistance [57] and changing behaviour [8, 9] of the malaria vectors. For example, a study conducted in Papua New Guinea showed a shift in mosquito biting from night to earlier hours in the evening after a nationwide distribution of LLINs [10]. Similar changes in the behaviour of Anopheles funestus have been observed in Benin and Senegal after LLIN distribution achieved a high level of coverage [9, 10]. Furthermore, studies suggest that the scaling up of LLIN distribution and indoor residual spraying (IRS) have led to more outdoor biting by Anopheles gambiae sensu lato (s.l.), commonly considered endophagic mosquitoes [1113]. A recent study in the Cascades region of Burkina Faso showed a high level of insecticide resistance [14] where more than 50% of the major vector, An. gambiae s.l., were collected biting outdoors [15]. These altered patterns of outdoors, early evening and morning biting, by anophelines, combined with resistance to insecticides appear to be caused by the mass distribution of LLINs and imply the inexorable loss of efficacy of these interventions [16, 17]. A recent study highlighted that an increase in early evening biting could increase transmission not only because people are unprotected by nets, but also because there is a higher chance of malaria vectors becoming infectious [18]. The development of new vector control tools, in addition to LLINs, is therefore necessary to protect people, when they are not under a bed net.\nTopical repellents could play an important role in addressing this problem if they are effective and accepted by the population. A systematic review of repellent interventions and mathematical modeling has shown that user compliance is indeed one of the most decisive factors for the success of this intervention [19]. In sub-Saharan Africa, ointments are used primarily by mothers and children to moisturize their skins. In Burkina Faso, ointments are applied to 80% of children every evening, when mosquitoes start biting (Kadidia Ouedraogo et al., in prep). Maa Africa, a company based in Burkina Faso, has developed a mosquito repellent ointment, MAA, uniquely designed with local mothers, to be used daily within their families. The underlying idea is to leverage the existing habits of mothers to protect their families from infectious bites whenever they are not under a net. Affordability is a key criterion for the products adoption and use. MAA has been industrially produced since June 2020 in Cte dIvoire and integrates a large share of ingredients sourced in West Africa. The ointment was officially launched in August 2020 in Burkina Faso and over 50,000 units were sold in the first four months. In March 2021, over 500 points of sales (mainly general stores, pharmacies and kiosks) distributed the product in the country. If MAA proves that it is both effective and accepted by the population, it could play a key role in reducing the probability of children experiencing infectious bites during the evening and be positioned as a complementary intervention to LLINs.\nThe aim of this study is to evaluate the effectiveness of MAA in both laboratory and field conditions, especially the median complete protection time (CPT) offered by the product. Results from these evaluations are important for validating how effective this new repellent is; behavioral responses to repellent differ between wild mosquito and laboratory-reared mosquito populations [20].\n\nMethods:\nStudy area Laboratory tests were conducted in May 2019 in the insectary of Centre National de Recherche et de Formation sur le Paludisme (CNRFP) in Ouagadougou, Burkina Faso. Field tests were carried out at Goden (1225N, 120W), a site located at 15km northeast of Ouagadougou, the capital city of Burkina Faso (Fig.1). Goden is a rural village with a Sudanian savanna climate and rainfall under 900 mm annually. The ~800 inhabitants mainly belong to the Mossi ethnic group, and are mostly devoted to agriculture and raising pigs, dogs, goats, and chickens within their compounds. LLINs were distributed in 2016 to ~90% of the population. Goden is known for its high density of malaria vectors due to its proximity to the Massili River. The field study was carried out during the rainy season (August to November 2019) corresponding to high vector density and high malaria transmission. A preliminary assessment of the mosquito density on the collection site was carried out using human landing catches (HLCs) before the tests started.\n\nFig. 1Study area\nStudy area\nLaboratory tests were conducted in May 2019 in the insectary of Centre National de Recherche et de Formation sur le Paludisme (CNRFP) in Ouagadougou, Burkina Faso. Field tests were carried out at Goden (1225N, 120W), a site located at 15km northeast of Ouagadougou, the capital city of Burkina Faso (Fig.1). Goden is a rural village with a Sudanian savanna climate and rainfall under 900 mm annually. The ~800 inhabitants mainly belong to the Mossi ethnic group, and are mostly devoted to agriculture and raising pigs, dogs, goats, and chickens within their compounds. LLINs were distributed in 2016 to ~90% of the population. Goden is known for its high density of malaria vectors due to its proximity to the Massili River. The field study was carried out during the rainy season (August to November 2019) corresponding to high vector density and high malaria transmission. A preliminary assessment of the mosquito density on the collection site was carried out using human landing catches (HLCs) before the tests started.\n\nFig. 1Study area\nStudy area\nHuman volunteer preparation Healthy adult male volunteers aged between 18 and 40 years were enrolled in this study. The volunteers were instructed not to use fragranced soaps, perfume, tobacco, or alcohol 12h before the start and throughout their participation. To establish the amount of repellent required for application, the surface area of the arm (for laboratory tests) or the leg (for field tests) of volunteers was determined using the following formula: Area= (Cw+ Ce) Dwe.\nHere Cw is the circumference of the wrist or ankle in cm, Ce is the elbow cubital fossa or the knee circumference in cm, and Dwe is the distance in cm between Ce and Cw [21]. The amount of ointment needed for each volunteer was determined depending on the area of their forearm or leg length. The quantity of product left in bottles was weighed using a precision weighing balance (KERN & SOHN GmbH, Balingen, Germany) to determine the amount applied by each volunteer.\nHealthy adult male volunteers aged between 18 and 40 years were enrolled in this study. The volunteers were instructed not to use fragranced soaps, perfume, tobacco, or alcohol 12h before the start and throughout their participation. To establish the amount of repellent required for application, the surface area of the arm (for laboratory tests) or the leg (for field tests) of volunteers was determined using the following formula: Area= (Cw+ Ce) Dwe.\nHere Cw is the circumference of the wrist or ankle in cm, Ce is the elbow cubital fossa or the knee circumference in cm, and Dwe is the distance in cm between Ce and Cw [21]. The amount of ointment needed for each volunteer was determined depending on the area of their forearm or leg length. The quantity of product left in bottles was weighed using a precision weighing balance (KERN & SOHN GmbH, Balingen, Germany) to determine the amount applied by each volunteer.\nRepellents MAA, a shea butter-based ointment containing 15% DEET (N, N-diethyl-3-methylbenzamide) was received from Maa Africa SAS. It was tested against an ethanolic solution of 20% DEET as positive control and a negative control of 70% ethanol. DEET is known as the standard repellent reference.\nMAA, a shea butter-based ointment containing 15% DEET (N, N-diethyl-3-methylbenzamide) was received from Maa Africa SAS. It was tested against an ethanolic solution of 20% DEET as positive control and a negative control of 70% ethanol. DEET is known as the standard repellent reference.\nStrains of mosquitoes Four strains of mosquitoes were used in the laboratory tests, including Kisumu F57 and Bora bora F58 susceptible strains of respectively An. gambiae and Aedes aegypti. In addition, local strains laboratory-colonized from rural areas of Goden, in Burkina Faso, were used: hereafter named An-Goden (An. gambiae local strain, F418) and Loc-Aedes (local Ae. aegypti, F318). These species were maintained under a 12:12h (light: dark) photoperiod. During rearing, larvae were fed on fish food while glucose was used for adults. The temperature and relative humidity in the rearing room were 2528C and 6080%, respectively. Individual mosquitoes used in these experiments were 5 to 10 days old nulliparous females starved from sugar solution for 12h before the experiment.\nFour strains of mosquitoes were used in the laboratory tests, including Kisumu F57 and Bora bora F58 susceptible strains of respectively An. gambiae and Aedes aegypti. In addition, local strains laboratory-colonized from rural areas of Goden, in Burkina Faso, were used: hereafter named An-Goden (An. gambiae local strain, F418) and Loc-Aedes (local Ae. aegypti, F318). These species were maintained under a 12:12h (light: dark) photoperiod. During rearing, larvae were fed on fish food while glucose was used for adults. The temperature and relative humidity in the rearing room were 2528C and 6080%, respectively. Individual mosquitoes used in these experiments were 5 to 10 days old nulliparous females starved from sugar solution for 12h before the experiment.\nEvaluation in the laboratory The laboratory experiments were conducted following WHO guidelines for the arm-in-cage test [21]. Cages were 454545cm screen enclosures. Two test cages were used, one for the repellent candidate and the other for the positive control. The test cages contained 200 females aged 5 to 10 days of one of the four mosquito strains: Kisumu, An-Goden, Bora bora and loc-Aedes. The experiment in the laboratory was carried out at temperatures ranging between 25 and 28C, with relative humidity between 60 and 80%.\nOverall, 2mg of ointment were applied per sq cm on the left forearm of each volunteer, a concentration estimated from the average of 5 volunteers from the laboratory asked to apply ointment on their left forearm as they would normally do in real life. In all subsequent repellent trials, volunteers were then supplied with a total volume that would achieve this concentration over the surface area of their forearms and/or legs (determined as described above). A steel spatula was used to apply the ointment on the forearm of each volunteer prior to each experiment to avoid absorbing ointment on non-targeted areas of skin. Positive control consisted of 1 ml of 20% DEET solution applied to the right forearms of volunteers.\nNegative controls and MAA test arms were prepared by first washing left forearms with odorless soap, drying and rinsing with 70% ethanol solution and then drying again. All volunteers wore latex gloves to protect their hands from mosquitoes. To assess the readiness of the mosquitoes to land, both left and right cleaned forearms of volunteers were exposed in the experimental cages for 30s (or until 10 landings of mosquito were counted). Then, for each volunteer, the right forearm was treated from wrist to elbow using 1 ml of the 20% DEET solution whilst the left forearm was treated from the wrist to elbow with MAA ointment. Thirty mins after application of the repellents, the volunteer exposed their treated forearm in the test cage for 3min. The procedure was repeated every 30min until the first bite occurred and the elapsed time to the first bite was recorded. The test was performed three times for each volunteer per mosquito species. Considering the difference in the relative periods of biting activity of each mosquito species, the tests using Ae. aegypti strains were carried out between 09:00 and 18:00, whereas those for An. gambiae were conducted between 17:00 and 05:00 [21].\nThe laboratory experiments were conducted following WHO guidelines for the arm-in-cage test [21]. Cages were 454545cm screen enclosures. Two test cages were used, one for the repellent candidate and the other for the positive control. The test cages contained 200 females aged 5 to 10 days of one of the four mosquito strains: Kisumu, An-Goden, Bora bora and loc-Aedes. The experiment in the laboratory was carried out at temperatures ranging between 25 and 28C, with relative humidity between 60 and 80%.\nOverall, 2mg of ointment were applied per sq cm on the left forearm of each volunteer, a concentration estimated from the average of 5 volunteers from the laboratory asked to apply ointment on their left forearm as they would normally do in real life. In all subsequent repellent trials, volunteers were then supplied with a total volume that would achieve this concentration over the surface area of their forearms and/or legs (determined as described above). A steel spatula was used to apply the ointment on the forearm of each volunteer prior to each experiment to avoid absorbing ointment on non-targeted areas of skin. Positive control consisted of 1 ml of 20% DEET solution applied to the right forearms of volunteers.\nNegative controls and MAA test arms were prepared by first washing left forearms with odorless soap, drying and rinsing with 70% ethanol solution and then drying again. All volunteers wore latex gloves to protect their hands from mosquitoes. To assess the readiness of the mosquitoes to land, both left and right cleaned forearms of volunteers were exposed in the experimental cages for 30s (or until 10 landings of mosquito were counted). Then, for each volunteer, the right forearm was treated from wrist to elbow using 1 ml of the 20% DEET solution whilst the left forearm was treated from the wrist to elbow with MAA ointment. Thirty mins after application of the repellents, the volunteer exposed their treated forearm in the test cage for 3min. The procedure was repeated every 30min until the first bite occurred and the elapsed time to the first bite was recorded. The test was performed three times for each volunteer per mosquito species. Considering the difference in the relative periods of biting activity of each mosquito species, the tests using Ae. aegypti strains were carried out between 09:00 and 18:00, whereas those for An. gambiae were conducted between 17:00 and 05:00 [21].\nField evaluation The lower legs of volunteers were washed with neutral soap, rinsed with 70% of ethanol solution and naturally dried. Once their legs were treated volunteers were asked to avoid rubbing, touching or wetting the repellent-treated area. Two mg of MAA per sq cm (2.40.2g per 118979.2 sq cm) and 2 ml per sq cm of 20% DEET (2 ml0.1 ml per 118979.2 sq cm, as a positive control) were applied to volunteers lower legs, from knee to ankle. A total of 20 volunteers were recruited from Goden village and trained for nocturnal mosquito collection using HLC. Each volunteer was later randomly allocated to one of the five groups (2 for MAIA, 2 for positive control, 1 for negative control) of four volunteers according to the repellent received. Each night of collection, the experiment took place at five different households, located at least 20m apart as per WHO guidelines [21], in order to avoid biases in attractiveness to the mosquitoes.\nMosquito collection started 30min following treatments. Volunteers acting as bait, sat on a chair in pairs (one indoor and one outdoor) and actively collected mosquitoes that landed on their treated lower leg using mouth aspirator and flash torch [22] for 45min, followed by a 15-mins break. Volunteers wore long-sleeved shirts, buttoned at the wrist, long trousers, closed shoes and latex gloves with a hat on their head, but with the treated lower leg to be exposed to mosquitoes by rolling up trousers to the knee. During these experiments, mosquitoes were collected simultaneously indoors and outdoors between 19:00 and 06:00. To avoid biases introduced by individual attractiveness and skills [23, 24] volunteers at the same household rotated between indoors and outdoors hourly. In each household two groups of two people rotated collecting from 18:00 to 24:00 and from 00:00 to 06:00, following the Williams balanced Latin Square design.\nCollected mosquitoes were transferred into plastic cups, covered with a piece of untreated net, with a small hole at the bottom to allow mosquitoes to be easily aspirated into them. After collection mosquitoes were brought to the entomological laboratory of CNRFP and morphologically identified using a stereo microscope and identification keys [25].\nThe lower legs of volunteers were washed with neutral soap, rinsed with 70% of ethanol solution and naturally dried. Once their legs were treated volunteers were asked to avoid rubbing, touching or wetting the repellent-treated area. Two mg of MAA per sq cm (2.40.2g per 118979.2 sq cm) and 2 ml per sq cm of 20% DEET (2 ml0.1 ml per 118979.2 sq cm, as a positive control) were applied to volunteers lower legs, from knee to ankle. A total of 20 volunteers were recruited from Goden village and trained for nocturnal mosquito collection using HLC. Each volunteer was later randomly allocated to one of the five groups (2 for MAIA, 2 for positive control, 1 for negative control) of four volunteers according to the repellent received. Each night of collection, the experiment took place at five different households, located at least 20m apart as per WHO guidelines [21], in order to avoid biases in attractiveness to the mosquitoes.\nMosquito collection started 30min following treatments. Volunteers acting as bait, sat on a chair in pairs (one indoor and one outdoor) and actively collected mosquitoes that landed on their treated lower leg using mouth aspirator and flash torch [22] for 45min, followed by a 15-mins break. Volunteers wore long-sleeved shirts, buttoned at the wrist, long trousers, closed shoes and latex gloves with a hat on their head, but with the treated lower leg to be exposed to mosquitoes by rolling up trousers to the knee. During these experiments, mosquitoes were collected simultaneously indoors and outdoors between 19:00 and 06:00. To avoid biases introduced by individual attractiveness and skills [23, 24] volunteers at the same household rotated between indoors and outdoors hourly. In each household two groups of two people rotated collecting from 18:00 to 24:00 and from 00:00 to 06:00, following the Williams balanced Latin Square design.\nCollected mosquitoes were transferred into plastic cups, covered with a piece of untreated net, with a small hole at the bottom to allow mosquitoes to be easily aspirated into them. After collection mosquitoes were brought to the entomological laboratory of CNRFP and morphologically identified using a stereo microscope and identification keys [25].\nSide effects No side effects were observed or reported by any of the volunteers throughout the period of tests both in the laboratory and in the field.\nNo side effects were observed or reported by any of the volunteers throughout the period of tests both in the laboratory and in the field.\nEthical clearance Written informed consent was obtained from all volunteers and household owners recruited in this study. The study was approved by the institutional ethic committee of CNRFP under 2019/000008/MS/SG/CNRFP/CIB.\nWritten informed consent was obtained from all volunteers and household owners recruited in this study. The study was approved by the institutional ethic committee of CNRFP under 2019/000008/MS/SG/CNRFP/CIB.\nData analysis All data were collected on standard forms and entered twice in a database by different people. Databases have been compared using Epi Info 3.5.3, and inconsistencies were verified using printed and corrected forms. The performance of the repellent was measured by calculating the repulsive efficiency and the median full protection time. A generalized linear mixed model (GLMM) was used to further analyse the effect of the location (indoors vs. outdoors) on the performance of the treatments. Variation in the average number of bites received between treatments was also assessed.\nThe median CPT is defined as the interval of time between the beginning of collection/test and the first mosquito landing. To estimate the median CPT of each treatment, a Kaplan-Meier survival analysis was performed for each vector species and strain used in the laboratory experiments and on field data through survival function from R software-version 3.5.0 (2018-04-23). However, for the field test, the analysis was performed on only An. gambiae s.l. as it was the most abundant species collected (~96% of the total collection). The analysis consisted of assessing the median CPT and the repulsive efficacy. The repulsive efficacy was calculated as a percentage of repulsion (% R) according to the formula % R = ((C T) / C) 100, where C is the number of mosquitoes collected on the treated legs of the two control treatments separately, and T is the total number of mosquito bite attempts on the volunteers legs treated with the test product [21].\nAll data were collected on standard forms and entered twice in a database by different people. Databases have been compared using Epi Info 3.5.3, and inconsistencies were verified using printed and corrected forms. The performance of the repellent was measured by calculating the repulsive efficiency and the median full protection time. A generalized linear mixed model (GLMM) was used to further analyse the effect of the location (indoors vs. outdoors) on the performance of the treatments. Variation in the average number of bites received between treatments was also assessed.\nThe median CPT is defined as the interval of time between the beginning of collection/test and the first mosquito landing. To estimate the median CPT of each treatment, a Kaplan-Meier survival analysis was performed for each vector species and strain used in the laboratory experiments and on field data through survival function from R software-version 3.5.0 (2018-04-23). However, for the field test, the analysis was performed on only An. gambiae s.l. as it was the most abundant species collected (~96% of the total collection). The analysis consisted of assessing the median CPT and the repulsive efficacy. The repulsive efficacy was calculated as a percentage of repulsion (% R) according to the formula % R = ((C T) / C) 100, where C is the number of mosquitoes collected on the treated legs of the two control treatments separately, and T is the total number of mosquito bite attempts on the volunteers legs treated with the test product [21].\n\nStudy area:\nLaboratory tests were conducted in May 2019 in the insectary of Centre National de Recherche et de Formation sur le Paludisme (CNRFP) in Ouagadougou, Burkina Faso. Field tests were carried out at Goden (1225N, 120W), a site located at 15km northeast of Ouagadougou, the capital city of Burkina Faso (Fig.1). Goden is a rural village with a Sudanian savanna climate and rainfall under 900 mm annually. The ~800 inhabitants mainly belong to the Mossi ethnic group, and are mostly devoted to agriculture and raising pigs, dogs, goats, and chickens within their compounds. LLINs were distributed in 2016 to ~90% of the population. Goden is known for its high density of malaria vectors due to its proximity to the Massili River. The field study was carried out during the rainy season (August to November 2019) corresponding to high vector density and high malaria transmission. A preliminary assessment of the mosquito density on the collection site was carried out using human landing catches (HLCs) before the tests started.\n\nFig. 1Study area\nStudy area\n\nHuman volunteer preparation:\nHealthy adult male volunteers aged between 18 and 40 years were enrolled in this study. The volunteers were instructed not to use fragranced soaps, perfume, tobacco, or alcohol 12h before the start and throughout their participation. To establish the amount of repellent required for application, the surface area of the arm (for laboratory tests) or the leg (for field tests) of volunteers was determined using the following formula: Area= (Cw+ Ce) Dwe.\nHere Cw is the circumference of the wrist or ankle in cm, Ce is the elbow cubital fossa or the knee circumference in cm, and Dwe is the distance in cm between Ce and Cw [21]. The amount of ointment needed for each volunteer was determined depending on the area of their forearm or leg length. The quantity of product left in bottles was weighed using a precision weighing balance (KERN & SOHN GmbH, Balingen, Germany) to determine the amount applied by each volunteer.\n\nRepellents:\nMAA, a shea butter-based ointment containing 15% DEET (N, N-diethyl-3-methylbenzamide) was received from Maa Africa SAS. It was tested against an ethanolic solution of 20% DEET as positive control and a negative control of 70% ethanol. DEET is known as the standard repellent reference.\n\nStrains of mosquitoes:\nFour strains of mosquitoes were used in the laboratory tests, including Kisumu F57 and Bora bora F58 susceptible strains of respectively An. gambiae and Aedes aegypti. In addition, local strains laboratory-colonized from rural areas of Goden, in Burkina Faso, were used: hereafter named An-Goden (An. gambiae local strain, F418) and Loc-Aedes (local Ae. aegypti, F318). These species were maintained under a 12:12h (light: dark) photoperiod. During rearing, larvae were fed on fish food while glucose was used for adults. The temperature and relative humidity in the rearing room were 2528C and 6080%, respectively. Individual mosquitoes used in these experiments were 5 to 10 days old nulliparous females starved from sugar solution for 12h before the experiment.\n\nEvaluation in the laboratory:\nThe laboratory experiments were conducted following WHO guidelines for the arm-in-cage test [21]. Cages were 454545cm screen enclosures. Two test cages were used, one for the repellent candidate and the other for the positive control. The test cages contained 200 females aged 5 to 10 days of one of the four mosquito strains: Kisumu, An-Goden, Bora bora and loc-Aedes. The experiment in the laboratory was carried out at temperatures ranging between 25 and 28C, with relative humidity between 60 and 80%.\nOverall, 2mg of ointment were applied per sq cm on the left forearm of each volunteer, a concentration estimated from the average of 5 volunteers from the laboratory asked to apply ointment on their left forearm as they would normally do in real life. In all subsequent repellent trials, volunteers were then supplied with a total volume that would achieve this concentration over the surface area of their forearms and/or legs (determined as described above). A steel spatula was used to apply the ointment on the forearm of each volunteer prior to each experiment to avoid absorbing ointment on non-targeted areas of skin. Positive control consisted of 1 ml of 20% DEET solution applied to the right forearms of volunteers.\nNegative controls and MAA test arms were prepared by first washing left forearms with odorless soap, drying and rinsing with 70% ethanol solution and then drying again. All volunteers wore latex gloves to protect their hands from mosquitoes. To assess the readiness of the mosquitoes to land, both left and right cleaned forearms of volunteers were exposed in the experimental cages for 30s (or until 10 landings of mosquito were counted). Then, for each volunteer, the right forearm was treated from wrist to elbow using 1 ml of the 20% DEET solution whilst the left forearm was treated from the wrist to elbow with MAA ointment. Thirty mins after application of the repellents, the volunteer exposed their treated forearm in the test cage for 3min. The procedure was repeated every 30min until the first bite occurred and the elapsed time to the first bite was recorded. The test was performed three times for each volunteer per mosquito species. Considering the difference in the relative periods of biting activity of each mosquito species, the tests using Ae. aegypti strains were carried out between 09:00 and 18:00, whereas those for An. gambiae were conducted between 17:00 and 05:00 [21].\n\nField evaluation:\nThe lower legs of volunteers were washed with neutral soap, rinsed with 70% of ethanol solution and naturally dried. Once their legs were treated volunteers were asked to avoid rubbing, touching or wetting the repellent-treated area. Two mg of MAA per sq cm (2.40.2g per 118979.2 sq cm) and 2 ml per sq cm of 20% DEET (2 ml0.1 ml per 118979.2 sq cm, as a positive control) were applied to volunteers lower legs, from knee to ankle. A total of 20 volunteers were recruited from Goden village and trained for nocturnal mosquito collection using HLC. Each volunteer was later randomly allocated to one of the five groups (2 for MAIA, 2 for positive control, 1 for negative control) of four volunteers according to the repellent received. Each night of collection, the experiment took place at five different households, located at least 20m apart as per WHO guidelines [21], in order to avoid biases in attractiveness to the mosquitoes.\nMosquito collection started 30min following treatments. Volunteers acting as bait, sat on a chair in pairs (one indoor and one outdoor) and actively collected mosquitoes that landed on their treated lower leg using mouth aspirator and flash torch [22] for 45min, followed by a 15-mins break. Volunteers wore long-sleeved shirts, buttoned at the wrist, long trousers, closed shoes and latex gloves with a hat on their head, but with the treated lower leg to be exposed to mosquitoes by rolling up trousers to the knee. During these experiments, mosquitoes were collected simultaneously indoors and outdoors between 19:00 and 06:00. To avoid biases introduced by individual attractiveness and skills [23, 24] volunteers at the same household rotated between indoors and outdoors hourly. In each household two groups of two people rotated collecting from 18:00 to 24:00 and from 00:00 to 06:00, following the Williams balanced Latin Square design.\nCollected mosquitoes were transferred into plastic cups, covered with a piece of untreated net, with a small hole at the bottom to allow mosquitoes to be easily aspirated into them. After collection mosquitoes were brought to the entomological laboratory of CNRFP and morphologically identified using a stereo microscope and identification keys [25].\n\nSide effects:\nNo side effects were observed or reported by any of the volunteers throughout the period of tests both in the laboratory and in the field.\n\nEthical clearance:\nWritten informed consent was obtained from all volunteers and household owners recruited in this study. The study was approved by the institutional ethic committee of CNRFP under 2019/000008/MS/SG/CNRFP/CIB.\n\nData analysis:\nAll data were collected on standard forms and entered twice in a database by different people. Databases have been compared using Epi Info 3.5.3, and inconsistencies were verified using printed and corrected forms. The performance of the repellent was measured by calculating the repulsive efficiency and the median full protection time. A generalized linear mixed model (GLMM) was used to further analyse the effect of the location (indoors vs. outdoors) on the performance of the treatments. Variation in the average number of bites received between treatments was also assessed.\nThe median CPT is defined as the interval of time between the beginning of collection/test and the first mosquito landing. To estimate the median CPT of each treatment, a Kaplan-Meier survival analysis was performed for each vector species and strain used in the laboratory experiments and on field data through survival function from R software-version 3.5.0 (2018-04-23). However, for the field test, the analysis was performed on only An. gambiae s.l. as it was the most abundant species collected (~96% of the total collection). The analysis consisted of assessing the median CPT and the repulsive efficacy. The repulsive efficacy was calculated as a percentage of repulsion (% R) according to the formula % R = ((C T) / C) 100, where C is the number of mosquitoes collected on the treated legs of the two control treatments separately, and T is the total number of mosquito bite attempts on the volunteers legs treated with the test product [21].\n\nResults:\nLaboratory tests Overall, under laboratory conditions the relative repellency (median CPT) was higher for both MAA and 20% DEET against An. gambiae compare to Ae. aegypti (Table1). MAA performed well in repelling the four mosquito species used in this study. The median CPTs were, respectively, 6.5h for An. gambiae (Kisumu), 5.5h for An. gambiae (Goden, local strain) and 4h for Ae. aegypti for both the local and sensitive strain. There was no significant difference between the two treatments for each of the experiment (Kisumu: 2=2.1, p value=0.14; Goden: 2=0.8, p value=0.36; Bora bora: 2=1.7, p value=0.19; Ae. aegypti (local strain): 2=0.9, p value=0. 35) indicating that both MAA and 20% DEET have equal repellency for these strains. The Kaplan-Meier curves for MAA and 20% DEET, respectively, for Kisumu, An-Goden, Bora bora and Loc-Aedes are shown in Fig.2.\n\nTable 1\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \nAn. gambiae\nKisumu\nAn. gambiae\nGoden\nAe. aegypti\nBora bora\nAe. aegypti\nLocalDEETMAADEETMAADEETMAADEETMAAMedian. CPT390390300330270240240240Lower CI368334272216252239212225Upper CI412446328444288241268255Fig. 2Kaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\n\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \nKaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\nOverall, under laboratory conditions the relative repellency (median CPT) was higher for both MAA and 20% DEET against An. gambiae compare to Ae. aegypti (Table1). MAA performed well in repelling the four mosquito species used in this study. The median CPTs were, respectively, 6.5h for An. gambiae (Kisumu), 5.5h for An. gambiae (Goden, local strain) and 4h for Ae. aegypti for both the local and sensitive strain. There was no significant difference between the two treatments for each of the experiment (Kisumu: 2=2.1, p value=0.14; Goden: 2=0.8, p value=0.36; Bora bora: 2=1.7, p value=0.19; Ae. aegypti (local strain): 2=0.9, p value=0. 35) indicating that both MAA and 20% DEET have equal repellency for these strains. The Kaplan-Meier curves for MAA and 20% DEET, respectively, for Kisumu, An-Goden, Bora bora and Loc-Aedes are shown in Fig.2.\n\nTable 1\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \nAn. gambiae\nKisumu\nAn. gambiae\nGoden\nAe. aegypti\nBora bora\nAe. aegypti\nLocalDEETMAADEETMAADEETMAADEETMAAMedian. CPT390390300330270240240240Lower CI368334272216252239212225Upper CI412446328444288241268255Fig. 2Kaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\n\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \nKaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\nField test Mosquito species composition and biting behaviours A total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\nAnopheles gambiae sensu lato (s.l.)\n6862660480\nAnopheles funestus\n121Other Anopheles193Culicines710623\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\nA total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\nAnopheles gambiae sensu lato (s.l.)\n6862660480\nAnopheles funestus\n121Other Anopheles193Culicines710623\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\nRepellency against mosquitoes Repellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\nRepellency of 20% DEET and MAA indoor and outdoor collection\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\nRepellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\nRepellency of 20% DEET and MAA indoor and outdoor collection\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\nComplete protection time The overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nAnopheles gambiae s.l.\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nAnopheles gambiae s.l\n\nIndoors \nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nThe overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nAnopheles gambiae s.l.\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nAnopheles gambiae s.l\n\nIndoors \nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nMosquito species composition and biting behaviours A total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\nAnopheles gambiae sensu lato (s.l.)\n6862660480\nAnopheles funestus\n121Other Anopheles193Culicines710623\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\nA total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\nAnopheles gambiae sensu lato (s.l.)\n6862660480\nAnopheles funestus\n121Other Anopheles193Culicines710623\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\nRepellency against mosquitoes Repellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\nRepellency of 20% DEET and MAA indoor and outdoor collection\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\nRepellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\nRepellency of 20% DEET and MAA indoor and outdoor collection\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\nComplete protection time The overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nAnopheles gambiae s.l.\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nAnopheles gambiae s.l\n\nIndoors \nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nThe overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nAnopheles gambiae s.l.\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nAnopheles gambiae s.l\n\nIndoors \nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\n\nLaboratory tests:\nOverall, under laboratory conditions the relative repellency (median CPT) was higher for both MAA and 20% DEET against An. gambiae compare to Ae. aegypti (Table1). MAA performed well in repelling the four mosquito species used in this study. The median CPTs were, respectively, 6.5h for An. gambiae (Kisumu), 5.5h for An. gambiae (Goden, local strain) and 4h for Ae. aegypti for both the local and sensitive strain. There was no significant difference between the two treatments for each of the experiment (Kisumu: 2=2.1, p value=0.14; Goden: 2=0.8, p value=0.36; Bora bora: 2=1.7, p value=0.19; Ae. aegypti (local strain): 2=0.9, p value=0. 35) indicating that both MAA and 20% DEET have equal repellency for these strains. The Kaplan-Meier curves for MAA and 20% DEET, respectively, for Kisumu, An-Goden, Bora bora and Loc-Aedes are shown in Fig.2.\n\nTable 1\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \nAn. gambiae\nKisumu\nAn. gambiae\nGoden\nAe. aegypti\nBora bora\nAe. aegypti\nLocalDEETMAADEETMAADEETMAADEETMAAMedian. CPT390390300330270240240240Lower CI368334272216252239212225Upper CI412446328444288241268255Fig. 2Kaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\n\nMedian complete protection times (CPT) in minutes and their 95% confidence intervals (CI) against mosquito strains, according to treatments 20% DEET and MAA, under laboratory conditions \nKaplan-Meier plots for 20% DEET and MAA tested against the four species on five volunteers\n\nField test:\nMosquito species composition and biting behaviours A total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\nAnopheles gambiae sensu lato (s.l.)\n6862660480\nAnopheles funestus\n121Other Anopheles193Culicines710623\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\nA total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\nAnopheles gambiae sensu lato (s.l.)\n6862660480\nAnopheles funestus\n121Other Anopheles193Culicines710623\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\nRepellency against mosquitoes Repellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\nRepellency of 20% DEET and MAA indoor and outdoor collection\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\nRepellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\nRepellency of 20% DEET and MAA indoor and outdoor collection\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\nComplete protection time The overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nAnopheles gambiae s.l.\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nAnopheles gambiae s.l\n\nIndoors \nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nThe overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nAnopheles gambiae s.l.\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nAnopheles gambiae s.l\n\nIndoors \nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\n\nMosquito species composition and biting behaviours:\nA total of 3,979 mosquitoes, stratified by treatment and species (Table2), were caught using HLC. Anophelines represented 98.5% of the total catch, with culicines (Aedes) making up the remaining 1.5%. Among anopheline species, 99.6% belonged to An. gambiae complex, followed by An. funestus (0.1%), and Anopheles pharoensis (0.3%). The frequency of mosquitoes landing on treated collectors, compared with control subjects, varied according to the repellent used (Table2). The hourly mosquito biting rate varied significantly between treatments (df=2, 2=426.22, p<0.0001). An average of 0.68 (95% CI: 0.510.91) mosquito bites were received per person per hour for MAA compared to 1.01 (95% CI: 0.761.33) for 20% DEET, and 8.98 (95% CI: 6.5612.29) for the 70% ethanol. In addition, there was no variation between treatments according to location (outdoors and indoors, df=2, 2=1.703, p=0.42). Overall, the ratio outdoors:indoors biting was 1.26 (95% CI: 1.251.27) showing that more bites were taking place outdoors compared to indoors (df=1, 2=5.79, p=0.016).Table 2Total number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%Mosquito speciesTreatmentDEETEthanolMAA\nAnopheles gambiae sensu lato (s.l.)\n6862660480\nAnopheles funestus\n121Other Anopheles193Culicines710623\nTotal number of common mosquitoes collected after treatment of 20% DEET, MAA and ethanol 70%\n\nRepellency against mosquitoes:\nRepellency against An. gambiae s.l. was stratified by time of collection. From 18:00 to 24:00 (6h after application), the percentage of repellency varied from 100 to 90% for MAA and DEET. Between 00:00 to 03:00 (9h after application), the percentage was between 90 and 80% (Fig.3). After 03:00 (10h after application), this percentage was under 80% for 20% DEET, but MAA was over 80%. MAAgave a high percentage of repellency thoughout, however during the first 9h after applications no difference in the repellency was observed between MAA and the positive control. Fig. 3Repellency of 20% DEET and MAA indoor and outdoor collection\nRepellency of 20% DEET and MAA indoor and outdoor collection\nWhen data were stratified by location of mosquito biting, the trend was the same for indoors and outdoors. No difference was observed during the first 9h between MAA and 20% DEET. These results show that MAA can protect both indoors and outdoors.\n\nComplete protection time:\nThe overall median CPTs of 20% DEET and MAA (Table3) were estimated at 480min (8h) against 120min (2h) for the negative control. For outdoor collections, the median CPTs were 480, 450 and 120min for 20% DEET, MAA and negative control respectively (Table4). For indoor collections, these estimates were 480, 480 and 60min, respectively, for 20% DEET, MAA and ethanol (Table4). Statistical analyses showed that there was no difference in the median CPT between 20% DEET and MAA (df=1, 2=0.2, p=0.7). However, there was a significant difference between median CPT as estimated for MAA and the negative control (df=2, 2=106, p<0.0001, Fig.4). Even when the collection was stratified by location this difference still occurred in both indoor (Fig.5A; df=2, 2=41.6, p<0.0001) and outdoor collections; df=2, 2=66.7, p<0.0001) Fig.5B). Table 3Estimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nAnopheles gambiae s.l.\nDEETEthanolMAAMedian CPT480120480Lower CI45491448Upper CI506149512Table 4The estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nAnopheles gambiae s.l\n\nIndoors \nOutdoors 20% DEETEthanolMAA20% DEETEthanolMAAMedian CPT48060480480120450Lower CI440<6044044795428Upper CI521NA521513145472Fig. 4Overall estimated probabilities of no mosquitoes landing for each treatment according to time of collectionsFig. 5Estimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\nEstimated complete protection time (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAAand ethanol 70%\nThe estimated complete protection times (mins) with 95% CI, against Anopheles gambiae s.l. for 20% DEET, MAA and ethanol 70%, indoors and outdoors\nOverall estimated probabilities of no mosquitoes landing for each treatment according to time of collections\nEstimated probabilities of no mosquitoes landing for each treatment according to the time at indoor (A) and outdoor locations (B)\n\nDiscussion:\nThe results of this study demonstrated that MAA, a shea butter-based ointment with 15% DEET provides high protection against mosquitoes in Goden, a rural area of Burkina Faso. Both tests under field and laboratory conditions suggested that MAA has equal repellency effect as 20% DEET ethanolic solution during the period of collection. Similar results were also found both indoors and outdoors. The percentage of repellency when using MAA varied between 100 and 80% over the major malaria vector biting period, which occurs between 18:00 and 06:00. The median CPT were also similar and estimated around 480min. Both MAA and the 20% DEET ethanolic solution were found to provide up to 90% of repellency during the first 6h after their applications. Furthermore, results suggested that using MAA, the average hourly bites received was significantly lower (less than 1 bite per hour) compared to that of both 20% DEET and negative control. However, one of the limitations is that biting activity on control arms was not checked at the end of laboratory experiments to validate consistency of mosquito avidity, but this did not affect the overall result. Overall, it can be argued that MAA could provide protection to people before they go to bed.\nPrevious studies in the same locality comparing repellency between three different repellents found that DEET, IR3535 and KBR 3023 were effective against An. gambiae s.l. and other Afrotropical vector mosquitoes [26]. In this study authors showed that protection from KBR 3023, DEET and IR3535 were still high against anophelines for up to 10h post-exposure. In contrast, results from the current study indicated that the relative repellency was 100% for ~8h. Results were similar to that from a recent study in Ethiopia comparing DEET (N, N-diethyl-1,3-methylbenzamide) and MyggA (p-methane diol) and other laboratory products (20% neem oil and 20% chinaberry oil), where the mean CPT was 8h for DEET whilst an estimated 6h for MyggA [27]. Eight hours of repellency may suffice to protect against earlier vector biting both indoors and outdoors before residents take protection from insecticide-treated nets deployed indoors.\nTo date, 11 countries across the world are classified as having a high burden of malaria (2). In these countries malaria vector control is still based on the use of insecticides, either in the form of indoor spraying or by promoting the large-scale distribution of LLINs. These strategies can effectively reduce the number of malaria cases [28], however the major challenge is the resistance of malaria vectors to different classes of insecticides and the shifts in their feeding and resting behaviours, with the tendency of biting and resting outdoors. For example, a study in the Cascades region in Burkina Faso indicate that in addition to insecticide resistance, more than 50% of the malaria vector biting were taking place outdoors. Therefore, new and supplementary methods are urgently needed to complement these tools in the perspective of malaria elimination [29]. In accordance with the spirit of locally adapted-integrated vector and disease control [30], repellents can usefully complement existing control strategies and provide an additional tool in the management of insecticide resistance. In the context of widespread resistant vectors to insecticide and the tendency of mosquitoes to bite outside houses, there is a need to add MAA ointment to vector control tools in sub-Saharan, malaria-burdened countries.\nThe originality of MAA comes from its formulation based on local butter extensively used in rural areas of West African, and which contributes to womens economic income. Promotion of use of local endogen strategies can sustain malaria control, and also improve the economic situation for African women. Additionally, it has been shown that shea butter is a source of anti-inflammatory and anti-tumour promoting compounds [31]. Another interesting compound in shea butter is cinnamic acid which is known for its antibacterial, antifungal and antiviral properties [32]. Shea butter both moisturizes and heals the skin. Clinical studies have shown it to be safe for skin [33]. MAA ointment will not only protect from mosquito-borne diseases but also against other micro-organisms.\nBesides the level of protection offered by repellents, daily compliance and appropriate use seem to be major obstacles to achieving the potential impact on malaria [34]. An efficacy study carried out in Tanzania has shown that volunteers preferred MAA ointment to a more classical 20% DEET solution [35]. Sales by Maa Africa SAS of 50,000 units in local stores in Burkina Faso between August and November 2021 further illustrate its desirability. However, more data are needed to understand who is likely to use the product and whether its usage is appropriate in terms of frequency and application in order to have an impact on mosquito-borne diseases, such as malaria.\n\nConclusions:\nMAA, a novel ointment formulated with shea butter widely used in West Africa to moisturize the skin of children, has shown high repellency against laboratory-reared and wild malaria vectors. In the context of widespread vector resistance to insecticide and growing tendency of mosquitoes to bite outside houses, there is a need to add MAA ointment to the vector control tools used in sub-Saharan countries with high malaria burden.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nMalaria vector control relies upon the use of insecticide-treated nets and indoor residual spraying. However, as the emergency of insecticide resistance in malaria vectors grows, the effectiveness of these measures could be limited. Alternative tools are needed. In this context, repellents can play an important role against exophagic and exophilic mosquitoes. This study evaluated the efficacy of MAÏA®, a novel repellent ointment, in laboratory and field conditions in Burkina Faso.\n\nMethods:\nFor laboratory and field assessment, 20 volunteers were enrolled and trained for nocturnal collection of mosquitoes using human landing catches (HLC). In the laboratory tests, 2 mg/sq cm of treatment (either MAIA® or 20 % DEET) were used to assess median complete protection time (CPT) against two species: Anopheles gambiae and Aedes aegypti, following WHO guidelines. For both species, two strains consisting of susceptible and local strains were used. The susceptible strains were Kisumu and Bora Bora for An. gambiae and Ae. aegypti, respectively. For the field test, the median CPT of MAÏA® was compared to that of a negative (70 % ethanol) and positive (20 % DEET) after carrying out HLCs in rural Burkina Faso in both indoor and outdoor settings.\n\nResults:\nLaboratory tests showed median Kaplan-Meier CPT of 6 h 30 min for An. gambiae (Kisumu), 5 h 30 min for An. gambiae (Goden, local strain), and 4 h for Ae. aegypti for both the local and sensitive strain. These laboratory results suggest that MAÏA® is a good repellent against the three mosquito species. During these field tests, a total of 3979 mosquitoes were caught. In this population, anophelines represented 98.5 %, with culicines (Aedes) making up the remaining 1.5 %. Among anopheline mosquitoes, 95 % belonged to the An. gambiae complex, followed by Anopheles funestus and Anopheles pharoensis. The median CPT of 20 % DEET and MAÏA® were similar (8 h) and much longer than that of the negative control (2 h).\n\nConclusions:\nResults from the present studies showed that MAÏA® offers high protection against anophelines biting indoors and outdoors and could play an important role in malaria prevention in Africa."},"background_conclusion_text":{"kind":"string","value":"Background:\nMalaria is one of the deadliest diseases in many low- and middle-income countries, affecting mainly children and pregnant women in sub-Saharan Africa [1]. Long-lasting insecticide-treated nets (LLINs) have been regarded as the most effective method for controlling mosquitoes transmitting malaria parasites. Since 2000, about one billion nets have been distributed in Africa, resulting in a significant decline in malaria-related deaths on the continent between 2000 and 2015 [24].\nHowever, the massive use of insecticides in public health in addition to that in agriculture causes concern regarding insecticide resistance [57] and changing behaviour [8, 9] of the malaria vectors. For example, a study conducted in Papua New Guinea showed a shift in mosquito biting from night to earlier hours in the evening after a nationwide distribution of LLINs [10]. Similar changes in the behaviour of Anopheles funestus have been observed in Benin and Senegal after LLIN distribution achieved a high level of coverage [9, 10]. Furthermore, studies suggest that the scaling up of LLIN distribution and indoor residual spraying (IRS) have led to more outdoor biting by Anopheles gambiae sensu lato (s.l.), commonly considered endophagic mosquitoes [1113]. A recent study in the Cascades region of Burkina Faso showed a high level of insecticide resistance [14] where more than 50% of the major vector, An. gambiae s.l., were collected biting outdoors [15]. These altered patterns of outdoors, early evening and morning biting, by anophelines, combined with resistance to insecticides appear to be caused by the mass distribution of LLINs and imply the inexorable loss of efficacy of these interventions [16, 17]. A recent study highlighted that an increase in early evening biting could increase transmission not only because people are unprotected by nets, but also because there is a higher chance of malaria vectors becoming infectious [18]. The development of new vector control tools, in addition to LLINs, is therefore necessary to protect people, when they are not under a bed net.\nTopical repellents could play an important role in addressing this problem if they are effective and accepted by the population. A systematic review of repellent interventions and mathematical modeling has shown that user compliance is indeed one of the most decisive factors for the success of this intervention [19]. In sub-Saharan Africa, ointments are used primarily by mothers and children to moisturize their skins. In Burkina Faso, ointments are applied to 80% of children every evening, when mosquitoes start biting (Kadidia Ouedraogo et al., in prep). Maa Africa, a company based in Burkina Faso, has developed a mosquito repellent ointment, MAA, uniquely designed with local mothers, to be used daily within their families. The underlying idea is to leverage the existing habits of mothers to protect their families from infectious bites whenever they are not under a net. Affordability is a key criterion for the products adoption and use. MAA has been industrially produced since June 2020 in Cte dIvoire and integrates a large share of ingredients sourced in West Africa. The ointment was officially launched in August 2020 in Burkina Faso and over 50,000 units were sold in the first four months. In March 2021, over 500 points of sales (mainly general stores, pharmacies and kiosks) distributed the product in the country. If MAA proves that it is both effective and accepted by the population, it could play a key role in reducing the probability of children experiencing infectious bites during the evening and be positioned as a complementary intervention to LLINs.\nThe aim of this study is to evaluate the effectiveness of MAA in both laboratory and field conditions, especially the median complete protection time (CPT) offered by the product. Results from these evaluations are important for validating how effective this new repellent is; behavioral responses to repellent differ between wild mosquito and laboratory-reared mosquito populations [20].\n\nConclusions:\nMAA, a novel ointment formulated with shea butter widely used in West Africa to moisturize the skin of children, has shown high repellency against laboratory-reared and wild malaria vectors. In the context of widespread vector resistance to insecticide and growing tendency of mosquitoes to bite outside houses, there is a need to add MAA ointment to the vector control tools used in sub-Saharan countries with high malaria burden."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nMalaria vector control relies upon the use of insecticide-treated nets and indoor residual spraying. However, as the emergency of insecticide resistance in malaria vectors grows, the effectiveness of these measures could be limited. Alternative tools are needed. In this context, repellents can play an important role against exophagic and exophilic mosquitoes. This study evaluated the efficacy of MAÏA®, a novel repellent ointment, in laboratory and field conditions in Burkina Faso.\n\nMethods:\nFor laboratory and field assessment, 20 volunteers were enrolled and trained for nocturnal collection of mosquitoes using human landing catches (HLC). In the laboratory tests, 2 mg/sq cm of treatment (either MAIA® or 20 % DEET) were used to assess median complete protection time (CPT) against two species: Anopheles gambiae and Aedes aegypti, following WHO guidelines. For both species, two strains consisting of susceptible and local strains were used. The susceptible strains were Kisumu and Bora Bora for An. gambiae and Ae. aegypti, respectively. For the field test, the median CPT of MAÏA® was compared to that of a negative (70 % ethanol) and positive (20 % DEET) after carrying out HLCs in rural Burkina Faso in both indoor and outdoor settings.\n\nResults:\nLaboratory tests showed median Kaplan-Meier CPT of 6 h 30 min for An. gambiae (Kisumu), 5 h 30 min for An. gambiae (Goden, local strain), and 4 h for Ae. aegypti for both the local and sensitive strain. These laboratory results suggest that MAÏA® is a good repellent against the three mosquito species. During these field tests, a total of 3979 mosquitoes were caught. In this population, anophelines represented 98.5 %, with culicines (Aedes) making up the remaining 1.5 %. Among anopheline mosquitoes, 95 % belonged to the An. gambiae complex, followed by Anopheles funestus and Anopheles pharoensis. The median CPT of 20 % DEET and MAÏA® were similar (8 h) and much longer than that of the negative control (2 h).\n\nConclusions:\nResults from the present studies showed that MAÏA® offers high protection against anophelines biting indoors and outdoors and could play an important role in malaria prevention in Africa."},"whole_article_text_length":{"kind":"number","value":14461,"string":"14,461"},"whole_article_abstract_length":{"kind":"number","value":447,"string":"447"},"other_sections_lengths":{"kind":"list like","value":[747,3692,202,182,60,148,458,420,26,38,293,327,1751,276,189,402],"string":"[\n 747,\n 3692,\n 202,\n 182,\n 60,\n 148,\n 458,\n 420,\n 26,\n 38,\n 293,\n 327,\n 1751,\n 276,\n 189,\n 402\n]","isConversational":false},"num_sections":{"kind":"number","value":19,"string":"19"},"most_frequent_words":{"kind":"list like","value":["maa","deet","20","20 deet","mosquitoes","gambiae","20 deet maa","deet maa","00","mosquito"],"string":"[\n \"maa\",\n \"deet\",\n \"20\",\n \"20 deet\",\n \"mosquitoes\",\n \"gambiae\",\n \"20 deet maa\",\n \"deet maa\",\n \"00\",\n \"mosquito\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["mosquitoes repellency gambiae","malaria vector control","impact malaria","biting anopheles gambiae","insecticide tendency mosquitoes"],"string":"[\n \"mosquitoes repellency gambiae\",\n \"malaria vector control\",\n \"impact malaria\",\n \"biting anopheles gambiae\",\n \"insecticide tendency mosquitoes\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Malaria | Mosquito | \nAnopheles gambiae\n | \nAedes aegypti\n | Repellent | MAA | Burkina Faso [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Malaria | Mosquito | \nAnopheles gambiae\n | \nAedes aegypti\n | Repellent | MAA | Burkina Faso [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Malaria | Mosquito | \nAnopheles gambiae\n | \nAedes aegypti\n | Repellent | MAA | Burkina Faso [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Malaria | Mosquito | \nAnopheles gambiae\n | \nAedes aegypti\n | Repellent | MAA | Burkina Faso [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Malaria | Mosquito | \nAnopheles gambiae\n | \nAedes aegypti\n | Repellent | MAA | Burkina Faso [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aedes | Animals | Anopheles | Burkina Faso | DEET | Female | Humans | Insect Repellents | Malaria | Male | Ointments | Young Adult [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aedes | Animals | Anopheles | Burkina Faso | DEET | Female | Humans | Insect Repellents | Malaria | Male | Ointments | Young Adult [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aedes | Animals | Anopheles | Burkina Faso | DEET | Female | Humans | Insect Repellents | Malaria | Male | Ointments | Young Adult [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aedes | Animals | Anopheles | Burkina Faso | DEET | Female | Humans | Insect Repellents | Malaria | Male | Ointments | Young Adult [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aedes | Animals | Anopheles | Burkina Faso | DEET | Female | Humans | Insect Repellents | Malaria | Male | Ointments | Young Adult [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] mosquitoes repellency gambiae | malaria vector control | impact malaria | biting 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gambiae | insecticide tendency mosquitoes [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] maa | deet | 20 | 20 deet | mosquitoes | gambiae | 20 deet maa | deet maa | 00 | mosquito [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] maa | deet | 20 | 20 deet | mosquitoes | gambiae | 20 deet maa | deet maa | 00 | mosquito [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] maa | deet | 20 | 20 deet | mosquitoes | gambiae | 20 deet maa | deet maa | 00 | mosquito [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] maa | deet | 20 | 20 deet | mosquitoes | gambiae | 20 deet maa | deet maa | 00 | mosquito [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] maa | deet | 20 | 20 deet | mosquitoes | gambiae | 20 deet maa | deet maa | 00 | mosquito [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] evening | llins | effective | distribution | children | malaria | africa | biting | mothers | infectious [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] deet | maa | 20 deet | 20 | deet maa | 20 deet maa | anopheles | 95 | ci | 95 ci [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] malaria | vector | ointment | high | butter widely west | malaria vectors context widespread | malaria vectors context | butter widely west africa | novel ointment formulated shea | west africa moisturize [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] maa | deet | 20 | 20 deet | volunteers | 00 | mosquitoes | deet maa | 20 deet maa | gambiae [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] maa | deet | 20 | 20 deet | volunteers | 00 | mosquitoes | deet maa | 20 deet maa | gambiae [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Malaria ||| ||| ||| ||| Burkina Faso [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 6 | 30 | 5 | 30 | Goden | 4 ||| MAÏA | three ||| 3979 ||| 98.5 % | 1.5 % ||| 95 % ||| 20 % | MAÏA | 2 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] MAÏA | Africa [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Malaria ||| ||| ||| ||| Burkina Faso ||| 20 | HLC ||| 2 | MAIA | 20 % | two | Aedes | WHO ||| two ||| Kisumu | Bora Bora ||| 70 % | 20 % | Burkina Faso ||| ||| 6 | 30 | 5 | 30 | Goden | 4 ||| MAÏA | three ||| 3979 ||| 98.5 % | 1.5 % ||| 95 % ||| 20 % | MAÏA | 2 ||| MAÏA | Africa [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Malaria ||| ||| ||| ||| Burkina Faso ||| 20 | HLC ||| 2 | MAIA | 20 % | two | Aedes | WHO ||| two ||| Kisumu | Bora Bora ||| 70 % | 20 % | Burkina Faso ||| ||| 6 | 30 | 5 | 30 | Goden | 4 ||| MAÏA | three ||| 3979 ||| 98.5 % | 1.5 % ||| 95 % ||| 20 % | MAÏA | 2 ||| MAÏA | Africa [SUMMARY]"}}},{"rowIdx":88,"cells":{"title":{"kind":"string","value":"The role of histological subtype in hormone receptor positive metastatic breast cancer: similar survival but different therapeutic approaches."},"pmid":{"kind":"string","value":"27121067"},"background_abstract":{"kind":"string","value":"This study describes the differences between the two largest histological breast cancer subtypes (invasive ductal carcinoma (IDC) and invasive (mixed) lobular carcinoma (ILC) with respect to patient and tumor characteristics, treatment-choices and outcome in metastatic breast cancer."},"background_abstract_label":{"kind":"string","value":"INTRODUCTION"},"methods_abstract":{"kind":"string","value":"We included 437 patients with hormone receptor-positive IDC and 131 patients with hormone receptor-positive ILC, all diagnosed with metastatic breast cancer between 2007-2009, irrespective of date of the primary diagnosis. Patient and tumor characteristics and data on treatment and outcome were collected. Survival curves were obtained using the Kaplan-Meier method."},"methods_abstract_label":{"kind":"string","value":"MATERIALS AND METHODS"},"results_abstract":{"kind":"string","value":"Patients with ILC were older at diagnosis of primary breast cancer and had more often initial bone metastasis (46.5% versus 34.8%, P = 0.01) and less often multiple metastatic sites compared to IDC (23.7% versus 30.9%, P = 0.11). Six months after diagnosis of metastatic breast cancer, 28.1% of patients with ILC and 39.8% of patients with IDC had received chemotherapy with a longer median time to first chemotherapy for those with ILC (P = 0.001). After six months 84.8% of patients with ILC had received endocrine therapy versus 72.5% of patients with IDC (P = 0.0001). Median overall survival was 29 months for ILC and 25 months for IDC (P = 0.53)."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Treatment strategies of hormone receptor-positive metastatic breast cancer were remarkably different for patients with ILC and IDC. Further research is required to understand tumor behavior and treatment-choices in real-life."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","Aged","Aged, 80 and over","Antineoplastic Agents","Breast Neoplasms","Carcinoma, Ductal, Breast","Carcinoma, Lobular","Disease-Free Survival","Female","Follow-Up Studies","Humans","Kaplan-Meier Estimate","Middle Aged","Receptor, ErbB-2","Receptors, Estrogen","Receptors, Progesterone","Treatment Outcome"],"string":"[\n \"Adult\",\n \"Aged\",\n \"Aged, 80 and over\",\n \"Antineoplastic Agents\",\n \"Breast Neoplasms\",\n \"Carcinoma, Ductal, Breast\",\n \"Carcinoma, Lobular\",\n \"Disease-Free Survival\",\n \"Female\",\n \"Follow-Up Studies\",\n \"Humans\",\n \"Kaplan-Meier Estimate\",\n \"Middle Aged\",\n \"Receptor, ErbB-2\",\n \"Receptors, Estrogen\",\n \"Receptors, Progesterone\",\n \"Treatment Outcome\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"5045405"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"The two most frequent histological subtypes of breast cancer are invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC), with IDC comprising 75–80% and ILC 5–15% of all breast cancer cases. ILC is being associated with larger tumor size at presentation, more bilateral and multifocal involvement and with a different pattern of metastatic spread compared with IDC [1, 2]. Furthermore, ILC is more often HR-positive, HER2-negative, with lower S-phase fraction and less often positive for the tumor suppressor gene p53, compared with IDC [1]. Also, treatment response is known to be different. In a combined analysis on a number of retrospective series, the pathological complete response rate of neo-adjuvant chemotherapy was significantly lower in ILC (1.7%) than in IDC (11.6%) [3]. In the adjuvant setting, retrospective data suggest a higher efficacy of endocrine therapy for ILC than IDC [4]. More recently, a large retrospective study strongly suggested that HR-positive breast cancer patients with ILC do not seem to benefit from adjuvant chemotherapy in addition to endocrine therapy [5].\nWhether these differences have a prognostic impact is controversial. Some studies found no effect of histology on survival [1, 6, 7], others found a better prognosis for patients with ILC compared to those with IDC [8–10] and some found a change in prognosis over time with a better prognosis for ILC during the first years of follow-up and a worse prognosis during later years [2, 11].\nSo far, studies on histological subtypes consider early breast cancer and not metastatic breast cancer. The aim of this study was to describe the differences between IDC and ILC with respect to patient and tumor characteristics, treatment-choices and outcome in metastatic breast cancer. In order to account for the effect of the hormone receptor (HR) on outcome and treatment-decision making, we only included patients with HR-positive breast cancer."},"methods_title":{"kind":"string","value":"MATERIALS AND METHODS"},"methods_text":{"kind":"string","value":" Patient selection We identified all patients diagnosed with metastatic breast cancer between 2007–2009 in eight hospitals in the South-East of the Netherlands. All patients with metastatic breast cancer were selected irrespective of the date of primary breast cancer diagnosis (also including patients with de novo metastatic breast cancer; 18.5% of patients with IDC and 19.9% of patients with ILC), with the exception of patients with a diagnosis of primary breast cancer before 1990, due to limitations in the availability of data on the primary tumor and initial treatment.\nHistology was classified according to the International Classification of Diseases for Oncology (ICD-O) [23]. ILC and mixed histology were defined as code 8520 and 8522. IDC was defined as code 8500. From the total of 815 metastatic breast cancer patients, we excluded 76 patients with either unknown histology or histological subtypes other than IDC or ILC. Of the remaining 739 metastatic breast cancer patients, we excluded 171 patients with HR-negative tumors (27% of patients with IDC and 8% of patients with ILC) in order to rule out the impact of HR status. In total, our study population consisted of 568 patients divided in two groups based on histology; one group of 437 patients with IDC and the other group of 131 patients with ILC.\nWe identified all patients diagnosed with metastatic breast cancer between 2007–2009 in eight hospitals in the South-East of the Netherlands. All patients with metastatic breast cancer were selected irrespective of the date of primary breast cancer diagnosis (also including patients with de novo metastatic breast cancer; 18.5% of patients with IDC and 19.9% of patients with ILC), with the exception of patients with a diagnosis of primary breast cancer before 1990, due to limitations in the availability of data on the primary tumor and initial treatment.\nHistology was classified according to the International Classification of Diseases for Oncology (ICD-O) [23]. ILC and mixed histology were defined as code 8520 and 8522. IDC was defined as code 8500. From the total of 815 metastatic breast cancer patients, we excluded 76 patients with either unknown histology or histological subtypes other than IDC or ILC. Of the remaining 739 metastatic breast cancer patients, we excluded 171 patients with HR-negative tumors (27% of patients with IDC and 8% of patients with ILC) in order to rule out the impact of HR status. In total, our study population consisted of 568 patients divided in two groups based on histology; one group of 437 patients with IDC and the other group of 131 patients with ILC.\n Data collection Information was collected on patient and tumor characteristics, treatment and outcome. Tumors were characterized by the sixth edition of the TNM classification of malignant tumors [24] and Scarff Bloom Richardson (SBR) histological grading [25]. Estrogen receptor (ER) and progesterone receptor (PR) positivity was defined as positive nuclear staining of ≥ 10%. HER2 positivity was defined as immunohistochemistry score of 3+ or 2+ with positive FISH. In case of missing HER2 status a dedicated pathologist centrally reviewed missing data when material was available. Initial sites of metastasis were categorized as: bone, visceral (including lung, liver, pleural, peritoneal, pericardial and lymphangitic carcinomatosis), brain (including leptomeningeal and CNS), skin and lymph nodes, and multiple metastases (more than one of the metastatic sites).\nInformation was collected on patient and tumor characteristics, treatment and outcome. Tumors were characterized by the sixth edition of the TNM classification of malignant tumors [24] and Scarff Bloom Richardson (SBR) histological grading [25]. Estrogen receptor (ER) and progesterone receptor (PR) positivity was defined as positive nuclear staining of ≥ 10%. HER2 positivity was defined as immunohistochemistry score of 3+ or 2+ with positive FISH. In case of missing HER2 status a dedicated pathologist centrally reviewed missing data when material was available. Initial sites of metastasis were categorized as: bone, visceral (including lung, liver, pleural, peritoneal, pericardial and lymphangitic carcinomatosis), brain (including leptomeningeal and CNS), skin and lymph nodes, and multiple metastases (more than one of the metastatic sites).\n Statistical analysis Baseline characteristics between the two histological groups were compared using chi-square tests for categorical variables and Wilcoxon rank sum tests for continuous variables.\nMetastatic-free interval was defined as time between date of primary diagnosis and date of first distant metastasis. Overall survival after diagnosis of metastatic breast cancer was defined as time between date of first distant metastasis and date of death. Survival curves and time to first palliative systemic therapy (either chemotherapy or endocrine therapy) were estimated using the Kaplan-Meier method and compared using log-rank tests. All patients still alive were censored at the date of last follow-up of each individual patient. Patients who died without palliative therapy were censored at the date of death in the analysis of time to first palliative therapy.\nTo explore the association of palliative systemic therapy with the survival of patients with metastatic breast cancer for both histological subtypes a Cox proportional hazards model was performed with palliative chemotherapy and endocrine therapy as a time-dependent covariate, since the administration of treatment can change over time and is dependent on the time available for each patient to receive the treatment. We did not explore the association between palliative targeted therapy, such as trastuzumab and bevacizumab, and survival since the number of patients with IDC and ILC receiving targeted therapy was very low. Since all these patients received targeted therapy with chemotherapy, these patients were included in the analysis on initial chemotherapy. In addition, the landmark method was used to estimate survival after a specific time-point, the so-called residual survival [26]. As we were interested to learn about the obtained survival in relation to the initial palliative treatment choices, we chose six months after diagnosis of metastatic breast cancer as landmark. Consequently, patients who already died within 6 months were excluded for the residual survival curves.\nAll analyses were performed using SAS version 9.2. All reported P-values are two-sided and P-value ≤ 0.05 was considered statistically significant.\nBaseline characteristics between the two histological groups were compared using chi-square tests for categorical variables and Wilcoxon rank sum tests for continuous variables.\nMetastatic-free interval was defined as time between date of primary diagnosis and date of first distant metastasis. Overall survival after diagnosis of metastatic breast cancer was defined as time between date of first distant metastasis and date of death. Survival curves and time to first palliative systemic therapy (either chemotherapy or endocrine therapy) were estimated using the Kaplan-Meier method and compared using log-rank tests. All patients still alive were censored at the date of last follow-up of each individual patient. Patients who died without palliative therapy were censored at the date of death in the analysis of time to first palliative therapy.\nTo explore the association of palliative systemic therapy with the survival of patients with metastatic breast cancer for both histological subtypes a Cox proportional hazards model was performed with palliative chemotherapy and endocrine therapy as a time-dependent covariate, since the administration of treatment can change over time and is dependent on the time available for each patient to receive the treatment. We did not explore the association between palliative targeted therapy, such as trastuzumab and bevacizumab, and survival since the number of patients with IDC and ILC receiving targeted therapy was very low. Since all these patients received targeted therapy with chemotherapy, these patients were included in the analysis on initial chemotherapy. In addition, the landmark method was used to estimate survival after a specific time-point, the so-called residual survival [26]. As we were interested to learn about the obtained survival in relation to the initial palliative treatment choices, we chose six months after diagnosis of metastatic breast cancer as landmark. Consequently, patients who already died within 6 months were excluded for the residual survival curves.\nAll analyses were performed using SAS version 9.2. All reported P-values are two-sided and P-value ≤ 0.05 was considered statistically significant."},"results_title":{"kind":"null"},"results_text":{"kind":"null"},"conclusions_title":{"kind":"string","value":"DISCUSSION"},"conclusions_text":{"kind":"string","value":"Baseline characteristics between the two histological groups were compared using chi-square tests for categorical variables and Wilcoxon rank sum tests for continuous variables.\nMetastatic-free interval was defined as time between date of primary diagnosis and date of first distant metastasis. Overall survival after diagnosis of metastatic breast cancer was defined as time between date of first distant metastasis and date of death. Survival curves and time to first palliative systemic therapy (either chemotherapy or endocrine therapy) were estimated using the Kaplan-Meier method and compared using log-rank tests. All patients still alive were censored at the date of last follow-up of each individual patient. Patients who died without palliative therapy were censored at the date of death in the analysis of time to first palliative therapy.\nTo explore the association of palliative systemic therapy with the survival of patients with metastatic breast cancer for both histological subtypes a Cox proportional hazards model was performed with palliative chemotherapy and endocrine therapy as a time-dependent covariate, since the administration of treatment can change over time and is dependent on the time available for each patient to receive the treatment. We did not explore the association between palliative targeted therapy, such as trastuzumab and bevacizumab, and survival since the number of patients with IDC and ILC receiving targeted therapy was very low. Since all these patients received targeted therapy with chemotherapy, these patients were included in the analysis on initial chemotherapy. In addition, the landmark method was used to estimate survival after a specific time-point, the so-called residual survival [26]. As we were interested to learn about the obtained survival in relation to the initial palliative treatment choices, we chose six months after diagnosis of metastatic breast cancer as landmark. Consequently, patients who already died within 6 months were excluded for the residual survival curves.\nAll analyses were performed using SAS version 9.2. All reported P-values are two-sided and P-value ≤ 0.05 was considered statistically significant."},"other_sections_titles":{"kind":"list like","value":["INTRODUCTION","RESULTS","Patient characteristics","Palliative systemic treatment","Outcome","Residual survival: six months after diagnosis","DISCUSSION"],"string":"[\n \"INTRODUCTION\",\n \"RESULTS\",\n \"Patient characteristics\",\n \"Palliative systemic treatment\",\n \"Outcome\",\n \"Residual survival: six months after diagnosis\",\n \"DISCUSSION\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["The two most frequent histological subtypes of breast cancer are invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC), with IDC comprising 75–80% and ILC 5–15% of all breast cancer cases. ILC is being associated with larger tumor size at presentation, more bilateral and multifocal involvement and with a different pattern of metastatic spread compared with IDC [1, 2]. Furthermore, ILC is more often HR-positive, HER2-negative, with lower S-phase fraction and less often positive for the tumor suppressor gene p53, compared with IDC [1]. Also, treatment response is known to be different. In a combined analysis on a number of retrospective series, the pathological complete response rate of neo-adjuvant chemotherapy was significantly lower in ILC (1.7%) than in IDC (11.6%) [3]. In the adjuvant setting, retrospective data suggest a higher efficacy of endocrine therapy for ILC than IDC [4]. More recently, a large retrospective study strongly suggested that HR-positive breast cancer patients with ILC do not seem to benefit from adjuvant chemotherapy in addition to endocrine therapy [5].\nWhether these differences have a prognostic impact is controversial. Some studies found no effect of histology on survival [1, 6, 7], others found a better prognosis for patients with ILC compared to those with IDC [8–10] and some found a change in prognosis over time with a better prognosis for ILC during the first years of follow-up and a worse prognosis during later years [2, 11].\nSo far, studies on histological subtypes consider early breast cancer and not metastatic breast cancer. The aim of this study was to describe the differences between IDC and ILC with respect to patient and tumor characteristics, treatment-choices and outcome in metastatic breast cancer. In order to account for the effect of the hormone receptor (HR) on outcome and treatment-decision making, we only included patients with HR-positive breast cancer."," Patient characteristics Of the 568 patients with HR-positive metastatic breast cancer, 23% had (mixed type) ILC (131 patients), hereafter referred to as ILC and 77% had IDC (437 patients) (Table 1).\nAbbreviations; HR+ hormone receptor positive, N number, SBR Scarff Bloom Richardson, MFI metastatic-free interval * ≥ 1 of the aforementioned initial sites of metastasis.\nMetastatic breast cancer patients with ILC were older at the time of primary breast cancer diagnosis, compared with patients with IDC (median age at diagnosis 62 years for ILC versus 58 years for IDC, P = 0.03). At initial presentation, patients with HR-positive ILC had larger tumors (T2-3, 62.5% versus 49.7%, P = 0.002) and slightly more node-positive disease (66% versus 56.9%, P = 0.10), but with less often a HER2 positive status (7.6% versus 15.3%, P = 0.02) and lower grade (not significant) compared with patients with HR-positive IDC. No differences were seen in use of adjuvant chemotherapy or endocrine therapy between the histological subtypes.\nBone was the most common initial site of distant metastasis in both HR-positive histological subtypes, although bone metastasis as initial site was more frequently observed for patients with ILC compared to those with IDC (46.5% versus 34.8%, P = 0.01). Fewer patients with ILC were diagnosed with multiple sites of distant metastasis compared to those with IDC (23.7% versus 30.9%, P = 0.11).\nOf the 568 patients with HR-positive metastatic breast cancer, 23% had (mixed type) ILC (131 patients), hereafter referred to as ILC and 77% had IDC (437 patients) (Table 1).\nAbbreviations; HR+ hormone receptor positive, N number, SBR Scarff Bloom Richardson, MFI metastatic-free interval * ≥ 1 of the aforementioned initial sites of metastasis.\nMetastatic breast cancer patients with ILC were older at the time of primary breast cancer diagnosis, compared with patients with IDC (median age at diagnosis 62 years for ILC versus 58 years for IDC, P = 0.03). At initial presentation, patients with HR-positive ILC had larger tumors (T2-3, 62.5% versus 49.7%, P = 0.002) and slightly more node-positive disease (66% versus 56.9%, P = 0.10), but with less often a HER2 positive status (7.6% versus 15.3%, P = 0.02) and lower grade (not significant) compared with patients with HR-positive IDC. No differences were seen in use of adjuvant chemotherapy or endocrine therapy between the histological subtypes.\nBone was the most common initial site of distant metastasis in both HR-positive histological subtypes, although bone metastasis as initial site was more frequently observed for patients with ILC compared to those with IDC (46.5% versus 34.8%, P = 0.01). Fewer patients with ILC were diagnosed with multiple sites of distant metastasis compared to those with IDC (23.7% versus 30.9%, P = 0.11).\n Palliative systemic treatment Median follow-up after diagnosis of metastatic disease was 37.1 months (range 5.2–54.6) with 239 patients (42%) alive at the end of the follow-up period.\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative chemotherapy was significantly longer for patients with ILC (median not yet reached) compared with IDC (median 16.9 months, 95% Confidence Interval (CI) 9.6-22.3, P = 0.001) (Figure 1A). Six months after diagnosis of HR-positive metastatic breast cancer less patients with ILC had received palliative chemotherapy compared with IDC (28.1% versus 39.8% respectively).\nTime to first palliative chemotherapy (A) and endocrine therapy (B) by histological subtype.\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative endocrine therapy was significantly shorter for patients with ILC (median, 0.6 months, 95% CI 0.5–0.8) compared with patients with IDC (median, 1.1 months, 95% CI 1.0–1.5, P = 0.0001) (Figure 1B). Six months after diagnosis of metastatic breast cancer 84.8% of patients with ILC had received palliative endocrine therapy compared with 72.5% of patients with IDC.\nMedian follow-up after diagnosis of metastatic disease was 37.1 months (range 5.2–54.6) with 239 patients (42%) alive at the end of the follow-up period.\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative chemotherapy was significantly longer for patients with ILC (median not yet reached) compared with IDC (median 16.9 months, 95% Confidence Interval (CI) 9.6-22.3, P = 0.001) (Figure 1A). Six months after diagnosis of HR-positive metastatic breast cancer less patients with ILC had received palliative chemotherapy compared with IDC (28.1% versus 39.8% respectively).\nTime to first palliative chemotherapy (A) and endocrine therapy (B) by histological subtype.\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative endocrine therapy was significantly shorter for patients with ILC (median, 0.6 months, 95% CI 0.5–0.8) compared with patients with IDC (median, 1.1 months, 95% CI 1.0–1.5, P = 0.0001) (Figure 1B). Six months after diagnosis of metastatic breast cancer 84.8% of patients with ILC had received palliative endocrine therapy compared with 72.5% of patients with IDC.\n Outcome Median overall survival was 29.4 months (95% CI 22.5–36.6) for patients with HR-positive ILC and 25.4 months (95% CI 21.8–31.7) for patients with HR-positive IDC (P = 0.53).\nIn multivariable analysis for patients with ILC with palliative endocrine therapy and palliative chemotherapy as time-dependent covariates, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.8, 95% CI 1.7–4.6, P < .0001) compared to no palliative chemotherapy during the observation period. Conversely, early initiation of palliative endocrine therapy was associated with a favorable survival (hazard ratio 0.4, 95% CI 0.2–0.8, P = 0.005) compared to no palliative endocrine therapy during the observation period.\nIn multivariable analysis for patients with IDC, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.1, 95% CI 1.6–2.7. P < .0001) when compared with no chemotherapy, whereas early treatment with palliative endocrine therapy was not associated with survival (hazard ratio 0.9, 95% CI 0.6–1.2, P = 0.4).\nMedian overall survival was 29.4 months (95% CI 22.5–36.6) for patients with HR-positive ILC and 25.4 months (95% CI 21.8–31.7) for patients with HR-positive IDC (P = 0.53).\nIn multivariable analysis for patients with ILC with palliative endocrine therapy and palliative chemotherapy as time-dependent covariates, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.8, 95% CI 1.7–4.6, P < .0001) compared to no palliative chemotherapy during the observation period. Conversely, early initiation of palliative endocrine therapy was associated with a favorable survival (hazard ratio 0.4, 95% CI 0.2–0.8, P = 0.005) compared to no palliative endocrine therapy during the observation period.\nIn multivariable analysis for patients with IDC, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.1, 95% CI 1.6–2.7. P < .0001) when compared with no chemotherapy, whereas early treatment with palliative endocrine therapy was not associated with survival (hazard ratio 0.9, 95% CI 0.6–1.2, P = 0.4).\n Residual survival: six months after diagnosis For ILC, the residual survival was significantly longer for patients not treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (median 44.0 months, 95% CI 30.2-not yet reached) versus patients treated with palliative chemotherapy (median 15.2 months, 95% CI 7.8–19.2) (Figure 2A). For IDC, residual survival when not treated with palliative chemotherapy was 41.8 (95% CI 33.3-not yet reached) compared with 16.8 months (95% CI 13.6–22.5) for patients treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (Figure 2B).\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative chemotherapy during the first six months after diagnosis of metastatic breast cancer.\nReversely, for ILC-patients with palliative endocrine treatment within the first six months after diagnosis of metastatic breast cancer residual survival was 37.5 months (95% CI 26.9-not yet reached) compared with 11.2 months (95% CI 3.0–20.4) for patients with HR-positive metastatic ILC without palliative endocrine therapy before the first six months (Figure 3A). For patients with HR-positive metastatic IDC treated with palliative endocrine therapy during the first six months, residual survival was 33.5 months (95% CI 25.7-not yet reached) compared with 18.9 months (95% CI 14.0-26.8) for patients with HR-positive metastatic IDC without palliative endocrine therapy in the first six months of metastatic disease (Figure 3B).\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative endocrine therapy during the first six months after diagnosis of metastatic breast cancer.\nFor ILC, the residual survival was significantly longer for patients not treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (median 44.0 months, 95% CI 30.2-not yet reached) versus patients treated with palliative chemotherapy (median 15.2 months, 95% CI 7.8–19.2) (Figure 2A). For IDC, residual survival when not treated with palliative chemotherapy was 41.8 (95% CI 33.3-not yet reached) compared with 16.8 months (95% CI 13.6–22.5) for patients treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (Figure 2B).\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative chemotherapy during the first six months after diagnosis of metastatic breast cancer.\nReversely, for ILC-patients with palliative endocrine treatment within the first six months after diagnosis of metastatic breast cancer residual survival was 37.5 months (95% CI 26.9-not yet reached) compared with 11.2 months (95% CI 3.0–20.4) for patients with HR-positive metastatic ILC without palliative endocrine therapy before the first six months (Figure 3A). For patients with HR-positive metastatic IDC treated with palliative endocrine therapy during the first six months, residual survival was 33.5 months (95% CI 25.7-not yet reached) compared with 18.9 months (95% CI 14.0-26.8) for patients with HR-positive metastatic IDC without palliative endocrine therapy in the first six months of metastatic disease (Figure 3B).\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative endocrine therapy during the first six months after diagnosis of metastatic breast cancer.","Of the 568 patients with HR-positive metastatic breast cancer, 23% had (mixed type) ILC (131 patients), hereafter referred to as ILC and 77% had IDC (437 patients) (Table 1).\nAbbreviations; HR+ hormone receptor positive, N number, SBR Scarff Bloom Richardson, MFI metastatic-free interval * ≥ 1 of the aforementioned initial sites of metastasis.\nMetastatic breast cancer patients with ILC were older at the time of primary breast cancer diagnosis, compared with patients with IDC (median age at diagnosis 62 years for ILC versus 58 years for IDC, P = 0.03). At initial presentation, patients with HR-positive ILC had larger tumors (T2-3, 62.5% versus 49.7%, P = 0.002) and slightly more node-positive disease (66% versus 56.9%, P = 0.10), but with less often a HER2 positive status (7.6% versus 15.3%, P = 0.02) and lower grade (not significant) compared with patients with HR-positive IDC. No differences were seen in use of adjuvant chemotherapy or endocrine therapy between the histological subtypes.\nBone was the most common initial site of distant metastasis in both HR-positive histological subtypes, although bone metastasis as initial site was more frequently observed for patients with ILC compared to those with IDC (46.5% versus 34.8%, P = 0.01). Fewer patients with ILC were diagnosed with multiple sites of distant metastasis compared to those with IDC (23.7% versus 30.9%, P = 0.11).","Median follow-up after diagnosis of metastatic disease was 37.1 months (range 5.2–54.6) with 239 patients (42%) alive at the end of the follow-up period.\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative chemotherapy was significantly longer for patients with ILC (median not yet reached) compared with IDC (median 16.9 months, 95% Confidence Interval (CI) 9.6-22.3, P = 0.001) (Figure 1A). Six months after diagnosis of HR-positive metastatic breast cancer less patients with ILC had received palliative chemotherapy compared with IDC (28.1% versus 39.8% respectively).\nTime to first palliative chemotherapy (A) and endocrine therapy (B) by histological subtype.\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative endocrine therapy was significantly shorter for patients with ILC (median, 0.6 months, 95% CI 0.5–0.8) compared with patients with IDC (median, 1.1 months, 95% CI 1.0–1.5, P = 0.0001) (Figure 1B). Six months after diagnosis of metastatic breast cancer 84.8% of patients with ILC had received palliative endocrine therapy compared with 72.5% of patients with IDC.","Median overall survival was 29.4 months (95% CI 22.5–36.6) for patients with HR-positive ILC and 25.4 months (95% CI 21.8–31.7) for patients with HR-positive IDC (P = 0.53).\nIn multivariable analysis for patients with ILC with palliative endocrine therapy and palliative chemotherapy as time-dependent covariates, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.8, 95% CI 1.7–4.6, P < .0001) compared to no palliative chemotherapy during the observation period. Conversely, early initiation of palliative endocrine therapy was associated with a favorable survival (hazard ratio 0.4, 95% CI 0.2–0.8, P = 0.005) compared to no palliative endocrine therapy during the observation period.\nIn multivariable analysis for patients with IDC, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.1, 95% CI 1.6–2.7. P < .0001) when compared with no chemotherapy, whereas early treatment with palliative endocrine therapy was not associated with survival (hazard ratio 0.9, 95% CI 0.6–1.2, P = 0.4).","For ILC, the residual survival was significantly longer for patients not treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (median 44.0 months, 95% CI 30.2-not yet reached) versus patients treated with palliative chemotherapy (median 15.2 months, 95% CI 7.8–19.2) (Figure 2A). For IDC, residual survival when not treated with palliative chemotherapy was 41.8 (95% CI 33.3-not yet reached) compared with 16.8 months (95% CI 13.6–22.5) for patients treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (Figure 2B).\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative chemotherapy during the first six months after diagnosis of metastatic breast cancer.\nReversely, for ILC-patients with palliative endocrine treatment within the first six months after diagnosis of metastatic breast cancer residual survival was 37.5 months (95% CI 26.9-not yet reached) compared with 11.2 months (95% CI 3.0–20.4) for patients with HR-positive metastatic ILC without palliative endocrine therapy before the first six months (Figure 3A). For patients with HR-positive metastatic IDC treated with palliative endocrine therapy during the first six months, residual survival was 33.5 months (95% CI 25.7-not yet reached) compared with 18.9 months (95% CI 14.0-26.8) for patients with HR-positive metastatic IDC without palliative endocrine therapy in the first six months of metastatic disease (Figure 3B).\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative endocrine therapy during the first six months after diagnosis of metastatic breast cancer.","To our knowledge, the role of histological subtype in palliative systemic treatment of metastatic breast cancer has not been studied before. In a cohort of 568 HR-positive metastatic breast cancer patients, we showed that patients with HR-positive ILC had more often bone and less often multiple sites as initial site of distant metastasis compared to those with HR-positive IDC. Six months after metastatic diagnosis, patients with HR-positive ILC had received significantly less often chemotherapy (28% versus 40%) and more often endocrine therapy (85% versus 73%) as compared to patients with HR-positive IDC. Patients starting with palliative chemotherapy during the first six months had a significantly shorter median residual survival thereafter as compared to those who did not (15 months versus 44 months for ILC, and 17 and 42 months for IDC, respectively). Reversely, patients treated with endocrine therapy in the first six months had a longer median residual survival compared to those who did not (37 months versus 11 months for ILC and 33 and 19 months for IDC, respectively). Our results confirm that, in addition to HR-status, histology correlates with presentation of metastatic disease by number and type of distant metastatic sites, and thereby affects treatment-decision making in real-life.\nThe intriguing scientific question is to determine how histological subtype influences the metastatic spread in HR-positive breast cancer. It is hypothesized that the loss of E-cadherin, a cell-cell adhesion molecule, in ILC may result in less adhesiveness of the tumor cells and therefore disseminate and infiltrate certain distant locations more easily [1].\nIn this HR-positive metastatic breast cancer cohort we also looked at the association of palliative systemic treatment with survival of patients with metastatic breast cancer for both histological subtypes. With the favorable presentation of distant metastasis by number and location of patients with HR-positive ILC as compared to HR-positive IDC, one would expect a more indolent disease course and a better outcome. However, we showed that for patients with HR-positive breast cancer, overall disease outcome was comparable irrespective of histology and irrespective of different treatment choices made in real-life, as discussed above. And interestingly, the efficacy of chemotherapy - once the treatment choice was made - was comparable irrespective of histology. However, treatment with palliative endocrine therapy was only associated with a favorable prognosis of patients with HR-positive ILC, whereas this was not seen in patients with HR-positive IDC, also suggesting the impact of histology over HR-status. This is in concordance with the early breast cancer setting, in which there is evidence suggesting differences in efficacy of systemic therapy between the two histological subtypes [3–5, 12].\nAlthough there is some proof that histology could be helpful in treatment decision-making [13–15], the current guidelines do not include histological subtype as an indicator for the use of systemic treatment in general, or for a specific regimen [16, 17]. Interestingly, in the adjuvant setting, histology has been shown to be of importance when deciding between the use of breast-conserving surgery or mastectomy after neo-adjuvant chemotherapy [18]. The current study shows that histological subtype can be of predictive value for HR-positive metastatic breast cancer with regards to the effectiveness of early initiation of palliative endocrine therapy. It may be possible that in patients with HR-positive lobular breast cancer, the higher incidence of bone metastases as initial metastatic site partly accounts for the initial choice of endocrine therapy. It underlines the relevance of acknowledging a different metastatic pattern, and thereby different initial treatment choices, between breast cancer patients with ductal versus lobular histology.\nMuch more than in the adjuvant setting, treatment decisions in the metastatic setting are based on the observed and anticipated clinical course of the disease, which is not only determined by the tumor characteristics as described earlier. Also age, performance status, previous therapies and toxicities, comorbidity and patient and doctor preferences play a role. The complexity of this process, together with the retrospective design of our study make it impossible to identify and rule out confounding by indication. Furthermore, localization of the metastases (visceral versus bone metastasis for instance) could be related to symptomatology and thereby timing of detection, and could therefore have introduced lead time bias.\nIn this study patients with pure lobular carcinoma and mixed lobular carcinomas were combined for the analyses. In other studies on histological subtypes of breast cancer mixed and lobular carcinoma had similar outcome [14, 19]. Even with combining these subgroups, the proportion of HER2-positive tumors was too low to further analyze anti-HER2 therapy. In the adjuvant treatment setting the magnitude of benefit from adjuvant trastuzumab between patients with ILC and IDC was shown not to be different [20]. Another limitation of our study is that patient numbers were too small to perform analysis on specific chemotherapy regimens. Microarray analysis have demonstrated that ILC and IDC can be distinguished on the basis of genomic and expression profiles [21]. The increasing knowledge on genomic differences between ILC and IDC can help to answer questions on in vivo chemosensitivity. For example, topoisomerase-IIα gene amplification is a predictive biomarker for response to anthracyclines and in ILC this gene amplification is lacking, which could help understand the poor responsiveness of ILC to neo-adjuvant chemotherapy, including anthracyclines [22]. This genetic information can help guide further research and may eventually be useful in making treatment decisions for histological breast cancer subtypes.\nIn conclusion, to our knowledge, this is the first study that investigates the role of histological subtype in HR-positive metastatic breast cancer. Although survival was comparable for the two histological subtypes, this was achieved with different treatment strategies. As patients with HR-positive ILC were less likely to receive chemotherapy than those with HR-positive IDC, histology may be a relevant factor in treatment-decision making. For a more definite conclusion on the role of histology, we recommend to incorporate histological subtype as a stratification factor in future clinical trials."],"string":"[\n \"The two most frequent histological subtypes of breast cancer are invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC), with IDC comprising 75–80% and ILC 5–15% of all breast cancer cases. ILC is being associated with larger tumor size at presentation, more bilateral and multifocal involvement and with a different pattern of metastatic spread compared with IDC [1, 2]. Furthermore, ILC is more often HR-positive, HER2-negative, with lower S-phase fraction and less often positive for the tumor suppressor gene p53, compared with IDC [1]. Also, treatment response is known to be different. In a combined analysis on a number of retrospective series, the pathological complete response rate of neo-adjuvant chemotherapy was significantly lower in ILC (1.7%) than in IDC (11.6%) [3]. In the adjuvant setting, retrospective data suggest a higher efficacy of endocrine therapy for ILC than IDC [4]. More recently, a large retrospective study strongly suggested that HR-positive breast cancer patients with ILC do not seem to benefit from adjuvant chemotherapy in addition to endocrine therapy [5].\\nWhether these differences have a prognostic impact is controversial. Some studies found no effect of histology on survival [1, 6, 7], others found a better prognosis for patients with ILC compared to those with IDC [8–10] and some found a change in prognosis over time with a better prognosis for ILC during the first years of follow-up and a worse prognosis during later years [2, 11].\\nSo far, studies on histological subtypes consider early breast cancer and not metastatic breast cancer. The aim of this study was to describe the differences between IDC and ILC with respect to patient and tumor characteristics, treatment-choices and outcome in metastatic breast cancer. In order to account for the effect of the hormone receptor (HR) on outcome and treatment-decision making, we only included patients with HR-positive breast cancer.\",\n \" Patient characteristics Of the 568 patients with HR-positive metastatic breast cancer, 23% had (mixed type) ILC (131 patients), hereafter referred to as ILC and 77% had IDC (437 patients) (Table 1).\\nAbbreviations; HR+ hormone receptor positive, N number, SBR Scarff Bloom Richardson, MFI metastatic-free interval * ≥ 1 of the aforementioned initial sites of metastasis.\\nMetastatic breast cancer patients with ILC were older at the time of primary breast cancer diagnosis, compared with patients with IDC (median age at diagnosis 62 years for ILC versus 58 years for IDC, P = 0.03). At initial presentation, patients with HR-positive ILC had larger tumors (T2-3, 62.5% versus 49.7%, P = 0.002) and slightly more node-positive disease (66% versus 56.9%, P = 0.10), but with less often a HER2 positive status (7.6% versus 15.3%, P = 0.02) and lower grade (not significant) compared with patients with HR-positive IDC. No differences were seen in use of adjuvant chemotherapy or endocrine therapy between the histological subtypes.\\nBone was the most common initial site of distant metastasis in both HR-positive histological subtypes, although bone metastasis as initial site was more frequently observed for patients with ILC compared to those with IDC (46.5% versus 34.8%, P = 0.01). Fewer patients with ILC were diagnosed with multiple sites of distant metastasis compared to those with IDC (23.7% versus 30.9%, P = 0.11).\\nOf the 568 patients with HR-positive metastatic breast cancer, 23% had (mixed type) ILC (131 patients), hereafter referred to as ILC and 77% had IDC (437 patients) (Table 1).\\nAbbreviations; HR+ hormone receptor positive, N number, SBR Scarff Bloom Richardson, MFI metastatic-free interval * ≥ 1 of the aforementioned initial sites of metastasis.\\nMetastatic breast cancer patients with ILC were older at the time of primary breast cancer diagnosis, compared with patients with IDC (median age at diagnosis 62 years for ILC versus 58 years for IDC, P = 0.03). At initial presentation, patients with HR-positive ILC had larger tumors (T2-3, 62.5% versus 49.7%, P = 0.002) and slightly more node-positive disease (66% versus 56.9%, P = 0.10), but with less often a HER2 positive status (7.6% versus 15.3%, P = 0.02) and lower grade (not significant) compared with patients with HR-positive IDC. No differences were seen in use of adjuvant chemotherapy or endocrine therapy between the histological subtypes.\\nBone was the most common initial site of distant metastasis in both HR-positive histological subtypes, although bone metastasis as initial site was more frequently observed for patients with ILC compared to those with IDC (46.5% versus 34.8%, P = 0.01). Fewer patients with ILC were diagnosed with multiple sites of distant metastasis compared to those with IDC (23.7% versus 30.9%, P = 0.11).\\n Palliative systemic treatment Median follow-up after diagnosis of metastatic disease was 37.1 months (range 5.2–54.6) with 239 patients (42%) alive at the end of the follow-up period.\\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative chemotherapy was significantly longer for patients with ILC (median not yet reached) compared with IDC (median 16.9 months, 95% Confidence Interval (CI) 9.6-22.3, P = 0.001) (Figure 1A). Six months after diagnosis of HR-positive metastatic breast cancer less patients with ILC had received palliative chemotherapy compared with IDC (28.1% versus 39.8% respectively).\\nTime to first palliative chemotherapy (A) and endocrine therapy (B) by histological subtype.\\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative endocrine therapy was significantly shorter for patients with ILC (median, 0.6 months, 95% CI 0.5–0.8) compared with patients with IDC (median, 1.1 months, 95% CI 1.0–1.5, P = 0.0001) (Figure 1B). Six months after diagnosis of metastatic breast cancer 84.8% of patients with ILC had received palliative endocrine therapy compared with 72.5% of patients with IDC.\\nMedian follow-up after diagnosis of metastatic disease was 37.1 months (range 5.2–54.6) with 239 patients (42%) alive at the end of the follow-up period.\\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative chemotherapy was significantly longer for patients with ILC (median not yet reached) compared with IDC (median 16.9 months, 95% Confidence Interval (CI) 9.6-22.3, P = 0.001) (Figure 1A). Six months after diagnosis of HR-positive metastatic breast cancer less patients with ILC had received palliative chemotherapy compared with IDC (28.1% versus 39.8% respectively).\\nTime to first palliative chemotherapy (A) and endocrine therapy (B) by histological subtype.\\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative endocrine therapy was significantly shorter for patients with ILC (median, 0.6 months, 95% CI 0.5–0.8) compared with patients with IDC (median, 1.1 months, 95% CI 1.0–1.5, P = 0.0001) (Figure 1B). Six months after diagnosis of metastatic breast cancer 84.8% of patients with ILC had received palliative endocrine therapy compared with 72.5% of patients with IDC.\\n Outcome Median overall survival was 29.4 months (95% CI 22.5–36.6) for patients with HR-positive ILC and 25.4 months (95% CI 21.8–31.7) for patients with HR-positive IDC (P = 0.53).\\nIn multivariable analysis for patients with ILC with palliative endocrine therapy and palliative chemotherapy as time-dependent covariates, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.8, 95% CI 1.7–4.6, P < .0001) compared to no palliative chemotherapy during the observation period. Conversely, early initiation of palliative endocrine therapy was associated with a favorable survival (hazard ratio 0.4, 95% CI 0.2–0.8, P = 0.005) compared to no palliative endocrine therapy during the observation period.\\nIn multivariable analysis for patients with IDC, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.1, 95% CI 1.6–2.7. P < .0001) when compared with no chemotherapy, whereas early treatment with palliative endocrine therapy was not associated with survival (hazard ratio 0.9, 95% CI 0.6–1.2, P = 0.4).\\nMedian overall survival was 29.4 months (95% CI 22.5–36.6) for patients with HR-positive ILC and 25.4 months (95% CI 21.8–31.7) for patients with HR-positive IDC (P = 0.53).\\nIn multivariable analysis for patients with ILC with palliative endocrine therapy and palliative chemotherapy as time-dependent covariates, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.8, 95% CI 1.7–4.6, P < .0001) compared to no palliative chemotherapy during the observation period. Conversely, early initiation of palliative endocrine therapy was associated with a favorable survival (hazard ratio 0.4, 95% CI 0.2–0.8, P = 0.005) compared to no palliative endocrine therapy during the observation period.\\nIn multivariable analysis for patients with IDC, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.1, 95% CI 1.6–2.7. P < .0001) when compared with no chemotherapy, whereas early treatment with palliative endocrine therapy was not associated with survival (hazard ratio 0.9, 95% CI 0.6–1.2, P = 0.4).\\n Residual survival: six months after diagnosis For ILC, the residual survival was significantly longer for patients not treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (median 44.0 months, 95% CI 30.2-not yet reached) versus patients treated with palliative chemotherapy (median 15.2 months, 95% CI 7.8–19.2) (Figure 2A). For IDC, residual survival when not treated with palliative chemotherapy was 41.8 (95% CI 33.3-not yet reached) compared with 16.8 months (95% CI 13.6–22.5) for patients treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (Figure 2B).\\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative chemotherapy during the first six months after diagnosis of metastatic breast cancer.\\nReversely, for ILC-patients with palliative endocrine treatment within the first six months after diagnosis of metastatic breast cancer residual survival was 37.5 months (95% CI 26.9-not yet reached) compared with 11.2 months (95% CI 3.0–20.4) for patients with HR-positive metastatic ILC without palliative endocrine therapy before the first six months (Figure 3A). For patients with HR-positive metastatic IDC treated with palliative endocrine therapy during the first six months, residual survival was 33.5 months (95% CI 25.7-not yet reached) compared with 18.9 months (95% CI 14.0-26.8) for patients with HR-positive metastatic IDC without palliative endocrine therapy in the first six months of metastatic disease (Figure 3B).\\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative endocrine therapy during the first six months after diagnosis of metastatic breast cancer.\\nFor ILC, the residual survival was significantly longer for patients not treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (median 44.0 months, 95% CI 30.2-not yet reached) versus patients treated with palliative chemotherapy (median 15.2 months, 95% CI 7.8–19.2) (Figure 2A). For IDC, residual survival when not treated with palliative chemotherapy was 41.8 (95% CI 33.3-not yet reached) compared with 16.8 months (95% CI 13.6–22.5) for patients treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (Figure 2B).\\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative chemotherapy during the first six months after diagnosis of metastatic breast cancer.\\nReversely, for ILC-patients with palliative endocrine treatment within the first six months after diagnosis of metastatic breast cancer residual survival was 37.5 months (95% CI 26.9-not yet reached) compared with 11.2 months (95% CI 3.0–20.4) for patients with HR-positive metastatic ILC without palliative endocrine therapy before the first six months (Figure 3A). For patients with HR-positive metastatic IDC treated with palliative endocrine therapy during the first six months, residual survival was 33.5 months (95% CI 25.7-not yet reached) compared with 18.9 months (95% CI 14.0-26.8) for patients with HR-positive metastatic IDC without palliative endocrine therapy in the first six months of metastatic disease (Figure 3B).\\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative endocrine therapy during the first six months after diagnosis of metastatic breast cancer.\",\n \"Of the 568 patients with HR-positive metastatic breast cancer, 23% had (mixed type) ILC (131 patients), hereafter referred to as ILC and 77% had IDC (437 patients) (Table 1).\\nAbbreviations; HR+ hormone receptor positive, N number, SBR Scarff Bloom Richardson, MFI metastatic-free interval * ≥ 1 of the aforementioned initial sites of metastasis.\\nMetastatic breast cancer patients with ILC were older at the time of primary breast cancer diagnosis, compared with patients with IDC (median age at diagnosis 62 years for ILC versus 58 years for IDC, P = 0.03). At initial presentation, patients with HR-positive ILC had larger tumors (T2-3, 62.5% versus 49.7%, P = 0.002) and slightly more node-positive disease (66% versus 56.9%, P = 0.10), but with less often a HER2 positive status (7.6% versus 15.3%, P = 0.02) and lower grade (not significant) compared with patients with HR-positive IDC. No differences were seen in use of adjuvant chemotherapy or endocrine therapy between the histological subtypes.\\nBone was the most common initial site of distant metastasis in both HR-positive histological subtypes, although bone metastasis as initial site was more frequently observed for patients with ILC compared to those with IDC (46.5% versus 34.8%, P = 0.01). Fewer patients with ILC were diagnosed with multiple sites of distant metastasis compared to those with IDC (23.7% versus 30.9%, P = 0.11).\",\n \"Median follow-up after diagnosis of metastatic disease was 37.1 months (range 5.2–54.6) with 239 patients (42%) alive at the end of the follow-up period.\\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative chemotherapy was significantly longer for patients with ILC (median not yet reached) compared with IDC (median 16.9 months, 95% Confidence Interval (CI) 9.6-22.3, P = 0.001) (Figure 1A). Six months after diagnosis of HR-positive metastatic breast cancer less patients with ILC had received palliative chemotherapy compared with IDC (28.1% versus 39.8% respectively).\\nTime to first palliative chemotherapy (A) and endocrine therapy (B) by histological subtype.\\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative endocrine therapy was significantly shorter for patients with ILC (median, 0.6 months, 95% CI 0.5–0.8) compared with patients with IDC (median, 1.1 months, 95% CI 1.0–1.5, P = 0.0001) (Figure 1B). Six months after diagnosis of metastatic breast cancer 84.8% of patients with ILC had received palliative endocrine therapy compared with 72.5% of patients with IDC.\",\n \"Median overall survival was 29.4 months (95% CI 22.5–36.6) for patients with HR-positive ILC and 25.4 months (95% CI 21.8–31.7) for patients with HR-positive IDC (P = 0.53).\\nIn multivariable analysis for patients with ILC with palliative endocrine therapy and palliative chemotherapy as time-dependent covariates, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.8, 95% CI 1.7–4.6, P < .0001) compared to no palliative chemotherapy during the observation period. Conversely, early initiation of palliative endocrine therapy was associated with a favorable survival (hazard ratio 0.4, 95% CI 0.2–0.8, P = 0.005) compared to no palliative endocrine therapy during the observation period.\\nIn multivariable analysis for patients with IDC, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.1, 95% CI 1.6–2.7. P < .0001) when compared with no chemotherapy, whereas early treatment with palliative endocrine therapy was not associated with survival (hazard ratio 0.9, 95% CI 0.6–1.2, P = 0.4).\",\n \"For ILC, the residual survival was significantly longer for patients not treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (median 44.0 months, 95% CI 30.2-not yet reached) versus patients treated with palliative chemotherapy (median 15.2 months, 95% CI 7.8–19.2) (Figure 2A). For IDC, residual survival when not treated with palliative chemotherapy was 41.8 (95% CI 33.3-not yet reached) compared with 16.8 months (95% CI 13.6–22.5) for patients treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (Figure 2B).\\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative chemotherapy during the first six months after diagnosis of metastatic breast cancer.\\nReversely, for ILC-patients with palliative endocrine treatment within the first six months after diagnosis of metastatic breast cancer residual survival was 37.5 months (95% CI 26.9-not yet reached) compared with 11.2 months (95% CI 3.0–20.4) for patients with HR-positive metastatic ILC without palliative endocrine therapy before the first six months (Figure 3A). For patients with HR-positive metastatic IDC treated with palliative endocrine therapy during the first six months, residual survival was 33.5 months (95% CI 25.7-not yet reached) compared with 18.9 months (95% CI 14.0-26.8) for patients with HR-positive metastatic IDC without palliative endocrine therapy in the first six months of metastatic disease (Figure 3B).\\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative endocrine therapy during the first six months after diagnosis of metastatic breast cancer.\",\n \"To our knowledge, the role of histological subtype in palliative systemic treatment of metastatic breast cancer has not been studied before. In a cohort of 568 HR-positive metastatic breast cancer patients, we showed that patients with HR-positive ILC had more often bone and less often multiple sites as initial site of distant metastasis compared to those with HR-positive IDC. Six months after metastatic diagnosis, patients with HR-positive ILC had received significantly less often chemotherapy (28% versus 40%) and more often endocrine therapy (85% versus 73%) as compared to patients with HR-positive IDC. Patients starting with palliative chemotherapy during the first six months had a significantly shorter median residual survival thereafter as compared to those who did not (15 months versus 44 months for ILC, and 17 and 42 months for IDC, respectively). Reversely, patients treated with endocrine therapy in the first six months had a longer median residual survival compared to those who did not (37 months versus 11 months for ILC and 33 and 19 months for IDC, respectively). Our results confirm that, in addition to HR-status, histology correlates with presentation of metastatic disease by number and type of distant metastatic sites, and thereby affects treatment-decision making in real-life.\\nThe intriguing scientific question is to determine how histological subtype influences the metastatic spread in HR-positive breast cancer. It is hypothesized that the loss of E-cadherin, a cell-cell adhesion molecule, in ILC may result in less adhesiveness of the tumor cells and therefore disseminate and infiltrate certain distant locations more easily [1].\\nIn this HR-positive metastatic breast cancer cohort we also looked at the association of palliative systemic treatment with survival of patients with metastatic breast cancer for both histological subtypes. With the favorable presentation of distant metastasis by number and location of patients with HR-positive ILC as compared to HR-positive IDC, one would expect a more indolent disease course and a better outcome. However, we showed that for patients with HR-positive breast cancer, overall disease outcome was comparable irrespective of histology and irrespective of different treatment choices made in real-life, as discussed above. And interestingly, the efficacy of chemotherapy - once the treatment choice was made - was comparable irrespective of histology. However, treatment with palliative endocrine therapy was only associated with a favorable prognosis of patients with HR-positive ILC, whereas this was not seen in patients with HR-positive IDC, also suggesting the impact of histology over HR-status. This is in concordance with the early breast cancer setting, in which there is evidence suggesting differences in efficacy of systemic therapy between the two histological subtypes [3–5, 12].\\nAlthough there is some proof that histology could be helpful in treatment decision-making [13–15], the current guidelines do not include histological subtype as an indicator for the use of systemic treatment in general, or for a specific regimen [16, 17]. Interestingly, in the adjuvant setting, histology has been shown to be of importance when deciding between the use of breast-conserving surgery or mastectomy after neo-adjuvant chemotherapy [18]. The current study shows that histological subtype can be of predictive value for HR-positive metastatic breast cancer with regards to the effectiveness of early initiation of palliative endocrine therapy. It may be possible that in patients with HR-positive lobular breast cancer, the higher incidence of bone metastases as initial metastatic site partly accounts for the initial choice of endocrine therapy. It underlines the relevance of acknowledging a different metastatic pattern, and thereby different initial treatment choices, between breast cancer patients with ductal versus lobular histology.\\nMuch more than in the adjuvant setting, treatment decisions in the metastatic setting are based on the observed and anticipated clinical course of the disease, which is not only determined by the tumor characteristics as described earlier. Also age, performance status, previous therapies and toxicities, comorbidity and patient and doctor preferences play a role. The complexity of this process, together with the retrospective design of our study make it impossible to identify and rule out confounding by indication. Furthermore, localization of the metastases (visceral versus bone metastasis for instance) could be related to symptomatology and thereby timing of detection, and could therefore have introduced lead time bias.\\nIn this study patients with pure lobular carcinoma and mixed lobular carcinomas were combined for the analyses. In other studies on histological subtypes of breast cancer mixed and lobular carcinoma had similar outcome [14, 19]. Even with combining these subgroups, the proportion of HER2-positive tumors was too low to further analyze anti-HER2 therapy. In the adjuvant treatment setting the magnitude of benefit from adjuvant trastuzumab between patients with ILC and IDC was shown not to be different [20]. Another limitation of our study is that patient numbers were too small to perform analysis on specific chemotherapy regimens. Microarray analysis have demonstrated that ILC and IDC can be distinguished on the basis of genomic and expression profiles [21]. The increasing knowledge on genomic differences between ILC and IDC can help to answer questions on in vivo chemosensitivity. For example, topoisomerase-IIα gene amplification is a predictive biomarker for response to anthracyclines and in ILC this gene amplification is lacking, which could help understand the poor responsiveness of ILC to neo-adjuvant chemotherapy, including anthracyclines [22]. This genetic information can help guide further research and may eventually be useful in making treatment decisions for histological breast cancer subtypes.\\nIn conclusion, to our knowledge, this is the first study that investigates the role of histological subtype in HR-positive metastatic breast cancer. Although survival was comparable for the two histological subtypes, this was achieved with different treatment strategies. As patients with HR-positive ILC were less likely to receive chemotherapy than those with HR-positive IDC, histology may be a relevant factor in treatment-decision making. For a more definite conclusion on the role of histology, we recommend to incorporate histological subtype as a stratification factor in future clinical trials.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","RESULTS","Patient characteristics","Palliative systemic treatment","Outcome","Residual survival: six months after diagnosis","DISCUSSION","MATERIALS AND METHODS"],"string":"[\n \"INTRODUCTION\",\n \"RESULTS\",\n \"Patient characteristics\",\n \"Palliative systemic treatment\",\n \"Outcome\",\n \"Residual survival: six months after diagnosis\",\n \"DISCUSSION\",\n \"MATERIALS AND METHODS\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["The two most frequent histological subtypes of breast cancer are invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC), with IDC comprising 75–80% and ILC 5–15% of all breast cancer cases. ILC is being associated with larger tumor size at presentation, more bilateral and multifocal involvement and with a different pattern of metastatic spread compared with IDC [1, 2]. Furthermore, ILC is more often HR-positive, HER2-negative, with lower S-phase fraction and less often positive for the tumor suppressor gene p53, compared with IDC [1]. Also, treatment response is known to be different. In a combined analysis on a number of retrospective series, the pathological complete response rate of neo-adjuvant chemotherapy was significantly lower in ILC (1.7%) than in IDC (11.6%) [3]. In the adjuvant setting, retrospective data suggest a higher efficacy of endocrine therapy for ILC than IDC [4]. More recently, a large retrospective study strongly suggested that HR-positive breast cancer patients with ILC do not seem to benefit from adjuvant chemotherapy in addition to endocrine therapy [5].\nWhether these differences have a prognostic impact is controversial. Some studies found no effect of histology on survival [1, 6, 7], others found a better prognosis for patients with ILC compared to those with IDC [8–10] and some found a change in prognosis over time with a better prognosis for ILC during the first years of follow-up and a worse prognosis during later years [2, 11].\nSo far, studies on histological subtypes consider early breast cancer and not metastatic breast cancer. The aim of this study was to describe the differences between IDC and ILC with respect to patient and tumor characteristics, treatment-choices and outcome in metastatic breast cancer. In order to account for the effect of the hormone receptor (HR) on outcome and treatment-decision making, we only included patients with HR-positive breast cancer."," Patient characteristics Of the 568 patients with HR-positive metastatic breast cancer, 23% had (mixed type) ILC (131 patients), hereafter referred to as ILC and 77% had IDC (437 patients) (Table 1).\nAbbreviations; HR+ hormone receptor positive, N number, SBR Scarff Bloom Richardson, MFI metastatic-free interval * ≥ 1 of the aforementioned initial sites of metastasis.\nMetastatic breast cancer patients with ILC were older at the time of primary breast cancer diagnosis, compared with patients with IDC (median age at diagnosis 62 years for ILC versus 58 years for IDC, P = 0.03). At initial presentation, patients with HR-positive ILC had larger tumors (T2-3, 62.5% versus 49.7%, P = 0.002) and slightly more node-positive disease (66% versus 56.9%, P = 0.10), but with less often a HER2 positive status (7.6% versus 15.3%, P = 0.02) and lower grade (not significant) compared with patients with HR-positive IDC. No differences were seen in use of adjuvant chemotherapy or endocrine therapy between the histological subtypes.\nBone was the most common initial site of distant metastasis in both HR-positive histological subtypes, although bone metastasis as initial site was more frequently observed for patients with ILC compared to those with IDC (46.5% versus 34.8%, P = 0.01). Fewer patients with ILC were diagnosed with multiple sites of distant metastasis compared to those with IDC (23.7% versus 30.9%, P = 0.11).\nOf the 568 patients with HR-positive metastatic breast cancer, 23% had (mixed type) ILC (131 patients), hereafter referred to as ILC and 77% had IDC (437 patients) (Table 1).\nAbbreviations; HR+ hormone receptor positive, N number, SBR Scarff Bloom Richardson, MFI metastatic-free interval * ≥ 1 of the aforementioned initial sites of metastasis.\nMetastatic breast cancer patients with ILC were older at the time of primary breast cancer diagnosis, compared with patients with IDC (median age at diagnosis 62 years for ILC versus 58 years for IDC, P = 0.03). At initial presentation, patients with HR-positive ILC had larger tumors (T2-3, 62.5% versus 49.7%, P = 0.002) and slightly more node-positive disease (66% versus 56.9%, P = 0.10), but with less often a HER2 positive status (7.6% versus 15.3%, P = 0.02) and lower grade (not significant) compared with patients with HR-positive IDC. No differences were seen in use of adjuvant chemotherapy or endocrine therapy between the histological subtypes.\nBone was the most common initial site of distant metastasis in both HR-positive histological subtypes, although bone metastasis as initial site was more frequently observed for patients with ILC compared to those with IDC (46.5% versus 34.8%, P = 0.01). Fewer patients with ILC were diagnosed with multiple sites of distant metastasis compared to those with IDC (23.7% versus 30.9%, P = 0.11).\n Palliative systemic treatment Median follow-up after diagnosis of metastatic disease was 37.1 months (range 5.2–54.6) with 239 patients (42%) alive at the end of the follow-up period.\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative chemotherapy was significantly longer for patients with ILC (median not yet reached) compared with IDC (median 16.9 months, 95% Confidence Interval (CI) 9.6-22.3, P = 0.001) (Figure 1A). Six months after diagnosis of HR-positive metastatic breast cancer less patients with ILC had received palliative chemotherapy compared with IDC (28.1% versus 39.8% respectively).\nTime to first palliative chemotherapy (A) and endocrine therapy (B) by histological subtype.\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative endocrine therapy was significantly shorter for patients with ILC (median, 0.6 months, 95% CI 0.5–0.8) compared with patients with IDC (median, 1.1 months, 95% CI 1.0–1.5, P = 0.0001) (Figure 1B). Six months after diagnosis of metastatic breast cancer 84.8% of patients with ILC had received palliative endocrine therapy compared with 72.5% of patients with IDC.\nMedian follow-up after diagnosis of metastatic disease was 37.1 months (range 5.2–54.6) with 239 patients (42%) alive at the end of the follow-up period.\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative chemotherapy was significantly longer for patients with ILC (median not yet reached) compared with IDC (median 16.9 months, 95% Confidence Interval (CI) 9.6-22.3, P = 0.001) (Figure 1A). Six months after diagnosis of HR-positive metastatic breast cancer less patients with ILC had received palliative chemotherapy compared with IDC (28.1% versus 39.8% respectively).\nTime to first palliative chemotherapy (A) and endocrine therapy (B) by histological subtype.\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative endocrine therapy was significantly shorter for patients with ILC (median, 0.6 months, 95% CI 0.5–0.8) compared with patients with IDC (median, 1.1 months, 95% CI 1.0–1.5, P = 0.0001) (Figure 1B). Six months after diagnosis of metastatic breast cancer 84.8% of patients with ILC had received palliative endocrine therapy compared with 72.5% of patients with IDC.\n Outcome Median overall survival was 29.4 months (95% CI 22.5–36.6) for patients with HR-positive ILC and 25.4 months (95% CI 21.8–31.7) for patients with HR-positive IDC (P = 0.53).\nIn multivariable analysis for patients with ILC with palliative endocrine therapy and palliative chemotherapy as time-dependent covariates, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.8, 95% CI 1.7–4.6, P < .0001) compared to no palliative chemotherapy during the observation period. Conversely, early initiation of palliative endocrine therapy was associated with a favorable survival (hazard ratio 0.4, 95% CI 0.2–0.8, P = 0.005) compared to no palliative endocrine therapy during the observation period.\nIn multivariable analysis for patients with IDC, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.1, 95% CI 1.6–2.7. P < .0001) when compared with no chemotherapy, whereas early treatment with palliative endocrine therapy was not associated with survival (hazard ratio 0.9, 95% CI 0.6–1.2, P = 0.4).\nMedian overall survival was 29.4 months (95% CI 22.5–36.6) for patients with HR-positive ILC and 25.4 months (95% CI 21.8–31.7) for patients with HR-positive IDC (P = 0.53).\nIn multivariable analysis for patients with ILC with palliative endocrine therapy and palliative chemotherapy as time-dependent covariates, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.8, 95% CI 1.7–4.6, P < .0001) compared to no palliative chemotherapy during the observation period. Conversely, early initiation of palliative endocrine therapy was associated with a favorable survival (hazard ratio 0.4, 95% CI 0.2–0.8, P = 0.005) compared to no palliative endocrine therapy during the observation period.\nIn multivariable analysis for patients with IDC, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.1, 95% CI 1.6–2.7. P < .0001) when compared with no chemotherapy, whereas early treatment with palliative endocrine therapy was not associated with survival (hazard ratio 0.9, 95% CI 0.6–1.2, P = 0.4).\n Residual survival: six months after diagnosis For ILC, the residual survival was significantly longer for patients not treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (median 44.0 months, 95% CI 30.2-not yet reached) versus patients treated with palliative chemotherapy (median 15.2 months, 95% CI 7.8–19.2) (Figure 2A). For IDC, residual survival when not treated with palliative chemotherapy was 41.8 (95% CI 33.3-not yet reached) compared with 16.8 months (95% CI 13.6–22.5) for patients treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (Figure 2B).\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative chemotherapy during the first six months after diagnosis of metastatic breast cancer.\nReversely, for ILC-patients with palliative endocrine treatment within the first six months after diagnosis of metastatic breast cancer residual survival was 37.5 months (95% CI 26.9-not yet reached) compared with 11.2 months (95% CI 3.0–20.4) for patients with HR-positive metastatic ILC without palliative endocrine therapy before the first six months (Figure 3A). For patients with HR-positive metastatic IDC treated with palliative endocrine therapy during the first six months, residual survival was 33.5 months (95% CI 25.7-not yet reached) compared with 18.9 months (95% CI 14.0-26.8) for patients with HR-positive metastatic IDC without palliative endocrine therapy in the first six months of metastatic disease (Figure 3B).\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative endocrine therapy during the first six months after diagnosis of metastatic breast cancer.\nFor ILC, the residual survival was significantly longer for patients not treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (median 44.0 months, 95% CI 30.2-not yet reached) versus patients treated with palliative chemotherapy (median 15.2 months, 95% CI 7.8–19.2) (Figure 2A). For IDC, residual survival when not treated with palliative chemotherapy was 41.8 (95% CI 33.3-not yet reached) compared with 16.8 months (95% CI 13.6–22.5) for patients treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (Figure 2B).\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative chemotherapy during the first six months after diagnosis of metastatic breast cancer.\nReversely, for ILC-patients with palliative endocrine treatment within the first six months after diagnosis of metastatic breast cancer residual survival was 37.5 months (95% CI 26.9-not yet reached) compared with 11.2 months (95% CI 3.0–20.4) for patients with HR-positive metastatic ILC without palliative endocrine therapy before the first six months (Figure 3A). For patients with HR-positive metastatic IDC treated with palliative endocrine therapy during the first six months, residual survival was 33.5 months (95% CI 25.7-not yet reached) compared with 18.9 months (95% CI 14.0-26.8) for patients with HR-positive metastatic IDC without palliative endocrine therapy in the first six months of metastatic disease (Figure 3B).\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative endocrine therapy during the first six months after diagnosis of metastatic breast cancer.","Of the 568 patients with HR-positive metastatic breast cancer, 23% had (mixed type) ILC (131 patients), hereafter referred to as ILC and 77% had IDC (437 patients) (Table 1).\nAbbreviations; HR+ hormone receptor positive, N number, SBR Scarff Bloom Richardson, MFI metastatic-free interval * ≥ 1 of the aforementioned initial sites of metastasis.\nMetastatic breast cancer patients with ILC were older at the time of primary breast cancer diagnosis, compared with patients with IDC (median age at diagnosis 62 years for ILC versus 58 years for IDC, P = 0.03). At initial presentation, patients with HR-positive ILC had larger tumors (T2-3, 62.5% versus 49.7%, P = 0.002) and slightly more node-positive disease (66% versus 56.9%, P = 0.10), but with less often a HER2 positive status (7.6% versus 15.3%, P = 0.02) and lower grade (not significant) compared with patients with HR-positive IDC. No differences were seen in use of adjuvant chemotherapy or endocrine therapy between the histological subtypes.\nBone was the most common initial site of distant metastasis in both HR-positive histological subtypes, although bone metastasis as initial site was more frequently observed for patients with ILC compared to those with IDC (46.5% versus 34.8%, P = 0.01). Fewer patients with ILC were diagnosed with multiple sites of distant metastasis compared to those with IDC (23.7% versus 30.9%, P = 0.11).","Median follow-up after diagnosis of metastatic disease was 37.1 months (range 5.2–54.6) with 239 patients (42%) alive at the end of the follow-up period.\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative chemotherapy was significantly longer for patients with ILC (median not yet reached) compared with IDC (median 16.9 months, 95% Confidence Interval (CI) 9.6-22.3, P = 0.001) (Figure 1A). Six months after diagnosis of HR-positive metastatic breast cancer less patients with ILC had received palliative chemotherapy compared with IDC (28.1% versus 39.8% respectively).\nTime to first palliative chemotherapy (A) and endocrine therapy (B) by histological subtype.\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative endocrine therapy was significantly shorter for patients with ILC (median, 0.6 months, 95% CI 0.5–0.8) compared with patients with IDC (median, 1.1 months, 95% CI 1.0–1.5, P = 0.0001) (Figure 1B). Six months after diagnosis of metastatic breast cancer 84.8% of patients with ILC had received palliative endocrine therapy compared with 72.5% of patients with IDC.","Median overall survival was 29.4 months (95% CI 22.5–36.6) for patients with HR-positive ILC and 25.4 months (95% CI 21.8–31.7) for patients with HR-positive IDC (P = 0.53).\nIn multivariable analysis for patients with ILC with palliative endocrine therapy and palliative chemotherapy as time-dependent covariates, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.8, 95% CI 1.7–4.6, P < .0001) compared to no palliative chemotherapy during the observation period. Conversely, early initiation of palliative endocrine therapy was associated with a favorable survival (hazard ratio 0.4, 95% CI 0.2–0.8, P = 0.005) compared to no palliative endocrine therapy during the observation period.\nIn multivariable analysis for patients with IDC, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.1, 95% CI 1.6–2.7. P < .0001) when compared with no chemotherapy, whereas early treatment with palliative endocrine therapy was not associated with survival (hazard ratio 0.9, 95% CI 0.6–1.2, P = 0.4).","For ILC, the residual survival was significantly longer for patients not treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (median 44.0 months, 95% CI 30.2-not yet reached) versus patients treated with palliative chemotherapy (median 15.2 months, 95% CI 7.8–19.2) (Figure 2A). For IDC, residual survival when not treated with palliative chemotherapy was 41.8 (95% CI 33.3-not yet reached) compared with 16.8 months (95% CI 13.6–22.5) for patients treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (Figure 2B).\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative chemotherapy during the first six months after diagnosis of metastatic breast cancer.\nReversely, for ILC-patients with palliative endocrine treatment within the first six months after diagnosis of metastatic breast cancer residual survival was 37.5 months (95% CI 26.9-not yet reached) compared with 11.2 months (95% CI 3.0–20.4) for patients with HR-positive metastatic ILC without palliative endocrine therapy before the first six months (Figure 3A). For patients with HR-positive metastatic IDC treated with palliative endocrine therapy during the first six months, residual survival was 33.5 months (95% CI 25.7-not yet reached) compared with 18.9 months (95% CI 14.0-26.8) for patients with HR-positive metastatic IDC without palliative endocrine therapy in the first six months of metastatic disease (Figure 3B).\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative endocrine therapy during the first six months after diagnosis of metastatic breast cancer.","To our knowledge, the role of histological subtype in palliative systemic treatment of metastatic breast cancer has not been studied before. In a cohort of 568 HR-positive metastatic breast cancer patients, we showed that patients with HR-positive ILC had more often bone and less often multiple sites as initial site of distant metastasis compared to those with HR-positive IDC. Six months after metastatic diagnosis, patients with HR-positive ILC had received significantly less often chemotherapy (28% versus 40%) and more often endocrine therapy (85% versus 73%) as compared to patients with HR-positive IDC. Patients starting with palliative chemotherapy during the first six months had a significantly shorter median residual survival thereafter as compared to those who did not (15 months versus 44 months for ILC, and 17 and 42 months for IDC, respectively). Reversely, patients treated with endocrine therapy in the first six months had a longer median residual survival compared to those who did not (37 months versus 11 months for ILC and 33 and 19 months for IDC, respectively). Our results confirm that, in addition to HR-status, histology correlates with presentation of metastatic disease by number and type of distant metastatic sites, and thereby affects treatment-decision making in real-life.\nThe intriguing scientific question is to determine how histological subtype influences the metastatic spread in HR-positive breast cancer. It is hypothesized that the loss of E-cadherin, a cell-cell adhesion molecule, in ILC may result in less adhesiveness of the tumor cells and therefore disseminate and infiltrate certain distant locations more easily [1].\nIn this HR-positive metastatic breast cancer cohort we also looked at the association of palliative systemic treatment with survival of patients with metastatic breast cancer for both histological subtypes. With the favorable presentation of distant metastasis by number and location of patients with HR-positive ILC as compared to HR-positive IDC, one would expect a more indolent disease course and a better outcome. However, we showed that for patients with HR-positive breast cancer, overall disease outcome was comparable irrespective of histology and irrespective of different treatment choices made in real-life, as discussed above. And interestingly, the efficacy of chemotherapy - once the treatment choice was made - was comparable irrespective of histology. However, treatment with palliative endocrine therapy was only associated with a favorable prognosis of patients with HR-positive ILC, whereas this was not seen in patients with HR-positive IDC, also suggesting the impact of histology over HR-status. This is in concordance with the early breast cancer setting, in which there is evidence suggesting differences in efficacy of systemic therapy between the two histological subtypes [3–5, 12].\nAlthough there is some proof that histology could be helpful in treatment decision-making [13–15], the current guidelines do not include histological subtype as an indicator for the use of systemic treatment in general, or for a specific regimen [16, 17]. Interestingly, in the adjuvant setting, histology has been shown to be of importance when deciding between the use of breast-conserving surgery or mastectomy after neo-adjuvant chemotherapy [18]. The current study shows that histological subtype can be of predictive value for HR-positive metastatic breast cancer with regards to the effectiveness of early initiation of palliative endocrine therapy. It may be possible that in patients with HR-positive lobular breast cancer, the higher incidence of bone metastases as initial metastatic site partly accounts for the initial choice of endocrine therapy. It underlines the relevance of acknowledging a different metastatic pattern, and thereby different initial treatment choices, between breast cancer patients with ductal versus lobular histology.\nMuch more than in the adjuvant setting, treatment decisions in the metastatic setting are based on the observed and anticipated clinical course of the disease, which is not only determined by the tumor characteristics as described earlier. Also age, performance status, previous therapies and toxicities, comorbidity and patient and doctor preferences play a role. The complexity of this process, together with the retrospective design of our study make it impossible to identify and rule out confounding by indication. Furthermore, localization of the metastases (visceral versus bone metastasis for instance) could be related to symptomatology and thereby timing of detection, and could therefore have introduced lead time bias.\nIn this study patients with pure lobular carcinoma and mixed lobular carcinomas were combined for the analyses. In other studies on histological subtypes of breast cancer mixed and lobular carcinoma had similar outcome [14, 19]. Even with combining these subgroups, the proportion of HER2-positive tumors was too low to further analyze anti-HER2 therapy. In the adjuvant treatment setting the magnitude of benefit from adjuvant trastuzumab between patients with ILC and IDC was shown not to be different [20]. Another limitation of our study is that patient numbers were too small to perform analysis on specific chemotherapy regimens. Microarray analysis have demonstrated that ILC and IDC can be distinguished on the basis of genomic and expression profiles [21]. The increasing knowledge on genomic differences between ILC and IDC can help to answer questions on in vivo chemosensitivity. For example, topoisomerase-IIα gene amplification is a predictive biomarker for response to anthracyclines and in ILC this gene amplification is lacking, which could help understand the poor responsiveness of ILC to neo-adjuvant chemotherapy, including anthracyclines [22]. This genetic information can help guide further research and may eventually be useful in making treatment decisions for histological breast cancer subtypes.\nIn conclusion, to our knowledge, this is the first study that investigates the role of histological subtype in HR-positive metastatic breast cancer. Although survival was comparable for the two histological subtypes, this was achieved with different treatment strategies. As patients with HR-positive ILC were less likely to receive chemotherapy than those with HR-positive IDC, histology may be a relevant factor in treatment-decision making. For a more definite conclusion on the role of histology, we recommend to incorporate histological subtype as a stratification factor in future clinical trials."," Patient selection We identified all patients diagnosed with metastatic breast cancer between 2007–2009 in eight hospitals in the South-East of the Netherlands. All patients with metastatic breast cancer were selected irrespective of the date of primary breast cancer diagnosis (also including patients with de novo metastatic breast cancer; 18.5% of patients with IDC and 19.9% of patients with ILC), with the exception of patients with a diagnosis of primary breast cancer before 1990, due to limitations in the availability of data on the primary tumor and initial treatment.\nHistology was classified according to the International Classification of Diseases for Oncology (ICD-O) [23]. ILC and mixed histology were defined as code 8520 and 8522. IDC was defined as code 8500. From the total of 815 metastatic breast cancer patients, we excluded 76 patients with either unknown histology or histological subtypes other than IDC or ILC. Of the remaining 739 metastatic breast cancer patients, we excluded 171 patients with HR-negative tumors (27% of patients with IDC and 8% of patients with ILC) in order to rule out the impact of HR status. In total, our study population consisted of 568 patients divided in two groups based on histology; one group of 437 patients with IDC and the other group of 131 patients with ILC.\nWe identified all patients diagnosed with metastatic breast cancer between 2007–2009 in eight hospitals in the South-East of the Netherlands. All patients with metastatic breast cancer were selected irrespective of the date of primary breast cancer diagnosis (also including patients with de novo metastatic breast cancer; 18.5% of patients with IDC and 19.9% of patients with ILC), with the exception of patients with a diagnosis of primary breast cancer before 1990, due to limitations in the availability of data on the primary tumor and initial treatment.\nHistology was classified according to the International Classification of Diseases for Oncology (ICD-O) [23]. ILC and mixed histology were defined as code 8520 and 8522. IDC was defined as code 8500. From the total of 815 metastatic breast cancer patients, we excluded 76 patients with either unknown histology or histological subtypes other than IDC or ILC. Of the remaining 739 metastatic breast cancer patients, we excluded 171 patients with HR-negative tumors (27% of patients with IDC and 8% of patients with ILC) in order to rule out the impact of HR status. In total, our study population consisted of 568 patients divided in two groups based on histology; one group of 437 patients with IDC and the other group of 131 patients with ILC.\n Data collection Information was collected on patient and tumor characteristics, treatment and outcome. Tumors were characterized by the sixth edition of the TNM classification of malignant tumors [24] and Scarff Bloom Richardson (SBR) histological grading [25]. Estrogen receptor (ER) and progesterone receptor (PR) positivity was defined as positive nuclear staining of ≥ 10%. HER2 positivity was defined as immunohistochemistry score of 3+ or 2+ with positive FISH. In case of missing HER2 status a dedicated pathologist centrally reviewed missing data when material was available. Initial sites of metastasis were categorized as: bone, visceral (including lung, liver, pleural, peritoneal, pericardial and lymphangitic carcinomatosis), brain (including leptomeningeal and CNS), skin and lymph nodes, and multiple metastases (more than one of the metastatic sites).\nInformation was collected on patient and tumor characteristics, treatment and outcome. Tumors were characterized by the sixth edition of the TNM classification of malignant tumors [24] and Scarff Bloom Richardson (SBR) histological grading [25]. Estrogen receptor (ER) and progesterone receptor (PR) positivity was defined as positive nuclear staining of ≥ 10%. HER2 positivity was defined as immunohistochemistry score of 3+ or 2+ with positive FISH. In case of missing HER2 status a dedicated pathologist centrally reviewed missing data when material was available. Initial sites of metastasis were categorized as: bone, visceral (including lung, liver, pleural, peritoneal, pericardial and lymphangitic carcinomatosis), brain (including leptomeningeal and CNS), skin and lymph nodes, and multiple metastases (more than one of the metastatic sites).\n Statistical analysis Baseline characteristics between the two histological groups were compared using chi-square tests for categorical variables and Wilcoxon rank sum tests for continuous variables.\nMetastatic-free interval was defined as time between date of primary diagnosis and date of first distant metastasis. Overall survival after diagnosis of metastatic breast cancer was defined as time between date of first distant metastasis and date of death. Survival curves and time to first palliative systemic therapy (either chemotherapy or endocrine therapy) were estimated using the Kaplan-Meier method and compared using log-rank tests. All patients still alive were censored at the date of last follow-up of each individual patient. Patients who died without palliative therapy were censored at the date of death in the analysis of time to first palliative therapy.\nTo explore the association of palliative systemic therapy with the survival of patients with metastatic breast cancer for both histological subtypes a Cox proportional hazards model was performed with palliative chemotherapy and endocrine therapy as a time-dependent covariate, since the administration of treatment can change over time and is dependent on the time available for each patient to receive the treatment. We did not explore the association between palliative targeted therapy, such as trastuzumab and bevacizumab, and survival since the number of patients with IDC and ILC receiving targeted therapy was very low. Since all these patients received targeted therapy with chemotherapy, these patients were included in the analysis on initial chemotherapy. In addition, the landmark method was used to estimate survival after a specific time-point, the so-called residual survival [26]. As we were interested to learn about the obtained survival in relation to the initial palliative treatment choices, we chose six months after diagnosis of metastatic breast cancer as landmark. Consequently, patients who already died within 6 months were excluded for the residual survival curves.\nAll analyses were performed using SAS version 9.2. All reported P-values are two-sided and P-value ≤ 0.05 was considered statistically significant.\nBaseline characteristics between the two histological groups were compared using chi-square tests for categorical variables and Wilcoxon rank sum tests for continuous variables.\nMetastatic-free interval was defined as time between date of primary diagnosis and date of first distant metastasis. Overall survival after diagnosis of metastatic breast cancer was defined as time between date of first distant metastasis and date of death. Survival curves and time to first palliative systemic therapy (either chemotherapy or endocrine therapy) were estimated using the Kaplan-Meier method and compared using log-rank tests. All patients still alive were censored at the date of last follow-up of each individual patient. Patients who died without palliative therapy were censored at the date of death in the analysis of time to first palliative therapy.\nTo explore the association of palliative systemic therapy with the survival of patients with metastatic breast cancer for both histological subtypes a Cox proportional hazards model was performed with palliative chemotherapy and endocrine therapy as a time-dependent covariate, since the administration of treatment can change over time and is dependent on the time available for each patient to receive the treatment. We did not explore the association between palliative targeted therapy, such as trastuzumab and bevacizumab, and survival since the number of patients with IDC and ILC receiving targeted therapy was very low. Since all these patients received targeted therapy with chemotherapy, these patients were included in the analysis on initial chemotherapy. In addition, the landmark method was used to estimate survival after a specific time-point, the so-called residual survival [26]. As we were interested to learn about the obtained survival in relation to the initial palliative treatment choices, we chose six months after diagnosis of metastatic breast cancer as landmark. Consequently, patients who already died within 6 months were excluded for the residual survival curves.\nAll analyses were performed using SAS version 9.2. All reported P-values are two-sided and P-value ≤ 0.05 was considered statistically significant."],"string":"[\n \"The two most frequent histological subtypes of breast cancer are invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC), with IDC comprising 75–80% and ILC 5–15% of all breast cancer cases. ILC is being associated with larger tumor size at presentation, more bilateral and multifocal involvement and with a different pattern of metastatic spread compared with IDC [1, 2]. Furthermore, ILC is more often HR-positive, HER2-negative, with lower S-phase fraction and less often positive for the tumor suppressor gene p53, compared with IDC [1]. Also, treatment response is known to be different. In a combined analysis on a number of retrospective series, the pathological complete response rate of neo-adjuvant chemotherapy was significantly lower in ILC (1.7%) than in IDC (11.6%) [3]. In the adjuvant setting, retrospective data suggest a higher efficacy of endocrine therapy for ILC than IDC [4]. More recently, a large retrospective study strongly suggested that HR-positive breast cancer patients with ILC do not seem to benefit from adjuvant chemotherapy in addition to endocrine therapy [5].\\nWhether these differences have a prognostic impact is controversial. Some studies found no effect of histology on survival [1, 6, 7], others found a better prognosis for patients with ILC compared to those with IDC [8–10] and some found a change in prognosis over time with a better prognosis for ILC during the first years of follow-up and a worse prognosis during later years [2, 11].\\nSo far, studies on histological subtypes consider early breast cancer and not metastatic breast cancer. The aim of this study was to describe the differences between IDC and ILC with respect to patient and tumor characteristics, treatment-choices and outcome in metastatic breast cancer. In order to account for the effect of the hormone receptor (HR) on outcome and treatment-decision making, we only included patients with HR-positive breast cancer.\",\n \" Patient characteristics Of the 568 patients with HR-positive metastatic breast cancer, 23% had (mixed type) ILC (131 patients), hereafter referred to as ILC and 77% had IDC (437 patients) (Table 1).\\nAbbreviations; HR+ hormone receptor positive, N number, SBR Scarff Bloom Richardson, MFI metastatic-free interval * ≥ 1 of the aforementioned initial sites of metastasis.\\nMetastatic breast cancer patients with ILC were older at the time of primary breast cancer diagnosis, compared with patients with IDC (median age at diagnosis 62 years for ILC versus 58 years for IDC, P = 0.03). At initial presentation, patients with HR-positive ILC had larger tumors (T2-3, 62.5% versus 49.7%, P = 0.002) and slightly more node-positive disease (66% versus 56.9%, P = 0.10), but with less often a HER2 positive status (7.6% versus 15.3%, P = 0.02) and lower grade (not significant) compared with patients with HR-positive IDC. No differences were seen in use of adjuvant chemotherapy or endocrine therapy between the histological subtypes.\\nBone was the most common initial site of distant metastasis in both HR-positive histological subtypes, although bone metastasis as initial site was more frequently observed for patients with ILC compared to those with IDC (46.5% versus 34.8%, P = 0.01). Fewer patients with ILC were diagnosed with multiple sites of distant metastasis compared to those with IDC (23.7% versus 30.9%, P = 0.11).\\nOf the 568 patients with HR-positive metastatic breast cancer, 23% had (mixed type) ILC (131 patients), hereafter referred to as ILC and 77% had IDC (437 patients) (Table 1).\\nAbbreviations; HR+ hormone receptor positive, N number, SBR Scarff Bloom Richardson, MFI metastatic-free interval * ≥ 1 of the aforementioned initial sites of metastasis.\\nMetastatic breast cancer patients with ILC were older at the time of primary breast cancer diagnosis, compared with patients with IDC (median age at diagnosis 62 years for ILC versus 58 years for IDC, P = 0.03). At initial presentation, patients with HR-positive ILC had larger tumors (T2-3, 62.5% versus 49.7%, P = 0.002) and slightly more node-positive disease (66% versus 56.9%, P = 0.10), but with less often a HER2 positive status (7.6% versus 15.3%, P = 0.02) and lower grade (not significant) compared with patients with HR-positive IDC. No differences were seen in use of adjuvant chemotherapy or endocrine therapy between the histological subtypes.\\nBone was the most common initial site of distant metastasis in both HR-positive histological subtypes, although bone metastasis as initial site was more frequently observed for patients with ILC compared to those with IDC (46.5% versus 34.8%, P = 0.01). Fewer patients with ILC were diagnosed with multiple sites of distant metastasis compared to those with IDC (23.7% versus 30.9%, P = 0.11).\\n Palliative systemic treatment Median follow-up after diagnosis of metastatic disease was 37.1 months (range 5.2–54.6) with 239 patients (42%) alive at the end of the follow-up period.\\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative chemotherapy was significantly longer for patients with ILC (median not yet reached) compared with IDC (median 16.9 months, 95% Confidence Interval (CI) 9.6-22.3, P = 0.001) (Figure 1A). Six months after diagnosis of HR-positive metastatic breast cancer less patients with ILC had received palliative chemotherapy compared with IDC (28.1% versus 39.8% respectively).\\nTime to first palliative chemotherapy (A) and endocrine therapy (B) by histological subtype.\\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative endocrine therapy was significantly shorter for patients with ILC (median, 0.6 months, 95% CI 0.5–0.8) compared with patients with IDC (median, 1.1 months, 95% CI 1.0–1.5, P = 0.0001) (Figure 1B). Six months after diagnosis of metastatic breast cancer 84.8% of patients with ILC had received palliative endocrine therapy compared with 72.5% of patients with IDC.\\nMedian follow-up after diagnosis of metastatic disease was 37.1 months (range 5.2–54.6) with 239 patients (42%) alive at the end of the follow-up period.\\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative chemotherapy was significantly longer for patients with ILC (median not yet reached) compared with IDC (median 16.9 months, 95% Confidence Interval (CI) 9.6-22.3, P = 0.001) (Figure 1A). Six months after diagnosis of HR-positive metastatic breast cancer less patients with ILC had received palliative chemotherapy compared with IDC (28.1% versus 39.8% respectively).\\nTime to first palliative chemotherapy (A) and endocrine therapy (B) by histological subtype.\\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative endocrine therapy was significantly shorter for patients with ILC (median, 0.6 months, 95% CI 0.5–0.8) compared with patients with IDC (median, 1.1 months, 95% CI 1.0–1.5, P = 0.0001) (Figure 1B). Six months after diagnosis of metastatic breast cancer 84.8% of patients with ILC had received palliative endocrine therapy compared with 72.5% of patients with IDC.\\n Outcome Median overall survival was 29.4 months (95% CI 22.5–36.6) for patients with HR-positive ILC and 25.4 months (95% CI 21.8–31.7) for patients with HR-positive IDC (P = 0.53).\\nIn multivariable analysis for patients with ILC with palliative endocrine therapy and palliative chemotherapy as time-dependent covariates, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.8, 95% CI 1.7–4.6, P < .0001) compared to no palliative chemotherapy during the observation period. Conversely, early initiation of palliative endocrine therapy was associated with a favorable survival (hazard ratio 0.4, 95% CI 0.2–0.8, P = 0.005) compared to no palliative endocrine therapy during the observation period.\\nIn multivariable analysis for patients with IDC, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.1, 95% CI 1.6–2.7. P < .0001) when compared with no chemotherapy, whereas early treatment with palliative endocrine therapy was not associated with survival (hazard ratio 0.9, 95% CI 0.6–1.2, P = 0.4).\\nMedian overall survival was 29.4 months (95% CI 22.5–36.6) for patients with HR-positive ILC and 25.4 months (95% CI 21.8–31.7) for patients with HR-positive IDC (P = 0.53).\\nIn multivariable analysis for patients with ILC with palliative endocrine therapy and palliative chemotherapy as time-dependent covariates, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.8, 95% CI 1.7–4.6, P < .0001) compared to no palliative chemotherapy during the observation period. Conversely, early initiation of palliative endocrine therapy was associated with a favorable survival (hazard ratio 0.4, 95% CI 0.2–0.8, P = 0.005) compared to no palliative endocrine therapy during the observation period.\\nIn multivariable analysis for patients with IDC, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.1, 95% CI 1.6–2.7. P < .0001) when compared with no chemotherapy, whereas early treatment with palliative endocrine therapy was not associated with survival (hazard ratio 0.9, 95% CI 0.6–1.2, P = 0.4).\\n Residual survival: six months after diagnosis For ILC, the residual survival was significantly longer for patients not treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (median 44.0 months, 95% CI 30.2-not yet reached) versus patients treated with palliative chemotherapy (median 15.2 months, 95% CI 7.8–19.2) (Figure 2A). For IDC, residual survival when not treated with palliative chemotherapy was 41.8 (95% CI 33.3-not yet reached) compared with 16.8 months (95% CI 13.6–22.5) for patients treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (Figure 2B).\\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative chemotherapy during the first six months after diagnosis of metastatic breast cancer.\\nReversely, for ILC-patients with palliative endocrine treatment within the first six months after diagnosis of metastatic breast cancer residual survival was 37.5 months (95% CI 26.9-not yet reached) compared with 11.2 months (95% CI 3.0–20.4) for patients with HR-positive metastatic ILC without palliative endocrine therapy before the first six months (Figure 3A). For patients with HR-positive metastatic IDC treated with palliative endocrine therapy during the first six months, residual survival was 33.5 months (95% CI 25.7-not yet reached) compared with 18.9 months (95% CI 14.0-26.8) for patients with HR-positive metastatic IDC without palliative endocrine therapy in the first six months of metastatic disease (Figure 3B).\\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative endocrine therapy during the first six months after diagnosis of metastatic breast cancer.\\nFor ILC, the residual survival was significantly longer for patients not treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (median 44.0 months, 95% CI 30.2-not yet reached) versus patients treated with palliative chemotherapy (median 15.2 months, 95% CI 7.8–19.2) (Figure 2A). For IDC, residual survival when not treated with palliative chemotherapy was 41.8 (95% CI 33.3-not yet reached) compared with 16.8 months (95% CI 13.6–22.5) for patients treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (Figure 2B).\\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative chemotherapy during the first six months after diagnosis of metastatic breast cancer.\\nReversely, for ILC-patients with palliative endocrine treatment within the first six months after diagnosis of metastatic breast cancer residual survival was 37.5 months (95% CI 26.9-not yet reached) compared with 11.2 months (95% CI 3.0–20.4) for patients with HR-positive metastatic ILC without palliative endocrine therapy before the first six months (Figure 3A). For patients with HR-positive metastatic IDC treated with palliative endocrine therapy during the first six months, residual survival was 33.5 months (95% CI 25.7-not yet reached) compared with 18.9 months (95% CI 14.0-26.8) for patients with HR-positive metastatic IDC without palliative endocrine therapy in the first six months of metastatic disease (Figure 3B).\\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative endocrine therapy during the first six months after diagnosis of metastatic breast cancer.\",\n \"Of the 568 patients with HR-positive metastatic breast cancer, 23% had (mixed type) ILC (131 patients), hereafter referred to as ILC and 77% had IDC (437 patients) (Table 1).\\nAbbreviations; HR+ hormone receptor positive, N number, SBR Scarff Bloom Richardson, MFI metastatic-free interval * ≥ 1 of the aforementioned initial sites of metastasis.\\nMetastatic breast cancer patients with ILC were older at the time of primary breast cancer diagnosis, compared with patients with IDC (median age at diagnosis 62 years for ILC versus 58 years for IDC, P = 0.03). At initial presentation, patients with HR-positive ILC had larger tumors (T2-3, 62.5% versus 49.7%, P = 0.002) and slightly more node-positive disease (66% versus 56.9%, P = 0.10), but with less often a HER2 positive status (7.6% versus 15.3%, P = 0.02) and lower grade (not significant) compared with patients with HR-positive IDC. No differences were seen in use of adjuvant chemotherapy or endocrine therapy between the histological subtypes.\\nBone was the most common initial site of distant metastasis in both HR-positive histological subtypes, although bone metastasis as initial site was more frequently observed for patients with ILC compared to those with IDC (46.5% versus 34.8%, P = 0.01). Fewer patients with ILC were diagnosed with multiple sites of distant metastasis compared to those with IDC (23.7% versus 30.9%, P = 0.11).\",\n \"Median follow-up after diagnosis of metastatic disease was 37.1 months (range 5.2–54.6) with 239 patients (42%) alive at the end of the follow-up period.\\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative chemotherapy was significantly longer for patients with ILC (median not yet reached) compared with IDC (median 16.9 months, 95% Confidence Interval (CI) 9.6-22.3, P = 0.001) (Figure 1A). Six months after diagnosis of HR-positive metastatic breast cancer less patients with ILC had received palliative chemotherapy compared with IDC (28.1% versus 39.8% respectively).\\nTime to first palliative chemotherapy (A) and endocrine therapy (B) by histological subtype.\\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative endocrine therapy was significantly shorter for patients with ILC (median, 0.6 months, 95% CI 0.5–0.8) compared with patients with IDC (median, 1.1 months, 95% CI 1.0–1.5, P = 0.0001) (Figure 1B). Six months after diagnosis of metastatic breast cancer 84.8% of patients with ILC had received palliative endocrine therapy compared with 72.5% of patients with IDC.\",\n \"Median overall survival was 29.4 months (95% CI 22.5–36.6) for patients with HR-positive ILC and 25.4 months (95% CI 21.8–31.7) for patients with HR-positive IDC (P = 0.53).\\nIn multivariable analysis for patients with ILC with palliative endocrine therapy and palliative chemotherapy as time-dependent covariates, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.8, 95% CI 1.7–4.6, P < .0001) compared to no palliative chemotherapy during the observation period. Conversely, early initiation of palliative endocrine therapy was associated with a favorable survival (hazard ratio 0.4, 95% CI 0.2–0.8, P = 0.005) compared to no palliative endocrine therapy during the observation period.\\nIn multivariable analysis for patients with IDC, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.1, 95% CI 1.6–2.7. P < .0001) when compared with no chemotherapy, whereas early treatment with palliative endocrine therapy was not associated with survival (hazard ratio 0.9, 95% CI 0.6–1.2, P = 0.4).\",\n \"For ILC, the residual survival was significantly longer for patients not treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (median 44.0 months, 95% CI 30.2-not yet reached) versus patients treated with palliative chemotherapy (median 15.2 months, 95% CI 7.8–19.2) (Figure 2A). For IDC, residual survival when not treated with palliative chemotherapy was 41.8 (95% CI 33.3-not yet reached) compared with 16.8 months (95% CI 13.6–22.5) for patients treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (Figure 2B).\\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative chemotherapy during the first six months after diagnosis of metastatic breast cancer.\\nReversely, for ILC-patients with palliative endocrine treatment within the first six months after diagnosis of metastatic breast cancer residual survival was 37.5 months (95% CI 26.9-not yet reached) compared with 11.2 months (95% CI 3.0–20.4) for patients with HR-positive metastatic ILC without palliative endocrine therapy before the first six months (Figure 3A). For patients with HR-positive metastatic IDC treated with palliative endocrine therapy during the first six months, residual survival was 33.5 months (95% CI 25.7-not yet reached) compared with 18.9 months (95% CI 14.0-26.8) for patients with HR-positive metastatic IDC without palliative endocrine therapy in the first six months of metastatic disease (Figure 3B).\\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative endocrine therapy during the first six months after diagnosis of metastatic breast cancer.\",\n \"To our knowledge, the role of histological subtype in palliative systemic treatment of metastatic breast cancer has not been studied before. In a cohort of 568 HR-positive metastatic breast cancer patients, we showed that patients with HR-positive ILC had more often bone and less often multiple sites as initial site of distant metastasis compared to those with HR-positive IDC. Six months after metastatic diagnosis, patients with HR-positive ILC had received significantly less often chemotherapy (28% versus 40%) and more often endocrine therapy (85% versus 73%) as compared to patients with HR-positive IDC. Patients starting with palliative chemotherapy during the first six months had a significantly shorter median residual survival thereafter as compared to those who did not (15 months versus 44 months for ILC, and 17 and 42 months for IDC, respectively). Reversely, patients treated with endocrine therapy in the first six months had a longer median residual survival compared to those who did not (37 months versus 11 months for ILC and 33 and 19 months for IDC, respectively). Our results confirm that, in addition to HR-status, histology correlates with presentation of metastatic disease by number and type of distant metastatic sites, and thereby affects treatment-decision making in real-life.\\nThe intriguing scientific question is to determine how histological subtype influences the metastatic spread in HR-positive breast cancer. It is hypothesized that the loss of E-cadherin, a cell-cell adhesion molecule, in ILC may result in less adhesiveness of the tumor cells and therefore disseminate and infiltrate certain distant locations more easily [1].\\nIn this HR-positive metastatic breast cancer cohort we also looked at the association of palliative systemic treatment with survival of patients with metastatic breast cancer for both histological subtypes. With the favorable presentation of distant metastasis by number and location of patients with HR-positive ILC as compared to HR-positive IDC, one would expect a more indolent disease course and a better outcome. However, we showed that for patients with HR-positive breast cancer, overall disease outcome was comparable irrespective of histology and irrespective of different treatment choices made in real-life, as discussed above. And interestingly, the efficacy of chemotherapy - once the treatment choice was made - was comparable irrespective of histology. However, treatment with palliative endocrine therapy was only associated with a favorable prognosis of patients with HR-positive ILC, whereas this was not seen in patients with HR-positive IDC, also suggesting the impact of histology over HR-status. This is in concordance with the early breast cancer setting, in which there is evidence suggesting differences in efficacy of systemic therapy between the two histological subtypes [3–5, 12].\\nAlthough there is some proof that histology could be helpful in treatment decision-making [13–15], the current guidelines do not include histological subtype as an indicator for the use of systemic treatment in general, or for a specific regimen [16, 17]. Interestingly, in the adjuvant setting, histology has been shown to be of importance when deciding between the use of breast-conserving surgery or mastectomy after neo-adjuvant chemotherapy [18]. The current study shows that histological subtype can be of predictive value for HR-positive metastatic breast cancer with regards to the effectiveness of early initiation of palliative endocrine therapy. It may be possible that in patients with HR-positive lobular breast cancer, the higher incidence of bone metastases as initial metastatic site partly accounts for the initial choice of endocrine therapy. It underlines the relevance of acknowledging a different metastatic pattern, and thereby different initial treatment choices, between breast cancer patients with ductal versus lobular histology.\\nMuch more than in the adjuvant setting, treatment decisions in the metastatic setting are based on the observed and anticipated clinical course of the disease, which is not only determined by the tumor characteristics as described earlier. Also age, performance status, previous therapies and toxicities, comorbidity and patient and doctor preferences play a role. The complexity of this process, together with the retrospective design of our study make it impossible to identify and rule out confounding by indication. Furthermore, localization of the metastases (visceral versus bone metastasis for instance) could be related to symptomatology and thereby timing of detection, and could therefore have introduced lead time bias.\\nIn this study patients with pure lobular carcinoma and mixed lobular carcinomas were combined for the analyses. In other studies on histological subtypes of breast cancer mixed and lobular carcinoma had similar outcome [14, 19]. Even with combining these subgroups, the proportion of HER2-positive tumors was too low to further analyze anti-HER2 therapy. In the adjuvant treatment setting the magnitude of benefit from adjuvant trastuzumab between patients with ILC and IDC was shown not to be different [20]. Another limitation of our study is that patient numbers were too small to perform analysis on specific chemotherapy regimens. Microarray analysis have demonstrated that ILC and IDC can be distinguished on the basis of genomic and expression profiles [21]. The increasing knowledge on genomic differences between ILC and IDC can help to answer questions on in vivo chemosensitivity. For example, topoisomerase-IIα gene amplification is a predictive biomarker for response to anthracyclines and in ILC this gene amplification is lacking, which could help understand the poor responsiveness of ILC to neo-adjuvant chemotherapy, including anthracyclines [22]. This genetic information can help guide further research and may eventually be useful in making treatment decisions for histological breast cancer subtypes.\\nIn conclusion, to our knowledge, this is the first study that investigates the role of histological subtype in HR-positive metastatic breast cancer. Although survival was comparable for the two histological subtypes, this was achieved with different treatment strategies. As patients with HR-positive ILC were less likely to receive chemotherapy than those with HR-positive IDC, histology may be a relevant factor in treatment-decision making. For a more definite conclusion on the role of histology, we recommend to incorporate histological subtype as a stratification factor in future clinical trials.\",\n \" Patient selection We identified all patients diagnosed with metastatic breast cancer between 2007–2009 in eight hospitals in the South-East of the Netherlands. All patients with metastatic breast cancer were selected irrespective of the date of primary breast cancer diagnosis (also including patients with de novo metastatic breast cancer; 18.5% of patients with IDC and 19.9% of patients with ILC), with the exception of patients with a diagnosis of primary breast cancer before 1990, due to limitations in the availability of data on the primary tumor and initial treatment.\\nHistology was classified according to the International Classification of Diseases for Oncology (ICD-O) [23]. ILC and mixed histology were defined as code 8520 and 8522. IDC was defined as code 8500. From the total of 815 metastatic breast cancer patients, we excluded 76 patients with either unknown histology or histological subtypes other than IDC or ILC. Of the remaining 739 metastatic breast cancer patients, we excluded 171 patients with HR-negative tumors (27% of patients with IDC and 8% of patients with ILC) in order to rule out the impact of HR status. In total, our study population consisted of 568 patients divided in two groups based on histology; one group of 437 patients with IDC and the other group of 131 patients with ILC.\\nWe identified all patients diagnosed with metastatic breast cancer between 2007–2009 in eight hospitals in the South-East of the Netherlands. All patients with metastatic breast cancer were selected irrespective of the date of primary breast cancer diagnosis (also including patients with de novo metastatic breast cancer; 18.5% of patients with IDC and 19.9% of patients with ILC), with the exception of patients with a diagnosis of primary breast cancer before 1990, due to limitations in the availability of data on the primary tumor and initial treatment.\\nHistology was classified according to the International Classification of Diseases for Oncology (ICD-O) [23]. ILC and mixed histology were defined as code 8520 and 8522. IDC was defined as code 8500. From the total of 815 metastatic breast cancer patients, we excluded 76 patients with either unknown histology or histological subtypes other than IDC or ILC. Of the remaining 739 metastatic breast cancer patients, we excluded 171 patients with HR-negative tumors (27% of patients with IDC and 8% of patients with ILC) in order to rule out the impact of HR status. In total, our study population consisted of 568 patients divided in two groups based on histology; one group of 437 patients with IDC and the other group of 131 patients with ILC.\\n Data collection Information was collected on patient and tumor characteristics, treatment and outcome. Tumors were characterized by the sixth edition of the TNM classification of malignant tumors [24] and Scarff Bloom Richardson (SBR) histological grading [25]. Estrogen receptor (ER) and progesterone receptor (PR) positivity was defined as positive nuclear staining of ≥ 10%. HER2 positivity was defined as immunohistochemistry score of 3+ or 2+ with positive FISH. In case of missing HER2 status a dedicated pathologist centrally reviewed missing data when material was available. Initial sites of metastasis were categorized as: bone, visceral (including lung, liver, pleural, peritoneal, pericardial and lymphangitic carcinomatosis), brain (including leptomeningeal and CNS), skin and lymph nodes, and multiple metastases (more than one of the metastatic sites).\\nInformation was collected on patient and tumor characteristics, treatment and outcome. Tumors were characterized by the sixth edition of the TNM classification of malignant tumors [24] and Scarff Bloom Richardson (SBR) histological grading [25]. Estrogen receptor (ER) and progesterone receptor (PR) positivity was defined as positive nuclear staining of ≥ 10%. HER2 positivity was defined as immunohistochemistry score of 3+ or 2+ with positive FISH. In case of missing HER2 status a dedicated pathologist centrally reviewed missing data when material was available. Initial sites of metastasis were categorized as: bone, visceral (including lung, liver, pleural, peritoneal, pericardial and lymphangitic carcinomatosis), brain (including leptomeningeal and CNS), skin and lymph nodes, and multiple metastases (more than one of the metastatic sites).\\n Statistical analysis Baseline characteristics between the two histological groups were compared using chi-square tests for categorical variables and Wilcoxon rank sum tests for continuous variables.\\nMetastatic-free interval was defined as time between date of primary diagnosis and date of first distant metastasis. Overall survival after diagnosis of metastatic breast cancer was defined as time between date of first distant metastasis and date of death. Survival curves and time to first palliative systemic therapy (either chemotherapy or endocrine therapy) were estimated using the Kaplan-Meier method and compared using log-rank tests. All patients still alive were censored at the date of last follow-up of each individual patient. Patients who died without palliative therapy were censored at the date of death in the analysis of time to first palliative therapy.\\nTo explore the association of palliative systemic therapy with the survival of patients with metastatic breast cancer for both histological subtypes a Cox proportional hazards model was performed with palliative chemotherapy and endocrine therapy as a time-dependent covariate, since the administration of treatment can change over time and is dependent on the time available for each patient to receive the treatment. We did not explore the association between palliative targeted therapy, such as trastuzumab and bevacizumab, and survival since the number of patients with IDC and ILC receiving targeted therapy was very low. Since all these patients received targeted therapy with chemotherapy, these patients were included in the analysis on initial chemotherapy. In addition, the landmark method was used to estimate survival after a specific time-point, the so-called residual survival [26]. As we were interested to learn about the obtained survival in relation to the initial palliative treatment choices, we chose six months after diagnosis of metastatic breast cancer as landmark. Consequently, patients who already died within 6 months were excluded for the residual survival curves.\\nAll analyses were performed using SAS version 9.2. All reported P-values are two-sided and P-value ≤ 0.05 was considered statistically significant.\\nBaseline characteristics between the two histological groups were compared using chi-square tests for categorical variables and Wilcoxon rank sum tests for continuous variables.\\nMetastatic-free interval was defined as time between date of primary diagnosis and date of first distant metastasis. Overall survival after diagnosis of metastatic breast cancer was defined as time between date of first distant metastasis and date of death. Survival curves and time to first palliative systemic therapy (either chemotherapy or endocrine therapy) were estimated using the Kaplan-Meier method and compared using log-rank tests. All patients still alive were censored at the date of last follow-up of each individual patient. Patients who died without palliative therapy were censored at the date of death in the analysis of time to first palliative therapy.\\nTo explore the association of palliative systemic therapy with the survival of patients with metastatic breast cancer for both histological subtypes a Cox proportional hazards model was performed with palliative chemotherapy and endocrine therapy as a time-dependent covariate, since the administration of treatment can change over time and is dependent on the time available for each patient to receive the treatment. We did not explore the association between palliative targeted therapy, such as trastuzumab and bevacizumab, and survival since the number of patients with IDC and ILC receiving targeted therapy was very low. Since all these patients received targeted therapy with chemotherapy, these patients were included in the analysis on initial chemotherapy. In addition, the landmark method was used to estimate survival after a specific time-point, the so-called residual survival [26]. As we were interested to learn about the obtained survival in relation to the initial palliative treatment choices, we chose six months after diagnosis of metastatic breast cancer as landmark. Consequently, patients who already died within 6 months were excluded for the residual survival curves.\\nAll analyses were performed using SAS version 9.2. All reported P-values are two-sided and P-value ≤ 0.05 was considered statistically significant.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,"methods"],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"methods\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["metastatic breast cancer","histology","invasive lobular carcinoma","invasive ductal carcinoma","treatment"],"string":"[\n \"metastatic breast cancer\",\n \"histology\",\n \"invasive lobular carcinoma\",\n \"invasive ductal carcinoma\",\n \"treatment\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nThe two most frequent histological subtypes of breast cancer are invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC), with IDC comprising 75–80% and ILC 5–15% of all breast cancer cases. ILC is being associated with larger tumor size at presentation, more bilateral and multifocal involvement and with a different pattern of metastatic spread compared with IDC [1, 2]. Furthermore, ILC is more often HR-positive, HER2-negative, with lower S-phase fraction and less often positive for the tumor suppressor gene p53, compared with IDC [1]. Also, treatment response is known to be different. In a combined analysis on a number of retrospective series, the pathological complete response rate of neo-adjuvant chemotherapy was significantly lower in ILC (1.7%) than in IDC (11.6%) [3]. In the adjuvant setting, retrospective data suggest a higher efficacy of endocrine therapy for ILC than IDC [4]. More recently, a large retrospective study strongly suggested that HR-positive breast cancer patients with ILC do not seem to benefit from adjuvant chemotherapy in addition to endocrine therapy [5].\nWhether these differences have a prognostic impact is controversial. Some studies found no effect of histology on survival [1, 6, 7], others found a better prognosis for patients with ILC compared to those with IDC [8–10] and some found a change in prognosis over time with a better prognosis for ILC during the first years of follow-up and a worse prognosis during later years [2, 11].\nSo far, studies on histological subtypes consider early breast cancer and not metastatic breast cancer. The aim of this study was to describe the differences between IDC and ILC with respect to patient and tumor characteristics, treatment-choices and outcome in metastatic breast cancer. In order to account for the effect of the hormone receptor (HR) on outcome and treatment-decision making, we only included patients with HR-positive breast cancer.\n\nRESULTS:\n Patient characteristics Of the 568 patients with HR-positive metastatic breast cancer, 23% had (mixed type) ILC (131 patients), hereafter referred to as ILC and 77% had IDC (437 patients) (Table 1).\nAbbreviations; HR+ hormone receptor positive, N number, SBR Scarff Bloom Richardson, MFI metastatic-free interval * ≥ 1 of the aforementioned initial sites of metastasis.\nMetastatic breast cancer patients with ILC were older at the time of primary breast cancer diagnosis, compared with patients with IDC (median age at diagnosis 62 years for ILC versus 58 years for IDC, P = 0.03). At initial presentation, patients with HR-positive ILC had larger tumors (T2-3, 62.5% versus 49.7%, P = 0.002) and slightly more node-positive disease (66% versus 56.9%, P = 0.10), but with less often a HER2 positive status (7.6% versus 15.3%, P = 0.02) and lower grade (not significant) compared with patients with HR-positive IDC. No differences were seen in use of adjuvant chemotherapy or endocrine therapy between the histological subtypes.\nBone was the most common initial site of distant metastasis in both HR-positive histological subtypes, although bone metastasis as initial site was more frequently observed for patients with ILC compared to those with IDC (46.5% versus 34.8%, P = 0.01). Fewer patients with ILC were diagnosed with multiple sites of distant metastasis compared to those with IDC (23.7% versus 30.9%, P = 0.11).\nOf the 568 patients with HR-positive metastatic breast cancer, 23% had (mixed type) ILC (131 patients), hereafter referred to as ILC and 77% had IDC (437 patients) (Table 1).\nAbbreviations; HR+ hormone receptor positive, N number, SBR Scarff Bloom Richardson, MFI metastatic-free interval * ≥ 1 of the aforementioned initial sites of metastasis.\nMetastatic breast cancer patients with ILC were older at the time of primary breast cancer diagnosis, compared with patients with IDC (median age at diagnosis 62 years for ILC versus 58 years for IDC, P = 0.03). At initial presentation, patients with HR-positive ILC had larger tumors (T2-3, 62.5% versus 49.7%, P = 0.002) and slightly more node-positive disease (66% versus 56.9%, P = 0.10), but with less often a HER2 positive status (7.6% versus 15.3%, P = 0.02) and lower grade (not significant) compared with patients with HR-positive IDC. No differences were seen in use of adjuvant chemotherapy or endocrine therapy between the histological subtypes.\nBone was the most common initial site of distant metastasis in both HR-positive histological subtypes, although bone metastasis as initial site was more frequently observed for patients with ILC compared to those with IDC (46.5% versus 34.8%, P = 0.01). Fewer patients with ILC were diagnosed with multiple sites of distant metastasis compared to those with IDC (23.7% versus 30.9%, P = 0.11).\n Palliative systemic treatment Median follow-up after diagnosis of metastatic disease was 37.1 months (range 5.2–54.6) with 239 patients (42%) alive at the end of the follow-up period.\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative chemotherapy was significantly longer for patients with ILC (median not yet reached) compared with IDC (median 16.9 months, 95% Confidence Interval (CI) 9.6-22.3, P = 0.001) (Figure 1A). Six months after diagnosis of HR-positive metastatic breast cancer less patients with ILC had received palliative chemotherapy compared with IDC (28.1% versus 39.8% respectively).\nTime to first palliative chemotherapy (A) and endocrine therapy (B) by histological subtype.\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative endocrine therapy was significantly shorter for patients with ILC (median, 0.6 months, 95% CI 0.5–0.8) compared with patients with IDC (median, 1.1 months, 95% CI 1.0–1.5, P = 0.0001) (Figure 1B). Six months after diagnosis of metastatic breast cancer 84.8% of patients with ILC had received palliative endocrine therapy compared with 72.5% of patients with IDC.\nMedian follow-up after diagnosis of metastatic disease was 37.1 months (range 5.2–54.6) with 239 patients (42%) alive at the end of the follow-up period.\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative chemotherapy was significantly longer for patients with ILC (median not yet reached) compared with IDC (median 16.9 months, 95% Confidence Interval (CI) 9.6-22.3, P = 0.001) (Figure 1A). Six months after diagnosis of HR-positive metastatic breast cancer less patients with ILC had received palliative chemotherapy compared with IDC (28.1% versus 39.8% respectively).\nTime to first palliative chemotherapy (A) and endocrine therapy (B) by histological subtype.\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative endocrine therapy was significantly shorter for patients with ILC (median, 0.6 months, 95% CI 0.5–0.8) compared with patients with IDC (median, 1.1 months, 95% CI 1.0–1.5, P = 0.0001) (Figure 1B). Six months after diagnosis of metastatic breast cancer 84.8% of patients with ILC had received palliative endocrine therapy compared with 72.5% of patients with IDC.\n Outcome Median overall survival was 29.4 months (95% CI 22.5–36.6) for patients with HR-positive ILC and 25.4 months (95% CI 21.8–31.7) for patients with HR-positive IDC (P = 0.53).\nIn multivariable analysis for patients with ILC with palliative endocrine therapy and palliative chemotherapy as time-dependent covariates, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.8, 95% CI 1.7–4.6, P < .0001) compared to no palliative chemotherapy during the observation period. Conversely, early initiation of palliative endocrine therapy was associated with a favorable survival (hazard ratio 0.4, 95% CI 0.2–0.8, P = 0.005) compared to no palliative endocrine therapy during the observation period.\nIn multivariable analysis for patients with IDC, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.1, 95% CI 1.6–2.7. P < .0001) when compared with no chemotherapy, whereas early treatment with palliative endocrine therapy was not associated with survival (hazard ratio 0.9, 95% CI 0.6–1.2, P = 0.4).\nMedian overall survival was 29.4 months (95% CI 22.5–36.6) for patients with HR-positive ILC and 25.4 months (95% CI 21.8–31.7) for patients with HR-positive IDC (P = 0.53).\nIn multivariable analysis for patients with ILC with palliative endocrine therapy and palliative chemotherapy as time-dependent covariates, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.8, 95% CI 1.7–4.6, P < .0001) compared to no palliative chemotherapy during the observation period. Conversely, early initiation of palliative endocrine therapy was associated with a favorable survival (hazard ratio 0.4, 95% CI 0.2–0.8, P = 0.005) compared to no palliative endocrine therapy during the observation period.\nIn multivariable analysis for patients with IDC, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.1, 95% CI 1.6–2.7. P < .0001) when compared with no chemotherapy, whereas early treatment with palliative endocrine therapy was not associated with survival (hazard ratio 0.9, 95% CI 0.6–1.2, P = 0.4).\n Residual survival: six months after diagnosis For ILC, the residual survival was significantly longer for patients not treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (median 44.0 months, 95% CI 30.2-not yet reached) versus patients treated with palliative chemotherapy (median 15.2 months, 95% CI 7.8–19.2) (Figure 2A). For IDC, residual survival when not treated with palliative chemotherapy was 41.8 (95% CI 33.3-not yet reached) compared with 16.8 months (95% CI 13.6–22.5) for patients treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (Figure 2B).\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative chemotherapy during the first six months after diagnosis of metastatic breast cancer.\nReversely, for ILC-patients with palliative endocrine treatment within the first six months after diagnosis of metastatic breast cancer residual survival was 37.5 months (95% CI 26.9-not yet reached) compared with 11.2 months (95% CI 3.0–20.4) for patients with HR-positive metastatic ILC without palliative endocrine therapy before the first six months (Figure 3A). For patients with HR-positive metastatic IDC treated with palliative endocrine therapy during the first six months, residual survival was 33.5 months (95% CI 25.7-not yet reached) compared with 18.9 months (95% CI 14.0-26.8) for patients with HR-positive metastatic IDC without palliative endocrine therapy in the first six months of metastatic disease (Figure 3B).\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative endocrine therapy during the first six months after diagnosis of metastatic breast cancer.\nFor ILC, the residual survival was significantly longer for patients not treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (median 44.0 months, 95% CI 30.2-not yet reached) versus patients treated with palliative chemotherapy (median 15.2 months, 95% CI 7.8–19.2) (Figure 2A). For IDC, residual survival when not treated with palliative chemotherapy was 41.8 (95% CI 33.3-not yet reached) compared with 16.8 months (95% CI 13.6–22.5) for patients treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (Figure 2B).\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative chemotherapy during the first six months after diagnosis of metastatic breast cancer.\nReversely, for ILC-patients with palliative endocrine treatment within the first six months after diagnosis of metastatic breast cancer residual survival was 37.5 months (95% CI 26.9-not yet reached) compared with 11.2 months (95% CI 3.0–20.4) for patients with HR-positive metastatic ILC without palliative endocrine therapy before the first six months (Figure 3A). For patients with HR-positive metastatic IDC treated with palliative endocrine therapy during the first six months, residual survival was 33.5 months (95% CI 25.7-not yet reached) compared with 18.9 months (95% CI 14.0-26.8) for patients with HR-positive metastatic IDC without palliative endocrine therapy in the first six months of metastatic disease (Figure 3B).\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative endocrine therapy during the first six months after diagnosis of metastatic breast cancer.\n\nPatient characteristics:\nOf the 568 patients with HR-positive metastatic breast cancer, 23% had (mixed type) ILC (131 patients), hereafter referred to as ILC and 77% had IDC (437 patients) (Table 1).\nAbbreviations; HR+ hormone receptor positive, N number, SBR Scarff Bloom Richardson, MFI metastatic-free interval * ≥ 1 of the aforementioned initial sites of metastasis.\nMetastatic breast cancer patients with ILC were older at the time of primary breast cancer diagnosis, compared with patients with IDC (median age at diagnosis 62 years for ILC versus 58 years for IDC, P = 0.03). At initial presentation, patients with HR-positive ILC had larger tumors (T2-3, 62.5% versus 49.7%, P = 0.002) and slightly more node-positive disease (66% versus 56.9%, P = 0.10), but with less often a HER2 positive status (7.6% versus 15.3%, P = 0.02) and lower grade (not significant) compared with patients with HR-positive IDC. No differences were seen in use of adjuvant chemotherapy or endocrine therapy between the histological subtypes.\nBone was the most common initial site of distant metastasis in both HR-positive histological subtypes, although bone metastasis as initial site was more frequently observed for patients with ILC compared to those with IDC (46.5% versus 34.8%, P = 0.01). Fewer patients with ILC were diagnosed with multiple sites of distant metastasis compared to those with IDC (23.7% versus 30.9%, P = 0.11).\n\nPalliative systemic treatment:\nMedian follow-up after diagnosis of metastatic disease was 37.1 months (range 5.2–54.6) with 239 patients (42%) alive at the end of the follow-up period.\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative chemotherapy was significantly longer for patients with ILC (median not yet reached) compared with IDC (median 16.9 months, 95% Confidence Interval (CI) 9.6-22.3, P = 0.001) (Figure 1A). Six months after diagnosis of HR-positive metastatic breast cancer less patients with ILC had received palliative chemotherapy compared with IDC (28.1% versus 39.8% respectively).\nTime to first palliative chemotherapy (A) and endocrine therapy (B) by histological subtype.\nTime between diagnosis of HR-positive metastatic breast cancer and start of palliative endocrine therapy was significantly shorter for patients with ILC (median, 0.6 months, 95% CI 0.5–0.8) compared with patients with IDC (median, 1.1 months, 95% CI 1.0–1.5, P = 0.0001) (Figure 1B). Six months after diagnosis of metastatic breast cancer 84.8% of patients with ILC had received palliative endocrine therapy compared with 72.5% of patients with IDC.\n\nOutcome:\nMedian overall survival was 29.4 months (95% CI 22.5–36.6) for patients with HR-positive ILC and 25.4 months (95% CI 21.8–31.7) for patients with HR-positive IDC (P = 0.53).\nIn multivariable analysis for patients with ILC with palliative endocrine therapy and palliative chemotherapy as time-dependent covariates, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.8, 95% CI 1.7–4.6, P < .0001) compared to no palliative chemotherapy during the observation period. Conversely, early initiation of palliative endocrine therapy was associated with a favorable survival (hazard ratio 0.4, 95% CI 0.2–0.8, P = 0.005) compared to no palliative endocrine therapy during the observation period.\nIn multivariable analysis for patients with IDC, early initiation of palliative chemotherapy was associated with an unfavorable survival (hazard ratio 2.1, 95% CI 1.6–2.7. P < .0001) when compared with no chemotherapy, whereas early treatment with palliative endocrine therapy was not associated with survival (hazard ratio 0.9, 95% CI 0.6–1.2, P = 0.4).\n\nResidual survival: six months after diagnosis:\nFor ILC, the residual survival was significantly longer for patients not treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (median 44.0 months, 95% CI 30.2-not yet reached) versus patients treated with palliative chemotherapy (median 15.2 months, 95% CI 7.8–19.2) (Figure 2A). For IDC, residual survival when not treated with palliative chemotherapy was 41.8 (95% CI 33.3-not yet reached) compared with 16.8 months (95% CI 13.6–22.5) for patients treated with palliative chemotherapy within the first six months after diagnosis of metastatic breast cancer (Figure 2B).\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative chemotherapy during the first six months after diagnosis of metastatic breast cancer.\nReversely, for ILC-patients with palliative endocrine treatment within the first six months after diagnosis of metastatic breast cancer residual survival was 37.5 months (95% CI 26.9-not yet reached) compared with 11.2 months (95% CI 3.0–20.4) for patients with HR-positive metastatic ILC without palliative endocrine therapy before the first six months (Figure 3A). For patients with HR-positive metastatic IDC treated with palliative endocrine therapy during the first six months, residual survival was 33.5 months (95% CI 25.7-not yet reached) compared with 18.9 months (95% CI 14.0-26.8) for patients with HR-positive metastatic IDC without palliative endocrine therapy in the first six months of metastatic disease (Figure 3B).\nResidual survival for patients with HR-positive ILC (A) and HR-positive IDC (B) treated with or without any palliative endocrine therapy during the first six months after diagnosis of metastatic breast cancer.\n\nDISCUSSION:\nTo our knowledge, the role of histological subtype in palliative systemic treatment of metastatic breast cancer has not been studied before. In a cohort of 568 HR-positive metastatic breast cancer patients, we showed that patients with HR-positive ILC had more often bone and less often multiple sites as initial site of distant metastasis compared to those with HR-positive IDC. Six months after metastatic diagnosis, patients with HR-positive ILC had received significantly less often chemotherapy (28% versus 40%) and more often endocrine therapy (85% versus 73%) as compared to patients with HR-positive IDC. Patients starting with palliative chemotherapy during the first six months had a significantly shorter median residual survival thereafter as compared to those who did not (15 months versus 44 months for ILC, and 17 and 42 months for IDC, respectively). Reversely, patients treated with endocrine therapy in the first six months had a longer median residual survival compared to those who did not (37 months versus 11 months for ILC and 33 and 19 months for IDC, respectively). Our results confirm that, in addition to HR-status, histology correlates with presentation of metastatic disease by number and type of distant metastatic sites, and thereby affects treatment-decision making in real-life.\nThe intriguing scientific question is to determine how histological subtype influences the metastatic spread in HR-positive breast cancer. It is hypothesized that the loss of E-cadherin, a cell-cell adhesion molecule, in ILC may result in less adhesiveness of the tumor cells and therefore disseminate and infiltrate certain distant locations more easily [1].\nIn this HR-positive metastatic breast cancer cohort we also looked at the association of palliative systemic treatment with survival of patients with metastatic breast cancer for both histological subtypes. With the favorable presentation of distant metastasis by number and location of patients with HR-positive ILC as compared to HR-positive IDC, one would expect a more indolent disease course and a better outcome. However, we showed that for patients with HR-positive breast cancer, overall disease outcome was comparable irrespective of histology and irrespective of different treatment choices made in real-life, as discussed above. And interestingly, the efficacy of chemotherapy - once the treatment choice was made - was comparable irrespective of histology. However, treatment with palliative endocrine therapy was only associated with a favorable prognosis of patients with HR-positive ILC, whereas this was not seen in patients with HR-positive IDC, also suggesting the impact of histology over HR-status. This is in concordance with the early breast cancer setting, in which there is evidence suggesting differences in efficacy of systemic therapy between the two histological subtypes [3–5, 12].\nAlthough there is some proof that histology could be helpful in treatment decision-making [13–15], the current guidelines do not include histological subtype as an indicator for the use of systemic treatment in general, or for a specific regimen [16, 17]. Interestingly, in the adjuvant setting, histology has been shown to be of importance when deciding between the use of breast-conserving surgery or mastectomy after neo-adjuvant chemotherapy [18]. The current study shows that histological subtype can be of predictive value for HR-positive metastatic breast cancer with regards to the effectiveness of early initiation of palliative endocrine therapy. It may be possible that in patients with HR-positive lobular breast cancer, the higher incidence of bone metastases as initial metastatic site partly accounts for the initial choice of endocrine therapy. It underlines the relevance of acknowledging a different metastatic pattern, and thereby different initial treatment choices, between breast cancer patients with ductal versus lobular histology.\nMuch more than in the adjuvant setting, treatment decisions in the metastatic setting are based on the observed and anticipated clinical course of the disease, which is not only determined by the tumor characteristics as described earlier. Also age, performance status, previous therapies and toxicities, comorbidity and patient and doctor preferences play a role. The complexity of this process, together with the retrospective design of our study make it impossible to identify and rule out confounding by indication. Furthermore, localization of the metastases (visceral versus bone metastasis for instance) could be related to symptomatology and thereby timing of detection, and could therefore have introduced lead time bias.\nIn this study patients with pure lobular carcinoma and mixed lobular carcinomas were combined for the analyses. In other studies on histological subtypes of breast cancer mixed and lobular carcinoma had similar outcome [14, 19]. Even with combining these subgroups, the proportion of HER2-positive tumors was too low to further analyze anti-HER2 therapy. In the adjuvant treatment setting the magnitude of benefit from adjuvant trastuzumab between patients with ILC and IDC was shown not to be different [20]. Another limitation of our study is that patient numbers were too small to perform analysis on specific chemotherapy regimens. Microarray analysis have demonstrated that ILC and IDC can be distinguished on the basis of genomic and expression profiles [21]. The increasing knowledge on genomic differences between ILC and IDC can help to answer questions on in vivo chemosensitivity. For example, topoisomerase-IIα gene amplification is a predictive biomarker for response to anthracyclines and in ILC this gene amplification is lacking, which could help understand the poor responsiveness of ILC to neo-adjuvant chemotherapy, including anthracyclines [22]. This genetic information can help guide further research and may eventually be useful in making treatment decisions for histological breast cancer subtypes.\nIn conclusion, to our knowledge, this is the first study that investigates the role of histological subtype in HR-positive metastatic breast cancer. Although survival was comparable for the two histological subtypes, this was achieved with different treatment strategies. As patients with HR-positive ILC were less likely to receive chemotherapy than those with HR-positive IDC, histology may be a relevant factor in treatment-decision making. For a more definite conclusion on the role of histology, we recommend to incorporate histological subtype as a stratification factor in future clinical trials.\n\nMATERIALS AND METHODS:\n Patient selection We identified all patients diagnosed with metastatic breast cancer between 2007–2009 in eight hospitals in the South-East of the Netherlands. All patients with metastatic breast cancer were selected irrespective of the date of primary breast cancer diagnosis (also including patients with de novo metastatic breast cancer; 18.5% of patients with IDC and 19.9% of patients with ILC), with the exception of patients with a diagnosis of primary breast cancer before 1990, due to limitations in the availability of data on the primary tumor and initial treatment.\nHistology was classified according to the International Classification of Diseases for Oncology (ICD-O) [23]. ILC and mixed histology were defined as code 8520 and 8522. IDC was defined as code 8500. From the total of 815 metastatic breast cancer patients, we excluded 76 patients with either unknown histology or histological subtypes other than IDC or ILC. Of the remaining 739 metastatic breast cancer patients, we excluded 171 patients with HR-negative tumors (27% of patients with IDC and 8% of patients with ILC) in order to rule out the impact of HR status. In total, our study population consisted of 568 patients divided in two groups based on histology; one group of 437 patients with IDC and the other group of 131 patients with ILC.\nWe identified all patients diagnosed with metastatic breast cancer between 2007–2009 in eight hospitals in the South-East of the Netherlands. All patients with metastatic breast cancer were selected irrespective of the date of primary breast cancer diagnosis (also including patients with de novo metastatic breast cancer; 18.5% of patients with IDC and 19.9% of patients with ILC), with the exception of patients with a diagnosis of primary breast cancer before 1990, due to limitations in the availability of data on the primary tumor and initial treatment.\nHistology was classified according to the International Classification of Diseases for Oncology (ICD-O) [23]. ILC and mixed histology were defined as code 8520 and 8522. IDC was defined as code 8500. From the total of 815 metastatic breast cancer patients, we excluded 76 patients with either unknown histology or histological subtypes other than IDC or ILC. Of the remaining 739 metastatic breast cancer patients, we excluded 171 patients with HR-negative tumors (27% of patients with IDC and 8% of patients with ILC) in order to rule out the impact of HR status. In total, our study population consisted of 568 patients divided in two groups based on histology; one group of 437 patients with IDC and the other group of 131 patients with ILC.\n Data collection Information was collected on patient and tumor characteristics, treatment and outcome. Tumors were characterized by the sixth edition of the TNM classification of malignant tumors [24] and Scarff Bloom Richardson (SBR) histological grading [25]. Estrogen receptor (ER) and progesterone receptor (PR) positivity was defined as positive nuclear staining of ≥ 10%. HER2 positivity was defined as immunohistochemistry score of 3+ or 2+ with positive FISH. In case of missing HER2 status a dedicated pathologist centrally reviewed missing data when material was available. Initial sites of metastasis were categorized as: bone, visceral (including lung, liver, pleural, peritoneal, pericardial and lymphangitic carcinomatosis), brain (including leptomeningeal and CNS), skin and lymph nodes, and multiple metastases (more than one of the metastatic sites).\nInformation was collected on patient and tumor characteristics, treatment and outcome. Tumors were characterized by the sixth edition of the TNM classification of malignant tumors [24] and Scarff Bloom Richardson (SBR) histological grading [25]. Estrogen receptor (ER) and progesterone receptor (PR) positivity was defined as positive nuclear staining of ≥ 10%. HER2 positivity was defined as immunohistochemistry score of 3+ or 2+ with positive FISH. In case of missing HER2 status a dedicated pathologist centrally reviewed missing data when material was available. Initial sites of metastasis were categorized as: bone, visceral (including lung, liver, pleural, peritoneal, pericardial and lymphangitic carcinomatosis), brain (including leptomeningeal and CNS), skin and lymph nodes, and multiple metastases (more than one of the metastatic sites).\n Statistical analysis Baseline characteristics between the two histological groups were compared using chi-square tests for categorical variables and Wilcoxon rank sum tests for continuous variables.\nMetastatic-free interval was defined as time between date of primary diagnosis and date of first distant metastasis. Overall survival after diagnosis of metastatic breast cancer was defined as time between date of first distant metastasis and date of death. Survival curves and time to first palliative systemic therapy (either chemotherapy or endocrine therapy) were estimated using the Kaplan-Meier method and compared using log-rank tests. All patients still alive were censored at the date of last follow-up of each individual patient. Patients who died without palliative therapy were censored at the date of death in the analysis of time to first palliative therapy.\nTo explore the association of palliative systemic therapy with the survival of patients with metastatic breast cancer for both histological subtypes a Cox proportional hazards model was performed with palliative chemotherapy and endocrine therapy as a time-dependent covariate, since the administration of treatment can change over time and is dependent on the time available for each patient to receive the treatment. We did not explore the association between palliative targeted therapy, such as trastuzumab and bevacizumab, and survival since the number of patients with IDC and ILC receiving targeted therapy was very low. Since all these patients received targeted therapy with chemotherapy, these patients were included in the analysis on initial chemotherapy. In addition, the landmark method was used to estimate survival after a specific time-point, the so-called residual survival [26]. As we were interested to learn about the obtained survival in relation to the initial palliative treatment choices, we chose six months after diagnosis of metastatic breast cancer as landmark. Consequently, patients who already died within 6 months were excluded for the residual survival curves.\nAll analyses were performed using SAS version 9.2. All reported P-values are two-sided and P-value ≤ 0.05 was considered statistically significant.\nBaseline characteristics between the two histological groups were compared using chi-square tests for categorical variables and Wilcoxon rank sum tests for continuous variables.\nMetastatic-free interval was defined as time between date of primary diagnosis and date of first distant metastasis. Overall survival after diagnosis of metastatic breast cancer was defined as time between date of first distant metastasis and date of death. Survival curves and time to first palliative systemic therapy (either chemotherapy or endocrine therapy) were estimated using the Kaplan-Meier method and compared using log-rank tests. All patients still alive were censored at the date of last follow-up of each individual patient. Patients who died without palliative therapy were censored at the date of death in the analysis of time to first palliative therapy.\nTo explore the association of palliative systemic therapy with the survival of patients with metastatic breast cancer for both histological subtypes a Cox proportional hazards model was performed with palliative chemotherapy and endocrine therapy as a time-dependent covariate, since the administration of treatment can change over time and is dependent on the time available for each patient to receive the treatment. We did not explore the association between palliative targeted therapy, such as trastuzumab and bevacizumab, and survival since the number of patients with IDC and ILC receiving targeted therapy was very low. Since all these patients received targeted therapy with chemotherapy, these patients were included in the analysis on initial chemotherapy. In addition, the landmark method was used to estimate survival after a specific time-point, the so-called residual survival [26]. As we were interested to learn about the obtained survival in relation to the initial palliative treatment choices, we chose six months after diagnosis of metastatic breast cancer as landmark. Consequently, patients who already died within 6 months were excluded for the residual survival curves.\nAll analyses were performed using SAS version 9.2. All reported P-values are two-sided and P-value ≤ 0.05 was considered statistically significant.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThis study describes the differences between the two largest histological breast cancer subtypes (invasive ductal carcinoma (IDC) and invasive (mixed) lobular carcinoma (ILC) with respect to patient and tumor characteristics, treatment-choices and outcome in metastatic breast cancer.\n\nMethods:\nWe included 437 patients with hormone receptor-positive IDC and 131 patients with hormone receptor-positive ILC, all diagnosed with metastatic breast cancer between 2007-2009, irrespective of date of the primary diagnosis. Patient and tumor characteristics and data on treatment and outcome were collected. Survival curves were obtained using the Kaplan-Meier method.\n\nResults:\nPatients with ILC were older at diagnosis of primary breast cancer and had more often initial bone metastasis (46.5% versus 34.8%, P = 0.01) and less often multiple metastatic sites compared to IDC (23.7% versus 30.9%, P = 0.11). Six months after diagnosis of metastatic breast cancer, 28.1% of patients with ILC and 39.8% of patients with IDC had received chemotherapy with a longer median time to first chemotherapy for those with ILC (P = 0.001). After six months 84.8% of patients with ILC had received endocrine therapy versus 72.5% of patients with IDC (P = 0.0001). Median overall survival was 29 months for ILC and 25 months for IDC (P = 0.53).\n\nConclusions:\nTreatment strategies of hormone receptor-positive metastatic breast cancer were remarkably different for patients with ILC and IDC. Further research is required to understand tumor behavior and treatment-choices in real-life."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nThe two most frequent histological subtypes of breast cancer are invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC), with IDC comprising 75–80% and ILC 5–15% of all breast cancer cases. ILC is being associated with larger tumor size at presentation, more bilateral and multifocal involvement and with a different pattern of metastatic spread compared with IDC [1, 2]. Furthermore, ILC is more often HR-positive, HER2-negative, with lower S-phase fraction and less often positive for the tumor suppressor gene p53, compared with IDC [1]. Also, treatment response is known to be different. In a combined analysis on a number of retrospective series, the pathological complete response rate of neo-adjuvant chemotherapy was significantly lower in ILC (1.7%) than in IDC (11.6%) [3]. In the adjuvant setting, retrospective data suggest a higher efficacy of endocrine therapy for ILC than IDC [4]. More recently, a large retrospective study strongly suggested that HR-positive breast cancer patients with ILC do not seem to benefit from adjuvant chemotherapy in addition to endocrine therapy [5].\nWhether these differences have a prognostic impact is controversial. Some studies found no effect of histology on survival [1, 6, 7], others found a better prognosis for patients with ILC compared to those with IDC [8–10] and some found a change in prognosis over time with a better prognosis for ILC during the first years of follow-up and a worse prognosis during later years [2, 11].\nSo far, studies on histological subtypes consider early breast cancer and not metastatic breast cancer. The aim of this study was to describe the differences between IDC and ILC with respect to patient and tumor characteristics, treatment-choices and outcome in metastatic breast cancer. In order to account for the effect of the hormone receptor (HR) on outcome and treatment-decision making, we only included patients with HR-positive breast cancer.\n\nDISCUSSION:\nBaseline characteristics between the two histological groups were compared using chi-square tests for categorical variables and Wilcoxon rank sum tests for continuous variables.\nMetastatic-free interval was defined as time between date of primary diagnosis and date of first distant metastasis. Overall survival after diagnosis of metastatic breast cancer was defined as time between date of first distant metastasis and date of death. Survival curves and time to first palliative systemic therapy (either chemotherapy or endocrine therapy) were estimated using the Kaplan-Meier method and compared using log-rank tests. All patients still alive were censored at the date of last follow-up of each individual patient. Patients who died without palliative therapy were censored at the date of death in the analysis of time to first palliative therapy.\nTo explore the association of palliative systemic therapy with the survival of patients with metastatic breast cancer for both histological subtypes a Cox proportional hazards model was performed with palliative chemotherapy and endocrine therapy as a time-dependent covariate, since the administration of treatment can change over time and is dependent on the time available for each patient to receive the treatment. We did not explore the association between palliative targeted therapy, such as trastuzumab and bevacizumab, and survival since the number of patients with IDC and ILC receiving targeted therapy was very low. Since all these patients received targeted therapy with chemotherapy, these patients were included in the analysis on initial chemotherapy. In addition, the landmark method was used to estimate survival after a specific time-point, the so-called residual survival [26]. As we were interested to learn about the obtained survival in relation to the initial palliative treatment choices, we chose six months after diagnosis of metastatic breast cancer as landmark. Consequently, patients who already died within 6 months were excluded for the residual survival curves.\nAll analyses were performed using SAS version 9.2. All reported P-values are two-sided and P-value ≤ 0.05 was considered statistically significant."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nThis study describes the differences between the two largest histological breast cancer subtypes (invasive ductal carcinoma (IDC) and invasive (mixed) lobular carcinoma (ILC) with respect to patient and tumor characteristics, treatment-choices and outcome in metastatic breast cancer.\n\nMethods:\nWe included 437 patients with hormone receptor-positive IDC and 131 patients with hormone receptor-positive ILC, all diagnosed with metastatic breast cancer between 2007-2009, irrespective of date of the primary diagnosis. Patient and tumor characteristics and data on treatment and outcome were collected. Survival curves were obtained using the Kaplan-Meier method.\n\nResults:\nPatients with ILC were older at diagnosis of primary breast cancer and had more often initial bone metastasis (46.5% versus 34.8%, P = 0.01) and less often multiple metastatic sites compared to IDC (23.7% versus 30.9%, P = 0.11). Six months after diagnosis of metastatic breast cancer, 28.1% of patients with ILC and 39.8% of patients with IDC had received chemotherapy with a longer median time to first chemotherapy for those with ILC (P = 0.001). After six months 84.8% of patients with ILC had received endocrine therapy versus 72.5% of patients with IDC (P = 0.0001). Median overall survival was 29 months for ILC and 25 months for IDC (P = 0.53).\n\nConclusions:\nTreatment strategies of hormone receptor-positive metastatic breast cancer were remarkably different for patients with ILC and IDC. Further research is required to understand tumor behavior and treatment-choices in real-life."},"whole_article_text_length":{"kind":"number","value":6360,"string":"6,360"},"whole_article_abstract_length":{"kind":"number","value":301,"string":"301"},"other_sections_lengths":{"kind":"list like","value":[382,2159,297,229,203,338,1154],"string":"[\n 382,\n 2159,\n 297,\n 229,\n 203,\n 338,\n 1154\n]","isConversational":false},"num_sections":{"kind":"number","value":8,"string":"8"},"most_frequent_words":{"kind":"list like","value":["patients","palliative","ilc","metastatic","months","positive","idc","hr","breast","breast cancer"],"string":"[\n \"patients\",\n \"palliative\",\n \"ilc\",\n \"metastatic\",\n \"months\",\n \"positive\",\n \"idc\",\n \"hr\",\n \"breast\",\n \"breast cancer\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["adjuvant chemotherapy significantly","lobular carcinoma ilc","metastatic breast cancer","breast cancer subtypes","chemotherapy compared idc"],"string":"[\n \"adjuvant chemotherapy significantly\",\n \"lobular carcinoma ilc\",\n \"metastatic breast cancer\",\n \"breast cancer subtypes\",\n \"chemotherapy compared idc\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"null"},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] metastatic breast cancer | histology | invasive lobular carcinoma | invasive ductal carcinoma | treatment [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] metastatic breast cancer | histology | invasive lobular carcinoma | invasive ductal carcinoma | treatment [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"null"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] metastatic breast cancer | histology | invasive lobular carcinoma | invasive ductal carcinoma | treatment [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] metastatic breast cancer | histology | invasive lobular carcinoma | invasive ductal carcinoma | treatment [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] metastatic breast cancer | histology | invasive lobular carcinoma | invasive ductal carcinoma | treatment [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Aged, 80 and over | Antineoplastic Agents | Breast Neoplasms | Carcinoma, Ductal, Breast | Carcinoma, Lobular | Disease-Free Survival | Female | Follow-Up Studies | Humans | Kaplan-Meier Estimate | Middle Aged | Receptor, ErbB-2 | Receptors, Estrogen | Receptors, Progesterone | Treatment Outcome [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Aged, 80 and over | Antineoplastic Agents | Breast Neoplasms | Carcinoma, Ductal, Breast | Carcinoma, Lobular | Disease-Free Survival | Female | Follow-Up Studies | Humans | Kaplan-Meier Estimate | Middle Aged | Receptor, ErbB-2 | Receptors, Estrogen | Receptors, Progesterone | Treatment Outcome [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"null"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Aged, 80 and over | Antineoplastic Agents | Breast Neoplasms | Carcinoma, Ductal, Breast | Carcinoma, Lobular | Disease-Free Survival | Female | Follow-Up Studies | Humans | Kaplan-Meier Estimate | Middle Aged | Receptor, ErbB-2 | Receptors, Estrogen | Receptors, Progesterone | Treatment Outcome [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Aged, 80 and over | Antineoplastic Agents | Breast Neoplasms | Carcinoma, Ductal, Breast | Carcinoma, Lobular | Disease-Free Survival | Female | Follow-Up Studies | Humans | Kaplan-Meier Estimate | Middle Aged | Receptor, ErbB-2 | Receptors, Estrogen | Receptors, Progesterone | Treatment Outcome [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adult | Aged | Aged, 80 and over | Antineoplastic Agents | Breast Neoplasms | Carcinoma, Ductal, Breast | Carcinoma, Lobular | Disease-Free Survival | Female | Follow-Up Studies | Humans | Kaplan-Meier Estimate | Middle Aged | Receptor, ErbB-2 | Receptors, Estrogen | Receptors, Progesterone | Treatment Outcome [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] adjuvant chemotherapy significantly | lobular carcinoma ilc | metastatic breast cancer | breast cancer subtypes | chemotherapy compared idc [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] adjuvant chemotherapy significantly | lobular carcinoma ilc | metastatic breast cancer | breast cancer subtypes | chemotherapy compared idc [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"null"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] adjuvant chemotherapy significantly | lobular carcinoma ilc | metastatic breast cancer | breast cancer subtypes | chemotherapy compared idc [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] adjuvant chemotherapy significantly | lobular carcinoma ilc | metastatic breast cancer | breast cancer subtypes | chemotherapy compared idc [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] adjuvant chemotherapy significantly | lobular carcinoma ilc | metastatic breast cancer | breast cancer subtypes | chemotherapy compared idc [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | palliative | ilc | metastatic | months | positive | idc | hr | breast | breast cancer [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | palliative | ilc | metastatic | months | positive | idc | hr | breast | breast cancer [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"null"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | palliative | ilc | metastatic | months | positive | idc | hr | breast | breast cancer [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | palliative | ilc | metastatic | months | positive | idc | hr | breast | breast cancer [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | palliative | ilc | metastatic | months | positive | idc | hr | breast | breast cancer [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ilc | prognosis | idc | breast cancer | breast | cancer | found | retrospective | ilc idc | tumor [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | date | defined | cancer | metastatic | breast | breast cancer | survival | therapy | metastatic breast [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"null"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] hr positive | hr | positive | treatment | histology | patients | breast | cancer | metastatic | breast cancer [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | palliative | months | ilc | metastatic | positive | idc | breast | breast cancer | cancer [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | palliative | months | ilc | metastatic | positive | idc | breast | breast cancer | cancer [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] two [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 437 | 131 | ILC | between 2007-2009 ||| ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"null"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] ILC | IDC ||| [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] two ||| 437 | 131 | ILC | between 2007-2009 ||| ||| ||| ILC | 46.5% | 34.8% | 0.01 | IDC | 23.7% | 30.9% | 0.11 ||| Six months | 28.1% | ILC | 39.8% | IDC | first | ILC | 0.001 ||| six months | 84.8% | ILC | 72.5% | 0.0001 ||| Median | 29 months | ILC | 25 months | 0.53 ||| ILC | IDC ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] two ||| 437 | 131 | ILC | between 2007-2009 ||| ||| ||| ILC | 46.5% | 34.8% | 0.01 | IDC | 23.7% | 30.9% | 0.11 ||| Six months | 28.1% | ILC | 39.8% | IDC | first | ILC | 0.001 ||| six months | 84.8% | ILC | 72.5% | 0.0001 ||| Median | 29 months | ILC | 25 months | 0.53 ||| ILC | IDC ||| [SUMMARY]"}}},{"rowIdx":89,"cells":{"title":{"kind":"string","value":"The Impact of Inflammatory Profile on Selenium Levels in Hemodialysis Patients."},"pmid":{"kind":"string","value":"30666918"},"background_abstract":{"kind":"string","value":"Hemodialysis stands out as an eligible treatment for patients with chronic kidney disease. The subsequent inflammatory process resulting from this disease and hemodialysis per se is exacerbated in this therapy. Selenium (Se) is an essential trace element that can participate in the inhibition of pro-oxidant and pro-inflammatory processes and could be considered a measurement that indicates the progression of chronic kidney disease and inflammation."},"background_abstract_label":{"kind":"string","value":"INTRODUCTION"},"methods_abstract":{"kind":"string","value":"This is an observational cross-sectional study of the Faculdade de Medicina do ABC in patients submitted to hemodialysis three times a week for at least six months. The eligible group composed of 21 patients, who filled out forms and underwent biochemical tests (colorimetric enzyme methods, flow cytometer, turbidimetric method and mass spectrometry)."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The study population showed, women (70%), men (30%) with a mean age of 47 ± 17 years, Caucasians (36%) and non-Caucasian (64%), hypertensive (68%), smokers (53%) and non-smokers (64%). There was a hegemonic prevalence of systolic arterial hypertension (SAH) 68.1% in relation to diabetes mellitus (DM) (50%). Pre and post hemodialysis (HD) selenemia showed statistical significance, which did not occur with Creactive protein. There was a predominance of females in our sample; the pre- and post- HD selenemia were within the normal range of the reference values; there was a statistically significant correlation between pre and post-HD selenemia; there was no correlation with statistical significance between values of pre and post-HD C-reactive protein."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Our data showed that there was no direct relationship between pre- and post- HD inflammation and pre- and post-HD selenemia."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Adult","C-Reactive Protein","Female","Humans","Inflammation","Male","Middle Aged","Renal Dialysis","Renal Insufficiency, Chronic","Selenium"],"string":"[\n \"Adult\",\n \"C-Reactive Protein\",\n \"Female\",\n \"Humans\",\n \"Inflammation\",\n \"Male\",\n \"Middle Aged\",\n \"Renal Dialysis\",\n \"Renal Insufficiency, Chronic\",\n \"Selenium\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"7460750"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Current evidence suggests that systemic arterial hypertension (SAH) and type II diabetes mellitus (DM) are the main causes of chronic kidney disease (CKD) worldwide [1-3]. CKD has a direct relationship with cardiovascular diseases and is considered a high risk factor for death and comorbidities [4-6]. Therefore, renal dysfunction is an additional target for intervention and prevention of cardiopathies [5].\nBoth SAH and DM are progressive, and in relation to renal function, are the main causes that contribute to the high risk of progression to end-stage renal disease. This condition requires long-term maintenance of survival therapies such as hemodialysis or renal transplantation [6].\nStudies that evaluated the prevalence and incidence of CKD in the world are based on patients who started renal replacement therapy (RRT) [6-8]. In America, a 1.1% increase in incidence was detected, representing 116.395 new cases in the terminal stage [9]. In Japan, the European Union, India and sub-Saharan Africa, the number of TS patients reaches 2000, 800 and 100 per million of the population, respectively [1]. In Brazil, prevalence and incidence increase each year, concurrent with the increase in the number of patients in RRT [8, 9]. In the last Brazilian census of dialysis of 2011-2014, dialytics accounted for 112,004 [10].\nPatients with inflammatory CKD constitute an independent risk factor for morbidity and mortality. Studies have shown that inflammation increases cardiovascular risk and mortality in patients with terminal CKD [11, 12]. Inflammation is characterized by elevated levels of C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), albumin and pre-albumin that constitute common findings in dialysis patients. It is also postulated that the traditional risk factors for cardiovascular disease such as hypertension, DM, dyslipidemia and obesity would not be sufficient to explain the high incidence of cardiovascular complications in terminal CKD [12, 13].\nRecent findings in the literature attribute to inflammation, oxidative stress, malnutrition, accelerated atherosclerosis as well as renin angiotensin aldosterone system imbalance the responsibility for worse prognosis in kidney diseases. These findings have established an association between inflammation, malnutrition and atherosclerosis in terminal CKD [14, 15]. Beneficial effects are attributed to selenium (Se) in areas such as immuno-competence, cardio-protection and cancer prevention. And low selenium status has been associated with increased risk of mortality, and cognitive decline [16, 17].\nThe tissue levels depend on daily intake and the form in which Se is consumed, as well as on the chemical form found and metabolism [18, 19]. Associated with this is also the case that selenemia in people from seleniferous regions surpasses those of non-seleniferous areas, especially when observed with antitumor actions [20-22].\nThe biological effect of Se is due to the amino acid selenocysteine, an active component of glutathione peroxidase (GPX), whose main function is to neutralize hydrogen peroxide and oxidative damage [23-25].\nThe chronic renal patient has deficient intestinal absorption of Se, on the other hand, there is increased urinary and dialytic loss, in addition to abnormal binding to the proteins involved in its transport. In addition to that, there is a reduction in the intake of Se associated with restricted consumption of protein foods and the urinary loss of albumin. The kidneys are responsible for the synthesis of the enzyme glutathione peroxidase (GSH-Px), a factor that contributes to the low plasma levels of this enzyme in the more advanced stages of CKF [26-28].\nZachara and collaborators have shown that reduced blood levels of Se in association with the decrease of plasma GSH-Px enzyme are correlated with the progression of the kidney disease to the terminal stage [24]. Oxidative stress, a classical risk factor for cardiovascular events and deficiency of selenium antioxidant mechanisms, compose reports from the literature on dialysis. Reduced levels of Se can contribute to endothelial dysfunction, affect coronary flow, and promote accelerated atherosclerosis. In addition to these findings, serum levels of Se are known to be inversely associated with the risk of mortality in patients in HD [17, 24, 25, 27]."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"In this study, the following variables were evaluated: blood urea, CRP and plasma Se before and after hemodialysis. The magnitude of inflammatory impairment was assessed through CRP levels and of the renal impairment through the levels of urea, besides the variation in the levels of Se.\nTable 1 shows the baseline characteristics of the individuals evaluated in this study, the percentage values were: women (70%), hypertensive (68%), non-Caucasian (64%) and non-smokers (64%), with a mean age of 47 ± 17 years, contrasting with men (30%), non-caucasian, smokers. There was a hegemonic prevalence of SAH 68.1% in relation to DM (50%).\nWe observed a statistical reduction in plasma hemoglobin and selenium levels after the hemodialysis session post HD compared to pre HD as shown in Table 2. We did not observe an alteration in the other biochemical parameters studied."},"conclusions_title":{"kind":"string","value":"CONCLUSION"},"conclusions_text":{"kind":"string","value":"Summing up the data of this study, we verified that there was a predominance of females in our sample. Pre- and post-HD selenemia values were within the normal range of the literature reference values. There is a correlation between pre- and post-HD selenemia, but not between selenemia pre-HD and post-HD with CRP values. Therefore, this suggest that the concentration of selenium does not affect CRP levels in pre- and post-HD patients."},"other_sections_titles":{"kind":"list like","value":["Inclusion Criteria","Exclusion Criteria"],"string":"[\n \"Inclusion Criteria\",\n \"Exclusion Criteria\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Patients over 21 years of age who underwent hemodialysis 3 times a week for at least 6 months and who signed the informed consent form were included.","Patients undergoing antibiotic therapy, with chronic and acute inflammatory disorders, zinc supplementation, who were discharged from the hospital 30 days prior to collection, patients with human immunodeficiency virus or acquired immunodeficiency syndrome, and those with viral hepatitis B and C, who had rheumatoid arthritis, scleroderma, systemic lupus erythematosus and who were undergoing continuous corticosteroid therapy were excluded.\nThe sample size was calculated for convenience because it did not have the expected value for the difference between patients with dialytic renal disease.\nDuplicate, individualized blood samples were collected from eligible patients for the analysis of routine parameters: urea, creatinine, serum albumin, complete blood count, in addition to serum Se concentration and C-reactive protein (CRP).\nThe samples were kept in a freezer at -20°C and later processed in automatic analyzers from the Laboratory of Clinical Analysis of the School of Medicine.\nUrea, creatinine and albumin were evaluated by using colorimetric enzyme methods in Cobas 6000® apparatus (Roche®). The total blood counting cells were performed using flow cytometer in ABX Penta 120® apparatus (HORIBA®). CRP ultrasensitive was measured by a turbidimetric method in Immulite 1000® (Siemens®). Finally, selenium concentration was determined by ICP-MS plasma inductively coupled to mass spectrometer.\nAll parameters were performed following a practical clinical laboratory analysis.\nResults are shown as mean ± standard deviation of the mean. The data were analyzed by unpaired T test by STATA 15 software. The level of statistical significance was defined as p<0.05."],"string":"[\n \"Patients over 21 years of age who underwent hemodialysis 3 times a week for at least 6 months and who signed the informed consent form were included.\",\n \"Patients undergoing antibiotic therapy, with chronic and acute inflammatory disorders, zinc supplementation, who were discharged from the hospital 30 days prior to collection, patients with human immunodeficiency virus or acquired immunodeficiency syndrome, and those with viral hepatitis B and C, who had rheumatoid arthritis, scleroderma, systemic lupus erythematosus and who were undergoing continuous corticosteroid therapy were excluded.\\nThe sample size was calculated for convenience because it did not have the expected value for the difference between patients with dialytic renal disease.\\nDuplicate, individualized blood samples were collected from eligible patients for the analysis of routine parameters: urea, creatinine, serum albumin, complete blood count, in addition to serum Se concentration and C-reactive protein (CRP).\\nThe samples were kept in a freezer at -20°C and later processed in automatic analyzers from the Laboratory of Clinical Analysis of the School of Medicine.\\nUrea, creatinine and albumin were evaluated by using colorimetric enzyme methods in Cobas 6000® apparatus (Roche®). The total blood counting cells were performed using flow cytometer in ABX Penta 120® apparatus (HORIBA®). CRP ultrasensitive was measured by a turbidimetric method in Immulite 1000® (Siemens®). Finally, selenium concentration was determined by ICP-MS plasma inductively coupled to mass spectrometer.\\nAll parameters were performed following a practical clinical laboratory analysis.\\nResults are shown as mean ± standard deviation of the mean. The data were analyzed by unpaired T test by STATA 15 software. The level of statistical significance was defined as p<0.05.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null],"string":"[\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","OBJECTIVES","MATERIALS AND METHODS","Inclusion Criteria","Exclusion Criteria","RESULTS","DISCUSSION","CONCLUSION","ETHICS APPROVAL AND CONSENT TO PARTICIPATE","HUMAN AND ANIMAL RIGHTS","CONSENT FOR PUBLICATION","AVAILABILITY OF DATA AND MATERIALS","FUNDING"],"string":"[\n \"INTRODUCTION\",\n \"OBJECTIVES\",\n \"MATERIALS AND METHODS\",\n \"Inclusion Criteria\",\n \"Exclusion Criteria\",\n \"RESULTS\",\n \"DISCUSSION\",\n \"CONCLUSION\",\n \"ETHICS APPROVAL AND CONSENT TO PARTICIPATE\",\n \"HUMAN AND ANIMAL RIGHTS\",\n \"CONSENT FOR PUBLICATION\",\n \"AVAILABILITY OF DATA AND MATERIALS\",\n \"FUNDING\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Current evidence suggests that systemic arterial hypertension (SAH) and type II diabetes mellitus (DM) are the main causes of chronic kidney disease (CKD) worldwide [1-3]. CKD has a direct relationship with cardiovascular diseases and is considered a high risk factor for death and comorbidities [4-6]. Therefore, renal dysfunction is an additional target for intervention and prevention of cardiopathies [5].\nBoth SAH and DM are progressive, and in relation to renal function, are the main causes that contribute to the high risk of progression to end-stage renal disease. This condition requires long-term maintenance of survival therapies such as hemodialysis or renal transplantation [6].\nStudies that evaluated the prevalence and incidence of CKD in the world are based on patients who started renal replacement therapy (RRT) [6-8]. In America, a 1.1% increase in incidence was detected, representing 116.395 new cases in the terminal stage [9]. In Japan, the European Union, India and sub-Saharan Africa, the number of TS patients reaches 2000, 800 and 100 per million of the population, respectively [1]. In Brazil, prevalence and incidence increase each year, concurrent with the increase in the number of patients in RRT [8, 9]. In the last Brazilian census of dialysis of 2011-2014, dialytics accounted for 112,004 [10].\nPatients with inflammatory CKD constitute an independent risk factor for morbidity and mortality. Studies have shown that inflammation increases cardiovascular risk and mortality in patients with terminal CKD [11, 12]. Inflammation is characterized by elevated levels of C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), albumin and pre-albumin that constitute common findings in dialysis patients. It is also postulated that the traditional risk factors for cardiovascular disease such as hypertension, DM, dyslipidemia and obesity would not be sufficient to explain the high incidence of cardiovascular complications in terminal CKD [12, 13].\nRecent findings in the literature attribute to inflammation, oxidative stress, malnutrition, accelerated atherosclerosis as well as renin angiotensin aldosterone system imbalance the responsibility for worse prognosis in kidney diseases. These findings have established an association between inflammation, malnutrition and atherosclerosis in terminal CKD [14, 15]. Beneficial effects are attributed to selenium (Se) in areas such as immuno-competence, cardio-protection and cancer prevention. And low selenium status has been associated with increased risk of mortality, and cognitive decline [16, 17].\nThe tissue levels depend on daily intake and the form in which Se is consumed, as well as on the chemical form found and metabolism [18, 19]. Associated with this is also the case that selenemia in people from seleniferous regions surpasses those of non-seleniferous areas, especially when observed with antitumor actions [20-22].\nThe biological effect of Se is due to the amino acid selenocysteine, an active component of glutathione peroxidase (GPX), whose main function is to neutralize hydrogen peroxide and oxidative damage [23-25].\nThe chronic renal patient has deficient intestinal absorption of Se, on the other hand, there is increased urinary and dialytic loss, in addition to abnormal binding to the proteins involved in its transport. In addition to that, there is a reduction in the intake of Se associated with restricted consumption of protein foods and the urinary loss of albumin. The kidneys are responsible for the synthesis of the enzyme glutathione peroxidase (GSH-Px), a factor that contributes to the low plasma levels of this enzyme in the more advanced stages of CKF [26-28].\nZachara and collaborators have shown that reduced blood levels of Se in association with the decrease of plasma GSH-Px enzyme are correlated with the progression of the kidney disease to the terminal stage [24]. Oxidative stress, a classical risk factor for cardiovascular events and deficiency of selenium antioxidant mechanisms, compose reports from the literature on dialysis. Reduced levels of Se can contribute to endothelial dysfunction, affect coronary flow, and promote accelerated atherosclerosis. In addition to these findings, serum levels of Se are known to be inversely associated with the risk of mortality in patients in HD [17, 24, 25, 27].","Our aim was to investigate serum levels of selenium in hemodialysis patients and to establish the correlation between the inflammatory marker and selenemia in hemodialysis patients.","This is an observational cross-sectional study of patients at the Reference Center for Nephrology at the Mário Covas State Hospital of the Faculdade de Medicina do ABC submitted to hemodialysis 3 times a week for at least 6 months to investigate the correlation between serum Se levels and inflammatory markers.\nThe eligible group was comprised of 22 patients who filled out forms and submitted to laboratory tests. Individual blood samples were collected for the analysis of inflammatory markers of the serum concentration of Se.\n Inclusion Criteria Patients over 21 years of age who underwent hemodialysis 3 times a week for at least 6 months and who signed the informed consent form were included.\nPatients over 21 years of age who underwent hemodialysis 3 times a week for at least 6 months and who signed the informed consent form were included.\n Exclusion Criteria Patients undergoing antibiotic therapy, with chronic and acute inflammatory disorders, zinc supplementation, who were discharged from the hospital 30 days prior to collection, patients with human immunodeficiency virus or acquired immunodeficiency syndrome, and those with viral hepatitis B and C, who had rheumatoid arthritis, scleroderma, systemic lupus erythematosus and who were undergoing continuous corticosteroid therapy were excluded.\nThe sample size was calculated for convenience because it did not have the expected value for the difference between patients with dialytic renal disease.\nDuplicate, individualized blood samples were collected from eligible patients for the analysis of routine parameters: urea, creatinine, serum albumin, complete blood count, in addition to serum Se concentration and C-reactive protein (CRP).\nThe samples were kept in a freezer at -20°C and later processed in automatic analyzers from the Laboratory of Clinical Analysis of the School of Medicine.\nUrea, creatinine and albumin were evaluated by using colorimetric enzyme methods in Cobas 6000® apparatus (Roche®). The total blood counting cells were performed using flow cytometer in ABX Penta 120® apparatus (HORIBA®). CRP ultrasensitive was measured by a turbidimetric method in Immulite 1000® (Siemens®). Finally, selenium concentration was determined by ICP-MS plasma inductively coupled to mass spectrometer.\nAll parameters were performed following a practical clinical laboratory analysis.\nResults are shown as mean ± standard deviation of the mean. The data were analyzed by unpaired T test by STATA 15 software. The level of statistical significance was defined as p<0.05.\nPatients undergoing antibiotic therapy, with chronic and acute inflammatory disorders, zinc supplementation, who were discharged from the hospital 30 days prior to collection, patients with human immunodeficiency virus or acquired immunodeficiency syndrome, and those with viral hepatitis B and C, who had rheumatoid arthritis, scleroderma, systemic lupus erythematosus and who were undergoing continuous corticosteroid therapy were excluded.\nThe sample size was calculated for convenience because it did not have the expected value for the difference between patients with dialytic renal disease.\nDuplicate, individualized blood samples were collected from eligible patients for the analysis of routine parameters: urea, creatinine, serum albumin, complete blood count, in addition to serum Se concentration and C-reactive protein (CRP).\nThe samples were kept in a freezer at -20°C and later processed in automatic analyzers from the Laboratory of Clinical Analysis of the School of Medicine.\nUrea, creatinine and albumin were evaluated by using colorimetric enzyme methods in Cobas 6000® apparatus (Roche®). The total blood counting cells were performed using flow cytometer in ABX Penta 120® apparatus (HORIBA®). CRP ultrasensitive was measured by a turbidimetric method in Immulite 1000® (Siemens®). Finally, selenium concentration was determined by ICP-MS plasma inductively coupled to mass spectrometer.\nAll parameters were performed following a practical clinical laboratory analysis.\nResults are shown as mean ± standard deviation of the mean. The data were analyzed by unpaired T test by STATA 15 software. The level of statistical significance was defined as p<0.05.","Patients over 21 years of age who underwent hemodialysis 3 times a week for at least 6 months and who signed the informed consent form were included.","Patients undergoing antibiotic therapy, with chronic and acute inflammatory disorders, zinc supplementation, who were discharged from the hospital 30 days prior to collection, patients with human immunodeficiency virus or acquired immunodeficiency syndrome, and those with viral hepatitis B and C, who had rheumatoid arthritis, scleroderma, systemic lupus erythematosus and who were undergoing continuous corticosteroid therapy were excluded.\nThe sample size was calculated for convenience because it did not have the expected value for the difference between patients with dialytic renal disease.\nDuplicate, individualized blood samples were collected from eligible patients for the analysis of routine parameters: urea, creatinine, serum albumin, complete blood count, in addition to serum Se concentration and C-reactive protein (CRP).\nThe samples were kept in a freezer at -20°C and later processed in automatic analyzers from the Laboratory of Clinical Analysis of the School of Medicine.\nUrea, creatinine and albumin were evaluated by using colorimetric enzyme methods in Cobas 6000® apparatus (Roche®). The total blood counting cells were performed using flow cytometer in ABX Penta 120® apparatus (HORIBA®). CRP ultrasensitive was measured by a turbidimetric method in Immulite 1000® (Siemens®). Finally, selenium concentration was determined by ICP-MS plasma inductively coupled to mass spectrometer.\nAll parameters were performed following a practical clinical laboratory analysis.\nResults are shown as mean ± standard deviation of the mean. The data were analyzed by unpaired T test by STATA 15 software. The level of statistical significance was defined as p<0.05.","In this study, the following variables were evaluated: blood urea, CRP and plasma Se before and after hemodialysis. The magnitude of inflammatory impairment was assessed through CRP levels and of the renal impairment through the levels of urea, besides the variation in the levels of Se.\nTable 1 shows the baseline characteristics of the individuals evaluated in this study, the percentage values were: women (70%), hypertensive (68%), non-Caucasian (64%) and non-smokers (64%), with a mean age of 47 ± 17 years, contrasting with men (30%), non-caucasian, smokers. There was a hegemonic prevalence of SAH 68.1% in relation to DM (50%).\nWe observed a statistical reduction in plasma hemoglobin and selenium levels after the hemodialysis session post HD compared to pre HD as shown in Table 2. We did not observe an alteration in the other biochemical parameters studied.","The demographic characteristics of the study differed from those of other studies conducted in other countries [28-34].\nA prevalence of 68% of SAH was found in this study, a result that was lower than several published studies such as Portolés et al. [34], which showed a percentage of 75.8% in Spain within a decade, the study by Ohsawa et al. [33] in Japan (87%), and the study by Agarwal et al. [35] in the USA, in which 86% of the patients were hypertensive.\nTwo meta-analyses emphasize the importance of treating hypertension in hemodialysis patients, with the reduction of all-cause mortality and morbidity in those treated with antihypertensive drugs [36, 37].\nA prevalence of 50% of DM in this study was higher than the USRDS report, which showed that 38.5% of patients undergoing hemodialysis in the USA are diabetic [38]. Other study [39], this prevalence ranged from 26% to 43%, with the exception of the CHOICE study [40] with a prevalence of 54% of diabetics. Among the individual cardiovascular risk factors, smoking has a significant association with the incidence of cardiac events.\nIn our study, the prevalence of smoking was 36%, differing from others in the literature which ranged from 40% to 61% in CHOICE [40] and 65% in ANSWER [39]. It is possible that this difference between the data found in this study and others already described present different population, in diet and period studied. We did not identify differences in selenium or CRP levels when we separated gender.\nIn the population sample of ABC, pre-HD selenemia was at the lower limit, wherein the selenemia reduced before hemodialysis. The variability of these values stems from innumerable factors, which can be due to the selenium content in the soil of each region, which determines the concentration of this element in foods of plant origin and subsequently those of animal origin, both terrestrial and aquatic [21].\nThe values in the northeastern region has consubstantiated to the consumption of Brazil nuts, which contain high amounts of selenium due to the high concentration of this micronutrient in the soil. Stockler-Pinto et al. [20-22], determined the selenium content in foods consumed in Brazil from samples taken from the retail trade in several states and showed higher levels of selenium in plasma of hemodialysis patients. Selenium contents in foods of plant origin were observed to be generally below 5.0μg/100g. In Brazil, the presence of fish, mainly, and other products of animal origin in the diet is important to guarantee the recommended levels of selenium. This variability evidences the lack of homogeneity of the results due to the factors, already reported, that influence the concentration of Se, especially food habits.\nThe normal values of Se are needed to saturate GPX, resulting in maximum activity of the enzyme [41-43]. What remains unknown in this sample is lack of information about the eating habits that are not contextualized in the applied questionnaire. There was a statistically significant reduction of selenium between pre- and post-HD, which is consistent with the literature showing that HD promotes a decrease in this micronutrient. Selenium deficiency is a constant in dialysis patients (Table 3).\nSerum selenium levels are markedly low in hemodialytics [44] compared to non-dialytics; in view of the essential role of selenium in GPX activity, selenium deficiency is associated with increased cancer [45] and coronary artery disease [46] in the general population. Selenium is an essential micronutrient with known antioxidant properties [42, 43]. Prabhu et al. (2002) suggested that Se supplementation dietary can be used to prevent and/or treat inflammatory diseases mediated by oxidative stress, considering that HD patients present an oxidative imbalance and an increase of inflammation. It is possible that selenium supplementation is a viable treatment in this case [42]. Furthermore, strongly positive correlations between selenium levels and nutritional markers such as serum albumin have been reported [44, 47].\nTherefore, selenium deficiency may also contribute to malnutrition in patients with HD [48]. The decrease in post-HD levels of selenium did not promote any clinically detectable infectious alterations during the observational period, since pre- and post-HD values of C-reactive protein did not show statistical significance. Thus, our data paradoxically showed no correlation between selenemia and inflammation.","Summing up the data of this study, we verified that there was a predominance of females in our sample. Pre- and post-HD selenemia values were within the normal range of the literature reference values. There is a correlation between pre- and post-HD selenemia, but not between selenemia pre-HD and post-HD with CRP values. Therefore, this suggest that the concentration of selenium does not affect CRP levels in pre- and post-HD patients.","This study was approved by the Institutional Research Ethics Committee of Faculdade de Medicina do ABC, Brazil, under Protocol no. 2.302.284.","The present study was carried out in accordance with the ethical standards of the committee responsible for human experimentation (institutional and national), and with the Helsinki Declaration of 1975, as revised in 2013.","Patients who agreed to participate in the study were given a free and informed consent form (FICF) with the appropriate explanations of the adopted protocols and the informed consent forms were signed by the participants.","The data that support the findings of this study are available from the corresponding author, [G.L. da V.] upon reasonable request.","None."],"string":"[\n \"Current evidence suggests that systemic arterial hypertension (SAH) and type II diabetes mellitus (DM) are the main causes of chronic kidney disease (CKD) worldwide [1-3]. CKD has a direct relationship with cardiovascular diseases and is considered a high risk factor for death and comorbidities [4-6]. Therefore, renal dysfunction is an additional target for intervention and prevention of cardiopathies [5].\\nBoth SAH and DM are progressive, and in relation to renal function, are the main causes that contribute to the high risk of progression to end-stage renal disease. This condition requires long-term maintenance of survival therapies such as hemodialysis or renal transplantation [6].\\nStudies that evaluated the prevalence and incidence of CKD in the world are based on patients who started renal replacement therapy (RRT) [6-8]. In America, a 1.1% increase in incidence was detected, representing 116.395 new cases in the terminal stage [9]. In Japan, the European Union, India and sub-Saharan Africa, the number of TS patients reaches 2000, 800 and 100 per million of the population, respectively [1]. In Brazil, prevalence and incidence increase each year, concurrent with the increase in the number of patients in RRT [8, 9]. In the last Brazilian census of dialysis of 2011-2014, dialytics accounted for 112,004 [10].\\nPatients with inflammatory CKD constitute an independent risk factor for morbidity and mortality. Studies have shown that inflammation increases cardiovascular risk and mortality in patients with terminal CKD [11, 12]. Inflammation is characterized by elevated levels of C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), albumin and pre-albumin that constitute common findings in dialysis patients. It is also postulated that the traditional risk factors for cardiovascular disease such as hypertension, DM, dyslipidemia and obesity would not be sufficient to explain the high incidence of cardiovascular complications in terminal CKD [12, 13].\\nRecent findings in the literature attribute to inflammation, oxidative stress, malnutrition, accelerated atherosclerosis as well as renin angiotensin aldosterone system imbalance the responsibility for worse prognosis in kidney diseases. These findings have established an association between inflammation, malnutrition and atherosclerosis in terminal CKD [14, 15]. Beneficial effects are attributed to selenium (Se) in areas such as immuno-competence, cardio-protection and cancer prevention. And low selenium status has been associated with increased risk of mortality, and cognitive decline [16, 17].\\nThe tissue levels depend on daily intake and the form in which Se is consumed, as well as on the chemical form found and metabolism [18, 19]. Associated with this is also the case that selenemia in people from seleniferous regions surpasses those of non-seleniferous areas, especially when observed with antitumor actions [20-22].\\nThe biological effect of Se is due to the amino acid selenocysteine, an active component of glutathione peroxidase (GPX), whose main function is to neutralize hydrogen peroxide and oxidative damage [23-25].\\nThe chronic renal patient has deficient intestinal absorption of Se, on the other hand, there is increased urinary and dialytic loss, in addition to abnormal binding to the proteins involved in its transport. In addition to that, there is a reduction in the intake of Se associated with restricted consumption of protein foods and the urinary loss of albumin. The kidneys are responsible for the synthesis of the enzyme glutathione peroxidase (GSH-Px), a factor that contributes to the low plasma levels of this enzyme in the more advanced stages of CKF [26-28].\\nZachara and collaborators have shown that reduced blood levels of Se in association with the decrease of plasma GSH-Px enzyme are correlated with the progression of the kidney disease to the terminal stage [24]. Oxidative stress, a classical risk factor for cardiovascular events and deficiency of selenium antioxidant mechanisms, compose reports from the literature on dialysis. Reduced levels of Se can contribute to endothelial dysfunction, affect coronary flow, and promote accelerated atherosclerosis. In addition to these findings, serum levels of Se are known to be inversely associated with the risk of mortality in patients in HD [17, 24, 25, 27].\",\n \"Our aim was to investigate serum levels of selenium in hemodialysis patients and to establish the correlation between the inflammatory marker and selenemia in hemodialysis patients.\",\n \"This is an observational cross-sectional study of patients at the Reference Center for Nephrology at the Mário Covas State Hospital of the Faculdade de Medicina do ABC submitted to hemodialysis 3 times a week for at least 6 months to investigate the correlation between serum Se levels and inflammatory markers.\\nThe eligible group was comprised of 22 patients who filled out forms and submitted to laboratory tests. Individual blood samples were collected for the analysis of inflammatory markers of the serum concentration of Se.\\n Inclusion Criteria Patients over 21 years of age who underwent hemodialysis 3 times a week for at least 6 months and who signed the informed consent form were included.\\nPatients over 21 years of age who underwent hemodialysis 3 times a week for at least 6 months and who signed the informed consent form were included.\\n Exclusion Criteria Patients undergoing antibiotic therapy, with chronic and acute inflammatory disorders, zinc supplementation, who were discharged from the hospital 30 days prior to collection, patients with human immunodeficiency virus or acquired immunodeficiency syndrome, and those with viral hepatitis B and C, who had rheumatoid arthritis, scleroderma, systemic lupus erythematosus and who were undergoing continuous corticosteroid therapy were excluded.\\nThe sample size was calculated for convenience because it did not have the expected value for the difference between patients with dialytic renal disease.\\nDuplicate, individualized blood samples were collected from eligible patients for the analysis of routine parameters: urea, creatinine, serum albumin, complete blood count, in addition to serum Se concentration and C-reactive protein (CRP).\\nThe samples were kept in a freezer at -20°C and later processed in automatic analyzers from the Laboratory of Clinical Analysis of the School of Medicine.\\nUrea, creatinine and albumin were evaluated by using colorimetric enzyme methods in Cobas 6000® apparatus (Roche®). The total blood counting cells were performed using flow cytometer in ABX Penta 120® apparatus (HORIBA®). CRP ultrasensitive was measured by a turbidimetric method in Immulite 1000® (Siemens®). Finally, selenium concentration was determined by ICP-MS plasma inductively coupled to mass spectrometer.\\nAll parameters were performed following a practical clinical laboratory analysis.\\nResults are shown as mean ± standard deviation of the mean. The data were analyzed by unpaired T test by STATA 15 software. The level of statistical significance was defined as p<0.05.\\nPatients undergoing antibiotic therapy, with chronic and acute inflammatory disorders, zinc supplementation, who were discharged from the hospital 30 days prior to collection, patients with human immunodeficiency virus or acquired immunodeficiency syndrome, and those with viral hepatitis B and C, who had rheumatoid arthritis, scleroderma, systemic lupus erythematosus and who were undergoing continuous corticosteroid therapy were excluded.\\nThe sample size was calculated for convenience because it did not have the expected value for the difference between patients with dialytic renal disease.\\nDuplicate, individualized blood samples were collected from eligible patients for the analysis of routine parameters: urea, creatinine, serum albumin, complete blood count, in addition to serum Se concentration and C-reactive protein (CRP).\\nThe samples were kept in a freezer at -20°C and later processed in automatic analyzers from the Laboratory of Clinical Analysis of the School of Medicine.\\nUrea, creatinine and albumin were evaluated by using colorimetric enzyme methods in Cobas 6000® apparatus (Roche®). The total blood counting cells were performed using flow cytometer in ABX Penta 120® apparatus (HORIBA®). CRP ultrasensitive was measured by a turbidimetric method in Immulite 1000® (Siemens®). Finally, selenium concentration was determined by ICP-MS plasma inductively coupled to mass spectrometer.\\nAll parameters were performed following a practical clinical laboratory analysis.\\nResults are shown as mean ± standard deviation of the mean. The data were analyzed by unpaired T test by STATA 15 software. The level of statistical significance was defined as p<0.05.\",\n \"Patients over 21 years of age who underwent hemodialysis 3 times a week for at least 6 months and who signed the informed consent form were included.\",\n \"Patients undergoing antibiotic therapy, with chronic and acute inflammatory disorders, zinc supplementation, who were discharged from the hospital 30 days prior to collection, patients with human immunodeficiency virus or acquired immunodeficiency syndrome, and those with viral hepatitis B and C, who had rheumatoid arthritis, scleroderma, systemic lupus erythematosus and who were undergoing continuous corticosteroid therapy were excluded.\\nThe sample size was calculated for convenience because it did not have the expected value for the difference between patients with dialytic renal disease.\\nDuplicate, individualized blood samples were collected from eligible patients for the analysis of routine parameters: urea, creatinine, serum albumin, complete blood count, in addition to serum Se concentration and C-reactive protein (CRP).\\nThe samples were kept in a freezer at -20°C and later processed in automatic analyzers from the Laboratory of Clinical Analysis of the School of Medicine.\\nUrea, creatinine and albumin were evaluated by using colorimetric enzyme methods in Cobas 6000® apparatus (Roche®). The total blood counting cells were performed using flow cytometer in ABX Penta 120® apparatus (HORIBA®). CRP ultrasensitive was measured by a turbidimetric method in Immulite 1000® (Siemens®). Finally, selenium concentration was determined by ICP-MS plasma inductively coupled to mass spectrometer.\\nAll parameters were performed following a practical clinical laboratory analysis.\\nResults are shown as mean ± standard deviation of the mean. The data were analyzed by unpaired T test by STATA 15 software. The level of statistical significance was defined as p<0.05.\",\n \"In this study, the following variables were evaluated: blood urea, CRP and plasma Se before and after hemodialysis. The magnitude of inflammatory impairment was assessed through CRP levels and of the renal impairment through the levels of urea, besides the variation in the levels of Se.\\nTable 1 shows the baseline characteristics of the individuals evaluated in this study, the percentage values were: women (70%), hypertensive (68%), non-Caucasian (64%) and non-smokers (64%), with a mean age of 47 ± 17 years, contrasting with men (30%), non-caucasian, smokers. There was a hegemonic prevalence of SAH 68.1% in relation to DM (50%).\\nWe observed a statistical reduction in plasma hemoglobin and selenium levels after the hemodialysis session post HD compared to pre HD as shown in Table 2. We did not observe an alteration in the other biochemical parameters studied.\",\n \"The demographic characteristics of the study differed from those of other studies conducted in other countries [28-34].\\nA prevalence of 68% of SAH was found in this study, a result that was lower than several published studies such as Portolés et al. [34], which showed a percentage of 75.8% in Spain within a decade, the study by Ohsawa et al. [33] in Japan (87%), and the study by Agarwal et al. [35] in the USA, in which 86% of the patients were hypertensive.\\nTwo meta-analyses emphasize the importance of treating hypertension in hemodialysis patients, with the reduction of all-cause mortality and morbidity in those treated with antihypertensive drugs [36, 37].\\nA prevalence of 50% of DM in this study was higher than the USRDS report, which showed that 38.5% of patients undergoing hemodialysis in the USA are diabetic [38]. Other study [39], this prevalence ranged from 26% to 43%, with the exception of the CHOICE study [40] with a prevalence of 54% of diabetics. Among the individual cardiovascular risk factors, smoking has a significant association with the incidence of cardiac events.\\nIn our study, the prevalence of smoking was 36%, differing from others in the literature which ranged from 40% to 61% in CHOICE [40] and 65% in ANSWER [39]. It is possible that this difference between the data found in this study and others already described present different population, in diet and period studied. We did not identify differences in selenium or CRP levels when we separated gender.\\nIn the population sample of ABC, pre-HD selenemia was at the lower limit, wherein the selenemia reduced before hemodialysis. The variability of these values stems from innumerable factors, which can be due to the selenium content in the soil of each region, which determines the concentration of this element in foods of plant origin and subsequently those of animal origin, both terrestrial and aquatic [21].\\nThe values in the northeastern region has consubstantiated to the consumption of Brazil nuts, which contain high amounts of selenium due to the high concentration of this micronutrient in the soil. Stockler-Pinto et al. [20-22], determined the selenium content in foods consumed in Brazil from samples taken from the retail trade in several states and showed higher levels of selenium in plasma of hemodialysis patients. Selenium contents in foods of plant origin were observed to be generally below 5.0μg/100g. In Brazil, the presence of fish, mainly, and other products of animal origin in the diet is important to guarantee the recommended levels of selenium. This variability evidences the lack of homogeneity of the results due to the factors, already reported, that influence the concentration of Se, especially food habits.\\nThe normal values of Se are needed to saturate GPX, resulting in maximum activity of the enzyme [41-43]. What remains unknown in this sample is lack of information about the eating habits that are not contextualized in the applied questionnaire. There was a statistically significant reduction of selenium between pre- and post-HD, which is consistent with the literature showing that HD promotes a decrease in this micronutrient. Selenium deficiency is a constant in dialysis patients (Table 3).\\nSerum selenium levels are markedly low in hemodialytics [44] compared to non-dialytics; in view of the essential role of selenium in GPX activity, selenium deficiency is associated with increased cancer [45] and coronary artery disease [46] in the general population. Selenium is an essential micronutrient with known antioxidant properties [42, 43]. Prabhu et al. (2002) suggested that Se supplementation dietary can be used to prevent and/or treat inflammatory diseases mediated by oxidative stress, considering that HD patients present an oxidative imbalance and an increase of inflammation. It is possible that selenium supplementation is a viable treatment in this case [42]. Furthermore, strongly positive correlations between selenium levels and nutritional markers such as serum albumin have been reported [44, 47].\\nTherefore, selenium deficiency may also contribute to malnutrition in patients with HD [48]. The decrease in post-HD levels of selenium did not promote any clinically detectable infectious alterations during the observational period, since pre- and post-HD values of C-reactive protein did not show statistical significance. Thus, our data paradoxically showed no correlation between selenemia and inflammation.\",\n \"Summing up the data of this study, we verified that there was a predominance of females in our sample. Pre- and post-HD selenemia values were within the normal range of the literature reference values. There is a correlation between pre- and post-HD selenemia, but not between selenemia pre-HD and post-HD with CRP values. Therefore, this suggest that the concentration of selenium does not affect CRP levels in pre- and post-HD patients.\",\n \"This study was approved by the Institutional Research Ethics Committee of Faculdade de Medicina do ABC, Brazil, under Protocol no. 2.302.284.\",\n \"The present study was carried out in accordance with the ethical standards of the committee responsible for human experimentation (institutional and national), and with the Helsinki Declaration of 1975, as revised in 2013.\",\n \"Patients who agreed to participate in the study were given a free and informed consent form (FICF) with the appropriate explanations of the adopted protocols and the informed consent forms were signed by the participants.\",\n \"The data that support the findings of this study are available from the corresponding author, [G.L. da V.] upon reasonable request.\",\n \"None.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["intro","other1","materials|methods",null,null,"results","discussion","conclusions","other2","other3","other4","data-availability","other5"],"string":"[\n \"intro\",\n \"other1\",\n \"materials|methods\",\n null,\n null,\n \"results\",\n \"discussion\",\n \"conclusions\",\n \"other2\",\n \"other3\",\n \"other4\",\n \"data-availability\",\n \"other5\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["Chronic kidney disease","hemodialysis","selenium","pro-oxidant","inflammation","systemic arterial hypertension"],"string":"[\n \"Chronic kidney disease\",\n \"hemodialysis\",\n \"selenium\",\n \"pro-oxidant\",\n \"inflammation\",\n \"systemic arterial hypertension\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nCurrent evidence suggests that systemic arterial hypertension (SAH) and type II diabetes mellitus (DM) are the main causes of chronic kidney disease (CKD) worldwide [1-3]. CKD has a direct relationship with cardiovascular diseases and is considered a high risk factor for death and comorbidities [4-6]. Therefore, renal dysfunction is an additional target for intervention and prevention of cardiopathies [5].\nBoth SAH and DM are progressive, and in relation to renal function, are the main causes that contribute to the high risk of progression to end-stage renal disease. This condition requires long-term maintenance of survival therapies such as hemodialysis or renal transplantation [6].\nStudies that evaluated the prevalence and incidence of CKD in the world are based on patients who started renal replacement therapy (RRT) [6-8]. In America, a 1.1% increase in incidence was detected, representing 116.395 new cases in the terminal stage [9]. In Japan, the European Union, India and sub-Saharan Africa, the number of TS patients reaches 2000, 800 and 100 per million of the population, respectively [1]. In Brazil, prevalence and incidence increase each year, concurrent with the increase in the number of patients in RRT [8, 9]. In the last Brazilian census of dialysis of 2011-2014, dialytics accounted for 112,004 [10].\nPatients with inflammatory CKD constitute an independent risk factor for morbidity and mortality. Studies have shown that inflammation increases cardiovascular risk and mortality in patients with terminal CKD [11, 12]. Inflammation is characterized by elevated levels of C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), albumin and pre-albumin that constitute common findings in dialysis patients. It is also postulated that the traditional risk factors for cardiovascular disease such as hypertension, DM, dyslipidemia and obesity would not be sufficient to explain the high incidence of cardiovascular complications in terminal CKD [12, 13].\nRecent findings in the literature attribute to inflammation, oxidative stress, malnutrition, accelerated atherosclerosis as well as renin angiotensin aldosterone system imbalance the responsibility for worse prognosis in kidney diseases. These findings have established an association between inflammation, malnutrition and atherosclerosis in terminal CKD [14, 15]. Beneficial effects are attributed to selenium (Se) in areas such as immuno-competence, cardio-protection and cancer prevention. And low selenium status has been associated with increased risk of mortality, and cognitive decline [16, 17].\nThe tissue levels depend on daily intake and the form in which Se is consumed, as well as on the chemical form found and metabolism [18, 19]. Associated with this is also the case that selenemia in people from seleniferous regions surpasses those of non-seleniferous areas, especially when observed with antitumor actions [20-22].\nThe biological effect of Se is due to the amino acid selenocysteine, an active component of glutathione peroxidase (GPX), whose main function is to neutralize hydrogen peroxide and oxidative damage [23-25].\nThe chronic renal patient has deficient intestinal absorption of Se, on the other hand, there is increased urinary and dialytic loss, in addition to abnormal binding to the proteins involved in its transport. In addition to that, there is a reduction in the intake of Se associated with restricted consumption of protein foods and the urinary loss of albumin. The kidneys are responsible for the synthesis of the enzyme glutathione peroxidase (GSH-Px), a factor that contributes to the low plasma levels of this enzyme in the more advanced stages of CKF [26-28].\nZachara and collaborators have shown that reduced blood levels of Se in association with the decrease of plasma GSH-Px enzyme are correlated with the progression of the kidney disease to the terminal stage [24]. Oxidative stress, a classical risk factor for cardiovascular events and deficiency of selenium antioxidant mechanisms, compose reports from the literature on dialysis. Reduced levels of Se can contribute to endothelial dysfunction, affect coronary flow, and promote accelerated atherosclerosis. In addition to these findings, serum levels of Se are known to be inversely associated with the risk of mortality in patients in HD [17, 24, 25, 27].\n\nOBJECTIVES:\nOur aim was to investigate serum levels of selenium in hemodialysis patients and to establish the correlation between the inflammatory marker and selenemia in hemodialysis patients.\n\nMATERIALS AND METHODS:\nThis is an observational cross-sectional study of patients at the Reference Center for Nephrology at the Mário Covas State Hospital of the Faculdade de Medicina do ABC submitted to hemodialysis 3 times a week for at least 6 months to investigate the correlation between serum Se levels and inflammatory markers.\nThe eligible group was comprised of 22 patients who filled out forms and submitted to laboratory tests. Individual blood samples were collected for the analysis of inflammatory markers of the serum concentration of Se.\n Inclusion Criteria Patients over 21 years of age who underwent hemodialysis 3 times a week for at least 6 months and who signed the informed consent form were included.\nPatients over 21 years of age who underwent hemodialysis 3 times a week for at least 6 months and who signed the informed consent form were included.\n Exclusion Criteria Patients undergoing antibiotic therapy, with chronic and acute inflammatory disorders, zinc supplementation, who were discharged from the hospital 30 days prior to collection, patients with human immunodeficiency virus or acquired immunodeficiency syndrome, and those with viral hepatitis B and C, who had rheumatoid arthritis, scleroderma, systemic lupus erythematosus and who were undergoing continuous corticosteroid therapy were excluded.\nThe sample size was calculated for convenience because it did not have the expected value for the difference between patients with dialytic renal disease.\nDuplicate, individualized blood samples were collected from eligible patients for the analysis of routine parameters: urea, creatinine, serum albumin, complete blood count, in addition to serum Se concentration and C-reactive protein (CRP).\nThe samples were kept in a freezer at -20°C and later processed in automatic analyzers from the Laboratory of Clinical Analysis of the School of Medicine.\nUrea, creatinine and albumin were evaluated by using colorimetric enzyme methods in Cobas 6000® apparatus (Roche®). The total blood counting cells were performed using flow cytometer in ABX Penta 120® apparatus (HORIBA®). CRP ultrasensitive was measured by a turbidimetric method in Immulite 1000® (Siemens®). Finally, selenium concentration was determined by ICP-MS plasma inductively coupled to mass spectrometer.\nAll parameters were performed following a practical clinical laboratory analysis.\nResults are shown as mean ± standard deviation of the mean. The data were analyzed by unpaired T test by STATA 15 software. The level of statistical significance was defined as p<0.05.\nPatients undergoing antibiotic therapy, with chronic and acute inflammatory disorders, zinc supplementation, who were discharged from the hospital 30 days prior to collection, patients with human immunodeficiency virus or acquired immunodeficiency syndrome, and those with viral hepatitis B and C, who had rheumatoid arthritis, scleroderma, systemic lupus erythematosus and who were undergoing continuous corticosteroid therapy were excluded.\nThe sample size was calculated for convenience because it did not have the expected value for the difference between patients with dialytic renal disease.\nDuplicate, individualized blood samples were collected from eligible patients for the analysis of routine parameters: urea, creatinine, serum albumin, complete blood count, in addition to serum Se concentration and C-reactive protein (CRP).\nThe samples were kept in a freezer at -20°C and later processed in automatic analyzers from the Laboratory of Clinical Analysis of the School of Medicine.\nUrea, creatinine and albumin were evaluated by using colorimetric enzyme methods in Cobas 6000® apparatus (Roche®). The total blood counting cells were performed using flow cytometer in ABX Penta 120® apparatus (HORIBA®). CRP ultrasensitive was measured by a turbidimetric method in Immulite 1000® (Siemens®). Finally, selenium concentration was determined by ICP-MS plasma inductively coupled to mass spectrometer.\nAll parameters were performed following a practical clinical laboratory analysis.\nResults are shown as mean ± standard deviation of the mean. The data were analyzed by unpaired T test by STATA 15 software. The level of statistical significance was defined as p<0.05.\n\nInclusion Criteria:\nPatients over 21 years of age who underwent hemodialysis 3 times a week for at least 6 months and who signed the informed consent form were included.\n\nExclusion Criteria:\nPatients undergoing antibiotic therapy, with chronic and acute inflammatory disorders, zinc supplementation, who were discharged from the hospital 30 days prior to collection, patients with human immunodeficiency virus or acquired immunodeficiency syndrome, and those with viral hepatitis B and C, who had rheumatoid arthritis, scleroderma, systemic lupus erythematosus and who were undergoing continuous corticosteroid therapy were excluded.\nThe sample size was calculated for convenience because it did not have the expected value for the difference between patients with dialytic renal disease.\nDuplicate, individualized blood samples were collected from eligible patients for the analysis of routine parameters: urea, creatinine, serum albumin, complete blood count, in addition to serum Se concentration and C-reactive protein (CRP).\nThe samples were kept in a freezer at -20°C and later processed in automatic analyzers from the Laboratory of Clinical Analysis of the School of Medicine.\nUrea, creatinine and albumin were evaluated by using colorimetric enzyme methods in Cobas 6000® apparatus (Roche®). The total blood counting cells were performed using flow cytometer in ABX Penta 120® apparatus (HORIBA®). CRP ultrasensitive was measured by a turbidimetric method in Immulite 1000® (Siemens®). Finally, selenium concentration was determined by ICP-MS plasma inductively coupled to mass spectrometer.\nAll parameters were performed following a practical clinical laboratory analysis.\nResults are shown as mean ± standard deviation of the mean. The data were analyzed by unpaired T test by STATA 15 software. The level of statistical significance was defined as p<0.05.\n\nRESULTS:\nIn this study, the following variables were evaluated: blood urea, CRP and plasma Se before and after hemodialysis. The magnitude of inflammatory impairment was assessed through CRP levels and of the renal impairment through the levels of urea, besides the variation in the levels of Se.\nTable 1 shows the baseline characteristics of the individuals evaluated in this study, the percentage values were: women (70%), hypertensive (68%), non-Caucasian (64%) and non-smokers (64%), with a mean age of 47 ± 17 years, contrasting with men (30%), non-caucasian, smokers. There was a hegemonic prevalence of SAH 68.1% in relation to DM (50%).\nWe observed a statistical reduction in plasma hemoglobin and selenium levels after the hemodialysis session post HD compared to pre HD as shown in Table 2. We did not observe an alteration in the other biochemical parameters studied.\n\nDISCUSSION:\nThe demographic characteristics of the study differed from those of other studies conducted in other countries [28-34].\nA prevalence of 68% of SAH was found in this study, a result that was lower than several published studies such as Portolés et al. [34], which showed a percentage of 75.8% in Spain within a decade, the study by Ohsawa et al. [33] in Japan (87%), and the study by Agarwal et al. [35] in the USA, in which 86% of the patients were hypertensive.\nTwo meta-analyses emphasize the importance of treating hypertension in hemodialysis patients, with the reduction of all-cause mortality and morbidity in those treated with antihypertensive drugs [36, 37].\nA prevalence of 50% of DM in this study was higher than the USRDS report, which showed that 38.5% of patients undergoing hemodialysis in the USA are diabetic [38]. Other study [39], this prevalence ranged from 26% to 43%, with the exception of the CHOICE study [40] with a prevalence of 54% of diabetics. Among the individual cardiovascular risk factors, smoking has a significant association with the incidence of cardiac events.\nIn our study, the prevalence of smoking was 36%, differing from others in the literature which ranged from 40% to 61% in CHOICE [40] and 65% in ANSWER [39]. It is possible that this difference between the data found in this study and others already described present different population, in diet and period studied. We did not identify differences in selenium or CRP levels when we separated gender.\nIn the population sample of ABC, pre-HD selenemia was at the lower limit, wherein the selenemia reduced before hemodialysis. The variability of these values stems from innumerable factors, which can be due to the selenium content in the soil of each region, which determines the concentration of this element in foods of plant origin and subsequently those of animal origin, both terrestrial and aquatic [21].\nThe values in the northeastern region has consubstantiated to the consumption of Brazil nuts, which contain high amounts of selenium due to the high concentration of this micronutrient in the soil. Stockler-Pinto et al. [20-22], determined the selenium content in foods consumed in Brazil from samples taken from the retail trade in several states and showed higher levels of selenium in plasma of hemodialysis patients. Selenium contents in foods of plant origin were observed to be generally below 5.0μg/100g. In Brazil, the presence of fish, mainly, and other products of animal origin in the diet is important to guarantee the recommended levels of selenium. This variability evidences the lack of homogeneity of the results due to the factors, already reported, that influence the concentration of Se, especially food habits.\nThe normal values of Se are needed to saturate GPX, resulting in maximum activity of the enzyme [41-43]. What remains unknown in this sample is lack of information about the eating habits that are not contextualized in the applied questionnaire. There was a statistically significant reduction of selenium between pre- and post-HD, which is consistent with the literature showing that HD promotes a decrease in this micronutrient. Selenium deficiency is a constant in dialysis patients (Table 3).\nSerum selenium levels are markedly low in hemodialytics [44] compared to non-dialytics; in view of the essential role of selenium in GPX activity, selenium deficiency is associated with increased cancer [45] and coronary artery disease [46] in the general population. Selenium is an essential micronutrient with known antioxidant properties [42, 43]. Prabhu et al. (2002) suggested that Se supplementation dietary can be used to prevent and/or treat inflammatory diseases mediated by oxidative stress, considering that HD patients present an oxidative imbalance and an increase of inflammation. It is possible that selenium supplementation is a viable treatment in this case [42]. Furthermore, strongly positive correlations between selenium levels and nutritional markers such as serum albumin have been reported [44, 47].\nTherefore, selenium deficiency may also contribute to malnutrition in patients with HD [48]. The decrease in post-HD levels of selenium did not promote any clinically detectable infectious alterations during the observational period, since pre- and post-HD values of C-reactive protein did not show statistical significance. Thus, our data paradoxically showed no correlation between selenemia and inflammation.\n\nCONCLUSION:\nSumming up the data of this study, we verified that there was a predominance of females in our sample. Pre- and post-HD selenemia values were within the normal range of the literature reference values. There is a correlation between pre- and post-HD selenemia, but not between selenemia pre-HD and post-HD with CRP values. Therefore, this suggest that the concentration of selenium does not affect CRP levels in pre- and post-HD patients.\n\nETHICS APPROVAL AND CONSENT TO PARTICIPATE:\nThis study was approved by the Institutional Research Ethics Committee of Faculdade de Medicina do ABC, Brazil, under Protocol no. 2.302.284.\n\nHUMAN AND ANIMAL RIGHTS:\nThe present study was carried out in accordance with the ethical standards of the committee responsible for human experimentation (institutional and national), and with the Helsinki Declaration of 1975, as revised in 2013.\n\nCONSENT FOR PUBLICATION:\nPatients who agreed to participate in the study were given a free and informed consent form (FICF) with the appropriate explanations of the adopted protocols and the informed consent forms were signed by the participants.\n\nAVAILABILITY OF DATA AND MATERIALS:\nThe data that support the findings of this study are available from the corresponding author, [G.L. da V.] upon reasonable request.\n\nFUNDING:\nNone.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nHemodialysis stands out as an eligible treatment for patients with chronic kidney disease. The subsequent inflammatory process resulting from this disease and hemodialysis per se is exacerbated in this therapy. Selenium (Se) is an essential trace element that can participate in the inhibition of pro-oxidant and pro-inflammatory processes and could be considered a measurement that indicates the progression of chronic kidney disease and inflammation.\n\nMethods:\nThis is an observational cross-sectional study of the Faculdade de Medicina do ABC in patients submitted to hemodialysis three times a week for at least six months. The eligible group composed of 21 patients, who filled out forms and underwent biochemical tests (colorimetric enzyme methods, flow cytometer, turbidimetric method and mass spectrometry).\n\nResults:\nThe study population showed, women (70%), men (30%) with a mean age of 47 ± 17 years, Caucasians (36%) and non-Caucasian (64%), hypertensive (68%), smokers (53%) and non-smokers (64%). There was a hegemonic prevalence of systolic arterial hypertension (SAH) 68.1% in relation to diabetes mellitus (DM) (50%). Pre and post hemodialysis (HD) selenemia showed statistical significance, which did not occur with Creactive protein. There was a predominance of females in our sample; the pre- and post- HD selenemia were within the normal range of the reference values; there was a statistically significant correlation between pre and post-HD selenemia; there was no correlation with statistical significance between values of pre and post-HD C-reactive protein.\n\nConclusions:\nOur data showed that there was no direct relationship between pre- and post- HD inflammation and pre- and post-HD selenemia."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nCurrent evidence suggests that systemic arterial hypertension (SAH) and type II diabetes mellitus (DM) are the main causes of chronic kidney disease (CKD) worldwide [1-3]. CKD has a direct relationship with cardiovascular diseases and is considered a high risk factor for death and comorbidities [4-6]. Therefore, renal dysfunction is an additional target for intervention and prevention of cardiopathies [5].\nBoth SAH and DM are progressive, and in relation to renal function, are the main causes that contribute to the high risk of progression to end-stage renal disease. This condition requires long-term maintenance of survival therapies such as hemodialysis or renal transplantation [6].\nStudies that evaluated the prevalence and incidence of CKD in the world are based on patients who started renal replacement therapy (RRT) [6-8]. In America, a 1.1% increase in incidence was detected, representing 116.395 new cases in the terminal stage [9]. In Japan, the European Union, India and sub-Saharan Africa, the number of TS patients reaches 2000, 800 and 100 per million of the population, respectively [1]. In Brazil, prevalence and incidence increase each year, concurrent with the increase in the number of patients in RRT [8, 9]. In the last Brazilian census of dialysis of 2011-2014, dialytics accounted for 112,004 [10].\nPatients with inflammatory CKD constitute an independent risk factor for morbidity and mortality. Studies have shown that inflammation increases cardiovascular risk and mortality in patients with terminal CKD [11, 12]. Inflammation is characterized by elevated levels of C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), albumin and pre-albumin that constitute common findings in dialysis patients. It is also postulated that the traditional risk factors for cardiovascular disease such as hypertension, DM, dyslipidemia and obesity would not be sufficient to explain the high incidence of cardiovascular complications in terminal CKD [12, 13].\nRecent findings in the literature attribute to inflammation, oxidative stress, malnutrition, accelerated atherosclerosis as well as renin angiotensin aldosterone system imbalance the responsibility for worse prognosis in kidney diseases. These findings have established an association between inflammation, malnutrition and atherosclerosis in terminal CKD [14, 15]. Beneficial effects are attributed to selenium (Se) in areas such as immuno-competence, cardio-protection and cancer prevention. And low selenium status has been associated with increased risk of mortality, and cognitive decline [16, 17].\nThe tissue levels depend on daily intake and the form in which Se is consumed, as well as on the chemical form found and metabolism [18, 19]. Associated with this is also the case that selenemia in people from seleniferous regions surpasses those of non-seleniferous areas, especially when observed with antitumor actions [20-22].\nThe biological effect of Se is due to the amino acid selenocysteine, an active component of glutathione peroxidase (GPX), whose main function is to neutralize hydrogen peroxide and oxidative damage [23-25].\nThe chronic renal patient has deficient intestinal absorption of Se, on the other hand, there is increased urinary and dialytic loss, in addition to abnormal binding to the proteins involved in its transport. In addition to that, there is a reduction in the intake of Se associated with restricted consumption of protein foods and the urinary loss of albumin. The kidneys are responsible for the synthesis of the enzyme glutathione peroxidase (GSH-Px), a factor that contributes to the low plasma levels of this enzyme in the more advanced stages of CKF [26-28].\nZachara and collaborators have shown that reduced blood levels of Se in association with the decrease of plasma GSH-Px enzyme are correlated with the progression of the kidney disease to the terminal stage [24]. Oxidative stress, a classical risk factor for cardiovascular events and deficiency of selenium antioxidant mechanisms, compose reports from the literature on dialysis. Reduced levels of Se can contribute to endothelial dysfunction, affect coronary flow, and promote accelerated atherosclerosis. In addition to these findings, serum levels of Se are known to be inversely associated with the risk of mortality in patients in HD [17, 24, 25, 27].\n\nCONCLUSION:\nSumming up the data of this study, we verified that there was a predominance of females in our sample. Pre- and post-HD selenemia values were within the normal range of the literature reference values. There is a correlation between pre- and post-HD selenemia, but not between selenemia pre-HD and post-HD with CRP values. Therefore, this suggest that the concentration of selenium does not affect CRP levels in pre- and post-HD patients."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nHemodialysis stands out as an eligible treatment for patients with chronic kidney disease. The subsequent inflammatory process resulting from this disease and hemodialysis per se is exacerbated in this therapy. Selenium (Se) is an essential trace element that can participate in the inhibition of pro-oxidant and pro-inflammatory processes and could be considered a measurement that indicates the progression of chronic kidney disease and inflammation.\n\nMethods:\nThis is an observational cross-sectional study of the Faculdade de Medicina do ABC in patients submitted to hemodialysis three times a week for at least six months. The eligible group composed of 21 patients, who filled out forms and underwent biochemical tests (colorimetric enzyme methods, flow cytometer, turbidimetric method and mass spectrometry).\n\nResults:\nThe study population showed, women (70%), men (30%) with a mean age of 47 ± 17 years, Caucasians (36%) and non-Caucasian (64%), hypertensive (68%), smokers (53%) and non-smokers (64%). There was a hegemonic prevalence of systolic arterial hypertension (SAH) 68.1% in relation to diabetes mellitus (DM) (50%). Pre and post hemodialysis (HD) selenemia showed statistical significance, which did not occur with Creactive protein. There was a predominance of females in our sample; the pre- and post- HD selenemia were within the normal range of the reference values; there was a statistically significant correlation between pre and post-HD selenemia; there was no correlation with statistical significance between values of pre and post-HD C-reactive protein.\n\nConclusions:\nOur data showed that there was no direct relationship between pre- and post- HD inflammation and pre- and post-HD selenemia."},"whole_article_text_length":{"kind":"number","value":3244,"string":"3,244"},"whole_article_abstract_length":{"kind":"number","value":339,"string":"339"},"other_sections_lengths":{"kind":"list like","value":[28,292],"string":"[\n 28,\n 292\n]","isConversational":false},"num_sections":{"kind":"number","value":13,"string":"13"},"most_frequent_words":{"kind":"list like","value":["patients","selenium","levels","se","study","hd","hemodialysis","crp","blood","serum"],"string":"[\n \"patients\",\n \"selenium\",\n \"levels\",\n 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[SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] hemodialysis usa diabetic | causes chronic kidney | comorbidities renal dysfunction | treating hypertension hemodialysis | prognosis kidney diseases [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] hemodialysis usa diabetic | causes chronic kidney | comorbidities renal dysfunction | treating hypertension hemodialysis | prognosis kidney diseases [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] hemodialysis usa diabetic | causes chronic kidney | comorbidities renal dysfunction | treating hypertension hemodialysis | prognosis kidney diseases [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] hemodialysis usa diabetic | causes chronic kidney | comorbidities renal dysfunction | treating hypertension hemodialysis | prognosis kidney diseases [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] hemodialysis usa diabetic | causes chronic kidney | comorbidities renal dysfunction | treating hypertension hemodialysis | prognosis kidney diseases [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | selenium | levels | se | study | hd | hemodialysis | crp | blood | serum [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | selenium | levels | se | study | hd | hemodialysis | crp | blood | serum [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | selenium | levels | se | study | hd | hemodialysis | crp | blood | serum 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selenemia | pre post hd selenemia [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | study | hd | selenium | hemodialysis | levels | consent | informed | informed consent | post hd [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | study | hd | selenium | hemodialysis | levels | consent | informed | informed consent | post hd [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 70% | 30% | 17 years | Caucasians | 36% | non-Caucasian | 64% | 68% | 53% | 64% ||| SAH | 68.1% | 50% ||| Creactive ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| the Faculdade de Medicina | ABC | three | at least six months ||| 21 ||| ||| 70% | 30% | 17 years | Caucasians | 36% | non-Caucasian | 64% | 68% | 53% | 64% ||| SAH | 68.1% | 50% ||| Creactive ||| ||| [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| ||| ||| the Faculdade de Medicina | ABC | three | at least six months ||| 21 ||| ||| 70% | 30% | 17 years | Caucasians | 36% | non-Caucasian | 64% | 68% | 53% | 64% ||| SAH | 68.1% | 50% ||| Creactive ||| ||| [SUMMARY]"}}},{"rowIdx":90,"cells":{"title":{"kind":"string","value":"Nutrition Interventions for Children with Cerebral Palsy in Low- and Middle-Income Countries: A Scoping Review."},"pmid":{"kind":"string","value":"35334869"},"background_abstract":{"kind":"string","value":"Malnutrition is substantially higher among children with cerebral palsy (CP) in low- and middle-income countries (LMICs) when compared with the general population. Access to appropriate interventions is crucial for better management of malnutrition and nutritional outcomes of those children. We aimed to review the existing evidence on nutrition interventions for children with CP in LMICs."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Online databases, i.e., PubMed and Scopus, and Google Scholar were searched up to 10 January 2022, to identify peer-reviewed publications/evidence on LMIC focused nutritional management guidelines/interventions. Following title screening and abstract review, full articles that met the inclusion/exclusion criteria were retained for data charting. Information about the study characteristics, nutrition interventions, and their effectiveness were extracted. Descriptive data were reported."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Eight articles published between 2008 and 2019 were included with data from a total of n = 252 children with CP (age range: 1 y 0 m-18 y 7 m, 42% female). Five studies followed experimental design; n = 6 were conducted in hospital/clinic/center-based settings. Four studies focused on parental/caregiver training; n = 2 studies had surgical interventions (i.e., gastrostomy) and n = 1 provided neurodevelopmental therapy feeding intervention. Dietary modification as an intervention (or component) was reported in n = 5 studies and had better effect on the nutritional outcomes of children with CP compared to interventions focused on feeding skills or other behavioral modifications. Surgical interventions improved nutritional outcomes in both studies; however, none documented any adverse consequences of the surgical interventions."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"There is a substantial knowledge gap on nutrition interventions for children with CP in LMICs. This hinders the development of best practice guidelines for the nutritional management of children with CP in those settings. Findings suggest interventions directly related to growth/feeding of children had a better outcome than behavioral interventions. This should be considered in planning of nutrition-focused intervention or comprehensive services for children with CP in LMICs."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Cerebral Palsy","Child","Developing Countries","Female","Humans","Income","Infant","Male","Malnutrition","Poverty"],"string":"[\n \"Cerebral Palsy\",\n \"Child\",\n \"Developing Countries\",\n \"Female\",\n \"Humans\",\n \"Income\",\n \"Infant\",\n \"Male\",\n \"Malnutrition\",\n \"Poverty\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"8951851"},"background_title":{"kind":"string","value":"1. Introduction"},"background_text":{"kind":"string","value":"Undernutrition is a major global public health challenge, and children with disability, such as cerebral palsy (CP), often suffer from undernutrition, especially in low- and middle-income countries (LMICs). The proportion of malnutrition is substantially higher among children with CP in LMICs compared to the general population [1,2,3,4,5]. Ensuring availability and accessibility to evidence-based nutrition interventions is essential to avert adverse outcomes of malnutrition among those vulnerable children [6].\nNutritional management of children with CP is complex as several interlinked factors interfere with their growth directly and indirectly [7]. While a few factors have been identified as common predictors of malnutrition (e.g., gross motor and oromotor function limitations) [1,8,9], the conceptual framework of malnutrition in children with CP is not yet clearly understood, especially in LMICs. To date, several clinical nutrition guidelines based on current evidence on surgical and non-surgical intervention outcomes have been published [6,10]. However, most of those studies were conducted in high-income countries (HICs) [6], whereas 85% of children with disabilities live in LMICs and the majority have no or limited access to any rehabilitation services [11,12]. With the extreme shortage of trained professionals including dietitians, in addition to the limited availability and accessibility to institutionalized intervention programs in LMICs [13], it is likely that not all the interventions found effective in HIC settings may be applicable in LMIC settings. In absence of optimal management, these children are at high risk of malnutrition, which can in turn impact their functional outcomes, quality of life, and survival [14,15,16].\nAny available evidence in the context of LMICs could guide resource mobilization, assist in understanding the need and cost-effectiveness as well as to identify interventions that may improve the nutritional status of children with CP in low-resource settings. This is important to establish a platform for developing and implementing best practice CP-specific nutrition intervention guidelines in LMICs. In this scoping review, we aimed to systematically map the existing evidence on nutritional interventions for children with CP living in LMIC settings. The following research questions were explored: (i) What is known about the available nutrition interventions for children with CP in LMICs? (ii) What are the outcomes of those interventions on the nutritional status of participating children with CP in LMICs?"},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"3. Results"},"results_text":{"kind":"string","value":"A total of n = 4885 citations were identified from the databases after deduplication. Following title screening, n = 132 abstracts were reviewed; of those, n = 26 articles were selected for full review. However, of those, n = 5 full articles were not available and an additional n = 4 were identified from handsearching the bibliographies of selected articles. So, a total of n = 25 full articles met the inclusion criteria. However, of those n = 13 were conducted in high-income settings, n = 1 did not report information for children with CP separately, thus the outcome could not be differentiated from other study participants, and n = 3 were conducted in HICs and did not report data on CP separately. Hence, those n = 17 were excluded. Finally, n = 8 articles published between the years 2008 and 2019 were included in the review for data charting. A flow diagram of the study selection procedure is shown in Figure 1. A summary of the excluded studies is also available in Supplementary Table S2.\nThe details about study characteristics, study participants, intervention details, and outcomes of the included studies are summarized in the subsequent sections.\n3.1. Study Characteristics The characteristics of the included studies have been summarized in Table 1.\n3.1.1. Study Design Five out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\nCharacteristics of the included study.\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\nFive out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\nCharacteristics of the included study.\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\n3.1.2. Study Location Of the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\nOf the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\n3.1.3. Study Settings Six of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\nSix of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\n3.1.4. Study Participants and Sample Size Five studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\nFive studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\nThe characteristics of the included studies have been summarized in Table 1.\n3.1.1. Study Design Five out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\nCharacteristics of the included study.\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\nFive out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\nCharacteristics of the included study.\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\n3.1.2. Study Location Of the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\nOf the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\n3.1.3. Study Settings Six of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\nSix of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\n3.1.4. Study Participants and Sample Size Five studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\nFive studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\n3.2. Intervention Details Of the eight studies, n = 6 had single type interventions [20,21,23,24,25,27] and the remaining n = 2 had multiple interventions [22,26]. Of all, n = 4 provided training to parents/caregivers [20,21,24,27] and n = 2 involved surgical interventions (e.g., gastrostomy or nasogastric tube feeding) [25,26]. Furthermore, dietary modification (e.g., modification of calorie and nutrient density/balanced diet/frequency/consistency/nutritional adequacy) was an intervention component in n = 5 studies [20,21,22,23,26]. Both studies with surgical interventions were descriptive in design (i.e., comparative study, before-after study, case series) and were conducted in Brazil [25] and Mexico [26]. The studies that emphasized parental/caregiver training covered a wide range of content, such as therapy, feeding skills, dietary modifications, position, and carrying [20,21,22,24,27]. The number of sessions varied between 5–11 [20,21,22,24], and the follow-up duration ranged between 1–18 months [20,21,22,23,24,26,27]. The study implementation team was documented only in one study by Pike et al. (2016) where a team of physiotherapists, occupational therapists, and speech-language therapists provided need-based neurodevelopmental therapy (NDT) feeding intervention to children with CP, trained the caregivers on recommended NDT feeding intervention and dietary modifications for children with CP in a hospital-based/institution-based setting in South Africa [22].\nThe details of different interventions provided in each of the studies included in this review are summarized in Table 2.\nOf the eight studies, n = 6 had single type interventions [20,21,23,24,25,27] and the remaining n = 2 had multiple interventions [22,26]. Of all, n = 4 provided training to parents/caregivers [20,21,24,27] and n = 2 involved surgical interventions (e.g., gastrostomy or nasogastric tube feeding) [25,26]. Furthermore, dietary modification (e.g., modification of calorie and nutrient density/balanced diet/frequency/consistency/nutritional adequacy) was an intervention component in n = 5 studies [20,21,22,23,26]. Both studies with surgical interventions were descriptive in design (i.e., comparative study, before-after study, case series) and were conducted in Brazil [25] and Mexico [26]. The studies that emphasized parental/caregiver training covered a wide range of content, such as therapy, feeding skills, dietary modifications, position, and carrying [20,21,22,24,27]. The number of sessions varied between 5–11 [20,21,22,24], and the follow-up duration ranged between 1–18 months [20,21,22,23,24,26,27]. The study implementation team was documented only in one study by Pike et al. (2016) where a team of physiotherapists, occupational therapists, and speech-language therapists provided need-based neurodevelopmental therapy (NDT) feeding intervention to children with CP, trained the caregivers on recommended NDT feeding intervention and dietary modifications for children with CP in a hospital-based/institution-based setting in South Africa [22].\nThe details of different interventions provided in each of the studies included in this review are summarized in Table 2.\n3.3. Outcome Measures Different anthropometric measurements were used to evaluate changes in the nutritional status of participating children. The most commonly used anthropometric measurements in the selected articles were weight (n = 8) [20,21,22,23,24,25,26,27], length/height (n = 7) [21,22,23,24,25,26,27], mid-upper arm circumference (MUAC) (n = 7) [20,21,22,23,24,26,27], and at least one skin-fold thickness measurement (n = 4) [22,23,25,26].\nIn all studies, nutritional status was determined by comparing the anthropometric measurements of children with CP to national standards/general population/WHO reference population [20,21,22,23,24,25,26,27]. However, one study also compared the anthropometric measurements with CP specific growth charts [23].\nAll but one article had other outcome measures in addition to the nutritional assessment; these include (i) dietary intake practices (n = 3) [20,25,27], feeding skills/feeding practices/feeding profiles (n = 5) [20,21,22,26,27], outcomes related to caregivers’ quality of life, knowledge and confidence about child care, perception about child’s health, feelings about child’s feeding difficulties, and compliance with the training [20,24], and child’s health-related quality of life [22]. Any adverse outcomes related to intervention were reported in n = 3 articles [20,23,24]; these outcomes included infections (one study) [23], chest health (one study) [20], and mortality (two studies) [20,24] (Table 3).\nDifferent anthropometric measurements were used to evaluate changes in the nutritional status of participating children. The most commonly used anthropometric measurements in the selected articles were weight (n = 8) [20,21,22,23,24,25,26,27], length/height (n = 7) [21,22,23,24,25,26,27], mid-upper arm circumference (MUAC) (n = 7) [20,21,22,23,24,26,27], and at least one skin-fold thickness measurement (n = 4) [22,23,25,26].\nIn all studies, nutritional status was determined by comparing the anthropometric measurements of children with CP to national standards/general population/WHO reference population [20,21,22,23,24,25,26,27]. However, one study also compared the anthropometric measurements with CP specific growth charts [23].\nAll but one article had other outcome measures in addition to the nutritional assessment; these include (i) dietary intake practices (n = 3) [20,25,27], feeding skills/feeding practices/feeding profiles (n = 5) [20,21,22,26,27], outcomes related to caregivers’ quality of life, knowledge and confidence about child care, perception about child’s health, feelings about child’s feeding difficulties, and compliance with the training [20,24], and child’s health-related quality of life [22]. Any adverse outcomes related to intervention were reported in n = 3 articles [20,23,24]; these outcomes included infections (one study) [23], chest health (one study) [20], and mortality (two studies) [20,24] (Table 3).\n3.4. Effect of Different Interventions on Nutritional Status of Children with CP Of the eight studies, seven included follow-up data of the participating children with CP (i.e., two or more data points for each participant) [20,21,22,23,24,26,27] and the other one compared between two groups (i.e., intervention vs. control group) [25]. Of those n = 7 studies with longitudinal data [20,21,22,23,24,26,27], n = 4 showed improvement in nutritional status among children with CP following intervention [20,22,23,26], one showed no change [21], whereas two showed deterioration [24,27] in the nutritional status following respective interventions among children with CP in selected LMICs.\nFour out of the five studies that focused on dietary modifications showed improvement in nutritional status of participating children [20,22,23,26]. Studies that focused on improving feeding skills (n = 3) had both positive and negative nutritional outcomes [20,21,24].\nBoth studies with GTF as an intervention showed a positive effect of GTF on the nutritional outcome of children with CP [25,26], however, of those none reported any adverse outcome of GTF [25,26]. (Table 3)\nOf the eight studies, seven included follow-up data of the participating children with CP (i.e., two or more data points for each participant) [20,21,22,23,24,26,27] and the other one compared between two groups (i.e., intervention vs. control group) [25]. Of those n = 7 studies with longitudinal data [20,21,22,23,24,26,27], n = 4 showed improvement in nutritional status among children with CP following intervention [20,22,23,26], one showed no change [21], whereas two showed deterioration [24,27] in the nutritional status following respective interventions among children with CP in selected LMICs.\nFour out of the five studies that focused on dietary modifications showed improvement in nutritional status of participating children [20,22,23,26]. Studies that focused on improving feeding skills (n = 3) had both positive and negative nutritional outcomes [20,21,24].\nBoth studies with GTF as an intervention showed a positive effect of GTF on the nutritional outcome of children with CP [25,26], however, of those none reported any adverse outcome of GTF [25,26]. (Table 3)"},"conclusions_title":{"kind":"string","value":"5. Conclusions"},"conclusions_text":{"kind":"string","value":"The current review highlights the knowledge gap and lack of available evidence on nutrition interventions for children with CP in LMICs. Strong evidence is essential to identify and determine the best practices and nutritional management guidelines for children with CP, especially for optimal utilization of the limited available resources in LMICs. National and international stakeholders, therefore, should make this a priority for future research and services. Considering the high proportion of children with CP with undernutrition in LMICs, existing interventions and services for children with CP should integrate a nutrition component. The current limited evidence suggests that nutrition-specific programs have more of a positive effect on the growth of children with CP than other strategies. This should be taken under consideration when planning for any nutrition focus and other comprehensive services for children with CP in LMICs."},"other_sections_titles":{"kind":"list like","value":["2. Materials and Methods","2.1. Study Design","2.2. Database Searching","2.3. Inclusion and Exclusion Criteria","2.4. Data Charting Process","2.5. Assessment of Risk of Bias and Synthesis of Results","2.6. Ethics","3.1. Study Characteristics","3.1.1. Study Design","3.1.2. Study Location","3.1.3. Study Settings","3.2. Intervention Details","3.3. Outcome Measures","3.4. Effect of Different Interventions on Nutritional Status of Children with CP"],"string":"[\n \"2. Materials and Methods\",\n \"2.1. Study Design\",\n \"2.2. Database Searching\",\n \"2.3. Inclusion and Exclusion Criteria\",\n \"2.4. Data Charting Process\",\n \"2.5. Assessment of Risk of Bias and Synthesis of Results\",\n \"2.6. Ethics\",\n \"3.1. Study Characteristics\",\n \"3.1.1. Study Design\",\n \"3.1.2. Study Location\",\n \"3.1.3. Study Settings\",\n \"3.2. Intervention Details\",\n \"3.3. Outcome Measures\",\n \"3.4. Effect of Different Interventions on Nutritional Status of Children with CP\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["2.1. Study Design We conducted a scoping review to summarize the available evidence and provide an overview of different intervention programs and their outcomes related to the nutritional status of children with CP in LMICs. A protocol was developed following the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) extension for scoping reviews guideline [17] and is available upon request from the corresponding author.\nWe conducted a scoping review to summarize the available evidence and provide an overview of different intervention programs and their outcomes related to the nutritional status of children with CP in LMICs. A protocol was developed following the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) extension for scoping reviews guideline [17] and is available upon request from the corresponding author.\n2.2. Database Searching The key words and search strategy were developed by two of the authors (I.J. and G.K.) and pretested prior to the initiation of the search. PubMed (up to 15 December 2021) and Scopus (up to 10 January 2022) were searched to identify relevant articles. Google Scholar was searched for any additional relevant articles or scientific guidelines. The search was not restricted to any specific language. The key terms used included “cerebral palsy”/”neurodevelopmental disorder”; children/adolescent/infant; parent/mother/caregiver; intervention/trial/outcome; training/education/feeding/technique/surgical/teaching; nutrition/malnutrition/growth/health; “low- and middle income countries (LMICs)”, and individual country listed as LMICs according to the 2020 World Bank country classifications by income level (i.e., low-income, lower middle-income, and upper-middle income countries) [18]. The electronic search strategy for Scopus is outlined in Supplementary Table S1. Two reviewers (I.J. and R.S.) independently searched the databases and included articles following the inclusion and exclusion criteria. Any disagreement was resolved by discussion between the reviewers and in consultation with a third reviewer (G.K.).\nThe key words and search strategy were developed by two of the authors (I.J. and G.K.) and pretested prior to the initiation of the search. PubMed (up to 15 December 2021) and Scopus (up to 10 January 2022) were searched to identify relevant articles. Google Scholar was searched for any additional relevant articles or scientific guidelines. The search was not restricted to any specific language. The key terms used included “cerebral palsy”/”neurodevelopmental disorder”; children/adolescent/infant; parent/mother/caregiver; intervention/trial/outcome; training/education/feeding/technique/surgical/teaching; nutrition/malnutrition/growth/health; “low- and middle income countries (LMICs)”, and individual country listed as LMICs according to the 2020 World Bank country classifications by income level (i.e., low-income, lower middle-income, and upper-middle income countries) [18]. The electronic search strategy for Scopus is outlined in Supplementary Table S1. Two reviewers (I.J. and R.S.) independently searched the databases and included articles following the inclusion and exclusion criteria. Any disagreement was resolved by discussion between the reviewers and in consultation with a third reviewer (G.K.).\n2.3. Inclusion and Exclusion Criteria Articles that met the following criteria were included in the review: (i) the study participants were children with CP, (ii) the outcome measures included nutritional status as determined by anthropometric measurements, e.g., weight, height/length of children with CP and/or body composition, and (iii) followed analytical study design (e.g., experimental/quasi-experimental, pre- and post-intervention study) or descriptive study with a control group or comparison group (e.g., children with gastrostomy versus children fed orally). Articles were excluded (i) if the study was conducted in HICs, (ii) if data about children with CP could not be differentiated and was reported together with children with other form of impairments, and (iii) if they were not peer-reviewed publications and were protocols, guidelines, book chapters, conference presentations, forewords, or replies to commentaries.\nArticles that met the following criteria were included in the review: (i) the study participants were children with CP, (ii) the outcome measures included nutritional status as determined by anthropometric measurements, e.g., weight, height/length of children with CP and/or body composition, and (iii) followed analytical study design (e.g., experimental/quasi-experimental, pre- and post-intervention study) or descriptive study with a control group or comparison group (e.g., children with gastrostomy versus children fed orally). Articles were excluded (i) if the study was conducted in HICs, (ii) if data about children with CP could not be differentiated and was reported together with children with other form of impairments, and (iii) if they were not peer-reviewed publications and were protocols, guidelines, book chapters, conference presentations, forewords, or replies to commentaries.\n2.4. Data Charting Process Data charting was primarily completed by two independent reviewers (I.J. and R.S.) using an a priori template developed by I.J. and G.K. Information on study design, settings, country, study participants (age, sex, motor function severity, sample size), intervention provided (type, settings, intervention contents, number of sessions as reported, follow-up period), and outcome (measures used and outcome reported) were extracted as available. Any missing information was documented as “not reported”.\nData charting was primarily completed by two independent reviewers (I.J. and R.S.) using an a priori template developed by I.J. and G.K. Information on study design, settings, country, study participants (age, sex, motor function severity, sample size), intervention provided (type, settings, intervention contents, number of sessions as reported, follow-up period), and outcome (measures used and outcome reported) were extracted as available. Any missing information was documented as “not reported”.\n2.5. Assessment of Risk of Bias and Synthesis of Results The risk of bias of the included studies were not assessed. Descriptive findings from individual studies including study characteristics (name and economic classification of the country according to the World Bank definitions in 2020, study locations, settings, study design, and study period), participants (sample size, age, sex, gross motor function classification system (GMFCS) level [19]), and outcome measures were reported. The different types of interventions were reported under broad headings, e.g., “training to parents/caregivers”, “gastrostomy tube placement/feeding/nasogastric tube feeding”, “nutritional rehabilitation/therapy”, and “dietary modification”. No statistical test was used considering the study objectives.\nThe risk of bias of the included studies were not assessed. Descriptive findings from individual studies including study characteristics (name and economic classification of the country according to the World Bank definitions in 2020, study locations, settings, study design, and study period), participants (sample size, age, sex, gross motor function classification system (GMFCS) level [19]), and outcome measures were reported. The different types of interventions were reported under broad headings, e.g., “training to parents/caregivers”, “gastrostomy tube placement/feeding/nasogastric tube feeding”, “nutritional rehabilitation/therapy”, and “dietary modification”. No statistical test was used considering the study objectives.\n2.6. Ethics This study did not require ethics approval as data were collected from existing publications (i.e., secondary data) and no humans were directly contacted to collect/gather any information.\nThis study did not require ethics approval as data were collected from existing publications (i.e., secondary data) and no humans were directly contacted to collect/gather any information.","We conducted a scoping review to summarize the available evidence and provide an overview of different intervention programs and their outcomes related to the nutritional status of children with CP in LMICs. A protocol was developed following the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) extension for scoping reviews guideline [17] and is available upon request from the corresponding author.","The key words and search strategy were developed by two of the authors (I.J. and G.K.) and pretested prior to the initiation of the search. PubMed (up to 15 December 2021) and Scopus (up to 10 January 2022) were searched to identify relevant articles. Google Scholar was searched for any additional relevant articles or scientific guidelines. The search was not restricted to any specific language. The key terms used included “cerebral palsy”/”neurodevelopmental disorder”; children/adolescent/infant; parent/mother/caregiver; intervention/trial/outcome; training/education/feeding/technique/surgical/teaching; nutrition/malnutrition/growth/health; “low- and middle income countries (LMICs)”, and individual country listed as LMICs according to the 2020 World Bank country classifications by income level (i.e., low-income, lower middle-income, and upper-middle income countries) [18]. The electronic search strategy for Scopus is outlined in Supplementary Table S1. Two reviewers (I.J. and R.S.) independently searched the databases and included articles following the inclusion and exclusion criteria. Any disagreement was resolved by discussion between the reviewers and in consultation with a third reviewer (G.K.).","Articles that met the following criteria were included in the review: (i) the study participants were children with CP, (ii) the outcome measures included nutritional status as determined by anthropometric measurements, e.g., weight, height/length of children with CP and/or body composition, and (iii) followed analytical study design (e.g., experimental/quasi-experimental, pre- and post-intervention study) or descriptive study with a control group or comparison group (e.g., children with gastrostomy versus children fed orally). Articles were excluded (i) if the study was conducted in HICs, (ii) if data about children with CP could not be differentiated and was reported together with children with other form of impairments, and (iii) if they were not peer-reviewed publications and were protocols, guidelines, book chapters, conference presentations, forewords, or replies to commentaries.","Data charting was primarily completed by two independent reviewers (I.J. and R.S.) using an a priori template developed by I.J. and G.K. Information on study design, settings, country, study participants (age, sex, motor function severity, sample size), intervention provided (type, settings, intervention contents, number of sessions as reported, follow-up period), and outcome (measures used and outcome reported) were extracted as available. Any missing information was documented as “not reported”.","The risk of bias of the included studies were not assessed. Descriptive findings from individual studies including study characteristics (name and economic classification of the country according to the World Bank definitions in 2020, study locations, settings, study design, and study period), participants (sample size, age, sex, gross motor function classification system (GMFCS) level [19]), and outcome measures were reported. The different types of interventions were reported under broad headings, e.g., “training to parents/caregivers”, “gastrostomy tube placement/feeding/nasogastric tube feeding”, “nutritional rehabilitation/therapy”, and “dietary modification”. No statistical test was used considering the study objectives.","This study did not require ethics approval as data were collected from existing publications (i.e., secondary data) and no humans were directly contacted to collect/gather any information.","The characteristics of the included studies have been summarized in Table 1.\n3.1.1. Study Design Five out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\nCharacteristics of the included study.\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\nFive out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\nCharacteristics of the included study.\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\n3.1.2. Study Location Of the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\nOf the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\n3.1.3. Study Settings Six of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\nSix of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\n3.1.4. Study Participants and Sample Size Five studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\nFive studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.","Five out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\nCharacteristics of the included study.\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.","Of the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].","Six of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].","Of the eight studies, n = 6 had single type interventions [20,21,23,24,25,27] and the remaining n = 2 had multiple interventions [22,26]. Of all, n = 4 provided training to parents/caregivers [20,21,24,27] and n = 2 involved surgical interventions (e.g., gastrostomy or nasogastric tube feeding) [25,26]. Furthermore, dietary modification (e.g., modification of calorie and nutrient density/balanced diet/frequency/consistency/nutritional adequacy) was an intervention component in n = 5 studies [20,21,22,23,26]. Both studies with surgical interventions were descriptive in design (i.e., comparative study, before-after study, case series) and were conducted in Brazil [25] and Mexico [26]. The studies that emphasized parental/caregiver training covered a wide range of content, such as therapy, feeding skills, dietary modifications, position, and carrying [20,21,22,24,27]. The number of sessions varied between 5–11 [20,21,22,24], and the follow-up duration ranged between 1–18 months [20,21,22,23,24,26,27]. The study implementation team was documented only in one study by Pike et al. (2016) where a team of physiotherapists, occupational therapists, and speech-language therapists provided need-based neurodevelopmental therapy (NDT) feeding intervention to children with CP, trained the caregivers on recommended NDT feeding intervention and dietary modifications for children with CP in a hospital-based/institution-based setting in South Africa [22].\nThe details of different interventions provided in each of the studies included in this review are summarized in Table 2.","Different anthropometric measurements were used to evaluate changes in the nutritional status of participating children. The most commonly used anthropometric measurements in the selected articles were weight (n = 8) [20,21,22,23,24,25,26,27], length/height (n = 7) [21,22,23,24,25,26,27], mid-upper arm circumference (MUAC) (n = 7) [20,21,22,23,24,26,27], and at least one skin-fold thickness measurement (n = 4) [22,23,25,26].\nIn all studies, nutritional status was determined by comparing the anthropometric measurements of children with CP to national standards/general population/WHO reference population [20,21,22,23,24,25,26,27]. However, one study also compared the anthropometric measurements with CP specific growth charts [23].\nAll but one article had other outcome measures in addition to the nutritional assessment; these include (i) dietary intake practices (n = 3) [20,25,27], feeding skills/feeding practices/feeding profiles (n = 5) [20,21,22,26,27], outcomes related to caregivers’ quality of life, knowledge and confidence about child care, perception about child’s health, feelings about child’s feeding difficulties, and compliance with the training [20,24], and child’s health-related quality of life [22]. Any adverse outcomes related to intervention were reported in n = 3 articles [20,23,24]; these outcomes included infections (one study) [23], chest health (one study) [20], and mortality (two studies) [20,24] (Table 3).","Of the eight studies, seven included follow-up data of the participating children with CP (i.e., two or more data points for each participant) [20,21,22,23,24,26,27] and the other one compared between two groups (i.e., intervention vs. control group) [25]. Of those n = 7 studies with longitudinal data [20,21,22,23,24,26,27], n = 4 showed improvement in nutritional status among children with CP following intervention [20,22,23,26], one showed no change [21], whereas two showed deterioration [24,27] in the nutritional status following respective interventions among children with CP in selected LMICs.\nFour out of the five studies that focused on dietary modifications showed improvement in nutritional status of participating children [20,22,23,26]. Studies that focused on improving feeding skills (n = 3) had both positive and negative nutritional outcomes [20,21,24].\nBoth studies with GTF as an intervention showed a positive effect of GTF on the nutritional outcome of children with CP [25,26], however, of those none reported any adverse outcome of GTF [25,26]. (Table 3)"],"string":"[\n \"2.1. Study Design We conducted a scoping review to summarize the available evidence and provide an overview of different intervention programs and their outcomes related to the nutritional status of children with CP in LMICs. A protocol was developed following the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) extension for scoping reviews guideline [17] and is available upon request from the corresponding author.\\nWe conducted a scoping review to summarize the available evidence and provide an overview of different intervention programs and their outcomes related to the nutritional status of children with CP in LMICs. A protocol was developed following the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) extension for scoping reviews guideline [17] and is available upon request from the corresponding author.\\n2.2. Database Searching The key words and search strategy were developed by two of the authors (I.J. and G.K.) and pretested prior to the initiation of the search. PubMed (up to 15 December 2021) and Scopus (up to 10 January 2022) were searched to identify relevant articles. Google Scholar was searched for any additional relevant articles or scientific guidelines. The search was not restricted to any specific language. The key terms used included “cerebral palsy”/”neurodevelopmental disorder”; children/adolescent/infant; parent/mother/caregiver; intervention/trial/outcome; training/education/feeding/technique/surgical/teaching; nutrition/malnutrition/growth/health; “low- and middle income countries (LMICs)”, and individual country listed as LMICs according to the 2020 World Bank country classifications by income level (i.e., low-income, lower middle-income, and upper-middle income countries) [18]. The electronic search strategy for Scopus is outlined in Supplementary Table S1. Two reviewers (I.J. and R.S.) independently searched the databases and included articles following the inclusion and exclusion criteria. Any disagreement was resolved by discussion between the reviewers and in consultation with a third reviewer (G.K.).\\nThe key words and search strategy were developed by two of the authors (I.J. and G.K.) and pretested prior to the initiation of the search. PubMed (up to 15 December 2021) and Scopus (up to 10 January 2022) were searched to identify relevant articles. Google Scholar was searched for any additional relevant articles or scientific guidelines. The search was not restricted to any specific language. The key terms used included “cerebral palsy”/”neurodevelopmental disorder”; children/adolescent/infant; parent/mother/caregiver; intervention/trial/outcome; training/education/feeding/technique/surgical/teaching; nutrition/malnutrition/growth/health; “low- and middle income countries (LMICs)”, and individual country listed as LMICs according to the 2020 World Bank country classifications by income level (i.e., low-income, lower middle-income, and upper-middle income countries) [18]. The electronic search strategy for Scopus is outlined in Supplementary Table S1. Two reviewers (I.J. and R.S.) independently searched the databases and included articles following the inclusion and exclusion criteria. Any disagreement was resolved by discussion between the reviewers and in consultation with a third reviewer (G.K.).\\n2.3. Inclusion and Exclusion Criteria Articles that met the following criteria were included in the review: (i) the study participants were children with CP, (ii) the outcome measures included nutritional status as determined by anthropometric measurements, e.g., weight, height/length of children with CP and/or body composition, and (iii) followed analytical study design (e.g., experimental/quasi-experimental, pre- and post-intervention study) or descriptive study with a control group or comparison group (e.g., children with gastrostomy versus children fed orally). Articles were excluded (i) if the study was conducted in HICs, (ii) if data about children with CP could not be differentiated and was reported together with children with other form of impairments, and (iii) if they were not peer-reviewed publications and were protocols, guidelines, book chapters, conference presentations, forewords, or replies to commentaries.\\nArticles that met the following criteria were included in the review: (i) the study participants were children with CP, (ii) the outcome measures included nutritional status as determined by anthropometric measurements, e.g., weight, height/length of children with CP and/or body composition, and (iii) followed analytical study design (e.g., experimental/quasi-experimental, pre- and post-intervention study) or descriptive study with a control group or comparison group (e.g., children with gastrostomy versus children fed orally). Articles were excluded (i) if the study was conducted in HICs, (ii) if data about children with CP could not be differentiated and was reported together with children with other form of impairments, and (iii) if they were not peer-reviewed publications and were protocols, guidelines, book chapters, conference presentations, forewords, or replies to commentaries.\\n2.4. Data Charting Process Data charting was primarily completed by two independent reviewers (I.J. and R.S.) using an a priori template developed by I.J. and G.K. Information on study design, settings, country, study participants (age, sex, motor function severity, sample size), intervention provided (type, settings, intervention contents, number of sessions as reported, follow-up period), and outcome (measures used and outcome reported) were extracted as available. Any missing information was documented as “not reported”.\\nData charting was primarily completed by two independent reviewers (I.J. and R.S.) using an a priori template developed by I.J. and G.K. Information on study design, settings, country, study participants (age, sex, motor function severity, sample size), intervention provided (type, settings, intervention contents, number of sessions as reported, follow-up period), and outcome (measures used and outcome reported) were extracted as available. Any missing information was documented as “not reported”.\\n2.5. Assessment of Risk of Bias and Synthesis of Results The risk of bias of the included studies were not assessed. Descriptive findings from individual studies including study characteristics (name and economic classification of the country according to the World Bank definitions in 2020, study locations, settings, study design, and study period), participants (sample size, age, sex, gross motor function classification system (GMFCS) level [19]), and outcome measures were reported. The different types of interventions were reported under broad headings, e.g., “training to parents/caregivers”, “gastrostomy tube placement/feeding/nasogastric tube feeding”, “nutritional rehabilitation/therapy”, and “dietary modification”. No statistical test was used considering the study objectives.\\nThe risk of bias of the included studies were not assessed. Descriptive findings from individual studies including study characteristics (name and economic classification of the country according to the World Bank definitions in 2020, study locations, settings, study design, and study period), participants (sample size, age, sex, gross motor function classification system (GMFCS) level [19]), and outcome measures were reported. The different types of interventions were reported under broad headings, e.g., “training to parents/caregivers”, “gastrostomy tube placement/feeding/nasogastric tube feeding”, “nutritional rehabilitation/therapy”, and “dietary modification”. No statistical test was used considering the study objectives.\\n2.6. Ethics This study did not require ethics approval as data were collected from existing publications (i.e., secondary data) and no humans were directly contacted to collect/gather any information.\\nThis study did not require ethics approval as data were collected from existing publications (i.e., secondary data) and no humans were directly contacted to collect/gather any information.\",\n \"We conducted a scoping review to summarize the available evidence and provide an overview of different intervention programs and their outcomes related to the nutritional status of children with CP in LMICs. A protocol was developed following the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) extension for scoping reviews guideline [17] and is available upon request from the corresponding author.\",\n \"The key words and search strategy were developed by two of the authors (I.J. and G.K.) and pretested prior to the initiation of the search. PubMed (up to 15 December 2021) and Scopus (up to 10 January 2022) were searched to identify relevant articles. Google Scholar was searched for any additional relevant articles or scientific guidelines. The search was not restricted to any specific language. The key terms used included “cerebral palsy”/”neurodevelopmental disorder”; children/adolescent/infant; parent/mother/caregiver; intervention/trial/outcome; training/education/feeding/technique/surgical/teaching; nutrition/malnutrition/growth/health; “low- and middle income countries (LMICs)”, and individual country listed as LMICs according to the 2020 World Bank country classifications by income level (i.e., low-income, lower middle-income, and upper-middle income countries) [18]. The electronic search strategy for Scopus is outlined in Supplementary Table S1. Two reviewers (I.J. and R.S.) independently searched the databases and included articles following the inclusion and exclusion criteria. Any disagreement was resolved by discussion between the reviewers and in consultation with a third reviewer (G.K.).\",\n \"Articles that met the following criteria were included in the review: (i) the study participants were children with CP, (ii) the outcome measures included nutritional status as determined by anthropometric measurements, e.g., weight, height/length of children with CP and/or body composition, and (iii) followed analytical study design (e.g., experimental/quasi-experimental, pre- and post-intervention study) or descriptive study with a control group or comparison group (e.g., children with gastrostomy versus children fed orally). Articles were excluded (i) if the study was conducted in HICs, (ii) if data about children with CP could not be differentiated and was reported together with children with other form of impairments, and (iii) if they were not peer-reviewed publications and were protocols, guidelines, book chapters, conference presentations, forewords, or replies to commentaries.\",\n \"Data charting was primarily completed by two independent reviewers (I.J. and R.S.) using an a priori template developed by I.J. and G.K. Information on study design, settings, country, study participants (age, sex, motor function severity, sample size), intervention provided (type, settings, intervention contents, number of sessions as reported, follow-up period), and outcome (measures used and outcome reported) were extracted as available. Any missing information was documented as “not reported”.\",\n \"The risk of bias of the included studies were not assessed. Descriptive findings from individual studies including study characteristics (name and economic classification of the country according to the World Bank definitions in 2020, study locations, settings, study design, and study period), participants (sample size, age, sex, gross motor function classification system (GMFCS) level [19]), and outcome measures were reported. The different types of interventions were reported under broad headings, e.g., “training to parents/caregivers”, “gastrostomy tube placement/feeding/nasogastric tube feeding”, “nutritional rehabilitation/therapy”, and “dietary modification”. No statistical test was used considering the study objectives.\",\n \"This study did not require ethics approval as data were collected from existing publications (i.e., secondary data) and no humans were directly contacted to collect/gather any information.\",\n \"The characteristics of the included studies have been summarized in Table 1.\\n3.1.1. Study Design Five out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\\nCharacteristics of the included study.\\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\\nFive out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\\nCharacteristics of the included study.\\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\\n3.1.2. Study Location Of the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\\nOf the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\\n3.1.3. Study Settings Six of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\\nSix of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\\n3.1.4. Study Participants and Sample Size Five studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\\nFive studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\",\n \"Five out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\\nCharacteristics of the included study.\\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\",\n \"Of the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\",\n \"Six of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\",\n \"Of the eight studies, n = 6 had single type interventions [20,21,23,24,25,27] and the remaining n = 2 had multiple interventions [22,26]. Of all, n = 4 provided training to parents/caregivers [20,21,24,27] and n = 2 involved surgical interventions (e.g., gastrostomy or nasogastric tube feeding) [25,26]. Furthermore, dietary modification (e.g., modification of calorie and nutrient density/balanced diet/frequency/consistency/nutritional adequacy) was an intervention component in n = 5 studies [20,21,22,23,26]. Both studies with surgical interventions were descriptive in design (i.e., comparative study, before-after study, case series) and were conducted in Brazil [25] and Mexico [26]. The studies that emphasized parental/caregiver training covered a wide range of content, such as therapy, feeding skills, dietary modifications, position, and carrying [20,21,22,24,27]. The number of sessions varied between 5–11 [20,21,22,24], and the follow-up duration ranged between 1–18 months [20,21,22,23,24,26,27]. The study implementation team was documented only in one study by Pike et al. (2016) where a team of physiotherapists, occupational therapists, and speech-language therapists provided need-based neurodevelopmental therapy (NDT) feeding intervention to children with CP, trained the caregivers on recommended NDT feeding intervention and dietary modifications for children with CP in a hospital-based/institution-based setting in South Africa [22].\\nThe details of different interventions provided in each of the studies included in this review are summarized in Table 2.\",\n \"Different anthropometric measurements were used to evaluate changes in the nutritional status of participating children. The most commonly used anthropometric measurements in the selected articles were weight (n = 8) [20,21,22,23,24,25,26,27], length/height (n = 7) [21,22,23,24,25,26,27], mid-upper arm circumference (MUAC) (n = 7) [20,21,22,23,24,26,27], and at least one skin-fold thickness measurement (n = 4) [22,23,25,26].\\nIn all studies, nutritional status was determined by comparing the anthropometric measurements of children with CP to national standards/general population/WHO reference population [20,21,22,23,24,25,26,27]. However, one study also compared the anthropometric measurements with CP specific growth charts [23].\\nAll but one article had other outcome measures in addition to the nutritional assessment; these include (i) dietary intake practices (n = 3) [20,25,27], feeding skills/feeding practices/feeding profiles (n = 5) [20,21,22,26,27], outcomes related to caregivers’ quality of life, knowledge and confidence about child care, perception about child’s health, feelings about child’s feeding difficulties, and compliance with the training [20,24], and child’s health-related quality of life [22]. Any adverse outcomes related to intervention were reported in n = 3 articles [20,23,24]; these outcomes included infections (one study) [23], chest health (one study) [20], and mortality (two studies) [20,24] (Table 3).\",\n \"Of the eight studies, seven included follow-up data of the participating children with CP (i.e., two or more data points for each participant) [20,21,22,23,24,26,27] and the other one compared between two groups (i.e., intervention vs. control group) [25]. Of those n = 7 studies with longitudinal data [20,21,22,23,24,26,27], n = 4 showed improvement in nutritional status among children with CP following intervention [20,22,23,26], one showed no change [21], whereas two showed deterioration [24,27] in the nutritional status following respective interventions among children with CP in selected LMICs.\\nFour out of the five studies that focused on dietary modifications showed improvement in nutritional status of participating children [20,22,23,26]. Studies that focused on improving feeding skills (n = 3) had both positive and negative nutritional outcomes [20,21,24].\\nBoth studies with GTF as an intervention showed a positive effect of GTF on the nutritional outcome of children with CP [25,26], however, of those none reported any adverse outcome of GTF [25,26]. (Table 3)\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["1. Introduction","2. Materials and Methods","2.1. Study Design","2.2. Database Searching","2.3. Inclusion and Exclusion Criteria","2.4. Data Charting Process","2.5. Assessment of Risk of Bias and Synthesis of Results","2.6. Ethics","3. Results","3.1. Study Characteristics","3.1.1. Study Design","3.1.2. Study Location","3.1.3. Study Settings","3.1.4. Study Participants and Sample Size","3.2. Intervention Details","3.3. Outcome Measures","3.4. Effect of Different Interventions on Nutritional Status of Children with CP","4. Discussion","5. Conclusions"],"string":"[\n \"1. Introduction\",\n \"2. Materials and Methods\",\n \"2.1. Study Design\",\n \"2.2. Database Searching\",\n \"2.3. Inclusion and Exclusion Criteria\",\n \"2.4. Data Charting Process\",\n \"2.5. Assessment of Risk of Bias and Synthesis of Results\",\n \"2.6. Ethics\",\n \"3. Results\",\n \"3.1. Study Characteristics\",\n \"3.1.1. Study Design\",\n \"3.1.2. Study Location\",\n \"3.1.3. Study Settings\",\n \"3.1.4. Study Participants and Sample Size\",\n \"3.2. Intervention Details\",\n \"3.3. Outcome Measures\",\n \"3.4. Effect of Different Interventions on Nutritional Status of Children with CP\",\n \"4. Discussion\",\n \"5. Conclusions\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Undernutrition is a major global public health challenge, and children with disability, such as cerebral palsy (CP), often suffer from undernutrition, especially in low- and middle-income countries (LMICs). The proportion of malnutrition is substantially higher among children with CP in LMICs compared to the general population [1,2,3,4,5]. Ensuring availability and accessibility to evidence-based nutrition interventions is essential to avert adverse outcomes of malnutrition among those vulnerable children [6].\nNutritional management of children with CP is complex as several interlinked factors interfere with their growth directly and indirectly [7]. While a few factors have been identified as common predictors of malnutrition (e.g., gross motor and oromotor function limitations) [1,8,9], the conceptual framework of malnutrition in children with CP is not yet clearly understood, especially in LMICs. To date, several clinical nutrition guidelines based on current evidence on surgical and non-surgical intervention outcomes have been published [6,10]. However, most of those studies were conducted in high-income countries (HICs) [6], whereas 85% of children with disabilities live in LMICs and the majority have no or limited access to any rehabilitation services [11,12]. With the extreme shortage of trained professionals including dietitians, in addition to the limited availability and accessibility to institutionalized intervention programs in LMICs [13], it is likely that not all the interventions found effective in HIC settings may be applicable in LMIC settings. In absence of optimal management, these children are at high risk of malnutrition, which can in turn impact their functional outcomes, quality of life, and survival [14,15,16].\nAny available evidence in the context of LMICs could guide resource mobilization, assist in understanding the need and cost-effectiveness as well as to identify interventions that may improve the nutritional status of children with CP in low-resource settings. This is important to establish a platform for developing and implementing best practice CP-specific nutrition intervention guidelines in LMICs. In this scoping review, we aimed to systematically map the existing evidence on nutritional interventions for children with CP living in LMIC settings. The following research questions were explored: (i) What is known about the available nutrition interventions for children with CP in LMICs? (ii) What are the outcomes of those interventions on the nutritional status of participating children with CP in LMICs?","2.1. Study Design We conducted a scoping review to summarize the available evidence and provide an overview of different intervention programs and their outcomes related to the nutritional status of children with CP in LMICs. A protocol was developed following the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) extension for scoping reviews guideline [17] and is available upon request from the corresponding author.\nWe conducted a scoping review to summarize the available evidence and provide an overview of different intervention programs and their outcomes related to the nutritional status of children with CP in LMICs. A protocol was developed following the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) extension for scoping reviews guideline [17] and is available upon request from the corresponding author.\n2.2. Database Searching The key words and search strategy were developed by two of the authors (I.J. and G.K.) and pretested prior to the initiation of the search. PubMed (up to 15 December 2021) and Scopus (up to 10 January 2022) were searched to identify relevant articles. Google Scholar was searched for any additional relevant articles or scientific guidelines. The search was not restricted to any specific language. The key terms used included “cerebral palsy”/”neurodevelopmental disorder”; children/adolescent/infant; parent/mother/caregiver; intervention/trial/outcome; training/education/feeding/technique/surgical/teaching; nutrition/malnutrition/growth/health; “low- and middle income countries (LMICs)”, and individual country listed as LMICs according to the 2020 World Bank country classifications by income level (i.e., low-income, lower middle-income, and upper-middle income countries) [18]. The electronic search strategy for Scopus is outlined in Supplementary Table S1. Two reviewers (I.J. and R.S.) independently searched the databases and included articles following the inclusion and exclusion criteria. Any disagreement was resolved by discussion between the reviewers and in consultation with a third reviewer (G.K.).\nThe key words and search strategy were developed by two of the authors (I.J. and G.K.) and pretested prior to the initiation of the search. PubMed (up to 15 December 2021) and Scopus (up to 10 January 2022) were searched to identify relevant articles. Google Scholar was searched for any additional relevant articles or scientific guidelines. The search was not restricted to any specific language. The key terms used included “cerebral palsy”/”neurodevelopmental disorder”; children/adolescent/infant; parent/mother/caregiver; intervention/trial/outcome; training/education/feeding/technique/surgical/teaching; nutrition/malnutrition/growth/health; “low- and middle income countries (LMICs)”, and individual country listed as LMICs according to the 2020 World Bank country classifications by income level (i.e., low-income, lower middle-income, and upper-middle income countries) [18]. The electronic search strategy for Scopus is outlined in Supplementary Table S1. Two reviewers (I.J. and R.S.) independently searched the databases and included articles following the inclusion and exclusion criteria. Any disagreement was resolved by discussion between the reviewers and in consultation with a third reviewer (G.K.).\n2.3. Inclusion and Exclusion Criteria Articles that met the following criteria were included in the review: (i) the study participants were children with CP, (ii) the outcome measures included nutritional status as determined by anthropometric measurements, e.g., weight, height/length of children with CP and/or body composition, and (iii) followed analytical study design (e.g., experimental/quasi-experimental, pre- and post-intervention study) or descriptive study with a control group or comparison group (e.g., children with gastrostomy versus children fed orally). Articles were excluded (i) if the study was conducted in HICs, (ii) if data about children with CP could not be differentiated and was reported together with children with other form of impairments, and (iii) if they were not peer-reviewed publications and were protocols, guidelines, book chapters, conference presentations, forewords, or replies to commentaries.\nArticles that met the following criteria were included in the review: (i) the study participants were children with CP, (ii) the outcome measures included nutritional status as determined by anthropometric measurements, e.g., weight, height/length of children with CP and/or body composition, and (iii) followed analytical study design (e.g., experimental/quasi-experimental, pre- and post-intervention study) or descriptive study with a control group or comparison group (e.g., children with gastrostomy versus children fed orally). Articles were excluded (i) if the study was conducted in HICs, (ii) if data about children with CP could not be differentiated and was reported together with children with other form of impairments, and (iii) if they were not peer-reviewed publications and were protocols, guidelines, book chapters, conference presentations, forewords, or replies to commentaries.\n2.4. Data Charting Process Data charting was primarily completed by two independent reviewers (I.J. and R.S.) using an a priori template developed by I.J. and G.K. Information on study design, settings, country, study participants (age, sex, motor function severity, sample size), intervention provided (type, settings, intervention contents, number of sessions as reported, follow-up period), and outcome (measures used and outcome reported) were extracted as available. Any missing information was documented as “not reported”.\nData charting was primarily completed by two independent reviewers (I.J. and R.S.) using an a priori template developed by I.J. and G.K. Information on study design, settings, country, study participants (age, sex, motor function severity, sample size), intervention provided (type, settings, intervention contents, number of sessions as reported, follow-up period), and outcome (measures used and outcome reported) were extracted as available. Any missing information was documented as “not reported”.\n2.5. Assessment of Risk of Bias and Synthesis of Results The risk of bias of the included studies were not assessed. Descriptive findings from individual studies including study characteristics (name and economic classification of the country according to the World Bank definitions in 2020, study locations, settings, study design, and study period), participants (sample size, age, sex, gross motor function classification system (GMFCS) level [19]), and outcome measures were reported. The different types of interventions were reported under broad headings, e.g., “training to parents/caregivers”, “gastrostomy tube placement/feeding/nasogastric tube feeding”, “nutritional rehabilitation/therapy”, and “dietary modification”. No statistical test was used considering the study objectives.\nThe risk of bias of the included studies were not assessed. Descriptive findings from individual studies including study characteristics (name and economic classification of the country according to the World Bank definitions in 2020, study locations, settings, study design, and study period), participants (sample size, age, sex, gross motor function classification system (GMFCS) level [19]), and outcome measures were reported. The different types of interventions were reported under broad headings, e.g., “training to parents/caregivers”, “gastrostomy tube placement/feeding/nasogastric tube feeding”, “nutritional rehabilitation/therapy”, and “dietary modification”. No statistical test was used considering the study objectives.\n2.6. Ethics This study did not require ethics approval as data were collected from existing publications (i.e., secondary data) and no humans were directly contacted to collect/gather any information.\nThis study did not require ethics approval as data were collected from existing publications (i.e., secondary data) and no humans were directly contacted to collect/gather any information.","We conducted a scoping review to summarize the available evidence and provide an overview of different intervention programs and their outcomes related to the nutritional status of children with CP in LMICs. A protocol was developed following the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) extension for scoping reviews guideline [17] and is available upon request from the corresponding author.","The key words and search strategy were developed by two of the authors (I.J. and G.K.) and pretested prior to the initiation of the search. PubMed (up to 15 December 2021) and Scopus (up to 10 January 2022) were searched to identify relevant articles. Google Scholar was searched for any additional relevant articles or scientific guidelines. The search was not restricted to any specific language. The key terms used included “cerebral palsy”/”neurodevelopmental disorder”; children/adolescent/infant; parent/mother/caregiver; intervention/trial/outcome; training/education/feeding/technique/surgical/teaching; nutrition/malnutrition/growth/health; “low- and middle income countries (LMICs)”, and individual country listed as LMICs according to the 2020 World Bank country classifications by income level (i.e., low-income, lower middle-income, and upper-middle income countries) [18]. The electronic search strategy for Scopus is outlined in Supplementary Table S1. Two reviewers (I.J. and R.S.) independently searched the databases and included articles following the inclusion and exclusion criteria. Any disagreement was resolved by discussion between the reviewers and in consultation with a third reviewer (G.K.).","Articles that met the following criteria were included in the review: (i) the study participants were children with CP, (ii) the outcome measures included nutritional status as determined by anthropometric measurements, e.g., weight, height/length of children with CP and/or body composition, and (iii) followed analytical study design (e.g., experimental/quasi-experimental, pre- and post-intervention study) or descriptive study with a control group or comparison group (e.g., children with gastrostomy versus children fed orally). Articles were excluded (i) if the study was conducted in HICs, (ii) if data about children with CP could not be differentiated and was reported together with children with other form of impairments, and (iii) if they were not peer-reviewed publications and were protocols, guidelines, book chapters, conference presentations, forewords, or replies to commentaries.","Data charting was primarily completed by two independent reviewers (I.J. and R.S.) using an a priori template developed by I.J. and G.K. Information on study design, settings, country, study participants (age, sex, motor function severity, sample size), intervention provided (type, settings, intervention contents, number of sessions as reported, follow-up period), and outcome (measures used and outcome reported) were extracted as available. Any missing information was documented as “not reported”.","The risk of bias of the included studies were not assessed. Descriptive findings from individual studies including study characteristics (name and economic classification of the country according to the World Bank definitions in 2020, study locations, settings, study design, and study period), participants (sample size, age, sex, gross motor function classification system (GMFCS) level [19]), and outcome measures were reported. The different types of interventions were reported under broad headings, e.g., “training to parents/caregivers”, “gastrostomy tube placement/feeding/nasogastric tube feeding”, “nutritional rehabilitation/therapy”, and “dietary modification”. No statistical test was used considering the study objectives.","This study did not require ethics approval as data were collected from existing publications (i.e., secondary data) and no humans were directly contacted to collect/gather any information.","A total of n = 4885 citations were identified from the databases after deduplication. Following title screening, n = 132 abstracts were reviewed; of those, n = 26 articles were selected for full review. However, of those, n = 5 full articles were not available and an additional n = 4 were identified from handsearching the bibliographies of selected articles. So, a total of n = 25 full articles met the inclusion criteria. However, of those n = 13 were conducted in high-income settings, n = 1 did not report information for children with CP separately, thus the outcome could not be differentiated from other study participants, and n = 3 were conducted in HICs and did not report data on CP separately. Hence, those n = 17 were excluded. Finally, n = 8 articles published between the years 2008 and 2019 were included in the review for data charting. A flow diagram of the study selection procedure is shown in Figure 1. A summary of the excluded studies is also available in Supplementary Table S2.\nThe details about study characteristics, study participants, intervention details, and outcomes of the included studies are summarized in the subsequent sections.\n3.1. Study Characteristics The characteristics of the included studies have been summarized in Table 1.\n3.1.1. Study Design Five out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\nCharacteristics of the included study.\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\nFive out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\nCharacteristics of the included study.\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\n3.1.2. Study Location Of the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\nOf the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\n3.1.3. Study Settings Six of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\nSix of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\n3.1.4. Study Participants and Sample Size Five studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\nFive studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\nThe characteristics of the included studies have been summarized in Table 1.\n3.1.1. Study Design Five out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\nCharacteristics of the included study.\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\nFive out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\nCharacteristics of the included study.\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\n3.1.2. Study Location Of the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\nOf the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\n3.1.3. Study Settings Six of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\nSix of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\n3.1.4. Study Participants and Sample Size Five studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\nFive studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\n3.2. Intervention Details Of the eight studies, n = 6 had single type interventions [20,21,23,24,25,27] and the remaining n = 2 had multiple interventions [22,26]. Of all, n = 4 provided training to parents/caregivers [20,21,24,27] and n = 2 involved surgical interventions (e.g., gastrostomy or nasogastric tube feeding) [25,26]. Furthermore, dietary modification (e.g., modification of calorie and nutrient density/balanced diet/frequency/consistency/nutritional adequacy) was an intervention component in n = 5 studies [20,21,22,23,26]. Both studies with surgical interventions were descriptive in design (i.e., comparative study, before-after study, case series) and were conducted in Brazil [25] and Mexico [26]. The studies that emphasized parental/caregiver training covered a wide range of content, such as therapy, feeding skills, dietary modifications, position, and carrying [20,21,22,24,27]. The number of sessions varied between 5–11 [20,21,22,24], and the follow-up duration ranged between 1–18 months [20,21,22,23,24,26,27]. The study implementation team was documented only in one study by Pike et al. (2016) where a team of physiotherapists, occupational therapists, and speech-language therapists provided need-based neurodevelopmental therapy (NDT) feeding intervention to children with CP, trained the caregivers on recommended NDT feeding intervention and dietary modifications for children with CP in a hospital-based/institution-based setting in South Africa [22].\nThe details of different interventions provided in each of the studies included in this review are summarized in Table 2.\nOf the eight studies, n = 6 had single type interventions [20,21,23,24,25,27] and the remaining n = 2 had multiple interventions [22,26]. Of all, n = 4 provided training to parents/caregivers [20,21,24,27] and n = 2 involved surgical interventions (e.g., gastrostomy or nasogastric tube feeding) [25,26]. Furthermore, dietary modification (e.g., modification of calorie and nutrient density/balanced diet/frequency/consistency/nutritional adequacy) was an intervention component in n = 5 studies [20,21,22,23,26]. Both studies with surgical interventions were descriptive in design (i.e., comparative study, before-after study, case series) and were conducted in Brazil [25] and Mexico [26]. The studies that emphasized parental/caregiver training covered a wide range of content, such as therapy, feeding skills, dietary modifications, position, and carrying [20,21,22,24,27]. The number of sessions varied between 5–11 [20,21,22,24], and the follow-up duration ranged between 1–18 months [20,21,22,23,24,26,27]. The study implementation team was documented only in one study by Pike et al. (2016) where a team of physiotherapists, occupational therapists, and speech-language therapists provided need-based neurodevelopmental therapy (NDT) feeding intervention to children with CP, trained the caregivers on recommended NDT feeding intervention and dietary modifications for children with CP in a hospital-based/institution-based setting in South Africa [22].\nThe details of different interventions provided in each of the studies included in this review are summarized in Table 2.\n3.3. Outcome Measures Different anthropometric measurements were used to evaluate changes in the nutritional status of participating children. The most commonly used anthropometric measurements in the selected articles were weight (n = 8) [20,21,22,23,24,25,26,27], length/height (n = 7) [21,22,23,24,25,26,27], mid-upper arm circumference (MUAC) (n = 7) [20,21,22,23,24,26,27], and at least one skin-fold thickness measurement (n = 4) [22,23,25,26].\nIn all studies, nutritional status was determined by comparing the anthropometric measurements of children with CP to national standards/general population/WHO reference population [20,21,22,23,24,25,26,27]. However, one study also compared the anthropometric measurements with CP specific growth charts [23].\nAll but one article had other outcome measures in addition to the nutritional assessment; these include (i) dietary intake practices (n = 3) [20,25,27], feeding skills/feeding practices/feeding profiles (n = 5) [20,21,22,26,27], outcomes related to caregivers’ quality of life, knowledge and confidence about child care, perception about child’s health, feelings about child’s feeding difficulties, and compliance with the training [20,24], and child’s health-related quality of life [22]. Any adverse outcomes related to intervention were reported in n = 3 articles [20,23,24]; these outcomes included infections (one study) [23], chest health (one study) [20], and mortality (two studies) [20,24] (Table 3).\nDifferent anthropometric measurements were used to evaluate changes in the nutritional status of participating children. The most commonly used anthropometric measurements in the selected articles were weight (n = 8) [20,21,22,23,24,25,26,27], length/height (n = 7) [21,22,23,24,25,26,27], mid-upper arm circumference (MUAC) (n = 7) [20,21,22,23,24,26,27], and at least one skin-fold thickness measurement (n = 4) [22,23,25,26].\nIn all studies, nutritional status was determined by comparing the anthropometric measurements of children with CP to national standards/general population/WHO reference population [20,21,22,23,24,25,26,27]. However, one study also compared the anthropometric measurements with CP specific growth charts [23].\nAll but one article had other outcome measures in addition to the nutritional assessment; these include (i) dietary intake practices (n = 3) [20,25,27], feeding skills/feeding practices/feeding profiles (n = 5) [20,21,22,26,27], outcomes related to caregivers’ quality of life, knowledge and confidence about child care, perception about child’s health, feelings about child’s feeding difficulties, and compliance with the training [20,24], and child’s health-related quality of life [22]. Any adverse outcomes related to intervention were reported in n = 3 articles [20,23,24]; these outcomes included infections (one study) [23], chest health (one study) [20], and mortality (two studies) [20,24] (Table 3).\n3.4. Effect of Different Interventions on Nutritional Status of Children with CP Of the eight studies, seven included follow-up data of the participating children with CP (i.e., two or more data points for each participant) [20,21,22,23,24,26,27] and the other one compared between two groups (i.e., intervention vs. control group) [25]. Of those n = 7 studies with longitudinal data [20,21,22,23,24,26,27], n = 4 showed improvement in nutritional status among children with CP following intervention [20,22,23,26], one showed no change [21], whereas two showed deterioration [24,27] in the nutritional status following respective interventions among children with CP in selected LMICs.\nFour out of the five studies that focused on dietary modifications showed improvement in nutritional status of participating children [20,22,23,26]. Studies that focused on improving feeding skills (n = 3) had both positive and negative nutritional outcomes [20,21,24].\nBoth studies with GTF as an intervention showed a positive effect of GTF on the nutritional outcome of children with CP [25,26], however, of those none reported any adverse outcome of GTF [25,26]. (Table 3)\nOf the eight studies, seven included follow-up data of the participating children with CP (i.e., two or more data points for each participant) [20,21,22,23,24,26,27] and the other one compared between two groups (i.e., intervention vs. control group) [25]. Of those n = 7 studies with longitudinal data [20,21,22,23,24,26,27], n = 4 showed improvement in nutritional status among children with CP following intervention [20,22,23,26], one showed no change [21], whereas two showed deterioration [24,27] in the nutritional status following respective interventions among children with CP in selected LMICs.\nFour out of the five studies that focused on dietary modifications showed improvement in nutritional status of participating children [20,22,23,26]. Studies that focused on improving feeding skills (n = 3) had both positive and negative nutritional outcomes [20,21,24].\nBoth studies with GTF as an intervention showed a positive effect of GTF on the nutritional outcome of children with CP [25,26], however, of those none reported any adverse outcome of GTF [25,26]. (Table 3)","The characteristics of the included studies have been summarized in Table 1.\n3.1.1. Study Design Five out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\nCharacteristics of the included study.\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\nFive out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\nCharacteristics of the included study.\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\n3.1.2. Study Location Of the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\nOf the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\n3.1.3. Study Settings Six of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\nSix of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\n3.1.4. Study Participants and Sample Size Five studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\nFive studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.","Five out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\nCharacteristics of the included study.\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.","Of the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].","Six of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].","Five studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.","Of the eight studies, n = 6 had single type interventions [20,21,23,24,25,27] and the remaining n = 2 had multiple interventions [22,26]. Of all, n = 4 provided training to parents/caregivers [20,21,24,27] and n = 2 involved surgical interventions (e.g., gastrostomy or nasogastric tube feeding) [25,26]. Furthermore, dietary modification (e.g., modification of calorie and nutrient density/balanced diet/frequency/consistency/nutritional adequacy) was an intervention component in n = 5 studies [20,21,22,23,26]. Both studies with surgical interventions were descriptive in design (i.e., comparative study, before-after study, case series) and were conducted in Brazil [25] and Mexico [26]. The studies that emphasized parental/caregiver training covered a wide range of content, such as therapy, feeding skills, dietary modifications, position, and carrying [20,21,22,24,27]. The number of sessions varied between 5–11 [20,21,22,24], and the follow-up duration ranged between 1–18 months [20,21,22,23,24,26,27]. The study implementation team was documented only in one study by Pike et al. (2016) where a team of physiotherapists, occupational therapists, and speech-language therapists provided need-based neurodevelopmental therapy (NDT) feeding intervention to children with CP, trained the caregivers on recommended NDT feeding intervention and dietary modifications for children with CP in a hospital-based/institution-based setting in South Africa [22].\nThe details of different interventions provided in each of the studies included in this review are summarized in Table 2.","Different anthropometric measurements were used to evaluate changes in the nutritional status of participating children. The most commonly used anthropometric measurements in the selected articles were weight (n = 8) [20,21,22,23,24,25,26,27], length/height (n = 7) [21,22,23,24,25,26,27], mid-upper arm circumference (MUAC) (n = 7) [20,21,22,23,24,26,27], and at least one skin-fold thickness measurement (n = 4) [22,23,25,26].\nIn all studies, nutritional status was determined by comparing the anthropometric measurements of children with CP to national standards/general population/WHO reference population [20,21,22,23,24,25,26,27]. However, one study also compared the anthropometric measurements with CP specific growth charts [23].\nAll but one article had other outcome measures in addition to the nutritional assessment; these include (i) dietary intake practices (n = 3) [20,25,27], feeding skills/feeding practices/feeding profiles (n = 5) [20,21,22,26,27], outcomes related to caregivers’ quality of life, knowledge and confidence about child care, perception about child’s health, feelings about child’s feeding difficulties, and compliance with the training [20,24], and child’s health-related quality of life [22]. Any adverse outcomes related to intervention were reported in n = 3 articles [20,23,24]; these outcomes included infections (one study) [23], chest health (one study) [20], and mortality (two studies) [20,24] (Table 3).","Of the eight studies, seven included follow-up data of the participating children with CP (i.e., two or more data points for each participant) [20,21,22,23,24,26,27] and the other one compared between two groups (i.e., intervention vs. control group) [25]. Of those n = 7 studies with longitudinal data [20,21,22,23,24,26,27], n = 4 showed improvement in nutritional status among children with CP following intervention [20,22,23,26], one showed no change [21], whereas two showed deterioration [24,27] in the nutritional status following respective interventions among children with CP in selected LMICs.\nFour out of the five studies that focused on dietary modifications showed improvement in nutritional status of participating children [20,22,23,26]. Studies that focused on improving feeding skills (n = 3) had both positive and negative nutritional outcomes [20,21,24].\nBoth studies with GTF as an intervention showed a positive effect of GTF on the nutritional outcome of children with CP [25,26], however, of those none reported any adverse outcome of GTF [25,26]. (Table 3)","In this review we provide a summary of the available evidence on different nutrition interventions for children with CP in LMICs. Our search identified only a few relevant studies on the topic that were conducted in resource-constrained settings of LMICs. Only eight articles were identified, none of which had representation from low-income countries. Such an evidence gap from LMICs where the majority of children with CP reside is concerning [11]. Considering the high burden of malnourished children with CP in LMICs [1,2,3,4,5,8,9], there is a dire need to reduce this evidence gap, and integration of nutrition interventions in the existing service plan for children with CP is essential.\nThere are global efforts toward reducing the burden of malnutrition among children, especially in LMICs. The United Nations Sustainable Development Goals (UN SDGs) has an emphasis on childhood nutrition for human growth and development [28]. The SDGs also strongly advocate for disability inclusiveness, especially the SDGs which are related to growth, health, education, employment, and addressing inequality globally [29]. National and international partners are working together to develop standard guidelines, strategies, and interventions addressing the immediate determinants (i.e., nutrition-specific intervention) and the underlying or root causes (i.e., nutrition-sensitive interventions) to improve the overall nutritional status of children, especially in LMICs [30,31]. These interventions should be disability-inclusive, and priority should be given to reporting/documentation of the effectiveness of different nutrition interventions on growth and nutrition of children with disabilities, including CP.\nWhile reviewing the limited available studies, we observed that a majority of the interventions were trialed or implemented in institution-based settings. Though interventions implemented in the institution-based settings have been proven to result in better outcomes in the past [6], our evidence suggests the majority of children with CP in LMICs lack access to any rehabilitation services for numerous reasons, including limited availability of services in their neighborhoods, transport difficulties, and financial constraints [12,32]. Furthermore, the severe shortage of trained health professionals, e.g., rehabilitation service providers and dietitians, limits the scope to implement and scale-up different institution-based interventions for children with CP in LMICs [13]. In such a context, community-based approaches are highly recommended and there is a lack of evidence on community-based nutrition interventions for children with CP in LMICs. Nevertheless, in one recent study from Bangladesh, community-based parent-led intervention was found to be highly successful in improving functional outcomes of children with CP [33]. Emphasis should be given to the capacity development of mid-level service providers on growth monitoring and promotion, early identification, different blanket interventions, and referral to prevent and treat malnutrition among children with CP in LMICs.\nIn addition to the settings, the sample sizes of the studies were relatively low. Although the included studies provided valuable insights on different nutritional interventions, the small sample size limits the strength and generalizability of the study findings. Moreover, most children with CP in the selected studies had severe functional motor limitations. These could be due to selection bias related to more severe institutional samples, as well as the more severe end of the clinical spectrum of CP in LMICs, likely exacerbated by delayed age of diagnosis and lack of access to rehabilitation services [12,32,34]. Studies also show that the likelihood of receiving intervention and rehabilitation is higher among those with severe CP (e.g., GMFCS level III–V) compared to milder forms (e.g., GMFCS I–II) [12]. However, it is also known that severe functional motor limitations (e.g., GMFCS level III–V) in children with CP are often accompanied by severe oromotor dysfunction, e.g., dysphagia [35]. It remains unclear whether the interventions for children with more severe motor limitations are also appropriate for children with CP who are GMFCS I–II.\nMost of the interventions identified in this review improved the nutritional status of participating children with CP. Importantly, nutrition-specific interventions (i.e., interventions that aim to intervene in or correct the immediate causes of malnutrition, such as inadequate diet, disease severity and caring practices) had comparatively better outcomes than other intervention strategies [20,22,24,30]. We observed that the interventions that included dietary modifications (only, or as a component of multiple approaches) had better effect on nutritional outcomes of children with CP. Studies where participants received surgical interventions (e.g., GTF) also showed a positive effect, whereas studies that provided behavioral intervention on feeding skills only did not show any significant change.\nTo the best of our knowledge, this is one of the first reviews conducted on nutrition interventions for children with CP in LMICs. Although during our search we identified several studies that aimed to improve the nutritional status of children with CP in HICs, our search revealed only a few reviews (including one systematic review) that summarized findings from different nutrition intervention studies among children with CP in HICs [6,36,37]. The data provided in our study, therefore, contribute important information to the existing knowledge gap. Nevertheless, this review has several limitations. We focused our search on two databases, so a risk remains that there may have been other publications available through other databases. However, our search revealed a large number of duplicates; therefore, we are confident that we have covered all or almost all of the studies published online. Second, we only searched for articles that had our key words in title/abstracts/author specified key words in the articles. This may have reduced the number of articles initially identified for title screening. Third, we did not assess the risk of bias and quality of evidence of the articles finally included in the review, which may have posed bias in the information reported. Finally, due to the differences in reporting format and data availability, we could only include a few common variables and could not report on some of the important characteristics of the participants. For the same reason, we had to rely on reporting the descriptive findings only.","The current review highlights the knowledge gap and lack of available evidence on nutrition interventions for children with CP in LMICs. Strong evidence is essential to identify and determine the best practices and nutritional management guidelines for children with CP, especially for optimal utilization of the limited available resources in LMICs. National and international stakeholders, therefore, should make this a priority for future research and services. Considering the high proportion of children with CP with undernutrition in LMICs, existing interventions and services for children with CP should integrate a nutrition component. The current limited evidence suggests that nutrition-specific programs have more of a positive effect on the growth of children with CP than other strategies. This should be taken under consideration when planning for any nutrition focus and other comprehensive services for children with CP in LMICs."],"string":"[\n \"Undernutrition is a major global public health challenge, and children with disability, such as cerebral palsy (CP), often suffer from undernutrition, especially in low- and middle-income countries (LMICs). The proportion of malnutrition is substantially higher among children with CP in LMICs compared to the general population [1,2,3,4,5]. Ensuring availability and accessibility to evidence-based nutrition interventions is essential to avert adverse outcomes of malnutrition among those vulnerable children [6].\\nNutritional management of children with CP is complex as several interlinked factors interfere with their growth directly and indirectly [7]. While a few factors have been identified as common predictors of malnutrition (e.g., gross motor and oromotor function limitations) [1,8,9], the conceptual framework of malnutrition in children with CP is not yet clearly understood, especially in LMICs. To date, several clinical nutrition guidelines based on current evidence on surgical and non-surgical intervention outcomes have been published [6,10]. However, most of those studies were conducted in high-income countries (HICs) [6], whereas 85% of children with disabilities live in LMICs and the majority have no or limited access to any rehabilitation services [11,12]. With the extreme shortage of trained professionals including dietitians, in addition to the limited availability and accessibility to institutionalized intervention programs in LMICs [13], it is likely that not all the interventions found effective in HIC settings may be applicable in LMIC settings. In absence of optimal management, these children are at high risk of malnutrition, which can in turn impact their functional outcomes, quality of life, and survival [14,15,16].\\nAny available evidence in the context of LMICs could guide resource mobilization, assist in understanding the need and cost-effectiveness as well as to identify interventions that may improve the nutritional status of children with CP in low-resource settings. This is important to establish a platform for developing and implementing best practice CP-specific nutrition intervention guidelines in LMICs. In this scoping review, we aimed to systematically map the existing evidence on nutritional interventions for children with CP living in LMIC settings. The following research questions were explored: (i) What is known about the available nutrition interventions for children with CP in LMICs? (ii) What are the outcomes of those interventions on the nutritional status of participating children with CP in LMICs?\",\n \"2.1. Study Design We conducted a scoping review to summarize the available evidence and provide an overview of different intervention programs and their outcomes related to the nutritional status of children with CP in LMICs. A protocol was developed following the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) extension for scoping reviews guideline [17] and is available upon request from the corresponding author.\\nWe conducted a scoping review to summarize the available evidence and provide an overview of different intervention programs and their outcomes related to the nutritional status of children with CP in LMICs. A protocol was developed following the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) extension for scoping reviews guideline [17] and is available upon request from the corresponding author.\\n2.2. Database Searching The key words and search strategy were developed by two of the authors (I.J. and G.K.) and pretested prior to the initiation of the search. PubMed (up to 15 December 2021) and Scopus (up to 10 January 2022) were searched to identify relevant articles. Google Scholar was searched for any additional relevant articles or scientific guidelines. The search was not restricted to any specific language. The key terms used included “cerebral palsy”/”neurodevelopmental disorder”; children/adolescent/infant; parent/mother/caregiver; intervention/trial/outcome; training/education/feeding/technique/surgical/teaching; nutrition/malnutrition/growth/health; “low- and middle income countries (LMICs)”, and individual country listed as LMICs according to the 2020 World Bank country classifications by income level (i.e., low-income, lower middle-income, and upper-middle income countries) [18]. The electronic search strategy for Scopus is outlined in Supplementary Table S1. Two reviewers (I.J. and R.S.) independently searched the databases and included articles following the inclusion and exclusion criteria. Any disagreement was resolved by discussion between the reviewers and in consultation with a third reviewer (G.K.).\\nThe key words and search strategy were developed by two of the authors (I.J. and G.K.) and pretested prior to the initiation of the search. PubMed (up to 15 December 2021) and Scopus (up to 10 January 2022) were searched to identify relevant articles. Google Scholar was searched for any additional relevant articles or scientific guidelines. The search was not restricted to any specific language. The key terms used included “cerebral palsy”/”neurodevelopmental disorder”; children/adolescent/infant; parent/mother/caregiver; intervention/trial/outcome; training/education/feeding/technique/surgical/teaching; nutrition/malnutrition/growth/health; “low- and middle income countries (LMICs)”, and individual country listed as LMICs according to the 2020 World Bank country classifications by income level (i.e., low-income, lower middle-income, and upper-middle income countries) [18]. The electronic search strategy for Scopus is outlined in Supplementary Table S1. Two reviewers (I.J. and R.S.) independently searched the databases and included articles following the inclusion and exclusion criteria. Any disagreement was resolved by discussion between the reviewers and in consultation with a third reviewer (G.K.).\\n2.3. Inclusion and Exclusion Criteria Articles that met the following criteria were included in the review: (i) the study participants were children with CP, (ii) the outcome measures included nutritional status as determined by anthropometric measurements, e.g., weight, height/length of children with CP and/or body composition, and (iii) followed analytical study design (e.g., experimental/quasi-experimental, pre- and post-intervention study) or descriptive study with a control group or comparison group (e.g., children with gastrostomy versus children fed orally). Articles were excluded (i) if the study was conducted in HICs, (ii) if data about children with CP could not be differentiated and was reported together with children with other form of impairments, and (iii) if they were not peer-reviewed publications and were protocols, guidelines, book chapters, conference presentations, forewords, or replies to commentaries.\\nArticles that met the following criteria were included in the review: (i) the study participants were children with CP, (ii) the outcome measures included nutritional status as determined by anthropometric measurements, e.g., weight, height/length of children with CP and/or body composition, and (iii) followed analytical study design (e.g., experimental/quasi-experimental, pre- and post-intervention study) or descriptive study with a control group or comparison group (e.g., children with gastrostomy versus children fed orally). Articles were excluded (i) if the study was conducted in HICs, (ii) if data about children with CP could not be differentiated and was reported together with children with other form of impairments, and (iii) if they were not peer-reviewed publications and were protocols, guidelines, book chapters, conference presentations, forewords, or replies to commentaries.\\n2.4. Data Charting Process Data charting was primarily completed by two independent reviewers (I.J. and R.S.) using an a priori template developed by I.J. and G.K. Information on study design, settings, country, study participants (age, sex, motor function severity, sample size), intervention provided (type, settings, intervention contents, number of sessions as reported, follow-up period), and outcome (measures used and outcome reported) were extracted as available. Any missing information was documented as “not reported”.\\nData charting was primarily completed by two independent reviewers (I.J. and R.S.) using an a priori template developed by I.J. and G.K. Information on study design, settings, country, study participants (age, sex, motor function severity, sample size), intervention provided (type, settings, intervention contents, number of sessions as reported, follow-up period), and outcome (measures used and outcome reported) were extracted as available. Any missing information was documented as “not reported”.\\n2.5. Assessment of Risk of Bias and Synthesis of Results The risk of bias of the included studies were not assessed. Descriptive findings from individual studies including study characteristics (name and economic classification of the country according to the World Bank definitions in 2020, study locations, settings, study design, and study period), participants (sample size, age, sex, gross motor function classification system (GMFCS) level [19]), and outcome measures were reported. The different types of interventions were reported under broad headings, e.g., “training to parents/caregivers”, “gastrostomy tube placement/feeding/nasogastric tube feeding”, “nutritional rehabilitation/therapy”, and “dietary modification”. No statistical test was used considering the study objectives.\\nThe risk of bias of the included studies were not assessed. Descriptive findings from individual studies including study characteristics (name and economic classification of the country according to the World Bank definitions in 2020, study locations, settings, study design, and study period), participants (sample size, age, sex, gross motor function classification system (GMFCS) level [19]), and outcome measures were reported. The different types of interventions were reported under broad headings, e.g., “training to parents/caregivers”, “gastrostomy tube placement/feeding/nasogastric tube feeding”, “nutritional rehabilitation/therapy”, and “dietary modification”. No statistical test was used considering the study objectives.\\n2.6. Ethics This study did not require ethics approval as data were collected from existing publications (i.e., secondary data) and no humans were directly contacted to collect/gather any information.\\nThis study did not require ethics approval as data were collected from existing publications (i.e., secondary data) and no humans were directly contacted to collect/gather any information.\",\n \"We conducted a scoping review to summarize the available evidence and provide an overview of different intervention programs and their outcomes related to the nutritional status of children with CP in LMICs. A protocol was developed following the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) extension for scoping reviews guideline [17] and is available upon request from the corresponding author.\",\n \"The key words and search strategy were developed by two of the authors (I.J. and G.K.) and pretested prior to the initiation of the search. PubMed (up to 15 December 2021) and Scopus (up to 10 January 2022) were searched to identify relevant articles. Google Scholar was searched for any additional relevant articles or scientific guidelines. The search was not restricted to any specific language. The key terms used included “cerebral palsy”/”neurodevelopmental disorder”; children/adolescent/infant; parent/mother/caregiver; intervention/trial/outcome; training/education/feeding/technique/surgical/teaching; nutrition/malnutrition/growth/health; “low- and middle income countries (LMICs)”, and individual country listed as LMICs according to the 2020 World Bank country classifications by income level (i.e., low-income, lower middle-income, and upper-middle income countries) [18]. The electronic search strategy for Scopus is outlined in Supplementary Table S1. Two reviewers (I.J. and R.S.) independently searched the databases and included articles following the inclusion and exclusion criteria. Any disagreement was resolved by discussion between the reviewers and in consultation with a third reviewer (G.K.).\",\n \"Articles that met the following criteria were included in the review: (i) the study participants were children with CP, (ii) the outcome measures included nutritional status as determined by anthropometric measurements, e.g., weight, height/length of children with CP and/or body composition, and (iii) followed analytical study design (e.g., experimental/quasi-experimental, pre- and post-intervention study) or descriptive study with a control group or comparison group (e.g., children with gastrostomy versus children fed orally). Articles were excluded (i) if the study was conducted in HICs, (ii) if data about children with CP could not be differentiated and was reported together with children with other form of impairments, and (iii) if they were not peer-reviewed publications and were protocols, guidelines, book chapters, conference presentations, forewords, or replies to commentaries.\",\n \"Data charting was primarily completed by two independent reviewers (I.J. and R.S.) using an a priori template developed by I.J. and G.K. Information on study design, settings, country, study participants (age, sex, motor function severity, sample size), intervention provided (type, settings, intervention contents, number of sessions as reported, follow-up period), and outcome (measures used and outcome reported) were extracted as available. Any missing information was documented as “not reported”.\",\n \"The risk of bias of the included studies were not assessed. Descriptive findings from individual studies including study characteristics (name and economic classification of the country according to the World Bank definitions in 2020, study locations, settings, study design, and study period), participants (sample size, age, sex, gross motor function classification system (GMFCS) level [19]), and outcome measures were reported. The different types of interventions were reported under broad headings, e.g., “training to parents/caregivers”, “gastrostomy tube placement/feeding/nasogastric tube feeding”, “nutritional rehabilitation/therapy”, and “dietary modification”. No statistical test was used considering the study objectives.\",\n \"This study did not require ethics approval as data were collected from existing publications (i.e., secondary data) and no humans were directly contacted to collect/gather any information.\",\n \"A total of n = 4885 citations were identified from the databases after deduplication. Following title screening, n = 132 abstracts were reviewed; of those, n = 26 articles were selected for full review. However, of those, n = 5 full articles were not available and an additional n = 4 were identified from handsearching the bibliographies of selected articles. So, a total of n = 25 full articles met the inclusion criteria. However, of those n = 13 were conducted in high-income settings, n = 1 did not report information for children with CP separately, thus the outcome could not be differentiated from other study participants, and n = 3 were conducted in HICs and did not report data on CP separately. Hence, those n = 17 were excluded. Finally, n = 8 articles published between the years 2008 and 2019 were included in the review for data charting. A flow diagram of the study selection procedure is shown in Figure 1. A summary of the excluded studies is also available in Supplementary Table S2.\\nThe details about study characteristics, study participants, intervention details, and outcomes of the included studies are summarized in the subsequent sections.\\n3.1. Study Characteristics The characteristics of the included studies have been summarized in Table 1.\\n3.1.1. Study Design Five out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\\nCharacteristics of the included study.\\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\\nFive out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\\nCharacteristics of the included study.\\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\\n3.1.2. Study Location Of the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\\nOf the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\\n3.1.3. Study Settings Six of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\\nSix of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\\n3.1.4. Study Participants and Sample Size Five studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\\nFive studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\\nThe characteristics of the included studies have been summarized in Table 1.\\n3.1.1. Study Design Five out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\\nCharacteristics of the included study.\\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\\nFive out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\\nCharacteristics of the included study.\\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\\n3.1.2. Study Location Of the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\\nOf the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\\n3.1.3. Study Settings Six of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\\nSix of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\\n3.1.4. Study Participants and Sample Size Five studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\\nFive studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\\n3.2. Intervention Details Of the eight studies, n = 6 had single type interventions [20,21,23,24,25,27] and the remaining n = 2 had multiple interventions [22,26]. Of all, n = 4 provided training to parents/caregivers [20,21,24,27] and n = 2 involved surgical interventions (e.g., gastrostomy or nasogastric tube feeding) [25,26]. Furthermore, dietary modification (e.g., modification of calorie and nutrient density/balanced diet/frequency/consistency/nutritional adequacy) was an intervention component in n = 5 studies [20,21,22,23,26]. Both studies with surgical interventions were descriptive in design (i.e., comparative study, before-after study, case series) and were conducted in Brazil [25] and Mexico [26]. The studies that emphasized parental/caregiver training covered a wide range of content, such as therapy, feeding skills, dietary modifications, position, and carrying [20,21,22,24,27]. The number of sessions varied between 5–11 [20,21,22,24], and the follow-up duration ranged between 1–18 months [20,21,22,23,24,26,27]. The study implementation team was documented only in one study by Pike et al. (2016) where a team of physiotherapists, occupational therapists, and speech-language therapists provided need-based neurodevelopmental therapy (NDT) feeding intervention to children with CP, trained the caregivers on recommended NDT feeding intervention and dietary modifications for children with CP in a hospital-based/institution-based setting in South Africa [22].\\nThe details of different interventions provided in each of the studies included in this review are summarized in Table 2.\\nOf the eight studies, n = 6 had single type interventions [20,21,23,24,25,27] and the remaining n = 2 had multiple interventions [22,26]. Of all, n = 4 provided training to parents/caregivers [20,21,24,27] and n = 2 involved surgical interventions (e.g., gastrostomy or nasogastric tube feeding) [25,26]. Furthermore, dietary modification (e.g., modification of calorie and nutrient density/balanced diet/frequency/consistency/nutritional adequacy) was an intervention component in n = 5 studies [20,21,22,23,26]. Both studies with surgical interventions were descriptive in design (i.e., comparative study, before-after study, case series) and were conducted in Brazil [25] and Mexico [26]. The studies that emphasized parental/caregiver training covered a wide range of content, such as therapy, feeding skills, dietary modifications, position, and carrying [20,21,22,24,27]. The number of sessions varied between 5–11 [20,21,22,24], and the follow-up duration ranged between 1–18 months [20,21,22,23,24,26,27]. The study implementation team was documented only in one study by Pike et al. (2016) where a team of physiotherapists, occupational therapists, and speech-language therapists provided need-based neurodevelopmental therapy (NDT) feeding intervention to children with CP, trained the caregivers on recommended NDT feeding intervention and dietary modifications for children with CP in a hospital-based/institution-based setting in South Africa [22].\\nThe details of different interventions provided in each of the studies included in this review are summarized in Table 2.\\n3.3. Outcome Measures Different anthropometric measurements were used to evaluate changes in the nutritional status of participating children. The most commonly used anthropometric measurements in the selected articles were weight (n = 8) [20,21,22,23,24,25,26,27], length/height (n = 7) [21,22,23,24,25,26,27], mid-upper arm circumference (MUAC) (n = 7) [20,21,22,23,24,26,27], and at least one skin-fold thickness measurement (n = 4) [22,23,25,26].\\nIn all studies, nutritional status was determined by comparing the anthropometric measurements of children with CP to national standards/general population/WHO reference population [20,21,22,23,24,25,26,27]. However, one study also compared the anthropometric measurements with CP specific growth charts [23].\\nAll but one article had other outcome measures in addition to the nutritional assessment; these include (i) dietary intake practices (n = 3) [20,25,27], feeding skills/feeding practices/feeding profiles (n = 5) [20,21,22,26,27], outcomes related to caregivers’ quality of life, knowledge and confidence about child care, perception about child’s health, feelings about child’s feeding difficulties, and compliance with the training [20,24], and child’s health-related quality of life [22]. Any adverse outcomes related to intervention were reported in n = 3 articles [20,23,24]; these outcomes included infections (one study) [23], chest health (one study) [20], and mortality (two studies) [20,24] (Table 3).\\nDifferent anthropometric measurements were used to evaluate changes in the nutritional status of participating children. The most commonly used anthropometric measurements in the selected articles were weight (n = 8) [20,21,22,23,24,25,26,27], length/height (n = 7) [21,22,23,24,25,26,27], mid-upper arm circumference (MUAC) (n = 7) [20,21,22,23,24,26,27], and at least one skin-fold thickness measurement (n = 4) [22,23,25,26].\\nIn all studies, nutritional status was determined by comparing the anthropometric measurements of children with CP to national standards/general population/WHO reference population [20,21,22,23,24,25,26,27]. However, one study also compared the anthropometric measurements with CP specific growth charts [23].\\nAll but one article had other outcome measures in addition to the nutritional assessment; these include (i) dietary intake practices (n = 3) [20,25,27], feeding skills/feeding practices/feeding profiles (n = 5) [20,21,22,26,27], outcomes related to caregivers’ quality of life, knowledge and confidence about child care, perception about child’s health, feelings about child’s feeding difficulties, and compliance with the training [20,24], and child’s health-related quality of life [22]. Any adverse outcomes related to intervention were reported in n = 3 articles [20,23,24]; these outcomes included infections (one study) [23], chest health (one study) [20], and mortality (two studies) [20,24] (Table 3).\\n3.4. Effect of Different Interventions on Nutritional Status of Children with CP Of the eight studies, seven included follow-up data of the participating children with CP (i.e., two or more data points for each participant) [20,21,22,23,24,26,27] and the other one compared between two groups (i.e., intervention vs. control group) [25]. Of those n = 7 studies with longitudinal data [20,21,22,23,24,26,27], n = 4 showed improvement in nutritional status among children with CP following intervention [20,22,23,26], one showed no change [21], whereas two showed deterioration [24,27] in the nutritional status following respective interventions among children with CP in selected LMICs.\\nFour out of the five studies that focused on dietary modifications showed improvement in nutritional status of participating children [20,22,23,26]. Studies that focused on improving feeding skills (n = 3) had both positive and negative nutritional outcomes [20,21,24].\\nBoth studies with GTF as an intervention showed a positive effect of GTF on the nutritional outcome of children with CP [25,26], however, of those none reported any adverse outcome of GTF [25,26]. (Table 3)\\nOf the eight studies, seven included follow-up data of the participating children with CP (i.e., two or more data points for each participant) [20,21,22,23,24,26,27] and the other one compared between two groups (i.e., intervention vs. control group) [25]. Of those n = 7 studies with longitudinal data [20,21,22,23,24,26,27], n = 4 showed improvement in nutritional status among children with CP following intervention [20,22,23,26], one showed no change [21], whereas two showed deterioration [24,27] in the nutritional status following respective interventions among children with CP in selected LMICs.\\nFour out of the five studies that focused on dietary modifications showed improvement in nutritional status of participating children [20,22,23,26]. Studies that focused on improving feeding skills (n = 3) had both positive and negative nutritional outcomes [20,21,24].\\nBoth studies with GTF as an intervention showed a positive effect of GTF on the nutritional outcome of children with CP [25,26], however, of those none reported any adverse outcome of GTF [25,26]. (Table 3)\",\n \"The characteristics of the included studies have been summarized in Table 1.\\n3.1.1. Study Design Five out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\\nCharacteristics of the included study.\\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\\nFive out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\\nCharacteristics of the included study.\\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\\n3.1.2. Study Location Of the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\\nOf the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\\n3.1.3. Study Settings Six of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\\nSix of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\\n3.1.4. Study Participants and Sample Size Five studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\\nFive studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\",\n \"Five out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\\nCharacteristics of the included study.\\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\",\n \"Of the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\",\n \"Six of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\",\n \"Five studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\",\n \"Of the eight studies, n = 6 had single type interventions [20,21,23,24,25,27] and the remaining n = 2 had multiple interventions [22,26]. Of all, n = 4 provided training to parents/caregivers [20,21,24,27] and n = 2 involved surgical interventions (e.g., gastrostomy or nasogastric tube feeding) [25,26]. Furthermore, dietary modification (e.g., modification of calorie and nutrient density/balanced diet/frequency/consistency/nutritional adequacy) was an intervention component in n = 5 studies [20,21,22,23,26]. Both studies with surgical interventions were descriptive in design (i.e., comparative study, before-after study, case series) and were conducted in Brazil [25] and Mexico [26]. The studies that emphasized parental/caregiver training covered a wide range of content, such as therapy, feeding skills, dietary modifications, position, and carrying [20,21,22,24,27]. The number of sessions varied between 5–11 [20,21,22,24], and the follow-up duration ranged between 1–18 months [20,21,22,23,24,26,27]. The study implementation team was documented only in one study by Pike et al. (2016) where a team of physiotherapists, occupational therapists, and speech-language therapists provided need-based neurodevelopmental therapy (NDT) feeding intervention to children with CP, trained the caregivers on recommended NDT feeding intervention and dietary modifications for children with CP in a hospital-based/institution-based setting in South Africa [22].\\nThe details of different interventions provided in each of the studies included in this review are summarized in Table 2.\",\n \"Different anthropometric measurements were used to evaluate changes in the nutritional status of participating children. The most commonly used anthropometric measurements in the selected articles were weight (n = 8) [20,21,22,23,24,25,26,27], length/height (n = 7) [21,22,23,24,25,26,27], mid-upper arm circumference (MUAC) (n = 7) [20,21,22,23,24,26,27], and at least one skin-fold thickness measurement (n = 4) [22,23,25,26].\\nIn all studies, nutritional status was determined by comparing the anthropometric measurements of children with CP to national standards/general population/WHO reference population [20,21,22,23,24,25,26,27]. However, one study also compared the anthropometric measurements with CP specific growth charts [23].\\nAll but one article had other outcome measures in addition to the nutritional assessment; these include (i) dietary intake practices (n = 3) [20,25,27], feeding skills/feeding practices/feeding profiles (n = 5) [20,21,22,26,27], outcomes related to caregivers’ quality of life, knowledge and confidence about child care, perception about child’s health, feelings about child’s feeding difficulties, and compliance with the training [20,24], and child’s health-related quality of life [22]. Any adverse outcomes related to intervention were reported in n = 3 articles [20,23,24]; these outcomes included infections (one study) [23], chest health (one study) [20], and mortality (two studies) [20,24] (Table 3).\",\n \"Of the eight studies, seven included follow-up data of the participating children with CP (i.e., two or more data points for each participant) [20,21,22,23,24,26,27] and the other one compared between two groups (i.e., intervention vs. control group) [25]. Of those n = 7 studies with longitudinal data [20,21,22,23,24,26,27], n = 4 showed improvement in nutritional status among children with CP following intervention [20,22,23,26], one showed no change [21], whereas two showed deterioration [24,27] in the nutritional status following respective interventions among children with CP in selected LMICs.\\nFour out of the five studies that focused on dietary modifications showed improvement in nutritional status of participating children [20,22,23,26]. Studies that focused on improving feeding skills (n = 3) had both positive and negative nutritional outcomes [20,21,24].\\nBoth studies with GTF as an intervention showed a positive effect of GTF on the nutritional outcome of children with CP [25,26], however, of those none reported any adverse outcome of GTF [25,26]. (Table 3)\",\n \"In this review we provide a summary of the available evidence on different nutrition interventions for children with CP in LMICs. Our search identified only a few relevant studies on the topic that were conducted in resource-constrained settings of LMICs. Only eight articles were identified, none of which had representation from low-income countries. Such an evidence gap from LMICs where the majority of children with CP reside is concerning [11]. Considering the high burden of malnourished children with CP in LMICs [1,2,3,4,5,8,9], there is a dire need to reduce this evidence gap, and integration of nutrition interventions in the existing service plan for children with CP is essential.\\nThere are global efforts toward reducing the burden of malnutrition among children, especially in LMICs. The United Nations Sustainable Development Goals (UN SDGs) has an emphasis on childhood nutrition for human growth and development [28]. The SDGs also strongly advocate for disability inclusiveness, especially the SDGs which are related to growth, health, education, employment, and addressing inequality globally [29]. National and international partners are working together to develop standard guidelines, strategies, and interventions addressing the immediate determinants (i.e., nutrition-specific intervention) and the underlying or root causes (i.e., nutrition-sensitive interventions) to improve the overall nutritional status of children, especially in LMICs [30,31]. These interventions should be disability-inclusive, and priority should be given to reporting/documentation of the effectiveness of different nutrition interventions on growth and nutrition of children with disabilities, including CP.\\nWhile reviewing the limited available studies, we observed that a majority of the interventions were trialed or implemented in institution-based settings. Though interventions implemented in the institution-based settings have been proven to result in better outcomes in the past [6], our evidence suggests the majority of children with CP in LMICs lack access to any rehabilitation services for numerous reasons, including limited availability of services in their neighborhoods, transport difficulties, and financial constraints [12,32]. Furthermore, the severe shortage of trained health professionals, e.g., rehabilitation service providers and dietitians, limits the scope to implement and scale-up different institution-based interventions for children with CP in LMICs [13]. In such a context, community-based approaches are highly recommended and there is a lack of evidence on community-based nutrition interventions for children with CP in LMICs. Nevertheless, in one recent study from Bangladesh, community-based parent-led intervention was found to be highly successful in improving functional outcomes of children with CP [33]. Emphasis should be given to the capacity development of mid-level service providers on growth monitoring and promotion, early identification, different blanket interventions, and referral to prevent and treat malnutrition among children with CP in LMICs.\\nIn addition to the settings, the sample sizes of the studies were relatively low. Although the included studies provided valuable insights on different nutritional interventions, the small sample size limits the strength and generalizability of the study findings. Moreover, most children with CP in the selected studies had severe functional motor limitations. These could be due to selection bias related to more severe institutional samples, as well as the more severe end of the clinical spectrum of CP in LMICs, likely exacerbated by delayed age of diagnosis and lack of access to rehabilitation services [12,32,34]. Studies also show that the likelihood of receiving intervention and rehabilitation is higher among those with severe CP (e.g., GMFCS level III–V) compared to milder forms (e.g., GMFCS I–II) [12]. However, it is also known that severe functional motor limitations (e.g., GMFCS level III–V) in children with CP are often accompanied by severe oromotor dysfunction, e.g., dysphagia [35]. It remains unclear whether the interventions for children with more severe motor limitations are also appropriate for children with CP who are GMFCS I–II.\\nMost of the interventions identified in this review improved the nutritional status of participating children with CP. Importantly, nutrition-specific interventions (i.e., interventions that aim to intervene in or correct the immediate causes of malnutrition, such as inadequate diet, disease severity and caring practices) had comparatively better outcomes than other intervention strategies [20,22,24,30]. We observed that the interventions that included dietary modifications (only, or as a component of multiple approaches) had better effect on nutritional outcomes of children with CP. Studies where participants received surgical interventions (e.g., GTF) also showed a positive effect, whereas studies that provided behavioral intervention on feeding skills only did not show any significant change.\\nTo the best of our knowledge, this is one of the first reviews conducted on nutrition interventions for children with CP in LMICs. Although during our search we identified several studies that aimed to improve the nutritional status of children with CP in HICs, our search revealed only a few reviews (including one systematic review) that summarized findings from different nutrition intervention studies among children with CP in HICs [6,36,37]. The data provided in our study, therefore, contribute important information to the existing knowledge gap. Nevertheless, this review has several limitations. We focused our search on two databases, so a risk remains that there may have been other publications available through other databases. However, our search revealed a large number of duplicates; therefore, we are confident that we have covered all or almost all of the studies published online. Second, we only searched for articles that had our key words in title/abstracts/author specified key words in the articles. This may have reduced the number of articles initially identified for title screening. Third, we did not assess the risk of bias and quality of evidence of the articles finally included in the review, which may have posed bias in the information reported. Finally, due to the differences in reporting format and data availability, we could only include a few common variables and could not report on some of the important characteristics of the participants. For the same reason, we had to rely on reporting the descriptive findings only.\",\n \"The current review highlights the knowledge gap and lack of available evidence on nutrition interventions for children with CP in LMICs. Strong evidence is essential to identify and determine the best practices and nutritional management guidelines for children with CP, especially for optimal utilization of the limited available resources in LMICs. National and international stakeholders, therefore, should make this a priority for future research and services. Considering the high proportion of children with CP with undernutrition in LMICs, existing interventions and services for children with CP should integrate a nutrition component. The current limited evidence suggests that nutrition-specific programs have more of a positive effect on the growth of children with CP than other strategies. This should be taken under consideration when planning for any nutrition focus and other comprehensive services for children with CP in LMICs.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["intro",null,null,null,null,null,null,null,"results",null,null,null,null,"subjects",null,null,null,"discussion","conclusions"],"string":"[\n \"intro\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"subjects\",\n null,\n null,\n null,\n \"discussion\",\n \"conclusions\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["nutrition","intervention","children","cerebral palsy","disability","LMICs"],"string":"[\n \"nutrition\",\n \"intervention\",\n \"children\",\n \"cerebral palsy\",\n \"disability\",\n \"LMICs\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"1. Introduction:\nUndernutrition is a major global public health challenge, and children with disability, such as cerebral palsy (CP), often suffer from undernutrition, especially in low- and middle-income countries (LMICs). The proportion of malnutrition is substantially higher among children with CP in LMICs compared to the general population [1,2,3,4,5]. Ensuring availability and accessibility to evidence-based nutrition interventions is essential to avert adverse outcomes of malnutrition among those vulnerable children [6].\nNutritional management of children with CP is complex as several interlinked factors interfere with their growth directly and indirectly [7]. While a few factors have been identified as common predictors of malnutrition (e.g., gross motor and oromotor function limitations) [1,8,9], the conceptual framework of malnutrition in children with CP is not yet clearly understood, especially in LMICs. To date, several clinical nutrition guidelines based on current evidence on surgical and non-surgical intervention outcomes have been published [6,10]. However, most of those studies were conducted in high-income countries (HICs) [6], whereas 85% of children with disabilities live in LMICs and the majority have no or limited access to any rehabilitation services [11,12]. With the extreme shortage of trained professionals including dietitians, in addition to the limited availability and accessibility to institutionalized intervention programs in LMICs [13], it is likely that not all the interventions found effective in HIC settings may be applicable in LMIC settings. In absence of optimal management, these children are at high risk of malnutrition, which can in turn impact their functional outcomes, quality of life, and survival [14,15,16].\nAny available evidence in the context of LMICs could guide resource mobilization, assist in understanding the need and cost-effectiveness as well as to identify interventions that may improve the nutritional status of children with CP in low-resource settings. This is important to establish a platform for developing and implementing best practice CP-specific nutrition intervention guidelines in LMICs. In this scoping review, we aimed to systematically map the existing evidence on nutritional interventions for children with CP living in LMIC settings. The following research questions were explored: (i) What is known about the available nutrition interventions for children with CP in LMICs? (ii) What are the outcomes of those interventions on the nutritional status of participating children with CP in LMICs?\n\n2. Materials and Methods:\n2.1. Study Design We conducted a scoping review to summarize the available evidence and provide an overview of different intervention programs and their outcomes related to the nutritional status of children with CP in LMICs. A protocol was developed following the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) extension for scoping reviews guideline [17] and is available upon request from the corresponding author.\nWe conducted a scoping review to summarize the available evidence and provide an overview of different intervention programs and their outcomes related to the nutritional status of children with CP in LMICs. A protocol was developed following the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) extension for scoping reviews guideline [17] and is available upon request from the corresponding author.\n2.2. Database Searching The key words and search strategy were developed by two of the authors (I.J. and G.K.) and pretested prior to the initiation of the search. PubMed (up to 15 December 2021) and Scopus (up to 10 January 2022) were searched to identify relevant articles. Google Scholar was searched for any additional relevant articles or scientific guidelines. The search was not restricted to any specific language. The key terms used included “cerebral palsy”/”neurodevelopmental disorder”; children/adolescent/infant; parent/mother/caregiver; intervention/trial/outcome; training/education/feeding/technique/surgical/teaching; nutrition/malnutrition/growth/health; “low- and middle income countries (LMICs)”, and individual country listed as LMICs according to the 2020 World Bank country classifications by income level (i.e., low-income, lower middle-income, and upper-middle income countries) [18]. The electronic search strategy for Scopus is outlined in Supplementary Table S1. Two reviewers (I.J. and R.S.) independently searched the databases and included articles following the inclusion and exclusion criteria. Any disagreement was resolved by discussion between the reviewers and in consultation with a third reviewer (G.K.).\nThe key words and search strategy were developed by two of the authors (I.J. and G.K.) and pretested prior to the initiation of the search. PubMed (up to 15 December 2021) and Scopus (up to 10 January 2022) were searched to identify relevant articles. Google Scholar was searched for any additional relevant articles or scientific guidelines. The search was not restricted to any specific language. The key terms used included “cerebral palsy”/”neurodevelopmental disorder”; children/adolescent/infant; parent/mother/caregiver; intervention/trial/outcome; training/education/feeding/technique/surgical/teaching; nutrition/malnutrition/growth/health; “low- and middle income countries (LMICs)”, and individual country listed as LMICs according to the 2020 World Bank country classifications by income level (i.e., low-income, lower middle-income, and upper-middle income countries) [18]. The electronic search strategy for Scopus is outlined in Supplementary Table S1. Two reviewers (I.J. and R.S.) independently searched the databases and included articles following the inclusion and exclusion criteria. Any disagreement was resolved by discussion between the reviewers and in consultation with a third reviewer (G.K.).\n2.3. Inclusion and Exclusion Criteria Articles that met the following criteria were included in the review: (i) the study participants were children with CP, (ii) the outcome measures included nutritional status as determined by anthropometric measurements, e.g., weight, height/length of children with CP and/or body composition, and (iii) followed analytical study design (e.g., experimental/quasi-experimental, pre- and post-intervention study) or descriptive study with a control group or comparison group (e.g., children with gastrostomy versus children fed orally). Articles were excluded (i) if the study was conducted in HICs, (ii) if data about children with CP could not be differentiated and was reported together with children with other form of impairments, and (iii) if they were not peer-reviewed publications and were protocols, guidelines, book chapters, conference presentations, forewords, or replies to commentaries.\nArticles that met the following criteria were included in the review: (i) the study participants were children with CP, (ii) the outcome measures included nutritional status as determined by anthropometric measurements, e.g., weight, height/length of children with CP and/or body composition, and (iii) followed analytical study design (e.g., experimental/quasi-experimental, pre- and post-intervention study) or descriptive study with a control group or comparison group (e.g., children with gastrostomy versus children fed orally). Articles were excluded (i) if the study was conducted in HICs, (ii) if data about children with CP could not be differentiated and was reported together with children with other form of impairments, and (iii) if they were not peer-reviewed publications and were protocols, guidelines, book chapters, conference presentations, forewords, or replies to commentaries.\n2.4. Data Charting Process Data charting was primarily completed by two independent reviewers (I.J. and R.S.) using an a priori template developed by I.J. and G.K. Information on study design, settings, country, study participants (age, sex, motor function severity, sample size), intervention provided (type, settings, intervention contents, number of sessions as reported, follow-up period), and outcome (measures used and outcome reported) were extracted as available. Any missing information was documented as “not reported”.\nData charting was primarily completed by two independent reviewers (I.J. and R.S.) using an a priori template developed by I.J. and G.K. Information on study design, settings, country, study participants (age, sex, motor function severity, sample size), intervention provided (type, settings, intervention contents, number of sessions as reported, follow-up period), and outcome (measures used and outcome reported) were extracted as available. Any missing information was documented as “not reported”.\n2.5. Assessment of Risk of Bias and Synthesis of Results The risk of bias of the included studies were not assessed. Descriptive findings from individual studies including study characteristics (name and economic classification of the country according to the World Bank definitions in 2020, study locations, settings, study design, and study period), participants (sample size, age, sex, gross motor function classification system (GMFCS) level [19]), and outcome measures were reported. The different types of interventions were reported under broad headings, e.g., “training to parents/caregivers”, “gastrostomy tube placement/feeding/nasogastric tube feeding”, “nutritional rehabilitation/therapy”, and “dietary modification”. No statistical test was used considering the study objectives.\nThe risk of bias of the included studies were not assessed. Descriptive findings from individual studies including study characteristics (name and economic classification of the country according to the World Bank definitions in 2020, study locations, settings, study design, and study period), participants (sample size, age, sex, gross motor function classification system (GMFCS) level [19]), and outcome measures were reported. The different types of interventions were reported under broad headings, e.g., “training to parents/caregivers”, “gastrostomy tube placement/feeding/nasogastric tube feeding”, “nutritional rehabilitation/therapy”, and “dietary modification”. No statistical test was used considering the study objectives.\n2.6. Ethics This study did not require ethics approval as data were collected from existing publications (i.e., secondary data) and no humans were directly contacted to collect/gather any information.\nThis study did not require ethics approval as data were collected from existing publications (i.e., secondary data) and no humans were directly contacted to collect/gather any information.\n\n2.1. Study Design:\nWe conducted a scoping review to summarize the available evidence and provide an overview of different intervention programs and their outcomes related to the nutritional status of children with CP in LMICs. A protocol was developed following the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) extension for scoping reviews guideline [17] and is available upon request from the corresponding author.\n\n2.2. Database Searching:\nThe key words and search strategy were developed by two of the authors (I.J. and G.K.) and pretested prior to the initiation of the search. PubMed (up to 15 December 2021) and Scopus (up to 10 January 2022) were searched to identify relevant articles. Google Scholar was searched for any additional relevant articles or scientific guidelines. The search was not restricted to any specific language. The key terms used included “cerebral palsy”/”neurodevelopmental disorder”; children/adolescent/infant; parent/mother/caregiver; intervention/trial/outcome; training/education/feeding/technique/surgical/teaching; nutrition/malnutrition/growth/health; “low- and middle income countries (LMICs)”, and individual country listed as LMICs according to the 2020 World Bank country classifications by income level (i.e., low-income, lower middle-income, and upper-middle income countries) [18]. The electronic search strategy for Scopus is outlined in Supplementary Table S1. Two reviewers (I.J. and R.S.) independently searched the databases and included articles following the inclusion and exclusion criteria. Any disagreement was resolved by discussion between the reviewers and in consultation with a third reviewer (G.K.).\n\n2.3. Inclusion and Exclusion Criteria:\nArticles that met the following criteria were included in the review: (i) the study participants were children with CP, (ii) the outcome measures included nutritional status as determined by anthropometric measurements, e.g., weight, height/length of children with CP and/or body composition, and (iii) followed analytical study design (e.g., experimental/quasi-experimental, pre- and post-intervention study) or descriptive study with a control group or comparison group (e.g., children with gastrostomy versus children fed orally). Articles were excluded (i) if the study was conducted in HICs, (ii) if data about children with CP could not be differentiated and was reported together with children with other form of impairments, and (iii) if they were not peer-reviewed publications and were protocols, guidelines, book chapters, conference presentations, forewords, or replies to commentaries.\n\n2.4. Data Charting Process:\nData charting was primarily completed by two independent reviewers (I.J. and R.S.) using an a priori template developed by I.J. and G.K. Information on study design, settings, country, study participants (age, sex, motor function severity, sample size), intervention provided (type, settings, intervention contents, number of sessions as reported, follow-up period), and outcome (measures used and outcome reported) were extracted as available. Any missing information was documented as “not reported”.\n\n2.5. Assessment of Risk of Bias and Synthesis of Results:\nThe risk of bias of the included studies were not assessed. Descriptive findings from individual studies including study characteristics (name and economic classification of the country according to the World Bank definitions in 2020, study locations, settings, study design, and study period), participants (sample size, age, sex, gross motor function classification system (GMFCS) level [19]), and outcome measures were reported. The different types of interventions were reported under broad headings, e.g., “training to parents/caregivers”, “gastrostomy tube placement/feeding/nasogastric tube feeding”, “nutritional rehabilitation/therapy”, and “dietary modification”. No statistical test was used considering the study objectives.\n\n2.6. Ethics:\nThis study did not require ethics approval as data were collected from existing publications (i.e., secondary data) and no humans were directly contacted to collect/gather any information.\n\n3. Results:\nA total of n = 4885 citations were identified from the databases after deduplication. Following title screening, n = 132 abstracts were reviewed; of those, n = 26 articles were selected for full review. However, of those, n = 5 full articles were not available and an additional n = 4 were identified from handsearching the bibliographies of selected articles. So, a total of n = 25 full articles met the inclusion criteria. However, of those n = 13 were conducted in high-income settings, n = 1 did not report information for children with CP separately, thus the outcome could not be differentiated from other study participants, and n = 3 were conducted in HICs and did not report data on CP separately. Hence, those n = 17 were excluded. Finally, n = 8 articles published between the years 2008 and 2019 were included in the review for data charting. A flow diagram of the study selection procedure is shown in Figure 1. A summary of the excluded studies is also available in Supplementary Table S2.\nThe details about study characteristics, study participants, intervention details, and outcomes of the included studies are summarized in the subsequent sections.\n3.1. Study Characteristics The characteristics of the included studies have been summarized in Table 1.\n3.1.1. Study Design Five out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\nCharacteristics of the included study.\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\nFive out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\nCharacteristics of the included study.\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\n3.1.2. Study Location Of the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\nOf the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\n3.1.3. Study Settings Six of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\nSix of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\n3.1.4. Study Participants and Sample Size Five studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\nFive studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\nThe characteristics of the included studies have been summarized in Table 1.\n3.1.1. Study Design Five out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\nCharacteristics of the included study.\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\nFive out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\nCharacteristics of the included study.\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\n3.1.2. Study Location Of the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\nOf the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\n3.1.3. Study Settings Six of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\nSix of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\n3.1.4. Study Participants and Sample Size Five studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\nFive studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\n3.2. Intervention Details Of the eight studies, n = 6 had single type interventions [20,21,23,24,25,27] and the remaining n = 2 had multiple interventions [22,26]. Of all, n = 4 provided training to parents/caregivers [20,21,24,27] and n = 2 involved surgical interventions (e.g., gastrostomy or nasogastric tube feeding) [25,26]. Furthermore, dietary modification (e.g., modification of calorie and nutrient density/balanced diet/frequency/consistency/nutritional adequacy) was an intervention component in n = 5 studies [20,21,22,23,26]. Both studies with surgical interventions were descriptive in design (i.e., comparative study, before-after study, case series) and were conducted in Brazil [25] and Mexico [26]. The studies that emphasized parental/caregiver training covered a wide range of content, such as therapy, feeding skills, dietary modifications, position, and carrying [20,21,22,24,27]. The number of sessions varied between 5–11 [20,21,22,24], and the follow-up duration ranged between 1–18 months [20,21,22,23,24,26,27]. The study implementation team was documented only in one study by Pike et al. (2016) where a team of physiotherapists, occupational therapists, and speech-language therapists provided need-based neurodevelopmental therapy (NDT) feeding intervention to children with CP, trained the caregivers on recommended NDT feeding intervention and dietary modifications for children with CP in a hospital-based/institution-based setting in South Africa [22].\nThe details of different interventions provided in each of the studies included in this review are summarized in Table 2.\nOf the eight studies, n = 6 had single type interventions [20,21,23,24,25,27] and the remaining n = 2 had multiple interventions [22,26]. Of all, n = 4 provided training to parents/caregivers [20,21,24,27] and n = 2 involved surgical interventions (e.g., gastrostomy or nasogastric tube feeding) [25,26]. Furthermore, dietary modification (e.g., modification of calorie and nutrient density/balanced diet/frequency/consistency/nutritional adequacy) was an intervention component in n = 5 studies [20,21,22,23,26]. Both studies with surgical interventions were descriptive in design (i.e., comparative study, before-after study, case series) and were conducted in Brazil [25] and Mexico [26]. The studies that emphasized parental/caregiver training covered a wide range of content, such as therapy, feeding skills, dietary modifications, position, and carrying [20,21,22,24,27]. The number of sessions varied between 5–11 [20,21,22,24], and the follow-up duration ranged between 1–18 months [20,21,22,23,24,26,27]. The study implementation team was documented only in one study by Pike et al. (2016) where a team of physiotherapists, occupational therapists, and speech-language therapists provided need-based neurodevelopmental therapy (NDT) feeding intervention to children with CP, trained the caregivers on recommended NDT feeding intervention and dietary modifications for children with CP in a hospital-based/institution-based setting in South Africa [22].\nThe details of different interventions provided in each of the studies included in this review are summarized in Table 2.\n3.3. Outcome Measures Different anthropometric measurements were used to evaluate changes in the nutritional status of participating children. The most commonly used anthropometric measurements in the selected articles were weight (n = 8) [20,21,22,23,24,25,26,27], length/height (n = 7) [21,22,23,24,25,26,27], mid-upper arm circumference (MUAC) (n = 7) [20,21,22,23,24,26,27], and at least one skin-fold thickness measurement (n = 4) [22,23,25,26].\nIn all studies, nutritional status was determined by comparing the anthropometric measurements of children with CP to national standards/general population/WHO reference population [20,21,22,23,24,25,26,27]. However, one study also compared the anthropometric measurements with CP specific growth charts [23].\nAll but one article had other outcome measures in addition to the nutritional assessment; these include (i) dietary intake practices (n = 3) [20,25,27], feeding skills/feeding practices/feeding profiles (n = 5) [20,21,22,26,27], outcomes related to caregivers’ quality of life, knowledge and confidence about child care, perception about child’s health, feelings about child’s feeding difficulties, and compliance with the training [20,24], and child’s health-related quality of life [22]. Any adverse outcomes related to intervention were reported in n = 3 articles [20,23,24]; these outcomes included infections (one study) [23], chest health (one study) [20], and mortality (two studies) [20,24] (Table 3).\nDifferent anthropometric measurements were used to evaluate changes in the nutritional status of participating children. The most commonly used anthropometric measurements in the selected articles were weight (n = 8) [20,21,22,23,24,25,26,27], length/height (n = 7) [21,22,23,24,25,26,27], mid-upper arm circumference (MUAC) (n = 7) [20,21,22,23,24,26,27], and at least one skin-fold thickness measurement (n = 4) [22,23,25,26].\nIn all studies, nutritional status was determined by comparing the anthropometric measurements of children with CP to national standards/general population/WHO reference population [20,21,22,23,24,25,26,27]. However, one study also compared the anthropometric measurements with CP specific growth charts [23].\nAll but one article had other outcome measures in addition to the nutritional assessment; these include (i) dietary intake practices (n = 3) [20,25,27], feeding skills/feeding practices/feeding profiles (n = 5) [20,21,22,26,27], outcomes related to caregivers’ quality of life, knowledge and confidence about child care, perception about child’s health, feelings about child’s feeding difficulties, and compliance with the training [20,24], and child’s health-related quality of life [22]. Any adverse outcomes related to intervention were reported in n = 3 articles [20,23,24]; these outcomes included infections (one study) [23], chest health (one study) [20], and mortality (two studies) [20,24] (Table 3).\n3.4. Effect of Different Interventions on Nutritional Status of Children with CP Of the eight studies, seven included follow-up data of the participating children with CP (i.e., two or more data points for each participant) [20,21,22,23,24,26,27] and the other one compared between two groups (i.e., intervention vs. control group) [25]. Of those n = 7 studies with longitudinal data [20,21,22,23,24,26,27], n = 4 showed improvement in nutritional status among children with CP following intervention [20,22,23,26], one showed no change [21], whereas two showed deterioration [24,27] in the nutritional status following respective interventions among children with CP in selected LMICs.\nFour out of the five studies that focused on dietary modifications showed improvement in nutritional status of participating children [20,22,23,26]. Studies that focused on improving feeding skills (n = 3) had both positive and negative nutritional outcomes [20,21,24].\nBoth studies with GTF as an intervention showed a positive effect of GTF on the nutritional outcome of children with CP [25,26], however, of those none reported any adverse outcome of GTF [25,26]. (Table 3)\nOf the eight studies, seven included follow-up data of the participating children with CP (i.e., two or more data points for each participant) [20,21,22,23,24,26,27] and the other one compared between two groups (i.e., intervention vs. control group) [25]. Of those n = 7 studies with longitudinal data [20,21,22,23,24,26,27], n = 4 showed improvement in nutritional status among children with CP following intervention [20,22,23,26], one showed no change [21], whereas two showed deterioration [24,27] in the nutritional status following respective interventions among children with CP in selected LMICs.\nFour out of the five studies that focused on dietary modifications showed improvement in nutritional status of participating children [20,22,23,26]. Studies that focused on improving feeding skills (n = 3) had both positive and negative nutritional outcomes [20,21,24].\nBoth studies with GTF as an intervention showed a positive effect of GTF on the nutritional outcome of children with CP [25,26], however, of those none reported any adverse outcome of GTF [25,26]. (Table 3)\n\n3.1. Study Characteristics:\nThe characteristics of the included studies have been summarized in Table 1.\n3.1.1. Study Design Five out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\nCharacteristics of the included study.\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\nFive out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\nCharacteristics of the included study.\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\n3.1.2. Study Location Of the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\nOf the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\n3.1.3. Study Settings Six of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\nSix of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\n3.1.4. Study Participants and Sample Size Five studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\nFive studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\n\n3.1.1. Study Design:\nFive out of eight studies were experimental in design (three conducted in lower middle-income countries (lower MICs) and two in upper middle-income countries (UMICs)) [20,21,22,23,24], two followed a descriptive analytical study design [25,26], and the remaining one was a qualitative study [27].\nCharacteristics of the included study.\nCP, Cerebral Palsy; MIC, Middle-income country; GMFCS, Gross Motor Function Classification System.\n\n3.1.2. Study Location:\nOf the eight studies, n = 4 were from UMICs (Brazil, South Africa, Turkey, Mexico) [22,23,25,26], and n = 4 were from lower MICs (Bangladesh, Ghana × 2, Egypt) [20,21,24,27]; no studies from low-income countries (LICs) were identified. As per the geographical distribution, n = 3 studies were conducted in sub-Saharan Africa (Ghana × 2 and South Africa) [22,24,27], n = 2 were from Latin America and the Caribbean region (Brazil and Mexico) [25,26], n = 1 from the South Asia region (Bangladesh) [20], n = 1 from the Middle East and the North Africa region (Egypt) [21], and n = 1 from the Europe and Central Asia region (Turkey) [23].\n\n3.1.3. Study Settings:\nSix of the eight studies were conducted in hospital/clinic/institution-based settings [20,21,22,23,25,26]. The remaining two were conducted in community-based settings (both evaluated the impact of the same intervention model on different outcome measures) in Ghana; one followed an experimental study design and the other a qualitative approach [24,27]. Among the hospital/clinic/institution-based studies, four were experimental studies conducted in Bangladesh, Egypt, South Africa, and Turkey [20,21,22,23], and two were descriptive analytical studies conducted in Brazil and Mexico [25,26].\n\n3.1.4. Study Participants and Sample Size:\nFive studies had children with CP and their primary caregivers as study participants [20,21,22,24,27], and three included children with quadriplegic CP only [23,25,26].\nA total of 252 children with CP were included in the selected studies (age ranged between 1 year (y) and 18 y 7months (m)). The lowest sample size was n = 13 (a descriptive analytical study conducted in hospital/clinic/institution-based settings in Mexico [26], and the highest sample size was n = 64 (a community-based experimental study conducted in Ghana) [24].\nOverall, 12%, n = 23/198 children, had GMFCS level I–II and the 88%, n = 175/198 children, had GMFCS level III–V [20,21,22,23,26,27]; one study did not report the GMFCS level of the participants [24,25].\nA summary of individual study design, locations, settings, participants, and sample size is presented in Table 1.\n\n3.2. Intervention Details:\nOf the eight studies, n = 6 had single type interventions [20,21,23,24,25,27] and the remaining n = 2 had multiple interventions [22,26]. Of all, n = 4 provided training to parents/caregivers [20,21,24,27] and n = 2 involved surgical interventions (e.g., gastrostomy or nasogastric tube feeding) [25,26]. Furthermore, dietary modification (e.g., modification of calorie and nutrient density/balanced diet/frequency/consistency/nutritional adequacy) was an intervention component in n = 5 studies [20,21,22,23,26]. Both studies with surgical interventions were descriptive in design (i.e., comparative study, before-after study, case series) and were conducted in Brazil [25] and Mexico [26]. The studies that emphasized parental/caregiver training covered a wide range of content, such as therapy, feeding skills, dietary modifications, position, and carrying [20,21,22,24,27]. The number of sessions varied between 5–11 [20,21,22,24], and the follow-up duration ranged between 1–18 months [20,21,22,23,24,26,27]. The study implementation team was documented only in one study by Pike et al. (2016) where a team of physiotherapists, occupational therapists, and speech-language therapists provided need-based neurodevelopmental therapy (NDT) feeding intervention to children with CP, trained the caregivers on recommended NDT feeding intervention and dietary modifications for children with CP in a hospital-based/institution-based setting in South Africa [22].\nThe details of different interventions provided in each of the studies included in this review are summarized in Table 2.\n\n3.3. Outcome Measures:\nDifferent anthropometric measurements were used to evaluate changes in the nutritional status of participating children. The most commonly used anthropometric measurements in the selected articles were weight (n = 8) [20,21,22,23,24,25,26,27], length/height (n = 7) [21,22,23,24,25,26,27], mid-upper arm circumference (MUAC) (n = 7) [20,21,22,23,24,26,27], and at least one skin-fold thickness measurement (n = 4) [22,23,25,26].\nIn all studies, nutritional status was determined by comparing the anthropometric measurements of children with CP to national standards/general population/WHO reference population [20,21,22,23,24,25,26,27]. However, one study also compared the anthropometric measurements with CP specific growth charts [23].\nAll but one article had other outcome measures in addition to the nutritional assessment; these include (i) dietary intake practices (n = 3) [20,25,27], feeding skills/feeding practices/feeding profiles (n = 5) [20,21,22,26,27], outcomes related to caregivers’ quality of life, knowledge and confidence about child care, perception about child’s health, feelings about child’s feeding difficulties, and compliance with the training [20,24], and child’s health-related quality of life [22]. Any adverse outcomes related to intervention were reported in n = 3 articles [20,23,24]; these outcomes included infections (one study) [23], chest health (one study) [20], and mortality (two studies) [20,24] (Table 3).\n\n3.4. Effect of Different Interventions on Nutritional Status of Children with CP:\nOf the eight studies, seven included follow-up data of the participating children with CP (i.e., two or more data points for each participant) [20,21,22,23,24,26,27] and the other one compared between two groups (i.e., intervention vs. control group) [25]. Of those n = 7 studies with longitudinal data [20,21,22,23,24,26,27], n = 4 showed improvement in nutritional status among children with CP following intervention [20,22,23,26], one showed no change [21], whereas two showed deterioration [24,27] in the nutritional status following respective interventions among children with CP in selected LMICs.\nFour out of the five studies that focused on dietary modifications showed improvement in nutritional status of participating children [20,22,23,26]. Studies that focused on improving feeding skills (n = 3) had both positive and negative nutritional outcomes [20,21,24].\nBoth studies with GTF as an intervention showed a positive effect of GTF on the nutritional outcome of children with CP [25,26], however, of those none reported any adverse outcome of GTF [25,26]. (Table 3)\n\n4. Discussion:\nIn this review we provide a summary of the available evidence on different nutrition interventions for children with CP in LMICs. Our search identified only a few relevant studies on the topic that were conducted in resource-constrained settings of LMICs. Only eight articles were identified, none of which had representation from low-income countries. Such an evidence gap from LMICs where the majority of children with CP reside is concerning [11]. Considering the high burden of malnourished children with CP in LMICs [1,2,3,4,5,8,9], there is a dire need to reduce this evidence gap, and integration of nutrition interventions in the existing service plan for children with CP is essential.\nThere are global efforts toward reducing the burden of malnutrition among children, especially in LMICs. The United Nations Sustainable Development Goals (UN SDGs) has an emphasis on childhood nutrition for human growth and development [28]. The SDGs also strongly advocate for disability inclusiveness, especially the SDGs which are related to growth, health, education, employment, and addressing inequality globally [29]. National and international partners are working together to develop standard guidelines, strategies, and interventions addressing the immediate determinants (i.e., nutrition-specific intervention) and the underlying or root causes (i.e., nutrition-sensitive interventions) to improve the overall nutritional status of children, especially in LMICs [30,31]. These interventions should be disability-inclusive, and priority should be given to reporting/documentation of the effectiveness of different nutrition interventions on growth and nutrition of children with disabilities, including CP.\nWhile reviewing the limited available studies, we observed that a majority of the interventions were trialed or implemented in institution-based settings. Though interventions implemented in the institution-based settings have been proven to result in better outcomes in the past [6], our evidence suggests the majority of children with CP in LMICs lack access to any rehabilitation services for numerous reasons, including limited availability of services in their neighborhoods, transport difficulties, and financial constraints [12,32]. Furthermore, the severe shortage of trained health professionals, e.g., rehabilitation service providers and dietitians, limits the scope to implement and scale-up different institution-based interventions for children with CP in LMICs [13]. In such a context, community-based approaches are highly recommended and there is a lack of evidence on community-based nutrition interventions for children with CP in LMICs. Nevertheless, in one recent study from Bangladesh, community-based parent-led intervention was found to be highly successful in improving functional outcomes of children with CP [33]. Emphasis should be given to the capacity development of mid-level service providers on growth monitoring and promotion, early identification, different blanket interventions, and referral to prevent and treat malnutrition among children with CP in LMICs.\nIn addition to the settings, the sample sizes of the studies were relatively low. Although the included studies provided valuable insights on different nutritional interventions, the small sample size limits the strength and generalizability of the study findings. Moreover, most children with CP in the selected studies had severe functional motor limitations. These could be due to selection bias related to more severe institutional samples, as well as the more severe end of the clinical spectrum of CP in LMICs, likely exacerbated by delayed age of diagnosis and lack of access to rehabilitation services [12,32,34]. Studies also show that the likelihood of receiving intervention and rehabilitation is higher among those with severe CP (e.g., GMFCS level III–V) compared to milder forms (e.g., GMFCS I–II) [12]. However, it is also known that severe functional motor limitations (e.g., GMFCS level III–V) in children with CP are often accompanied by severe oromotor dysfunction, e.g., dysphagia [35]. It remains unclear whether the interventions for children with more severe motor limitations are also appropriate for children with CP who are GMFCS I–II.\nMost of the interventions identified in this review improved the nutritional status of participating children with CP. Importantly, nutrition-specific interventions (i.e., interventions that aim to intervene in or correct the immediate causes of malnutrition, such as inadequate diet, disease severity and caring practices) had comparatively better outcomes than other intervention strategies [20,22,24,30]. We observed that the interventions that included dietary modifications (only, or as a component of multiple approaches) had better effect on nutritional outcomes of children with CP. Studies where participants received surgical interventions (e.g., GTF) also showed a positive effect, whereas studies that provided behavioral intervention on feeding skills only did not show any significant change.\nTo the best of our knowledge, this is one of the first reviews conducted on nutrition interventions for children with CP in LMICs. Although during our search we identified several studies that aimed to improve the nutritional status of children with CP in HICs, our search revealed only a few reviews (including one systematic review) that summarized findings from different nutrition intervention studies among children with CP in HICs [6,36,37]. The data provided in our study, therefore, contribute important information to the existing knowledge gap. Nevertheless, this review has several limitations. We focused our search on two databases, so a risk remains that there may have been other publications available through other databases. However, our search revealed a large number of duplicates; therefore, we are confident that we have covered all or almost all of the studies published online. Second, we only searched for articles that had our key words in title/abstracts/author specified key words in the articles. This may have reduced the number of articles initially identified for title screening. Third, we did not assess the risk of bias and quality of evidence of the articles finally included in the review, which may have posed bias in the information reported. Finally, due to the differences in reporting format and data availability, we could only include a few common variables and could not report on some of the important characteristics of the participants. For the same reason, we had to rely on reporting the descriptive findings only.\n\n5. Conclusions:\nThe current review highlights the knowledge gap and lack of available evidence on nutrition interventions for children with CP in LMICs. Strong evidence is essential to identify and determine the best practices and nutritional management guidelines for children with CP, especially for optimal utilization of the limited available resources in LMICs. National and international stakeholders, therefore, should make this a priority for future research and services. Considering the high proportion of children with CP with undernutrition in LMICs, existing interventions and services for children with CP should integrate a nutrition component. The current limited evidence suggests that nutrition-specific programs have more of a positive effect on the growth of children with CP than other strategies. This should be taken under consideration when planning for any nutrition focus and other comprehensive services for children with CP in LMICs.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nMalnutrition is substantially higher among children with cerebral palsy (CP) in low- and middle-income countries (LMICs) when compared with the general population. Access to appropriate interventions is crucial for better management of malnutrition and nutritional outcomes of those children. We aimed to review the existing evidence on nutrition interventions for children with CP in LMICs.\n\nMethods:\nOnline databases, i.e., PubMed and Scopus, and Google Scholar were searched up to 10 January 2022, to identify peer-reviewed publications/evidence on LMIC focused nutritional management guidelines/interventions. Following title screening and abstract review, full articles that met the inclusion/exclusion criteria were retained for data charting. Information about the study characteristics, nutrition interventions, and their effectiveness were extracted. Descriptive data were reported.\n\nResults:\nEight articles published between 2008 and 2019 were included with data from a total of n = 252 children with CP (age range: 1 y 0 m-18 y 7 m, 42% female). Five studies followed experimental design; n = 6 were conducted in hospital/clinic/center-based settings. Four studies focused on parental/caregiver training; n = 2 studies had surgical interventions (i.e., gastrostomy) and n = 1 provided neurodevelopmental therapy feeding intervention. Dietary modification as an intervention (or component) was reported in n = 5 studies and had better effect on the nutritional outcomes of children with CP compared to interventions focused on feeding skills or other behavioral modifications. Surgical interventions improved nutritional outcomes in both studies; however, none documented any adverse consequences of the surgical interventions.\n\nConclusions:\nThere is a substantial knowledge gap on nutrition interventions for children with CP in LMICs. This hinders the development of best practice guidelines for the nutritional management of children with CP in those settings. Findings suggest interventions directly related to growth/feeding of children had a better outcome than behavioral interventions. This should be considered in planning of nutrition-focused intervention or comprehensive services for children with CP in LMICs."},"background_conclusion_text":{"kind":"string","value":"1. Introduction:\nUndernutrition is a major global public health challenge, and children with disability, such as cerebral palsy (CP), often suffer from undernutrition, especially in low- and middle-income countries (LMICs). The proportion of malnutrition is substantially higher among children with CP in LMICs compared to the general population [1,2,3,4,5]. Ensuring availability and accessibility to evidence-based nutrition interventions is essential to avert adverse outcomes of malnutrition among those vulnerable children [6].\nNutritional management of children with CP is complex as several interlinked factors interfere with their growth directly and indirectly [7]. While a few factors have been identified as common predictors of malnutrition (e.g., gross motor and oromotor function limitations) [1,8,9], the conceptual framework of malnutrition in children with CP is not yet clearly understood, especially in LMICs. To date, several clinical nutrition guidelines based on current evidence on surgical and non-surgical intervention outcomes have been published [6,10]. However, most of those studies were conducted in high-income countries (HICs) [6], whereas 85% of children with disabilities live in LMICs and the majority have no or limited access to any rehabilitation services [11,12]. With the extreme shortage of trained professionals including dietitians, in addition to the limited availability and accessibility to institutionalized intervention programs in LMICs [13], it is likely that not all the interventions found effective in HIC settings may be applicable in LMIC settings. In absence of optimal management, these children are at high risk of malnutrition, which can in turn impact their functional outcomes, quality of life, and survival [14,15,16].\nAny available evidence in the context of LMICs could guide resource mobilization, assist in understanding the need and cost-effectiveness as well as to identify interventions that may improve the nutritional status of children with CP in low-resource settings. This is important to establish a platform for developing and implementing best practice CP-specific nutrition intervention guidelines in LMICs. In this scoping review, we aimed to systematically map the existing evidence on nutritional interventions for children with CP living in LMIC settings. The following research questions were explored: (i) What is known about the available nutrition interventions for children with CP in LMICs? (ii) What are the outcomes of those interventions on the nutritional status of participating children with CP in LMICs?\n\n5. Conclusions:\nThe current review highlights the knowledge gap and lack of available evidence on nutrition interventions for children with CP in LMICs. Strong evidence is essential to identify and determine the best practices and nutritional management guidelines for children with CP, especially for optimal utilization of the limited available resources in LMICs. National and international stakeholders, therefore, should make this a priority for future research and services. Considering the high proportion of children with CP with undernutrition in LMICs, existing interventions and services for children with CP should integrate a nutrition component. The current limited evidence suggests that nutrition-specific programs have more of a positive effect on the growth of children with CP than other strategies. This should be taken under consideration when planning for any nutrition focus and other comprehensive services for children with CP in LMICs."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nMalnutrition is substantially higher among children with cerebral palsy (CP) in low- and middle-income countries (LMICs) when compared with the general population. Access to appropriate interventions is crucial for better management of malnutrition and nutritional outcomes of those children. We aimed to review the existing evidence on nutrition interventions for children with CP in LMICs.\n\nMethods:\nOnline databases, i.e., PubMed and Scopus, and Google Scholar were searched up to 10 January 2022, to identify peer-reviewed publications/evidence on LMIC focused nutritional management guidelines/interventions. Following title screening and abstract review, full articles that met the inclusion/exclusion criteria were retained for data charting. Information about the study characteristics, nutrition interventions, and their effectiveness were extracted. Descriptive data were reported.\n\nResults:\nEight articles published between 2008 and 2019 were included with data from a total of n = 252 children with CP (age range: 1 y 0 m-18 y 7 m, 42% female). Five studies followed experimental design; n = 6 were conducted in hospital/clinic/center-based settings. Four studies focused on parental/caregiver training; n = 2 studies had surgical interventions (i.e., gastrostomy) and n = 1 provided neurodevelopmental therapy feeding intervention. Dietary modification as an intervention (or component) was reported in n = 5 studies and had better effect on the nutritional outcomes of children with CP compared to interventions focused on feeding skills or other behavioral modifications. Surgical interventions improved nutritional outcomes in both studies; however, none documented any adverse consequences of the surgical interventions.\n\nConclusions:\nThere is a substantial knowledge gap on nutrition interventions for children with CP in LMICs. This hinders the development of best practice guidelines for the nutritional management of children with CP in those settings. Findings suggest interventions directly related to growth/feeding of children had a better outcome than behavioral interventions. This should be considered in planning of nutrition-focused intervention or comprehensive services for children with CP in LMICs."},"whole_article_text_length":{"kind":"number","value":10720,"string":"10,720"},"whole_article_abstract_length":{"kind":"number","value":385,"string":"385"},"other_sections_lengths":{"kind":"list like","value":[1506,70,229,169,95,135,33,1134,89,160,109,297,287,205],"string":"[\n 1506,\n 70,\n 229,\n 169,\n 95,\n 135,\n 33,\n 1134,\n 89,\n 160,\n 109,\n 297,\n 287,\n 205\n]","isConversational":false},"num_sections":{"kind":"number","value":19,"string":"19"},"most_frequent_words":{"kind":"list like","value":["study","studies","children","20","26","cp","22","23","24","21"],"string":"[\n \"study\",\n \"studies\",\n \"children\",\n \"20\",\n \"26\",\n \"cp\",\n \"22\",\n \"23\",\n \"24\",\n \"21\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["nutritional outcomes children","malnutrition vulnerable children","framework malnutrition 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We aimed at pursuing a clinical and pathological review of patients undergoing post chemotherapy retroperitoneal lymph node dissection (PC-RPLND) in order to identify variables that may help predict necrosis in the retroperitoneum."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"We performed a retrospective analysis of all patients who underwent PC-RPLND at the University Hospital of the University of São Paulo and Cancer Institute of Sao Paulo between January 2005 and September 2011. Clinical and pathological data were obtained and consisted basically of: measures of retroperitoneal masses, histology of the orchiectomy specimen, serum tumor marker and retroperitoneal nodal size before and after chemotherapy."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"We gathered a total of 32 patients with a mean age of 29.7; pathological analysis in our series demonstrated that 15 (47%) had necrosis in residual retroperitoneal masses, 15 had teratoma (47%) and 2 (6.4%) had viable germ cell tumors (GCT). The mean size of the retroperitoneal mass was 4.94 cm in our sample, without a difference between the groups (P = 0.176). From all studied variables, relative changes in retroperitoneal lymph node size (P = 0.04), the absence of teratoma in the orchiectomy specimen (P = 0.03) and the presence of choriocarcinoma in the testicular analysis after orchiectomy (P = 0.03) were statistically significant predictors of the presence of necrosis. A reduction level of 35% was therefore suggested to be the best cutoff for predicting the absence of tumor in the retroperitoneum with a sensitivity of 73.3% and specificity of 82.4%."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Even though retroperitoneal lymph node dissection remains the gold standard for patients with residual masses, those without teratoma in the primary tumor and a shrinkage of 35% or more in retroperitoneal mass have a considerably smaller chance of having viable GCT or teratoma in the retroperitoneum and a surveillance program could be considered."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adolescent","Adult","Humans","Lymph Node Excision","Male","Middle Aged","Necrosis","Neoplasms, Germ Cell and Embryonal","Retroperitoneal Neoplasms","Retrospective Studies","Teratoma","Testicular Neoplasms"],"string":"[\n \"Adolescent\",\n \"Adult\",\n \"Humans\",\n \"Lymph Node Excision\",\n \"Male\",\n \"Middle Aged\",\n \"Necrosis\",\n \"Neoplasms, Germ Cell and Embryonal\",\n \"Retroperitoneal Neoplasms\",\n \"Retrospective Studies\",\n \"Teratoma\",\n \"Testicular Neoplasms\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"3502267"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Testicular cancer has become one of the most curable solid neoplasms and serves as a paradigm for the multimodal treatment of malignancies. The appropriate integration of chemotherapy, retroperitoneal lymph node dissection (RPLND) and observation for the management of testis cancer has resulted in overall survival rates greater than 90% [1,2].\nRPLND plays an important role in the management of patients with testicular germ cell tumors (GCTs), especially in those with residual masses after chemotherapy [3]. To date, series have demonstrated that pathological analysis of these masses reveals findings of necrotic debris or fibrosis in 40% to 50% of patients, teratoma in 35% to 40% and viable malignant cells in 10% to 15% of patients [4].\nEven though early recognition and resection of teratoma in the retroperitoneum after chemotherapy have been accompanied by excellent prognosis, once it was initially thought to represent a benign course when present in the retroperitoneal space, but the untreated disease may have a lethal potential due to progressive local growth or malignant transformation, not to mention its classical unresponsiveness to conventional cisplatin-based chemotherapy regimens [3-6]. As for residual viable GCT, the consequence of its incomplete resection is certain disease progression [7]. Therefore, a more aggressive approach should always be considered when treating patients with teratoma or viable GCT in the retroperitoneum [7,8].\nThus, the appropriate approach to residual masses following chemotherapy remains a controversial issue, since the literature has shown that as many as half of resected masses are basically composed of necrosis or fibrotic tissue and any sort of adjuvant therapy could be waived [9]. In order to avoid a great number of apparently unnecessary post-chemotherapy RPLND (PC-RPLND), many studies have tried to develop algorithms to predict the presence of necrosis in the retroperitoneum. However, currently predictive models and imaging modalities cannot reliably predict the pathological finding of necrosis/fibrosis at PC-RPLND [9].\nSome authors have established that patients without teratoma in the primary tumor and a shrinkage of 90% or more in retroperitoneal mass had little chance of having viable GCT or teratoma and could be safely put under a surveillance program with periodic imaging scans [10]. However, prospective analyses have demonstrated that approximately 30% of patients will harbor teratoma or viable malignancy even with normal post-chemotherapy computed tomography (CT) results and no teratoma in the primary tumor [9].\nThe purpose of this work is to pursue a clinical and pathological review of patients undergoing PC-RPLND at a reference university oncology center in Brazil, in order to identify variables that may help predict the histological finding of necrosis in the retroperitoneum and perhaps establish a differentiated surveillance protocol."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":"We performed a retrospective analysis of all patients from our computerized database who underwent PC-RPLND at our service between January 2005 and May 2011. Patients were operated on after having undergone three to four cycles of primary chemotherapy with bleomycin, etoposide and cisplatin.\nClinical and pathological data were obtained and consisted basically of measures of retroperitoneal masses, serum tumor markers, histology of the orchiectomy specimen, tumor marker values and retroperitoneal nodal size before and after chemotherapy.\nThe presence of either immature or mature teratoma in the resected specimen, as well as teratoma with malignant transformation, was considered part of the same group. Choriocarcinoma, yolk sac tumors, embryonal carcinoma and seminoma were considered viable GCTs. All histological findings were submitted to quantitative and qualitative analysis.\nPost-chemotherapy alpha-fetoprotein (AFP) and lactate dehydrogenase (LDH) levels were registered as a continuous variable while human chorionic gonadotropin (hCG) levels were considered a categorical variable ranging from undetectable to detectable when serum concentrations were greater than 3 mIU.\nRetroperitoneal nodal size before and after chemotherapy was determined by the longest transverse diameter of the largest mass on CT imaging. Relative change in nodal size before and after chemotherapy was calculated by dividing post-chemotherapy nodal size by pre-chemotherapy nodal size and was analyzed as a continuous variable.\nMultiple variables were analyzed independently in order to establish any predictive value for finding necrotic tissue in the retroperitoneum. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS, version 12.0, Chicago, IL, USA), applying the Mann–Whitney U test for non-parametric variables and the Fisher's exact test for categorical variables, with the level of significance set at P < 0.05.\nA cut-off level for predicting necrosis at retroperitoneal residual mass analysis was sought by constructing a receiver operating characteristic (ROC) curve of all significant variables, which were generated using graphical visualization in our statistical software. This study was carried-out in accordance with the Ethics Committee regulations."},"results_title":{"kind":"null"},"results_text":{"kind":"null"},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"Even though RPLND remains the gold standard for patients with residual masses, those without teratoma in the primary tumor and a shrinkage of 35% or more in retroperitoneal mass have a considerably smaller chance of having viable GCTs or teratoma in the retroperitoneum."},"other_sections_titles":{"kind":"list like","value":["Background","Results and discussion","Abbreviations","Competing interests","Authors’ contributions"],"string":"[\n \"Background\",\n \"Results and discussion\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Testicular cancer has become one of the most curable solid neoplasms and serves as a paradigm for the multimodal treatment of malignancies. The appropriate integration of chemotherapy, retroperitoneal lymph node dissection (RPLND) and observation for the management of testis cancer has resulted in overall survival rates greater than 90% [1,2].\nRPLND plays an important role in the management of patients with testicular germ cell tumors (GCTs), especially in those with residual masses after chemotherapy [3]. To date, series have demonstrated that pathological analysis of these masses reveals findings of necrotic debris or fibrosis in 40% to 50% of patients, teratoma in 35% to 40% and viable malignant cells in 10% to 15% of patients [4].\nEven though early recognition and resection of teratoma in the retroperitoneum after chemotherapy have been accompanied by excellent prognosis, once it was initially thought to represent a benign course when present in the retroperitoneal space, but the untreated disease may have a lethal potential due to progressive local growth or malignant transformation, not to mention its classical unresponsiveness to conventional cisplatin-based chemotherapy regimens [3-6]. As for residual viable GCT, the consequence of its incomplete resection is certain disease progression [7]. Therefore, a more aggressive approach should always be considered when treating patients with teratoma or viable GCT in the retroperitoneum [7,8].\nThus, the appropriate approach to residual masses following chemotherapy remains a controversial issue, since the literature has shown that as many as half of resected masses are basically composed of necrosis or fibrotic tissue and any sort of adjuvant therapy could be waived [9]. In order to avoid a great number of apparently unnecessary post-chemotherapy RPLND (PC-RPLND), many studies have tried to develop algorithms to predict the presence of necrosis in the retroperitoneum. However, currently predictive models and imaging modalities cannot reliably predict the pathological finding of necrosis/fibrosis at PC-RPLND [9].\nSome authors have established that patients without teratoma in the primary tumor and a shrinkage of 90% or more in retroperitoneal mass had little chance of having viable GCT or teratoma and could be safely put under a surveillance program with periodic imaging scans [10]. However, prospective analyses have demonstrated that approximately 30% of patients will harbor teratoma or viable malignancy even with normal post-chemotherapy computed tomography (CT) results and no teratoma in the primary tumor [9].\nThe purpose of this work is to pursue a clinical and pathological review of patients undergoing PC-RPLND at a reference university oncology center in Brazil, in order to identify variables that may help predict the histological finding of necrosis in the retroperitoneum and perhaps establish a differentiated surveillance protocol.","We gathered information on a total of 32 patients, who were 18- to 49-years old (mean age 30.5) and harbored seminomatous, nonseminomatous or mixed tumors in the testis tissue in different clinical stages. Three patients (9.4%) had seminoma, five (16%) had pure nonseminoma (choriocarcinoma, yolk sac tumors, embryonal carcinoma, teratoma, teratocarcinoma) and 24 (75%) harbored mixed nonseminomatous GCT (NSGCT). None of the patients with seminoma had normal AFP levels, indicating that these patients may have had tumors with similar biological behavior to those with NSGCT.\nAt diagnosis, seven (22%) patients were classified as having a stage I disease, while 21 (66%) were stage II and four (12.5%) were stage III. Clinical stage I patients were individuals with longer follow-up, who presented with retroperitoneal disease nonresponsive to chemotherapy and ultimately underwent PC-RPLND.\nPathological analysis in our series demonstrated that 15 (47%) patients had necrosis in residual retroperitoneal masses, 15 had teratoma (47%) and two (6%) had viable GCT: one seminoma and one yolk sac tumor. For statistical reasons and in alignment with the aims of this study, we divided those patients into two groups, assembling patients with viable GCT and teratoma and analyzing them as one group. Mean size of the retroperitoneal mass was 4.94 cm in our sample, 3.79 cm in the group of necrosis and 5.96 cm in the group of teratoma and viable GCT (P = 0.176). There was also no difference between groups regarding stratification of nodal size, as shown in Table 1.\n\nDistribution according to nodal size in men undergoing PC-RPLND\nData are shown as number (%). GCT, germ cell tumor; PC-RPLND, post-chemotherapy retroperitoneal lymph node dissection.\nPrimary tumor histology revealed embryonal cell carcinoma in 56%, seminomatous elements in 16%, yolk sac tumor in 19% and teratomatous elements in 56% of the patients. Of the 18 patients with teratomatous elements in the primary tumor, 13 (72%) had teratoma in the retroperitoneum at PC-RPLND. Even in the absence of teratoma in the primary tumor, teratoma was present in the retroperitoneum in five patients (16%).\nWhen comparing pathological analysis of primary tumor specimens, we found a statistical difference when comparing the prevalence of teratoma and choriocarcinoma. Other findings, such as the presence of seminoma, yolk sac tumor, embryonal carcinoma, endodermal sinus tumor lymph vascular invasion (LVI), rete testis invasion and spermatic invasion were similar between groups.\nUnivariate analysis of quantitative components at orchiectomy histology, retroperitoneal node size with its relative measures and serum markers are shown in Table 2.\n\nUnivariate analysis predicting necrosis at PC-RPLND\n** unable to calculate. Data expressed as mean ± standard deviation. AFP, alpha-fetoprotein; GCT, germ cell tumor; hCG, human chorionic gonadotropin; LDH, lactate dehydrogenase; PC-RPLND, post-chemotherapy retroperitoneal lymph node dissection; RP, retroperitoneal.\nEven though the presence of teratoma in the primary tumor was an important negative predictor for the finding of necrosis, the quantitative analysis has proven to be statistically irrelevant.\nWhile relative reduction in mass size after chemotherapy has been shown to be an important predictor of necrosis, even when considering patients not responding to chemotherapy, absolute reduction and enlargement of the residual mass did not show a significant difference between groups.\nThere was a statistical difference in AFP levels between groups; however, when comparing relative changes in AFP levels after chemotherapy, no difference was found. Comparison of LDH levels and their relative changes was nonsignificant. We were unable to compare hCG levels between groups because we only had two patients whose hCG levels were not undetectable.\nOf all the studied variables, relative changes in retroperitoneal lymph node size (P =0.04), the absence of teratoma in the orchiectomy specimen (P =0.03) and the presence of choriocarcinoma in the testicular analysis after orchiectomy (P =0.03) were statistically significant predictors of the presence of necrosis in the retroperitoneum.\nROC curves were built for variables that were independent predictors of necrosis at PC-RPLND. Even though the size of the retroperitoneal mass after chemotherapy showed no statistical difference, we also built a curve for it. The variable relative reduction after chemotherapy was the only one with predictive value according to the Youlden index. An area under the curve (AUC) of 0.710 was obtained and a reduction level of 35% was therefore suggested to be the best cutoff for predicting the absence of tumor in the retroperitoneum with a sensitivity of 73% and a specificity of 82%. ROC curves of isolated size of retroperitoneal mass, absence of teratoma and presence of choriocarcinoma did not indicate significant findings.\nIn our institution, relative change in retroperitoneal node size, absence of teratoma in the orchiectomy specimen and the presence of choriocarcinoma in the testicular analysis after orchiectomy were statistically significant predictors of the presence of necrosis in the retroperitoneum and a surveillance program could be considered, given the uncertainty in predicting the histology of residual masses after chemotherapy in patients with metastatic testicular tumors.\nAnalysis of residual retroperitoneal masses after chemotherapy is being increasingly regarded as a fundamental issue, not only for orienting adjuvant therapies, but also because it has prognostic implications [11]. Outcome assessments in patients with NSGCT have demonstrated that incomplete resection of residual retroperitoneal masses, the size of residual retroperitoneal masses and the finding of teratoma and viable GCT at RPLND independently predict disease progression and relapse [11,12].\nThe size of residual retroperitoneal masses after chemotherapy has traditionally been considered when choosing the subsequent treatment modality [9]. Studies have shown that residual masses smaller than 2 cm are considered one of the most significant predictors for finding necrosis at PC-RPLND at logistic regression [4]. Furthermore, a number of investigators continue to base the decision to perform PC-RPLND on residual mass size alone, obviating PC-RPLND in patients with residual masses of 1 cm or less [13].\nHowever, recent studies have shown that after chemotherapy a third of retroperitoneal masses of <2 cm harbored either teratoma or viable GCT [14]. Even though we found a trend of having only necrosis in the retroperitoneum for masses <2 cm (P = 0.09), in the present study residual mass size alone has not appeared to be a good predictor of necrosis, with nonsignificant size differences between groups and an underrated AUC in ROC curves. In addition, one case of teratoma in a 1 cm mass was registered in our sample. Therefore, we advocate that the decision of not operating on patients cannot be based on mass size alone due to the lack of consensus in the literature [9,10,13,14].\nTraditional series report necrotic debris or fibrosis in 40% to 50%, teratoma in 35% to 40% and viable malignant cells in 10% to 15% of patients [3]. In more recent analyses, the incidence of residual microscopic teratoma in the retroperitoneum has decreased to approximately 20% to 25%, with an increase of necrotic tissue findings of up to 60% and stable rates of viable GCT [15]. It has been reported that an increase in the proportion of necrosis is generally attributed to stage migration and the use of more effective chemotherapy regimens, especially in patients achieving a complete response to chemotherapy [15].\nIn our series we found a distribution pattern similar to former studies, with almost 50% of patients harboring teratoma in the retroperitoneal histology at RPLND, which may suggest that our chemotherapy regimens have been having inferior rates of complete responders or simply because our group is composed of patients in more advanced stages.\nTeratoma-negative primary tumor and volumetric regression of at least 90% after chemotherapy have been described as being highly predictive of harboring necrosis only at PC-RPLND [9,16]. Other series include normal serum tumor markers, and node size < 2 cm, the presence of yolk sac tumor or embryonal carcinoma on primary tumor and lymph vascular invasion, among others [9,10,15-17]. On the other hand, some authors have been unable to identify variables to be highly predictive of harboring necrosis only at PC-RPLND [15,18]. Nevertheless, currently predictive models fail to accurately predict necrosis in the retroperitoneum, since almost 30% of patients will harbor teratoma or viable malignancy even with normal post-chemotherapy CT and no teratoma in the primary tumor [9]. A study carried out at the Memorial Sloan-Kettering Cancer Center revealed that 26% of patients had teratoma or viable GCT, even those with radiographically normal retroperitoneum who underwent PC-RPLND [9].\nThe relatively small number of patients when compared to other institutional retrospective studies is a limitation of the present study; however, other studies with similar sample sizes have also come to significant findings.\nIn the present study, relative changes in retroperitoneal node size stood out as the single best predictor of the presence of necrosis in the retroperitoneum, which is in accordance with actual logistic regression models [9,10]. While other series suggest 90% as a shrinkage cutoff value to reliably predict necrosis, our quantitative analysis have demonstrated a cutoff level of 35% with approximate sensitivity and specificity [9].\nThe absence of teratoma has also been demonstrated to be significant in qualitative analysis, but not in quantitative analysis. The presence of choriocarcinoma in the testicular histology predicted necrosis in the retroperitoneum, despite only four (12.5%) patients who had such a finding. This association is an interesting finding that has not been previously reported. We reviewed the literature and no possible explanations were found. We believe further investigation is necessary to confirm this finding, since the number of patients in our sample is relatively small.","AFP: Alphs-fetoprotein; AUC: Area under the curve; CT: Computed tomography; GCT: Germ cell tumor; hCG: Human chorionic gonadotropin; LDH: Lactate dehydrogenase; LVI: Lymph vascular invasion; NSGCT: Nonseminomatous germ cell tumor; PC-RPLND: Post chemotherapy retroperitoneal lymph node dissection; ROC: Receiver operating characteristic; RPLND: Retroperitoneal lymph node dissection.","The authors declare that they have no competing interests.","EPM and DKA gathered and compiled data and have been involved in drafting the manuscript. AJN and STL participated in the design of the study and performed the statistical analysis. ACS and MS revised the manuscript critically for important intellectual content. MS and MFD have given final approval of the version to be published. All authors read and approved the final manuscript."],"string":"[\n \"Testicular cancer has become one of the most curable solid neoplasms and serves as a paradigm for the multimodal treatment of malignancies. The appropriate integration of chemotherapy, retroperitoneal lymph node dissection (RPLND) and observation for the management of testis cancer has resulted in overall survival rates greater than 90% [1,2].\\nRPLND plays an important role in the management of patients with testicular germ cell tumors (GCTs), especially in those with residual masses after chemotherapy [3]. To date, series have demonstrated that pathological analysis of these masses reveals findings of necrotic debris or fibrosis in 40% to 50% of patients, teratoma in 35% to 40% and viable malignant cells in 10% to 15% of patients [4].\\nEven though early recognition and resection of teratoma in the retroperitoneum after chemotherapy have been accompanied by excellent prognosis, once it was initially thought to represent a benign course when present in the retroperitoneal space, but the untreated disease may have a lethal potential due to progressive local growth or malignant transformation, not to mention its classical unresponsiveness to conventional cisplatin-based chemotherapy regimens [3-6]. As for residual viable GCT, the consequence of its incomplete resection is certain disease progression [7]. Therefore, a more aggressive approach should always be considered when treating patients with teratoma or viable GCT in the retroperitoneum [7,8].\\nThus, the appropriate approach to residual masses following chemotherapy remains a controversial issue, since the literature has shown that as many as half of resected masses are basically composed of necrosis or fibrotic tissue and any sort of adjuvant therapy could be waived [9]. In order to avoid a great number of apparently unnecessary post-chemotherapy RPLND (PC-RPLND), many studies have tried to develop algorithms to predict the presence of necrosis in the retroperitoneum. However, currently predictive models and imaging modalities cannot reliably predict the pathological finding of necrosis/fibrosis at PC-RPLND [9].\\nSome authors have established that patients without teratoma in the primary tumor and a shrinkage of 90% or more in retroperitoneal mass had little chance of having viable GCT or teratoma and could be safely put under a surveillance program with periodic imaging scans [10]. However, prospective analyses have demonstrated that approximately 30% of patients will harbor teratoma or viable malignancy even with normal post-chemotherapy computed tomography (CT) results and no teratoma in the primary tumor [9].\\nThe purpose of this work is to pursue a clinical and pathological review of patients undergoing PC-RPLND at a reference university oncology center in Brazil, in order to identify variables that may help predict the histological finding of necrosis in the retroperitoneum and perhaps establish a differentiated surveillance protocol.\",\n \"We gathered information on a total of 32 patients, who were 18- to 49-years old (mean age 30.5) and harbored seminomatous, nonseminomatous or mixed tumors in the testis tissue in different clinical stages. Three patients (9.4%) had seminoma, five (16%) had pure nonseminoma (choriocarcinoma, yolk sac tumors, embryonal carcinoma, teratoma, teratocarcinoma) and 24 (75%) harbored mixed nonseminomatous GCT (NSGCT). None of the patients with seminoma had normal AFP levels, indicating that these patients may have had tumors with similar biological behavior to those with NSGCT.\\nAt diagnosis, seven (22%) patients were classified as having a stage I disease, while 21 (66%) were stage II and four (12.5%) were stage III. Clinical stage I patients were individuals with longer follow-up, who presented with retroperitoneal disease nonresponsive to chemotherapy and ultimately underwent PC-RPLND.\\nPathological analysis in our series demonstrated that 15 (47%) patients had necrosis in residual retroperitoneal masses, 15 had teratoma (47%) and two (6%) had viable GCT: one seminoma and one yolk sac tumor. For statistical reasons and in alignment with the aims of this study, we divided those patients into two groups, assembling patients with viable GCT and teratoma and analyzing them as one group. Mean size of the retroperitoneal mass was 4.94 cm in our sample, 3.79 cm in the group of necrosis and 5.96 cm in the group of teratoma and viable GCT (P = 0.176). There was also no difference between groups regarding stratification of nodal size, as shown in Table 1.\\n\\nDistribution according to nodal size in men undergoing PC-RPLND\\nData are shown as number (%). GCT, germ cell tumor; PC-RPLND, post-chemotherapy retroperitoneal lymph node dissection.\\nPrimary tumor histology revealed embryonal cell carcinoma in 56%, seminomatous elements in 16%, yolk sac tumor in 19% and teratomatous elements in 56% of the patients. Of the 18 patients with teratomatous elements in the primary tumor, 13 (72%) had teratoma in the retroperitoneum at PC-RPLND. Even in the absence of teratoma in the primary tumor, teratoma was present in the retroperitoneum in five patients (16%).\\nWhen comparing pathological analysis of primary tumor specimens, we found a statistical difference when comparing the prevalence of teratoma and choriocarcinoma. Other findings, such as the presence of seminoma, yolk sac tumor, embryonal carcinoma, endodermal sinus tumor lymph vascular invasion (LVI), rete testis invasion and spermatic invasion were similar between groups.\\nUnivariate analysis of quantitative components at orchiectomy histology, retroperitoneal node size with its relative measures and serum markers are shown in Table 2.\\n\\nUnivariate analysis predicting necrosis at PC-RPLND\\n** unable to calculate. Data expressed as mean ± standard deviation. AFP, alpha-fetoprotein; GCT, germ cell tumor; hCG, human chorionic gonadotropin; LDH, lactate dehydrogenase; PC-RPLND, post-chemotherapy retroperitoneal lymph node dissection; RP, retroperitoneal.\\nEven though the presence of teratoma in the primary tumor was an important negative predictor for the finding of necrosis, the quantitative analysis has proven to be statistically irrelevant.\\nWhile relative reduction in mass size after chemotherapy has been shown to be an important predictor of necrosis, even when considering patients not responding to chemotherapy, absolute reduction and enlargement of the residual mass did not show a significant difference between groups.\\nThere was a statistical difference in AFP levels between groups; however, when comparing relative changes in AFP levels after chemotherapy, no difference was found. Comparison of LDH levels and their relative changes was nonsignificant. We were unable to compare hCG levels between groups because we only had two patients whose hCG levels were not undetectable.\\nOf all the studied variables, relative changes in retroperitoneal lymph node size (P =0.04), the absence of teratoma in the orchiectomy specimen (P =0.03) and the presence of choriocarcinoma in the testicular analysis after orchiectomy (P =0.03) were statistically significant predictors of the presence of necrosis in the retroperitoneum.\\nROC curves were built for variables that were independent predictors of necrosis at PC-RPLND. Even though the size of the retroperitoneal mass after chemotherapy showed no statistical difference, we also built a curve for it. The variable relative reduction after chemotherapy was the only one with predictive value according to the Youlden index. An area under the curve (AUC) of 0.710 was obtained and a reduction level of 35% was therefore suggested to be the best cutoff for predicting the absence of tumor in the retroperitoneum with a sensitivity of 73% and a specificity of 82%. ROC curves of isolated size of retroperitoneal mass, absence of teratoma and presence of choriocarcinoma did not indicate significant findings.\\nIn our institution, relative change in retroperitoneal node size, absence of teratoma in the orchiectomy specimen and the presence of choriocarcinoma in the testicular analysis after orchiectomy were statistically significant predictors of the presence of necrosis in the retroperitoneum and a surveillance program could be considered, given the uncertainty in predicting the histology of residual masses after chemotherapy in patients with metastatic testicular tumors.\\nAnalysis of residual retroperitoneal masses after chemotherapy is being increasingly regarded as a fundamental issue, not only for orienting adjuvant therapies, but also because it has prognostic implications [11]. Outcome assessments in patients with NSGCT have demonstrated that incomplete resection of residual retroperitoneal masses, the size of residual retroperitoneal masses and the finding of teratoma and viable GCT at RPLND independently predict disease progression and relapse [11,12].\\nThe size of residual retroperitoneal masses after chemotherapy has traditionally been considered when choosing the subsequent treatment modality [9]. Studies have shown that residual masses smaller than 2 cm are considered one of the most significant predictors for finding necrosis at PC-RPLND at logistic regression [4]. Furthermore, a number of investigators continue to base the decision to perform PC-RPLND on residual mass size alone, obviating PC-RPLND in patients with residual masses of 1 cm or less [13].\\nHowever, recent studies have shown that after chemotherapy a third of retroperitoneal masses of <2 cm harbored either teratoma or viable GCT [14]. Even though we found a trend of having only necrosis in the retroperitoneum for masses <2 cm (P = 0.09), in the present study residual mass size alone has not appeared to be a good predictor of necrosis, with nonsignificant size differences between groups and an underrated AUC in ROC curves. In addition, one case of teratoma in a 1 cm mass was registered in our sample. Therefore, we advocate that the decision of not operating on patients cannot be based on mass size alone due to the lack of consensus in the literature [9,10,13,14].\\nTraditional series report necrotic debris or fibrosis in 40% to 50%, teratoma in 35% to 40% and viable malignant cells in 10% to 15% of patients [3]. In more recent analyses, the incidence of residual microscopic teratoma in the retroperitoneum has decreased to approximately 20% to 25%, with an increase of necrotic tissue findings of up to 60% and stable rates of viable GCT [15]. It has been reported that an increase in the proportion of necrosis is generally attributed to stage migration and the use of more effective chemotherapy regimens, especially in patients achieving a complete response to chemotherapy [15].\\nIn our series we found a distribution pattern similar to former studies, with almost 50% of patients harboring teratoma in the retroperitoneal histology at RPLND, which may suggest that our chemotherapy regimens have been having inferior rates of complete responders or simply because our group is composed of patients in more advanced stages.\\nTeratoma-negative primary tumor and volumetric regression of at least 90% after chemotherapy have been described as being highly predictive of harboring necrosis only at PC-RPLND [9,16]. Other series include normal serum tumor markers, and node size < 2 cm, the presence of yolk sac tumor or embryonal carcinoma on primary tumor and lymph vascular invasion, among others [9,10,15-17]. On the other hand, some authors have been unable to identify variables to be highly predictive of harboring necrosis only at PC-RPLND [15,18]. Nevertheless, currently predictive models fail to accurately predict necrosis in the retroperitoneum, since almost 30% of patients will harbor teratoma or viable malignancy even with normal post-chemotherapy CT and no teratoma in the primary tumor [9]. A study carried out at the Memorial Sloan-Kettering Cancer Center revealed that 26% of patients had teratoma or viable GCT, even those with radiographically normal retroperitoneum who underwent PC-RPLND [9].\\nThe relatively small number of patients when compared to other institutional retrospective studies is a limitation of the present study; however, other studies with similar sample sizes have also come to significant findings.\\nIn the present study, relative changes in retroperitoneal node size stood out as the single best predictor of the presence of necrosis in the retroperitoneum, which is in accordance with actual logistic regression models [9,10]. While other series suggest 90% as a shrinkage cutoff value to reliably predict necrosis, our quantitative analysis have demonstrated a cutoff level of 35% with approximate sensitivity and specificity [9].\\nThe absence of teratoma has also been demonstrated to be significant in qualitative analysis, but not in quantitative analysis. The presence of choriocarcinoma in the testicular histology predicted necrosis in the retroperitoneum, despite only four (12.5%) patients who had such a finding. This association is an interesting finding that has not been previously reported. We reviewed the literature and no possible explanations were found. We believe further investigation is necessary to confirm this finding, since the number of patients in our sample is relatively small.\",\n \"AFP: Alphs-fetoprotein; AUC: Area under the curve; CT: Computed tomography; GCT: Germ cell tumor; hCG: Human chorionic gonadotropin; LDH: Lactate dehydrogenase; LVI: Lymph vascular invasion; NSGCT: Nonseminomatous germ cell tumor; PC-RPLND: Post chemotherapy retroperitoneal lymph node dissection; ROC: Receiver operating characteristic; RPLND: Retroperitoneal lymph node dissection.\",\n \"The authors declare that they have no competing interests.\",\n \"EPM and DKA gathered and compiled data and have been involved in drafting the manuscript. AJN and STL participated in the design of the study and performed the statistical analysis. ACS and MS revised the manuscript critically for important intellectual content. MS and MFD have given final approval of the version to be published. All authors read and approved the final manuscript.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Results and discussion","Conclusions","Abbreviations","Competing interests","Authors’ contributions"],"string":"[\n \"Background\",\n \"Methods\",\n \"Results and discussion\",\n \"Conclusions\",\n \"Abbreviations\",\n \"Competing interests\",\n \"Authors’ contributions\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Testicular cancer has become one of the most curable solid neoplasms and serves as a paradigm for the multimodal treatment of malignancies. The appropriate integration of chemotherapy, retroperitoneal lymph node dissection (RPLND) and observation for the management of testis cancer has resulted in overall survival rates greater than 90% [1,2].\nRPLND plays an important role in the management of patients with testicular germ cell tumors (GCTs), especially in those with residual masses after chemotherapy [3]. To date, series have demonstrated that pathological analysis of these masses reveals findings of necrotic debris or fibrosis in 40% to 50% of patients, teratoma in 35% to 40% and viable malignant cells in 10% to 15% of patients [4].\nEven though early recognition and resection of teratoma in the retroperitoneum after chemotherapy have been accompanied by excellent prognosis, once it was initially thought to represent a benign course when present in the retroperitoneal space, but the untreated disease may have a lethal potential due to progressive local growth or malignant transformation, not to mention its classical unresponsiveness to conventional cisplatin-based chemotherapy regimens [3-6]. As for residual viable GCT, the consequence of its incomplete resection is certain disease progression [7]. Therefore, a more aggressive approach should always be considered when treating patients with teratoma or viable GCT in the retroperitoneum [7,8].\nThus, the appropriate approach to residual masses following chemotherapy remains a controversial issue, since the literature has shown that as many as half of resected masses are basically composed of necrosis or fibrotic tissue and any sort of adjuvant therapy could be waived [9]. In order to avoid a great number of apparently unnecessary post-chemotherapy RPLND (PC-RPLND), many studies have tried to develop algorithms to predict the presence of necrosis in the retroperitoneum. However, currently predictive models and imaging modalities cannot reliably predict the pathological finding of necrosis/fibrosis at PC-RPLND [9].\nSome authors have established that patients without teratoma in the primary tumor and a shrinkage of 90% or more in retroperitoneal mass had little chance of having viable GCT or teratoma and could be safely put under a surveillance program with periodic imaging scans [10]. However, prospective analyses have demonstrated that approximately 30% of patients will harbor teratoma or viable malignancy even with normal post-chemotherapy computed tomography (CT) results and no teratoma in the primary tumor [9].\nThe purpose of this work is to pursue a clinical and pathological review of patients undergoing PC-RPLND at a reference university oncology center in Brazil, in order to identify variables that may help predict the histological finding of necrosis in the retroperitoneum and perhaps establish a differentiated surveillance protocol.","We performed a retrospective analysis of all patients from our computerized database who underwent PC-RPLND at our service between January 2005 and May 2011. Patients were operated on after having undergone three to four cycles of primary chemotherapy with bleomycin, etoposide and cisplatin.\nClinical and pathological data were obtained and consisted basically of measures of retroperitoneal masses, serum tumor markers, histology of the orchiectomy specimen, tumor marker values and retroperitoneal nodal size before and after chemotherapy.\nThe presence of either immature or mature teratoma in the resected specimen, as well as teratoma with malignant transformation, was considered part of the same group. Choriocarcinoma, yolk sac tumors, embryonal carcinoma and seminoma were considered viable GCTs. All histological findings were submitted to quantitative and qualitative analysis.\nPost-chemotherapy alpha-fetoprotein (AFP) and lactate dehydrogenase (LDH) levels were registered as a continuous variable while human chorionic gonadotropin (hCG) levels were considered a categorical variable ranging from undetectable to detectable when serum concentrations were greater than 3 mIU.\nRetroperitoneal nodal size before and after chemotherapy was determined by the longest transverse diameter of the largest mass on CT imaging. Relative change in nodal size before and after chemotherapy was calculated by dividing post-chemotherapy nodal size by pre-chemotherapy nodal size and was analyzed as a continuous variable.\nMultiple variables were analyzed independently in order to establish any predictive value for finding necrotic tissue in the retroperitoneum. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS, version 12.0, Chicago, IL, USA), applying the Mann–Whitney U test for non-parametric variables and the Fisher's exact test for categorical variables, with the level of significance set at P < 0.05.\nA cut-off level for predicting necrosis at retroperitoneal residual mass analysis was sought by constructing a receiver operating characteristic (ROC) curve of all significant variables, which were generated using graphical visualization in our statistical software. This study was carried-out in accordance with the Ethics Committee regulations.","We gathered information on a total of 32 patients, who were 18- to 49-years old (mean age 30.5) and harbored seminomatous, nonseminomatous or mixed tumors in the testis tissue in different clinical stages. Three patients (9.4%) had seminoma, five (16%) had pure nonseminoma (choriocarcinoma, yolk sac tumors, embryonal carcinoma, teratoma, teratocarcinoma) and 24 (75%) harbored mixed nonseminomatous GCT (NSGCT). None of the patients with seminoma had normal AFP levels, indicating that these patients may have had tumors with similar biological behavior to those with NSGCT.\nAt diagnosis, seven (22%) patients were classified as having a stage I disease, while 21 (66%) were stage II and four (12.5%) were stage III. Clinical stage I patients were individuals with longer follow-up, who presented with retroperitoneal disease nonresponsive to chemotherapy and ultimately underwent PC-RPLND.\nPathological analysis in our series demonstrated that 15 (47%) patients had necrosis in residual retroperitoneal masses, 15 had teratoma (47%) and two (6%) had viable GCT: one seminoma and one yolk sac tumor. For statistical reasons and in alignment with the aims of this study, we divided those patients into two groups, assembling patients with viable GCT and teratoma and analyzing them as one group. Mean size of the retroperitoneal mass was 4.94 cm in our sample, 3.79 cm in the group of necrosis and 5.96 cm in the group of teratoma and viable GCT (P = 0.176). There was also no difference between groups regarding stratification of nodal size, as shown in Table 1.\n\nDistribution according to nodal size in men undergoing PC-RPLND\nData are shown as number (%). GCT, germ cell tumor; PC-RPLND, post-chemotherapy retroperitoneal lymph node dissection.\nPrimary tumor histology revealed embryonal cell carcinoma in 56%, seminomatous elements in 16%, yolk sac tumor in 19% and teratomatous elements in 56% of the patients. Of the 18 patients with teratomatous elements in the primary tumor, 13 (72%) had teratoma in the retroperitoneum at PC-RPLND. Even in the absence of teratoma in the primary tumor, teratoma was present in the retroperitoneum in five patients (16%).\nWhen comparing pathological analysis of primary tumor specimens, we found a statistical difference when comparing the prevalence of teratoma and choriocarcinoma. Other findings, such as the presence of seminoma, yolk sac tumor, embryonal carcinoma, endodermal sinus tumor lymph vascular invasion (LVI), rete testis invasion and spermatic invasion were similar between groups.\nUnivariate analysis of quantitative components at orchiectomy histology, retroperitoneal node size with its relative measures and serum markers are shown in Table 2.\n\nUnivariate analysis predicting necrosis at PC-RPLND\n** unable to calculate. Data expressed as mean ± standard deviation. AFP, alpha-fetoprotein; GCT, germ cell tumor; hCG, human chorionic gonadotropin; LDH, lactate dehydrogenase; PC-RPLND, post-chemotherapy retroperitoneal lymph node dissection; RP, retroperitoneal.\nEven though the presence of teratoma in the primary tumor was an important negative predictor for the finding of necrosis, the quantitative analysis has proven to be statistically irrelevant.\nWhile relative reduction in mass size after chemotherapy has been shown to be an important predictor of necrosis, even when considering patients not responding to chemotherapy, absolute reduction and enlargement of the residual mass did not show a significant difference between groups.\nThere was a statistical difference in AFP levels between groups; however, when comparing relative changes in AFP levels after chemotherapy, no difference was found. Comparison of LDH levels and their relative changes was nonsignificant. We were unable to compare hCG levels between groups because we only had two patients whose hCG levels were not undetectable.\nOf all the studied variables, relative changes in retroperitoneal lymph node size (P =0.04), the absence of teratoma in the orchiectomy specimen (P =0.03) and the presence of choriocarcinoma in the testicular analysis after orchiectomy (P =0.03) were statistically significant predictors of the presence of necrosis in the retroperitoneum.\nROC curves were built for variables that were independent predictors of necrosis at PC-RPLND. Even though the size of the retroperitoneal mass after chemotherapy showed no statistical difference, we also built a curve for it. The variable relative reduction after chemotherapy was the only one with predictive value according to the Youlden index. An area under the curve (AUC) of 0.710 was obtained and a reduction level of 35% was therefore suggested to be the best cutoff for predicting the absence of tumor in the retroperitoneum with a sensitivity of 73% and a specificity of 82%. ROC curves of isolated size of retroperitoneal mass, absence of teratoma and presence of choriocarcinoma did not indicate significant findings.\nIn our institution, relative change in retroperitoneal node size, absence of teratoma in the orchiectomy specimen and the presence of choriocarcinoma in the testicular analysis after orchiectomy were statistically significant predictors of the presence of necrosis in the retroperitoneum and a surveillance program could be considered, given the uncertainty in predicting the histology of residual masses after chemotherapy in patients with metastatic testicular tumors.\nAnalysis of residual retroperitoneal masses after chemotherapy is being increasingly regarded as a fundamental issue, not only for orienting adjuvant therapies, but also because it has prognostic implications [11]. Outcome assessments in patients with NSGCT have demonstrated that incomplete resection of residual retroperitoneal masses, the size of residual retroperitoneal masses and the finding of teratoma and viable GCT at RPLND independently predict disease progression and relapse [11,12].\nThe size of residual retroperitoneal masses after chemotherapy has traditionally been considered when choosing the subsequent treatment modality [9]. Studies have shown that residual masses smaller than 2 cm are considered one of the most significant predictors for finding necrosis at PC-RPLND at logistic regression [4]. Furthermore, a number of investigators continue to base the decision to perform PC-RPLND on residual mass size alone, obviating PC-RPLND in patients with residual masses of 1 cm or less [13].\nHowever, recent studies have shown that after chemotherapy a third of retroperitoneal masses of <2 cm harbored either teratoma or viable GCT [14]. Even though we found a trend of having only necrosis in the retroperitoneum for masses <2 cm (P = 0.09), in the present study residual mass size alone has not appeared to be a good predictor of necrosis, with nonsignificant size differences between groups and an underrated AUC in ROC curves. In addition, one case of teratoma in a 1 cm mass was registered in our sample. Therefore, we advocate that the decision of not operating on patients cannot be based on mass size alone due to the lack of consensus in the literature [9,10,13,14].\nTraditional series report necrotic debris or fibrosis in 40% to 50%, teratoma in 35% to 40% and viable malignant cells in 10% to 15% of patients [3]. In more recent analyses, the incidence of residual microscopic teratoma in the retroperitoneum has decreased to approximately 20% to 25%, with an increase of necrotic tissue findings of up to 60% and stable rates of viable GCT [15]. It has been reported that an increase in the proportion of necrosis is generally attributed to stage migration and the use of more effective chemotherapy regimens, especially in patients achieving a complete response to chemotherapy [15].\nIn our series we found a distribution pattern similar to former studies, with almost 50% of patients harboring teratoma in the retroperitoneal histology at RPLND, which may suggest that our chemotherapy regimens have been having inferior rates of complete responders or simply because our group is composed of patients in more advanced stages.\nTeratoma-negative primary tumor and volumetric regression of at least 90% after chemotherapy have been described as being highly predictive of harboring necrosis only at PC-RPLND [9,16]. Other series include normal serum tumor markers, and node size < 2 cm, the presence of yolk sac tumor or embryonal carcinoma on primary tumor and lymph vascular invasion, among others [9,10,15-17]. On the other hand, some authors have been unable to identify variables to be highly predictive of harboring necrosis only at PC-RPLND [15,18]. Nevertheless, currently predictive models fail to accurately predict necrosis in the retroperitoneum, since almost 30% of patients will harbor teratoma or viable malignancy even with normal post-chemotherapy CT and no teratoma in the primary tumor [9]. A study carried out at the Memorial Sloan-Kettering Cancer Center revealed that 26% of patients had teratoma or viable GCT, even those with radiographically normal retroperitoneum who underwent PC-RPLND [9].\nThe relatively small number of patients when compared to other institutional retrospective studies is a limitation of the present study; however, other studies with similar sample sizes have also come to significant findings.\nIn the present study, relative changes in retroperitoneal node size stood out as the single best predictor of the presence of necrosis in the retroperitoneum, which is in accordance with actual logistic regression models [9,10]. While other series suggest 90% as a shrinkage cutoff value to reliably predict necrosis, our quantitative analysis have demonstrated a cutoff level of 35% with approximate sensitivity and specificity [9].\nThe absence of teratoma has also been demonstrated to be significant in qualitative analysis, but not in quantitative analysis. The presence of choriocarcinoma in the testicular histology predicted necrosis in the retroperitoneum, despite only four (12.5%) patients who had such a finding. This association is an interesting finding that has not been previously reported. We reviewed the literature and no possible explanations were found. We believe further investigation is necessary to confirm this finding, since the number of patients in our sample is relatively small.","Even though RPLND remains the gold standard for patients with residual masses, those without teratoma in the primary tumor and a shrinkage of 35% or more in retroperitoneal mass have a considerably smaller chance of having viable GCTs or teratoma in the retroperitoneum.","AFP: Alphs-fetoprotein; AUC: Area under the curve; CT: Computed tomography; GCT: Germ cell tumor; hCG: Human chorionic gonadotropin; LDH: Lactate dehydrogenase; LVI: Lymph vascular invasion; NSGCT: Nonseminomatous germ cell tumor; PC-RPLND: Post chemotherapy retroperitoneal lymph node dissection; ROC: Receiver operating characteristic; RPLND: Retroperitoneal lymph node dissection.","The authors declare that they have no competing interests.","EPM and DKA gathered and compiled data and have been involved in drafting the manuscript. AJN and STL participated in the design of the study and performed the statistical analysis. ACS and MS revised the manuscript critically for important intellectual content. MS and MFD have given final approval of the version to be published. All authors read and approved the final manuscript."],"string":"[\n \"Testicular cancer has become one of the most curable solid neoplasms and serves as a paradigm for the multimodal treatment of malignancies. The appropriate integration of chemotherapy, retroperitoneal lymph node dissection (RPLND) and observation for the management of testis cancer has resulted in overall survival rates greater than 90% [1,2].\\nRPLND plays an important role in the management of patients with testicular germ cell tumors (GCTs), especially in those with residual masses after chemotherapy [3]. To date, series have demonstrated that pathological analysis of these masses reveals findings of necrotic debris or fibrosis in 40% to 50% of patients, teratoma in 35% to 40% and viable malignant cells in 10% to 15% of patients [4].\\nEven though early recognition and resection of teratoma in the retroperitoneum after chemotherapy have been accompanied by excellent prognosis, once it was initially thought to represent a benign course when present in the retroperitoneal space, but the untreated disease may have a lethal potential due to progressive local growth or malignant transformation, not to mention its classical unresponsiveness to conventional cisplatin-based chemotherapy regimens [3-6]. As for residual viable GCT, the consequence of its incomplete resection is certain disease progression [7]. Therefore, a more aggressive approach should always be considered when treating patients with teratoma or viable GCT in the retroperitoneum [7,8].\\nThus, the appropriate approach to residual masses following chemotherapy remains a controversial issue, since the literature has shown that as many as half of resected masses are basically composed of necrosis or fibrotic tissue and any sort of adjuvant therapy could be waived [9]. In order to avoid a great number of apparently unnecessary post-chemotherapy RPLND (PC-RPLND), many studies have tried to develop algorithms to predict the presence of necrosis in the retroperitoneum. However, currently predictive models and imaging modalities cannot reliably predict the pathological finding of necrosis/fibrosis at PC-RPLND [9].\\nSome authors have established that patients without teratoma in the primary tumor and a shrinkage of 90% or more in retroperitoneal mass had little chance of having viable GCT or teratoma and could be safely put under a surveillance program with periodic imaging scans [10]. However, prospective analyses have demonstrated that approximately 30% of patients will harbor teratoma or viable malignancy even with normal post-chemotherapy computed tomography (CT) results and no teratoma in the primary tumor [9].\\nThe purpose of this work is to pursue a clinical and pathological review of patients undergoing PC-RPLND at a reference university oncology center in Brazil, in order to identify variables that may help predict the histological finding of necrosis in the retroperitoneum and perhaps establish a differentiated surveillance protocol.\",\n \"We performed a retrospective analysis of all patients from our computerized database who underwent PC-RPLND at our service between January 2005 and May 2011. Patients were operated on after having undergone three to four cycles of primary chemotherapy with bleomycin, etoposide and cisplatin.\\nClinical and pathological data were obtained and consisted basically of measures of retroperitoneal masses, serum tumor markers, histology of the orchiectomy specimen, tumor marker values and retroperitoneal nodal size before and after chemotherapy.\\nThe presence of either immature or mature teratoma in the resected specimen, as well as teratoma with malignant transformation, was considered part of the same group. Choriocarcinoma, yolk sac tumors, embryonal carcinoma and seminoma were considered viable GCTs. All histological findings were submitted to quantitative and qualitative analysis.\\nPost-chemotherapy alpha-fetoprotein (AFP) and lactate dehydrogenase (LDH) levels were registered as a continuous variable while human chorionic gonadotropin (hCG) levels were considered a categorical variable ranging from undetectable to detectable when serum concentrations were greater than 3 mIU.\\nRetroperitoneal nodal size before and after chemotherapy was determined by the longest transverse diameter of the largest mass on CT imaging. Relative change in nodal size before and after chemotherapy was calculated by dividing post-chemotherapy nodal size by pre-chemotherapy nodal size and was analyzed as a continuous variable.\\nMultiple variables were analyzed independently in order to establish any predictive value for finding necrotic tissue in the retroperitoneum. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS, version 12.0, Chicago, IL, USA), applying the Mann–Whitney U test for non-parametric variables and the Fisher's exact test for categorical variables, with the level of significance set at P < 0.05.\\nA cut-off level for predicting necrosis at retroperitoneal residual mass analysis was sought by constructing a receiver operating characteristic (ROC) curve of all significant variables, which were generated using graphical visualization in our statistical software. This study was carried-out in accordance with the Ethics Committee regulations.\",\n \"We gathered information on a total of 32 patients, who were 18- to 49-years old (mean age 30.5) and harbored seminomatous, nonseminomatous or mixed tumors in the testis tissue in different clinical stages. Three patients (9.4%) had seminoma, five (16%) had pure nonseminoma (choriocarcinoma, yolk sac tumors, embryonal carcinoma, teratoma, teratocarcinoma) and 24 (75%) harbored mixed nonseminomatous GCT (NSGCT). None of the patients with seminoma had normal AFP levels, indicating that these patients may have had tumors with similar biological behavior to those with NSGCT.\\nAt diagnosis, seven (22%) patients were classified as having a stage I disease, while 21 (66%) were stage II and four (12.5%) were stage III. Clinical stage I patients were individuals with longer follow-up, who presented with retroperitoneal disease nonresponsive to chemotherapy and ultimately underwent PC-RPLND.\\nPathological analysis in our series demonstrated that 15 (47%) patients had necrosis in residual retroperitoneal masses, 15 had teratoma (47%) and two (6%) had viable GCT: one seminoma and one yolk sac tumor. For statistical reasons and in alignment with the aims of this study, we divided those patients into two groups, assembling patients with viable GCT and teratoma and analyzing them as one group. Mean size of the retroperitoneal mass was 4.94 cm in our sample, 3.79 cm in the group of necrosis and 5.96 cm in the group of teratoma and viable GCT (P = 0.176). There was also no difference between groups regarding stratification of nodal size, as shown in Table 1.\\n\\nDistribution according to nodal size in men undergoing PC-RPLND\\nData are shown as number (%). GCT, germ cell tumor; PC-RPLND, post-chemotherapy retroperitoneal lymph node dissection.\\nPrimary tumor histology revealed embryonal cell carcinoma in 56%, seminomatous elements in 16%, yolk sac tumor in 19% and teratomatous elements in 56% of the patients. Of the 18 patients with teratomatous elements in the primary tumor, 13 (72%) had teratoma in the retroperitoneum at PC-RPLND. Even in the absence of teratoma in the primary tumor, teratoma was present in the retroperitoneum in five patients (16%).\\nWhen comparing pathological analysis of primary tumor specimens, we found a statistical difference when comparing the prevalence of teratoma and choriocarcinoma. Other findings, such as the presence of seminoma, yolk sac tumor, embryonal carcinoma, endodermal sinus tumor lymph vascular invasion (LVI), rete testis invasion and spermatic invasion were similar between groups.\\nUnivariate analysis of quantitative components at orchiectomy histology, retroperitoneal node size with its relative measures and serum markers are shown in Table 2.\\n\\nUnivariate analysis predicting necrosis at PC-RPLND\\n** unable to calculate. Data expressed as mean ± standard deviation. AFP, alpha-fetoprotein; GCT, germ cell tumor; hCG, human chorionic gonadotropin; LDH, lactate dehydrogenase; PC-RPLND, post-chemotherapy retroperitoneal lymph node dissection; RP, retroperitoneal.\\nEven though the presence of teratoma in the primary tumor was an important negative predictor for the finding of necrosis, the quantitative analysis has proven to be statistically irrelevant.\\nWhile relative reduction in mass size after chemotherapy has been shown to be an important predictor of necrosis, even when considering patients not responding to chemotherapy, absolute reduction and enlargement of the residual mass did not show a significant difference between groups.\\nThere was a statistical difference in AFP levels between groups; however, when comparing relative changes in AFP levels after chemotherapy, no difference was found. Comparison of LDH levels and their relative changes was nonsignificant. We were unable to compare hCG levels between groups because we only had two patients whose hCG levels were not undetectable.\\nOf all the studied variables, relative changes in retroperitoneal lymph node size (P =0.04), the absence of teratoma in the orchiectomy specimen (P =0.03) and the presence of choriocarcinoma in the testicular analysis after orchiectomy (P =0.03) were statistically significant predictors of the presence of necrosis in the retroperitoneum.\\nROC curves were built for variables that were independent predictors of necrosis at PC-RPLND. Even though the size of the retroperitoneal mass after chemotherapy showed no statistical difference, we also built a curve for it. The variable relative reduction after chemotherapy was the only one with predictive value according to the Youlden index. An area under the curve (AUC) of 0.710 was obtained and a reduction level of 35% was therefore suggested to be the best cutoff for predicting the absence of tumor in the retroperitoneum with a sensitivity of 73% and a specificity of 82%. ROC curves of isolated size of retroperitoneal mass, absence of teratoma and presence of choriocarcinoma did not indicate significant findings.\\nIn our institution, relative change in retroperitoneal node size, absence of teratoma in the orchiectomy specimen and the presence of choriocarcinoma in the testicular analysis after orchiectomy were statistically significant predictors of the presence of necrosis in the retroperitoneum and a surveillance program could be considered, given the uncertainty in predicting the histology of residual masses after chemotherapy in patients with metastatic testicular tumors.\\nAnalysis of residual retroperitoneal masses after chemotherapy is being increasingly regarded as a fundamental issue, not only for orienting adjuvant therapies, but also because it has prognostic implications [11]. Outcome assessments in patients with NSGCT have demonstrated that incomplete resection of residual retroperitoneal masses, the size of residual retroperitoneal masses and the finding of teratoma and viable GCT at RPLND independently predict disease progression and relapse [11,12].\\nThe size of residual retroperitoneal masses after chemotherapy has traditionally been considered when choosing the subsequent treatment modality [9]. Studies have shown that residual masses smaller than 2 cm are considered one of the most significant predictors for finding necrosis at PC-RPLND at logistic regression [4]. Furthermore, a number of investigators continue to base the decision to perform PC-RPLND on residual mass size alone, obviating PC-RPLND in patients with residual masses of 1 cm or less [13].\\nHowever, recent studies have shown that after chemotherapy a third of retroperitoneal masses of <2 cm harbored either teratoma or viable GCT [14]. Even though we found a trend of having only necrosis in the retroperitoneum for masses <2 cm (P = 0.09), in the present study residual mass size alone has not appeared to be a good predictor of necrosis, with nonsignificant size differences between groups and an underrated AUC in ROC curves. In addition, one case of teratoma in a 1 cm mass was registered in our sample. Therefore, we advocate that the decision of not operating on patients cannot be based on mass size alone due to the lack of consensus in the literature [9,10,13,14].\\nTraditional series report necrotic debris or fibrosis in 40% to 50%, teratoma in 35% to 40% and viable malignant cells in 10% to 15% of patients [3]. In more recent analyses, the incidence of residual microscopic teratoma in the retroperitoneum has decreased to approximately 20% to 25%, with an increase of necrotic tissue findings of up to 60% and stable rates of viable GCT [15]. It has been reported that an increase in the proportion of necrosis is generally attributed to stage migration and the use of more effective chemotherapy regimens, especially in patients achieving a complete response to chemotherapy [15].\\nIn our series we found a distribution pattern similar to former studies, with almost 50% of patients harboring teratoma in the retroperitoneal histology at RPLND, which may suggest that our chemotherapy regimens have been having inferior rates of complete responders or simply because our group is composed of patients in more advanced stages.\\nTeratoma-negative primary tumor and volumetric regression of at least 90% after chemotherapy have been described as being highly predictive of harboring necrosis only at PC-RPLND [9,16]. Other series include normal serum tumor markers, and node size < 2 cm, the presence of yolk sac tumor or embryonal carcinoma on primary tumor and lymph vascular invasion, among others [9,10,15-17]. On the other hand, some authors have been unable to identify variables to be highly predictive of harboring necrosis only at PC-RPLND [15,18]. Nevertheless, currently predictive models fail to accurately predict necrosis in the retroperitoneum, since almost 30% of patients will harbor teratoma or viable malignancy even with normal post-chemotherapy CT and no teratoma in the primary tumor [9]. A study carried out at the Memorial Sloan-Kettering Cancer Center revealed that 26% of patients had teratoma or viable GCT, even those with radiographically normal retroperitoneum who underwent PC-RPLND [9].\\nThe relatively small number of patients when compared to other institutional retrospective studies is a limitation of the present study; however, other studies with similar sample sizes have also come to significant findings.\\nIn the present study, relative changes in retroperitoneal node size stood out as the single best predictor of the presence of necrosis in the retroperitoneum, which is in accordance with actual logistic regression models [9,10]. While other series suggest 90% as a shrinkage cutoff value to reliably predict necrosis, our quantitative analysis have demonstrated a cutoff level of 35% with approximate sensitivity and specificity [9].\\nThe absence of teratoma has also been demonstrated to be significant in qualitative analysis, but not in quantitative analysis. The presence of choriocarcinoma in the testicular histology predicted necrosis in the retroperitoneum, despite only four (12.5%) patients who had such a finding. This association is an interesting finding that has not been previously reported. We reviewed the literature and no possible explanations were found. We believe further investigation is necessary to confirm this finding, since the number of patients in our sample is relatively small.\",\n \"Even though RPLND remains the gold standard for patients with residual masses, those without teratoma in the primary tumor and a shrinkage of 35% or more in retroperitoneal mass have a considerably smaller chance of having viable GCTs or teratoma in the retroperitoneum.\",\n \"AFP: Alphs-fetoprotein; AUC: Area under the curve; CT: Computed tomography; GCT: Germ cell tumor; hCG: Human chorionic gonadotropin; LDH: Lactate dehydrogenase; LVI: Lymph vascular invasion; NSGCT: Nonseminomatous germ cell tumor; PC-RPLND: Post chemotherapy retroperitoneal lymph node dissection; ROC: Receiver operating characteristic; RPLND: Retroperitoneal lymph node dissection.\",\n \"The authors declare that they have no competing interests.\",\n \"EPM and DKA gathered and compiled data and have been involved in drafting the manuscript. AJN and STL participated in the design of the study and performed the statistical analysis. ACS and MS revised the manuscript critically for important intellectual content. MS and MFD have given final approval of the version to be published. All authors read and approved the final manuscript.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,"conclusions",null,null,null],"string":"[\n null,\n \"methods\",\n null,\n \"conclusions\",\n null,\n null,\n null\n]","isConversational":false},"keywords":{"kind":"list like","value":["Testicular cancer","Retroperitoneal lymph node dissection","Necrosis","Teratoma"],"string":"[\n \"Testicular cancer\",\n \"Retroperitoneal lymph node dissection\",\n \"Necrosis\",\n \"Teratoma\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Background:\nTesticular cancer has become one of the most curable solid neoplasms and serves as a paradigm for the multimodal treatment of malignancies. The appropriate integration of chemotherapy, retroperitoneal lymph node dissection (RPLND) and observation for the management of testis cancer has resulted in overall survival rates greater than 90% [1,2].\nRPLND plays an important role in the management of patients with testicular germ cell tumors (GCTs), especially in those with residual masses after chemotherapy [3]. To date, series have demonstrated that pathological analysis of these masses reveals findings of necrotic debris or fibrosis in 40% to 50% of patients, teratoma in 35% to 40% and viable malignant cells in 10% to 15% of patients [4].\nEven though early recognition and resection of teratoma in the retroperitoneum after chemotherapy have been accompanied by excellent prognosis, once it was initially thought to represent a benign course when present in the retroperitoneal space, but the untreated disease may have a lethal potential due to progressive local growth or malignant transformation, not to mention its classical unresponsiveness to conventional cisplatin-based chemotherapy regimens [3-6]. As for residual viable GCT, the consequence of its incomplete resection is certain disease progression [7]. Therefore, a more aggressive approach should always be considered when treating patients with teratoma or viable GCT in the retroperitoneum [7,8].\nThus, the appropriate approach to residual masses following chemotherapy remains a controversial issue, since the literature has shown that as many as half of resected masses are basically composed of necrosis or fibrotic tissue and any sort of adjuvant therapy could be waived [9]. In order to avoid a great number of apparently unnecessary post-chemotherapy RPLND (PC-RPLND), many studies have tried to develop algorithms to predict the presence of necrosis in the retroperitoneum. However, currently predictive models and imaging modalities cannot reliably predict the pathological finding of necrosis/fibrosis at PC-RPLND [9].\nSome authors have established that patients without teratoma in the primary tumor and a shrinkage of 90% or more in retroperitoneal mass had little chance of having viable GCT or teratoma and could be safely put under a surveillance program with periodic imaging scans [10]. However, prospective analyses have demonstrated that approximately 30% of patients will harbor teratoma or viable malignancy even with normal post-chemotherapy computed tomography (CT) results and no teratoma in the primary tumor [9].\nThe purpose of this work is to pursue a clinical and pathological review of patients undergoing PC-RPLND at a reference university oncology center in Brazil, in order to identify variables that may help predict the histological finding of necrosis in the retroperitoneum and perhaps establish a differentiated surveillance protocol.\n\nMethods:\nWe performed a retrospective analysis of all patients from our computerized database who underwent PC-RPLND at our service between January 2005 and May 2011. Patients were operated on after having undergone three to four cycles of primary chemotherapy with bleomycin, etoposide and cisplatin.\nClinical and pathological data were obtained and consisted basically of measures of retroperitoneal masses, serum tumor markers, histology of the orchiectomy specimen, tumor marker values and retroperitoneal nodal size before and after chemotherapy.\nThe presence of either immature or mature teratoma in the resected specimen, as well as teratoma with malignant transformation, was considered part of the same group. Choriocarcinoma, yolk sac tumors, embryonal carcinoma and seminoma were considered viable GCTs. All histological findings were submitted to quantitative and qualitative analysis.\nPost-chemotherapy alpha-fetoprotein (AFP) and lactate dehydrogenase (LDH) levels were registered as a continuous variable while human chorionic gonadotropin (hCG) levels were considered a categorical variable ranging from undetectable to detectable when serum concentrations were greater than 3 mIU.\nRetroperitoneal nodal size before and after chemotherapy was determined by the longest transverse diameter of the largest mass on CT imaging. Relative change in nodal size before and after chemotherapy was calculated by dividing post-chemotherapy nodal size by pre-chemotherapy nodal size and was analyzed as a continuous variable.\nMultiple variables were analyzed independently in order to establish any predictive value for finding necrotic tissue in the retroperitoneum. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS, version 12.0, Chicago, IL, USA), applying the Mann–Whitney U test for non-parametric variables and the Fisher's exact test for categorical variables, with the level of significance set at P < 0.05.\nA cut-off level for predicting necrosis at retroperitoneal residual mass analysis was sought by constructing a receiver operating characteristic (ROC) curve of all significant variables, which were generated using graphical visualization in our statistical software. This study was carried-out in accordance with the Ethics Committee regulations.\n\nResults and discussion:\nWe gathered information on a total of 32 patients, who were 18- to 49-years old (mean age 30.5) and harbored seminomatous, nonseminomatous or mixed tumors in the testis tissue in different clinical stages. Three patients (9.4%) had seminoma, five (16%) had pure nonseminoma (choriocarcinoma, yolk sac tumors, embryonal carcinoma, teratoma, teratocarcinoma) and 24 (75%) harbored mixed nonseminomatous GCT (NSGCT). None of the patients with seminoma had normal AFP levels, indicating that these patients may have had tumors with similar biological behavior to those with NSGCT.\nAt diagnosis, seven (22%) patients were classified as having a stage I disease, while 21 (66%) were stage II and four (12.5%) were stage III. Clinical stage I patients were individuals with longer follow-up, who presented with retroperitoneal disease nonresponsive to chemotherapy and ultimately underwent PC-RPLND.\nPathological analysis in our series demonstrated that 15 (47%) patients had necrosis in residual retroperitoneal masses, 15 had teratoma (47%) and two (6%) had viable GCT: one seminoma and one yolk sac tumor. For statistical reasons and in alignment with the aims of this study, we divided those patients into two groups, assembling patients with viable GCT and teratoma and analyzing them as one group. Mean size of the retroperitoneal mass was 4.94 cm in our sample, 3.79 cm in the group of necrosis and 5.96 cm in the group of teratoma and viable GCT (P = 0.176). There was also no difference between groups regarding stratification of nodal size, as shown in Table 1.\n\nDistribution according to nodal size in men undergoing PC-RPLND\nData are shown as number (%). GCT, germ cell tumor; PC-RPLND, post-chemotherapy retroperitoneal lymph node dissection.\nPrimary tumor histology revealed embryonal cell carcinoma in 56%, seminomatous elements in 16%, yolk sac tumor in 19% and teratomatous elements in 56% of the patients. Of the 18 patients with teratomatous elements in the primary tumor, 13 (72%) had teratoma in the retroperitoneum at PC-RPLND. Even in the absence of teratoma in the primary tumor, teratoma was present in the retroperitoneum in five patients (16%).\nWhen comparing pathological analysis of primary tumor specimens, we found a statistical difference when comparing the prevalence of teratoma and choriocarcinoma. Other findings, such as the presence of seminoma, yolk sac tumor, embryonal carcinoma, endodermal sinus tumor lymph vascular invasion (LVI), rete testis invasion and spermatic invasion were similar between groups.\nUnivariate analysis of quantitative components at orchiectomy histology, retroperitoneal node size with its relative measures and serum markers are shown in Table 2.\n\nUnivariate analysis predicting necrosis at PC-RPLND\n** unable to calculate. Data expressed as mean ± standard deviation. AFP, alpha-fetoprotein; GCT, germ cell tumor; hCG, human chorionic gonadotropin; LDH, lactate dehydrogenase; PC-RPLND, post-chemotherapy retroperitoneal lymph node dissection; RP, retroperitoneal.\nEven though the presence of teratoma in the primary tumor was an important negative predictor for the finding of necrosis, the quantitative analysis has proven to be statistically irrelevant.\nWhile relative reduction in mass size after chemotherapy has been shown to be an important predictor of necrosis, even when considering patients not responding to chemotherapy, absolute reduction and enlargement of the residual mass did not show a significant difference between groups.\nThere was a statistical difference in AFP levels between groups; however, when comparing relative changes in AFP levels after chemotherapy, no difference was found. Comparison of LDH levels and their relative changes was nonsignificant. We were unable to compare hCG levels between groups because we only had two patients whose hCG levels were not undetectable.\nOf all the studied variables, relative changes in retroperitoneal lymph node size (P =0.04), the absence of teratoma in the orchiectomy specimen (P =0.03) and the presence of choriocarcinoma in the testicular analysis after orchiectomy (P =0.03) were statistically significant predictors of the presence of necrosis in the retroperitoneum.\nROC curves were built for variables that were independent predictors of necrosis at PC-RPLND. Even though the size of the retroperitoneal mass after chemotherapy showed no statistical difference, we also built a curve for it. The variable relative reduction after chemotherapy was the only one with predictive value according to the Youlden index. An area under the curve (AUC) of 0.710 was obtained and a reduction level of 35% was therefore suggested to be the best cutoff for predicting the absence of tumor in the retroperitoneum with a sensitivity of 73% and a specificity of 82%. ROC curves of isolated size of retroperitoneal mass, absence of teratoma and presence of choriocarcinoma did not indicate significant findings.\nIn our institution, relative change in retroperitoneal node size, absence of teratoma in the orchiectomy specimen and the presence of choriocarcinoma in the testicular analysis after orchiectomy were statistically significant predictors of the presence of necrosis in the retroperitoneum and a surveillance program could be considered, given the uncertainty in predicting the histology of residual masses after chemotherapy in patients with metastatic testicular tumors.\nAnalysis of residual retroperitoneal masses after chemotherapy is being increasingly regarded as a fundamental issue, not only for orienting adjuvant therapies, but also because it has prognostic implications [11]. Outcome assessments in patients with NSGCT have demonstrated that incomplete resection of residual retroperitoneal masses, the size of residual retroperitoneal masses and the finding of teratoma and viable GCT at RPLND independently predict disease progression and relapse [11,12].\nThe size of residual retroperitoneal masses after chemotherapy has traditionally been considered when choosing the subsequent treatment modality [9]. Studies have shown that residual masses smaller than 2 cm are considered one of the most significant predictors for finding necrosis at PC-RPLND at logistic regression [4]. Furthermore, a number of investigators continue to base the decision to perform PC-RPLND on residual mass size alone, obviating PC-RPLND in patients with residual masses of 1 cm or less [13].\nHowever, recent studies have shown that after chemotherapy a third of retroperitoneal masses of <2 cm harbored either teratoma or viable GCT [14]. Even though we found a trend of having only necrosis in the retroperitoneum for masses <2 cm (P = 0.09), in the present study residual mass size alone has not appeared to be a good predictor of necrosis, with nonsignificant size differences between groups and an underrated AUC in ROC curves. In addition, one case of teratoma in a 1 cm mass was registered in our sample. Therefore, we advocate that the decision of not operating on patients cannot be based on mass size alone due to the lack of consensus in the literature [9,10,13,14].\nTraditional series report necrotic debris or fibrosis in 40% to 50%, teratoma in 35% to 40% and viable malignant cells in 10% to 15% of patients [3]. In more recent analyses, the incidence of residual microscopic teratoma in the retroperitoneum has decreased to approximately 20% to 25%, with an increase of necrotic tissue findings of up to 60% and stable rates of viable GCT [15]. It has been reported that an increase in the proportion of necrosis is generally attributed to stage migration and the use of more effective chemotherapy regimens, especially in patients achieving a complete response to chemotherapy [15].\nIn our series we found a distribution pattern similar to former studies, with almost 50% of patients harboring teratoma in the retroperitoneal histology at RPLND, which may suggest that our chemotherapy regimens have been having inferior rates of complete responders or simply because our group is composed of patients in more advanced stages.\nTeratoma-negative primary tumor and volumetric regression of at least 90% after chemotherapy have been described as being highly predictive of harboring necrosis only at PC-RPLND [9,16]. Other series include normal serum tumor markers, and node size < 2 cm, the presence of yolk sac tumor or embryonal carcinoma on primary tumor and lymph vascular invasion, among others [9,10,15-17]. On the other hand, some authors have been unable to identify variables to be highly predictive of harboring necrosis only at PC-RPLND [15,18]. Nevertheless, currently predictive models fail to accurately predict necrosis in the retroperitoneum, since almost 30% of patients will harbor teratoma or viable malignancy even with normal post-chemotherapy CT and no teratoma in the primary tumor [9]. A study carried out at the Memorial Sloan-Kettering Cancer Center revealed that 26% of patients had teratoma or viable GCT, even those with radiographically normal retroperitoneum who underwent PC-RPLND [9].\nThe relatively small number of patients when compared to other institutional retrospective studies is a limitation of the present study; however, other studies with similar sample sizes have also come to significant findings.\nIn the present study, relative changes in retroperitoneal node size stood out as the single best predictor of the presence of necrosis in the retroperitoneum, which is in accordance with actual logistic regression models [9,10]. While other series suggest 90% as a shrinkage cutoff value to reliably predict necrosis, our quantitative analysis have demonstrated a cutoff level of 35% with approximate sensitivity and specificity [9].\nThe absence of teratoma has also been demonstrated to be significant in qualitative analysis, but not in quantitative analysis. The presence of choriocarcinoma in the testicular histology predicted necrosis in the retroperitoneum, despite only four (12.5%) patients who had such a finding. This association is an interesting finding that has not been previously reported. We reviewed the literature and no possible explanations were found. We believe further investigation is necessary to confirm this finding, since the number of patients in our sample is relatively small.\n\nConclusions:\nEven though RPLND remains the gold standard for patients with residual masses, those without teratoma in the primary tumor and a shrinkage of 35% or more in retroperitoneal mass have a considerably smaller chance of having viable GCTs or teratoma in the retroperitoneum.\n\nAbbreviations:\nAFP: Alphs-fetoprotein; AUC: Area under the curve; CT: Computed tomography; GCT: Germ cell tumor; hCG: Human chorionic gonadotropin; LDH: Lactate dehydrogenase; LVI: Lymph vascular invasion; NSGCT: Nonseminomatous germ cell tumor; PC-RPLND: Post chemotherapy retroperitoneal lymph node dissection; ROC: Receiver operating characteristic; RPLND: Retroperitoneal lymph node dissection.\n\nCompeting interests:\nThe authors declare that they have no competing interests.\n\nAuthors’ contributions:\nEPM and DKA gathered and compiled data and have been involved in drafting the manuscript. AJN and STL participated in the design of the study and performed the statistical analysis. ACS and MS revised the manuscript critically for important intellectual content. MS and MFD have given final approval of the version to be published. All authors read and approved the final manuscript.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nRecent studies have demonstrated that pathological analysis of retroperitoneal residual masses of patients with testicular germ cell tumors revealed findings of necrotic debris or fibrosis in up to 50% of patients. We aimed at pursuing a clinical and pathological review of patients undergoing post chemotherapy retroperitoneal lymph node dissection (PC-RPLND) in order to identify variables that may help predict necrosis in the retroperitoneum.\n\nMethods:\nWe performed a retrospective analysis of all patients who underwent PC-RPLND at the University Hospital of the University of São Paulo and Cancer Institute of Sao Paulo between January 2005 and September 2011. Clinical and pathological data were obtained and consisted basically of: measures of retroperitoneal masses, histology of the orchiectomy specimen, serum tumor marker and retroperitoneal nodal size before and after chemotherapy.\n\nResults:\nWe gathered a total of 32 patients with a mean age of 29.7; pathological analysis in our series demonstrated that 15 (47%) had necrosis in residual retroperitoneal masses, 15 had teratoma (47%) and 2 (6.4%) had viable germ cell tumors (GCT). The mean size of the retroperitoneal mass was 4.94 cm in our sample, without a difference between the groups (P = 0.176). From all studied variables, relative changes in retroperitoneal lymph node size (P = 0.04), the absence of teratoma in the orchiectomy specimen (P = 0.03) and the presence of choriocarcinoma in the testicular analysis after orchiectomy (P = 0.03) were statistically significant predictors of the presence of necrosis. A reduction level of 35% was therefore suggested to be the best cutoff for predicting the absence of tumor in the retroperitoneum with a sensitivity of 73.3% and specificity of 82.4%.\n\nConclusions:\nEven though retroperitoneal lymph node dissection remains the gold standard for patients with residual masses, those without teratoma in the primary tumor and a shrinkage of 35% or more in retroperitoneal mass have a considerably smaller chance of having viable GCT or teratoma in the retroperitoneum and a surveillance program could be considered."},"background_conclusion_text":{"kind":"string","value":"Background:\nTesticular cancer has become one of the most curable solid neoplasms and serves as a paradigm for the multimodal treatment of malignancies. The appropriate integration of chemotherapy, retroperitoneal lymph node dissection (RPLND) and observation for the management of testis cancer has resulted in overall survival rates greater than 90% [1,2].\nRPLND plays an important role in the management of patients with testicular germ cell tumors (GCTs), especially in those with residual masses after chemotherapy [3]. To date, series have demonstrated that pathological analysis of these masses reveals findings of necrotic debris or fibrosis in 40% to 50% of patients, teratoma in 35% to 40% and viable malignant cells in 10% to 15% of patients [4].\nEven though early recognition and resection of teratoma in the retroperitoneum after chemotherapy have been accompanied by excellent prognosis, once it was initially thought to represent a benign course when present in the retroperitoneal space, but the untreated disease may have a lethal potential due to progressive local growth or malignant transformation, not to mention its classical unresponsiveness to conventional cisplatin-based chemotherapy regimens [3-6]. As for residual viable GCT, the consequence of its incomplete resection is certain disease progression [7]. Therefore, a more aggressive approach should always be considered when treating patients with teratoma or viable GCT in the retroperitoneum [7,8].\nThus, the appropriate approach to residual masses following chemotherapy remains a controversial issue, since the literature has shown that as many as half of resected masses are basically composed of necrosis or fibrotic tissue and any sort of adjuvant therapy could be waived [9]. In order to avoid a great number of apparently unnecessary post-chemotherapy RPLND (PC-RPLND), many studies have tried to develop algorithms to predict the presence of necrosis in the retroperitoneum. However, currently predictive models and imaging modalities cannot reliably predict the pathological finding of necrosis/fibrosis at PC-RPLND [9].\nSome authors have established that patients without teratoma in the primary tumor and a shrinkage of 90% or more in retroperitoneal mass had little chance of having viable GCT or teratoma and could be safely put under a surveillance program with periodic imaging scans [10]. However, prospective analyses have demonstrated that approximately 30% of patients will harbor teratoma or viable malignancy even with normal post-chemotherapy computed tomography (CT) results and no teratoma in the primary tumor [9].\nThe purpose of this work is to pursue a clinical and pathological review of patients undergoing PC-RPLND at a reference university oncology center in Brazil, in order to identify variables that may help predict the histological finding of necrosis in the retroperitoneum and perhaps establish a differentiated surveillance protocol.\n\nConclusions:\nEven though RPLND remains the gold standard for patients with residual masses, those without teratoma in the primary tumor and a shrinkage of 35% or more in retroperitoneal mass have a considerably smaller chance of having viable GCTs or teratoma in the retroperitoneum."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nRecent studies have demonstrated that pathological analysis of retroperitoneal residual masses of patients with testicular germ cell tumors revealed findings of necrotic debris or fibrosis in up to 50% of patients. We aimed at pursuing a clinical and pathological review of patients undergoing post chemotherapy retroperitoneal lymph node dissection (PC-RPLND) in order to identify variables that may help predict necrosis in the retroperitoneum.\n\nMethods:\nWe performed a retrospective analysis of all patients who underwent PC-RPLND at the University Hospital of the University of São Paulo and Cancer Institute of Sao Paulo between January 2005 and September 2011. Clinical and pathological data were obtained and consisted basically of: measures of retroperitoneal masses, histology of the orchiectomy specimen, serum tumor marker and retroperitoneal nodal size before and after chemotherapy.\n\nResults:\nWe gathered a total of 32 patients with a mean age of 29.7; pathological analysis in our series demonstrated that 15 (47%) had necrosis in residual retroperitoneal masses, 15 had teratoma (47%) and 2 (6.4%) had viable germ cell tumors (GCT). The mean size of the retroperitoneal mass was 4.94 cm in our sample, without a difference between the groups (P = 0.176). From all studied variables, relative changes in retroperitoneal lymph node size (P = 0.04), the absence of teratoma in the orchiectomy specimen (P = 0.03) and the presence of choriocarcinoma in the testicular analysis after orchiectomy (P = 0.03) were statistically significant predictors of the presence of necrosis. A reduction level of 35% was therefore suggested to be the best cutoff for predicting the absence of tumor in the retroperitoneum with a sensitivity of 73.3% and specificity of 82.4%.\n\nConclusions:\nEven though retroperitoneal lymph node dissection remains the gold standard for patients with residual masses, those without teratoma in the primary tumor and a shrinkage of 35% or more in retroperitoneal mass have a considerably smaller chance of having viable GCT or teratoma in the retroperitoneum and a surveillance program could be considered."},"whole_article_text_length":{"kind":"number","value":3022,"string":"3,022"},"whole_article_abstract_length":{"kind":"number","value":392,"string":"392"},"other_sections_lengths":{"kind":"list like","value":[520,1891,72,10,67],"string":"[\n 520,\n 1891,\n 72,\n 10,\n 67\n]","isConversational":false},"num_sections":{"kind":"number","value":7,"string":"7"},"most_frequent_words":{"kind":"list like","value":["patients","teratoma","chemotherapy","retroperitoneal","rplnd","tumor","necrosis","size","pc","pc rplnd"],"string":"[\n \"patients\",\n \"teratoma\",\n \"chemotherapy\",\n \"retroperitoneal\",\n \"rplnd\",\n \"tumor\",\n \"necrosis\",\n \"size\",\n \"pc\",\n \"pc rplnd\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["management testis cancer","tumors testis tissue","testicular cancer curable","metastatic testicular tumors","background testicular cancer"],"string":"[\n \"management testis cancer\",\n \"tumors testis tissue\",\n \"testicular cancer curable\",\n \"metastatic testicular tumors\",\n \"background testicular cancer\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"string","value":""},"annotated_base_results_abstract_prompt":{"kind":"null"},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Testicular cancer | Retroperitoneal lymph node dissection | Necrosis | Teratoma [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Testicular cancer | Retroperitoneal lymph node dissection | Necrosis | Teratoma [SUMMARY]"},"annotated_keywords_results_abstract_prompt":{"kind":"null"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Testicular cancer | Retroperitoneal lymph node dissection | Necrosis | Teratoma [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Testicular cancer | Retroperitoneal lymph node dissection | Necrosis | Teratoma [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Testicular cancer | Retroperitoneal lymph node dissection | Necrosis | Teratoma [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Humans | Lymph Node Excision | Male | Middle Aged | Necrosis | Neoplasms, Germ Cell and Embryonal | Retroperitoneal Neoplasms | Retrospective Studies | Teratoma | Testicular Neoplasms [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Humans | Lymph Node Excision | Male | Middle Aged | Necrosis | Neoplasms, Germ Cell and Embryonal | Retroperitoneal Neoplasms | Retrospective Studies | Teratoma | Testicular Neoplasms [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"null"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Humans | Lymph Node Excision | Male | Middle Aged | Necrosis | Neoplasms, Germ Cell and Embryonal | Retroperitoneal Neoplasms | Retrospective Studies | Teratoma | Testicular Neoplasms [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Humans | Lymph Node Excision | Male | Middle Aged | Necrosis | Neoplasms, Germ Cell and Embryonal | Retroperitoneal Neoplasms | Retrospective Studies | Teratoma | Testicular Neoplasms [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Humans | Lymph Node Excision | Male | Middle Aged | Necrosis | Neoplasms, Germ Cell and Embryonal | Retroperitoneal Neoplasms | Retrospective Studies | Teratoma | Testicular Neoplasms [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] management testis cancer | tumors testis tissue | testicular cancer curable | metastatic testicular tumors | background testicular cancer [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] management testis cancer | tumors testis tissue | testicular cancer curable | metastatic testicular tumors | background testicular cancer [SUMMARY]"},"annotated_keybert_results_abstract_prompt":{"kind":"null"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] management testis cancer | tumors testis tissue | testicular cancer curable | metastatic testicular tumors | background testicular cancer [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] management testis cancer | tumors testis tissue | testicular cancer curable | metastatic testicular tumors | background testicular cancer [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] management testis cancer | tumors testis tissue | testicular cancer curable | metastatic testicular tumors | background testicular cancer [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | teratoma | chemotherapy | retroperitoneal | rplnd | tumor | necrosis | size | pc | pc rplnd [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | teratoma | chemotherapy | retroperitoneal | rplnd | tumor | necrosis | size | pc | pc rplnd [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"null"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | teratoma | chemotherapy | retroperitoneal | rplnd | tumor | necrosis | size | pc | pc rplnd [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | teratoma | chemotherapy | retroperitoneal | rplnd | tumor | necrosis | size | pc | pc rplnd [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | teratoma | chemotherapy | retroperitoneal | rplnd | tumor | necrosis | size | pc | pc rplnd [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] patients | teratoma | chemotherapy | rplnd | viable | necrosis | patients teratoma | viable gct | predict | masses [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] chemotherapy | nodal size | nodal | size | nodal size chemotherapy | variables | variable | size chemotherapy | analysis | retroperitoneal [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"null"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] teratoma | having viable gcts teratoma | smaller chance having | mass considerably smaller chance | considerably smaller | considerably smaller chance | considerably smaller chance having | primary tumor shrinkage 35 | patients residual masses teratoma | retroperitoneal mass considerably smaller [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] teratoma | chemotherapy | patients | retroperitoneal | rplnd | tumor | declare competing interests | competing interests | competing | interests [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] teratoma | chemotherapy | patients | retroperitoneal | rplnd | tumor | declare competing interests | competing interests | competing | interests [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] up to 50% ||| [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] the University Hospital of the University of São Paulo | Cancer Institute of Sao Paulo | between January 2005 | September 2011 ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"null"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] node | 35% | GCT [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| up to 50% ||| ||| the University Hospital of the University of São Paulo | Cancer Institute of Sao Paulo | between January 2005 | September 2011 ||| ||| ||| 32 | 29.7 | 15 | 47% | 15 | 47% | 2 | 6.4% ||| 4.94 cm | 0.176 ||| 0.04 | 0.03 | 0.03 ||| 35% | 73.3% | 82.4% ||| node | 35% | GCT [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] ||| up to 50% ||| ||| the University Hospital of the University of São Paulo | Cancer Institute of Sao Paulo | between January 2005 | September 2011 ||| ||| ||| 32 | 29.7 | 15 | 47% | 15 | 47% | 2 | 6.4% ||| 4.94 cm | 0.176 ||| 0.04 | 0.03 | 0.03 ||| 35% | 73.3% | 82.4% ||| node | 35% | GCT [SUMMARY]"}}},{"rowIdx":92,"cells":{"title":{"kind":"string","value":"Uncovering the treatable burden of severe aortic stenosis in Australia: current and future projections within an ageing population."},"pmid":{"kind":"string","value":"34376198"},"background_abstract":{"kind":"string","value":"We aimed to address the paucity of information describing the treatable burden of disease associated with severe aortic stenosis (AS) within Australia's ageing population."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"A contemporary model of the population prevalence of symptomatic, severe AS and treatment pathways in Europe and North America was applied to the 2019 Australian population aged ≥ 55 years (7 million people) on an age-specific basis. Applying Australian-specific data, these estimates were used to further calculate the total number of associated deaths and incident cases of severe AS per annum."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"Based on an overall point prevalence of 1.48 % among those aged ≥ 55 years, we estimate that a minimum of 97,000 Australians are living with severe AS. With a 2-fold increased risk of mortality without undergoing aortic valve replacement (AVR), more than half of these individuals (∼56,000) will die within 5-years. From a clinical management perspective, among those with concurrent symptoms (68.3 %, 66,500 [95 % CI 59,000-74,000] cases) more than half (58.4 %, 38,800 [95 % CI 35,700 - 42,000] cases) would be potentially considered for surgical AVR (SAVR) - comprising 2,400, 5,400 and 31,000 cases assessed as high-, medium- or low peri-operative mortality risk, respectively. A further 17,000/27,700 (41.6 % [95 % CI 11,600 - 22,600]) of such individuals would be potentially considered to a transthoracic AVR (TAVR). During the subsequent 5-year period (2020-2024), each year, we estimate an additional 9,300 Australians aged ≥ 60 years will subsequently develop severe AS (6,300 of whom will experience concurrent symptoms). Of these symptomatic cases, an estimated 3,700 and 1,600 cases/annum, will be potentially suitable for SAVR and TAVR, respectively."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"These data suggest there is likely to be a substantive burden of individuals living with severe AS in Australia. Many of these cases may not have been diagnosed and/or received appropriate treatment (based on the evidence-based application of SAVR and TAVR) to reduce their high-risk of subsequent mortality."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Aging","Aortic Valve Stenosis","Australia","Heart Valve Prosthesis Implantation","Humans","Risk Factors","Transcatheter Aortic Valve Replacement","Treatment Outcome"],"string":"[\n \"Aging\",\n \"Aortic Valve Stenosis\",\n \"Australia\",\n \"Heart Valve Prosthesis Implantation\",\n \"Humans\",\n \"Risk Factors\",\n \"Transcatheter Aortic Valve Replacement\",\n \"Treatment Outcome\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"8356417"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"One of the most common cardiac conditions affecting the progressively aging populations of high-income countries such as Australia is aortic stenosis (AS) [1]. Without timely intervention, severe AS is associated with a very poor prognosis [2]. However, like many other countries, there has been a paucity of reports focusing on the overall prevalence and treatable burden of AS in Australia. A recent AS report from National Echocardiography Database of Australia (NEDA) [3] that assessed the severity of AS and subsequent pattern of survival among 122,809 men and 118,494 women with a mean age of 62 ± 18 years highlighted an urgent need to better understand the burden imposed by this potentially deadly condition. Overall, the indicative prevalence of severe low or high gradient AS among adults being investigated with echocardiography during the overall study period of 2000–2019, was 1.1 and 2.1 % (3.2 % combined), respectively. Actual 5-year mortality ranged from 56 to 67 % in those cases with a native aortic valve and no indication of surgical intervention [3].\nHistorically, surgical aortic valve replacement (SAVR) has been the preferred intervention for severe AS [4]. However, transcatheter aortic valve replacement (TAVR) has been successfully applied in those with severe AS with high/prohibitive surgical risk [5–7]. Moreover, two head-to-head randomized trials have now reported non-inferiority [8] and superiority [9] comparing TAVR to SAVR in low-risk patients with severe AS, in respect to mortality and subsequent risk of stroke. Consistent with these data, for most of the 6,050 cases within the NEDA cohort who underwent AVR, their post-procedure AV hemodynamic profile and survival outcomes were favourable [10].\nOverall, these data suggest more Australians might benefit from AVR. However, without reliable estimates of the treatable burden of AS, this critical number (for health service and resource planning) remains unknown. A series of modelled studies, first published in 2013 [11] and then an updated version in 2018 [12], applied the best available epidemiological and registry data to estimate the following for Europe and North America – 1) the overall proportion of older individuals affected by AS and more specifically severe AS; and 2) proportion of these individuals who had and/or would benefit from a valve replacement procedure (SAVR or TAVR). Given the geographical focus and source data used, these estimates now provide these target regions with (moderate) reliable estimates of the treatable burden of AS. To date, there are no equivalent burden of disease estimates for Australia.\nStudy aims We sought, for the first time, to generate reliable (but inherently conservative) estimates on the prevalence and treatable patient population with severe, symptomatic AS in Australia. Specifically, our aim was to replicate the same robust models recently used to generate contemporary AS-specific projections for Europe and North America [12] as highlighted above with specific modifications relevant to the Australian context. Firstly, this included applying population profiling (denominator) data derived from the Australian Bureau of Statistics (ABS). Secondly, given the Australian context, we aimed to expand our burden of disease estimates to those aged ≥ 55 years (noting the original study applied a single estimate of the overall prevalence of severe AS (numerator) to those aged ≥ 75 years [12]) and by applying age- and sex-specific, incident, and prevalent estimates of AS informed by the recent NEDA Study of AS [3].\nWe sought, for the first time, to generate reliable (but inherently conservative) estimates on the prevalence and treatable patient population with severe, symptomatic AS in Australia. Specifically, our aim was to replicate the same robust models recently used to generate contemporary AS-specific projections for Europe and North America [12] as highlighted above with specific modifications relevant to the Australian context. Firstly, this included applying population profiling (denominator) data derived from the Australian Bureau of Statistics (ABS). Secondly, given the Australian context, we aimed to expand our burden of disease estimates to those aged ≥ 55 years (noting the original study applied a single estimate of the overall prevalence of severe AS (numerator) to those aged ≥ 75 years [12]) and by applying age- and sex-specific, incident, and prevalent estimates of AS informed by the recent NEDA Study of AS [3]."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Prevalent cases of severe AS As shown in Fig. 2, we conservatively estimate that around 97,300 Australians are living with severe AS (symptomatic or otherwise). Moreover, assuming just over two-thirds of these cases experience concurrent symptoms linked to the condition, according to contemporary clinical recommendations/ best practice around 66,500 (95 % CI 59,200 to 74,000) people might be considered for an AVR procedure at any one time.\nFig. 2Estimated Point Prevalence and Distribution of Severe AS in Australia\nEstimated Point Prevalence and Distribution of Severe AS in Australia\nAs shown in Fig. 2, we conservatively estimate that around 97,300 Australians are living with severe AS (symptomatic or otherwise). Moreover, assuming just over two-thirds of these cases experience concurrent symptoms linked to the condition, according to contemporary clinical recommendations/ best practice around 66,500 (95 % CI 59,200 to 74,000) people might be considered for an AVR procedure at any one time.\nFig. 2Estimated Point Prevalence and Distribution of Severe AS in Australia\nEstimated Point Prevalence and Distribution of Severe AS in Australia\nAortic valve replacement Figure 3 summarises the overall treatable burden/management of severe AS when assuming 58.4 % of cases with symptomatic, severe AS would be referred for SAVR (primary replacement or revision) and the remainder (41.6 %) for potential TAVR. On this basis, we estimate that around 38,800 (95 % CI 35,700 to 42,000) Australians aged ≥ 55 years with severe, symptomatic AS might be considered for SAVR; based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score [17]. An additional 17,000 cases (95 % CI 11,600 to 22,600) of the approximately 27,700 people not eligible for SAVR due to high peri-operative risk could be potentially considered for TAVR instead.\nFig. 3Estimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\nEstimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\nFigure 3 summarises the overall treatable burden/management of severe AS when assuming 58.4 % of cases with symptomatic, severe AS would be referred for SAVR (primary replacement or revision) and the remainder (41.6 %) for potential TAVR. On this basis, we estimate that around 38,800 (95 % CI 35,700 to 42,000) Australians aged ≥ 55 years with severe, symptomatic AS might be considered for SAVR; based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score [17]. An additional 17,000 cases (95 % CI 11,600 to 22,600) of the approximately 27,700 people not eligible for SAVR due to high peri-operative risk could be potentially considered for TAVR instead.\nFig. 3Estimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\nEstimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\nAS-related mortality Based on the age- and sex-specific rates of actual mortality (ranging from 17 to 84 % for those aged 55–64 years to ≥ 85 years) observed in the NEDA cohort, we estimate that 56,300 (95 % CI 56,100 to 56,500) of the prevalent population with severe AS of their native valve will subsequently die within the next 5 years - see Fig. 4.\nFig. 4Severe AS Related Mortality\nSevere AS Related Mortality\nBased on the age- and sex-specific rates of actual mortality (ranging from 17 to 84 % for those aged 55–64 years to ≥ 85 years) observed in the NEDA cohort, we estimate that 56,300 (95 % CI 56,100 to 56,500) of the prevalent population with severe AS of their native valve will subsequently die within the next 5 years - see Fig. 4.\nFig. 4Severe AS Related Mortality\nSevere AS Related Mortality\nIncident cases As shown in Fig. 5, each year, we further estimate that around 9,300 (95 % CI 9,000 to 9,500) more Australians aged ≥ 60 years will subsequently develop severe AS. Assuming the same pattern of potential AVR procedures (once again based on peri-operative risk status), 3,700 (95 % CI 3,400 to 4,000) and 2,600 (95 % CI 2,300 to 2,900) of the approximate de novo 6,300 (95 % CI 5,600 to 7,000) cases with concurrent symptoms linked to the condition, would be potentially managed with/eligible for a SAVR or TAVR procedure, respectively (see Supplementary Figure S2).\nFig. 5Annual Incident Cases of Severe AS among Australians Aged ≥55 years\nAnnual Incident Cases of Severe AS among Australians Aged ≥55 years\nAs shown in Fig. 5, each year, we further estimate that around 9,300 (95 % CI 9,000 to 9,500) more Australians aged ≥ 60 years will subsequently develop severe AS. Assuming the same pattern of potential AVR procedures (once again based on peri-operative risk status), 3,700 (95 % CI 3,400 to 4,000) and 2,600 (95 % CI 2,300 to 2,900) of the approximate de novo 6,300 (95 % CI 5,600 to 7,000) cases with concurrent symptoms linked to the condition, would be potentially managed with/eligible for a SAVR or TAVR procedure, respectively (see Supplementary Figure S2).\nFig. 5Annual Incident Cases of Severe AS among Australians Aged ≥55 years\nAnnual Incident Cases of Severe AS among Australians Aged ≥55 years"},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"These unique estimates provide an important insight into the current and future treatable burden of severe AS in Australia. At the most conservative level, the likely number of currently affected Australians aged 55 years and over will soon rise to 100,000 people. Moreover, the number of new cases with this potentially deadly condition is likely approaching 10,000 per annum. Based on current clinical practice/recommendations, around two-thirds of such cases should be actively managed due to their symptomatic status (predominantly with a combination of SAVR and TAVR) and a high-risk of mortality. However, it is unclear if that is truly the case. Whether there is sufficient clinical awareness of AS and pro-active referral patterns (particularly for Australian women) for active management is yet to be determined."},"other_sections_titles":{"kind":"list like","value":["Study aims","Methods","Study design","Study setting","Study data","Estimating the prevalence & treatable burden of severe AS","Mortality","Incident cases of severe AS","Statistical analyses","Prevalent cases of severe AS","Aortic valve replacement","AS-related mortality","Incident cases","Limitations",""],"string":"[\n \"Study aims\",\n \"Methods\",\n \"Study design\",\n \"Study setting\",\n \"Study data\",\n \"Estimating the prevalence & treatable burden of severe AS\",\n \"Mortality\",\n \"Incident cases of severe AS\",\n \"Statistical analyses\",\n \"Prevalent cases of severe AS\",\n \"Aortic valve replacement\",\n \"AS-related mortality\",\n \"Incident cases\",\n \"Limitations\",\n \"\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["We sought, for the first time, to generate reliable (but inherently conservative) estimates on the prevalence and treatable patient population with severe, symptomatic AS in Australia. Specifically, our aim was to replicate the same robust models recently used to generate contemporary AS-specific projections for Europe and North America [12] as highlighted above with specific modifications relevant to the Australian context. Firstly, this included applying population profiling (denominator) data derived from the Australian Bureau of Statistics (ABS). Secondly, given the Australian context, we aimed to expand our burden of disease estimates to those aged ≥ 55 years (noting the original study applied a single estimate of the overall prevalence of severe AS (numerator) to those aged ≥ 75 years [12]) and by applying age- and sex-specific, incident, and prevalent estimates of AS informed by the recent NEDA Study of AS [3].","Study design Consistent with previous reports of this type specifically focusing on atrial fibrillation [13] and heart failure [14], we combined official national population data with the best available epidemiological and clinical data to generate estimates of the treatable burden of disease relating to AS. Given the anonymous source and nature of the study data, no ethical approvals were required.\nConsistent with previous reports of this type specifically focusing on atrial fibrillation [13] and heart failure [14], we combined official national population data with the best available epidemiological and clinical data to generate estimates of the treatable burden of disease relating to AS. Given the anonymous source and nature of the study data, no ethical approvals were required.\nStudy setting Projections were applied to the entire Australian population and then each State and Territory according to their currently estimated demographic profile. ABS data projecting the future demographic structure and profile were also used to derive future projections on the treatable burden of AS. (https://www.abs.gov.au/Population - Accessed January 2021).\nProjections were applied to the entire Australian population and then each State and Territory according to their currently estimated demographic profile. ABS data projecting the future demographic structure and profile were also used to derive future projections on the treatable burden of AS. (https://www.abs.gov.au/Population - Accessed January 2021).\nStudy data The primary analyses presented in this report used age-, sex- and geographic-specific demography data for Australian men and women aged ≥ 55 years for the calendar year 2019. Specifically, the ABS currently provides population projections derived from the official 2016 Census - http://www.abs.gov.au/ausstats/abs@.nsf/mf/3101.0 - Accessed January 2021).\nAs indicated above, the primary basis for this report is the recently updated study of the number of treatable and treated patients with severe AS published by Durko and colleagues in 2018 [12]. Specifically, based on an expanded analysis of 37 relevant studies involving 26,402 cases/patients, this study provides a flow-chart (see Fig. 1) of the key estimates/parameters (with 95 % confidence interval, CI) that can be applied (as a primary analysis) to the latest Australian population data (on an age-, sex- and geographic-specific basis) to estimate the pool of treatable severe AS patients per annum. It can also be applied to broad population projections for an increasing pool of older Australians over time; noting that over a decade (from 2006 to 2016) the proportion of Australians aged > 65 years had increased by around 4 % and this proportion (and absolute numbers) will steadily increase.\nFig. 1Model/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\nModel/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\nSpecifically, the model developed and subsequently applied in this report provides overall estimates (based on a combination of European and North American cohort studies) on the proportion of patients with severe AS who – 1) are symptomatic and under current expert guidelines, are largely excluded from surgical management; 2) are symptomatic but unsuitable for SAVR but might safely undergo TAVR (the alternative being medical therapy); or 3) are symptomatic and will undergo SAVR; a proportion of whom (particularly due to high-to-medium surgical risk) may benefit from TAVR. The range of derived estimates for each parameter, despite efforts to smooth-out inevitable heterogeneity, are indicative of the methodological weaknesses/biases inherent to source data. It is important to note these treatment pathways are continually evolving as new, lower risk valve interventions are developed and lower the likely threshold of AVR. Without Australian-specific to correct the rates of intervention applied, it is generally acknowledged that intervention rates for severe AS broadly follow those applied in North America.\nWhere possible, we were able to improve our assumptions and therefore our projections by considering primary profiling and outcome data derived from (NEDA) [3]. This unique study has now captured echocardiographic data on > 750,000 Australians (with no exclusion criteria) being routinely investigated with echocardiography from > 25 centres Australia-wide. With individual data linkage, NEDA also generates real-world, short- to long-term survival data on those affected by common cardiac conditions including AS [3].\nThe primary analyses presented in this report used age-, sex- and geographic-specific demography data for Australian men and women aged ≥ 55 years for the calendar year 2019. Specifically, the ABS currently provides population projections derived from the official 2016 Census - http://www.abs.gov.au/ausstats/abs@.nsf/mf/3101.0 - Accessed January 2021).\nAs indicated above, the primary basis for this report is the recently updated study of the number of treatable and treated patients with severe AS published by Durko and colleagues in 2018 [12]. Specifically, based on an expanded analysis of 37 relevant studies involving 26,402 cases/patients, this study provides a flow-chart (see Fig. 1) of the key estimates/parameters (with 95 % confidence interval, CI) that can be applied (as a primary analysis) to the latest Australian population data (on an age-, sex- and geographic-specific basis) to estimate the pool of treatable severe AS patients per annum. It can also be applied to broad population projections for an increasing pool of older Australians over time; noting that over a decade (from 2006 to 2016) the proportion of Australians aged > 65 years had increased by around 4 % and this proportion (and absolute numbers) will steadily increase.\nFig. 1Model/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\nModel/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\nSpecifically, the model developed and subsequently applied in this report provides overall estimates (based on a combination of European and North American cohort studies) on the proportion of patients with severe AS who – 1) are symptomatic and under current expert guidelines, are largely excluded from surgical management; 2) are symptomatic but unsuitable for SAVR but might safely undergo TAVR (the alternative being medical therapy); or 3) are symptomatic and will undergo SAVR; a proportion of whom (particularly due to high-to-medium surgical risk) may benefit from TAVR. The range of derived estimates for each parameter, despite efforts to smooth-out inevitable heterogeneity, are indicative of the methodological weaknesses/biases inherent to source data. It is important to note these treatment pathways are continually evolving as new, lower risk valve interventions are developed and lower the likely threshold of AVR. Without Australian-specific to correct the rates of intervention applied, it is generally acknowledged that intervention rates for severe AS broadly follow those applied in North America.\nWhere possible, we were able to improve our assumptions and therefore our projections by considering primary profiling and outcome data derived from (NEDA) [3]. This unique study has now captured echocardiographic data on > 750,000 Australians (with no exclusion criteria) being routinely investigated with echocardiography from > 25 centres Australia-wide. With individual data linkage, NEDA also generates real-world, short- to long-term survival data on those affected by common cardiac conditions including AS [3].\nEstimating the prevalence & treatable burden of severe AS For these analyses, we applied a point prevalence of 3.5 % for severe AS among individuals aged ≥ 75 years, This small adjustment to the original European model [12] reflects recent reports of an increasing incidence of AS in other high-income countries such as the UK [15] and is only slightly higher than that of the NEDA cohort of actively investigated patients [3]. To derive specific prevalence rates for those aged < 75 years, we used the ratio of cases of severe AS observed in each age-band of the NEDA cohort [3]. This resulted in the following age-specific prevalence estimates being applied: ≥75 years, 3.5 %; 70–74 years, 1.2 %; 65–69 years, 0.7 %; 60–64 years, 0.5 %; and 55–59 years, 0.4 %. When applied to the Australian population aged ≥ 55 years (see Supplementary Figure S1), the overall estimated point prevalence of severe AS is 1.48 % for 2019; a figure that is broadly consistent with that published and applied previously [16]. The same rates were applied to men and women given that, on an age-specific basis, there were minimum differences based on sex within the NEDA cohort [3].\nFor these analyses, we applied a point prevalence of 3.5 % for severe AS among individuals aged ≥ 75 years, This small adjustment to the original European model [12] reflects recent reports of an increasing incidence of AS in other high-income countries such as the UK [15] and is only slightly higher than that of the NEDA cohort of actively investigated patients [3]. To derive specific prevalence rates for those aged < 75 years, we used the ratio of cases of severe AS observed in each age-band of the NEDA cohort [3]. This resulted in the following age-specific prevalence estimates being applied: ≥75 years, 3.5 %; 70–74 years, 1.2 %; 65–69 years, 0.7 %; 60–64 years, 0.5 %; and 55–59 years, 0.4 %. When applied to the Australian population aged ≥ 55 years (see Supplementary Figure S1), the overall estimated point prevalence of severe AS is 1.48 % for 2019; a figure that is broadly consistent with that published and applied previously [16]. The same rates were applied to men and women given that, on an age-specific basis, there were minimum differences based on sex within the NEDA cohort [3].\nMortality To understand the potential consequences of no AVR intervention in the setting of severe AS, age- and sex-specific, actual 5-year mortality rates were applied to the estimated case prevalence of severe AS by applying those observed rates within the equivalent patient cohort identified within NEDA who had a native AV throughout follow-up [3]. Although these data only reflect cases referred for echocardiography, they provide both a discussion point and means to validate prevalence estimates when combined with incidence rates (see below).\nTo understand the potential consequences of no AVR intervention in the setting of severe AS, age- and sex-specific, actual 5-year mortality rates were applied to the estimated case prevalence of severe AS by applying those observed rates within the equivalent patient cohort identified within NEDA who had a native AV throughout follow-up [3]. Although these data only reflect cases referred for echocardiography, they provide both a discussion point and means to validate prevalence estimates when combined with incidence rates (see below).\nIncident cases of severe AS In the absence of specific population incidence data, we used the differential in prevalence for each 5-year age group (i.e., how many more at risk individuals would develop severe AS over 5-years to reflect the number of prevalent cases in that older age group) to calculate how many individuals would develop severe AS each year. By necessity this means incident cases were only calculated for those aged ≥ 60 years. Applying conservatively derived data from the NEDA cohort [3], the following age-specific, annual incident rates, were applied to the Australian population aged ≥ 60 years for the subsequent (i.e. beyond 2019) 5-year period 2020-24: aged ≥ 75 years, 460 cases; 70–74 years, 40 cases; 65–69 years, 40 cases; and 60–64 years, 20 cases per 100,000 population per annum. When combined, these rates generated an annual incidence of severe AS of 182 cases per 100,000 population per annum within the Australian population aged ≥ 60 years.\nIn the absence of specific population incidence data, we used the differential in prevalence for each 5-year age group (i.e., how many more at risk individuals would develop severe AS over 5-years to reflect the number of prevalent cases in that older age group) to calculate how many individuals would develop severe AS each year. By necessity this means incident cases were only calculated for those aged ≥ 60 years. Applying conservatively derived data from the NEDA cohort [3], the following age-specific, annual incident rates, were applied to the Australian population aged ≥ 60 years for the subsequent (i.e. beyond 2019) 5-year period 2020-24: aged ≥ 75 years, 460 cases; 70–74 years, 40 cases; 65–69 years, 40 cases; and 60–64 years, 20 cases per 100,000 population per annum. When combined, these rates generated an annual incidence of severe AS of 182 cases per 100,000 population per annum within the Australian population aged ≥ 60 years.\nStatistical analyses All projections are reported as whole numbers and proportions with 95 % CI where appropriate. All statistical analyses are descriptive in nature and population-based; with no inferential statistics applied. Exact estimates are provided in the figures whilst rounded up (to the nearest hundred) figures are provided in text.\nAll projections are reported as whole numbers and proportions with 95 % CI where appropriate. All statistical analyses are descriptive in nature and population-based; with no inferential statistics applied. Exact estimates are provided in the figures whilst rounded up (to the nearest hundred) figures are provided in text.","Consistent with previous reports of this type specifically focusing on atrial fibrillation [13] and heart failure [14], we combined official national population data with the best available epidemiological and clinical data to generate estimates of the treatable burden of disease relating to AS. Given the anonymous source and nature of the study data, no ethical approvals were required.","Projections were applied to the entire Australian population and then each State and Territory according to their currently estimated demographic profile. ABS data projecting the future demographic structure and profile were also used to derive future projections on the treatable burden of AS. (https://www.abs.gov.au/Population - Accessed January 2021).","The primary analyses presented in this report used age-, sex- and geographic-specific demography data for Australian men and women aged ≥ 55 years for the calendar year 2019. Specifically, the ABS currently provides population projections derived from the official 2016 Census - http://www.abs.gov.au/ausstats/abs@.nsf/mf/3101.0 - Accessed January 2021).\nAs indicated above, the primary basis for this report is the recently updated study of the number of treatable and treated patients with severe AS published by Durko and colleagues in 2018 [12]. Specifically, based on an expanded analysis of 37 relevant studies involving 26,402 cases/patients, this study provides a flow-chart (see Fig. 1) of the key estimates/parameters (with 95 % confidence interval, CI) that can be applied (as a primary analysis) to the latest Australian population data (on an age-, sex- and geographic-specific basis) to estimate the pool of treatable severe AS patients per annum. It can also be applied to broad population projections for an increasing pool of older Australians over time; noting that over a decade (from 2006 to 2016) the proportion of Australians aged > 65 years had increased by around 4 % and this proportion (and absolute numbers) will steadily increase.\nFig. 1Model/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\nModel/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\nSpecifically, the model developed and subsequently applied in this report provides overall estimates (based on a combination of European and North American cohort studies) on the proportion of patients with severe AS who – 1) are symptomatic and under current expert guidelines, are largely excluded from surgical management; 2) are symptomatic but unsuitable for SAVR but might safely undergo TAVR (the alternative being medical therapy); or 3) are symptomatic and will undergo SAVR; a proportion of whom (particularly due to high-to-medium surgical risk) may benefit from TAVR. The range of derived estimates for each parameter, despite efforts to smooth-out inevitable heterogeneity, are indicative of the methodological weaknesses/biases inherent to source data. It is important to note these treatment pathways are continually evolving as new, lower risk valve interventions are developed and lower the likely threshold of AVR. Without Australian-specific to correct the rates of intervention applied, it is generally acknowledged that intervention rates for severe AS broadly follow those applied in North America.\nWhere possible, we were able to improve our assumptions and therefore our projections by considering primary profiling and outcome data derived from (NEDA) [3]. This unique study has now captured echocardiographic data on > 750,000 Australians (with no exclusion criteria) being routinely investigated with echocardiography from > 25 centres Australia-wide. With individual data linkage, NEDA also generates real-world, short- to long-term survival data on those affected by common cardiac conditions including AS [3].","For these analyses, we applied a point prevalence of 3.5 % for severe AS among individuals aged ≥ 75 years, This small adjustment to the original European model [12] reflects recent reports of an increasing incidence of AS in other high-income countries such as the UK [15] and is only slightly higher than that of the NEDA cohort of actively investigated patients [3]. To derive specific prevalence rates for those aged < 75 years, we used the ratio of cases of severe AS observed in each age-band of the NEDA cohort [3]. This resulted in the following age-specific prevalence estimates being applied: ≥75 years, 3.5 %; 70–74 years, 1.2 %; 65–69 years, 0.7 %; 60–64 years, 0.5 %; and 55–59 years, 0.4 %. When applied to the Australian population aged ≥ 55 years (see Supplementary Figure S1), the overall estimated point prevalence of severe AS is 1.48 % for 2019; a figure that is broadly consistent with that published and applied previously [16]. The same rates were applied to men and women given that, on an age-specific basis, there were minimum differences based on sex within the NEDA cohort [3].","To understand the potential consequences of no AVR intervention in the setting of severe AS, age- and sex-specific, actual 5-year mortality rates were applied to the estimated case prevalence of severe AS by applying those observed rates within the equivalent patient cohort identified within NEDA who had a native AV throughout follow-up [3]. Although these data only reflect cases referred for echocardiography, they provide both a discussion point and means to validate prevalence estimates when combined with incidence rates (see below).","In the absence of specific population incidence data, we used the differential in prevalence for each 5-year age group (i.e., how many more at risk individuals would develop severe AS over 5-years to reflect the number of prevalent cases in that older age group) to calculate how many individuals would develop severe AS each year. By necessity this means incident cases were only calculated for those aged ≥ 60 years. Applying conservatively derived data from the NEDA cohort [3], the following age-specific, annual incident rates, were applied to the Australian population aged ≥ 60 years for the subsequent (i.e. beyond 2019) 5-year period 2020-24: aged ≥ 75 years, 460 cases; 70–74 years, 40 cases; 65–69 years, 40 cases; and 60–64 years, 20 cases per 100,000 population per annum. When combined, these rates generated an annual incidence of severe AS of 182 cases per 100,000 population per annum within the Australian population aged ≥ 60 years.","All projections are reported as whole numbers and proportions with 95 % CI where appropriate. All statistical analyses are descriptive in nature and population-based; with no inferential statistics applied. Exact estimates are provided in the figures whilst rounded up (to the nearest hundred) figures are provided in text.","As shown in Fig. 2, we conservatively estimate that around 97,300 Australians are living with severe AS (symptomatic or otherwise). Moreover, assuming just over two-thirds of these cases experience concurrent symptoms linked to the condition, according to contemporary clinical recommendations/ best practice around 66,500 (95 % CI 59,200 to 74,000) people might be considered for an AVR procedure at any one time.\nFig. 2Estimated Point Prevalence and Distribution of Severe AS in Australia\nEstimated Point Prevalence and Distribution of Severe AS in Australia","Figure 3 summarises the overall treatable burden/management of severe AS when assuming 58.4 % of cases with symptomatic, severe AS would be referred for SAVR (primary replacement or revision) and the remainder (41.6 %) for potential TAVR. On this basis, we estimate that around 38,800 (95 % CI 35,700 to 42,000) Australians aged ≥ 55 years with severe, symptomatic AS might be considered for SAVR; based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score [17]. An additional 17,000 cases (95 % CI 11,600 to 22,600) of the approximately 27,700 people not eligible for SAVR due to high peri-operative risk could be potentially considered for TAVR instead.\nFig. 3Estimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\nEstimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)","Based on the age- and sex-specific rates of actual mortality (ranging from 17 to 84 % for those aged 55–64 years to ≥ 85 years) observed in the NEDA cohort, we estimate that 56,300 (95 % CI 56,100 to 56,500) of the prevalent population with severe AS of their native valve will subsequently die within the next 5 years - see Fig. 4.\nFig. 4Severe AS Related Mortality\nSevere AS Related Mortality","As shown in Fig. 5, each year, we further estimate that around 9,300 (95 % CI 9,000 to 9,500) more Australians aged ≥ 60 years will subsequently develop severe AS. Assuming the same pattern of potential AVR procedures (once again based on peri-operative risk status), 3,700 (95 % CI 3,400 to 4,000) and 2,600 (95 % CI 2,300 to 2,900) of the approximate de novo 6,300 (95 % CI 5,600 to 7,000) cases with concurrent symptoms linked to the condition, would be potentially managed with/eligible for a SAVR or TAVR procedure, respectively (see Supplementary Figure S2).\nFig. 5Annual Incident Cases of Severe AS among Australians Aged ≥55 years\nAnnual Incident Cases of Severe AS among Australians Aged ≥55 years","As noted, these data do not completely rely on Australian specific data (other than population estimates/demographic structure and NEDA-derived adjustments to original estimates). To partially address this, we have used a conservative estimate of the point prevalence of severe AS and provide 95 % CI for lower and higher and estimates. However, we acknowledge the dynamics of medical management of severe AS (i.e., conservative treatment versus TAVR versus SAVR) in Australia is likely to be different than reported in Europe/North America. The differential clinical uptake and reimbursement of different procedures from a public health to private health perspective within Australia’s increasingly complex and hybrid health care system, is particularly relevant - as is the variable population dynamics of each jurisdiction across the country. Beyond the broader demographic features of Australia and its major jurisdictions, we did not consider other important factors such as socio-economic status and the concentration of particularly high-risk groups (e.g. predominantly younger Indigenous Australians in Central Australia who experience much higher levels of valvular disease and heart failure [25]); nor did we consider the cost-burden of treating severe AS. Applying the growing resources of NEDA, we hope to address most of these issues/limitations in the future, in order to provide greater clarity around the full spectrum of AS in Australia. Finally, it is important to acknowledge that our mortality estimates (even when considering that they are focussed on those with native valves) are discordant with the low rates of mortality reported in trials such as the PARTNER 3 Trial [9]. This discordance only reinforces the benefits of early recognition and expert management of this otherwise deadly condition.","\nAdditional file 1: Supplementary Figure 1. Population Distribution of Australians Aged ≥55 years. Legend: Australia is a federated country, comprising the main populated States (Northern Territory and Capital Territory not shown) of Western Australian (WA), South Australia (SA), Victoria (VIC), Tasmania (TAS), New South Wales (NSW) and Queensland (QLD).\nAdditional file 2: Supplementary Figure 2. Estimated Treatable Burden/Management of Severe AS in Australia (Based on Incident Cases per Annum).\n\nAdditional file 1: Supplementary Figure 1. Population Distribution of Australians Aged ≥55 years. Legend: Australia is a federated country, comprising the main populated States (Northern Territory and Capital Territory not shown) of Western Australian (WA), South Australia (SA), Victoria (VIC), Tasmania (TAS), New South Wales (NSW) and Queensland (QLD).\nAdditional file 2: Supplementary Figure 2. Estimated Treatable Burden/Management of Severe AS in Australia (Based on Incident Cases per Annum)."],"string":"[\n \"We sought, for the first time, to generate reliable (but inherently conservative) estimates on the prevalence and treatable patient population with severe, symptomatic AS in Australia. Specifically, our aim was to replicate the same robust models recently used to generate contemporary AS-specific projections for Europe and North America [12] as highlighted above with specific modifications relevant to the Australian context. Firstly, this included applying population profiling (denominator) data derived from the Australian Bureau of Statistics (ABS). Secondly, given the Australian context, we aimed to expand our burden of disease estimates to those aged ≥ 55 years (noting the original study applied a single estimate of the overall prevalence of severe AS (numerator) to those aged ≥ 75 years [12]) and by applying age- and sex-specific, incident, and prevalent estimates of AS informed by the recent NEDA Study of AS [3].\",\n \"Study design Consistent with previous reports of this type specifically focusing on atrial fibrillation [13] and heart failure [14], we combined official national population data with the best available epidemiological and clinical data to generate estimates of the treatable burden of disease relating to AS. Given the anonymous source and nature of the study data, no ethical approvals were required.\\nConsistent with previous reports of this type specifically focusing on atrial fibrillation [13] and heart failure [14], we combined official national population data with the best available epidemiological and clinical data to generate estimates of the treatable burden of disease relating to AS. Given the anonymous source and nature of the study data, no ethical approvals were required.\\nStudy setting Projections were applied to the entire Australian population and then each State and Territory according to their currently estimated demographic profile. ABS data projecting the future demographic structure and profile were also used to derive future projections on the treatable burden of AS. (https://www.abs.gov.au/Population - Accessed January 2021).\\nProjections were applied to the entire Australian population and then each State and Territory according to their currently estimated demographic profile. ABS data projecting the future demographic structure and profile were also used to derive future projections on the treatable burden of AS. (https://www.abs.gov.au/Population - Accessed January 2021).\\nStudy data The primary analyses presented in this report used age-, sex- and geographic-specific demography data for Australian men and women aged ≥ 55 years for the calendar year 2019. Specifically, the ABS currently provides population projections derived from the official 2016 Census - http://www.abs.gov.au/ausstats/abs@.nsf/mf/3101.0 - Accessed January 2021).\\nAs indicated above, the primary basis for this report is the recently updated study of the number of treatable and treated patients with severe AS published by Durko and colleagues in 2018 [12]. Specifically, based on an expanded analysis of 37 relevant studies involving 26,402 cases/patients, this study provides a flow-chart (see Fig. 1) of the key estimates/parameters (with 95 % confidence interval, CI) that can be applied (as a primary analysis) to the latest Australian population data (on an age-, sex- and geographic-specific basis) to estimate the pool of treatable severe AS patients per annum. It can also be applied to broad population projections for an increasing pool of older Australians over time; noting that over a decade (from 2006 to 2016) the proportion of Australians aged > 65 years had increased by around 4 % and this proportion (and absolute numbers) will steadily increase.\\nFig. 1Model/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\\nModel/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\\nSpecifically, the model developed and subsequently applied in this report provides overall estimates (based on a combination of European and North American cohort studies) on the proportion of patients with severe AS who – 1) are symptomatic and under current expert guidelines, are largely excluded from surgical management; 2) are symptomatic but unsuitable for SAVR but might safely undergo TAVR (the alternative being medical therapy); or 3) are symptomatic and will undergo SAVR; a proportion of whom (particularly due to high-to-medium surgical risk) may benefit from TAVR. The range of derived estimates for each parameter, despite efforts to smooth-out inevitable heterogeneity, are indicative of the methodological weaknesses/biases inherent to source data. It is important to note these treatment pathways are continually evolving as new, lower risk valve interventions are developed and lower the likely threshold of AVR. Without Australian-specific to correct the rates of intervention applied, it is generally acknowledged that intervention rates for severe AS broadly follow those applied in North America.\\nWhere possible, we were able to improve our assumptions and therefore our projections by considering primary profiling and outcome data derived from (NEDA) [3]. This unique study has now captured echocardiographic data on > 750,000 Australians (with no exclusion criteria) being routinely investigated with echocardiography from > 25 centres Australia-wide. With individual data linkage, NEDA also generates real-world, short- to long-term survival data on those affected by common cardiac conditions including AS [3].\\nThe primary analyses presented in this report used age-, sex- and geographic-specific demography data for Australian men and women aged ≥ 55 years for the calendar year 2019. Specifically, the ABS currently provides population projections derived from the official 2016 Census - http://www.abs.gov.au/ausstats/abs@.nsf/mf/3101.0 - Accessed January 2021).\\nAs indicated above, the primary basis for this report is the recently updated study of the number of treatable and treated patients with severe AS published by Durko and colleagues in 2018 [12]. Specifically, based on an expanded analysis of 37 relevant studies involving 26,402 cases/patients, this study provides a flow-chart (see Fig. 1) of the key estimates/parameters (with 95 % confidence interval, CI) that can be applied (as a primary analysis) to the latest Australian population data (on an age-, sex- and geographic-specific basis) to estimate the pool of treatable severe AS patients per annum. It can also be applied to broad population projections for an increasing pool of older Australians over time; noting that over a decade (from 2006 to 2016) the proportion of Australians aged > 65 years had increased by around 4 % and this proportion (and absolute numbers) will steadily increase.\\nFig. 1Model/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\\nModel/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\\nSpecifically, the model developed and subsequently applied in this report provides overall estimates (based on a combination of European and North American cohort studies) on the proportion of patients with severe AS who – 1) are symptomatic and under current expert guidelines, are largely excluded from surgical management; 2) are symptomatic but unsuitable for SAVR but might safely undergo TAVR (the alternative being medical therapy); or 3) are symptomatic and will undergo SAVR; a proportion of whom (particularly due to high-to-medium surgical risk) may benefit from TAVR. The range of derived estimates for each parameter, despite efforts to smooth-out inevitable heterogeneity, are indicative of the methodological weaknesses/biases inherent to source data. It is important to note these treatment pathways are continually evolving as new, lower risk valve interventions are developed and lower the likely threshold of AVR. Without Australian-specific to correct the rates of intervention applied, it is generally acknowledged that intervention rates for severe AS broadly follow those applied in North America.\\nWhere possible, we were able to improve our assumptions and therefore our projections by considering primary profiling and outcome data derived from (NEDA) [3]. This unique study has now captured echocardiographic data on > 750,000 Australians (with no exclusion criteria) being routinely investigated with echocardiography from > 25 centres Australia-wide. With individual data linkage, NEDA also generates real-world, short- to long-term survival data on those affected by common cardiac conditions including AS [3].\\nEstimating the prevalence & treatable burden of severe AS For these analyses, we applied a point prevalence of 3.5 % for severe AS among individuals aged ≥ 75 years, This small adjustment to the original European model [12] reflects recent reports of an increasing incidence of AS in other high-income countries such as the UK [15] and is only slightly higher than that of the NEDA cohort of actively investigated patients [3]. To derive specific prevalence rates for those aged < 75 years, we used the ratio of cases of severe AS observed in each age-band of the NEDA cohort [3]. This resulted in the following age-specific prevalence estimates being applied: ≥75 years, 3.5 %; 70–74 years, 1.2 %; 65–69 years, 0.7 %; 60–64 years, 0.5 %; and 55–59 years, 0.4 %. When applied to the Australian population aged ≥ 55 years (see Supplementary Figure S1), the overall estimated point prevalence of severe AS is 1.48 % for 2019; a figure that is broadly consistent with that published and applied previously [16]. The same rates were applied to men and women given that, on an age-specific basis, there were minimum differences based on sex within the NEDA cohort [3].\\nFor these analyses, we applied a point prevalence of 3.5 % for severe AS among individuals aged ≥ 75 years, This small adjustment to the original European model [12] reflects recent reports of an increasing incidence of AS in other high-income countries such as the UK [15] and is only slightly higher than that of the NEDA cohort of actively investigated patients [3]. To derive specific prevalence rates for those aged < 75 years, we used the ratio of cases of severe AS observed in each age-band of the NEDA cohort [3]. This resulted in the following age-specific prevalence estimates being applied: ≥75 years, 3.5 %; 70–74 years, 1.2 %; 65–69 years, 0.7 %; 60–64 years, 0.5 %; and 55–59 years, 0.4 %. When applied to the Australian population aged ≥ 55 years (see Supplementary Figure S1), the overall estimated point prevalence of severe AS is 1.48 % for 2019; a figure that is broadly consistent with that published and applied previously [16]. The same rates were applied to men and women given that, on an age-specific basis, there were minimum differences based on sex within the NEDA cohort [3].\\nMortality To understand the potential consequences of no AVR intervention in the setting of severe AS, age- and sex-specific, actual 5-year mortality rates were applied to the estimated case prevalence of severe AS by applying those observed rates within the equivalent patient cohort identified within NEDA who had a native AV throughout follow-up [3]. Although these data only reflect cases referred for echocardiography, they provide both a discussion point and means to validate prevalence estimates when combined with incidence rates (see below).\\nTo understand the potential consequences of no AVR intervention in the setting of severe AS, age- and sex-specific, actual 5-year mortality rates were applied to the estimated case prevalence of severe AS by applying those observed rates within the equivalent patient cohort identified within NEDA who had a native AV throughout follow-up [3]. Although these data only reflect cases referred for echocardiography, they provide both a discussion point and means to validate prevalence estimates when combined with incidence rates (see below).\\nIncident cases of severe AS In the absence of specific population incidence data, we used the differential in prevalence for each 5-year age group (i.e., how many more at risk individuals would develop severe AS over 5-years to reflect the number of prevalent cases in that older age group) to calculate how many individuals would develop severe AS each year. By necessity this means incident cases were only calculated for those aged ≥ 60 years. Applying conservatively derived data from the NEDA cohort [3], the following age-specific, annual incident rates, were applied to the Australian population aged ≥ 60 years for the subsequent (i.e. beyond 2019) 5-year period 2020-24: aged ≥ 75 years, 460 cases; 70–74 years, 40 cases; 65–69 years, 40 cases; and 60–64 years, 20 cases per 100,000 population per annum. When combined, these rates generated an annual incidence of severe AS of 182 cases per 100,000 population per annum within the Australian population aged ≥ 60 years.\\nIn the absence of specific population incidence data, we used the differential in prevalence for each 5-year age group (i.e., how many more at risk individuals would develop severe AS over 5-years to reflect the number of prevalent cases in that older age group) to calculate how many individuals would develop severe AS each year. By necessity this means incident cases were only calculated for those aged ≥ 60 years. Applying conservatively derived data from the NEDA cohort [3], the following age-specific, annual incident rates, were applied to the Australian population aged ≥ 60 years for the subsequent (i.e. beyond 2019) 5-year period 2020-24: aged ≥ 75 years, 460 cases; 70–74 years, 40 cases; 65–69 years, 40 cases; and 60–64 years, 20 cases per 100,000 population per annum. When combined, these rates generated an annual incidence of severe AS of 182 cases per 100,000 population per annum within the Australian population aged ≥ 60 years.\\nStatistical analyses All projections are reported as whole numbers and proportions with 95 % CI where appropriate. All statistical analyses are descriptive in nature and population-based; with no inferential statistics applied. Exact estimates are provided in the figures whilst rounded up (to the nearest hundred) figures are provided in text.\\nAll projections are reported as whole numbers and proportions with 95 % CI where appropriate. All statistical analyses are descriptive in nature and population-based; with no inferential statistics applied. Exact estimates are provided in the figures whilst rounded up (to the nearest hundred) figures are provided in text.\",\n \"Consistent with previous reports of this type specifically focusing on atrial fibrillation [13] and heart failure [14], we combined official national population data with the best available epidemiological and clinical data to generate estimates of the treatable burden of disease relating to AS. Given the anonymous source and nature of the study data, no ethical approvals were required.\",\n \"Projections were applied to the entire Australian population and then each State and Territory according to their currently estimated demographic profile. ABS data projecting the future demographic structure and profile were also used to derive future projections on the treatable burden of AS. (https://www.abs.gov.au/Population - Accessed January 2021).\",\n \"The primary analyses presented in this report used age-, sex- and geographic-specific demography data for Australian men and women aged ≥ 55 years for the calendar year 2019. Specifically, the ABS currently provides population projections derived from the official 2016 Census - http://www.abs.gov.au/ausstats/abs@.nsf/mf/3101.0 - Accessed January 2021).\\nAs indicated above, the primary basis for this report is the recently updated study of the number of treatable and treated patients with severe AS published by Durko and colleagues in 2018 [12]. Specifically, based on an expanded analysis of 37 relevant studies involving 26,402 cases/patients, this study provides a flow-chart (see Fig. 1) of the key estimates/parameters (with 95 % confidence interval, CI) that can be applied (as a primary analysis) to the latest Australian population data (on an age-, sex- and geographic-specific basis) to estimate the pool of treatable severe AS patients per annum. It can also be applied to broad population projections for an increasing pool of older Australians over time; noting that over a decade (from 2006 to 2016) the proportion of Australians aged > 65 years had increased by around 4 % and this proportion (and absolute numbers) will steadily increase.\\nFig. 1Model/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\\nModel/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\\nSpecifically, the model developed and subsequently applied in this report provides overall estimates (based on a combination of European and North American cohort studies) on the proportion of patients with severe AS who – 1) are symptomatic and under current expert guidelines, are largely excluded from surgical management; 2) are symptomatic but unsuitable for SAVR but might safely undergo TAVR (the alternative being medical therapy); or 3) are symptomatic and will undergo SAVR; a proportion of whom (particularly due to high-to-medium surgical risk) may benefit from TAVR. The range of derived estimates for each parameter, despite efforts to smooth-out inevitable heterogeneity, are indicative of the methodological weaknesses/biases inherent to source data. It is important to note these treatment pathways are continually evolving as new, lower risk valve interventions are developed and lower the likely threshold of AVR. Without Australian-specific to correct the rates of intervention applied, it is generally acknowledged that intervention rates for severe AS broadly follow those applied in North America.\\nWhere possible, we were able to improve our assumptions and therefore our projections by considering primary profiling and outcome data derived from (NEDA) [3]. This unique study has now captured echocardiographic data on > 750,000 Australians (with no exclusion criteria) being routinely investigated with echocardiography from > 25 centres Australia-wide. With individual data linkage, NEDA also generates real-world, short- to long-term survival data on those affected by common cardiac conditions including AS [3].\",\n \"For these analyses, we applied a point prevalence of 3.5 % for severe AS among individuals aged ≥ 75 years, This small adjustment to the original European model [12] reflects recent reports of an increasing incidence of AS in other high-income countries such as the UK [15] and is only slightly higher than that of the NEDA cohort of actively investigated patients [3]. To derive specific prevalence rates for those aged < 75 years, we used the ratio of cases of severe AS observed in each age-band of the NEDA cohort [3]. This resulted in the following age-specific prevalence estimates being applied: ≥75 years, 3.5 %; 70–74 years, 1.2 %; 65–69 years, 0.7 %; 60–64 years, 0.5 %; and 55–59 years, 0.4 %. When applied to the Australian population aged ≥ 55 years (see Supplementary Figure S1), the overall estimated point prevalence of severe AS is 1.48 % for 2019; a figure that is broadly consistent with that published and applied previously [16]. The same rates were applied to men and women given that, on an age-specific basis, there were minimum differences based on sex within the NEDA cohort [3].\",\n \"To understand the potential consequences of no AVR intervention in the setting of severe AS, age- and sex-specific, actual 5-year mortality rates were applied to the estimated case prevalence of severe AS by applying those observed rates within the equivalent patient cohort identified within NEDA who had a native AV throughout follow-up [3]. Although these data only reflect cases referred for echocardiography, they provide both a discussion point and means to validate prevalence estimates when combined with incidence rates (see below).\",\n \"In the absence of specific population incidence data, we used the differential in prevalence for each 5-year age group (i.e., how many more at risk individuals would develop severe AS over 5-years to reflect the number of prevalent cases in that older age group) to calculate how many individuals would develop severe AS each year. By necessity this means incident cases were only calculated for those aged ≥ 60 years. Applying conservatively derived data from the NEDA cohort [3], the following age-specific, annual incident rates, were applied to the Australian population aged ≥ 60 years for the subsequent (i.e. beyond 2019) 5-year period 2020-24: aged ≥ 75 years, 460 cases; 70–74 years, 40 cases; 65–69 years, 40 cases; and 60–64 years, 20 cases per 100,000 population per annum. When combined, these rates generated an annual incidence of severe AS of 182 cases per 100,000 population per annum within the Australian population aged ≥ 60 years.\",\n \"All projections are reported as whole numbers and proportions with 95 % CI where appropriate. All statistical analyses are descriptive in nature and population-based; with no inferential statistics applied. Exact estimates are provided in the figures whilst rounded up (to the nearest hundred) figures are provided in text.\",\n \"As shown in Fig. 2, we conservatively estimate that around 97,300 Australians are living with severe AS (symptomatic or otherwise). Moreover, assuming just over two-thirds of these cases experience concurrent symptoms linked to the condition, according to contemporary clinical recommendations/ best practice around 66,500 (95 % CI 59,200 to 74,000) people might be considered for an AVR procedure at any one time.\\nFig. 2Estimated Point Prevalence and Distribution of Severe AS in Australia\\nEstimated Point Prevalence and Distribution of Severe AS in Australia\",\n \"Figure 3 summarises the overall treatable burden/management of severe AS when assuming 58.4 % of cases with symptomatic, severe AS would be referred for SAVR (primary replacement or revision) and the remainder (41.6 %) for potential TAVR. On this basis, we estimate that around 38,800 (95 % CI 35,700 to 42,000) Australians aged ≥ 55 years with severe, symptomatic AS might be considered for SAVR; based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score [17]. An additional 17,000 cases (95 % CI 11,600 to 22,600) of the approximately 27,700 people not eligible for SAVR due to high peri-operative risk could be potentially considered for TAVR instead.\\nFig. 3Estimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\\nEstimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\",\n \"Based on the age- and sex-specific rates of actual mortality (ranging from 17 to 84 % for those aged 55–64 years to ≥ 85 years) observed in the NEDA cohort, we estimate that 56,300 (95 % CI 56,100 to 56,500) of the prevalent population with severe AS of their native valve will subsequently die within the next 5 years - see Fig. 4.\\nFig. 4Severe AS Related Mortality\\nSevere AS Related Mortality\",\n \"As shown in Fig. 5, each year, we further estimate that around 9,300 (95 % CI 9,000 to 9,500) more Australians aged ≥ 60 years will subsequently develop severe AS. Assuming the same pattern of potential AVR procedures (once again based on peri-operative risk status), 3,700 (95 % CI 3,400 to 4,000) and 2,600 (95 % CI 2,300 to 2,900) of the approximate de novo 6,300 (95 % CI 5,600 to 7,000) cases with concurrent symptoms linked to the condition, would be potentially managed with/eligible for a SAVR or TAVR procedure, respectively (see Supplementary Figure S2).\\nFig. 5Annual Incident Cases of Severe AS among Australians Aged ≥55 years\\nAnnual Incident Cases of Severe AS among Australians Aged ≥55 years\",\n \"As noted, these data do not completely rely on Australian specific data (other than population estimates/demographic structure and NEDA-derived adjustments to original estimates). To partially address this, we have used a conservative estimate of the point prevalence of severe AS and provide 95 % CI for lower and higher and estimates. However, we acknowledge the dynamics of medical management of severe AS (i.e., conservative treatment versus TAVR versus SAVR) in Australia is likely to be different than reported in Europe/North America. The differential clinical uptake and reimbursement of different procedures from a public health to private health perspective within Australia’s increasingly complex and hybrid health care system, is particularly relevant - as is the variable population dynamics of each jurisdiction across the country. Beyond the broader demographic features of Australia and its major jurisdictions, we did not consider other important factors such as socio-economic status and the concentration of particularly high-risk groups (e.g. predominantly younger Indigenous Australians in Central Australia who experience much higher levels of valvular disease and heart failure [25]); nor did we consider the cost-burden of treating severe AS. Applying the growing resources of NEDA, we hope to address most of these issues/limitations in the future, in order to provide greater clarity around the full spectrum of AS in Australia. Finally, it is important to acknowledge that our mortality estimates (even when considering that they are focussed on those with native valves) are discordant with the low rates of mortality reported in trials such as the PARTNER 3 Trial [9]. This discordance only reinforces the benefits of early recognition and expert management of this otherwise deadly condition.\",\n \"\\nAdditional file 1: Supplementary Figure 1. Population Distribution of Australians Aged ≥55 years. Legend: Australia is a federated country, comprising the main populated States (Northern Territory and Capital Territory not shown) of Western Australian (WA), South Australia (SA), Victoria (VIC), Tasmania (TAS), New South Wales (NSW) and Queensland (QLD).\\nAdditional file 2: Supplementary Figure 2. Estimated Treatable Burden/Management of Severe AS in Australia (Based on Incident Cases per Annum).\\n\\nAdditional file 1: Supplementary Figure 1. Population Distribution of Australians Aged ≥55 years. Legend: Australia is a federated country, comprising the main populated States (Northern Territory and Capital Territory not shown) of Western Australian (WA), South Australia (SA), Victoria (VIC), Tasmania (TAS), New South Wales (NSW) and Queensland (QLD).\\nAdditional file 2: Supplementary Figure 2. Estimated Treatable Burden/Management of Severe AS in Australia (Based on Incident Cases per Annum).\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Introduction","Study aims","Methods","Study design","Study setting","Study data","Estimating the prevalence & treatable burden of severe AS","Mortality","Incident cases of severe AS","Statistical analyses","Results","Prevalent cases of severe AS","Aortic valve replacement","AS-related mortality","Incident cases","Discussion","Limitations","Conclusions","Supplementary Information",""],"string":"[\n \"Introduction\",\n \"Study aims\",\n \"Methods\",\n \"Study design\",\n \"Study setting\",\n \"Study data\",\n \"Estimating the prevalence & treatable burden of severe AS\",\n \"Mortality\",\n \"Incident cases of severe AS\",\n \"Statistical analyses\",\n \"Results\",\n \"Prevalent cases of severe AS\",\n \"Aortic valve replacement\",\n \"AS-related mortality\",\n \"Incident cases\",\n \"Discussion\",\n \"Limitations\",\n \"Conclusions\",\n \"Supplementary Information\",\n \"\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["One of the most common cardiac conditions affecting the progressively aging populations of high-income countries such as Australia is aortic stenosis (AS) [1]. Without timely intervention, severe AS is associated with a very poor prognosis [2]. However, like many other countries, there has been a paucity of reports focusing on the overall prevalence and treatable burden of AS in Australia. A recent AS report from National Echocardiography Database of Australia (NEDA) [3] that assessed the severity of AS and subsequent pattern of survival among 122,809 men and 118,494 women with a mean age of 62 ± 18 years highlighted an urgent need to better understand the burden imposed by this potentially deadly condition. Overall, the indicative prevalence of severe low or high gradient AS among adults being investigated with echocardiography during the overall study period of 2000–2019, was 1.1 and 2.1 % (3.2 % combined), respectively. Actual 5-year mortality ranged from 56 to 67 % in those cases with a native aortic valve and no indication of surgical intervention [3].\nHistorically, surgical aortic valve replacement (SAVR) has been the preferred intervention for severe AS [4]. However, transcatheter aortic valve replacement (TAVR) has been successfully applied in those with severe AS with high/prohibitive surgical risk [5–7]. Moreover, two head-to-head randomized trials have now reported non-inferiority [8] and superiority [9] comparing TAVR to SAVR in low-risk patients with severe AS, in respect to mortality and subsequent risk of stroke. Consistent with these data, for most of the 6,050 cases within the NEDA cohort who underwent AVR, their post-procedure AV hemodynamic profile and survival outcomes were favourable [10].\nOverall, these data suggest more Australians might benefit from AVR. However, without reliable estimates of the treatable burden of AS, this critical number (for health service and resource planning) remains unknown. A series of modelled studies, first published in 2013 [11] and then an updated version in 2018 [12], applied the best available epidemiological and registry data to estimate the following for Europe and North America – 1) the overall proportion of older individuals affected by AS and more specifically severe AS; and 2) proportion of these individuals who had and/or would benefit from a valve replacement procedure (SAVR or TAVR). Given the geographical focus and source data used, these estimates now provide these target regions with (moderate) reliable estimates of the treatable burden of AS. To date, there are no equivalent burden of disease estimates for Australia.\nStudy aims We sought, for the first time, to generate reliable (but inherently conservative) estimates on the prevalence and treatable patient population with severe, symptomatic AS in Australia. Specifically, our aim was to replicate the same robust models recently used to generate contemporary AS-specific projections for Europe and North America [12] as highlighted above with specific modifications relevant to the Australian context. Firstly, this included applying population profiling (denominator) data derived from the Australian Bureau of Statistics (ABS). Secondly, given the Australian context, we aimed to expand our burden of disease estimates to those aged ≥ 55 years (noting the original study applied a single estimate of the overall prevalence of severe AS (numerator) to those aged ≥ 75 years [12]) and by applying age- and sex-specific, incident, and prevalent estimates of AS informed by the recent NEDA Study of AS [3].\nWe sought, for the first time, to generate reliable (but inherently conservative) estimates on the prevalence and treatable patient population with severe, symptomatic AS in Australia. Specifically, our aim was to replicate the same robust models recently used to generate contemporary AS-specific projections for Europe and North America [12] as highlighted above with specific modifications relevant to the Australian context. Firstly, this included applying population profiling (denominator) data derived from the Australian Bureau of Statistics (ABS). Secondly, given the Australian context, we aimed to expand our burden of disease estimates to those aged ≥ 55 years (noting the original study applied a single estimate of the overall prevalence of severe AS (numerator) to those aged ≥ 75 years [12]) and by applying age- and sex-specific, incident, and prevalent estimates of AS informed by the recent NEDA Study of AS [3].","We sought, for the first time, to generate reliable (but inherently conservative) estimates on the prevalence and treatable patient population with severe, symptomatic AS in Australia. Specifically, our aim was to replicate the same robust models recently used to generate contemporary AS-specific projections for Europe and North America [12] as highlighted above with specific modifications relevant to the Australian context. Firstly, this included applying population profiling (denominator) data derived from the Australian Bureau of Statistics (ABS). Secondly, given the Australian context, we aimed to expand our burden of disease estimates to those aged ≥ 55 years (noting the original study applied a single estimate of the overall prevalence of severe AS (numerator) to those aged ≥ 75 years [12]) and by applying age- and sex-specific, incident, and prevalent estimates of AS informed by the recent NEDA Study of AS [3].","Study design Consistent with previous reports of this type specifically focusing on atrial fibrillation [13] and heart failure [14], we combined official national population data with the best available epidemiological and clinical data to generate estimates of the treatable burden of disease relating to AS. Given the anonymous source and nature of the study data, no ethical approvals were required.\nConsistent with previous reports of this type specifically focusing on atrial fibrillation [13] and heart failure [14], we combined official national population data with the best available epidemiological and clinical data to generate estimates of the treatable burden of disease relating to AS. Given the anonymous source and nature of the study data, no ethical approvals were required.\nStudy setting Projections were applied to the entire Australian population and then each State and Territory according to their currently estimated demographic profile. ABS data projecting the future demographic structure and profile were also used to derive future projections on the treatable burden of AS. (https://www.abs.gov.au/Population - Accessed January 2021).\nProjections were applied to the entire Australian population and then each State and Territory according to their currently estimated demographic profile. ABS data projecting the future demographic structure and profile were also used to derive future projections on the treatable burden of AS. (https://www.abs.gov.au/Population - Accessed January 2021).\nStudy data The primary analyses presented in this report used age-, sex- and geographic-specific demography data for Australian men and women aged ≥ 55 years for the calendar year 2019. Specifically, the ABS currently provides population projections derived from the official 2016 Census - http://www.abs.gov.au/ausstats/abs@.nsf/mf/3101.0 - Accessed January 2021).\nAs indicated above, the primary basis for this report is the recently updated study of the number of treatable and treated patients with severe AS published by Durko and colleagues in 2018 [12]. Specifically, based on an expanded analysis of 37 relevant studies involving 26,402 cases/patients, this study provides a flow-chart (see Fig. 1) of the key estimates/parameters (with 95 % confidence interval, CI) that can be applied (as a primary analysis) to the latest Australian population data (on an age-, sex- and geographic-specific basis) to estimate the pool of treatable severe AS patients per annum. It can also be applied to broad population projections for an increasing pool of older Australians over time; noting that over a decade (from 2006 to 2016) the proportion of Australians aged > 65 years had increased by around 4 % and this proportion (and absolute numbers) will steadily increase.\nFig. 1Model/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\nModel/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\nSpecifically, the model developed and subsequently applied in this report provides overall estimates (based on a combination of European and North American cohort studies) on the proportion of patients with severe AS who – 1) are symptomatic and under current expert guidelines, are largely excluded from surgical management; 2) are symptomatic but unsuitable for SAVR but might safely undergo TAVR (the alternative being medical therapy); or 3) are symptomatic and will undergo SAVR; a proportion of whom (particularly due to high-to-medium surgical risk) may benefit from TAVR. The range of derived estimates for each parameter, despite efforts to smooth-out inevitable heterogeneity, are indicative of the methodological weaknesses/biases inherent to source data. It is important to note these treatment pathways are continually evolving as new, lower risk valve interventions are developed and lower the likely threshold of AVR. Without Australian-specific to correct the rates of intervention applied, it is generally acknowledged that intervention rates for severe AS broadly follow those applied in North America.\nWhere possible, we were able to improve our assumptions and therefore our projections by considering primary profiling and outcome data derived from (NEDA) [3]. This unique study has now captured echocardiographic data on > 750,000 Australians (with no exclusion criteria) being routinely investigated with echocardiography from > 25 centres Australia-wide. With individual data linkage, NEDA also generates real-world, short- to long-term survival data on those affected by common cardiac conditions including AS [3].\nThe primary analyses presented in this report used age-, sex- and geographic-specific demography data for Australian men and women aged ≥ 55 years for the calendar year 2019. Specifically, the ABS currently provides population projections derived from the official 2016 Census - http://www.abs.gov.au/ausstats/abs@.nsf/mf/3101.0 - Accessed January 2021).\nAs indicated above, the primary basis for this report is the recently updated study of the number of treatable and treated patients with severe AS published by Durko and colleagues in 2018 [12]. Specifically, based on an expanded analysis of 37 relevant studies involving 26,402 cases/patients, this study provides a flow-chart (see Fig. 1) of the key estimates/parameters (with 95 % confidence interval, CI) that can be applied (as a primary analysis) to the latest Australian population data (on an age-, sex- and geographic-specific basis) to estimate the pool of treatable severe AS patients per annum. It can also be applied to broad population projections for an increasing pool of older Australians over time; noting that over a decade (from 2006 to 2016) the proportion of Australians aged > 65 years had increased by around 4 % and this proportion (and absolute numbers) will steadily increase.\nFig. 1Model/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\nModel/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\nSpecifically, the model developed and subsequently applied in this report provides overall estimates (based on a combination of European and North American cohort studies) on the proportion of patients with severe AS who – 1) are symptomatic and under current expert guidelines, are largely excluded from surgical management; 2) are symptomatic but unsuitable for SAVR but might safely undergo TAVR (the alternative being medical therapy); or 3) are symptomatic and will undergo SAVR; a proportion of whom (particularly due to high-to-medium surgical risk) may benefit from TAVR. The range of derived estimates for each parameter, despite efforts to smooth-out inevitable heterogeneity, are indicative of the methodological weaknesses/biases inherent to source data. It is important to note these treatment pathways are continually evolving as new, lower risk valve interventions are developed and lower the likely threshold of AVR. Without Australian-specific to correct the rates of intervention applied, it is generally acknowledged that intervention rates for severe AS broadly follow those applied in North America.\nWhere possible, we were able to improve our assumptions and therefore our projections by considering primary profiling and outcome data derived from (NEDA) [3]. This unique study has now captured echocardiographic data on > 750,000 Australians (with no exclusion criteria) being routinely investigated with echocardiography from > 25 centres Australia-wide. With individual data linkage, NEDA also generates real-world, short- to long-term survival data on those affected by common cardiac conditions including AS [3].\nEstimating the prevalence & treatable burden of severe AS For these analyses, we applied a point prevalence of 3.5 % for severe AS among individuals aged ≥ 75 years, This small adjustment to the original European model [12] reflects recent reports of an increasing incidence of AS in other high-income countries such as the UK [15] and is only slightly higher than that of the NEDA cohort of actively investigated patients [3]. To derive specific prevalence rates for those aged < 75 years, we used the ratio of cases of severe AS observed in each age-band of the NEDA cohort [3]. This resulted in the following age-specific prevalence estimates being applied: ≥75 years, 3.5 %; 70–74 years, 1.2 %; 65–69 years, 0.7 %; 60–64 years, 0.5 %; and 55–59 years, 0.4 %. When applied to the Australian population aged ≥ 55 years (see Supplementary Figure S1), the overall estimated point prevalence of severe AS is 1.48 % for 2019; a figure that is broadly consistent with that published and applied previously [16]. The same rates were applied to men and women given that, on an age-specific basis, there were minimum differences based on sex within the NEDA cohort [3].\nFor these analyses, we applied a point prevalence of 3.5 % for severe AS among individuals aged ≥ 75 years, This small adjustment to the original European model [12] reflects recent reports of an increasing incidence of AS in other high-income countries such as the UK [15] and is only slightly higher than that of the NEDA cohort of actively investigated patients [3]. To derive specific prevalence rates for those aged < 75 years, we used the ratio of cases of severe AS observed in each age-band of the NEDA cohort [3]. This resulted in the following age-specific prevalence estimates being applied: ≥75 years, 3.5 %; 70–74 years, 1.2 %; 65–69 years, 0.7 %; 60–64 years, 0.5 %; and 55–59 years, 0.4 %. When applied to the Australian population aged ≥ 55 years (see Supplementary Figure S1), the overall estimated point prevalence of severe AS is 1.48 % for 2019; a figure that is broadly consistent with that published and applied previously [16]. The same rates were applied to men and women given that, on an age-specific basis, there were minimum differences based on sex within the NEDA cohort [3].\nMortality To understand the potential consequences of no AVR intervention in the setting of severe AS, age- and sex-specific, actual 5-year mortality rates were applied to the estimated case prevalence of severe AS by applying those observed rates within the equivalent patient cohort identified within NEDA who had a native AV throughout follow-up [3]. Although these data only reflect cases referred for echocardiography, they provide both a discussion point and means to validate prevalence estimates when combined with incidence rates (see below).\nTo understand the potential consequences of no AVR intervention in the setting of severe AS, age- and sex-specific, actual 5-year mortality rates were applied to the estimated case prevalence of severe AS by applying those observed rates within the equivalent patient cohort identified within NEDA who had a native AV throughout follow-up [3]. Although these data only reflect cases referred for echocardiography, they provide both a discussion point and means to validate prevalence estimates when combined with incidence rates (see below).\nIncident cases of severe AS In the absence of specific population incidence data, we used the differential in prevalence for each 5-year age group (i.e., how many more at risk individuals would develop severe AS over 5-years to reflect the number of prevalent cases in that older age group) to calculate how many individuals would develop severe AS each year. By necessity this means incident cases were only calculated for those aged ≥ 60 years. Applying conservatively derived data from the NEDA cohort [3], the following age-specific, annual incident rates, were applied to the Australian population aged ≥ 60 years for the subsequent (i.e. beyond 2019) 5-year period 2020-24: aged ≥ 75 years, 460 cases; 70–74 years, 40 cases; 65–69 years, 40 cases; and 60–64 years, 20 cases per 100,000 population per annum. When combined, these rates generated an annual incidence of severe AS of 182 cases per 100,000 population per annum within the Australian population aged ≥ 60 years.\nIn the absence of specific population incidence data, we used the differential in prevalence for each 5-year age group (i.e., how many more at risk individuals would develop severe AS over 5-years to reflect the number of prevalent cases in that older age group) to calculate how many individuals would develop severe AS each year. By necessity this means incident cases were only calculated for those aged ≥ 60 years. Applying conservatively derived data from the NEDA cohort [3], the following age-specific, annual incident rates, were applied to the Australian population aged ≥ 60 years for the subsequent (i.e. beyond 2019) 5-year period 2020-24: aged ≥ 75 years, 460 cases; 70–74 years, 40 cases; 65–69 years, 40 cases; and 60–64 years, 20 cases per 100,000 population per annum. When combined, these rates generated an annual incidence of severe AS of 182 cases per 100,000 population per annum within the Australian population aged ≥ 60 years.\nStatistical analyses All projections are reported as whole numbers and proportions with 95 % CI where appropriate. All statistical analyses are descriptive in nature and population-based; with no inferential statistics applied. Exact estimates are provided in the figures whilst rounded up (to the nearest hundred) figures are provided in text.\nAll projections are reported as whole numbers and proportions with 95 % CI where appropriate. All statistical analyses are descriptive in nature and population-based; with no inferential statistics applied. Exact estimates are provided in the figures whilst rounded up (to the nearest hundred) figures are provided in text.","Consistent with previous reports of this type specifically focusing on atrial fibrillation [13] and heart failure [14], we combined official national population data with the best available epidemiological and clinical data to generate estimates of the treatable burden of disease relating to AS. Given the anonymous source and nature of the study data, no ethical approvals were required.","Projections were applied to the entire Australian population and then each State and Territory according to their currently estimated demographic profile. ABS data projecting the future demographic structure and profile were also used to derive future projections on the treatable burden of AS. (https://www.abs.gov.au/Population - Accessed January 2021).","The primary analyses presented in this report used age-, sex- and geographic-specific demography data for Australian men and women aged ≥ 55 years for the calendar year 2019. Specifically, the ABS currently provides population projections derived from the official 2016 Census - http://www.abs.gov.au/ausstats/abs@.nsf/mf/3101.0 - Accessed January 2021).\nAs indicated above, the primary basis for this report is the recently updated study of the number of treatable and treated patients with severe AS published by Durko and colleagues in 2018 [12]. Specifically, based on an expanded analysis of 37 relevant studies involving 26,402 cases/patients, this study provides a flow-chart (see Fig. 1) of the key estimates/parameters (with 95 % confidence interval, CI) that can be applied (as a primary analysis) to the latest Australian population data (on an age-, sex- and geographic-specific basis) to estimate the pool of treatable severe AS patients per annum. It can also be applied to broad population projections for an increasing pool of older Australians over time; noting that over a decade (from 2006 to 2016) the proportion of Australians aged > 65 years had increased by around 4 % and this proportion (and absolute numbers) will steadily increase.\nFig. 1Model/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\nModel/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\nSpecifically, the model developed and subsequently applied in this report provides overall estimates (based on a combination of European and North American cohort studies) on the proportion of patients with severe AS who – 1) are symptomatic and under current expert guidelines, are largely excluded from surgical management; 2) are symptomatic but unsuitable for SAVR but might safely undergo TAVR (the alternative being medical therapy); or 3) are symptomatic and will undergo SAVR; a proportion of whom (particularly due to high-to-medium surgical risk) may benefit from TAVR. The range of derived estimates for each parameter, despite efforts to smooth-out inevitable heterogeneity, are indicative of the methodological weaknesses/biases inherent to source data. It is important to note these treatment pathways are continually evolving as new, lower risk valve interventions are developed and lower the likely threshold of AVR. Without Australian-specific to correct the rates of intervention applied, it is generally acknowledged that intervention rates for severe AS broadly follow those applied in North America.\nWhere possible, we were able to improve our assumptions and therefore our projections by considering primary profiling and outcome data derived from (NEDA) [3]. This unique study has now captured echocardiographic data on > 750,000 Australians (with no exclusion criteria) being routinely investigated with echocardiography from > 25 centres Australia-wide. With individual data linkage, NEDA also generates real-world, short- to long-term survival data on those affected by common cardiac conditions including AS [3].","For these analyses, we applied a point prevalence of 3.5 % for severe AS among individuals aged ≥ 75 years, This small adjustment to the original European model [12] reflects recent reports of an increasing incidence of AS in other high-income countries such as the UK [15] and is only slightly higher than that of the NEDA cohort of actively investigated patients [3]. To derive specific prevalence rates for those aged < 75 years, we used the ratio of cases of severe AS observed in each age-band of the NEDA cohort [3]. This resulted in the following age-specific prevalence estimates being applied: ≥75 years, 3.5 %; 70–74 years, 1.2 %; 65–69 years, 0.7 %; 60–64 years, 0.5 %; and 55–59 years, 0.4 %. When applied to the Australian population aged ≥ 55 years (see Supplementary Figure S1), the overall estimated point prevalence of severe AS is 1.48 % for 2019; a figure that is broadly consistent with that published and applied previously [16]. The same rates were applied to men and women given that, on an age-specific basis, there were minimum differences based on sex within the NEDA cohort [3].","To understand the potential consequences of no AVR intervention in the setting of severe AS, age- and sex-specific, actual 5-year mortality rates were applied to the estimated case prevalence of severe AS by applying those observed rates within the equivalent patient cohort identified within NEDA who had a native AV throughout follow-up [3]. Although these data only reflect cases referred for echocardiography, they provide both a discussion point and means to validate prevalence estimates when combined with incidence rates (see below).","In the absence of specific population incidence data, we used the differential in prevalence for each 5-year age group (i.e., how many more at risk individuals would develop severe AS over 5-years to reflect the number of prevalent cases in that older age group) to calculate how many individuals would develop severe AS each year. By necessity this means incident cases were only calculated for those aged ≥ 60 years. Applying conservatively derived data from the NEDA cohort [3], the following age-specific, annual incident rates, were applied to the Australian population aged ≥ 60 years for the subsequent (i.e. beyond 2019) 5-year period 2020-24: aged ≥ 75 years, 460 cases; 70–74 years, 40 cases; 65–69 years, 40 cases; and 60–64 years, 20 cases per 100,000 population per annum. When combined, these rates generated an annual incidence of severe AS of 182 cases per 100,000 population per annum within the Australian population aged ≥ 60 years.","All projections are reported as whole numbers and proportions with 95 % CI where appropriate. All statistical analyses are descriptive in nature and population-based; with no inferential statistics applied. Exact estimates are provided in the figures whilst rounded up (to the nearest hundred) figures are provided in text.","Prevalent cases of severe AS As shown in Fig. 2, we conservatively estimate that around 97,300 Australians are living with severe AS (symptomatic or otherwise). Moreover, assuming just over two-thirds of these cases experience concurrent symptoms linked to the condition, according to contemporary clinical recommendations/ best practice around 66,500 (95 % CI 59,200 to 74,000) people might be considered for an AVR procedure at any one time.\nFig. 2Estimated Point Prevalence and Distribution of Severe AS in Australia\nEstimated Point Prevalence and Distribution of Severe AS in Australia\nAs shown in Fig. 2, we conservatively estimate that around 97,300 Australians are living with severe AS (symptomatic or otherwise). Moreover, assuming just over two-thirds of these cases experience concurrent symptoms linked to the condition, according to contemporary clinical recommendations/ best practice around 66,500 (95 % CI 59,200 to 74,000) people might be considered for an AVR procedure at any one time.\nFig. 2Estimated Point Prevalence and Distribution of Severe AS in Australia\nEstimated Point Prevalence and Distribution of Severe AS in Australia\nAortic valve replacement Figure 3 summarises the overall treatable burden/management of severe AS when assuming 58.4 % of cases with symptomatic, severe AS would be referred for SAVR (primary replacement or revision) and the remainder (41.6 %) for potential TAVR. On this basis, we estimate that around 38,800 (95 % CI 35,700 to 42,000) Australians aged ≥ 55 years with severe, symptomatic AS might be considered for SAVR; based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score [17]. An additional 17,000 cases (95 % CI 11,600 to 22,600) of the approximately 27,700 people not eligible for SAVR due to high peri-operative risk could be potentially considered for TAVR instead.\nFig. 3Estimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\nEstimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\nFigure 3 summarises the overall treatable burden/management of severe AS when assuming 58.4 % of cases with symptomatic, severe AS would be referred for SAVR (primary replacement or revision) and the remainder (41.6 %) for potential TAVR. On this basis, we estimate that around 38,800 (95 % CI 35,700 to 42,000) Australians aged ≥ 55 years with severe, symptomatic AS might be considered for SAVR; based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score [17]. An additional 17,000 cases (95 % CI 11,600 to 22,600) of the approximately 27,700 people not eligible for SAVR due to high peri-operative risk could be potentially considered for TAVR instead.\nFig. 3Estimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\nEstimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\nAS-related mortality Based on the age- and sex-specific rates of actual mortality (ranging from 17 to 84 % for those aged 55–64 years to ≥ 85 years) observed in the NEDA cohort, we estimate that 56,300 (95 % CI 56,100 to 56,500) of the prevalent population with severe AS of their native valve will subsequently die within the next 5 years - see Fig. 4.\nFig. 4Severe AS Related Mortality\nSevere AS Related Mortality\nBased on the age- and sex-specific rates of actual mortality (ranging from 17 to 84 % for those aged 55–64 years to ≥ 85 years) observed in the NEDA cohort, we estimate that 56,300 (95 % CI 56,100 to 56,500) of the prevalent population with severe AS of their native valve will subsequently die within the next 5 years - see Fig. 4.\nFig. 4Severe AS Related Mortality\nSevere AS Related Mortality\nIncident cases As shown in Fig. 5, each year, we further estimate that around 9,300 (95 % CI 9,000 to 9,500) more Australians aged ≥ 60 years will subsequently develop severe AS. Assuming the same pattern of potential AVR procedures (once again based on peri-operative risk status), 3,700 (95 % CI 3,400 to 4,000) and 2,600 (95 % CI 2,300 to 2,900) of the approximate de novo 6,300 (95 % CI 5,600 to 7,000) cases with concurrent symptoms linked to the condition, would be potentially managed with/eligible for a SAVR or TAVR procedure, respectively (see Supplementary Figure S2).\nFig. 5Annual Incident Cases of Severe AS among Australians Aged ≥55 years\nAnnual Incident Cases of Severe AS among Australians Aged ≥55 years\nAs shown in Fig. 5, each year, we further estimate that around 9,300 (95 % CI 9,000 to 9,500) more Australians aged ≥ 60 years will subsequently develop severe AS. Assuming the same pattern of potential AVR procedures (once again based on peri-operative risk status), 3,700 (95 % CI 3,400 to 4,000) and 2,600 (95 % CI 2,300 to 2,900) of the approximate de novo 6,300 (95 % CI 5,600 to 7,000) cases with concurrent symptoms linked to the condition, would be potentially managed with/eligible for a SAVR or TAVR procedure, respectively (see Supplementary Figure S2).\nFig. 5Annual Incident Cases of Severe AS among Australians Aged ≥55 years\nAnnual Incident Cases of Severe AS among Australians Aged ≥55 years","As shown in Fig. 2, we conservatively estimate that around 97,300 Australians are living with severe AS (symptomatic or otherwise). Moreover, assuming just over two-thirds of these cases experience concurrent symptoms linked to the condition, according to contemporary clinical recommendations/ best practice around 66,500 (95 % CI 59,200 to 74,000) people might be considered for an AVR procedure at any one time.\nFig. 2Estimated Point Prevalence and Distribution of Severe AS in Australia\nEstimated Point Prevalence and Distribution of Severe AS in Australia","Figure 3 summarises the overall treatable burden/management of severe AS when assuming 58.4 % of cases with symptomatic, severe AS would be referred for SAVR (primary replacement or revision) and the remainder (41.6 %) for potential TAVR. On this basis, we estimate that around 38,800 (95 % CI 35,700 to 42,000) Australians aged ≥ 55 years with severe, symptomatic AS might be considered for SAVR; based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score [17]. An additional 17,000 cases (95 % CI 11,600 to 22,600) of the approximately 27,700 people not eligible for SAVR due to high peri-operative risk could be potentially considered for TAVR instead.\nFig. 3Estimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\nEstimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)","Based on the age- and sex-specific rates of actual mortality (ranging from 17 to 84 % for those aged 55–64 years to ≥ 85 years) observed in the NEDA cohort, we estimate that 56,300 (95 % CI 56,100 to 56,500) of the prevalent population with severe AS of their native valve will subsequently die within the next 5 years - see Fig. 4.\nFig. 4Severe AS Related Mortality\nSevere AS Related Mortality","As shown in Fig. 5, each year, we further estimate that around 9,300 (95 % CI 9,000 to 9,500) more Australians aged ≥ 60 years will subsequently develop severe AS. Assuming the same pattern of potential AVR procedures (once again based on peri-operative risk status), 3,700 (95 % CI 3,400 to 4,000) and 2,600 (95 % CI 2,300 to 2,900) of the approximate de novo 6,300 (95 % CI 5,600 to 7,000) cases with concurrent symptoms linked to the condition, would be potentially managed with/eligible for a SAVR or TAVR procedure, respectively (see Supplementary Figure S2).\nFig. 5Annual Incident Cases of Severe AS among Australians Aged ≥55 years\nAnnual Incident Cases of Severe AS among Australians Aged ≥55 years","In the setting of specific surgical registry reports [18] and recent insightful data generated by the NEDA Study [3] but little else, we present a unique analysis of the potential treatable burden of severe AS in Australia. As with many substantive public health issues that routinely affect a large proportion of the population and is associated with costly treatment and historically poor health outcomes [13, 14], unfortunately there is a paucity of specific burden of disease data to guide health resources and clinical practice. Indeed, worldwide, the natural history and impact of AS remains poorly characterised [19]. This relative lack of data is exacerbated by the rapid progression of disease and high mortality in those affected [20]. It was on this basis we chose to use the best available modelling and projections on the population prevalence and treatable pattern/burden of severe AS [12] and then applied them to the Australian population aged ≥ 55 years with further adjustments/ improvements based on NEDA Study data [3]. Overall, our analyses suggest that close to 100,000 Australians in this at-risk age-group are currently living with this potentially deadly condition. Accordingly, in the next 5 years, more than half of these individuals will die without having undergone an AVR procedure – their risk of dying being two-fold higher on an adjusted basis than their counterparts without severe AS [3]. Without any change in its natural history (there being strong evidence that its prevalence will rise within our increasingly sedentary and obese population [15, 21], this number will likely rise substantially within Australia’s ageing population. At minimum, we estimate that an additional 9,000 Australians aged ≥ 60 years and over will develop severe AS each year.\nOverall, based on contemporary management practices in Europe and North America (noting this remains a highly evolving field), we estimated that just under 56,000 prevalent cases would be potentially eligible for a SAVR or TAVR procedure. As also shown by the NEDA Study, successful restoration of AV function with AVR is associated with markedly improved survival [10]. Due to population dynamics (including greater longevity among Australian women) more women than men are likely affected by severe AS, but many of these are aged > 80 years and may have comorbidities that will favour more conservative management options.\nRegardless, of the relative accuracy of these projections (noting our critical corrections of the original model [12] based on the very large and robust data derived from the NEDA cohort [3] – see below), these data provide an important context to the largely hidden but substantive burden of disease imposed by AS in Australia. From an individual to societal perspective, it seems clear that due to Australia’s progressively ageing population, a clear strategy to detect and then optimally manage an increasing burden of AS is urgently required. Outcome data derived from close to 350,000 Australians investigated with echocardiography and collectively followed-up > 1 million person-years as part of the NEDA Study [3] further reinforces the need to prioritise AS management. In that study we reaffirmed that severe AS had very poor survival rates (two-thirds dead within 5-years). We also confirmed that when applied, AVR was largely associated with optimal AV hemodynamic profiles and lower mortality [10].\nA recent analysis of 18,147 patients (mean age 72 years and 64 % men) with AS who underwent SAVR captured by the Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) database during 2002–2015, showed that this procedure accounted for 20 % of all adult cardiac surgeries by the end of the study period [18]. In recent years, TAVR has been successfully applied to patients with severe AS and with high/prohibitive surgical risk [5–7]; with two randomized trials reporting the non-inferiority [8] and superiority [9] of TAVR in respect to mortality and subsequent risk of stroke, respectively, when compared to SAVR in low-risk patients with severe AS. A recently reported Australian trial of TAVR applied to 199 intermediate-risk cases of severe AS, is indicative of the changing clinical landscape in this regard [22].\nDespite the type of data described above, determining the actual proportion of Australians at risk of poor outcomes associated with AS and then actively treated with SAVR or TAVR, remains problematic; even when reconciling the largely concordant data around the size of the likely active patient population derived from the NEDA Study and those formal projections. However, there does appear to be a disconnect between the number of Australians with severe AS who might benefit either SAVR or TAVR and their subsequent access to these procedures. A recent report from the ANZSCTS database recorded ~ 4,000 SAVR procedures overall in Australia during the period 2009–2015 [18]. Even when accounting for the fact that registry did become truly national until 2015, there appears to be a large shortfall in the expected number of such procedures per annum relative to our projections. This may well be explained by the demographic profile of those undergoing SAVR in Australia. As reported [18], the mean age of SAVR cases is around 72 years of age and only 37 % were female. Alternatively, the mean age of those with severe AS (during a similar timeframe of surveillance) within the NEDA cohort is around 80 years of age and, consistent with this report, more than half of cases were women. Anecdotally, referral for SAVR in the Australian population has typically paralleled North American trends and the presumptions of the modelling (shown in Fig. 1) are based on historical data from a combination of North American and European centres. Given the lag in referral behaviour to new low-risk interventions (e.g., TAVR), by definition, all these assumptions for the modelling will be conservative. Critically, the availability of these new valve technologies has developed rapidly in Australia in the last 5 years and, other than NEDA, there is a paucity of resources to track these changes in real-time.\nThe recently reported NEDA Study data suggesting that even mild-to-moderate forms of AS are associated with high-levels of mortality approaching that of severe AS within 5-years of follow-up [3], when combined with other contemporary reports [23, 24], are likely to change the landscape of AS management. Specifically, this will likely reflect a recognition that a “watchful wait” approach to determine the transition from moderate to severe AS and also from asymptomatic to symptomatic status [4] may be associated with unacceptably high mortality rates [5]. For example, reflective of concerns around intermediate-to-high risk of surgical mortality (> 4 %) and the evolving efficacy of TAVR versus the more costly and invasive SAVR, in the current analyses, it was estimated that around 9,000 SAVR cases could potentially be replaced with TAVR. However, determining how and where to invest in dedicated screening programs and apply the latest evidence to prolong the lives of those affected in Australia (noting our estimate of approximately 9,000 new cases per annum – of whom > 4,000 would be aged < 75 years) is futile without firm evidence of the number of individuals involved. The noted “disconnect” between the potential and actual number of Australians who derive survival benefits from an AVR, therefore, is likely to increase over time.\nLimitations As noted, these data do not completely rely on Australian specific data (other than population estimates/demographic structure and NEDA-derived adjustments to original estimates). To partially address this, we have used a conservative estimate of the point prevalence of severe AS and provide 95 % CI for lower and higher and estimates. However, we acknowledge the dynamics of medical management of severe AS (i.e., conservative treatment versus TAVR versus SAVR) in Australia is likely to be different than reported in Europe/North America. The differential clinical uptake and reimbursement of different procedures from a public health to private health perspective within Australia’s increasingly complex and hybrid health care system, is particularly relevant - as is the variable population dynamics of each jurisdiction across the country. Beyond the broader demographic features of Australia and its major jurisdictions, we did not consider other important factors such as socio-economic status and the concentration of particularly high-risk groups (e.g. predominantly younger Indigenous Australians in Central Australia who experience much higher levels of valvular disease and heart failure [25]); nor did we consider the cost-burden of treating severe AS. Applying the growing resources of NEDA, we hope to address most of these issues/limitations in the future, in order to provide greater clarity around the full spectrum of AS in Australia. Finally, it is important to acknowledge that our mortality estimates (even when considering that they are focussed on those with native valves) are discordant with the low rates of mortality reported in trials such as the PARTNER 3 Trial [9]. This discordance only reinforces the benefits of early recognition and expert management of this otherwise deadly condition.\nAs noted, these data do not completely rely on Australian specific data (other than population estimates/demographic structure and NEDA-derived adjustments to original estimates). To partially address this, we have used a conservative estimate of the point prevalence of severe AS and provide 95 % CI for lower and higher and estimates. However, we acknowledge the dynamics of medical management of severe AS (i.e., conservative treatment versus TAVR versus SAVR) in Australia is likely to be different than reported in Europe/North America. The differential clinical uptake and reimbursement of different procedures from a public health to private health perspective within Australia’s increasingly complex and hybrid health care system, is particularly relevant - as is the variable population dynamics of each jurisdiction across the country. Beyond the broader demographic features of Australia and its major jurisdictions, we did not consider other important factors such as socio-economic status and the concentration of particularly high-risk groups (e.g. predominantly younger Indigenous Australians in Central Australia who experience much higher levels of valvular disease and heart failure [25]); nor did we consider the cost-burden of treating severe AS. Applying the growing resources of NEDA, we hope to address most of these issues/limitations in the future, in order to provide greater clarity around the full spectrum of AS in Australia. Finally, it is important to acknowledge that our mortality estimates (even when considering that they are focussed on those with native valves) are discordant with the low rates of mortality reported in trials such as the PARTNER 3 Trial [9]. This discordance only reinforces the benefits of early recognition and expert management of this otherwise deadly condition.","As noted, these data do not completely rely on Australian specific data (other than population estimates/demographic structure and NEDA-derived adjustments to original estimates). To partially address this, we have used a conservative estimate of the point prevalence of severe AS and provide 95 % CI for lower and higher and estimates. However, we acknowledge the dynamics of medical management of severe AS (i.e., conservative treatment versus TAVR versus SAVR) in Australia is likely to be different than reported in Europe/North America. The differential clinical uptake and reimbursement of different procedures from a public health to private health perspective within Australia’s increasingly complex and hybrid health care system, is particularly relevant - as is the variable population dynamics of each jurisdiction across the country. Beyond the broader demographic features of Australia and its major jurisdictions, we did not consider other important factors such as socio-economic status and the concentration of particularly high-risk groups (e.g. predominantly younger Indigenous Australians in Central Australia who experience much higher levels of valvular disease and heart failure [25]); nor did we consider the cost-burden of treating severe AS. Applying the growing resources of NEDA, we hope to address most of these issues/limitations in the future, in order to provide greater clarity around the full spectrum of AS in Australia. Finally, it is important to acknowledge that our mortality estimates (even when considering that they are focussed on those with native valves) are discordant with the low rates of mortality reported in trials such as the PARTNER 3 Trial [9]. This discordance only reinforces the benefits of early recognition and expert management of this otherwise deadly condition.","These unique estimates provide an important insight into the current and future treatable burden of severe AS in Australia. At the most conservative level, the likely number of currently affected Australians aged 55 years and over will soon rise to 100,000 people. Moreover, the number of new cases with this potentially deadly condition is likely approaching 10,000 per annum. Based on current clinical practice/recommendations, around two-thirds of such cases should be actively managed due to their symptomatic status (predominantly with a combination of SAVR and TAVR) and a high-risk of mortality. However, it is unclear if that is truly the case. Whether there is sufficient clinical awareness of AS and pro-active referral patterns (particularly for Australian women) for active management is yet to be determined."," \nAdditional file 1: Supplementary Figure 1. Population Distribution of Australians Aged ≥55 years. Legend: Australia is a federated country, comprising the main populated States (Northern Territory and Capital Territory not shown) of Western Australian (WA), South Australia (SA), Victoria (VIC), Tasmania (TAS), New South Wales (NSW) and Queensland (QLD).\nAdditional file 2: Supplementary Figure 2. Estimated Treatable Burden/Management of Severe AS in Australia (Based on Incident Cases per Annum).\n\nAdditional file 1: Supplementary Figure 1. Population Distribution of Australians Aged ≥55 years. Legend: Australia is a federated country, comprising the main populated States (Northern Territory and Capital Territory not shown) of Western Australian (WA), South Australia (SA), Victoria (VIC), Tasmania (TAS), New South Wales (NSW) and Queensland (QLD).\nAdditional file 2: Supplementary Figure 2. Estimated Treatable Burden/Management of Severe AS in Australia (Based on Incident Cases per Annum).\n\nAdditional file 1: Supplementary Figure 1. Population Distribution of Australians Aged ≥55 years. Legend: Australia is a federated country, comprising the main populated States (Northern Territory and Capital Territory not shown) of Western Australian (WA), South Australia (SA), Victoria (VIC), Tasmania (TAS), New South Wales (NSW) and Queensland (QLD).\nAdditional file 2: Supplementary Figure 2. Estimated Treatable Burden/Management of Severe AS in Australia (Based on Incident Cases per Annum).\n\nAdditional file 1: Supplementary Figure 1. Population Distribution of Australians Aged ≥55 years. Legend: Australia is a federated country, comprising the main populated States (Northern Territory and Capital Territory not shown) of Western Australian (WA), South Australia (SA), Victoria (VIC), Tasmania (TAS), New South Wales (NSW) and Queensland (QLD).\nAdditional file 2: Supplementary Figure 2. Estimated Treatable Burden/Management of Severe AS in Australia (Based on Incident Cases per Annum).","\nAdditional file 1: Supplementary Figure 1. Population Distribution of Australians Aged ≥55 years. Legend: Australia is a federated country, comprising the main populated States (Northern Territory and Capital Territory not shown) of Western Australian (WA), South Australia (SA), Victoria (VIC), Tasmania (TAS), New South Wales (NSW) and Queensland (QLD).\nAdditional file 2: Supplementary Figure 2. Estimated Treatable Burden/Management of Severe AS in Australia (Based on Incident Cases per Annum).\n\nAdditional file 1: Supplementary Figure 1. Population Distribution of Australians Aged ≥55 years. Legend: Australia is a federated country, comprising the main populated States (Northern Territory and Capital Territory not shown) of Western Australian (WA), South Australia (SA), Victoria (VIC), Tasmania (TAS), New South Wales (NSW) and Queensland (QLD).\nAdditional file 2: Supplementary Figure 2. Estimated Treatable Burden/Management of Severe AS in Australia (Based on Incident Cases per Annum)."],"string":"[\n \"One of the most common cardiac conditions affecting the progressively aging populations of high-income countries such as Australia is aortic stenosis (AS) [1]. Without timely intervention, severe AS is associated with a very poor prognosis [2]. However, like many other countries, there has been a paucity of reports focusing on the overall prevalence and treatable burden of AS in Australia. A recent AS report from National Echocardiography Database of Australia (NEDA) [3] that assessed the severity of AS and subsequent pattern of survival among 122,809 men and 118,494 women with a mean age of 62 ± 18 years highlighted an urgent need to better understand the burden imposed by this potentially deadly condition. Overall, the indicative prevalence of severe low or high gradient AS among adults being investigated with echocardiography during the overall study period of 2000–2019, was 1.1 and 2.1 % (3.2 % combined), respectively. Actual 5-year mortality ranged from 56 to 67 % in those cases with a native aortic valve and no indication of surgical intervention [3].\\nHistorically, surgical aortic valve replacement (SAVR) has been the preferred intervention for severe AS [4]. However, transcatheter aortic valve replacement (TAVR) has been successfully applied in those with severe AS with high/prohibitive surgical risk [5–7]. Moreover, two head-to-head randomized trials have now reported non-inferiority [8] and superiority [9] comparing TAVR to SAVR in low-risk patients with severe AS, in respect to mortality and subsequent risk of stroke. Consistent with these data, for most of the 6,050 cases within the NEDA cohort who underwent AVR, their post-procedure AV hemodynamic profile and survival outcomes were favourable [10].\\nOverall, these data suggest more Australians might benefit from AVR. However, without reliable estimates of the treatable burden of AS, this critical number (for health service and resource planning) remains unknown. A series of modelled studies, first published in 2013 [11] and then an updated version in 2018 [12], applied the best available epidemiological and registry data to estimate the following for Europe and North America – 1) the overall proportion of older individuals affected by AS and more specifically severe AS; and 2) proportion of these individuals who had and/or would benefit from a valve replacement procedure (SAVR or TAVR). Given the geographical focus and source data used, these estimates now provide these target regions with (moderate) reliable estimates of the treatable burden of AS. To date, there are no equivalent burden of disease estimates for Australia.\\nStudy aims We sought, for the first time, to generate reliable (but inherently conservative) estimates on the prevalence and treatable patient population with severe, symptomatic AS in Australia. Specifically, our aim was to replicate the same robust models recently used to generate contemporary AS-specific projections for Europe and North America [12] as highlighted above with specific modifications relevant to the Australian context. Firstly, this included applying population profiling (denominator) data derived from the Australian Bureau of Statistics (ABS). Secondly, given the Australian context, we aimed to expand our burden of disease estimates to those aged ≥ 55 years (noting the original study applied a single estimate of the overall prevalence of severe AS (numerator) to those aged ≥ 75 years [12]) and by applying age- and sex-specific, incident, and prevalent estimates of AS informed by the recent NEDA Study of AS [3].\\nWe sought, for the first time, to generate reliable (but inherently conservative) estimates on the prevalence and treatable patient population with severe, symptomatic AS in Australia. Specifically, our aim was to replicate the same robust models recently used to generate contemporary AS-specific projections for Europe and North America [12] as highlighted above with specific modifications relevant to the Australian context. Firstly, this included applying population profiling (denominator) data derived from the Australian Bureau of Statistics (ABS). Secondly, given the Australian context, we aimed to expand our burden of disease estimates to those aged ≥ 55 years (noting the original study applied a single estimate of the overall prevalence of severe AS (numerator) to those aged ≥ 75 years [12]) and by applying age- and sex-specific, incident, and prevalent estimates of AS informed by the recent NEDA Study of AS [3].\",\n \"We sought, for the first time, to generate reliable (but inherently conservative) estimates on the prevalence and treatable patient population with severe, symptomatic AS in Australia. Specifically, our aim was to replicate the same robust models recently used to generate contemporary AS-specific projections for Europe and North America [12] as highlighted above with specific modifications relevant to the Australian context. Firstly, this included applying population profiling (denominator) data derived from the Australian Bureau of Statistics (ABS). Secondly, given the Australian context, we aimed to expand our burden of disease estimates to those aged ≥ 55 years (noting the original study applied a single estimate of the overall prevalence of severe AS (numerator) to those aged ≥ 75 years [12]) and by applying age- and sex-specific, incident, and prevalent estimates of AS informed by the recent NEDA Study of AS [3].\",\n \"Study design Consistent with previous reports of this type specifically focusing on atrial fibrillation [13] and heart failure [14], we combined official national population data with the best available epidemiological and clinical data to generate estimates of the treatable burden of disease relating to AS. Given the anonymous source and nature of the study data, no ethical approvals were required.\\nConsistent with previous reports of this type specifically focusing on atrial fibrillation [13] and heart failure [14], we combined official national population data with the best available epidemiological and clinical data to generate estimates of the treatable burden of disease relating to AS. Given the anonymous source and nature of the study data, no ethical approvals were required.\\nStudy setting Projections were applied to the entire Australian population and then each State and Territory according to their currently estimated demographic profile. ABS data projecting the future demographic structure and profile were also used to derive future projections on the treatable burden of AS. (https://www.abs.gov.au/Population - Accessed January 2021).\\nProjections were applied to the entire Australian population and then each State and Territory according to their currently estimated demographic profile. ABS data projecting the future demographic structure and profile were also used to derive future projections on the treatable burden of AS. (https://www.abs.gov.au/Population - Accessed January 2021).\\nStudy data The primary analyses presented in this report used age-, sex- and geographic-specific demography data for Australian men and women aged ≥ 55 years for the calendar year 2019. Specifically, the ABS currently provides population projections derived from the official 2016 Census - http://www.abs.gov.au/ausstats/abs@.nsf/mf/3101.0 - Accessed January 2021).\\nAs indicated above, the primary basis for this report is the recently updated study of the number of treatable and treated patients with severe AS published by Durko and colleagues in 2018 [12]. Specifically, based on an expanded analysis of 37 relevant studies involving 26,402 cases/patients, this study provides a flow-chart (see Fig. 1) of the key estimates/parameters (with 95 % confidence interval, CI) that can be applied (as a primary analysis) to the latest Australian population data (on an age-, sex- and geographic-specific basis) to estimate the pool of treatable severe AS patients per annum. It can also be applied to broad population projections for an increasing pool of older Australians over time; noting that over a decade (from 2006 to 2016) the proportion of Australians aged > 65 years had increased by around 4 % and this proportion (and absolute numbers) will steadily increase.\\nFig. 1Model/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\\nModel/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\\nSpecifically, the model developed and subsequently applied in this report provides overall estimates (based on a combination of European and North American cohort studies) on the proportion of patients with severe AS who – 1) are symptomatic and under current expert guidelines, are largely excluded from surgical management; 2) are symptomatic but unsuitable for SAVR but might safely undergo TAVR (the alternative being medical therapy); or 3) are symptomatic and will undergo SAVR; a proportion of whom (particularly due to high-to-medium surgical risk) may benefit from TAVR. The range of derived estimates for each parameter, despite efforts to smooth-out inevitable heterogeneity, are indicative of the methodological weaknesses/biases inherent to source data. It is important to note these treatment pathways are continually evolving as new, lower risk valve interventions are developed and lower the likely threshold of AVR. Without Australian-specific to correct the rates of intervention applied, it is generally acknowledged that intervention rates for severe AS broadly follow those applied in North America.\\nWhere possible, we were able to improve our assumptions and therefore our projections by considering primary profiling and outcome data derived from (NEDA) [3]. This unique study has now captured echocardiographic data on > 750,000 Australians (with no exclusion criteria) being routinely investigated with echocardiography from > 25 centres Australia-wide. With individual data linkage, NEDA also generates real-world, short- to long-term survival data on those affected by common cardiac conditions including AS [3].\\nThe primary analyses presented in this report used age-, sex- and geographic-specific demography data for Australian men and women aged ≥ 55 years for the calendar year 2019. Specifically, the ABS currently provides population projections derived from the official 2016 Census - http://www.abs.gov.au/ausstats/abs@.nsf/mf/3101.0 - Accessed January 2021).\\nAs indicated above, the primary basis for this report is the recently updated study of the number of treatable and treated patients with severe AS published by Durko and colleagues in 2018 [12]. Specifically, based on an expanded analysis of 37 relevant studies involving 26,402 cases/patients, this study provides a flow-chart (see Fig. 1) of the key estimates/parameters (with 95 % confidence interval, CI) that can be applied (as a primary analysis) to the latest Australian population data (on an age-, sex- and geographic-specific basis) to estimate the pool of treatable severe AS patients per annum. It can also be applied to broad population projections for an increasing pool of older Australians over time; noting that over a decade (from 2006 to 2016) the proportion of Australians aged > 65 years had increased by around 4 % and this proportion (and absolute numbers) will steadily increase.\\nFig. 1Model/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\\nModel/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\\nSpecifically, the model developed and subsequently applied in this report provides overall estimates (based on a combination of European and North American cohort studies) on the proportion of patients with severe AS who – 1) are symptomatic and under current expert guidelines, are largely excluded from surgical management; 2) are symptomatic but unsuitable for SAVR but might safely undergo TAVR (the alternative being medical therapy); or 3) are symptomatic and will undergo SAVR; a proportion of whom (particularly due to high-to-medium surgical risk) may benefit from TAVR. The range of derived estimates for each parameter, despite efforts to smooth-out inevitable heterogeneity, are indicative of the methodological weaknesses/biases inherent to source data. It is important to note these treatment pathways are continually evolving as new, lower risk valve interventions are developed and lower the likely threshold of AVR. Without Australian-specific to correct the rates of intervention applied, it is generally acknowledged that intervention rates for severe AS broadly follow those applied in North America.\\nWhere possible, we were able to improve our assumptions and therefore our projections by considering primary profiling and outcome data derived from (NEDA) [3]. This unique study has now captured echocardiographic data on > 750,000 Australians (with no exclusion criteria) being routinely investigated with echocardiography from > 25 centres Australia-wide. With individual data linkage, NEDA also generates real-world, short- to long-term survival data on those affected by common cardiac conditions including AS [3].\\nEstimating the prevalence & treatable burden of severe AS For these analyses, we applied a point prevalence of 3.5 % for severe AS among individuals aged ≥ 75 years, This small adjustment to the original European model [12] reflects recent reports of an increasing incidence of AS in other high-income countries such as the UK [15] and is only slightly higher than that of the NEDA cohort of actively investigated patients [3]. To derive specific prevalence rates for those aged < 75 years, we used the ratio of cases of severe AS observed in each age-band of the NEDA cohort [3]. This resulted in the following age-specific prevalence estimates being applied: ≥75 years, 3.5 %; 70–74 years, 1.2 %; 65–69 years, 0.7 %; 60–64 years, 0.5 %; and 55–59 years, 0.4 %. When applied to the Australian population aged ≥ 55 years (see Supplementary Figure S1), the overall estimated point prevalence of severe AS is 1.48 % for 2019; a figure that is broadly consistent with that published and applied previously [16]. The same rates were applied to men and women given that, on an age-specific basis, there were minimum differences based on sex within the NEDA cohort [3].\\nFor these analyses, we applied a point prevalence of 3.5 % for severe AS among individuals aged ≥ 75 years, This small adjustment to the original European model [12] reflects recent reports of an increasing incidence of AS in other high-income countries such as the UK [15] and is only slightly higher than that of the NEDA cohort of actively investigated patients [3]. To derive specific prevalence rates for those aged < 75 years, we used the ratio of cases of severe AS observed in each age-band of the NEDA cohort [3]. This resulted in the following age-specific prevalence estimates being applied: ≥75 years, 3.5 %; 70–74 years, 1.2 %; 65–69 years, 0.7 %; 60–64 years, 0.5 %; and 55–59 years, 0.4 %. When applied to the Australian population aged ≥ 55 years (see Supplementary Figure S1), the overall estimated point prevalence of severe AS is 1.48 % for 2019; a figure that is broadly consistent with that published and applied previously [16]. The same rates were applied to men and women given that, on an age-specific basis, there were minimum differences based on sex within the NEDA cohort [3].\\nMortality To understand the potential consequences of no AVR intervention in the setting of severe AS, age- and sex-specific, actual 5-year mortality rates were applied to the estimated case prevalence of severe AS by applying those observed rates within the equivalent patient cohort identified within NEDA who had a native AV throughout follow-up [3]. Although these data only reflect cases referred for echocardiography, they provide both a discussion point and means to validate prevalence estimates when combined with incidence rates (see below).\\nTo understand the potential consequences of no AVR intervention in the setting of severe AS, age- and sex-specific, actual 5-year mortality rates were applied to the estimated case prevalence of severe AS by applying those observed rates within the equivalent patient cohort identified within NEDA who had a native AV throughout follow-up [3]. Although these data only reflect cases referred for echocardiography, they provide both a discussion point and means to validate prevalence estimates when combined with incidence rates (see below).\\nIncident cases of severe AS In the absence of specific population incidence data, we used the differential in prevalence for each 5-year age group (i.e., how many more at risk individuals would develop severe AS over 5-years to reflect the number of prevalent cases in that older age group) to calculate how many individuals would develop severe AS each year. By necessity this means incident cases were only calculated for those aged ≥ 60 years. Applying conservatively derived data from the NEDA cohort [3], the following age-specific, annual incident rates, were applied to the Australian population aged ≥ 60 years for the subsequent (i.e. beyond 2019) 5-year period 2020-24: aged ≥ 75 years, 460 cases; 70–74 years, 40 cases; 65–69 years, 40 cases; and 60–64 years, 20 cases per 100,000 population per annum. When combined, these rates generated an annual incidence of severe AS of 182 cases per 100,000 population per annum within the Australian population aged ≥ 60 years.\\nIn the absence of specific population incidence data, we used the differential in prevalence for each 5-year age group (i.e., how many more at risk individuals would develop severe AS over 5-years to reflect the number of prevalent cases in that older age group) to calculate how many individuals would develop severe AS each year. By necessity this means incident cases were only calculated for those aged ≥ 60 years. Applying conservatively derived data from the NEDA cohort [3], the following age-specific, annual incident rates, were applied to the Australian population aged ≥ 60 years for the subsequent (i.e. beyond 2019) 5-year period 2020-24: aged ≥ 75 years, 460 cases; 70–74 years, 40 cases; 65–69 years, 40 cases; and 60–64 years, 20 cases per 100,000 population per annum. When combined, these rates generated an annual incidence of severe AS of 182 cases per 100,000 population per annum within the Australian population aged ≥ 60 years.\\nStatistical analyses All projections are reported as whole numbers and proportions with 95 % CI where appropriate. All statistical analyses are descriptive in nature and population-based; with no inferential statistics applied. Exact estimates are provided in the figures whilst rounded up (to the nearest hundred) figures are provided in text.\\nAll projections are reported as whole numbers and proportions with 95 % CI where appropriate. All statistical analyses are descriptive in nature and population-based; with no inferential statistics applied. Exact estimates are provided in the figures whilst rounded up (to the nearest hundred) figures are provided in text.\",\n \"Consistent with previous reports of this type specifically focusing on atrial fibrillation [13] and heart failure [14], we combined official national population data with the best available epidemiological and clinical data to generate estimates of the treatable burden of disease relating to AS. Given the anonymous source and nature of the study data, no ethical approvals were required.\",\n \"Projections were applied to the entire Australian population and then each State and Territory according to their currently estimated demographic profile. ABS data projecting the future demographic structure and profile were also used to derive future projections on the treatable burden of AS. (https://www.abs.gov.au/Population - Accessed January 2021).\",\n \"The primary analyses presented in this report used age-, sex- and geographic-specific demography data for Australian men and women aged ≥ 55 years for the calendar year 2019. Specifically, the ABS currently provides population projections derived from the official 2016 Census - http://www.abs.gov.au/ausstats/abs@.nsf/mf/3101.0 - Accessed January 2021).\\nAs indicated above, the primary basis for this report is the recently updated study of the number of treatable and treated patients with severe AS published by Durko and colleagues in 2018 [12]. Specifically, based on an expanded analysis of 37 relevant studies involving 26,402 cases/patients, this study provides a flow-chart (see Fig. 1) of the key estimates/parameters (with 95 % confidence interval, CI) that can be applied (as a primary analysis) to the latest Australian population data (on an age-, sex- and geographic-specific basis) to estimate the pool of treatable severe AS patients per annum. It can also be applied to broad population projections for an increasing pool of older Australians over time; noting that over a decade (from 2006 to 2016) the proportion of Australians aged > 65 years had increased by around 4 % and this proportion (and absolute numbers) will steadily increase.\\nFig. 1Model/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\\nModel/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\\nSpecifically, the model developed and subsequently applied in this report provides overall estimates (based on a combination of European and North American cohort studies) on the proportion of patients with severe AS who – 1) are symptomatic and under current expert guidelines, are largely excluded from surgical management; 2) are symptomatic but unsuitable for SAVR but might safely undergo TAVR (the alternative being medical therapy); or 3) are symptomatic and will undergo SAVR; a proportion of whom (particularly due to high-to-medium surgical risk) may benefit from TAVR. The range of derived estimates for each parameter, despite efforts to smooth-out inevitable heterogeneity, are indicative of the methodological weaknesses/biases inherent to source data. It is important to note these treatment pathways are continually evolving as new, lower risk valve interventions are developed and lower the likely threshold of AVR. Without Australian-specific to correct the rates of intervention applied, it is generally acknowledged that intervention rates for severe AS broadly follow those applied in North America.\\nWhere possible, we were able to improve our assumptions and therefore our projections by considering primary profiling and outcome data derived from (NEDA) [3]. This unique study has now captured echocardiographic data on > 750,000 Australians (with no exclusion criteria) being routinely investigated with echocardiography from > 25 centres Australia-wide. With individual data linkage, NEDA also generates real-world, short- to long-term survival data on those affected by common cardiac conditions including AS [3].\",\n \"For these analyses, we applied a point prevalence of 3.5 % for severe AS among individuals aged ≥ 75 years, This small adjustment to the original European model [12] reflects recent reports of an increasing incidence of AS in other high-income countries such as the UK [15] and is only slightly higher than that of the NEDA cohort of actively investigated patients [3]. To derive specific prevalence rates for those aged < 75 years, we used the ratio of cases of severe AS observed in each age-band of the NEDA cohort [3]. This resulted in the following age-specific prevalence estimates being applied: ≥75 years, 3.5 %; 70–74 years, 1.2 %; 65–69 years, 0.7 %; 60–64 years, 0.5 %; and 55–59 years, 0.4 %. When applied to the Australian population aged ≥ 55 years (see Supplementary Figure S1), the overall estimated point prevalence of severe AS is 1.48 % for 2019; a figure that is broadly consistent with that published and applied previously [16]. The same rates were applied to men and women given that, on an age-specific basis, there were minimum differences based on sex within the NEDA cohort [3].\",\n \"To understand the potential consequences of no AVR intervention in the setting of severe AS, age- and sex-specific, actual 5-year mortality rates were applied to the estimated case prevalence of severe AS by applying those observed rates within the equivalent patient cohort identified within NEDA who had a native AV throughout follow-up [3]. Although these data only reflect cases referred for echocardiography, they provide both a discussion point and means to validate prevalence estimates when combined with incidence rates (see below).\",\n \"In the absence of specific population incidence data, we used the differential in prevalence for each 5-year age group (i.e., how many more at risk individuals would develop severe AS over 5-years to reflect the number of prevalent cases in that older age group) to calculate how many individuals would develop severe AS each year. By necessity this means incident cases were only calculated for those aged ≥ 60 years. Applying conservatively derived data from the NEDA cohort [3], the following age-specific, annual incident rates, were applied to the Australian population aged ≥ 60 years for the subsequent (i.e. beyond 2019) 5-year period 2020-24: aged ≥ 75 years, 460 cases; 70–74 years, 40 cases; 65–69 years, 40 cases; and 60–64 years, 20 cases per 100,000 population per annum. When combined, these rates generated an annual incidence of severe AS of 182 cases per 100,000 population per annum within the Australian population aged ≥ 60 years.\",\n \"All projections are reported as whole numbers and proportions with 95 % CI where appropriate. All statistical analyses are descriptive in nature and population-based; with no inferential statistics applied. Exact estimates are provided in the figures whilst rounded up (to the nearest hundred) figures are provided in text.\",\n \"Prevalent cases of severe AS As shown in Fig. 2, we conservatively estimate that around 97,300 Australians are living with severe AS (symptomatic or otherwise). Moreover, assuming just over two-thirds of these cases experience concurrent symptoms linked to the condition, according to contemporary clinical recommendations/ best practice around 66,500 (95 % CI 59,200 to 74,000) people might be considered for an AVR procedure at any one time.\\nFig. 2Estimated Point Prevalence and Distribution of Severe AS in Australia\\nEstimated Point Prevalence and Distribution of Severe AS in Australia\\nAs shown in Fig. 2, we conservatively estimate that around 97,300 Australians are living with severe AS (symptomatic or otherwise). Moreover, assuming just over two-thirds of these cases experience concurrent symptoms linked to the condition, according to contemporary clinical recommendations/ best practice around 66,500 (95 % CI 59,200 to 74,000) people might be considered for an AVR procedure at any one time.\\nFig. 2Estimated Point Prevalence and Distribution of Severe AS in Australia\\nEstimated Point Prevalence and Distribution of Severe AS in Australia\\nAortic valve replacement Figure 3 summarises the overall treatable burden/management of severe AS when assuming 58.4 % of cases with symptomatic, severe AS would be referred for SAVR (primary replacement or revision) and the remainder (41.6 %) for potential TAVR. On this basis, we estimate that around 38,800 (95 % CI 35,700 to 42,000) Australians aged ≥ 55 years with severe, symptomatic AS might be considered for SAVR; based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score [17]. An additional 17,000 cases (95 % CI 11,600 to 22,600) of the approximately 27,700 people not eligible for SAVR due to high peri-operative risk could be potentially considered for TAVR instead.\\nFig. 3Estimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\\nEstimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\\nFigure 3 summarises the overall treatable burden/management of severe AS when assuming 58.4 % of cases with symptomatic, severe AS would be referred for SAVR (primary replacement or revision) and the remainder (41.6 %) for potential TAVR. On this basis, we estimate that around 38,800 (95 % CI 35,700 to 42,000) Australians aged ≥ 55 years with severe, symptomatic AS might be considered for SAVR; based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score [17]. An additional 17,000 cases (95 % CI 11,600 to 22,600) of the approximately 27,700 people not eligible for SAVR due to high peri-operative risk could be potentially considered for TAVR instead.\\nFig. 3Estimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\\nEstimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\\nAS-related mortality Based on the age- and sex-specific rates of actual mortality (ranging from 17 to 84 % for those aged 55–64 years to ≥ 85 years) observed in the NEDA cohort, we estimate that 56,300 (95 % CI 56,100 to 56,500) of the prevalent population with severe AS of their native valve will subsequently die within the next 5 years - see Fig. 4.\\nFig. 4Severe AS Related Mortality\\nSevere AS Related Mortality\\nBased on the age- and sex-specific rates of actual mortality (ranging from 17 to 84 % for those aged 55–64 years to ≥ 85 years) observed in the NEDA cohort, we estimate that 56,300 (95 % CI 56,100 to 56,500) of the prevalent population with severe AS of their native valve will subsequently die within the next 5 years - see Fig. 4.\\nFig. 4Severe AS Related Mortality\\nSevere AS Related Mortality\\nIncident cases As shown in Fig. 5, each year, we further estimate that around 9,300 (95 % CI 9,000 to 9,500) more Australians aged ≥ 60 years will subsequently develop severe AS. Assuming the same pattern of potential AVR procedures (once again based on peri-operative risk status), 3,700 (95 % CI 3,400 to 4,000) and 2,600 (95 % CI 2,300 to 2,900) of the approximate de novo 6,300 (95 % CI 5,600 to 7,000) cases with concurrent symptoms linked to the condition, would be potentially managed with/eligible for a SAVR or TAVR procedure, respectively (see Supplementary Figure S2).\\nFig. 5Annual Incident Cases of Severe AS among Australians Aged ≥55 years\\nAnnual Incident Cases of Severe AS among Australians Aged ≥55 years\\nAs shown in Fig. 5, each year, we further estimate that around 9,300 (95 % CI 9,000 to 9,500) more Australians aged ≥ 60 years will subsequently develop severe AS. Assuming the same pattern of potential AVR procedures (once again based on peri-operative risk status), 3,700 (95 % CI 3,400 to 4,000) and 2,600 (95 % CI 2,300 to 2,900) of the approximate de novo 6,300 (95 % CI 5,600 to 7,000) cases with concurrent symptoms linked to the condition, would be potentially managed with/eligible for a SAVR or TAVR procedure, respectively (see Supplementary Figure S2).\\nFig. 5Annual Incident Cases of Severe AS among Australians Aged ≥55 years\\nAnnual Incident Cases of Severe AS among Australians Aged ≥55 years\",\n \"As shown in Fig. 2, we conservatively estimate that around 97,300 Australians are living with severe AS (symptomatic or otherwise). Moreover, assuming just over two-thirds of these cases experience concurrent symptoms linked to the condition, according to contemporary clinical recommendations/ best practice around 66,500 (95 % CI 59,200 to 74,000) people might be considered for an AVR procedure at any one time.\\nFig. 2Estimated Point Prevalence and Distribution of Severe AS in Australia\\nEstimated Point Prevalence and Distribution of Severe AS in Australia\",\n \"Figure 3 summarises the overall treatable burden/management of severe AS when assuming 58.4 % of cases with symptomatic, severe AS would be referred for SAVR (primary replacement or revision) and the remainder (41.6 %) for potential TAVR. On this basis, we estimate that around 38,800 (95 % CI 35,700 to 42,000) Australians aged ≥ 55 years with severe, symptomatic AS might be considered for SAVR; based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score [17]. An additional 17,000 cases (95 % CI 11,600 to 22,600) of the approximately 27,700 people not eligible for SAVR due to high peri-operative risk could be potentially considered for TAVR instead.\\nFig. 3Estimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\\nEstimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\",\n \"Based on the age- and sex-specific rates of actual mortality (ranging from 17 to 84 % for those aged 55–64 years to ≥ 85 years) observed in the NEDA cohort, we estimate that 56,300 (95 % CI 56,100 to 56,500) of the prevalent population with severe AS of their native valve will subsequently die within the next 5 years - see Fig. 4.\\nFig. 4Severe AS Related Mortality\\nSevere AS Related Mortality\",\n \"As shown in Fig. 5, each year, we further estimate that around 9,300 (95 % CI 9,000 to 9,500) more Australians aged ≥ 60 years will subsequently develop severe AS. Assuming the same pattern of potential AVR procedures (once again based on peri-operative risk status), 3,700 (95 % CI 3,400 to 4,000) and 2,600 (95 % CI 2,300 to 2,900) of the approximate de novo 6,300 (95 % CI 5,600 to 7,000) cases with concurrent symptoms linked to the condition, would be potentially managed with/eligible for a SAVR or TAVR procedure, respectively (see Supplementary Figure S2).\\nFig. 5Annual Incident Cases of Severe AS among Australians Aged ≥55 years\\nAnnual Incident Cases of Severe AS among Australians Aged ≥55 years\",\n \"In the setting of specific surgical registry reports [18] and recent insightful data generated by the NEDA Study [3] but little else, we present a unique analysis of the potential treatable burden of severe AS in Australia. As with many substantive public health issues that routinely affect a large proportion of the population and is associated with costly treatment and historically poor health outcomes [13, 14], unfortunately there is a paucity of specific burden of disease data to guide health resources and clinical practice. Indeed, worldwide, the natural history and impact of AS remains poorly characterised [19]. This relative lack of data is exacerbated by the rapid progression of disease and high mortality in those affected [20]. It was on this basis we chose to use the best available modelling and projections on the population prevalence and treatable pattern/burden of severe AS [12] and then applied them to the Australian population aged ≥ 55 years with further adjustments/ improvements based on NEDA Study data [3]. Overall, our analyses suggest that close to 100,000 Australians in this at-risk age-group are currently living with this potentially deadly condition. Accordingly, in the next 5 years, more than half of these individuals will die without having undergone an AVR procedure – their risk of dying being two-fold higher on an adjusted basis than their counterparts without severe AS [3]. Without any change in its natural history (there being strong evidence that its prevalence will rise within our increasingly sedentary and obese population [15, 21], this number will likely rise substantially within Australia’s ageing population. At minimum, we estimate that an additional 9,000 Australians aged ≥ 60 years and over will develop severe AS each year.\\nOverall, based on contemporary management practices in Europe and North America (noting this remains a highly evolving field), we estimated that just under 56,000 prevalent cases would be potentially eligible for a SAVR or TAVR procedure. As also shown by the NEDA Study, successful restoration of AV function with AVR is associated with markedly improved survival [10]. Due to population dynamics (including greater longevity among Australian women) more women than men are likely affected by severe AS, but many of these are aged > 80 years and may have comorbidities that will favour more conservative management options.\\nRegardless, of the relative accuracy of these projections (noting our critical corrections of the original model [12] based on the very large and robust data derived from the NEDA cohort [3] – see below), these data provide an important context to the largely hidden but substantive burden of disease imposed by AS in Australia. From an individual to societal perspective, it seems clear that due to Australia’s progressively ageing population, a clear strategy to detect and then optimally manage an increasing burden of AS is urgently required. Outcome data derived from close to 350,000 Australians investigated with echocardiography and collectively followed-up > 1 million person-years as part of the NEDA Study [3] further reinforces the need to prioritise AS management. In that study we reaffirmed that severe AS had very poor survival rates (two-thirds dead within 5-years). We also confirmed that when applied, AVR was largely associated with optimal AV hemodynamic profiles and lower mortality [10].\\nA recent analysis of 18,147 patients (mean age 72 years and 64 % men) with AS who underwent SAVR captured by the Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) database during 2002–2015, showed that this procedure accounted for 20 % of all adult cardiac surgeries by the end of the study period [18]. In recent years, TAVR has been successfully applied to patients with severe AS and with high/prohibitive surgical risk [5–7]; with two randomized trials reporting the non-inferiority [8] and superiority [9] of TAVR in respect to mortality and subsequent risk of stroke, respectively, when compared to SAVR in low-risk patients with severe AS. A recently reported Australian trial of TAVR applied to 199 intermediate-risk cases of severe AS, is indicative of the changing clinical landscape in this regard [22].\\nDespite the type of data described above, determining the actual proportion of Australians at risk of poor outcomes associated with AS and then actively treated with SAVR or TAVR, remains problematic; even when reconciling the largely concordant data around the size of the likely active patient population derived from the NEDA Study and those formal projections. However, there does appear to be a disconnect between the number of Australians with severe AS who might benefit either SAVR or TAVR and their subsequent access to these procedures. A recent report from the ANZSCTS database recorded ~ 4,000 SAVR procedures overall in Australia during the period 2009–2015 [18]. Even when accounting for the fact that registry did become truly national until 2015, there appears to be a large shortfall in the expected number of such procedures per annum relative to our projections. This may well be explained by the demographic profile of those undergoing SAVR in Australia. As reported [18], the mean age of SAVR cases is around 72 years of age and only 37 % were female. Alternatively, the mean age of those with severe AS (during a similar timeframe of surveillance) within the NEDA cohort is around 80 years of age and, consistent with this report, more than half of cases were women. Anecdotally, referral for SAVR in the Australian population has typically paralleled North American trends and the presumptions of the modelling (shown in Fig. 1) are based on historical data from a combination of North American and European centres. Given the lag in referral behaviour to new low-risk interventions (e.g., TAVR), by definition, all these assumptions for the modelling will be conservative. Critically, the availability of these new valve technologies has developed rapidly in Australia in the last 5 years and, other than NEDA, there is a paucity of resources to track these changes in real-time.\\nThe recently reported NEDA Study data suggesting that even mild-to-moderate forms of AS are associated with high-levels of mortality approaching that of severe AS within 5-years of follow-up [3], when combined with other contemporary reports [23, 24], are likely to change the landscape of AS management. Specifically, this will likely reflect a recognition that a “watchful wait” approach to determine the transition from moderate to severe AS and also from asymptomatic to symptomatic status [4] may be associated with unacceptably high mortality rates [5]. For example, reflective of concerns around intermediate-to-high risk of surgical mortality (> 4 %) and the evolving efficacy of TAVR versus the more costly and invasive SAVR, in the current analyses, it was estimated that around 9,000 SAVR cases could potentially be replaced with TAVR. However, determining how and where to invest in dedicated screening programs and apply the latest evidence to prolong the lives of those affected in Australia (noting our estimate of approximately 9,000 new cases per annum – of whom > 4,000 would be aged < 75 years) is futile without firm evidence of the number of individuals involved. The noted “disconnect” between the potential and actual number of Australians who derive survival benefits from an AVR, therefore, is likely to increase over time.\\nLimitations As noted, these data do not completely rely on Australian specific data (other than population estimates/demographic structure and NEDA-derived adjustments to original estimates). To partially address this, we have used a conservative estimate of the point prevalence of severe AS and provide 95 % CI for lower and higher and estimates. However, we acknowledge the dynamics of medical management of severe AS (i.e., conservative treatment versus TAVR versus SAVR) in Australia is likely to be different than reported in Europe/North America. The differential clinical uptake and reimbursement of different procedures from a public health to private health perspective within Australia’s increasingly complex and hybrid health care system, is particularly relevant - as is the variable population dynamics of each jurisdiction across the country. Beyond the broader demographic features of Australia and its major jurisdictions, we did not consider other important factors such as socio-economic status and the concentration of particularly high-risk groups (e.g. predominantly younger Indigenous Australians in Central Australia who experience much higher levels of valvular disease and heart failure [25]); nor did we consider the cost-burden of treating severe AS. Applying the growing resources of NEDA, we hope to address most of these issues/limitations in the future, in order to provide greater clarity around the full spectrum of AS in Australia. Finally, it is important to acknowledge that our mortality estimates (even when considering that they are focussed on those with native valves) are discordant with the low rates of mortality reported in trials such as the PARTNER 3 Trial [9]. This discordance only reinforces the benefits of early recognition and expert management of this otherwise deadly condition.\\nAs noted, these data do not completely rely on Australian specific data (other than population estimates/demographic structure and NEDA-derived adjustments to original estimates). To partially address this, we have used a conservative estimate of the point prevalence of severe AS and provide 95 % CI for lower and higher and estimates. However, we acknowledge the dynamics of medical management of severe AS (i.e., conservative treatment versus TAVR versus SAVR) in Australia is likely to be different than reported in Europe/North America. The differential clinical uptake and reimbursement of different procedures from a public health to private health perspective within Australia’s increasingly complex and hybrid health care system, is particularly relevant - as is the variable population dynamics of each jurisdiction across the country. Beyond the broader demographic features of Australia and its major jurisdictions, we did not consider other important factors such as socio-economic status and the concentration of particularly high-risk groups (e.g. predominantly younger Indigenous Australians in Central Australia who experience much higher levels of valvular disease and heart failure [25]); nor did we consider the cost-burden of treating severe AS. Applying the growing resources of NEDA, we hope to address most of these issues/limitations in the future, in order to provide greater clarity around the full spectrum of AS in Australia. Finally, it is important to acknowledge that our mortality estimates (even when considering that they are focussed on those with native valves) are discordant with the low rates of mortality reported in trials such as the PARTNER 3 Trial [9]. This discordance only reinforces the benefits of early recognition and expert management of this otherwise deadly condition.\",\n \"As noted, these data do not completely rely on Australian specific data (other than population estimates/demographic structure and NEDA-derived adjustments to original estimates). To partially address this, we have used a conservative estimate of the point prevalence of severe AS and provide 95 % CI for lower and higher and estimates. However, we acknowledge the dynamics of medical management of severe AS (i.e., conservative treatment versus TAVR versus SAVR) in Australia is likely to be different than reported in Europe/North America. The differential clinical uptake and reimbursement of different procedures from a public health to private health perspective within Australia’s increasingly complex and hybrid health care system, is particularly relevant - as is the variable population dynamics of each jurisdiction across the country. Beyond the broader demographic features of Australia and its major jurisdictions, we did not consider other important factors such as socio-economic status and the concentration of particularly high-risk groups (e.g. predominantly younger Indigenous Australians in Central Australia who experience much higher levels of valvular disease and heart failure [25]); nor did we consider the cost-burden of treating severe AS. Applying the growing resources of NEDA, we hope to address most of these issues/limitations in the future, in order to provide greater clarity around the full spectrum of AS in Australia. Finally, it is important to acknowledge that our mortality estimates (even when considering that they are focussed on those with native valves) are discordant with the low rates of mortality reported in trials such as the PARTNER 3 Trial [9]. This discordance only reinforces the benefits of early recognition and expert management of this otherwise deadly condition.\",\n \"These unique estimates provide an important insight into the current and future treatable burden of severe AS in Australia. At the most conservative level, the likely number of currently affected Australians aged 55 years and over will soon rise to 100,000 people. Moreover, the number of new cases with this potentially deadly condition is likely approaching 10,000 per annum. Based on current clinical practice/recommendations, around two-thirds of such cases should be actively managed due to their symptomatic status (predominantly with a combination of SAVR and TAVR) and a high-risk of mortality. However, it is unclear if that is truly the case. Whether there is sufficient clinical awareness of AS and pro-active referral patterns (particularly for Australian women) for active management is yet to be determined.\",\n \" \\nAdditional file 1: Supplementary Figure 1. Population Distribution of Australians Aged ≥55 years. Legend: Australia is a federated country, comprising the main populated States (Northern Territory and Capital Territory not shown) of Western Australian (WA), South Australia (SA), Victoria (VIC), Tasmania (TAS), New South Wales (NSW) and Queensland (QLD).\\nAdditional file 2: Supplementary Figure 2. Estimated Treatable Burden/Management of Severe AS in Australia (Based on Incident Cases per Annum).\\n\\nAdditional file 1: Supplementary Figure 1. Population Distribution of Australians Aged ≥55 years. Legend: Australia is a federated country, comprising the main populated States (Northern Territory and Capital Territory not shown) of Western Australian (WA), South Australia (SA), Victoria (VIC), Tasmania (TAS), New South Wales (NSW) and Queensland (QLD).\\nAdditional file 2: Supplementary Figure 2. Estimated Treatable Burden/Management of Severe AS in Australia (Based on Incident Cases per Annum).\\n\\nAdditional file 1: Supplementary Figure 1. Population Distribution of Australians Aged ≥55 years. Legend: Australia is a federated country, comprising the main populated States (Northern Territory and Capital Territory not shown) of Western Australian (WA), South Australia (SA), Victoria (VIC), Tasmania (TAS), New South Wales (NSW) and Queensland (QLD).\\nAdditional file 2: Supplementary Figure 2. Estimated Treatable Burden/Management of Severe AS in Australia (Based on Incident Cases per Annum).\\n\\nAdditional file 1: Supplementary Figure 1. Population Distribution of Australians Aged ≥55 years. Legend: Australia is a federated country, comprising the main populated States (Northern Territory and Capital Territory not shown) of Western Australian (WA), South Australia (SA), Victoria (VIC), Tasmania (TAS), New South Wales (NSW) and Queensland (QLD).\\nAdditional file 2: Supplementary Figure 2. Estimated Treatable Burden/Management of Severe AS in Australia (Based on Incident Cases per Annum).\",\n \"\\nAdditional file 1: Supplementary Figure 1. Population Distribution of Australians Aged ≥55 years. Legend: Australia is a federated country, comprising the main populated States (Northern Territory and Capital Territory not shown) of Western Australian (WA), South Australia (SA), Victoria (VIC), Tasmania (TAS), New South Wales (NSW) and Queensland (QLD).\\nAdditional file 2: Supplementary Figure 2. Estimated Treatable Burden/Management of Severe AS in Australia (Based on Incident Cases per Annum).\\n\\nAdditional file 1: Supplementary Figure 1. Population Distribution of Australians Aged ≥55 years. Legend: Australia is a federated country, comprising the main populated States (Northern Territory and Capital Territory not shown) of Western Australian (WA), South Australia (SA), Victoria (VIC), Tasmania (TAS), New South Wales (NSW) and Queensland (QLD).\\nAdditional file 2: Supplementary Figure 2. Estimated Treatable Burden/Management of Severe AS in Australia (Based on Incident Cases per Annum).\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["introduction",null,null,null,null,null,null,null,null,null,"results",null,null,null,null,"discussion",null,"conclusion","supplementary-material",null],"string":"[\n \"introduction\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n null,\n null,\n null,\n null,\n \"discussion\",\n null,\n \"conclusion\",\n \"supplementary-material\",\n null\n]","isConversational":false},"keywords":{"kind":"list like","value":["Aortic stenosis","Aortic valve replacement","Health-services"],"string":"[\n \"Aortic stenosis\",\n \"Aortic valve replacement\",\n \"Health-services\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Introduction:\nOne of the most common cardiac conditions affecting the progressively aging populations of high-income countries such as Australia is aortic stenosis (AS) [1]. Without timely intervention, severe AS is associated with a very poor prognosis [2]. However, like many other countries, there has been a paucity of reports focusing on the overall prevalence and treatable burden of AS in Australia. A recent AS report from National Echocardiography Database of Australia (NEDA) [3] that assessed the severity of AS and subsequent pattern of survival among 122,809 men and 118,494 women with a mean age of 62 ± 18 years highlighted an urgent need to better understand the burden imposed by this potentially deadly condition. Overall, the indicative prevalence of severe low or high gradient AS among adults being investigated with echocardiography during the overall study period of 2000–2019, was 1.1 and 2.1 % (3.2 % combined), respectively. Actual 5-year mortality ranged from 56 to 67 % in those cases with a native aortic valve and no indication of surgical intervention [3].\nHistorically, surgical aortic valve replacement (SAVR) has been the preferred intervention for severe AS [4]. However, transcatheter aortic valve replacement (TAVR) has been successfully applied in those with severe AS with high/prohibitive surgical risk [5–7]. Moreover, two head-to-head randomized trials have now reported non-inferiority [8] and superiority [9] comparing TAVR to SAVR in low-risk patients with severe AS, in respect to mortality and subsequent risk of stroke. Consistent with these data, for most of the 6,050 cases within the NEDA cohort who underwent AVR, their post-procedure AV hemodynamic profile and survival outcomes were favourable [10].\nOverall, these data suggest more Australians might benefit from AVR. However, without reliable estimates of the treatable burden of AS, this critical number (for health service and resource planning) remains unknown. A series of modelled studies, first published in 2013 [11] and then an updated version in 2018 [12], applied the best available epidemiological and registry data to estimate the following for Europe and North America – 1) the overall proportion of older individuals affected by AS and more specifically severe AS; and 2) proportion of these individuals who had and/or would benefit from a valve replacement procedure (SAVR or TAVR). Given the geographical focus and source data used, these estimates now provide these target regions with (moderate) reliable estimates of the treatable burden of AS. To date, there are no equivalent burden of disease estimates for Australia.\nStudy aims We sought, for the first time, to generate reliable (but inherently conservative) estimates on the prevalence and treatable patient population with severe, symptomatic AS in Australia. Specifically, our aim was to replicate the same robust models recently used to generate contemporary AS-specific projections for Europe and North America [12] as highlighted above with specific modifications relevant to the Australian context. Firstly, this included applying population profiling (denominator) data derived from the Australian Bureau of Statistics (ABS). Secondly, given the Australian context, we aimed to expand our burden of disease estimates to those aged ≥ 55 years (noting the original study applied a single estimate of the overall prevalence of severe AS (numerator) to those aged ≥ 75 years [12]) and by applying age- and sex-specific, incident, and prevalent estimates of AS informed by the recent NEDA Study of AS [3].\nWe sought, for the first time, to generate reliable (but inherently conservative) estimates on the prevalence and treatable patient population with severe, symptomatic AS in Australia. Specifically, our aim was to replicate the same robust models recently used to generate contemporary AS-specific projections for Europe and North America [12] as highlighted above with specific modifications relevant to the Australian context. Firstly, this included applying population profiling (denominator) data derived from the Australian Bureau of Statistics (ABS). Secondly, given the Australian context, we aimed to expand our burden of disease estimates to those aged ≥ 55 years (noting the original study applied a single estimate of the overall prevalence of severe AS (numerator) to those aged ≥ 75 years [12]) and by applying age- and sex-specific, incident, and prevalent estimates of AS informed by the recent NEDA Study of AS [3].\n\nStudy aims:\nWe sought, for the first time, to generate reliable (but inherently conservative) estimates on the prevalence and treatable patient population with severe, symptomatic AS in Australia. Specifically, our aim was to replicate the same robust models recently used to generate contemporary AS-specific projections for Europe and North America [12] as highlighted above with specific modifications relevant to the Australian context. Firstly, this included applying population profiling (denominator) data derived from the Australian Bureau of Statistics (ABS). Secondly, given the Australian context, we aimed to expand our burden of disease estimates to those aged ≥ 55 years (noting the original study applied a single estimate of the overall prevalence of severe AS (numerator) to those aged ≥ 75 years [12]) and by applying age- and sex-specific, incident, and prevalent estimates of AS informed by the recent NEDA Study of AS [3].\n\nMethods:\nStudy design Consistent with previous reports of this type specifically focusing on atrial fibrillation [13] and heart failure [14], we combined official national population data with the best available epidemiological and clinical data to generate estimates of the treatable burden of disease relating to AS. Given the anonymous source and nature of the study data, no ethical approvals were required.\nConsistent with previous reports of this type specifically focusing on atrial fibrillation [13] and heart failure [14], we combined official national population data with the best available epidemiological and clinical data to generate estimates of the treatable burden of disease relating to AS. Given the anonymous source and nature of the study data, no ethical approvals were required.\nStudy setting Projections were applied to the entire Australian population and then each State and Territory according to their currently estimated demographic profile. ABS data projecting the future demographic structure and profile were also used to derive future projections on the treatable burden of AS. (https://www.abs.gov.au/Population - Accessed January 2021).\nProjections were applied to the entire Australian population and then each State and Territory according to their currently estimated demographic profile. ABS data projecting the future demographic structure and profile were also used to derive future projections on the treatable burden of AS. (https://www.abs.gov.au/Population - Accessed January 2021).\nStudy data The primary analyses presented in this report used age-, sex- and geographic-specific demography data for Australian men and women aged ≥ 55 years for the calendar year 2019. Specifically, the ABS currently provides population projections derived from the official 2016 Census - http://www.abs.gov.au/ausstats/abs@.nsf/mf/3101.0 - Accessed January 2021).\nAs indicated above, the primary basis for this report is the recently updated study of the number of treatable and treated patients with severe AS published by Durko and colleagues in 2018 [12]. Specifically, based on an expanded analysis of 37 relevant studies involving 26,402 cases/patients, this study provides a flow-chart (see Fig. 1) of the key estimates/parameters (with 95 % confidence interval, CI) that can be applied (as a primary analysis) to the latest Australian population data (on an age-, sex- and geographic-specific basis) to estimate the pool of treatable severe AS patients per annum. It can also be applied to broad population projections for an increasing pool of older Australians over time; noting that over a decade (from 2006 to 2016) the proportion of Australians aged > 65 years had increased by around 4 % and this proportion (and absolute numbers) will steadily increase.\nFig. 1Model/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\nModel/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\nSpecifically, the model developed and subsequently applied in this report provides overall estimates (based on a combination of European and North American cohort studies) on the proportion of patients with severe AS who – 1) are symptomatic and under current expert guidelines, are largely excluded from surgical management; 2) are symptomatic but unsuitable for SAVR but might safely undergo TAVR (the alternative being medical therapy); or 3) are symptomatic and will undergo SAVR; a proportion of whom (particularly due to high-to-medium surgical risk) may benefit from TAVR. The range of derived estimates for each parameter, despite efforts to smooth-out inevitable heterogeneity, are indicative of the methodological weaknesses/biases inherent to source data. It is important to note these treatment pathways are continually evolving as new, lower risk valve interventions are developed and lower the likely threshold of AVR. Without Australian-specific to correct the rates of intervention applied, it is generally acknowledged that intervention rates for severe AS broadly follow those applied in North America.\nWhere possible, we were able to improve our assumptions and therefore our projections by considering primary profiling and outcome data derived from (NEDA) [3]. This unique study has now captured echocardiographic data on > 750,000 Australians (with no exclusion criteria) being routinely investigated with echocardiography from > 25 centres Australia-wide. With individual data linkage, NEDA also generates real-world, short- to long-term survival data on those affected by common cardiac conditions including AS [3].\nThe primary analyses presented in this report used age-, sex- and geographic-specific demography data for Australian men and women aged ≥ 55 years for the calendar year 2019. Specifically, the ABS currently provides population projections derived from the official 2016 Census - http://www.abs.gov.au/ausstats/abs@.nsf/mf/3101.0 - Accessed January 2021).\nAs indicated above, the primary basis for this report is the recently updated study of the number of treatable and treated patients with severe AS published by Durko and colleagues in 2018 [12]. Specifically, based on an expanded analysis of 37 relevant studies involving 26,402 cases/patients, this study provides a flow-chart (see Fig. 1) of the key estimates/parameters (with 95 % confidence interval, CI) that can be applied (as a primary analysis) to the latest Australian population data (on an age-, sex- and geographic-specific basis) to estimate the pool of treatable severe AS patients per annum. It can also be applied to broad population projections for an increasing pool of older Australians over time; noting that over a decade (from 2006 to 2016) the proportion of Australians aged > 65 years had increased by around 4 % and this proportion (and absolute numbers) will steadily increase.\nFig. 1Model/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\nModel/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\nSpecifically, the model developed and subsequently applied in this report provides overall estimates (based on a combination of European and North American cohort studies) on the proportion of patients with severe AS who – 1) are symptomatic and under current expert guidelines, are largely excluded from surgical management; 2) are symptomatic but unsuitable for SAVR but might safely undergo TAVR (the alternative being medical therapy); or 3) are symptomatic and will undergo SAVR; a proportion of whom (particularly due to high-to-medium surgical risk) may benefit from TAVR. The range of derived estimates for each parameter, despite efforts to smooth-out inevitable heterogeneity, are indicative of the methodological weaknesses/biases inherent to source data. It is important to note these treatment pathways are continually evolving as new, lower risk valve interventions are developed and lower the likely threshold of AVR. Without Australian-specific to correct the rates of intervention applied, it is generally acknowledged that intervention rates for severe AS broadly follow those applied in North America.\nWhere possible, we were able to improve our assumptions and therefore our projections by considering primary profiling and outcome data derived from (NEDA) [3]. This unique study has now captured echocardiographic data on > 750,000 Australians (with no exclusion criteria) being routinely investigated with echocardiography from > 25 centres Australia-wide. With individual data linkage, NEDA also generates real-world, short- to long-term survival data on those affected by common cardiac conditions including AS [3].\nEstimating the prevalence & treatable burden of severe AS For these analyses, we applied a point prevalence of 3.5 % for severe AS among individuals aged ≥ 75 years, This small adjustment to the original European model [12] reflects recent reports of an increasing incidence of AS in other high-income countries such as the UK [15] and is only slightly higher than that of the NEDA cohort of actively investigated patients [3]. To derive specific prevalence rates for those aged < 75 years, we used the ratio of cases of severe AS observed in each age-band of the NEDA cohort [3]. This resulted in the following age-specific prevalence estimates being applied: ≥75 years, 3.5 %; 70–74 years, 1.2 %; 65–69 years, 0.7 %; 60–64 years, 0.5 %; and 55–59 years, 0.4 %. When applied to the Australian population aged ≥ 55 years (see Supplementary Figure S1), the overall estimated point prevalence of severe AS is 1.48 % for 2019; a figure that is broadly consistent with that published and applied previously [16]. The same rates were applied to men and women given that, on an age-specific basis, there were minimum differences based on sex within the NEDA cohort [3].\nFor these analyses, we applied a point prevalence of 3.5 % for severe AS among individuals aged ≥ 75 years, This small adjustment to the original European model [12] reflects recent reports of an increasing incidence of AS in other high-income countries such as the UK [15] and is only slightly higher than that of the NEDA cohort of actively investigated patients [3]. To derive specific prevalence rates for those aged < 75 years, we used the ratio of cases of severe AS observed in each age-band of the NEDA cohort [3]. This resulted in the following age-specific prevalence estimates being applied: ≥75 years, 3.5 %; 70–74 years, 1.2 %; 65–69 years, 0.7 %; 60–64 years, 0.5 %; and 55–59 years, 0.4 %. When applied to the Australian population aged ≥ 55 years (see Supplementary Figure S1), the overall estimated point prevalence of severe AS is 1.48 % for 2019; a figure that is broadly consistent with that published and applied previously [16]. The same rates were applied to men and women given that, on an age-specific basis, there were minimum differences based on sex within the NEDA cohort [3].\nMortality To understand the potential consequences of no AVR intervention in the setting of severe AS, age- and sex-specific, actual 5-year mortality rates were applied to the estimated case prevalence of severe AS by applying those observed rates within the equivalent patient cohort identified within NEDA who had a native AV throughout follow-up [3]. Although these data only reflect cases referred for echocardiography, they provide both a discussion point and means to validate prevalence estimates when combined with incidence rates (see below).\nTo understand the potential consequences of no AVR intervention in the setting of severe AS, age- and sex-specific, actual 5-year mortality rates were applied to the estimated case prevalence of severe AS by applying those observed rates within the equivalent patient cohort identified within NEDA who had a native AV throughout follow-up [3]. Although these data only reflect cases referred for echocardiography, they provide both a discussion point and means to validate prevalence estimates when combined with incidence rates (see below).\nIncident cases of severe AS In the absence of specific population incidence data, we used the differential in prevalence for each 5-year age group (i.e., how many more at risk individuals would develop severe AS over 5-years to reflect the number of prevalent cases in that older age group) to calculate how many individuals would develop severe AS each year. By necessity this means incident cases were only calculated for those aged ≥ 60 years. Applying conservatively derived data from the NEDA cohort [3], the following age-specific, annual incident rates, were applied to the Australian population aged ≥ 60 years for the subsequent (i.e. beyond 2019) 5-year period 2020-24: aged ≥ 75 years, 460 cases; 70–74 years, 40 cases; 65–69 years, 40 cases; and 60–64 years, 20 cases per 100,000 population per annum. When combined, these rates generated an annual incidence of severe AS of 182 cases per 100,000 population per annum within the Australian population aged ≥ 60 years.\nIn the absence of specific population incidence data, we used the differential in prevalence for each 5-year age group (i.e., how many more at risk individuals would develop severe AS over 5-years to reflect the number of prevalent cases in that older age group) to calculate how many individuals would develop severe AS each year. By necessity this means incident cases were only calculated for those aged ≥ 60 years. Applying conservatively derived data from the NEDA cohort [3], the following age-specific, annual incident rates, were applied to the Australian population aged ≥ 60 years for the subsequent (i.e. beyond 2019) 5-year period 2020-24: aged ≥ 75 years, 460 cases; 70–74 years, 40 cases; 65–69 years, 40 cases; and 60–64 years, 20 cases per 100,000 population per annum. When combined, these rates generated an annual incidence of severe AS of 182 cases per 100,000 population per annum within the Australian population aged ≥ 60 years.\nStatistical analyses All projections are reported as whole numbers and proportions with 95 % CI where appropriate. All statistical analyses are descriptive in nature and population-based; with no inferential statistics applied. Exact estimates are provided in the figures whilst rounded up (to the nearest hundred) figures are provided in text.\nAll projections are reported as whole numbers and proportions with 95 % CI where appropriate. All statistical analyses are descriptive in nature and population-based; with no inferential statistics applied. Exact estimates are provided in the figures whilst rounded up (to the nearest hundred) figures are provided in text.\n\nStudy design:\nConsistent with previous reports of this type specifically focusing on atrial fibrillation [13] and heart failure [14], we combined official national population data with the best available epidemiological and clinical data to generate estimates of the treatable burden of disease relating to AS. Given the anonymous source and nature of the study data, no ethical approvals were required.\n\nStudy setting:\nProjections were applied to the entire Australian population and then each State and Territory according to their currently estimated demographic profile. ABS data projecting the future demographic structure and profile were also used to derive future projections on the treatable burden of AS. (https://www.abs.gov.au/Population - Accessed January 2021).\n\nStudy data:\nThe primary analyses presented in this report used age-, sex- and geographic-specific demography data for Australian men and women aged ≥ 55 years for the calendar year 2019. Specifically, the ABS currently provides population projections derived from the official 2016 Census - http://www.abs.gov.au/ausstats/abs@.nsf/mf/3101.0 - Accessed January 2021).\nAs indicated above, the primary basis for this report is the recently updated study of the number of treatable and treated patients with severe AS published by Durko and colleagues in 2018 [12]. Specifically, based on an expanded analysis of 37 relevant studies involving 26,402 cases/patients, this study provides a flow-chart (see Fig. 1) of the key estimates/parameters (with 95 % confidence interval, CI) that can be applied (as a primary analysis) to the latest Australian population data (on an age-, sex- and geographic-specific basis) to estimate the pool of treatable severe AS patients per annum. It can also be applied to broad population projections for an increasing pool of older Australians over time; noting that over a decade (from 2006 to 2016) the proportion of Australians aged > 65 years had increased by around 4 % and this proportion (and absolute numbers) will steadily increase.\nFig. 1Model/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\nModel/Decision Tree to Determine Treatable Burden of Severe AS in Australia (Adapted from original [12]). Legend: AS = Aortic Stenosis, SAVR = Surgical Aortic Valve Replacement, TAVR = Transcatheter Aortic Valve Replacement, High, Medium and Low Risk based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score of >8%, 4-8% and <4% for SAVR-related mortality, respectively\nSpecifically, the model developed and subsequently applied in this report provides overall estimates (based on a combination of European and North American cohort studies) on the proportion of patients with severe AS who – 1) are symptomatic and under current expert guidelines, are largely excluded from surgical management; 2) are symptomatic but unsuitable for SAVR but might safely undergo TAVR (the alternative being medical therapy); or 3) are symptomatic and will undergo SAVR; a proportion of whom (particularly due to high-to-medium surgical risk) may benefit from TAVR. The range of derived estimates for each parameter, despite efforts to smooth-out inevitable heterogeneity, are indicative of the methodological weaknesses/biases inherent to source data. It is important to note these treatment pathways are continually evolving as new, lower risk valve interventions are developed and lower the likely threshold of AVR. Without Australian-specific to correct the rates of intervention applied, it is generally acknowledged that intervention rates for severe AS broadly follow those applied in North America.\nWhere possible, we were able to improve our assumptions and therefore our projections by considering primary profiling and outcome data derived from (NEDA) [3]. This unique study has now captured echocardiographic data on > 750,000 Australians (with no exclusion criteria) being routinely investigated with echocardiography from > 25 centres Australia-wide. With individual data linkage, NEDA also generates real-world, short- to long-term survival data on those affected by common cardiac conditions including AS [3].\n\nEstimating the prevalence & treatable burden of severe AS:\nFor these analyses, we applied a point prevalence of 3.5 % for severe AS among individuals aged ≥ 75 years, This small adjustment to the original European model [12] reflects recent reports of an increasing incidence of AS in other high-income countries such as the UK [15] and is only slightly higher than that of the NEDA cohort of actively investigated patients [3]. To derive specific prevalence rates for those aged < 75 years, we used the ratio of cases of severe AS observed in each age-band of the NEDA cohort [3]. This resulted in the following age-specific prevalence estimates being applied: ≥75 years, 3.5 %; 70–74 years, 1.2 %; 65–69 years, 0.7 %; 60–64 years, 0.5 %; and 55–59 years, 0.4 %. When applied to the Australian population aged ≥ 55 years (see Supplementary Figure S1), the overall estimated point prevalence of severe AS is 1.48 % for 2019; a figure that is broadly consistent with that published and applied previously [16]. The same rates were applied to men and women given that, on an age-specific basis, there were minimum differences based on sex within the NEDA cohort [3].\n\nMortality:\nTo understand the potential consequences of no AVR intervention in the setting of severe AS, age- and sex-specific, actual 5-year mortality rates were applied to the estimated case prevalence of severe AS by applying those observed rates within the equivalent patient cohort identified within NEDA who had a native AV throughout follow-up [3]. Although these data only reflect cases referred for echocardiography, they provide both a discussion point and means to validate prevalence estimates when combined with incidence rates (see below).\n\nIncident cases of severe AS:\nIn the absence of specific population incidence data, we used the differential in prevalence for each 5-year age group (i.e., how many more at risk individuals would develop severe AS over 5-years to reflect the number of prevalent cases in that older age group) to calculate how many individuals would develop severe AS each year. By necessity this means incident cases were only calculated for those aged ≥ 60 years. Applying conservatively derived data from the NEDA cohort [3], the following age-specific, annual incident rates, were applied to the Australian population aged ≥ 60 years for the subsequent (i.e. beyond 2019) 5-year period 2020-24: aged ≥ 75 years, 460 cases; 70–74 years, 40 cases; 65–69 years, 40 cases; and 60–64 years, 20 cases per 100,000 population per annum. When combined, these rates generated an annual incidence of severe AS of 182 cases per 100,000 population per annum within the Australian population aged ≥ 60 years.\n\nStatistical analyses:\nAll projections are reported as whole numbers and proportions with 95 % CI where appropriate. All statistical analyses are descriptive in nature and population-based; with no inferential statistics applied. Exact estimates are provided in the figures whilst rounded up (to the nearest hundred) figures are provided in text.\n\nResults:\nPrevalent cases of severe AS As shown in Fig. 2, we conservatively estimate that around 97,300 Australians are living with severe AS (symptomatic or otherwise). Moreover, assuming just over two-thirds of these cases experience concurrent symptoms linked to the condition, according to contemporary clinical recommendations/ best practice around 66,500 (95 % CI 59,200 to 74,000) people might be considered for an AVR procedure at any one time.\nFig. 2Estimated Point Prevalence and Distribution of Severe AS in Australia\nEstimated Point Prevalence and Distribution of Severe AS in Australia\nAs shown in Fig. 2, we conservatively estimate that around 97,300 Australians are living with severe AS (symptomatic or otherwise). Moreover, assuming just over two-thirds of these cases experience concurrent symptoms linked to the condition, according to contemporary clinical recommendations/ best practice around 66,500 (95 % CI 59,200 to 74,000) people might be considered for an AVR procedure at any one time.\nFig. 2Estimated Point Prevalence and Distribution of Severe AS in Australia\nEstimated Point Prevalence and Distribution of Severe AS in Australia\nAortic valve replacement Figure 3 summarises the overall treatable burden/management of severe AS when assuming 58.4 % of cases with symptomatic, severe AS would be referred for SAVR (primary replacement or revision) and the remainder (41.6 %) for potential TAVR. On this basis, we estimate that around 38,800 (95 % CI 35,700 to 42,000) Australians aged ≥ 55 years with severe, symptomatic AS might be considered for SAVR; based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score [17]. An additional 17,000 cases (95 % CI 11,600 to 22,600) of the approximately 27,700 people not eligible for SAVR due to high peri-operative risk could be potentially considered for TAVR instead.\nFig. 3Estimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\nEstimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\nFigure 3 summarises the overall treatable burden/management of severe AS when assuming 58.4 % of cases with symptomatic, severe AS would be referred for SAVR (primary replacement or revision) and the remainder (41.6 %) for potential TAVR. On this basis, we estimate that around 38,800 (95 % CI 35,700 to 42,000) Australians aged ≥ 55 years with severe, symptomatic AS might be considered for SAVR; based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score [17]. An additional 17,000 cases (95 % CI 11,600 to 22,600) of the approximately 27,700 people not eligible for SAVR due to high peri-operative risk could be potentially considered for TAVR instead.\nFig. 3Estimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\nEstimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\nAS-related mortality Based on the age- and sex-specific rates of actual mortality (ranging from 17 to 84 % for those aged 55–64 years to ≥ 85 years) observed in the NEDA cohort, we estimate that 56,300 (95 % CI 56,100 to 56,500) of the prevalent population with severe AS of their native valve will subsequently die within the next 5 years - see Fig. 4.\nFig. 4Severe AS Related Mortality\nSevere AS Related Mortality\nBased on the age- and sex-specific rates of actual mortality (ranging from 17 to 84 % for those aged 55–64 years to ≥ 85 years) observed in the NEDA cohort, we estimate that 56,300 (95 % CI 56,100 to 56,500) of the prevalent population with severe AS of their native valve will subsequently die within the next 5 years - see Fig. 4.\nFig. 4Severe AS Related Mortality\nSevere AS Related Mortality\nIncident cases As shown in Fig. 5, each year, we further estimate that around 9,300 (95 % CI 9,000 to 9,500) more Australians aged ≥ 60 years will subsequently develop severe AS. Assuming the same pattern of potential AVR procedures (once again based on peri-operative risk status), 3,700 (95 % CI 3,400 to 4,000) and 2,600 (95 % CI 2,300 to 2,900) of the approximate de novo 6,300 (95 % CI 5,600 to 7,000) cases with concurrent symptoms linked to the condition, would be potentially managed with/eligible for a SAVR or TAVR procedure, respectively (see Supplementary Figure S2).\nFig. 5Annual Incident Cases of Severe AS among Australians Aged ≥55 years\nAnnual Incident Cases of Severe AS among Australians Aged ≥55 years\nAs shown in Fig. 5, each year, we further estimate that around 9,300 (95 % CI 9,000 to 9,500) more Australians aged ≥ 60 years will subsequently develop severe AS. Assuming the same pattern of potential AVR procedures (once again based on peri-operative risk status), 3,700 (95 % CI 3,400 to 4,000) and 2,600 (95 % CI 2,300 to 2,900) of the approximate de novo 6,300 (95 % CI 5,600 to 7,000) cases with concurrent symptoms linked to the condition, would be potentially managed with/eligible for a SAVR or TAVR procedure, respectively (see Supplementary Figure S2).\nFig. 5Annual Incident Cases of Severe AS among Australians Aged ≥55 years\nAnnual Incident Cases of Severe AS among Australians Aged ≥55 years\n\nPrevalent cases of severe AS:\nAs shown in Fig. 2, we conservatively estimate that around 97,300 Australians are living with severe AS (symptomatic or otherwise). Moreover, assuming just over two-thirds of these cases experience concurrent symptoms linked to the condition, according to contemporary clinical recommendations/ best practice around 66,500 (95 % CI 59,200 to 74,000) people might be considered for an AVR procedure at any one time.\nFig. 2Estimated Point Prevalence and Distribution of Severe AS in Australia\nEstimated Point Prevalence and Distribution of Severe AS in Australia\n\nAortic valve replacement:\nFigure 3 summarises the overall treatable burden/management of severe AS when assuming 58.4 % of cases with symptomatic, severe AS would be referred for SAVR (primary replacement or revision) and the remainder (41.6 %) for potential TAVR. On this basis, we estimate that around 38,800 (95 % CI 35,700 to 42,000) Australians aged ≥ 55 years with severe, symptomatic AS might be considered for SAVR; based on the Society of Thoracic Surgeons Predicted Risk of Mortality Score [17]. An additional 17,000 cases (95 % CI 11,600 to 22,600) of the approximately 27,700 people not eligible for SAVR due to high peri-operative risk could be potentially considered for TAVR instead.\nFig. 3Estimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\nEstimated Treatable Burden/Management of Severe AS in Australia (Based on Prevalent Cases)\n\nAS-related mortality:\nBased on the age- and sex-specific rates of actual mortality (ranging from 17 to 84 % for those aged 55–64 years to ≥ 85 years) observed in the NEDA cohort, we estimate that 56,300 (95 % CI 56,100 to 56,500) of the prevalent population with severe AS of their native valve will subsequently die within the next 5 years - see Fig. 4.\nFig. 4Severe AS Related Mortality\nSevere AS Related Mortality\n\nIncident cases:\nAs shown in Fig. 5, each year, we further estimate that around 9,300 (95 % CI 9,000 to 9,500) more Australians aged ≥ 60 years will subsequently develop severe AS. Assuming the same pattern of potential AVR procedures (once again based on peri-operative risk status), 3,700 (95 % CI 3,400 to 4,000) and 2,600 (95 % CI 2,300 to 2,900) of the approximate de novo 6,300 (95 % CI 5,600 to 7,000) cases with concurrent symptoms linked to the condition, would be potentially managed with/eligible for a SAVR or TAVR procedure, respectively (see Supplementary Figure S2).\nFig. 5Annual Incident Cases of Severe AS among Australians Aged ≥55 years\nAnnual Incident Cases of Severe AS among Australians Aged ≥55 years\n\nDiscussion:\nIn the setting of specific surgical registry reports [18] and recent insightful data generated by the NEDA Study [3] but little else, we present a unique analysis of the potential treatable burden of severe AS in Australia. As with many substantive public health issues that routinely affect a large proportion of the population and is associated with costly treatment and historically poor health outcomes [13, 14], unfortunately there is a paucity of specific burden of disease data to guide health resources and clinical practice. Indeed, worldwide, the natural history and impact of AS remains poorly characterised [19]. This relative lack of data is exacerbated by the rapid progression of disease and high mortality in those affected [20]. It was on this basis we chose to use the best available modelling and projections on the population prevalence and treatable pattern/burden of severe AS [12] and then applied them to the Australian population aged ≥ 55 years with further adjustments/ improvements based on NEDA Study data [3]. Overall, our analyses suggest that close to 100,000 Australians in this at-risk age-group are currently living with this potentially deadly condition. Accordingly, in the next 5 years, more than half of these individuals will die without having undergone an AVR procedure – their risk of dying being two-fold higher on an adjusted basis than their counterparts without severe AS [3]. Without any change in its natural history (there being strong evidence that its prevalence will rise within our increasingly sedentary and obese population [15, 21], this number will likely rise substantially within Australia’s ageing population. At minimum, we estimate that an additional 9,000 Australians aged ≥ 60 years and over will develop severe AS each year.\nOverall, based on contemporary management practices in Europe and North America (noting this remains a highly evolving field), we estimated that just under 56,000 prevalent cases would be potentially eligible for a SAVR or TAVR procedure. As also shown by the NEDA Study, successful restoration of AV function with AVR is associated with markedly improved survival [10]. Due to population dynamics (including greater longevity among Australian women) more women than men are likely affected by severe AS, but many of these are aged > 80 years and may have comorbidities that will favour more conservative management options.\nRegardless, of the relative accuracy of these projections (noting our critical corrections of the original model [12] based on the very large and robust data derived from the NEDA cohort [3] – see below), these data provide an important context to the largely hidden but substantive burden of disease imposed by AS in Australia. From an individual to societal perspective, it seems clear that due to Australia’s progressively ageing population, a clear strategy to detect and then optimally manage an increasing burden of AS is urgently required. Outcome data derived from close to 350,000 Australians investigated with echocardiography and collectively followed-up > 1 million person-years as part of the NEDA Study [3] further reinforces the need to prioritise AS management. In that study we reaffirmed that severe AS had very poor survival rates (two-thirds dead within 5-years). We also confirmed that when applied, AVR was largely associated with optimal AV hemodynamic profiles and lower mortality [10].\nA recent analysis of 18,147 patients (mean age 72 years and 64 % men) with AS who underwent SAVR captured by the Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) database during 2002–2015, showed that this procedure accounted for 20 % of all adult cardiac surgeries by the end of the study period [18]. In recent years, TAVR has been successfully applied to patients with severe AS and with high/prohibitive surgical risk [5–7]; with two randomized trials reporting the non-inferiority [8] and superiority [9] of TAVR in respect to mortality and subsequent risk of stroke, respectively, when compared to SAVR in low-risk patients with severe AS. A recently reported Australian trial of TAVR applied to 199 intermediate-risk cases of severe AS, is indicative of the changing clinical landscape in this regard [22].\nDespite the type of data described above, determining the actual proportion of Australians at risk of poor outcomes associated with AS and then actively treated with SAVR or TAVR, remains problematic; even when reconciling the largely concordant data around the size of the likely active patient population derived from the NEDA Study and those formal projections. However, there does appear to be a disconnect between the number of Australians with severe AS who might benefit either SAVR or TAVR and their subsequent access to these procedures. A recent report from the ANZSCTS database recorded ~ 4,000 SAVR procedures overall in Australia during the period 2009–2015 [18]. Even when accounting for the fact that registry did become truly national until 2015, there appears to be a large shortfall in the expected number of such procedures per annum relative to our projections. This may well be explained by the demographic profile of those undergoing SAVR in Australia. As reported [18], the mean age of SAVR cases is around 72 years of age and only 37 % were female. Alternatively, the mean age of those with severe AS (during a similar timeframe of surveillance) within the NEDA cohort is around 80 years of age and, consistent with this report, more than half of cases were women. Anecdotally, referral for SAVR in the Australian population has typically paralleled North American trends and the presumptions of the modelling (shown in Fig. 1) are based on historical data from a combination of North American and European centres. Given the lag in referral behaviour to new low-risk interventions (e.g., TAVR), by definition, all these assumptions for the modelling will be conservative. Critically, the availability of these new valve technologies has developed rapidly in Australia in the last 5 years and, other than NEDA, there is a paucity of resources to track these changes in real-time.\nThe recently reported NEDA Study data suggesting that even mild-to-moderate forms of AS are associated with high-levels of mortality approaching that of severe AS within 5-years of follow-up [3], when combined with other contemporary reports [23, 24], are likely to change the landscape of AS management. Specifically, this will likely reflect a recognition that a “watchful wait” approach to determine the transition from moderate to severe AS and also from asymptomatic to symptomatic status [4] may be associated with unacceptably high mortality rates [5]. For example, reflective of concerns around intermediate-to-high risk of surgical mortality (> 4 %) and the evolving efficacy of TAVR versus the more costly and invasive SAVR, in the current analyses, it was estimated that around 9,000 SAVR cases could potentially be replaced with TAVR. However, determining how and where to invest in dedicated screening programs and apply the latest evidence to prolong the lives of those affected in Australia (noting our estimate of approximately 9,000 new cases per annum – of whom > 4,000 would be aged < 75 years) is futile without firm evidence of the number of individuals involved. The noted “disconnect” between the potential and actual number of Australians who derive survival benefits from an AVR, therefore, is likely to increase over time.\nLimitations As noted, these data do not completely rely on Australian specific data (other than population estimates/demographic structure and NEDA-derived adjustments to original estimates). To partially address this, we have used a conservative estimate of the point prevalence of severe AS and provide 95 % CI for lower and higher and estimates. However, we acknowledge the dynamics of medical management of severe AS (i.e., conservative treatment versus TAVR versus SAVR) in Australia is likely to be different than reported in Europe/North America. The differential clinical uptake and reimbursement of different procedures from a public health to private health perspective within Australia’s increasingly complex and hybrid health care system, is particularly relevant - as is the variable population dynamics of each jurisdiction across the country. Beyond the broader demographic features of Australia and its major jurisdictions, we did not consider other important factors such as socio-economic status and the concentration of particularly high-risk groups (e.g. predominantly younger Indigenous Australians in Central Australia who experience much higher levels of valvular disease and heart failure [25]); nor did we consider the cost-burden of treating severe AS. Applying the growing resources of NEDA, we hope to address most of these issues/limitations in the future, in order to provide greater clarity around the full spectrum of AS in Australia. Finally, it is important to acknowledge that our mortality estimates (even when considering that they are focussed on those with native valves) are discordant with the low rates of mortality reported in trials such as the PARTNER 3 Trial [9]. This discordance only reinforces the benefits of early recognition and expert management of this otherwise deadly condition.\nAs noted, these data do not completely rely on Australian specific data (other than population estimates/demographic structure and NEDA-derived adjustments to original estimates). To partially address this, we have used a conservative estimate of the point prevalence of severe AS and provide 95 % CI for lower and higher and estimates. However, we acknowledge the dynamics of medical management of severe AS (i.e., conservative treatment versus TAVR versus SAVR) in Australia is likely to be different than reported in Europe/North America. The differential clinical uptake and reimbursement of different procedures from a public health to private health perspective within Australia’s increasingly complex and hybrid health care system, is particularly relevant - as is the variable population dynamics of each jurisdiction across the country. Beyond the broader demographic features of Australia and its major jurisdictions, we did not consider other important factors such as socio-economic status and the concentration of particularly high-risk groups (e.g. predominantly younger Indigenous Australians in Central Australia who experience much higher levels of valvular disease and heart failure [25]); nor did we consider the cost-burden of treating severe AS. Applying the growing resources of NEDA, we hope to address most of these issues/limitations in the future, in order to provide greater clarity around the full spectrum of AS in Australia. Finally, it is important to acknowledge that our mortality estimates (even when considering that they are focussed on those with native valves) are discordant with the low rates of mortality reported in trials such as the PARTNER 3 Trial [9]. This discordance only reinforces the benefits of early recognition and expert management of this otherwise deadly condition.\n\nLimitations:\nAs noted, these data do not completely rely on Australian specific data (other than population estimates/demographic structure and NEDA-derived adjustments to original estimates). To partially address this, we have used a conservative estimate of the point prevalence of severe AS and provide 95 % CI for lower and higher and estimates. However, we acknowledge the dynamics of medical management of severe AS (i.e., conservative treatment versus TAVR versus SAVR) in Australia is likely to be different than reported in Europe/North America. The differential clinical uptake and reimbursement of different procedures from a public health to private health perspective within Australia’s increasingly complex and hybrid health care system, is particularly relevant - as is the variable population dynamics of each jurisdiction across the country. Beyond the broader demographic features of Australia and its major jurisdictions, we did not consider other important factors such as socio-economic status and the concentration of particularly high-risk groups (e.g. predominantly younger Indigenous Australians in Central Australia who experience much higher levels of valvular disease and heart failure [25]); nor did we consider the cost-burden of treating severe AS. Applying the growing resources of NEDA, we hope to address most of these issues/limitations in the future, in order to provide greater clarity around the full spectrum of AS in Australia. Finally, it is important to acknowledge that our mortality estimates (even when considering that they are focussed on those with native valves) are discordant with the low rates of mortality reported in trials such as the PARTNER 3 Trial [9]. This discordance only reinforces the benefits of early recognition and expert management of this otherwise deadly condition.\n\nConclusions:\nThese unique estimates provide an important insight into the current and future treatable burden of severe AS in Australia. At the most conservative level, the likely number of currently affected Australians aged 55 years and over will soon rise to 100,000 people. Moreover, the number of new cases with this potentially deadly condition is likely approaching 10,000 per annum. Based on current clinical practice/recommendations, around two-thirds of such cases should be actively managed due to their symptomatic status (predominantly with a combination of SAVR and TAVR) and a high-risk of mortality. However, it is unclear if that is truly the case. Whether there is sufficient clinical awareness of AS and pro-active referral patterns (particularly for Australian women) for active management is yet to be determined.\n\nSupplementary Information:\n \nAdditional file 1: Supplementary Figure 1. Population Distribution of Australians Aged ≥55 years. Legend: Australia is a federated country, comprising the main populated States (Northern Territory and Capital Territory not shown) of Western Australian (WA), South Australia (SA), Victoria (VIC), Tasmania (TAS), New South Wales (NSW) and Queensland (QLD).\nAdditional file 2: Supplementary Figure 2. Estimated Treatable Burden/Management of Severe AS in Australia (Based on Incident Cases per Annum).\n\nAdditional file 1: Supplementary Figure 1. Population Distribution of Australians Aged ≥55 years. Legend: Australia is a federated country, comprising the main populated States (Northern Territory and Capital Territory not shown) of Western Australian (WA), South Australia (SA), Victoria (VIC), Tasmania (TAS), New South Wales (NSW) and Queensland (QLD).\nAdditional file 2: Supplementary Figure 2. Estimated Treatable Burden/Management of Severe AS in Australia (Based on Incident Cases per Annum).\n\nAdditional file 1: Supplementary Figure 1. Population Distribution of Australians Aged ≥55 years. Legend: Australia is a federated country, comprising the main populated States (Northern Territory and Capital Territory not shown) of Western Australian (WA), South Australia (SA), Victoria (VIC), Tasmania (TAS), New South Wales (NSW) and Queensland (QLD).\nAdditional file 2: Supplementary Figure 2. Estimated Treatable Burden/Management of Severe AS in Australia (Based on Incident Cases per Annum).\n\nAdditional file 1: Supplementary Figure 1. Population Distribution of Australians Aged ≥55 years. Legend: Australia is a federated country, comprising the main populated States (Northern Territory and Capital Territory not shown) of Western Australian (WA), South Australia (SA), Victoria (VIC), Tasmania (TAS), New South Wales (NSW) and Queensland (QLD).\nAdditional file 2: Supplementary Figure 2. Estimated Treatable Burden/Management of Severe AS in Australia (Based on Incident Cases per Annum).\n\n:\n\nAdditional file 1: Supplementary Figure 1. Population Distribution of Australians Aged ≥55 years. Legend: Australia is a federated country, comprising the main populated States (Northern Territory and Capital Territory not shown) of Western Australian (WA), South Australia (SA), Victoria (VIC), Tasmania (TAS), New South Wales (NSW) and Queensland (QLD).\nAdditional file 2: Supplementary Figure 2. Estimated Treatable Burden/Management of Severe AS in Australia (Based on Incident Cases per Annum).\n\nAdditional file 1: Supplementary Figure 1. Population Distribution of Australians Aged ≥55 years. Legend: Australia is a federated country, comprising the main populated States (Northern Territory and Capital Territory not shown) of Western Australian (WA), South Australia (SA), Victoria (VIC), Tasmania (TAS), New South Wales (NSW) and Queensland (QLD).\nAdditional file 2: Supplementary Figure 2. Estimated Treatable Burden/Management of Severe AS in Australia (Based on Incident Cases per Annum).\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nWe aimed to address the paucity of information describing the treatable burden of disease associated with severe aortic stenosis (AS) within Australia's ageing population.\n\nMethods:\nA contemporary model of the population prevalence of symptomatic, severe AS and treatment pathways in Europe and North America was applied to the 2019 Australian population aged ≥ 55 years (7 million people) on an age-specific basis. Applying Australian-specific data, these estimates were used to further calculate the total number of associated deaths and incident cases of severe AS per annum.\n\nResults:\nBased on an overall point prevalence of 1.48 % among those aged ≥ 55 years, we estimate that a minimum of 97,000 Australians are living with severe AS. With a 2-fold increased risk of mortality without undergoing aortic valve replacement (AVR), more than half of these individuals (∼56,000) will die within 5-years. From a clinical management perspective, among those with concurrent symptoms (68.3 %, 66,500 [95 % CI 59,000-74,000] cases) more than half (58.4 %, 38,800 [95 % CI 35,700 - 42,000] cases) would be potentially considered for surgical AVR (SAVR) - comprising 2,400, 5,400 and 31,000 cases assessed as high-, medium- or low peri-operative mortality risk, respectively. A further 17,000/27,700 (41.6 % [95 % CI 11,600 - 22,600]) of such individuals would be potentially considered to a transthoracic AVR (TAVR). During the subsequent 5-year period (2020-2024), each year, we estimate an additional 9,300 Australians aged ≥ 60 years will subsequently develop severe AS (6,300 of whom will experience concurrent symptoms). Of these symptomatic cases, an estimated 3,700 and 1,600 cases/annum, will be potentially suitable for SAVR and TAVR, respectively.\n\nConclusions:\nThese data suggest there is likely to be a substantive burden of individuals living with severe AS in Australia. Many of these cases may not have been diagnosed and/or received appropriate treatment (based on the evidence-based application of SAVR and TAVR) to reduce their high-risk of subsequent mortality."},"background_conclusion_text":{"kind":"string","value":"Introduction:\nOne of the most common cardiac conditions affecting the progressively aging populations of high-income countries such as Australia is aortic stenosis (AS) [1]. Without timely intervention, severe AS is associated with a very poor prognosis [2]. However, like many other countries, there has been a paucity of reports focusing on the overall prevalence and treatable burden of AS in Australia. A recent AS report from National Echocardiography Database of Australia (NEDA) [3] that assessed the severity of AS and subsequent pattern of survival among 122,809 men and 118,494 women with a mean age of 62 ± 18 years highlighted an urgent need to better understand the burden imposed by this potentially deadly condition. Overall, the indicative prevalence of severe low or high gradient AS among adults being investigated with echocardiography during the overall study period of 2000–2019, was 1.1 and 2.1 % (3.2 % combined), respectively. Actual 5-year mortality ranged from 56 to 67 % in those cases with a native aortic valve and no indication of surgical intervention [3].\nHistorically, surgical aortic valve replacement (SAVR) has been the preferred intervention for severe AS [4]. However, transcatheter aortic valve replacement (TAVR) has been successfully applied in those with severe AS with high/prohibitive surgical risk [5–7]. Moreover, two head-to-head randomized trials have now reported non-inferiority [8] and superiority [9] comparing TAVR to SAVR in low-risk patients with severe AS, in respect to mortality and subsequent risk of stroke. Consistent with these data, for most of the 6,050 cases within the NEDA cohort who underwent AVR, their post-procedure AV hemodynamic profile and survival outcomes were favourable [10].\nOverall, these data suggest more Australians might benefit from AVR. However, without reliable estimates of the treatable burden of AS, this critical number (for health service and resource planning) remains unknown. A series of modelled studies, first published in 2013 [11] and then an updated version in 2018 [12], applied the best available epidemiological and registry data to estimate the following for Europe and North America – 1) the overall proportion of older individuals affected by AS and more specifically severe AS; and 2) proportion of these individuals who had and/or would benefit from a valve replacement procedure (SAVR or TAVR). Given the geographical focus and source data used, these estimates now provide these target regions with (moderate) reliable estimates of the treatable burden of AS. To date, there are no equivalent burden of disease estimates for Australia.\nStudy aims We sought, for the first time, to generate reliable (but inherently conservative) estimates on the prevalence and treatable patient population with severe, symptomatic AS in Australia. Specifically, our aim was to replicate the same robust models recently used to generate contemporary AS-specific projections for Europe and North America [12] as highlighted above with specific modifications relevant to the Australian context. Firstly, this included applying population profiling (denominator) data derived from the Australian Bureau of Statistics (ABS). Secondly, given the Australian context, we aimed to expand our burden of disease estimates to those aged ≥ 55 years (noting the original study applied a single estimate of the overall prevalence of severe AS (numerator) to those aged ≥ 75 years [12]) and by applying age- and sex-specific, incident, and prevalent estimates of AS informed by the recent NEDA Study of AS [3].\nWe sought, for the first time, to generate reliable (but inherently conservative) estimates on the prevalence and treatable patient population with severe, symptomatic AS in Australia. Specifically, our aim was to replicate the same robust models recently used to generate contemporary AS-specific projections for Europe and North America [12] as highlighted above with specific modifications relevant to the Australian context. Firstly, this included applying population profiling (denominator) data derived from the Australian Bureau of Statistics (ABS). Secondly, given the Australian context, we aimed to expand our burden of disease estimates to those aged ≥ 55 years (noting the original study applied a single estimate of the overall prevalence of severe AS (numerator) to those aged ≥ 75 years [12]) and by applying age- and sex-specific, incident, and prevalent estimates of AS informed by the recent NEDA Study of AS [3].\n\nConclusions:\nThese unique estimates provide an important insight into the current and future treatable burden of severe AS in Australia. At the most conservative level, the likely number of currently affected Australians aged 55 years and over will soon rise to 100,000 people. Moreover, the number of new cases with this potentially deadly condition is likely approaching 10,000 per annum. Based on current clinical practice/recommendations, around two-thirds of such cases should be actively managed due to their symptomatic status (predominantly with a combination of SAVR and TAVR) and a high-risk of mortality. However, it is unclear if that is truly the case. Whether there is sufficient clinical awareness of AS and pro-active referral patterns (particularly for Australian women) for active management is yet to be determined."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nWe aimed to address the paucity of information describing the treatable burden of disease associated with severe aortic stenosis (AS) within Australia's ageing population.\n\nMethods:\nA contemporary model of the population prevalence of symptomatic, severe AS and treatment pathways in Europe and North America was applied to the 2019 Australian population aged ≥ 55 years (7 million people) on an age-specific basis. Applying Australian-specific data, these estimates were used to further calculate the total number of associated deaths and incident cases of severe AS per annum.\n\nResults:\nBased on an overall point prevalence of 1.48 % among those aged ≥ 55 years, we estimate that a minimum of 97,000 Australians are living with severe AS. With a 2-fold increased risk of mortality without undergoing aortic valve replacement (AVR), more than half of these individuals (∼56,000) will die within 5-years. From a clinical management perspective, among those with concurrent symptoms (68.3 %, 66,500 [95 % CI 59,000-74,000] cases) more than half (58.4 %, 38,800 [95 % CI 35,700 - 42,000] cases) would be potentially considered for surgical AVR (SAVR) - comprising 2,400, 5,400 and 31,000 cases assessed as high-, medium- or low peri-operative mortality risk, respectively. A further 17,000/27,700 (41.6 % [95 % CI 11,600 - 22,600]) of such individuals would be potentially considered to a transthoracic AVR (TAVR). During the subsequent 5-year period (2020-2024), each year, we estimate an additional 9,300 Australians aged ≥ 60 years will subsequently develop severe AS (6,300 of whom will experience concurrent symptoms). Of these symptomatic cases, an estimated 3,700 and 1,600 cases/annum, will be potentially suitable for SAVR and TAVR, respectively.\n\nConclusions:\nThese data suggest there is likely to be a substantive burden of individuals living with severe AS in Australia. Many of these cases may not have been diagnosed and/or received appropriate treatment (based on the evidence-based application of SAVR and TAVR) to reduce their high-risk of subsequent mortality."},"whole_article_text_length":{"kind":"number","value":10123,"string":"10,123"},"whole_article_abstract_length":{"kind":"number","value":425,"string":"425"},"other_sections_lengths":{"kind":"list like","value":[177,2864,66,53,698,247,96,197,57,100,174,88,153,316,206],"string":"[\n 177,\n 2864,\n 66,\n 53,\n 698,\n 247,\n 96,\n 197,\n 57,\n 100,\n 174,\n 88,\n 153,\n 316,\n 206\n]","isConversational":false},"num_sections":{"kind":"number","value":20,"string":"20"},"most_frequent_words":{"kind":"list like","value":["severe","years","cases","australia","population","data","aged","applied","savr","estimates"],"string":"[\n \"severe\",\n \"years\",\n \"cases\",\n \"australia\",\n \"population\",\n \"data\",\n \"aged\",\n \"applied\",\n \"savr\",\n 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Aortic valve replacement | Health-services [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Aortic stenosis | Aortic valve replacement | Health-services [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Aortic stenosis | Aortic valve replacement | Health-services [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Aortic stenosis | Aortic valve replacement | Health-services [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Aortic stenosis | Aortic valve replacement | Health-services [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Aging | Aortic Valve Stenosis | Australia | Heart Valve Prosthesis Implantation | Humans | Risk Factors | Transcatheter Aortic Valve Replacement | Treatment Outcome 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mortality unclear | awareness pro active referral | awareness pro active | awareness pro [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] severe | years | cases | australia | data | population | aged | estimates | applied | prevalence [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] severe | years | cases | australia | data | population | aged | estimates | applied | prevalence [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Australia [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 1.48 % | ≥ | 55 years | 97,000 | Australians ||| 2-fold | AVR | more than half | ∼56,000 | 5-years ||| 68.3 % | 66,500 | 95 % | 59,000-74,000 | more than half | 58.4 % | 38,800 | 95 % | CI | 35,700 | 42,000 | 2,400 | 5,400 | 31,000 ||| 17,000/27,700 | 41.6 % ||| 95 % | CI | 11,600 | 22,600 | AVR ||| 5-year | 2020-2024 | each year | Australians | ≥ | 60 years | 6,300 ||| an estimated 3,700 | 1,600 | SAVR | TAVR [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Australia ||| SAVR [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Australia ||| Europe | North America | 2019 | Australian | ≥ | 55 years | 7 million ||| Australian ||| 1.48 % | ≥ | 55 years | 97,000 | Australians ||| 2-fold | AVR | more than half | ∼56,000 | 5-years ||| 68.3 % | 66,500 | 95 % | 59,000-74,000 | more than half | 58.4 % | 38,800 | 95 % | CI | 35,700 | 42,000 | 2,400 | 5,400 | 31,000 ||| 17,000/27,700 | 41.6 % ||| 95 % | CI | 11,600 | 22,600 | AVR ||| 5-year | 2020-2024 | each year | Australians | ≥ | 60 years | 6,300 ||| an estimated 3,700 | 1,600 | SAVR | TAVR ||| Australia ||| SAVR [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Australia ||| Europe | North America | 2019 | Australian | ≥ | 55 years | 7 million ||| Australian ||| 1.48 % | ≥ | 55 years | 97,000 | Australians ||| 2-fold | AVR | more than half | ∼56,000 | 5-years ||| 68.3 % | 66,500 | 95 % | 59,000-74,000 | more than half | 58.4 % | 38,800 | 95 % | CI | 35,700 | 42,000 | 2,400 | 5,400 | 31,000 ||| 17,000/27,700 | 41.6 % ||| 95 % | CI | 11,600 | 22,600 | AVR ||| 5-year | 2020-2024 | each year | Australians | ≥ | 60 years | 6,300 ||| an estimated 3,700 | 1,600 | SAVR | TAVR ||| Australia ||| SAVR [SUMMARY]"}}},{"rowIdx":93,"cells":{"title":{"kind":"string","value":"Using CF11 cellulose columns to inexpensively and effectively remove human DNA from Plasmodium falciparum-infected whole blood samples."},"pmid":{"kind":"string","value":"22321373"},"background_abstract":{"kind":"string","value":"Genome and transcriptome studies of Plasmodium nucleic acids obtained from parasitized whole blood are greatly improved by depletion of human DNA or enrichment of parasite DNA prior to next-generation sequencing and microarray hybridization. The most effective method currently used is a two-step procedure to deplete leukocytes: centrifugation using density gradient media followed by filtration through expensive, commercially available columns. This method is not easily implemented in field studies that collect hundreds of samples and simultaneously process samples for multiple laboratory analyses. Inexpensive syringes, hand-packed with CF11 cellulose powder, were recently shown to improve ex vivo cultivation of Plasmodium vivax obtained from parasitized whole blood. This study was undertaken to determine whether CF11 columns could be adapted to isolate Plasmodium falciparum DNA from parasitized whole blood and achieve current quantity and purity requirements for Illumina sequencing."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"The CF11 procedure was compared with the current two-step standard of leukocyte depletion using parasitized red blood cells cultured in vitro and parasitized blood obtained ex vivo from Cambodian patients with malaria. Procedural variations in centrifugation and column size were tested, along with a range of blood volumes and parasite densities."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"CF11 filtration reliably produces 500 nanograms of DNA with less than 50% human DNA contamination, which is comparable to that obtained by the two-step method and falls within the current quality control requirements for Illumina sequencing. In addition, a centrifuge-free version of the CF11 filtration method to isolate P. falciparum DNA at remote and minimally equipped field sites in malaria-endemic areas was validated."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"CF11 filtration is a cost-effective, scalable, one-step approach to remove human DNA from P. falciparum-infected whole blood samples."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSIONS"},"mesh_descriptor_names":{"kind":"list like","value":["Adolescent","Adult","Aged","Aged, 80 and over","Blood","Cambodia","Child","Child, Preschool","Chromatography","DNA, Protozoan","Humans","Infant","Malaria","Middle Aged","Parasitology","Plasmodium falciparum","Sensitivity and Specificity","Specimen Handling","Young Adult"],"string":"[\n \"Adolescent\",\n \"Adult\",\n \"Aged\",\n \"Aged, 80 and over\",\n \"Blood\",\n \"Cambodia\",\n \"Child\",\n \"Child, Preschool\",\n \"Chromatography\",\n \"DNA, Protozoan\",\n \"Humans\",\n \"Infant\",\n \"Malaria\",\n \"Middle Aged\",\n \"Parasitology\",\n \"Plasmodium falciparum\",\n \"Sensitivity and Specificity\",\n \"Specimen Handling\",\n \"Young Adult\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"3295709"},"background_title":{"kind":"string","value":"Background"},"background_text":{"kind":"string","value":"Recent technological advances have enabled next-generation genomic and transcriptomic analysis of Plasmodium falciparum from parasitized whole blood samples without the need for culturing. High-density genotyping of parasites obtained directly from patients with malaria has improved our understanding of population structure and genomic and transcriptional variation [1,2]. Highly parallel sequencing is currently being used to identify the genetic determinants of clinically relevant phenotypes including drug resistance, vaccine escape and disease severity. Importantly, genomic characterization of parasite populations in patients captures intra-host diversity and prevents the potential loss of sequence data from phenotype-conferring parasite isolates during their culture adaptation.\nThe performance of highly parallel sequencing platforms, such as Illumina, is greatly enhanced in sample preparations with a high parasite-to-human nucleic acid ratio [3]. Such high ratios can be achieved by either selectively capturing parasite nucleic acids or by removing human material from the sample. Hybrid selection [4] using RNA 'baits' complementary to the P. falciparum genome can achieve over 40-fold enrichment of parasite DNA and can be performed at any time following DNA extraction [5]; however, at $250 USD per sample, this technique may be prohibitively costly for epidemiological or population-level studies.\nThe alternative, leukocyte depletion, must be performed in field sites soon after blood collection and before transport and storage. Commercially available magnetic columns have been used to rapidly isolate parasitized red blood cells (RBCs) from uninfected RBCs and leukocytes [6]. However, magnetic purification depends on short-term culturing of patient blood to transform ring-stage parasites to haemozoin-rich trophozoites and schizonts, which requires equipment for in vitro parasite cultivation. Furthermore, parasites obtained directly from patients mature at different rates in culture, resulting in inconvenient time-points for purification. The current standard for leukocyte depletion of parasitized blood for P. falciparum nucleotide sequencing is a two-step process: centrifugation using Lymphoprep or another sucrose density gradient solution, followed by filtration using Plasmodipur filters [3]. This 'LP' method is effective but difficult to scale-up in field settings because it requires extensive handling and transfer of blood, training to perform sensitive steps, and costly commercial reagents and materials.\nFiltration with hand-made columns has been used as an inexpensive and less time-consuming alternative for leukocyte depletion of Plasmodium-infected blood for decades [7,8]. Recently, non-woven fabric filters [9] and plastic syringes filled with CF11 cellulose powder (Whatman) [10] were shown to effectively remove leukocytes and platelets from Plasmodium vivax-infected blood, where their phagocytic and degranulating activities may interfere with some ex vivo studies. CF11 filtration has also been used to improve microarray-based transcriptome analysis of P. vivax-infected blood [2,11]. CF11 cellulose is thought to work by trapping leukocytes by size exclusion and/or interactions between cellulose hydroxyl groups and leukocyte surface molecules. In this study, laboratory-adapted P. falciparum clones were used to adapt CF11 columns to remove human DNA from P. falciparum-infected blood for genomic studies, compare CF11 filtration to the established LP method, and validate a centrifuge-free option for CF11 filtration. Both methods were also compared in a field setting using P. falciparum isolates obtained directly from patients with malaria. Furthermore, routine CF11 filtration of parasitized blood collected from patients in a second field site with minimal facilities was successfully implemented."},"methods_title":{"kind":"string","value":"Methods"},"methods_text":{"kind":"string","value":" Blood samples Experiments using laboratory-adapted P. falciparum clones were conducted at the University of Maryland, Baltimore. Blood obtained from healthy volunteers was mixed with purified P. falciparum parasites (3D7 or Dd2 clones) to achieve parasite densities of 10,000/μl and 5,000/μl. These samples were stored at 4°C for up to two hours prior to CF11 or LP filtration.\nExperiments using non-adapted P. falciparum isolates were conducted in August 2010 at Sampov Meas Referral Hospital, Pursat, Cambodia. Blood samples for these experiments were collected from patients with falciparum malaria who were ≥ 10 years old and had a wide range of parasite densities (≥ 10,000/μl). These samples were held at 4°C for up to six hours prior to CF11 or LP filtration. Additional blood samples were collected in November to December 2010 at Lumphat District Health Centre, Ratanakiri, Cambodia. Blood samples were obtained from patients with falciparum malaria who were ≥ 1 year old and had a wide range of parasite densities ≥ 10,000/μl. These samples were held at 4°C for up to six hours prior to CF11 filtration. Following CF11 filtration, samples were stored on ice for up to six hours and transported on ice for up to 12 hours to Phnom Penh for centrifugation and RBC pellet storage.\nAll blood samples were collected in EDTA or CPDA-1 tubes. Heparin was not used as an anticoagulant as it thought to inhibit Taq polymerase during DNA amplification.\nBlood samples from healthy volunteers were collected under a protocol approved by the Institutional Review Board of the University of Maryland School of Medicine. Blood samples from patients with falciparum malaria were collected under protocols approved by the Institutional Review Board of the National Institute for Allergy and Infectious Diseases and the Cambodian National Ethics Committee for Health Research (http://ClinicalTrials.gov identifiers: NCT00341003 and NCT01240603). Written, informed consent was obtained from all study participants or their parents or guardians.\nExperiments using laboratory-adapted P. falciparum clones were conducted at the University of Maryland, Baltimore. Blood obtained from healthy volunteers was mixed with purified P. falciparum parasites (3D7 or Dd2 clones) to achieve parasite densities of 10,000/μl and 5,000/μl. These samples were stored at 4°C for up to two hours prior to CF11 or LP filtration.\nExperiments using non-adapted P. falciparum isolates were conducted in August 2010 at Sampov Meas Referral Hospital, Pursat, Cambodia. Blood samples for these experiments were collected from patients with falciparum malaria who were ≥ 10 years old and had a wide range of parasite densities (≥ 10,000/μl). These samples were held at 4°C for up to six hours prior to CF11 or LP filtration. Additional blood samples were collected in November to December 2010 at Lumphat District Health Centre, Ratanakiri, Cambodia. Blood samples were obtained from patients with falciparum malaria who were ≥ 1 year old and had a wide range of parasite densities ≥ 10,000/μl. These samples were held at 4°C for up to six hours prior to CF11 filtration. Following CF11 filtration, samples were stored on ice for up to six hours and transported on ice for up to 12 hours to Phnom Penh for centrifugation and RBC pellet storage.\nAll blood samples were collected in EDTA or CPDA-1 tubes. Heparin was not used as an anticoagulant as it thought to inhibit Taq polymerase during DNA amplification.\nBlood samples from healthy volunteers were collected under a protocol approved by the Institutional Review Board of the University of Maryland School of Medicine. Blood samples from patients with falciparum malaria were collected under protocols approved by the Institutional Review Board of the National Institute for Allergy and Infectious Diseases and the Cambodian National Ethics Committee for Health Research (http://ClinicalTrials.gov identifiers: NCT00341003 and NCT01240603). Written, informed consent was obtained from all study participants or their parents or guardians.\n Leukocyte depletion Lymphoprep (Axis-shield, Oslo, Norway) + Plasmodipur (Euro-diagnostica, Arnhem, Netherlands) filtration ('LP') was performed as previously described [3]. CF11 columns were loosely filled to the 10-ml mark, packed to the 5.5-ml mark with CF11 cellulose powder (Whatman, Kent, UK), and wetted with isotonic phosphate buffered saline (PBS), as described [10]. Initial tests indicated that plasma separation prior to CF11 filtration of blood did not affect human DNA depletion and that washing with PBS after CF11 filtration improved recovery of parasite DNA. CF11 filtration was therefore carried out as follows. Parasitized blood or blood/parasite mixtures were diluted with an equal volume of PBS. These samples were added to the CF11 column and allowed to flow through by gravity. To wash the sample, 5 ml PBS was added to the column and allowed to pass through by gravity. A plunger was then inserted into the top of the column and the final few drops pushed through the column. The filtrates were centrifuged for 10 minutes at 1,000 x g and the supernatants removed. RBC pellets from all filtrates in all experiments were stored at -20°C until DNA extraction. A packed CF11 column and filtration of blood diluted with PBS are shown in Figures 1A and 1B. The full CF11 filtration procedure is available on the WWARN website [12].\nPacked CF11 column ready for storage, shipping or use (A) and filtration of blood-PBS mixture through CF11 column into collection tube (B).\nLymphoprep (Axis-shield, Oslo, Norway) + Plasmodipur (Euro-diagnostica, Arnhem, Netherlands) filtration ('LP') was performed as previously described [3]. CF11 columns were loosely filled to the 10-ml mark, packed to the 5.5-ml mark with CF11 cellulose powder (Whatman, Kent, UK), and wetted with isotonic phosphate buffered saline (PBS), as described [10]. Initial tests indicated that plasma separation prior to CF11 filtration of blood did not affect human DNA depletion and that washing with PBS after CF11 filtration improved recovery of parasite DNA. CF11 filtration was therefore carried out as follows. Parasitized blood or blood/parasite mixtures were diluted with an equal volume of PBS. These samples were added to the CF11 column and allowed to flow through by gravity. To wash the sample, 5 ml PBS was added to the column and allowed to pass through by gravity. A plunger was then inserted into the top of the column and the final few drops pushed through the column. The filtrates were centrifuged for 10 minutes at 1,000 x g and the supernatants removed. RBC pellets from all filtrates in all experiments were stored at -20°C until DNA extraction. A packed CF11 column and filtration of blood diluted with PBS are shown in Figures 1A and 1B. The full CF11 filtration procedure is available on the WWARN website [12].\nPacked CF11 column ready for storage, shipping or use (A) and filtration of blood-PBS mixture through CF11 column into collection tube (B).\n Experimental design CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\n CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\n Experimental design CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\n CF11 filtration, with procedural modifications To test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\nTo test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\n Modifications in blood volume and parasite density To investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\nTo investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\n Routine blood collection for further validation and sequencing To determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\nTo determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\n CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\n CF11 filtration, with procedural modifications To test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\nTo test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\n Modifications in blood volume and parasite density To investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\nTo investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\n Routine blood collection for further validation and sequencing To determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\nTo determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\n DNA quantification DNA was extracted from leukocyte-depleted and unfiltered control samples using Qiamp DNA Blood Midi Kits (Qiagen, Venlo, Netherlands) according to the manufacturer's protocol. Total DNA from experiments using P. falciparum clones was estimated with a SpectraMax M2 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) using the Quant-IT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). The standard curve ranged from 0.20 to 50 ng/μl. Total DNA from experiments using P. falciparum isolates was estimated with a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) using both the dsDNA HS and BR Assay kits.\nQuantitative real-time PCR (qPCR) was used to measure the relative amounts of human and parasite DNA in each sample. Primers targeting the human LRAP (F: 5'-ACGTTGGATGAATTTTCCACTGGATTCCAT-'3; R: 5'-ACGTTGGATGTGAACCATGCTCCTTGCATC-'3) and TLR9 (F: 5'-ACGTTGGATGCAAAGGGCTGGCTGTTGTAG-'3; R: 5'- ACGTTGGATGTCTACCACGAGCACTCATTC-'3) genes and the P. falciparum AMA-1 gene (F: 5'-ACGTTGGATGGATTCTCTTTCGATTTCTTTC-'3; R: 5'-ACGTTGGATGTGCTACTACTGCTTTGTCCC-'3) were used to amplify DNA from samples, negative controls and standards in duplicate. Amplification occurred in an Applied Biosystems 7300 or StepOne real-time PCR machine. Human placental and P. falciparum (3D7 clone) genomic DNA standards ranged from 0.20 to 50 ng/μl. For each 25 μl reaction, 12.5 μl SYBR Green qPCR Master Mix (Applied Biosystems, Foster City, CA, USA) was mixed with 1.5 μl of each forward and reverse primers at 2 μM concentrations, 7.5-8.5 μl water, and 1-2 μl of template DNA. Reaction conditions were previously reported [3]: an initial denaturing step of 95°C for 10 minutes, five cycles of 94°C for 45 seconds, 56°C for 45 seconds, 72°C for 45 seconds, followed by 30 cycles of 94°C for 45 seconds, 65°C for 45 seconds and 72°C for 45 seconds and one dissociation cycle (conditions vary by real-time PCR instrument). Results were analysed using Applied Biosystems 7300 System SDS or StepOne v2.0 software.\nqPCR quantification of AMA-1 and TLR9 or LRAP was used to determine the proportions of P. falciparum and human DNA in the sample. Total DNA was estimated by multiplying the concentration given by Spectramax or Qubit by the extracted sample volume.\nDNA was extracted from leukocyte-depleted and unfiltered control samples using Qiamp DNA Blood Midi Kits (Qiagen, Venlo, Netherlands) according to the manufacturer's protocol. Total DNA from experiments using P. falciparum clones was estimated with a SpectraMax M2 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) using the Quant-IT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). The standard curve ranged from 0.20 to 50 ng/μl. Total DNA from experiments using P. falciparum isolates was estimated with a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) using both the dsDNA HS and BR Assay kits.\nQuantitative real-time PCR (qPCR) was used to measure the relative amounts of human and parasite DNA in each sample. Primers targeting the human LRAP (F: 5'-ACGTTGGATGAATTTTCCACTGGATTCCAT-'3; R: 5'-ACGTTGGATGTGAACCATGCTCCTTGCATC-'3) and TLR9 (F: 5'-ACGTTGGATGCAAAGGGCTGGCTGTTGTAG-'3; R: 5'- ACGTTGGATGTCTACCACGAGCACTCATTC-'3) genes and the P. falciparum AMA-1 gene (F: 5'-ACGTTGGATGGATTCTCTTTCGATTTCTTTC-'3; R: 5'-ACGTTGGATGTGCTACTACTGCTTTGTCCC-'3) were used to amplify DNA from samples, negative controls and standards in duplicate. Amplification occurred in an Applied Biosystems 7300 or StepOne real-time PCR machine. Human placental and P. falciparum (3D7 clone) genomic DNA standards ranged from 0.20 to 50 ng/μl. For each 25 μl reaction, 12.5 μl SYBR Green qPCR Master Mix (Applied Biosystems, Foster City, CA, USA) was mixed with 1.5 μl of each forward and reverse primers at 2 μM concentrations, 7.5-8.5 μl water, and 1-2 μl of template DNA. Reaction conditions were previously reported [3]: an initial denaturing step of 95°C for 10 minutes, five cycles of 94°C for 45 seconds, 56°C for 45 seconds, 72°C for 45 seconds, followed by 30 cycles of 94°C for 45 seconds, 65°C for 45 seconds and 72°C for 45 seconds and one dissociation cycle (conditions vary by real-time PCR instrument). Results were analysed using Applied Biosystems 7300 System SDS or StepOne v2.0 software.\nqPCR quantification of AMA-1 and TLR9 or LRAP was used to determine the proportions of P. falciparum and human DNA in the sample. Total DNA was estimated by multiplying the concentration given by Spectramax or Qubit by the extracted sample volume.\n Statistical comparisons Means, variance, standard errors and statistical comparisons of means were calculated using Stata 11 software. Boxplots indicating medians, interquartile ranges and outliers were also generated using Stata 11.\nMeans, variance, standard errors and statistical comparisons of means were calculated using Stata 11 software. Boxplots indicating medians, interquartile ranges and outliers were also generated using Stata 11."},"results_title":{"kind":"null"},"results_text":{"kind":"null"},"conclusions_title":{"kind":"string","value":"Conclusions"},"conclusions_text":{"kind":"string","value":"MV, CA, SC, OK, PL, SA and SU designed the study and collected the data. MV, CA and SC analysed the data. DS, DPK, RMF and CVP provided guidance and coordination for study design, data collection and analysis. MV wrote the first draft of the manuscript. RMF, CA, SC and CVP edited and revised the manuscript. All authors read and approved the final manuscript."},"other_sections_titles":{"kind":"list like","value":["Background","Blood samples","Leukocyte depletion","Experimental design","CF11 versus LP filtration, without modifications","Experimental design","CF11 versus LP filtration, without modifications","CF11 filtration, with procedural modifications","Modifications in blood volume and parasite density","Routine blood collection for further validation and sequencing","DNA quantification","Statistical comparisons","Results","Comparison of unmodified LP and CF11 filtration for leukocyte depletion","Procedural modifications of CF11 filtration method","Blood volume and parasite density","Validation of routine blood collection for illumina sequencing","Discussion","Conclusions"],"string":"[\n \"Background\",\n \"Blood samples\",\n \"Leukocyte depletion\",\n \"Experimental design\",\n \"CF11 versus LP filtration, without modifications\",\n \"Experimental design\",\n \"CF11 versus LP filtration, without modifications\",\n \"CF11 filtration, with procedural modifications\",\n \"Modifications in blood volume and parasite density\",\n \"Routine blood collection for further validation and sequencing\",\n \"DNA quantification\",\n \"Statistical comparisons\",\n \"Results\",\n \"Comparison of unmodified LP and CF11 filtration for leukocyte depletion\",\n \"Procedural modifications of CF11 filtration method\",\n \"Blood volume and parasite density\",\n \"Validation of routine blood collection for illumina sequencing\",\n \"Discussion\",\n \"Conclusions\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Recent technological advances have enabled next-generation genomic and transcriptomic analysis of Plasmodium falciparum from parasitized whole blood samples without the need for culturing. High-density genotyping of parasites obtained directly from patients with malaria has improved our understanding of population structure and genomic and transcriptional variation [1,2]. Highly parallel sequencing is currently being used to identify the genetic determinants of clinically relevant phenotypes including drug resistance, vaccine escape and disease severity. Importantly, genomic characterization of parasite populations in patients captures intra-host diversity and prevents the potential loss of sequence data from phenotype-conferring parasite isolates during their culture adaptation.\nThe performance of highly parallel sequencing platforms, such as Illumina, is greatly enhanced in sample preparations with a high parasite-to-human nucleic acid ratio [3]. Such high ratios can be achieved by either selectively capturing parasite nucleic acids or by removing human material from the sample. Hybrid selection [4] using RNA 'baits' complementary to the P. falciparum genome can achieve over 40-fold enrichment of parasite DNA and can be performed at any time following DNA extraction [5]; however, at $250 USD per sample, this technique may be prohibitively costly for epidemiological or population-level studies.\nThe alternative, leukocyte depletion, must be performed in field sites soon after blood collection and before transport and storage. Commercially available magnetic columns have been used to rapidly isolate parasitized red blood cells (RBCs) from uninfected RBCs and leukocytes [6]. However, magnetic purification depends on short-term culturing of patient blood to transform ring-stage parasites to haemozoin-rich trophozoites and schizonts, which requires equipment for in vitro parasite cultivation. Furthermore, parasites obtained directly from patients mature at different rates in culture, resulting in inconvenient time-points for purification. The current standard for leukocyte depletion of parasitized blood for P. falciparum nucleotide sequencing is a two-step process: centrifugation using Lymphoprep or another sucrose density gradient solution, followed by filtration using Plasmodipur filters [3]. This 'LP' method is effective but difficult to scale-up in field settings because it requires extensive handling and transfer of blood, training to perform sensitive steps, and costly commercial reagents and materials.\nFiltration with hand-made columns has been used as an inexpensive and less time-consuming alternative for leukocyte depletion of Plasmodium-infected blood for decades [7,8]. Recently, non-woven fabric filters [9] and plastic syringes filled with CF11 cellulose powder (Whatman) [10] were shown to effectively remove leukocytes and platelets from Plasmodium vivax-infected blood, where their phagocytic and degranulating activities may interfere with some ex vivo studies. CF11 filtration has also been used to improve microarray-based transcriptome analysis of P. vivax-infected blood [2,11]. CF11 cellulose is thought to work by trapping leukocytes by size exclusion and/or interactions between cellulose hydroxyl groups and leukocyte surface molecules. In this study, laboratory-adapted P. falciparum clones were used to adapt CF11 columns to remove human DNA from P. falciparum-infected blood for genomic studies, compare CF11 filtration to the established LP method, and validate a centrifuge-free option for CF11 filtration. Both methods were also compared in a field setting using P. falciparum isolates obtained directly from patients with malaria. Furthermore, routine CF11 filtration of parasitized blood collected from patients in a second field site with minimal facilities was successfully implemented.","Experiments using laboratory-adapted P. falciparum clones were conducted at the University of Maryland, Baltimore. Blood obtained from healthy volunteers was mixed with purified P. falciparum parasites (3D7 or Dd2 clones) to achieve parasite densities of 10,000/μl and 5,000/μl. These samples were stored at 4°C for up to two hours prior to CF11 or LP filtration.\nExperiments using non-adapted P. falciparum isolates were conducted in August 2010 at Sampov Meas Referral Hospital, Pursat, Cambodia. Blood samples for these experiments were collected from patients with falciparum malaria who were ≥ 10 years old and had a wide range of parasite densities (≥ 10,000/μl). These samples were held at 4°C for up to six hours prior to CF11 or LP filtration. Additional blood samples were collected in November to December 2010 at Lumphat District Health Centre, Ratanakiri, Cambodia. Blood samples were obtained from patients with falciparum malaria who were ≥ 1 year old and had a wide range of parasite densities ≥ 10,000/μl. These samples were held at 4°C for up to six hours prior to CF11 filtration. Following CF11 filtration, samples were stored on ice for up to six hours and transported on ice for up to 12 hours to Phnom Penh for centrifugation and RBC pellet storage.\nAll blood samples were collected in EDTA or CPDA-1 tubes. Heparin was not used as an anticoagulant as it thought to inhibit Taq polymerase during DNA amplification.\nBlood samples from healthy volunteers were collected under a protocol approved by the Institutional Review Board of the University of Maryland School of Medicine. Blood samples from patients with falciparum malaria were collected under protocols approved by the Institutional Review Board of the National Institute for Allergy and Infectious Diseases and the Cambodian National Ethics Committee for Health Research (http://ClinicalTrials.gov identifiers: NCT00341003 and NCT01240603). Written, informed consent was obtained from all study participants or their parents or guardians.","Lymphoprep (Axis-shield, Oslo, Norway) + Plasmodipur (Euro-diagnostica, Arnhem, Netherlands) filtration ('LP') was performed as previously described [3]. CF11 columns were loosely filled to the 10-ml mark, packed to the 5.5-ml mark with CF11 cellulose powder (Whatman, Kent, UK), and wetted with isotonic phosphate buffered saline (PBS), as described [10]. Initial tests indicated that plasma separation prior to CF11 filtration of blood did not affect human DNA depletion and that washing with PBS after CF11 filtration improved recovery of parasite DNA. CF11 filtration was therefore carried out as follows. Parasitized blood or blood/parasite mixtures were diluted with an equal volume of PBS. These samples were added to the CF11 column and allowed to flow through by gravity. To wash the sample, 5 ml PBS was added to the column and allowed to pass through by gravity. A plunger was then inserted into the top of the column and the final few drops pushed through the column. The filtrates were centrifuged for 10 minutes at 1,000 x g and the supernatants removed. RBC pellets from all filtrates in all experiments were stored at -20°C until DNA extraction. A packed CF11 column and filtration of blood diluted with PBS are shown in Figures 1A and 1B. The full CF11 filtration procedure is available on the WWARN website [12].\nPacked CF11 column ready for storage, shipping or use (A) and filtration of blood-PBS mixture through CF11 column into collection tube (B)."," CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method","To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method"," CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\n CF11 filtration, with procedural modifications To test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\nTo test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\n Modifications in blood volume and parasite density To investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\nTo investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\n Routine blood collection for further validation and sequencing To determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\nTo determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.","To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.","To test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.","To investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.","To determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.","DNA was extracted from leukocyte-depleted and unfiltered control samples using Qiamp DNA Blood Midi Kits (Qiagen, Venlo, Netherlands) according to the manufacturer's protocol. Total DNA from experiments using P. falciparum clones was estimated with a SpectraMax M2 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) using the Quant-IT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). The standard curve ranged from 0.20 to 50 ng/μl. Total DNA from experiments using P. falciparum isolates was estimated with a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) using both the dsDNA HS and BR Assay kits.\nQuantitative real-time PCR (qPCR) was used to measure the relative amounts of human and parasite DNA in each sample. Primers targeting the human LRAP (F: 5'-ACGTTGGATGAATTTTCCACTGGATTCCAT-'3; R: 5'-ACGTTGGATGTGAACCATGCTCCTTGCATC-'3) and TLR9 (F: 5'-ACGTTGGATGCAAAGGGCTGGCTGTTGTAG-'3; R: 5'- ACGTTGGATGTCTACCACGAGCACTCATTC-'3) genes and the P. falciparum AMA-1 gene (F: 5'-ACGTTGGATGGATTCTCTTTCGATTTCTTTC-'3; R: 5'-ACGTTGGATGTGCTACTACTGCTTTGTCCC-'3) were used to amplify DNA from samples, negative controls and standards in duplicate. Amplification occurred in an Applied Biosystems 7300 or StepOne real-time PCR machine. Human placental and P. falciparum (3D7 clone) genomic DNA standards ranged from 0.20 to 50 ng/μl. For each 25 μl reaction, 12.5 μl SYBR Green qPCR Master Mix (Applied Biosystems, Foster City, CA, USA) was mixed with 1.5 μl of each forward and reverse primers at 2 μM concentrations, 7.5-8.5 μl water, and 1-2 μl of template DNA. Reaction conditions were previously reported [3]: an initial denaturing step of 95°C for 10 minutes, five cycles of 94°C for 45 seconds, 56°C for 45 seconds, 72°C for 45 seconds, followed by 30 cycles of 94°C for 45 seconds, 65°C for 45 seconds and 72°C for 45 seconds and one dissociation cycle (conditions vary by real-time PCR instrument). Results were analysed using Applied Biosystems 7300 System SDS or StepOne v2.0 software.\nqPCR quantification of AMA-1 and TLR9 or LRAP was used to determine the proportions of P. falciparum and human DNA in the sample. Total DNA was estimated by multiplying the concentration given by Spectramax or Qubit by the extracted sample volume.","Means, variance, standard errors and statistical comparisons of means were calculated using Stata 11 software. Boxplots indicating medians, interquartile ranges and outliers were also generated using Stata 11."," Comparison of unmodified LP and CF11 filtration for leukocyte depletion Filtration of blood-parasite mixtures using unmodified LP and CF11 methods in parallel successfully depleted human DNA to 1% and 6% of total DNA, respectively (Figure 2A), well below the current Illumina sequencing threshold of < 50%. CF11 was more effective than LP in recovering total DNA from blood-parasite mixtures (mean 1.11 μg vs. 0.37 μg; p = 0.03) (Figure 2B). All 15 CF11-filtered parasitized blood samples and 12 of 14 LP-filtered parasitized blood samples yielded < 50% human DNA contamination (Figure 3A). Human DNA contamination was lower for parasitized blood filtered by CF11 than by LP (mean 2.4% vs. 15.9%; p = 0.03) (Figure 3B). Recovery of total DNA was comparably high in both sets of samples (mean 4.64 μg for CF11 vs. 2.86 μg for LP; p = 0.46) One LP-filtered sample failed to produce a human DNA estimate by qPCR and was omitted from the analysis.\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of blood-parasite mixtures. The volume and parasite density of blood-parasite mixtures were 3 ml and 10,000/μl except where stated otherwise. Three replicates for each filtration method were performed. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column, CF11 O/N = CF11 filtrate held overnight prior to removal of supernatant, CF11 4 ml = filtration using a 4-ml CF11 column, CF11 low vol = filtration of a 1.5-ml blood-parasite mixture containing 10,000 parasites/μl, CF11 low para = filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl, NF = no filtration, NF low para = no filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl. Box plots show median and interquartile range.\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of Plasmodium falciparum-infected blood samples that vary in parasite density. Light gray shading indicates samples with < 50,000 parasites/μl, medium gray shading indicates samples with 50,000-100,000 parasites/μl, and dark gray shading indicates samples with parasite density > 100,000 parasites/μl. All sample volumes were 4 ml. n = sample size for each method within each range of parasite density. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column. Box plots show median and interquartile range. Whiskers span the range of data points within 1.5 times the interquartile range of the lower and upper quartiles. Outliers are shown as points.\nFiltration of blood-parasite mixtures using unmodified LP and CF11 methods in parallel successfully depleted human DNA to 1% and 6% of total DNA, respectively (Figure 2A), well below the current Illumina sequencing threshold of < 50%. CF11 was more effective than LP in recovering total DNA from blood-parasite mixtures (mean 1.11 μg vs. 0.37 μg; p = 0.03) (Figure 2B). All 15 CF11-filtered parasitized blood samples and 12 of 14 LP-filtered parasitized blood samples yielded < 50% human DNA contamination (Figure 3A). Human DNA contamination was lower for parasitized blood filtered by CF11 than by LP (mean 2.4% vs. 15.9%; p = 0.03) (Figure 3B). Recovery of total DNA was comparably high in both sets of samples (mean 4.64 μg for CF11 vs. 2.86 μg for LP; p = 0.46) One LP-filtered sample failed to produce a human DNA estimate by qPCR and was omitted from the analysis.\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of blood-parasite mixtures. The volume and parasite density of blood-parasite mixtures were 3 ml and 10,000/μl except where stated otherwise. Three replicates for each filtration method were performed. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column, CF11 O/N = CF11 filtrate held overnight prior to removal of supernatant, CF11 4 ml = filtration using a 4-ml CF11 column, CF11 low vol = filtration of a 1.5-ml blood-parasite mixture containing 10,000 parasites/μl, CF11 low para = filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl, NF = no filtration, NF low para = no filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl. Box plots show median and interquartile range.\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of Plasmodium falciparum-infected blood samples that vary in parasite density. Light gray shading indicates samples with < 50,000 parasites/μl, medium gray shading indicates samples with 50,000-100,000 parasites/μl, and dark gray shading indicates samples with parasite density > 100,000 parasites/μl. All sample volumes were 4 ml. n = sample size for each method within each range of parasite density. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column. Box plots show median and interquartile range. Whiskers span the range of data points within 1.5 times the interquartile range of the lower and upper quartiles. Outliers are shown as points.\n Procedural modifications of CF11 filtration method To explore whether CF11 filtrates could be processed without centrifugation, CF11-filtered blood-parasite mixtures were refrigerated overnight. The RBC pellet formed during overnight settling was not as tightly packed as that formed during centrifugation and required careful handling when removing supernatant to prevent disruption. This modification resulted in adequate human DNA depletion (mean contamination 11%) (Figure 2A) and total DNA recovery (mean 1.22 μg) (Figure 2B). Columns with less CF11 powder, packed to the 4-ml mark rather than to the 5.5-ml mark, also yielded high total DNA (mean 1.56 μg) and a sufficiently low proportion of human DNA (mean 30%) (Figure 2A and 2B).\nTo explore whether CF11 filtrates could be processed without centrifugation, CF11-filtered blood-parasite mixtures were refrigerated overnight. The RBC pellet formed during overnight settling was not as tightly packed as that formed during centrifugation and required careful handling when removing supernatant to prevent disruption. This modification resulted in adequate human DNA depletion (mean contamination 11%) (Figure 2A) and total DNA recovery (mean 1.22 μg) (Figure 2B). Columns with less CF11 powder, packed to the 4-ml mark rather than to the 5.5-ml mark, also yielded high total DNA (mean 1.56 μg) and a sufficiently low proportion of human DNA (mean 30%) (Figure 2A and 2B).\n Blood volume and parasite density All CF11-filtered blood-parasite mixtures (3 ml volume, 10,000/μl parasite density) consistently yielded samples with < 30% human DNA contamination and > 500 ng total DNA (Figure 2A and 2B). However, the ex vivo study of P. falciparum isolates with interesting phenotypes can be difficult if blood volumes are limited and parasite densities are low. To determine whether such limitations might significantly reduce the quality of DNA samples, CF11 filtrations were repeated using blood-parasite mixtures of lower volume (1.5 ml) or lower parasite densities (5,000/μl). CF11 filtration of low-volume samples (1.5 ml; 10,000 parasites/μl) produced acceptable human DNA contamination (mean 28%) and mean total DNA (0.51 μg), but individual yields of total DNA were highly variable, ranging from 0.15 to 1.09 μg (Figure 2A and 2B). CF11 filtration of low parasite density samples (3 ml; 5,000 parasites/μl) produced adequate amounts of total DNA (mean 2.68 μg) but unacceptable human DNA contamination (mean 77%) (Figure 2A and 2B). Thus, reducing sample volumes or parasite densities by half failed to reliably produce DNA samples acceptable for Illumina sequencing.\nFiltration of P. falciparum-infected blood obtained from patients with malaria (Pursat, Cambodia) using the CF11 and LP methods consistently achieved high-quality results at parasite densities ranging from 20,000 to 475,000/μl (Figure 3A and 3B). At parasite densities < 50,000/μl and 50,000-100,000/μl, both filtration methods depleted human DNA contamination to ≤ 30% and produced mean DNA yields > 1 μg. Samples with > 100,000 parasites/μl yielded several micrograms of DNA with < 5% human DNA contamination.\nAll CF11-filtered blood-parasite mixtures (3 ml volume, 10,000/μl parasite density) consistently yielded samples with < 30% human DNA contamination and > 500 ng total DNA (Figure 2A and 2B). However, the ex vivo study of P. falciparum isolates with interesting phenotypes can be difficult if blood volumes are limited and parasite densities are low. To determine whether such limitations might significantly reduce the quality of DNA samples, CF11 filtrations were repeated using blood-parasite mixtures of lower volume (1.5 ml) or lower parasite densities (5,000/μl). CF11 filtration of low-volume samples (1.5 ml; 10,000 parasites/μl) produced acceptable human DNA contamination (mean 28%) and mean total DNA (0.51 μg), but individual yields of total DNA were highly variable, ranging from 0.15 to 1.09 μg (Figure 2A and 2B). CF11 filtration of low parasite density samples (3 ml; 5,000 parasites/μl) produced adequate amounts of total DNA (mean 2.68 μg) but unacceptable human DNA contamination (mean 77%) (Figure 2A and 2B). Thus, reducing sample volumes or parasite densities by half failed to reliably produce DNA samples acceptable for Illumina sequencing.\nFiltration of P. falciparum-infected blood obtained from patients with malaria (Pursat, Cambodia) using the CF11 and LP methods consistently achieved high-quality results at parasite densities ranging from 20,000 to 475,000/μl (Figure 3A and 3B). At parasite densities < 50,000/μl and 50,000-100,000/μl, both filtration methods depleted human DNA contamination to ≤ 30% and produced mean DNA yields > 1 μg. Samples with > 100,000 parasites/μl yielded several micrograms of DNA with < 5% human DNA contamination.\n Validation of routine blood collection for illumina sequencing The 51 parasitized blood samples that were collected and CF11-filtered in Ratanakiri, Cambodia, ranged in parasite density from 15,000 to 290,000/μl and yielded a mean 0.64 μg total DNA with 34% human DNA contamination. All 51 samples met quality standards and have been successfully sequenced on the Illumina platform at the Wellcome Trust Sanger Institute (Hinxton, UK).\nThe 51 parasitized blood samples that were collected and CF11-filtered in Ratanakiri, Cambodia, ranged in parasite density from 15,000 to 290,000/μl and yielded a mean 0.64 μg total DNA with 34% human DNA contamination. All 51 samples met quality standards and have been successfully sequenced on the Illumina platform at the Wellcome Trust Sanger Institute (Hinxton, UK).","Filtration of blood-parasite mixtures using unmodified LP and CF11 methods in parallel successfully depleted human DNA to 1% and 6% of total DNA, respectively (Figure 2A), well below the current Illumina sequencing threshold of < 50%. CF11 was more effective than LP in recovering total DNA from blood-parasite mixtures (mean 1.11 μg vs. 0.37 μg; p = 0.03) (Figure 2B). All 15 CF11-filtered parasitized blood samples and 12 of 14 LP-filtered parasitized blood samples yielded < 50% human DNA contamination (Figure 3A). Human DNA contamination was lower for parasitized blood filtered by CF11 than by LP (mean 2.4% vs. 15.9%; p = 0.03) (Figure 3B). Recovery of total DNA was comparably high in both sets of samples (mean 4.64 μg for CF11 vs. 2.86 μg for LP; p = 0.46) One LP-filtered sample failed to produce a human DNA estimate by qPCR and was omitted from the analysis.\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of blood-parasite mixtures. The volume and parasite density of blood-parasite mixtures were 3 ml and 10,000/μl except where stated otherwise. Three replicates for each filtration method were performed. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column, CF11 O/N = CF11 filtrate held overnight prior to removal of supernatant, CF11 4 ml = filtration using a 4-ml CF11 column, CF11 low vol = filtration of a 1.5-ml blood-parasite mixture containing 10,000 parasites/μl, CF11 low para = filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl, NF = no filtration, NF low para = no filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl. Box plots show median and interquartile range.\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of Plasmodium falciparum-infected blood samples that vary in parasite density. Light gray shading indicates samples with < 50,000 parasites/μl, medium gray shading indicates samples with 50,000-100,000 parasites/μl, and dark gray shading indicates samples with parasite density > 100,000 parasites/μl. All sample volumes were 4 ml. n = sample size for each method within each range of parasite density. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column. Box plots show median and interquartile range. Whiskers span the range of data points within 1.5 times the interquartile range of the lower and upper quartiles. Outliers are shown as points.","To explore whether CF11 filtrates could be processed without centrifugation, CF11-filtered blood-parasite mixtures were refrigerated overnight. The RBC pellet formed during overnight settling was not as tightly packed as that formed during centrifugation and required careful handling when removing supernatant to prevent disruption. This modification resulted in adequate human DNA depletion (mean contamination 11%) (Figure 2A) and total DNA recovery (mean 1.22 μg) (Figure 2B). Columns with less CF11 powder, packed to the 4-ml mark rather than to the 5.5-ml mark, also yielded high total DNA (mean 1.56 μg) and a sufficiently low proportion of human DNA (mean 30%) (Figure 2A and 2B).","All CF11-filtered blood-parasite mixtures (3 ml volume, 10,000/μl parasite density) consistently yielded samples with < 30% human DNA contamination and > 500 ng total DNA (Figure 2A and 2B). However, the ex vivo study of P. falciparum isolates with interesting phenotypes can be difficult if blood volumes are limited and parasite densities are low. To determine whether such limitations might significantly reduce the quality of DNA samples, CF11 filtrations were repeated using blood-parasite mixtures of lower volume (1.5 ml) or lower parasite densities (5,000/μl). CF11 filtration of low-volume samples (1.5 ml; 10,000 parasites/μl) produced acceptable human DNA contamination (mean 28%) and mean total DNA (0.51 μg), but individual yields of total DNA were highly variable, ranging from 0.15 to 1.09 μg (Figure 2A and 2B). CF11 filtration of low parasite density samples (3 ml; 5,000 parasites/μl) produced adequate amounts of total DNA (mean 2.68 μg) but unacceptable human DNA contamination (mean 77%) (Figure 2A and 2B). Thus, reducing sample volumes or parasite densities by half failed to reliably produce DNA samples acceptable for Illumina sequencing.\nFiltration of P. falciparum-infected blood obtained from patients with malaria (Pursat, Cambodia) using the CF11 and LP methods consistently achieved high-quality results at parasite densities ranging from 20,000 to 475,000/μl (Figure 3A and 3B). At parasite densities < 50,000/μl and 50,000-100,000/μl, both filtration methods depleted human DNA contamination to ≤ 30% and produced mean DNA yields > 1 μg. Samples with > 100,000 parasites/μl yielded several micrograms of DNA with < 5% human DNA contamination.","The 51 parasitized blood samples that were collected and CF11-filtered in Ratanakiri, Cambodia, ranged in parasite density from 15,000 to 290,000/μl and yielded a mean 0.64 μg total DNA with 34% human DNA contamination. All 51 samples met quality standards and have been successfully sequenced on the Illumina platform at the Wellcome Trust Sanger Institute (Hinxton, UK).","As next-generation sequencing technologies improve, both costs and stringency of DNA sample quality requirements continue to fall. Between 2009 and 2011, requirements for Illumina sequencing of P. falciparum DNA by the Wellcome Trust Sanger Institute have relaxed from 30% to 50% human DNA contamination. Moreover, the possibility of sequencing multiple DNA samples in a single lane of a flow cell ('multiplex sequencing') has decreased costs immensely while yielding ~2 GB of P. falciparum genomic sequence data per sample, with further improvements anticipated in coming years. To take advantage of these increasingly accessible tools, researchers need scalable procedures for DNA sample preparation in minimally equipped field sites.\nThis study shows that CF11 columns can be used to effectively deplete human DNA from parasitized blood and achieve Illumina sequencing requirements for total DNA yield and percent human DNA contamination, using sample volumes and parasite densities comparable to those used in LP filtration experiments [3]. A major advantage of CF11 over LP filtration is cost-effectiveness. CF11 columns cost approximately one USD each, compared to 10-50 USD for the Plasmodipur filter alone. Additionally, CF11 filtration is a very simple one-step procedure that does not require specialized equipment. A centrifugation step is only used to pellet RBCs after CF11 filtration and can be replaced with a convenient overnight period of refrigeration to allow the RBCs to pellet by gravity. These advantages should enable a large share of the malaria research community to produce parasite DNA samples appropriate for highly parallel sequencing and microarray technologies.\nBecause CF11 columns are hand-made rather than commercially produced, variation in the length of the column is likely to occur. Samples filtered in columns packed with less CF11 powder exhibited higher human DNA contamination but still met the threshold level for sequencing, indicating that some variation in column preparation can be tolerated. Samples with low blood volumes (1.5 ml) or parasite densities (5,000/μl) did not consistently achieve sufficient human DNA depletion or total DNA recovery when filtered with either the CF11 or LP method. Whole-genome amplification (WGA) after DNA isolation [13] could boost the amount of starting material in cases where human DNA removal is satisfactory but total DNA yield is low. WGA may therefore be useful in enabling genomic characterization of CF11-filtered blood samples with low starting volume, such as those collected from very young children and patients with severe malarial anaemia. It may also be useful in processing blood samples with low parasite densities, as is often the case in patients with a recrudescent parasitaemia after anti-malarial treatment and those with high levels of naturally acquired immunity.\nAlthough specific parameters have not been rigorously tested, observations in the field have indicated that CF11 and parasitized blood can be effectively stored and used in a variety of environmental conditions. CF11 kept in an airtight container with regularly replaced desiccant and stored in a cool place can be used for at least three months, even in climates with high heat and humidity (unpublished observation). EDTA-treated blood stored at 4°C for up to 24 hours prior to CF11 filtration was successfully processed for Illumina DNA sequencing (unpublished observation), but for RNA analysis, no more than six hours of refrigeration is suggested (ZB Bozdech, personal communication). Updates to the range of storage conditions will be made to the WWARN protocol [12] as more information becomes available.","CF11 filtration currently offers the best cost-effective, one-step approach to remove human DNA from P. falciparum-infected whole blood samples and can be successfully implemented in large genome-wide sequencing studies of P. falciparum isolates from patients with malaria."],"string":"[\n \"Recent technological advances have enabled next-generation genomic and transcriptomic analysis of Plasmodium falciparum from parasitized whole blood samples without the need for culturing. High-density genotyping of parasites obtained directly from patients with malaria has improved our understanding of population structure and genomic and transcriptional variation [1,2]. Highly parallel sequencing is currently being used to identify the genetic determinants of clinically relevant phenotypes including drug resistance, vaccine escape and disease severity. Importantly, genomic characterization of parasite populations in patients captures intra-host diversity and prevents the potential loss of sequence data from phenotype-conferring parasite isolates during their culture adaptation.\\nThe performance of highly parallel sequencing platforms, such as Illumina, is greatly enhanced in sample preparations with a high parasite-to-human nucleic acid ratio [3]. Such high ratios can be achieved by either selectively capturing parasite nucleic acids or by removing human material from the sample. Hybrid selection [4] using RNA 'baits' complementary to the P. falciparum genome can achieve over 40-fold enrichment of parasite DNA and can be performed at any time following DNA extraction [5]; however, at $250 USD per sample, this technique may be prohibitively costly for epidemiological or population-level studies.\\nThe alternative, leukocyte depletion, must be performed in field sites soon after blood collection and before transport and storage. Commercially available magnetic columns have been used to rapidly isolate parasitized red blood cells (RBCs) from uninfected RBCs and leukocytes [6]. However, magnetic purification depends on short-term culturing of patient blood to transform ring-stage parasites to haemozoin-rich trophozoites and schizonts, which requires equipment for in vitro parasite cultivation. Furthermore, parasites obtained directly from patients mature at different rates in culture, resulting in inconvenient time-points for purification. The current standard for leukocyte depletion of parasitized blood for P. falciparum nucleotide sequencing is a two-step process: centrifugation using Lymphoprep or another sucrose density gradient solution, followed by filtration using Plasmodipur filters [3]. This 'LP' method is effective but difficult to scale-up in field settings because it requires extensive handling and transfer of blood, training to perform sensitive steps, and costly commercial reagents and materials.\\nFiltration with hand-made columns has been used as an inexpensive and less time-consuming alternative for leukocyte depletion of Plasmodium-infected blood for decades [7,8]. Recently, non-woven fabric filters [9] and plastic syringes filled with CF11 cellulose powder (Whatman) [10] were shown to effectively remove leukocytes and platelets from Plasmodium vivax-infected blood, where their phagocytic and degranulating activities may interfere with some ex vivo studies. CF11 filtration has also been used to improve microarray-based transcriptome analysis of P. vivax-infected blood [2,11]. CF11 cellulose is thought to work by trapping leukocytes by size exclusion and/or interactions between cellulose hydroxyl groups and leukocyte surface molecules. In this study, laboratory-adapted P. falciparum clones were used to adapt CF11 columns to remove human DNA from P. falciparum-infected blood for genomic studies, compare CF11 filtration to the established LP method, and validate a centrifuge-free option for CF11 filtration. Both methods were also compared in a field setting using P. falciparum isolates obtained directly from patients with malaria. Furthermore, routine CF11 filtration of parasitized blood collected from patients in a second field site with minimal facilities was successfully implemented.\",\n \"Experiments using laboratory-adapted P. falciparum clones were conducted at the University of Maryland, Baltimore. Blood obtained from healthy volunteers was mixed with purified P. falciparum parasites (3D7 or Dd2 clones) to achieve parasite densities of 10,000/μl and 5,000/μl. These samples were stored at 4°C for up to two hours prior to CF11 or LP filtration.\\nExperiments using non-adapted P. falciparum isolates were conducted in August 2010 at Sampov Meas Referral Hospital, Pursat, Cambodia. Blood samples for these experiments were collected from patients with falciparum malaria who were ≥ 10 years old and had a wide range of parasite densities (≥ 10,000/μl). These samples were held at 4°C for up to six hours prior to CF11 or LP filtration. Additional blood samples were collected in November to December 2010 at Lumphat District Health Centre, Ratanakiri, Cambodia. Blood samples were obtained from patients with falciparum malaria who were ≥ 1 year old and had a wide range of parasite densities ≥ 10,000/μl. These samples were held at 4°C for up to six hours prior to CF11 filtration. Following CF11 filtration, samples were stored on ice for up to six hours and transported on ice for up to 12 hours to Phnom Penh for centrifugation and RBC pellet storage.\\nAll blood samples were collected in EDTA or CPDA-1 tubes. Heparin was not used as an anticoagulant as it thought to inhibit Taq polymerase during DNA amplification.\\nBlood samples from healthy volunteers were collected under a protocol approved by the Institutional Review Board of the University of Maryland School of Medicine. Blood samples from patients with falciparum malaria were collected under protocols approved by the Institutional Review Board of the National Institute for Allergy and Infectious Diseases and the Cambodian National Ethics Committee for Health Research (http://ClinicalTrials.gov identifiers: NCT00341003 and NCT01240603). Written, informed consent was obtained from all study participants or their parents or guardians.\",\n \"Lymphoprep (Axis-shield, Oslo, Norway) + Plasmodipur (Euro-diagnostica, Arnhem, Netherlands) filtration ('LP') was performed as previously described [3]. CF11 columns were loosely filled to the 10-ml mark, packed to the 5.5-ml mark with CF11 cellulose powder (Whatman, Kent, UK), and wetted with isotonic phosphate buffered saline (PBS), as described [10]. Initial tests indicated that plasma separation prior to CF11 filtration of blood did not affect human DNA depletion and that washing with PBS after CF11 filtration improved recovery of parasite DNA. CF11 filtration was therefore carried out as follows. Parasitized blood or blood/parasite mixtures were diluted with an equal volume of PBS. These samples were added to the CF11 column and allowed to flow through by gravity. To wash the sample, 5 ml PBS was added to the column and allowed to pass through by gravity. A plunger was then inserted into the top of the column and the final few drops pushed through the column. The filtrates were centrifuged for 10 minutes at 1,000 x g and the supernatants removed. RBC pellets from all filtrates in all experiments were stored at -20°C until DNA extraction. A packed CF11 column and filtration of blood diluted with PBS are shown in Figures 1A and 1B. The full CF11 filtration procedure is available on the WWARN website [12].\\nPacked CF11 column ready for storage, shipping or use (A) and filtration of blood-PBS mixture through CF11 column into collection tube (B).\",\n \" CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\",\n \"To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\",\n \" CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\\n CF11 filtration, with procedural modifications To test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\\nTo test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\\n Modifications in blood volume and parasite density To investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\\nTo investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\\n Routine blood collection for further validation and sequencing To determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\\nTo determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\",\n \"To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\",\n \"To test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\",\n \"To investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\",\n \"To determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\",\n \"DNA was extracted from leukocyte-depleted and unfiltered control samples using Qiamp DNA Blood Midi Kits (Qiagen, Venlo, Netherlands) according to the manufacturer's protocol. Total DNA from experiments using P. falciparum clones was estimated with a SpectraMax M2 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) using the Quant-IT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). The standard curve ranged from 0.20 to 50 ng/μl. Total DNA from experiments using P. falciparum isolates was estimated with a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) using both the dsDNA HS and BR Assay kits.\\nQuantitative real-time PCR (qPCR) was used to measure the relative amounts of human and parasite DNA in each sample. Primers targeting the human LRAP (F: 5'-ACGTTGGATGAATTTTCCACTGGATTCCAT-'3; R: 5'-ACGTTGGATGTGAACCATGCTCCTTGCATC-'3) and TLR9 (F: 5'-ACGTTGGATGCAAAGGGCTGGCTGTTGTAG-'3; R: 5'- ACGTTGGATGTCTACCACGAGCACTCATTC-'3) genes and the P. falciparum AMA-1 gene (F: 5'-ACGTTGGATGGATTCTCTTTCGATTTCTTTC-'3; R: 5'-ACGTTGGATGTGCTACTACTGCTTTGTCCC-'3) were used to amplify DNA from samples, negative controls and standards in duplicate. Amplification occurred in an Applied Biosystems 7300 or StepOne real-time PCR machine. Human placental and P. falciparum (3D7 clone) genomic DNA standards ranged from 0.20 to 50 ng/μl. For each 25 μl reaction, 12.5 μl SYBR Green qPCR Master Mix (Applied Biosystems, Foster City, CA, USA) was mixed with 1.5 μl of each forward and reverse primers at 2 μM concentrations, 7.5-8.5 μl water, and 1-2 μl of template DNA. Reaction conditions were previously reported [3]: an initial denaturing step of 95°C for 10 minutes, five cycles of 94°C for 45 seconds, 56°C for 45 seconds, 72°C for 45 seconds, followed by 30 cycles of 94°C for 45 seconds, 65°C for 45 seconds and 72°C for 45 seconds and one dissociation cycle (conditions vary by real-time PCR instrument). Results were analysed using Applied Biosystems 7300 System SDS or StepOne v2.0 software.\\nqPCR quantification of AMA-1 and TLR9 or LRAP was used to determine the proportions of P. falciparum and human DNA in the sample. Total DNA was estimated by multiplying the concentration given by Spectramax or Qubit by the extracted sample volume.\",\n \"Means, variance, standard errors and statistical comparisons of means were calculated using Stata 11 software. Boxplots indicating medians, interquartile ranges and outliers were also generated using Stata 11.\",\n \" Comparison of unmodified LP and CF11 filtration for leukocyte depletion Filtration of blood-parasite mixtures using unmodified LP and CF11 methods in parallel successfully depleted human DNA to 1% and 6% of total DNA, respectively (Figure 2A), well below the current Illumina sequencing threshold of < 50%. CF11 was more effective than LP in recovering total DNA from blood-parasite mixtures (mean 1.11 μg vs. 0.37 μg; p = 0.03) (Figure 2B). All 15 CF11-filtered parasitized blood samples and 12 of 14 LP-filtered parasitized blood samples yielded < 50% human DNA contamination (Figure 3A). Human DNA contamination was lower for parasitized blood filtered by CF11 than by LP (mean 2.4% vs. 15.9%; p = 0.03) (Figure 3B). Recovery of total DNA was comparably high in both sets of samples (mean 4.64 μg for CF11 vs. 2.86 μg for LP; p = 0.46) One LP-filtered sample failed to produce a human DNA estimate by qPCR and was omitted from the analysis.\\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of blood-parasite mixtures. The volume and parasite density of blood-parasite mixtures were 3 ml and 10,000/μl except where stated otherwise. Three replicates for each filtration method were performed. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column, CF11 O/N = CF11 filtrate held overnight prior to removal of supernatant, CF11 4 ml = filtration using a 4-ml CF11 column, CF11 low vol = filtration of a 1.5-ml blood-parasite mixture containing 10,000 parasites/μl, CF11 low para = filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl, NF = no filtration, NF low para = no filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl. Box plots show median and interquartile range.\\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of Plasmodium falciparum-infected blood samples that vary in parasite density. Light gray shading indicates samples with < 50,000 parasites/μl, medium gray shading indicates samples with 50,000-100,000 parasites/μl, and dark gray shading indicates samples with parasite density > 100,000 parasites/μl. All sample volumes were 4 ml. n = sample size for each method within each range of parasite density. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column. Box plots show median and interquartile range. Whiskers span the range of data points within 1.5 times the interquartile range of the lower and upper quartiles. Outliers are shown as points.\\nFiltration of blood-parasite mixtures using unmodified LP and CF11 methods in parallel successfully depleted human DNA to 1% and 6% of total DNA, respectively (Figure 2A), well below the current Illumina sequencing threshold of < 50%. CF11 was more effective than LP in recovering total DNA from blood-parasite mixtures (mean 1.11 μg vs. 0.37 μg; p = 0.03) (Figure 2B). All 15 CF11-filtered parasitized blood samples and 12 of 14 LP-filtered parasitized blood samples yielded < 50% human DNA contamination (Figure 3A). Human DNA contamination was lower for parasitized blood filtered by CF11 than by LP (mean 2.4% vs. 15.9%; p = 0.03) (Figure 3B). Recovery of total DNA was comparably high in both sets of samples (mean 4.64 μg for CF11 vs. 2.86 μg for LP; p = 0.46) One LP-filtered sample failed to produce a human DNA estimate by qPCR and was omitted from the analysis.\\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of blood-parasite mixtures. The volume and parasite density of blood-parasite mixtures were 3 ml and 10,000/μl except where stated otherwise. Three replicates for each filtration method were performed. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column, CF11 O/N = CF11 filtrate held overnight prior to removal of supernatant, CF11 4 ml = filtration using a 4-ml CF11 column, CF11 low vol = filtration of a 1.5-ml blood-parasite mixture containing 10,000 parasites/μl, CF11 low para = filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl, NF = no filtration, NF low para = no filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl. Box plots show median and interquartile range.\\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of Plasmodium falciparum-infected blood samples that vary in parasite density. Light gray shading indicates samples with < 50,000 parasites/μl, medium gray shading indicates samples with 50,000-100,000 parasites/μl, and dark gray shading indicates samples with parasite density > 100,000 parasites/μl. All sample volumes were 4 ml. n = sample size for each method within each range of parasite density. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column. Box plots show median and interquartile range. Whiskers span the range of data points within 1.5 times the interquartile range of the lower and upper quartiles. Outliers are shown as points.\\n Procedural modifications of CF11 filtration method To explore whether CF11 filtrates could be processed without centrifugation, CF11-filtered blood-parasite mixtures were refrigerated overnight. The RBC pellet formed during overnight settling was not as tightly packed as that formed during centrifugation and required careful handling when removing supernatant to prevent disruption. This modification resulted in adequate human DNA depletion (mean contamination 11%) (Figure 2A) and total DNA recovery (mean 1.22 μg) (Figure 2B). Columns with less CF11 powder, packed to the 4-ml mark rather than to the 5.5-ml mark, also yielded high total DNA (mean 1.56 μg) and a sufficiently low proportion of human DNA (mean 30%) (Figure 2A and 2B).\\nTo explore whether CF11 filtrates could be processed without centrifugation, CF11-filtered blood-parasite mixtures were refrigerated overnight. The RBC pellet formed during overnight settling was not as tightly packed as that formed during centrifugation and required careful handling when removing supernatant to prevent disruption. This modification resulted in adequate human DNA depletion (mean contamination 11%) (Figure 2A) and total DNA recovery (mean 1.22 μg) (Figure 2B). Columns with less CF11 powder, packed to the 4-ml mark rather than to the 5.5-ml mark, also yielded high total DNA (mean 1.56 μg) and a sufficiently low proportion of human DNA (mean 30%) (Figure 2A and 2B).\\n Blood volume and parasite density All CF11-filtered blood-parasite mixtures (3 ml volume, 10,000/μl parasite density) consistently yielded samples with < 30% human DNA contamination and > 500 ng total DNA (Figure 2A and 2B). However, the ex vivo study of P. falciparum isolates with interesting phenotypes can be difficult if blood volumes are limited and parasite densities are low. To determine whether such limitations might significantly reduce the quality of DNA samples, CF11 filtrations were repeated using blood-parasite mixtures of lower volume (1.5 ml) or lower parasite densities (5,000/μl). CF11 filtration of low-volume samples (1.5 ml; 10,000 parasites/μl) produced acceptable human DNA contamination (mean 28%) and mean total DNA (0.51 μg), but individual yields of total DNA were highly variable, ranging from 0.15 to 1.09 μg (Figure 2A and 2B). CF11 filtration of low parasite density samples (3 ml; 5,000 parasites/μl) produced adequate amounts of total DNA (mean 2.68 μg) but unacceptable human DNA contamination (mean 77%) (Figure 2A and 2B). Thus, reducing sample volumes or parasite densities by half failed to reliably produce DNA samples acceptable for Illumina sequencing.\\nFiltration of P. falciparum-infected blood obtained from patients with malaria (Pursat, Cambodia) using the CF11 and LP methods consistently achieved high-quality results at parasite densities ranging from 20,000 to 475,000/μl (Figure 3A and 3B). At parasite densities < 50,000/μl and 50,000-100,000/μl, both filtration methods depleted human DNA contamination to ≤ 30% and produced mean DNA yields > 1 μg. Samples with > 100,000 parasites/μl yielded several micrograms of DNA with < 5% human DNA contamination.\\nAll CF11-filtered blood-parasite mixtures (3 ml volume, 10,000/μl parasite density) consistently yielded samples with < 30% human DNA contamination and > 500 ng total DNA (Figure 2A and 2B). However, the ex vivo study of P. falciparum isolates with interesting phenotypes can be difficult if blood volumes are limited and parasite densities are low. To determine whether such limitations might significantly reduce the quality of DNA samples, CF11 filtrations were repeated using blood-parasite mixtures of lower volume (1.5 ml) or lower parasite densities (5,000/μl). CF11 filtration of low-volume samples (1.5 ml; 10,000 parasites/μl) produced acceptable human DNA contamination (mean 28%) and mean total DNA (0.51 μg), but individual yields of total DNA were highly variable, ranging from 0.15 to 1.09 μg (Figure 2A and 2B). CF11 filtration of low parasite density samples (3 ml; 5,000 parasites/μl) produced adequate amounts of total DNA (mean 2.68 μg) but unacceptable human DNA contamination (mean 77%) (Figure 2A and 2B). Thus, reducing sample volumes or parasite densities by half failed to reliably produce DNA samples acceptable for Illumina sequencing.\\nFiltration of P. falciparum-infected blood obtained from patients with malaria (Pursat, Cambodia) using the CF11 and LP methods consistently achieved high-quality results at parasite densities ranging from 20,000 to 475,000/μl (Figure 3A and 3B). At parasite densities < 50,000/μl and 50,000-100,000/μl, both filtration methods depleted human DNA contamination to ≤ 30% and produced mean DNA yields > 1 μg. Samples with > 100,000 parasites/μl yielded several micrograms of DNA with < 5% human DNA contamination.\\n Validation of routine blood collection for illumina sequencing The 51 parasitized blood samples that were collected and CF11-filtered in Ratanakiri, Cambodia, ranged in parasite density from 15,000 to 290,000/μl and yielded a mean 0.64 μg total DNA with 34% human DNA contamination. All 51 samples met quality standards and have been successfully sequenced on the Illumina platform at the Wellcome Trust Sanger Institute (Hinxton, UK).\\nThe 51 parasitized blood samples that were collected and CF11-filtered in Ratanakiri, Cambodia, ranged in parasite density from 15,000 to 290,000/μl and yielded a mean 0.64 μg total DNA with 34% human DNA contamination. All 51 samples met quality standards and have been successfully sequenced on the Illumina platform at the Wellcome Trust Sanger Institute (Hinxton, UK).\",\n \"Filtration of blood-parasite mixtures using unmodified LP and CF11 methods in parallel successfully depleted human DNA to 1% and 6% of total DNA, respectively (Figure 2A), well below the current Illumina sequencing threshold of < 50%. CF11 was more effective than LP in recovering total DNA from blood-parasite mixtures (mean 1.11 μg vs. 0.37 μg; p = 0.03) (Figure 2B). All 15 CF11-filtered parasitized blood samples and 12 of 14 LP-filtered parasitized blood samples yielded < 50% human DNA contamination (Figure 3A). Human DNA contamination was lower for parasitized blood filtered by CF11 than by LP (mean 2.4% vs. 15.9%; p = 0.03) (Figure 3B). Recovery of total DNA was comparably high in both sets of samples (mean 4.64 μg for CF11 vs. 2.86 μg for LP; p = 0.46) One LP-filtered sample failed to produce a human DNA estimate by qPCR and was omitted from the analysis.\\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of blood-parasite mixtures. The volume and parasite density of blood-parasite mixtures were 3 ml and 10,000/μl except where stated otherwise. Three replicates for each filtration method were performed. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column, CF11 O/N = CF11 filtrate held overnight prior to removal of supernatant, CF11 4 ml = filtration using a 4-ml CF11 column, CF11 low vol = filtration of a 1.5-ml blood-parasite mixture containing 10,000 parasites/μl, CF11 low para = filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl, NF = no filtration, NF low para = no filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl. Box plots show median and interquartile range.\\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of Plasmodium falciparum-infected blood samples that vary in parasite density. Light gray shading indicates samples with < 50,000 parasites/μl, medium gray shading indicates samples with 50,000-100,000 parasites/μl, and dark gray shading indicates samples with parasite density > 100,000 parasites/μl. All sample volumes were 4 ml. n = sample size for each method within each range of parasite density. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column. Box plots show median and interquartile range. Whiskers span the range of data points within 1.5 times the interquartile range of the lower and upper quartiles. Outliers are shown as points.\",\n \"To explore whether CF11 filtrates could be processed without centrifugation, CF11-filtered blood-parasite mixtures were refrigerated overnight. The RBC pellet formed during overnight settling was not as tightly packed as that formed during centrifugation and required careful handling when removing supernatant to prevent disruption. This modification resulted in adequate human DNA depletion (mean contamination 11%) (Figure 2A) and total DNA recovery (mean 1.22 μg) (Figure 2B). Columns with less CF11 powder, packed to the 4-ml mark rather than to the 5.5-ml mark, also yielded high total DNA (mean 1.56 μg) and a sufficiently low proportion of human DNA (mean 30%) (Figure 2A and 2B).\",\n \"All CF11-filtered blood-parasite mixtures (3 ml volume, 10,000/μl parasite density) consistently yielded samples with < 30% human DNA contamination and > 500 ng total DNA (Figure 2A and 2B). However, the ex vivo study of P. falciparum isolates with interesting phenotypes can be difficult if blood volumes are limited and parasite densities are low. To determine whether such limitations might significantly reduce the quality of DNA samples, CF11 filtrations were repeated using blood-parasite mixtures of lower volume (1.5 ml) or lower parasite densities (5,000/μl). CF11 filtration of low-volume samples (1.5 ml; 10,000 parasites/μl) produced acceptable human DNA contamination (mean 28%) and mean total DNA (0.51 μg), but individual yields of total DNA were highly variable, ranging from 0.15 to 1.09 μg (Figure 2A and 2B). CF11 filtration of low parasite density samples (3 ml; 5,000 parasites/μl) produced adequate amounts of total DNA (mean 2.68 μg) but unacceptable human DNA contamination (mean 77%) (Figure 2A and 2B). Thus, reducing sample volumes or parasite densities by half failed to reliably produce DNA samples acceptable for Illumina sequencing.\\nFiltration of P. falciparum-infected blood obtained from patients with malaria (Pursat, Cambodia) using the CF11 and LP methods consistently achieved high-quality results at parasite densities ranging from 20,000 to 475,000/μl (Figure 3A and 3B). At parasite densities < 50,000/μl and 50,000-100,000/μl, both filtration methods depleted human DNA contamination to ≤ 30% and produced mean DNA yields > 1 μg. Samples with > 100,000 parasites/μl yielded several micrograms of DNA with < 5% human DNA contamination.\",\n \"The 51 parasitized blood samples that were collected and CF11-filtered in Ratanakiri, Cambodia, ranged in parasite density from 15,000 to 290,000/μl and yielded a mean 0.64 μg total DNA with 34% human DNA contamination. All 51 samples met quality standards and have been successfully sequenced on the Illumina platform at the Wellcome Trust Sanger Institute (Hinxton, UK).\",\n \"As next-generation sequencing technologies improve, both costs and stringency of DNA sample quality requirements continue to fall. Between 2009 and 2011, requirements for Illumina sequencing of P. falciparum DNA by the Wellcome Trust Sanger Institute have relaxed from 30% to 50% human DNA contamination. Moreover, the possibility of sequencing multiple DNA samples in a single lane of a flow cell ('multiplex sequencing') has decreased costs immensely while yielding ~2 GB of P. falciparum genomic sequence data per sample, with further improvements anticipated in coming years. To take advantage of these increasingly accessible tools, researchers need scalable procedures for DNA sample preparation in minimally equipped field sites.\\nThis study shows that CF11 columns can be used to effectively deplete human DNA from parasitized blood and achieve Illumina sequencing requirements for total DNA yield and percent human DNA contamination, using sample volumes and parasite densities comparable to those used in LP filtration experiments [3]. A major advantage of CF11 over LP filtration is cost-effectiveness. CF11 columns cost approximately one USD each, compared to 10-50 USD for the Plasmodipur filter alone. Additionally, CF11 filtration is a very simple one-step procedure that does not require specialized equipment. A centrifugation step is only used to pellet RBCs after CF11 filtration and can be replaced with a convenient overnight period of refrigeration to allow the RBCs to pellet by gravity. These advantages should enable a large share of the malaria research community to produce parasite DNA samples appropriate for highly parallel sequencing and microarray technologies.\\nBecause CF11 columns are hand-made rather than commercially produced, variation in the length of the column is likely to occur. Samples filtered in columns packed with less CF11 powder exhibited higher human DNA contamination but still met the threshold level for sequencing, indicating that some variation in column preparation can be tolerated. Samples with low blood volumes (1.5 ml) or parasite densities (5,000/μl) did not consistently achieve sufficient human DNA depletion or total DNA recovery when filtered with either the CF11 or LP method. Whole-genome amplification (WGA) after DNA isolation [13] could boost the amount of starting material in cases where human DNA removal is satisfactory but total DNA yield is low. WGA may therefore be useful in enabling genomic characterization of CF11-filtered blood samples with low starting volume, such as those collected from very young children and patients with severe malarial anaemia. It may also be useful in processing blood samples with low parasite densities, as is often the case in patients with a recrudescent parasitaemia after anti-malarial treatment and those with high levels of naturally acquired immunity.\\nAlthough specific parameters have not been rigorously tested, observations in the field have indicated that CF11 and parasitized blood can be effectively stored and used in a variety of environmental conditions. CF11 kept in an airtight container with regularly replaced desiccant and stored in a cool place can be used for at least three months, even in climates with high heat and humidity (unpublished observation). EDTA-treated blood stored at 4°C for up to 24 hours prior to CF11 filtration was successfully processed for Illumina DNA sequencing (unpublished observation), but for RNA analysis, no more than six hours of refrigeration is suggested (ZB Bozdech, personal communication). Updates to the range of storage conditions will be made to the WWARN protocol [12] as more information becomes available.\",\n \"CF11 filtration currently offers the best cost-effective, one-step approach to remove human DNA from P. falciparum-infected whole blood samples and can be successfully implemented in large genome-wide sequencing studies of P. falciparum isolates from patients with malaria.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Background","Methods","Blood samples","Leukocyte depletion","Experimental design","CF11 versus LP filtration, without modifications","Experimental design","CF11 versus LP filtration, without modifications","CF11 filtration, with procedural modifications","Modifications in blood volume and parasite density","Routine blood collection for further validation and sequencing","DNA quantification","Statistical comparisons","Results","Comparison of unmodified LP and CF11 filtration for leukocyte depletion","Procedural modifications of CF11 filtration method","Blood volume and parasite density","Validation of routine blood collection for illumina sequencing","Discussion","Conclusions"],"string":"[\n \"Background\",\n \"Methods\",\n \"Blood samples\",\n \"Leukocyte depletion\",\n \"Experimental design\",\n \"CF11 versus LP filtration, without modifications\",\n \"Experimental design\",\n \"CF11 versus LP filtration, without modifications\",\n \"CF11 filtration, with procedural modifications\",\n \"Modifications in blood volume and parasite density\",\n \"Routine blood collection for further validation and sequencing\",\n \"DNA quantification\",\n \"Statistical comparisons\",\n \"Results\",\n \"Comparison of unmodified LP and CF11 filtration for leukocyte depletion\",\n \"Procedural modifications of CF11 filtration method\",\n \"Blood volume and parasite density\",\n \"Validation of routine blood collection for illumina sequencing\",\n \"Discussion\",\n \"Conclusions\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Recent technological advances have enabled next-generation genomic and transcriptomic analysis of Plasmodium falciparum from parasitized whole blood samples without the need for culturing. High-density genotyping of parasites obtained directly from patients with malaria has improved our understanding of population structure and genomic and transcriptional variation [1,2]. Highly parallel sequencing is currently being used to identify the genetic determinants of clinically relevant phenotypes including drug resistance, vaccine escape and disease severity. Importantly, genomic characterization of parasite populations in patients captures intra-host diversity and prevents the potential loss of sequence data from phenotype-conferring parasite isolates during their culture adaptation.\nThe performance of highly parallel sequencing platforms, such as Illumina, is greatly enhanced in sample preparations with a high parasite-to-human nucleic acid ratio [3]. Such high ratios can be achieved by either selectively capturing parasite nucleic acids or by removing human material from the sample. Hybrid selection [4] using RNA 'baits' complementary to the P. falciparum genome can achieve over 40-fold enrichment of parasite DNA and can be performed at any time following DNA extraction [5]; however, at $250 USD per sample, this technique may be prohibitively costly for epidemiological or population-level studies.\nThe alternative, leukocyte depletion, must be performed in field sites soon after blood collection and before transport and storage. Commercially available magnetic columns have been used to rapidly isolate parasitized red blood cells (RBCs) from uninfected RBCs and leukocytes [6]. However, magnetic purification depends on short-term culturing of patient blood to transform ring-stage parasites to haemozoin-rich trophozoites and schizonts, which requires equipment for in vitro parasite cultivation. Furthermore, parasites obtained directly from patients mature at different rates in culture, resulting in inconvenient time-points for purification. The current standard for leukocyte depletion of parasitized blood for P. falciparum nucleotide sequencing is a two-step process: centrifugation using Lymphoprep or another sucrose density gradient solution, followed by filtration using Plasmodipur filters [3]. This 'LP' method is effective but difficult to scale-up in field settings because it requires extensive handling and transfer of blood, training to perform sensitive steps, and costly commercial reagents and materials.\nFiltration with hand-made columns has been used as an inexpensive and less time-consuming alternative for leukocyte depletion of Plasmodium-infected blood for decades [7,8]. Recently, non-woven fabric filters [9] and plastic syringes filled with CF11 cellulose powder (Whatman) [10] were shown to effectively remove leukocytes and platelets from Plasmodium vivax-infected blood, where their phagocytic and degranulating activities may interfere with some ex vivo studies. CF11 filtration has also been used to improve microarray-based transcriptome analysis of P. vivax-infected blood [2,11]. CF11 cellulose is thought to work by trapping leukocytes by size exclusion and/or interactions between cellulose hydroxyl groups and leukocyte surface molecules. In this study, laboratory-adapted P. falciparum clones were used to adapt CF11 columns to remove human DNA from P. falciparum-infected blood for genomic studies, compare CF11 filtration to the established LP method, and validate a centrifuge-free option for CF11 filtration. Both methods were also compared in a field setting using P. falciparum isolates obtained directly from patients with malaria. Furthermore, routine CF11 filtration of parasitized blood collected from patients in a second field site with minimal facilities was successfully implemented."," Blood samples Experiments using laboratory-adapted P. falciparum clones were conducted at the University of Maryland, Baltimore. Blood obtained from healthy volunteers was mixed with purified P. falciparum parasites (3D7 or Dd2 clones) to achieve parasite densities of 10,000/μl and 5,000/μl. These samples were stored at 4°C for up to two hours prior to CF11 or LP filtration.\nExperiments using non-adapted P. falciparum isolates were conducted in August 2010 at Sampov Meas Referral Hospital, Pursat, Cambodia. Blood samples for these experiments were collected from patients with falciparum malaria who were ≥ 10 years old and had a wide range of parasite densities (≥ 10,000/μl). These samples were held at 4°C for up to six hours prior to CF11 or LP filtration. Additional blood samples were collected in November to December 2010 at Lumphat District Health Centre, Ratanakiri, Cambodia. Blood samples were obtained from patients with falciparum malaria who were ≥ 1 year old and had a wide range of parasite densities ≥ 10,000/μl. These samples were held at 4°C for up to six hours prior to CF11 filtration. Following CF11 filtration, samples were stored on ice for up to six hours and transported on ice for up to 12 hours to Phnom Penh for centrifugation and RBC pellet storage.\nAll blood samples were collected in EDTA or CPDA-1 tubes. Heparin was not used as an anticoagulant as it thought to inhibit Taq polymerase during DNA amplification.\nBlood samples from healthy volunteers were collected under a protocol approved by the Institutional Review Board of the University of Maryland School of Medicine. Blood samples from patients with falciparum malaria were collected under protocols approved by the Institutional Review Board of the National Institute for Allergy and Infectious Diseases and the Cambodian National Ethics Committee for Health Research (http://ClinicalTrials.gov identifiers: NCT00341003 and NCT01240603). Written, informed consent was obtained from all study participants or their parents or guardians.\nExperiments using laboratory-adapted P. falciparum clones were conducted at the University of Maryland, Baltimore. Blood obtained from healthy volunteers was mixed with purified P. falciparum parasites (3D7 or Dd2 clones) to achieve parasite densities of 10,000/μl and 5,000/μl. These samples were stored at 4°C for up to two hours prior to CF11 or LP filtration.\nExperiments using non-adapted P. falciparum isolates were conducted in August 2010 at Sampov Meas Referral Hospital, Pursat, Cambodia. Blood samples for these experiments were collected from patients with falciparum malaria who were ≥ 10 years old and had a wide range of parasite densities (≥ 10,000/μl). These samples were held at 4°C for up to six hours prior to CF11 or LP filtration. Additional blood samples were collected in November to December 2010 at Lumphat District Health Centre, Ratanakiri, Cambodia. Blood samples were obtained from patients with falciparum malaria who were ≥ 1 year old and had a wide range of parasite densities ≥ 10,000/μl. These samples were held at 4°C for up to six hours prior to CF11 filtration. Following CF11 filtration, samples were stored on ice for up to six hours and transported on ice for up to 12 hours to Phnom Penh for centrifugation and RBC pellet storage.\nAll blood samples were collected in EDTA or CPDA-1 tubes. Heparin was not used as an anticoagulant as it thought to inhibit Taq polymerase during DNA amplification.\nBlood samples from healthy volunteers were collected under a protocol approved by the Institutional Review Board of the University of Maryland School of Medicine. Blood samples from patients with falciparum malaria were collected under protocols approved by the Institutional Review Board of the National Institute for Allergy and Infectious Diseases and the Cambodian National Ethics Committee for Health Research (http://ClinicalTrials.gov identifiers: NCT00341003 and NCT01240603). Written, informed consent was obtained from all study participants or their parents or guardians.\n Leukocyte depletion Lymphoprep (Axis-shield, Oslo, Norway) + Plasmodipur (Euro-diagnostica, Arnhem, Netherlands) filtration ('LP') was performed as previously described [3]. CF11 columns were loosely filled to the 10-ml mark, packed to the 5.5-ml mark with CF11 cellulose powder (Whatman, Kent, UK), and wetted with isotonic phosphate buffered saline (PBS), as described [10]. Initial tests indicated that plasma separation prior to CF11 filtration of blood did not affect human DNA depletion and that washing with PBS after CF11 filtration improved recovery of parasite DNA. CF11 filtration was therefore carried out as follows. Parasitized blood or blood/parasite mixtures were diluted with an equal volume of PBS. These samples were added to the CF11 column and allowed to flow through by gravity. To wash the sample, 5 ml PBS was added to the column and allowed to pass through by gravity. A plunger was then inserted into the top of the column and the final few drops pushed through the column. The filtrates were centrifuged for 10 minutes at 1,000 x g and the supernatants removed. RBC pellets from all filtrates in all experiments were stored at -20°C until DNA extraction. A packed CF11 column and filtration of blood diluted with PBS are shown in Figures 1A and 1B. The full CF11 filtration procedure is available on the WWARN website [12].\nPacked CF11 column ready for storage, shipping or use (A) and filtration of blood-PBS mixture through CF11 column into collection tube (B).\nLymphoprep (Axis-shield, Oslo, Norway) + Plasmodipur (Euro-diagnostica, Arnhem, Netherlands) filtration ('LP') was performed as previously described [3]. CF11 columns were loosely filled to the 10-ml mark, packed to the 5.5-ml mark with CF11 cellulose powder (Whatman, Kent, UK), and wetted with isotonic phosphate buffered saline (PBS), as described [10]. Initial tests indicated that plasma separation prior to CF11 filtration of blood did not affect human DNA depletion and that washing with PBS after CF11 filtration improved recovery of parasite DNA. CF11 filtration was therefore carried out as follows. Parasitized blood or blood/parasite mixtures were diluted with an equal volume of PBS. These samples were added to the CF11 column and allowed to flow through by gravity. To wash the sample, 5 ml PBS was added to the column and allowed to pass through by gravity. A plunger was then inserted into the top of the column and the final few drops pushed through the column. The filtrates were centrifuged for 10 minutes at 1,000 x g and the supernatants removed. RBC pellets from all filtrates in all experiments were stored at -20°C until DNA extraction. A packed CF11 column and filtration of blood diluted with PBS are shown in Figures 1A and 1B. The full CF11 filtration procedure is available on the WWARN website [12].\nPacked CF11 column ready for storage, shipping or use (A) and filtration of blood-PBS mixture through CF11 column into collection tube (B).\n Experimental design CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\n CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\n Experimental design CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\n CF11 filtration, with procedural modifications To test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\nTo test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\n Modifications in blood volume and parasite density To investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\nTo investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\n Routine blood collection for further validation and sequencing To determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\nTo determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\n CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\n CF11 filtration, with procedural modifications To test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\nTo test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\n Modifications in blood volume and parasite density To investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\nTo investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\n Routine blood collection for further validation and sequencing To determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\nTo determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\n DNA quantification DNA was extracted from leukocyte-depleted and unfiltered control samples using Qiamp DNA Blood Midi Kits (Qiagen, Venlo, Netherlands) according to the manufacturer's protocol. Total DNA from experiments using P. falciparum clones was estimated with a SpectraMax M2 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) using the Quant-IT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). The standard curve ranged from 0.20 to 50 ng/μl. Total DNA from experiments using P. falciparum isolates was estimated with a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) using both the dsDNA HS and BR Assay kits.\nQuantitative real-time PCR (qPCR) was used to measure the relative amounts of human and parasite DNA in each sample. Primers targeting the human LRAP (F: 5'-ACGTTGGATGAATTTTCCACTGGATTCCAT-'3; R: 5'-ACGTTGGATGTGAACCATGCTCCTTGCATC-'3) and TLR9 (F: 5'-ACGTTGGATGCAAAGGGCTGGCTGTTGTAG-'3; R: 5'- ACGTTGGATGTCTACCACGAGCACTCATTC-'3) genes and the P. falciparum AMA-1 gene (F: 5'-ACGTTGGATGGATTCTCTTTCGATTTCTTTC-'3; R: 5'-ACGTTGGATGTGCTACTACTGCTTTGTCCC-'3) were used to amplify DNA from samples, negative controls and standards in duplicate. Amplification occurred in an Applied Biosystems 7300 or StepOne real-time PCR machine. Human placental and P. falciparum (3D7 clone) genomic DNA standards ranged from 0.20 to 50 ng/μl. For each 25 μl reaction, 12.5 μl SYBR Green qPCR Master Mix (Applied Biosystems, Foster City, CA, USA) was mixed with 1.5 μl of each forward and reverse primers at 2 μM concentrations, 7.5-8.5 μl water, and 1-2 μl of template DNA. Reaction conditions were previously reported [3]: an initial denaturing step of 95°C for 10 minutes, five cycles of 94°C for 45 seconds, 56°C for 45 seconds, 72°C for 45 seconds, followed by 30 cycles of 94°C for 45 seconds, 65°C for 45 seconds and 72°C for 45 seconds and one dissociation cycle (conditions vary by real-time PCR instrument). Results were analysed using Applied Biosystems 7300 System SDS or StepOne v2.0 software.\nqPCR quantification of AMA-1 and TLR9 or LRAP was used to determine the proportions of P. falciparum and human DNA in the sample. Total DNA was estimated by multiplying the concentration given by Spectramax or Qubit by the extracted sample volume.\nDNA was extracted from leukocyte-depleted and unfiltered control samples using Qiamp DNA Blood Midi Kits (Qiagen, Venlo, Netherlands) according to the manufacturer's protocol. Total DNA from experiments using P. falciparum clones was estimated with a SpectraMax M2 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) using the Quant-IT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). The standard curve ranged from 0.20 to 50 ng/μl. Total DNA from experiments using P. falciparum isolates was estimated with a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) using both the dsDNA HS and BR Assay kits.\nQuantitative real-time PCR (qPCR) was used to measure the relative amounts of human and parasite DNA in each sample. Primers targeting the human LRAP (F: 5'-ACGTTGGATGAATTTTCCACTGGATTCCAT-'3; R: 5'-ACGTTGGATGTGAACCATGCTCCTTGCATC-'3) and TLR9 (F: 5'-ACGTTGGATGCAAAGGGCTGGCTGTTGTAG-'3; R: 5'- ACGTTGGATGTCTACCACGAGCACTCATTC-'3) genes and the P. falciparum AMA-1 gene (F: 5'-ACGTTGGATGGATTCTCTTTCGATTTCTTTC-'3; R: 5'-ACGTTGGATGTGCTACTACTGCTTTGTCCC-'3) were used to amplify DNA from samples, negative controls and standards in duplicate. Amplification occurred in an Applied Biosystems 7300 or StepOne real-time PCR machine. Human placental and P. falciparum (3D7 clone) genomic DNA standards ranged from 0.20 to 50 ng/μl. For each 25 μl reaction, 12.5 μl SYBR Green qPCR Master Mix (Applied Biosystems, Foster City, CA, USA) was mixed with 1.5 μl of each forward and reverse primers at 2 μM concentrations, 7.5-8.5 μl water, and 1-2 μl of template DNA. Reaction conditions were previously reported [3]: an initial denaturing step of 95°C for 10 minutes, five cycles of 94°C for 45 seconds, 56°C for 45 seconds, 72°C for 45 seconds, followed by 30 cycles of 94°C for 45 seconds, 65°C for 45 seconds and 72°C for 45 seconds and one dissociation cycle (conditions vary by real-time PCR instrument). Results were analysed using Applied Biosystems 7300 System SDS or StepOne v2.0 software.\nqPCR quantification of AMA-1 and TLR9 or LRAP was used to determine the proportions of P. falciparum and human DNA in the sample. Total DNA was estimated by multiplying the concentration given by Spectramax or Qubit by the extracted sample volume.\n Statistical comparisons Means, variance, standard errors and statistical comparisons of means were calculated using Stata 11 software. Boxplots indicating medians, interquartile ranges and outliers were also generated using Stata 11.\nMeans, variance, standard errors and statistical comparisons of means were calculated using Stata 11 software. Boxplots indicating medians, interquartile ranges and outliers were also generated using Stata 11.","Experiments using laboratory-adapted P. falciparum clones were conducted at the University of Maryland, Baltimore. Blood obtained from healthy volunteers was mixed with purified P. falciparum parasites (3D7 or Dd2 clones) to achieve parasite densities of 10,000/μl and 5,000/μl. These samples were stored at 4°C for up to two hours prior to CF11 or LP filtration.\nExperiments using non-adapted P. falciparum isolates were conducted in August 2010 at Sampov Meas Referral Hospital, Pursat, Cambodia. Blood samples for these experiments were collected from patients with falciparum malaria who were ≥ 10 years old and had a wide range of parasite densities (≥ 10,000/μl). These samples were held at 4°C for up to six hours prior to CF11 or LP filtration. Additional blood samples were collected in November to December 2010 at Lumphat District Health Centre, Ratanakiri, Cambodia. Blood samples were obtained from patients with falciparum malaria who were ≥ 1 year old and had a wide range of parasite densities ≥ 10,000/μl. These samples were held at 4°C for up to six hours prior to CF11 filtration. Following CF11 filtration, samples were stored on ice for up to six hours and transported on ice for up to 12 hours to Phnom Penh for centrifugation and RBC pellet storage.\nAll blood samples were collected in EDTA or CPDA-1 tubes. Heparin was not used as an anticoagulant as it thought to inhibit Taq polymerase during DNA amplification.\nBlood samples from healthy volunteers were collected under a protocol approved by the Institutional Review Board of the University of Maryland School of Medicine. Blood samples from patients with falciparum malaria were collected under protocols approved by the Institutional Review Board of the National Institute for Allergy and Infectious Diseases and the Cambodian National Ethics Committee for Health Research (http://ClinicalTrials.gov identifiers: NCT00341003 and NCT01240603). Written, informed consent was obtained from all study participants or their parents or guardians.","Lymphoprep (Axis-shield, Oslo, Norway) + Plasmodipur (Euro-diagnostica, Arnhem, Netherlands) filtration ('LP') was performed as previously described [3]. CF11 columns were loosely filled to the 10-ml mark, packed to the 5.5-ml mark with CF11 cellulose powder (Whatman, Kent, UK), and wetted with isotonic phosphate buffered saline (PBS), as described [10]. Initial tests indicated that plasma separation prior to CF11 filtration of blood did not affect human DNA depletion and that washing with PBS after CF11 filtration improved recovery of parasite DNA. CF11 filtration was therefore carried out as follows. Parasitized blood or blood/parasite mixtures were diluted with an equal volume of PBS. These samples were added to the CF11 column and allowed to flow through by gravity. To wash the sample, 5 ml PBS was added to the column and allowed to pass through by gravity. A plunger was then inserted into the top of the column and the final few drops pushed through the column. The filtrates were centrifuged for 10 minutes at 1,000 x g and the supernatants removed. RBC pellets from all filtrates in all experiments were stored at -20°C until DNA extraction. A packed CF11 column and filtration of blood diluted with PBS are shown in Figures 1A and 1B. The full CF11 filtration procedure is available on the WWARN website [12].\nPacked CF11 column ready for storage, shipping or use (A) and filtration of blood-PBS mixture through CF11 column into collection tube (B)."," CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method","To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method"," CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\n CF11 filtration, with procedural modifications To test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\nTo test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\n Modifications in blood volume and parasite density To investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\nTo investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\n Routine blood collection for further validation and sequencing To determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\nTo determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.","To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.","To test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.","To investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.","To determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.","DNA was extracted from leukocyte-depleted and unfiltered control samples using Qiamp DNA Blood Midi Kits (Qiagen, Venlo, Netherlands) according to the manufacturer's protocol. Total DNA from experiments using P. falciparum clones was estimated with a SpectraMax M2 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) using the Quant-IT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). The standard curve ranged from 0.20 to 50 ng/μl. Total DNA from experiments using P. falciparum isolates was estimated with a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) using both the dsDNA HS and BR Assay kits.\nQuantitative real-time PCR (qPCR) was used to measure the relative amounts of human and parasite DNA in each sample. Primers targeting the human LRAP (F: 5'-ACGTTGGATGAATTTTCCACTGGATTCCAT-'3; R: 5'-ACGTTGGATGTGAACCATGCTCCTTGCATC-'3) and TLR9 (F: 5'-ACGTTGGATGCAAAGGGCTGGCTGTTGTAG-'3; R: 5'- ACGTTGGATGTCTACCACGAGCACTCATTC-'3) genes and the P. falciparum AMA-1 gene (F: 5'-ACGTTGGATGGATTCTCTTTCGATTTCTTTC-'3; R: 5'-ACGTTGGATGTGCTACTACTGCTTTGTCCC-'3) were used to amplify DNA from samples, negative controls and standards in duplicate. Amplification occurred in an Applied Biosystems 7300 or StepOne real-time PCR machine. Human placental and P. falciparum (3D7 clone) genomic DNA standards ranged from 0.20 to 50 ng/μl. For each 25 μl reaction, 12.5 μl SYBR Green qPCR Master Mix (Applied Biosystems, Foster City, CA, USA) was mixed with 1.5 μl of each forward and reverse primers at 2 μM concentrations, 7.5-8.5 μl water, and 1-2 μl of template DNA. Reaction conditions were previously reported [3]: an initial denaturing step of 95°C for 10 minutes, five cycles of 94°C for 45 seconds, 56°C for 45 seconds, 72°C for 45 seconds, followed by 30 cycles of 94°C for 45 seconds, 65°C for 45 seconds and 72°C for 45 seconds and one dissociation cycle (conditions vary by real-time PCR instrument). Results were analysed using Applied Biosystems 7300 System SDS or StepOne v2.0 software.\nqPCR quantification of AMA-1 and TLR9 or LRAP was used to determine the proportions of P. falciparum and human DNA in the sample. Total DNA was estimated by multiplying the concentration given by Spectramax or Qubit by the extracted sample volume.","Means, variance, standard errors and statistical comparisons of means were calculated using Stata 11 software. Boxplots indicating medians, interquartile ranges and outliers were also generated using Stata 11."," Comparison of unmodified LP and CF11 filtration for leukocyte depletion Filtration of blood-parasite mixtures using unmodified LP and CF11 methods in parallel successfully depleted human DNA to 1% and 6% of total DNA, respectively (Figure 2A), well below the current Illumina sequencing threshold of < 50%. CF11 was more effective than LP in recovering total DNA from blood-parasite mixtures (mean 1.11 μg vs. 0.37 μg; p = 0.03) (Figure 2B). All 15 CF11-filtered parasitized blood samples and 12 of 14 LP-filtered parasitized blood samples yielded < 50% human DNA contamination (Figure 3A). Human DNA contamination was lower for parasitized blood filtered by CF11 than by LP (mean 2.4% vs. 15.9%; p = 0.03) (Figure 3B). Recovery of total DNA was comparably high in both sets of samples (mean 4.64 μg for CF11 vs. 2.86 μg for LP; p = 0.46) One LP-filtered sample failed to produce a human DNA estimate by qPCR and was omitted from the analysis.\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of blood-parasite mixtures. The volume and parasite density of blood-parasite mixtures were 3 ml and 10,000/μl except where stated otherwise. Three replicates for each filtration method were performed. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column, CF11 O/N = CF11 filtrate held overnight prior to removal of supernatant, CF11 4 ml = filtration using a 4-ml CF11 column, CF11 low vol = filtration of a 1.5-ml blood-parasite mixture containing 10,000 parasites/μl, CF11 low para = filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl, NF = no filtration, NF low para = no filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl. Box plots show median and interquartile range.\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of Plasmodium falciparum-infected blood samples that vary in parasite density. Light gray shading indicates samples with < 50,000 parasites/μl, medium gray shading indicates samples with 50,000-100,000 parasites/μl, and dark gray shading indicates samples with parasite density > 100,000 parasites/μl. All sample volumes were 4 ml. n = sample size for each method within each range of parasite density. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column. Box plots show median and interquartile range. Whiskers span the range of data points within 1.5 times the interquartile range of the lower and upper quartiles. Outliers are shown as points.\nFiltration of blood-parasite mixtures using unmodified LP and CF11 methods in parallel successfully depleted human DNA to 1% and 6% of total DNA, respectively (Figure 2A), well below the current Illumina sequencing threshold of < 50%. CF11 was more effective than LP in recovering total DNA from blood-parasite mixtures (mean 1.11 μg vs. 0.37 μg; p = 0.03) (Figure 2B). All 15 CF11-filtered parasitized blood samples and 12 of 14 LP-filtered parasitized blood samples yielded < 50% human DNA contamination (Figure 3A). Human DNA contamination was lower for parasitized blood filtered by CF11 than by LP (mean 2.4% vs. 15.9%; p = 0.03) (Figure 3B). Recovery of total DNA was comparably high in both sets of samples (mean 4.64 μg for CF11 vs. 2.86 μg for LP; p = 0.46) One LP-filtered sample failed to produce a human DNA estimate by qPCR and was omitted from the analysis.\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of blood-parasite mixtures. The volume and parasite density of blood-parasite mixtures were 3 ml and 10,000/μl except where stated otherwise. Three replicates for each filtration method were performed. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column, CF11 O/N = CF11 filtrate held overnight prior to removal of supernatant, CF11 4 ml = filtration using a 4-ml CF11 column, CF11 low vol = filtration of a 1.5-ml blood-parasite mixture containing 10,000 parasites/μl, CF11 low para = filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl, NF = no filtration, NF low para = no filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl. Box plots show median and interquartile range.\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of Plasmodium falciparum-infected blood samples that vary in parasite density. Light gray shading indicates samples with < 50,000 parasites/μl, medium gray shading indicates samples with 50,000-100,000 parasites/μl, and dark gray shading indicates samples with parasite density > 100,000 parasites/μl. All sample volumes were 4 ml. n = sample size for each method within each range of parasite density. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column. Box plots show median and interquartile range. Whiskers span the range of data points within 1.5 times the interquartile range of the lower and upper quartiles. Outliers are shown as points.\n Procedural modifications of CF11 filtration method To explore whether CF11 filtrates could be processed without centrifugation, CF11-filtered blood-parasite mixtures were refrigerated overnight. The RBC pellet formed during overnight settling was not as tightly packed as that formed during centrifugation and required careful handling when removing supernatant to prevent disruption. This modification resulted in adequate human DNA depletion (mean contamination 11%) (Figure 2A) and total DNA recovery (mean 1.22 μg) (Figure 2B). Columns with less CF11 powder, packed to the 4-ml mark rather than to the 5.5-ml mark, also yielded high total DNA (mean 1.56 μg) and a sufficiently low proportion of human DNA (mean 30%) (Figure 2A and 2B).\nTo explore whether CF11 filtrates could be processed without centrifugation, CF11-filtered blood-parasite mixtures were refrigerated overnight. The RBC pellet formed during overnight settling was not as tightly packed as that formed during centrifugation and required careful handling when removing supernatant to prevent disruption. This modification resulted in adequate human DNA depletion (mean contamination 11%) (Figure 2A) and total DNA recovery (mean 1.22 μg) (Figure 2B). Columns with less CF11 powder, packed to the 4-ml mark rather than to the 5.5-ml mark, also yielded high total DNA (mean 1.56 μg) and a sufficiently low proportion of human DNA (mean 30%) (Figure 2A and 2B).\n Blood volume and parasite density All CF11-filtered blood-parasite mixtures (3 ml volume, 10,000/μl parasite density) consistently yielded samples with < 30% human DNA contamination and > 500 ng total DNA (Figure 2A and 2B). However, the ex vivo study of P. falciparum isolates with interesting phenotypes can be difficult if blood volumes are limited and parasite densities are low. To determine whether such limitations might significantly reduce the quality of DNA samples, CF11 filtrations were repeated using blood-parasite mixtures of lower volume (1.5 ml) or lower parasite densities (5,000/μl). CF11 filtration of low-volume samples (1.5 ml; 10,000 parasites/μl) produced acceptable human DNA contamination (mean 28%) and mean total DNA (0.51 μg), but individual yields of total DNA were highly variable, ranging from 0.15 to 1.09 μg (Figure 2A and 2B). CF11 filtration of low parasite density samples (3 ml; 5,000 parasites/μl) produced adequate amounts of total DNA (mean 2.68 μg) but unacceptable human DNA contamination (mean 77%) (Figure 2A and 2B). Thus, reducing sample volumes or parasite densities by half failed to reliably produce DNA samples acceptable for Illumina sequencing.\nFiltration of P. falciparum-infected blood obtained from patients with malaria (Pursat, Cambodia) using the CF11 and LP methods consistently achieved high-quality results at parasite densities ranging from 20,000 to 475,000/μl (Figure 3A and 3B). At parasite densities < 50,000/μl and 50,000-100,000/μl, both filtration methods depleted human DNA contamination to ≤ 30% and produced mean DNA yields > 1 μg. Samples with > 100,000 parasites/μl yielded several micrograms of DNA with < 5% human DNA contamination.\nAll CF11-filtered blood-parasite mixtures (3 ml volume, 10,000/μl parasite density) consistently yielded samples with < 30% human DNA contamination and > 500 ng total DNA (Figure 2A and 2B). However, the ex vivo study of P. falciparum isolates with interesting phenotypes can be difficult if blood volumes are limited and parasite densities are low. To determine whether such limitations might significantly reduce the quality of DNA samples, CF11 filtrations were repeated using blood-parasite mixtures of lower volume (1.5 ml) or lower parasite densities (5,000/μl). CF11 filtration of low-volume samples (1.5 ml; 10,000 parasites/μl) produced acceptable human DNA contamination (mean 28%) and mean total DNA (0.51 μg), but individual yields of total DNA were highly variable, ranging from 0.15 to 1.09 μg (Figure 2A and 2B). CF11 filtration of low parasite density samples (3 ml; 5,000 parasites/μl) produced adequate amounts of total DNA (mean 2.68 μg) but unacceptable human DNA contamination (mean 77%) (Figure 2A and 2B). Thus, reducing sample volumes or parasite densities by half failed to reliably produce DNA samples acceptable for Illumina sequencing.\nFiltration of P. falciparum-infected blood obtained from patients with malaria (Pursat, Cambodia) using the CF11 and LP methods consistently achieved high-quality results at parasite densities ranging from 20,000 to 475,000/μl (Figure 3A and 3B). At parasite densities < 50,000/μl and 50,000-100,000/μl, both filtration methods depleted human DNA contamination to ≤ 30% and produced mean DNA yields > 1 μg. Samples with > 100,000 parasites/μl yielded several micrograms of DNA with < 5% human DNA contamination.\n Validation of routine blood collection for illumina sequencing The 51 parasitized blood samples that were collected and CF11-filtered in Ratanakiri, Cambodia, ranged in parasite density from 15,000 to 290,000/μl and yielded a mean 0.64 μg total DNA with 34% human DNA contamination. All 51 samples met quality standards and have been successfully sequenced on the Illumina platform at the Wellcome Trust Sanger Institute (Hinxton, UK).\nThe 51 parasitized blood samples that were collected and CF11-filtered in Ratanakiri, Cambodia, ranged in parasite density from 15,000 to 290,000/μl and yielded a mean 0.64 μg total DNA with 34% human DNA contamination. All 51 samples met quality standards and have been successfully sequenced on the Illumina platform at the Wellcome Trust Sanger Institute (Hinxton, UK).","Filtration of blood-parasite mixtures using unmodified LP and CF11 methods in parallel successfully depleted human DNA to 1% and 6% of total DNA, respectively (Figure 2A), well below the current Illumina sequencing threshold of < 50%. CF11 was more effective than LP in recovering total DNA from blood-parasite mixtures (mean 1.11 μg vs. 0.37 μg; p = 0.03) (Figure 2B). All 15 CF11-filtered parasitized blood samples and 12 of 14 LP-filtered parasitized blood samples yielded < 50% human DNA contamination (Figure 3A). Human DNA contamination was lower for parasitized blood filtered by CF11 than by LP (mean 2.4% vs. 15.9%; p = 0.03) (Figure 3B). Recovery of total DNA was comparably high in both sets of samples (mean 4.64 μg for CF11 vs. 2.86 μg for LP; p = 0.46) One LP-filtered sample failed to produce a human DNA estimate by qPCR and was omitted from the analysis.\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of blood-parasite mixtures. The volume and parasite density of blood-parasite mixtures were 3 ml and 10,000/μl except where stated otherwise. Three replicates for each filtration method were performed. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column, CF11 O/N = CF11 filtrate held overnight prior to removal of supernatant, CF11 4 ml = filtration using a 4-ml CF11 column, CF11 low vol = filtration of a 1.5-ml blood-parasite mixture containing 10,000 parasites/μl, CF11 low para = filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl, NF = no filtration, NF low para = no filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl. Box plots show median and interquartile range.\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of Plasmodium falciparum-infected blood samples that vary in parasite density. Light gray shading indicates samples with < 50,000 parasites/μl, medium gray shading indicates samples with 50,000-100,000 parasites/μl, and dark gray shading indicates samples with parasite density > 100,000 parasites/μl. All sample volumes were 4 ml. n = sample size for each method within each range of parasite density. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column. Box plots show median and interquartile range. Whiskers span the range of data points within 1.5 times the interquartile range of the lower and upper quartiles. Outliers are shown as points.","To explore whether CF11 filtrates could be processed without centrifugation, CF11-filtered blood-parasite mixtures were refrigerated overnight. The RBC pellet formed during overnight settling was not as tightly packed as that formed during centrifugation and required careful handling when removing supernatant to prevent disruption. This modification resulted in adequate human DNA depletion (mean contamination 11%) (Figure 2A) and total DNA recovery (mean 1.22 μg) (Figure 2B). Columns with less CF11 powder, packed to the 4-ml mark rather than to the 5.5-ml mark, also yielded high total DNA (mean 1.56 μg) and a sufficiently low proportion of human DNA (mean 30%) (Figure 2A and 2B).","All CF11-filtered blood-parasite mixtures (3 ml volume, 10,000/μl parasite density) consistently yielded samples with < 30% human DNA contamination and > 500 ng total DNA (Figure 2A and 2B). However, the ex vivo study of P. falciparum isolates with interesting phenotypes can be difficult if blood volumes are limited and parasite densities are low. To determine whether such limitations might significantly reduce the quality of DNA samples, CF11 filtrations were repeated using blood-parasite mixtures of lower volume (1.5 ml) or lower parasite densities (5,000/μl). CF11 filtration of low-volume samples (1.5 ml; 10,000 parasites/μl) produced acceptable human DNA contamination (mean 28%) and mean total DNA (0.51 μg), but individual yields of total DNA were highly variable, ranging from 0.15 to 1.09 μg (Figure 2A and 2B). CF11 filtration of low parasite density samples (3 ml; 5,000 parasites/μl) produced adequate amounts of total DNA (mean 2.68 μg) but unacceptable human DNA contamination (mean 77%) (Figure 2A and 2B). Thus, reducing sample volumes or parasite densities by half failed to reliably produce DNA samples acceptable for Illumina sequencing.\nFiltration of P. falciparum-infected blood obtained from patients with malaria (Pursat, Cambodia) using the CF11 and LP methods consistently achieved high-quality results at parasite densities ranging from 20,000 to 475,000/μl (Figure 3A and 3B). At parasite densities < 50,000/μl and 50,000-100,000/μl, both filtration methods depleted human DNA contamination to ≤ 30% and produced mean DNA yields > 1 μg. Samples with > 100,000 parasites/μl yielded several micrograms of DNA with < 5% human DNA contamination.","The 51 parasitized blood samples that were collected and CF11-filtered in Ratanakiri, Cambodia, ranged in parasite density from 15,000 to 290,000/μl and yielded a mean 0.64 μg total DNA with 34% human DNA contamination. All 51 samples met quality standards and have been successfully sequenced on the Illumina platform at the Wellcome Trust Sanger Institute (Hinxton, UK).","As next-generation sequencing technologies improve, both costs and stringency of DNA sample quality requirements continue to fall. Between 2009 and 2011, requirements for Illumina sequencing of P. falciparum DNA by the Wellcome Trust Sanger Institute have relaxed from 30% to 50% human DNA contamination. Moreover, the possibility of sequencing multiple DNA samples in a single lane of a flow cell ('multiplex sequencing') has decreased costs immensely while yielding ~2 GB of P. falciparum genomic sequence data per sample, with further improvements anticipated in coming years. To take advantage of these increasingly accessible tools, researchers need scalable procedures for DNA sample preparation in minimally equipped field sites.\nThis study shows that CF11 columns can be used to effectively deplete human DNA from parasitized blood and achieve Illumina sequencing requirements for total DNA yield and percent human DNA contamination, using sample volumes and parasite densities comparable to those used in LP filtration experiments [3]. A major advantage of CF11 over LP filtration is cost-effectiveness. CF11 columns cost approximately one USD each, compared to 10-50 USD for the Plasmodipur filter alone. Additionally, CF11 filtration is a very simple one-step procedure that does not require specialized equipment. A centrifugation step is only used to pellet RBCs after CF11 filtration and can be replaced with a convenient overnight period of refrigeration to allow the RBCs to pellet by gravity. These advantages should enable a large share of the malaria research community to produce parasite DNA samples appropriate for highly parallel sequencing and microarray technologies.\nBecause CF11 columns are hand-made rather than commercially produced, variation in the length of the column is likely to occur. Samples filtered in columns packed with less CF11 powder exhibited higher human DNA contamination but still met the threshold level for sequencing, indicating that some variation in column preparation can be tolerated. Samples with low blood volumes (1.5 ml) or parasite densities (5,000/μl) did not consistently achieve sufficient human DNA depletion or total DNA recovery when filtered with either the CF11 or LP method. Whole-genome amplification (WGA) after DNA isolation [13] could boost the amount of starting material in cases where human DNA removal is satisfactory but total DNA yield is low. WGA may therefore be useful in enabling genomic characterization of CF11-filtered blood samples with low starting volume, such as those collected from very young children and patients with severe malarial anaemia. It may also be useful in processing blood samples with low parasite densities, as is often the case in patients with a recrudescent parasitaemia after anti-malarial treatment and those with high levels of naturally acquired immunity.\nAlthough specific parameters have not been rigorously tested, observations in the field have indicated that CF11 and parasitized blood can be effectively stored and used in a variety of environmental conditions. CF11 kept in an airtight container with regularly replaced desiccant and stored in a cool place can be used for at least three months, even in climates with high heat and humidity (unpublished observation). EDTA-treated blood stored at 4°C for up to 24 hours prior to CF11 filtration was successfully processed for Illumina DNA sequencing (unpublished observation), but for RNA analysis, no more than six hours of refrigeration is suggested (ZB Bozdech, personal communication). Updates to the range of storage conditions will be made to the WWARN protocol [12] as more information becomes available.","CF11 filtration currently offers the best cost-effective, one-step approach to remove human DNA from P. falciparum-infected whole blood samples and can be successfully implemented in large genome-wide sequencing studies of P. falciparum isolates from patients with malaria."],"string":"[\n \"Recent technological advances have enabled next-generation genomic and transcriptomic analysis of Plasmodium falciparum from parasitized whole blood samples without the need for culturing. High-density genotyping of parasites obtained directly from patients with malaria has improved our understanding of population structure and genomic and transcriptional variation [1,2]. Highly parallel sequencing is currently being used to identify the genetic determinants of clinically relevant phenotypes including drug resistance, vaccine escape and disease severity. Importantly, genomic characterization of parasite populations in patients captures intra-host diversity and prevents the potential loss of sequence data from phenotype-conferring parasite isolates during their culture adaptation.\\nThe performance of highly parallel sequencing platforms, such as Illumina, is greatly enhanced in sample preparations with a high parasite-to-human nucleic acid ratio [3]. Such high ratios can be achieved by either selectively capturing parasite nucleic acids or by removing human material from the sample. Hybrid selection [4] using RNA 'baits' complementary to the P. falciparum genome can achieve over 40-fold enrichment of parasite DNA and can be performed at any time following DNA extraction [5]; however, at $250 USD per sample, this technique may be prohibitively costly for epidemiological or population-level studies.\\nThe alternative, leukocyte depletion, must be performed in field sites soon after blood collection and before transport and storage. Commercially available magnetic columns have been used to rapidly isolate parasitized red blood cells (RBCs) from uninfected RBCs and leukocytes [6]. However, magnetic purification depends on short-term culturing of patient blood to transform ring-stage parasites to haemozoin-rich trophozoites and schizonts, which requires equipment for in vitro parasite cultivation. Furthermore, parasites obtained directly from patients mature at different rates in culture, resulting in inconvenient time-points for purification. The current standard for leukocyte depletion of parasitized blood for P. falciparum nucleotide sequencing is a two-step process: centrifugation using Lymphoprep or another sucrose density gradient solution, followed by filtration using Plasmodipur filters [3]. This 'LP' method is effective but difficult to scale-up in field settings because it requires extensive handling and transfer of blood, training to perform sensitive steps, and costly commercial reagents and materials.\\nFiltration with hand-made columns has been used as an inexpensive and less time-consuming alternative for leukocyte depletion of Plasmodium-infected blood for decades [7,8]. Recently, non-woven fabric filters [9] and plastic syringes filled with CF11 cellulose powder (Whatman) [10] were shown to effectively remove leukocytes and platelets from Plasmodium vivax-infected blood, where their phagocytic and degranulating activities may interfere with some ex vivo studies. CF11 filtration has also been used to improve microarray-based transcriptome analysis of P. vivax-infected blood [2,11]. CF11 cellulose is thought to work by trapping leukocytes by size exclusion and/or interactions between cellulose hydroxyl groups and leukocyte surface molecules. In this study, laboratory-adapted P. falciparum clones were used to adapt CF11 columns to remove human DNA from P. falciparum-infected blood for genomic studies, compare CF11 filtration to the established LP method, and validate a centrifuge-free option for CF11 filtration. Both methods were also compared in a field setting using P. falciparum isolates obtained directly from patients with malaria. Furthermore, routine CF11 filtration of parasitized blood collected from patients in a second field site with minimal facilities was successfully implemented.\",\n \" Blood samples Experiments using laboratory-adapted P. falciparum clones were conducted at the University of Maryland, Baltimore. Blood obtained from healthy volunteers was mixed with purified P. falciparum parasites (3D7 or Dd2 clones) to achieve parasite densities of 10,000/μl and 5,000/μl. These samples were stored at 4°C for up to two hours prior to CF11 or LP filtration.\\nExperiments using non-adapted P. falciparum isolates were conducted in August 2010 at Sampov Meas Referral Hospital, Pursat, Cambodia. Blood samples for these experiments were collected from patients with falciparum malaria who were ≥ 10 years old and had a wide range of parasite densities (≥ 10,000/μl). These samples were held at 4°C for up to six hours prior to CF11 or LP filtration. Additional blood samples were collected in November to December 2010 at Lumphat District Health Centre, Ratanakiri, Cambodia. Blood samples were obtained from patients with falciparum malaria who were ≥ 1 year old and had a wide range of parasite densities ≥ 10,000/μl. These samples were held at 4°C for up to six hours prior to CF11 filtration. Following CF11 filtration, samples were stored on ice for up to six hours and transported on ice for up to 12 hours to Phnom Penh for centrifugation and RBC pellet storage.\\nAll blood samples were collected in EDTA or CPDA-1 tubes. Heparin was not used as an anticoagulant as it thought to inhibit Taq polymerase during DNA amplification.\\nBlood samples from healthy volunteers were collected under a protocol approved by the Institutional Review Board of the University of Maryland School of Medicine. Blood samples from patients with falciparum malaria were collected under protocols approved by the Institutional Review Board of the National Institute for Allergy and Infectious Diseases and the Cambodian National Ethics Committee for Health Research (http://ClinicalTrials.gov identifiers: NCT00341003 and NCT01240603). Written, informed consent was obtained from all study participants or their parents or guardians.\\nExperiments using laboratory-adapted P. falciparum clones were conducted at the University of Maryland, Baltimore. Blood obtained from healthy volunteers was mixed with purified P. falciparum parasites (3D7 or Dd2 clones) to achieve parasite densities of 10,000/μl and 5,000/μl. These samples were stored at 4°C for up to two hours prior to CF11 or LP filtration.\\nExperiments using non-adapted P. falciparum isolates were conducted in August 2010 at Sampov Meas Referral Hospital, Pursat, Cambodia. Blood samples for these experiments were collected from patients with falciparum malaria who were ≥ 10 years old and had a wide range of parasite densities (≥ 10,000/μl). These samples were held at 4°C for up to six hours prior to CF11 or LP filtration. Additional blood samples were collected in November to December 2010 at Lumphat District Health Centre, Ratanakiri, Cambodia. Blood samples were obtained from patients with falciparum malaria who were ≥ 1 year old and had a wide range of parasite densities ≥ 10,000/μl. These samples were held at 4°C for up to six hours prior to CF11 filtration. Following CF11 filtration, samples were stored on ice for up to six hours and transported on ice for up to 12 hours to Phnom Penh for centrifugation and RBC pellet storage.\\nAll blood samples were collected in EDTA or CPDA-1 tubes. Heparin was not used as an anticoagulant as it thought to inhibit Taq polymerase during DNA amplification.\\nBlood samples from healthy volunteers were collected under a protocol approved by the Institutional Review Board of the University of Maryland School of Medicine. Blood samples from patients with falciparum malaria were collected under protocols approved by the Institutional Review Board of the National Institute for Allergy and Infectious Diseases and the Cambodian National Ethics Committee for Health Research (http://ClinicalTrials.gov identifiers: NCT00341003 and NCT01240603). Written, informed consent was obtained from all study participants or their parents or guardians.\\n Leukocyte depletion Lymphoprep (Axis-shield, Oslo, Norway) + Plasmodipur (Euro-diagnostica, Arnhem, Netherlands) filtration ('LP') was performed as previously described [3]. CF11 columns were loosely filled to the 10-ml mark, packed to the 5.5-ml mark with CF11 cellulose powder (Whatman, Kent, UK), and wetted with isotonic phosphate buffered saline (PBS), as described [10]. Initial tests indicated that plasma separation prior to CF11 filtration of blood did not affect human DNA depletion and that washing with PBS after CF11 filtration improved recovery of parasite DNA. CF11 filtration was therefore carried out as follows. Parasitized blood or blood/parasite mixtures were diluted with an equal volume of PBS. These samples were added to the CF11 column and allowed to flow through by gravity. To wash the sample, 5 ml PBS was added to the column and allowed to pass through by gravity. A plunger was then inserted into the top of the column and the final few drops pushed through the column. The filtrates were centrifuged for 10 minutes at 1,000 x g and the supernatants removed. RBC pellets from all filtrates in all experiments were stored at -20°C until DNA extraction. A packed CF11 column and filtration of blood diluted with PBS are shown in Figures 1A and 1B. The full CF11 filtration procedure is available on the WWARN website [12].\\nPacked CF11 column ready for storage, shipping or use (A) and filtration of blood-PBS mixture through CF11 column into collection tube (B).\\nLymphoprep (Axis-shield, Oslo, Norway) + Plasmodipur (Euro-diagnostica, Arnhem, Netherlands) filtration ('LP') was performed as previously described [3]. CF11 columns were loosely filled to the 10-ml mark, packed to the 5.5-ml mark with CF11 cellulose powder (Whatman, Kent, UK), and wetted with isotonic phosphate buffered saline (PBS), as described [10]. Initial tests indicated that plasma separation prior to CF11 filtration of blood did not affect human DNA depletion and that washing with PBS after CF11 filtration improved recovery of parasite DNA. CF11 filtration was therefore carried out as follows. Parasitized blood or blood/parasite mixtures were diluted with an equal volume of PBS. These samples were added to the CF11 column and allowed to flow through by gravity. To wash the sample, 5 ml PBS was added to the column and allowed to pass through by gravity. A plunger was then inserted into the top of the column and the final few drops pushed through the column. The filtrates were centrifuged for 10 minutes at 1,000 x g and the supernatants removed. RBC pellets from all filtrates in all experiments were stored at -20°C until DNA extraction. A packed CF11 column and filtration of blood diluted with PBS are shown in Figures 1A and 1B. The full CF11 filtration procedure is available on the WWARN website [12].\\nPacked CF11 column ready for storage, shipping or use (A) and filtration of blood-PBS mixture through CF11 column into collection tube (B).\\n Experimental design CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\\n CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\\n Experimental design CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\\n CF11 filtration, with procedural modifications To test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\\nTo test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\\n Modifications in blood volume and parasite density To investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\\nTo investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\\n Routine blood collection for further validation and sequencing To determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\\nTo determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\\n CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\\n CF11 filtration, with procedural modifications To test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\\nTo test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\\n Modifications in blood volume and parasite density To investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\\nTo investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\\n Routine blood collection for further validation and sequencing To determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\\nTo determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\\n DNA quantification DNA was extracted from leukocyte-depleted and unfiltered control samples using Qiamp DNA Blood Midi Kits (Qiagen, Venlo, Netherlands) according to the manufacturer's protocol. Total DNA from experiments using P. falciparum clones was estimated with a SpectraMax M2 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) using the Quant-IT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). The standard curve ranged from 0.20 to 50 ng/μl. Total DNA from experiments using P. falciparum isolates was estimated with a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) using both the dsDNA HS and BR Assay kits.\\nQuantitative real-time PCR (qPCR) was used to measure the relative amounts of human and parasite DNA in each sample. Primers targeting the human LRAP (F: 5'-ACGTTGGATGAATTTTCCACTGGATTCCAT-'3; R: 5'-ACGTTGGATGTGAACCATGCTCCTTGCATC-'3) and TLR9 (F: 5'-ACGTTGGATGCAAAGGGCTGGCTGTTGTAG-'3; R: 5'- ACGTTGGATGTCTACCACGAGCACTCATTC-'3) genes and the P. falciparum AMA-1 gene (F: 5'-ACGTTGGATGGATTCTCTTTCGATTTCTTTC-'3; R: 5'-ACGTTGGATGTGCTACTACTGCTTTGTCCC-'3) were used to amplify DNA from samples, negative controls and standards in duplicate. Amplification occurred in an Applied Biosystems 7300 or StepOne real-time PCR machine. Human placental and P. falciparum (3D7 clone) genomic DNA standards ranged from 0.20 to 50 ng/μl. For each 25 μl reaction, 12.5 μl SYBR Green qPCR Master Mix (Applied Biosystems, Foster City, CA, USA) was mixed with 1.5 μl of each forward and reverse primers at 2 μM concentrations, 7.5-8.5 μl water, and 1-2 μl of template DNA. Reaction conditions were previously reported [3]: an initial denaturing step of 95°C for 10 minutes, five cycles of 94°C for 45 seconds, 56°C for 45 seconds, 72°C for 45 seconds, followed by 30 cycles of 94°C for 45 seconds, 65°C for 45 seconds and 72°C for 45 seconds and one dissociation cycle (conditions vary by real-time PCR instrument). Results were analysed using Applied Biosystems 7300 System SDS or StepOne v2.0 software.\\nqPCR quantification of AMA-1 and TLR9 or LRAP was used to determine the proportions of P. falciparum and human DNA in the sample. Total DNA was estimated by multiplying the concentration given by Spectramax or Qubit by the extracted sample volume.\\nDNA was extracted from leukocyte-depleted and unfiltered control samples using Qiamp DNA Blood Midi Kits (Qiagen, Venlo, Netherlands) according to the manufacturer's protocol. Total DNA from experiments using P. falciparum clones was estimated with a SpectraMax M2 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) using the Quant-IT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). The standard curve ranged from 0.20 to 50 ng/μl. Total DNA from experiments using P. falciparum isolates was estimated with a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) using both the dsDNA HS and BR Assay kits.\\nQuantitative real-time PCR (qPCR) was used to measure the relative amounts of human and parasite DNA in each sample. Primers targeting the human LRAP (F: 5'-ACGTTGGATGAATTTTCCACTGGATTCCAT-'3; R: 5'-ACGTTGGATGTGAACCATGCTCCTTGCATC-'3) and TLR9 (F: 5'-ACGTTGGATGCAAAGGGCTGGCTGTTGTAG-'3; R: 5'- ACGTTGGATGTCTACCACGAGCACTCATTC-'3) genes and the P. falciparum AMA-1 gene (F: 5'-ACGTTGGATGGATTCTCTTTCGATTTCTTTC-'3; R: 5'-ACGTTGGATGTGCTACTACTGCTTTGTCCC-'3) were used to amplify DNA from samples, negative controls and standards in duplicate. Amplification occurred in an Applied Biosystems 7300 or StepOne real-time PCR machine. Human placental and P. falciparum (3D7 clone) genomic DNA standards ranged from 0.20 to 50 ng/μl. For each 25 μl reaction, 12.5 μl SYBR Green qPCR Master Mix (Applied Biosystems, Foster City, CA, USA) was mixed with 1.5 μl of each forward and reverse primers at 2 μM concentrations, 7.5-8.5 μl water, and 1-2 μl of template DNA. Reaction conditions were previously reported [3]: an initial denaturing step of 95°C for 10 minutes, five cycles of 94°C for 45 seconds, 56°C for 45 seconds, 72°C for 45 seconds, followed by 30 cycles of 94°C for 45 seconds, 65°C for 45 seconds and 72°C for 45 seconds and one dissociation cycle (conditions vary by real-time PCR instrument). Results were analysed using Applied Biosystems 7300 System SDS or StepOne v2.0 software.\\nqPCR quantification of AMA-1 and TLR9 or LRAP was used to determine the proportions of P. falciparum and human DNA in the sample. Total DNA was estimated by multiplying the concentration given by Spectramax or Qubit by the extracted sample volume.\\n Statistical comparisons Means, variance, standard errors and statistical comparisons of means were calculated using Stata 11 software. Boxplots indicating medians, interquartile ranges and outliers were also generated using Stata 11.\\nMeans, variance, standard errors and statistical comparisons of means were calculated using Stata 11 software. Boxplots indicating medians, interquartile ranges and outliers were also generated using Stata 11.\",\n \"Experiments using laboratory-adapted P. falciparum clones were conducted at the University of Maryland, Baltimore. Blood obtained from healthy volunteers was mixed with purified P. falciparum parasites (3D7 or Dd2 clones) to achieve parasite densities of 10,000/μl and 5,000/μl. These samples were stored at 4°C for up to two hours prior to CF11 or LP filtration.\\nExperiments using non-adapted P. falciparum isolates were conducted in August 2010 at Sampov Meas Referral Hospital, Pursat, Cambodia. Blood samples for these experiments were collected from patients with falciparum malaria who were ≥ 10 years old and had a wide range of parasite densities (≥ 10,000/μl). These samples were held at 4°C for up to six hours prior to CF11 or LP filtration. Additional blood samples were collected in November to December 2010 at Lumphat District Health Centre, Ratanakiri, Cambodia. Blood samples were obtained from patients with falciparum malaria who were ≥ 1 year old and had a wide range of parasite densities ≥ 10,000/μl. These samples were held at 4°C for up to six hours prior to CF11 filtration. Following CF11 filtration, samples were stored on ice for up to six hours and transported on ice for up to 12 hours to Phnom Penh for centrifugation and RBC pellet storage.\\nAll blood samples were collected in EDTA or CPDA-1 tubes. Heparin was not used as an anticoagulant as it thought to inhibit Taq polymerase during DNA amplification.\\nBlood samples from healthy volunteers were collected under a protocol approved by the Institutional Review Board of the University of Maryland School of Medicine. Blood samples from patients with falciparum malaria were collected under protocols approved by the Institutional Review Board of the National Institute for Allergy and Infectious Diseases and the Cambodian National Ethics Committee for Health Research (http://ClinicalTrials.gov identifiers: NCT00341003 and NCT01240603). Written, informed consent was obtained from all study participants or their parents or guardians.\",\n \"Lymphoprep (Axis-shield, Oslo, Norway) + Plasmodipur (Euro-diagnostica, Arnhem, Netherlands) filtration ('LP') was performed as previously described [3]. CF11 columns were loosely filled to the 10-ml mark, packed to the 5.5-ml mark with CF11 cellulose powder (Whatman, Kent, UK), and wetted with isotonic phosphate buffered saline (PBS), as described [10]. Initial tests indicated that plasma separation prior to CF11 filtration of blood did not affect human DNA depletion and that washing with PBS after CF11 filtration improved recovery of parasite DNA. CF11 filtration was therefore carried out as follows. Parasitized blood or blood/parasite mixtures were diluted with an equal volume of PBS. These samples were added to the CF11 column and allowed to flow through by gravity. To wash the sample, 5 ml PBS was added to the column and allowed to pass through by gravity. A plunger was then inserted into the top of the column and the final few drops pushed through the column. The filtrates were centrifuged for 10 minutes at 1,000 x g and the supernatants removed. RBC pellets from all filtrates in all experiments were stored at -20°C until DNA extraction. A packed CF11 column and filtration of blood diluted with PBS are shown in Figures 1A and 1B. The full CF11 filtration procedure is available on the WWARN website [12].\\nPacked CF11 column ready for storage, shipping or use (A) and filtration of blood-PBS mixture through CF11 column into collection tube (B).\",\n \" CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\",\n \"To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\",\n \" CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\\n CF11 filtration, with procedural modifications To test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\\nTo test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\\n Modifications in blood volume and parasite density To investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\\nTo investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\\n Routine blood collection for further validation and sequencing To determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\\nTo determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\",\n \"To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\",\n \"To test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\",\n \"To investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\",\n \"To determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\",\n \"DNA was extracted from leukocyte-depleted and unfiltered control samples using Qiamp DNA Blood Midi Kits (Qiagen, Venlo, Netherlands) according to the manufacturer's protocol. Total DNA from experiments using P. falciparum clones was estimated with a SpectraMax M2 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) using the Quant-IT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). The standard curve ranged from 0.20 to 50 ng/μl. Total DNA from experiments using P. falciparum isolates was estimated with a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) using both the dsDNA HS and BR Assay kits.\\nQuantitative real-time PCR (qPCR) was used to measure the relative amounts of human and parasite DNA in each sample. Primers targeting the human LRAP (F: 5'-ACGTTGGATGAATTTTCCACTGGATTCCAT-'3; R: 5'-ACGTTGGATGTGAACCATGCTCCTTGCATC-'3) and TLR9 (F: 5'-ACGTTGGATGCAAAGGGCTGGCTGTTGTAG-'3; R: 5'- ACGTTGGATGTCTACCACGAGCACTCATTC-'3) genes and the P. falciparum AMA-1 gene (F: 5'-ACGTTGGATGGATTCTCTTTCGATTTCTTTC-'3; R: 5'-ACGTTGGATGTGCTACTACTGCTTTGTCCC-'3) were used to amplify DNA from samples, negative controls and standards in duplicate. Amplification occurred in an Applied Biosystems 7300 or StepOne real-time PCR machine. Human placental and P. falciparum (3D7 clone) genomic DNA standards ranged from 0.20 to 50 ng/μl. For each 25 μl reaction, 12.5 μl SYBR Green qPCR Master Mix (Applied Biosystems, Foster City, CA, USA) was mixed with 1.5 μl of each forward and reverse primers at 2 μM concentrations, 7.5-8.5 μl water, and 1-2 μl of template DNA. Reaction conditions were previously reported [3]: an initial denaturing step of 95°C for 10 minutes, five cycles of 94°C for 45 seconds, 56°C for 45 seconds, 72°C for 45 seconds, followed by 30 cycles of 94°C for 45 seconds, 65°C for 45 seconds and 72°C for 45 seconds and one dissociation cycle (conditions vary by real-time PCR instrument). Results were analysed using Applied Biosystems 7300 System SDS or StepOne v2.0 software.\\nqPCR quantification of AMA-1 and TLR9 or LRAP was used to determine the proportions of P. falciparum and human DNA in the sample. Total DNA was estimated by multiplying the concentration given by Spectramax or Qubit by the extracted sample volume.\",\n \"Means, variance, standard errors and statistical comparisons of means were calculated using Stata 11 software. Boxplots indicating medians, interquartile ranges and outliers were also generated using Stata 11.\",\n \" Comparison of unmodified LP and CF11 filtration for leukocyte depletion Filtration of blood-parasite mixtures using unmodified LP and CF11 methods in parallel successfully depleted human DNA to 1% and 6% of total DNA, respectively (Figure 2A), well below the current Illumina sequencing threshold of < 50%. CF11 was more effective than LP in recovering total DNA from blood-parasite mixtures (mean 1.11 μg vs. 0.37 μg; p = 0.03) (Figure 2B). All 15 CF11-filtered parasitized blood samples and 12 of 14 LP-filtered parasitized blood samples yielded < 50% human DNA contamination (Figure 3A). Human DNA contamination was lower for parasitized blood filtered by CF11 than by LP (mean 2.4% vs. 15.9%; p = 0.03) (Figure 3B). Recovery of total DNA was comparably high in both sets of samples (mean 4.64 μg for CF11 vs. 2.86 μg for LP; p = 0.46) One LP-filtered sample failed to produce a human DNA estimate by qPCR and was omitted from the analysis.\\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of blood-parasite mixtures. The volume and parasite density of blood-parasite mixtures were 3 ml and 10,000/μl except where stated otherwise. Three replicates for each filtration method were performed. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column, CF11 O/N = CF11 filtrate held overnight prior to removal of supernatant, CF11 4 ml = filtration using a 4-ml CF11 column, CF11 low vol = filtration of a 1.5-ml blood-parasite mixture containing 10,000 parasites/μl, CF11 low para = filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl, NF = no filtration, NF low para = no filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl. Box plots show median and interquartile range.\\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of Plasmodium falciparum-infected blood samples that vary in parasite density. Light gray shading indicates samples with < 50,000 parasites/μl, medium gray shading indicates samples with 50,000-100,000 parasites/μl, and dark gray shading indicates samples with parasite density > 100,000 parasites/μl. All sample volumes were 4 ml. n = sample size for each method within each range of parasite density. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column. Box plots show median and interquartile range. Whiskers span the range of data points within 1.5 times the interquartile range of the lower and upper quartiles. Outliers are shown as points.\\nFiltration of blood-parasite mixtures using unmodified LP and CF11 methods in parallel successfully depleted human DNA to 1% and 6% of total DNA, respectively (Figure 2A), well below the current Illumina sequencing threshold of < 50%. CF11 was more effective than LP in recovering total DNA from blood-parasite mixtures (mean 1.11 μg vs. 0.37 μg; p = 0.03) (Figure 2B). All 15 CF11-filtered parasitized blood samples and 12 of 14 LP-filtered parasitized blood samples yielded < 50% human DNA contamination (Figure 3A). Human DNA contamination was lower for parasitized blood filtered by CF11 than by LP (mean 2.4% vs. 15.9%; p = 0.03) (Figure 3B). Recovery of total DNA was comparably high in both sets of samples (mean 4.64 μg for CF11 vs. 2.86 μg for LP; p = 0.46) One LP-filtered sample failed to produce a human DNA estimate by qPCR and was omitted from the analysis.\\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of blood-parasite mixtures. The volume and parasite density of blood-parasite mixtures were 3 ml and 10,000/μl except where stated otherwise. Three replicates for each filtration method were performed. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column, CF11 O/N = CF11 filtrate held overnight prior to removal of supernatant, CF11 4 ml = filtration using a 4-ml CF11 column, CF11 low vol = filtration of a 1.5-ml blood-parasite mixture containing 10,000 parasites/μl, CF11 low para = filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl, NF = no filtration, NF low para = no filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl. Box plots show median and interquartile range.\\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of Plasmodium falciparum-infected blood samples that vary in parasite density. Light gray shading indicates samples with < 50,000 parasites/μl, medium gray shading indicates samples with 50,000-100,000 parasites/μl, and dark gray shading indicates samples with parasite density > 100,000 parasites/μl. All sample volumes were 4 ml. n = sample size for each method within each range of parasite density. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column. Box plots show median and interquartile range. Whiskers span the range of data points within 1.5 times the interquartile range of the lower and upper quartiles. Outliers are shown as points.\\n Procedural modifications of CF11 filtration method To explore whether CF11 filtrates could be processed without centrifugation, CF11-filtered blood-parasite mixtures were refrigerated overnight. The RBC pellet formed during overnight settling was not as tightly packed as that formed during centrifugation and required careful handling when removing supernatant to prevent disruption. This modification resulted in adequate human DNA depletion (mean contamination 11%) (Figure 2A) and total DNA recovery (mean 1.22 μg) (Figure 2B). Columns with less CF11 powder, packed to the 4-ml mark rather than to the 5.5-ml mark, also yielded high total DNA (mean 1.56 μg) and a sufficiently low proportion of human DNA (mean 30%) (Figure 2A and 2B).\\nTo explore whether CF11 filtrates could be processed without centrifugation, CF11-filtered blood-parasite mixtures were refrigerated overnight. The RBC pellet formed during overnight settling was not as tightly packed as that formed during centrifugation and required careful handling when removing supernatant to prevent disruption. This modification resulted in adequate human DNA depletion (mean contamination 11%) (Figure 2A) and total DNA recovery (mean 1.22 μg) (Figure 2B). Columns with less CF11 powder, packed to the 4-ml mark rather than to the 5.5-ml mark, also yielded high total DNA (mean 1.56 μg) and a sufficiently low proportion of human DNA (mean 30%) (Figure 2A and 2B).\\n Blood volume and parasite density All CF11-filtered blood-parasite mixtures (3 ml volume, 10,000/μl parasite density) consistently yielded samples with < 30% human DNA contamination and > 500 ng total DNA (Figure 2A and 2B). However, the ex vivo study of P. falciparum isolates with interesting phenotypes can be difficult if blood volumes are limited and parasite densities are low. To determine whether such limitations might significantly reduce the quality of DNA samples, CF11 filtrations were repeated using blood-parasite mixtures of lower volume (1.5 ml) or lower parasite densities (5,000/μl). CF11 filtration of low-volume samples (1.5 ml; 10,000 parasites/μl) produced acceptable human DNA contamination (mean 28%) and mean total DNA (0.51 μg), but individual yields of total DNA were highly variable, ranging from 0.15 to 1.09 μg (Figure 2A and 2B). CF11 filtration of low parasite density samples (3 ml; 5,000 parasites/μl) produced adequate amounts of total DNA (mean 2.68 μg) but unacceptable human DNA contamination (mean 77%) (Figure 2A and 2B). Thus, reducing sample volumes or parasite densities by half failed to reliably produce DNA samples acceptable for Illumina sequencing.\\nFiltration of P. falciparum-infected blood obtained from patients with malaria (Pursat, Cambodia) using the CF11 and LP methods consistently achieved high-quality results at parasite densities ranging from 20,000 to 475,000/μl (Figure 3A and 3B). At parasite densities < 50,000/μl and 50,000-100,000/μl, both filtration methods depleted human DNA contamination to ≤ 30% and produced mean DNA yields > 1 μg. Samples with > 100,000 parasites/μl yielded several micrograms of DNA with < 5% human DNA contamination.\\nAll CF11-filtered blood-parasite mixtures (3 ml volume, 10,000/μl parasite density) consistently yielded samples with < 30% human DNA contamination and > 500 ng total DNA (Figure 2A and 2B). However, the ex vivo study of P. falciparum isolates with interesting phenotypes can be difficult if blood volumes are limited and parasite densities are low. To determine whether such limitations might significantly reduce the quality of DNA samples, CF11 filtrations were repeated using blood-parasite mixtures of lower volume (1.5 ml) or lower parasite densities (5,000/μl). CF11 filtration of low-volume samples (1.5 ml; 10,000 parasites/μl) produced acceptable human DNA contamination (mean 28%) and mean total DNA (0.51 μg), but individual yields of total DNA were highly variable, ranging from 0.15 to 1.09 μg (Figure 2A and 2B). CF11 filtration of low parasite density samples (3 ml; 5,000 parasites/μl) produced adequate amounts of total DNA (mean 2.68 μg) but unacceptable human DNA contamination (mean 77%) (Figure 2A and 2B). Thus, reducing sample volumes or parasite densities by half failed to reliably produce DNA samples acceptable for Illumina sequencing.\\nFiltration of P. falciparum-infected blood obtained from patients with malaria (Pursat, Cambodia) using the CF11 and LP methods consistently achieved high-quality results at parasite densities ranging from 20,000 to 475,000/μl (Figure 3A and 3B). At parasite densities < 50,000/μl and 50,000-100,000/μl, both filtration methods depleted human DNA contamination to ≤ 30% and produced mean DNA yields > 1 μg. Samples with > 100,000 parasites/μl yielded several micrograms of DNA with < 5% human DNA contamination.\\n Validation of routine blood collection for illumina sequencing The 51 parasitized blood samples that were collected and CF11-filtered in Ratanakiri, Cambodia, ranged in parasite density from 15,000 to 290,000/μl and yielded a mean 0.64 μg total DNA with 34% human DNA contamination. All 51 samples met quality standards and have been successfully sequenced on the Illumina platform at the Wellcome Trust Sanger Institute (Hinxton, UK).\\nThe 51 parasitized blood samples that were collected and CF11-filtered in Ratanakiri, Cambodia, ranged in parasite density from 15,000 to 290,000/μl and yielded a mean 0.64 μg total DNA with 34% human DNA contamination. All 51 samples met quality standards and have been successfully sequenced on the Illumina platform at the Wellcome Trust Sanger Institute (Hinxton, UK).\",\n \"Filtration of blood-parasite mixtures using unmodified LP and CF11 methods in parallel successfully depleted human DNA to 1% and 6% of total DNA, respectively (Figure 2A), well below the current Illumina sequencing threshold of < 50%. CF11 was more effective than LP in recovering total DNA from blood-parasite mixtures (mean 1.11 μg vs. 0.37 μg; p = 0.03) (Figure 2B). All 15 CF11-filtered parasitized blood samples and 12 of 14 LP-filtered parasitized blood samples yielded < 50% human DNA contamination (Figure 3A). Human DNA contamination was lower for parasitized blood filtered by CF11 than by LP (mean 2.4% vs. 15.9%; p = 0.03) (Figure 3B). Recovery of total DNA was comparably high in both sets of samples (mean 4.64 μg for CF11 vs. 2.86 μg for LP; p = 0.46) One LP-filtered sample failed to produce a human DNA estimate by qPCR and was omitted from the analysis.\\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of blood-parasite mixtures. The volume and parasite density of blood-parasite mixtures were 3 ml and 10,000/μl except where stated otherwise. Three replicates for each filtration method were performed. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column, CF11 O/N = CF11 filtrate held overnight prior to removal of supernatant, CF11 4 ml = filtration using a 4-ml CF11 column, CF11 low vol = filtration of a 1.5-ml blood-parasite mixture containing 10,000 parasites/μl, CF11 low para = filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl, NF = no filtration, NF low para = no filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl. Box plots show median and interquartile range.\\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of Plasmodium falciparum-infected blood samples that vary in parasite density. Light gray shading indicates samples with < 50,000 parasites/μl, medium gray shading indicates samples with 50,000-100,000 parasites/μl, and dark gray shading indicates samples with parasite density > 100,000 parasites/μl. All sample volumes were 4 ml. n = sample size for each method within each range of parasite density. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column. Box plots show median and interquartile range. Whiskers span the range of data points within 1.5 times the interquartile range of the lower and upper quartiles. Outliers are shown as points.\",\n \"To explore whether CF11 filtrates could be processed without centrifugation, CF11-filtered blood-parasite mixtures were refrigerated overnight. The RBC pellet formed during overnight settling was not as tightly packed as that formed during centrifugation and required careful handling when removing supernatant to prevent disruption. This modification resulted in adequate human DNA depletion (mean contamination 11%) (Figure 2A) and total DNA recovery (mean 1.22 μg) (Figure 2B). Columns with less CF11 powder, packed to the 4-ml mark rather than to the 5.5-ml mark, also yielded high total DNA (mean 1.56 μg) and a sufficiently low proportion of human DNA (mean 30%) (Figure 2A and 2B).\",\n \"All CF11-filtered blood-parasite mixtures (3 ml volume, 10,000/μl parasite density) consistently yielded samples with < 30% human DNA contamination and > 500 ng total DNA (Figure 2A and 2B). However, the ex vivo study of P. falciparum isolates with interesting phenotypes can be difficult if blood volumes are limited and parasite densities are low. To determine whether such limitations might significantly reduce the quality of DNA samples, CF11 filtrations were repeated using blood-parasite mixtures of lower volume (1.5 ml) or lower parasite densities (5,000/μl). CF11 filtration of low-volume samples (1.5 ml; 10,000 parasites/μl) produced acceptable human DNA contamination (mean 28%) and mean total DNA (0.51 μg), but individual yields of total DNA were highly variable, ranging from 0.15 to 1.09 μg (Figure 2A and 2B). CF11 filtration of low parasite density samples (3 ml; 5,000 parasites/μl) produced adequate amounts of total DNA (mean 2.68 μg) but unacceptable human DNA contamination (mean 77%) (Figure 2A and 2B). Thus, reducing sample volumes or parasite densities by half failed to reliably produce DNA samples acceptable for Illumina sequencing.\\nFiltration of P. falciparum-infected blood obtained from patients with malaria (Pursat, Cambodia) using the CF11 and LP methods consistently achieved high-quality results at parasite densities ranging from 20,000 to 475,000/μl (Figure 3A and 3B). At parasite densities < 50,000/μl and 50,000-100,000/μl, both filtration methods depleted human DNA contamination to ≤ 30% and produced mean DNA yields > 1 μg. Samples with > 100,000 parasites/μl yielded several micrograms of DNA with < 5% human DNA contamination.\",\n \"The 51 parasitized blood samples that were collected and CF11-filtered in Ratanakiri, Cambodia, ranged in parasite density from 15,000 to 290,000/μl and yielded a mean 0.64 μg total DNA with 34% human DNA contamination. All 51 samples met quality standards and have been successfully sequenced on the Illumina platform at the Wellcome Trust Sanger Institute (Hinxton, UK).\",\n \"As next-generation sequencing technologies improve, both costs and stringency of DNA sample quality requirements continue to fall. Between 2009 and 2011, requirements for Illumina sequencing of P. falciparum DNA by the Wellcome Trust Sanger Institute have relaxed from 30% to 50% human DNA contamination. Moreover, the possibility of sequencing multiple DNA samples in a single lane of a flow cell ('multiplex sequencing') has decreased costs immensely while yielding ~2 GB of P. falciparum genomic sequence data per sample, with further improvements anticipated in coming years. To take advantage of these increasingly accessible tools, researchers need scalable procedures for DNA sample preparation in minimally equipped field sites.\\nThis study shows that CF11 columns can be used to effectively deplete human DNA from parasitized blood and achieve Illumina sequencing requirements for total DNA yield and percent human DNA contamination, using sample volumes and parasite densities comparable to those used in LP filtration experiments [3]. A major advantage of CF11 over LP filtration is cost-effectiveness. CF11 columns cost approximately one USD each, compared to 10-50 USD for the Plasmodipur filter alone. Additionally, CF11 filtration is a very simple one-step procedure that does not require specialized equipment. A centrifugation step is only used to pellet RBCs after CF11 filtration and can be replaced with a convenient overnight period of refrigeration to allow the RBCs to pellet by gravity. These advantages should enable a large share of the malaria research community to produce parasite DNA samples appropriate for highly parallel sequencing and microarray technologies.\\nBecause CF11 columns are hand-made rather than commercially produced, variation in the length of the column is likely to occur. Samples filtered in columns packed with less CF11 powder exhibited higher human DNA contamination but still met the threshold level for sequencing, indicating that some variation in column preparation can be tolerated. Samples with low blood volumes (1.5 ml) or parasite densities (5,000/μl) did not consistently achieve sufficient human DNA depletion or total DNA recovery when filtered with either the CF11 or LP method. Whole-genome amplification (WGA) after DNA isolation [13] could boost the amount of starting material in cases where human DNA removal is satisfactory but total DNA yield is low. WGA may therefore be useful in enabling genomic characterization of CF11-filtered blood samples with low starting volume, such as those collected from very young children and patients with severe malarial anaemia. It may also be useful in processing blood samples with low parasite densities, as is often the case in patients with a recrudescent parasitaemia after anti-malarial treatment and those with high levels of naturally acquired immunity.\\nAlthough specific parameters have not been rigorously tested, observations in the field have indicated that CF11 and parasitized blood can be effectively stored and used in a variety of environmental conditions. CF11 kept in an airtight container with regularly replaced desiccant and stored in a cool place can be used for at least three months, even in climates with high heat and humidity (unpublished observation). EDTA-treated blood stored at 4°C for up to 24 hours prior to CF11 filtration was successfully processed for Illumina DNA sequencing (unpublished observation), but for RNA analysis, no more than six hours of refrigeration is suggested (ZB Bozdech, personal communication). Updates to the range of storage conditions will be made to the WWARN protocol [12] as more information becomes available.\",\n \"CF11 filtration currently offers the best cost-effective, one-step approach to remove human DNA from P. falciparum-infected whole blood samples and can be successfully implemented in large genome-wide sequencing studies of P. falciparum isolates from patients with malaria.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":[null,"methods",null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"keywords":{"kind":"list like","value":["CF11","Cellulose powder","Leukocyte depletion","\nPlasmodium falciparum\n","Malaria","Next-generation sequencing"],"string":"[\n \"CF11\",\n \"Cellulose powder\",\n \"Leukocyte depletion\",\n \"\\nPlasmodium falciparum\\n\",\n \"Malaria\",\n \"Next-generation sequencing\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Background:\nRecent technological advances have enabled next-generation genomic and transcriptomic analysis of Plasmodium falciparum from parasitized whole blood samples without the need for culturing. High-density genotyping of parasites obtained directly from patients with malaria has improved our understanding of population structure and genomic and transcriptional variation [1,2]. Highly parallel sequencing is currently being used to identify the genetic determinants of clinically relevant phenotypes including drug resistance, vaccine escape and disease severity. Importantly, genomic characterization of parasite populations in patients captures intra-host diversity and prevents the potential loss of sequence data from phenotype-conferring parasite isolates during their culture adaptation.\nThe performance of highly parallel sequencing platforms, such as Illumina, is greatly enhanced in sample preparations with a high parasite-to-human nucleic acid ratio [3]. Such high ratios can be achieved by either selectively capturing parasite nucleic acids or by removing human material from the sample. Hybrid selection [4] using RNA 'baits' complementary to the P. falciparum genome can achieve over 40-fold enrichment of parasite DNA and can be performed at any time following DNA extraction [5]; however, at $250 USD per sample, this technique may be prohibitively costly for epidemiological or population-level studies.\nThe alternative, leukocyte depletion, must be performed in field sites soon after blood collection and before transport and storage. Commercially available magnetic columns have been used to rapidly isolate parasitized red blood cells (RBCs) from uninfected RBCs and leukocytes [6]. However, magnetic purification depends on short-term culturing of patient blood to transform ring-stage parasites to haemozoin-rich trophozoites and schizonts, which requires equipment for in vitro parasite cultivation. Furthermore, parasites obtained directly from patients mature at different rates in culture, resulting in inconvenient time-points for purification. The current standard for leukocyte depletion of parasitized blood for P. falciparum nucleotide sequencing is a two-step process: centrifugation using Lymphoprep or another sucrose density gradient solution, followed by filtration using Plasmodipur filters [3]. This 'LP' method is effective but difficult to scale-up in field settings because it requires extensive handling and transfer of blood, training to perform sensitive steps, and costly commercial reagents and materials.\nFiltration with hand-made columns has been used as an inexpensive and less time-consuming alternative for leukocyte depletion of Plasmodium-infected blood for decades [7,8]. Recently, non-woven fabric filters [9] and plastic syringes filled with CF11 cellulose powder (Whatman) [10] were shown to effectively remove leukocytes and platelets from Plasmodium vivax-infected blood, where their phagocytic and degranulating activities may interfere with some ex vivo studies. CF11 filtration has also been used to improve microarray-based transcriptome analysis of P. vivax-infected blood [2,11]. CF11 cellulose is thought to work by trapping leukocytes by size exclusion and/or interactions between cellulose hydroxyl groups and leukocyte surface molecules. In this study, laboratory-adapted P. falciparum clones were used to adapt CF11 columns to remove human DNA from P. falciparum-infected blood for genomic studies, compare CF11 filtration to the established LP method, and validate a centrifuge-free option for CF11 filtration. Both methods were also compared in a field setting using P. falciparum isolates obtained directly from patients with malaria. Furthermore, routine CF11 filtration of parasitized blood collected from patients in a second field site with minimal facilities was successfully implemented.\n\nMethods:\n Blood samples Experiments using laboratory-adapted P. falciparum clones were conducted at the University of Maryland, Baltimore. Blood obtained from healthy volunteers was mixed with purified P. falciparum parasites (3D7 or Dd2 clones) to achieve parasite densities of 10,000/μl and 5,000/μl. These samples were stored at 4°C for up to two hours prior to CF11 or LP filtration.\nExperiments using non-adapted P. falciparum isolates were conducted in August 2010 at Sampov Meas Referral Hospital, Pursat, Cambodia. Blood samples for these experiments were collected from patients with falciparum malaria who were ≥ 10 years old and had a wide range of parasite densities (≥ 10,000/μl). These samples were held at 4°C for up to six hours prior to CF11 or LP filtration. Additional blood samples were collected in November to December 2010 at Lumphat District Health Centre, Ratanakiri, Cambodia. Blood samples were obtained from patients with falciparum malaria who were ≥ 1 year old and had a wide range of parasite densities ≥ 10,000/μl. These samples were held at 4°C for up to six hours prior to CF11 filtration. Following CF11 filtration, samples were stored on ice for up to six hours and transported on ice for up to 12 hours to Phnom Penh for centrifugation and RBC pellet storage.\nAll blood samples were collected in EDTA or CPDA-1 tubes. Heparin was not used as an anticoagulant as it thought to inhibit Taq polymerase during DNA amplification.\nBlood samples from healthy volunteers were collected under a protocol approved by the Institutional Review Board of the University of Maryland School of Medicine. Blood samples from patients with falciparum malaria were collected under protocols approved by the Institutional Review Board of the National Institute for Allergy and Infectious Diseases and the Cambodian National Ethics Committee for Health Research (http://ClinicalTrials.gov identifiers: NCT00341003 and NCT01240603). Written, informed consent was obtained from all study participants or their parents or guardians.\nExperiments using laboratory-adapted P. falciparum clones were conducted at the University of Maryland, Baltimore. Blood obtained from healthy volunteers was mixed with purified P. falciparum parasites (3D7 or Dd2 clones) to achieve parasite densities of 10,000/μl and 5,000/μl. These samples were stored at 4°C for up to two hours prior to CF11 or LP filtration.\nExperiments using non-adapted P. falciparum isolates were conducted in August 2010 at Sampov Meas Referral Hospital, Pursat, Cambodia. Blood samples for these experiments were collected from patients with falciparum malaria who were ≥ 10 years old and had a wide range of parasite densities (≥ 10,000/μl). These samples were held at 4°C for up to six hours prior to CF11 or LP filtration. Additional blood samples were collected in November to December 2010 at Lumphat District Health Centre, Ratanakiri, Cambodia. Blood samples were obtained from patients with falciparum malaria who were ≥ 1 year old and had a wide range of parasite densities ≥ 10,000/μl. These samples were held at 4°C for up to six hours prior to CF11 filtration. Following CF11 filtration, samples were stored on ice for up to six hours and transported on ice for up to 12 hours to Phnom Penh for centrifugation and RBC pellet storage.\nAll blood samples were collected in EDTA or CPDA-1 tubes. Heparin was not used as an anticoagulant as it thought to inhibit Taq polymerase during DNA amplification.\nBlood samples from healthy volunteers were collected under a protocol approved by the Institutional Review Board of the University of Maryland School of Medicine. Blood samples from patients with falciparum malaria were collected under protocols approved by the Institutional Review Board of the National Institute for Allergy and Infectious Diseases and the Cambodian National Ethics Committee for Health Research (http://ClinicalTrials.gov identifiers: NCT00341003 and NCT01240603). Written, informed consent was obtained from all study participants or their parents or guardians.\n Leukocyte depletion Lymphoprep (Axis-shield, Oslo, Norway) + Plasmodipur (Euro-diagnostica, Arnhem, Netherlands) filtration ('LP') was performed as previously described [3]. CF11 columns were loosely filled to the 10-ml mark, packed to the 5.5-ml mark with CF11 cellulose powder (Whatman, Kent, UK), and wetted with isotonic phosphate buffered saline (PBS), as described [10]. Initial tests indicated that plasma separation prior to CF11 filtration of blood did not affect human DNA depletion and that washing with PBS after CF11 filtration improved recovery of parasite DNA. CF11 filtration was therefore carried out as follows. Parasitized blood or blood/parasite mixtures were diluted with an equal volume of PBS. These samples were added to the CF11 column and allowed to flow through by gravity. To wash the sample, 5 ml PBS was added to the column and allowed to pass through by gravity. A plunger was then inserted into the top of the column and the final few drops pushed through the column. The filtrates were centrifuged for 10 minutes at 1,000 x g and the supernatants removed. RBC pellets from all filtrates in all experiments were stored at -20°C until DNA extraction. A packed CF11 column and filtration of blood diluted with PBS are shown in Figures 1A and 1B. The full CF11 filtration procedure is available on the WWARN website [12].\nPacked CF11 column ready for storage, shipping or use (A) and filtration of blood-PBS mixture through CF11 column into collection tube (B).\nLymphoprep (Axis-shield, Oslo, Norway) + Plasmodipur (Euro-diagnostica, Arnhem, Netherlands) filtration ('LP') was performed as previously described [3]. CF11 columns were loosely filled to the 10-ml mark, packed to the 5.5-ml mark with CF11 cellulose powder (Whatman, Kent, UK), and wetted with isotonic phosphate buffered saline (PBS), as described [10]. Initial tests indicated that plasma separation prior to CF11 filtration of blood did not affect human DNA depletion and that washing with PBS after CF11 filtration improved recovery of parasite DNA. CF11 filtration was therefore carried out as follows. Parasitized blood or blood/parasite mixtures were diluted with an equal volume of PBS. These samples were added to the CF11 column and allowed to flow through by gravity. To wash the sample, 5 ml PBS was added to the column and allowed to pass through by gravity. A plunger was then inserted into the top of the column and the final few drops pushed through the column. The filtrates were centrifuged for 10 minutes at 1,000 x g and the supernatants removed. RBC pellets from all filtrates in all experiments were stored at -20°C until DNA extraction. A packed CF11 column and filtration of blood diluted with PBS are shown in Figures 1A and 1B. The full CF11 filtration procedure is available on the WWARN website [12].\nPacked CF11 column ready for storage, shipping or use (A) and filtration of blood-PBS mixture through CF11 column into collection tube (B).\n Experimental design CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\n CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\n Experimental design CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\n CF11 filtration, with procedural modifications To test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\nTo test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\n Modifications in blood volume and parasite density To investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\nTo investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\n Routine blood collection for further validation and sequencing To determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\nTo determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\n CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\n CF11 filtration, with procedural modifications To test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\nTo test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\n Modifications in blood volume and parasite density To investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\nTo investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\n Routine blood collection for further validation and sequencing To determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\nTo determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\n DNA quantification DNA was extracted from leukocyte-depleted and unfiltered control samples using Qiamp DNA Blood Midi Kits (Qiagen, Venlo, Netherlands) according to the manufacturer's protocol. Total DNA from experiments using P. falciparum clones was estimated with a SpectraMax M2 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) using the Quant-IT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). The standard curve ranged from 0.20 to 50 ng/μl. Total DNA from experiments using P. falciparum isolates was estimated with a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) using both the dsDNA HS and BR Assay kits.\nQuantitative real-time PCR (qPCR) was used to measure the relative amounts of human and parasite DNA in each sample. Primers targeting the human LRAP (F: 5'-ACGTTGGATGAATTTTCCACTGGATTCCAT-'3; R: 5'-ACGTTGGATGTGAACCATGCTCCTTGCATC-'3) and TLR9 (F: 5'-ACGTTGGATGCAAAGGGCTGGCTGTTGTAG-'3; R: 5'- ACGTTGGATGTCTACCACGAGCACTCATTC-'3) genes and the P. falciparum AMA-1 gene (F: 5'-ACGTTGGATGGATTCTCTTTCGATTTCTTTC-'3; R: 5'-ACGTTGGATGTGCTACTACTGCTTTGTCCC-'3) were used to amplify DNA from samples, negative controls and standards in duplicate. Amplification occurred in an Applied Biosystems 7300 or StepOne real-time PCR machine. Human placental and P. falciparum (3D7 clone) genomic DNA standards ranged from 0.20 to 50 ng/μl. For each 25 μl reaction, 12.5 μl SYBR Green qPCR Master Mix (Applied Biosystems, Foster City, CA, USA) was mixed with 1.5 μl of each forward and reverse primers at 2 μM concentrations, 7.5-8.5 μl water, and 1-2 μl of template DNA. Reaction conditions were previously reported [3]: an initial denaturing step of 95°C for 10 minutes, five cycles of 94°C for 45 seconds, 56°C for 45 seconds, 72°C for 45 seconds, followed by 30 cycles of 94°C for 45 seconds, 65°C for 45 seconds and 72°C for 45 seconds and one dissociation cycle (conditions vary by real-time PCR instrument). Results were analysed using Applied Biosystems 7300 System SDS or StepOne v2.0 software.\nqPCR quantification of AMA-1 and TLR9 or LRAP was used to determine the proportions of P. falciparum and human DNA in the sample. Total DNA was estimated by multiplying the concentration given by Spectramax or Qubit by the extracted sample volume.\nDNA was extracted from leukocyte-depleted and unfiltered control samples using Qiamp DNA Blood Midi Kits (Qiagen, Venlo, Netherlands) according to the manufacturer's protocol. Total DNA from experiments using P. falciparum clones was estimated with a SpectraMax M2 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) using the Quant-IT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). The standard curve ranged from 0.20 to 50 ng/μl. Total DNA from experiments using P. falciparum isolates was estimated with a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) using both the dsDNA HS and BR Assay kits.\nQuantitative real-time PCR (qPCR) was used to measure the relative amounts of human and parasite DNA in each sample. Primers targeting the human LRAP (F: 5'-ACGTTGGATGAATTTTCCACTGGATTCCAT-'3; R: 5'-ACGTTGGATGTGAACCATGCTCCTTGCATC-'3) and TLR9 (F: 5'-ACGTTGGATGCAAAGGGCTGGCTGTTGTAG-'3; R: 5'- ACGTTGGATGTCTACCACGAGCACTCATTC-'3) genes and the P. falciparum AMA-1 gene (F: 5'-ACGTTGGATGGATTCTCTTTCGATTTCTTTC-'3; R: 5'-ACGTTGGATGTGCTACTACTGCTTTGTCCC-'3) were used to amplify DNA from samples, negative controls and standards in duplicate. Amplification occurred in an Applied Biosystems 7300 or StepOne real-time PCR machine. Human placental and P. falciparum (3D7 clone) genomic DNA standards ranged from 0.20 to 50 ng/μl. For each 25 μl reaction, 12.5 μl SYBR Green qPCR Master Mix (Applied Biosystems, Foster City, CA, USA) was mixed with 1.5 μl of each forward and reverse primers at 2 μM concentrations, 7.5-8.5 μl water, and 1-2 μl of template DNA. Reaction conditions were previously reported [3]: an initial denaturing step of 95°C for 10 minutes, five cycles of 94°C for 45 seconds, 56°C for 45 seconds, 72°C for 45 seconds, followed by 30 cycles of 94°C for 45 seconds, 65°C for 45 seconds and 72°C for 45 seconds and one dissociation cycle (conditions vary by real-time PCR instrument). Results were analysed using Applied Biosystems 7300 System SDS or StepOne v2.0 software.\nqPCR quantification of AMA-1 and TLR9 or LRAP was used to determine the proportions of P. falciparum and human DNA in the sample. Total DNA was estimated by multiplying the concentration given by Spectramax or Qubit by the extracted sample volume.\n Statistical comparisons Means, variance, standard errors and statistical comparisons of means were calculated using Stata 11 software. Boxplots indicating medians, interquartile ranges and outliers were also generated using Stata 11.\nMeans, variance, standard errors and statistical comparisons of means were calculated using Stata 11 software. Boxplots indicating medians, interquartile ranges and outliers were also generated using Stata 11.\n\nBlood samples:\nExperiments using laboratory-adapted P. falciparum clones were conducted at the University of Maryland, Baltimore. Blood obtained from healthy volunteers was mixed with purified P. falciparum parasites (3D7 or Dd2 clones) to achieve parasite densities of 10,000/μl and 5,000/μl. These samples were stored at 4°C for up to two hours prior to CF11 or LP filtration.\nExperiments using non-adapted P. falciparum isolates were conducted in August 2010 at Sampov Meas Referral Hospital, Pursat, Cambodia. Blood samples for these experiments were collected from patients with falciparum malaria who were ≥ 10 years old and had a wide range of parasite densities (≥ 10,000/μl). These samples were held at 4°C for up to six hours prior to CF11 or LP filtration. Additional blood samples were collected in November to December 2010 at Lumphat District Health Centre, Ratanakiri, Cambodia. Blood samples were obtained from patients with falciparum malaria who were ≥ 1 year old and had a wide range of parasite densities ≥ 10,000/μl. These samples were held at 4°C for up to six hours prior to CF11 filtration. Following CF11 filtration, samples were stored on ice for up to six hours and transported on ice for up to 12 hours to Phnom Penh for centrifugation and RBC pellet storage.\nAll blood samples were collected in EDTA or CPDA-1 tubes. Heparin was not used as an anticoagulant as it thought to inhibit Taq polymerase during DNA amplification.\nBlood samples from healthy volunteers were collected under a protocol approved by the Institutional Review Board of the University of Maryland School of Medicine. Blood samples from patients with falciparum malaria were collected under protocols approved by the Institutional Review Board of the National Institute for Allergy and Infectious Diseases and the Cambodian National Ethics Committee for Health Research (http://ClinicalTrials.gov identifiers: NCT00341003 and NCT01240603). Written, informed consent was obtained from all study participants or their parents or guardians.\n\nLeukocyte depletion:\nLymphoprep (Axis-shield, Oslo, Norway) + Plasmodipur (Euro-diagnostica, Arnhem, Netherlands) filtration ('LP') was performed as previously described [3]. CF11 columns were loosely filled to the 10-ml mark, packed to the 5.5-ml mark with CF11 cellulose powder (Whatman, Kent, UK), and wetted with isotonic phosphate buffered saline (PBS), as described [10]. Initial tests indicated that plasma separation prior to CF11 filtration of blood did not affect human DNA depletion and that washing with PBS after CF11 filtration improved recovery of parasite DNA. CF11 filtration was therefore carried out as follows. Parasitized blood or blood/parasite mixtures were diluted with an equal volume of PBS. These samples were added to the CF11 column and allowed to flow through by gravity. To wash the sample, 5 ml PBS was added to the column and allowed to pass through by gravity. A plunger was then inserted into the top of the column and the final few drops pushed through the column. The filtrates were centrifuged for 10 minutes at 1,000 x g and the supernatants removed. RBC pellets from all filtrates in all experiments were stored at -20°C until DNA extraction. A packed CF11 column and filtration of blood diluted with PBS are shown in Figures 1A and 1B. The full CF11 filtration procedure is available on the WWARN website [12].\nPacked CF11 column ready for storage, shipping or use (A) and filtration of blood-PBS mixture through CF11 column into collection tube (B).\n\nExperimental design:\n CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\n\nCF11 versus LP filtration, without modifications:\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method\n\nExperimental design:\n CF11 versus LP filtration, without modifications To compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\n CF11 filtration, with procedural modifications To test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\nTo test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\n Modifications in blood volume and parasite density To investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\nTo investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\n Routine blood collection for further validation and sequencing To determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\nTo determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\n\nCF11 versus LP filtration, without modifications:\nTo compare the CF11 and LP methods for leukocyte depletion in a laboratory setting, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared and filtered in parallel using each method. Unfiltered blood-parasite mixtures were prepared as control samples. To compare the two methods in a field setting (Pursat, Cambodia), 8 ml blood from 15 patients with falciparum malaria were obtained and split into two 4-ml aliquots. Each aliquot was processed by either the CF11 or LP method.\n\nCF11 filtration, with procedural modifications:\nTo test a centrifuge-free method of CF11 filtration, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were prepared, filtered through CF11 columns, and held overnight at 4°C to allow RBCs pellet by gravity. Supernatants were removed the following day. To investigate the effect of decreased CF11 column length on leukocyte depletion, three replicates of blood-parasite mixtures (3-ml volume; 10,000 parasites/μl) were filtered through columns loosely filled with CF11 to the 8-ml mark and packed to the 4-ml mark.\n\nModifications in blood volume and parasite density:\nTo investigate the effect of low sample volume on CF11 filtration, three replicates of 1.5-ml blood-parasite mixtures containing 10,000 parasites/μl were prepared and filtered in parallel through CF11 columns loosely filled to the 5-ml mark and packed to the 2.5-ml mark. To investigate the effect of low parasite density on CF11 filtration, three replicates of 3-ml blood-parasite mixtures containing 5,000 parasites/μl were prepared and filtered in parallel through CF11 columns packed to the 5.5-ml mark. In both experiments, unfiltered blood-parasite mixtures were prepared as control samples. The effect of parasite density on CF11 and LP filtration was tested in Pursat, Cambodia, using P. falciparum isolates obtained from patients with malaria who had parasite densities ranging from 20,000-475,000/μl.\n\nRoutine blood collection for further validation and sequencing:\nTo determine how CF11 filtration performed during a routine blood collection protocol, 3 ml blood were obtained from 51 patients presenting with falciparum malaria in Ratanakiri, Cambodia. Blood samples were passed through CF11 columns and the filtrates were transported on ice to the laboratory in Phnom Penh, where the samples were centrifuged to obtain RBC pellets.\n\nDNA quantification:\nDNA was extracted from leukocyte-depleted and unfiltered control samples using Qiamp DNA Blood Midi Kits (Qiagen, Venlo, Netherlands) according to the manufacturer's protocol. Total DNA from experiments using P. falciparum clones was estimated with a SpectraMax M2 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) using the Quant-IT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). The standard curve ranged from 0.20 to 50 ng/μl. Total DNA from experiments using P. falciparum isolates was estimated with a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) using both the dsDNA HS and BR Assay kits.\nQuantitative real-time PCR (qPCR) was used to measure the relative amounts of human and parasite DNA in each sample. Primers targeting the human LRAP (F: 5'-ACGTTGGATGAATTTTCCACTGGATTCCAT-'3; R: 5'-ACGTTGGATGTGAACCATGCTCCTTGCATC-'3) and TLR9 (F: 5'-ACGTTGGATGCAAAGGGCTGGCTGTTGTAG-'3; R: 5'- ACGTTGGATGTCTACCACGAGCACTCATTC-'3) genes and the P. falciparum AMA-1 gene (F: 5'-ACGTTGGATGGATTCTCTTTCGATTTCTTTC-'3; R: 5'-ACGTTGGATGTGCTACTACTGCTTTGTCCC-'3) were used to amplify DNA from samples, negative controls and standards in duplicate. Amplification occurred in an Applied Biosystems 7300 or StepOne real-time PCR machine. Human placental and P. falciparum (3D7 clone) genomic DNA standards ranged from 0.20 to 50 ng/μl. For each 25 μl reaction, 12.5 μl SYBR Green qPCR Master Mix (Applied Biosystems, Foster City, CA, USA) was mixed with 1.5 μl of each forward and reverse primers at 2 μM concentrations, 7.5-8.5 μl water, and 1-2 μl of template DNA. Reaction conditions were previously reported [3]: an initial denaturing step of 95°C for 10 minutes, five cycles of 94°C for 45 seconds, 56°C for 45 seconds, 72°C for 45 seconds, followed by 30 cycles of 94°C for 45 seconds, 65°C for 45 seconds and 72°C for 45 seconds and one dissociation cycle (conditions vary by real-time PCR instrument). Results were analysed using Applied Biosystems 7300 System SDS or StepOne v2.0 software.\nqPCR quantification of AMA-1 and TLR9 or LRAP was used to determine the proportions of P. falciparum and human DNA in the sample. Total DNA was estimated by multiplying the concentration given by Spectramax or Qubit by the extracted sample volume.\n\nStatistical comparisons:\nMeans, variance, standard errors and statistical comparisons of means were calculated using Stata 11 software. Boxplots indicating medians, interquartile ranges and outliers were also generated using Stata 11.\n\nResults:\n Comparison of unmodified LP and CF11 filtration for leukocyte depletion Filtration of blood-parasite mixtures using unmodified LP and CF11 methods in parallel successfully depleted human DNA to 1% and 6% of total DNA, respectively (Figure 2A), well below the current Illumina sequencing threshold of < 50%. CF11 was more effective than LP in recovering total DNA from blood-parasite mixtures (mean 1.11 μg vs. 0.37 μg; p = 0.03) (Figure 2B). All 15 CF11-filtered parasitized blood samples and 12 of 14 LP-filtered parasitized blood samples yielded < 50% human DNA contamination (Figure 3A). Human DNA contamination was lower for parasitized blood filtered by CF11 than by LP (mean 2.4% vs. 15.9%; p = 0.03) (Figure 3B). Recovery of total DNA was comparably high in both sets of samples (mean 4.64 μg for CF11 vs. 2.86 μg for LP; p = 0.46) One LP-filtered sample failed to produce a human DNA estimate by qPCR and was omitted from the analysis.\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of blood-parasite mixtures. The volume and parasite density of blood-parasite mixtures were 3 ml and 10,000/μl except where stated otherwise. Three replicates for each filtration method were performed. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column, CF11 O/N = CF11 filtrate held overnight prior to removal of supernatant, CF11 4 ml = filtration using a 4-ml CF11 column, CF11 low vol = filtration of a 1.5-ml blood-parasite mixture containing 10,000 parasites/μl, CF11 low para = filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl, NF = no filtration, NF low para = no filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl. Box plots show median and interquartile range.\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of Plasmodium falciparum-infected blood samples that vary in parasite density. Light gray shading indicates samples with < 50,000 parasites/μl, medium gray shading indicates samples with 50,000-100,000 parasites/μl, and dark gray shading indicates samples with parasite density > 100,000 parasites/μl. All sample volumes were 4 ml. n = sample size for each method within each range of parasite density. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column. Box plots show median and interquartile range. Whiskers span the range of data points within 1.5 times the interquartile range of the lower and upper quartiles. Outliers are shown as points.\nFiltration of blood-parasite mixtures using unmodified LP and CF11 methods in parallel successfully depleted human DNA to 1% and 6% of total DNA, respectively (Figure 2A), well below the current Illumina sequencing threshold of < 50%. CF11 was more effective than LP in recovering total DNA from blood-parasite mixtures (mean 1.11 μg vs. 0.37 μg; p = 0.03) (Figure 2B). All 15 CF11-filtered parasitized blood samples and 12 of 14 LP-filtered parasitized blood samples yielded < 50% human DNA contamination (Figure 3A). Human DNA contamination was lower for parasitized blood filtered by CF11 than by LP (mean 2.4% vs. 15.9%; p = 0.03) (Figure 3B). Recovery of total DNA was comparably high in both sets of samples (mean 4.64 μg for CF11 vs. 2.86 μg for LP; p = 0.46) One LP-filtered sample failed to produce a human DNA estimate by qPCR and was omitted from the analysis.\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of blood-parasite mixtures. The volume and parasite density of blood-parasite mixtures were 3 ml and 10,000/μl except where stated otherwise. Three replicates for each filtration method were performed. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column, CF11 O/N = CF11 filtrate held overnight prior to removal of supernatant, CF11 4 ml = filtration using a 4-ml CF11 column, CF11 low vol = filtration of a 1.5-ml blood-parasite mixture containing 10,000 parasites/μl, CF11 low para = filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl, NF = no filtration, NF low para = no filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl. Box plots show median and interquartile range.\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of Plasmodium falciparum-infected blood samples that vary in parasite density. Light gray shading indicates samples with < 50,000 parasites/μl, medium gray shading indicates samples with 50,000-100,000 parasites/μl, and dark gray shading indicates samples with parasite density > 100,000 parasites/μl. All sample volumes were 4 ml. n = sample size for each method within each range of parasite density. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column. Box plots show median and interquartile range. Whiskers span the range of data points within 1.5 times the interquartile range of the lower and upper quartiles. Outliers are shown as points.\n Procedural modifications of CF11 filtration method To explore whether CF11 filtrates could be processed without centrifugation, CF11-filtered blood-parasite mixtures were refrigerated overnight. The RBC pellet formed during overnight settling was not as tightly packed as that formed during centrifugation and required careful handling when removing supernatant to prevent disruption. This modification resulted in adequate human DNA depletion (mean contamination 11%) (Figure 2A) and total DNA recovery (mean 1.22 μg) (Figure 2B). Columns with less CF11 powder, packed to the 4-ml mark rather than to the 5.5-ml mark, also yielded high total DNA (mean 1.56 μg) and a sufficiently low proportion of human DNA (mean 30%) (Figure 2A and 2B).\nTo explore whether CF11 filtrates could be processed without centrifugation, CF11-filtered blood-parasite mixtures were refrigerated overnight. The RBC pellet formed during overnight settling was not as tightly packed as that formed during centrifugation and required careful handling when removing supernatant to prevent disruption. This modification resulted in adequate human DNA depletion (mean contamination 11%) (Figure 2A) and total DNA recovery (mean 1.22 μg) (Figure 2B). Columns with less CF11 powder, packed to the 4-ml mark rather than to the 5.5-ml mark, also yielded high total DNA (mean 1.56 μg) and a sufficiently low proportion of human DNA (mean 30%) (Figure 2A and 2B).\n Blood volume and parasite density All CF11-filtered blood-parasite mixtures (3 ml volume, 10,000/μl parasite density) consistently yielded samples with < 30% human DNA contamination and > 500 ng total DNA (Figure 2A and 2B). However, the ex vivo study of P. falciparum isolates with interesting phenotypes can be difficult if blood volumes are limited and parasite densities are low. To determine whether such limitations might significantly reduce the quality of DNA samples, CF11 filtrations were repeated using blood-parasite mixtures of lower volume (1.5 ml) or lower parasite densities (5,000/μl). CF11 filtration of low-volume samples (1.5 ml; 10,000 parasites/μl) produced acceptable human DNA contamination (mean 28%) and mean total DNA (0.51 μg), but individual yields of total DNA were highly variable, ranging from 0.15 to 1.09 μg (Figure 2A and 2B). CF11 filtration of low parasite density samples (3 ml; 5,000 parasites/μl) produced adequate amounts of total DNA (mean 2.68 μg) but unacceptable human DNA contamination (mean 77%) (Figure 2A and 2B). Thus, reducing sample volumes or parasite densities by half failed to reliably produce DNA samples acceptable for Illumina sequencing.\nFiltration of P. falciparum-infected blood obtained from patients with malaria (Pursat, Cambodia) using the CF11 and LP methods consistently achieved high-quality results at parasite densities ranging from 20,000 to 475,000/μl (Figure 3A and 3B). At parasite densities < 50,000/μl and 50,000-100,000/μl, both filtration methods depleted human DNA contamination to ≤ 30% and produced mean DNA yields > 1 μg. Samples with > 100,000 parasites/μl yielded several micrograms of DNA with < 5% human DNA contamination.\nAll CF11-filtered blood-parasite mixtures (3 ml volume, 10,000/μl parasite density) consistently yielded samples with < 30% human DNA contamination and > 500 ng total DNA (Figure 2A and 2B). However, the ex vivo study of P. falciparum isolates with interesting phenotypes can be difficult if blood volumes are limited and parasite densities are low. To determine whether such limitations might significantly reduce the quality of DNA samples, CF11 filtrations were repeated using blood-parasite mixtures of lower volume (1.5 ml) or lower parasite densities (5,000/μl). CF11 filtration of low-volume samples (1.5 ml; 10,000 parasites/μl) produced acceptable human DNA contamination (mean 28%) and mean total DNA (0.51 μg), but individual yields of total DNA were highly variable, ranging from 0.15 to 1.09 μg (Figure 2A and 2B). CF11 filtration of low parasite density samples (3 ml; 5,000 parasites/μl) produced adequate amounts of total DNA (mean 2.68 μg) but unacceptable human DNA contamination (mean 77%) (Figure 2A and 2B). Thus, reducing sample volumes or parasite densities by half failed to reliably produce DNA samples acceptable for Illumina sequencing.\nFiltration of P. falciparum-infected blood obtained from patients with malaria (Pursat, Cambodia) using the CF11 and LP methods consistently achieved high-quality results at parasite densities ranging from 20,000 to 475,000/μl (Figure 3A and 3B). At parasite densities < 50,000/μl and 50,000-100,000/μl, both filtration methods depleted human DNA contamination to ≤ 30% and produced mean DNA yields > 1 μg. Samples with > 100,000 parasites/μl yielded several micrograms of DNA with < 5% human DNA contamination.\n Validation of routine blood collection for illumina sequencing The 51 parasitized blood samples that were collected and CF11-filtered in Ratanakiri, Cambodia, ranged in parasite density from 15,000 to 290,000/μl and yielded a mean 0.64 μg total DNA with 34% human DNA contamination. All 51 samples met quality standards and have been successfully sequenced on the Illumina platform at the Wellcome Trust Sanger Institute (Hinxton, UK).\nThe 51 parasitized blood samples that were collected and CF11-filtered in Ratanakiri, Cambodia, ranged in parasite density from 15,000 to 290,000/μl and yielded a mean 0.64 μg total DNA with 34% human DNA contamination. All 51 samples met quality standards and have been successfully sequenced on the Illumina platform at the Wellcome Trust Sanger Institute (Hinxton, UK).\n\nComparison of unmodified LP and CF11 filtration for leukocyte depletion:\nFiltration of blood-parasite mixtures using unmodified LP and CF11 methods in parallel successfully depleted human DNA to 1% and 6% of total DNA, respectively (Figure 2A), well below the current Illumina sequencing threshold of < 50%. CF11 was more effective than LP in recovering total DNA from blood-parasite mixtures (mean 1.11 μg vs. 0.37 μg; p = 0.03) (Figure 2B). All 15 CF11-filtered parasitized blood samples and 12 of 14 LP-filtered parasitized blood samples yielded < 50% human DNA contamination (Figure 3A). Human DNA contamination was lower for parasitized blood filtered by CF11 than by LP (mean 2.4% vs. 15.9%; p = 0.03) (Figure 3B). Recovery of total DNA was comparably high in both sets of samples (mean 4.64 μg for CF11 vs. 2.86 μg for LP; p = 0.46) One LP-filtered sample failed to produce a human DNA estimate by qPCR and was omitted from the analysis.\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of blood-parasite mixtures. The volume and parasite density of blood-parasite mixtures were 3 ml and 10,000/μl except where stated otherwise. Three replicates for each filtration method were performed. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column, CF11 O/N = CF11 filtrate held overnight prior to removal of supernatant, CF11 4 ml = filtration using a 4-ml CF11 column, CF11 low vol = filtration of a 1.5-ml blood-parasite mixture containing 10,000 parasites/μl, CF11 low para = filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl, NF = no filtration, NF low para = no filtration of a 3-ml blood-parasite mixture containing 5,000 parasites/μl. Box plots show median and interquartile range.\nPercent human DNA (A) and total DNA (B) as estimated by qPCR after leukocyte depletion of Plasmodium falciparum-infected blood samples that vary in parasite density. Light gray shading indicates samples with < 50,000 parasites/μl, medium gray shading indicates samples with 50,000-100,000 parasites/μl, and dark gray shading indicates samples with parasite density > 100,000 parasites/μl. All sample volumes were 4 ml. n = sample size for each method within each range of parasite density. Dashed lines indicate current criteria for Illumina sequencing: < 50% human DNA and > 500 ng total DNA. LP = Lymphoprep + Plasmodipur, CF11 = filtration using a 5.5-ml CF11 column. Box plots show median and interquartile range. Whiskers span the range of data points within 1.5 times the interquartile range of the lower and upper quartiles. Outliers are shown as points.\n\nProcedural modifications of CF11 filtration method:\nTo explore whether CF11 filtrates could be processed without centrifugation, CF11-filtered blood-parasite mixtures were refrigerated overnight. The RBC pellet formed during overnight settling was not as tightly packed as that formed during centrifugation and required careful handling when removing supernatant to prevent disruption. This modification resulted in adequate human DNA depletion (mean contamination 11%) (Figure 2A) and total DNA recovery (mean 1.22 μg) (Figure 2B). Columns with less CF11 powder, packed to the 4-ml mark rather than to the 5.5-ml mark, also yielded high total DNA (mean 1.56 μg) and a sufficiently low proportion of human DNA (mean 30%) (Figure 2A and 2B).\n\nBlood volume and parasite density:\nAll CF11-filtered blood-parasite mixtures (3 ml volume, 10,000/μl parasite density) consistently yielded samples with < 30% human DNA contamination and > 500 ng total DNA (Figure 2A and 2B). However, the ex vivo study of P. falciparum isolates with interesting phenotypes can be difficult if blood volumes are limited and parasite densities are low. To determine whether such limitations might significantly reduce the quality of DNA samples, CF11 filtrations were repeated using blood-parasite mixtures of lower volume (1.5 ml) or lower parasite densities (5,000/μl). CF11 filtration of low-volume samples (1.5 ml; 10,000 parasites/μl) produced acceptable human DNA contamination (mean 28%) and mean total DNA (0.51 μg), but individual yields of total DNA were highly variable, ranging from 0.15 to 1.09 μg (Figure 2A and 2B). CF11 filtration of low parasite density samples (3 ml; 5,000 parasites/μl) produced adequate amounts of total DNA (mean 2.68 μg) but unacceptable human DNA contamination (mean 77%) (Figure 2A and 2B). Thus, reducing sample volumes or parasite densities by half failed to reliably produce DNA samples acceptable for Illumina sequencing.\nFiltration of P. falciparum-infected blood obtained from patients with malaria (Pursat, Cambodia) using the CF11 and LP methods consistently achieved high-quality results at parasite densities ranging from 20,000 to 475,000/μl (Figure 3A and 3B). At parasite densities < 50,000/μl and 50,000-100,000/μl, both filtration methods depleted human DNA contamination to ≤ 30% and produced mean DNA yields > 1 μg. Samples with > 100,000 parasites/μl yielded several micrograms of DNA with < 5% human DNA contamination.\n\nValidation of routine blood collection for illumina sequencing:\nThe 51 parasitized blood samples that were collected and CF11-filtered in Ratanakiri, Cambodia, ranged in parasite density from 15,000 to 290,000/μl and yielded a mean 0.64 μg total DNA with 34% human DNA contamination. All 51 samples met quality standards and have been successfully sequenced on the Illumina platform at the Wellcome Trust Sanger Institute (Hinxton, UK).\n\nDiscussion:\nAs next-generation sequencing technologies improve, both costs and stringency of DNA sample quality requirements continue to fall. Between 2009 and 2011, requirements for Illumina sequencing of P. falciparum DNA by the Wellcome Trust Sanger Institute have relaxed from 30% to 50% human DNA contamination. Moreover, the possibility of sequencing multiple DNA samples in a single lane of a flow cell ('multiplex sequencing') has decreased costs immensely while yielding ~2 GB of P. falciparum genomic sequence data per sample, with further improvements anticipated in coming years. To take advantage of these increasingly accessible tools, researchers need scalable procedures for DNA sample preparation in minimally equipped field sites.\nThis study shows that CF11 columns can be used to effectively deplete human DNA from parasitized blood and achieve Illumina sequencing requirements for total DNA yield and percent human DNA contamination, using sample volumes and parasite densities comparable to those used in LP filtration experiments [3]. A major advantage of CF11 over LP filtration is cost-effectiveness. CF11 columns cost approximately one USD each, compared to 10-50 USD for the Plasmodipur filter alone. Additionally, CF11 filtration is a very simple one-step procedure that does not require specialized equipment. A centrifugation step is only used to pellet RBCs after CF11 filtration and can be replaced with a convenient overnight period of refrigeration to allow the RBCs to pellet by gravity. These advantages should enable a large share of the malaria research community to produce parasite DNA samples appropriate for highly parallel sequencing and microarray technologies.\nBecause CF11 columns are hand-made rather than commercially produced, variation in the length of the column is likely to occur. Samples filtered in columns packed with less CF11 powder exhibited higher human DNA contamination but still met the threshold level for sequencing, indicating that some variation in column preparation can be tolerated. Samples with low blood volumes (1.5 ml) or parasite densities (5,000/μl) did not consistently achieve sufficient human DNA depletion or total DNA recovery when filtered with either the CF11 or LP method. Whole-genome amplification (WGA) after DNA isolation [13] could boost the amount of starting material in cases where human DNA removal is satisfactory but total DNA yield is low. WGA may therefore be useful in enabling genomic characterization of CF11-filtered blood samples with low starting volume, such as those collected from very young children and patients with severe malarial anaemia. It may also be useful in processing blood samples with low parasite densities, as is often the case in patients with a recrudescent parasitaemia after anti-malarial treatment and those with high levels of naturally acquired immunity.\nAlthough specific parameters have not been rigorously tested, observations in the field have indicated that CF11 and parasitized blood can be effectively stored and used in a variety of environmental conditions. CF11 kept in an airtight container with regularly replaced desiccant and stored in a cool place can be used for at least three months, even in climates with high heat and humidity (unpublished observation). EDTA-treated blood stored at 4°C for up to 24 hours prior to CF11 filtration was successfully processed for Illumina DNA sequencing (unpublished observation), but for RNA analysis, no more than six hours of refrigeration is suggested (ZB Bozdech, personal communication). Updates to the range of storage conditions will be made to the WWARN protocol [12] as more information becomes available.\n\nConclusions:\nCF11 filtration currently offers the best cost-effective, one-step approach to remove human DNA from P. falciparum-infected whole blood samples and can be successfully implemented in large genome-wide sequencing studies of P. falciparum isolates from patients with malaria.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nGenome and transcriptome studies of Plasmodium nucleic acids obtained from parasitized whole blood are greatly improved by depletion of human DNA or enrichment of parasite DNA prior to next-generation sequencing and microarray hybridization. The most effective method currently used is a two-step procedure to deplete leukocytes: centrifugation using density gradient media followed by filtration through expensive, commercially available columns. This method is not easily implemented in field studies that collect hundreds of samples and simultaneously process samples for multiple laboratory analyses. Inexpensive syringes, hand-packed with CF11 cellulose powder, were recently shown to improve ex vivo cultivation of Plasmodium vivax obtained from parasitized whole blood. This study was undertaken to determine whether CF11 columns could be adapted to isolate Plasmodium falciparum DNA from parasitized whole blood and achieve current quantity and purity requirements for Illumina sequencing.\n\nMethods:\nThe CF11 procedure was compared with the current two-step standard of leukocyte depletion using parasitized red blood cells cultured in vitro and parasitized blood obtained ex vivo from Cambodian patients with malaria. Procedural variations in centrifugation and column size were tested, along with a range of blood volumes and parasite densities.\n\nResults:\nCF11 filtration reliably produces 500 nanograms of DNA with less than 50% human DNA contamination, which is comparable to that obtained by the two-step method and falls within the current quality control requirements for Illumina sequencing. In addition, a centrifuge-free version of the CF11 filtration method to isolate P. falciparum DNA at remote and minimally equipped field sites in malaria-endemic areas was validated.\n\nConclusions:\nCF11 filtration is a cost-effective, scalable, one-step approach to remove human DNA from P. falciparum-infected whole blood samples."},"background_conclusion_text":{"kind":"string","value":"Background:\nRecent technological advances have enabled next-generation genomic and transcriptomic analysis of Plasmodium falciparum from parasitized whole blood samples without the need for culturing. High-density genotyping of parasites obtained directly from patients with malaria has improved our understanding of population structure and genomic and transcriptional variation [1,2]. Highly parallel sequencing is currently being used to identify the genetic determinants of clinically relevant phenotypes including drug resistance, vaccine escape and disease severity. Importantly, genomic characterization of parasite populations in patients captures intra-host diversity and prevents the potential loss of sequence data from phenotype-conferring parasite isolates during their culture adaptation.\nThe performance of highly parallel sequencing platforms, such as Illumina, is greatly enhanced in sample preparations with a high parasite-to-human nucleic acid ratio [3]. Such high ratios can be achieved by either selectively capturing parasite nucleic acids or by removing human material from the sample. Hybrid selection [4] using RNA 'baits' complementary to the P. falciparum genome can achieve over 40-fold enrichment of parasite DNA and can be performed at any time following DNA extraction [5]; however, at $250 USD per sample, this technique may be prohibitively costly for epidemiological or population-level studies.\nThe alternative, leukocyte depletion, must be performed in field sites soon after blood collection and before transport and storage. Commercially available magnetic columns have been used to rapidly isolate parasitized red blood cells (RBCs) from uninfected RBCs and leukocytes [6]. However, magnetic purification depends on short-term culturing of patient blood to transform ring-stage parasites to haemozoin-rich trophozoites and schizonts, which requires equipment for in vitro parasite cultivation. Furthermore, parasites obtained directly from patients mature at different rates in culture, resulting in inconvenient time-points for purification. The current standard for leukocyte depletion of parasitized blood for P. falciparum nucleotide sequencing is a two-step process: centrifugation using Lymphoprep or another sucrose density gradient solution, followed by filtration using Plasmodipur filters [3]. This 'LP' method is effective but difficult to scale-up in field settings because it requires extensive handling and transfer of blood, training to perform sensitive steps, and costly commercial reagents and materials.\nFiltration with hand-made columns has been used as an inexpensive and less time-consuming alternative for leukocyte depletion of Plasmodium-infected blood for decades [7,8]. Recently, non-woven fabric filters [9] and plastic syringes filled with CF11 cellulose powder (Whatman) [10] were shown to effectively remove leukocytes and platelets from Plasmodium vivax-infected blood, where their phagocytic and degranulating activities may interfere with some ex vivo studies. CF11 filtration has also been used to improve microarray-based transcriptome analysis of P. vivax-infected blood [2,11]. CF11 cellulose is thought to work by trapping leukocytes by size exclusion and/or interactions between cellulose hydroxyl groups and leukocyte surface molecules. In this study, laboratory-adapted P. falciparum clones were used to adapt CF11 columns to remove human DNA from P. falciparum-infected blood for genomic studies, compare CF11 filtration to the established LP method, and validate a centrifuge-free option for CF11 filtration. Both methods were also compared in a field setting using P. falciparum isolates obtained directly from patients with malaria. Furthermore, routine CF11 filtration of parasitized blood collected from patients in a second field site with minimal facilities was successfully implemented.\n\nConclusions:\nMV, CA, SC, OK, PL, SA and SU designed the study and collected the data. MV, CA and SC analysed the data. DS, DPK, RMF and CVP provided guidance and coordination for study design, data collection and analysis. MV wrote the first draft of the manuscript. RMF, CA, SC and CVP edited and revised the manuscript. All authors read and approved the final manuscript."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nGenome and transcriptome studies of Plasmodium nucleic acids obtained from parasitized whole blood are greatly improved by depletion of human DNA or enrichment of parasite DNA prior to next-generation sequencing and microarray hybridization. The most effective method currently used is a two-step procedure to deplete leukocytes: centrifugation using density gradient media followed by filtration through expensive, commercially available columns. This method is not easily implemented in field studies that collect hundreds of samples and simultaneously process samples for multiple laboratory analyses. Inexpensive syringes, hand-packed with CF11 cellulose powder, were recently shown to improve ex vivo cultivation of Plasmodium vivax obtained from parasitized whole blood. This study was undertaken to determine whether CF11 columns could be adapted to isolate Plasmodium falciparum DNA from parasitized whole blood and achieve current quantity and purity requirements for Illumina sequencing.\n\nMethods:\nThe CF11 procedure was compared with the current two-step standard of leukocyte depletion using parasitized red blood cells cultured in vitro and parasitized blood obtained ex vivo from Cambodian patients with malaria. Procedural variations in centrifugation and column size were tested, along with a range of blood volumes and parasite densities.\n\nResults:\nCF11 filtration reliably produces 500 nanograms of DNA with less than 50% human DNA contamination, which is comparable to that obtained by the two-step method and falls within the current quality control requirements for Illumina sequencing. In addition, a centrifuge-free version of the CF11 filtration method to isolate P. falciparum DNA at remote and minimally equipped field sites in malaria-endemic areas was validated.\n\nConclusions:\nCF11 filtration is a cost-effective, scalable, one-step approach to remove human DNA from P. falciparum-infected whole blood samples."},"whole_article_text_length":{"kind":"number","value":11595,"string":"11,595"},"whole_article_abstract_length":{"kind":"number","value":322,"string":"322"},"other_sections_lengths":{"kind":"list like","value":[645,361,297,94,42,888,101,112,150,61,438,33,2215,551,134,333,69,639,47],"string":"[\n 645,\n 361,\n 297,\n 94,\n 42,\n 888,\n 101,\n 112,\n 150,\n 61,\n 438,\n 33,\n 2215,\n 551,\n 134,\n 333,\n 69,\n 639,\n 47\n]","isConversational":false},"num_sections":{"kind":"number","value":20,"string":"20"},"most_frequent_words":{"kind":"list like","value":["cf11","blood","parasite","dna","ml","filtration","000","μl","samples","blood parasite"],"string":"[\n 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falciparum | Sensitivity and Specificity | Specimen Handling | Young Adult [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Aged | Aged, 80 and over | Blood | Cambodia | Child | Child, Preschool | Chromatography | DNA, Protozoan | Humans | Infant | Malaria | Middle Aged | Parasitology | Plasmodium falciparum | Sensitivity and Specificity | Specimen Handling | Young Adult [SUMMARY]"},"annotated_mesh_results_abstract_prompt":{"kind":"null"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Aged | Aged, 80 and over | Blood | Cambodia | Child | Child, Preschool | Chromatography | DNA, Protozoan | Humans | Infant | Malaria | Middle Aged | Parasitology | Plasmodium falciparum | Sensitivity and Specificity | Specimen Handling | Young Adult [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Adolescent | Adult | Aged | Aged, 80 and over | 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[SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Plasmodium ||| two ||| hundreds ||| Plasmodium ||| Plasmodium | Illumina ||| two | Cambodian ||| ||| 500 | less than 50% | two | Illumina ||| ||| one [SUMMARY]"}}},{"rowIdx":94,"cells":{"title":{"kind":"string","value":"Undiagnosed Depression among Hypertensive Individuals in Gaza: A Cross-sectional Survey from Palestine."},"pmid":{"kind":"string","value":"34158786"},"background_abstract":{"kind":"string","value":"The aim of this study was to estimate the prevalence and to determine the associated factors of undiagnosed depression amongst hypertensive patients (HTNP) at primary health care centers (PHCC) in Gaza."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"A cross-sectional survey was conducted including 538 HTNP as a recruitment phase of a clustered randomized controlled trial. Data were collected through face-to-face structured interview, and depression status was assessed by Beck's Depression Inventory (BDI-II). Data were analyzed by STATA version 14 using standard complex survey analyses, accounted for unresponsiveness and clustering approach. Generalized linear regression analysis was performed to assess associations."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"The prevalence of undiagnosed clinical depression was 11.6% (95% confidence interval [CI]: 8.1, 16.3). Moreover, prevalence of 15.4% (95% CI: 10.8, 21.6) was found for mild depression symptoms. We found that non-adherence to antihypertensive medications (AHTNM) (β = 0.9, 95% CI: 0.17, 1.7), having more health-care system support (β = 2.8, 95% CI: 1.6, 3.9) and number of AHTNM (β = 1.5, 95% CI: 0.6, 2.5) remain significantly positively associated with BDI-II score. On the other hand, older age (β = -0.1, 95% CI: -0.2, -0.02), having better social support (β = -6.8, 95% CI: -8.9, -4.7) and having stronger patient-doctor relationship (β = -4.1, 95% CI: -6.9, -1.2) kept significantly negative association."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The prevalence of undiagnosed depression was about one-quarter of all cases; half of them were moderate to severe. Routine screening of depression status should be a part of the care of HTNP in PHCC."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Aged","Antihypertensive Agents","Cross-Sectional Studies","Depression","Depressive Disorder","Humans","Hypertension","Prevalence"],"string":"[\n \"Aged\",\n \"Antihypertensive Agents\",\n \"Cross-Sectional Studies\",\n \"Depression\",\n \"Depressive Disorder\",\n \"Humans\",\n \"Hypertension\",\n \"Prevalence\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"8188075"},"background_title":{"kind":"string","value":"Introduction"},"background_text":{"kind":"string","value":"Globally, depression affects 350 million people around the world; it is strongly contributes to the burden of disease, and is expected to increase by 5.7% of global burden of disease and become the second after ischemic heart disease by 2020 (1). Notably, episodes of depression are accompanied with other chronic diseases especially hypertension (2,3). Hypertension affects globally one fourth of adults, and is likely to increase to one third by 2025 (4). People with hypertension and subclinical depression are at extra risk of complications such as cerebrovascular stroke, cardiovascular and kidney diseases (3, 5–7). Moreover, depression represents an important predictor among hypertensive individual whom are non-adherence to their treatment (8).\nMany studies demonstrated an increased co-occurrence of depression with hypertension in different countries (9); however, little is known about depression prevalence among hypertensive patients in Gaza Strip (GS). This study seems to be the first study aimed to estimate the prevalence and to determine the associated factors of undiagnosed depression among hypertensive patients attending primary healthcare clinics in GS."},"methods_title":{"kind":"string","value":"Methods and Participants"},"methods_text":{"kind":"string","value":"A cross-sectional survey as the recruitment phase of a clustered randomized controlled trial was conducted between 1st August and 30th December, 2018. We recruited 538 hypertensive persons seeking primary health care by two stages cluster random sampling from ten primary health centers. Initially, centers were randomly selected by stratified simple random sampling approach to get two centers from each governorate. Then, participants from each center were proportionally selected through a systematic random sampling method based on eligibility criteria and agreement to take part in the study.\nThis cross-sectional survey was conducted as the recruitment phase of a clustered randomized controlled trial. All eligible participants who agreed to participate in the study were enrolled regardless of their antihypertensive medication adherence status. Psychological status was initially classified according to the BDI-II score. Later, adherent status to antihypertensive medication was determined. Subsequently, the intervention of the clustered randomized controlled trial was implemented based on the adherent status results.\nMeasures and data collection: Face-to-face interview was used to collect data from participants using a structured questionnaire. The interviews lasted fifteen minutes during the clinic hours (8 am to 2 pm, five days a week). The exposure variables of the study included participants' characteristics (age, gender, marital status, employment, education level), as well as participants' health status variables including smoking status, comorbidities, body mass index (BMI), blood pressure (BP) measurement, and medication adherence status. Other predictors of interest such as social support, relationships between patients and physician and health system support were also assessed. Moreover, depressive symptoms as the outcome variable were assessed using the validated Arabic language Beck's Depression Inventory (BDI-II) (10).\nBlood pressure was measured on the right arm using mercury sphygmomanometer after completing the interview; the value was recorded in terms of mm of mercury. BMI was calculated by using the WHO chart based on weight and height. Weight and height were measured by mechanical weighing machine with height rod (Health o meter 402LB Physician Beam Scale, Height Rod).\nInstrument: The questionnaire consisted of items about the baseline characteristics of participants, clinical health history, adherence status, patient-doctor relationship, healthcare system support, perceived social support and psychological status of depressive symptoms.\nDepressive symptoms were assessed by the aid of BDI-II. A 21-item self-report inventory was designed to assess the presence and severity of depressive symptoms. Each item is rated on a four-point Likert-type scale ranging from 0 to 3, based on the severity in the last two weeks. The total score ranges from 0 to 63, with higher scores indicating more severe depressive symptoms. The results were stratified into four groups; BDI-II: 1–13 these ups and downs are considered normal, BDI-II: 14–19 are mood disturbance or mild depression, BDI-II: 20–28 are moderate depression, and BDI-II: more than 29 are severe depression (11). We provided psychological counseling by psychotherapist for those who scored from 11 to 19. Special precautions were taken for those individuals who endorsed having suicidal ideation. Mainly, we involved the family and coordinated for urgent counseling in the related psychological health center. Moreover, all cases scored 20 and above were referred to psychology health centers. Consequently, stratification into two groups was done; considering BDI-II ≥20 who have clinical depression and BDI-II <20 who do not have clinical depression (12,13).\nAdherence status was assessed by Morisky, Green, and Levine Adherence Scale (MGL), a known validated and reliable self-report medication adherence score (14). Similarly, medical comorbidities were assessed using Charlson comorbidity index, a validated and widely used weighted-index designed scoring as low and high comorbidities (15). Likewise, social support was assessed by multidimensional Scale of Perceived Social Support (MSPSS); a known valid and reliable questionnaire which measures perceptions of support from three sources: family, friends, and a significant other (16). Arabic translation was performed based on WHO five steps for process of translation and adaptation of instrument (17).\nAn Arabic validated and reliable version of patient-doctor relationship questionnaire-9 (PDRQ-9) was used to assess the relationship between patients and physicians (6,18). Likewise, healthcare system support questionnaire was used with little modifications (18, 19). The whole Arabic questionnaire content and face validity were reviewed by panel of experts. Required changes were made to clarify any ambiguity and to ensure comprehension of Palestinian participants after pilot study, detailed information about the questionnaire are available in our published article elsewhere (20).\nEligibility criteria: Palestinian citizens attending Gaza governmental primary health centers, aged above 18 years, registered as hypertension patient since at least one year and taking at least one antihypertensive medication were eligible to participate in the study. Patients with a diagnosis of cognitive impairment, history of depression or being currently on antidepressants as reported by their primary care physician were excluded from the study.\nData analysis: Standard complex survey data analysis method was performed by STATA version 14. We accounted for clustering and unresponsiveness using STATA PSU option and unequal probability of selection using sample weight variable analysis and post stratification weight for each age and sex group strata. Furthermore, since the BDI-II score did not follow normality assumption, Generalized Linear Model (GLM) with Gaussian family and log link was run. Moreover, linearized standard error which is quite robust to non-uniformity of variance was used.\nData were described using descriptive statistics; categorical variables were compared using the Chi-squared test. After checking assumption of linear regression, univariable analysis followed by multiple linear regression were performed to assess the association between depression status and all other independent variables including participants' characteristic. Statistically significant variables were included in multiple regression analysis model based on 0.1 level. However, variables had been excluded by backward stepwise elimination method.\nEthics approval and consent to participate: Prior to conducting this research study, ethical approval from Tehran University of Medical Sciences Ethical Committee (code number: IR.TUMS.SPH.REC.1396.4828) was obtained. Approvals from the Palestinian Health Research Council(PHRC/HC/322/18) and Research Committee at the Palestinian Ministry of Health were also obtained. Purposes of the study were explained to participants, and they were reassured about confidentiality of data. Each participant was asked to sign a consent paper prior to participation."},"results_title":{"kind":"string","value":"Results"},"results_text":{"kind":"string","value":"Participants' characteristics: Five hundred and thirty-eight participants were included in the survey from the five governorates of Gaza Strip by response rate of 96%. The overall mean age was 57.1 years (95% confidence interval [CI]: 55.9, 58.2) and more than half of them (60.9%) were females. In addition, the majority of them were literate (90.2%), married (90.4%), unemployed (86.5%), and not smokers (81.4%). Since obesity is known to have BMI more than 30 kg/m2, it was most common among study population as BMI mean reported 32.3 (95% CI: 31.9, 32.6). Moreover, more than half of the participants (57.2%) had been diagnosed with hypertension for more than five years with a mean of 8.44 years (95% CI: 7.3, 9.4). Almost, two third of them (64.4%) were treated with only one antihypertensive medication once a day (64.8%); however, 35.6% were treated with two or more medications with a frequency of twice or more (35.2%). Only 14.43% of them were classified with two or more comorbidities. In addition, the participants reported a mean of depression status based on BDI-II score of 10 (95%CI: 8.4, 11.6%) (Table 1).\nParticipants characteristics (n= 538)\nDepression status: The BDI-II mean score for all participants was 10 (95% CI: 8.4, 11.6); and they were categorized into three groups: 1) normal (BDI-II 0–13) which had a prevalence proportion of 73% (95% CI: 65.6, 79.3), 2), mild clinical depression (BDI-II: 14–19) with a prevalence proportion of 15.4% (95% CI: 10.8, 21.6), in which they received psychological counseling in the same clinic by psychotherapist; and 3) clinical depression BDI-II ≥20 with a prevalence of 11.6% (95% CI: 8.1, 16.3) of undiagnosed depression cases which all were referred to psychology health centers for further evaluation and treatment (Table 2).\nDepression status prevalence proportion (n= 538)\n\nRelationship of depression status and predictors\n\nUnder standard complex survey data setting, the Chi-squared test was used to compare categorical variables of depression status and other explanatory variables. Table 3 shows that depression status had relations with the categorical variables: level of education and BMI categories.\nRelation between depression status and participants characteristics\nUnivariable linear regression analysis presented in Table 4 shows that increased BMI (β = 0.11, 95% CI: 0.01, 0.21), being non-adherent to antihypertensive medications (β = 1.3, 95% CI: 0.5, 2.1) and having better healthcare system support (β = 2.8, 95% CI: 1.2, 4.4) were significantly associated with increased BDI-II score. In contrast, older age (β = -1.5, 95% CI: - 0.29, -0.01), longer duration of hypertension (β = -0.12, 95% CI: -0.19, -0.04), stronger patient-doctor relationship (β = -3.7, 95% CI: -6.4, -1.1), and having superior social support (β = -7.1, 95% CI: -9.3, -4.9) were significantly associated with reduced BDI-II score.\nUnivariate linear regression analysis of depression status and predictors\nStatistically significant variables for multivariable analysis\nMultivariable linear regression analysis in Table 5 revealed similar results except for increased BMI and duration of hypertension. Thus, non-adherence to antihypertensive medications (β = 0.9, 95% CI: 0.2, 1.7), having more healthcare system support (β = 2.8, 95% CI: 1.6, 3.9) and number of antihypertensive medications (β = 1.5, 95% CI: 0.6, 2.5) remain significantly positively associated with BDI-II score. On the other hand, older age (β = -0.11, 95% CI: -0.20, -0.02), having better social support (β = -6.8, 95% CI: -8.9, -4.7) and stronger relation with physician (β = -4.1, 95% CI: -6.9, -1.2) remain significantly negatively associated with BDI-II score.\nMultivariable linear regression analysis of depression status and predictors"},"conclusions_title":{"kind":"null"},"conclusions_text":{"kind":"null"},"other_sections_titles":{"kind":"list like","value":[],"string":"[]","isConversational":false},"other_sections_texts":{"kind":"list like","value":[],"string":"[]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[],"string":"[]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["Introduction","Methods and Participants","Results","Discussion"],"string":"[\n \"Introduction\",\n \"Methods and Participants\",\n \"Results\",\n \"Discussion\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Globally, depression affects 350 million people around the world; it is strongly contributes to the burden of disease, and is expected to increase by 5.7% of global burden of disease and become the second after ischemic heart disease by 2020 (1). Notably, episodes of depression are accompanied with other chronic diseases especially hypertension (2,3). Hypertension affects globally one fourth of adults, and is likely to increase to one third by 2025 (4). People with hypertension and subclinical depression are at extra risk of complications such as cerebrovascular stroke, cardiovascular and kidney diseases (3, 5–7). Moreover, depression represents an important predictor among hypertensive individual whom are non-adherence to their treatment (8).\nMany studies demonstrated an increased co-occurrence of depression with hypertension in different countries (9); however, little is known about depression prevalence among hypertensive patients in Gaza Strip (GS). This study seems to be the first study aimed to estimate the prevalence and to determine the associated factors of undiagnosed depression among hypertensive patients attending primary healthcare clinics in GS.","A cross-sectional survey as the recruitment phase of a clustered randomized controlled trial was conducted between 1st August and 30th December, 2018. We recruited 538 hypertensive persons seeking primary health care by two stages cluster random sampling from ten primary health centers. Initially, centers were randomly selected by stratified simple random sampling approach to get two centers from each governorate. Then, participants from each center were proportionally selected through a systematic random sampling method based on eligibility criteria and agreement to take part in the study.\nThis cross-sectional survey was conducted as the recruitment phase of a clustered randomized controlled trial. All eligible participants who agreed to participate in the study were enrolled regardless of their antihypertensive medication adherence status. Psychological status was initially classified according to the BDI-II score. Later, adherent status to antihypertensive medication was determined. Subsequently, the intervention of the clustered randomized controlled trial was implemented based on the adherent status results.\nMeasures and data collection: Face-to-face interview was used to collect data from participants using a structured questionnaire. The interviews lasted fifteen minutes during the clinic hours (8 am to 2 pm, five days a week). The exposure variables of the study included participants' characteristics (age, gender, marital status, employment, education level), as well as participants' health status variables including smoking status, comorbidities, body mass index (BMI), blood pressure (BP) measurement, and medication adherence status. Other predictors of interest such as social support, relationships between patients and physician and health system support were also assessed. Moreover, depressive symptoms as the outcome variable were assessed using the validated Arabic language Beck's Depression Inventory (BDI-II) (10).\nBlood pressure was measured on the right arm using mercury sphygmomanometer after completing the interview; the value was recorded in terms of mm of mercury. BMI was calculated by using the WHO chart based on weight and height. Weight and height were measured by mechanical weighing machine with height rod (Health o meter 402LB Physician Beam Scale, Height Rod).\nInstrument: The questionnaire consisted of items about the baseline characteristics of participants, clinical health history, adherence status, patient-doctor relationship, healthcare system support, perceived social support and psychological status of depressive symptoms.\nDepressive symptoms were assessed by the aid of BDI-II. A 21-item self-report inventory was designed to assess the presence and severity of depressive symptoms. Each item is rated on a four-point Likert-type scale ranging from 0 to 3, based on the severity in the last two weeks. The total score ranges from 0 to 63, with higher scores indicating more severe depressive symptoms. The results were stratified into four groups; BDI-II: 1–13 these ups and downs are considered normal, BDI-II: 14–19 are mood disturbance or mild depression, BDI-II: 20–28 are moderate depression, and BDI-II: more than 29 are severe depression (11). We provided psychological counseling by psychotherapist for those who scored from 11 to 19. Special precautions were taken for those individuals who endorsed having suicidal ideation. Mainly, we involved the family and coordinated for urgent counseling in the related psychological health center. Moreover, all cases scored 20 and above were referred to psychology health centers. Consequently, stratification into two groups was done; considering BDI-II ≥20 who have clinical depression and BDI-II <20 who do not have clinical depression (12,13).\nAdherence status was assessed by Morisky, Green, and Levine Adherence Scale (MGL), a known validated and reliable self-report medication adherence score (14). Similarly, medical comorbidities were assessed using Charlson comorbidity index, a validated and widely used weighted-index designed scoring as low and high comorbidities (15). Likewise, social support was assessed by multidimensional Scale of Perceived Social Support (MSPSS); a known valid and reliable questionnaire which measures perceptions of support from three sources: family, friends, and a significant other (16). Arabic translation was performed based on WHO five steps for process of translation and adaptation of instrument (17).\nAn Arabic validated and reliable version of patient-doctor relationship questionnaire-9 (PDRQ-9) was used to assess the relationship between patients and physicians (6,18). Likewise, healthcare system support questionnaire was used with little modifications (18, 19). The whole Arabic questionnaire content and face validity were reviewed by panel of experts. Required changes were made to clarify any ambiguity and to ensure comprehension of Palestinian participants after pilot study, detailed information about the questionnaire are available in our published article elsewhere (20).\nEligibility criteria: Palestinian citizens attending Gaza governmental primary health centers, aged above 18 years, registered as hypertension patient since at least one year and taking at least one antihypertensive medication were eligible to participate in the study. Patients with a diagnosis of cognitive impairment, history of depression or being currently on antidepressants as reported by their primary care physician were excluded from the study.\nData analysis: Standard complex survey data analysis method was performed by STATA version 14. We accounted for clustering and unresponsiveness using STATA PSU option and unequal probability of selection using sample weight variable analysis and post stratification weight for each age and sex group strata. Furthermore, since the BDI-II score did not follow normality assumption, Generalized Linear Model (GLM) with Gaussian family and log link was run. Moreover, linearized standard error which is quite robust to non-uniformity of variance was used.\nData were described using descriptive statistics; categorical variables were compared using the Chi-squared test. After checking assumption of linear regression, univariable analysis followed by multiple linear regression were performed to assess the association between depression status and all other independent variables including participants' characteristic. Statistically significant variables were included in multiple regression analysis model based on 0.1 level. However, variables had been excluded by backward stepwise elimination method.\nEthics approval and consent to participate: Prior to conducting this research study, ethical approval from Tehran University of Medical Sciences Ethical Committee (code number: IR.TUMS.SPH.REC.1396.4828) was obtained. Approvals from the Palestinian Health Research Council(PHRC/HC/322/18) and Research Committee at the Palestinian Ministry of Health were also obtained. Purposes of the study were explained to participants, and they were reassured about confidentiality of data. Each participant was asked to sign a consent paper prior to participation.","Participants' characteristics: Five hundred and thirty-eight participants were included in the survey from the five governorates of Gaza Strip by response rate of 96%. The overall mean age was 57.1 years (95% confidence interval [CI]: 55.9, 58.2) and more than half of them (60.9%) were females. In addition, the majority of them were literate (90.2%), married (90.4%), unemployed (86.5%), and not smokers (81.4%). Since obesity is known to have BMI more than 30 kg/m2, it was most common among study population as BMI mean reported 32.3 (95% CI: 31.9, 32.6). Moreover, more than half of the participants (57.2%) had been diagnosed with hypertension for more than five years with a mean of 8.44 years (95% CI: 7.3, 9.4). Almost, two third of them (64.4%) were treated with only one antihypertensive medication once a day (64.8%); however, 35.6% were treated with two or more medications with a frequency of twice or more (35.2%). Only 14.43% of them were classified with two or more comorbidities. In addition, the participants reported a mean of depression status based on BDI-II score of 10 (95%CI: 8.4, 11.6%) (Table 1).\nParticipants characteristics (n= 538)\nDepression status: The BDI-II mean score for all participants was 10 (95% CI: 8.4, 11.6); and they were categorized into three groups: 1) normal (BDI-II 0–13) which had a prevalence proportion of 73% (95% CI: 65.6, 79.3), 2), mild clinical depression (BDI-II: 14–19) with a prevalence proportion of 15.4% (95% CI: 10.8, 21.6), in which they received psychological counseling in the same clinic by psychotherapist; and 3) clinical depression BDI-II ≥20 with a prevalence of 11.6% (95% CI: 8.1, 16.3) of undiagnosed depression cases which all were referred to psychology health centers for further evaluation and treatment (Table 2).\nDepression status prevalence proportion (n= 538)\n\nRelationship of depression status and predictors\n\nUnder standard complex survey data setting, the Chi-squared test was used to compare categorical variables of depression status and other explanatory variables. Table 3 shows that depression status had relations with the categorical variables: level of education and BMI categories.\nRelation between depression status and participants characteristics\nUnivariable linear regression analysis presented in Table 4 shows that increased BMI (β = 0.11, 95% CI: 0.01, 0.21), being non-adherent to antihypertensive medications (β = 1.3, 95% CI: 0.5, 2.1) and having better healthcare system support (β = 2.8, 95% CI: 1.2, 4.4) were significantly associated with increased BDI-II score. In contrast, older age (β = -1.5, 95% CI: - 0.29, -0.01), longer duration of hypertension (β = -0.12, 95% CI: -0.19, -0.04), stronger patient-doctor relationship (β = -3.7, 95% CI: -6.4, -1.1), and having superior social support (β = -7.1, 95% CI: -9.3, -4.9) were significantly associated with reduced BDI-II score.\nUnivariate linear regression analysis of depression status and predictors\nStatistically significant variables for multivariable analysis\nMultivariable linear regression analysis in Table 5 revealed similar results except for increased BMI and duration of hypertension. Thus, non-adherence to antihypertensive medications (β = 0.9, 95% CI: 0.2, 1.7), having more healthcare system support (β = 2.8, 95% CI: 1.6, 3.9) and number of antihypertensive medications (β = 1.5, 95% CI: 0.6, 2.5) remain significantly positively associated with BDI-II score. On the other hand, older age (β = -0.11, 95% CI: -0.20, -0.02), having better social support (β = -6.8, 95% CI: -8.9, -4.7) and stronger relation with physician (β = -4.1, 95% CI: -6.9, -1.2) remain significantly negatively associated with BDI-II score.\nMultivariable linear regression analysis of depression status and predictors","To best of our knowledge, this is the first cross-sectional survey from Gaza to document the prevalence of undiagnosed depression among hypertensive patients. We found that the prevalence of undiagnosed clinical depression in our hypertensive population is almost higher than what is observed in Norway (6.2%) and South Africa (6%), and lower than in the US(44.9%), China (44.2%), Mexico (57.5%), Pakistan (40.1%), Croatia (29.6%) and Nigeria (26.7%). However, it was similar to that observed in Brazil (12.1%), Ghana (10.5%) and Netherlands (11.4%) (9, 21). In addition, this prevalence was lower than the rate reported in a systematic review and meta-analysis (26.8%) which summarized the prevalence of depression among hypertensive individuals in 41 studies (9).\nUnfortunately, very limited data is available from Arabic regional countries. One study from Saudi Arabia found a prevalence of 20.7% (22). Still, no local prevalence was found from other parts of Palestine. Our study showed that the prevalence of depression among hypertensive patients was lower than the prevalence of patients with type two diabetic in the West Bank of Palestine (40%) (23).\nThe associated factors found by multiple linear regression in this study were: age, number of antihypertensive medication, adherent status, healthcare system support, patient-doctor relationship and perceived social support. Increasing age was found to be an associated factor with increased depressive symptom in other studies (21, 22, 24, 25), although, our results predicted a negative relationship between age and depressive status. An explanation for this could be related to the nature of aged people in Gaza, since they have more acceptance and are adapted to their disease more than younger adults.\nHowever, gender and smoking (21, 22, 24, 26) found to be associated factors in previous studies, did not reach statistical significant level in our study. Longer hypertension duration has been previously found to be a significantly associated factor in an Indian study (26); and was associated factor in our univariable analysis, although it could not reach significant level in multivariable analysis. In addition, in line with what has been found on a systematic review (8), we found a negative relationship between depression and adherence to antihypertensive medications. Furthermore, statistically significant association with number of antihypertensive medication and depression was observed in our study, which was supported by previous findings (24, 27).\nIn this study, we have investigated the relation of depression status with healthcare system support, patient-doctor relationship and perceived social support. Interestingly, a positive relationship between depression and healthcare system support was found. This could be explained by the frequent visit of the patient to the primary healthcare clinic, and his increased need to its supportive aids. On the other hand, it was a negative relationship with patient-doctor relationship and social support. It is highly believed to be negative relationship between depression and perceived social support since social support is a significant predictor of depression and hypertension treatment programs (28, 29).\nWe shed the light on the undiagnosed depression cases among hypertensive persons receiving their usual primary health care in primary healthcare clinics. The estimated prevalence proportion of undiagnosed subclinical depression cases was about one-quarter of all cases, in which half of them had moderate to severe depression status. Furthermore, age, number of antihypertensive medication, adherence status, patient-physician relationship, health-care system support and social support were associated with depression status. We suggest periodically screening of depression and adherence status as a part of routine care for hypertensive primary healthcare seekers, particularly younger hypertensive patients. We also call for exploring the ways to promote social support among them.\nHowever, since this is the first study in Gaza Strip which assessed the psychological status among hypertensive patients, several limitations were observed. First, alcohol consumption is an important factor related to depression and adherence and may confound the reported relationships, although it has not been measured in this study. Our justification is that alcohol consumption is illegal and prohibited to be used in Gaza as Muslim citizens, and so it was difficult to get trustful answers from the participants. Thus, more than 98% of them answered no. Indeed, even if he/she was a user he/she would not tell the truth because of fear of legal prosecutions in spite of assurance about data confidentiality. As a result, we decided to omit this variable as a predictor from this study.\nThe second limitation is that some antihypertensive medications may be related with depression. However, we were concerned only about the number of antihypertensive medications and the frequency and did not discuss the type and classes of the medications.\nAnother limitation of this study is that the prevalence of depression would be underestimated because we excluded the already known patients with a history of depression or currently on antidepressant medications. In addition, BDI-II is a depression screening tool only and not a diagnostic tool. This will surely affect the true prevalence of depression and therefore this could be another limitation regarding the estimation of the true prevalence. However, BDI-II as a screening tool gave a good impression of the depression prevalence among hypertensive patients and was able to discover many unknown cases that were referred for further diagnostic procedures. Despite the limitations, the findings support the routine screening of depression and medication adherence status as a part of care for hypertensive patients in PHCCs."],"string":"[\n \"Globally, depression affects 350 million people around the world; it is strongly contributes to the burden of disease, and is expected to increase by 5.7% of global burden of disease and become the second after ischemic heart disease by 2020 (1). Notably, episodes of depression are accompanied with other chronic diseases especially hypertension (2,3). Hypertension affects globally one fourth of adults, and is likely to increase to one third by 2025 (4). People with hypertension and subclinical depression are at extra risk of complications such as cerebrovascular stroke, cardiovascular and kidney diseases (3, 5–7). Moreover, depression represents an important predictor among hypertensive individual whom are non-adherence to their treatment (8).\\nMany studies demonstrated an increased co-occurrence of depression with hypertension in different countries (9); however, little is known about depression prevalence among hypertensive patients in Gaza Strip (GS). This study seems to be the first study aimed to estimate the prevalence and to determine the associated factors of undiagnosed depression among hypertensive patients attending primary healthcare clinics in GS.\",\n \"A cross-sectional survey as the recruitment phase of a clustered randomized controlled trial was conducted between 1st August and 30th December, 2018. We recruited 538 hypertensive persons seeking primary health care by two stages cluster random sampling from ten primary health centers. Initially, centers were randomly selected by stratified simple random sampling approach to get two centers from each governorate. Then, participants from each center were proportionally selected through a systematic random sampling method based on eligibility criteria and agreement to take part in the study.\\nThis cross-sectional survey was conducted as the recruitment phase of a clustered randomized controlled trial. All eligible participants who agreed to participate in the study were enrolled regardless of their antihypertensive medication adherence status. Psychological status was initially classified according to the BDI-II score. Later, adherent status to antihypertensive medication was determined. Subsequently, the intervention of the clustered randomized controlled trial was implemented based on the adherent status results.\\nMeasures and data collection: Face-to-face interview was used to collect data from participants using a structured questionnaire. The interviews lasted fifteen minutes during the clinic hours (8 am to 2 pm, five days a week). The exposure variables of the study included participants' characteristics (age, gender, marital status, employment, education level), as well as participants' health status variables including smoking status, comorbidities, body mass index (BMI), blood pressure (BP) measurement, and medication adherence status. Other predictors of interest such as social support, relationships between patients and physician and health system support were also assessed. Moreover, depressive symptoms as the outcome variable were assessed using the validated Arabic language Beck's Depression Inventory (BDI-II) (10).\\nBlood pressure was measured on the right arm using mercury sphygmomanometer after completing the interview; the value was recorded in terms of mm of mercury. BMI was calculated by using the WHO chart based on weight and height. Weight and height were measured by mechanical weighing machine with height rod (Health o meter 402LB Physician Beam Scale, Height Rod).\\nInstrument: The questionnaire consisted of items about the baseline characteristics of participants, clinical health history, adherence status, patient-doctor relationship, healthcare system support, perceived social support and psychological status of depressive symptoms.\\nDepressive symptoms were assessed by the aid of BDI-II. A 21-item self-report inventory was designed to assess the presence and severity of depressive symptoms. Each item is rated on a four-point Likert-type scale ranging from 0 to 3, based on the severity in the last two weeks. The total score ranges from 0 to 63, with higher scores indicating more severe depressive symptoms. The results were stratified into four groups; BDI-II: 1–13 these ups and downs are considered normal, BDI-II: 14–19 are mood disturbance or mild depression, BDI-II: 20–28 are moderate depression, and BDI-II: more than 29 are severe depression (11). We provided psychological counseling by psychotherapist for those who scored from 11 to 19. Special precautions were taken for those individuals who endorsed having suicidal ideation. Mainly, we involved the family and coordinated for urgent counseling in the related psychological health center. Moreover, all cases scored 20 and above were referred to psychology health centers. Consequently, stratification into two groups was done; considering BDI-II ≥20 who have clinical depression and BDI-II <20 who do not have clinical depression (12,13).\\nAdherence status was assessed by Morisky, Green, and Levine Adherence Scale (MGL), a known validated and reliable self-report medication adherence score (14). Similarly, medical comorbidities were assessed using Charlson comorbidity index, a validated and widely used weighted-index designed scoring as low and high comorbidities (15). Likewise, social support was assessed by multidimensional Scale of Perceived Social Support (MSPSS); a known valid and reliable questionnaire which measures perceptions of support from three sources: family, friends, and a significant other (16). Arabic translation was performed based on WHO five steps for process of translation and adaptation of instrument (17).\\nAn Arabic validated and reliable version of patient-doctor relationship questionnaire-9 (PDRQ-9) was used to assess the relationship between patients and physicians (6,18). Likewise, healthcare system support questionnaire was used with little modifications (18, 19). The whole Arabic questionnaire content and face validity were reviewed by panel of experts. Required changes were made to clarify any ambiguity and to ensure comprehension of Palestinian participants after pilot study, detailed information about the questionnaire are available in our published article elsewhere (20).\\nEligibility criteria: Palestinian citizens attending Gaza governmental primary health centers, aged above 18 years, registered as hypertension patient since at least one year and taking at least one antihypertensive medication were eligible to participate in the study. Patients with a diagnosis of cognitive impairment, history of depression or being currently on antidepressants as reported by their primary care physician were excluded from the study.\\nData analysis: Standard complex survey data analysis method was performed by STATA version 14. We accounted for clustering and unresponsiveness using STATA PSU option and unequal probability of selection using sample weight variable analysis and post stratification weight for each age and sex group strata. Furthermore, since the BDI-II score did not follow normality assumption, Generalized Linear Model (GLM) with Gaussian family and log link was run. Moreover, linearized standard error which is quite robust to non-uniformity of variance was used.\\nData were described using descriptive statistics; categorical variables were compared using the Chi-squared test. After checking assumption of linear regression, univariable analysis followed by multiple linear regression were performed to assess the association between depression status and all other independent variables including participants' characteristic. Statistically significant variables were included in multiple regression analysis model based on 0.1 level. However, variables had been excluded by backward stepwise elimination method.\\nEthics approval and consent to participate: Prior to conducting this research study, ethical approval from Tehran University of Medical Sciences Ethical Committee (code number: IR.TUMS.SPH.REC.1396.4828) was obtained. Approvals from the Palestinian Health Research Council(PHRC/HC/322/18) and Research Committee at the Palestinian Ministry of Health were also obtained. Purposes of the study were explained to participants, and they were reassured about confidentiality of data. Each participant was asked to sign a consent paper prior to participation.\",\n \"Participants' characteristics: Five hundred and thirty-eight participants were included in the survey from the five governorates of Gaza Strip by response rate of 96%. The overall mean age was 57.1 years (95% confidence interval [CI]: 55.9, 58.2) and more than half of them (60.9%) were females. In addition, the majority of them were literate (90.2%), married (90.4%), unemployed (86.5%), and not smokers (81.4%). Since obesity is known to have BMI more than 30 kg/m2, it was most common among study population as BMI mean reported 32.3 (95% CI: 31.9, 32.6). Moreover, more than half of the participants (57.2%) had been diagnosed with hypertension for more than five years with a mean of 8.44 years (95% CI: 7.3, 9.4). Almost, two third of them (64.4%) were treated with only one antihypertensive medication once a day (64.8%); however, 35.6% were treated with two or more medications with a frequency of twice or more (35.2%). Only 14.43% of them were classified with two or more comorbidities. In addition, the participants reported a mean of depression status based on BDI-II score of 10 (95%CI: 8.4, 11.6%) (Table 1).\\nParticipants characteristics (n= 538)\\nDepression status: The BDI-II mean score for all participants was 10 (95% CI: 8.4, 11.6); and they were categorized into three groups: 1) normal (BDI-II 0–13) which had a prevalence proportion of 73% (95% CI: 65.6, 79.3), 2), mild clinical depression (BDI-II: 14–19) with a prevalence proportion of 15.4% (95% CI: 10.8, 21.6), in which they received psychological counseling in the same clinic by psychotherapist; and 3) clinical depression BDI-II ≥20 with a prevalence of 11.6% (95% CI: 8.1, 16.3) of undiagnosed depression cases which all were referred to psychology health centers for further evaluation and treatment (Table 2).\\nDepression status prevalence proportion (n= 538)\\n\\nRelationship of depression status and predictors\\n\\nUnder standard complex survey data setting, the Chi-squared test was used to compare categorical variables of depression status and other explanatory variables. Table 3 shows that depression status had relations with the categorical variables: level of education and BMI categories.\\nRelation between depression status and participants characteristics\\nUnivariable linear regression analysis presented in Table 4 shows that increased BMI (β = 0.11, 95% CI: 0.01, 0.21), being non-adherent to antihypertensive medications (β = 1.3, 95% CI: 0.5, 2.1) and having better healthcare system support (β = 2.8, 95% CI: 1.2, 4.4) were significantly associated with increased BDI-II score. In contrast, older age (β = -1.5, 95% CI: - 0.29, -0.01), longer duration of hypertension (β = -0.12, 95% CI: -0.19, -0.04), stronger patient-doctor relationship (β = -3.7, 95% CI: -6.4, -1.1), and having superior social support (β = -7.1, 95% CI: -9.3, -4.9) were significantly associated with reduced BDI-II score.\\nUnivariate linear regression analysis of depression status and predictors\\nStatistically significant variables for multivariable analysis\\nMultivariable linear regression analysis in Table 5 revealed similar results except for increased BMI and duration of hypertension. Thus, non-adherence to antihypertensive medications (β = 0.9, 95% CI: 0.2, 1.7), having more healthcare system support (β = 2.8, 95% CI: 1.6, 3.9) and number of antihypertensive medications (β = 1.5, 95% CI: 0.6, 2.5) remain significantly positively associated with BDI-II score. On the other hand, older age (β = -0.11, 95% CI: -0.20, -0.02), having better social support (β = -6.8, 95% CI: -8.9, -4.7) and stronger relation with physician (β = -4.1, 95% CI: -6.9, -1.2) remain significantly negatively associated with BDI-II score.\\nMultivariable linear regression analysis of depression status and predictors\",\n \"To best of our knowledge, this is the first cross-sectional survey from Gaza to document the prevalence of undiagnosed depression among hypertensive patients. We found that the prevalence of undiagnosed clinical depression in our hypertensive population is almost higher than what is observed in Norway (6.2%) and South Africa (6%), and lower than in the US(44.9%), China (44.2%), Mexico (57.5%), Pakistan (40.1%), Croatia (29.6%) and Nigeria (26.7%). However, it was similar to that observed in Brazil (12.1%), Ghana (10.5%) and Netherlands (11.4%) (9, 21). In addition, this prevalence was lower than the rate reported in a systematic review and meta-analysis (26.8%) which summarized the prevalence of depression among hypertensive individuals in 41 studies (9).\\nUnfortunately, very limited data is available from Arabic regional countries. One study from Saudi Arabia found a prevalence of 20.7% (22). Still, no local prevalence was found from other parts of Palestine. Our study showed that the prevalence of depression among hypertensive patients was lower than the prevalence of patients with type two diabetic in the West Bank of Palestine (40%) (23).\\nThe associated factors found by multiple linear regression in this study were: age, number of antihypertensive medication, adherent status, healthcare system support, patient-doctor relationship and perceived social support. Increasing age was found to be an associated factor with increased depressive symptom in other studies (21, 22, 24, 25), although, our results predicted a negative relationship between age and depressive status. An explanation for this could be related to the nature of aged people in Gaza, since they have more acceptance and are adapted to their disease more than younger adults.\\nHowever, gender and smoking (21, 22, 24, 26) found to be associated factors in previous studies, did not reach statistical significant level in our study. Longer hypertension duration has been previously found to be a significantly associated factor in an Indian study (26); and was associated factor in our univariable analysis, although it could not reach significant level in multivariable analysis. In addition, in line with what has been found on a systematic review (8), we found a negative relationship between depression and adherence to antihypertensive medications. Furthermore, statistically significant association with number of antihypertensive medication and depression was observed in our study, which was supported by previous findings (24, 27).\\nIn this study, we have investigated the relation of depression status with healthcare system support, patient-doctor relationship and perceived social support. Interestingly, a positive relationship between depression and healthcare system support was found. This could be explained by the frequent visit of the patient to the primary healthcare clinic, and his increased need to its supportive aids. On the other hand, it was a negative relationship with patient-doctor relationship and social support. It is highly believed to be negative relationship between depression and perceived social support since social support is a significant predictor of depression and hypertension treatment programs (28, 29).\\nWe shed the light on the undiagnosed depression cases among hypertensive persons receiving their usual primary health care in primary healthcare clinics. The estimated prevalence proportion of undiagnosed subclinical depression cases was about one-quarter of all cases, in which half of them had moderate to severe depression status. Furthermore, age, number of antihypertensive medication, adherence status, patient-physician relationship, health-care system support and social support were associated with depression status. We suggest periodically screening of depression and adherence status as a part of routine care for hypertensive primary healthcare seekers, particularly younger hypertensive patients. We also call for exploring the ways to promote social support among them.\\nHowever, since this is the first study in Gaza Strip which assessed the psychological status among hypertensive patients, several limitations were observed. First, alcohol consumption is an important factor related to depression and adherence and may confound the reported relationships, although it has not been measured in this study. Our justification is that alcohol consumption is illegal and prohibited to be used in Gaza as Muslim citizens, and so it was difficult to get trustful answers from the participants. Thus, more than 98% of them answered no. Indeed, even if he/she was a user he/she would not tell the truth because of fear of legal prosecutions in spite of assurance about data confidentiality. As a result, we decided to omit this variable as a predictor from this study.\\nThe second limitation is that some antihypertensive medications may be related with depression. However, we were concerned only about the number of antihypertensive medications and the frequency and did not discuss the type and classes of the medications.\\nAnother limitation of this study is that the prevalence of depression would be underestimated because we excluded the already known patients with a history of depression or currently on antidepressant medications. In addition, BDI-II is a depression screening tool only and not a diagnostic tool. This will surely affect the true prevalence of depression and therefore this could be another limitation regarding the estimation of the true prevalence. However, BDI-II as a screening tool gave a good impression of the depression prevalence among hypertensive patients and was able to discover many unknown cases that were referred for further diagnostic procedures. Despite the limitations, the findings support the routine screening of depression and medication adherence status as a part of care for hypertensive patients in PHCCs.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["intro","methods","results","discussion"],"string":"[\n \"intro\",\n \"methods\",\n \"results\",\n \"discussion\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["Hypertension","Depression","Prevalence","Medication adherence"],"string":"[\n \"Hypertension\",\n \"Depression\",\n \"Prevalence\",\n \"Medication adherence\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"Introduction:\nGlobally, depression affects 350 million people around the world; it is strongly contributes to the burden of disease, and is expected to increase by 5.7% of global burden of disease and become the second after ischemic heart disease by 2020 (1). Notably, episodes of depression are accompanied with other chronic diseases especially hypertension (2,3). Hypertension affects globally one fourth of adults, and is likely to increase to one third by 2025 (4). People with hypertension and subclinical depression are at extra risk of complications such as cerebrovascular stroke, cardiovascular and kidney diseases (3, 5–7). Moreover, depression represents an important predictor among hypertensive individual whom are non-adherence to their treatment (8).\nMany studies demonstrated an increased co-occurrence of depression with hypertension in different countries (9); however, little is known about depression prevalence among hypertensive patients in Gaza Strip (GS). This study seems to be the first study aimed to estimate the prevalence and to determine the associated factors of undiagnosed depression among hypertensive patients attending primary healthcare clinics in GS.\n\nMethods and Participants:\nA cross-sectional survey as the recruitment phase of a clustered randomized controlled trial was conducted between 1st August and 30th December, 2018. We recruited 538 hypertensive persons seeking primary health care by two stages cluster random sampling from ten primary health centers. Initially, centers were randomly selected by stratified simple random sampling approach to get two centers from each governorate. Then, participants from each center were proportionally selected through a systematic random sampling method based on eligibility criteria and agreement to take part in the study.\nThis cross-sectional survey was conducted as the recruitment phase of a clustered randomized controlled trial. All eligible participants who agreed to participate in the study were enrolled regardless of their antihypertensive medication adherence status. Psychological status was initially classified according to the BDI-II score. Later, adherent status to antihypertensive medication was determined. Subsequently, the intervention of the clustered randomized controlled trial was implemented based on the adherent status results.\nMeasures and data collection: Face-to-face interview was used to collect data from participants using a structured questionnaire. The interviews lasted fifteen minutes during the clinic hours (8 am to 2 pm, five days a week). The exposure variables of the study included participants' characteristics (age, gender, marital status, employment, education level), as well as participants' health status variables including smoking status, comorbidities, body mass index (BMI), blood pressure (BP) measurement, and medication adherence status. Other predictors of interest such as social support, relationships between patients and physician and health system support were also assessed. Moreover, depressive symptoms as the outcome variable were assessed using the validated Arabic language Beck's Depression Inventory (BDI-II) (10).\nBlood pressure was measured on the right arm using mercury sphygmomanometer after completing the interview; the value was recorded in terms of mm of mercury. BMI was calculated by using the WHO chart based on weight and height. Weight and height were measured by mechanical weighing machine with height rod (Health o meter 402LB Physician Beam Scale, Height Rod).\nInstrument: The questionnaire consisted of items about the baseline characteristics of participants, clinical health history, adherence status, patient-doctor relationship, healthcare system support, perceived social support and psychological status of depressive symptoms.\nDepressive symptoms were assessed by the aid of BDI-II. A 21-item self-report inventory was designed to assess the presence and severity of depressive symptoms. Each item is rated on a four-point Likert-type scale ranging from 0 to 3, based on the severity in the last two weeks. The total score ranges from 0 to 63, with higher scores indicating more severe depressive symptoms. The results were stratified into four groups; BDI-II: 1–13 these ups and downs are considered normal, BDI-II: 14–19 are mood disturbance or mild depression, BDI-II: 20–28 are moderate depression, and BDI-II: more than 29 are severe depression (11). We provided psychological counseling by psychotherapist for those who scored from 11 to 19. Special precautions were taken for those individuals who endorsed having suicidal ideation. Mainly, we involved the family and coordinated for urgent counseling in the related psychological health center. Moreover, all cases scored 20 and above were referred to psychology health centers. Consequently, stratification into two groups was done; considering BDI-II ≥20 who have clinical depression and BDI-II <20 who do not have clinical depression (12,13).\nAdherence status was assessed by Morisky, Green, and Levine Adherence Scale (MGL), a known validated and reliable self-report medication adherence score (14). Similarly, medical comorbidities were assessed using Charlson comorbidity index, a validated and widely used weighted-index designed scoring as low and high comorbidities (15). Likewise, social support was assessed by multidimensional Scale of Perceived Social Support (MSPSS); a known valid and reliable questionnaire which measures perceptions of support from three sources: family, friends, and a significant other (16). Arabic translation was performed based on WHO five steps for process of translation and adaptation of instrument (17).\nAn Arabic validated and reliable version of patient-doctor relationship questionnaire-9 (PDRQ-9) was used to assess the relationship between patients and physicians (6,18). Likewise, healthcare system support questionnaire was used with little modifications (18, 19). The whole Arabic questionnaire content and face validity were reviewed by panel of experts. Required changes were made to clarify any ambiguity and to ensure comprehension of Palestinian participants after pilot study, detailed information about the questionnaire are available in our published article elsewhere (20).\nEligibility criteria: Palestinian citizens attending Gaza governmental primary health centers, aged above 18 years, registered as hypertension patient since at least one year and taking at least one antihypertensive medication were eligible to participate in the study. Patients with a diagnosis of cognitive impairment, history of depression or being currently on antidepressants as reported by their primary care physician were excluded from the study.\nData analysis: Standard complex survey data analysis method was performed by STATA version 14. We accounted for clustering and unresponsiveness using STATA PSU option and unequal probability of selection using sample weight variable analysis and post stratification weight for each age and sex group strata. Furthermore, since the BDI-II score did not follow normality assumption, Generalized Linear Model (GLM) with Gaussian family and log link was run. Moreover, linearized standard error which is quite robust to non-uniformity of variance was used.\nData were described using descriptive statistics; categorical variables were compared using the Chi-squared test. After checking assumption of linear regression, univariable analysis followed by multiple linear regression were performed to assess the association between depression status and all other independent variables including participants' characteristic. Statistically significant variables were included in multiple regression analysis model based on 0.1 level. However, variables had been excluded by backward stepwise elimination method.\nEthics approval and consent to participate: Prior to conducting this research study, ethical approval from Tehran University of Medical Sciences Ethical Committee (code number: IR.TUMS.SPH.REC.1396.4828) was obtained. Approvals from the Palestinian Health Research Council(PHRC/HC/322/18) and Research Committee at the Palestinian Ministry of Health were also obtained. Purposes of the study were explained to participants, and they were reassured about confidentiality of data. Each participant was asked to sign a consent paper prior to participation.\n\nResults:\nParticipants' characteristics: Five hundred and thirty-eight participants were included in the survey from the five governorates of Gaza Strip by response rate of 96%. The overall mean age was 57.1 years (95% confidence interval [CI]: 55.9, 58.2) and more than half of them (60.9%) were females. In addition, the majority of them were literate (90.2%), married (90.4%), unemployed (86.5%), and not smokers (81.4%). Since obesity is known to have BMI more than 30 kg/m2, it was most common among study population as BMI mean reported 32.3 (95% CI: 31.9, 32.6). Moreover, more than half of the participants (57.2%) had been diagnosed with hypertension for more than five years with a mean of 8.44 years (95% CI: 7.3, 9.4). Almost, two third of them (64.4%) were treated with only one antihypertensive medication once a day (64.8%); however, 35.6% were treated with two or more medications with a frequency of twice or more (35.2%). Only 14.43% of them were classified with two or more comorbidities. In addition, the participants reported a mean of depression status based on BDI-II score of 10 (95%CI: 8.4, 11.6%) (Table 1).\nParticipants characteristics (n= 538)\nDepression status: The BDI-II mean score for all participants was 10 (95% CI: 8.4, 11.6); and they were categorized into three groups: 1) normal (BDI-II 0–13) which had a prevalence proportion of 73% (95% CI: 65.6, 79.3), 2), mild clinical depression (BDI-II: 14–19) with a prevalence proportion of 15.4% (95% CI: 10.8, 21.6), in which they received psychological counseling in the same clinic by psychotherapist; and 3) clinical depression BDI-II ≥20 with a prevalence of 11.6% (95% CI: 8.1, 16.3) of undiagnosed depression cases which all were referred to psychology health centers for further evaluation and treatment (Table 2).\nDepression status prevalence proportion (n= 538)\n\nRelationship of depression status and predictors\n\nUnder standard complex survey data setting, the Chi-squared test was used to compare categorical variables of depression status and other explanatory variables. Table 3 shows that depression status had relations with the categorical variables: level of education and BMI categories.\nRelation between depression status and participants characteristics\nUnivariable linear regression analysis presented in Table 4 shows that increased BMI (β = 0.11, 95% CI: 0.01, 0.21), being non-adherent to antihypertensive medications (β = 1.3, 95% CI: 0.5, 2.1) and having better healthcare system support (β = 2.8, 95% CI: 1.2, 4.4) were significantly associated with increased BDI-II score. In contrast, older age (β = -1.5, 95% CI: - 0.29, -0.01), longer duration of hypertension (β = -0.12, 95% CI: -0.19, -0.04), stronger patient-doctor relationship (β = -3.7, 95% CI: -6.4, -1.1), and having superior social support (β = -7.1, 95% CI: -9.3, -4.9) were significantly associated with reduced BDI-II score.\nUnivariate linear regression analysis of depression status and predictors\nStatistically significant variables for multivariable analysis\nMultivariable linear regression analysis in Table 5 revealed similar results except for increased BMI and duration of hypertension. Thus, non-adherence to antihypertensive medications (β = 0.9, 95% CI: 0.2, 1.7), having more healthcare system support (β = 2.8, 95% CI: 1.6, 3.9) and number of antihypertensive medications (β = 1.5, 95% CI: 0.6, 2.5) remain significantly positively associated with BDI-II score. On the other hand, older age (β = -0.11, 95% CI: -0.20, -0.02), having better social support (β = -6.8, 95% CI: -8.9, -4.7) and stronger relation with physician (β = -4.1, 95% CI: -6.9, -1.2) remain significantly negatively associated with BDI-II score.\nMultivariable linear regression analysis of depression status and predictors\n\nDiscussion:\nTo best of our knowledge, this is the first cross-sectional survey from Gaza to document the prevalence of undiagnosed depression among hypertensive patients. We found that the prevalence of undiagnosed clinical depression in our hypertensive population is almost higher than what is observed in Norway (6.2%) and South Africa (6%), and lower than in the US(44.9%), China (44.2%), Mexico (57.5%), Pakistan (40.1%), Croatia (29.6%) and Nigeria (26.7%). However, it was similar to that observed in Brazil (12.1%), Ghana (10.5%) and Netherlands (11.4%) (9, 21). In addition, this prevalence was lower than the rate reported in a systematic review and meta-analysis (26.8%) which summarized the prevalence of depression among hypertensive individuals in 41 studies (9).\nUnfortunately, very limited data is available from Arabic regional countries. One study from Saudi Arabia found a prevalence of 20.7% (22). Still, no local prevalence was found from other parts of Palestine. Our study showed that the prevalence of depression among hypertensive patients was lower than the prevalence of patients with type two diabetic in the West Bank of Palestine (40%) (23).\nThe associated factors found by multiple linear regression in this study were: age, number of antihypertensive medication, adherent status, healthcare system support, patient-doctor relationship and perceived social support. Increasing age was found to be an associated factor with increased depressive symptom in other studies (21, 22, 24, 25), although, our results predicted a negative relationship between age and depressive status. An explanation for this could be related to the nature of aged people in Gaza, since they have more acceptance and are adapted to their disease more than younger adults.\nHowever, gender and smoking (21, 22, 24, 26) found to be associated factors in previous studies, did not reach statistical significant level in our study. Longer hypertension duration has been previously found to be a significantly associated factor in an Indian study (26); and was associated factor in our univariable analysis, although it could not reach significant level in multivariable analysis. In addition, in line with what has been found on a systematic review (8), we found a negative relationship between depression and adherence to antihypertensive medications. Furthermore, statistically significant association with number of antihypertensive medication and depression was observed in our study, which was supported by previous findings (24, 27).\nIn this study, we have investigated the relation of depression status with healthcare system support, patient-doctor relationship and perceived social support. Interestingly, a positive relationship between depression and healthcare system support was found. This could be explained by the frequent visit of the patient to the primary healthcare clinic, and his increased need to its supportive aids. On the other hand, it was a negative relationship with patient-doctor relationship and social support. It is highly believed to be negative relationship between depression and perceived social support since social support is a significant predictor of depression and hypertension treatment programs (28, 29).\nWe shed the light on the undiagnosed depression cases among hypertensive persons receiving their usual primary health care in primary healthcare clinics. The estimated prevalence proportion of undiagnosed subclinical depression cases was about one-quarter of all cases, in which half of them had moderate to severe depression status. Furthermore, age, number of antihypertensive medication, adherence status, patient-physician relationship, health-care system support and social support were associated with depression status. We suggest periodically screening of depression and adherence status as a part of routine care for hypertensive primary healthcare seekers, particularly younger hypertensive patients. We also call for exploring the ways to promote social support among them.\nHowever, since this is the first study in Gaza Strip which assessed the psychological status among hypertensive patients, several limitations were observed. First, alcohol consumption is an important factor related to depression and adherence and may confound the reported relationships, although it has not been measured in this study. Our justification is that alcohol consumption is illegal and prohibited to be used in Gaza as Muslim citizens, and so it was difficult to get trustful answers from the participants. Thus, more than 98% of them answered no. Indeed, even if he/she was a user he/she would not tell the truth because of fear of legal prosecutions in spite of assurance about data confidentiality. As a result, we decided to omit this variable as a predictor from this study.\nThe second limitation is that some antihypertensive medications may be related with depression. However, we were concerned only about the number of antihypertensive medications and the frequency and did not discuss the type and classes of the medications.\nAnother limitation of this study is that the prevalence of depression would be underestimated because we excluded the already known patients with a history of depression or currently on antidepressant medications. In addition, BDI-II is a depression screening tool only and not a diagnostic tool. This will surely affect the true prevalence of depression and therefore this could be another limitation regarding the estimation of the true prevalence. However, BDI-II as a screening tool gave a good impression of the depression prevalence among hypertensive patients and was able to discover many unknown cases that were referred for further diagnostic procedures. Despite the limitations, the findings support the routine screening of depression and medication adherence status as a part of care for hypertensive patients in PHCCs.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nThe aim of this study was to estimate the prevalence and to determine the associated factors of undiagnosed depression amongst hypertensive patients (HTNP) at primary health care centers (PHCC) in Gaza.\n\nMethods:\nA cross-sectional survey was conducted including 538 HTNP as a recruitment phase of a clustered randomized controlled trial. Data were collected through face-to-face structured interview, and depression status was assessed by Beck's Depression Inventory (BDI-II). Data were analyzed by STATA version 14 using standard complex survey analyses, accounted for unresponsiveness and clustering approach. Generalized linear regression analysis was performed to assess associations.\n\nResults:\nThe prevalence of undiagnosed clinical depression was 11.6% (95% confidence interval [CI]: 8.1, 16.3). Moreover, prevalence of 15.4% (95% CI: 10.8, 21.6) was found for mild depression symptoms. We found that non-adherence to antihypertensive medications (AHTNM) (β = 0.9, 95% CI: 0.17, 1.7), having more health-care system support (β = 2.8, 95% CI: 1.6, 3.9) and number of AHTNM (β = 1.5, 95% CI: 0.6, 2.5) remain significantly positively associated with BDI-II score. On the other hand, older age (β = -0.1, 95% CI: -0.2, -0.02), having better social support (β = -6.8, 95% CI: -8.9, -4.7) and having stronger patient-doctor relationship (β = -4.1, 95% CI: -6.9, -1.2) kept significantly negative association.\n\nConclusions:\nThe prevalence of undiagnosed depression was about one-quarter of all cases; half of them were moderate to severe. Routine screening of depression status should be a part of the care of HTNP in PHCC."},"background_conclusion_text":{"kind":"null"},"background_conclusion_abstract":{"kind":"null"},"whole_article_text_length":{"kind":"number","value":3348,"string":"3,348"},"whole_article_abstract_length":{"kind":"number","value":350,"string":"350"},"other_sections_lengths":{"kind":"list like","value":[],"string":"[]","isConversational":false},"num_sections":{"kind":"number","value":4,"string":"4"},"most_frequent_words":{"kind":"list like","value":["depression","status","support","study","ii","95","bdi ii","bdi","ci","95 ci"],"string":"[\n \"depression\",\n \"status\",\n \"support\",\n \"study\",\n \"ii\",\n \"95\",\n \"bdi ii\",\n \"bdi\",\n \"ci\",\n \"95 ci\"\n]","isConversational":false},"keybert_topics":{"kind":"list like","value":["clinical depression hypertensive","predictor depression hypertension","depression hypertension different","occurrence depression hypertension","prevalence depression 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status | support | study | ii | 95 | bdi ii | bdi | ci | 95 ci [SUMMARY]"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] depression | status | support | study | ii | 95 | bdi ii | bdi | ci | 95 ci [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"null"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] depression | status | support | study | ii | 95 | bdi ii | bdi | ci | 95 ci [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] depression | disease | hypertension | gs | diseases | burden disease | burden | increase | globally | affects [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] status | health | questionnaire | bdi ii | bdi | ii | participants | support | depressive symptoms | symptoms [SUMMARY]"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] ci | 95 | 95 ci | depression | status | ii | bdi | bdi ii | depression status | mean [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"null"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] depression | ci | 95 | 95 ci | status | support | prevalence | study | bdi | bdi ii [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"null"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Gaza [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"string","value":"[CONTENT] 538 ||| Beck's Depression Inventory | BDI-II ||| 14 ||| [SUMMARY]"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 11.6% | 95% ||| CI | 8.1 | 16.3 ||| 15.4% | 95% | CI | 10.8 | 21.6 ||| 0.9 | 95% | CI | 0.17 | 1.7 | 2.8 | 95% | CI | 1.6 | 3.9 | AHTNM | 1.5 | 95% | CI | 0.6 | 2.5 | BDI-II ||| 95% | CI | 95% | CI | 95% | CI ||| [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"null"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Gaza ||| 538 ||| Beck's Depression Inventory | BDI-II ||| 14 ||| ||| ||| 11.6% | 95% ||| CI | 8.1 | 16.3 ||| 15.4% | 95% | CI | 10.8 | 21.6 ||| 0.9 | 95% | CI | 0.17 | 1.7 | 2.8 | 95% | CI | 1.6 | 3.9 | AHTNM | 1.5 | 95% | CI | 0.6 | 2.5 | BDI-II ||| 95% | CI | 95% | CI | 95% | CI ||| ||| one-quarter | half ||| PHCC [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"null"}}},{"rowIdx":95,"cells":{"title":{"kind":"string","value":"The magnitude of syphilis: from prevalence to vertical transmission."},"pmid":{"kind":"string","value":"29267586"},"background_abstract":{"kind":"string","value":"In 2013, the World Health Organization (WHO) reported that 1.9 million pregnant women were infected with syphilis worldwide, of which 66.5% had adverse fetal effects in cases of untreated syphilis. Congenital syphilis contributes significantly to infant mortality, accounting for 305,000 perinatal deaths worldwide annually."},"background_abstract_label":{"kind":"string","value":"INTRODUCTION"},"methods_abstract":{"kind":"string","value":"a cross-sectional study with data collected from 2041 parturients who had undergone treatment between 2012 and 2014 in the maternity section of the Pedro Ernesto Hospital of the State University of Rio de Janeiro, in the metropolitan area of Rio de Janeiro. The inclusion criterion was positive VDRL and treponemal test in a hospital environment."},"methods_abstract_label":{"kind":"string","value":"MATERIAL AND METHODS"},"results_abstract":{"kind":"string","value":"the prevalence of syphilis in pregnant women was 4.1% in 2012, 3.1% in 2013 and 5% in 2014, with official reporting of 15.6%, 25.0% and 48.1%, respectively. The incidence of congenital syphilis (CS) was 22/1,000 in live births (LB) in 2012; 17/1,000 LB in 2013 and 44.8/1,000 LB in 2014. CS underreporting during the period was 6.7%. Vertical transmission occurred in 65.8% of infants from infected mothers. It was concluded that, in 34.6% of the CS cases, maternal VDRL titers were = 1/4."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"Results demonstrate the magnitude of the disease, fragility of the reporting system in the assessment of the actual prevalence, impact on perinatal outcomes, and they are a warning about the real situation of syphilis, which is still underestimated in the State."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Brazil","Cross-Sectional Studies","Female","Humans","Incidence","Infant, Newborn","Infectious Disease Transmission, Vertical","Pregnancy","Pregnancy Complications, Infectious","Prevalence","Syphilis","Syphilis, Congenital"],"string":"[\n \"Brazil\",\n \"Cross-Sectional Studies\",\n \"Female\",\n \"Humans\",\n \"Incidence\",\n \"Infant, Newborn\",\n \"Infectious Disease Transmission, Vertical\",\n \"Pregnancy\",\n \"Pregnancy Complications, Infectious\",\n \"Prevalence\",\n \"Syphilis\",\n \"Syphilis, Congenital\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"5738763"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Syphilis is a sexually transmitted disease caused by the bacterium Treponema pallidum. Infection can be transmitted to the unborn child through the placenta at any gestational stage. In exceptional cases, syphilis can be transmitted at birth through the child's contact with the birth canal, when there are maternal genital lesions (direct transmission). Syphilis may only be transmitted through breastfeeding if there are breast syphilis lesions\n1\n.\nCongenital infection is associated with adverse outcomes, including: perinatal death, preterm birth, low birth weight, congenital anomalies, active syphilis in the newborn (NB), and long-term sequelae, such as deafness and neurological impairment\n2\n.\nThere is a significant global increase in the prevalence of syphilis in pregnant women, despite all efforts from governments to control the disease. In 2013, the WHO reported 1.9 million pregnant women infected with syphilis worldwide, with 66.5% of adverse fetal outcomes occurring in cases of untreated syphilis\n3\n. Congenital syphilis significantly contributes to infant mortality, accounting for 305,000 perinatal deaths per year worldwide\n4\n. There are more newborns affected by syphilis than by any other neonatal infection. It is estimated that 520,000 adverse fetal outcomes occur worldwide each year, making congenital syphilis more common than congenital HIV infection\n5\n. In areas where syphilis is prevalent, about half of the stillbirth rate may have been caused solely by this infection\n6\n. The direct medical cost associated with adverse fetal outcomes resulting from syphilis is U$ 309 million dollars a year\n4\n.\nHigh rates of syphilis in parturients mean a high incidence of congenital syphilis and millions of missed opportunities to save lives during pre-natal care. Over the past ten years, the infant mortality rate due to syphilis increased 150% in Brazil, from 2.2 per 100,000 live births (LB) in 2004 to 5.5 per 100,000 LB in 2013. By 2015, the State of Rio de Janeiro had the highest rate of syphilis during pregnancy (2.2%) and of congenital syphilis in the country (16 cases per 1,000 LB)\n7\n\n,\n\n8\n.\nThe last syphilis monitoring study in pregnant women performed in Brazil in 2014, with a statistical sample from public and private hospitals, estimated the prevalence of syphilis at 1.02%, with no significant regional differences\n9\n.\nAccording to the prediction curve of the disease, the Ministry of Health (MH) expected 40,000 cases in pregnant women in Brazil in 2016. An epidemic of the disease in the country was confirmed by the Ministry of Health in 2016. The increased incidence of syphilis in pregnant women is alarming\n8\n. In 2015, according to the MH, the States of Rio de Janeiro and Mato Grosso do Sul were the most affected in the country, reaching a syphilis rate in pregnant women of 2.2%\n8\n.\nThe national studies show differences in the prevalence of the disease according to the region studied, with rates ranging around 0.4% in Vitória (Espírito Santo)\n10\n, Itajaí (Santa Catarina)\n11\n and 7.7% in Fortaleza (Ceará)\n12\n.\nThe analysis of international studies on the subject in countries such as Haiti\n13\n shows that the prevalence of syphilis in pregnant women reaches 7.6%, while in other countries, such as Canada, syphilis has already been eliminated\n14\n. The WHO certified Cuba in 2013 as the first country to eliminate the vertical transmission of HIV and syphilis during pregnancy. Six other countries are currently able to request the WHO to validate the double elimination, including Canada and the United States\n15\n.\n Aim To estimate the prevalence and coverage of syphilis reporting in parturients, the incidence of congenital syphilis and its corresponding underreporting, as well as the rate of vertical transmission of infection.\nTo estimate the prevalence and coverage of syphilis reporting in parturients, the incidence of congenital syphilis and its corresponding underreporting, as well as the rate of vertical transmission of infection."},"methods_title":{"kind":"null"},"methods_text":{"kind":"null"},"results_title":{"kind":"string","value":"RESULTS"},"results_text":{"kind":"string","value":"A total of 2,041 parturients were studied in HUPE: 735 in 2012, 664 in 2013 and 642 in 2014. After the exclusion of 19 pregnant women due to serological scarring or false-positive results (treponemal negative test), 79 cases of parturients with syphilis were identified: 32 cases in 2012, 20 in 2013 and 27 in 2014. Access to the result of the treponemal and non-treponemal tests for syphilis was obtained from all the pregnant women admitted during the period.\nThe search on SINASC, considering HUPE as the place of birth, totaled 1,956 live births in HUPE for the three years studied, with 773 live births in 2012, 647 in 2013 and 536 in 2014. The prevalence of syphilis in the HUPE, using the number of live births officially described in SINASC as the denominator, was 4.1% in 2012, 3.1% in 2013 and 5.0% in 2014. Between 2012 and 2014, there was an increase on the prevalence of the disease (Table 1).\nLegend: live births (LB); live birth information system (SINASC); confidence limit (CL)\nThe search on SINAN came up with 23 cases of syphilis in HUPE for the three years studied (2012 = 5, 2013 = 5 and 2014 = 13). The research showed 79 cases in the three years (2012 = 32, 2013 = 20 and 2014 = 27).\nTherefore, 15.6% of cases in 2012, 25.0% in 2013 and 48.1% in 2014 were reported, which shows a significant increase for the period.\nThree parturients (3.8%) from HUPE were double counted in the prevalence calculation, when they were admitted with syphilis at the time of delivery of subsequent pregnancies. One of the three parturients had VDRL titer =1/4 and progressed to 1/128 in the next gestation. The other two had 1/32 titers in the first gestation and had titers of 1/32 and 1/128, respectively, in subsequent pregnancies.\nThe incidence of CS was 22/1,000 LB in 2012, 17/1,000 LB in 2013 and 44.8/ 1,000 LB in 2014. The notification of congenital syphilis reached 92.3% of the cases and only four cases were not officially reported. The present study demonstrated a vertical transmission rate of 65.8%, with 52 newborns affected by congenital syphilis.\nFrom the group of parturients with vertical transmission of the disease, 18 mothers had VDRL titers = 1/4, corresponding to 34.6% of the cases of congenital syphilis. The vertical transmission occurred in two consecutive pregnancies in one case that was within the low titers group (= 1/4)."},"conclusions_title":{"kind":"string","value":"CONCLUSIONS"},"conclusions_text":{"kind":"string","value":"The estimated prevalence of syphilis in pregnant women in Rio de Janeiro is at least twice as high as previously reported by regional studies and epidemiological surveillance data in the State, reaching levels found in African countries and higher than the figures for other countries in Latin America. These values are a warning on the magnitude of the disease in the State of Rio de Janeiro, its upward curve and its consequences. Our reporting system has proven to be fragile and unable to assess the current prevalence of syphilis in Rio de Janeiro, which is underestimated. Due to the high prevalence of syphilis among parturients, there was a high rate of CS and vertical transmission that occurred even in pregnant women with low VDRL titers, emphasizing the importance of evaluating low titers in the management of these patients.\nIt is a responsibility of the health professionals, who daily handle this sad reality, to strive for excellence in caring for pregnant women. Even in the 21st century, the high rates of syphilis in parturients and CS in Brazil are still ignored and underestimated."},"other_sections_titles":{"kind":"list like","value":["Aim","Study design","Studied population","Data collection","Inclusion criterion","Exclusion criterion","Variable definition","Epidemiological definitions","Data analysis","Considerations on ethical aspects"],"string":"[\n \"Aim\",\n \"Study design\",\n \"Studied population\",\n \"Data collection\",\n \"Inclusion criterion\",\n \"Exclusion criterion\",\n \"Variable definition\",\n \"Epidemiological definitions\",\n \"Data analysis\",\n \"Considerations on ethical aspects\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["To estimate the prevalence and coverage of syphilis reporting in parturients, the incidence of congenital syphilis and its corresponding underreporting, as well as the rate of vertical transmission of infection.","Cross-sectional study.","A total of 2,041 pregnant women were recruited after admission to a public hospital in the metropolitan region of Rio de Janeiro, between January 2012 and December 2014. The institution involved in the study was the Pedro Ernesto Hospital of the State University of Rio de Janeiro (HUPE / UERJ).","At HUPE, blood samples are routinely collected for syphilis investigation upon admission, with both non-treponemal testing (VDRL-Venereal Disease Research Laboratory) and confirmatory treponemal test (TPHA-Treponema Pallidum Hemagglutination). The confirmation via the treponemal test is important due to the possibility of a false-positive result. All the tests were performed in the clinical analysis laboratory of HUPE. According to the protocol of the Brazilian Ministry of Health, blood samples are collected for syphilis testing from all parturients. An active search of syphilis serology results (treponemal and non-treponemal tests) with the respective titrations was conducted by accessing the database of the hospital laboratory. A thorough review of the medical records of pregnant women and their newborns was also conducted. Data from the HUPE epidemiology service were evaluated in order to identify the total number of infected pregnant women and newborns, in order to avoid underestimating the results. A survey was conducted on the website of the State's Department of Health website to collect data on syphilis reports during pregnancy, as well as reports of congenital syphilis on the SINAN (National Disease Notification System) and the number of live births in the SINASC (Live Births Notification System).","The following parturients were eligible for the study: pregnant women admitted for delivery with a live fetus of any gestational age and weight, stillbirths with gestational age equal to22 weeks or weight equal to 500 grams.\nThe following cases were defined as syphilis during pregnancy: all cases in which the parturient was admitted with laboratory evidence of positive VDRL (any titer) collected at the time of admission and confirmed by the treponemal test; all cases in which the parturient's newborn (stillbirth or live birth) has been reported as a case of congenital syphilis (CS), identified in any of the information systems consulted.","The following parturients were excluded: the ones with positive VDRL resulting from adequately treated previous syphilis (complete treatment with benzathine penicillin, according to the clinical stage of the disease, complete treatment of the partner, documentation confirming the couple's treatment, drop in VDRL titers after adequate treatment, treatment completed more than 30 days before delivery).","The following situations were included as CS cases: all the gestation products (live births or stillborn) identified in any of the information systems as premature congenital syphilis; all newborns with VDRL titers higher than the maternal ones; all newborns with clinical manifestations suggestive of syphilis or complementary tests pointing to CS\n7\n.","According to the recommendation of the strategic management of the MH, the prevalence of syphilis in pregnant women is the ratio of the number of cases of syphilis detected in pregnant women to every 1,000 live births, within a geographical space and for a given period. For the purpose of calculating the prevalence, we used as the numerator the number of cases of syphilis detected in pregnant women, in a given notification year and place of residence multiplied by 1,000 and divided by the total number of live births in the same place and in the same reported year\n16\n. The numerator could be found in the SINAN (national disease notification system) and the denominator in the SINASC (live births information system).\nFor the calculation of the prevalence of parturients as a percentage, the number of cases of syphilis in parturients identified in the study was used as the numerator; the number of live births in that place and period, multiplied by 100 as the denominator.\nTo evaluate underreporting, we divided the number of parturients with syphilis in SINAN by the number of parturients with syphilis in hospitals x 100.\nFollowing the MH recommendation for the calculation of the CS incidence, the number of CS cases identified in the study was used as the numerator and the number of live births at that location and period, multiplied by 1000, as the denominator.\nIn order to evaluate the underreporting of congenital syphilis, we divided the number of CS cases by the number of cases reported in SINAN x 100.","The sample size was calculated considering an expected prevalence of syphilis in pregnant women according to the MH in public hospitals of 1.1% in 2006, in Brazil\n9\n. To perform this calculation, assuming an error of 5% and a confidence interval (CI) of 95% the sample size was calculated to be 17 for 95% CI and 46 for 99,9% CI.\nCollected variables were used in the comparative analyzes to identify prevalence factors. The results were expressed as percentages. The process of entering and analyzing statistical data was performed through the EPIINFO 3.5.2 version 3.0.1 computing program.","The research project was carried out within the standards required by the Declaration of Helsinki and Resolution 466 of December 12, 2012, and approved by the Research Ethics Committee of the UERJ (COEP) in July 2012, process N° 034.3.2012 and sponsored by FAPERJ, process N° E-26/110.351/2012."],"string":"[\n \"To estimate the prevalence and coverage of syphilis reporting in parturients, the incidence of congenital syphilis and its corresponding underreporting, as well as the rate of vertical transmission of infection.\",\n \"Cross-sectional study.\",\n \"A total of 2,041 pregnant women were recruited after admission to a public hospital in the metropolitan region of Rio de Janeiro, between January 2012 and December 2014. The institution involved in the study was the Pedro Ernesto Hospital of the State University of Rio de Janeiro (HUPE / UERJ).\",\n \"At HUPE, blood samples are routinely collected for syphilis investigation upon admission, with both non-treponemal testing (VDRL-Venereal Disease Research Laboratory) and confirmatory treponemal test (TPHA-Treponema Pallidum Hemagglutination). The confirmation via the treponemal test is important due to the possibility of a false-positive result. All the tests were performed in the clinical analysis laboratory of HUPE. According to the protocol of the Brazilian Ministry of Health, blood samples are collected for syphilis testing from all parturients. An active search of syphilis serology results (treponemal and non-treponemal tests) with the respective titrations was conducted by accessing the database of the hospital laboratory. A thorough review of the medical records of pregnant women and their newborns was also conducted. Data from the HUPE epidemiology service were evaluated in order to identify the total number of infected pregnant women and newborns, in order to avoid underestimating the results. A survey was conducted on the website of the State's Department of Health website to collect data on syphilis reports during pregnancy, as well as reports of congenital syphilis on the SINAN (National Disease Notification System) and the number of live births in the SINASC (Live Births Notification System).\",\n \"The following parturients were eligible for the study: pregnant women admitted for delivery with a live fetus of any gestational age and weight, stillbirths with gestational age equal to22 weeks or weight equal to 500 grams.\\nThe following cases were defined as syphilis during pregnancy: all cases in which the parturient was admitted with laboratory evidence of positive VDRL (any titer) collected at the time of admission and confirmed by the treponemal test; all cases in which the parturient's newborn (stillbirth or live birth) has been reported as a case of congenital syphilis (CS), identified in any of the information systems consulted.\",\n \"The following parturients were excluded: the ones with positive VDRL resulting from adequately treated previous syphilis (complete treatment with benzathine penicillin, according to the clinical stage of the disease, complete treatment of the partner, documentation confirming the couple's treatment, drop in VDRL titers after adequate treatment, treatment completed more than 30 days before delivery).\",\n \"The following situations were included as CS cases: all the gestation products (live births or stillborn) identified in any of the information systems as premature congenital syphilis; all newborns with VDRL titers higher than the maternal ones; all newborns with clinical manifestations suggestive of syphilis or complementary tests pointing to CS\\n7\\n.\",\n \"According to the recommendation of the strategic management of the MH, the prevalence of syphilis in pregnant women is the ratio of the number of cases of syphilis detected in pregnant women to every 1,000 live births, within a geographical space and for a given period. For the purpose of calculating the prevalence, we used as the numerator the number of cases of syphilis detected in pregnant women, in a given notification year and place of residence multiplied by 1,000 and divided by the total number of live births in the same place and in the same reported year\\n16\\n. The numerator could be found in the SINAN (national disease notification system) and the denominator in the SINASC (live births information system).\\nFor the calculation of the prevalence of parturients as a percentage, the number of cases of syphilis in parturients identified in the study was used as the numerator; the number of live births in that place and period, multiplied by 100 as the denominator.\\nTo evaluate underreporting, we divided the number of parturients with syphilis in SINAN by the number of parturients with syphilis in hospitals x 100.\\nFollowing the MH recommendation for the calculation of the CS incidence, the number of CS cases identified in the study was used as the numerator and the number of live births at that location and period, multiplied by 1000, as the denominator.\\nIn order to evaluate the underreporting of congenital syphilis, we divided the number of CS cases by the number of cases reported in SINAN x 100.\",\n \"The sample size was calculated considering an expected prevalence of syphilis in pregnant women according to the MH in public hospitals of 1.1% in 2006, in Brazil\\n9\\n. To perform this calculation, assuming an error of 5% and a confidence interval (CI) of 95% the sample size was calculated to be 17 for 95% CI and 46 for 99,9% CI.\\nCollected variables were used in the comparative analyzes to identify prevalence factors. The results were expressed as percentages. The process of entering and analyzing statistical data was performed through the EPIINFO 3.5.2 version 3.0.1 computing program.\",\n \"The research project was carried out within the standards required by the Declaration of Helsinki and Resolution 466 of December 12, 2012, and approved by the Research Ethics Committee of the UERJ (COEP) in July 2012, process N° 034.3.2012 and sponsored by FAPERJ, process N° E-26/110.351/2012.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","Aim","MATERIALS AND METHODS","Study design","Studied population","Data collection","Inclusion criterion","Exclusion criterion","Variable definition","Epidemiological definitions","Data analysis","Considerations on ethical aspects","RESULTS","DISCUSSION","CONCLUSIONS"],"string":"[\n \"INTRODUCTION\",\n \"Aim\",\n \"MATERIALS AND METHODS\",\n \"Study design\",\n \"Studied population\",\n \"Data collection\",\n \"Inclusion criterion\",\n \"Exclusion criterion\",\n \"Variable definition\",\n \"Epidemiological definitions\",\n \"Data analysis\",\n \"Considerations on ethical aspects\",\n \"RESULTS\",\n \"DISCUSSION\",\n \"CONCLUSIONS\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Syphilis is a sexually transmitted disease caused by the bacterium Treponema pallidum. Infection can be transmitted to the unborn child through the placenta at any gestational stage. In exceptional cases, syphilis can be transmitted at birth through the child's contact with the birth canal, when there are maternal genital lesions (direct transmission). Syphilis may only be transmitted through breastfeeding if there are breast syphilis lesions\n1\n.\nCongenital infection is associated with adverse outcomes, including: perinatal death, preterm birth, low birth weight, congenital anomalies, active syphilis in the newborn (NB), and long-term sequelae, such as deafness and neurological impairment\n2\n.\nThere is a significant global increase in the prevalence of syphilis in pregnant women, despite all efforts from governments to control the disease. In 2013, the WHO reported 1.9 million pregnant women infected with syphilis worldwide, with 66.5% of adverse fetal outcomes occurring in cases of untreated syphilis\n3\n. Congenital syphilis significantly contributes to infant mortality, accounting for 305,000 perinatal deaths per year worldwide\n4\n. There are more newborns affected by syphilis than by any other neonatal infection. It is estimated that 520,000 adverse fetal outcomes occur worldwide each year, making congenital syphilis more common than congenital HIV infection\n5\n. In areas where syphilis is prevalent, about half of the stillbirth rate may have been caused solely by this infection\n6\n. The direct medical cost associated with adverse fetal outcomes resulting from syphilis is U$ 309 million dollars a year\n4\n.\nHigh rates of syphilis in parturients mean a high incidence of congenital syphilis and millions of missed opportunities to save lives during pre-natal care. Over the past ten years, the infant mortality rate due to syphilis increased 150% in Brazil, from 2.2 per 100,000 live births (LB) in 2004 to 5.5 per 100,000 LB in 2013. By 2015, the State of Rio de Janeiro had the highest rate of syphilis during pregnancy (2.2%) and of congenital syphilis in the country (16 cases per 1,000 LB)\n7\n\n,\n\n8\n.\nThe last syphilis monitoring study in pregnant women performed in Brazil in 2014, with a statistical sample from public and private hospitals, estimated the prevalence of syphilis at 1.02%, with no significant regional differences\n9\n.\nAccording to the prediction curve of the disease, the Ministry of Health (MH) expected 40,000 cases in pregnant women in Brazil in 2016. An epidemic of the disease in the country was confirmed by the Ministry of Health in 2016. The increased incidence of syphilis in pregnant women is alarming\n8\n. In 2015, according to the MH, the States of Rio de Janeiro and Mato Grosso do Sul were the most affected in the country, reaching a syphilis rate in pregnant women of 2.2%\n8\n.\nThe national studies show differences in the prevalence of the disease according to the region studied, with rates ranging around 0.4% in Vitória (Espírito Santo)\n10\n, Itajaí (Santa Catarina)\n11\n and 7.7% in Fortaleza (Ceará)\n12\n.\nThe analysis of international studies on the subject in countries such as Haiti\n13\n shows that the prevalence of syphilis in pregnant women reaches 7.6%, while in other countries, such as Canada, syphilis has already been eliminated\n14\n. The WHO certified Cuba in 2013 as the first country to eliminate the vertical transmission of HIV and syphilis during pregnancy. Six other countries are currently able to request the WHO to validate the double elimination, including Canada and the United States\n15\n.\n Aim To estimate the prevalence and coverage of syphilis reporting in parturients, the incidence of congenital syphilis and its corresponding underreporting, as well as the rate of vertical transmission of infection.\nTo estimate the prevalence and coverage of syphilis reporting in parturients, the incidence of congenital syphilis and its corresponding underreporting, as well as the rate of vertical transmission of infection.","To estimate the prevalence and coverage of syphilis reporting in parturients, the incidence of congenital syphilis and its corresponding underreporting, as well as the rate of vertical transmission of infection."," Study design Cross-sectional study.\nCross-sectional study.\n Studied population A total of 2,041 pregnant women were recruited after admission to a public hospital in the metropolitan region of Rio de Janeiro, between January 2012 and December 2014. The institution involved in the study was the Pedro Ernesto Hospital of the State University of Rio de Janeiro (HUPE / UERJ).\nA total of 2,041 pregnant women were recruited after admission to a public hospital in the metropolitan region of Rio de Janeiro, between January 2012 and December 2014. The institution involved in the study was the Pedro Ernesto Hospital of the State University of Rio de Janeiro (HUPE / UERJ).\n Data collection At HUPE, blood samples are routinely collected for syphilis investigation upon admission, with both non-treponemal testing (VDRL-Venereal Disease Research Laboratory) and confirmatory treponemal test (TPHA-Treponema Pallidum Hemagglutination). The confirmation via the treponemal test is important due to the possibility of a false-positive result. All the tests were performed in the clinical analysis laboratory of HUPE. According to the protocol of the Brazilian Ministry of Health, blood samples are collected for syphilis testing from all parturients. An active search of syphilis serology results (treponemal and non-treponemal tests) with the respective titrations was conducted by accessing the database of the hospital laboratory. A thorough review of the medical records of pregnant women and their newborns was also conducted. Data from the HUPE epidemiology service were evaluated in order to identify the total number of infected pregnant women and newborns, in order to avoid underestimating the results. A survey was conducted on the website of the State's Department of Health website to collect data on syphilis reports during pregnancy, as well as reports of congenital syphilis on the SINAN (National Disease Notification System) and the number of live births in the SINASC (Live Births Notification System).\nAt HUPE, blood samples are routinely collected for syphilis investigation upon admission, with both non-treponemal testing (VDRL-Venereal Disease Research Laboratory) and confirmatory treponemal test (TPHA-Treponema Pallidum Hemagglutination). The confirmation via the treponemal test is important due to the possibility of a false-positive result. All the tests were performed in the clinical analysis laboratory of HUPE. According to the protocol of the Brazilian Ministry of Health, blood samples are collected for syphilis testing from all parturients. An active search of syphilis serology results (treponemal and non-treponemal tests) with the respective titrations was conducted by accessing the database of the hospital laboratory. A thorough review of the medical records of pregnant women and their newborns was also conducted. Data from the HUPE epidemiology service were evaluated in order to identify the total number of infected pregnant women and newborns, in order to avoid underestimating the results. A survey was conducted on the website of the State's Department of Health website to collect data on syphilis reports during pregnancy, as well as reports of congenital syphilis on the SINAN (National Disease Notification System) and the number of live births in the SINASC (Live Births Notification System).\n Inclusion criterion The following parturients were eligible for the study: pregnant women admitted for delivery with a live fetus of any gestational age and weight, stillbirths with gestational age equal to22 weeks or weight equal to 500 grams.\nThe following cases were defined as syphilis during pregnancy: all cases in which the parturient was admitted with laboratory evidence of positive VDRL (any titer) collected at the time of admission and confirmed by the treponemal test; all cases in which the parturient's newborn (stillbirth or live birth) has been reported as a case of congenital syphilis (CS), identified in any of the information systems consulted.\nThe following parturients were eligible for the study: pregnant women admitted for delivery with a live fetus of any gestational age and weight, stillbirths with gestational age equal to22 weeks or weight equal to 500 grams.\nThe following cases were defined as syphilis during pregnancy: all cases in which the parturient was admitted with laboratory evidence of positive VDRL (any titer) collected at the time of admission and confirmed by the treponemal test; all cases in which the parturient's newborn (stillbirth or live birth) has been reported as a case of congenital syphilis (CS), identified in any of the information systems consulted.\n Exclusion criterion The following parturients were excluded: the ones with positive VDRL resulting from adequately treated previous syphilis (complete treatment with benzathine penicillin, according to the clinical stage of the disease, complete treatment of the partner, documentation confirming the couple's treatment, drop in VDRL titers after adequate treatment, treatment completed more than 30 days before delivery).\nThe following parturients were excluded: the ones with positive VDRL resulting from adequately treated previous syphilis (complete treatment with benzathine penicillin, according to the clinical stage of the disease, complete treatment of the partner, documentation confirming the couple's treatment, drop in VDRL titers after adequate treatment, treatment completed more than 30 days before delivery).\n Variable definition The following situations were included as CS cases: all the gestation products (live births or stillborn) identified in any of the information systems as premature congenital syphilis; all newborns with VDRL titers higher than the maternal ones; all newborns with clinical manifestations suggestive of syphilis or complementary tests pointing to CS\n7\n.\nThe following situations were included as CS cases: all the gestation products (live births or stillborn) identified in any of the information systems as premature congenital syphilis; all newborns with VDRL titers higher than the maternal ones; all newborns with clinical manifestations suggestive of syphilis or complementary tests pointing to CS\n7\n.\n Epidemiological definitions According to the recommendation of the strategic management of the MH, the prevalence of syphilis in pregnant women is the ratio of the number of cases of syphilis detected in pregnant women to every 1,000 live births, within a geographical space and for a given period. For the purpose of calculating the prevalence, we used as the numerator the number of cases of syphilis detected in pregnant women, in a given notification year and place of residence multiplied by 1,000 and divided by the total number of live births in the same place and in the same reported year\n16\n. The numerator could be found in the SINAN (national disease notification system) and the denominator in the SINASC (live births information system).\nFor the calculation of the prevalence of parturients as a percentage, the number of cases of syphilis in parturients identified in the study was used as the numerator; the number of live births in that place and period, multiplied by 100 as the denominator.\nTo evaluate underreporting, we divided the number of parturients with syphilis in SINAN by the number of parturients with syphilis in hospitals x 100.\nFollowing the MH recommendation for the calculation of the CS incidence, the number of CS cases identified in the study was used as the numerator and the number of live births at that location and period, multiplied by 1000, as the denominator.\nIn order to evaluate the underreporting of congenital syphilis, we divided the number of CS cases by the number of cases reported in SINAN x 100.\nAccording to the recommendation of the strategic management of the MH, the prevalence of syphilis in pregnant women is the ratio of the number of cases of syphilis detected in pregnant women to every 1,000 live births, within a geographical space and for a given period. For the purpose of calculating the prevalence, we used as the numerator the number of cases of syphilis detected in pregnant women, in a given notification year and place of residence multiplied by 1,000 and divided by the total number of live births in the same place and in the same reported year\n16\n. The numerator could be found in the SINAN (national disease notification system) and the denominator in the SINASC (live births information system).\nFor the calculation of the prevalence of parturients as a percentage, the number of cases of syphilis in parturients identified in the study was used as the numerator; the number of live births in that place and period, multiplied by 100 as the denominator.\nTo evaluate underreporting, we divided the number of parturients with syphilis in SINAN by the number of parturients with syphilis in hospitals x 100.\nFollowing the MH recommendation for the calculation of the CS incidence, the number of CS cases identified in the study was used as the numerator and the number of live births at that location and period, multiplied by 1000, as the denominator.\nIn order to evaluate the underreporting of congenital syphilis, we divided the number of CS cases by the number of cases reported in SINAN x 100.\n Data analysis The sample size was calculated considering an expected prevalence of syphilis in pregnant women according to the MH in public hospitals of 1.1% in 2006, in Brazil\n9\n. To perform this calculation, assuming an error of 5% and a confidence interval (CI) of 95% the sample size was calculated to be 17 for 95% CI and 46 for 99,9% CI.\nCollected variables were used in the comparative analyzes to identify prevalence factors. The results were expressed as percentages. The process of entering and analyzing statistical data was performed through the EPIINFO 3.5.2 version 3.0.1 computing program.\nThe sample size was calculated considering an expected prevalence of syphilis in pregnant women according to the MH in public hospitals of 1.1% in 2006, in Brazil\n9\n. To perform this calculation, assuming an error of 5% and a confidence interval (CI) of 95% the sample size was calculated to be 17 for 95% CI and 46 for 99,9% CI.\nCollected variables were used in the comparative analyzes to identify prevalence factors. The results were expressed as percentages. The process of entering and analyzing statistical data was performed through the EPIINFO 3.5.2 version 3.0.1 computing program.\n Considerations on ethical aspects The research project was carried out within the standards required by the Declaration of Helsinki and Resolution 466 of December 12, 2012, and approved by the Research Ethics Committee of the UERJ (COEP) in July 2012, process N° 034.3.2012 and sponsored by FAPERJ, process N° E-26/110.351/2012.\nThe research project was carried out within the standards required by the Declaration of Helsinki and Resolution 466 of December 12, 2012, and approved by the Research Ethics Committee of the UERJ (COEP) in July 2012, process N° 034.3.2012 and sponsored by FAPERJ, process N° E-26/110.351/2012.","Cross-sectional study.","A total of 2,041 pregnant women were recruited after admission to a public hospital in the metropolitan region of Rio de Janeiro, between January 2012 and December 2014. The institution involved in the study was the Pedro Ernesto Hospital of the State University of Rio de Janeiro (HUPE / UERJ).","At HUPE, blood samples are routinely collected for syphilis investigation upon admission, with both non-treponemal testing (VDRL-Venereal Disease Research Laboratory) and confirmatory treponemal test (TPHA-Treponema Pallidum Hemagglutination). The confirmation via the treponemal test is important due to the possibility of a false-positive result. All the tests were performed in the clinical analysis laboratory of HUPE. According to the protocol of the Brazilian Ministry of Health, blood samples are collected for syphilis testing from all parturients. An active search of syphilis serology results (treponemal and non-treponemal tests) with the respective titrations was conducted by accessing the database of the hospital laboratory. A thorough review of the medical records of pregnant women and their newborns was also conducted. Data from the HUPE epidemiology service were evaluated in order to identify the total number of infected pregnant women and newborns, in order to avoid underestimating the results. A survey was conducted on the website of the State's Department of Health website to collect data on syphilis reports during pregnancy, as well as reports of congenital syphilis on the SINAN (National Disease Notification System) and the number of live births in the SINASC (Live Births Notification System).","The following parturients were eligible for the study: pregnant women admitted for delivery with a live fetus of any gestational age and weight, stillbirths with gestational age equal to22 weeks or weight equal to 500 grams.\nThe following cases were defined as syphilis during pregnancy: all cases in which the parturient was admitted with laboratory evidence of positive VDRL (any titer) collected at the time of admission and confirmed by the treponemal test; all cases in which the parturient's newborn (stillbirth or live birth) has been reported as a case of congenital syphilis (CS), identified in any of the information systems consulted.","The following parturients were excluded: the ones with positive VDRL resulting from adequately treated previous syphilis (complete treatment with benzathine penicillin, according to the clinical stage of the disease, complete treatment of the partner, documentation confirming the couple's treatment, drop in VDRL titers after adequate treatment, treatment completed more than 30 days before delivery).","The following situations were included as CS cases: all the gestation products (live births or stillborn) identified in any of the information systems as premature congenital syphilis; all newborns with VDRL titers higher than the maternal ones; all newborns with clinical manifestations suggestive of syphilis or complementary tests pointing to CS\n7\n.","According to the recommendation of the strategic management of the MH, the prevalence of syphilis in pregnant women is the ratio of the number of cases of syphilis detected in pregnant women to every 1,000 live births, within a geographical space and for a given period. For the purpose of calculating the prevalence, we used as the numerator the number of cases of syphilis detected in pregnant women, in a given notification year and place of residence multiplied by 1,000 and divided by the total number of live births in the same place and in the same reported year\n16\n. The numerator could be found in the SINAN (national disease notification system) and the denominator in the SINASC (live births information system).\nFor the calculation of the prevalence of parturients as a percentage, the number of cases of syphilis in parturients identified in the study was used as the numerator; the number of live births in that place and period, multiplied by 100 as the denominator.\nTo evaluate underreporting, we divided the number of parturients with syphilis in SINAN by the number of parturients with syphilis in hospitals x 100.\nFollowing the MH recommendation for the calculation of the CS incidence, the number of CS cases identified in the study was used as the numerator and the number of live births at that location and period, multiplied by 1000, as the denominator.\nIn order to evaluate the underreporting of congenital syphilis, we divided the number of CS cases by the number of cases reported in SINAN x 100.","The sample size was calculated considering an expected prevalence of syphilis in pregnant women according to the MH in public hospitals of 1.1% in 2006, in Brazil\n9\n. To perform this calculation, assuming an error of 5% and a confidence interval (CI) of 95% the sample size was calculated to be 17 for 95% CI and 46 for 99,9% CI.\nCollected variables were used in the comparative analyzes to identify prevalence factors. The results were expressed as percentages. The process of entering and analyzing statistical data was performed through the EPIINFO 3.5.2 version 3.0.1 computing program.","The research project was carried out within the standards required by the Declaration of Helsinki and Resolution 466 of December 12, 2012, and approved by the Research Ethics Committee of the UERJ (COEP) in July 2012, process N° 034.3.2012 and sponsored by FAPERJ, process N° E-26/110.351/2012.","A total of 2,041 parturients were studied in HUPE: 735 in 2012, 664 in 2013 and 642 in 2014. After the exclusion of 19 pregnant women due to serological scarring or false-positive results (treponemal negative test), 79 cases of parturients with syphilis were identified: 32 cases in 2012, 20 in 2013 and 27 in 2014. Access to the result of the treponemal and non-treponemal tests for syphilis was obtained from all the pregnant women admitted during the period.\nThe search on SINASC, considering HUPE as the place of birth, totaled 1,956 live births in HUPE for the three years studied, with 773 live births in 2012, 647 in 2013 and 536 in 2014. The prevalence of syphilis in the HUPE, using the number of live births officially described in SINASC as the denominator, was 4.1% in 2012, 3.1% in 2013 and 5.0% in 2014. Between 2012 and 2014, there was an increase on the prevalence of the disease (Table 1).\nLegend: live births (LB); live birth information system (SINASC); confidence limit (CL)\nThe search on SINAN came up with 23 cases of syphilis in HUPE for the three years studied (2012 = 5, 2013 = 5 and 2014 = 13). The research showed 79 cases in the three years (2012 = 32, 2013 = 20 and 2014 = 27).\nTherefore, 15.6% of cases in 2012, 25.0% in 2013 and 48.1% in 2014 were reported, which shows a significant increase for the period.\nThree parturients (3.8%) from HUPE were double counted in the prevalence calculation, when they were admitted with syphilis at the time of delivery of subsequent pregnancies. One of the three parturients had VDRL titer =1/4 and progressed to 1/128 in the next gestation. The other two had 1/32 titers in the first gestation and had titers of 1/32 and 1/128, respectively, in subsequent pregnancies.\nThe incidence of CS was 22/1,000 LB in 2012, 17/1,000 LB in 2013 and 44.8/ 1,000 LB in 2014. The notification of congenital syphilis reached 92.3% of the cases and only four cases were not officially reported. The present study demonstrated a vertical transmission rate of 65.8%, with 52 newborns affected by congenital syphilis.\nFrom the group of parturients with vertical transmission of the disease, 18 mothers had VDRL titers = 1/4, corresponding to 34.6% of the cases of congenital syphilis. The vertical transmission occurred in two consecutive pregnancies in one case that was within the low titers group (= 1/4).","The latest national studies in public or public-private hospitals that monitor the prevalence of syphilis in the country demonstrated a reduction in the prevalence of syphilis among parturients in Brazil. Sentinel studies revealed a prevalence of 1.6%\n17\n in 2004 and 1.02%\n9\n in 2014.\nHowever, the present study does not confirm this trend and demonstrates that the disease is not under control in the State of Rio de Janeiro. In 2014, the prevalence rate of 5.0% was four-fold higher than the one registered in the country in 2014 by Domingues et al.\n\n19\n and twice higher than the one registered in the State of Rio de Janeiro in 2008 by the same author\n18\n.\nThe results are similar to those described by the MH regarding the ascending curve of the disease in recent years, but they express a significantly higher prevalence when compared with data from the epidemiological surveillance agencies for the same period. In 2014, although the State of Rio de Janeiro had the highest syphilis rate in pregnant women in the country (2.2%), the prevalence registered at HUPE was 2.3 times higher than the official one\n10\n.\nThe prevalence registered in 2012 was similar to the one of the Republic of Congo in Africa (4.2%)\n19\n. It was superior to levels in other Latin American countries in the three years studied and it is a warning regarding the epidemiological situation of syphilis in the State\n20\n.\nFor some authors, the increase in the disease prevalence in the country may be associated with the improvement in the diagnosis and reporting systems, and not necessarily with the rise in the total number of cases. However, for some infectologists, the rising prevalence is due to the increasing practice of unprotected sex, which would intensify the number of cases of syphilis in the adult population and, consequently, during pregnancy. For others, there are flaws in the gestation treatment of prenatal services, in both diagnosis and treatment of the pregnant women and their partners\n7\n\n,\n\n21\n.\nA possible limitation of the present study is the selection bias, since the prevalence can be overestimated by including parturients with serological scarring (<1/8) in their calculation, even after the meticulous evaluation of the medical records and considering epidemiological history. The MH considers low VDRL titers when diagnosing syphilis, due to the variations in readings and the lack of detection by the tests. All the national studies consulted, including the sentinel studies in parturients, used any VDRL title for the calculation of the prevalence in its methodology\n9\n\n,\n\n22\n. Furthermore, several authors have already described the clinical significance of low titers of VDRL in the diagnosis of congenital syphilis\n23\n.\nAlthough there has been a substantial increase in the reporting of syphilis in parturients in HUPE between 2012 and 2014, it has not reached half of the cases, which demonstrates how fragile the epidemiological surveillance system is in identifying and reporting all cases. The considerable increase in the coverage of notifications follows the increased number of notifications observed in Brazil, which, according to the MH, is related to the Rede Cegonha program, which increased the coverage of pregnant women testing and follow-up of cases\n24\n. In Brazil, between 2012 and 2013, there was a 25% increase in the number of syphilis reports in pregnant women, and 23% if only the Southeast region is considered, which is responsible for 45.9% of the country's notifications. The increase of the number of notifications in the State of Rio de Janeiro was 16%. In the country, São Paulo is the State with the highest number of notifications, followed by the State of Rio de Janeiro\n25\n.\nAlthough the surveillance agencies considered underreporting for the purposes of disease monitoring, the present study shows that the level of underreporting was higher than estimated and should not be used as a basis for calculation. Without the proper notification, there is a tendency to underestimate the problem. However, the underreporting found in HUPE in 2013 is similar to the one described by Cavalcante et al.\n\n26\n in Fortaleza and Domingues et al.\n\n18\n in SUS units (state health clinics and hospitals) in the city of Rio de Janeiro, where underreporting was 73%, 70.3% and 76.1%, respectively, demonstrating the fragility of the information services in Brazil to work as a basis for promoting measures to combat the disease.\nThe high prevalence of the disease among the parturients of the present study resulted in an incidence of congenital syphilis (26.6 per 1,000 LB) greater than that reported in the State of Rio de Janeiro in 2015 (16 per 1,000 LB)\n10\n and at least five-fold greater than the incidence of the disease in Brazil, according to the Nascer Study (3.51 per 1,000 LB)\n27\n.\nRegarding the notification of congenital syphilis, the present study reached more than 90%, a result far superior to that reported by the MH, which estimates notification at only 17.4% of the cases of CS in the country\n24\n. HUPE is located in the capital of the State of Rio de Janeiro and, according to the epidemiological bulletin of the State Health Department, it is the city with the highest number of notifications of CS in the State (63.5%)\n25\n.\nThe high vertical transmission rate of syphilis in this study (65.8%) shows the high prevalence of syphilis among parturients, since among the various diseases that can be transmitted during the puerperal-pregnancy cycle, syphilis is the one that presents the greater chances of transmission\n28\n. Kupek et al.\n\n11\n showed similar rates (68.9%). Domingues et al.\n18\n and the MH\n7\n, however, estimated lower rates of vertical transmission, 34.8% in Rio de Janeiro and 25% in Brazil, respectively.\nThe occurrence of maternal syphilis in a subsequent gestation demonstrated that a prior gestation with syphilis did not eliminate the risk of the disease in future pregnancies. The recurrence of syphilis in two distinct pregnancies is the major sign of the inefficiency of the health system in properly conducting these cases.\nFetal infection occurred even in the presence of low VDRL maternal titers. The lack of proper consideration of low titers of VDRL by the health professionals is still a barrier to the control of congenital syphilis. In a study with health professionals, it was observed that only 48% had adequate knowledge about the management of syphilis, especially regarding the treatment of pregnant women with low titration\n29\n.\nSince syphilis is an easily diagnosed disease that has an established treatment and generates low costs for testing and treatment during pregnancy, it should be possible to control the disease. However, the disease continues to advance in Brazil in alarming numbers.","The estimated prevalence of syphilis in pregnant women in Rio de Janeiro is at least twice as high as previously reported by regional studies and epidemiological surveillance data in the State, reaching levels found in African countries and higher than the figures for other countries in Latin America. These values are a warning on the magnitude of the disease in the State of Rio de Janeiro, its upward curve and its consequences. Our reporting system has proven to be fragile and unable to assess the current prevalence of syphilis in Rio de Janeiro, which is underestimated. Due to the high prevalence of syphilis among parturients, there was a high rate of CS and vertical transmission that occurred even in pregnant women with low VDRL titers, emphasizing the importance of evaluating low titers in the management of these patients.\nIt is a responsibility of the health professionals, who daily handle this sad reality, to strive for excellence in caring for pregnant women. Even in the 21st century, the high rates of syphilis in parturients and CS in Brazil are still ignored and underestimated."],"string":"[\n \"Syphilis is a sexually transmitted disease caused by the bacterium Treponema pallidum. Infection can be transmitted to the unborn child through the placenta at any gestational stage. In exceptional cases, syphilis can be transmitted at birth through the child's contact with the birth canal, when there are maternal genital lesions (direct transmission). Syphilis may only be transmitted through breastfeeding if there are breast syphilis lesions\\n1\\n.\\nCongenital infection is associated with adverse outcomes, including: perinatal death, preterm birth, low birth weight, congenital anomalies, active syphilis in the newborn (NB), and long-term sequelae, such as deafness and neurological impairment\\n2\\n.\\nThere is a significant global increase in the prevalence of syphilis in pregnant women, despite all efforts from governments to control the disease. In 2013, the WHO reported 1.9 million pregnant women infected with syphilis worldwide, with 66.5% of adverse fetal outcomes occurring in cases of untreated syphilis\\n3\\n. Congenital syphilis significantly contributes to infant mortality, accounting for 305,000 perinatal deaths per year worldwide\\n4\\n. There are more newborns affected by syphilis than by any other neonatal infection. It is estimated that 520,000 adverse fetal outcomes occur worldwide each year, making congenital syphilis more common than congenital HIV infection\\n5\\n. In areas where syphilis is prevalent, about half of the stillbirth rate may have been caused solely by this infection\\n6\\n. The direct medical cost associated with adverse fetal outcomes resulting from syphilis is U$ 309 million dollars a year\\n4\\n.\\nHigh rates of syphilis in parturients mean a high incidence of congenital syphilis and millions of missed opportunities to save lives during pre-natal care. Over the past ten years, the infant mortality rate due to syphilis increased 150% in Brazil, from 2.2 per 100,000 live births (LB) in 2004 to 5.5 per 100,000 LB in 2013. By 2015, the State of Rio de Janeiro had the highest rate of syphilis during pregnancy (2.2%) and of congenital syphilis in the country (16 cases per 1,000 LB)\\n7\\n\\n,\\n\\n8\\n.\\nThe last syphilis monitoring study in pregnant women performed in Brazil in 2014, with a statistical sample from public and private hospitals, estimated the prevalence of syphilis at 1.02%, with no significant regional differences\\n9\\n.\\nAccording to the prediction curve of the disease, the Ministry of Health (MH) expected 40,000 cases in pregnant women in Brazil in 2016. An epidemic of the disease in the country was confirmed by the Ministry of Health in 2016. The increased incidence of syphilis in pregnant women is alarming\\n8\\n. In 2015, according to the MH, the States of Rio de Janeiro and Mato Grosso do Sul were the most affected in the country, reaching a syphilis rate in pregnant women of 2.2%\\n8\\n.\\nThe national studies show differences in the prevalence of the disease according to the region studied, with rates ranging around 0.4% in Vitória (Espírito Santo)\\n10\\n, Itajaí (Santa Catarina)\\n11\\n and 7.7% in Fortaleza (Ceará)\\n12\\n.\\nThe analysis of international studies on the subject in countries such as Haiti\\n13\\n shows that the prevalence of syphilis in pregnant women reaches 7.6%, while in other countries, such as Canada, syphilis has already been eliminated\\n14\\n. The WHO certified Cuba in 2013 as the first country to eliminate the vertical transmission of HIV and syphilis during pregnancy. Six other countries are currently able to request the WHO to validate the double elimination, including Canada and the United States\\n15\\n.\\n Aim To estimate the prevalence and coverage of syphilis reporting in parturients, the incidence of congenital syphilis and its corresponding underreporting, as well as the rate of vertical transmission of infection.\\nTo estimate the prevalence and coverage of syphilis reporting in parturients, the incidence of congenital syphilis and its corresponding underreporting, as well as the rate of vertical transmission of infection.\",\n \"To estimate the prevalence and coverage of syphilis reporting in parturients, the incidence of congenital syphilis and its corresponding underreporting, as well as the rate of vertical transmission of infection.\",\n \" Study design Cross-sectional study.\\nCross-sectional study.\\n Studied population A total of 2,041 pregnant women were recruited after admission to a public hospital in the metropolitan region of Rio de Janeiro, between January 2012 and December 2014. The institution involved in the study was the Pedro Ernesto Hospital of the State University of Rio de Janeiro (HUPE / UERJ).\\nA total of 2,041 pregnant women were recruited after admission to a public hospital in the metropolitan region of Rio de Janeiro, between January 2012 and December 2014. The institution involved in the study was the Pedro Ernesto Hospital of the State University of Rio de Janeiro (HUPE / UERJ).\\n Data collection At HUPE, blood samples are routinely collected for syphilis investigation upon admission, with both non-treponemal testing (VDRL-Venereal Disease Research Laboratory) and confirmatory treponemal test (TPHA-Treponema Pallidum Hemagglutination). The confirmation via the treponemal test is important due to the possibility of a false-positive result. All the tests were performed in the clinical analysis laboratory of HUPE. According to the protocol of the Brazilian Ministry of Health, blood samples are collected for syphilis testing from all parturients. An active search of syphilis serology results (treponemal and non-treponemal tests) with the respective titrations was conducted by accessing the database of the hospital laboratory. A thorough review of the medical records of pregnant women and their newborns was also conducted. Data from the HUPE epidemiology service were evaluated in order to identify the total number of infected pregnant women and newborns, in order to avoid underestimating the results. A survey was conducted on the website of the State's Department of Health website to collect data on syphilis reports during pregnancy, as well as reports of congenital syphilis on the SINAN (National Disease Notification System) and the number of live births in the SINASC (Live Births Notification System).\\nAt HUPE, blood samples are routinely collected for syphilis investigation upon admission, with both non-treponemal testing (VDRL-Venereal Disease Research Laboratory) and confirmatory treponemal test (TPHA-Treponema Pallidum Hemagglutination). The confirmation via the treponemal test is important due to the possibility of a false-positive result. All the tests were performed in the clinical analysis laboratory of HUPE. According to the protocol of the Brazilian Ministry of Health, blood samples are collected for syphilis testing from all parturients. An active search of syphilis serology results (treponemal and non-treponemal tests) with the respective titrations was conducted by accessing the database of the hospital laboratory. A thorough review of the medical records of pregnant women and their newborns was also conducted. Data from the HUPE epidemiology service were evaluated in order to identify the total number of infected pregnant women and newborns, in order to avoid underestimating the results. A survey was conducted on the website of the State's Department of Health website to collect data on syphilis reports during pregnancy, as well as reports of congenital syphilis on the SINAN (National Disease Notification System) and the number of live births in the SINASC (Live Births Notification System).\\n Inclusion criterion The following parturients were eligible for the study: pregnant women admitted for delivery with a live fetus of any gestational age and weight, stillbirths with gestational age equal to22 weeks or weight equal to 500 grams.\\nThe following cases were defined as syphilis during pregnancy: all cases in which the parturient was admitted with laboratory evidence of positive VDRL (any titer) collected at the time of admission and confirmed by the treponemal test; all cases in which the parturient's newborn (stillbirth or live birth) has been reported as a case of congenital syphilis (CS), identified in any of the information systems consulted.\\nThe following parturients were eligible for the study: pregnant women admitted for delivery with a live fetus of any gestational age and weight, stillbirths with gestational age equal to22 weeks or weight equal to 500 grams.\\nThe following cases were defined as syphilis during pregnancy: all cases in which the parturient was admitted with laboratory evidence of positive VDRL (any titer) collected at the time of admission and confirmed by the treponemal test; all cases in which the parturient's newborn (stillbirth or live birth) has been reported as a case of congenital syphilis (CS), identified in any of the information systems consulted.\\n Exclusion criterion The following parturients were excluded: the ones with positive VDRL resulting from adequately treated previous syphilis (complete treatment with benzathine penicillin, according to the clinical stage of the disease, complete treatment of the partner, documentation confirming the couple's treatment, drop in VDRL titers after adequate treatment, treatment completed more than 30 days before delivery).\\nThe following parturients were excluded: the ones with positive VDRL resulting from adequately treated previous syphilis (complete treatment with benzathine penicillin, according to the clinical stage of the disease, complete treatment of the partner, documentation confirming the couple's treatment, drop in VDRL titers after adequate treatment, treatment completed more than 30 days before delivery).\\n Variable definition The following situations were included as CS cases: all the gestation products (live births or stillborn) identified in any of the information systems as premature congenital syphilis; all newborns with VDRL titers higher than the maternal ones; all newborns with clinical manifestations suggestive of syphilis or complementary tests pointing to CS\\n7\\n.\\nThe following situations were included as CS cases: all the gestation products (live births or stillborn) identified in any of the information systems as premature congenital syphilis; all newborns with VDRL titers higher than the maternal ones; all newborns with clinical manifestations suggestive of syphilis or complementary tests pointing to CS\\n7\\n.\\n Epidemiological definitions According to the recommendation of the strategic management of the MH, the prevalence of syphilis in pregnant women is the ratio of the number of cases of syphilis detected in pregnant women to every 1,000 live births, within a geographical space and for a given period. For the purpose of calculating the prevalence, we used as the numerator the number of cases of syphilis detected in pregnant women, in a given notification year and place of residence multiplied by 1,000 and divided by the total number of live births in the same place and in the same reported year\\n16\\n. The numerator could be found in the SINAN (national disease notification system) and the denominator in the SINASC (live births information system).\\nFor the calculation of the prevalence of parturients as a percentage, the number of cases of syphilis in parturients identified in the study was used as the numerator; the number of live births in that place and period, multiplied by 100 as the denominator.\\nTo evaluate underreporting, we divided the number of parturients with syphilis in SINAN by the number of parturients with syphilis in hospitals x 100.\\nFollowing the MH recommendation for the calculation of the CS incidence, the number of CS cases identified in the study was used as the numerator and the number of live births at that location and period, multiplied by 1000, as the denominator.\\nIn order to evaluate the underreporting of congenital syphilis, we divided the number of CS cases by the number of cases reported in SINAN x 100.\\nAccording to the recommendation of the strategic management of the MH, the prevalence of syphilis in pregnant women is the ratio of the number of cases of syphilis detected in pregnant women to every 1,000 live births, within a geographical space and for a given period. For the purpose of calculating the prevalence, we used as the numerator the number of cases of syphilis detected in pregnant women, in a given notification year and place of residence multiplied by 1,000 and divided by the total number of live births in the same place and in the same reported year\\n16\\n. The numerator could be found in the SINAN (national disease notification system) and the denominator in the SINASC (live births information system).\\nFor the calculation of the prevalence of parturients as a percentage, the number of cases of syphilis in parturients identified in the study was used as the numerator; the number of live births in that place and period, multiplied by 100 as the denominator.\\nTo evaluate underreporting, we divided the number of parturients with syphilis in SINAN by the number of parturients with syphilis in hospitals x 100.\\nFollowing the MH recommendation for the calculation of the CS incidence, the number of CS cases identified in the study was used as the numerator and the number of live births at that location and period, multiplied by 1000, as the denominator.\\nIn order to evaluate the underreporting of congenital syphilis, we divided the number of CS cases by the number of cases reported in SINAN x 100.\\n Data analysis The sample size was calculated considering an expected prevalence of syphilis in pregnant women according to the MH in public hospitals of 1.1% in 2006, in Brazil\\n9\\n. To perform this calculation, assuming an error of 5% and a confidence interval (CI) of 95% the sample size was calculated to be 17 for 95% CI and 46 for 99,9% CI.\\nCollected variables were used in the comparative analyzes to identify prevalence factors. The results were expressed as percentages. The process of entering and analyzing statistical data was performed through the EPIINFO 3.5.2 version 3.0.1 computing program.\\nThe sample size was calculated considering an expected prevalence of syphilis in pregnant women according to the MH in public hospitals of 1.1% in 2006, in Brazil\\n9\\n. To perform this calculation, assuming an error of 5% and a confidence interval (CI) of 95% the sample size was calculated to be 17 for 95% CI and 46 for 99,9% CI.\\nCollected variables were used in the comparative analyzes to identify prevalence factors. The results were expressed as percentages. The process of entering and analyzing statistical data was performed through the EPIINFO 3.5.2 version 3.0.1 computing program.\\n Considerations on ethical aspects The research project was carried out within the standards required by the Declaration of Helsinki and Resolution 466 of December 12, 2012, and approved by the Research Ethics Committee of the UERJ (COEP) in July 2012, process N° 034.3.2012 and sponsored by FAPERJ, process N° E-26/110.351/2012.\\nThe research project was carried out within the standards required by the Declaration of Helsinki and Resolution 466 of December 12, 2012, and approved by the Research Ethics Committee of the UERJ (COEP) in July 2012, process N° 034.3.2012 and sponsored by FAPERJ, process N° E-26/110.351/2012.\",\n \"Cross-sectional study.\",\n \"A total of 2,041 pregnant women were recruited after admission to a public hospital in the metropolitan region of Rio de Janeiro, between January 2012 and December 2014. The institution involved in the study was the Pedro Ernesto Hospital of the State University of Rio de Janeiro (HUPE / UERJ).\",\n \"At HUPE, blood samples are routinely collected for syphilis investigation upon admission, with both non-treponemal testing (VDRL-Venereal Disease Research Laboratory) and confirmatory treponemal test (TPHA-Treponema Pallidum Hemagglutination). The confirmation via the treponemal test is important due to the possibility of a false-positive result. All the tests were performed in the clinical analysis laboratory of HUPE. According to the protocol of the Brazilian Ministry of Health, blood samples are collected for syphilis testing from all parturients. An active search of syphilis serology results (treponemal and non-treponemal tests) with the respective titrations was conducted by accessing the database of the hospital laboratory. A thorough review of the medical records of pregnant women and their newborns was also conducted. Data from the HUPE epidemiology service were evaluated in order to identify the total number of infected pregnant women and newborns, in order to avoid underestimating the results. A survey was conducted on the website of the State's Department of Health website to collect data on syphilis reports during pregnancy, as well as reports of congenital syphilis on the SINAN (National Disease Notification System) and the number of live births in the SINASC (Live Births Notification System).\",\n \"The following parturients were eligible for the study: pregnant women admitted for delivery with a live fetus of any gestational age and weight, stillbirths with gestational age equal to22 weeks or weight equal to 500 grams.\\nThe following cases were defined as syphilis during pregnancy: all cases in which the parturient was admitted with laboratory evidence of positive VDRL (any titer) collected at the time of admission and confirmed by the treponemal test; all cases in which the parturient's newborn (stillbirth or live birth) has been reported as a case of congenital syphilis (CS), identified in any of the information systems consulted.\",\n \"The following parturients were excluded: the ones with positive VDRL resulting from adequately treated previous syphilis (complete treatment with benzathine penicillin, according to the clinical stage of the disease, complete treatment of the partner, documentation confirming the couple's treatment, drop in VDRL titers after adequate treatment, treatment completed more than 30 days before delivery).\",\n \"The following situations were included as CS cases: all the gestation products (live births or stillborn) identified in any of the information systems as premature congenital syphilis; all newborns with VDRL titers higher than the maternal ones; all newborns with clinical manifestations suggestive of syphilis or complementary tests pointing to CS\\n7\\n.\",\n \"According to the recommendation of the strategic management of the MH, the prevalence of syphilis in pregnant women is the ratio of the number of cases of syphilis detected in pregnant women to every 1,000 live births, within a geographical space and for a given period. For the purpose of calculating the prevalence, we used as the numerator the number of cases of syphilis detected in pregnant women, in a given notification year and place of residence multiplied by 1,000 and divided by the total number of live births in the same place and in the same reported year\\n16\\n. The numerator could be found in the SINAN (national disease notification system) and the denominator in the SINASC (live births information system).\\nFor the calculation of the prevalence of parturients as a percentage, the number of cases of syphilis in parturients identified in the study was used as the numerator; the number of live births in that place and period, multiplied by 100 as the denominator.\\nTo evaluate underreporting, we divided the number of parturients with syphilis in SINAN by the number of parturients with syphilis in hospitals x 100.\\nFollowing the MH recommendation for the calculation of the CS incidence, the number of CS cases identified in the study was used as the numerator and the number of live births at that location and period, multiplied by 1000, as the denominator.\\nIn order to evaluate the underreporting of congenital syphilis, we divided the number of CS cases by the number of cases reported in SINAN x 100.\",\n \"The sample size was calculated considering an expected prevalence of syphilis in pregnant women according to the MH in public hospitals of 1.1% in 2006, in Brazil\\n9\\n. To perform this calculation, assuming an error of 5% and a confidence interval (CI) of 95% the sample size was calculated to be 17 for 95% CI and 46 for 99,9% CI.\\nCollected variables were used in the comparative analyzes to identify prevalence factors. The results were expressed as percentages. The process of entering and analyzing statistical data was performed through the EPIINFO 3.5.2 version 3.0.1 computing program.\",\n \"The research project was carried out within the standards required by the Declaration of Helsinki and Resolution 466 of December 12, 2012, and approved by the Research Ethics Committee of the UERJ (COEP) in July 2012, process N° 034.3.2012 and sponsored by FAPERJ, process N° E-26/110.351/2012.\",\n \"A total of 2,041 parturients were studied in HUPE: 735 in 2012, 664 in 2013 and 642 in 2014. After the exclusion of 19 pregnant women due to serological scarring or false-positive results (treponemal negative test), 79 cases of parturients with syphilis were identified: 32 cases in 2012, 20 in 2013 and 27 in 2014. Access to the result of the treponemal and non-treponemal tests for syphilis was obtained from all the pregnant women admitted during the period.\\nThe search on SINASC, considering HUPE as the place of birth, totaled 1,956 live births in HUPE for the three years studied, with 773 live births in 2012, 647 in 2013 and 536 in 2014. The prevalence of syphilis in the HUPE, using the number of live births officially described in SINASC as the denominator, was 4.1% in 2012, 3.1% in 2013 and 5.0% in 2014. Between 2012 and 2014, there was an increase on the prevalence of the disease (Table 1).\\nLegend: live births (LB); live birth information system (SINASC); confidence limit (CL)\\nThe search on SINAN came up with 23 cases of syphilis in HUPE for the three years studied (2012 = 5, 2013 = 5 and 2014 = 13). The research showed 79 cases in the three years (2012 = 32, 2013 = 20 and 2014 = 27).\\nTherefore, 15.6% of cases in 2012, 25.0% in 2013 and 48.1% in 2014 were reported, which shows a significant increase for the period.\\nThree parturients (3.8%) from HUPE were double counted in the prevalence calculation, when they were admitted with syphilis at the time of delivery of subsequent pregnancies. One of the three parturients had VDRL titer =1/4 and progressed to 1/128 in the next gestation. The other two had 1/32 titers in the first gestation and had titers of 1/32 and 1/128, respectively, in subsequent pregnancies.\\nThe incidence of CS was 22/1,000 LB in 2012, 17/1,000 LB in 2013 and 44.8/ 1,000 LB in 2014. The notification of congenital syphilis reached 92.3% of the cases and only four cases were not officially reported. The present study demonstrated a vertical transmission rate of 65.8%, with 52 newborns affected by congenital syphilis.\\nFrom the group of parturients with vertical transmission of the disease, 18 mothers had VDRL titers = 1/4, corresponding to 34.6% of the cases of congenital syphilis. The vertical transmission occurred in two consecutive pregnancies in one case that was within the low titers group (= 1/4).\",\n \"The latest national studies in public or public-private hospitals that monitor the prevalence of syphilis in the country demonstrated a reduction in the prevalence of syphilis among parturients in Brazil. Sentinel studies revealed a prevalence of 1.6%\\n17\\n in 2004 and 1.02%\\n9\\n in 2014.\\nHowever, the present study does not confirm this trend and demonstrates that the disease is not under control in the State of Rio de Janeiro. In 2014, the prevalence rate of 5.0% was four-fold higher than the one registered in the country in 2014 by Domingues et al.\\n\\n19\\n and twice higher than the one registered in the State of Rio de Janeiro in 2008 by the same author\\n18\\n.\\nThe results are similar to those described by the MH regarding the ascending curve of the disease in recent years, but they express a significantly higher prevalence when compared with data from the epidemiological surveillance agencies for the same period. In 2014, although the State of Rio de Janeiro had the highest syphilis rate in pregnant women in the country (2.2%), the prevalence registered at HUPE was 2.3 times higher than the official one\\n10\\n.\\nThe prevalence registered in 2012 was similar to the one of the Republic of Congo in Africa (4.2%)\\n19\\n. It was superior to levels in other Latin American countries in the three years studied and it is a warning regarding the epidemiological situation of syphilis in the State\\n20\\n.\\nFor some authors, the increase in the disease prevalence in the country may be associated with the improvement in the diagnosis and reporting systems, and not necessarily with the rise in the total number of cases. However, for some infectologists, the rising prevalence is due to the increasing practice of unprotected sex, which would intensify the number of cases of syphilis in the adult population and, consequently, during pregnancy. For others, there are flaws in the gestation treatment of prenatal services, in both diagnosis and treatment of the pregnant women and their partners\\n7\\n\\n,\\n\\n21\\n.\\nA possible limitation of the present study is the selection bias, since the prevalence can be overestimated by including parturients with serological scarring (<1/8) in their calculation, even after the meticulous evaluation of the medical records and considering epidemiological history. The MH considers low VDRL titers when diagnosing syphilis, due to the variations in readings and the lack of detection by the tests. All the national studies consulted, including the sentinel studies in parturients, used any VDRL title for the calculation of the prevalence in its methodology\\n9\\n\\n,\\n\\n22\\n. Furthermore, several authors have already described the clinical significance of low titers of VDRL in the diagnosis of congenital syphilis\\n23\\n.\\nAlthough there has been a substantial increase in the reporting of syphilis in parturients in HUPE between 2012 and 2014, it has not reached half of the cases, which demonstrates how fragile the epidemiological surveillance system is in identifying and reporting all cases. The considerable increase in the coverage of notifications follows the increased number of notifications observed in Brazil, which, according to the MH, is related to the Rede Cegonha program, which increased the coverage of pregnant women testing and follow-up of cases\\n24\\n. In Brazil, between 2012 and 2013, there was a 25% increase in the number of syphilis reports in pregnant women, and 23% if only the Southeast region is considered, which is responsible for 45.9% of the country's notifications. The increase of the number of notifications in the State of Rio de Janeiro was 16%. In the country, São Paulo is the State with the highest number of notifications, followed by the State of Rio de Janeiro\\n25\\n.\\nAlthough the surveillance agencies considered underreporting for the purposes of disease monitoring, the present study shows that the level of underreporting was higher than estimated and should not be used as a basis for calculation. Without the proper notification, there is a tendency to underestimate the problem. However, the underreporting found in HUPE in 2013 is similar to the one described by Cavalcante et al.\\n\\n26\\n in Fortaleza and Domingues et al.\\n\\n18\\n in SUS units (state health clinics and hospitals) in the city of Rio de Janeiro, where underreporting was 73%, 70.3% and 76.1%, respectively, demonstrating the fragility of the information services in Brazil to work as a basis for promoting measures to combat the disease.\\nThe high prevalence of the disease among the parturients of the present study resulted in an incidence of congenital syphilis (26.6 per 1,000 LB) greater than that reported in the State of Rio de Janeiro in 2015 (16 per 1,000 LB)\\n10\\n and at least five-fold greater than the incidence of the disease in Brazil, according to the Nascer Study (3.51 per 1,000 LB)\\n27\\n.\\nRegarding the notification of congenital syphilis, the present study reached more than 90%, a result far superior to that reported by the MH, which estimates notification at only 17.4% of the cases of CS in the country\\n24\\n. HUPE is located in the capital of the State of Rio de Janeiro and, according to the epidemiological bulletin of the State Health Department, it is the city with the highest number of notifications of CS in the State (63.5%)\\n25\\n.\\nThe high vertical transmission rate of syphilis in this study (65.8%) shows the high prevalence of syphilis among parturients, since among the various diseases that can be transmitted during the puerperal-pregnancy cycle, syphilis is the one that presents the greater chances of transmission\\n28\\n. Kupek et al.\\n\\n11\\n showed similar rates (68.9%). Domingues et al.\\n18\\n and the MH\\n7\\n, however, estimated lower rates of vertical transmission, 34.8% in Rio de Janeiro and 25% in Brazil, respectively.\\nThe occurrence of maternal syphilis in a subsequent gestation demonstrated that a prior gestation with syphilis did not eliminate the risk of the disease in future pregnancies. The recurrence of syphilis in two distinct pregnancies is the major sign of the inefficiency of the health system in properly conducting these cases.\\nFetal infection occurred even in the presence of low VDRL maternal titers. The lack of proper consideration of low titers of VDRL by the health professionals is still a barrier to the control of congenital syphilis. In a study with health professionals, it was observed that only 48% had adequate knowledge about the management of syphilis, especially regarding the treatment of pregnant women with low titration\\n29\\n.\\nSince syphilis is an easily diagnosed disease that has an established treatment and generates low costs for testing and treatment during pregnancy, it should be possible to control the disease. However, the disease continues to advance in Brazil in alarming numbers.\",\n \"The estimated prevalence of syphilis in pregnant women in Rio de Janeiro is at least twice as high as previously reported by regional studies and epidemiological surveillance data in the State, reaching levels found in African countries and higher than the figures for other countries in Latin America. These values are a warning on the magnitude of the disease in the State of Rio de Janeiro, its upward curve and its consequences. Our reporting system has proven to be fragile and unable to assess the current prevalence of syphilis in Rio de Janeiro, which is underestimated. Due to the high prevalence of syphilis among parturients, there was a high rate of CS and vertical transmission that occurred even in pregnant women with low VDRL titers, emphasizing the importance of evaluating low titers in the management of these patients.\\nIt is a responsibility of the health professionals, who daily handle this sad reality, to strive for excellence in caring for pregnant women. Even in the 21st century, the high rates of syphilis in parturients and CS in Brazil are still ignored and underestimated.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":["intro",null,"materials|methods",null,null,null,null,null,null,null,null,null,"results","discussion","conclusions"],"string":"[\n \"intro\",\n null,\n \"materials|methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n \"results\",\n \"discussion\",\n \"conclusions\"\n]","isConversational":false},"keywords":{"kind":"list like","value":["Syphilis","Gestation","Congenital syphilis","Prevalence","VDRL","Treponemal tests","Treponema pallidum"],"string":"[\n \"Syphilis\",\n \"Gestation\",\n \"Congenital syphilis\",\n \"Prevalence\",\n \"VDRL\",\n \"Treponemal tests\",\n \"Treponema pallidum\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nSyphilis is a sexually transmitted disease caused by the bacterium Treponema pallidum. Infection can be transmitted to the unborn child through the placenta at any gestational stage. In exceptional cases, syphilis can be transmitted at birth through the child's contact with the birth canal, when there are maternal genital lesions (direct transmission). Syphilis may only be transmitted through breastfeeding if there are breast syphilis lesions\n1\n.\nCongenital infection is associated with adverse outcomes, including: perinatal death, preterm birth, low birth weight, congenital anomalies, active syphilis in the newborn (NB), and long-term sequelae, such as deafness and neurological impairment\n2\n.\nThere is a significant global increase in the prevalence of syphilis in pregnant women, despite all efforts from governments to control the disease. In 2013, the WHO reported 1.9 million pregnant women infected with syphilis worldwide, with 66.5% of adverse fetal outcomes occurring in cases of untreated syphilis\n3\n. Congenital syphilis significantly contributes to infant mortality, accounting for 305,000 perinatal deaths per year worldwide\n4\n. There are more newborns affected by syphilis than by any other neonatal infection. It is estimated that 520,000 adverse fetal outcomes occur worldwide each year, making congenital syphilis more common than congenital HIV infection\n5\n. In areas where syphilis is prevalent, about half of the stillbirth rate may have been caused solely by this infection\n6\n. The direct medical cost associated with adverse fetal outcomes resulting from syphilis is U$ 309 million dollars a year\n4\n.\nHigh rates of syphilis in parturients mean a high incidence of congenital syphilis and millions of missed opportunities to save lives during pre-natal care. Over the past ten years, the infant mortality rate due to syphilis increased 150% in Brazil, from 2.2 per 100,000 live births (LB) in 2004 to 5.5 per 100,000 LB in 2013. By 2015, the State of Rio de Janeiro had the highest rate of syphilis during pregnancy (2.2%) and of congenital syphilis in the country (16 cases per 1,000 LB)\n7\n\n,\n\n8\n.\nThe last syphilis monitoring study in pregnant women performed in Brazil in 2014, with a statistical sample from public and private hospitals, estimated the prevalence of syphilis at 1.02%, with no significant regional differences\n9\n.\nAccording to the prediction curve of the disease, the Ministry of Health (MH) expected 40,000 cases in pregnant women in Brazil in 2016. An epidemic of the disease in the country was confirmed by the Ministry of Health in 2016. The increased incidence of syphilis in pregnant women is alarming\n8\n. In 2015, according to the MH, the States of Rio de Janeiro and Mato Grosso do Sul were the most affected in the country, reaching a syphilis rate in pregnant women of 2.2%\n8\n.\nThe national studies show differences in the prevalence of the disease according to the region studied, with rates ranging around 0.4% in Vitória (Espírito Santo)\n10\n, Itajaí (Santa Catarina)\n11\n and 7.7% in Fortaleza (Ceará)\n12\n.\nThe analysis of international studies on the subject in countries such as Haiti\n13\n shows that the prevalence of syphilis in pregnant women reaches 7.6%, while in other countries, such as Canada, syphilis has already been eliminated\n14\n. The WHO certified Cuba in 2013 as the first country to eliminate the vertical transmission of HIV and syphilis during pregnancy. Six other countries are currently able to request the WHO to validate the double elimination, including Canada and the United States\n15\n.\n Aim To estimate the prevalence and coverage of syphilis reporting in parturients, the incidence of congenital syphilis and its corresponding underreporting, as well as the rate of vertical transmission of infection.\nTo estimate the prevalence and coverage of syphilis reporting in parturients, the incidence of congenital syphilis and its corresponding underreporting, as well as the rate of vertical transmission of infection.\n\nAim:\nTo estimate the prevalence and coverage of syphilis reporting in parturients, the incidence of congenital syphilis and its corresponding underreporting, as well as the rate of vertical transmission of infection.\n\nMATERIALS AND METHODS:\n Study design Cross-sectional study.\nCross-sectional study.\n Studied population A total of 2,041 pregnant women were recruited after admission to a public hospital in the metropolitan region of Rio de Janeiro, between January 2012 and December 2014. The institution involved in the study was the Pedro Ernesto Hospital of the State University of Rio de Janeiro (HUPE / UERJ).\nA total of 2,041 pregnant women were recruited after admission to a public hospital in the metropolitan region of Rio de Janeiro, between January 2012 and December 2014. The institution involved in the study was the Pedro Ernesto Hospital of the State University of Rio de Janeiro (HUPE / UERJ).\n Data collection At HUPE, blood samples are routinely collected for syphilis investigation upon admission, with both non-treponemal testing (VDRL-Venereal Disease Research Laboratory) and confirmatory treponemal test (TPHA-Treponema Pallidum Hemagglutination). The confirmation via the treponemal test is important due to the possibility of a false-positive result. All the tests were performed in the clinical analysis laboratory of HUPE. According to the protocol of the Brazilian Ministry of Health, blood samples are collected for syphilis testing from all parturients. An active search of syphilis serology results (treponemal and non-treponemal tests) with the respective titrations was conducted by accessing the database of the hospital laboratory. A thorough review of the medical records of pregnant women and their newborns was also conducted. Data from the HUPE epidemiology service were evaluated in order to identify the total number of infected pregnant women and newborns, in order to avoid underestimating the results. A survey was conducted on the website of the State's Department of Health website to collect data on syphilis reports during pregnancy, as well as reports of congenital syphilis on the SINAN (National Disease Notification System) and the number of live births in the SINASC (Live Births Notification System).\nAt HUPE, blood samples are routinely collected for syphilis investigation upon admission, with both non-treponemal testing (VDRL-Venereal Disease Research Laboratory) and confirmatory treponemal test (TPHA-Treponema Pallidum Hemagglutination). The confirmation via the treponemal test is important due to the possibility of a false-positive result. All the tests were performed in the clinical analysis laboratory of HUPE. According to the protocol of the Brazilian Ministry of Health, blood samples are collected for syphilis testing from all parturients. An active search of syphilis serology results (treponemal and non-treponemal tests) with the respective titrations was conducted by accessing the database of the hospital laboratory. A thorough review of the medical records of pregnant women and their newborns was also conducted. Data from the HUPE epidemiology service were evaluated in order to identify the total number of infected pregnant women and newborns, in order to avoid underestimating the results. A survey was conducted on the website of the State's Department of Health website to collect data on syphilis reports during pregnancy, as well as reports of congenital syphilis on the SINAN (National Disease Notification System) and the number of live births in the SINASC (Live Births Notification System).\n Inclusion criterion The following parturients were eligible for the study: pregnant women admitted for delivery with a live fetus of any gestational age and weight, stillbirths with gestational age equal to22 weeks or weight equal to 500 grams.\nThe following cases were defined as syphilis during pregnancy: all cases in which the parturient was admitted with laboratory evidence of positive VDRL (any titer) collected at the time of admission and confirmed by the treponemal test; all cases in which the parturient's newborn (stillbirth or live birth) has been reported as a case of congenital syphilis (CS), identified in any of the information systems consulted.\nThe following parturients were eligible for the study: pregnant women admitted for delivery with a live fetus of any gestational age and weight, stillbirths with gestational age equal to22 weeks or weight equal to 500 grams.\nThe following cases were defined as syphilis during pregnancy: all cases in which the parturient was admitted with laboratory evidence of positive VDRL (any titer) collected at the time of admission and confirmed by the treponemal test; all cases in which the parturient's newborn (stillbirth or live birth) has been reported as a case of congenital syphilis (CS), identified in any of the information systems consulted.\n Exclusion criterion The following parturients were excluded: the ones with positive VDRL resulting from adequately treated previous syphilis (complete treatment with benzathine penicillin, according to the clinical stage of the disease, complete treatment of the partner, documentation confirming the couple's treatment, drop in VDRL titers after adequate treatment, treatment completed more than 30 days before delivery).\nThe following parturients were excluded: the ones with positive VDRL resulting from adequately treated previous syphilis (complete treatment with benzathine penicillin, according to the clinical stage of the disease, complete treatment of the partner, documentation confirming the couple's treatment, drop in VDRL titers after adequate treatment, treatment completed more than 30 days before delivery).\n Variable definition The following situations were included as CS cases: all the gestation products (live births or stillborn) identified in any of the information systems as premature congenital syphilis; all newborns with VDRL titers higher than the maternal ones; all newborns with clinical manifestations suggestive of syphilis or complementary tests pointing to CS\n7\n.\nThe following situations were included as CS cases: all the gestation products (live births or stillborn) identified in any of the information systems as premature congenital syphilis; all newborns with VDRL titers higher than the maternal ones; all newborns with clinical manifestations suggestive of syphilis or complementary tests pointing to CS\n7\n.\n Epidemiological definitions According to the recommendation of the strategic management of the MH, the prevalence of syphilis in pregnant women is the ratio of the number of cases of syphilis detected in pregnant women to every 1,000 live births, within a geographical space and for a given period. For the purpose of calculating the prevalence, we used as the numerator the number of cases of syphilis detected in pregnant women, in a given notification year and place of residence multiplied by 1,000 and divided by the total number of live births in the same place and in the same reported year\n16\n. The numerator could be found in the SINAN (national disease notification system) and the denominator in the SINASC (live births information system).\nFor the calculation of the prevalence of parturients as a percentage, the number of cases of syphilis in parturients identified in the study was used as the numerator; the number of live births in that place and period, multiplied by 100 as the denominator.\nTo evaluate underreporting, we divided the number of parturients with syphilis in SINAN by the number of parturients with syphilis in hospitals x 100.\nFollowing the MH recommendation for the calculation of the CS incidence, the number of CS cases identified in the study was used as the numerator and the number of live births at that location and period, multiplied by 1000, as the denominator.\nIn order to evaluate the underreporting of congenital syphilis, we divided the number of CS cases by the number of cases reported in SINAN x 100.\nAccording to the recommendation of the strategic management of the MH, the prevalence of syphilis in pregnant women is the ratio of the number of cases of syphilis detected in pregnant women to every 1,000 live births, within a geographical space and for a given period. For the purpose of calculating the prevalence, we used as the numerator the number of cases of syphilis detected in pregnant women, in a given notification year and place of residence multiplied by 1,000 and divided by the total number of live births in the same place and in the same reported year\n16\n. The numerator could be found in the SINAN (national disease notification system) and the denominator in the SINASC (live births information system).\nFor the calculation of the prevalence of parturients as a percentage, the number of cases of syphilis in parturients identified in the study was used as the numerator; the number of live births in that place and period, multiplied by 100 as the denominator.\nTo evaluate underreporting, we divided the number of parturients with syphilis in SINAN by the number of parturients with syphilis in hospitals x 100.\nFollowing the MH recommendation for the calculation of the CS incidence, the number of CS cases identified in the study was used as the numerator and the number of live births at that location and period, multiplied by 1000, as the denominator.\nIn order to evaluate the underreporting of congenital syphilis, we divided the number of CS cases by the number of cases reported in SINAN x 100.\n Data analysis The sample size was calculated considering an expected prevalence of syphilis in pregnant women according to the MH in public hospitals of 1.1% in 2006, in Brazil\n9\n. To perform this calculation, assuming an error of 5% and a confidence interval (CI) of 95% the sample size was calculated to be 17 for 95% CI and 46 for 99,9% CI.\nCollected variables were used in the comparative analyzes to identify prevalence factors. The results were expressed as percentages. The process of entering and analyzing statistical data was performed through the EPIINFO 3.5.2 version 3.0.1 computing program.\nThe sample size was calculated considering an expected prevalence of syphilis in pregnant women according to the MH in public hospitals of 1.1% in 2006, in Brazil\n9\n. To perform this calculation, assuming an error of 5% and a confidence interval (CI) of 95% the sample size was calculated to be 17 for 95% CI and 46 for 99,9% CI.\nCollected variables were used in the comparative analyzes to identify prevalence factors. The results were expressed as percentages. The process of entering and analyzing statistical data was performed through the EPIINFO 3.5.2 version 3.0.1 computing program.\n Considerations on ethical aspects The research project was carried out within the standards required by the Declaration of Helsinki and Resolution 466 of December 12, 2012, and approved by the Research Ethics Committee of the UERJ (COEP) in July 2012, process N° 034.3.2012 and sponsored by FAPERJ, process N° E-26/110.351/2012.\nThe research project was carried out within the standards required by the Declaration of Helsinki and Resolution 466 of December 12, 2012, and approved by the Research Ethics Committee of the UERJ (COEP) in July 2012, process N° 034.3.2012 and sponsored by FAPERJ, process N° E-26/110.351/2012.\n\nStudy design:\nCross-sectional study.\n\nStudied population:\nA total of 2,041 pregnant women were recruited after admission to a public hospital in the metropolitan region of Rio de Janeiro, between January 2012 and December 2014. The institution involved in the study was the Pedro Ernesto Hospital of the State University of Rio de Janeiro (HUPE / UERJ).\n\nData collection:\nAt HUPE, blood samples are routinely collected for syphilis investigation upon admission, with both non-treponemal testing (VDRL-Venereal Disease Research Laboratory) and confirmatory treponemal test (TPHA-Treponema Pallidum Hemagglutination). The confirmation via the treponemal test is important due to the possibility of a false-positive result. All the tests were performed in the clinical analysis laboratory of HUPE. According to the protocol of the Brazilian Ministry of Health, blood samples are collected for syphilis testing from all parturients. An active search of syphilis serology results (treponemal and non-treponemal tests) with the respective titrations was conducted by accessing the database of the hospital laboratory. A thorough review of the medical records of pregnant women and their newborns was also conducted. Data from the HUPE epidemiology service were evaluated in order to identify the total number of infected pregnant women and newborns, in order to avoid underestimating the results. A survey was conducted on the website of the State's Department of Health website to collect data on syphilis reports during pregnancy, as well as reports of congenital syphilis on the SINAN (National Disease Notification System) and the number of live births in the SINASC (Live Births Notification System).\n\nInclusion criterion:\nThe following parturients were eligible for the study: pregnant women admitted for delivery with a live fetus of any gestational age and weight, stillbirths with gestational age equal to22 weeks or weight equal to 500 grams.\nThe following cases were defined as syphilis during pregnancy: all cases in which the parturient was admitted with laboratory evidence of positive VDRL (any titer) collected at the time of admission and confirmed by the treponemal test; all cases in which the parturient's newborn (stillbirth or live birth) has been reported as a case of congenital syphilis (CS), identified in any of the information systems consulted.\n\nExclusion criterion:\nThe following parturients were excluded: the ones with positive VDRL resulting from adequately treated previous syphilis (complete treatment with benzathine penicillin, according to the clinical stage of the disease, complete treatment of the partner, documentation confirming the couple's treatment, drop in VDRL titers after adequate treatment, treatment completed more than 30 days before delivery).\n\nVariable definition:\nThe following situations were included as CS cases: all the gestation products (live births or stillborn) identified in any of the information systems as premature congenital syphilis; all newborns with VDRL titers higher than the maternal ones; all newborns with clinical manifestations suggestive of syphilis or complementary tests pointing to CS\n7\n.\n\nEpidemiological definitions:\nAccording to the recommendation of the strategic management of the MH, the prevalence of syphilis in pregnant women is the ratio of the number of cases of syphilis detected in pregnant women to every 1,000 live births, within a geographical space and for a given period. For the purpose of calculating the prevalence, we used as the numerator the number of cases of syphilis detected in pregnant women, in a given notification year and place of residence multiplied by 1,000 and divided by the total number of live births in the same place and in the same reported year\n16\n. The numerator could be found in the SINAN (national disease notification system) and the denominator in the SINASC (live births information system).\nFor the calculation of the prevalence of parturients as a percentage, the number of cases of syphilis in parturients identified in the study was used as the numerator; the number of live births in that place and period, multiplied by 100 as the denominator.\nTo evaluate underreporting, we divided the number of parturients with syphilis in SINAN by the number of parturients with syphilis in hospitals x 100.\nFollowing the MH recommendation for the calculation of the CS incidence, the number of CS cases identified in the study was used as the numerator and the number of live births at that location and period, multiplied by 1000, as the denominator.\nIn order to evaluate the underreporting of congenital syphilis, we divided the number of CS cases by the number of cases reported in SINAN x 100.\n\nData analysis:\nThe sample size was calculated considering an expected prevalence of syphilis in pregnant women according to the MH in public hospitals of 1.1% in 2006, in Brazil\n9\n. To perform this calculation, assuming an error of 5% and a confidence interval (CI) of 95% the sample size was calculated to be 17 for 95% CI and 46 for 99,9% CI.\nCollected variables were used in the comparative analyzes to identify prevalence factors. The results were expressed as percentages. The process of entering and analyzing statistical data was performed through the EPIINFO 3.5.2 version 3.0.1 computing program.\n\nConsiderations on ethical aspects:\nThe research project was carried out within the standards required by the Declaration of Helsinki and Resolution 466 of December 12, 2012, and approved by the Research Ethics Committee of the UERJ (COEP) in July 2012, process N° 034.3.2012 and sponsored by FAPERJ, process N° E-26/110.351/2012.\n\nRESULTS:\nA total of 2,041 parturients were studied in HUPE: 735 in 2012, 664 in 2013 and 642 in 2014. After the exclusion of 19 pregnant women due to serological scarring or false-positive results (treponemal negative test), 79 cases of parturients with syphilis were identified: 32 cases in 2012, 20 in 2013 and 27 in 2014. Access to the result of the treponemal and non-treponemal tests for syphilis was obtained from all the pregnant women admitted during the period.\nThe search on SINASC, considering HUPE as the place of birth, totaled 1,956 live births in HUPE for the three years studied, with 773 live births in 2012, 647 in 2013 and 536 in 2014. The prevalence of syphilis in the HUPE, using the number of live births officially described in SINASC as the denominator, was 4.1% in 2012, 3.1% in 2013 and 5.0% in 2014. Between 2012 and 2014, there was an increase on the prevalence of the disease (Table 1).\nLegend: live births (LB); live birth information system (SINASC); confidence limit (CL)\nThe search on SINAN came up with 23 cases of syphilis in HUPE for the three years studied (2012 = 5, 2013 = 5 and 2014 = 13). The research showed 79 cases in the three years (2012 = 32, 2013 = 20 and 2014 = 27).\nTherefore, 15.6% of cases in 2012, 25.0% in 2013 and 48.1% in 2014 were reported, which shows a significant increase for the period.\nThree parturients (3.8%) from HUPE were double counted in the prevalence calculation, when they were admitted with syphilis at the time of delivery of subsequent pregnancies. One of the three parturients had VDRL titer =1/4 and progressed to 1/128 in the next gestation. The other two had 1/32 titers in the first gestation and had titers of 1/32 and 1/128, respectively, in subsequent pregnancies.\nThe incidence of CS was 22/1,000 LB in 2012, 17/1,000 LB in 2013 and 44.8/ 1,000 LB in 2014. The notification of congenital syphilis reached 92.3% of the cases and only four cases were not officially reported. The present study demonstrated a vertical transmission rate of 65.8%, with 52 newborns affected by congenital syphilis.\nFrom the group of parturients with vertical transmission of the disease, 18 mothers had VDRL titers = 1/4, corresponding to 34.6% of the cases of congenital syphilis. The vertical transmission occurred in two consecutive pregnancies in one case that was within the low titers group (= 1/4).\n\nDISCUSSION:\nThe latest national studies in public or public-private hospitals that monitor the prevalence of syphilis in the country demonstrated a reduction in the prevalence of syphilis among parturients in Brazil. Sentinel studies revealed a prevalence of 1.6%\n17\n in 2004 and 1.02%\n9\n in 2014.\nHowever, the present study does not confirm this trend and demonstrates that the disease is not under control in the State of Rio de Janeiro. In 2014, the prevalence rate of 5.0% was four-fold higher than the one registered in the country in 2014 by Domingues et al.\n\n19\n and twice higher than the one registered in the State of Rio de Janeiro in 2008 by the same author\n18\n.\nThe results are similar to those described by the MH regarding the ascending curve of the disease in recent years, but they express a significantly higher prevalence when compared with data from the epidemiological surveillance agencies for the same period. In 2014, although the State of Rio de Janeiro had the highest syphilis rate in pregnant women in the country (2.2%), the prevalence registered at HUPE was 2.3 times higher than the official one\n10\n.\nThe prevalence registered in 2012 was similar to the one of the Republic of Congo in Africa (4.2%)\n19\n. It was superior to levels in other Latin American countries in the three years studied and it is a warning regarding the epidemiological situation of syphilis in the State\n20\n.\nFor some authors, the increase in the disease prevalence in the country may be associated with the improvement in the diagnosis and reporting systems, and not necessarily with the rise in the total number of cases. However, for some infectologists, the rising prevalence is due to the increasing practice of unprotected sex, which would intensify the number of cases of syphilis in the adult population and, consequently, during pregnancy. For others, there are flaws in the gestation treatment of prenatal services, in both diagnosis and treatment of the pregnant women and their partners\n7\n\n,\n\n21\n.\nA possible limitation of the present study is the selection bias, since the prevalence can be overestimated by including parturients with serological scarring (<1/8) in their calculation, even after the meticulous evaluation of the medical records and considering epidemiological history. The MH considers low VDRL titers when diagnosing syphilis, due to the variations in readings and the lack of detection by the tests. All the national studies consulted, including the sentinel studies in parturients, used any VDRL title for the calculation of the prevalence in its methodology\n9\n\n,\n\n22\n. Furthermore, several authors have already described the clinical significance of low titers of VDRL in the diagnosis of congenital syphilis\n23\n.\nAlthough there has been a substantial increase in the reporting of syphilis in parturients in HUPE between 2012 and 2014, it has not reached half of the cases, which demonstrates how fragile the epidemiological surveillance system is in identifying and reporting all cases. The considerable increase in the coverage of notifications follows the increased number of notifications observed in Brazil, which, according to the MH, is related to the Rede Cegonha program, which increased the coverage of pregnant women testing and follow-up of cases\n24\n. In Brazil, between 2012 and 2013, there was a 25% increase in the number of syphilis reports in pregnant women, and 23% if only the Southeast region is considered, which is responsible for 45.9% of the country's notifications. The increase of the number of notifications in the State of Rio de Janeiro was 16%. In the country, São Paulo is the State with the highest number of notifications, followed by the State of Rio de Janeiro\n25\n.\nAlthough the surveillance agencies considered underreporting for the purposes of disease monitoring, the present study shows that the level of underreporting was higher than estimated and should not be used as a basis for calculation. Without the proper notification, there is a tendency to underestimate the problem. However, the underreporting found in HUPE in 2013 is similar to the one described by Cavalcante et al.\n\n26\n in Fortaleza and Domingues et al.\n\n18\n in SUS units (state health clinics and hospitals) in the city of Rio de Janeiro, where underreporting was 73%, 70.3% and 76.1%, respectively, demonstrating the fragility of the information services in Brazil to work as a basis for promoting measures to combat the disease.\nThe high prevalence of the disease among the parturients of the present study resulted in an incidence of congenital syphilis (26.6 per 1,000 LB) greater than that reported in the State of Rio de Janeiro in 2015 (16 per 1,000 LB)\n10\n and at least five-fold greater than the incidence of the disease in Brazil, according to the Nascer Study (3.51 per 1,000 LB)\n27\n.\nRegarding the notification of congenital syphilis, the present study reached more than 90%, a result far superior to that reported by the MH, which estimates notification at only 17.4% of the cases of CS in the country\n24\n. HUPE is located in the capital of the State of Rio de Janeiro and, according to the epidemiological bulletin of the State Health Department, it is the city with the highest number of notifications of CS in the State (63.5%)\n25\n.\nThe high vertical transmission rate of syphilis in this study (65.8%) shows the high prevalence of syphilis among parturients, since among the various diseases that can be transmitted during the puerperal-pregnancy cycle, syphilis is the one that presents the greater chances of transmission\n28\n. Kupek et al.\n\n11\n showed similar rates (68.9%). Domingues et al.\n18\n and the MH\n7\n, however, estimated lower rates of vertical transmission, 34.8% in Rio de Janeiro and 25% in Brazil, respectively.\nThe occurrence of maternal syphilis in a subsequent gestation demonstrated that a prior gestation with syphilis did not eliminate the risk of the disease in future pregnancies. The recurrence of syphilis in two distinct pregnancies is the major sign of the inefficiency of the health system in properly conducting these cases.\nFetal infection occurred even in the presence of low VDRL maternal titers. The lack of proper consideration of low titers of VDRL by the health professionals is still a barrier to the control of congenital syphilis. In a study with health professionals, it was observed that only 48% had adequate knowledge about the management of syphilis, especially regarding the treatment of pregnant women with low titration\n29\n.\nSince syphilis is an easily diagnosed disease that has an established treatment and generates low costs for testing and treatment during pregnancy, it should be possible to control the disease. However, the disease continues to advance in Brazil in alarming numbers.\n\nCONCLUSIONS:\nThe estimated prevalence of syphilis in pregnant women in Rio de Janeiro is at least twice as high as previously reported by regional studies and epidemiological surveillance data in the State, reaching levels found in African countries and higher than the figures for other countries in Latin America. These values are a warning on the magnitude of the disease in the State of Rio de Janeiro, its upward curve and its consequences. Our reporting system has proven to be fragile and unable to assess the current prevalence of syphilis in Rio de Janeiro, which is underestimated. Due to the high prevalence of syphilis among parturients, there was a high rate of CS and vertical transmission that occurred even in pregnant women with low VDRL titers, emphasizing the importance of evaluating low titers in the management of these patients.\nIt is a responsibility of the health professionals, who daily handle this sad reality, to strive for excellence in caring for pregnant women. Even in the 21st century, the high rates of syphilis in parturients and CS in Brazil are still ignored and underestimated.\n\n"},"whole_article_abstract":{"kind":"string","value":"Background:\nIn 2013, the World Health Organization (WHO) reported that 1.9 million pregnant women were infected with syphilis worldwide, of which 66.5% had adverse fetal effects in cases of untreated syphilis. Congenital syphilis contributes significantly to infant mortality, accounting for 305,000 perinatal deaths worldwide annually.\n\nMethods:\na cross-sectional study with data collected from 2041 parturients who had undergone treatment between 2012 and 2014 in the maternity section of the Pedro Ernesto Hospital of the State University of Rio de Janeiro, in the metropolitan area of Rio de Janeiro. The inclusion criterion was positive VDRL and treponemal test in a hospital environment.\n\nResults:\nthe prevalence of syphilis in pregnant women was 4.1% in 2012, 3.1% in 2013 and 5% in 2014, with official reporting of 15.6%, 25.0% and 48.1%, respectively. The incidence of congenital syphilis (CS) was 22/1,000 in live births (LB) in 2012; 17/1,000 LB in 2013 and 44.8/1,000 LB in 2014. CS underreporting during the period was 6.7%. Vertical transmission occurred in 65.8% of infants from infected mothers. It was concluded that, in 34.6% of the CS cases, maternal VDRL titers were = 1/4.\n\nConclusions:\nResults demonstrate the magnitude of the disease, fragility of the reporting system in the assessment of the actual prevalence, impact on perinatal outcomes, and they are a warning about the real situation of syphilis, which is still underestimated in the State."},"background_conclusion_text":{"kind":"string","value":"INTRODUCTION:\nSyphilis is a sexually transmitted disease caused by the bacterium Treponema pallidum. Infection can be transmitted to the unborn child through the placenta at any gestational stage. In exceptional cases, syphilis can be transmitted at birth through the child's contact with the birth canal, when there are maternal genital lesions (direct transmission). Syphilis may only be transmitted through breastfeeding if there are breast syphilis lesions\n1\n.\nCongenital infection is associated with adverse outcomes, including: perinatal death, preterm birth, low birth weight, congenital anomalies, active syphilis in the newborn (NB), and long-term sequelae, such as deafness and neurological impairment\n2\n.\nThere is a significant global increase in the prevalence of syphilis in pregnant women, despite all efforts from governments to control the disease. In 2013, the WHO reported 1.9 million pregnant women infected with syphilis worldwide, with 66.5% of adverse fetal outcomes occurring in cases of untreated syphilis\n3\n. Congenital syphilis significantly contributes to infant mortality, accounting for 305,000 perinatal deaths per year worldwide\n4\n. There are more newborns affected by syphilis than by any other neonatal infection. It is estimated that 520,000 adverse fetal outcomes occur worldwide each year, making congenital syphilis more common than congenital HIV infection\n5\n. In areas where syphilis is prevalent, about half of the stillbirth rate may have been caused solely by this infection\n6\n. The direct medical cost associated with adverse fetal outcomes resulting from syphilis is U$ 309 million dollars a year\n4\n.\nHigh rates of syphilis in parturients mean a high incidence of congenital syphilis and millions of missed opportunities to save lives during pre-natal care. Over the past ten years, the infant mortality rate due to syphilis increased 150% in Brazil, from 2.2 per 100,000 live births (LB) in 2004 to 5.5 per 100,000 LB in 2013. By 2015, the State of Rio de Janeiro had the highest rate of syphilis during pregnancy (2.2%) and of congenital syphilis in the country (16 cases per 1,000 LB)\n7\n\n,\n\n8\n.\nThe last syphilis monitoring study in pregnant women performed in Brazil in 2014, with a statistical sample from public and private hospitals, estimated the prevalence of syphilis at 1.02%, with no significant regional differences\n9\n.\nAccording to the prediction curve of the disease, the Ministry of Health (MH) expected 40,000 cases in pregnant women in Brazil in 2016. An epidemic of the disease in the country was confirmed by the Ministry of Health in 2016. The increased incidence of syphilis in pregnant women is alarming\n8\n. In 2015, according to the MH, the States of Rio de Janeiro and Mato Grosso do Sul were the most affected in the country, reaching a syphilis rate in pregnant women of 2.2%\n8\n.\nThe national studies show differences in the prevalence of the disease according to the region studied, with rates ranging around 0.4% in Vitória (Espírito Santo)\n10\n, Itajaí (Santa Catarina)\n11\n and 7.7% in Fortaleza (Ceará)\n12\n.\nThe analysis of international studies on the subject in countries such as Haiti\n13\n shows that the prevalence of syphilis in pregnant women reaches 7.6%, while in other countries, such as Canada, syphilis has already been eliminated\n14\n. The WHO certified Cuba in 2013 as the first country to eliminate the vertical transmission of HIV and syphilis during pregnancy. Six other countries are currently able to request the WHO to validate the double elimination, including Canada and the United States\n15\n.\n Aim To estimate the prevalence and coverage of syphilis reporting in parturients, the incidence of congenital syphilis and its corresponding underreporting, as well as the rate of vertical transmission of infection.\nTo estimate the prevalence and coverage of syphilis reporting in parturients, the incidence of congenital syphilis and its corresponding underreporting, as well as the rate of vertical transmission of infection.\n\nCONCLUSIONS:\nThe estimated prevalence of syphilis in pregnant women in Rio de Janeiro is at least twice as high as previously reported by regional studies and epidemiological surveillance data in the State, reaching levels found in African countries and higher than the figures for other countries in Latin America. These values are a warning on the magnitude of the disease in the State of Rio de Janeiro, its upward curve and its consequences. Our reporting system has proven to be fragile and unable to assess the current prevalence of syphilis in Rio de Janeiro, which is underestimated. Due to the high prevalence of syphilis among parturients, there was a high rate of CS and vertical transmission that occurred even in pregnant women with low VDRL titers, emphasizing the importance of evaluating low titers in the management of these patients.\nIt is a responsibility of the health professionals, who daily handle this sad reality, to strive for excellence in caring for pregnant women. Even in the 21st century, the high rates of syphilis in parturients and CS in Brazil are still ignored and underestimated."},"background_conclusion_abstract":{"kind":"string","value":"Background:\nIn 2013, the World Health Organization (WHO) reported that 1.9 million pregnant women were infected with syphilis worldwide, of which 66.5% had adverse fetal effects in cases of untreated syphilis. Congenital syphilis contributes significantly to infant mortality, accounting for 305,000 perinatal deaths worldwide annually.\n\nMethods:\na cross-sectional study with data collected from 2041 parturients who had undergone treatment between 2012 and 2014 in the maternity section of the Pedro Ernesto Hospital of the State University of Rio de Janeiro, in the metropolitan area of Rio de Janeiro. The inclusion criterion was positive VDRL and treponemal test in a hospital environment.\n\nResults:\nthe prevalence of syphilis in pregnant women was 4.1% in 2012, 3.1% in 2013 and 5% in 2014, with official reporting of 15.6%, 25.0% and 48.1%, respectively. The incidence of congenital syphilis (CS) was 22/1,000 in live births (LB) in 2012; 17/1,000 LB in 2013 and 44.8/1,000 LB in 2014. CS underreporting during the period was 6.7%. Vertical transmission occurred in 65.8% of infants from infected mothers. It was concluded that, in 34.6% of the CS cases, maternal VDRL titers were = 1/4.\n\nConclusions:\nResults demonstrate the magnitude of the disease, fragility of the reporting system in the assessment of the actual prevalence, impact on perinatal outcomes, and they are a warning about the real situation of syphilis, which is still underestimated in the State."},"whole_article_text_length":{"kind":"number","value":5870,"string":"5,870"},"whole_article_abstract_length":{"kind":"number","value":281,"string":"281"},"other_sections_lengths":{"kind":"list like","value":[33,5,55,227,117,64,60,287,113,55],"string":"[\n 33,\n 5,\n 55,\n 227,\n 117,\n 64,\n 60,\n 287,\n 113,\n 55\n]","isConversational":false},"num_sections":{"kind":"number","value":15,"string":"15"},"most_frequent_words":{"kind":"list like","value":["syphilis","cases","number","prevalence","pregnant","pregnant women","women","parturients","live","disease"],"string":"[\n \"syphilis\",\n \"cases\",\n \"number\",\n 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syphilis\"\n]","isConversational":false},"annotated_base_background_abstract_prompt":{"kind":"string","value":""},"annotated_base_methods_abstract_prompt":{"kind":"null"},"annotated_base_results_abstract_prompt":{"kind":"string","value":""},"annotated_base_conclusions_abstract_prompt":{"kind":"string","value":""},"annotated_base_whole_article_abstract_prompt":{"kind":"string","value":""},"annotated_base_background_conclusion_abstract_prompt":{"kind":"string","value":""},"annotated_keywords_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Syphilis | Gestation | Congenital syphilis | Prevalence | VDRL | Treponemal tests | Treponema pallidum [SUMMARY]"},"annotated_keywords_methods_abstract_prompt":{"kind":"null"},"annotated_keywords_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Syphilis | Gestation | Congenital syphilis | Prevalence | VDRL | Treponemal tests | Treponema pallidum [SUMMARY]"},"annotated_keywords_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Syphilis | Gestation | Congenital syphilis | Prevalence | VDRL | Treponemal tests | Treponema pallidum [SUMMARY]"},"annotated_keywords_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Syphilis | Gestation | Congenital syphilis | Prevalence | VDRL | Treponemal tests | Treponema pallidum [SUMMARY]"},"annotated_keywords_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Syphilis | Gestation | Congenital syphilis | Prevalence | VDRL | Treponemal tests | Treponema pallidum [SUMMARY]"},"annotated_mesh_background_abstract_prompt":{"kind":"string","value":"[CONTENT] Brazil | Cross-Sectional Studies | Female | Humans | Incidence | Infant, Newborn | Infectious Disease Transmission, Vertical | Pregnancy | Pregnancy Complications, Infectious | Prevalence | Syphilis | Syphilis, Congenital [SUMMARY]"},"annotated_mesh_methods_abstract_prompt":{"kind":"null"},"annotated_mesh_results_abstract_prompt":{"kind":"string","value":"[CONTENT] Brazil | Cross-Sectional Studies | Female | Humans | Incidence | Infant, Newborn | Infectious Disease Transmission, Vertical | Pregnancy | Pregnancy Complications, Infectious | Prevalence | Syphilis | Syphilis, Congenital [SUMMARY]"},"annotated_mesh_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] Brazil | Cross-Sectional Studies | Female | Humans | Incidence | Infant, Newborn | Infectious Disease Transmission, Vertical | Pregnancy | Pregnancy Complications, Infectious | Prevalence | Syphilis | Syphilis, Congenital [SUMMARY]"},"annotated_mesh_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] Brazil | Cross-Sectional Studies | Female | Humans | Incidence | Infant, Newborn | Infectious Disease Transmission, Vertical | Pregnancy | Pregnancy Complications, Infectious | Prevalence | Syphilis | Syphilis, Congenital [SUMMARY]"},"annotated_mesh_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] Brazil | Cross-Sectional Studies | Female | Humans | Incidence | Infant, Newborn | Infectious Disease Transmission, Vertical | Pregnancy | Pregnancy Complications, Infectious | Prevalence | Syphilis | Syphilis, Congenital [SUMMARY]"},"annotated_keybert_background_abstract_prompt":{"kind":"string","value":"[CONTENT] maternal syphilis | affected syphilis neonatal | underreporting congenital syphilis | syphilis transmitted birth | newborns affected syphilis [SUMMARY]"},"annotated_keybert_methods_abstract_prompt":{"kind":"null"},"annotated_keybert_results_abstract_prompt":{"kind":"string","value":"[CONTENT] maternal syphilis | affected syphilis neonatal | underreporting congenital syphilis | syphilis transmitted birth | newborns affected syphilis [SUMMARY]"},"annotated_keybert_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] maternal syphilis | affected syphilis neonatal | underreporting congenital syphilis | syphilis transmitted birth | newborns affected syphilis [SUMMARY]"},"annotated_keybert_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] maternal syphilis | affected syphilis neonatal | underreporting congenital syphilis | syphilis transmitted birth | newborns affected syphilis [SUMMARY]"},"annotated_keybert_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] maternal syphilis | affected syphilis neonatal | underreporting congenital syphilis | syphilis transmitted birth | newborns affected syphilis [SUMMARY]"},"annotated_most_frequent_background_abstract_prompt":{"kind":"string","value":"[CONTENT] syphilis | cases | number | prevalence | pregnant | pregnant women | women | parturients | live | disease [SUMMARY]"},"annotated_most_frequent_methods_abstract_prompt":{"kind":"null"},"annotated_most_frequent_results_abstract_prompt":{"kind":"string","value":"[CONTENT] syphilis | cases | number | prevalence | pregnant | pregnant women | women | parturients | live | disease [SUMMARY]"},"annotated_most_frequent_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] syphilis | cases | number | prevalence | pregnant | pregnant women | women | parturients | live | disease [SUMMARY]"},"annotated_most_frequent_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] syphilis | cases | number | prevalence | pregnant | pregnant women | women | parturients | live | disease [SUMMARY]"},"annotated_most_frequent_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] syphilis | cases | number | prevalence | pregnant | pregnant women | women | parturients | live | disease [SUMMARY]"},"annotated_tf_idf_background_abstract_prompt":{"kind":"string","value":"[CONTENT] syphilis | infection | congenital | adverse | outcomes | 000 | rate | transmitted | country | pregnant women [SUMMARY]"},"annotated_tf_idf_methods_abstract_prompt":{"kind":"null"},"annotated_tf_idf_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 2013 | 2014 | 2012 | cases | 32 | hupe | syphilis | lb | live | pregnancies [SUMMARY]"},"annotated_tf_idf_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] high | underestimated | janeiro | de | rio | rio de janeiro | rio de | de janeiro | prevalence syphilis | syphilis [SUMMARY]"},"annotated_tf_idf_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] syphilis | cases | number | 2012 | prevalence | study | live | treatment | parturients | pregnant women [SUMMARY]"},"annotated_tf_idf_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] syphilis | cases | number | 2012 | prevalence | study | live | treatment | parturients | pregnant women [SUMMARY]"},"annotated_entity_plan_background_abstract_prompt":{"kind":"string","value":"[CONTENT] 2013 | the World Health Organization (WHO | 1.9 million | 66.5% ||| 305,000 | annually [SUMMARY]"},"annotated_entity_plan_methods_abstract_prompt":{"kind":"null"},"annotated_entity_plan_results_abstract_prompt":{"kind":"string","value":"[CONTENT] 4.1% | 2012 | 3.1% | 2013 | 5% | 2014 | 15.6% | 25.0% | 48.1% ||| CS | 22/1,000 | LB | 2012 | LB | 2013 | 44.8/1,000 | LB | 2014 ||| CS | 6.7% ||| 65.8% ||| 34.6% | CS | VDRL | 1/4 [SUMMARY]"},"annotated_entity_plan_conclusions_abstract_prompt":{"kind":"string","value":"[CONTENT] State [SUMMARY]"},"annotated_entity_plan_whole_article_abstract_prompt":{"kind":"string","value":"[CONTENT] 2013 | the World Health Organization (WHO | 1.9 million | 66.5% ||| 305,000 | annually ||| 2041 | between 2012 and 2014 | the Pedro Ernesto Hospital | the State University of Rio de Janeiro | Rio de Janeiro ||| VDRL ||| 4.1% | 2012 | 3.1% | 2013 | 5% | 2014 | 15.6% | 25.0% | 48.1% ||| CS | 22/1,000 | LB | 2012 | LB | 2013 | 44.8/1,000 | LB | 2014 ||| CS | 6.7% ||| 65.8% ||| 34.6% | CS | VDRL | 1/4 ||| State [SUMMARY]"},"annotated_entity_plan_background_conclusion_abstract_prompt":{"kind":"string","value":"[CONTENT] 2013 | the World Health Organization (WHO | 1.9 million | 66.5% ||| 305,000 | annually ||| 2041 | between 2012 and 2014 | the Pedro Ernesto Hospital | the State University of Rio de Janeiro | Rio de Janeiro ||| VDRL ||| 4.1% | 2012 | 3.1% | 2013 | 5% | 2014 | 15.6% | 25.0% | 48.1% ||| CS | 22/1,000 | LB | 2012 | LB | 2013 | 44.8/1,000 | LB | 2014 ||| CS | 6.7% ||| 65.8% ||| 34.6% | CS | VDRL | 1/4 ||| State [SUMMARY]"}}},{"rowIdx":96,"cells":{"title":{"kind":"null"},"pmid":{"kind":"string","value":"34712033"},"background_abstract":{"kind":"string","value":"Helicobacter pylori (H. pylori) is a spiral-shaped bacterium responsible for the development of chronic gastritis, gastric ulcer, gastric cancer (GC), and MALT-lymphoma of the stomach. H. pylori can be present in the gastric mucosa (GM) in both spiral and coccoid forms. However, it is not known whether the severity of GM contamination by various vegetative forms of H. pylori is associated with clinical and morphological characteristics and long-term results of GC treatment."},"background_abstract_label":{"kind":"string","value":"BACKGROUND"},"methods_abstract":{"kind":"string","value":"Of 109 patients with GC were included in a prospective cohort study. H. pylori in the GM and tumor was determined by rapid urease test and by immunohistochemically using the antibody to H. pylori. The results obtained were compared with the clinical and morphological characteristics and prognosis of GC. Statistical analysis was performed using the Statistica 10.0 software."},"methods_abstract_label":{"kind":"string","value":"METHODS"},"results_abstract":{"kind":"string","value":"H. pylori was detected in the adjacent to the tumor GM in 84.5% of cases, of which a high degree of contamination was noted in 50.4% of the samples. Coccoid forms of H. pylori were detected in 93.4% of infected patients, and only coccoid-in 68.9%. It was found that a high degree of GM contamination by the coccoid forms of H. pylori was observed significantly more often in diffuse type of GC (P = 0.024), in poorly differentiated GC (P = 0.011), in stage T3-4 (P = 0.04) and in N1 (P = 0.011). In cases of moderate and marked concentrations of H. pylori in GM, a decrease in 10-year relapse free and overall survival from 55.6% to 26.3% was observed (P = 0.02 and P = 0.07, respectively). The relationship between the severity of the GM contamination by the spiral-shaped forms of H. pylori and the clinical and morphological characteristics and prognosis of GC was not revealed."},"results_abstract_label":{"kind":"string","value":"RESULTS"},"conclusions_abstract":{"kind":"string","value":"The data obtained indicates that H. pylori may be associated not only with induction but also with the progression of GC."},"conclusions_abstract_label":{"kind":"string","value":"CONCLUSION"},"mesh_descriptor_names":{"kind":"list like","value":["Gastric Mucosa","Helicobacter Infections","Helicobacter pylori","Humans","Neoplasm Recurrence, Local","Prospective Studies","Stomach Neoplasms"],"string":"[\n \"Gastric Mucosa\",\n \"Helicobacter Infections\",\n \"Helicobacter pylori\",\n \"Humans\",\n \"Neoplasm Recurrence, Local\",\n \"Prospective Studies\",\n \"Stomach Neoplasms\"\n]","isConversational":false},"pmcid":{"kind":"string","value":"8515796"},"background_title":{"kind":"string","value":"INTRODUCTION"},"background_text":{"kind":"string","value":"Gastric cancer (GC) continues to be one of the most common malignant diseases in the world[1,2]. Despite a decreasing trend in the incidence of GC in most countries of the world, the treatment results of this pathology cannot be considered satisfactory. In the structure of mortality from malignant neoplasms, this pathology firmly occupies 2nd place in most developed countries of the world, and the 5-year survival rate of radically operated patients does not exceed 15%-30%[3,4]. \nIt is important to note that it is impossible to improve the long-term results of malignant neoplasms treatment without knowledge of the mechanisms associated with their progression[3]. Clinical studies in recent years indicate that inflammatory infiltration of the tumor stroma and surrounding tissues can have an important prognostic value and affect the long-term results of malignant neoplasm treatment[5-9]. A number of studies have shown that inflammatory infiltration of the tumor stroma is associated with the body’s adequate immune response to the tumor, and may be a favorable prognosis factor in various malignant neoplasms[10,11], including GC[12,13]. At the same time, the data obtained by other researchers indicates that pronounced inflammatory infiltration of the tumor stroma, especially T-reg lymphocytes and macrophages, may be a factor contributing to the progression of malignant neoplasms[14,15]. There was a decrease in the overall survival (OS) and relapse-free survival (RFS) of GC patients with a high content of Foxp3 + T-reg in the tumor stroma and regional metastases[16-18] and macrophages[16,19]. It is believed that inflammatory infiltration of the tumor stroma can contribute to tumor progression by activating the mechanisms of angiogenesis, expression of E- and L-selectins, formation of the products of lipid peroxidation and free radicals, destruction of connective tissue matrix and basement membranes of epithelia by proteolytic enzymes, and activation of epithelial-mesenchymal transformation[20-23].\nWhen studying the role of inflammation in the progression of GC, it is impossible to ignore the problem of Helicobacter pylori (H. pylori) infection. H. pylori is a gram-negative, spiral-shaped bacterium, the habitat of which is the gastric mucosa (GM) and duodenum. H. pylori differs from other bacteria in a set of properties that make it possible to colonize the GM and persist for a long time under conditions that are unfavorable for other microorganisms[24,25]. These include: (1) The ability to produce a special enzyme-urease; (2) Synthesis of lytic enzymes that cause the depolymerization and dissolution of gastric mucus, consisting mainly of mucin; (3) The mobility of the bacterium, which is ensured by the presence of 5-6 flagella; (4) The high adhesiveness of bacteria to GM epithelial cells of the GM and elements of connective tissue due to the interaction of bacterial ligands with the corresponding cells receptors; (5) Production of various exotoxins (VacA, CagA, and others); (6) Instability of the H. pylori genome; (7) The presence of vegetative and coccoid forms of bacteria; and (8) possibility of intracellular persistence and translocation outside the GM [26-29].\nIt should be noted that despite the huge number of studies devoted to H. pylori, it is still not clear whether H. pylori is involved only in the initiation of the tumor process in the stomach, or whether it can affect the mechanisms of tumor progression. The relationship between the severity of H. pylori infection and the clinical and morphological characteristics of GC and long-term results of this pathology treatment remains poorly studied, and in this connection, the question of the expediency of anti-Helicobacter therapy in patients with invasive GC remains open.\nObjective To assess the features of H. pylori infection in patients with Stage I-IIIB GC and their correlation with the clinical and morphological characteristics of the disease, the presence of antibiotic therapy (AT) before surgery, and long-term treatment results.\nTo assess the features of H. pylori infection in patients with Stage I-IIIB GC and their correlation with the clinical and morphological characteristics of the disease, the presence of antibiotic therapy (AT) before surgery, and long-term treatment results."},"methods_title":{"kind":"string","value":"MATERIALS AND METHODS"},"methods_text":{"kind":"string","value":"The patients One hundred and nine patients with GC who had undergone radical surgery (R0) between May 2007 and March 2010 at the Orenburg Regional Clinical Oncology Center, were included in this prospective cohort pilot study. Study inclusion criteria were: Histologically proven invasive GC; no evidence of distant metastases; radical surgery (R0); no prior gastric surgery; no previous chemotherapy or radiotherapy. The study did not include patients with decompensation of cardiovascular and renal diseases, exacerbation of chronic inflammatory processes, severe allergic processes, or who received glucocorticoids, antihistamines, and non-steroidal anti-inflammatory drugs. The study was performed in accordance with the Helsinki Declaration and internationally recognized guidelines, and the privacy of patients was protected by decoding the data according to the privacy regulations of the Orenburg Regional Clinical Oncologic Center (Russia, Orenburg). All patients provided written informed consent. The protocol was approved by the Institutional Review Board of the Orenburg State Medical University (Russia, Orenburg).\nClinical and pathological data including age, tumor localization, stage, type of surgery, histology, the presence of AT before surgery, postoperative therapy, and long-term results of treatment were retrieved from the routine reports for analyses. The distribution of patients according to the clinical and pathological characteristics of GC is presented in Table 1.\nThe distribution of patients according to the clinical and pathological characteristics of gastric cancer\nGC: Gastric cancer.\nWhen interviewing patients, it was found that 45 patients received AT before admission to the clinic due to a preliminary wrong diagnosis of gastric ulcer or chronic gastritis. The following combinations of antibacterial drugs were most commonly ordered: Amoxicillin + clarithromycin (34 patients), amoxicillin + clarithromycin + metronidazole (6 patients), amoxicillin + metronidazole (3 patients), other drugs (2 patients). Information about the antibacterial drugs, the timing and duration of their intake was entered into the primary patient documentation and then taken into account in the analysis. We considered only those patients who underwent AT in the period from 1 to 1.5 mo before the operation, lasting at least seven days, and using two or more antibacterial drugs.\nThe long-term results of treatment were assessed for the period from May 12, 2007 to April 12, 2021. The median follow-up period was 86.2 mo. As of April 12, 2021, 26 (24.5%) patients were alive, 54 (50.9%) had died from the progression of GC, 20 (18.9%) had died from causes other than GC, and six (5.6%) left the region at different follow-up periods. Malignant tumors of other localizations were diagnosed in 8 patients at different times after the operation: Non-Hodgkin's lymphomas-in three, prostate cancer-in one, lung cancer-in two, laryngeal cancer-in one, and breast cancer-in one patient. With the exception of one patient with non-Hodgkin's lymphoma, the other patients died from the progression of these diseases. The causes of death were not associated with malignant tumors for the other patients. During the period 2020-2021, seven patients contracted coronavirus disease-19, one of whom died from the disease, but the rest are alive.\nOne hundred and nine patients with GC who had undergone radical surgery (R0) between May 2007 and March 2010 at the Orenburg Regional Clinical Oncology Center, were included in this prospective cohort pilot study. Study inclusion criteria were: Histologically proven invasive GC; no evidence of distant metastases; radical surgery (R0); no prior gastric surgery; no previous chemotherapy or radiotherapy. The study did not include patients with decompensation of cardiovascular and renal diseases, exacerbation of chronic inflammatory processes, severe allergic processes, or who received glucocorticoids, antihistamines, and non-steroidal anti-inflammatory drugs. The study was performed in accordance with the Helsinki Declaration and internationally recognized guidelines, and the privacy of patients was protected by decoding the data according to the privacy regulations of the Orenburg Regional Clinical Oncologic Center (Russia, Orenburg). All patients provided written informed consent. The protocol was approved by the Institutional Review Board of the Orenburg State Medical University (Russia, Orenburg).\nClinical and pathological data including age, tumor localization, stage, type of surgery, histology, the presence of AT before surgery, postoperative therapy, and long-term results of treatment were retrieved from the routine reports for analyses. The distribution of patients according to the clinical and pathological characteristics of GC is presented in Table 1.\nThe distribution of patients according to the clinical and pathological characteristics of gastric cancer\nGC: Gastric cancer.\nWhen interviewing patients, it was found that 45 patients received AT before admission to the clinic due to a preliminary wrong diagnosis of gastric ulcer or chronic gastritis. The following combinations of antibacterial drugs were most commonly ordered: Amoxicillin + clarithromycin (34 patients), amoxicillin + clarithromycin + metronidazole (6 patients), amoxicillin + metronidazole (3 patients), other drugs (2 patients). Information about the antibacterial drugs, the timing and duration of their intake was entered into the primary patient documentation and then taken into account in the analysis. We considered only those patients who underwent AT in the period from 1 to 1.5 mo before the operation, lasting at least seven days, and using two or more antibacterial drugs.\nThe long-term results of treatment were assessed for the period from May 12, 2007 to April 12, 2021. The median follow-up period was 86.2 mo. As of April 12, 2021, 26 (24.5%) patients were alive, 54 (50.9%) had died from the progression of GC, 20 (18.9%) had died from causes other than GC, and six (5.6%) left the region at different follow-up periods. Malignant tumors of other localizations were diagnosed in 8 patients at different times after the operation: Non-Hodgkin's lymphomas-in three, prostate cancer-in one, lung cancer-in two, laryngeal cancer-in one, and breast cancer-in one patient. With the exception of one patient with non-Hodgkin's lymphoma, the other patients died from the progression of these diseases. The causes of death were not associated with malignant tumors for the other patients. During the period 2020-2021, seven patients contracted coronavirus disease-19, one of whom died from the disease, but the rest are alive.\nDetection H. pylori infection \nH. pylori in the GM and tumor was determined by rapid urease test (RUT) and by immunohistochemically (IHC) using the antibody to H. pylori.\n\nH. pylori in the GM and tumor was determined by rapid urease test (RUT) and by immunohistochemically (IHC) using the antibody to H. pylori.\nRUT After removal of the stomach (within 30 min) a greater curvature of the organ was opened and biopsy samples were taken from the tumor and the adjacent macroscopically non-tumorous GM at a distance of 3-5 cm from the tumor margin. The samples were placed on test strips (HELPIL–test, “АМА”, Russia) for three minutes. The presence and the severity of the infection were evaluated by the color change of the indicator from yellow to blue. \nAccording to the intensity and the time of the appearance of a blue color, we distinguished three degrees of infection: 3+: Marked (+++)-bright staining in the first minute of the study; 2+: Moderate (++)–for an average intensity of staining for 2 min and 1+ mild (+)-weak staining for three minutes. If the color of the indicator did not change, or became dirty gray, and if after repeated research the same result was received, the test was evaluated as negative. The same samples were later used for histological analysis and IGH. \nAfter removal of the stomach (within 30 min) a greater curvature of the organ was opened and biopsy samples were taken from the tumor and the adjacent macroscopically non-tumorous GM at a distance of 3-5 cm from the tumor margin. The samples were placed on test strips (HELPIL–test, “АМА”, Russia) for three minutes. The presence and the severity of the infection were evaluated by the color change of the indicator from yellow to blue. \nAccording to the intensity and the time of the appearance of a blue color, we distinguished three degrees of infection: 3+: Marked (+++)-bright staining in the first minute of the study; 2+: Moderate (++)–for an average intensity of staining for 2 min and 1+ mild (+)-weak staining for three minutes. If the color of the indicator did not change, or became dirty gray, and if after repeated research the same result was received, the test was evaluated as negative. The same samples were later used for histological analysis and IGH. \nImmunohistochemistry The presence and features of H. pylori infection were studied in samples of the GM adjacent to the tumor, in the tumor tissue, in the omentum, and regional lymph nodes of 46 patients, by immunohistochemistry.\nThe sections for IGH were dewaxed and rehydrated by sequential immersion in xylene and graded ethanol and water. For antigen retrieval, the sections boiling for 10 min in citrate buffer (pH 6) and endogenous peroxidase activity was blocked with 30 mL/L hydrogen peroxide solution. Slides were incubated at room temperature with the anti-H. pylori (RB-9070, Thermo Fisher Scientific, the immunogen is purified H. pylori) rabbit polyclonal antibodies in diluted at 1:1000 for 30 min. \nThe visualization system included DAB (UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen) and hematoxylin counterstaining. For negative control sections, the primary antibody was replaced with phosphate-buffered saline and processed in the same manner.\nThe concentration of H. pylori in the GM detected by IGH was graded as 1+ for mild, 2+ for moderate, and 3+ for marked according to the Sydney system[30]. The presence of point inclusions giving a positive reaction with antibodies to H. pylori in the cytoplasm of epithelial cells of deep gastric glands and of the lymphoid cells of the lamina propria of GM as well as in omentum and lymph node, were taken into account. \nAll sections were carefully and completely scanned by two of the authors (MS and OT) without knowledge of the clinical and pathological data. \nThe data obtained was compared with clinical features of GC: Stage, localization, histology, the presence of AT before surgery, and 10-year OS and RFS.\nThe presence and features of H. pylori infection were studied in samples of the GM adjacent to the tumor, in the tumor tissue, in the omentum, and regional lymph nodes of 46 patients, by immunohistochemistry.\nThe sections for IGH were dewaxed and rehydrated by sequential immersion in xylene and graded ethanol and water. For antigen retrieval, the sections boiling for 10 min in citrate buffer (pH 6) and endogenous peroxidase activity was blocked with 30 mL/L hydrogen peroxide solution. Slides were incubated at room temperature with the anti-H. pylori (RB-9070, Thermo Fisher Scientific, the immunogen is purified H. pylori) rabbit polyclonal antibodies in diluted at 1:1000 for 30 min. \nThe visualization system included DAB (UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen) and hematoxylin counterstaining. For negative control sections, the primary antibody was replaced with phosphate-buffered saline and processed in the same manner.\nThe concentration of H. pylori in the GM detected by IGH was graded as 1+ for mild, 2+ for moderate, and 3+ for marked according to the Sydney system[30]. The presence of point inclusions giving a positive reaction with antibodies to H. pylori in the cytoplasm of epithelial cells of deep gastric glands and of the lymphoid cells of the lamina propria of GM as well as in omentum and lymph node, were taken into account. \nAll sections were carefully and completely scanned by two of the authors (MS and OT) without knowledge of the clinical and pathological data. \nThe data obtained was compared with clinical features of GC: Stage, localization, histology, the presence of AT before surgery, and 10-year OS and RFS.\nStatistics Statistical analysis was performed using the Statistica 10.0 software. The correlations between different data were evaluated using the nonparametric Spearman's rank correlation or gamma correlation. Chi-square tests were carried out to analyze the difference of distribution among the categorized data. Mann–Whitney U nonparametric test was used to compare the value of the quantitative data. The survival was analyzed using the Kaplan-Meier method. The log-rank test was used to compare survival curves between subgroups of patients. A value of P < 0.05 was considered statistically significant.\nStatistical analysis was performed using the Statistica 10.0 software. The correlations between different data were evaluated using the nonparametric Spearman's rank correlation or gamma correlation. Chi-square tests were carried out to analyze the difference of distribution among the categorized data. Mann–Whitney U nonparametric test was used to compare the value of the quantitative data. The survival was analyzed using the Kaplan-Meier method. The log-rank test was used to compare survival curves between subgroups of patients. A value of P < 0.05 was considered statistically significant."},"results_title":{"kind":"null"},"results_text":{"kind":"null"},"conclusions_title":{"kind":"string","value":"CONCLUSION"},"conclusions_text":{"kind":"string","value":"The results obtained do not allow one to draw unambiguous conclusions about the role of H. pylori in the progression of GC. Further appropriate prospective studies regarding the role of H. pylori in the progression of GC are obviously advisable."},"other_sections_titles":{"kind":"list like","value":["INTRODUCTION","Objective","The patients","Detection H. pylori infection","RUT","Immunohistochemistry ","Statistics","RESULTS","The features of H. pylori infection in GC","Correlations of clinical and pathological characteristics of GC with the severity of H. pylori infection according to RUT data ","Correlations of clinical and pathological characteristics of GC with the concentration of spiral and coccoid forms of H. pylori in the GM ","DISCUSSION","CONCLUSION"],"string":"[\n \"INTRODUCTION\",\n \"Objective\",\n \"The patients\",\n \"Detection H. pylori infection\",\n \"RUT\",\n \"Immunohistochemistry \",\n \"Statistics\",\n \"RESULTS\",\n \"The features of H. pylori infection in GC\",\n \"Correlations of clinical and pathological characteristics of GC with the severity of H. pylori infection according to RUT data \",\n \"Correlations of clinical and pathological characteristics of GC with the concentration of spiral and coccoid forms of H. pylori in the GM \",\n \"DISCUSSION\",\n \"CONCLUSION\"\n]","isConversational":false},"other_sections_texts":{"kind":"list like","value":["Gastric cancer (GC) continues to be one of the most common malignant diseases in the world[1,2]. Despite a decreasing trend in the incidence of GC in most countries of the world, the treatment results of this pathology cannot be considered satisfactory. In the structure of mortality from malignant neoplasms, this pathology firmly occupies 2nd place in most developed countries of the world, and the 5-year survival rate of radically operated patients does not exceed 15%-30%[3,4]. \nIt is important to note that it is impossible to improve the long-term results of malignant neoplasms treatment without knowledge of the mechanisms associated with their progression[3]. Clinical studies in recent years indicate that inflammatory infiltration of the tumor stroma and surrounding tissues can have an important prognostic value and affect the long-term results of malignant neoplasm treatment[5-9]. A number of studies have shown that inflammatory infiltration of the tumor stroma is associated with the body’s adequate immune response to the tumor, and may be a favorable prognosis factor in various malignant neoplasms[10,11], including GC[12,13]. At the same time, the data obtained by other researchers indicates that pronounced inflammatory infiltration of the tumor stroma, especially T-reg lymphocytes and macrophages, may be a factor contributing to the progression of malignant neoplasms[14,15]. There was a decrease in the overall survival (OS) and relapse-free survival (RFS) of GC patients with a high content of Foxp3 + T-reg in the tumor stroma and regional metastases[16-18] and macrophages[16,19]. It is believed that inflammatory infiltration of the tumor stroma can contribute to tumor progression by activating the mechanisms of angiogenesis, expression of E- and L-selectins, formation of the products of lipid peroxidation and free radicals, destruction of connective tissue matrix and basement membranes of epithelia by proteolytic enzymes, and activation of epithelial-mesenchymal transformation[20-23].\nWhen studying the role of inflammation in the progression of GC, it is impossible to ignore the problem of Helicobacter pylori (H. pylori) infection. H. pylori is a gram-negative, spiral-shaped bacterium, the habitat of which is the gastric mucosa (GM) and duodenum. H. pylori differs from other bacteria in a set of properties that make it possible to colonize the GM and persist for a long time under conditions that are unfavorable for other microorganisms[24,25]. These include: (1) The ability to produce a special enzyme-urease; (2) Synthesis of lytic enzymes that cause the depolymerization and dissolution of gastric mucus, consisting mainly of mucin; (3) The mobility of the bacterium, which is ensured by the presence of 5-6 flagella; (4) The high adhesiveness of bacteria to GM epithelial cells of the GM and elements of connective tissue due to the interaction of bacterial ligands with the corresponding cells receptors; (5) Production of various exotoxins (VacA, CagA, and others); (6) Instability of the H. pylori genome; (7) The presence of vegetative and coccoid forms of bacteria; and (8) possibility of intracellular persistence and translocation outside the GM [26-29].\nIt should be noted that despite the huge number of studies devoted to H. pylori, it is still not clear whether H. pylori is involved only in the initiation of the tumor process in the stomach, or whether it can affect the mechanisms of tumor progression. The relationship between the severity of H. pylori infection and the clinical and morphological characteristics of GC and long-term results of this pathology treatment remains poorly studied, and in this connection, the question of the expediency of anti-Helicobacter therapy in patients with invasive GC remains open.\nObjective To assess the features of H. pylori infection in patients with Stage I-IIIB GC and their correlation with the clinical and morphological characteristics of the disease, the presence of antibiotic therapy (AT) before surgery, and long-term treatment results.\nTo assess the features of H. pylori infection in patients with Stage I-IIIB GC and their correlation with the clinical and morphological characteristics of the disease, the presence of antibiotic therapy (AT) before surgery, and long-term treatment results.","To assess the features of H. pylori infection in patients with Stage I-IIIB GC and their correlation with the clinical and morphological characteristics of the disease, the presence of antibiotic therapy (AT) before surgery, and long-term treatment results.","One hundred and nine patients with GC who had undergone radical surgery (R0) between May 2007 and March 2010 at the Orenburg Regional Clinical Oncology Center, were included in this prospective cohort pilot study. Study inclusion criteria were: Histologically proven invasive GC; no evidence of distant metastases; radical surgery (R0); no prior gastric surgery; no previous chemotherapy or radiotherapy. The study did not include patients with decompensation of cardiovascular and renal diseases, exacerbation of chronic inflammatory processes, severe allergic processes, or who received glucocorticoids, antihistamines, and non-steroidal anti-inflammatory drugs. The study was performed in accordance with the Helsinki Declaration and internationally recognized guidelines, and the privacy of patients was protected by decoding the data according to the privacy regulations of the Orenburg Regional Clinical Oncologic Center (Russia, Orenburg). All patients provided written informed consent. The protocol was approved by the Institutional Review Board of the Orenburg State Medical University (Russia, Orenburg).\nClinical and pathological data including age, tumor localization, stage, type of surgery, histology, the presence of AT before surgery, postoperative therapy, and long-term results of treatment were retrieved from the routine reports for analyses. The distribution of patients according to the clinical and pathological characteristics of GC is presented in Table 1.\nThe distribution of patients according to the clinical and pathological characteristics of gastric cancer\nGC: Gastric cancer.\nWhen interviewing patients, it was found that 45 patients received AT before admission to the clinic due to a preliminary wrong diagnosis of gastric ulcer or chronic gastritis. The following combinations of antibacterial drugs were most commonly ordered: Amoxicillin + clarithromycin (34 patients), amoxicillin + clarithromycin + metronidazole (6 patients), amoxicillin + metronidazole (3 patients), other drugs (2 patients). Information about the antibacterial drugs, the timing and duration of their intake was entered into the primary patient documentation and then taken into account in the analysis. We considered only those patients who underwent AT in the period from 1 to 1.5 mo before the operation, lasting at least seven days, and using two or more antibacterial drugs.\nThe long-term results of treatment were assessed for the period from May 12, 2007 to April 12, 2021. The median follow-up period was 86.2 mo. As of April 12, 2021, 26 (24.5%) patients were alive, 54 (50.9%) had died from the progression of GC, 20 (18.9%) had died from causes other than GC, and six (5.6%) left the region at different follow-up periods. Malignant tumors of other localizations were diagnosed in 8 patients at different times after the operation: Non-Hodgkin's lymphomas-in three, prostate cancer-in one, lung cancer-in two, laryngeal cancer-in one, and breast cancer-in one patient. With the exception of one patient with non-Hodgkin's lymphoma, the other patients died from the progression of these diseases. The causes of death were not associated with malignant tumors for the other patients. During the period 2020-2021, seven patients contracted coronavirus disease-19, one of whom died from the disease, but the rest are alive.","\nH. pylori in the GM and tumor was determined by rapid urease test (RUT) and by immunohistochemically (IHC) using the antibody to H. pylori.","After removal of the stomach (within 30 min) a greater curvature of the organ was opened and biopsy samples were taken from the tumor and the adjacent macroscopically non-tumorous GM at a distance of 3-5 cm from the tumor margin. The samples were placed on test strips (HELPIL–test, “АМА”, Russia) for three minutes. The presence and the severity of the infection were evaluated by the color change of the indicator from yellow to blue. \nAccording to the intensity and the time of the appearance of a blue color, we distinguished three degrees of infection: 3+: Marked (+++)-bright staining in the first minute of the study; 2+: Moderate (++)–for an average intensity of staining for 2 min and 1+ mild (+)-weak staining for three minutes. If the color of the indicator did not change, or became dirty gray, and if after repeated research the same result was received, the test was evaluated as negative. The same samples were later used for histological analysis and IGH. ","The presence and features of H. pylori infection were studied in samples of the GM adjacent to the tumor, in the tumor tissue, in the omentum, and regional lymph nodes of 46 patients, by immunohistochemistry.\nThe sections for IGH were dewaxed and rehydrated by sequential immersion in xylene and graded ethanol and water. For antigen retrieval, the sections boiling for 10 min in citrate buffer (pH 6) and endogenous peroxidase activity was blocked with 30 mL/L hydrogen peroxide solution. Slides were incubated at room temperature with the anti-H. pylori (RB-9070, Thermo Fisher Scientific, the immunogen is purified H. pylori) rabbit polyclonal antibodies in diluted at 1:1000 for 30 min. \nThe visualization system included DAB (UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen) and hematoxylin counterstaining. For negative control sections, the primary antibody was replaced with phosphate-buffered saline and processed in the same manner.\nThe concentration of H. pylori in the GM detected by IGH was graded as 1+ for mild, 2+ for moderate, and 3+ for marked according to the Sydney system[30]. The presence of point inclusions giving a positive reaction with antibodies to H. pylori in the cytoplasm of epithelial cells of deep gastric glands and of the lymphoid cells of the lamina propria of GM as well as in omentum and lymph node, were taken into account. \nAll sections were carefully and completely scanned by two of the authors (MS and OT) without knowledge of the clinical and pathological data. \nThe data obtained was compared with clinical features of GC: Stage, localization, histology, the presence of AT before surgery, and 10-year OS and RFS.","Statistical analysis was performed using the Statistica 10.0 software. The correlations between different data were evaluated using the nonparametric Spearman's rank correlation or gamma correlation. Chi-square tests were carried out to analyze the difference of distribution among the categorized data. Mann–Whitney U nonparametric test was used to compare the value of the quantitative data. The survival was analyzed using the Kaplan-Meier method. The log-rank test was used to compare survival curves between subgroups of patients. A value of P < 0.05 was considered statistically significant.","The features of H. pylori infection in GC Of 93 patients (84.5%) demonstrated positive RUT. According to RUT the concentration of H. pylori in GM was mild (1+) in 38 (34.8%) patients, moderate (2+)-in 37 (33.9%) and marked (3+)–in 8 (16.5%). RUT was negative in 16 patients (14.7%).\nIt was found that urease activity 2+ and 3+ were significantly more frequent in GM than in tumors (in 55.5% and 13.3% of cases, respectively, P = 0.01). In more than half the cases (53.3%), the urease activity in the tumor was lower than in the adjacent GM.\nForty-six samples of GM were stained by IHC. The coccoid forms of H. pylori were found to prevail in GM adjacent to the tumor (Figure 1A). Coccoid forms of H. pylori only were identified in 31 (63.1%) patients, and coccoid and spiral in 12 (30.4%) patients (Figure 1B). No signs of infection were found in 3 (6.5%) patients. The concentration of coccoid and spiral forms of H. pylori in GM according to IGH was 1+ in 24 (52.2%) and 1 (19.6%) patients, 2+-in 11 (23.9%) and 4 (8.7%), and 3+-in 8 (17.4%) and 1 (2.2%) patients. A positive correlation between the concentration of coccoid and spiral forms of H. pylori (gamma = 0.642, P < 0.0001) was noted.\n\nThe features of Helicobacter pylori localization in gastric mucosa in patients with gastric cancer. A: Coccoid forms of Helicobacter pylori (H. pylori) in the gastric pit. The some bacteria within the cytoplasm of epithelium cells (arrows); B: The spiral (black arrows) and coccoid (orange arrows) forms of H. pylori on the surface of superficial-foveolar gastric epithelium; C: The bacteria in the surface mucous gel layer of stomach (arrows); D: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of epithelial cells of deep gastric glands (arrows); E: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of the immune cells of the lamina propria of gastric mucosa (arrows); F: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of intraepithelial lymphocytes (arrows). Immunoperoxidase staining with anti-H. pylori antibody, immersion. Bars: A: 20 μm; B: 10 μm; C: 20 μm; D-F: 10 μm. \nThe localization of H. pylori in GM was observed not only in the surface mucous gel layer of the stomach (Figure 1C) but also within the cytoplasm of the gastric superficial-foveolar epithelium (Figure 1D). The point inclusions giving a positive reaction with antibodies to H. pylori were also revealed in the cytoplasm of the epithelial cells of deep gastric glands (in 41.3% samples) and of the immune cells (Figure 1E) of the lamina propria of GM (in 43.5% samples), as well as of the intraepithelial lymphocytes (Figure 1F). The close relationship between the presence of point inclusions in immune cells and epithelial cells (gamma = 0.642, P < 0.0001) was noted. The individual cocci or their small clusters, and sometimes the short rod bacterium, were also often detected in the lamina propria of the GM. We also found bacteria in the omentum and lymph nodes. In the omentum the bacteria were presented predominantly by cocci between 0.5 and 1 μm in diameter (Figure 2A). Cocci were arranged most commonly by the small compact groups up to 10-15 cells in the immediate vicinity of the LN capsule. The bacteria were located mainly between the adipocytes. However, it was not clear whether bacteria were outside of the cells or in a narrow rim of cytoplasm of the fat cells. \n\nThe features of Helicobacter pylori localization in omentum and lymph node in patients with gastric cancer. A: The small group of cocci located in the central part of the omentum adipocyte; B: The congestions of bacteria around the nucleus of lymphocytes in the paracortical area of lymph node. Immunoperoxidase staining with anti-Helicobacter pylori antibody, immersion, Bars: 10 μm. \nIn the tissue of lymph nodes we usually observed the small accumulations of cocci between 10 and 15 cells (Figure 2B). Bacteria were located between lymphocytes of the paracortical area, but not so compact, as in the omentum. Quite often the concentration of bacteria was observed around the nuclei of cells that can testify to their intracellular localization.\nOf 93 patients (84.5%) demonstrated positive RUT. According to RUT the concentration of H. pylori in GM was mild (1+) in 38 (34.8%) patients, moderate (2+)-in 37 (33.9%) and marked (3+)–in 8 (16.5%). RUT was negative in 16 patients (14.7%).\nIt was found that urease activity 2+ and 3+ were significantly more frequent in GM than in tumors (in 55.5% and 13.3% of cases, respectively, P = 0.01). In more than half the cases (53.3%), the urease activity in the tumor was lower than in the adjacent GM.\nForty-six samples of GM were stained by IHC. The coccoid forms of H. pylori were found to prevail in GM adjacent to the tumor (Figure 1A). Coccoid forms of H. pylori only were identified in 31 (63.1%) patients, and coccoid and spiral in 12 (30.4%) patients (Figure 1B). No signs of infection were found in 3 (6.5%) patients. The concentration of coccoid and spiral forms of H. pylori in GM according to IGH was 1+ in 24 (52.2%) and 1 (19.6%) patients, 2+-in 11 (23.9%) and 4 (8.7%), and 3+-in 8 (17.4%) and 1 (2.2%) patients. A positive correlation between the concentration of coccoid and spiral forms of H. pylori (gamma = 0.642, P < 0.0001) was noted.\n\nThe features of Helicobacter pylori localization in gastric mucosa in patients with gastric cancer. A: Coccoid forms of Helicobacter pylori (H. pylori) in the gastric pit. The some bacteria within the cytoplasm of epithelium cells (arrows); B: The spiral (black arrows) and coccoid (orange arrows) forms of H. pylori on the surface of superficial-foveolar gastric epithelium; C: The bacteria in the surface mucous gel layer of stomach (arrows); D: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of epithelial cells of deep gastric glands (arrows); E: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of the immune cells of the lamina propria of gastric mucosa (arrows); F: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of intraepithelial lymphocytes (arrows). Immunoperoxidase staining with anti-H. pylori antibody, immersion. Bars: A: 20 μm; B: 10 μm; C: 20 μm; D-F: 10 μm. \nThe localization of H. pylori in GM was observed not only in the surface mucous gel layer of the stomach (Figure 1C) but also within the cytoplasm of the gastric superficial-foveolar epithelium (Figure 1D). The point inclusions giving a positive reaction with antibodies to H. pylori were also revealed in the cytoplasm of the epithelial cells of deep gastric glands (in 41.3% samples) and of the immune cells (Figure 1E) of the lamina propria of GM (in 43.5% samples), as well as of the intraepithelial lymphocytes (Figure 1F). The close relationship between the presence of point inclusions in immune cells and epithelial cells (gamma = 0.642, P < 0.0001) was noted. The individual cocci or their small clusters, and sometimes the short rod bacterium, were also often detected in the lamina propria of the GM. We also found bacteria in the omentum and lymph nodes. In the omentum the bacteria were presented predominantly by cocci between 0.5 and 1 μm in diameter (Figure 2A). Cocci were arranged most commonly by the small compact groups up to 10-15 cells in the immediate vicinity of the LN capsule. The bacteria were located mainly between the adipocytes. However, it was not clear whether bacteria were outside of the cells or in a narrow rim of cytoplasm of the fat cells. \n\nThe features of Helicobacter pylori localization in omentum and lymph node in patients with gastric cancer. A: The small group of cocci located in the central part of the omentum adipocyte; B: The congestions of bacteria around the nucleus of lymphocytes in the paracortical area of lymph node. Immunoperoxidase staining with anti-Helicobacter pylori antibody, immersion, Bars: 10 μm. \nIn the tissue of lymph nodes we usually observed the small accumulations of cocci between 10 and 15 cells (Figure 2B). Bacteria were located between lymphocytes of the paracortical area, but not so compact, as in the omentum. Quite often the concentration of bacteria was observed around the nuclei of cells that can testify to their intracellular localization.\nCorrelations of clinical and pathological characteristics of GC with the severity of H. pylori infection according to RUT data The gamma correlation coefficient test (gamma) showed that the severity of H. pylori in GM according to RUT positively correlated with the T status (gamma = 0.537, P < 0.00001), N status (gamma = 0.371, P = 0.0007) and stage (gamma = 0.520, P < 0.00001), and negatively correlated with the presence of AT in anamnesis (gamma = -0.418, P = 0.003). The marked (+++) and moderate (++) degrees of H. pylori infection were more often observed in Grade 2 and Grade 3, in T3-4 status, in N1 status, in the T3-4N1-2 stage, and in the absence of AT in anamnesis (Table 2). Correlations of H. pylori concentration in GM according to RUT with 10-year OS and RFS of GC patients were not determined. \nClinical and pathological characteristics of gastric cancer depending on the data rapid urease test\nRUT: Rapid urease test; GC: Gastric cancer.\nIt is important to note that the presence in AT 1-1.5 mo before surgery was associated with a significant improvement in RFS and OS (Figure 3), however, this applied only to patients with local GC (T1-3N0). In advanced GC (T3-4N1 and T3-4N2) there were no significant differences in patient survival (Figure 4).\n\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\n\n10-year overall surviving and relapse-free surviving of patients with T3-4N1-2M0 stages of gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.78). A: 10-year overall surviving; B: Relapse-free surviving.\nThe gamma correlation coefficient test (gamma) showed that the severity of H. pylori in GM according to RUT positively correlated with the T status (gamma = 0.537, P < 0.00001), N status (gamma = 0.371, P = 0.0007) and stage (gamma = 0.520, P < 0.00001), and negatively correlated with the presence of AT in anamnesis (gamma = -0.418, P = 0.003). The marked (+++) and moderate (++) degrees of H. pylori infection were more often observed in Grade 2 and Grade 3, in T3-4 status, in N1 status, in the T3-4N1-2 stage, and in the absence of AT in anamnesis (Table 2). Correlations of H. pylori concentration in GM according to RUT with 10-year OS and RFS of GC patients were not determined. \nClinical and pathological characteristics of gastric cancer depending on the data rapid urease test\nRUT: Rapid urease test; GC: Gastric cancer.\nIt is important to note that the presence in AT 1-1.5 mo before surgery was associated with a significant improvement in RFS and OS (Figure 3), however, this applied only to patients with local GC (T1-3N0). In advanced GC (T3-4N1 and T3-4N2) there were no significant differences in patient survival (Figure 4).\n\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\n\n10-year overall surviving and relapse-free surviving of patients with T3-4N1-2M0 stages of gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.78). A: 10-year overall surviving; B: Relapse-free surviving.\nCorrelations of clinical and pathological characteristics of GC with the concentration of spiral and coccoid forms of H. pylori in the GM It is important to note that correlations between the clinical and pathological characteristics of GC and the concentration of H. pylori spiral forms in GM were not found. However, the concentration of H. pylori coccoid forms correlated with age (ρ = -0.502, P = 0.0006), histology (gamma = 0.550, P = 0.0004), T status (gamma = 0.709, P = 0.0001), N status (gamma = 0.509, P = 0.002) stage (gamma = 0.636, P = 0.0002), and 10-year RFS (gamma = -0.521, P = 0.008) and OS (gamma = -0.500, P = 0.044). In cases with a moderate and marked concentration (2+ or 3+) of H. pylori coccoid forms in GM compared to cases with a low concentration (1+ or without infection) the patients were younger (57.9 ± 2.5 years vs 66.2 ± 1.4 years, respectively, P = 0.004) and the diffuse type of GC, poorly differentiated tumors (G3), T3-4 stage and N1 stage of GC were more often observed (Table 3). In cases of moderate and marked concentrations of H. pylori in GM, a decrease in 10-year progression-free survival (PFS) and OS survival from 55.6% to 26.3% was observed (P = 0.02 and P = 0.07, respectively). PFS and OS curves, depending on the concentration of coccoid forms of H. pylori in GM, are presented in Figure 5.\n\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\nClinical and pathological characteristics of gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa\nRUT: Rapid urease test; GC: Gastric cancer.\nThere were no correlations between the presence of point inclusions in the cytoplasm of epithelial cells of deep gastric glands, in the stroma immune cells, and in the intraepithelial lymphocytes with the clinical and pathological characteristics of GC.\nIt is important to note that correlations between the clinical and pathological characteristics of GC and the concentration of H. pylori spiral forms in GM were not found. However, the concentration of H. pylori coccoid forms correlated with age (ρ = -0.502, P = 0.0006), histology (gamma = 0.550, P = 0.0004), T status (gamma = 0.709, P = 0.0001), N status (gamma = 0.509, P = 0.002) stage (gamma = 0.636, P = 0.0002), and 10-year RFS (gamma = -0.521, P = 0.008) and OS (gamma = -0.500, P = 0.044). In cases with a moderate and marked concentration (2+ or 3+) of H. pylori coccoid forms in GM compared to cases with a low concentration (1+ or without infection) the patients were younger (57.9 ± 2.5 years vs 66.2 ± 1.4 years, respectively, P = 0.004) and the diffuse type of GC, poorly differentiated tumors (G3), T3-4 stage and N1 stage of GC were more often observed (Table 3). In cases of moderate and marked concentrations of H. pylori in GM, a decrease in 10-year progression-free survival (PFS) and OS survival from 55.6% to 26.3% was observed (P = 0.02 and P = 0.07, respectively). PFS and OS curves, depending on the concentration of coccoid forms of H. pylori in GM, are presented in Figure 5.\n\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\nClinical and pathological characteristics of gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa\nRUT: Rapid urease test; GC: Gastric cancer.\nThere were no correlations between the presence of point inclusions in the cytoplasm of epithelial cells of deep gastric glands, in the stroma immune cells, and in the intraepithelial lymphocytes with the clinical and pathological characteristics of GC.","Of 93 patients (84.5%) demonstrated positive RUT. According to RUT the concentration of H. pylori in GM was mild (1+) in 38 (34.8%) patients, moderate (2+)-in 37 (33.9%) and marked (3+)–in 8 (16.5%). RUT was negative in 16 patients (14.7%).\nIt was found that urease activity 2+ and 3+ were significantly more frequent in GM than in tumors (in 55.5% and 13.3% of cases, respectively, P = 0.01). In more than half the cases (53.3%), the urease activity in the tumor was lower than in the adjacent GM.\nForty-six samples of GM were stained by IHC. The coccoid forms of H. pylori were found to prevail in GM adjacent to the tumor (Figure 1A). Coccoid forms of H. pylori only were identified in 31 (63.1%) patients, and coccoid and spiral in 12 (30.4%) patients (Figure 1B). No signs of infection were found in 3 (6.5%) patients. The concentration of coccoid and spiral forms of H. pylori in GM according to IGH was 1+ in 24 (52.2%) and 1 (19.6%) patients, 2+-in 11 (23.9%) and 4 (8.7%), and 3+-in 8 (17.4%) and 1 (2.2%) patients. A positive correlation between the concentration of coccoid and spiral forms of H. pylori (gamma = 0.642, P < 0.0001) was noted.\n\nThe features of Helicobacter pylori localization in gastric mucosa in patients with gastric cancer. A: Coccoid forms of Helicobacter pylori (H. pylori) in the gastric pit. The some bacteria within the cytoplasm of epithelium cells (arrows); B: The spiral (black arrows) and coccoid (orange arrows) forms of H. pylori on the surface of superficial-foveolar gastric epithelium; C: The bacteria in the surface mucous gel layer of stomach (arrows); D: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of epithelial cells of deep gastric glands (arrows); E: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of the immune cells of the lamina propria of gastric mucosa (arrows); F: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of intraepithelial lymphocytes (arrows). Immunoperoxidase staining with anti-H. pylori antibody, immersion. Bars: A: 20 μm; B: 10 μm; C: 20 μm; D-F: 10 μm. \nThe localization of H. pylori in GM was observed not only in the surface mucous gel layer of the stomach (Figure 1C) but also within the cytoplasm of the gastric superficial-foveolar epithelium (Figure 1D). The point inclusions giving a positive reaction with antibodies to H. pylori were also revealed in the cytoplasm of the epithelial cells of deep gastric glands (in 41.3% samples) and of the immune cells (Figure 1E) of the lamina propria of GM (in 43.5% samples), as well as of the intraepithelial lymphocytes (Figure 1F). The close relationship between the presence of point inclusions in immune cells and epithelial cells (gamma = 0.642, P < 0.0001) was noted. The individual cocci or their small clusters, and sometimes the short rod bacterium, were also often detected in the lamina propria of the GM. We also found bacteria in the omentum and lymph nodes. In the omentum the bacteria were presented predominantly by cocci between 0.5 and 1 μm in diameter (Figure 2A). Cocci were arranged most commonly by the small compact groups up to 10-15 cells in the immediate vicinity of the LN capsule. The bacteria were located mainly between the adipocytes. However, it was not clear whether bacteria were outside of the cells or in a narrow rim of cytoplasm of the fat cells. \n\nThe features of Helicobacter pylori localization in omentum and lymph node in patients with gastric cancer. A: The small group of cocci located in the central part of the omentum adipocyte; B: The congestions of bacteria around the nucleus of lymphocytes in the paracortical area of lymph node. Immunoperoxidase staining with anti-Helicobacter pylori antibody, immersion, Bars: 10 μm. \nIn the tissue of lymph nodes we usually observed the small accumulations of cocci between 10 and 15 cells (Figure 2B). Bacteria were located between lymphocytes of the paracortical area, but not so compact, as in the omentum. Quite often the concentration of bacteria was observed around the nuclei of cells that can testify to their intracellular localization.","The gamma correlation coefficient test (gamma) showed that the severity of H. pylori in GM according to RUT positively correlated with the T status (gamma = 0.537, P < 0.00001), N status (gamma = 0.371, P = 0.0007) and stage (gamma = 0.520, P < 0.00001), and negatively correlated with the presence of AT in anamnesis (gamma = -0.418, P = 0.003). The marked (+++) and moderate (++) degrees of H. pylori infection were more often observed in Grade 2 and Grade 3, in T3-4 status, in N1 status, in the T3-4N1-2 stage, and in the absence of AT in anamnesis (Table 2). Correlations of H. pylori concentration in GM according to RUT with 10-year OS and RFS of GC patients were not determined. \nClinical and pathological characteristics of gastric cancer depending on the data rapid urease test\nRUT: Rapid urease test; GC: Gastric cancer.\nIt is important to note that the presence in AT 1-1.5 mo before surgery was associated with a significant improvement in RFS and OS (Figure 3), however, this applied only to patients with local GC (T1-3N0). In advanced GC (T3-4N1 and T3-4N2) there were no significant differences in patient survival (Figure 4).\n\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\n\n10-year overall surviving and relapse-free surviving of patients with T3-4N1-2M0 stages of gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.78). A: 10-year overall surviving; B: Relapse-free surviving.","It is important to note that correlations between the clinical and pathological characteristics of GC and the concentration of H. pylori spiral forms in GM were not found. However, the concentration of H. pylori coccoid forms correlated with age (ρ = -0.502, P = 0.0006), histology (gamma = 0.550, P = 0.0004), T status (gamma = 0.709, P = 0.0001), N status (gamma = 0.509, P = 0.002) stage (gamma = 0.636, P = 0.0002), and 10-year RFS (gamma = -0.521, P = 0.008) and OS (gamma = -0.500, P = 0.044). In cases with a moderate and marked concentration (2+ or 3+) of H. pylori coccoid forms in GM compared to cases with a low concentration (1+ or without infection) the patients were younger (57.9 ± 2.5 years vs 66.2 ± 1.4 years, respectively, P = 0.004) and the diffuse type of GC, poorly differentiated tumors (G3), T3-4 stage and N1 stage of GC were more often observed (Table 3). In cases of moderate and marked concentrations of H. pylori in GM, a decrease in 10-year progression-free survival (PFS) and OS survival from 55.6% to 26.3% was observed (P = 0.02 and P = 0.07, respectively). PFS and OS curves, depending on the concentration of coccoid forms of H. pylori in GM, are presented in Figure 5.\n\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\nClinical and pathological characteristics of gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa\nRUT: Rapid urease test; GC: Gastric cancer.\nThere were no correlations between the presence of point inclusions in the cytoplasm of epithelial cells of deep gastric glands, in the stroma immune cells, and in the intraepithelial lymphocytes with the clinical and pathological characteristics of GC.","A large amount of clinical and experimental data testifies to the important role of H. pylori in the occurrence of GC[31-34], but, there is less research into the features of H. pylori infection in patients with GC and its role in tumor progression, and the results are quite contradictory. These contradictions relate to many aspects, such as: (1) The frequency of infection in patients with GC. This data varies widely and ranges from 36% to 100%[35-40]; (2) The relation of infection with GC prognosis. Some researchers have noted an improvement in the long-term results of GC treatment in infected patients[41,42], while others, on the contrary, found that the presence of H. pylori infection was associated with a decrease in patient survival[43,44]; and (3) The connection between the infection and a histologic type of GC. In some studies, it was noted that patients with an intestinal type of GC are more often infected with H. pylori than patients with the diffuse type of GC[45,46]. Other researchers did not find a difference in H. pylori infection in patients with different histological types of tumors[47].\nIt is believed that the differences noted are associated with the fact that to reveal the infection the authors used the methods that were significantly different in their sensitivity, and primarily, to coccoid forms of bacteria. Most of the studies were carried out without considering coccoid forms of H. pylori and the concentration of bacteria in GM. The use of the biochemical method for the detection of urease activity and immunohistochemistry for visualization of bacteria in our study allowed us not only to assess the presence of infection in patients with GC, but also to mark some of its features associated with the localization of bacteria in the stomach, with a ratio of cocci and spiral forms, and the degree of bacterial contamination of GM.\nOur data for the Orenburg region recorded a high rate of H. pylori infection in patients with GC (84.5%). The coccoid forms of H. pylori, preserving a high degree of urease activity, dominated in GM in patients with GC. They were found in 93.4% of infected patients, with only coccoid forms of H. pylori-in 68.9%. \nIt is known that the coccoid forms of H. pylori can arise in response to unfavorable environmental factors, such as AT [47,48]. These forms are resistant to AT[49,50] and are able to form biofilms[51] and avoid the immune system[50]. They express a higher rate of cagE mRNA than their spiral counterparts[52], and by increasing the synthesis of tumor necrosis factor-alpha (TNF-α)-inducing protein (Hps), which is introduced into the cytosol and cell nuclei, they can activate nuclear factor-kappaB (NF-κB) and the expression of TNF-α and other cytokines involved in carcinogenesis[53,54]. The effect on the proliferation of gastric epithelial cells in the H. pylori coccoid forms is also stronger than in the helical forms[55], and they can induce the expression of VEGF-A and transforming growth factor-β[56]. The ability to transform into coccoid forms was also found to be characteristic of the most virulent strains of H. pylori[50,54].\nIt should be noted that the higher infection by coccoid forms of H. pylori in patients with GC, compared to patients with gastritis or gastric ulcer, had been mentioned by other researchers[57]. A number of studies have shown that coccoid forms of H. pylori retained urease activity[58] and the expression of such antigens as CagA, UreA, porin, components of the Cag type IV secretion system (TFSS), antigen-binding adhesin of the blood group BabA and others[59,60].\nThe use of immunohistochemistry in this study made it possible to detect the bacteria not only in the gastric mucus and on the surface of epithelial cells, but also within the cytoplasm of epithelial and immune cells of GM. Such intracellular expression was characterized by point inclusions giving a specific reaction with antibodies to H. pylori.\nThe intracellular persistence of H. pylori has been demonstrated by many investigators. They found H. pylori in the cytoplasm of epithelial cells, intercellular spaces, in the lamina propria of GM, and in the lumen of small vessels[61-63]. We assume that the point inclusions in the cytoplasm of epithelial and immune cells giving a positive reaction with antibodies to H. pylori is similar to those particle-rich cytoplasmic structure (PaCS) described earlier in the human superficial-foveolar epithelium and its metaplastic or dysplastic foci[64]. The authors found that the PaCS are a colocalization of VacA, CagA, urease, and outer membrane proteins with NOD1 receptor, ubiquitin-activating enzyme E1, polyubiquitinated proteins, proteasome components, and potentially oncogenic proteins like SHP2 and ERKs[64]. They believe that PaCS is a novel, proteasome-enriched structure arising in ribosome-rich cytoplasm at sites of H. pylori product accumulation. \nWe believe that the immune cells with point inclusions in the lamina propria of GM are likely to be macrophages. The data obtained by several researchers suggests this conclusion[65,66]. It is noted that even the absorbed bacteria retains their viability in macrophages, which may be associated with the violation of the phagosome maturation[66-68]. The use of confocal microscopy enabled the localization of the bacteria within the cells to be associated with the endosomal and lysosomal markers, and found that H. pylori could use the vesicles of autophagosomes (autophagic vesicles) for its own replication[63,69]. \nThe study found that the concentration of H. pylori coccoid forms in GM was the most significant clinical factor. This factor was associated with the tumor histology, T status, N status, stage, 10-year PFS, and OS. The moderate and marked concentrations of coccoid forms of H. pylori were more often found in the diffuse type of GC (P = 0.024) and T3-4 (P = 0.04) stage. Interestingly, the high concentration of H. pylori is more frequent in Stage N1 than in N2 (at 90.0% and 53.1%, respectively, P = 0.024).\nThe moderate and marked concentrations of coccoid forms of H. pylori represented a prognostic factor associated with the decrease of 10-year RFS and OS from 55.6% to 26.3% (P = 0.02 and P = 0.07, respectively).\nIt should be noted that the results of this study do not allow us to unambiguously judge the effect of H. pylori on GC progression. A decrease in OS and DFS in patients with moderate and marked concentrations of H. pylori coccoid forms in the GM may be due to the fact that these patients had more advanced stages and more aggressive forms of GC. Meanwhile, there are more and more studies showing that H. pylori infection can promote GC progression by activating the NF-κB signaling pathway and induction of interleukin-8 secretion[70], the activation of epithelial-mesenchymal transformation[71-74] and angiogenesis[75,76], as well as increasing the invasive properties of tumor cells[77]. It can be assumed that the administration of AT before surgery contributes to the reduction of the inflammatory process activity and normalization of the adhesive properties of tumour cells, which in turn decreases metastasis risk and improves the long-term results of the treatment of GC. The data literature on the improvement of the long-term results of malignant tumours treatment when using antibacterial drugs testify in favour of this hypothesis[78-80].","The data obtained indicates that H. pylori may be associated not only with induction but also with the progression of GC. It can be assumed that the prevalence of coccoid forms of bacteria and their intracellular persistence can affect the mechanisms of tumor progression. Further appropriate studies regarding the role of H. pylori in the progression of GC are obviously advisable."],"string":"[\n \"Gastric cancer (GC) continues to be one of the most common malignant diseases in the world[1,2]. Despite a decreasing trend in the incidence of GC in most countries of the world, the treatment results of this pathology cannot be considered satisfactory. In the structure of mortality from malignant neoplasms, this pathology firmly occupies 2nd place in most developed countries of the world, and the 5-year survival rate of radically operated patients does not exceed 15%-30%[3,4]. \\nIt is important to note that it is impossible to improve the long-term results of malignant neoplasms treatment without knowledge of the mechanisms associated with their progression[3]. Clinical studies in recent years indicate that inflammatory infiltration of the tumor stroma and surrounding tissues can have an important prognostic value and affect the long-term results of malignant neoplasm treatment[5-9]. A number of studies have shown that inflammatory infiltration of the tumor stroma is associated with the body’s adequate immune response to the tumor, and may be a favorable prognosis factor in various malignant neoplasms[10,11], including GC[12,13]. At the same time, the data obtained by other researchers indicates that pronounced inflammatory infiltration of the tumor stroma, especially T-reg lymphocytes and macrophages, may be a factor contributing to the progression of malignant neoplasms[14,15]. There was a decrease in the overall survival (OS) and relapse-free survival (RFS) of GC patients with a high content of Foxp3 + T-reg in the tumor stroma and regional metastases[16-18] and macrophages[16,19]. It is believed that inflammatory infiltration of the tumor stroma can contribute to tumor progression by activating the mechanisms of angiogenesis, expression of E- and L-selectins, formation of the products of lipid peroxidation and free radicals, destruction of connective tissue matrix and basement membranes of epithelia by proteolytic enzymes, and activation of epithelial-mesenchymal transformation[20-23].\\nWhen studying the role of inflammation in the progression of GC, it is impossible to ignore the problem of Helicobacter pylori (H. pylori) infection. H. pylori is a gram-negative, spiral-shaped bacterium, the habitat of which is the gastric mucosa (GM) and duodenum. H. pylori differs from other bacteria in a set of properties that make it possible to colonize the GM and persist for a long time under conditions that are unfavorable for other microorganisms[24,25]. These include: (1) The ability to produce a special enzyme-urease; (2) Synthesis of lytic enzymes that cause the depolymerization and dissolution of gastric mucus, consisting mainly of mucin; (3) The mobility of the bacterium, which is ensured by the presence of 5-6 flagella; (4) The high adhesiveness of bacteria to GM epithelial cells of the GM and elements of connective tissue due to the interaction of bacterial ligands with the corresponding cells receptors; (5) Production of various exotoxins (VacA, CagA, and others); (6) Instability of the H. pylori genome; (7) The presence of vegetative and coccoid forms of bacteria; and (8) possibility of intracellular persistence and translocation outside the GM [26-29].\\nIt should be noted that despite the huge number of studies devoted to H. pylori, it is still not clear whether H. pylori is involved only in the initiation of the tumor process in the stomach, or whether it can affect the mechanisms of tumor progression. The relationship between the severity of H. pylori infection and the clinical and morphological characteristics of GC and long-term results of this pathology treatment remains poorly studied, and in this connection, the question of the expediency of anti-Helicobacter therapy in patients with invasive GC remains open.\\nObjective To assess the features of H. pylori infection in patients with Stage I-IIIB GC and their correlation with the clinical and morphological characteristics of the disease, the presence of antibiotic therapy (AT) before surgery, and long-term treatment results.\\nTo assess the features of H. pylori infection in patients with Stage I-IIIB GC and their correlation with the clinical and morphological characteristics of the disease, the presence of antibiotic therapy (AT) before surgery, and long-term treatment results.\",\n \"To assess the features of H. pylori infection in patients with Stage I-IIIB GC and their correlation with the clinical and morphological characteristics of the disease, the presence of antibiotic therapy (AT) before surgery, and long-term treatment results.\",\n \"One hundred and nine patients with GC who had undergone radical surgery (R0) between May 2007 and March 2010 at the Orenburg Regional Clinical Oncology Center, were included in this prospective cohort pilot study. Study inclusion criteria were: Histologically proven invasive GC; no evidence of distant metastases; radical surgery (R0); no prior gastric surgery; no previous chemotherapy or radiotherapy. The study did not include patients with decompensation of cardiovascular and renal diseases, exacerbation of chronic inflammatory processes, severe allergic processes, or who received glucocorticoids, antihistamines, and non-steroidal anti-inflammatory drugs. The study was performed in accordance with the Helsinki Declaration and internationally recognized guidelines, and the privacy of patients was protected by decoding the data according to the privacy regulations of the Orenburg Regional Clinical Oncologic Center (Russia, Orenburg). All patients provided written informed consent. The protocol was approved by the Institutional Review Board of the Orenburg State Medical University (Russia, Orenburg).\\nClinical and pathological data including age, tumor localization, stage, type of surgery, histology, the presence of AT before surgery, postoperative therapy, and long-term results of treatment were retrieved from the routine reports for analyses. The distribution of patients according to the clinical and pathological characteristics of GC is presented in Table 1.\\nThe distribution of patients according to the clinical and pathological characteristics of gastric cancer\\nGC: Gastric cancer.\\nWhen interviewing patients, it was found that 45 patients received AT before admission to the clinic due to a preliminary wrong diagnosis of gastric ulcer or chronic gastritis. The following combinations of antibacterial drugs were most commonly ordered: Amoxicillin + clarithromycin (34 patients), amoxicillin + clarithromycin + metronidazole (6 patients), amoxicillin + metronidazole (3 patients), other drugs (2 patients). Information about the antibacterial drugs, the timing and duration of their intake was entered into the primary patient documentation and then taken into account in the analysis. We considered only those patients who underwent AT in the period from 1 to 1.5 mo before the operation, lasting at least seven days, and using two or more antibacterial drugs.\\nThe long-term results of treatment were assessed for the period from May 12, 2007 to April 12, 2021. The median follow-up period was 86.2 mo. As of April 12, 2021, 26 (24.5%) patients were alive, 54 (50.9%) had died from the progression of GC, 20 (18.9%) had died from causes other than GC, and six (5.6%) left the region at different follow-up periods. Malignant tumors of other localizations were diagnosed in 8 patients at different times after the operation: Non-Hodgkin's lymphomas-in three, prostate cancer-in one, lung cancer-in two, laryngeal cancer-in one, and breast cancer-in one patient. With the exception of one patient with non-Hodgkin's lymphoma, the other patients died from the progression of these diseases. The causes of death were not associated with malignant tumors for the other patients. During the period 2020-2021, seven patients contracted coronavirus disease-19, one of whom died from the disease, but the rest are alive.\",\n \"\\nH. pylori in the GM and tumor was determined by rapid urease test (RUT) and by immunohistochemically (IHC) using the antibody to H. pylori.\",\n \"After removal of the stomach (within 30 min) a greater curvature of the organ was opened and biopsy samples were taken from the tumor and the adjacent macroscopically non-tumorous GM at a distance of 3-5 cm from the tumor margin. The samples were placed on test strips (HELPIL–test, “АМА”, Russia) for three minutes. The presence and the severity of the infection were evaluated by the color change of the indicator from yellow to blue. \\nAccording to the intensity and the time of the appearance of a blue color, we distinguished three degrees of infection: 3+: Marked (+++)-bright staining in the first minute of the study; 2+: Moderate (++)–for an average intensity of staining for 2 min and 1+ mild (+)-weak staining for three minutes. If the color of the indicator did not change, or became dirty gray, and if after repeated research the same result was received, the test was evaluated as negative. The same samples were later used for histological analysis and IGH. \",\n \"The presence and features of H. pylori infection were studied in samples of the GM adjacent to the tumor, in the tumor tissue, in the omentum, and regional lymph nodes of 46 patients, by immunohistochemistry.\\nThe sections for IGH were dewaxed and rehydrated by sequential immersion in xylene and graded ethanol and water. For antigen retrieval, the sections boiling for 10 min in citrate buffer (pH 6) and endogenous peroxidase activity was blocked with 30 mL/L hydrogen peroxide solution. Slides were incubated at room temperature with the anti-H. pylori (RB-9070, Thermo Fisher Scientific, the immunogen is purified H. pylori) rabbit polyclonal antibodies in diluted at 1:1000 for 30 min. \\nThe visualization system included DAB (UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen) and hematoxylin counterstaining. For negative control sections, the primary antibody was replaced with phosphate-buffered saline and processed in the same manner.\\nThe concentration of H. pylori in the GM detected by IGH was graded as 1+ for mild, 2+ for moderate, and 3+ for marked according to the Sydney system[30]. The presence of point inclusions giving a positive reaction with antibodies to H. pylori in the cytoplasm of epithelial cells of deep gastric glands and of the lymphoid cells of the lamina propria of GM as well as in omentum and lymph node, were taken into account. \\nAll sections were carefully and completely scanned by two of the authors (MS and OT) without knowledge of the clinical and pathological data. \\nThe data obtained was compared with clinical features of GC: Stage, localization, histology, the presence of AT before surgery, and 10-year OS and RFS.\",\n \"Statistical analysis was performed using the Statistica 10.0 software. The correlations between different data were evaluated using the nonparametric Spearman's rank correlation or gamma correlation. Chi-square tests were carried out to analyze the difference of distribution among the categorized data. Mann–Whitney U nonparametric test was used to compare the value of the quantitative data. The survival was analyzed using the Kaplan-Meier method. The log-rank test was used to compare survival curves between subgroups of patients. A value of P < 0.05 was considered statistically significant.\",\n \"The features of H. pylori infection in GC Of 93 patients (84.5%) demonstrated positive RUT. According to RUT the concentration of H. pylori in GM was mild (1+) in 38 (34.8%) patients, moderate (2+)-in 37 (33.9%) and marked (3+)–in 8 (16.5%). RUT was negative in 16 patients (14.7%).\\nIt was found that urease activity 2+ and 3+ were significantly more frequent in GM than in tumors (in 55.5% and 13.3% of cases, respectively, P = 0.01). In more than half the cases (53.3%), the urease activity in the tumor was lower than in the adjacent GM.\\nForty-six samples of GM were stained by IHC. The coccoid forms of H. pylori were found to prevail in GM adjacent to the tumor (Figure 1A). Coccoid forms of H. pylori only were identified in 31 (63.1%) patients, and coccoid and spiral in 12 (30.4%) patients (Figure 1B). No signs of infection were found in 3 (6.5%) patients. The concentration of coccoid and spiral forms of H. pylori in GM according to IGH was 1+ in 24 (52.2%) and 1 (19.6%) patients, 2+-in 11 (23.9%) and 4 (8.7%), and 3+-in 8 (17.4%) and 1 (2.2%) patients. A positive correlation between the concentration of coccoid and spiral forms of H. pylori (gamma = 0.642, P < 0.0001) was noted.\\n\\nThe features of Helicobacter pylori localization in gastric mucosa in patients with gastric cancer. A: Coccoid forms of Helicobacter pylori (H. pylori) in the gastric pit. The some bacteria within the cytoplasm of epithelium cells (arrows); B: The spiral (black arrows) and coccoid (orange arrows) forms of H. pylori on the surface of superficial-foveolar gastric epithelium; C: The bacteria in the surface mucous gel layer of stomach (arrows); D: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of epithelial cells of deep gastric glands (arrows); E: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of the immune cells of the lamina propria of gastric mucosa (arrows); F: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of intraepithelial lymphocytes (arrows). Immunoperoxidase staining with anti-H. pylori antibody, immersion. Bars: A: 20 μm; B: 10 μm; C: 20 μm; D-F: 10 μm. \\nThe localization of H. pylori in GM was observed not only in the surface mucous gel layer of the stomach (Figure 1C) but also within the cytoplasm of the gastric superficial-foveolar epithelium (Figure 1D). The point inclusions giving a positive reaction with antibodies to H. pylori were also revealed in the cytoplasm of the epithelial cells of deep gastric glands (in 41.3% samples) and of the immune cells (Figure 1E) of the lamina propria of GM (in 43.5% samples), as well as of the intraepithelial lymphocytes (Figure 1F). The close relationship between the presence of point inclusions in immune cells and epithelial cells (gamma = 0.642, P < 0.0001) was noted. The individual cocci or their small clusters, and sometimes the short rod bacterium, were also often detected in the lamina propria of the GM. We also found bacteria in the omentum and lymph nodes. In the omentum the bacteria were presented predominantly by cocci between 0.5 and 1 μm in diameter (Figure 2A). Cocci were arranged most commonly by the small compact groups up to 10-15 cells in the immediate vicinity of the LN capsule. The bacteria were located mainly between the adipocytes. However, it was not clear whether bacteria were outside of the cells or in a narrow rim of cytoplasm of the fat cells. \\n\\nThe features of Helicobacter pylori localization in omentum and lymph node in patients with gastric cancer. A: The small group of cocci located in the central part of the omentum adipocyte; B: The congestions of bacteria around the nucleus of lymphocytes in the paracortical area of lymph node. Immunoperoxidase staining with anti-Helicobacter pylori antibody, immersion, Bars: 10 μm. \\nIn the tissue of lymph nodes we usually observed the small accumulations of cocci between 10 and 15 cells (Figure 2B). Bacteria were located between lymphocytes of the paracortical area, but not so compact, as in the omentum. Quite often the concentration of bacteria was observed around the nuclei of cells that can testify to their intracellular localization.\\nOf 93 patients (84.5%) demonstrated positive RUT. According to RUT the concentration of H. pylori in GM was mild (1+) in 38 (34.8%) patients, moderate (2+)-in 37 (33.9%) and marked (3+)–in 8 (16.5%). RUT was negative in 16 patients (14.7%).\\nIt was found that urease activity 2+ and 3+ were significantly more frequent in GM than in tumors (in 55.5% and 13.3% of cases, respectively, P = 0.01). In more than half the cases (53.3%), the urease activity in the tumor was lower than in the adjacent GM.\\nForty-six samples of GM were stained by IHC. The coccoid forms of H. pylori were found to prevail in GM adjacent to the tumor (Figure 1A). Coccoid forms of H. pylori only were identified in 31 (63.1%) patients, and coccoid and spiral in 12 (30.4%) patients (Figure 1B). No signs of infection were found in 3 (6.5%) patients. The concentration of coccoid and spiral forms of H. pylori in GM according to IGH was 1+ in 24 (52.2%) and 1 (19.6%) patients, 2+-in 11 (23.9%) and 4 (8.7%), and 3+-in 8 (17.4%) and 1 (2.2%) patients. A positive correlation between the concentration of coccoid and spiral forms of H. pylori (gamma = 0.642, P < 0.0001) was noted.\\n\\nThe features of Helicobacter pylori localization in gastric mucosa in patients with gastric cancer. A: Coccoid forms of Helicobacter pylori (H. pylori) in the gastric pit. The some bacteria within the cytoplasm of epithelium cells (arrows); B: The spiral (black arrows) and coccoid (orange arrows) forms of H. pylori on the surface of superficial-foveolar gastric epithelium; C: The bacteria in the surface mucous gel layer of stomach (arrows); D: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of epithelial cells of deep gastric glands (arrows); E: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of the immune cells of the lamina propria of gastric mucosa (arrows); F: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of intraepithelial lymphocytes (arrows). Immunoperoxidase staining with anti-H. pylori antibody, immersion. Bars: A: 20 μm; B: 10 μm; C: 20 μm; D-F: 10 μm. \\nThe localization of H. pylori in GM was observed not only in the surface mucous gel layer of the stomach (Figure 1C) but also within the cytoplasm of the gastric superficial-foveolar epithelium (Figure 1D). The point inclusions giving a positive reaction with antibodies to H. pylori were also revealed in the cytoplasm of the epithelial cells of deep gastric glands (in 41.3% samples) and of the immune cells (Figure 1E) of the lamina propria of GM (in 43.5% samples), as well as of the intraepithelial lymphocytes (Figure 1F). The close relationship between the presence of point inclusions in immune cells and epithelial cells (gamma = 0.642, P < 0.0001) was noted. The individual cocci or their small clusters, and sometimes the short rod bacterium, were also often detected in the lamina propria of the GM. We also found bacteria in the omentum and lymph nodes. In the omentum the bacteria were presented predominantly by cocci between 0.5 and 1 μm in diameter (Figure 2A). Cocci were arranged most commonly by the small compact groups up to 10-15 cells in the immediate vicinity of the LN capsule. The bacteria were located mainly between the adipocytes. However, it was not clear whether bacteria were outside of the cells or in a narrow rim of cytoplasm of the fat cells. \\n\\nThe features of Helicobacter pylori localization in omentum and lymph node in patients with gastric cancer. A: The small group of cocci located in the central part of the omentum adipocyte; B: The congestions of bacteria around the nucleus of lymphocytes in the paracortical area of lymph node. Immunoperoxidase staining with anti-Helicobacter pylori antibody, immersion, Bars: 10 μm. \\nIn the tissue of lymph nodes we usually observed the small accumulations of cocci between 10 and 15 cells (Figure 2B). Bacteria were located between lymphocytes of the paracortical area, but not so compact, as in the omentum. Quite often the concentration of bacteria was observed around the nuclei of cells that can testify to their intracellular localization.\\nCorrelations of clinical and pathological characteristics of GC with the severity of H. pylori infection according to RUT data The gamma correlation coefficient test (gamma) showed that the severity of H. pylori in GM according to RUT positively correlated with the T status (gamma = 0.537, P < 0.00001), N status (gamma = 0.371, P = 0.0007) and stage (gamma = 0.520, P < 0.00001), and negatively correlated with the presence of AT in anamnesis (gamma = -0.418, P = 0.003). The marked (+++) and moderate (++) degrees of H. pylori infection were more often observed in Grade 2 and Grade 3, in T3-4 status, in N1 status, in the T3-4N1-2 stage, and in the absence of AT in anamnesis (Table 2). Correlations of H. pylori concentration in GM according to RUT with 10-year OS and RFS of GC patients were not determined. \\nClinical and pathological characteristics of gastric cancer depending on the data rapid urease test\\nRUT: Rapid urease test; GC: Gastric cancer.\\nIt is important to note that the presence in AT 1-1.5 mo before surgery was associated with a significant improvement in RFS and OS (Figure 3), however, this applied only to patients with local GC (T1-3N0). In advanced GC (T3-4N1 and T3-4N2) there were no significant differences in patient survival (Figure 4).\\n\\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\\n\\n10-year overall surviving and relapse-free surviving of patients with T3-4N1-2M0 stages of gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.78). A: 10-year overall surviving; B: Relapse-free surviving.\\nThe gamma correlation coefficient test (gamma) showed that the severity of H. pylori in GM according to RUT positively correlated with the T status (gamma = 0.537, P < 0.00001), N status (gamma = 0.371, P = 0.0007) and stage (gamma = 0.520, P < 0.00001), and negatively correlated with the presence of AT in anamnesis (gamma = -0.418, P = 0.003). The marked (+++) and moderate (++) degrees of H. pylori infection were more often observed in Grade 2 and Grade 3, in T3-4 status, in N1 status, in the T3-4N1-2 stage, and in the absence of AT in anamnesis (Table 2). Correlations of H. pylori concentration in GM according to RUT with 10-year OS and RFS of GC patients were not determined. \\nClinical and pathological characteristics of gastric cancer depending on the data rapid urease test\\nRUT: Rapid urease test; GC: Gastric cancer.\\nIt is important to note that the presence in AT 1-1.5 mo before surgery was associated with a significant improvement in RFS and OS (Figure 3), however, this applied only to patients with local GC (T1-3N0). In advanced GC (T3-4N1 and T3-4N2) there were no significant differences in patient survival (Figure 4).\\n\\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\\n\\n10-year overall surviving and relapse-free surviving of patients with T3-4N1-2M0 stages of gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.78). A: 10-year overall surviving; B: Relapse-free surviving.\\nCorrelations of clinical and pathological characteristics of GC with the concentration of spiral and coccoid forms of H. pylori in the GM It is important to note that correlations between the clinical and pathological characteristics of GC and the concentration of H. pylori spiral forms in GM were not found. However, the concentration of H. pylori coccoid forms correlated with age (ρ = -0.502, P = 0.0006), histology (gamma = 0.550, P = 0.0004), T status (gamma = 0.709, P = 0.0001), N status (gamma = 0.509, P = 0.002) stage (gamma = 0.636, P = 0.0002), and 10-year RFS (gamma = -0.521, P = 0.008) and OS (gamma = -0.500, P = 0.044). In cases with a moderate and marked concentration (2+ or 3+) of H. pylori coccoid forms in GM compared to cases with a low concentration (1+ or without infection) the patients were younger (57.9 ± 2.5 years vs 66.2 ± 1.4 years, respectively, P = 0.004) and the diffuse type of GC, poorly differentiated tumors (G3), T3-4 stage and N1 stage of GC were more often observed (Table 3). In cases of moderate and marked concentrations of H. pylori in GM, a decrease in 10-year progression-free survival (PFS) and OS survival from 55.6% to 26.3% was observed (P = 0.02 and P = 0.07, respectively). PFS and OS curves, depending on the concentration of coccoid forms of H. pylori in GM, are presented in Figure 5.\\n\\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\\nClinical and pathological characteristics of gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa\\nRUT: Rapid urease test; GC: Gastric cancer.\\nThere were no correlations between the presence of point inclusions in the cytoplasm of epithelial cells of deep gastric glands, in the stroma immune cells, and in the intraepithelial lymphocytes with the clinical and pathological characteristics of GC.\\nIt is important to note that correlations between the clinical and pathological characteristics of GC and the concentration of H. pylori spiral forms in GM were not found. However, the concentration of H. pylori coccoid forms correlated with age (ρ = -0.502, P = 0.0006), histology (gamma = 0.550, P = 0.0004), T status (gamma = 0.709, P = 0.0001), N status (gamma = 0.509, P = 0.002) stage (gamma = 0.636, P = 0.0002), and 10-year RFS (gamma = -0.521, P = 0.008) and OS (gamma = -0.500, P = 0.044). In cases with a moderate and marked concentration (2+ or 3+) of H. pylori coccoid forms in GM compared to cases with a low concentration (1+ or without infection) the patients were younger (57.9 ± 2.5 years vs 66.2 ± 1.4 years, respectively, P = 0.004) and the diffuse type of GC, poorly differentiated tumors (G3), T3-4 stage and N1 stage of GC were more often observed (Table 3). In cases of moderate and marked concentrations of H. pylori in GM, a decrease in 10-year progression-free survival (PFS) and OS survival from 55.6% to 26.3% was observed (P = 0.02 and P = 0.07, respectively). PFS and OS curves, depending on the concentration of coccoid forms of H. pylori in GM, are presented in Figure 5.\\n\\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\\nClinical and pathological characteristics of gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa\\nRUT: Rapid urease test; GC: Gastric cancer.\\nThere were no correlations between the presence of point inclusions in the cytoplasm of epithelial cells of deep gastric glands, in the stroma immune cells, and in the intraepithelial lymphocytes with the clinical and pathological characteristics of GC.\",\n \"Of 93 patients (84.5%) demonstrated positive RUT. According to RUT the concentration of H. pylori in GM was mild (1+) in 38 (34.8%) patients, moderate (2+)-in 37 (33.9%) and marked (3+)–in 8 (16.5%). RUT was negative in 16 patients (14.7%).\\nIt was found that urease activity 2+ and 3+ were significantly more frequent in GM than in tumors (in 55.5% and 13.3% of cases, respectively, P = 0.01). In more than half the cases (53.3%), the urease activity in the tumor was lower than in the adjacent GM.\\nForty-six samples of GM were stained by IHC. The coccoid forms of H. pylori were found to prevail in GM adjacent to the tumor (Figure 1A). Coccoid forms of H. pylori only were identified in 31 (63.1%) patients, and coccoid and spiral in 12 (30.4%) patients (Figure 1B). No signs of infection were found in 3 (6.5%) patients. The concentration of coccoid and spiral forms of H. pylori in GM according to IGH was 1+ in 24 (52.2%) and 1 (19.6%) patients, 2+-in 11 (23.9%) and 4 (8.7%), and 3+-in 8 (17.4%) and 1 (2.2%) patients. A positive correlation between the concentration of coccoid and spiral forms of H. pylori (gamma = 0.642, P < 0.0001) was noted.\\n\\nThe features of Helicobacter pylori localization in gastric mucosa in patients with gastric cancer. A: Coccoid forms of Helicobacter pylori (H. pylori) in the gastric pit. The some bacteria within the cytoplasm of epithelium cells (arrows); B: The spiral (black arrows) and coccoid (orange arrows) forms of H. pylori on the surface of superficial-foveolar gastric epithelium; C: The bacteria in the surface mucous gel layer of stomach (arrows); D: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of epithelial cells of deep gastric glands (arrows); E: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of the immune cells of the lamina propria of gastric mucosa (arrows); F: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of intraepithelial lymphocytes (arrows). Immunoperoxidase staining with anti-H. pylori antibody, immersion. Bars: A: 20 μm; B: 10 μm; C: 20 μm; D-F: 10 μm. \\nThe localization of H. pylori in GM was observed not only in the surface mucous gel layer of the stomach (Figure 1C) but also within the cytoplasm of the gastric superficial-foveolar epithelium (Figure 1D). The point inclusions giving a positive reaction with antibodies to H. pylori were also revealed in the cytoplasm of the epithelial cells of deep gastric glands (in 41.3% samples) and of the immune cells (Figure 1E) of the lamina propria of GM (in 43.5% samples), as well as of the intraepithelial lymphocytes (Figure 1F). The close relationship between the presence of point inclusions in immune cells and epithelial cells (gamma = 0.642, P < 0.0001) was noted. The individual cocci or their small clusters, and sometimes the short rod bacterium, were also often detected in the lamina propria of the GM. We also found bacteria in the omentum and lymph nodes. In the omentum the bacteria were presented predominantly by cocci between 0.5 and 1 μm in diameter (Figure 2A). Cocci were arranged most commonly by the small compact groups up to 10-15 cells in the immediate vicinity of the LN capsule. The bacteria were located mainly between the adipocytes. However, it was not clear whether bacteria were outside of the cells or in a narrow rim of cytoplasm of the fat cells. \\n\\nThe features of Helicobacter pylori localization in omentum and lymph node in patients with gastric cancer. A: The small group of cocci located in the central part of the omentum adipocyte; B: The congestions of bacteria around the nucleus of lymphocytes in the paracortical area of lymph node. Immunoperoxidase staining with anti-Helicobacter pylori antibody, immersion, Bars: 10 μm. \\nIn the tissue of lymph nodes we usually observed the small accumulations of cocci between 10 and 15 cells (Figure 2B). Bacteria were located between lymphocytes of the paracortical area, but not so compact, as in the omentum. Quite often the concentration of bacteria was observed around the nuclei of cells that can testify to their intracellular localization.\",\n \"The gamma correlation coefficient test (gamma) showed that the severity of H. pylori in GM according to RUT positively correlated with the T status (gamma = 0.537, P < 0.00001), N status (gamma = 0.371, P = 0.0007) and stage (gamma = 0.520, P < 0.00001), and negatively correlated with the presence of AT in anamnesis (gamma = -0.418, P = 0.003). The marked (+++) and moderate (++) degrees of H. pylori infection were more often observed in Grade 2 and Grade 3, in T3-4 status, in N1 status, in the T3-4N1-2 stage, and in the absence of AT in anamnesis (Table 2). Correlations of H. pylori concentration in GM according to RUT with 10-year OS and RFS of GC patients were not determined. \\nClinical and pathological characteristics of gastric cancer depending on the data rapid urease test\\nRUT: Rapid urease test; GC: Gastric cancer.\\nIt is important to note that the presence in AT 1-1.5 mo before surgery was associated with a significant improvement in RFS and OS (Figure 3), however, this applied only to patients with local GC (T1-3N0). In advanced GC (T3-4N1 and T3-4N2) there were no significant differences in patient survival (Figure 4).\\n\\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\\n\\n10-year overall surviving and relapse-free surviving of patients with T3-4N1-2M0 stages of gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.78). A: 10-year overall surviving; B: Relapse-free surviving.\",\n \"It is important to note that correlations between the clinical and pathological characteristics of GC and the concentration of H. pylori spiral forms in GM were not found. However, the concentration of H. pylori coccoid forms correlated with age (ρ = -0.502, P = 0.0006), histology (gamma = 0.550, P = 0.0004), T status (gamma = 0.709, P = 0.0001), N status (gamma = 0.509, P = 0.002) stage (gamma = 0.636, P = 0.0002), and 10-year RFS (gamma = -0.521, P = 0.008) and OS (gamma = -0.500, P = 0.044). In cases with a moderate and marked concentration (2+ or 3+) of H. pylori coccoid forms in GM compared to cases with a low concentration (1+ or without infection) the patients were younger (57.9 ± 2.5 years vs 66.2 ± 1.4 years, respectively, P = 0.004) and the diffuse type of GC, poorly differentiated tumors (G3), T3-4 stage and N1 stage of GC were more often observed (Table 3). In cases of moderate and marked concentrations of H. pylori in GM, a decrease in 10-year progression-free survival (PFS) and OS survival from 55.6% to 26.3% was observed (P = 0.02 and P = 0.07, respectively). PFS and OS curves, depending on the concentration of coccoid forms of H. pylori in GM, are presented in Figure 5.\\n\\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\\nClinical and pathological characteristics of gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa\\nRUT: Rapid urease test; GC: Gastric cancer.\\nThere were no correlations between the presence of point inclusions in the cytoplasm of epithelial cells of deep gastric glands, in the stroma immune cells, and in the intraepithelial lymphocytes with the clinical and pathological characteristics of GC.\",\n \"A large amount of clinical and experimental data testifies to the important role of H. pylori in the occurrence of GC[31-34], but, there is less research into the features of H. pylori infection in patients with GC and its role in tumor progression, and the results are quite contradictory. These contradictions relate to many aspects, such as: (1) The frequency of infection in patients with GC. This data varies widely and ranges from 36% to 100%[35-40]; (2) The relation of infection with GC prognosis. Some researchers have noted an improvement in the long-term results of GC treatment in infected patients[41,42], while others, on the contrary, found that the presence of H. pylori infection was associated with a decrease in patient survival[43,44]; and (3) The connection between the infection and a histologic type of GC. In some studies, it was noted that patients with an intestinal type of GC are more often infected with H. pylori than patients with the diffuse type of GC[45,46]. Other researchers did not find a difference in H. pylori infection in patients with different histological types of tumors[47].\\nIt is believed that the differences noted are associated with the fact that to reveal the infection the authors used the methods that were significantly different in their sensitivity, and primarily, to coccoid forms of bacteria. Most of the studies were carried out without considering coccoid forms of H. pylori and the concentration of bacteria in GM. The use of the biochemical method for the detection of urease activity and immunohistochemistry for visualization of bacteria in our study allowed us not only to assess the presence of infection in patients with GC, but also to mark some of its features associated with the localization of bacteria in the stomach, with a ratio of cocci and spiral forms, and the degree of bacterial contamination of GM.\\nOur data for the Orenburg region recorded a high rate of H. pylori infection in patients with GC (84.5%). The coccoid forms of H. pylori, preserving a high degree of urease activity, dominated in GM in patients with GC. They were found in 93.4% of infected patients, with only coccoid forms of H. pylori-in 68.9%. \\nIt is known that the coccoid forms of H. pylori can arise in response to unfavorable environmental factors, such as AT [47,48]. These forms are resistant to AT[49,50] and are able to form biofilms[51] and avoid the immune system[50]. They express a higher rate of cagE mRNA than their spiral counterparts[52], and by increasing the synthesis of tumor necrosis factor-alpha (TNF-α)-inducing protein (Hps), which is introduced into the cytosol and cell nuclei, they can activate nuclear factor-kappaB (NF-κB) and the expression of TNF-α and other cytokines involved in carcinogenesis[53,54]. The effect on the proliferation of gastric epithelial cells in the H. pylori coccoid forms is also stronger than in the helical forms[55], and they can induce the expression of VEGF-A and transforming growth factor-β[56]. The ability to transform into coccoid forms was also found to be characteristic of the most virulent strains of H. pylori[50,54].\\nIt should be noted that the higher infection by coccoid forms of H. pylori in patients with GC, compared to patients with gastritis or gastric ulcer, had been mentioned by other researchers[57]. A number of studies have shown that coccoid forms of H. pylori retained urease activity[58] and the expression of such antigens as CagA, UreA, porin, components of the Cag type IV secretion system (TFSS), antigen-binding adhesin of the blood group BabA and others[59,60].\\nThe use of immunohistochemistry in this study made it possible to detect the bacteria not only in the gastric mucus and on the surface of epithelial cells, but also within the cytoplasm of epithelial and immune cells of GM. Such intracellular expression was characterized by point inclusions giving a specific reaction with antibodies to H. pylori.\\nThe intracellular persistence of H. pylori has been demonstrated by many investigators. They found H. pylori in the cytoplasm of epithelial cells, intercellular spaces, in the lamina propria of GM, and in the lumen of small vessels[61-63]. We assume that the point inclusions in the cytoplasm of epithelial and immune cells giving a positive reaction with antibodies to H. pylori is similar to those particle-rich cytoplasmic structure (PaCS) described earlier in the human superficial-foveolar epithelium and its metaplastic or dysplastic foci[64]. The authors found that the PaCS are a colocalization of VacA, CagA, urease, and outer membrane proteins with NOD1 receptor, ubiquitin-activating enzyme E1, polyubiquitinated proteins, proteasome components, and potentially oncogenic proteins like SHP2 and ERKs[64]. They believe that PaCS is a novel, proteasome-enriched structure arising in ribosome-rich cytoplasm at sites of H. pylori product accumulation. \\nWe believe that the immune cells with point inclusions in the lamina propria of GM are likely to be macrophages. The data obtained by several researchers suggests this conclusion[65,66]. It is noted that even the absorbed bacteria retains their viability in macrophages, which may be associated with the violation of the phagosome maturation[66-68]. The use of confocal microscopy enabled the localization of the bacteria within the cells to be associated with the endosomal and lysosomal markers, and found that H. pylori could use the vesicles of autophagosomes (autophagic vesicles) for its own replication[63,69]. \\nThe study found that the concentration of H. pylori coccoid forms in GM was the most significant clinical factor. This factor was associated with the tumor histology, T status, N status, stage, 10-year PFS, and OS. The moderate and marked concentrations of coccoid forms of H. pylori were more often found in the diffuse type of GC (P = 0.024) and T3-4 (P = 0.04) stage. Interestingly, the high concentration of H. pylori is more frequent in Stage N1 than in N2 (at 90.0% and 53.1%, respectively, P = 0.024).\\nThe moderate and marked concentrations of coccoid forms of H. pylori represented a prognostic factor associated with the decrease of 10-year RFS and OS from 55.6% to 26.3% (P = 0.02 and P = 0.07, respectively).\\nIt should be noted that the results of this study do not allow us to unambiguously judge the effect of H. pylori on GC progression. A decrease in OS and DFS in patients with moderate and marked concentrations of H. pylori coccoid forms in the GM may be due to the fact that these patients had more advanced stages and more aggressive forms of GC. Meanwhile, there are more and more studies showing that H. pylori infection can promote GC progression by activating the NF-κB signaling pathway and induction of interleukin-8 secretion[70], the activation of epithelial-mesenchymal transformation[71-74] and angiogenesis[75,76], as well as increasing the invasive properties of tumor cells[77]. It can be assumed that the administration of AT before surgery contributes to the reduction of the inflammatory process activity and normalization of the adhesive properties of tumour cells, which in turn decreases metastasis risk and improves the long-term results of the treatment of GC. The data literature on the improvement of the long-term results of malignant tumours treatment when using antibacterial drugs testify in favour of this hypothesis[78-80].\",\n \"The data obtained indicates that H. pylori may be associated not only with induction but also with the progression of GC. It can be assumed that the prevalence of coccoid forms of bacteria and their intracellular persistence can affect the mechanisms of tumor progression. Further appropriate studies regarding the role of H. pylori in the progression of GC are obviously advisable.\"\n]","isConversational":false},"other_sections_sec_types":{"kind":"list like","value":[null,null,null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"all_sections_titles":{"kind":"list like","value":["INTRODUCTION","Objective","MATERIALS AND METHODS","The patients","Detection H. pylori infection","RUT","Immunohistochemistry ","Statistics","RESULTS","The features of H. pylori infection in GC","Correlations of clinical and pathological characteristics of GC with the severity of H. pylori infection according to RUT data ","Correlations of clinical and pathological characteristics of GC with the concentration of spiral and coccoid forms of H. pylori in the GM ","DISCUSSION","CONCLUSION"],"string":"[\n \"INTRODUCTION\",\n \"Objective\",\n \"MATERIALS AND METHODS\",\n \"The patients\",\n \"Detection H. pylori infection\",\n \"RUT\",\n \"Immunohistochemistry \",\n \"Statistics\",\n \"RESULTS\",\n \"The features of H. pylori infection in GC\",\n \"Correlations of clinical and pathological characteristics of GC with the severity of H. pylori infection according to RUT data \",\n \"Correlations of clinical and pathological characteristics of GC with the concentration of spiral and coccoid forms of H. pylori in the GM \",\n \"DISCUSSION\",\n \"CONCLUSION\"\n]","isConversational":false},"all_sections_texts":{"kind":"list like","value":["Gastric cancer (GC) continues to be one of the most common malignant diseases in the world[1,2]. Despite a decreasing trend in the incidence of GC in most countries of the world, the treatment results of this pathology cannot be considered satisfactory. In the structure of mortality from malignant neoplasms, this pathology firmly occupies 2nd place in most developed countries of the world, and the 5-year survival rate of radically operated patients does not exceed 15%-30%[3,4]. \nIt is important to note that it is impossible to improve the long-term results of malignant neoplasms treatment without knowledge of the mechanisms associated with their progression[3]. Clinical studies in recent years indicate that inflammatory infiltration of the tumor stroma and surrounding tissues can have an important prognostic value and affect the long-term results of malignant neoplasm treatment[5-9]. A number of studies have shown that inflammatory infiltration of the tumor stroma is associated with the body’s adequate immune response to the tumor, and may be a favorable prognosis factor in various malignant neoplasms[10,11], including GC[12,13]. At the same time, the data obtained by other researchers indicates that pronounced inflammatory infiltration of the tumor stroma, especially T-reg lymphocytes and macrophages, may be a factor contributing to the progression of malignant neoplasms[14,15]. There was a decrease in the overall survival (OS) and relapse-free survival (RFS) of GC patients with a high content of Foxp3 + T-reg in the tumor stroma and regional metastases[16-18] and macrophages[16,19]. It is believed that inflammatory infiltration of the tumor stroma can contribute to tumor progression by activating the mechanisms of angiogenesis, expression of E- and L-selectins, formation of the products of lipid peroxidation and free radicals, destruction of connective tissue matrix and basement membranes of epithelia by proteolytic enzymes, and activation of epithelial-mesenchymal transformation[20-23].\nWhen studying the role of inflammation in the progression of GC, it is impossible to ignore the problem of Helicobacter pylori (H. pylori) infection. H. pylori is a gram-negative, spiral-shaped bacterium, the habitat of which is the gastric mucosa (GM) and duodenum. H. pylori differs from other bacteria in a set of properties that make it possible to colonize the GM and persist for a long time under conditions that are unfavorable for other microorganisms[24,25]. These include: (1) The ability to produce a special enzyme-urease; (2) Synthesis of lytic enzymes that cause the depolymerization and dissolution of gastric mucus, consisting mainly of mucin; (3) The mobility of the bacterium, which is ensured by the presence of 5-6 flagella; (4) The high adhesiveness of bacteria to GM epithelial cells of the GM and elements of connective tissue due to the interaction of bacterial ligands with the corresponding cells receptors; (5) Production of various exotoxins (VacA, CagA, and others); (6) Instability of the H. pylori genome; (7) The presence of vegetative and coccoid forms of bacteria; and (8) possibility of intracellular persistence and translocation outside the GM [26-29].\nIt should be noted that despite the huge number of studies devoted to H. pylori, it is still not clear whether H. pylori is involved only in the initiation of the tumor process in the stomach, or whether it can affect the mechanisms of tumor progression. The relationship between the severity of H. pylori infection and the clinical and morphological characteristics of GC and long-term results of this pathology treatment remains poorly studied, and in this connection, the question of the expediency of anti-Helicobacter therapy in patients with invasive GC remains open.\nObjective To assess the features of H. pylori infection in patients with Stage I-IIIB GC and their correlation with the clinical and morphological characteristics of the disease, the presence of antibiotic therapy (AT) before surgery, and long-term treatment results.\nTo assess the features of H. pylori infection in patients with Stage I-IIIB GC and their correlation with the clinical and morphological characteristics of the disease, the presence of antibiotic therapy (AT) before surgery, and long-term treatment results.","To assess the features of H. pylori infection in patients with Stage I-IIIB GC and their correlation with the clinical and morphological characteristics of the disease, the presence of antibiotic therapy (AT) before surgery, and long-term treatment results.","The patients One hundred and nine patients with GC who had undergone radical surgery (R0) between May 2007 and March 2010 at the Orenburg Regional Clinical Oncology Center, were included in this prospective cohort pilot study. Study inclusion criteria were: Histologically proven invasive GC; no evidence of distant metastases; radical surgery (R0); no prior gastric surgery; no previous chemotherapy or radiotherapy. The study did not include patients with decompensation of cardiovascular and renal diseases, exacerbation of chronic inflammatory processes, severe allergic processes, or who received glucocorticoids, antihistamines, and non-steroidal anti-inflammatory drugs. The study was performed in accordance with the Helsinki Declaration and internationally recognized guidelines, and the privacy of patients was protected by decoding the data according to the privacy regulations of the Orenburg Regional Clinical Oncologic Center (Russia, Orenburg). All patients provided written informed consent. The protocol was approved by the Institutional Review Board of the Orenburg State Medical University (Russia, Orenburg).\nClinical and pathological data including age, tumor localization, stage, type of surgery, histology, the presence of AT before surgery, postoperative therapy, and long-term results of treatment were retrieved from the routine reports for analyses. The distribution of patients according to the clinical and pathological characteristics of GC is presented in Table 1.\nThe distribution of patients according to the clinical and pathological characteristics of gastric cancer\nGC: Gastric cancer.\nWhen interviewing patients, it was found that 45 patients received AT before admission to the clinic due to a preliminary wrong diagnosis of gastric ulcer or chronic gastritis. The following combinations of antibacterial drugs were most commonly ordered: Amoxicillin + clarithromycin (34 patients), amoxicillin + clarithromycin + metronidazole (6 patients), amoxicillin + metronidazole (3 patients), other drugs (2 patients). Information about the antibacterial drugs, the timing and duration of their intake was entered into the primary patient documentation and then taken into account in the analysis. We considered only those patients who underwent AT in the period from 1 to 1.5 mo before the operation, lasting at least seven days, and using two or more antibacterial drugs.\nThe long-term results of treatment were assessed for the period from May 12, 2007 to April 12, 2021. The median follow-up period was 86.2 mo. As of April 12, 2021, 26 (24.5%) patients were alive, 54 (50.9%) had died from the progression of GC, 20 (18.9%) had died from causes other than GC, and six (5.6%) left the region at different follow-up periods. Malignant tumors of other localizations were diagnosed in 8 patients at different times after the operation: Non-Hodgkin's lymphomas-in three, prostate cancer-in one, lung cancer-in two, laryngeal cancer-in one, and breast cancer-in one patient. With the exception of one patient with non-Hodgkin's lymphoma, the other patients died from the progression of these diseases. The causes of death were not associated with malignant tumors for the other patients. During the period 2020-2021, seven patients contracted coronavirus disease-19, one of whom died from the disease, but the rest are alive.\nOne hundred and nine patients with GC who had undergone radical surgery (R0) between May 2007 and March 2010 at the Orenburg Regional Clinical Oncology Center, were included in this prospective cohort pilot study. Study inclusion criteria were: Histologically proven invasive GC; no evidence of distant metastases; radical surgery (R0); no prior gastric surgery; no previous chemotherapy or radiotherapy. The study did not include patients with decompensation of cardiovascular and renal diseases, exacerbation of chronic inflammatory processes, severe allergic processes, or who received glucocorticoids, antihistamines, and non-steroidal anti-inflammatory drugs. The study was performed in accordance with the Helsinki Declaration and internationally recognized guidelines, and the privacy of patients was protected by decoding the data according to the privacy regulations of the Orenburg Regional Clinical Oncologic Center (Russia, Orenburg). All patients provided written informed consent. The protocol was approved by the Institutional Review Board of the Orenburg State Medical University (Russia, Orenburg).\nClinical and pathological data including age, tumor localization, stage, type of surgery, histology, the presence of AT before surgery, postoperative therapy, and long-term results of treatment were retrieved from the routine reports for analyses. The distribution of patients according to the clinical and pathological characteristics of GC is presented in Table 1.\nThe distribution of patients according to the clinical and pathological characteristics of gastric cancer\nGC: Gastric cancer.\nWhen interviewing patients, it was found that 45 patients received AT before admission to the clinic due to a preliminary wrong diagnosis of gastric ulcer or chronic gastritis. The following combinations of antibacterial drugs were most commonly ordered: Amoxicillin + clarithromycin (34 patients), amoxicillin + clarithromycin + metronidazole (6 patients), amoxicillin + metronidazole (3 patients), other drugs (2 patients). Information about the antibacterial drugs, the timing and duration of their intake was entered into the primary patient documentation and then taken into account in the analysis. We considered only those patients who underwent AT in the period from 1 to 1.5 mo before the operation, lasting at least seven days, and using two or more antibacterial drugs.\nThe long-term results of treatment were assessed for the period from May 12, 2007 to April 12, 2021. The median follow-up period was 86.2 mo. As of April 12, 2021, 26 (24.5%) patients were alive, 54 (50.9%) had died from the progression of GC, 20 (18.9%) had died from causes other than GC, and six (5.6%) left the region at different follow-up periods. Malignant tumors of other localizations were diagnosed in 8 patients at different times after the operation: Non-Hodgkin's lymphomas-in three, prostate cancer-in one, lung cancer-in two, laryngeal cancer-in one, and breast cancer-in one patient. With the exception of one patient with non-Hodgkin's lymphoma, the other patients died from the progression of these diseases. The causes of death were not associated with malignant tumors for the other patients. During the period 2020-2021, seven patients contracted coronavirus disease-19, one of whom died from the disease, but the rest are alive.\nDetection H. pylori infection \nH. pylori in the GM and tumor was determined by rapid urease test (RUT) and by immunohistochemically (IHC) using the antibody to H. pylori.\n\nH. pylori in the GM and tumor was determined by rapid urease test (RUT) and by immunohistochemically (IHC) using the antibody to H. pylori.\nRUT After removal of the stomach (within 30 min) a greater curvature of the organ was opened and biopsy samples were taken from the tumor and the adjacent macroscopically non-tumorous GM at a distance of 3-5 cm from the tumor margin. The samples were placed on test strips (HELPIL–test, “АМА”, Russia) for three minutes. The presence and the severity of the infection were evaluated by the color change of the indicator from yellow to blue. \nAccording to the intensity and the time of the appearance of a blue color, we distinguished three degrees of infection: 3+: Marked (+++)-bright staining in the first minute of the study; 2+: Moderate (++)–for an average intensity of staining for 2 min and 1+ mild (+)-weak staining for three minutes. If the color of the indicator did not change, or became dirty gray, and if after repeated research the same result was received, the test was evaluated as negative. The same samples were later used for histological analysis and IGH. \nAfter removal of the stomach (within 30 min) a greater curvature of the organ was opened and biopsy samples were taken from the tumor and the adjacent macroscopically non-tumorous GM at a distance of 3-5 cm from the tumor margin. The samples were placed on test strips (HELPIL–test, “АМА”, Russia) for three minutes. The presence and the severity of the infection were evaluated by the color change of the indicator from yellow to blue. \nAccording to the intensity and the time of the appearance of a blue color, we distinguished three degrees of infection: 3+: Marked (+++)-bright staining in the first minute of the study; 2+: Moderate (++)–for an average intensity of staining for 2 min and 1+ mild (+)-weak staining for three minutes. If the color of the indicator did not change, or became dirty gray, and if after repeated research the same result was received, the test was evaluated as negative. The same samples were later used for histological analysis and IGH. \nImmunohistochemistry The presence and features of H. pylori infection were studied in samples of the GM adjacent to the tumor, in the tumor tissue, in the omentum, and regional lymph nodes of 46 patients, by immunohistochemistry.\nThe sections for IGH were dewaxed and rehydrated by sequential immersion in xylene and graded ethanol and water. For antigen retrieval, the sections boiling for 10 min in citrate buffer (pH 6) and endogenous peroxidase activity was blocked with 30 mL/L hydrogen peroxide solution. Slides were incubated at room temperature with the anti-H. pylori (RB-9070, Thermo Fisher Scientific, the immunogen is purified H. pylori) rabbit polyclonal antibodies in diluted at 1:1000 for 30 min. \nThe visualization system included DAB (UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen) and hematoxylin counterstaining. For negative control sections, the primary antibody was replaced with phosphate-buffered saline and processed in the same manner.\nThe concentration of H. pylori in the GM detected by IGH was graded as 1+ for mild, 2+ for moderate, and 3+ for marked according to the Sydney system[30]. The presence of point inclusions giving a positive reaction with antibodies to H. pylori in the cytoplasm of epithelial cells of deep gastric glands and of the lymphoid cells of the lamina propria of GM as well as in omentum and lymph node, were taken into account. \nAll sections were carefully and completely scanned by two of the authors (MS and OT) without knowledge of the clinical and pathological data. \nThe data obtained was compared with clinical features of GC: Stage, localization, histology, the presence of AT before surgery, and 10-year OS and RFS.\nThe presence and features of H. pylori infection were studied in samples of the GM adjacent to the tumor, in the tumor tissue, in the omentum, and regional lymph nodes of 46 patients, by immunohistochemistry.\nThe sections for IGH were dewaxed and rehydrated by sequential immersion in xylene and graded ethanol and water. For antigen retrieval, the sections boiling for 10 min in citrate buffer (pH 6) and endogenous peroxidase activity was blocked with 30 mL/L hydrogen peroxide solution. Slides were incubated at room temperature with the anti-H. pylori (RB-9070, Thermo Fisher Scientific, the immunogen is purified H. pylori) rabbit polyclonal antibodies in diluted at 1:1000 for 30 min. \nThe visualization system included DAB (UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen) and hematoxylin counterstaining. For negative control sections, the primary antibody was replaced with phosphate-buffered saline and processed in the same manner.\nThe concentration of H. pylori in the GM detected by IGH was graded as 1+ for mild, 2+ for moderate, and 3+ for marked according to the Sydney system[30]. The presence of point inclusions giving a positive reaction with antibodies to H. pylori in the cytoplasm of epithelial cells of deep gastric glands and of the lymphoid cells of the lamina propria of GM as well as in omentum and lymph node, were taken into account. \nAll sections were carefully and completely scanned by two of the authors (MS and OT) without knowledge of the clinical and pathological data. \nThe data obtained was compared with clinical features of GC: Stage, localization, histology, the presence of AT before surgery, and 10-year OS and RFS.\nStatistics Statistical analysis was performed using the Statistica 10.0 software. The correlations between different data were evaluated using the nonparametric Spearman's rank correlation or gamma correlation. Chi-square tests were carried out to analyze the difference of distribution among the categorized data. Mann–Whitney U nonparametric test was used to compare the value of the quantitative data. The survival was analyzed using the Kaplan-Meier method. The log-rank test was used to compare survival curves between subgroups of patients. A value of P < 0.05 was considered statistically significant.\nStatistical analysis was performed using the Statistica 10.0 software. The correlations between different data were evaluated using the nonparametric Spearman's rank correlation or gamma correlation. Chi-square tests were carried out to analyze the difference of distribution among the categorized data. Mann–Whitney U nonparametric test was used to compare the value of the quantitative data. The survival was analyzed using the Kaplan-Meier method. The log-rank test was used to compare survival curves between subgroups of patients. A value of P < 0.05 was considered statistically significant.","One hundred and nine patients with GC who had undergone radical surgery (R0) between May 2007 and March 2010 at the Orenburg Regional Clinical Oncology Center, were included in this prospective cohort pilot study. Study inclusion criteria were: Histologically proven invasive GC; no evidence of distant metastases; radical surgery (R0); no prior gastric surgery; no previous chemotherapy or radiotherapy. The study did not include patients with decompensation of cardiovascular and renal diseases, exacerbation of chronic inflammatory processes, severe allergic processes, or who received glucocorticoids, antihistamines, and non-steroidal anti-inflammatory drugs. The study was performed in accordance with the Helsinki Declaration and internationally recognized guidelines, and the privacy of patients was protected by decoding the data according to the privacy regulations of the Orenburg Regional Clinical Oncologic Center (Russia, Orenburg). All patients provided written informed consent. The protocol was approved by the Institutional Review Board of the Orenburg State Medical University (Russia, Orenburg).\nClinical and pathological data including age, tumor localization, stage, type of surgery, histology, the presence of AT before surgery, postoperative therapy, and long-term results of treatment were retrieved from the routine reports for analyses. The distribution of patients according to the clinical and pathological characteristics of GC is presented in Table 1.\nThe distribution of patients according to the clinical and pathological characteristics of gastric cancer\nGC: Gastric cancer.\nWhen interviewing patients, it was found that 45 patients received AT before admission to the clinic due to a preliminary wrong diagnosis of gastric ulcer or chronic gastritis. The following combinations of antibacterial drugs were most commonly ordered: Amoxicillin + clarithromycin (34 patients), amoxicillin + clarithromycin + metronidazole (6 patients), amoxicillin + metronidazole (3 patients), other drugs (2 patients). Information about the antibacterial drugs, the timing and duration of their intake was entered into the primary patient documentation and then taken into account in the analysis. We considered only those patients who underwent AT in the period from 1 to 1.5 mo before the operation, lasting at least seven days, and using two or more antibacterial drugs.\nThe long-term results of treatment were assessed for the period from May 12, 2007 to April 12, 2021. The median follow-up period was 86.2 mo. As of April 12, 2021, 26 (24.5%) patients were alive, 54 (50.9%) had died from the progression of GC, 20 (18.9%) had died from causes other than GC, and six (5.6%) left the region at different follow-up periods. Malignant tumors of other localizations were diagnosed in 8 patients at different times after the operation: Non-Hodgkin's lymphomas-in three, prostate cancer-in one, lung cancer-in two, laryngeal cancer-in one, and breast cancer-in one patient. With the exception of one patient with non-Hodgkin's lymphoma, the other patients died from the progression of these diseases. The causes of death were not associated with malignant tumors for the other patients. During the period 2020-2021, seven patients contracted coronavirus disease-19, one of whom died from the disease, but the rest are alive.","\nH. pylori in the GM and tumor was determined by rapid urease test (RUT) and by immunohistochemically (IHC) using the antibody to H. pylori.","After removal of the stomach (within 30 min) a greater curvature of the organ was opened and biopsy samples were taken from the tumor and the adjacent macroscopically non-tumorous GM at a distance of 3-5 cm from the tumor margin. The samples were placed on test strips (HELPIL–test, “АМА”, Russia) for three minutes. The presence and the severity of the infection were evaluated by the color change of the indicator from yellow to blue. \nAccording to the intensity and the time of the appearance of a blue color, we distinguished three degrees of infection: 3+: Marked (+++)-bright staining in the first minute of the study; 2+: Moderate (++)–for an average intensity of staining for 2 min and 1+ mild (+)-weak staining for three minutes. If the color of the indicator did not change, or became dirty gray, and if after repeated research the same result was received, the test was evaluated as negative. The same samples were later used for histological analysis and IGH. ","The presence and features of H. pylori infection were studied in samples of the GM adjacent to the tumor, in the tumor tissue, in the omentum, and regional lymph nodes of 46 patients, by immunohistochemistry.\nThe sections for IGH were dewaxed and rehydrated by sequential immersion in xylene and graded ethanol and water. For antigen retrieval, the sections boiling for 10 min in citrate buffer (pH 6) and endogenous peroxidase activity was blocked with 30 mL/L hydrogen peroxide solution. Slides were incubated at room temperature with the anti-H. pylori (RB-9070, Thermo Fisher Scientific, the immunogen is purified H. pylori) rabbit polyclonal antibodies in diluted at 1:1000 for 30 min. \nThe visualization system included DAB (UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen) and hematoxylin counterstaining. For negative control sections, the primary antibody was replaced with phosphate-buffered saline and processed in the same manner.\nThe concentration of H. pylori in the GM detected by IGH was graded as 1+ for mild, 2+ for moderate, and 3+ for marked according to the Sydney system[30]. The presence of point inclusions giving a positive reaction with antibodies to H. pylori in the cytoplasm of epithelial cells of deep gastric glands and of the lymphoid cells of the lamina propria of GM as well as in omentum and lymph node, were taken into account. \nAll sections were carefully and completely scanned by two of the authors (MS and OT) without knowledge of the clinical and pathological data. \nThe data obtained was compared with clinical features of GC: Stage, localization, histology, the presence of AT before surgery, and 10-year OS and RFS.","Statistical analysis was performed using the Statistica 10.0 software. The correlations between different data were evaluated using the nonparametric Spearman's rank correlation or gamma correlation. Chi-square tests were carried out to analyze the difference of distribution among the categorized data. Mann–Whitney U nonparametric test was used to compare the value of the quantitative data. The survival was analyzed using the Kaplan-Meier method. The log-rank test was used to compare survival curves between subgroups of patients. A value of P < 0.05 was considered statistically significant.","The features of H. pylori infection in GC Of 93 patients (84.5%) demonstrated positive RUT. According to RUT the concentration of H. pylori in GM was mild (1+) in 38 (34.8%) patients, moderate (2+)-in 37 (33.9%) and marked (3+)–in 8 (16.5%). RUT was negative in 16 patients (14.7%).\nIt was found that urease activity 2+ and 3+ were significantly more frequent in GM than in tumors (in 55.5% and 13.3% of cases, respectively, P = 0.01). In more than half the cases (53.3%), the urease activity in the tumor was lower than in the adjacent GM.\nForty-six samples of GM were stained by IHC. The coccoid forms of H. pylori were found to prevail in GM adjacent to the tumor (Figure 1A). Coccoid forms of H. pylori only were identified in 31 (63.1%) patients, and coccoid and spiral in 12 (30.4%) patients (Figure 1B). No signs of infection were found in 3 (6.5%) patients. The concentration of coccoid and spiral forms of H. pylori in GM according to IGH was 1+ in 24 (52.2%) and 1 (19.6%) patients, 2+-in 11 (23.9%) and 4 (8.7%), and 3+-in 8 (17.4%) and 1 (2.2%) patients. A positive correlation between the concentration of coccoid and spiral forms of H. pylori (gamma = 0.642, P < 0.0001) was noted.\n\nThe features of Helicobacter pylori localization in gastric mucosa in patients with gastric cancer. A: Coccoid forms of Helicobacter pylori (H. pylori) in the gastric pit. The some bacteria within the cytoplasm of epithelium cells (arrows); B: The spiral (black arrows) and coccoid (orange arrows) forms of H. pylori on the surface of superficial-foveolar gastric epithelium; C: The bacteria in the surface mucous gel layer of stomach (arrows); D: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of epithelial cells of deep gastric glands (arrows); E: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of the immune cells of the lamina propria of gastric mucosa (arrows); F: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of intraepithelial lymphocytes (arrows). Immunoperoxidase staining with anti-H. pylori antibody, immersion. Bars: A: 20 μm; B: 10 μm; C: 20 μm; D-F: 10 μm. \nThe localization of H. pylori in GM was observed not only in the surface mucous gel layer of the stomach (Figure 1C) but also within the cytoplasm of the gastric superficial-foveolar epithelium (Figure 1D). The point inclusions giving a positive reaction with antibodies to H. pylori were also revealed in the cytoplasm of the epithelial cells of deep gastric glands (in 41.3% samples) and of the immune cells (Figure 1E) of the lamina propria of GM (in 43.5% samples), as well as of the intraepithelial lymphocytes (Figure 1F). The close relationship between the presence of point inclusions in immune cells and epithelial cells (gamma = 0.642, P < 0.0001) was noted. The individual cocci or their small clusters, and sometimes the short rod bacterium, were also often detected in the lamina propria of the GM. We also found bacteria in the omentum and lymph nodes. In the omentum the bacteria were presented predominantly by cocci between 0.5 and 1 μm in diameter (Figure 2A). Cocci were arranged most commonly by the small compact groups up to 10-15 cells in the immediate vicinity of the LN capsule. The bacteria were located mainly between the adipocytes. However, it was not clear whether bacteria were outside of the cells or in a narrow rim of cytoplasm of the fat cells. \n\nThe features of Helicobacter pylori localization in omentum and lymph node in patients with gastric cancer. A: The small group of cocci located in the central part of the omentum adipocyte; B: The congestions of bacteria around the nucleus of lymphocytes in the paracortical area of lymph node. Immunoperoxidase staining with anti-Helicobacter pylori antibody, immersion, Bars: 10 μm. \nIn the tissue of lymph nodes we usually observed the small accumulations of cocci between 10 and 15 cells (Figure 2B). Bacteria were located between lymphocytes of the paracortical area, but not so compact, as in the omentum. Quite often the concentration of bacteria was observed around the nuclei of cells that can testify to their intracellular localization.\nOf 93 patients (84.5%) demonstrated positive RUT. According to RUT the concentration of H. pylori in GM was mild (1+) in 38 (34.8%) patients, moderate (2+)-in 37 (33.9%) and marked (3+)–in 8 (16.5%). RUT was negative in 16 patients (14.7%).\nIt was found that urease activity 2+ and 3+ were significantly more frequent in GM than in tumors (in 55.5% and 13.3% of cases, respectively, P = 0.01). In more than half the cases (53.3%), the urease activity in the tumor was lower than in the adjacent GM.\nForty-six samples of GM were stained by IHC. The coccoid forms of H. pylori were found to prevail in GM adjacent to the tumor (Figure 1A). Coccoid forms of H. pylori only were identified in 31 (63.1%) patients, and coccoid and spiral in 12 (30.4%) patients (Figure 1B). No signs of infection were found in 3 (6.5%) patients. The concentration of coccoid and spiral forms of H. pylori in GM according to IGH was 1+ in 24 (52.2%) and 1 (19.6%) patients, 2+-in 11 (23.9%) and 4 (8.7%), and 3+-in 8 (17.4%) and 1 (2.2%) patients. A positive correlation between the concentration of coccoid and spiral forms of H. pylori (gamma = 0.642, P < 0.0001) was noted.\n\nThe features of Helicobacter pylori localization in gastric mucosa in patients with gastric cancer. A: Coccoid forms of Helicobacter pylori (H. pylori) in the gastric pit. The some bacteria within the cytoplasm of epithelium cells (arrows); B: The spiral (black arrows) and coccoid (orange arrows) forms of H. pylori on the surface of superficial-foveolar gastric epithelium; C: The bacteria in the surface mucous gel layer of stomach (arrows); D: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of epithelial cells of deep gastric glands (arrows); E: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of the immune cells of the lamina propria of gastric mucosa (arrows); F: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of intraepithelial lymphocytes (arrows). Immunoperoxidase staining with anti-H. pylori antibody, immersion. Bars: A: 20 μm; B: 10 μm; C: 20 μm; D-F: 10 μm. \nThe localization of H. pylori in GM was observed not only in the surface mucous gel layer of the stomach (Figure 1C) but also within the cytoplasm of the gastric superficial-foveolar epithelium (Figure 1D). The point inclusions giving a positive reaction with antibodies to H. pylori were also revealed in the cytoplasm of the epithelial cells of deep gastric glands (in 41.3% samples) and of the immune cells (Figure 1E) of the lamina propria of GM (in 43.5% samples), as well as of the intraepithelial lymphocytes (Figure 1F). The close relationship between the presence of point inclusions in immune cells and epithelial cells (gamma = 0.642, P < 0.0001) was noted. The individual cocci or their small clusters, and sometimes the short rod bacterium, were also often detected in the lamina propria of the GM. We also found bacteria in the omentum and lymph nodes. In the omentum the bacteria were presented predominantly by cocci between 0.5 and 1 μm in diameter (Figure 2A). Cocci were arranged most commonly by the small compact groups up to 10-15 cells in the immediate vicinity of the LN capsule. The bacteria were located mainly between the adipocytes. However, it was not clear whether bacteria were outside of the cells or in a narrow rim of cytoplasm of the fat cells. \n\nThe features of Helicobacter pylori localization in omentum and lymph node in patients with gastric cancer. A: The small group of cocci located in the central part of the omentum adipocyte; B: The congestions of bacteria around the nucleus of lymphocytes in the paracortical area of lymph node. Immunoperoxidase staining with anti-Helicobacter pylori antibody, immersion, Bars: 10 μm. \nIn the tissue of lymph nodes we usually observed the small accumulations of cocci between 10 and 15 cells (Figure 2B). Bacteria were located between lymphocytes of the paracortical area, but not so compact, as in the omentum. Quite often the concentration of bacteria was observed around the nuclei of cells that can testify to their intracellular localization.\nCorrelations of clinical and pathological characteristics of GC with the severity of H. pylori infection according to RUT data The gamma correlation coefficient test (gamma) showed that the severity of H. pylori in GM according to RUT positively correlated with the T status (gamma = 0.537, P < 0.00001), N status (gamma = 0.371, P = 0.0007) and stage (gamma = 0.520, P < 0.00001), and negatively correlated with the presence of AT in anamnesis (gamma = -0.418, P = 0.003). The marked (+++) and moderate (++) degrees of H. pylori infection were more often observed in Grade 2 and Grade 3, in T3-4 status, in N1 status, in the T3-4N1-2 stage, and in the absence of AT in anamnesis (Table 2). Correlations of H. pylori concentration in GM according to RUT with 10-year OS and RFS of GC patients were not determined. \nClinical and pathological characteristics of gastric cancer depending on the data rapid urease test\nRUT: Rapid urease test; GC: Gastric cancer.\nIt is important to note that the presence in AT 1-1.5 mo before surgery was associated with a significant improvement in RFS and OS (Figure 3), however, this applied only to patients with local GC (T1-3N0). In advanced GC (T3-4N1 and T3-4N2) there were no significant differences in patient survival (Figure 4).\n\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\n\n10-year overall surviving and relapse-free surviving of patients with T3-4N1-2M0 stages of gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.78). A: 10-year overall surviving; B: Relapse-free surviving.\nThe gamma correlation coefficient test (gamma) showed that the severity of H. pylori in GM according to RUT positively correlated with the T status (gamma = 0.537, P < 0.00001), N status (gamma = 0.371, P = 0.0007) and stage (gamma = 0.520, P < 0.00001), and negatively correlated with the presence of AT in anamnesis (gamma = -0.418, P = 0.003). The marked (+++) and moderate (++) degrees of H. pylori infection were more often observed in Grade 2 and Grade 3, in T3-4 status, in N1 status, in the T3-4N1-2 stage, and in the absence of AT in anamnesis (Table 2). Correlations of H. pylori concentration in GM according to RUT with 10-year OS and RFS of GC patients were not determined. \nClinical and pathological characteristics of gastric cancer depending on the data rapid urease test\nRUT: Rapid urease test; GC: Gastric cancer.\nIt is important to note that the presence in AT 1-1.5 mo before surgery was associated with a significant improvement in RFS and OS (Figure 3), however, this applied only to patients with local GC (T1-3N0). In advanced GC (T3-4N1 and T3-4N2) there were no significant differences in patient survival (Figure 4).\n\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\n\n10-year overall surviving and relapse-free surviving of patients with T3-4N1-2M0 stages of gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.78). A: 10-year overall surviving; B: Relapse-free surviving.\nCorrelations of clinical and pathological characteristics of GC with the concentration of spiral and coccoid forms of H. pylori in the GM It is important to note that correlations between the clinical and pathological characteristics of GC and the concentration of H. pylori spiral forms in GM were not found. However, the concentration of H. pylori coccoid forms correlated with age (ρ = -0.502, P = 0.0006), histology (gamma = 0.550, P = 0.0004), T status (gamma = 0.709, P = 0.0001), N status (gamma = 0.509, P = 0.002) stage (gamma = 0.636, P = 0.0002), and 10-year RFS (gamma = -0.521, P = 0.008) and OS (gamma = -0.500, P = 0.044). In cases with a moderate and marked concentration (2+ or 3+) of H. pylori coccoid forms in GM compared to cases with a low concentration (1+ or without infection) the patients were younger (57.9 ± 2.5 years vs 66.2 ± 1.4 years, respectively, P = 0.004) and the diffuse type of GC, poorly differentiated tumors (G3), T3-4 stage and N1 stage of GC were more often observed (Table 3). In cases of moderate and marked concentrations of H. pylori in GM, a decrease in 10-year progression-free survival (PFS) and OS survival from 55.6% to 26.3% was observed (P = 0.02 and P = 0.07, respectively). PFS and OS curves, depending on the concentration of coccoid forms of H. pylori in GM, are presented in Figure 5.\n\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\nClinical and pathological characteristics of gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa\nRUT: Rapid urease test; GC: Gastric cancer.\nThere were no correlations between the presence of point inclusions in the cytoplasm of epithelial cells of deep gastric glands, in the stroma immune cells, and in the intraepithelial lymphocytes with the clinical and pathological characteristics of GC.\nIt is important to note that correlations between the clinical and pathological characteristics of GC and the concentration of H. pylori spiral forms in GM were not found. However, the concentration of H. pylori coccoid forms correlated with age (ρ = -0.502, P = 0.0006), histology (gamma = 0.550, P = 0.0004), T status (gamma = 0.709, P = 0.0001), N status (gamma = 0.509, P = 0.002) stage (gamma = 0.636, P = 0.0002), and 10-year RFS (gamma = -0.521, P = 0.008) and OS (gamma = -0.500, P = 0.044). In cases with a moderate and marked concentration (2+ or 3+) of H. pylori coccoid forms in GM compared to cases with a low concentration (1+ or without infection) the patients were younger (57.9 ± 2.5 years vs 66.2 ± 1.4 years, respectively, P = 0.004) and the diffuse type of GC, poorly differentiated tumors (G3), T3-4 stage and N1 stage of GC were more often observed (Table 3). In cases of moderate and marked concentrations of H. pylori in GM, a decrease in 10-year progression-free survival (PFS) and OS survival from 55.6% to 26.3% was observed (P = 0.02 and P = 0.07, respectively). PFS and OS curves, depending on the concentration of coccoid forms of H. pylori in GM, are presented in Figure 5.\n\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\nClinical and pathological characteristics of gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa\nRUT: Rapid urease test; GC: Gastric cancer.\nThere were no correlations between the presence of point inclusions in the cytoplasm of epithelial cells of deep gastric glands, in the stroma immune cells, and in the intraepithelial lymphocytes with the clinical and pathological characteristics of GC.","Of 93 patients (84.5%) demonstrated positive RUT. According to RUT the concentration of H. pylori in GM was mild (1+) in 38 (34.8%) patients, moderate (2+)-in 37 (33.9%) and marked (3+)–in 8 (16.5%). RUT was negative in 16 patients (14.7%).\nIt was found that urease activity 2+ and 3+ were significantly more frequent in GM than in tumors (in 55.5% and 13.3% of cases, respectively, P = 0.01). In more than half the cases (53.3%), the urease activity in the tumor was lower than in the adjacent GM.\nForty-six samples of GM were stained by IHC. The coccoid forms of H. pylori were found to prevail in GM adjacent to the tumor (Figure 1A). Coccoid forms of H. pylori only were identified in 31 (63.1%) patients, and coccoid and spiral in 12 (30.4%) patients (Figure 1B). No signs of infection were found in 3 (6.5%) patients. The concentration of coccoid and spiral forms of H. pylori in GM according to IGH was 1+ in 24 (52.2%) and 1 (19.6%) patients, 2+-in 11 (23.9%) and 4 (8.7%), and 3+-in 8 (17.4%) and 1 (2.2%) patients. A positive correlation between the concentration of coccoid and spiral forms of H. pylori (gamma = 0.642, P < 0.0001) was noted.\n\nThe features of Helicobacter pylori localization in gastric mucosa in patients with gastric cancer. A: Coccoid forms of Helicobacter pylori (H. pylori) in the gastric pit. The some bacteria within the cytoplasm of epithelium cells (arrows); B: The spiral (black arrows) and coccoid (orange arrows) forms of H. pylori on the surface of superficial-foveolar gastric epithelium; C: The bacteria in the surface mucous gel layer of stomach (arrows); D: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of epithelial cells of deep gastric glands (arrows); E: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of the immune cells of the lamina propria of gastric mucosa (arrows); F: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of intraepithelial lymphocytes (arrows). Immunoperoxidase staining with anti-H. pylori antibody, immersion. Bars: A: 20 μm; B: 10 μm; C: 20 μm; D-F: 10 μm. \nThe localization of H. pylori in GM was observed not only in the surface mucous gel layer of the stomach (Figure 1C) but also within the cytoplasm of the gastric superficial-foveolar epithelium (Figure 1D). The point inclusions giving a positive reaction with antibodies to H. pylori were also revealed in the cytoplasm of the epithelial cells of deep gastric glands (in 41.3% samples) and of the immune cells (Figure 1E) of the lamina propria of GM (in 43.5% samples), as well as of the intraepithelial lymphocytes (Figure 1F). The close relationship between the presence of point inclusions in immune cells and epithelial cells (gamma = 0.642, P < 0.0001) was noted. The individual cocci or their small clusters, and sometimes the short rod bacterium, were also often detected in the lamina propria of the GM. We also found bacteria in the omentum and lymph nodes. In the omentum the bacteria were presented predominantly by cocci between 0.5 and 1 μm in diameter (Figure 2A). Cocci were arranged most commonly by the small compact groups up to 10-15 cells in the immediate vicinity of the LN capsule. The bacteria were located mainly between the adipocytes. However, it was not clear whether bacteria were outside of the cells or in a narrow rim of cytoplasm of the fat cells. \n\nThe features of Helicobacter pylori localization in omentum and lymph node in patients with gastric cancer. A: The small group of cocci located in the central part of the omentum adipocyte; B: The congestions of bacteria around the nucleus of lymphocytes in the paracortical area of lymph node. Immunoperoxidase staining with anti-Helicobacter pylori antibody, immersion, Bars: 10 μm. \nIn the tissue of lymph nodes we usually observed the small accumulations of cocci between 10 and 15 cells (Figure 2B). Bacteria were located between lymphocytes of the paracortical area, but not so compact, as in the omentum. Quite often the concentration of bacteria was observed around the nuclei of cells that can testify to their intracellular localization.","The gamma correlation coefficient test (gamma) showed that the severity of H. pylori in GM according to RUT positively correlated with the T status (gamma = 0.537, P < 0.00001), N status (gamma = 0.371, P = 0.0007) and stage (gamma = 0.520, P < 0.00001), and negatively correlated with the presence of AT in anamnesis (gamma = -0.418, P = 0.003). The marked (+++) and moderate (++) degrees of H. pylori infection were more often observed in Grade 2 and Grade 3, in T3-4 status, in N1 status, in the T3-4N1-2 stage, and in the absence of AT in anamnesis (Table 2). Correlations of H. pylori concentration in GM according to RUT with 10-year OS and RFS of GC patients were not determined. \nClinical and pathological characteristics of gastric cancer depending on the data rapid urease test\nRUT: Rapid urease test; GC: Gastric cancer.\nIt is important to note that the presence in AT 1-1.5 mo before surgery was associated with a significant improvement in RFS and OS (Figure 3), however, this applied only to patients with local GC (T1-3N0). In advanced GC (T3-4N1 and T3-4N2) there were no significant differences in patient survival (Figure 4).\n\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\n\n10-year overall surviving and relapse-free surviving of patients with T3-4N1-2M0 stages of gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.78). A: 10-year overall surviving; B: Relapse-free surviving.","It is important to note that correlations between the clinical and pathological characteristics of GC and the concentration of H. pylori spiral forms in GM were not found. However, the concentration of H. pylori coccoid forms correlated with age (ρ = -0.502, P = 0.0006), histology (gamma = 0.550, P = 0.0004), T status (gamma = 0.709, P = 0.0001), N status (gamma = 0.509, P = 0.002) stage (gamma = 0.636, P = 0.0002), and 10-year RFS (gamma = -0.521, P = 0.008) and OS (gamma = -0.500, P = 0.044). In cases with a moderate and marked concentration (2+ or 3+) of H. pylori coccoid forms in GM compared to cases with a low concentration (1+ or without infection) the patients were younger (57.9 ± 2.5 years vs 66.2 ± 1.4 years, respectively, P = 0.004) and the diffuse type of GC, poorly differentiated tumors (G3), T3-4 stage and N1 stage of GC were more often observed (Table 3). In cases of moderate and marked concentrations of H. pylori in GM, a decrease in 10-year progression-free survival (PFS) and OS survival from 55.6% to 26.3% was observed (P = 0.02 and P = 0.07, respectively). PFS and OS curves, depending on the concentration of coccoid forms of H. pylori in GM, are presented in Figure 5.\n\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\nClinical and pathological characteristics of gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa\nRUT: Rapid urease test; GC: Gastric cancer.\nThere were no correlations between the presence of point inclusions in the cytoplasm of epithelial cells of deep gastric glands, in the stroma immune cells, and in the intraepithelial lymphocytes with the clinical and pathological characteristics of GC.","A large amount of clinical and experimental data testifies to the important role of H. pylori in the occurrence of GC[31-34], but, there is less research into the features of H. pylori infection in patients with GC and its role in tumor progression, and the results are quite contradictory. These contradictions relate to many aspects, such as: (1) The frequency of infection in patients with GC. This data varies widely and ranges from 36% to 100%[35-40]; (2) The relation of infection with GC prognosis. Some researchers have noted an improvement in the long-term results of GC treatment in infected patients[41,42], while others, on the contrary, found that the presence of H. pylori infection was associated with a decrease in patient survival[43,44]; and (3) The connection between the infection and a histologic type of GC. In some studies, it was noted that patients with an intestinal type of GC are more often infected with H. pylori than patients with the diffuse type of GC[45,46]. Other researchers did not find a difference in H. pylori infection in patients with different histological types of tumors[47].\nIt is believed that the differences noted are associated with the fact that to reveal the infection the authors used the methods that were significantly different in their sensitivity, and primarily, to coccoid forms of bacteria. Most of the studies were carried out without considering coccoid forms of H. pylori and the concentration of bacteria in GM. The use of the biochemical method for the detection of urease activity and immunohistochemistry for visualization of bacteria in our study allowed us not only to assess the presence of infection in patients with GC, but also to mark some of its features associated with the localization of bacteria in the stomach, with a ratio of cocci and spiral forms, and the degree of bacterial contamination of GM.\nOur data for the Orenburg region recorded a high rate of H. pylori infection in patients with GC (84.5%). The coccoid forms of H. pylori, preserving a high degree of urease activity, dominated in GM in patients with GC. They were found in 93.4% of infected patients, with only coccoid forms of H. pylori-in 68.9%. \nIt is known that the coccoid forms of H. pylori can arise in response to unfavorable environmental factors, such as AT [47,48]. These forms are resistant to AT[49,50] and are able to form biofilms[51] and avoid the immune system[50]. They express a higher rate of cagE mRNA than their spiral counterparts[52], and by increasing the synthesis of tumor necrosis factor-alpha (TNF-α)-inducing protein (Hps), which is introduced into the cytosol and cell nuclei, they can activate nuclear factor-kappaB (NF-κB) and the expression of TNF-α and other cytokines involved in carcinogenesis[53,54]. The effect on the proliferation of gastric epithelial cells in the H. pylori coccoid forms is also stronger than in the helical forms[55], and they can induce the expression of VEGF-A and transforming growth factor-β[56]. The ability to transform into coccoid forms was also found to be characteristic of the most virulent strains of H. pylori[50,54].\nIt should be noted that the higher infection by coccoid forms of H. pylori in patients with GC, compared to patients with gastritis or gastric ulcer, had been mentioned by other researchers[57]. A number of studies have shown that coccoid forms of H. pylori retained urease activity[58] and the expression of such antigens as CagA, UreA, porin, components of the Cag type IV secretion system (TFSS), antigen-binding adhesin of the blood group BabA and others[59,60].\nThe use of immunohistochemistry in this study made it possible to detect the bacteria not only in the gastric mucus and on the surface of epithelial cells, but also within the cytoplasm of epithelial and immune cells of GM. Such intracellular expression was characterized by point inclusions giving a specific reaction with antibodies to H. pylori.\nThe intracellular persistence of H. pylori has been demonstrated by many investigators. They found H. pylori in the cytoplasm of epithelial cells, intercellular spaces, in the lamina propria of GM, and in the lumen of small vessels[61-63]. We assume that the point inclusions in the cytoplasm of epithelial and immune cells giving a positive reaction with antibodies to H. pylori is similar to those particle-rich cytoplasmic structure (PaCS) described earlier in the human superficial-foveolar epithelium and its metaplastic or dysplastic foci[64]. The authors found that the PaCS are a colocalization of VacA, CagA, urease, and outer membrane proteins with NOD1 receptor, ubiquitin-activating enzyme E1, polyubiquitinated proteins, proteasome components, and potentially oncogenic proteins like SHP2 and ERKs[64]. They believe that PaCS is a novel, proteasome-enriched structure arising in ribosome-rich cytoplasm at sites of H. pylori product accumulation. \nWe believe that the immune cells with point inclusions in the lamina propria of GM are likely to be macrophages. The data obtained by several researchers suggests this conclusion[65,66]. It is noted that even the absorbed bacteria retains their viability in macrophages, which may be associated with the violation of the phagosome maturation[66-68]. The use of confocal microscopy enabled the localization of the bacteria within the cells to be associated with the endosomal and lysosomal markers, and found that H. pylori could use the vesicles of autophagosomes (autophagic vesicles) for its own replication[63,69]. \nThe study found that the concentration of H. pylori coccoid forms in GM was the most significant clinical factor. This factor was associated with the tumor histology, T status, N status, stage, 10-year PFS, and OS. The moderate and marked concentrations of coccoid forms of H. pylori were more often found in the diffuse type of GC (P = 0.024) and T3-4 (P = 0.04) stage. Interestingly, the high concentration of H. pylori is more frequent in Stage N1 than in N2 (at 90.0% and 53.1%, respectively, P = 0.024).\nThe moderate and marked concentrations of coccoid forms of H. pylori represented a prognostic factor associated with the decrease of 10-year RFS and OS from 55.6% to 26.3% (P = 0.02 and P = 0.07, respectively).\nIt should be noted that the results of this study do not allow us to unambiguously judge the effect of H. pylori on GC progression. A decrease in OS and DFS in patients with moderate and marked concentrations of H. pylori coccoid forms in the GM may be due to the fact that these patients had more advanced stages and more aggressive forms of GC. Meanwhile, there are more and more studies showing that H. pylori infection can promote GC progression by activating the NF-κB signaling pathway and induction of interleukin-8 secretion[70], the activation of epithelial-mesenchymal transformation[71-74] and angiogenesis[75,76], as well as increasing the invasive properties of tumor cells[77]. It can be assumed that the administration of AT before surgery contributes to the reduction of the inflammatory process activity and normalization of the adhesive properties of tumour cells, which in turn decreases metastasis risk and improves the long-term results of the treatment of GC. The data literature on the improvement of the long-term results of malignant tumours treatment when using antibacterial drugs testify in favour of this hypothesis[78-80].","The data obtained indicates that H. pylori may be associated not only with induction but also with the progression of GC. It can be assumed that the prevalence of coccoid forms of bacteria and their intracellular persistence can affect the mechanisms of tumor progression. Further appropriate studies regarding the role of H. pylori in the progression of GC are obviously advisable."],"string":"[\n \"Gastric cancer (GC) continues to be one of the most common malignant diseases in the world[1,2]. Despite a decreasing trend in the incidence of GC in most countries of the world, the treatment results of this pathology cannot be considered satisfactory. In the structure of mortality from malignant neoplasms, this pathology firmly occupies 2nd place in most developed countries of the world, and the 5-year survival rate of radically operated patients does not exceed 15%-30%[3,4]. \\nIt is important to note that it is impossible to improve the long-term results of malignant neoplasms treatment without knowledge of the mechanisms associated with their progression[3]. Clinical studies in recent years indicate that inflammatory infiltration of the tumor stroma and surrounding tissues can have an important prognostic value and affect the long-term results of malignant neoplasm treatment[5-9]. A number of studies have shown that inflammatory infiltration of the tumor stroma is associated with the body’s adequate immune response to the tumor, and may be a favorable prognosis factor in various malignant neoplasms[10,11], including GC[12,13]. At the same time, the data obtained by other researchers indicates that pronounced inflammatory infiltration of the tumor stroma, especially T-reg lymphocytes and macrophages, may be a factor contributing to the progression of malignant neoplasms[14,15]. There was a decrease in the overall survival (OS) and relapse-free survival (RFS) of GC patients with a high content of Foxp3 + T-reg in the tumor stroma and regional metastases[16-18] and macrophages[16,19]. It is believed that inflammatory infiltration of the tumor stroma can contribute to tumor progression by activating the mechanisms of angiogenesis, expression of E- and L-selectins, formation of the products of lipid peroxidation and free radicals, destruction of connective tissue matrix and basement membranes of epithelia by proteolytic enzymes, and activation of epithelial-mesenchymal transformation[20-23].\\nWhen studying the role of inflammation in the progression of GC, it is impossible to ignore the problem of Helicobacter pylori (H. pylori) infection. H. pylori is a gram-negative, spiral-shaped bacterium, the habitat of which is the gastric mucosa (GM) and duodenum. H. pylori differs from other bacteria in a set of properties that make it possible to colonize the GM and persist for a long time under conditions that are unfavorable for other microorganisms[24,25]. These include: (1) The ability to produce a special enzyme-urease; (2) Synthesis of lytic enzymes that cause the depolymerization and dissolution of gastric mucus, consisting mainly of mucin; (3) The mobility of the bacterium, which is ensured by the presence of 5-6 flagella; (4) The high adhesiveness of bacteria to GM epithelial cells of the GM and elements of connective tissue due to the interaction of bacterial ligands with the corresponding cells receptors; (5) Production of various exotoxins (VacA, CagA, and others); (6) Instability of the H. pylori genome; (7) The presence of vegetative and coccoid forms of bacteria; and (8) possibility of intracellular persistence and translocation outside the GM [26-29].\\nIt should be noted that despite the huge number of studies devoted to H. pylori, it is still not clear whether H. pylori is involved only in the initiation of the tumor process in the stomach, or whether it can affect the mechanisms of tumor progression. The relationship between the severity of H. pylori infection and the clinical and morphological characteristics of GC and long-term results of this pathology treatment remains poorly studied, and in this connection, the question of the expediency of anti-Helicobacter therapy in patients with invasive GC remains open.\\nObjective To assess the features of H. pylori infection in patients with Stage I-IIIB GC and their correlation with the clinical and morphological characteristics of the disease, the presence of antibiotic therapy (AT) before surgery, and long-term treatment results.\\nTo assess the features of H. pylori infection in patients with Stage I-IIIB GC and their correlation with the clinical and morphological characteristics of the disease, the presence of antibiotic therapy (AT) before surgery, and long-term treatment results.\",\n \"To assess the features of H. pylori infection in patients with Stage I-IIIB GC and their correlation with the clinical and morphological characteristics of the disease, the presence of antibiotic therapy (AT) before surgery, and long-term treatment results.\",\n \"The patients One hundred and nine patients with GC who had undergone radical surgery (R0) between May 2007 and March 2010 at the Orenburg Regional Clinical Oncology Center, were included in this prospective cohort pilot study. Study inclusion criteria were: Histologically proven invasive GC; no evidence of distant metastases; radical surgery (R0); no prior gastric surgery; no previous chemotherapy or radiotherapy. The study did not include patients with decompensation of cardiovascular and renal diseases, exacerbation of chronic inflammatory processes, severe allergic processes, or who received glucocorticoids, antihistamines, and non-steroidal anti-inflammatory drugs. The study was performed in accordance with the Helsinki Declaration and internationally recognized guidelines, and the privacy of patients was protected by decoding the data according to the privacy regulations of the Orenburg Regional Clinical Oncologic Center (Russia, Orenburg). All patients provided written informed consent. The protocol was approved by the Institutional Review Board of the Orenburg State Medical University (Russia, Orenburg).\\nClinical and pathological data including age, tumor localization, stage, type of surgery, histology, the presence of AT before surgery, postoperative therapy, and long-term results of treatment were retrieved from the routine reports for analyses. The distribution of patients according to the clinical and pathological characteristics of GC is presented in Table 1.\\nThe distribution of patients according to the clinical and pathological characteristics of gastric cancer\\nGC: Gastric cancer.\\nWhen interviewing patients, it was found that 45 patients received AT before admission to the clinic due to a preliminary wrong diagnosis of gastric ulcer or chronic gastritis. The following combinations of antibacterial drugs were most commonly ordered: Amoxicillin + clarithromycin (34 patients), amoxicillin + clarithromycin + metronidazole (6 patients), amoxicillin + metronidazole (3 patients), other drugs (2 patients). Information about the antibacterial drugs, the timing and duration of their intake was entered into the primary patient documentation and then taken into account in the analysis. We considered only those patients who underwent AT in the period from 1 to 1.5 mo before the operation, lasting at least seven days, and using two or more antibacterial drugs.\\nThe long-term results of treatment were assessed for the period from May 12, 2007 to April 12, 2021. The median follow-up period was 86.2 mo. As of April 12, 2021, 26 (24.5%) patients were alive, 54 (50.9%) had died from the progression of GC, 20 (18.9%) had died from causes other than GC, and six (5.6%) left the region at different follow-up periods. Malignant tumors of other localizations were diagnosed in 8 patients at different times after the operation: Non-Hodgkin's lymphomas-in three, prostate cancer-in one, lung cancer-in two, laryngeal cancer-in one, and breast cancer-in one patient. With the exception of one patient with non-Hodgkin's lymphoma, the other patients died from the progression of these diseases. The causes of death were not associated with malignant tumors for the other patients. During the period 2020-2021, seven patients contracted coronavirus disease-19, one of whom died from the disease, but the rest are alive.\\nOne hundred and nine patients with GC who had undergone radical surgery (R0) between May 2007 and March 2010 at the Orenburg Regional Clinical Oncology Center, were included in this prospective cohort pilot study. Study inclusion criteria were: Histologically proven invasive GC; no evidence of distant metastases; radical surgery (R0); no prior gastric surgery; no previous chemotherapy or radiotherapy. The study did not include patients with decompensation of cardiovascular and renal diseases, exacerbation of chronic inflammatory processes, severe allergic processes, or who received glucocorticoids, antihistamines, and non-steroidal anti-inflammatory drugs. The study was performed in accordance with the Helsinki Declaration and internationally recognized guidelines, and the privacy of patients was protected by decoding the data according to the privacy regulations of the Orenburg Regional Clinical Oncologic Center (Russia, Orenburg). All patients provided written informed consent. The protocol was approved by the Institutional Review Board of the Orenburg State Medical University (Russia, Orenburg).\\nClinical and pathological data including age, tumor localization, stage, type of surgery, histology, the presence of AT before surgery, postoperative therapy, and long-term results of treatment were retrieved from the routine reports for analyses. The distribution of patients according to the clinical and pathological characteristics of GC is presented in Table 1.\\nThe distribution of patients according to the clinical and pathological characteristics of gastric cancer\\nGC: Gastric cancer.\\nWhen interviewing patients, it was found that 45 patients received AT before admission to the clinic due to a preliminary wrong diagnosis of gastric ulcer or chronic gastritis. The following combinations of antibacterial drugs were most commonly ordered: Amoxicillin + clarithromycin (34 patients), amoxicillin + clarithromycin + metronidazole (6 patients), amoxicillin + metronidazole (3 patients), other drugs (2 patients). Information about the antibacterial drugs, the timing and duration of their intake was entered into the primary patient documentation and then taken into account in the analysis. We considered only those patients who underwent AT in the period from 1 to 1.5 mo before the operation, lasting at least seven days, and using two or more antibacterial drugs.\\nThe long-term results of treatment were assessed for the period from May 12, 2007 to April 12, 2021. The median follow-up period was 86.2 mo. As of April 12, 2021, 26 (24.5%) patients were alive, 54 (50.9%) had died from the progression of GC, 20 (18.9%) had died from causes other than GC, and six (5.6%) left the region at different follow-up periods. Malignant tumors of other localizations were diagnosed in 8 patients at different times after the operation: Non-Hodgkin's lymphomas-in three, prostate cancer-in one, lung cancer-in two, laryngeal cancer-in one, and breast cancer-in one patient. With the exception of one patient with non-Hodgkin's lymphoma, the other patients died from the progression of these diseases. The causes of death were not associated with malignant tumors for the other patients. During the period 2020-2021, seven patients contracted coronavirus disease-19, one of whom died from the disease, but the rest are alive.\\nDetection H. pylori infection \\nH. pylori in the GM and tumor was determined by rapid urease test (RUT) and by immunohistochemically (IHC) using the antibody to H. pylori.\\n\\nH. pylori in the GM and tumor was determined by rapid urease test (RUT) and by immunohistochemically (IHC) using the antibody to H. pylori.\\nRUT After removal of the stomach (within 30 min) a greater curvature of the organ was opened and biopsy samples were taken from the tumor and the adjacent macroscopically non-tumorous GM at a distance of 3-5 cm from the tumor margin. The samples were placed on test strips (HELPIL–test, “АМА”, Russia) for three minutes. The presence and the severity of the infection were evaluated by the color change of the indicator from yellow to blue. \\nAccording to the intensity and the time of the appearance of a blue color, we distinguished three degrees of infection: 3+: Marked (+++)-bright staining in the first minute of the study; 2+: Moderate (++)–for an average intensity of staining for 2 min and 1+ mild (+)-weak staining for three minutes. If the color of the indicator did not change, or became dirty gray, and if after repeated research the same result was received, the test was evaluated as negative. The same samples were later used for histological analysis and IGH. \\nAfter removal of the stomach (within 30 min) a greater curvature of the organ was opened and biopsy samples were taken from the tumor and the adjacent macroscopically non-tumorous GM at a distance of 3-5 cm from the tumor margin. The samples were placed on test strips (HELPIL–test, “АМА”, Russia) for three minutes. The presence and the severity of the infection were evaluated by the color change of the indicator from yellow to blue. \\nAccording to the intensity and the time of the appearance of a blue color, we distinguished three degrees of infection: 3+: Marked (+++)-bright staining in the first minute of the study; 2+: Moderate (++)–for an average intensity of staining for 2 min and 1+ mild (+)-weak staining for three minutes. If the color of the indicator did not change, or became dirty gray, and if after repeated research the same result was received, the test was evaluated as negative. The same samples were later used for histological analysis and IGH. \\nImmunohistochemistry The presence and features of H. pylori infection were studied in samples of the GM adjacent to the tumor, in the tumor tissue, in the omentum, and regional lymph nodes of 46 patients, by immunohistochemistry.\\nThe sections for IGH were dewaxed and rehydrated by sequential immersion in xylene and graded ethanol and water. For antigen retrieval, the sections boiling for 10 min in citrate buffer (pH 6) and endogenous peroxidase activity was blocked with 30 mL/L hydrogen peroxide solution. Slides were incubated at room temperature with the anti-H. pylori (RB-9070, Thermo Fisher Scientific, the immunogen is purified H. pylori) rabbit polyclonal antibodies in diluted at 1:1000 for 30 min. \\nThe visualization system included DAB (UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen) and hematoxylin counterstaining. For negative control sections, the primary antibody was replaced with phosphate-buffered saline and processed in the same manner.\\nThe concentration of H. pylori in the GM detected by IGH was graded as 1+ for mild, 2+ for moderate, and 3+ for marked according to the Sydney system[30]. The presence of point inclusions giving a positive reaction with antibodies to H. pylori in the cytoplasm of epithelial cells of deep gastric glands and of the lymphoid cells of the lamina propria of GM as well as in omentum and lymph node, were taken into account. \\nAll sections were carefully and completely scanned by two of the authors (MS and OT) without knowledge of the clinical and pathological data. \\nThe data obtained was compared with clinical features of GC: Stage, localization, histology, the presence of AT before surgery, and 10-year OS and RFS.\\nThe presence and features of H. pylori infection were studied in samples of the GM adjacent to the tumor, in the tumor tissue, in the omentum, and regional lymph nodes of 46 patients, by immunohistochemistry.\\nThe sections for IGH were dewaxed and rehydrated by sequential immersion in xylene and graded ethanol and water. For antigen retrieval, the sections boiling for 10 min in citrate buffer (pH 6) and endogenous peroxidase activity was blocked with 30 mL/L hydrogen peroxide solution. Slides were incubated at room temperature with the anti-H. pylori (RB-9070, Thermo Fisher Scientific, the immunogen is purified H. pylori) rabbit polyclonal antibodies in diluted at 1:1000 for 30 min. \\nThe visualization system included DAB (UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen) and hematoxylin counterstaining. For negative control sections, the primary antibody was replaced with phosphate-buffered saline and processed in the same manner.\\nThe concentration of H. pylori in the GM detected by IGH was graded as 1+ for mild, 2+ for moderate, and 3+ for marked according to the Sydney system[30]. The presence of point inclusions giving a positive reaction with antibodies to H. pylori in the cytoplasm of epithelial cells of deep gastric glands and of the lymphoid cells of the lamina propria of GM as well as in omentum and lymph node, were taken into account. \\nAll sections were carefully and completely scanned by two of the authors (MS and OT) without knowledge of the clinical and pathological data. \\nThe data obtained was compared with clinical features of GC: Stage, localization, histology, the presence of AT before surgery, and 10-year OS and RFS.\\nStatistics Statistical analysis was performed using the Statistica 10.0 software. The correlations between different data were evaluated using the nonparametric Spearman's rank correlation or gamma correlation. Chi-square tests were carried out to analyze the difference of distribution among the categorized data. Mann–Whitney U nonparametric test was used to compare the value of the quantitative data. The survival was analyzed using the Kaplan-Meier method. The log-rank test was used to compare survival curves between subgroups of patients. A value of P < 0.05 was considered statistically significant.\\nStatistical analysis was performed using the Statistica 10.0 software. The correlations between different data were evaluated using the nonparametric Spearman's rank correlation or gamma correlation. Chi-square tests were carried out to analyze the difference of distribution among the categorized data. Mann–Whitney U nonparametric test was used to compare the value of the quantitative data. The survival was analyzed using the Kaplan-Meier method. The log-rank test was used to compare survival curves between subgroups of patients. A value of P < 0.05 was considered statistically significant.\",\n \"One hundred and nine patients with GC who had undergone radical surgery (R0) between May 2007 and March 2010 at the Orenburg Regional Clinical Oncology Center, were included in this prospective cohort pilot study. Study inclusion criteria were: Histologically proven invasive GC; no evidence of distant metastases; radical surgery (R0); no prior gastric surgery; no previous chemotherapy or radiotherapy. The study did not include patients with decompensation of cardiovascular and renal diseases, exacerbation of chronic inflammatory processes, severe allergic processes, or who received glucocorticoids, antihistamines, and non-steroidal anti-inflammatory drugs. The study was performed in accordance with the Helsinki Declaration and internationally recognized guidelines, and the privacy of patients was protected by decoding the data according to the privacy regulations of the Orenburg Regional Clinical Oncologic Center (Russia, Orenburg). All patients provided written informed consent. The protocol was approved by the Institutional Review Board of the Orenburg State Medical University (Russia, Orenburg).\\nClinical and pathological data including age, tumor localization, stage, type of surgery, histology, the presence of AT before surgery, postoperative therapy, and long-term results of treatment were retrieved from the routine reports for analyses. The distribution of patients according to the clinical and pathological characteristics of GC is presented in Table 1.\\nThe distribution of patients according to the clinical and pathological characteristics of gastric cancer\\nGC: Gastric cancer.\\nWhen interviewing patients, it was found that 45 patients received AT before admission to the clinic due to a preliminary wrong diagnosis of gastric ulcer or chronic gastritis. The following combinations of antibacterial drugs were most commonly ordered: Amoxicillin + clarithromycin (34 patients), amoxicillin + clarithromycin + metronidazole (6 patients), amoxicillin + metronidazole (3 patients), other drugs (2 patients). Information about the antibacterial drugs, the timing and duration of their intake was entered into the primary patient documentation and then taken into account in the analysis. We considered only those patients who underwent AT in the period from 1 to 1.5 mo before the operation, lasting at least seven days, and using two or more antibacterial drugs.\\nThe long-term results of treatment were assessed for the period from May 12, 2007 to April 12, 2021. The median follow-up period was 86.2 mo. As of April 12, 2021, 26 (24.5%) patients were alive, 54 (50.9%) had died from the progression of GC, 20 (18.9%) had died from causes other than GC, and six (5.6%) left the region at different follow-up periods. Malignant tumors of other localizations were diagnosed in 8 patients at different times after the operation: Non-Hodgkin's lymphomas-in three, prostate cancer-in one, lung cancer-in two, laryngeal cancer-in one, and breast cancer-in one patient. With the exception of one patient with non-Hodgkin's lymphoma, the other patients died from the progression of these diseases. The causes of death were not associated with malignant tumors for the other patients. During the period 2020-2021, seven patients contracted coronavirus disease-19, one of whom died from the disease, but the rest are alive.\",\n \"\\nH. pylori in the GM and tumor was determined by rapid urease test (RUT) and by immunohistochemically (IHC) using the antibody to H. pylori.\",\n \"After removal of the stomach (within 30 min) a greater curvature of the organ was opened and biopsy samples were taken from the tumor and the adjacent macroscopically non-tumorous GM at a distance of 3-5 cm from the tumor margin. The samples were placed on test strips (HELPIL–test, “АМА”, Russia) for three minutes. The presence and the severity of the infection were evaluated by the color change of the indicator from yellow to blue. \\nAccording to the intensity and the time of the appearance of a blue color, we distinguished three degrees of infection: 3+: Marked (+++)-bright staining in the first minute of the study; 2+: Moderate (++)–for an average intensity of staining for 2 min and 1+ mild (+)-weak staining for three minutes. If the color of the indicator did not change, or became dirty gray, and if after repeated research the same result was received, the test was evaluated as negative. The same samples were later used for histological analysis and IGH. \",\n \"The presence and features of H. pylori infection were studied in samples of the GM adjacent to the tumor, in the tumor tissue, in the omentum, and regional lymph nodes of 46 patients, by immunohistochemistry.\\nThe sections for IGH were dewaxed and rehydrated by sequential immersion in xylene and graded ethanol and water. For antigen retrieval, the sections boiling for 10 min in citrate buffer (pH 6) and endogenous peroxidase activity was blocked with 30 mL/L hydrogen peroxide solution. Slides were incubated at room temperature with the anti-H. pylori (RB-9070, Thermo Fisher Scientific, the immunogen is purified H. pylori) rabbit polyclonal antibodies in diluted at 1:1000 for 30 min. \\nThe visualization system included DAB (UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen) and hematoxylin counterstaining. For negative control sections, the primary antibody was replaced with phosphate-buffered saline and processed in the same manner.\\nThe concentration of H. pylori in the GM detected by IGH was graded as 1+ for mild, 2+ for moderate, and 3+ for marked according to the Sydney system[30]. The presence of point inclusions giving a positive reaction with antibodies to H. pylori in the cytoplasm of epithelial cells of deep gastric glands and of the lymphoid cells of the lamina propria of GM as well as in omentum and lymph node, were taken into account. \\nAll sections were carefully and completely scanned by two of the authors (MS and OT) without knowledge of the clinical and pathological data. \\nThe data obtained was compared with clinical features of GC: Stage, localization, histology, the presence of AT before surgery, and 10-year OS and RFS.\",\n \"Statistical analysis was performed using the Statistica 10.0 software. The correlations between different data were evaluated using the nonparametric Spearman's rank correlation or gamma correlation. Chi-square tests were carried out to analyze the difference of distribution among the categorized data. Mann–Whitney U nonparametric test was used to compare the value of the quantitative data. The survival was analyzed using the Kaplan-Meier method. The log-rank test was used to compare survival curves between subgroups of patients. A value of P < 0.05 was considered statistically significant.\",\n \"The features of H. pylori infection in GC Of 93 patients (84.5%) demonstrated positive RUT. According to RUT the concentration of H. pylori in GM was mild (1+) in 38 (34.8%) patients, moderate (2+)-in 37 (33.9%) and marked (3+)–in 8 (16.5%). RUT was negative in 16 patients (14.7%).\\nIt was found that urease activity 2+ and 3+ were significantly more frequent in GM than in tumors (in 55.5% and 13.3% of cases, respectively, P = 0.01). In more than half the cases (53.3%), the urease activity in the tumor was lower than in the adjacent GM.\\nForty-six samples of GM were stained by IHC. The coccoid forms of H. pylori were found to prevail in GM adjacent to the tumor (Figure 1A). Coccoid forms of H. pylori only were identified in 31 (63.1%) patients, and coccoid and spiral in 12 (30.4%) patients (Figure 1B). No signs of infection were found in 3 (6.5%) patients. The concentration of coccoid and spiral forms of H. pylori in GM according to IGH was 1+ in 24 (52.2%) and 1 (19.6%) patients, 2+-in 11 (23.9%) and 4 (8.7%), and 3+-in 8 (17.4%) and 1 (2.2%) patients. A positive correlation between the concentration of coccoid and spiral forms of H. pylori (gamma = 0.642, P < 0.0001) was noted.\\n\\nThe features of Helicobacter pylori localization in gastric mucosa in patients with gastric cancer. A: Coccoid forms of Helicobacter pylori (H. pylori) in the gastric pit. The some bacteria within the cytoplasm of epithelium cells (arrows); B: The spiral (black arrows) and coccoid (orange arrows) forms of H. pylori on the surface of superficial-foveolar gastric epithelium; C: The bacteria in the surface mucous gel layer of stomach (arrows); D: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of epithelial cells of deep gastric glands (arrows); E: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of the immune cells of the lamina propria of gastric mucosa (arrows); F: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of intraepithelial lymphocytes (arrows). Immunoperoxidase staining with anti-H. pylori antibody, immersion. Bars: A: 20 μm; B: 10 μm; C: 20 μm; D-F: 10 μm. \\nThe localization of H. pylori in GM was observed not only in the surface mucous gel layer of the stomach (Figure 1C) but also within the cytoplasm of the gastric superficial-foveolar epithelium (Figure 1D). The point inclusions giving a positive reaction with antibodies to H. pylori were also revealed in the cytoplasm of the epithelial cells of deep gastric glands (in 41.3% samples) and of the immune cells (Figure 1E) of the lamina propria of GM (in 43.5% samples), as well as of the intraepithelial lymphocytes (Figure 1F). The close relationship between the presence of point inclusions in immune cells and epithelial cells (gamma = 0.642, P < 0.0001) was noted. The individual cocci or their small clusters, and sometimes the short rod bacterium, were also often detected in the lamina propria of the GM. We also found bacteria in the omentum and lymph nodes. In the omentum the bacteria were presented predominantly by cocci between 0.5 and 1 μm in diameter (Figure 2A). Cocci were arranged most commonly by the small compact groups up to 10-15 cells in the immediate vicinity of the LN capsule. The bacteria were located mainly between the adipocytes. However, it was not clear whether bacteria were outside of the cells or in a narrow rim of cytoplasm of the fat cells. \\n\\nThe features of Helicobacter pylori localization in omentum and lymph node in patients with gastric cancer. A: The small group of cocci located in the central part of the omentum adipocyte; B: The congestions of bacteria around the nucleus of lymphocytes in the paracortical area of lymph node. Immunoperoxidase staining with anti-Helicobacter pylori antibody, immersion, Bars: 10 μm. \\nIn the tissue of lymph nodes we usually observed the small accumulations of cocci between 10 and 15 cells (Figure 2B). Bacteria were located between lymphocytes of the paracortical area, but not so compact, as in the omentum. Quite often the concentration of bacteria was observed around the nuclei of cells that can testify to their intracellular localization.\\nOf 93 patients (84.5%) demonstrated positive RUT. According to RUT the concentration of H. pylori in GM was mild (1+) in 38 (34.8%) patients, moderate (2+)-in 37 (33.9%) and marked (3+)–in 8 (16.5%). RUT was negative in 16 patients (14.7%).\\nIt was found that urease activity 2+ and 3+ were significantly more frequent in GM than in tumors (in 55.5% and 13.3% of cases, respectively, P = 0.01). In more than half the cases (53.3%), the urease activity in the tumor was lower than in the adjacent GM.\\nForty-six samples of GM were stained by IHC. The coccoid forms of H. pylori were found to prevail in GM adjacent to the tumor (Figure 1A). Coccoid forms of H. pylori only were identified in 31 (63.1%) patients, and coccoid and spiral in 12 (30.4%) patients (Figure 1B). No signs of infection were found in 3 (6.5%) patients. The concentration of coccoid and spiral forms of H. pylori in GM according to IGH was 1+ in 24 (52.2%) and 1 (19.6%) patients, 2+-in 11 (23.9%) and 4 (8.7%), and 3+-in 8 (17.4%) and 1 (2.2%) patients. A positive correlation between the concentration of coccoid and spiral forms of H. pylori (gamma = 0.642, P < 0.0001) was noted.\\n\\nThe features of Helicobacter pylori localization in gastric mucosa in patients with gastric cancer. A: Coccoid forms of Helicobacter pylori (H. pylori) in the gastric pit. The some bacteria within the cytoplasm of epithelium cells (arrows); B: The spiral (black arrows) and coccoid (orange arrows) forms of H. pylori on the surface of superficial-foveolar gastric epithelium; C: The bacteria in the surface mucous gel layer of stomach (arrows); D: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of epithelial cells of deep gastric glands (arrows); E: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of the immune cells of the lamina propria of gastric mucosa (arrows); F: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of intraepithelial lymphocytes (arrows). Immunoperoxidase staining with anti-H. pylori antibody, immersion. Bars: A: 20 μm; B: 10 μm; C: 20 μm; D-F: 10 μm. \\nThe localization of H. pylori in GM was observed not only in the surface mucous gel layer of the stomach (Figure 1C) but also within the cytoplasm of the gastric superficial-foveolar epithelium (Figure 1D). The point inclusions giving a positive reaction with antibodies to H. pylori were also revealed in the cytoplasm of the epithelial cells of deep gastric glands (in 41.3% samples) and of the immune cells (Figure 1E) of the lamina propria of GM (in 43.5% samples), as well as of the intraepithelial lymphocytes (Figure 1F). The close relationship between the presence of point inclusions in immune cells and epithelial cells (gamma = 0.642, P < 0.0001) was noted. The individual cocci or their small clusters, and sometimes the short rod bacterium, were also often detected in the lamina propria of the GM. We also found bacteria in the omentum and lymph nodes. In the omentum the bacteria were presented predominantly by cocci between 0.5 and 1 μm in diameter (Figure 2A). Cocci were arranged most commonly by the small compact groups up to 10-15 cells in the immediate vicinity of the LN capsule. The bacteria were located mainly between the adipocytes. However, it was not clear whether bacteria were outside of the cells or in a narrow rim of cytoplasm of the fat cells. \\n\\nThe features of Helicobacter pylori localization in omentum and lymph node in patients with gastric cancer. A: The small group of cocci located in the central part of the omentum adipocyte; B: The congestions of bacteria around the nucleus of lymphocytes in the paracortical area of lymph node. Immunoperoxidase staining with anti-Helicobacter pylori antibody, immersion, Bars: 10 μm. \\nIn the tissue of lymph nodes we usually observed the small accumulations of cocci between 10 and 15 cells (Figure 2B). Bacteria were located between lymphocytes of the paracortical area, but not so compact, as in the omentum. Quite often the concentration of bacteria was observed around the nuclei of cells that can testify to their intracellular localization.\\nCorrelations of clinical and pathological characteristics of GC with the severity of H. pylori infection according to RUT data The gamma correlation coefficient test (gamma) showed that the severity of H. pylori in GM according to RUT positively correlated with the T status (gamma = 0.537, P < 0.00001), N status (gamma = 0.371, P = 0.0007) and stage (gamma = 0.520, P < 0.00001), and negatively correlated with the presence of AT in anamnesis (gamma = -0.418, P = 0.003). The marked (+++) and moderate (++) degrees of H. pylori infection were more often observed in Grade 2 and Grade 3, in T3-4 status, in N1 status, in the T3-4N1-2 stage, and in the absence of AT in anamnesis (Table 2). Correlations of H. pylori concentration in GM according to RUT with 10-year OS and RFS of GC patients were not determined. \\nClinical and pathological characteristics of gastric cancer depending on the data rapid urease test\\nRUT: Rapid urease test; GC: Gastric cancer.\\nIt is important to note that the presence in AT 1-1.5 mo before surgery was associated with a significant improvement in RFS and OS (Figure 3), however, this applied only to patients with local GC (T1-3N0). In advanced GC (T3-4N1 and T3-4N2) there were no significant differences in patient survival (Figure 4).\\n\\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\\n\\n10-year overall surviving and relapse-free surviving of patients with T3-4N1-2M0 stages of gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.78). A: 10-year overall surviving; B: Relapse-free surviving.\\nThe gamma correlation coefficient test (gamma) showed that the severity of H. pylori in GM according to RUT positively correlated with the T status (gamma = 0.537, P < 0.00001), N status (gamma = 0.371, P = 0.0007) and stage (gamma = 0.520, P < 0.00001), and negatively correlated with the presence of AT in anamnesis (gamma = -0.418, P = 0.003). The marked (+++) and moderate (++) degrees of H. pylori infection were more often observed in Grade 2 and Grade 3, in T3-4 status, in N1 status, in the T3-4N1-2 stage, and in the absence of AT in anamnesis (Table 2). Correlations of H. pylori concentration in GM according to RUT with 10-year OS and RFS of GC patients were not determined. \\nClinical and pathological characteristics of gastric cancer depending on the data rapid urease test\\nRUT: Rapid urease test; GC: Gastric cancer.\\nIt is important to note that the presence in AT 1-1.5 mo before surgery was associated with a significant improvement in RFS and OS (Figure 3), however, this applied only to patients with local GC (T1-3N0). In advanced GC (T3-4N1 and T3-4N2) there were no significant differences in patient survival (Figure 4).\\n\\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\\n\\n10-year overall surviving and relapse-free surviving of patients with T3-4N1-2M0 stages of gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.78). A: 10-year overall surviving; B: Relapse-free surviving.\\nCorrelations of clinical and pathological characteristics of GC with the concentration of spiral and coccoid forms of H. pylori in the GM It is important to note that correlations between the clinical and pathological characteristics of GC and the concentration of H. pylori spiral forms in GM were not found. However, the concentration of H. pylori coccoid forms correlated with age (ρ = -0.502, P = 0.0006), histology (gamma = 0.550, P = 0.0004), T status (gamma = 0.709, P = 0.0001), N status (gamma = 0.509, P = 0.002) stage (gamma = 0.636, P = 0.0002), and 10-year RFS (gamma = -0.521, P = 0.008) and OS (gamma = -0.500, P = 0.044). In cases with a moderate and marked concentration (2+ or 3+) of H. pylori coccoid forms in GM compared to cases with a low concentration (1+ or without infection) the patients were younger (57.9 ± 2.5 years vs 66.2 ± 1.4 years, respectively, P = 0.004) and the diffuse type of GC, poorly differentiated tumors (G3), T3-4 stage and N1 stage of GC were more often observed (Table 3). In cases of moderate and marked concentrations of H. pylori in GM, a decrease in 10-year progression-free survival (PFS) and OS survival from 55.6% to 26.3% was observed (P = 0.02 and P = 0.07, respectively). PFS and OS curves, depending on the concentration of coccoid forms of H. pylori in GM, are presented in Figure 5.\\n\\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\\nClinical and pathological characteristics of gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa\\nRUT: Rapid urease test; GC: Gastric cancer.\\nThere were no correlations between the presence of point inclusions in the cytoplasm of epithelial cells of deep gastric glands, in the stroma immune cells, and in the intraepithelial lymphocytes with the clinical and pathological characteristics of GC.\\nIt is important to note that correlations between the clinical and pathological characteristics of GC and the concentration of H. pylori spiral forms in GM were not found. However, the concentration of H. pylori coccoid forms correlated with age (ρ = -0.502, P = 0.0006), histology (gamma = 0.550, P = 0.0004), T status (gamma = 0.709, P = 0.0001), N status (gamma = 0.509, P = 0.002) stage (gamma = 0.636, P = 0.0002), and 10-year RFS (gamma = -0.521, P = 0.008) and OS (gamma = -0.500, P = 0.044). In cases with a moderate and marked concentration (2+ or 3+) of H. pylori coccoid forms in GM compared to cases with a low concentration (1+ or without infection) the patients were younger (57.9 ± 2.5 years vs 66.2 ± 1.4 years, respectively, P = 0.004) and the diffuse type of GC, poorly differentiated tumors (G3), T3-4 stage and N1 stage of GC were more often observed (Table 3). In cases of moderate and marked concentrations of H. pylori in GM, a decrease in 10-year progression-free survival (PFS) and OS survival from 55.6% to 26.3% was observed (P = 0.02 and P = 0.07, respectively). PFS and OS curves, depending on the concentration of coccoid forms of H. pylori in GM, are presented in Figure 5.\\n\\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\\nClinical and pathological characteristics of gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa\\nRUT: Rapid urease test; GC: Gastric cancer.\\nThere were no correlations between the presence of point inclusions in the cytoplasm of epithelial cells of deep gastric glands, in the stroma immune cells, and in the intraepithelial lymphocytes with the clinical and pathological characteristics of GC.\",\n \"Of 93 patients (84.5%) demonstrated positive RUT. According to RUT the concentration of H. pylori in GM was mild (1+) in 38 (34.8%) patients, moderate (2+)-in 37 (33.9%) and marked (3+)–in 8 (16.5%). RUT was negative in 16 patients (14.7%).\\nIt was found that urease activity 2+ and 3+ were significantly more frequent in GM than in tumors (in 55.5% and 13.3% of cases, respectively, P = 0.01). In more than half the cases (53.3%), the urease activity in the tumor was lower than in the adjacent GM.\\nForty-six samples of GM were stained by IHC. The coccoid forms of H. pylori were found to prevail in GM adjacent to the tumor (Figure 1A). Coccoid forms of H. pylori only were identified in 31 (63.1%) patients, and coccoid and spiral in 12 (30.4%) patients (Figure 1B). No signs of infection were found in 3 (6.5%) patients. The concentration of coccoid and spiral forms of H. pylori in GM according to IGH was 1+ in 24 (52.2%) and 1 (19.6%) patients, 2+-in 11 (23.9%) and 4 (8.7%), and 3+-in 8 (17.4%) and 1 (2.2%) patients. A positive correlation between the concentration of coccoid and spiral forms of H. pylori (gamma = 0.642, P < 0.0001) was noted.\\n\\nThe features of Helicobacter pylori localization in gastric mucosa in patients with gastric cancer. A: Coccoid forms of Helicobacter pylori (H. pylori) in the gastric pit. The some bacteria within the cytoplasm of epithelium cells (arrows); B: The spiral (black arrows) and coccoid (orange arrows) forms of H. pylori on the surface of superficial-foveolar gastric epithelium; C: The bacteria in the surface mucous gel layer of stomach (arrows); D: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of epithelial cells of deep gastric glands (arrows); E: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of the immune cells of the lamina propria of gastric mucosa (arrows); F: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of intraepithelial lymphocytes (arrows). Immunoperoxidase staining with anti-H. pylori antibody, immersion. Bars: A: 20 μm; B: 10 μm; C: 20 μm; D-F: 10 μm. \\nThe localization of H. pylori in GM was observed not only in the surface mucous gel layer of the stomach (Figure 1C) but also within the cytoplasm of the gastric superficial-foveolar epithelium (Figure 1D). The point inclusions giving a positive reaction with antibodies to H. pylori were also revealed in the cytoplasm of the epithelial cells of deep gastric glands (in 41.3% samples) and of the immune cells (Figure 1E) of the lamina propria of GM (in 43.5% samples), as well as of the intraepithelial lymphocytes (Figure 1F). The close relationship between the presence of point inclusions in immune cells and epithelial cells (gamma = 0.642, P < 0.0001) was noted. The individual cocci or their small clusters, and sometimes the short rod bacterium, were also often detected in the lamina propria of the GM. We also found bacteria in the omentum and lymph nodes. In the omentum the bacteria were presented predominantly by cocci between 0.5 and 1 μm in diameter (Figure 2A). Cocci were arranged most commonly by the small compact groups up to 10-15 cells in the immediate vicinity of the LN capsule. The bacteria were located mainly between the adipocytes. However, it was not clear whether bacteria were outside of the cells or in a narrow rim of cytoplasm of the fat cells. \\n\\nThe features of Helicobacter pylori localization in omentum and lymph node in patients with gastric cancer. A: The small group of cocci located in the central part of the omentum adipocyte; B: The congestions of bacteria around the nucleus of lymphocytes in the paracortical area of lymph node. Immunoperoxidase staining with anti-Helicobacter pylori antibody, immersion, Bars: 10 μm. \\nIn the tissue of lymph nodes we usually observed the small accumulations of cocci between 10 and 15 cells (Figure 2B). Bacteria were located between lymphocytes of the paracortical area, but not so compact, as in the omentum. Quite often the concentration of bacteria was observed around the nuclei of cells that can testify to their intracellular localization.\",\n \"The gamma correlation coefficient test (gamma) showed that the severity of H. pylori in GM according to RUT positively correlated with the T status (gamma = 0.537, P < 0.00001), N status (gamma = 0.371, P = 0.0007) and stage (gamma = 0.520, P < 0.00001), and negatively correlated with the presence of AT in anamnesis (gamma = -0.418, P = 0.003). The marked (+++) and moderate (++) degrees of H. pylori infection were more often observed in Grade 2 and Grade 3, in T3-4 status, in N1 status, in the T3-4N1-2 stage, and in the absence of AT in anamnesis (Table 2). Correlations of H. pylori concentration in GM according to RUT with 10-year OS and RFS of GC patients were not determined. \\nClinical and pathological characteristics of gastric cancer depending on the data rapid urease test\\nRUT: Rapid urease test; GC: Gastric cancer.\\nIt is important to note that the presence in AT 1-1.5 mo before surgery was associated with a significant improvement in RFS and OS (Figure 3), however, this applied only to patients with local GC (T1-3N0). In advanced GC (T3-4N1 and T3-4N2) there were no significant differences in patient survival (Figure 4).\\n\\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\\n\\n10-year overall surviving and relapse-free surviving of patients with T3-4N1-2M0 stages of gastric cancer depending on the presence of antibiotic therapy 1-1.5 mo before surgery (P = 0.78). A: 10-year overall surviving; B: Relapse-free surviving.\",\n \"It is important to note that correlations between the clinical and pathological characteristics of GC and the concentration of H. pylori spiral forms in GM were not found. However, the concentration of H. pylori coccoid forms correlated with age (ρ = -0.502, P = 0.0006), histology (gamma = 0.550, P = 0.0004), T status (gamma = 0.709, P = 0.0001), N status (gamma = 0.509, P = 0.002) stage (gamma = 0.636, P = 0.0002), and 10-year RFS (gamma = -0.521, P = 0.008) and OS (gamma = -0.500, P = 0.044). In cases with a moderate and marked concentration (2+ or 3+) of H. pylori coccoid forms in GM compared to cases with a low concentration (1+ or without infection) the patients were younger (57.9 ± 2.5 years vs 66.2 ± 1.4 years, respectively, P = 0.004) and the diffuse type of GC, poorly differentiated tumors (G3), T3-4 stage and N1 stage of GC were more often observed (Table 3). In cases of moderate and marked concentrations of H. pylori in GM, a decrease in 10-year progression-free survival (PFS) and OS survival from 55.6% to 26.3% was observed (P = 0.02 and P = 0.07, respectively). PFS and OS curves, depending on the concentration of coccoid forms of H. pylori in GM, are presented in Figure 5.\\n\\n10-year overall surviving and relapse-free surviving of patients with gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa (P = 0.02). A: 10-year overall surviving; B: Relapse-free surviving.\\nClinical and pathological characteristics of gastric cancer depending on the concentration of Helicobacter pylori coccoid forms in the gastric mucosa\\nRUT: Rapid urease test; GC: Gastric cancer.\\nThere were no correlations between the presence of point inclusions in the cytoplasm of epithelial cells of deep gastric glands, in the stroma immune cells, and in the intraepithelial lymphocytes with the clinical and pathological characteristics of GC.\",\n \"A large amount of clinical and experimental data testifies to the important role of H. pylori in the occurrence of GC[31-34], but, there is less research into the features of H. pylori infection in patients with GC and its role in tumor progression, and the results are quite contradictory. These contradictions relate to many aspects, such as: (1) The frequency of infection in patients with GC. This data varies widely and ranges from 36% to 100%[35-40]; (2) The relation of infection with GC prognosis. Some researchers have noted an improvement in the long-term results of GC treatment in infected patients[41,42], while others, on the contrary, found that the presence of H. pylori infection was associated with a decrease in patient survival[43,44]; and (3) The connection between the infection and a histologic type of GC. In some studies, it was noted that patients with an intestinal type of GC are more often infected with H. pylori than patients with the diffuse type of GC[45,46]. Other researchers did not find a difference in H. pylori infection in patients with different histological types of tumors[47].\\nIt is believed that the differences noted are associated with the fact that to reveal the infection the authors used the methods that were significantly different in their sensitivity, and primarily, to coccoid forms of bacteria. Most of the studies were carried out without considering coccoid forms of H. pylori and the concentration of bacteria in GM. The use of the biochemical method for the detection of urease activity and immunohistochemistry for visualization of bacteria in our study allowed us not only to assess the presence of infection in patients with GC, but also to mark some of its features associated with the localization of bacteria in the stomach, with a ratio of cocci and spiral forms, and the degree of bacterial contamination of GM.\\nOur data for the Orenburg region recorded a high rate of H. pylori infection in patients with GC (84.5%). The coccoid forms of H. pylori, preserving a high degree of urease activity, dominated in GM in patients with GC. They were found in 93.4% of infected patients, with only coccoid forms of H. pylori-in 68.9%. \\nIt is known that the coccoid forms of H. pylori can arise in response to unfavorable environmental factors, such as AT [47,48]. These forms are resistant to AT[49,50] and are able to form biofilms[51] and avoid the immune system[50]. They express a higher rate of cagE mRNA than their spiral counterparts[52], and by increasing the synthesis of tumor necrosis factor-alpha (TNF-α)-inducing protein (Hps), which is introduced into the cytosol and cell nuclei, they can activate nuclear factor-kappaB (NF-κB) and the expression of TNF-α and other cytokines involved in carcinogenesis[53,54]. The effect on the proliferation of gastric epithelial cells in the H. pylori coccoid forms is also stronger than in the helical forms[55], and they can induce the expression of VEGF-A and transforming growth factor-β[56]. The ability to transform into coccoid forms was also found to be characteristic of the most virulent strains of H. pylori[50,54].\\nIt should be noted that the higher infection by coccoid forms of H. pylori in patients with GC, compared to patients with gastritis or gastric ulcer, had been mentioned by other researchers[57]. A number of studies have shown that coccoid forms of H. pylori retained urease activity[58] and the expression of such antigens as CagA, UreA, porin, components of the Cag type IV secretion system (TFSS), antigen-binding adhesin of the blood group BabA and others[59,60].\\nThe use of immunohistochemistry in this study made it possible to detect the bacteria not only in the gastric mucus and on the surface of epithelial cells, but also within the cytoplasm of epithelial and immune cells of GM. Such intracellular expression was characterized by point inclusions giving a specific reaction with antibodies to H. pylori.\\nThe intracellular persistence of H. pylori has been demonstrated by many investigators. They found H. pylori in the cytoplasm of epithelial cells, intercellular spaces, in the lamina propria of GM, and in the lumen of small vessels[61-63]. We assume that the point inclusions in the cytoplasm of epithelial and immune cells giving a positive reaction with antibodies to H. pylori is similar to those particle-rich cytoplasmic structure (PaCS) described earlier in the human superficial-foveolar epithelium and its metaplastic or dysplastic foci[64]. The authors found that the PaCS are a colocalization of VacA, CagA, urease, and outer membrane proteins with NOD1 receptor, ubiquitin-activating enzyme E1, polyubiquitinated proteins, proteasome components, and potentially oncogenic proteins like SHP2 and ERKs[64]. They believe that PaCS is a novel, proteasome-enriched structure arising in ribosome-rich cytoplasm at sites of H. pylori product accumulation. \\nWe believe that the immune cells with point inclusions in the lamina propria of GM are likely to be macrophages. The data obtained by several researchers suggests this conclusion[65,66]. It is noted that even the absorbed bacteria retains their viability in macrophages, which may be associated with the violation of the phagosome maturation[66-68]. The use of confocal microscopy enabled the localization of the bacteria within the cells to be associated with the endosomal and lysosomal markers, and found that H. pylori could use the vesicles of autophagosomes (autophagic vesicles) for its own replication[63,69]. \\nThe study found that the concentration of H. pylori coccoid forms in GM was the most significant clinical factor. This factor was associated with the tumor histology, T status, N status, stage, 10-year PFS, and OS. The moderate and marked concentrations of coccoid forms of H. pylori were more often found in the diffuse type of GC (P = 0.024) and T3-4 (P = 0.04) stage. Interestingly, the high concentration of H. pylori is more frequent in Stage N1 than in N2 (at 90.0% and 53.1%, respectively, P = 0.024).\\nThe moderate and marked concentrations of coccoid forms of H. pylori represented a prognostic factor associated with the decrease of 10-year RFS and OS from 55.6% to 26.3% (P = 0.02 and P = 0.07, respectively).\\nIt should be noted that the results of this study do not allow us to unambiguously judge the effect of H. pylori on GC progression. A decrease in OS and DFS in patients with moderate and marked concentrations of H. pylori coccoid forms in the GM may be due to the fact that these patients had more advanced stages and more aggressive forms of GC. Meanwhile, there are more and more studies showing that H. pylori infection can promote GC progression by activating the NF-κB signaling pathway and induction of interleukin-8 secretion[70], the activation of epithelial-mesenchymal transformation[71-74] and angiogenesis[75,76], as well as increasing the invasive properties of tumor cells[77]. It can be assumed that the administration of AT before surgery contributes to the reduction of the inflammatory process activity and normalization of the adhesive properties of tumour cells, which in turn decreases metastasis risk and improves the long-term results of the treatment of GC. The data literature on the improvement of the long-term results of malignant tumours treatment when using antibacterial drugs testify in favour of this hypothesis[78-80].\",\n \"The data obtained indicates that H. pylori may be associated not only with induction but also with the progression of GC. It can be assumed that the prevalence of coccoid forms of bacteria and their intracellular persistence can affect the mechanisms of tumor progression. Further appropriate studies regarding the role of H. pylori in the progression of GC are obviously advisable.\"\n]","isConversational":false},"all_sections_sec_types":{"kind":"list like","value":[null,null,"methods",null,null,null,null,null,null,null,null,null,null,null],"string":"[\n null,\n null,\n \"methods\",\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null,\n null\n]","isConversational":false},"keywords":{"kind":"list like","value":["Gastric cancer","\nHelicobacter pylori\n","Coccoid and spiral forms of bacteria","Rapid urease test","Relapse free survival","Overall survival"],"string":"[\n \"Gastric cancer\",\n \"\\nHelicobacter pylori\\n\",\n \"Coccoid and spiral forms of bacteria\",\n \"Rapid urease test\",\n \"Relapse free survival\",\n \"Overall survival\"\n]","isConversational":false},"whole_article_text":{"kind":"string","value":"INTRODUCTION:\nGastric cancer (GC) continues to be one of the most common malignant diseases in the world[1,2]. Despite a decreasing trend in the incidence of GC in most countries of the world, the treatment results of this pathology cannot be considered satisfactory. In the structure of mortality from malignant neoplasms, this pathology firmly occupies 2nd place in most developed countries of the world, and the 5-year survival rate of radically operated patients does not exceed 15%-30%[3,4]. \nIt is important to note that it is impossible to improve the long-term results of malignant neoplasms treatment without knowledge of the mechanisms associated with their progression[3]. Clinical studies in recent years indicate that inflammatory infiltration of the tumor stroma and surrounding tissues can have an important prognostic value and affect the long-term results of malignant neoplasm treatment[5-9]. A number of studies have shown that inflammatory infiltration of the tumor stroma is associated with the body’s adequate immune response to the tumor, and may be a favorable prognosis factor in various malignant neoplasms[10,11], including GC[12,13]. At the same time, the data obtained by other researchers indicates that pronounced inflammatory infiltration of the tumor stroma, especially T-reg lymphocytes and macrophages, may be a factor contributing to the progression of malignant neoplasms[14,15]. There was a decrease in the overall survival (OS) and relapse-free survival (RFS) of GC patients with a high content of Foxp3 + T-reg in the tumor stroma and regional metastases[16-18] and macrophages[16,19]. It is believed that inflammatory infiltration of the tumor stroma can contribute to tumor progression by activating the mechanisms of angiogenesis, expression of E- and L-selectins, formation of the products of lipid peroxidation and free radicals, destruction of connective tissue matrix and basement membranes of epithelia by proteolytic enzymes, and activation of epithelial-mesenchymal transformation[20-23].\nWhen studying the role of inflammation in the progression of GC, it is impossible to ignore the problem of Helicobacter pylori (H. pylori) infection. H. pylori is a gram-negative, spiral-shaped bacterium, the habitat of which is the gastric mucosa (GM) and duodenum. H. pylori differs from other bacteria in a set of properties that make it possible to colonize the GM and persist for a long time under conditions that are unfavorable for other microorganisms[24,25]. These include: (1) The ability to produce a special enzyme-urease; (2) Synthesis of lytic enzymes that cause the depolymerization and dissolution of gastric mucus, consisting mainly of mucin; (3) The mobility of the bacterium, which is ensured by the presence of 5-6 flagella; (4) The high adhesiveness of bacteria to GM epithelial cells of the GM and elements of connective tissue due to the interaction of bacterial ligands with the corresponding cells receptors; (5) Production of various exotoxins (VacA, CagA, and others); (6) Instability of the H. pylori genome; (7) The presence of vegetative and coccoid forms of bacteria; and (8) possibility of intracellular persistence and translocation outside the GM [26-29].\nIt should be noted that despite the huge number of studies devoted to H. pylori, it is still not clear whether H. pylori is involved only in the initiation of the tumor process in the stomach, or whether it can affect the mechanisms of tumor progression. The relationship between the severity of H. pylori infection and the clinical and morphological characteristics of GC and long-term results of this pathology treatment remains poorly studied, and in this connection, the question of the expediency of anti-Helicobacter therapy in patients with invasive GC remains open.\nObjective To assess the features of H. pylori infection in patients with Stage I-IIIB GC and their correlation with the clinical and morphological characteristics of the disease, the presence of antibiotic therapy (AT) before surgery, and long-term treatment results.\nTo assess the features of H. pylori infection in patients with Stage I-IIIB GC and their correlation with the clinical and morphological characteristics of the disease, the presence of antibiotic therapy (AT) before surgery, and long-term treatment results.\n\nObjective:\nTo assess the features of H. pylori infection in patients with Stage I-IIIB GC and their correlation with the clinical and morphological characteristics of the disease, the presence of antibiotic therapy (AT) before surgery, and long-term treatment results.\n\nMATERIALS AND METHODS:\nThe patients One hundred and nine patients with GC who had undergone radical surgery (R0) between May 2007 and March 2010 at the Orenburg Regional Clinical Oncology Center, were included in this prospective cohort pilot study. Study inclusion criteria were: Histologically proven invasive GC; no evidence of distant metastases; radical surgery (R0); no prior gastric surgery; no previous chemotherapy or radiotherapy. The study did not include patients with decompensation of cardiovascular and renal diseases, exacerbation of chronic inflammatory processes, severe allergic processes, or who received glucocorticoids, antihistamines, and non-steroidal anti-inflammatory drugs. The study was performed in accordance with the Helsinki Declaration and internationally recognized guidelines, and the privacy of patients was protected by decoding the data according to the privacy regulations of the Orenburg Regional Clinical Oncologic Center (Russia, Orenburg). All patients provided written informed consent. The protocol was approved by the Institutional Review Board of the Orenburg State Medical University (Russia, Orenburg).\nClinical and pathological data including age, tumor localization, stage, type of surgery, histology, the presence of AT before surgery, postoperative therapy, and long-term results of treatment were retrieved from the routine reports for analyses. The distribution of patients according to the clinical and pathological characteristics of GC is presented in Table 1.\nThe distribution of patients according to the clinical and pathological characteristics of gastric cancer\nGC: Gastric cancer.\nWhen interviewing patients, it was found that 45 patients received AT before admission to the clinic due to a preliminary wrong diagnosis of gastric ulcer or chronic gastritis. The following combinations of antibacterial drugs were most commonly ordered: Amoxicillin + clarithromycin (34 patients), amoxicillin + clarithromycin + metronidazole (6 patients), amoxicillin + metronidazole (3 patients), other drugs (2 patients). Information about the antibacterial drugs, the timing and duration of their intake was entered into the primary patient documentation and then taken into account in the analysis. We considered only those patients who underwent AT in the period from 1 to 1.5 mo before the operation, lasting at least seven days, and using two or more antibacterial drugs.\nThe long-term results of treatment were assessed for the period from May 12, 2007 to April 12, 2021. The median follow-up period was 86.2 mo. As of April 12, 2021, 26 (24.5%) patients were alive, 54 (50.9%) had died from the progression of GC, 20 (18.9%) had died from causes other than GC, and six (5.6%) left the region at different follow-up periods. Malignant tumors of other localizations were diagnosed in 8 patients at different times after the operation: Non-Hodgkin's lymphomas-in three, prostate cancer-in one, lung cancer-in two, laryngeal cancer-in one, and breast cancer-in one patient. With the exception of one patient with non-Hodgkin's lymphoma, the other patients died from the progression of these diseases. The causes of death were not associated with malignant tumors for the other patients. During the period 2020-2021, seven patients contracted coronavirus disease-19, one of whom died from the disease, but the rest are alive.\nOne hundred and nine patients with GC who had undergone radical surgery (R0) between May 2007 and March 2010 at the Orenburg Regional Clinical Oncology Center, were included in this prospective cohort pilot study. Study inclusion criteria were: Histologically proven invasive GC; no evidence of distant metastases; radical surgery (R0); no prior gastric surgery; no previous chemotherapy or radiotherapy. The study did not include patients with decompensation of cardiovascular and renal diseases, exacerbation of chronic inflammatory processes, severe allergic processes, or who received glucocorticoids, antihistamines, and non-steroidal anti-inflammatory drugs. The study was performed in accordance with the Helsinki Declaration and internationally recognized guidelines, and the privacy of patients was protected by decoding the data according to the privacy regulations of the Orenburg Regional Clinical Oncologic Center (Russia, Orenburg). All patients provided written informed consent. The protocol was approved by the Institutional Review Board of the Orenburg State Medical University (Russia, Orenburg).\nClinical and pathological data including age, tumor localization, stage, type of surgery, histology, the presence of AT before surgery, postoperative therapy, and long-term results of treatment were retrieved from the routine reports for analyses. The distribution of patients according to the clinical and pathological characteristics of GC is presented in Table 1.\nThe distribution of patients according to the clinical and pathological characteristics of gastric cancer\nGC: Gastric cancer.\nWhen interviewing patients, it was found that 45 patients received AT before admission to the clinic due to a preliminary wrong diagnosis of gastric ulcer or chronic gastritis. The following combinations of antibacterial drugs were most commonly ordered: Amoxicillin + clarithromycin (34 patients), amoxicillin + clarithromycin + metronidazole (6 patients), amoxicillin + metronidazole (3 patients), other drugs (2 patients). Information about the antibacterial drugs, the timing and duration of their intake was entered into the primary patient documentation and then taken into account in the analysis. We considered only those patients who underwent AT in the period from 1 to 1.5 mo before the operation, lasting at least seven days, and using two or more antibacterial drugs.\nThe long-term results of treatment were assessed for the period from May 12, 2007 to April 12, 2021. The median follow-up period was 86.2 mo. As of April 12, 2021, 26 (24.5%) patients were alive, 54 (50.9%) had died from the progression of GC, 20 (18.9%) had died from causes other than GC, and six (5.6%) left the region at different follow-up periods. Malignant tumors of other localizations were diagnosed in 8 patients at different times after the operation: Non-Hodgkin's lymphomas-in three, prostate cancer-in one, lung cancer-in two, laryngeal cancer-in one, and breast cancer-in one patient. With the exception of one patient with non-Hodgkin's lymphoma, the other patients died from the progression of these diseases. The causes of death were not associated with malignant tumors for the other patients. During the period 2020-2021, seven patients contracted coronavirus disease-19, one of whom died from the disease, but the rest are alive.\nDetection H. pylori infection \nH. pylori in the GM and tumor was determined by rapid urease test (RUT) and by immunohistochemically (IHC) using the antibody to H. pylori.\n\nH. pylori in the GM and tumor was determined by rapid urease test (RUT) and by immunohistochemically (IHC) using the antibody to H. pylori.\nRUT After removal of the stomach (within 30 min) a greater curvature of the organ was opened and biopsy samples were taken from the tumor and the adjacent macroscopically non-tumorous GM at a distance of 3-5 cm from the tumor margin. The samples were placed on test strips (HELPIL–test, “АМА”, Russia) for three minutes. The presence and the severity of the infection were evaluated by the color change of the indicator from yellow to blue. \nAccording to the intensity and the time of the appearance of a blue color, we distinguished three degrees of infection: 3+: Marked (+++)-bright staining in the first minute of the study; 2+: Moderate (++)–for an average intensity of staining for 2 min and 1+ mild (+)-weak staining for three minutes. If the color of the indicator did not change, or became dirty gray, and if after repeated research the same result was received, the test was evaluated as negative. The same samples were later used for histological analysis and IGH. \nAfter removal of the stomach (within 30 min) a greater curvature of the organ was opened and biopsy samples were taken from the tumor and the adjacent macroscopically non-tumorous GM at a distance of 3-5 cm from the tumor margin. The samples were placed on test strips (HELPIL–test, “АМА”, Russia) for three minutes. The presence and the severity of the infection were evaluated by the color change of the indicator from yellow to blue. \nAccording to the intensity and the time of the appearance of a blue color, we distinguished three degrees of infection: 3+: Marked (+++)-bright staining in the first minute of the study; 2+: Moderate (++)–for an average intensity of staining for 2 min and 1+ mild (+)-weak staining for three minutes. If the color of the indicator did not change, or became dirty gray, and if after repeated research the same result was received, the test was evaluated as negative. The same samples were later used for histological analysis and IGH. \nImmunohistochemistry The presence and features of H. pylori infection were studied in samples of the GM adjacent to the tumor, in the tumor tissue, in the omentum, and regional lymph nodes of 46 patients, by immunohistochemistry.\nThe sections for IGH were dewaxed and rehydrated by sequential immersion in xylene and graded ethanol and water. For antigen retrieval, the sections boiling for 10 min in citrate buffer (pH 6) and endogenous peroxidase activity was blocked with 30 mL/L hydrogen peroxide solution. Slides were incubated at room temperature with the anti-H. pylori (RB-9070, Thermo Fisher Scientific, the immunogen is purified H. pylori) rabbit polyclonal antibodies in diluted at 1:1000 for 30 min. \nThe visualization system included DAB (UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen) and hematoxylin counterstaining. For negative control sections, the primary antibody was replaced with phosphate-buffered saline and processed in the same manner.\nThe concentration of H. pylori in the GM detected by IGH was graded as 1+ for mild, 2+ for moderate, and 3+ for marked according to the Sydney system[30]. The presence of point inclusions giving a positive reaction with antibodies to H. pylori in the cytoplasm of epithelial cells of deep gastric glands and of the lymphoid cells of the lamina propria of GM as well as in omentum and lymph node, were taken into account. \nAll sections were carefully and completely scanned by two of the authors (MS and OT) without knowledge of the clinical and pathological data. \nThe data obtained was compared with clinical features of GC: Stage, localization, histology, the presence of AT before surgery, and 10-year OS and RFS.\nThe presence and features of H. pylori infection were studied in samples of the GM adjacent to the tumor, in the tumor tissue, in the omentum, and regional lymph nodes of 46 patients, by immunohistochemistry.\nThe sections for IGH were dewaxed and rehydrated by sequential immersion in xylene and graded ethanol and water. For antigen retrieval, the sections boiling for 10 min in citrate buffer (pH 6) and endogenous peroxidase activity was blocked with 30 mL/L hydrogen peroxide solution. Slides were incubated at room temperature with the anti-H. pylori (RB-9070, Thermo Fisher Scientific, the immunogen is purified H. pylori) rabbit polyclonal antibodies in diluted at 1:1000 for 30 min. \nThe visualization system included DAB (UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen) and hematoxylin counterstaining. For negative control sections, the primary antibody was replaced with phosphate-buffered saline and processed in the same manner.\nThe concentration of H. pylori in the GM detected by IGH was graded as 1+ for mild, 2+ for moderate, and 3+ for marked according to the Sydney system[30]. The presence of point inclusions giving a positive reaction with antibodies to H. pylori in the cytoplasm of epithelial cells of deep gastric glands and of the lymphoid cells of the lamina propria of GM as well as in omentum and lymph node, were taken into account. \nAll sections were carefully and completely scanned by two of the authors (MS and OT) without knowledge of the clinical and pathological data. \nThe data obtained was compared with clinical features of GC: Stage, localization, histology, the presence of AT before surgery, and 10-year OS and RFS.\nStatistics Statistical analysis was performed using the Statistica 10.0 software. The correlations between different data were evaluated using the nonparametric Spearman's rank correlation or gamma correlation. Chi-square tests were carried out to analyze the difference of distribution among the categorized data. Mann–Whitney U nonparametric test was used to compare the value of the quantitative data. The survival was analyzed using the Kaplan-Meier method. The log-rank test was used to compare survival curves between subgroups of patients. A value of P < 0.05 was considered statistically significant.\nStatistical analysis was performed using the Statistica 10.0 software. The correlations between different data were evaluated using the nonparametric Spearman's rank correlation or gamma correlation. Chi-square tests were carried out to analyze the difference of distribution among the categorized data. Mann–Whitney U nonparametric test was used to compare the value of the quantitative data. The survival was analyzed using the Kaplan-Meier method. The log-rank test was used to compare survival curves between subgroups of patients. A value of P < 0.05 was considered statistically significant.\n\nThe patients:\nOne hundred and nine patients with GC who had undergone radical surgery (R0) between May 2007 and March 2010 at the Orenburg Regional Clinical Oncology Center, were included in this prospective cohort pilot study. Study inclusion criteria were: Histologically proven invasive GC; no evidence of distant metastases; radical surgery (R0); no prior gastric surgery; no previous chemotherapy or radiotherapy. The study did not include patients with decompensation of cardiovascular and renal diseases, exacerbation of chronic inflammatory processes, severe allergic processes, or who received glucocorticoids, antihistamines, and non-steroidal anti-inflammatory drugs. The study was performed in accordance with the Helsinki Declaration and internationally recognized guidelines, and the privacy of patients was protected by decoding the data according to the privacy regulations of the Orenburg Regional Clinical Oncologic Center (Russia, Orenburg). All patients provided written informed consent. The protocol was approved by the Institutional Review Board of the Orenburg State Medical University (Russia, Orenburg).\nClinical and pathological data including age, tumor localization, stage, type of surgery, histology, the presence of AT before surgery, postoperative therapy, and long-term results of treatment were retrieved from the routine reports for analyses. The distribution of patients according to the clinical and pathological characteristics of GC is presented in Table 1.\nThe distribution of patients according to the clinical and pathological characteristics of gastric cancer\nGC: Gastric cancer.\nWhen interviewing patients, it was found that 45 patients received AT before admission to the clinic due to a preliminary wrong diagnosis of gastric ulcer or chronic gastritis. The following combinations of antibacterial drugs were most commonly ordered: Amoxicillin + clarithromycin (34 patients), amoxicillin + clarithromycin + metronidazole (6 patients), amoxicillin + metronidazole (3 patients), other drugs (2 patients). Information about the antibacterial drugs, the timing and duration of their intake was entered into the primary patient documentation and then taken into account in the analysis. We considered only those patients who underwent AT in the period from 1 to 1.5 mo before the operation, lasting at least seven days, and using two or more antibacterial drugs.\nThe long-term results of treatment were assessed for the period from May 12, 2007 to April 12, 2021. The median follow-up period was 86.2 mo. As of April 12, 2021, 26 (24.5%) patients were alive, 54 (50.9%) had died from the progression of GC, 20 (18.9%) had died from causes other than GC, and six (5.6%) left the region at different follow-up periods. Malignant tumors of other localizations were diagnosed in 8 patients at different times after the operation: Non-Hodgkin's lymphomas-in three, prostate cancer-in one, lung cancer-in two, laryngeal cancer-in one, and breast cancer-in one patient. With the exception of one patient with non-Hodgkin's lymphoma, the other patients died from the progression of these diseases. The causes of death were not associated with malignant tumors for the other patients. During the period 2020-2021, seven patients contracted coronavirus disease-19, one of whom died from the disease, but the rest are alive.\n\nDetection H. pylori infection:\n\nH. pylori in the GM and tumor was determined by rapid urease test (RUT) and by immunohistochemically (IHC) using the antibody to H. pylori.\n\nRUT:\nAfter removal of the stomach (within 30 min) a greater curvature of the organ was opened and biopsy samples were taken from the tumor and the adjacent macroscopically non-tumorous GM at a distance of 3-5 cm from the tumor margin. The samples were placed on test strips (HELPIL–test, “АМА”, Russia) for three minutes. The presence and the severity of the infection were evaluated by the color change of the indicator from yellow to blue. \nAccording to the intensity and the time of the appearance of a blue color, we distinguished three degrees of infection: 3+: Marked (+++)-bright staining in the first minute of the study; 2+: Moderate (++)–for an average intensity of staining for 2 min and 1+ mild (+)-weak staining for three minutes. If the color of the indicator did not change, or became dirty gray, and if after repeated research the same result was received, the test was evaluated as negative. The same samples were later used for histological analysis and IGH. \n\nImmunohistochemistry :\nThe presence and features of H. pylori infection were studied in samples of the GM adjacent to the tumor, in the tumor tissue, in the omentum, and regional lymph nodes of 46 patients, by immunohistochemistry.\nThe sections for IGH were dewaxed and rehydrated by sequential immersion in xylene and graded ethanol and water. For antigen retrieval, the sections boiling for 10 min in citrate buffer (pH 6) and endogenous peroxidase activity was blocked with 30 mL/L hydrogen peroxide solution. Slides were incubated at room temperature with the anti-H. pylori (RB-9070, Thermo Fisher Scientific, the immunogen is purified H. pylori) rabbit polyclonal antibodies in diluted at 1:1000 for 30 min. \nThe visualization system included DAB (UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen) and hematoxylin counterstaining. For negative control sections, the primary antibody was replaced with phosphate-buffered saline and processed in the same manner.\nThe concentration of H. pylori in the GM detected by IGH was graded as 1+ for mild, 2+ for moderate, and 3+ for marked according to the Sydney system[30]. The presence of point inclusions giving a positive reaction with antibodies to H. pylori in the cytoplasm of epithelial cells of deep gastric glands and of the lymphoid cells of the lamina propria of GM as well as in omentum and lymph node, were taken into account. \nAll sections were carefully and completely scanned by two of the authors (MS and OT) without knowledge of the clinical and pathological data. \nThe data obtained was compared with clinical features of GC: Stage, localization, histology, the presence of AT before surgery, and 10-year OS and RFS.\n\nStatistics:\nStatistical analysis was performed using the Statistica 10.0 software. The correlations between different data were evaluated using the nonparametric Spearman's rank correlation or gamma correlation. Chi-square tests were carried out to analyze the difference of distribution among the categorized data. Mann–Whitney U nonparametric test was used to compare the value of the quantitative data. The survival was analyzed using the Kaplan-Meier method. The log-rank test was used to compare survival curves between subgroups of patients. A value of P < 0.05 was considered statistically significant.\n\nRESULTS:\nThe features of H. pylori infection in GC Of 93 patients (84.5%) demonstrated positive RUT. According to RUT the concentration of H. pylori in GM was mild (1+) in 38 (34.8%) patients, moderate (2+)-in 37 (33.9%) and marked (3+)–in 8 (16.5%). RUT was negative in 16 patients (14.7%).\nIt was found that urease activity 2+ and 3+ were significantly more frequent in GM than in tumors (in 55.5% and 13.3% of cases, respectively, P = 0.01). In more than half the cases (53.3%), the urease activity in the tumor was lower than in the adjacent GM.\nForty-six samples of GM were stained by IHC. The coccoid forms of H. pylori were found to prevail in GM adjacent to the tumor (Figure 1A). Coccoid forms of H. pylori only were identified in 31 (63.1%) patients, and coccoid and spiral in 12 (30.4%) patients (Figure 1B). No signs of infection were found in 3 (6.5%) patients. The concentration of coccoid and spiral forms of H. pylori in GM according to IGH was 1+ in 24 (52.2%) and 1 (19.6%) patients, 2+-in 11 (23.9%) and 4 (8.7%), and 3+-in 8 (17.4%) and 1 (2.2%) patients. A positive correlation between the concentration of coccoid and spiral forms of H. pylori (gamma = 0.642, P < 0.0001) was noted.\n\nThe features of Helicobacter pylori localization in gastric mucosa in patients with gastric cancer. A: Coccoid forms of Helicobacter pylori (H. pylori) in the gastric pit. The some bacteria within the cytoplasm of epithelium cells (arrows); B: The spiral (black arrows) and coccoid (orange arrows) forms of H. pylori on the surface of superficial-foveolar gastric epithelium; C: The bacteria in the surface mucous gel layer of stomach (arrows); D: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of epithelial cells of deep gastric glands (arrows); E: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of the immune cells of the lamina propria of gastric mucosa (arrows); F: The point inclusions giving a positive reaction with antibodies to H. pylori within the cytoplasm of intraepithelial lymphocytes (arrows). Immunoperoxidase staining with anti-H. pylori antibody, immersion. Bars: A: 20 μm; B: 10 μm; C: 20 μm; D-F: 10 μm. \nThe localization of H. pylori in GM was observed not only in the surface mucous gel layer of the stomach (Figure 1C) but also within the cytoplasm of the gastric superficial-foveolar epithelium (Figure 1D). The point inclusions giving a positive reaction with antibodies to H. pylori were also revealed in the cytoplasm of the epithelial cells of deep gastric glands (in 41.3% samples) and of the immune cells (Figure 1E) of the lamina propria of GM (in 43.5% samples), as well as of the intraepithelial lymphocytes (Figure 1F). The close relationship between the presence of point inclusions in immune cells and epithelial cells (gamma = 0.642, P <